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Combinatorial synthesis of β-peptides with microwave irradiation: Toward the discovery and development of protein -protein interaction inhibitors

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Combinatorial Synthesis of P-Peptides
with Microwave Irradiation: Toward the
Discovery and Development of
Protein-Protein Interaction Inhibitors
by
Justin K. Murray
A dissertation submitted in partial fulfillment o f the requirements for the degree o f
Doctor of Philosophy
(Chemistry)
at the
University of Wisconsin - Madison
2006
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
UMI Number: 3234764
Copyright 2006 by
Murray, Justin K.
All rights reserved.
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© Copyright by Justin K. Murray 2006
All Rights Reserved
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
A dissertation entitled
Combinatorial Synthesis of p-Peptides
with Microwave Irradiation:
Toward the Discovery and Development of
Protein-Protein Interaction Inhibitors
submitted to the Graduate School of the
University of Wisconsin-Madison
in partial fulfillment of the requirements for the
Degree of Doctor of Philosophy
by
JUSTIN K. MURRAY
Date of Final Oral Examination:
Month & Year Degree to be awarded:
June 5, 2006
December
May
August 2006
Approval Signatures/of Dissertation Committee
Signature, Dean of Graduate School
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1
To Becky
fo r her eternal love, devotion, and long-suffering.
I love you forever.
To Alyssa, Keith, and Camille
fo r showing me the excitement o f discovery and making me laugh.
I am proud to be your father.
To Mom and Dad
fo r encouraging me to always do my best.
Another day, Another “A. ”
To Gary Burgoyne
fo r reminding me why I go to work each day.
‘Til we meet again.
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Acknowledgements
I am especially grateful to my advisor, Sam Gellman, for giving me the freedom to pursue the
avenues of research that I found most interesting and for challenging me to become a better
scientist. I am also indebted to Professor Daniel H. Rich for his friendship and informal
mentoring. I thank Professor Helen E. Blackwell for her help and advice on combinatorial and
microwave-assisted chemistry. I am grateful to Dr. Mate Erdelyi and Jonathan Collins for useful
discussions on microwave-assisted peptide synthesis. I am grateful for my many wonderful
scientific friends from the Gellman group, past and present, especially Dr. Tim Peelen, Dr.
Joseph Langenhan, Dr. Matt Woll, Jack Sadowsky, Josh Price, Soo Hyuk Choi, and Emily
English. Finally, I could not have done this work without so many outstanding collaborators.
LC-MS/MS Peptide Sequencing
Mark Scalf
Professor Lloyd Smith
Undergraduate Researchers
Wesley Freund
Kevin Beier
Corbin Occhino
P53/MDM2
Dr. Jiandong Chen
Bilal Farooqi
Chemistry Mass Spectrometry Facility
Dr. Martha Vestling
Bcl-xr/Bak
Jack Sadowsky
Chemistry NMR Facility
Dr. Charlie Fry
TGFB/TBRII
Professor F. Michael Hoffmann
Dr. Andrew Hinck
Editorial Assistance
Emily English
Will Pomerantz
Dr. Seth Home
Josh Price
Neurokinin-B
Dr. Saumen Pal
Professor Emery Bresnick
Genentech
Dr. Nick Skelton
Dr. Andrea Cochran
Funding
NIH Biotechnology Training Program
Wisconsin Alumni Research Foundation
CEM Academic Grant
McElvain and Vilas Travel Award
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Ill
Table of Contents
1
Chapter 1: Targeting Protein-Protein Interactions: Lessons from p53/MDM2
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1.0 B rief Statement of Research Purpose
1.1 Protein-Protein Interactions
1.2 p53/MDM2: A Model Study in Protein-Protein Interaction Inhibition
1.2.1 p53 Biology
1.2.2 Early Efforts Toward Target Validation: Antibodies and Oligonucleotides
1.2.3 Crystal Structure o f the p53/MDM2 Complex
1.2.4 In Vitro Assays for the p53/MDM2 Interaction:ELISA, FP, and SPR
1.2.5 Drug Discovery Paradigm
1.3 a-Peptide Antagonists o f the p53/MDM2 Interaction
1.4 Natural Product Antagonists o f the p53/MDM2 Interaction
1.4.1 The Chalcones
1.4.2 Chlorofusin
1.4.3 Hexylitaconic Acid
1.5 Small Molecule Inhibitors o f p53/MDM2
1.5.1 The First: syc-7
1.5.2 Sulfonamides
1.5.3 Nutlins (cA-Imidazolines)
1.5.4 Benzodiazepinediones
1.5.5 Spiro-oxindoles
1.5.6 Summary o f Small Molecule Inhibitors
1.6 Oligomeric Scaffolds for a-H elix Mimicry and p53/MDM2 Inhibition
1.6.1 Retroinverso Peptides
1.6.2 Peptoids
1.6.2.1 Structure-Based Design
1.6.2.2 Combinatorial Approach
1.6.3 Terphenyls
1.6.4 P-Hairpin Protein Epitope Mimetics
1.6.5 /?-01igobenzamides
1.6.6 P-Peptides
1.6.6.1 12-Helical P-Peptides
1.6.6.2 14-Helical P-Peptides
1.6.7 Miniproteins
1.6.8 Summary o f Proteomimetic Inhibitors
1.7 Other Strategies for Activation o f the p53 Pathway
1.7.1 Reactivation of Mutant p53
1.7.2 Small Molecule Ligand for p53
1.8 Structural Insights
1.9 Conclusions
1.10 References
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Chapter 2: Application of Microwave Irradiation to the Synthesis of 14-Helical (3Peptides
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2.0 Brief Summary o f Chapter
2.1 Background
2.1.1 14-Helical p-Peptides
2.1.2 P-Peptide Synthesis
2.1.3 Microwave-Assisted Peptide Synthesis
2.2 Synthetic Optimization of P-Peptide Solid-Phase Synthesis with Microwave
Irradiation
2.2.1 Hexa-P-Peptide Test Case
2.2.2 Experimental Set-Up for Microwave-Assisted P-Peptide Synthesis
2.2.3 Microwave-Assisted Solid-Phase P-Peptide Synthesis
2.2.4 Further Attempts at Synthetic Optimization
2.2.5 Chaotropic Salts
2.2.6 Comparison with Conventional Heating
'j
2.2.7 Epimerization o f P -Amino Acids
2.2.8 Further Investigation of the Effects o f the NMP/LiCl Condition
2.3 Conclusions
2.4 Experimental Procedures
2.4.1 General Procedures
2.4.2 First Residue Loading
2.4.3 Microwave P-Peptide Synthesis
2.4.4 Manual P-Peptide Synthesis
2.4.5 Oil Bath p-Peptide Synthesis
2.4.6. P-Peptide Cleavage, Work-Up and HPLC
2.4.7 P-Peptide Characterization Data
2.4.8 Yield Calculation
2.4.8.1 p-Peptide 2-1
2.4.8.2 p-peptide 2-4
2.4.9 Additional Data
2.4.10 Step-by-Step Microwave-Assisted P-Peptide Synthesis Protocol for CEM
Discover
2.5 References
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Standard SPPS
Conditions
Oil Bath,
LiCI in NMP
Microwave Irradiation,
LiCI in NMP
21%
53%
88%
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Chapter 3: Efficient Synthesis of a Split-and-Mix p-Peptide Combinatorial Library
with Microwave Irradiation
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3.0 Brief Summary o f the Chapter
3.1 Background
3.2 Combinatorial P-Peptide Synthesis
3.2.1 Split-and-Mix Synthesis
3.2.2 Synthetic Optimization o f 14-Helical P-Peptides with Microwave
Irradiation
3.2.3 Polystyrene Macrobeads for Split-and-Mix Synthesis
3.2.4 P-Peptide Synthesis on PS Macrobeads
3.2.5 Microwave-Assisted P-Peptide Synthesis on PS Macrobeads
3.2.6 Comparison o f Microwave Irradiation to Conventional Heating
3.2.7 Discussion o f Effects o f Microwave Irradiation on P-Peptide Synthesis
3.3 Synthesis and Characterization o f a P-Peptide Combinatorial Library
3.3.1 P-Peptide Library Design
3.3.2 p-Peptide Library Synthesis on PS Macrobeads with Microwave Irradiation
3.3.3 Analytical Characterization o f P-Peptide Library
3.3.4 Library Screening for Inhibition o f the p53-MDM2 Interaction
3.3.5 Discussion of P-Peptide Inhibitors o f the p53/MDM2 Interaction
3.3.5.1 14-Helical Scaffold
3.3.5.2 Charge-Charge Interactions
3.3.5.3 Comparison to Positive Controls
3.4 Conclusions
3.5 Experimental Methods
3.5.1 General Procedures
3.5.2 First Residue Loading
3.5.3 p-Peptide Synthesis on PS Macrobeads
3.5.3.1 Microwave p-Peptide Synthesis on PS Macrobeads Using
Ramp/Cool Cycles
3.5.3.2 Manual P-Peptide Synthesis on PS Macrobeads
3.5.3.3 Oil Bath P-Peptide Synthesis on PS Macrobeads
3.5.4 P-Peptide Cleavage and HPLC Analysis
3.5.5 p-Peptide Characterization Data
3.5.6 Calculation o f p-Peptide Yield from a Single Macrobead
3.5.7 P-Peptide Library Synthesis on PS Macrobeads with Microwave Irradiation
3.5.8 Analytical Characterization o f Representative Library Members
3.5.9 HPLC ESI-MS/MS Analysis o f P-Peptides from Library
3.5.10 p53/MDM2 ELISA Procedure
3.5.11 Synthesis o f Peptides for ELISA Using Microwave Irradiation
3.5.12 Additional ELISA Data
3.6 References
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VI
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Chapter 4: Microwave-Assisted Parallel Synthesis of a 14-Helical P-Peptide
Library
4.0 Brief Summary of the Chapter
4.1 Background
4.1.1 Microwave-Assisted Combinatorial Chemistry
4.1.1.1 Automated Sequential Synthesis
4.1.1.2 Reaction Vessel for Microwave-Assisted Parallel Synthesis
4.1.2 P-Peptide Synthesis
4.1.3 Microwave-Assisted Synthesis o f a Split-and-Mix P-Peptide Library
4.2 Microwave-Assisted Parallel Synthesis o f a P-Peptide Library
4.2.1 Synthetic Optimization in Multimode Microwave Reactor
4.2.2 Microwave-Assisted Synthesis in 96-Well Filter Plate
4.2.3 Design o f 96-Membered P-Peptide Combinatorial Library
4.2.4 Parallel Synthesis o f a P-Peptide Combinatorial Library with Microwave
Irradiation
4.2.5 Homogeneity of Heating during Library Synthesis
4.2.6 Characterization o f Side Products from Library Synthesis
4.3 Conclusions
4.4 Experimental Methods
4.4.1 General Procedures
4.4.2 First Residue Loading
4.4.3 Multimode Microwave P-Peptide Synthesis
4.4.4 P-Peptide Cleavage, Work-Up and HPLC
4.4.5 P-Peptide Characterization Data
4.4.6 Microwave-Assisted Parallel Synthesis o f p-Peptide 4-1 in a 96-Well Filter
Plate
4.4.7 Microwave-Assisted Parallel p-Peptide Library Synthesis
4.4.8 Analytical Characterization o f P-Peptide Library
4.4.9 Yield Calculation o f p-Peptide 4-1
4.4.10 Step-by-Step Microwave-Assisted P-Peptide Synthesis Protocol
4.4.11 Screening Data for p-Peptide Library
4.5 References
GO 2H
".««L5A L a>z:^ i j . nXa
R
H
H
H
R
H
H
Parallel
A
Multimode Microwave Reactor
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Chapter 5: Optimization o f Chimeric (a/p + a)-Peptide Ligands for Bcl-xL via
Microwave-Assisted Combinatorial Synthesis
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5.0 Brief Summary o f Chapter
5.1 Background
5.1.1 Chimeric (a /p + a)-Peptide Ligands for B c1-xl
5.1.2 Microwave-Assisted Synthetic Methods for Rapid Lead Optimization
5.2 Optimization o f Chimeric (a /p + a)-Peptide Ligands for Bcl-xL
5.2.1 One-Bead-One-Compound Library for Side Chain Optimization
5.2.2 One-Bead-One-Compound Library for Backbone Modification
5.2.3 Parallel Synthesis o f the Combinatorial p-Scan Library
5.2.4 Second Generation Parallel Library
5.2.5 Structure-Based Design
5.2.6 Proteolytic Stability
5.3 Conclusions
5.4 Experimental Methods
5.4.1 General Procedures
5.4.2 Split-and-Mix Library Synthesis on PS Macrobeads with Microwave
Irradiation
5.4.3 Split-and-Mix Synthesis o f the P-Scan Library Using Microwave
Irradiation
5.4.4 Microwave-Assisted Parallel Synthesis o f the P-Scan Library
5.4.5 Synthesis of the Second Generation Parallel Library Using Microwave
Irradiation
5.4.6 Solid-Phase Oligomer Synthesis Using Microwave Irradiation
5.4.7 Oligomer Stability in Serum
5.5 References
Microwave
Irradiation
Split-and-Mix
vs.
Parallel Synthesis
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Chapter 6: Efforts Toward the Discovery of Proteomimetic Inhibitors for the
Transforming Growth Factor p/Type II Receptor Interaction
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6.0 Brief Summary o f the Chapter
6.1 Background
6.1.1 TGFp in Breast Cancer
6.1.2 Interactions and Signaling o f TGFp
6.1.3 TGFp3/TpRII Assay
6.1.4 Structure o f the TGFP3/TPRII Complex
6.2 Attempted Development ofp-Hairpin a-Peptide Inhibitors
6.2.1 P-Hairpin Library Design
6.2.2 Synthetic Optimization o f p-Hairpin
6.2.3 P-Hairpin Library Screening
6.2.4 Summary of P-Hairpin Designs
6.3 Development o f Foldamer Inhibitors
6.3.1 Structure-Based Design o f 14-Helical P-Peptide Inhibitors
6.3.2 First Generation 14-Helical P-Peptide Library Design
6.3.3 Screening Results for Foldamer Compound Collection
6.3.4 Second Generation 14-Helical P-Peptide Library
6.3.5 Screening Results for Second Generation 14-Helical P-Peptide Library
6.3.6 HPLC Purification o f Hydrophobic/Anionic P-Peptides
6.3.7 Evaluation o f Hits from Second Generation 14-Helical p-Peptide Library
6.3.8 Summary o f 14-Helical P-Peptide Designs
6.4 Conclusions and Future Directions
6.5 Experimental Methods
6.5.1 General Procedures
6.5.2 Multimode Microwave a -a n d P-Peptide Synthesis
6.5.3 Peptide Cleavage, Work-Up and HPLC
6.5.4 Peptide Characterization Data
6.5.5 Microwave-Assisted Parallel P-Hairpin Library Synthesis
6.5.6 Characterization o f P-Hairpin Library
6.5.7 First Generation 14-Helical p-Peptide Library Synthesis and
Characterization
6.5.8 Second Generation 14-Helical P-Peptide Library Synthesis and
Characterization
6.5.9 12-Helical P-Peptide Library Synthesis and Characterization
6.5.10 Experimental Procedure for TGFp3/TpRII HTRF Assay
6.6 References
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Chapter 7: Conclusions and Future Directions
7.1 Conclusions and Future Directions
7.2 References
398
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IX
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Appendix A: Scalable, Asymmetric Synthesis of 14-Helix-Promoting p-Amino Acids
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A.O Brief Summary of the Appendix
A .l Background
A.2 Preparation o f 14-Helix-Promoting P-Amino Acids
A.2.1 Synthesis o f APiC
A.2.2 Synthesis o f ACHC
A.3 Conclusions
A.4 Experimental Methods
A.4.1 General Procedures
A.4.2 Synthesis o f Fmoc-APIC(Boc)-OH
A.4.2.1 EnamineA-2
A.4.2.2 HC1 Salt A-3
A.4.2.3 (7?,/?)-Fmoc-APiC(Boc)-OH (A-4)
A.4.2.4 Chiral HPLC Assay o f A-6 and ent-A-6
A.4.3 Synthesis o f <7?,/?)-Fmoc-ACHC-OH
A.4.3.1 Enamine A-8
A.4.3.2 cis p-Amino ester A-9
A.4.3.3 HC1 Salt A-10
A.4.3.4 (R,7?)-Fmoc-ACHC-OH (A -ll)
A.4.3.5 Chiral HPLC Assay o f A-12 and ent-A-12
A.4.4 Large-Scale Synthesis o f (5,5)-Fmoc-ACHC-OH
A.4.4.1 Enamine ent-A-7
A.4.4.2 cis P-Amino ester ent-A-8
A.4.4.3 HC1 Salt ent-A-9
A.4.4.4 (S, 5)-Fmoc-ACHC-OH (ent-A-10)
A.5 References
FmocHN
o
•C 0 2Et
£ 0 2H
X'
X = NBrvHCI
= CH2
X = NBoc, Fmoc-APiC(Boc)-OH
= CH2) Fmoc-ACHC-OH, 182 g, 17%
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443
Appendix B: Efforts Toward Stabilization of the y-Peptide 14-Helix via Monomer
Cyclic Constraint
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B.O Brief Summary o f the Appendix
B .l Background
B.1.1 Foldamers
B .l.2 y-Peptides
B.2 Preparation and Characterization o f y-Peptide Oligomers
B.2.1 Synthesis of Ring Constrained y-Amino Acids
B.2.2 Synthesis and Characterization o f y-ACPC-Containing Oligomers
B.3 Conclusions
B.4 Experimental Methods
B.4.1 General Procedures
B.4.2 Synthesis o f Boc-y-ACPC-OH
B.4.3 Synthesis of Boc-(y-ACPC)2 -OBn (B-6)
B.4.4 Synthesis of Boc-(y-ACPC)4-OBn (B-7)
B.4.5 Synthesis of Boc-(y-ACPC)6-OBn (B-8)
B.4.6 Synthesis of Fmoc-y-ACPC-OH
B.4.7 Synthesis of Fmoc-y-APC(Boc)-OH
B.4.8 Preparation o f Ac-(y-APC-y-ACPC)3 -NH 2 (B-9)
B.4.9 Synthesis of Fmoc-y-APiC(Boc)-OH
B.4.10 Synthesis o f Boc-y-ACHC-OH
B.4.11 Synthesis o f Boc-(y-ACHC)2 -OH
B.5 References
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Appendix C: Toward a/p-Peptide Agonists of the Neurokinin-B/Receptor
Interaction
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C.O Brief Summary o f the Appendix
C .l Background
C.1.1 The Tachykinins and Their Receptors
C.1.2 Anti-Angiogenic Activity o f Neurokinin-B
C .l.3 Peptide Agonists o f the NK-B/NK Receptor Interactions
C.2 Development o f Proteolytically Stable NK Receptor a/p-Peptide Agonists
C.2.1 p-Scan Approach
C.2.2 Proteolytic Stability o f a/p-Peptide Agonists
C.3 Conclusions and Future Work
C.4 Experimental Methods
C.4.1 General Procedures
C.4.2 Solid-Phase Peptide Synthesis with Microwave Irradiation
C.4.3 Peptide Stability in Serum
C.5 References
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Chapter 1
Targeting Protein-Protein Interactions:
Lessons from p53/MDM2
Figure adapted from Kussie, P. H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau, J.;
Levine, A. J.; Pavletich, N. P. Science 1996, 274, 948. PDB code 1YCR.
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2
1.0 Brief Statement of Research Purpose
The desire to develop a treatment or cure for human illness drives both science
and scientists throughout the world.
The tremendous challenge o f inhibiting protein-
protein interactions in vivo requires new strategies that depart from traditional medicinal
chemistry,1 as learned through investigation o f the p53/MDM2 interaction described in
Chapter 1. One successful strategy has been the creation o f proteomimetic scaffolds
called foldamers, which include |3-pcptides.2 The predictable relationship between 13amino acid sequence and folding has inspired several biological applications o f 13peptides, including the inhibition o f protein-protein interactions.
For many such
applications it would be desirable to prepare and screen large P-peptide libraries.
However, standard solid-phase peptide synthesis (SPPS) protocols are not efficient
enough to support a library approach for some types o f P-peptides. The work described
in this Thesis demonstrates how the development o f microwave-assisted combinatorial
synthesis o f P-peptides has facilitated the discovery and development o f biologically
active foldamers. Chapter 2 evaluates the effects o f microwave irradiation on the solidphase synthesis o f P-peptides, identifying a clear benefit from microwave irradiation for
longer P-peptides and achieving a 10-fold reduction in synthesis time. Using microwaveassisted methodology, we rapidly prepared high-quality P-peptide combinatorial libraries
via both split-and-mix techniques on polystyrene (PS) macrobeads (Chapter 3) and in
parallel using 96-well filter plates (Chapter 4).
Libraries are designed, prepared, and
screened as part o f an effort to identify and optimize foldamer inhibitors o f several
protein-protein interactions, including p53/MDM2 (Chapter 3), Bcl-xi/Bak in (Chapter
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3
5), and transforming growth factor [33/type II receptor in (Chapter 6) and the neurokinin
B/neurokinin receptor peptide/protein interaction (Appendix C). The utility o f [3-amino
acids in medicinal chemistry and “|3-scanning” is discussed (Chapter 5 and Appendix C).
A comparison o f split-and-mix and parallel synthesis is also provided to determine when
and how to apply each technique (Chapter 5).
Large-scale synthesis o f important [3-
amino acids (Appendix A) and stabilization o f potential proteomimetic scaffold
(Appendix B) are described. In summary, this thesis describes initial efforts toward the
discovery and development o f foldamer inhibitors for protein-protein interactions using
structure-guided design as a starting point for the rapid and efficient searching o f
chemical space via microwave-assisted combinatorial synthesis.
1.1 Protein-Protein Interactions
Sequencing o f the human genome has greatly expanded our knowledge o f the
linear sequence o f human proteins.
What we have learned from this remarkable
achievement is how little we actually understand about how the human body works.
Scientists have consequently turned their attention to proteomics, in an attempt to
determine the structure and function o f every protein.
These efforts have shown that
regulation o f protein function is as important as the function itself, adding another
dimension of complexity to an already complicated picture.
Proteins are produced,
degraded, proteolytically processed, and post-translationally modified to varying extents
and at different rates, all in response to environmental stimuli.
Each o f these events
requires that proteins communicate with one another. Signals are continually sent and
received via transient, specific physical contacts between different proteins. We are just
beginning to learn about the roles o f these protein-protein interactions in living systems.
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However, we do know that disruption o f these signaling pathways as a result o f a small
mutational change in one of the protein partners or deregulation o f protein production can
result in a disease state. Chemical intervention, in the fonn o f new therapeutic agents,
could potentially restore the delicate balance and help relieve human suffering.
Unfortunately, it has not been easy to identify suitable inhibitors o f protein-protein
interactions.1,3 Targeting the large, broad, flat, malleable, and polar surface o f a protein
is fundamentally different from filling the small, sequestered, concave, rigid, and
hydrophobic pocket o f an enzymatic target with a small molecule.13 New strategies that
challenge or break the accepted rules o f medicinal chemistry may be required to find a
general approach for the development o f inhibitors o f this new and emerging class of
therapeutic targets.lb
1.2 p53/MDM2: A Model Study in Protein-Protein Interaction Inhibition
The biological importance o f p53 (“p” for protein and “53” for its apparent
molecular weight o f 53 kDa)4 was known long before chemists took an active interest in
this protein.5 The complexity o f the p53’s role within the cell and the question o f
whether it was even a “druggable” target were sufficient deterrents to prevent heavy
involvement by the pharmaceutical industry, until the publication o f the p53/MDM2 co­
crystal structure in 1996.6 Since then, a wealth o f knowledge resulting from the efforts of
both industrial and academic researchers has emerged.7 Just as the development o f HIV
protease inhibitors taught us about peptidomimetics for enzyme inhibition,8 p53/MDM2
has served in many respects as a model system for the inhibition o f protein-protein
interactions. The highly focused nature o f the p53/MDM2 interface means that it cannot
be considered a prototypical protein/protein interaction9 but has made it amenable to the
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5
development o f a variety o f approaches that can be extended to other, more challenging
protein-protein interactions. From p53/MDM2, we have learned much about the nature
o f protein/protein interactions, even if some o f the lessons may not be generally
applicable. The opportunity of inhibiting the p53/MDM2 interaction has inspired novel
strategies that may result in a viable clinical treatment for cancer, but beyond that, we can
hope that application o f these methods to other targets will eventually produce therapies
for a variety of diseases. Discussion o f these methods will illustrate the advantages and
disadvantages o f each approach, highlight important achievements, and identify
remaining challenges.
1.2.1 p53 Biology
The tumor suppressor protein p53 is a transcription factor that controls cellular
response to stress (i.e., DNA damage, hypoxia, etc.) through the induction o f cell cycle
arrest (by activating transcription of the W A Fl/C ipl gene, which leads to expression o f
the cyclin-dependent kinase inhibitor p 2 l)10 or apoptosis.11 The murine double minute 2
protein, or MDM2 (The literature often refers to the analogous human protein, sometimes
called hDM2, as MDM2 also, whose nomenclature has been adopted here.),12
downregulates p53 activity13 through a negative feedback loop14 by binding to the a helical transactivation domain near the N-terminus o f p53, blocking its DNA-binding
activity.133 MDM2 also exports p53 from the nucleus15 and acts as an E3 ubiquitin
protein ligase,16 thereby targeting p53 for proteosomal degradation.17
In response to stress, p53 is phosphorylated on specific serine residues near the
MDM2 binding domain, decreasing its affinity for MDM2 and activating it as a
transcription factor.18 Overproduction o f MDM2 inhibits activation o f the p53 pathway,
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6
leading to uncontrolled cell proliferation. MDM2 amplification is observed in about 7%
o f human tumors and is most commonly found in soft-tissue tumors (20%),
osteosarcomas (16%), and esophageal carcinomas (13% ).19 This amplification makes
tumors less susceptible to natural and chemotherapeutic signals to undergo programmed
cell death, or apoptosis, and generally results in a poor patient prognosis. According to
Vassilev,20 treatment o f cells overproducing MDM2 with an inhibitor o f the p53/MDM2
interaction should result in: 1) the stabilization and accumulation o f p53 resulting from
the blockage o f its export and degradation; 2) activation o f MDM2 production; and 3)
activation of other p53-regulated genes (i.e., the W A Fl/C ipl gene to produce p21) and
the p53 pathway, thereby causing cell cycle arrest in the Gi and G 2 phases and/or
apoptosis. Disruption o f the p53-MDM2 interaction is therefore a therapeutic target for
the treatment o f cancer.
21
Extensive efforts toward this goal have eventually yielded
tightly-binding a-peptides, several a-helix mimetics, and potent bioactive small molecule
antagonists o f the p53-MDM2 interaction. However, cell cycle arrest and/or apoptosis
resulting from treatment with a p53/MDM2 interaction inhibitor should only occur in
cells with wild-type p53 and not in cells with transcriptionally inactive, mutant p53.
Since p53 is mutated in approximately 50% o f human cancers,22 a different strategy for
reactivation o f the p53 pathway in this context is desirable.
1.2.2 Early Efforts Toward Target Validation: Antibodies and Oligonucleotides
Before medicinal chemists became involved in targeting the p53/MDM2
interaction, two important techniques from molecular biology, antibodies and RNA
interference (RNAi), were applied to the system resulting in validation o f the target. In
an early proof o f principal experiment, Blaydes et al.
found that microinjection o f an
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7
antibody for the p53-binding domain of MDM224 caused a marked increase in p53dependent transcription. While this strategy worked in cells, it was not a viable option
for treatment in vivo because MDM2 is an intracellular protein, and scientists have not
yet developed a method to deliver antibodies across cell membranes to the cytoplasm.
Nevertheless, monoclonal antibodies are arguably the most successful class o f proteinprotein interaction inhibitors.25 Antibodies for a number o f extracellular protein targets
are already either FDA-approved drugs or in clinical trials. Many more are in pre-clinical
development. While highly potent and selective antibodies can be raised to many protein
targets,26 the high costs and difficulties associated with manufacturing,27 the necessity of
administration via injection,28 and the potential for immunogenicity remain significant
disadvantages when compared to orally bioavailable small molecules.
When such a
small molecule cannot be identified, then an antibody is a more than acceptable solution,
although antibodies still do not work against intracellular targets. It will be interesting to
see in the years to come how many antibodies are replaced by small molecules or other
competing strategies.
Antisense oligonucleotides have been used to inhibit MDM2 expression, thereby
reducing MDM2 protein levels, diminishing the MDM2 negative feedback inhibition o f
p53, and increasing the levels o f functional p53.29 Knock-out o f the MDM2 gene also
resulted in p53 accumulation, p53-dependent gene expression, and growth inhibition o f
1 ft
colon
-5 i
and prostate cancer cells.
RNA interference via antisense oligonucleotides or
small interfering RNA (siRNA) has great potential for the treatment o f diseases resulting
from aberrant protein signaling.
'X')
By targeting and degrading the mRNA that encodes
the particular protein that is being overproduced, such as MDM2, the amount o f the
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8
protein within the cell is decreased, thereby preventing its association with its partner,
such as p53, and restoring the normal activity.33 Despite numerous modifications o f the
oligonucleotide backbone to improve stability in vivo,34 delivery o f an oligonucleotide
across both the cellular and nuclear membranes has proven to be a significant challenge,
although very recently lipid nanoparticles have been used successfully for this purpose in
non-human primates.35
Much remains to be done, including investigation o f the
threshold effect, or the actual amount by which transcription must be reduced to affect
protein levels and signaling.
“Xf
*
1.2.3 Crystal Structure of the p53/MDM2 Complex
For chemists, intense interest in the p53/MDM2 interaction started in 1996 with
the publication o f a co-crystal structure o f the complex between the MDM2 protein and a
peptide corresponding to p53 residues 17-29 by Pavletich and coworkers.6 The structure
revealed that the N-terminal transactivation domain o f p53 binds in an amphipathic a helical conformation within a hydrophobic cleft on the surface o f MDM2 (Figure 1).
Even more interesting was the positioning o f three hydrophobic residues from p53,
Phel9, Trp23, and Leu26, along one face o f the helix to make direct contacts with the
MDM2 protein. It seemed likely that the bulk o f the binding energy for this proteinprotein interaction was constituted by the van der Waals interactions o f these three
residues with the surface o f MDM2.
Similar observations that the intermolecular
interactions important for protein recognition were focused at a few high energy “hot
spots” within the binding interface rather than as many low energy interactions spread
broadly across the proteins’ surfaces had been made for a number o f different
protein/protein interactions.37 According to Pavletich et al.,6 the leucine pocket is defined
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9
Figure 1. Crystal structure of the p53/MDM2 protein-protein interaction (ref. 6). MDM2 rendered as a
surface using MOLCAD (Sybyl) and colored according to cavity depth, and the p53-peptide pictured as
sticks.
by MDM2 residues TyrlOO, ThrlO l, and Val53. The tryptophan pocket is defined by
Ser92, Val93, Leu54, Gly58, Tyr60, Val93, and Phe91.
The phenylalanine pocket is
composed by Arg65, Tyr67, Glu69, His73, Ile74, Val75, Met62, and Val93.
The
backbone o f the p53 peptide forms 2.5 turns o f a-helix (residues 18-26) with residues at
either end adopting an extended conformation. The C-terminal end o f the helix is less
tightly wound, leading some to classify the last turn o f helix between Trp23 and Leu26 as
a type I P-tum rather than an a-helix.
surface area, about 808
A2 and
690
The p53/MDM2 interface buries 1498
A2
on p53 and MDM2, respectively.
A2 of
The
hydrophobic contacts are augmented by only two intermolecular hydrogen bonds: one
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between the P hel9 backbone amide N-H o f p53 and the carbonyl o f the Gln72 side
chain of MDM2 at the entrance o f the cleft, and another between the p53 Trp23 indole NH and the MDM2 Leu54 backbone carbonyl deep inside the cleft.
The crystal structure revealed that p53/MDM2 is not a prototypical proteinprotein interaction.
Rather than finding a broad, flat interface with a few disperse
hydrophobic patches, MDM2 has a continuous, narrow hydrophobic cleft that is almost
deep enough to be called a “pocket,” and the complex can be reduced to a protein-peptide
interaction. However, since the cleft is long and is located on the surface rather than
within the interior o f the MDM2 protein, at least part of the inherent challenge o f
inhibiting a protein-protein interaction remained.
Medicinal chemists hoped that a
synthetic molecule capable of displaying three hydrophobic groups in an appropriate
orientation to mimic the presentation o f the Phel9, Trp23, and Leu26 side chains by p53
would be capable o f inhibiting the p53/MDM2 interaction.
Indeed, potent small molecule p53/MDM2 antagonists have been developed via
traditional structure-based design and/or combinatorial synthetic techniques. However,
these results were initially very slow in coming;38 the delay presented the opportunity to
develop alternative approaches to protein-protein interaction inhibition.
A second
strategy for the inhibition of the p53/MDM2 interaction was to develop a modular
scaffold that could in theory mimic any a-helical protein structure. Since p53/MDM2
was so amenable to these two approaches, as will be discussed, some o f the other more
creative and effective strategies for the inhibition o f protein/protein interactions, such as
fragment-based design, were never used to target p53/MDM2.
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Fragment-based designs for the development o f protein-protein interaction
inhibitors have been successful against a growing number o f challenging protein
targets.39 By screening very small organic molecules (average molecular weight o f 100
to 300 Da) via “SAR by NMR”40 or “Tethering,”41 several low-affinity ligands for
distinct sites on the targeted protein surface (hot spots) can be identified. Chemically
linking the fragments has generated potent ligands for Bcl-xL,42 interleukin-2, and others
but has not been applied to the p53/MDM2 interaction.lb
1.2.4 In Vitro Assays for the p53/MDM2 Interaction: ELISA, FP, and SPR
Before any protein-protein interaction inhibitor can identified, a suitable assay
must be developed. Several biochemical assays have been used to measure inhibition o f
the p53/MDM2 interaction, including an enzyme-linked immunosorbent assay (ELISA),
fluorescence polarization (FP), and surface plasmon resonance (SPR).
In the ELISA
format (Figure 2), recombinant MDM2 is adsorbed to the inner surface o f the assay
N O P -f 111i n r p s r p n t
Substrate
Fluorescent
Product
vessel (i.e., well o f a microtiter
plate).
Enzyme
Recombinant p53 and the
potential antagonist are added to the
well and allowed to bind to MDM2.
Secondantibody
Unbound protein and inhibitor are
washed away, and an antibody for
First antibody
p53 is added. A second antibody that
binds to the first antibody and is
MDM2
Figure 2. Schematic for p53/MDM2
ELISA. Figure adapted from Dr. H.-S. Lee.
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conjugated to an enzyme such as horseradish peroxidase is added next. A substrate for
the enzyme is added, and reaction converts this substrate to a UV-absorbent product, the
concentration of which is read out colorimetrically. If the potential antagonist inhibits the
interaction o f p53 with MDM2, then p53 remains in solution and is washed away. The
first antibody cannot bind to p53 if it is not present, decreasing the signal in the assay.
The FP assay format is more amenable to high-throughput screening than the
ELISA format because FP is solution-based and requires fewer steps than does ELISA.43
Recombinant MDM2 is mixed with a fluorescently labeled p53 peptide (probe) and the
potential inhibitor. The sample is then irradiated with polarized light. If the labeled
peptide binds to the protein, then it will tumble very slowly, and the fluorescence will be
highly polarized. However, if the inhibitor binds to MDM2 and displaces the probe, then
the fluorescent peptide will tumble very quickly in solution because o f its low molecular
weight, and the emission will be significantly depolarized.
A larger decrease in
fluorescence polarization indicates the presence o f a stronger inhibitor. One limitation o f
the FP format is the requirement for a low-molecular weight but tightly binding probe.
SPR can also measure the affinity o f ligand binding to a protein target in a
competition format.44 Biotinylated p53 peptide is immobilized on a streptavidin-coated
biosensor chip. A solution of MDM2 protein and the potential inhibitor is flowed over
the surface. With increasing concentrations o f the antagonist, the binding o f MDM2 to
the surface-bound peptide is increasingly inhibited, which leads to a decrease in the
biosensor response. These experiments require a longer time (about 1 hr per assay) than
do FP, but SPR assays have been made more high-throughput through introduction o f
parallel channels. Modifying the format o f the assay by adsorbing MDM2 to the chip and
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flowing over a solution o f the inhibitor can reveal the kinetics o f the binding event.
Development of a reliable assay is critical for drug discovery, as protein-protein
interactions are prone to the identification o f false positive hits, i.e., small molecules that
cause non-specific inhibition at low micromolar concentrations via aggregation o f the
inhibitor and/or the protein target.45
1.2.5 Drug Discovery Paradigm
With a new protein target identified and an assay developed, the stage is set for
the modem drug discovery process (Figure 3). In the first step, a number o f potential
inhibitors are proposed based on structure-based design. Those compounds are produced
via organic synthesis in the second step. The identity and purity o f the molecules is
Target:
p53/MDM2
Advance
Drug
Candidate
Structure-Based
Design
Bioassay
(in vitro or
in vivo)
Organic
Synthesis
Analytical
Characterization
and Purification
Figure 3. The modem drug discovery paradigm.
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confirmed using analytical techniques in step three. The compounds are then screened
for the biological property o f interest in step four. If no active compounds are identified,
then a new set o f designs must be created, synthesized, and tested. However, if there is a
“hit” within the set of compounds, then a second iteration o f the process is used to refine
the design and improve potency. Thus, the structure/activity relationship o f the active
compound and its analogues is elucidated incrementally, and the cycle continues until the
molecule meets all the criteria (i.e., potency, selectivity, pharmacokinetics, etc.) required
for advancement as a drug candidate. The development o f potent p53/MDM2 inhibitors
has closely followed this paradigm.
1.3 q-Peptide Antagonists of the p53/MDM2 Interaction
Inhibition of the p53/MDM2 interaction with a synthetic molecule was validated
as a viable strategy through the exceptional work o f a group o f Novartis researchers. As
part o f their drug discovery program to establish a pharmacophore model, the Novartis
team determined the specificity o f the amino acid-binding pockets on MDM2 and
developed a series o f highly potent peptide antagonists.46 Even before publication o f the
crystal structure, they studied the p53/MDM2 complex with a series o f monoclonal
antibodies to identify the interacting domains on the respective proteins.47 They mapped
the MDM2-binding site on p53 using synthetic peptide libraries derived from the Nterminal region o f p53. The active peptides defined the consensus MDM2-binding site
on p53 to be Thrl8-Phe-Ser-Asp-Leu-Trp23, but this hexapeptide had a low binding
affinity for MDM2 with an IC50 value, or concentration required for the inhibition o f 50%
of binding, o f 700 pM in an ELISA format.48 To find novel high-affinity ligands for
MDM2 that were able to block the interaction o f MDM2 with p53, the Novartis team
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screened phage display peptide libraries 49 Phage display is a powerful biological
technique where a randomized set o f peptides are displayed on the surface of filamentous
phage. Phage particles displaying peptides that bind to the target protein are isolated and
allowed to infect E. coli bacteria to amplify the subset o f phage that bind the target. After
several rounds o f selection and amplification, the phage population is highly enriched in
phage displaying peptides that bind to the target. By sequencing the phages’ DNA, the
peptide sequence is revealed.
The most active peptide obtained in this way by the
Novartis researchers (1-2, Figure 4) showed a 28-fold greater inhibition o f the
p53/MDM2 interaction than the wild-type p53-derived peptide (1-1). Peptide 1-2 was
effective at inhibiting the p53/MDM2 interaction in cells;50 it was also active when
expressed either with a glutathione ^-transferase (GST) tag51 or in the active-site loop o f
thioredoxin.52
More detailed information on the amino acid requirements for a potent peptide
inhibitor was obtained by synthesizing truncated versions o f peptide 1-2 and testing these
peptides in the ELISA. The truncation series included sequences containing from 6 to 11
residues.
While the 6- and 7-mer peptides were poor inhibitors o f the p53/MDM2
interaction, an 8-mer peptide (1-3) was identified as the minimal sequence retaining
micromolar affinity for MDM2. This peptide served as the starting point for optimization
via structure-based design.
Guided by the X-ray crystal structure o f the p53/MDM2 complex, the Novartis
researchers substituted the residues on p53-derived peptide 1-3 that do not interact with
the MDM2 protein in the bound conformation with structure-promoting a,a-disubstituted
residues 46 Examination o f 1-3 by circular dichroism (CD) and nuclear magnetic
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16
h2h^ o
M
H
-= M
H
yn
1-1: 8673 nM
K
I
HO
O
M
H2N^NH
O^NH
HN.
A
H O . ^ .0
|
1-2: 313 nM
OH
HN
H O . s.O
1-3:8949 nM
i
H
]
i
;
H
]
i
;
H
V
U " %<
:
j
i
t
-S o
H O . ^O
1-4: 2210 nM
H O -P
H O . ^.O
1-5:
1-6:
1-7:
1-8:
R = H, 314
R = F, 14
R = Me,10
R = Cl, 5
nM
nM
nM
nM
Y !A V / . V
--.
H
I!
£
H
II
/ \
H
Ij
Figure 4. IC50 concentration of MDM2-binding peptides in a p53/MDM2 competition ELISA from ref. 46
(Aib = a-aminoisobutyric acid; Ac3c = 1-aminocyclopropanecarboxylic acid; Pmp =
phosphonomethylphenylalanine).
resonance (NMR) spectroscopy revealed that this peptide adopts a random coil
conformation in aqueous solution. Residues 20, 21, 24, and 25 (p53 numbering) were
systematically replaced with a,a-disubstituted residues, which are known to promote a -
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17
and 3io-helices in short peptide motifs.53 The Novartis team hypothesized that these
substitutions would bias the peptide toward a helical confonnation, similar to that which
it adopts when bound to MDM2, thus reducing the entropic cost associated with binding
and increasing the affinity o f the peptide ligand for the protein. The best combination
was achieved by replacing Asp21 and Gly25 a-aminoisobutyric acid (Aib) and 1aminopropanecarboxylic acid (Ac3c), respectively (peptide 1-4). Specifically, Ac3c was
substituted for Gly25 because o f the marked structural preference o f Ac3c for the i+2
position in a type-I p~tum,54 which is similar to the local secondary structure observed for
Trp23-Leu26 in p53. These modifications result in only a 4-fold increase in MDM2binding affinity o f the peptide, but subsequent NMR experiments under physiological
conditions confirmed that the conformational restrictions imposed by the a ,a disubstituted amino acids in the peptide sequence induce a conformation similar to that
observed for the N-terminal segment o f p53 in the MDM2-bound state.
The next round o f optimization was focused on increasing the binding affinity o f
the peptide via chemical modification o f the tyrosine side chain.46 Examination o f a
model of peptide 1-2 in complex with MDM2 revealed that the hydroxyl group o f Tyr22
was positioned outside o f the canonical p53-binding pocket and near the side chain o f
MDM2 residue Lys94. The tyrosine was replaced with phosphonomethylphenylalanine
(peptide 1-5) in order to form a stabilizing salt bridge interaction with the s-amino group
o f Lys94, resulting in a 7-fold increase in binding affinity. The phosphonate moiety also
increased the water solubility o f the otherwise highly hydrophobic sequence.
Modification o f the Trp23 side chain resulted in a major increase in binding
affinity, an important finding that has been taken advantage o f by numerous subsequent
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18
researchers, as will be discussed.
In the X-ray crystal structure o f the p53/MDM2
complex, the side chain o f p53 residue Trp23 is embedded in a deep hydrophobic pocket
formed by MDM2 residues Leu57, Phe86, Ile99, and lie 103. Although the indole fits
rather tightly, careful inspection revealed that the pocket is not completely filled.
Specifically, an empty space near the 6 position o f the indole ring remains at the bottom
o f the pocket. The space corresponds to the volume of a methyl group or a chlorine
atom, and the possibility o f increasing the binding affinity by establishing additional van
der Waals interactions with the protein motivated the synthesis o f peptides 1-6, 1-7, and
1-8.
Replacement o f Trp23 with 6-substituted tryptophans resulted in a substantial
increase in binding affinity, which nicely correlated with the size o f the substituent and
therefore the occupancy o f the pocket.
The most active compound, peptide 1-8,
contained a 6-chloro-tryptophan and had an IC50 value o f 6 nM in their p53/MDM2
ELISA, a 63-fold increase in binding affinity relative to peptide 1-5 with the
unsubstituted tryptophan. The discovery o f this pocket has been exploited through the
incorporation o f />chlorophenyl groups in a variety o f small molecule inhibitors as will
be discussed.
The seminal contributions o f the Novartis researchers46 demonstrated that a
number o f the principles identified through the development o f peptidomimetics for
enzyme inhibition apply to the discovery o f protein-protein interaction inhibitors.
Structure-based design was employed to optimize the MDM2-binding affinity o f short
peptide motifs derived from the N-terminal domain o f p53 by combining conformational
constraints o f the backbone with side chain functional group modifications that establish
additional electrostatic and van der Waals interactions both inside and outside the natural
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binding pocket. Novartis’ effort was also an excellent example o f the integration of
multiple techniques in drug discovery for the identification o f compounds capable o f
inhibiting a therapeutically relevant protein-protein interaction. Armed with a refined
pharmacophore model and a strategy for the optimization o f non-peptidic leads, the
pharmaceutical industry began its assault on the p53/MDM2 interaction in earnest.
Although initial progress was slow, this effort has ultimately been rewarded with the
discovery o f small molecule p53/MDM2 inhibitors.
1.4 Natural Product Antagonists of the p53/MDM2 Interaction
About 60% o f drugs on the market today (excluding biologies) are o f natural
origin,55 but to date only three natural products have been reported as having any
inhibitory activity against the p53/MDM2 interaction.57,61,68 Chalcone-based inhibitors
were reported first and have been the most extensively studied.57 60 Chlorofusin was the
second natural product inhibitor o f p53/MDM2 to be reported.61 Efforts toward the total
synthesis o f chlorofusin are underway in a number of laboratories and will lead to further
investigation o f the structure/activity relationship o f this molecule and its analogues.63"66
Hexylitaconic acid has also recently been reported to inhibit the p53/MDM2 interaction.68
Thus, natural products have not been a fertile source o f lead compounds for the inhibition
of the p53/MDM2 protein/protein interaction.
While natural products are effective
inhibitors for a few protein-protein interaction targets, such as paclitaxel, the epothilones,
and discodermolide for tubulin polymerization, it can be difficult to find natural product
lead compounds for a new class o f protein-protein interaction targets.
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1.4.1 The Chalcones
The broad antitumor activities o f natural product-inspired chalcones (1,3diphenyl-2-propen-l-ones ) 5 6 led Holak and co-workers to examine the possible
interaction o f chalcones with the p53/MDM2 system in 2001 . 5 7 Compound 1-9 (Figure
5) had an ELISA IC 5 0 value o f 206 pM and caused a shift in the pattern o f the 'H -^N
HSQC NMR spectrum o f the l5N enriched MDM2 protein, consistent with chalcone
binding in the tryptophan pocket.
extended
71
Their proposed binding model assumed that the
-system o f the molecule is rigid and planar and that the monochlorophenyl
group resides in the tryptophan binding site. Based on their NMR data, Nolak et al.
proposed that the second aromatic ring o f the chalcone contacts residues Phe55 and
Tyr56 o f MDM2 outside o f the canonical p53-binding pocket. In this orientation the
carboxylic acid and ene-one carbonyl are both solvent-exposed, but the carboxylic acid
may be positioned correctly to engage MDM2 residue Lys51 in a salt bridge interaction.
Rz
H
Base
1-9: R1 =CI, R2 =0CH 2 C02H
IC5 0 = 206 pM ELISA
220 pM NMR
1-10: R1=l, R2 =OCH2 B(OH) 2
Figure 5. Claisen-Schmidt aldol condensation to form chalcone p53/MDM2 antagonists 1-9 (ref. 57) and
1-10 (ref. 59).
Despite their early identification as p53/MDM2 interaction inhibitors and their
straightforward combinatorial synthesis using Claisen-Schmidt aldol condensation
protocols,58 chalcones have been the subject o f only a few subsequent reports, perhaps
because this class o f compounds was not very selective for the target protein.
Dichlorophenyl derivatives were equally toxic to both normal and malignant breast
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epithelial cells, possibly due to p53/MDM2 independent mechanisms . 5 9
Other
members o f the original set o f chalcones were observed to denature or facilitate the
aggregation o f the MDM2 protein during testing . 5 7
A gel shift assay revealed that the
p53 released from the p53/MDM2 complex by treatment with 1-9 was unable to bind
DNA, suggesting an additional influence o f the chalcone on the p53 protein. One could
speculate on the potential o f chalcones as Michael acceptors at the ene-one functionality,
and that covalent modification resulting from attack by a protein nucleophile would affect
conformation and activity. A series of boronic chalcone analogues was prepared by Khan
and coworkers to address the lack o f specificity in the carboxylic acid-containing
chalcones . 5 9 They reported a modest improvement; compound 1-10 was 2.5- to 10-fold
more toxic to human breast cancer cell lines than to a normal breast epithelial cell line at
concentrations from 10-40 pM. Isoliquiritigenin (4,2’,4’-trihydroxychalcone), a natural
chalcone that is isolated from licorice root and shallot, has been shown to induce cell
cycle arrest and apoptosis in liver cancer cells via the p53 pathway at 10-20 pg/mL, but
its binding to MDM2 was not characterized . 6 0
1.4.2 Chlorofusin
Williams and coworkers identified chlorofusin as an inhibitor o f p53/MDM2
binding (1-11, Figure
6
) after testing over 53,000 extracts from the fermentations o f a
diverse collection o f microorganisms for this activity . 61 This novel metabolite from the
fungus Microdochium caespitosum, which in a purified form, had an IC 5 0 o f 4.6 pM in a
p53/MDM2 ELISA. Further studies using SPR confirmed that chlorofusin binds to the
N-terminal region of MDM2 (IQ = 4.7 pM ) . 6 2 Mass spectrometry, amino acid analysis,
and NMR spectroscopy revealed that chlorofusin contained a densely functionalized
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chromophore with an azaphilone core linked through the terminal amine o f an
ornithine residue to a cyclic peptide o f nine a-am ino acid residues.61 Two o f the amino
acids contain a nonstandard side chain (ornithine and 2-aminodecanoic acid), and four
possess the D-configuration. The relative stereochemistry of only C-4, C-8, and C-9 on
the chromophore and Asn3 and Asn4 in the cyclic peptide could be assigned.
The Boger and Searcey groups simultaneously reported syntheses o f the cyclic
peptide portion o f chlorofusin. Boger and co-workers reported a convergent solutionphase synthesis o f both the L-Asn3/ D-Asn4 and D-Asn3/L-Asn4 diastereomers.63 NMR
experiments identified the absolute stereochemistry o f asparagine residues 3 and 4 as
being
L
and D , respectively. Searcey et al. published a solid-phase synthesis via on-bead
Cl
.N-
C)
HN—
'/— NH
\ = 0
y j y \
r
O
'/— NH V -O H
HN—^
/
HN
O
/
)= 0
=0
HO — I
HN—<
/
HN
O O
n
\
H2 N.
V -—NmHu
O
Asn4^
H N -^
1-11: Chlorofusin
/
IC5 0 = 4.6 pM, ELISA
H2 N—
Asn3
Figure 6. Structure of chlorofusin (ref. 61) and hexylitaconic acid (ref.
1-12: Hexylitaconic Acid
IC5 0 = 230 pM, ELISA
68
).
cyclization.64 Both groups reported that the cyclic peptide alone had no inhibitory effect
on the p53/MDM2 interaction. A racemic synthesis o f the azaphilone core o f chlorofusin
has been recently reported by Porco and coworkers.65 Preparation o f the correct pure
enantiomer followed by coupling to the cyclic peptide will afford chlorofusin. The future
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23
synthesis o f chlorofusin analogues will help to elucidate structure/activity relationships
o f this class o f natural products and may afford more potent ligands for MDM2.
Williams et al. are investigating the biosynthesis of chlorofusin,66 which in the long term
could potentially provide a number o f analogues through manipulation o f the polyketide
and non-ribosomal peptide biosynthetic machineries.67
1.4.3 Hexylitaconic Acid
Recently, another natural product inhibitor o f the p53/MDM2 interaction, (-)hexylitaconic acid (1-12, Figure 6), was reported.68 Isolated from the fermentation
culture of a fungus, Arthrinium sp., which was separated from a marine sponge, (-)hexylitaconic acid had an IC 5o of approximately 230 pM in a p53/MDM2 ELISA. Both
enantiomers of hexylitaconic
acid were reported previously, but the
absolute
stereochemistry o f the active compound is unknown.69 Four derivatives o f 1-12, the
monomethyl ester, a dihydro derivative, a dihydro derivative o f the monomethyl ester,
and itaconic acid, were prepared but showed no inhibitory activity. This compound is
structurally distinct from other p53/MDM2 inhibitors, and more investigation o f its
mechanism of action is needed.
1.5 Small Molecule Inhibitors of p53/MDM2
1.5.1 The First: syc-7
The first de novo designed non-peptidic small molecule inhibitor o f the
p53/MDM2 interaction was reported by Zhao et al. in 2002.70 Using computer-aided
design and their knowledge o f the p53/MDM2 co-crystal structure, these workers
appended a variety o f aromatic rings on a [2.2.1]bicyclic scaffold (Figure 7) in hopes of
mimicking the presentation o f p53 residues Phel9 and Trp23 within the binding cleft o f
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24
MDM2.
Five o f their twenty-three compounds showed activity in a p53/MDM2
ELISA at micromolar concentrations. The most active compound, 1-13, was tested in a
variety o f cell-based assays (10 pM concentration), and Zhao et al. observed a timedependent accumulation o f the p53, MDM2, and p21 proteins and cytotoxicity via
induction o f apoptosis. While the potency o f 1-13 is rather low for a drug, this molecule
provided inspiration for the designs o f other researchers. Future compounds removed the
flexibility o f the chains bearing the aromatic groups in order to reduce the entropic cost
associated with binding and increase the affinity.
HN
.OH
NH
1-13: syc-7
Figure 7. Structure of the first non-peptidic small molecule inhibitor of the p53/MDM2 interaction (ref.
70).
1.5.2 Sulfonamides
Galatin and Abraham used a pharmacophore model and in silico screening to
identify sulfonamide 1-14 as a novel lead compound.71 It had an IC 5 0 of 32 pM in vitro
and yielded a 20% increase in p53-dependent transcription at 100 pM in a cell-based
assay. The authors claimed that the molecular weight o f the compound could likely be
reduced by removing one o f the phenyl substituents from the pyrazolidindione ring
without affecting binding affinity, but no further investigations were reported.
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Figure 8 . Structure of sulfonamide 1-14 (ref. 71).
1.5.3 Nutlins (t/s-Imidazolines)
The first potent and selective small molecule antagonists o f the p53/MDM2
interaction with both in vitro and in vivo activity were reported by Roche in 2004.72
While historically it had been difficult to identify small molecule protein-protein
interaction inhibitors, the focused nature o f the p53/MDM2 binding interface and deep,
well-defined hydrophobic pockets on the surface o f MDM2 raised expectations that a
small molecule could be found to do the job. A high-throughput screen o f the Roche
compound collection produced several lead structures.
One class was a series o f cis-
imidazoline analogues that were named Nutlins (for Nutley, NJ inhibitors). As racemic
mixtures, compounds 1-15, 1-16, and 1-17 had IC 5 0 values between 100-300 nM by SPR
in a competition format.
The racemic mixture o f compound 1-17 was resolved on a
chiral column, and the enantiomers were assayed individually. The enantiomer shown in
Figure 9 had an IC 5 0 o f 90 nM, while the other enantiomer was 150 times less potent
(IC50 = 13.6 pM).
O
o
r-O H
Cl
/
V N\ - - /
l\
K1
V Nw /
K > °x
•
NH
Q
Cl
1-15: 2 6 0 nM
1 -1 6 :1 4 0 nM
1-17: 90 nM
Figure 9. IC 50 concentration of Nutlins analyzed by Biacore’s surface plasmon resonance technology in a
solution competition format (ref. 72).
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26
The binding mode o f the Nutlins was revealed by the co-crystal structure o f 116 with MDM2 (Figure 1 0 ).72 The small molecule mimics the interactions o f the p53peptide, with one bromophenyl ring sitting deeply in the tryptophan binding pocket, the
other bromophenyl substituent occupying the leucine binding site, and the ethyl ether side
chain directed toward the phenylalanine binding pocket.
The imidazoline scaffold
replaces the a-helical backbone and is able to direct, in a fairly rigid fashion, the
projection of the three hydrophobic substituents into their respective binding pockets.
Most of the small molecule inhibitors discussed in section 1.5 share this common theme
o f a rigid heterocyclic scaffold with /?-halophenyl appendages.
Figure 10. Crystal structure of 1-16 in complex with MDM2 (ref. 73, PDB code 1RV1).
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27
To test whether inhibition o f p53 binding to MDM2 would translate into
activation of the p53 pathway, a battery o f cell-based and animal experiments was
performed.72 The Nutlins inhibited the growth and viability of cultured cancer cells with
IC50 values between 1.4-1.8 pM. As expected, treatment of cells having functional p53
with the Nutlins led to increased levels o f the p53, MDM2, and p21 proteins, but this
treatment had no effect on cells with mutant p53.
Additionally, the Roche workers
showed that accumulation o f p53 was due to reduced degradation rather than increased
production via measurement of the mRNA levels with PCR. Cell cycle analysis revealed
that treatment with the Nutlins arrested cells in the Gi and G 2 phases, preventing mitosis.
The Roche group found that the Nutlins could induce apoptosis via caspase activation.
Treatment of cells with the inactive enantiomer o f 1-17 showed that the effects were
specific to p53/MDM2 inhibition and that the compound class is not intrinsically
cytotoxic. Finally, this group found that oral administration of 1-17 to nude mice with an
established tumor xenograft resulted in 90% inhibition of tumor growth, relative to
controls. These small molecule antagonists are being used to investigate p53 signaling
(i.e., the role o f phosphorylation o f p53 in transcriptional activation)73 and have been
advanced for treatment o f acute myeloid leukemia74 and multiple myeloma.75
Despite these exciting results, the Roche researchers were only cautiously
optimistic about the future o f p53/MDM2 inhibitors. While the Nutlins have excellent in
vitro activity, they are about 100-fold less active in vivo. It is possible that the Roche
effort has not yet identified the optimal molecule or that the signaling pathway
downstream o f p53 is not completely intact, making p53 activation ineffective.76 A more
likely explanation for limited Nutlin activity is obtained through careful consideration of
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28
p53 regulation. In cancer cells containing wild-type p53 and overproducing MDM2,
inhibition o f the p53/MDM2 interaction leads to an increase in cellular levels o f p53.
Increased levels of transcriptionally active p53 lead to even higher levels o f production of
MDM2. Therefore, because o f the autoregulatory nature of the negative feedback loop
between p53 and MDM2, the inhibitor works against itself by raising the intracellular
levels of its target protein as a direct result o f its mechanism o f action. Thus a relatively
high steady-state concentration o f the inhibitor is required to have a significant sustained
effect. It will be interesting to see how this complex issue is resolved in the fixture.
The researchers at Roche have devised a clever strategy to expand the use o f their
Nutlin p53/MDM2 inhibitors from treatment o f the 7% o f human cancers with aberrant
production o f MDM2 and functional p53 to a therapy for the 50% o f cancers with an
inactive p53 pathway due to p53 mutation. Pretreatment with the Nutlin induces cell
cycle arrest in normal proliferating cells but does not affect the cancer cells with mutant
p53.
Subsequent treatment with a mitotic inhibitor, such as paclitaxel, causes mitotic
arrest and apoptosis o f the cancer cells (because they are still proliferating) but does not
cause any cytotoxic effects in the normal cells.77 The clinical application o f this method
for selective chemotherapy deserves further investigation.
1.5.4 Benzodiazepinediones
Grasberger and coworkers at Johnson & Johnson Pharmaceuticals developed the
benzodiazepinedione class o f small molecule p53/MDM2 inhibitors.78
More than
338,000 compounds were screened for MDM2 binding using their ThermoFluor® assay.79
This assay uses fluorescent dyes to monitor protein unfolding as a function o f
temperature, with the idea that compounds that bind the target protein will increase the
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29
protein’s
thermal
stability.
The
hits
included
116
compounds
from
a
benzodiazepinedione combinatorial library that had been synthesized via a highly
efficient Ugi four-component condensation.
80
An anthranilic acid, an a-am ino ester, an
aldehyde, and 1-isocyanocyclohexene81 were combined, followed by acid-catalyzed
cyclization, producing the desired benzodiazepinediones in good yield and purity. 82 The
discovery o f this class o f inhibitors is an excellent example of a successful application o f
combinatorial chemistry using a multi-component reaction. After careful investigation o f
the structure/activity relationship o f this compound class,803 an optimized inhibitor, 1-18
(IC5o = 80 nM by FP and 30 pM in cells), was co-crystallized with the MDM2 protein
(Figure 12).
■70
In the bound state, the compound projects the /7-chlorophenyl groups into
the Trp23 and Leu26 binding pockets on MDM2.
The iodobenzene portion o f the
benzodiazepinedione occupies the phenylalanine binding site.
NHBoc
XX
HN
OH
CN^
Ql
1-18: IC5 0 = 80 nM, FP
IC5 0 = 30 nM, colls
Figure 11. Ugi four component condensation to form benzodiazepinedione 1-18 (ref. 78)
To address the observed 375-fold lower potency in cells relative to in vitro
measurements, 1-18 was optimized for greater cellular permeability and then applied in
vivo as a sensitizing agent in conjunction with doxorubcin.
o->
The carboxylic acid
functionality o f 1-18 was replaced with a methyl group, and a valeryl acid (1-19) or
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Figure 12. Crystal structure of 1-18 in complex with MDM2 (ref.78, PDB code 1T4E).
piperazine (1-20) solubilizing moiety was added, yielding compounds with similar
potency in vitro and improved cell-based activity.
Compound 1-19 was 3-fold more
potent, on average, than 1-18 in cells, but 1-20 was approximately 22-fold more potent
than 1-19, with an average IC 5 0 o f 700 nM in cells expressing wild-type p53.
The
Johnson & Johnson team then tested whether activating p53 in combination with
chemotherapy would result in enhanced antitumor activity.
They found that
administration o f doxorubicin alone resulted in a 4- to 6-fold increase in p53 levels, but a
synergistic effect was achieved by treating cells with a combination o f 1-20 and
doxorubicin, resulting in an 18-fold increase in p53 levels. These workers found that
treating mice having established tumor xenografts with 1.5 mg/kg doxorubicin and 100
mg/kg 1-20 had a similar effect on tumor size as a larger dose o f doxorubicin alone (3
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31
mg/kg). However, the mice treated with the combination therapy experienced a lesser
degree o f weight loss than those on doxorubicin alone, suggesting a reduction in the side
effects of the chemotherapy. The prospect o f p53/MDM2 inhibitors as sensitizing agents
during chemotherapy needs to be investigated in the clinic. The primary concern is the
degree o f selectivity that can be achieved between normal and cancer tissue in order to
reduce side effects.
Cl
1-19: IC5 0 = 708 nM, FP
1-20: IC5 0 = 704 nM, FP
Figure 13. Structures of benzodiazepinediones optimized for cell-based activity (ref. 83).
1.5.5 Spiro-oxindoles
Wang and co-workers used structure-based design to develop a new class o f small
molecule p53/MDM2 interaction antagonists based on a spiro-oxindole core.84 These
workers focused on the indole ring o f p53 residue Trp23 not only because it is buried
deep within a hydrophobic pocket on the surface o f MDM2 but also because the indole
N-H also forms a hydrogen bond with the backbone carbonyl o f MDM2 residue Leu54.
Wang et al. decided that an oxindole could mimic the tryptophan side chain and
performed a sub-structure search for natural products containing an oxindole ring.
Several natural alkaloids that possessed a spiro-oxindole core structure were identified,
but modeling suggested that none would be a suitable inhibitor due to predicted steric
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32
clashes with the MDM2 protein.
Nevertheless, Wang et al. adopted the
spiro(oxindole-3,3’-pyrrolidine) core structure for their design, reasoning that the
spiropyrrolidine ring provided a rigid scaffold from which two hydrophobic substituents
could project into the P hel9 and Leu26 binding sites on MDM2. A chlorine was added
at the 6-position o f the oxindole based on the earlier peptide work.46 Several analogs
were rapidly constructed using an asymmetric 1,3 dipolar reaction85 as the key step.
Compound 1-21 had an IC 5 0 of 8.46 pM in their competition FP assay.
Modeling
suggested that additional space remained in the phenylalanine and leucine binding
pockets, so a chlorine was incorporated at the meta-position o f the phenyl ring, and the
isobutyl group was replaced with a 2,2-dimethylpropyl group resulting in compound 122, which was 98 times more potent than the original lead compound. Furthermore, 1-22
was a highly effective inhibitor o f human prostate cancer cells with an IC50 o f 0.86 pM,
13 times more toxic than to normal cells with wild-type p53.84
1-21: R 1 = H , R 2=H
IC 5 0 = 8 4 6 0 nM, F P
) mol. sieves
2 ) M e 2NH
1
r2
1-2 2 : R 1 =CI, R2= M e
IC 5 0 =
Cl
Cl
86
nM , FP
IC 5 0 = 8 3 0 nM , cells
H
Figure 14. Synthesis and structure of spiro-oxindole small molecule p53/MDM2 inhibitors (ref. 84).
1.5.6 Summary of Small Molecule Inhibitors
Potent and selective small molecule inhibitors o f the p53/MDM2 interaction have
now been developed.
Taking advantage o f the pharmacophore established by the
published co-crystal structure and earlier peptide work, several groups used a
combination o f structure-based design and combinatorial chemistry to identify a lead
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33
compound. The designs have typically incorporated a heterocyclic core that projects
p-haloaromatic appendages toward the P hel9 and Trp23 binding pockets on the surface
o f MDM2. It took several years for the first reports to surface,70’72 but once the drug
companies knew what type of molecule they were looking for, several similar reports
have followed in quick succession.
The current challenge is to obtain molecules that are
as potent in vivo as they are in vitro, perhaps due to the negative feedback loop o f p53
regulation by MDM2.
New strategies for using p53/MDM2 inhibitors as sensitizing
agents for malignant cells or selective protection o f normal cells during chemotherapy
77 83
may emerge as important therapies for the clinical treatment o f cancer. ’
1.6 Oligomeric Scaffolds for ra-Helix Mimicry and p53/MDM2 Inhibition
During the eight years between publication o f the p53/MDM2 co-crystal structure
in 1996 and report o f the Nutlins in 2004, many researchers initiated investigations o f
alternative strategies for the inhibition o f the p53/MDM2 interaction. Since the target
was a protein-protein interaction, these workers believed that p53/MDM2 may prove to
be intractable using traditional small molecule therapeutics.
This movement was
temporarily bolstered by the lack o f reports o f small molecule p53/MDM2 inhibitors
from the pharmaceutical companies during this time. The unifying theme o f these nontraditional approaches was to develop an oligomeric scaffold capable o f mimicking the
three-dimensional display o f side chains from an a-helix. Such a proteomimetic scaffold
could be applied not only to p53/MDM2 but also to other protein-protein interactions
modulated by a-helical binding epitopes, such as the Bcl-2 family/BH3 domain
interaction. A number o f successful scaffolds for inhibition of p53/MDM2 are described
herein. Though the potency is not usually as high as obtained with the small molecules,
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34
especially in vivo, it should be kept in mind that the true standard for comparison was
the potent a-peptide antagonists46 and not the small molecules because the latter had not
yet been reported. Thus, a number o f the strategies were aimed at improving upon the
disadvantages o f peptide therapeutics, such as their proteolytic and metabolic instability.
1.6.1 Retroinverso Peptides
Kahne et al. tested a series o f peptides to understand how structural modifications
(i.e., chirality and sequence reversal) o f the p53-peptide backbone would affect its
interaction with MDM2.86
Since the backbone makes no contacts with the protein
surface except for one hydrogen bond, Kahne et al. hypothesized that the left-handed a helix merely provided a scaffold for the correct spacing and orientation o f the interacting
side chains. These workers synthesized a number o f analogues o f the natural L-a-peptide
1-23, including the enantiomer (D-a-peptide 1-24), the L-a-retropeptide 1-25 (a peptide
with the opposite order o f residues), and the D-a-retropeptide or “retroinverso” peptide 126. The side chains o f peptides 1-24 and 1-25 are not superimposable with those o f 1-23
because o f their mirror image and N->C vs. C->N relationships, respectively. In the
preferred left-handed helical conformation formed by oligopeptides o f D-amino acids, the
side chains o f peptide 1-26 aiso do not align with those displayed by the natural peptide
in the right-handed a-helical form. However, if retroinverso peptide 1-26 could adopt a
right-handed helix, then its side chains would overlay with those presented by the natural
peptide. While peptides 1-24 and 1-25 had no measurable affinity for MDM2 in their
ELISA, the retroinverso isomer 1-26 interacted with MDM2 with potency comparable to
that of the natural peptide 1-23. In spite o f the reversed positions o f the nitrogen and
oxygen atoms in the backbone, the switched orientation of the backbone hydrogen bonds,
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35
and the inverted chirality o f the side chains, the retroinverso peptide is still able to
mimic the natural peptide. These workers speculate that the retroinverso peptide is being
forced to adopt a disfavored right-handed helical conformation, but no structural evidence
was provided. Due to their proteolytic stability, this class of peptide mimetics might have
some utility if active against extracellular protein targets.
1-23: (L)-a-peptide
IC s o -1 7 .4 pM
1-24: (D)-a~peptide
1C5Q > 1000 pM
o
Figure 15. Development o f retroinverso peptide p53/MDM2 antagonists (ref.
86
).
1.6.2 Peptoids
The development o f peptoids (oligomers o f iV-alkyl glycine) as ligands for
MDM2 has been pursued by both the Appella93 and Kodadek95,97 research groups.
Peptoid monomers are similar to regular a-am ino acids, except that the side chain is
attached to the backbone nitrogen instead o f the a-carbon.
07
Peptoids are one class o f
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36
foldamers, or unnatural oligomers that adopt a predictable conformation;
the
backbone can be sterically biased to form a helical conformation by incorporating chiral
side chains into the oligomer.88 This feature facilitates the structure-based design o f
peptoids that display side chains in a predictable three-dimensional array.89 Through the
sub-monomer synthetic approach,90 a wide variety o f side chains can easily be
incorporated.91 Peptoids are resistant to proteolytic degradation, and several biological
applications o f peptoids have been reported.92
1.6.2.1 Structure-Based Design Approach
Appella and coworkers attempted to mimic the display o f p53 side chains Phel9,
Trp23, and Leu26 on a helical peptoid scaffold.93 The helical conformation o f chiral
peptoids is similar to the type-I polyproline helix, in which the amide bonds are cis and
the carbonyl oxygens point toward the N-terminus, resulting in a helix macrodipole that
is opposite that o f an a-helix, relative to the backbone orientation.
In spite o f these
structural differences, the peptoid helix was superimposed upon the a-helix o f the p53
peptide via molecular modeling. Since the peptoid helix has only three residues per turn,
the interacting residues were displayed along one face at the i, i+3, and the i+6 positions.
It was noticed that the side chains project from the peptoid and a-helical scaffolds at
different angles. To compensate, an additional methylene group was inserted in the side
chain of the peptoid monomers between the backbone and hydrophobic group. To ensure
the peptoid’s aqueous solubility, three achiral peptoid glutamic acid residues were
incorporated at positions expected to be solvent-exposed.
Since the conformational
stability of the peptoid helix (and the validity o f the underlying model for the structurebased design) relies upon steric constraint o f the backbone via incorporation o f chiral
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37
monomers, four such residues were included in the design, resulting in 1-27 (Figure
16), whose CD spectrum in water showed the minima at 219 and 205 nm and maximum
at 193 nm characteristic o f the peptoid helix. However, this peptoid was completely
inactive in the p53/MDM2 FP assay.
1-27: no binding
helical by CD
1-28: IC 5 0 = 188 gM
weakly helical by CD
1-29: IC 5 0 = 17.8 gM
not helical by C D
Figure 16. Structures of peptoid ligands for MDM2.
Replacement o f the carboxylate side chains with phosphonate groups reduced
helical stability, increased water solubility, and gave rise to weak binding to MDM2 (128, IC 5o = 1 8 8 pM). Appella et al. proposed that the increased binding affinity resulted
from greater electrostatic attraction o f the phosphonates, relative to the carboxylates o f 127, to MDM2 residues Lys94, His96, and Lys51 positioned around the periphery o f the
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38
pocket. In a continued effort to make the peptoid more hydrophilic, p-nitro groups
were added to the phenyl rings of the chiral a-methylbenyl monomers.
One o f the
phosphonates was replaced with a sulfonamide to reduce the net charge o f the peptoid.
These changes generated 1-29, which did not display a helical signature in the CD
spectrum but showed improved binding affinity (10-fold) with an IC 5 0 o f 17.8 pM. Since
the reason for the improvement in binding affinity was unclear, a series o f peptoids was
prepared in which the side chains presumed to interact with MDM2 were removed (1-30,
1-31, and 1-32 in Figure 17). The IC 5 0 values for these compounds were only 1.4- to 3.4fold higher.
HCXP \
HO
o
1-30: IC5 0 = 59.8 |iM
2N
^
\
o 2n .
/V
O
T
s\'
0
S.
^
H N ^ |f
A
2
V
>
/V
0
NA
o
o2n^
°
o
o2n^
J o
X
2
?
,
HO
A
2
° = ' =°
NH2
^
o
° :a
o
1-31: IC5 0 = 28.3
o 2n
O
J o
O
° :a
\
- n ^ ^ NX
J o
HV
/
— NH
o'
N^ ^ NH2
- ^—
o 2n
^—
-
o=s=o
i H2
H0 . I ^
ho' p1
o 2n ^
.
o 2n
HO
1-32: IC5 0 = 25.3 pM
H N ^f
fX
JC<J
o2n^ ^
0
N
rX
J^-iJ
o 2n^ ^
0
A.
” ” Jr
1
0
J
o=s=o
^ h2
'
Figure 17. Structure and IC 50 values for peptoids with V-methyl replacement of the hydrophobic residues.
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39
Scrambling the relative positions o f the hydrophobic achiral side chains had a
similarly negligible effect (1-33 and 1-34, Figure 18). Preparation o f the choroindole
derivative at position 5 (1-35) reduced the IC5o by only 2-fold, to 9.9 pM. In contrast, as
discussed above, replacing Trp with 6-chlorotryptophan in the peptide series had reduced
the IC50 value by 63-fold.46
/
-N H
o
^
HCXp ^
o
2n
O
1-33: IC 5 0 = 26.8 uM
nh2
A
o=s=o
J
HCV °
o 2n
o
^
Q
^
o
o
1-34: IC 5 0 = 29.7 nM
o
O
^
JU
o
J o
J o
HO
o=s=o
NH2
O
HO .
/P
o 2n
HO
O
O
''
\'X
o
\
]
0
1-35: IC 5 0 = 9.9 hM
'n h
O
2
o
o=s=o
nh2
Figure 18. Structure and IC50 values of peptoids with scrambled sequence of hydrophobic residues (1-33
and 1-34) and 6-chloroindole side chain at position 5 (1-35).
Next Appella et al. investigated how varying the conformational stability o f the
backbone would affect the binding affinity o f the peptoid (Figure 19). Scrambling the
chirality o f the helix-promoting A-(/>nitrophenylethyl)-gIycine subunits (1-36) and even
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40
replacing them with achiral (V-(p-nitrobenzyl)-glycine (1-37) had virtually no effect on
activity. Substituting the 6-chloroindole at position 5 o f the achiral backbone resulted in
the best inhibitor in this series (1-38), with an IC 5o o f 6.6 pM.
A final peptoid was
prepared to determine the contribution o f the />nitrobenzyl group at position 3 by
replacing it with hydroxyl ethyl side chain (1-39).
The resulting 2-fold decrease in
activity (IC 5o = 12.7 pM) suggested that the />nitrobenzyl group contributes to binding.
H 07 <
HO 1
o 2n
o 2n
O
1-36: IC50 = 19.5 »M
hN
^
'
N^
o
N
' ^
N^
N
^
N' ^ N
J o
/L o
|
^ Y
J o
N^ N
H
2
nh2
HO'rtp
P.
I
H
N
^ N^
o 2n
I
N
^
N^
N
^
I
°
Tl
N^
N
^
°
I
N" - ^ ^ Y
N^
I nh2
o=s=o
o 2n
nh2
H O .//0
P.
I
1-38: IC50 = 6.6 pM
^ 'Y
J o
o=s=o
o 2n
1-37: IC50 = 18.2 pM
°
N^ N
H
N
^ N^
o 2n
v
I
N
^
N^
N
/ ^
I
0
N^
N
/
^
o 2n .
I
0
N^
>
J
I
-
^
N^
I nh2
^
N^ N H 2
o=s=o
H O ' \\
nh2
HO^P
I
1-39: IC50 = 12.7 pM
H
N
1
o 2n
o 2n
I
^
1
N^
N
^
'
I
0
N^
' N
1
' ^
1 o
H O ' \\
I
0
N^
N
I
'
^J o
o=s=o
NH2
Figure 19. Structure and IC 50 values o f scrambled chirality (1-36), achiral backbone (1-37), most potent
(1-38), and p-nitrobenzyl A hydroxyl ethyl (1-39) peptoid ligands.
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41
In summary, Appella and coworkers began with a peptoid that was rationally
designed to adopt a helical conformation and mimic the display o f p53 side chains Phel9,
Trp23, and Leu26 on the a-helix. However, this peptoid had no affinity for the MDM2
protein.
Removing the conformational constraint o f the backbone through the
incorporation of achiral monomers increased binding affinity, showing that canonical
peptoid helix is not quite correct for p53 mimicry, but a helical (or distorted helical)
conformation may nevertheless be required. Information on the structure o f the peptoid
in complex with the protein would facilitate future designs.
The modification o f the
hydrophobic side chains intended to interact with the protein surface resulted in very
small changes in binding affinity, suggesting that these groups are not positioned deeply
in their proposed binding sites. The greatest incremental increases in peptoid binding
were obtained through substitution o f the carboxylates in the original design with
phosphonates and the addition o f a p-nitro group to the phenylethyl side chains.
Observing such dramatic effects from manipulation o f the hydrophilic residues is unusual
and suggests that they are the greatest contributors to the interaction. In the end, low
micromolar binders were prepared, and further development o f peptoid inhibitors o f the
p53/MDM2 interaction would be greatly facilitated by structural characterization o f the
peptoid bound to MDM2.
1.6.2.2 Combinatorial Approach
Kodadek and coworkers took a completely different approach in both purpose and
method to the development o f peptoid ligands for MDM2. Recognizing the potential of
proteomics for medicine and biology,94 Kodadek et al. have utilized peptoids as highaffinity protein capture reagents.95 Using microwave-assisted96 combinatorial chemistry
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42
and on-bead screening to construct and assay large peptoid libraries, they identified a
peptoid (1-40, Figure 20) that binds to a MDM2-maltose binding protein (MBP) fusion
protein.
To validate the hit, a re-synthesized and purified sample was tested by
isothermal titration calorimetry (ITC) and found to have an IC5o o f 37 pM. Peptoid 1-40
did not interact with the MBP alone, but Appella found that it also had no activity in a
p53/MDM2 FP assay,93 suggesting that this peptoid binds to MDM2 at a site distinct
from the p53 peptide.
While attached to the TentaGel macrobeads, weakly binding
ligand 1-40 could selectively capture MDM2 in a complex mixture o f proteins.96
NH2
0=S=0
o=s=o
nh
nh
1-41:
2
o=s=o
2
nh
nh
2
2
s.
Kd = 1.3 nM, ITC
N
H 2N
N ^ jj^
OH
O
NR
Figure 20. Peptoid (1-40) and chimeric peptoid-chalcone (1-41) ligands for MDM2 (refs. 95 and 97).
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43
In a slightly different approach, a bivalent high-affmity ligand for MDM2 was
developed by capping a different peptoid library with a chalcone.97
The resulting
chimeric chalcone-peptoid ligand 1-41 showed greater affinity for the MDM2 protein (Ka
= 1.3 pM) than either o f its constituents (peptoid or chalcone alone). Immobilization of
1-41 on a surface created a protein-detecting array that was able to bind MDM2
selectively. These results may facilitate protein profiling experiments that could perhaps
quantify the amount o f MDM2 in a biological sample and determine if MDM2 is
overproduced in a particular cancer, which would be helpful in “personalizing” treatment
with p53/MDM2 inhibitors.
1.6.3 Terphenyls
Hamilton et al. used a terphenyl derivative to mimic the a-helical region o f the
p53 peptide and disrupt p53/MDM2 complexation.98 These workers proposed that, in the
preferred staggered conformation o f the terphenyl backbone, substituting appropriate
alkyl or aryl substituents at the three ortho positions o f the terphenyl scaffold would
cause these substituents to be projected with a similar distance and angular relationship to
the i, i + 4, and / + 7 side chains along one face o f an a -h e lix ." A series o f potential
terphenyl inhibitors was prepared via a modular synthesis by sequential Suzuki couplings
of the
appropriate
or/7zo-substituted
methoxyphenylboronate
and
phenyltriflate.
Screening in a p53/MDM2 FP assay identified 1-42 (Figure 21) as the most potent
inhibitor with a Kj = 182 nM. Further studies revealed that the choice o f side chain,
relative positions o f the side chains on the scaffold, and length o f the scaffold were all
critical to the interaction. The binding mode o f the terphenyls was investigated using 'H15N HSQC NMR, and the compounds consistently produced chemical shift changes for
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44
the residues within the p53-binding pocket on MDM2.
Compound 1-42 caused a
significant change in the chemical shift o f MDM2 residue L85 deep within the
tryptophan binding pocket, possibly indicating that the 2-naphthylmethylene side chain
inserts into this pocket.
One concern about the terphenyl scaffold is its extreme
hydrophobicity, which may result in non-specific interactions. To address this, Hamilton
and coworkers tested an underivatized terphenyl and found that it did not measurably
interact with MDM2. The selectivity o f compound 1-42 was assessed by comparison
with 1-43, a potent inhibitor o f the Bcl-xi7Bak protein-protein interaction.100 Compound
1-42 was found to bind to MDM2 100-fold more tightly than 1-43, and 1-42 was 14- and
82- fold selective for MDM2 over
B c 1 -X l
and Bcl-2, respectively. However, compound
1-42 was inactive in cells.101 Another terphenyl derivative, 1-44, had modest affinity in
the FP assay and similar activity in a p53/MDM2 ELISA (IC50 = 20 pM).
This
compound was able to activate p53 in cells at a 30 pM concentration.
The
pharmacokinetics o f this class o f a-helix mimetics needs considerable investigation.
C 0 2h
C 0 2h
1 -4 2 : Ki = 0 .1 8 2 pM , p 5 3 /M D M 2
c o
2h
1-4 3 : Kj = 2 5 .7 pM , p 5 3 /M D M 2
c o
2h
1-44: K; = 3 .8 3 pM , p 5 3 /M D M 2
K| = 2 .5 0 p M , Bcl-xL/B a k
K| = 0 .1 1 4 pM , Bcl-xL/B a k
IC 5 0 = 2 0 pM , p 5 3 /M D M 2 E L IS A
K| = 1 5 .0 p M , B cl-2 /B a k
Kj = 0 .1 2 1 p M , B cl-2 /B a k
active in cells a t 3 0 p M
Figure 21. Terphenyl inhibitors of the p53/MDM2 interaction.
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45
The terphenyl scaffold has been widely applied to the mimicry o f a-helical
protein recognition surfaces, including the C-helical region of gp41, which is important
for HIV viral fusion.102
The scaffold has recently been improved through the
incorporation o f a 1,6-disubustitued indane at the central position o f the terphenyl to
mimic the i + 3 position of the a-helix as well (1-45, Figure 22).
IOT
The solubility o f the
scaffold has been increased by preparation o f a terpyridine derivative (1-46).104 Other
similar scaffolds have been proposed (1-47)105 and utilized (1-48)106 as a-helical
mimetics.
c o 2h
c o 2h
/ + 7 R-
i+7 R t X 0 2H
/+ 3
R2
/ + 4 R2
N„
i + 4 R3
/ R!
/ R
ISk
c o 2h
1-46
1-47
1-48
Figure 22. Structures of a-helix mimetics: diphenylindane (1-45, ref. 103), terpyridine (1-46, ref. 104),
biphenyl (1-47, ref. 105), and terephthalamide (1-48, ref. 106).
1.6.4 P-Hairpin Protein Epitope Mimetics
Robinson and coworkers have successfully mimicked the a-helix o f p53 with a
cyclic P-hairpin to generate inhibitors o f the p53/MDM2 interaction.107 They noticed that
the distance between the Ca atoms o f the Phel9 and Trp23 residues on one face o f the
MDM2-bound p53 a-helix was similar to the distance between the C'„ atoms o f two
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46
residues (i and / + 2) along one strand o f a P-hairpin. A cyclic P-hairpin (1-49, Figure
23) was designed as a scaffold to hold the side chains of a phenylalanine and a tryptophan
residue in the correct relative positions so that they could interact with their respective
binding sites on MDM2.
These workers used a D-Pro-L-Pro dipeptide turn unit as a
template to stabilize the P-hairpin conformation.108 The peptide was synthesized on 2chlorotrityl chloride resin, released with 1% TFA, and cyclized via coupling o f Asn5 and
Leu4.
Compound 1-49 was identified as a weak lead with an IC5o o f 125 pM in a
solution-phase competition SPR assay.
Phe8
(6C I)-T rp 3
1-49: IC50 = 125 pM, SPR
\a
1-50: IC50 = 140 nM, SPR
Figure 23. P-Hairpin inhibitors of the p53/MDM2 interaction (ref. 107).
Using parallel synthesis,109 Robinson et al. elucidated the structure/activity relationships
among compounds related to 1-49. The optimized inhibitor 1-50, with an IC50 o f 140
nM, was nearly 900-fold more potent than the lead.
A P-hairpin conformation was
observed for the free ligand by 2D-NMR spectroscopy, and the *H-15N HSQC NM R data
for the complex showed that the P-hairpin binds in the p53-binding site on MDM2. A
crystal structure o f the MDM2/1-50 complex was obtained (Figure 24).110 Residues
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47
P hel, (6-Cl)Trp3, and Leu4 o f 1-50 were observed to bind in the hydrophobic p53binding cleft on MDM2. The side chain o f the (6-Cl)Trp3 residue inserts deeply into the
Trp23 binding pocket on MDM2. These three side chains are found on one face o f the Phairpin together with Trp6 and Phe8; this cluster o f hydrophobic residues stabilizes the P-
Figure 24. Crystal structure o f 1-50 in complex with MDM2 (ref.l 10, PDB code 2AX1).
hairpin conformation. Interestingly, the side chains o f Trp6 and Phe8 in the second Pstrand stack on both sides of MDM2 residue Phe55 on the side o f the cleft. Thus, these
residues help to preorganize the binding conformation o f the ligand and make additional
favorable van der Waals contacts, both o f which contribute to the high potency o f 1-50.
This class of compounds has been applied to a broad range of targets, demonstrating the
utility of these protein epitope mimetics.111 The greatest challenge will be to achieve
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48
activity in vivo against an intracellular target like p53/MDM2, because these hairpin
peptides will likely exhibit poor cell permeability. It is truly remarkable that a-helix and
p-sheet protein architectures are found to be interchangeable in this context, lending
credence to the idea o f oligomeric scaffolds as proteomimetics.
1.6.5 p-Oligobenzamides
Guy and coworkers used computational methods to design de novo a modular
scaffold (1-51, Figure 25) that mimics the projection o f the /, i+4, and i+7 positions o f an
a-helix.112 These researchers synthesized a combinatorial library in which the size and
nature of the side chains were varied. Compound 1-52 was identified as the most potent
molecule, with a Kd o f 12 pM. Binding o f 1-52 in the p53 pocket on the surface o f
MDM2 was observed by NMR. O f note is the fact that 1-52 only contains two o f the side
chains from the original design, and mimetics containing an additional monomer unit
were much less active.
F
NC
HN
OH
HN
HN
1-51: scaffold
1-52: Kd = 12 pM
Figure 25. Structure of p-oligobenzamide scaffold.
1.6.6 P-Peptides
Inhibitors o f the p53/MDM2 protein-protein interaction based on the P-peptide
scaffold have been investigated in both the Gellman
117
and Schepartz
177 177
'
laboratories.
p-Peptides (oligomers of p-amino acids) are a well-characterized class o f foldamers that
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49
can adopt a wide variety o f discrete secondary structures.2 It is interesting to note that
(3-peptides arose from fundamental studies directed toward understanding the folding of
oligomers rather than from a desire to develop a scaffold for a-helix mimicry. (3-Peptides
were extensively characterized structurally long before their application to the inhibition
of protein-protein interactions.
The most intensively studied (3-peptide secondary
structure is the 14-helix, which is defined by 14-membered ring N-H(—>0=C/+2 hydrogen
bonds between backbone amide groups.
Seebach et al. discovered that P-peptides
composed exclusively o f p3-residues (Figure 26) can form the 14-helix,114 and Gellman
and coworkers have shown that use o f p-amino acids with a six-membered ring
constraint, such as ^ram-2-aminocyclohexanecarboxylic acid (ACHC) or tram -4aminopiperidine-3-carboxylic acid (APiC), lead to a dramatic enhancement in 14-helix
stability relative to p3-amino acids.115 On the other hand, five-membered ring constraint
(as in fram-2-aminocyclopentanecarboxylic acid (ACPC) or tram-3-aminopyrrolidine-4carboxylic acid (APC)) leads to the 12-helix, defined by 12-membered ring C=0,—»HN /+ 3 hydrogen bonds.116 Combining constrained and acyclic residues allows one to
prepare P-peptides that display specific arrays o f diverse side chains on a stable threedimensional scaffold.117 Derivatization o f p-peptide monomers gives access to more
diversity than is available with a-am ino acids, since the position o f the substituent can be
varied between the a and p. carbons o f the p-amino acid residue, to generate p - or p amino acids, respectively. The predictable relationship between p-amino acid sequence
and folding at short oligomer lengths raises the prospect of endowing P-peptides with
useful functions.
A number o f applications have been reported for P-peptides.118
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Proteolytic119 and metabolic120 stability and the prospect of intracellular delivery118’
make P-peptides very attractive from a biomedical perspective. Both 12- and 14-helical
P-peptide scaffolds (Figure 27) have been used for the design o f p53/MDM2 inhibitors.
p3-amino acid
p2-amino acid
ACHC
APiC
ACPC
Figure 26. Structures of P-amino acids.
a-Peptide
a-Helix
p-Peptide
14-Helix
12-Helix
Figure 27. Structures of a - and P-peptide helices.
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APC
51
1.6.6.1 12-Helical B-Peptides
Gellman and coworkers sought to employ the P-peptide 12-helix for development
o f p53 mimics that would inhibit the p53/MDM2 interaction.121 They believed that the
12-helix was particularly well-suited to mimicking the a-helix.2 Both have an N->C
helical dipole.
The two helices also have a similar pitch despite a difference in the
number of residues per turn, 5.4
A
and 3.6 for the a-helix and 5.3
A
and 2.5 for the P-
peptide 12-helix, respectively. The internal diameter o f the P-peptide 12-helix is only
slightly larger than that o f the a-helix, 2.3
A
and 2.2
A,
respectively.
However,
incorporation o f the 12-helix-promoting ACPC residues makes the external diameter of
the P-peptide 12-helix considerably wider, so that its fit into the cleft on MDM2 is much
tighter than for an a-peptide a-helix.
Using a helical wheel diagram and molecular
modeling with the crystal structure, both right- and left-handed helical designs,
corresponding to the (S,S) and (R,R) configurations o f ACPC respectively, were
investigated. After several iterations o f the design, synthesis, and testing process, the
most active derivative (1-53, Figure 28) had an IC50 o f approximately 250 pM in a
p53/MDM2 ELISA.
COoH
1-53: IC50 - 2 5 0 |iM, ELISA
C 0 2H
Figure 28. 12-Helical P-peptide inhibitor.
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52
1.6.6.2 14-Helical B-Peptides
Schepartz et al. explored the P-peptide 14-helix as a scaffold for displaying a set
of side chains in a protein-mimetic manner.
.2 2
(3-Peptides were designed to mimic the
projection o f the three hydrophobic side chains (Phel9, Trp23, Leu26) from the a-helical
segment o f p53.123 Since there are three residues per turn of 14-helix, (33-hPhe, (33-hTrp,
and |33-hLeu were incorporated at positions 3, 6, and 9 o f deca-P-peptide 1-54 (Figure 29)
in order to be arrayed along one helical face in the folded conformation. The 14-helical
1-54: IC 5 0 = 94 nM, FP
1-55: IC 5 0 = 8 0 (.iM, FP
1-56: Not active
1-57: IC 5 0 = 13 p.M, FP
F ig u r e 2 9 .
14-Helical P-peptide inhibitors o f the p53/MDM2 interaction.
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53
■>
conformation was stabilized by p -hOm/p -hGlu side chain/side chain salt bridges
along the solvent-exposed second face of the helix.124 The third face, which was not
designed to interact with the protein in the bound state, was composed o f p3-hVal
residues. The overall design is unusual from a structure-based design standpoint because
a left-handed P-peptide helix is being used to mimic a right-handed a-helical structure.
Furthermore, P-peptide 1-54 is designed to bind in an opposite N->C orientation than the
p53 a-peptide. In spite o f these and other structural differences between the P-peptide
14-helix and the a-helix, these compounds were shown to bind with modest affinity to
MDM2. P-Peptides containing alternative arrangements o f the p3-hPhe, p3-hTrp, and p3hLeu residues or p3-hAla-mutants thereof were not active.
Deca-P-peptide 1-54 was reported to inhibit the interaction between MDM2 and a
fluorescently labeled a-peptide corresponding to residues 15-31 o f wild type p53 with an
IC 5 0 o f approximately 94 pM in a FP competition assay.122
Molecular modeling
suggested a potential steric clash between the C-terminus o f the P-peptide decamer with
the MDM2 protein.
Truncation o f the original design produced octa-P-peptide 1-55,
which was more potent (IC 5 0 = 80 pM) but less selective. Investigation by 2D NMR
spectroscopy suggested that P-peptide 1-54 adopts a slightly distorted 14-helical
conformation in methanol.
19 T
Schepartz and coworkers have proposed that P-peptides
composed entirely o f p3-residues can depart slightly from an idealized 14-helical
conformation, as expected based on the well-known flexibility and low intrinsic 14helical propensity of p3-residues,125 and they have speculated that such distortion is
necessary for optimal binding to the MDM2 cleft. Further efforts to optimize the binding
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54
affinity o f P-peptide 1-54 using a combinatorial approach identified 1-57 as a 7-fold
more potent inhibitor (IC 5 0 = 1 3 pM), approaching the activity o f the p53-derived a peptide.126 The triple p3-hVal to p3-hlle substitution makes that face o f the helix more
hydrophobic. Since this triple change improves the binding affinity, we suspect that the
all P -lie face of 14-helical p-peptide 1-57 interacts with the MDM2 surface.
These proof-of-principle experiments demonstrate the potential biological
applications of p-peptides.
177
These studies show also that structure-based design is of
only limited utility for the initial discovery o f p-peptide inhibitors o f protein-protein
interactions. The difference in activities o f the 12- and 14-helical designs demonstrates
the importance of scaffold choice for a particular target, and hence, the need for a large
set o f foldamer backbones with distinct conformations. Improved methods for the rapid
and efficient exploration of foldamers as potential inhibitors o f protein-protein
interactions is very important and will be discussed in the following chapters.
1.6.7 Miniproteins
Schepartz and coworkers have developed a miniature protein capable o f inhibiting
the p53/MDM2 interaction.128 The three critical MDM2 contact residues from p53 (Phe,
Trp and Leu) were grafted onto the a-helical segment o f the avian pancreatic polypeptide
(aPP).
17 0
aPP is a well-folded 37-residue polypeptide consisting o f an 8-residue
polyproline II helix linked through a type I P-tum to an 18-residue a-helix.130 Screening
a phage library that varied five presumed non-contact residues in the a-helix o f aPP
identified peptide 1-58 with a low micromolar IC50 value (Table 1). Further optimization
was achieved by varying the residues designed to contact MDM2 in the bound state (1-
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55
59) but not through an attempt to increase the conformational stability o f the structure
(1-60). Since these sequences are genetically encodable, they could be used as probes of
the p53 pathway in cells.
Table 1. Sequence and activity of miniature protein p53/MDM2 inhibitors.
No.
aPP
1-58
1-59
1-60
Sequence
GPSQPTYP
GPSQPTYP
GPSQPTYP
GKSWMTVP
ICsn(uM)
GDDAPVEDLIRF
GDDAPVEDLIRF
GDDAPVEDLIRF
GDDAPVEDLIRF
YNDLQQ YLNVVTRHRY C
KFLLQ WYLLALTRHRY A A AC
KFLLQ WYLLALSLRNY A AAC
KFLLQ WYLLALTRHRY A A AC
3.2
1.6
>150
1.6.8 Summary of Proteomimetic Inhibitors
A number o f oligomeric scaffolds have been successfully developed for structural
mimicry o f the a-helix.
These structures are typically much larger than the small
molecule inhibitors discussed previously, which allows the oligomeric molecules to
access spatially separated binding pockets on the protein surface. The larger size o f the
proteomimetics, relative to small molecule inhibitors, allows the oligomers to bury more
surface area to increase their binding affinity.
However, the potency o f the
proteomimetics is often less responsive to side chain substitution relative to small
molecules, indicating that the hydrophobic groups are not necessarily binding within the
pockets on the surface of the protein as designed. Another common shortcoming o f the
oligomeric scaffolds is poor cell permeability, which in general makes them unsuitable
for intracellular targets like MDM2. Nevertheless, important advances have been made
that will be applicable to the large number o f protein-protein interaction targets that,
unlike p53/MDM2, have been intractable to small molecule approaches. Many o f these
protein-protein interactions exist on the cell surface, obviating the need for proteomimetic
inhibitors to be cell permeable.
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56
1.7 Other Strategies for Activation of the p53 Pathway
While a majority o f the research effort has focused on inhibiting the p53/MDM2
interaction, a variety of other approaches to activation o f the p53 pathway have been
reported. Molecules that reactivate mutant p53 through stabilization o f the wild-type
conformation have been proposed.131"142
A ligand for p53 that acts as a potential
allosteric inhibitor of the p53/MDM2 interaction has also been reported.143 Another
common strategy, inhibition o f homooligomerization, would not be therapeutically useful
in this case because p53 is only active as the tetramer. While these ideas demonstrate
creativity, a number of conflicting reports and poorly characterized mechanisms o f action
have hindered progress.
1.7.1 Reactivation of Mutant p53
Strategies for the reactivation o f mutant p53 in vivo would be useful for treatment
of approximately 50% of cancers. For a tumor to escape the consequences o f functional
p53, the protein may be mutated at a number o f positions to prevent signaling either
through destabilization o f the protein’s tertiary structure or the protein-protein or DNAprotein signaling complex.131
A variety o f molecules that bind to mutant p53 and
stabilize its wild-type conformation, thus restoring its function, have been described but
not elaborated. A short peptide (REDEDEIEW-NH 2 ) that binds to and stabilizes the core
domain o f p53 has been proposed to act as a chaperone to maintain existing or newly
synthesized destabilized p53 mutants in a native conformation.132 The p53 protein is
active when it is tetrameric,133 so Giralt and coworkers developed a tetraguanadinium
ligand (1-61, Figure 30) that binds to the surface o f the tetramerization domain to
stabilize the active oligomeric state o f the protein.134
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57
1-62: R = H, PRIMA-1
1-64
1-65: MIRA-1
1-66: RITA
1-63: R = Me, PRIM A-1MET
Figure 30. Structures of molecules designed to activate p53.
A small molecule named PRIMA-1 (p53 reactivation and induction o f massive
apoptosis, 1-62) was reported by Selivanova et al. to be capable o f restoring wild-type
function to mutant p53.
I3S
However, more recent investigations have shown that while
PRIMA-1 and derivatives thereof (1-64) do selectively eliminate cells expressing mutant
p53, there is no evidence o f restoration o f wild-type p53 properties.136 In fact, a more
potent derivative named PRIMA-1met (1-63) did not affect mutant p53 protein levels but
did upregulate the expression o f PUM A,137 a BH3-only pro-apoptotic factor that is
regulated in both p53-dependent and -independent manners.138 While the idea continues
to be investigated with small molecules (1-65)139 and peptides,140 the molecular
mechanism by which a small organic molecule can induce refolding o f a mutant protein
remains unclear.
Efforts to date have been reminiscent o f the chemical genetics
approach,141 where a small molecule that produces the desired phenotype is identified
(i.e., apoptosis via the p53 pathway), but the actual molecular target is difficult to
discern.142
1.7.2 Small Molecule Ligand for p53
Selivanova and coworkers have identified a small molecule named RITA
(reactivation o f p53 and induction o f tumor cell apoptosis, 1-66), which they believed to
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58
bind to p53 rather than MDM2, thereby inhibiting the p53/MDM2 interaction, perhaps
by altering the conformation of p53.143 They studied the interaction o f RITA with p53
using fluorescence correlation spectroscopy and measured a IQ of 1.5 nM. RITA also
showed activation o f the p53 pathway in cells at a 10 pM concentration. The authors
proposed that RITA binds to p53 and induces a conformational change in the protein that
disrupts its interaction with MDM2, stabilizing p53 and causing its accumulation in cells.
However, Holak and coworkers investigated the binding o f RITA to p53, MDM2, and the
complex by NMR and found no evidence to support the idea that RITA inhibits the
p53/MDM2 interaction.144 The true mechanism o f action remains to be elucidated.145
1.8 Structural Insights
Recent elucidation o f the uncomplexed MDM2 protein structure by NMR
spectroscopy has revealed a number o f conformational adjustments that accompany the
binding o f p53 (Figure 31 and Figure 32).146 As had been suggested previously,147
MDM2 residues 18-24 form a flexible “lid” over part o f the binding site that must be
displaced upon binding o f the p53 peptide. The two MDM2 sub-domains that form the
walls of the hydrophobic cleft must swing apart to open the groove between them and
expose the deeper hydrophobic pockets for the p53 residues Phel9, Trp23, and Leu26.
Conformational plasticity o f the binding groove is a common theme among proteinprotein interactions that makes them such difficult targets.148 In this case, the other
reported crystal structures o f MDM2 in complex with peptides and small molecules are
very similar, and the most productive conformation to target has been the bound state.
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Figure 31. NMR structure of free MDM2 (ref. 146, PDB code 1Z1M).
Figure 32. Comparison of MDM2 structure in the free (left) and bound (right) states.
1.9 Conclusions
For a long time p53/MDM2 was thought to be an “undruggable” protein-protein
interaction. The community’s eyes were opened by the solution o f the p53/MDM2 co­
crystal structure, which revealed a focused binding interface.
Ten years have passed
since then, and still the race is on to advance a p53/MDM2 inhibitor into clinical trials.
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Industrial researchers have been forced to revise their structure-based design and
combinatorial approaches for targeting protein-protein interactions rather than enzyme
active sites, though the 6-chloroindole pharmacophore has served them well. Only now
are medicinal chemists refining their small molecule inhibitors for potency, selectivity,
and pharmacokinetics. This considerable lag time opened a window o f opportunity for
academic researchers to develop a whole new class o f compounds known as
proteomimetics. In the process, much is being learned about protein-protein interfaces
and what type of molecule is needed to inhibit a protein-protein interaction. While these
proteomimetics may not be the molecules that go to the clinic, there are many other
protein-protein interactions that are proving more intractable to small molecule
approaches than p53/MDM2, to which these lessons will be applied.
A class of
molecules, like foldamers, that is able to mimic a variety of protein structures through
variation o f the scaffold, that displays conformational stability, and that allows side chain
diversity, could be broadly applicable to the inhibition o f protein-protein interactions if
sufficient combinatorial synthetic techniques were developed to allow rapid exploration
o f potential inhibitors for new protein targets.
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73
Chapter 2
Application of Microwave Irradiation to the
Synthesis of 14-Helical P-Peptides
NH
nh
2
c o
2h
o
H 2N
v
NZ^T7
Purity =
N
H
^
N
H
Standard SPPS
Conditions
Oil Bath,
LiCI in NMP
Microwave Irradiation,
LiCI in NMP
21%
53%
88%
Portions of this chapter have been published as:
Murray, J. K.; Gellman, S. H. “Application of Microwave Irradiation to the
Synthesis o f 14-Helical [3-Peptides,” Organic Letters 2005, 7 (8), 517-520.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
74
2.0 Brief Summary of Chapter
We have evaluated the effects o f microwave irradiation on the solid-phase synthesis o f P~
peptides. Sequences designed to adopt the 14-helix, especially those containing the structurepromoting residue, /ra«.v-2-aminocyclohexanecarboxylic acid (ACHC), suffer from poor
synthetic efficiency under standard solid-phase peptide synthesis (SPPS) conditions.
A
comparison o f microwave and conventional heating shows that both provide excellent synthetic
results for shorter sequences; however, we identify a clear benefit from microwave irradiation for
longer p-peptides, including a 10-fold reduction in synthesis time.
2.1 Background
Microwave irradiation has been applied to a large and expanding range o f chemical
transformations in recent years, and this method o f energy transfer to a reaction mixture can
provide impressive enhancements in product yield, selectivity and/or reaction rate.1 However,
there have been only a handful o f reports on the application o f microwave methods to SPPS, and,
as discussed below, these reports all have limitations.2 This paucity of attention presumably
results from the fact that most peptide sequences, particularly those comprised o f conventional
a-am ino acid residues, are readily available via standard SPPS methods.3 We have been
motivated to explore microwave effects on carboxamide bond-forming reactions by our need to
streamline the synthesis o f p-amino acid oligomers (P-peptides).4 Interest in these unnatural
oligomers is growing because they can adopt a wide variety o f discrete secondary structures; the
predictable relationship between p-amino acid sequence and folding opens the prospect o f
endowing p-peptides with useful functions.
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75
2.1.1 14-Helical P-Peptides
The most widely studied P-peptide secondary structure is the 14-helix, which is defined
by 14-membered ring N-H;—>0=C,+2 hydrogen bonds between backbone amide groups.4
Seebach et al. discovered that P-peptides composed exclusively of p3-residues can form the 14helix,5 and we have shown that use o f P-amino acids with a six-membered ring constraint, such
as /‘ram ,-2-aminocyclohexanecarboxylic acid (ACHC) or ?r<my-4-aminopiperidine-3-carboxylic
3
acid (APiC), leads to a dramatic enhancement in 14-helix stability relative to P -amino acids.
6
Combining constrained and p3-residues allows one to prepare P-peptides that display specific
constellations o f diverse side chains on a stable three-dimensional scaffold.
A variety o f
applications o f 14-helical P-peptides have been reported,7 including the inhibition o f proteinprotein interactions.74 For many such applications it would be desirable to prepare and screen
large p-peptide libraries. Such libraries are most useful if the compounds are generated with
sufficient purity for direct evaluation. However, standard SPPS protocols are not efficient
enough to support a library approach for some types o f p-peptides, especially those with a
propensity to adopt the 14-helical conformation.
NH2 O
NH2 O
h, n A
A
r
oh
P3-amino acid
ACHC
N
H
^ ' OH
APiC
2.1.2 p-Peptide Synthesis
14-Helical P-peptides can be prepared via the manual or automated methods originally
developed for a-peptide synthesis, but efficiency is greatly diminished for these P-peptides
relative to typical a-peptides. Solid-phase P-peptide synthesis (Figure 1) is accomplished by: 1)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
76
loading the C-terminal (3-amino acid residue onto the solid support via reaction o f the
carboxylic acid with a suitable linker; 2) removing the base-labile 9-fluorenylmethoxycarbonyl
(Fmoc) group from the main chain nitrogen by treatment with piperidine; 3) coupling the next
monomer in the chain by addition o f the appropriate activated P-amino acid residue (steps 2 and
3 are repeated until the desired oligomer length is achieved); 4) cleavage o f the peptide from the
solid support with simultaneous global side chain deprotection by treatment with trifluoroacetic
acid(TFA). Seebach et al. have reported profound difficulties in the preparation o f relatively
Fmoc A
O
Loading
N '^ 'O H
+ HO
R
Deprotection
F m o c H N '^ ^ ^ O
piperidine,
15 min
O
FmocHN'j'^"OH ■t.* u
kX J L C
H pN
HBTU, HOBt, DIEA
1.5 h
h2n^
pg-r2
^X >
r1
q
PG .
Fm o cH N
R2
O
^ N
R1
1) piperidine, 15 min
O
^ O
- O
r
2
o
Q
q
-o -Q
H
C leavage
,_o
r1 o
FmocHN/ ^x ^ ^ s'0
PG .
Coupling
R1 o
MSNT, Melm
I X
r
1
o
2),ritlU0r0aCet'C aC ia2-
n3
Onh
M elm
pip erid in e
DIEA
no
="°-{J
HO
W a n g Linker
^
¥
o=s=o
2
/ PFe,
-N .
P o ly s ty re n e
R e s in o r B e a d
9 0 p m d ia m e te r
8 0 -1 0 0 pm ol
\
/
N UAN
\
O'
M SN T
HBTU
Figure 1. Solid-phase synthesis of (3-peptides.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
OH
HOBt
77
short sequences composed exclusively of p3-residues, with problems in both the Fmocdeprotection and amide bond formation steps, usually starting with the sixth residue from the Cterminus.8 For example, their optimized stepwise SPPS conditions provided a nona-(3-peptide in
only 12% purity.8c We have observed even more severe problems with analogous sequences that
contain mixtures o f p3-residues and ACFIC residues.70
2.1.3 Microwave-Assisted Peptide Synthesis
We contemplated the application o f microwave methodology to improve the synthesis of
P-peptides, but we were surprised to learn how little precedent was available for microwaveenhanced peptide coupling reactions. The first report o f microwave-assisted solid-phase
synthesis of an a-peptide was performed in a domestic microwave oven, making the conditions
difficult to reproduce.23 A more recent report describes the synthesis o f only very short a peptides (< three residues) using conditions that we quickly found to be counterproductive (>
110°C, closed vessel).2b In both cases, no comparison was made between conventional heating
and microwave irradiation, i.e., it was not clear from the available literature whether microwave
irradiation provides any advantage relative to more traditional methods o f reaction rate
enhancement for SPPS.
Recently, an automated microwave peptide synthesizer became
available, though neither reaction conditions nor synthetic results have been published.20 The
microwave-assisted synthesis o f glycopeptides has also been reported.2d
2.2 Synthetic Optimization of B-Peptide Solid-Phase Synthesis with Microwave Irradiation
The ability o f Schepartz and coworkers to identify moderate 14-helical p-peptide
inhibitors of the p53/MDM2 interaction suggested that we include this scaffold in our efforts to
discover foldamer proteomimetics.7d As described in Chapter 1, deca-P-peptide 1-54 was
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78
reported to have moderate activity against the p53/MDM2 interaction (IC 5o value o f 94 pM ).7d
We wondered whether truncation would relieve a potential steric clash with the MDM2 protein
at the N-terminus o f the oligmer and if substituting ACHC for p3-hVal on the supposedly non­
interacting face o f the 14-helix would increase the binding affinity of the oligomer by
preorganizing the ligand in the proposed binding conformation and reducing the entropic cost
associated with binding (2-1 and 2-4, Figure 2). This experiment would be a first step toward
testing whether these P-peptides are binding in a 14-helical conformation and if the p3-Val face
of the 14-helix makes important contacts with the MDM2 protein in the bound state. Synthesis
and purification o f the proposed ACHC-containing analogues was rather challenging, and we
came to the conclusion that optimization of the solid-phase synthesis o f 14-helical P-peptides
was critical to our ability to explore their biological application.
2-1
2 -2
2 -3
R = H
9
r = h2n£ ^ >
R = Fm oc
o
o
Figure 2. 14-helical P-peptides for synthetic optimization.
2.2.1 Hexa-P-Peptide Test Case
Hexamer 2-1 was selected as our initial target for synthetic optimization because this ppeptide contains a variety o f side chain functionalities and the minimum proportion o f ACHC
residues necessary for high 14-helicity.6e Oligomer 2-1 is just long enough to present a synthetic
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
challenge, allowing us to focus on the efficient incoporation o f a single monomer.
o
79
In
retrospect, our choice of hexa-P-peptide 2-1 as the test case sequence was essential to all the
work described in the following chapters. Because the synthesis of 2-1 was so difficult, we were
forced to develop new, highly effective methods that have since proven to be broadly applicable
across several foldamer classes and sequences. HPLC analysis of the crude products was a rapid
and reliable assay for monitoring our progress toward the goal through quantification o f each
component in the mixture.
Solid-phase synthesis o f 2-1 began by anchoring Fmoc-(S)- p3-homoglutamic acid8a to
polystyrene (PS) resin via a Wang linker.8b’9 Polystyrene resin was chosen because o f our
ultimate interest in preparing P-peptide libraries on polystyrene macrobeads using split-and-pool
methods (Chapter 3).10 The manual SPPS o f p-peptide 2-1 was performed under standard
conditions, with double coupling and double deprotection o f the N-terminal ACHC residue (i.e.
ACHC1, in standard peptide numbering). Although the penta-p-peptide precursor was > 95%
pure, hexamer 2-1 produced in this way was only 55% pure (Figure 3 and Figure 4A). The two
major impurities were the unreacted pentamer (33%) and the Fmoc-protected hexamer 2-2 (8%).
We focused our efforts on increasing the purity o f P-peptide 2-1 by optimizing the coupling and
deprotection reactions o f ACHC1 using microwave irradiation in order to reduce the amounts o f
the ACHC-deletion and Fmoc-protected side products.
2.2.2 Experimental Set-Up for Microwave-Assisted P-Peptide Synthesis
An experimental set-up compatible with both SPPS and the CEM Discover monomode
microwave reactor (the same reactor supplied with the automated microwave peptide
synthesizer, the CEM Liberty)20 was designed and constructed (Figure 5). After a brief
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
80
100
%
p-Peptide 2-1
^ ACHC1 -Deletion
3 P-Peptide 2-2
p-Peptide 2-3
Peak
Area
Percent
NMP
LiCI
Manual
DMF
NMP
LiCI
Microwave
DMF
NMP
LiCI
Oil Bath
Reaction Conditions
Figure 3. Amount of P-peptide 2-1 and major impurities (peak area percent, from analytical reverse-phase (RP)
HPLC monitored via UV absorbance at 220 nm) resulting from different synthetic conditions. All coupling and
deprotection reactions in the synthesis of the hexamer were conducted under the given reaction condition, i.e.,
manual, microwave, or oil bath, as described below. The given solvent refers only to the coupling o f ACHC1; all
other coupling reactions were performed in DMF. ACHC1 was double coupled and double deprotected in all cases.
Manual: 15 min deprotection, 1.5 hr coupling, RT; Microwave: 4 min deprotection at 60°C; all couplings were 6 min
at 50°C in DMF, except for 6 min at 45°C in 0.8 M LiCI in NMP for ACHC1 where noted; Oil Bath: 15 min
deprotection, 1.5 hr coupling, 60°C.
Table 1. Data for Figure 3.
HPLC Peak Area Percent
Reaction Condition
Compound
Manual
Microwave
Oil Bath
DMF
NMP
DMF
NMP
DMF
NMP
LiCI
LiCI
LiCI
JKM V 291 JKM V 199 JKM V 083 JKM VI 003 JKM VI 067 JKM VI 037
Notebook
p-Peptide 2-1
55
43
80
94
80
85
ACHC1-Deletion
33
31
5
2
7
0
p-Peptide 2-2
8
21
0
0
2
12
p-Peptide 2-3
0
0
6
0
5
0
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
81
4000
ACHCdeletion
A! Manual-DMF
B) Manual-NMP/LiCl
i
3000
C) Microwave-DMF
2000
D) Microwave-NMP/LiCl
Abs (mV)
1000
E) Oil Bath-DMF
F) Oil Bath-NMP/LiCl
20
30
40
50
Time (min)
Figure 4. Analytical RP-HPLC chromatograms (UV absorbance at 220 nm) of P-peptide 2-1 prepared under
different conditions. All coupling and deprotection reactions in the synthesis of hexamer 2-1 were conducted under
the given reaction condition, i.e., manual, microwave, or oil bath, as described below. The given solvent refers only
to the coupling of ACHC1; all other coupling reactions were performed in DMF. ACHC1 was double coupled and
double deprotected in all cases.
A) Manual-DMF: All couplings for 1.5 hr in DMF at RT; 15 min deprotection at RT.
B) Manual-NMP/LiCl: All couplings for 1.5 hr in DMF at RT, except for double coupling ACHC1 in 0.8
NMP at RT; 15 min deprotection at RT.
M
LiCI in
C) Microwave-DMF: All couplings for 6 min in the microwave at 50°C in DMF; 4 min deprotection in the
microwave at 60°C.
D) Microwave-NMP/LiCl: All couplings for 6 min in the microwave at 50°C in DMF, except for double coupling
ACHC1 in 0.8 M LiCI in NMP at 45°C in the microwave; 4 min deprotection in the microwave at 60°C.
E) Oil Bath-DMF: All couplings for 1.5 hr in DMF at 60°C in the oil bath; 15 min deprotection at 60°C in the oil
bath.
F) Oil Bath-NMP/LiCl: All couplings for 1.5 hr in DMF at 60°C in the oil bath, except for double coupling ACHC1
in 0.8 M LiCI in NMP at 60°C in the oil bath; 15 min deprotection at 60°C.
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82
pasteur pipette
for N2 agitation
^ 10 mL glass vial
^^•'4 mL polypropylene
SPE tube
^
DMF or 0.8 m
LiCI in NMP
coupling solution
resin
polyethylene frit
luer-lock cap
1
alttliSgBSIrmi
IR sensor
■HR
Figure 5. Left) Experimental set-up for small-scale microwave SPPS of P-peptides (SPE = solid-phase extraction)
and Right) CEM Discover microwave (http://www.cem.com/synthesis/discover_s.asp).
optimization o f the power, temperature and time variables (Table 2), we adopted the following
reaction conditions: 50 W maximum power, 6 min coupling at 50°C, and 4 min deprotection at
60°C.
Using the built-in IR temperature sensor with our reaction vessel gave reproducible
results with the empirically-derived set temperatures, but the temperature measurements were not
accurate.11 At the end o f the deprotection reaction, the actual temperature o f the mixture was
measured using a thermometer and found to be 71 - 74°C, higher than the 60°C target
temperature. The actual temperature o f the coupling reaction was found to be 61 - 65°C, higher
than the 50°C target temperature. These discrepancies were later resolved via use o f a fiber optic
temperature probe (Chapter 4).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
83
Table 2. Time, temperature, and power optimization for microwave-assisted P-peptide synthesis. ACHC 1 was
double coupled at 50W for 6 min at the given temperature, unless otherwise noted.
Notebook
Temperature (°C)
JKM IV 157
JKM IV 177
JKM IV 211
JKM IV 213
JKM V 083
JKM VI 219
40
45
50
50
50
60
2-1
61
69
62
6 6
80
73
Percent HPLC Peak Area
ACHC2-2
2-3
Deletion
27
0
0
7
2
0
9
1
2
7
2
3
5
0
6
0
0
23
Comments
15 min, single couple
30 W
2.2.3 Microwave-Assisted Solid-Phase P-Peptide Synthesis
Synthesis in the microwave reactor gave the pentamer in high purity (similar to room
temperature synthesis conditions), but now p-peptide 2-1 was provided in 80% purity with only
5% o f the ACHC 1-deletion impurity and complete elimination o f the Fmoc-protected P-peptide
2-2 (Figure 3 and Figure 4C). This dramatic improvement relative to the room temperature
synthesis led us to extend our effort to the more difficult deca-p-peptide 2-4. Under standard
non-microwave conditions, the synthesis o f 2-4 is very inefficient (21% product purity; Figure 6
and Figure 7A). Use of microwave irradiation for all reactions gave only 57% purity (JKM V
181), indicating the challenge of coupling the final five residues in 2-4. Though the microwaveassisted synthesis was a great improvement relative to non-microwave conditions, we did not
believe that this level o f purity would be sufficient for the reliable screening o f crude P-peptide
product mixtures for future biological applications.
2.2.4 Further Attempts at Synthetic Optimization
We returned our focus to the N-terminal ACHC residue of hexa-P-peptide 2-1, reasoning
that further optimization o f this difficult coupling reaction would lead to improvements that
could be applied to the preparation o f longer targets such as 2-4. Altering microwave parameters
did not provide any benefit: increasing power, time o f irradiation or temperature led to an
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84
100-1
p-Peptlde 2-4
p3-hOrn1-Deletion
ACHC2-Deletion
80-
p3-hG!u4-Deletion
60-
Peak
Area
Percent
40-
20-
Manual
Microwave
Oil Bath
Reaction Conditions
Figure 6. Amount o f P-peptide 2-4 and major impurities resulting from different synthetic conditions. All coupling
and deprotection reactions in the synthesis of the decamer were conducted under the given reaction condition, i.e.,
manual, microwave, or oil bath, as described in Figure 3. ACHC2 and ACHC5 were double coupled and double
deprotected in all cases. ACHC2 and ACHC5 were coupled in 0.8 M LiCI in NMP in the microwave and oil bath
syntheses. All other coupling reactions were performed in DMF. (p3-hOm = p3-(5)-homoomithine; p3-hGlu = p3(.Sj-homoglutamic acid)
Table 3. Data for Figure 6.
HPLC Peak Area Percent
Compound
Reaction Condition
Manual
Microwave
Oil Bath
JKM V 235 JKM VI 013 JKM VI 009
Notebook
p-Peptide 2-4
21
88
53
p3-hOrn1-Deletion
0
1
6
10
1
7
ACHC2-Deletion
p3-hGlu4-Deletion
9
1
6
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85
1400
A) Manual
U J W^“ W
1000
B) Microwave
A b s (mV)
400
C) Oil Bath
20
30
40
50
Time (min)
Figure 7. Analytical RP-HPLC chromatograms (UV absorbance at 220 nm) of P-peptide 2-4 prepared under
different synthetic conditions. All coupling and deprotection reactions in the synthesis of the decamer were
conducted under the given reaction condition, i.e., manual, microwave, or oil bath, as described below. ACHC2 and
ACHC5 were double coupled and double deprotected in all cases. ACHC2 and ACHC5 were coupled in 0.8 M LiCI
in NMP in the microwave and oil bath syntheses. All other coupling reactions were performed in DMF.
A) Manual: All couplings for 1.5 hr in DMF at RT; 15 min deprotection at RT.
B) Microwave: All couplings for 6 min in the microwave at 50°C in DMF, except for double coupling ACHC2 and
ACHC5 in 0.8 M LiCI in NMP at 45°C in the microwave; 4 min deprotection in the microwave at 60°C.
C) Oil Bath-NMP/LiCl: All couplings for 1.5 hr in DMF at 60°C in the oil bath, except for double coupling ACHC2
and ACHC5 in 0.8 M LiCI in NMP at 60°C in the oil bath; 15 min deprotection at 60°C.
increase in ACHC-addition impurity 2-3 (Table 2), which presumably results from premature
Fmoc-deprotection during the double coupling o f ACHC1.
We then screened ca. 35
combinations o f coupling reagents (HBTU, HATU, PyBOP, BOP, PyBrOP, DIC, and DCC),
additives (HOBt, HOAt, and DMAP), bases (/PoEtN, NMM, and collidine), and solvents (DMF,
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86
NMP, CH2C12, DMSO, DMPU, THF, and mixtures thereof) with microwave irradiation (Table
4) . In general, HBTU was the best coupling reagent, although PyBOP gave similar results.
HATU12 did not outperform either of these two less expensive reagents. The additive had little
effect on the reaction. DIEA was the superior base, and DMF was generally the best solvent.
None o f the variations completely eliminated the ACHC 1-deletion impurity, which suggests that
the difficult coupling cannot be improved by increasing the reactivity of the activated P-amino
acid.
Table 4. Synthetic optimization o f P-peptide 2-1. The pentamer was synthesized under standard microwave
conditions, and ACHC1 was double coupled under with the noted procedural modifications. (Magic Mixture =
(DCM/DMF/NMP (1:1:1) with 1% Triton X100 and 2 M ethylenecarbonate)13
N otebook
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
IV 175
IV 177
IV 179
IV 181
IV 183
IV 185
IV 187
IV 189
IV 191
IV 193
IV 195
IV 199
IV 211
IV 213
IV 215
IV 217
IV 221
IV 223
IV 225
IV 227
IV 229
IV 231
IV 233
IV 235
IV 237
IV 239
IV 241
IV 243
IV 245
IV 247
IV 249
IV 251
IV 253
IV 255
IV 257
IV 259
IV 285
S o lv e n t
DMF
DMF
DMF
DMF
DMF
DMF
DMF
NMP
4:1 NMP:DCM
DMF
1:1 DMF:DCM
DMF
DMF
DMF
25% DMSO In DMF
1:1 DMF:DCM
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
THF
DMPU
DMF
DMF
DMF
DCM
DCM
DMF
DMF
THF
DCM
M agic Mixture
C oupling
R eagen t
HBTU
HBTU
HBTU
HBTU
HBTU
HBTU
DIC
HBTU
HBTU
HBTU
DIC
HBTU
HBTU
HBTU
HBTU
HBTU
PyBOP
BOP
BOP
HATU
HATU
PyBOP
HBTU
HBTU
PyBrOP
HBTU
HBTU
BOP-CI
DCC
DCC
PyBrOP
BOP-CI
DIC
HBTU
HBTU
DIC
HBTU
A dditive
HOBt
HOBt
HOBt
HOBt
HOBt
none
HOBt
HOBt
HOBt
HOBt
HOBt
HOBt
HOBt
HOBt
HOBt
HOBt
none
HOBt
none
no n e
HOAt
HOBt
HOAt
HOBt
none
HOBt
HOBt
none
HOBt
DMAP
none
none
DMAP
HOBt
HOBt
DMAP
HOBt
B a se
DIEA
DIEA
NMM
DIEA
DIEA
DIEA
n on e
DIEA
DIEA
collidin e
n on e
DIEA
DIEA
DIEA
DIEA
DIEA
DIEA
DIEA
DIEA
DIEA
DIEA
DIEA
DIEA
DIEA
n on e
DIEA
DIEA
n on e
none
none
n on e
n on e
n on e
DIEA
DIEA
none
DIEA
2-1
73
69
44
57
1
70
65
70
67
35
67
62
65
66
62
60
64
65
64
61
63
64
52
17
65
58
24
61
3
50
16
0
27
41
45
42
P ercen t HPLC Peak Area
ACHC-Deletion
2-3
5
7
34
7
17
6
2
1
86
6
9
4
2
0
5
3
1
3
37
1
2
90
9
7
7
2.5
3
12
6
6
2
6
10
8
6
12
3
4
3
4
3
3
4
3
9
10
8
3
7
7
1
14
85
27
67
3
4
8
38
5
32
9
4
86
1
3
1
0
2
C om m en ts
2-2
0
0
0
0
0
0
0
0
2
0
0
1
1
0
1
0
0
0
0
0
0
2
4
Repurified ACHC
45°C
3 eq . DIEA in ste a d o f 6
0 eq. DIEA in ste a d o f 6
pentam er
sin g le c o u p le 15 min hold
30W, lo n g ramp
oil bath, 60°C
2
2
0
0
0
0
0
0
addition p eptide
13
0
0
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87
2.2.5 Chaotropic Salts
Difficulties arising at intermediate lengths in the SPPS o f a-peptides have been attributed
to aggregation and/or folding o f resin-bound intermediates,14 and salt additives, such as LiBr,
LiCl, KSCN, and NaC 1 0 4 , have been reported to alleviate such problems.15 We observed that
the deletion impurity was almost completely eliminated by double coupling the N-terminal
ACHC residue of 2-1 in a 0.8
M
solution o f LiCl in DMF with microwave irradiation (JKM IV
205, Table 5). This improvement, however, was accompanied by a substantial increase in the
amount of ACHC-addition impurity, 2-3 (JKM V 115). Further optimization was achieved by
switching the solvent from DMF to l-methyl-2-pyrrolidinone (NMP) for the double coupling o f
ACHC1, producing hexamer 2-1 in 94% purity (Figure 3 and Figure 4D) and 81% yield. (Yield
was quantified by correlation o f peak area in analytical RP-HPLC (UV absorbance at 220 nm) to
concentration of the injected sample.16) Application o f these conditions to the couplings o f
ACHC2 and ACHC5 in deca-(3-peptide 2-4 provided the desired product in 88% purity and 65%
yield (Figure 6).16 The HPLC comparison o f crude products from the standard and microwaveenhanced solid-phase syntheses o f 2-4 (Figure 7) reveals the extent o f improvement in the latter
case.
2.2.6 Comparison with Conventional Heating
Is microwave irradiation necessary for the results outlined above, or would conventional
heating offer similar benefits?14 For the synthesis o f hexamer 2-1, we found that conventional
heating (oil bath) to 60°C for 90 min per coupling step and 15 min per Fmoc removal step
provided product purities comparable to those obtained upon microwave irradiation for 6 min per
coupling step and 4 min per Fmoc removal step. (Conventional heating for shorter periods led to
diminished synthetic efficacy, JKM V 233.) When the final ACHC coupling reactions were
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88
Table 5. Use of chaotropic salts for synthetic optimization of (3-peptide 2-1. The pentamer was synthesized
under standard microwave conditions, and ACHC1 was double coupled under with the noted procedural
modifications.
N o tebook
JKM IV 2 0 3
JKM IV 2 0 5
JK M IV 2 0 7
JK M
JK M
JKM
JK M
IV 2 0 9
VI 187
IV 261
IV 2 6 3
JK M IV 26 5
JK M IV 2 6 7
JK M IV 2 6 9
JK M IV 271
JK M IV 2 7 3
JK M IV 2 7 5
JK M IV 2 7 7
JKM IV 2 7 9
JKM IV 281
JKM IV 2 8 3
JKM V 001
JK M V 0 0 3
JK M V 0 0 5
JKM V 0 0 7
JKM V 0 0 9
JKM V 011
JK M V 0 1 3
JK M V 01 5
JK M V 01 7
JK M V 0 1 9
JK M V 021
JK M V .0 2 3
JK M V 0 2 5
JK M V 02 7
JK M V 0 2 9
JK M V 031
JKM V 0 3 3
JK M V 0 3 5
JKM V 0 3 7
JKM V 0 3 9
S o lv e n t
0.8M LiBr in DMF
0.8M LiCl in DMF
0.8M N aC I0 4 in DMF
0.8M KSCN in DMF
0.8M LiCl in DMF
0.8M LiBr in DMF
0.8M LiCl in DMF
0.8M NaCIO„ in DMF
0.8M KSCN in DMF
0.8M LiBr in DMF
0.8M LiCl in DMF
0.8M N aC I0 4 in DMF
0.8M KSCN in DMF
0.8M LiBr in DMF
0.8M LiCl in DMF
0.8M N aC !0 4 in DMF
0.8M KSCN in DMF
0.8M LiBr in DMF
0.8M LiCl in DMF
0.8M N aC I0 4 in DMF
0.8M KSCN in DMF
0.4M LiBr in 1:1 DMF/DCE
0.4M LiCl in 1:1 DMF/DCE
0.4M N aC I04 in 1:1 DMF/DCE
0.4M KSCN in 1:1 DMF/DCE
0.8M LiBr in NMP
0.8M LiCl in NMP
0.8M N aC I0 4 in NMP
0.8M KSCN in NMP
0.6M LiBr in 3:1 NMP/DCE
0.6M LiCl in 3:1 NMP/DCE
0.6M N aC !04 in 3:1 NMP/DCE
0.6M KSCN in 3:1 NMP/DCE
0.4M LiBr in 1:1 DMF/DCE
0.4M LiCl in 1:1 DMF/DCE
0.4M N aC I04 in 1:1 DMF/DCE
0.4M KSCN in 1:1 DMF/DCE
C o u p lin g
R eagent
A dditive
HBTU
HBTU
HO B t
HO B t
HBTU
HOBt
DIEA
58
HBTU
HBTU
HBTU
HBTU
HO B t
HOBt
HOBt
HOBt
DIEA
DIEA
DIEA
DIEA
56
82
34
63
HBTU
HOBt
DIEA
53
3
3
3
HBTU
HBTU
HBTU
HOBt
HOBt
HOBt
DIEA
DIEA
DIEA
58
53
62
2
1
3
2
9
2
7
1
0
3
6
1
0
12
43
13
2
1
6
0
0
B ase
2-1
DIEA
DIEA
P e rc e n t HPLC P e a k Area
A C HC -D eletion
2-3
19
70
0
1
C o m m e n ts
2-2
40
4
0
1
1
6
15
0
0
5
5
7
3
2
2
13
0
0
1
HBTU
HOB t
DIEA
64
HBTU
HBTU
HBTU
HOBt
HOBt
HOBt
DIEA
DIEA
DIEA
57
' 66
40
HBTU
HOBt
DIEA
29
50
3
0
HBTU
HO B t
HO B t
HOBt
DIEA
DIEA
DIEA
56
23
53
22
4
28
2
18
1
0
0
1
HO B t
DIEA
54
0
7
13
HO B t
HO B t
HO B t
DIEA
DIEA
DIEA
13
39
31
0
27
50
44
6
2
2
0
0
HO B t
DIEA
49
20
3
1
HO B t
HO B t
HOB t
DIEA
DIEA
DIEA
63
14
68
4
0
1
3
5
5
1
0
0
HBTU
HO B t
DIEA
26
0
11
2
HBTU
HBTU
HBTU
HOBt
HOBt
HOBt
DIEA
DIEA
DIEA
17
37
70
0
7
2
17
10
2
0
0
0
HBTU
HOBt
DIEA
35
0
0
3
HBTU
HBTU
HBTU
HOBt
HOBt
HOBt
DIEA
DIEA
DIEA
59
53
57
1
7
6
4
11
3
8
0
4
HBTU
HOBt
DIEA
59
6
4
0
HBTU
HOBt
DIEA
68
4
5
0
DIC
DIC
DIC
DIC
DIC
DIC
DIC
DIC
HBTU
HBTU
DMF/LiCI in in n e r a n d o u te r tu b e s
s in g le c o u p le
sin g le c o u p le
s in g le c o u p le
s in g le c o u p le
d o u b le c o u p le , 40°C
d o u b le c o u p le , 40°C
d o u b le c o u p le , 40°C
d o u b le c o u p le , 40°C
d o u b le c o u p le , m an u a l
d o u b le c o u p le , m an u a l
d o u b le c o u p le , m an u a l
d o u b le c o u p le , m an u a l
DIC = A/.A/’-diisopropyf c a rb o d iim id e
D C E = d ic h lo ro e th a n e
carried out in NMP containing LiCl, the oil bath approach provided 2-1 in 85% purity, which
nearly matches the results from microwave irradiation; extending the final deprotection o f the oil
bath synthesis to 1 hr completely eliminates the Fmoc-protected impurity 2-2, providing the
product in 94% purity, equivalent to our best result with microwave irradiation (JKM VI 001).
I
n
Thus, in this case, the principal advantage o f microwave irradiation is diminished reaction time.
Further investigation of ACHC1 coupling (Figure 8) and Fmoc-deprotection (Figure 9) in the oil
bath showed that these reactions go to completion in approximately 60 and 45 min, respectively,
about 10 times longer than required for the microwave-assisted synthesis. The heat and LiCl
work synergistically, because use o f LiCl/NMP for the double ACHC1 coupling at room
temperature provides 2-1 in only 43% purity (Figure 3).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
89
100
90
C
Q>
O
s_
o
80
70
CL
60
(0
CD
50
P en tam er
40
2-3
03
<D
Q.
30
20
10
0
0
30
60
90
120
150
Time (min)
Figure 8. Time course of ACHC1 coupling reaction at 60°C in the oil bath. Double coupling occurred at 90 min.
100
C
0)
o
v_
0)
CL­
IO
a>
<
XL
(0
a>
a.
0
10
20
30
40
50
Time (min)
Figure 9. Time course of ACHC1 Fmoc-deprotection reaction at 60°C in the oil bath. Double deprotection
occurred at 15 min.
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90
A clear benefit from microwave irradiation became evident, however, in the synthesis
o f decamer 2-4.
Oil bath synthesis provided this [3-peptide in only 53% purity (Figure 6),
substantially lower than the 88% purity achieved with microwave irradiation.
18
We speculate
that the microwave advantage for 2-4 reflects the increasing difficulty o f couplings and
deprotections after the fifth residue, which may arise from aggregation and/or folding during
growth of the protected P-peptide chain.80,14 Perhaps an even longer period o f conventional
heating for each coupling/deprotection cycle for the final five residues would eventually have
allowed us to produce P-peptide 2-4 in purity comparable to that o f the microwave synthesis, as
was the case for P-peptide 2-1, but at the cost o f more time. Synthesis o f other p-peptides in the
following chapters using our optimized microwave-assisted conditions indicates that these
conditions are o f general utility.
2.2.7 Epim erization of p2-Amino Acids
One concern when applying microwave irradiation to an amide bond-forming reaction is
the potential for epimerization o f the activated amino acid at the position a to the carbonyl. We
had not observed this event since in p3-amino acids the side chain is attached to Cp, and ACHC
prefers the diequatorial trans configuration over the cis. However, we wondered if microwaveassisted P-peptide synthsis would facilitate epimerization during the coupling o f p -amino acids.
Two derivatives o f hexa-p-peptide 2-1 containing either (S)- or (f?)~P -hPhe at position 4 were
prepared. The two diastereomers were resolved by approximately two minutes in the HPLC
analysis (Figure 10). Careful examination o f the chromatograms showed little or no evidence o f
the undesired diastereomer in each authentic sample.
We concluded that epimerization is
negligible under our microwave-assisted reaction conditions. In fact, these methods have proven
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91
useful for difficult a-peptide sequences as well (Chapter 6), where epimerization is o f even
greater concern.
2500
2000
1500
A bs
(mV)
1000-1
500
16
18
20
22
24
26
Time (min)
28
30
32
34
Figure 10. HPLC traces (UV absorbance at 220 nm) of derivatives of 2-1 containing (32-hPhe at position 4.
2.2.8 Further Investigation of the Effects of the NMP/LiCl Condition
We performed a few follow-up experiments in an effort to understand why NMP with 0.8
M
LiCl is so effective as a solvent for the difficult coupling o f ACHC1 in P-peptide 2-1. The
addition o f LiCl is necessary, as coupling in NMP is very similar to coupling in DMF alone
(JKM V 117).
We found that using NMP/LiCl as the solvent for all coupling steps in the
synthesis of hexamer 2-1 was ineffective, giving an initial product purity o f only 61% and a
much lower yield than obtained with using NMP/LiCl for the coupling o f ACHC (JKM V 079)
and DMF for all other reactions. This observation supports the idea that the NMP/LiCl improves
the coupling efficiency through the disruption o f on-bead self-association o f the growing P~
peptide chains during the reaction. However, irradiating the resin in NMP/LiCl alone (without
activated amino acid present) followed by the coupling o f ACHC1 in a DMF solution yielded P-
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
92
peptide 2-1 in 78% purity (JKM VI 189).
Attempts to detect the on-bead aggregation
phenomenon by NMR spectroscopy with the nanoprobe were prohibited by the already broad
features associated with on-bead spectra (data not shown).
The enhanced microwave absorption properties o f the NMP/LiCl solution relative to
D M F19 result in a rapid initial increase in reaction temperature, probably contributing to the
structure-disruption effect. Under the optimized conditions, the target temperature was set to
only 45°C, but the high ionic strength o f the 0.8 M LiCl in NMP solution results in a greater
efficiency of energy transfer than for NMP or DMF alone, so the final temperature o f 56-61°C is
similar to that observed for a coupling reaction in DMF set at 50°C.
Microwave-assisted
couplings in NMP/LiCl reach the set reaction temperature in 30-45 sec as opposed to about 1.5
min for DMF. In summary, the use o f NMP/LiCl is recommended for only every third coupling
step during the synthesis o f an aggregation prone sequence. While the use o f NMP/LiCl for the
coupling of ACHC may be rather specific for the Gellman reasearch group, the microwaveassisted SPPS conditions are much more general. Rather than spend more time investigating the
origin o f the NMP/LiCl benefit, we concluded that it was more important to pursue the synthesis
o f more and different sequences in a combinatorial fashion using our microwave irradiation
techniques.
2.3 Conclusions
Through judicious choice o f test cases and careful comparisons among reaction
conditions, we have shown that the difficulty o f synthesizing 14-helical P-peptides can be
alleviated by using microwave irradiation.
Further, we have identified a synergy between
microwave irradiation and the use o f a salt additive. It is conceivable that the salt does double
duty under microwave conditions, disrupting the folding and/or aggregation o f resin-bound
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93
intennediates, as proposed for conventional a-peptide synthesis,15 and increasing the
efficiency o f microwave energy transfer, as proposed for microwave enhancements o f other
reaction types.
20
For short fl-peptidcs, the advantage o f microwave irradiation relative to
conventional heating is modest, principally an economy in reaction time (> 10-fold). For longer
P-peptides, however, microwave irradiation displays a clear superiority relative to conventional
heating. The results described here have provided a foundation for the rapid synthesis o f highpurity p-peptide libraries, via both parallel and split-and-mix combinatorial methods, which have
been used to discover P-peptides with useful biological properties.
2.4 Experimental Procedures
2.4.1 General Procedures
Fmoc-GS%')-ACHC (182 g, 17% yield overall, Appendix A) was prepared by the method
of Schinnerl et al.6d
Fmoc-(S)-p3-hGlu(fBu)-OH, Fmoc-(5)-p3-hPhe-OH, Fmoc-(5)-p3-
hOm(Boc)-OFI, Fmoc-(5)-p3-hTrp(Boc)-OH and Fmoc-(A)-p3-hLeu-01I were prepared from
their corresponding Fmoc-L-a-amino acids (Novabiochem) as described previously.83 1-Methyl2-pyrrolidinone was purchased from Advanced ChemTech. Methanol, CH 2 CI2 and acetonitrile
were
purchased
from
Burdick
&
Jackson.
1-Methylimidazole,
piperidine,
1-
hydroxybenzotriazole hydrate, zPr2EtN, trifluoroacetic acid, triethylsilane and DMSO were
purchased from Aldrich.
resin
(100-200
l-(2-Mesitylenesulfonyl)-3-nitro-l,2,4-triazole,
mesh)
and
polystyrene Wang
O-benzotriazol-1-yl-A N, N \N ’-tetramethyluronium
hexaflurorophosphate were purchased from Novabiochem. DMF (biotech grade solvent, 99.9+
%) was purchased from Aldrich and stored over Dowex ion exchange resin. Dry CH 2 CI2 and
zP^EtN were distilled from calcium hydride.
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94
2.4.2 First Residue Loading
First residue loading was accomplished as described.815’9 Fmoc-(>S)-p3-hGlu(/Bu)-OH
(1.06 g) was activated with 1-methylimidazole (144 pL) and l-(2-mesitylenesulfonyl)-3-nitro1,2,4-triazole (710 mg) in dry CFf2Cl2 (7.5 mL) and added to swollen polystyrene (PS) Wang
resin (500 mg, 100-200 mesh, initial loading: 0.96 mmol/g) in a polypropylene solid-phase
extraction (SPE) tube (25 mL, Alltech).
The tube was capped and placed on a wrist-action
shaker (Labquake, Bamstead/Thermolyne). After reaction for 12 hr at room temperature, the
resin was washed (5 x CH 2 CI2 , 5 x DMF, 5 x CFECI2 and 5 x MeOFI) using a vacuum manifold
(Vac-Man, Promega) connected to a water aspirator and then dried under a stream o f N 2 until
free-flowing. The yield was estimated by UV-quantification of the dibenzofulvene-piperidine
adduct at 290 nm in a known volume o f deprotection solution, as previously described (0.67
mmol/g, 70%).21
2.4.3 Microwave P-Peptide Synthesis
Loaded PS Wang resin (10 pmol, 14.9 mg) was placed in a modified polypropylene SPE
tube (4 mL, Alltech, top rim removed with a razor blade) and swelled with DMF for ~ 10 min.
The resin was washed (5 x DMF, 5 x CH 2 CI2 and 5 x DMF). Deprotection solution (750 pL o f
20% piperidine in DMF (v/v)) was added to the resin, and the tube was placed inside a glass 10
mL microwave reaction vessel containing ~ 2 mL o f DMF (Figure 5). A N 2 line was inserted for
agitation, and the vessel was placed in the microwave reactor (CEM Discover) and irradiated (50
W maximum power, 60°C, ramp 2 min, hold 2 min). All microwave irradiations were conducted
at atmospheric pressure, the temperature was measured via an IR sensor at the base o f the outer
reaction vessel, the temperature was controlled by modulation o f power, and the sample was
cooled with compressed air during the hold time. The tube was removed from the microwave
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95
reactor, and the resin was washed as before. In a separate vial, Fmoc-p-amino acid (30 pmol)
was
activated
by
adding
O bcnzotriazol-1-y\-N, /V. N ’,/V ’-tetramethyluronium
hexaflurorophosphate (HBTU, 60 pL o f 0.5 m solution in DMF), DMF (440 pL), 1hydroxybenzotriazole hydrate (HOBt, 60 pL o f 0.5 M solution in DMF), and /'PiyEtN (60 pL of
1.0 M solution in DMF). The mixture was vortexed and added to the resin. The sample was
irradiated in the microwave reactor (50 W maximum power, 50°C, ramp 2 min, hold 4 min).
Alternatively, the coupling reaction o f Fmoc-CyApACHC was performed by activating with
solutions of HBTU, HOBt, and iP^EtN in NMP and adding a solution of LiCl in NMP for a final
concentration o f 0.8 M LiCl (620 pL final volume), and then adding this solution to the resin.
The tube was placed in a microwave reaction vessel containing ~ 2 mL o f 0.8 M LiCl in NMP
and irradiated (50 W maximum power, 45°C, ramp 2 min, hold 4 min).
After the coupling
reaction, the resin was washed as before. The N-terminal ACHC residue in (3-peptide 2-1 and
ACHC2 and ACHC5 (standard peptide numbering, starting from the N-terminus o f the full
sequence) in P-peptide 2-4 were double coupled and double deprotected in all syntheses. All
other couplings were performed once with DMF as the solvent. The deprotection/coupling cycle
was repeated in a stepwise manner until the desired length o f the hexamer or decamer had been
reached.
2.4.4 Manual P-Peptide Synthesis
Loaded PS Wang resin (10 pmol, 14.9 mg) was placed in an SPE tube (4 mL, Alltech)
and swelled with DMF.
The resin was washed (5 x DMF, 5 x CH 2 CI2 and 5 x DMF).
Deprotection solution was added to the resin, and the tube was capped and placed on a shaker for
15 min at room temperature. The tube was removed from the shaker, and the resin was washed
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
96
as before. A solution o f activated Fmoc-P-amino acid (as described in section 2.4.3) was
added to the resin, and the tube was capped and placed on the shaker for 1.5 hr at room
temperature.
After the coupling reaction, the resin was washed as before.
The
deprotection/coupling cycle was repeated in a stepwise manner until the desired length o f the
hexamer or decamer had been reached.
2.4.5 Oil Bath p-Peptide Synthesis
Loaded PS Wang resin (10 pmol, 14.9 mg) was placed in a SPE tube (4 mL, Alltech) and
swelled with DMF. The resin was washed (5 x DMF, 5 x CH 2 CI2 and 5 x DMF). Deprotection
solution was added to the resin, a N 2 line was inserted for agitation, and the tube placed in an oil
bath at 60°C for 15 min. The tube was removed from the oil bath, and the resin was washed as
before. A solution o f activated Fmoc-P-amino acid (as described in section 2.4.3) was added to
the resin, a N 2 line was inserted for agitation, and the tube placed in an oil bath at 60°C for 1.5
hr. After the coupling reaction, the resin was washed as before. The deprotection/coupling cycle
was repeated in a stepwise manner until the desired length o f the hexamer or decamer had been
reached.
2.4.6 p-Peptide Cleavage, Work-Up and HPLC
After the final residue had been added and deprotected, the resin was washed (5 x DMF,
5 x CH2C12, 5 x DMF and 5 x CH2C12), and the P-peptide was cleaved from the solid support
with
simultaneous
side
chain
deprotection
(3
mL,
45:45:5:5
trifluoroacetic
acid
(TFA):CH2Cl2:triethylsilane:water, 2 h, RT, with rocking). The cleavage solution was drained
and concentrated under a stream o f N2. The crude P-peptide mixture was dissolved in 1.0 mL
DMSO, diluted (1 to 20 with DMSO) and analyzed by HPLC (10 pL injection, Shimadzu). The
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
97
C4 -silica reverse-phase analytical column (5 pm, 4 mm x 250 mm, Vydac) was eluted with a
gradient of acetonitrile in water (10 - 60%, 50 min, 0.1% TFA in each) at a flow rate o f 1
mL/min.
Product purity was determined as peak area percent by integration o f the UV
absorbance at 220 nm. Integration was performed over the 1 5 - 5 0 min time interval to exclude
the large absorbance o f DMSO that elutes from 5 - 1 5 min. The lower threshold o f integration
was set to exclude minor peaks whose areas were < 1% o f the peak area o f the major species. 13Peptide masses
were
measured
by
MALDI-TOF-MS
(Bruker Reflex
II,
a-cyano-4-
hydroxycinnamic acid matrix).
2.4.7 P-Peptide Characterization Data
OH
F m ocH N
p -P ep tid e 2-1
p-P eptide 2-2
^48^68^809
CsaHyaNsOn
Exact M ass: 900.51
Exact M ass: 1122.58
OH
h 2n
;
ACHC1-D eletion
p-P eptide 2-3
C-4lH57N708
C55H79N9OK)
Exact M ass: 775.43
Compound
Formula
Exact M ass: 1025.59
Calculated
Mass
MALDI-TOF MS
Observed Mass
RP-HPLC
Retention Time
(10-60%B, 50 min)
[M +Hf
TM+Naf
(min)
P-Peptide 2-1
ACHC1Deletion
C48H68Ng09
900.51
901.4
923.4
34.5
C 4 1 H5 7 N7 O8
775.43
.776.2
798.2
26.0
P-Peptide 2-2
C 6 3 H7 8 N8 O 11
1122.58
1123.5
1145.5
46.5
P-Peptide 2-3
C 5 5 H7 9 N9 O 10
1025.59
1026.3
1048.3
37.0
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
p-Peptide 2 -4
C 7 4 H 113N -I30i5
Exact Mass: 1 4 2 3.8 5
P -h O rn l-D e le tio n
C 6 8 H l0 lN llO -|4
Exact Mass: 1 2 9 5.7 5
C 0 2H
A C H C 2-D eletio n
C 6 7 H 102N 1 2 O l4
Exact Mass: 1 2 9 8.7 6 -
COoH
OH
P3-hG lu4-D eletion
C 6 8 H 104N 1 2 O l2
Exact Mass: 1 2 8 0.7 9
Compound
3-Peptide 2-4
33-hOrn1Deletion
ACHC2-Deletion
33-hGlu4Deletion
Formula
Calculated
Mass
MALDI-TOF MS
Observed Mass
RP-HPLC
Retention Time
(10-60%B, 50 min)
rM + H f
[M+Naf
(min)
C 74H 113N 13O 15
1423.85
1425.0
1447.0
41
C 68H 101N 11O 14
1295.75
1296.2
1318.2
45
C 67H 102N 12O 14
1298.76
1299.8
1321.7
33
1280.79
1282.1
1304.1
31
C
68H l 04N l 2O 12
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99
2.4.8 Yield Calculation
2.4.8.1 B-Peptide 2-1
Yields from p-peptide syntheses were quantified by the method o f Yan et al.16 A sample
of crude P-peptide 2-1 was purified by C4 -silica preparative reverse-phase HPLC (10 pm, 22 mm
x 250 mm, Vydac). The column was eluted with a gradient o f acetonitrile in water (25-55%, 30
min., 0.1% TFA in each) at a flow rate o f 15 mL/min. After lyophilization, a small sample (~
0.9 mg) of purified p-peptide 2-1 was dissolved in 1.0 mL o f DMSO to make a 0.74 mM stock
solution, the concentration being determined by p3-homotryptophan absorbance in 6 M guanidine
hydrochloride.22 The stock solution was then diluted to make a series o f calibration solutions
containing a fixed concentration o f Fmoc-L-phenylalanine-OH (retention time = 40 min) as an
external standard to compensate for instrumental fluctuation and other systematic errors. These
solutions were analyzed by analytical RP-HPLC (C 4 -silica reverse-phase analytical column (5
pm, 4 mm x 250 mm, Vydac) eluted with a gradient o f acetonitrile in water, 10 - 60%, 50 min,
0.1% TFA in each, at a flow rate o f 1 mL/min) and monitored by UV absorbance at 220 nm.
The peak area o f P-peptide 2-1 was divided by the peak area o f the external standard to give the
peak area ratio (peak area ratio = (peak area)p.peptide i / (peak area)cxtemai standard)- A plot o f peak
area ratio versus concentration yielded a calibration curve. The curve was fit by linear regression
((peak area ratio) = 6.435 (concentration) - 0.0134) with a correlation coefficient (R2) o f 0.9981.
The calibration curve was validated by preparing a solution o f P-peptide 2-1 o f a known
concentration and analyzing by HPLC with UV detection at 220 nm.
The determined
concentration from the calibration curve was accurate within 5%.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
To determine the yield of a 10 pmol synthesis, the crude P-peptide 2-1 mixture was
dissolved in 1.0 mL o f DMSO. This solution was diluted (40:1), external standard was added,
and the sample was analyzed by HPLC.
The peak area ratio was measured, and the
concentration was determined using the calibration curve. The yield was calculated based on the
theoretical yield ((10 pmol / 1.0 mL) = 10 h i m
).
Table 6. Data for yield calculation of p-peptide 2-1.
Reaction Condition
Calibration Samples
Manual
Microwave
Oil Bath
DMF
NMP/LiCl
DMF
NMP/LiCl
DMF
NMP/LiCl
Concentration
(mM)
0.0000
0.0185
0.0370
0.0555
0.0740
0.0925
0.1110
0.1295
0.1480
0.1665
0.1850
0.2035
0.2220
0.2405
0.2590
0.2775
0.2960
0.11
0.08
0.19
0.20
0.13
0.20
Peak Area
P-Peptide
External
2-1
Standard
0
8276989
1211257
9131124
1931141
8686859
2877937
7664868
3361122
8653703
7863561
4413160
5286486
7523855
7470489
6071957
7420454
6927605
7953161
7352056
8774908
7524649
7354814
9529455
10393326
7365035
11231814
7167190
7348024
12173026
7180157
12885869
13779014
7406739
10029684
6756388
7951137
4174374
7743152
9584578
12482129
9709881
11062174
9353486
10287614
8131450
Peak Area
Ratio
Yield
(%)
0
0.133
0.222
0.375
0.388
0.561
0.703
0.813
0.934
1.082
1.166
1.296
1.411
1.567
1.657
1.795
1.860
0.674
0.525
1.238
1.286
0.846
1.265
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43
33
78
81
53
79
101
y = 6 .4 3 5 x - 0.0134
R2 = 0.9981
I
I
Peak
Area
Ratio
0.6
0 .4
0.2
0.00
0.10
0.05
0.20
0.15
0.25
0 .3 0
0.35
Concentration (mM)
Figure 11. Calibration curve for yield calculation of P-peptide 2-1.
Yield (%)
i
DM F
NMP/LiCl
Manual
DM F
NMP/LiCl
Microwave
DM F
NMP/LiCl
Oil Bath
Reaction Conditions
Figure 12. Yield of P-peptide 2-1 under different reaction conditions.
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|
102
2.4.8.2 P-peptide 2-4
A sample o f crude P-peptide 2-4 was purified by C/t-silica preparative reverse-phase
HPLC (10 pm, 22 mm x
250 mm, Vydac).
The column was eluted with a gradient of
acetonitrile in water (30-60%, 30 min., 0.1% TFA in each) at a flow rate o f 15 mL/min. After
lyophilization, a small sample (~ 1.2 mg) o f purified P-peptide 2-4 was dissolved in 1.0 mL o f
DMSO to make a 1.58
mM
stock solution, the concentration being determined as above. The
stock solution was then diluted to make a series o f calibration solutions containing a fixed
concentration o f Fmoc-L-tryptophan(Boc)-OH (retention time = 48 min) as an external standard.
These solutions were analyzed by analytical RP-HPLC (C 4 -silica reverse-phase analytical
column (5 pm, 4 mm x 250 mm, Vydac) eluted with a gradient of acetonitrile in water, 10 - 60%,
50 min, 0.1% TFA in each, at a flow rate o f 1 mL/min) and monitored by UV absorbance at 220
nm. The peak area ratio was calculated as above. A plot o f peak area ratio versus concentration
yielded a calibration curve. The curve was fit by linear regression ((peak area ratio) = 2.4925
(concentration) + 0.0575) with a correlation coefficient (R2) o f 0.9929. The calibration curve
was validated by preparing a solution o f P-peptide 2-4 o f a known concentration and analyzing
by HPLC with UV detection at 220 nm. The determined concentration from the calibration
curve was accurate within 5%.
To determine the yield o f a 10 pmol synthesis, the crude p-peptide 2-4 mixture was
dissolved in 1.0 mL o f DMSO. This solution was diluted (40:1), external standard was added,
and the sample was analyzed by HPLC.
The peak area ratio was measured, and the
concentration was determined using the calibration curve. The yield was calculated as above.
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
Table 7. Data for yield calculation o f P-peptide 2-4.
Reaction Condition
Concentration
Calibration Samples
Manual
Microwave
Oil Bath
DMF
NMP/LiCl
NMP/LiCl
(mM)
0
0.0395
0.079
0.1185
0.158
0.1975
0.237
0.2765
0.316
0.3555
0.395
0.4345
0.474
0.5135
0.553
0.5925
0.632
0.035
0.161
0.090
Peak
p-Peptide
2-4
0
1164145
2378043
3774382
5262713
5916062
7474370
8832870
9444048
11094681
11329050
12544825
14227821
15018296
15310152
16534698
17415109
1508031
4990884
2983603
Area
External
Standard
10254832
10695257
10077136
11249881
10638793
10628323
11032064
10835896
10626986
11006756
10568910
10879760
11935025
11810970
10781026
10946512
10707317
10420135
10862338
10562164
Peak Area
Ratio
Yield
(%)
0
0.109
0.236
0.336
0.495
0.557
0.678
0.815
0.889
1.008
1.072
1.153
1.192
1.272
1.420
1.510
1.626
0.145
0.459
0.282
14
65
36
1.8
1.6
y = 2 .4 9 2 5 x + 0.0575
-■
R2 = 0.9929
1.4 -
1.2
- -
Peak 1
Area
Ratio 0-8
--
-
*
0.6
-- .--- -----------------------------
0 .4 -
0.2
- - ..... - -
^ --- -
0♦
0
0.1
0 .2
0 .3
0 .4
0 .5
Concentration (mM)
Figure 13. Calibration curve for yield calculation o f P-peptide 2-4.
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
0 .6
0 .7
10 4
70
60
50
40
Yield (%)
30
20
10
0
DMF
NMP/LiCl
NMP/LiCl
Manual
Microwave
Oil Bath
Reaction Conditions
Figure 14. Yield of P-peptide 2-4 under different reaction conditions.
2.4.9 A dditional D ata
Table 8. Full comparison of synthetic methods. While these are not the numbers reported in the publication of
these methods due to subsequent optimization/standardization of the HPLC assay,23 these were the initial data that
established the basic trends during the investigation. Oil bath “short” is 6 min coupling, 4 min deprotection. Oil
bath “long” is 1.5 hr coupling, 15 min deprotection.
Notebook
Condition
JKM IV 135
1x couple, 1x depro, DMF
JKM IV 137
1x couple, 2x depro, DMF
Manual
2x couple, 2x depro, DMF
JKM IV 139
2x couple, 2x depro, NMP + LiCl
JKM V 199
JKM IV 163
short, DMF
short, NMP + LiCl
JKM V 233
JKM IV 235
long, DMF
Oil Bath
JKM V 239
long, NMP
long, DMF + LiCl
JKM V 231
long, NMP + LiCl
JKM V 229
JKM IV 129
1x couple, 1x depro, DMF
1x couple, NMP + LiCl
JKM V 207
JKM V 083
2x couple, 2x depro, DMF
Microwave
JKM V 117
2x couple, 2x depro, NMP
2x couple, 2x depro, DMF + LiCl
JKM V 115
JKM V 113
2x couple, 2x depro, NMP + LiCl
2-1
27
35
46
43
46
49
52
45
55
60
66
67
72
75
74
91
ACHCDeletion
54
56
36
31
31
25
12
31
23
21
21
25
5
5
0
3
2-2
2-3
14
6
10
21
18
5
4
3
3
0
0
0
0
0
0
0
0
0
0
0
0
1
9
3
2
2
0
0
6
0
13
0
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105
2.4.10 Step-by-Step Microwave-Assisted P-Peptide Synthesis Protocol for CEM
Discover
For 25 nmol peptide on NovaSyn TGR resin:
1. Cut the top off of a 4.0 mL Alltech SPE tube with a razor blade.
2. Weigh 25 pmol o f resin (calculated from resin loading, i.e. 100 mg is 0.25 mmol/g) into
the Alltech tube.
3. Add 2 mL o f DMF and let swell for 5 min.
4. Transfer tube to wash station and wash 5 x DMF (attach aspirator to wash station, close
stopcock to fill tube with solvent, open to drain.)
5. Remove tube from wash station. Fix bottom cap on tube.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Weigh amino acid for coupling (3 equiv., 75 pmol) into a 1-dram vial. Add 150 pL o f
0.5 M solution of HBTU in DMF. Add 1.1 mL o f DMF and vortex until the amino acid
is dissolved.
Add 150 pL o f 0.5 M solution o f HOBt in DMF. Add 150 pL o f 1.0 M solution o f DIEA
in DMF. Vortex and allow to stand for 60 sec.
Add activated amino acid solution to resin.
Place vessel in 10 mL glass microwave vial containing approximately 2 mL o f DMF.
Insert the N 2 line (about 1 bubble per sec), and microwave (“B PE P C O U P L E ” method:
50W max, 50°C, ramp 2:00 min., hold 4:00 min).
Remove N 2 line and wipe clean. Remove vial from microwave. Remove Alltech tube
from vial. Remove bottom cap and quickly transfer tube to wash station.
Wash 5 x DMF.
Remove tube from wash station. Fix bottom cap on tube.
Add 1.5 mL deprotection solution (20% piperidine in DMF (v/v)).
Place vessel in 10 mL glass microwave vial containing approximately 2 mL o f room
temperature DMF. Insert the N 2 line, and microwave (“BPEP DEPRO” method: 50W
max, 60°C, ramp 2:00 min., hold 2:00 min).
Remove N 2 line and wipe clean. Remove vial from microwave. Remove Alltech tube
from vial. Remove bottom cap and quickly transfer tube to wash station.
Wash 5 x DMF. Remove tube from wash station. Fix bottom cap on tube.
Repeat steps 6 through 16 as necessary.
18. Wash the resin (5 x DMF and 5 x DCM).
19. Cleave the (3-peptide by adding (3 mL, 90:5:5 TFA:DCM:triethylsilane:H20 , 2 h., RT,
with rocking).
20. Drain the cleavage solution and concentrate under a stream o f N2.
21. Dissolve the crude |3-peptide in DMSO (2.5 mL) using the vortexer and/or sonication.
22. Analyze by C4 -silica reverse-phase analytical HPLC (5 pm, 4 mm x 250 mm, Vydac),
eluteing the column with a gradient of acetonitrile in water (10-60%, 50 min., 0.1% TFA
in each) at a flow rate o f 1 mL/min.
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106
2.5 References
1 Kappe, C. O. Angew. Chem. Int. Ed. 2004, 43, 6250.
2 (a) Yu,
Chen, S.-T.; Wang, K.-T. J. Org. Chem. 1992, 57, 4781. (b) Erdelyi, M.;
Gogoll, A. Synthesis 2002, 1592. (c) Ferguson, J. D. Mol. Div. 2003, 7, 281. (d) Matsushita, T.;
Hinou, H.; Kurogochi, M.; Shimizu, H.; Nishimura, S.-I. Org. Lett. 2005, 7, 877.
3 Albericio, F. Curr. Op. Chem. Biol. 2004, 8, 211.
4 (a) Gellman, S. H. Acc. Chem. Res. 1998, 31, 173. (b) Cheng, R. P.; Gellman, S. H.; DeGrado,
W. F. Chem. Rev. 2 0 0 1 ,101, 3219.
5 (a) Seebach, D.; Overhand, M.; Kuhnle, F. N. M.; Martinoni, B.; Oberer, L.; Hommel, U.;
Widmer, H Helv. Chim. Acta 1996, 79, 913. (b) Seebach, D.; Matthews, J. L. Chem. Commun.
1997,2015.
6 (a) Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.; Gellman, S. H. J. Am. Chem.
Soc. 1996,118, 13071. (b) Appella, D. H.; Barchi, J. J.; Durell, S. R.; Gellman, S. H. J. Am.
Chem. Soc. 1 999,121, 2309. (c) Appella, D. H.; Christianson, L.A.; Karle, I. L.; Powell, D. R.;
Gellman, S. H. J. Am. Chem. Soc. 19 9 9 ,121, 6206. (d) Schinnerl, M.; Murray, J. K.; Langenhan,
J. M.; Gellman, S. H. Eur. J. Org. Chem. 2003, 721. (e) Raguse, T. L.; Lai, J. R.; Gellman, S. FL;
. J. Am. Chem. Soc. 2 0 0 3 ,125, 5592.
7 (a) Werder, M.; Hauser, H.; Abele, S.; Seebach, D. Helv. Chim. Acta 1999, 82, 1774. (b)
Hamuro, Y.; Schneider, J. P.; DeGrado, W. F. J. Am. Chem. Soc. 1 9 9 9 ,121, 12200. (c) Raguse,
T. L.; Porter, E. A.; Weisblum, B.; Gellman, S. H. J. Am. Chem. Soc. 2 0 0 2 ,124, 12774. (d)
Kritzer, J. A.; Lear, J. D.; Hodson, M. E.; Schepartz, A. J. Am. Chem. Soc. 2 0 0 4 ,126, 9468.
8 (a) Guichard, G.; Abele, S.; Seebach, D. Helv. Chim. Acta 1998, 81, 187. (b) Arvidsson, P. L;
Rueping, M.; Seebach, D. Chem. Commun. 2001, 649. (c) Arvidsson, P. I.; Frackenpohl, J.;
Seebach, D. Helv. Chim. Acta 2003, 86, 1522.
9 Blankmeyer-Menge, B.; Nimitz, M.; Frank, R. Tetrahedron Lett. 1990, 31, 1701.
10 Blackwell, H. E.; Perez, L.; Stavenger, R. A.; Tallarico, J. A.; Eatough, E. C.; Foley, M. A.;
Schreiber, S. L. Chem. Biol. 2001, 8, 1167.
11 Niichter, M.; Ondruschka, B.; Bonrath, W.; Gum, A. Green Chem. 2004, 6, 128.
12 Carpino, L. A. J. Am. Chem. Soc. 1 9 9 3 ,115, 4397.
13
Zhang, L.; Goldammer, C.; Henkel, B.; Zuehl, F.; Panhaus, G.; Jung, G.; Bayer, E. Innovation
and Perspective in Solid Phase Synthesis, 3rd International Symposium; Epton, R., Ed.;
Mayflower Scientific Ltd.: Birmingham, UK, 1994; p 711-716.
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107
14 Tam, J. P.; Lu, Y. A. J. Am. Chem. Soc. 1 9 9 5 ,117, 12058.
15 (a) Thaler, A.; Seebach, D.; Cardinaux, F. Helv. Chim. Acta 1991, 74, 628. (b) Stewart, J. M.;
Klis, W. A. Innovation and Perspective in Solid Phase Synthesis: Peptides, Polypeptides and
Oligonucleotides', Epton, R., Ed.; SPCC: Birmingham, UK, 1990; pp 1-9. (c) Flendrix, J. C.;
Halverson, K. J.; Jarrett, J. T.; Lansbury, P. T. J. Org. Chem. 1990, 55, 4517.
16 Yan, B.; Fang, L.; Irving, M.; Zhang, S.; Boldi, A. M.; Woolard, F.; Johnson, C. R.;
Kshirsagar, T.; Figliozzi, G. M.; Krueger, C. A.; Collins, N. J. Comb. Chem. 2003, 5, 547.
17 (a) Kuhnert, N. Angew. Chem. Int. Ed. 2002, 41, 1863. (b) Olivos, H. J.; Alluri, P. G.; Reddy,
M. M.; Salony, D.; Kodadek, T. Org. Lett. 2002, 4, 4057.
18 Stadler, A.; Kappe, C. O. Eur. J. Org. Chem. 2001, 919.
19 (a) Gabriel, C.; Gabriel, S.; Grant, E. H.; Halstead, B. S. J.; Mingos, D. M. P. Chem. Soc. Rev.
1998,27,213.
20 Leadbeater, N. E.; Torenius, H. M ../. Org. Chem. 2002, 67, 3145.
21 Gude, M.; Ryf, J.; White, P. D. Lett. Pept. Sci. 2003, 9, 203.
22 Edelhoch, H. Biochemistry 1967, 6, 1948.
23 Murray, J. K.; Gellman, S. H. Org. Lett. 2005, 7, 517.
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108
Chapter 3
Efficient Synthesis of a Split-and-Mix
P-Peptide Combinatorial Library
with Microwave Irradiation
Microwave
Irradiation
Polystyrene
Macrobeads
c o 2h
c o 2h
o
O R 1 ©
X X.'N
HoN
H
N'
H
H
H
H
H
H
14 hr, 65% Average Purity of Major Product
Portions of this chapter have been published as:
Murray, J. K.; Farooqi, B.; Sadowsky, J. D.; Scalf, M.; Freund, W. A.; Smith, L.
M.; Chen, J.; Gellman, S. H. “Efficient Synthesis o f a P-Peptide Combinatorial
Library with Microwave Irradiation,” Journal o f the American Chemical Society
2005 , 727(38), 13271-13280.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
109
3.0 Brief Summary o f Chapter
The predictable relationship between (3-amino acid sequence and folding has inspired
several biological applications o f |3-peptides, including the inhibition o f protein-protein
interactions. For many such applications it would be desirable to prepare and screen P-peptide
libraries. Flowever, standard solid-phase peptide synthesis (SPPS) protocols are not efficient
enough to support a library approach for many types o f P-peptides. We optimized the solidphase synthesis o f P-peptides using microwave irradiation, as described in Chapter 2, and here
we describe how this approach has been adapted to synthesis on polystyrene macrobeads. We
rapidly prepared a high-quality p-peptide combinatorial library via a split-and-mix strategy.
This library was screened in search o f improved P-peptide antagonists o f the p53-MDM2
protein-protein interaction.
3.1 Background
Protein-protein interactions have emerged as an important new class o f therapeutic
targets. In many instances, traditional small molecule approaches have not been successful in
disrupting protein-protein interactions.1 A new generation o f “proteomimetic” strategies, or
oligomeric scaffolds that mimic the three-dimensional display of important side chains within a
protein, are showing great potential for the inhibition o f these interactions.2
Successfully
inhibiting a protein-protein interaction can be accomplished by selecting an appropriate class o f
molecules and then exploring the chosen scaffold through a combination o f structure-based
design and combinatorial chemistry. This chapter describes the development o f combinatorial 13peptide synthesis on polystyrene (PS) macrobeads using microwave irradiation in an attempt to
discover improved p53/MDM2 interaction inhibitors.
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110
Gellman
and
coworkers
have
pursued
the
development
o f (3-peptides
as
proteomimetics for the inhibition o f protein-protein interactions.3 Early efforts focused on the
p53/MDM2 interaction (Chapter 1). Guided by the crystal structure o f the complex, 12-helical
(3-peptides were designed to display a (33-hPhe, (33-hTrp, and p3-hFeu along one face o f an
amphipathic helix analogous to residues Phel9, Trp23, and Leu26 in the a-helical N-terminal
MDM2-binding domain o f p53. Following a traditional drug discovery cycle (Figure 1), a small
set o f P-peptides (approximately 10 members) was synthesized in parallel based on each design.
Following HPFC purification, the p-peptides were screened in a p53/MDM2 EFISA. Synthesis
and purification o f each set o f P-peptides required at least one week’s time, and screening took
another few weeks.
After several rounds, the most potent ligand had a median inhibitory
concentration (IC50) of approximately 250 pM.
Target:
Scaffold:
p53/M D M 2
12-Helix
A dvance to
in v iv o
Studies
S tructure-B ased
Design
p-P eptide
S ynthesis
(1 0 -m e m b er sets)
P53/M DM 2
E LIS A
HPLC P urification
Figure 1. Proteomimetic discovery paradigm.
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Ill
In the meantime, Schepartz et al. were also exploring P-peptides as a scaffold for
displaying a set o f side chains in a protein-mimetic manner.4 Instead o f using the 12-helix, they
designed 14-helical P-peptides to mimic the projection o f the three hydrophobic side chains from
the a-helical segment o f the protein p53.5 Two o f these compounds (1-54 and 1-55) were shown
to bind with modest affinity to MDM2.
These results had profound implications for our
research. First, the foldamer approach for development o f protein-protein interaction inhibitors
was validated, encouraging further studies toward this particular biological application. Second,
the 14-helix was shown to be an effective a-helix mimic. This contrasted with our observation
that the geometric characteristics (i.e., helix macrodipole, pitch, and internal diameter, etc.) o f
the different P-peptide helices had suggested that the 12-helix would be more suited to mimicry
of the a-helix than the 14-helix. A growing distrust o f molecular modeling for the structurebased design o f P-peptide proteomimetics and the need to rapidly explore the activity o f multiple
foldamer scaffolds against new protein-protein interaction targets caused us to refine our
paradigm for ligand discovery (Figure 2).
We reasoned that developing the combinatorial
synthesis of P-peptides could potentially accelerate the discovery cycle.
3.2 Combinatorial B-Peptide Synthesis
The discovery and optimization o f bioactive 14-helical P-peptides had been hindered by
the difficulty of their solid-phase synthesis.6,7 14-Helical P-peptides prepared using standard
o
SPPS protocols were usually not of sufficient purity for direct evaluation. The necessity of
HPLC purification prior to screening had limited synthetic efforts to small sets o f P-peptides.
Progress toward the biological application o f p-peptides could be more rapidly accomplished
through the synthesis and screening o f combinatorial libraries without HPLC purification.
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112
Scaffolds:
Target:
p53/MDM2
14-Helix
12-Helix
Advance to
in vivo
Structure-Based
Design
Studies
C om binatorial
p53/MDM2
p-Peptide
Synthesis
ELISA
HPLC Purification
Figure 2. Combinatorial discovery of foldamer proteomimetics.
3.2.1 Split-and-Mix Synthesis
Split-and-mix synthesis is an efficient method for generating hundreds to millions of
different compounds on only a few grams o f solid support.9 Through repeated rounds o f 1)
splitting the resin into different aliquots; 2) coupling a different building block to each resin
aliquot; and 3) recombining and mixing the resin, a complex mixture o f products is created, with
each bead containing a few nanomoles o f a single product (Figure 3). Screening the products
and identification o f the active molecules or “hits” are accomplished in a variety o f w ays.10 The
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113
preparation o f peptide libraries is straightforward; their oligomeric nature makes peptides
amenable to the incorporation o f functional group diversity (on side chains) and sequencing by
either LC-MS/MS or amino acid analysis.11
Building Blocks
A , B, C
Figure 3. Schematic for split-and-mix synthesis of a 27-membered library (3 x 3 x 3).
3.2.2 Synthetic Optimization of 14-Helical P-Peptides with Microwave Irradiation
As discussed in Chapter 2, we had optimized the SPPS o f 14-helical p-peptides using
microwave irradiation.12,13
During the solid-phase synthesis o f these oligomers, one often
observes a sudden onset o f problems as the chain length reaches ca. six residues.7 A t this length,
both removal of the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group and amide bond
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11 4
formation become difficult, perhaps because o f on-resin self-association o f the growing p~
peptide chain.7c’14 Incorporation o f ACHC tends to exacerbate these problems.6 We used Ppeptide 3-1 (Figure 4) as a test sequence for synthetic optimization because o f the extreme
challenge o f completely coupling and deprotecting the N-terminal ACHC residue.12
Both
microwave and conventional heating were found to provide p-peptide 3-1 in high purity, with
the advantage of microwave irradiation being a 10-fold reduction in synthesis time. Microwave
synthesis of the longer P-peptide 3-2 with a salt additive (LiCl)15 provided the product in much
higher purity than did other synthetic methods.
3-1
o
h2n £ ^ n
C 0 2H
NH
Figure 4. P-Peptides for synthetic optimization.
3.2.3 Polystyrene Macrobeads for Split-and-Mix Synthesis
We had used 100-200 mesh polystyrene (PS) resin (75-150 pm) as the solid support for
our previous synthetic optimization (Chapter 2) with the hope of identifying reaction conditions
that could be applied to the generation o f P-peptide combinatorial libraries on PS macrobeads
using split-and-mix methods. Polystyrene resin was selected because it is less expensive than is
polyethylene glycol-PS resin (TentaGel®), although the latter is generally more effective for a -
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115
and (3-peptide synthesis.16 Also, the largest commercially available macrobeads (500-600
pm) are composed of polystyrene. The high per-bead compound loading (80-200 nmol) o f PS
macrobeads is attractive for preparation o f one-bead-one-stock solution libraries; each bead
contains sufficient material for multiple
solution-based assays as well as analytical
characterization.17 TentaGel® macrobeads have a much lower loading, so their products cannot
be analyzed for purity but are merely identified by sensitive sequencing techniques.18 Because
they swell in water, TentaGel® macrobeads are most often employed in an on-bead assay format,
which can be prone to artifacts.18
Compound libraries are most useful if the compounds are generated with sufficient purity
to avoid ambiguity in biological assays. Unfortunately, reaction rate decreases with increasing
resin bead diameter, so library preparation on PS macrobeads can be hindered by slow reagent
diffusion into the polymer matrix and sluggish reaction rates.19 This problem is typically
resolved by using extremely long reaction times and large excesses (20 equivalents) o f reagents,
neither o f which is attractive, especially for P-peptide synthesis. 20 As microwave irradiation had
been found to reduce synthesis time,12 we applied this method to reactions on PS macrobeads to
address the inherent limitations o f this solid support.
3.2.4 p-Peptide Synthesis on PS Macrobeads
We sought to optimize synthesis on PS macrobeads as the next step towards the
preparation o f P-peptide combinatorial libraries. The high cost or synthesis time o f Fmoc-pamino acids makes it preferable to use as few monomer equivalents per coupling as possible; we
employed three equivalents. Manual synthesis o f P-peptides 3-1 and 3-2 on PS macrobeads
under extended reaction conditions at room temperature (6 hr per amide bond forming reaction
and 1 hr per Fmoc removal reaction) provided products with 69% and 56% purity, respectively
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116
(Figure 5).
The purities were improved to 87% and 70% by heating the coupling and
deprotection reactions to 65°C in an oil bath (Figure 5, Oil Bath A), but the 42 and 77 hr overall
synthesis times were cumbersome.
100,__
ji-P e p tid e
p -P e p tid e
3-1
3-2
HPLC
Peak
Area
Percent
A
Manual
|
B
|
C
Microwave
A
|
B
Oil Bath
R eaction Conditions
Figure 5. Initial purity of (1-peptides 3-1 and 3-2 (peak area percent, from analytical reverse-phase (RP) HPLC
monitored via UV absorbance at 220 nm) from synthesis on PS macrobeads under different conditions. All
coupling and deprotection reactions were conducted under the given reaction condition, i.e., manual, microwave, or
oil bath, as described below. ACHC-1 in (1-peptide 3-1 and ACHC-2 and ACHC-5 in P-peptide 3-2 were double­
coupled and double-deprotected in all syntheses. The coupling o f these residues was performed in 0.8 M LiCl in
NMP in the microwave and oil bath syntheses; all other coupling reactions were performed in DMF. Maximum
power for all microwave reactions was 50 W. Microwave-assisted deprotections were performed at 60°C, and
couplings were carried out at 50°C in DMF or 45°C in 0.8 M LiCl in NMP. Manual: 1 hr deprotection, 6 hr
coupling, RT; Microwave A: 1 hr deprotection at RT followed by 2 min ramp and 2 min hold, 2 hr coupling at RT
followed by 2 min ramp and 4 min hold; Microwave B: 2 min ramp and 1 hr hold for deprotection, 2 min ramp and
1 hr hold for coupling; Microwave C: 3 x (2 min ramp followed by 10 min cool at RT) for deprotection, 6 x (2 min
ramp followed by 10 min cool at RT) for coupling; Oil Bath A: 1 hr deprotection, 6 hr coupling, 65°C; Oil Bath B:
30 min deprotection, 1 hr coupling, 65°C.
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117
Table 1. Data for Figure 5.
Reaction Conditions
Manual
Microwave A
Microwave B
Microwave C
Oil Bath A
Oil Bath B
p-Peptide 3-1
Notebook Peak Area Percent
JKM V 253
69
JKM V 185
69
JKM V 243
70
JKM VI 069
81
JKM V 255
87
JKM VI 225
75
p-Peptide 3-2
Notebook Peak Area Percent
JKM V 285
56
JKM V 241
34
JKM V 263
54
JKM VI 079
70
JKM V 283
69
JKM VI 227
59
3000
Manila
2000
Abs
(mV)
Microwave A
Microwave B
1000
Microwave C
Oil Bath
Time (min)
Figure 6. Analytical RP-HPLC chromatograms o f p-peptide 3-1 (UV absorbance at 220 nm) prepared on PS
macrobeads under different conditions. All coupling and deprotection reactions were conducted under the given
reaction condition, i.e., manual, microwave, or oil bath, as described below. ACHC-1 in P-peptide 3-1 was double­
coupled and double-deprotected in all syntheses. The coupling of this residue was performed in 0.8 M LiCl in NMP
in the microwave and oil bath syntheses; all other coupling reactions were performed in DMF. Maximum power for
all microwave reactions was 50 W. Microwave-assisted deprotections were performed at 60°C, and couplings were
carried out at 50°C in DMF or 45°C in 0.8 M LiCl in NMP.
Manual: All couplings for 6 hr in DMF at RT; 1 hr deprotection at RT.
Microwave A: 1 hr deprotection at RT followed by 2 min ramp and 2 min hold; 2 hr coupling at RT followed by 2
min ramp and 4 min hold.
Microwave B: 2 min ramp and 1 hr hold for deprotection; 2 min ramp and 1 hr hold for coupling.
Microwave C: 3 * (2 min ramp followed by 10 min cool at RT) for deprotection; 6 x (2 min ramp followed by 10
min cool at RT) for coupling.
Oil Bath A: All couplings for 6 hr in DMF at 65°C in the oil bath, except for double-coupling ACHC-1 in 0.8 M
LiCl in NMP at 65°C in the oil bath; 1 hr deprotection at 65°C.
Oil Bath B: All couplings for 1 hr in DMF at 65°C in the oil bath, except for double-coupling ACHC-1 in 0.8 M
LiCl in NMP at 65°C in the oil bath; 30 min deprotection at 65°C.
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118
2000 -
3-2
Manual
M icrowave A
1500-----M icrowave B
Abs
1000
(m V )
V v __ I
-
M icrowave C
Oil Bath A „
Oil Bath B
10
o
20
30
40
50
T im e (m in)
Figure 7. Analytical RP-HPLC chromatograms of P-peptide 3-2 (UV absorbance at 220 nm) prepared on PS
macrobeads under different conditions. All coupling and deprotection reactions were conducted under the given
reaction condition, i.e., manual, microwave, or oil bath, as described below. ACHC-2 and ACHC-5 in p-peptide 32 were double-coupled and double-deprotected in all syntheses. The coupling of these residues was performed in
0.8 M LiCl in NMP in the microwave and oil bath syntheses; all other coupling reactions were performed in DMF.
Maximum power for all microwave reactions was 50 W. Microwave-assisted deprotections were performed at
60°C, and couplings were carried out at 50°C in DMF or 45°C in 0.8 M LiCl in NMP.
Manual: All couplings for 6 hr in DMF at RT; 1 hr deprotection at RT.
Microwave A: 1 hr deprotection at RT followed by 2 min ramp and 2 min hold; 2 hr coupling at RT followed by 2
min ramp and 4 min hold.
Microwave B: 2 min ramp and 1 hr hold for deprotection; 2 min ramp and 1 hr hold for coupling.
Microwave C: 3 x (2 min ramp followed by 10 min cool at RT) for deprotection; 6 x (2 min ramp followed by 10
min cool at RT) for coupling.
Oil Bath A: All couplings for 6 hr in DMF at 65°C in the oil bath, except for double-coupling ACHC-2 and ACHC5 in 0.8 M LiCl in NMP at 65°C in the oil bath; 1 hr deprotection at 65°C.
3.2.5 Microwave-Assisted (3-Peptide Synthesis on PS Macrobeads
Blackwell has shrewdly observed that microwave irradiation could be used to accelerate
reactions on PS macrobeads, thus overcoming the limitation of having to use long reaction
times.
21
At first it was unclear to us how to adapt the rapid heating o f microwave irradiation to
synthesis on PS macrobeads.
Our initial attempts were only moderately successful: direct
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119
application o f our microwave-assisted SPPS conditions (4 min deprotection and 6 min
coupling reactions) developed for 100-200 mesh PS resin to the synthesis of P-peptide 3-1 on PS
macrobeads provided the product in extremely low yield (JKM IV 159). We suspected that only
the surface o f the resin beads was being functionalized as the reagents were not likely to have
diffused very deeply into the polymer matrix during the short reaction time.
Allowing the
coupling to proceed for 2 hr at room temperature while the reagents diffused into the
macrobeads, followed by our typical 6 min microwave irradiation, gave P-peptide 3-1 in 69%
purity and P-peptide 3-2 in 34% purity (Figure 6 and Figure 7, Microwave A). Only marginal
enhancement was obtained by performing each reaction with 1 hr o f continuous microwave
irradiation (70% purity for 3-1 and 54% for 3-2; Figure 6 and Figure 7, Microwave B). (After
the initial 2 min ramp to the desired temperature, the power input was modulated to maintain that
temperature, resulting in a very low level o f microwave irradiation (< 5 W) being employed for a
majority o f the reaction time.) However, significant improvement was achieved by repeating a
cycle o f 2 min o f microwave irradiation to reach the desired temperature followed by 10 min o f
cooling (6 cycles per coupling, 3 cycles per deprotection). Subjecting the sample to several short
bursts o f intense microwave irradiation was found to be more effective than using a continuous
but lower level o f irradiation, as the former method provided P-peptide 3-1 on PS macrobeads in
high purity (81%, Figure 6, Microwave C). The reaction time Was significantly reduced relative
to oil bath heating (9 hr vs. 42 hr), p-peptide 3-2 was produced in 70% purity in only 16 hr
under these conditions (Figure 7, Microwave C). Thus, we observed a reduced reaction time for
microwave irradiation in comparison with conventional heating, but the two methods gave
comparable purities for both P-peptides.
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120
3.2.6 Comparison of Microwave Irradiation to Conventional Heating
To ascertain whether microwave irradiation gives a rate enhancement for synthesis on PS
macrobeads, we prepared p-peptides 3-1 and 3-2 using oil bath heating for short times (1 hr
coupling, 30 min deprotection).
We found that both 3-1 and 3-2 were produced in slightly
higher purities using conventional heating (75% and 59%; Figure 6 and Figure 7, Oil Bath B)
than with continuous microwave irradiation for the same reaction time (70% and 54%; Figure 6
and Figure 7, Microwave B). However, in neither case do the results match those obtained using
multiple cycles o f microwave irradiation (81% and 70%; Figure 6 and Figure 7, Microwave C),
which also require only 1 hr per coupling and 30 min per deprotection reaction. Therefore, our
optimized microwave irradiation conditions provide a moderate advantage for synthesis on PS
macrobeads, relative to the other conditions examined, as the optimized conditions provide the
product in the highest purity and shortest synthesis time. The temporal advantage is especially
important for oligomeric molecules, such as p-peptides, that require many sequential reactions.
3.2.7 Discussion of Effects of Microwave Irradiation on P-Peptide Synthesis
In our previous study o f P-peptide solid-phase synthesis on a different resin (PS 100-200
mesh), we found that both conventional heating and microwave irradiation gave similar results
for the shorter p-peptide 3-1 if reactions were heated 10 times .longer in the oil bath than in the
microwave reactor.12 However, synthesis o f the longer P-peptide 3-2 with microwave irradiation
had shown a significant advantage over conventional heating (beyond the decrease in time). We
had speculated that the microwave advantage for the synthesis o f 3-2 reflects the increasing
difficulty o f couplings and deprotections after the fifth residue, which may arise from
aggregation and/or folding during growth o f the p-peptide chain. Perhaps a longer period o f
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121
conventional heating for each coupling/deprotection cycle for the final five residues would
eventually have allowed us to produce p-peptide 3-2 in purity comparable to that o f the
microwave synthesis, as was the case for P-peptide 3-1.
An increasing number o f reports suggest that if microwave and conventional heating
reactions are carried out under identical conditions, then the results obtained with both methods
will be the same, i.e. there is no “non-thermal microwave effect.”
22
Although we went to great
lengths to carry out reactions under very similar experimental conditions (temperature,
atmospheric pressure, agitation with N 2 bubbling) for both methods o f energy input, we (and
others 23 ) found that conventional heating is unable to duplicate the rapid internal heating
provided by microwave irradiation.
(Heating a coupling solution o f DMF from room
temperature to 50°C with < 50 W o f microwave irradiation requires ~ 1.5 min; the same process
takes ~ 4 min in an oil bath.) We previously found also that the salt additive LiCl accentuates
the rapid heating and thus the advantage o f microwave synthesis for P-peptide 3-2.
12
(Heating a
coupling solution o f 0.8 M LiCl in NMP from room temperature to 50°C with < 50 W o f
microwave irradiation requires < 1 min.) Synthesis on PS macrobeads presents a new challenge
because the effectiveness o f rapid microwave heating is limited since the short irradiation cycle
is finished before the activated monomer can diffuse throughout the polymer matrix and react
with the free N-termini o f the growing oligomer chains. Employing a continuous, low level o f
microwave irradiation seems to exert a purely thermal effect, as synthesis under these conditions
(Microwave B) gives results similar to those obtained with oil bath heating for short times (Oil
Bath B). Therefore, the previously observed beneficial effects, obtained with 100-200 mesh PS
resin as a result of rapid internal heating with microwave irradiation,
12
are partially abrogated by
slow reagent diffusion and the long reaction times required with PS macrobeads.
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However,
122
using multiple cycles o f irradiation (Microwave C) re-establishes the advantage of
microwave heating by reducing the synthesis time needed to produce P-peptide 3-2 in similar
purity (~ 70%) to that achieved with conventional heating for longer periods o f time (Oil Bath
A). Perhaps the rapid heating associated with intense microwave irradiation is able to disrupt
on-bead P-peptide chain self-association more effectively than does conventional heating or
continuous lower levels o f microwave irradiation, thus driving the reaction to completion more
quickly. It has been suggested that microwave irradiation may enhance segmental motion in the
polymer matrix, thus assisting the diffusion o f reagents throughout the matrix.24
3.3 Synthesis and Characterization of a B-Peptide Combinatorial Library
Following our synthetic optimization on PS macrobeads, we used microwave irradiation
for rapid generation o f a 100-member P-peptide library via the split-and-pool technique (Figure
8). This library was based on octa-p-peptide 3-3, which was reported by Schepartz et al. to
block the interaction between the protein MDM2 and a 17-residue peptide from the N-terminal
region o f p53 4 The p53/MDM2 protein-protein interaction was discussed in Chapter 1.
3.3.1 P-Peptide Library Design
Octa-P-peptide 3-3 was reported to inhibit the interaction between MDM2 and a
fluorescently labeled a-peptide corresponding to residues 15-31 o f wild type p53 with a median
inhibitory concentration (IC 5 0 ) o f approximately 80 pM in a fluorescence polarization (FP)
competition assay 4 P-Peptide 3-3 was designed to adopt a 14-helical conformation that displays
three critical side chains, those of p3-hLeu-l, p3-hTrp-4, and p3-hPhe-7, along one side o f the
helix.5 These p-peptide side chains are intended to mimic those of Leu-26, Trp-23 and Phe-19 of
p53, which are observed to align along one side o f a distorted a-helix in a complex between
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123
MDM2 and an a-peptide corresponding to residues 15-29 o f p53 . 2 5 Since the unlabelled cxpeptide corresponding to residues 15-31 had an IC 5 0 o f ca. 2 pM in the FP competition assay,
roughly 40-fold lower than the IC 5 0 o f p-peptide 3-3, we wondered whether combinatorial
variation o f the sequence of 3-3 would generate improved inhibitors (Figure 8). Our library was
designed to probe the effect of conformational stability on binding affinity by incorporating both
structure-promoting (ACHC) and structure-destabilizing residues (P-hGly) at positions 3 and 6.
-j
o
The degree o f hydrophobicity at these positions was varied by including p -hLeu, P -hVal and
P -hAla as well. We also varied the nature o f the side chain at position 5: replacing p -hOm
with p3-hSer or p3-hGlu may better mimic the side chains displayed at this position in the p53 a helix, according to superimposition analysis.
NH
O
NH
O
N
N
H
OH
H
O
Figure 8. Octa-P-peptide library produced via split-and-pool synthesis on PS macrobeads with microwave
irradiation. Five different residues were incorporated at positions 3 and 6; four different residues were installed at
position 5 ( 5 x 4 x 5 = 100 members). The sequence of P-peptide 3-3 is from ref. 4.
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124
3.3.2 P-Peptide Library Synthesis on PS Macrobeads with Microwave Irradiation
We constructed the library on PS macrobeads in two days with multiple cycles of
microwave irradiation.
Reaction progress was monitored using 2,4,6-trinitrobenzenesulfonic
acid (TNBS), which stains beads containing unreacted amino groups.60 Incomplete coupling of
p3-hPhe-7 was detected at the end o f the standard reaction cycle, so the mixture was subjected to
an additional three microwave irradiation/cool cycles.
The APiC residue at position 5 was
double-coupled in DMF; ACHC-3 was double-coupled in NM P/LiCl.12 All other residues at
position 3 were double-coupled in DMF.
3.3.3 Analytical Characterization of P-Peptide Library
To assess the quality o f the library, 50 beads were selected at random, and the material
from them was characterized.
In all cases the major peak in the analytical RP-HPLC trace
corresponded to the molecular weight o f an expected library member, which indicates an
excellent library quality.
96
p-Peptides from 10 o f the randomly chosen beads were sequenced by
pLC-MS/MS by Dr. Mark Scalf.27 In each case the deduced sequence corresponded to an
expected member o f the library, with a matching parent molecular weight. We were especially
pleased that the sequence containing three cyclically-constrained residues (Figure 9A), expected
to be the most difficult library member to synthesize, was found among the 50 sequences via
identification o f its unique molecular weight. This P-peptide was produced in an acceptable
68% purity (Figure 9B), and sequencing confirmed its identity (Figure 9C-E).
The average
purity o f P-peptides from the library was 65% (Figure 10). The major impurity from each bead
had an average o f only 10% o f the total HPLC peak area. This high level o f initial purity makes
HPLC purification unnecessary and raises confidence in screening results obtained with initial
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125
products.
The lowest purity library members were still readily identified through pLC-
MS/MS sequencing and were each found to contain p -hSer at position 5. However, the major
impurities were not simply p3-hSer deletion sequences but were quite varied among the product
mixtures from different beads. This effort represents the first P-peptide combinatorial library,
and, to our knowledge, the first example o f a split-and-pool synthesis on PS macrobeads with
microwave irradiation.
A peptoid combinatorial library was synthesized on TentaGel®
macrobeads using microwave irradiation.18 M ost microwave-assisted syntheses o f combinatorial
libraries have been done in parallel using automated sequential irradiation.28 Subsequent to our
work, Schepartz et al. reported the preparation o f a P-peptide library on TentaGel® macrobeads,
although the purity o f the library was not ascertained. 29
A)
8 9 8 49
C alc. M a s s® 1 168.65
773.40
B)
1000
68%, product [M+H] ’
1169.4
Purity
4 4 7 .2 3
3 2 2 .1 5
CO*
d
ISH
Abs
(mV)
C 0 2H
3
C
O
0
n > v-
h 2n
soo
A
u.
OH
H
H
10
847.51
E)
7 2 2 .4 2
(M +H f
1169,9
C)
R elative 6 0
A b u n d a n ce
40
R elative
A b u n d a n ce
20
100
80
60
R elative
A b u n d a n ce
40
JM+2H]
586.3
20
20
40
60
80
Time (min)
100
i847.3
i
899.3
1084.3
iACHC
323.2
600 " 800 ~ 1000
mtz
±
so
30
40
Time (min)
774.3
1200 1400
44 8 .2
574.2 722.4
a i »c
0 200
[i .Tip
. .. .i l l .
400
600
J ..il
m/z
800
1000
Figure 9. Characterization a P-peptide library member. A) Deduced library member from MW and observed
fragmentation pattern (calculated masses). B) Analytical RP-HPLC trace (68% product purity) with MW from
MALDI-TOF MS of major peak. C) Base peak chromatogram of pLC (monitored by total ion count o f most
abundant ion in each scan). D) ESI-MS of bracketed region (57.5-62 min) in C. E) ESI-MS/MS of [M+2H]2+
parent ion in D with assigned P-amino acid fragments.
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126
18
Number 12
of
Beads g
6
3
0
6 0 -6 9
Product Purity (%)
Figure 10. Product purity of material from 50 randomly selected beads from the octa-P-peptide library, determined
as the area percent of the major peak in the analytical RP-HPLC chromatogram (UV absorbance at 220 nm). The
average purity was 65%.
Table 2. Data for Figure 10.
Product Purity (%) Number of Beads
5
40-49
8
50-59
17
60-69
70-79
20
3.3.4 Library Screening for Inhibition of the p53-MDM2 Interaction
We tested the library in a competition ELISA to determine whether any o f the members
was able to inhibit p53-MDM2 association with greater potency than P-peptide 3-3. This assay
was carried out by Bilal Farooqi in the laboratory o f Dr. Jiandong Chen at the University of
South Florida.
The ELISA format differs from the FP format used previously4 because the
ELISA tests for the inhibition o f a protein-protein interaction, while the FP assay reports on
inhibition of a protein-peptide interaction.
Plates were coated with full-length human p53
bearing a His6 tag. Crude P-peptide from a single macrobead and full-length human MDM2
fused to GST were added to each well. Screening at an approximate p-peptide concentration of
75 pM identified seven library members exhibiting roughly 80% inhibition, while P-peptide 3-3
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127
showed only about 15% inhibition at this concentration. The “hits” from the library were
identified via mass spectrometric sequencing, revealing seven different sequences (Table 3).
None of the “hits” was redundantly identified. Statistically, using 5 beads per compound ensures
adequate coverage o f this small library (between 95 and 99% o f the theoretical library members
will be represented),30 but does not guarantee that there will be multiple copies o f the same
compound within the library.
Furthermore, removal o f 50 beads for quality control, bead
breakage during synthesis (reducing the yield), an arbitrary definition o f what level o f percent
inhibition constitutes a “hit,” and the low sensitivity o f the assay for weakly binding compounds
all potentially explain why multiple copies o f the same compound were not identified.
The
seven active P-peptides were individually re-synthesized using our microwave conditions,
12
purified by HPLC, and evaluated in the ELISA. The identified compounds did show slightly
improved activity over P-peptide 3-3, validating them as “hits” and not false positives.
No
significant variation in activity was observed among these seven P-peptides, apparently because
the assay is relatively insensitive for such weakly binding compounds. Library member 3-4 was
investigated further and found to have an IC50 value o f ca. 250 pM (Figure 11); it was slightly
more effective at inhibiting the p53/MDM2 interaction than lead P-peptide 3-3 (IC50 > 600 pM).
Table 3. Octa-p-peptide hits from library screening and sequencing. Compound numbers correspond to list of
expected P-peptide library members in Tables 9 and 10.
Compound
Sequence
5
6
7
8
C
p3-hGlu
p3-hSer
p3-hGlu
ACHC
ACHC
p3-hLeu
p3-hPhe
p3-hPhe
p3-hPhe
p3-hGlu
p3-hGlu
p3-hGlu
OH
OH
OH
p3-hTrp
p3-hTrp
p3-hGlu
p3-hSer
p3-hLeu
p3-hLeu
p3-hPhe
p3-hPhe
p3-hGlu
p3-hGlu
OH
OH
p3-hT rp
p3-hTrp
p3-hSer
p3-hOrn
p3-hAla
p3-hGly
p3-hPhe
p3-hPhe
p3-hGlu
pJ-hGlu
OH
OH
N
1
2
3
4
3 - 4 /4
35
2
H
H
H
p3-hLeu
p3-hLeu
p3-hLeu
p3-hGlu
p3-hGlu
p3-hGlu
ACHC
ACHC
ACHC
p3-hT rp
p3-hT rp
p3-hT rp
1
26
H
H
p3-hLeu
p3-hLeu
p3-hGlu
p3-hGlu
p3-hLeu
p3-hLeu
79
73
H
H
p3-hLeu
p3-hLeu
p3-hGlu
p3-hGlu
p3-hVal
p3-hVal
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3-4
h 2n
OH
□ Nutlin-3
» p53 15-31
▼ |5-Peptide3-4
1,25-|
1 . 00 -
•
Cl
Cl
o
/
|S-Peptide3-3
nh
<
0,25'
il I
Q
0 . 00'
-
Cl
2
-
1
0
1
2
3
log [co m p etito r], pIVI
(±)-Nutlin-3
Figure 11. Binding data from the competition ELISA fitted with sigmoidal dose-response curves.
3.3.5 Discussion of P-Peptide Inhibitors o f the p53/MDM2 Interaction
Identifying a number o f library members with slightly higher potency than the lead
sequence provided useful information on the structure/activity relationship o f these compounds.
We were particularly interested in the substitution o f ACHC, which stabilizes the 14-helix, to
learn about the favored binding conformation o f these P-peptides. Molecular modeling was used
to visualize the p-peptide/protein interaction and formulate hypotheses for interpretation o f the
data. Finally, comparison to known inhibitors o f the p53/MDM2 interaction reveals that these
designs are only weakly active.
3.3.5.1 14-Helical Scaffold
P-Peptide 3-4 contains ACHC residues at positions 2 and 6, which should strongly
promote the 14-helical conformation.
However, stabilizing the 14-helix does not seem to
provide a benefit in terms o f interfering with p53-MDM2 binding, because other P-peptides
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12 9
Figure 12. Top view of co-crystal structure of p53-derived a-peptide (residues 17-29) bound to human MDM2
(ref. 25, PDB ID = 1YCR). MDM2 surface rendered using MOLCAD and colored by distance from the a-peptide
(red=close, gray=far). Figure produced using Sybyl (Tripos).
Figure 13. Representative low energy structure of P-peptide 3-3 computationally docked to rigid MDM2 crystal
structure using FlexiDock (25,000 generations, Sybyl). P-peptide side chains were allowed free rotation, but
backbone was locked in a 14-helical conformation.
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130
Figure 14. View of co-crystal structure down the axis of the a-helix (p53 residues 17-29) bound to human MDM2
(ref. 25, PDB ID = 1YCR). MDM2 surface rendered using MOLCAD and colored by distance from the a-peptide
(red=close, gray=far). Figure produced using Sybyl (Tripos).
Figure 15. View down the helix axis of p-Peptide 3-3 docked to MDM2. By comparison of the peptide structures
in Figure 14 and Figure 15, we observed that the internal and external diameters of the 14-helix are larger than those
of the a-helix and perhaps may be too large to fit deeply into the narrow hydrophobic cleft on the surface of
MDM2.
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131
among the seven active library members contain p3-hLeu in place o f one ACHC or both, or
even P-hGly at one o f these positions. These findings suggest that the P-peptide 14-helix may
not be an ideal scaffold for development o f p53-MDM2 antagonists.
Indeed, molecular
modeling with fixed backbone geometry indicates that the 14-helix is considerably wider than is
the a-helix, the secondary structure adopted by the N-terminal segment of p53 when bound to
MDM2 (Figure 12 and Figure 13). This may prevent the P-peptide scaffold from inserting as
deeply into the hydrophobic cleft on MDM2 as the a-helix o f p53 (Figure 14 and Figure 15).
Also, the 14-helix in this case (and the Schepartz case) has opposite handedness relative to the
p53-derived a-helix composed o f L-a-amino acid residues.
5
3
Schepartz and coworkers have suggested that P-peptides composed entirely o f p residues can depart slightly from an idealized 14-helical conformation, as expected based on the
well-known flexibility and low intrinsic 14-helical propensity of p3-residues,31 and they have
speculated that such distortion is necessary for optimal binding to the MDM2 cleft. The contrast
we observe between the lack o f significant variation in activity among the “hits” from our library
and the wide range o f expected 14-helical propensities among these compounds (arising from
variations in ACHC and P-hGly content) raises the possibility that small conformational
distortions are not important for MDM2 affinity, at least among the weakly binding P-peptides
reported to date.
3.3.5.2 Charge-Charge Interactions
Six o f the seven active library members contained a replacement for p -hOm, the original
residue at position 5.
The p3-hOm side chain should be cationic under assay conditions; a
positive charge at this position was dictated in the original design4 by the need for side chain ion
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132
pairing to stabilize the 14-helical conformation. We explored the effect o f anionic (|33-hGlu)
and neutral (p3-hSer) side chains at this position in the library, which may better mimic the Ser20 and Asp-21 residues in the p53-derived a-peptide. Although molecular modeling suggested
to us that the P-amino acid side chain at position 5 is solvent-exposed, the results imply that such
replacements o f p3-hOm-5 are at least modestly beneficial for p-peptide binding to MDM2.
3.3.5.3 Comparison to Positive Controls
Included in our ELISA as a positive control for inhibition o f the p53-MDM2 interaction
was a p53-derived a-peptide comprising residues 15-31.
This a-peptide contains all three
hydrophobic residues that are essential for binding to MDM2 (Leu-26, Trp-23, and Phe-19) and
has an IC50 o f ca. 25 pM in our ELISA, which is consistent with prior results.32 Thus, although
library member 3-4 is marginally improved relative to the original p-peptide sequence, 3-3, these
P-peptides are considerably less effective than the natural a-peptide sequence 15-31; comparable
findings were reported by Schepartz et al., based on FP competition assays.4 The deca-P-peptide
3-5, originally reported by Schepartz et al.,4 was also prepared and screened in the ELISA. It
had an IC50 value o f approximately 100 pM, similar to the reported 94 pM IC50 value reported
using the FP assay, but still four-fold less potent than the a-peptide control.
As a further positive control, we examined Nutlin-3, a synthetic small molecule that
effectively inhibits the p53-MDM2 interaction in vitro and is orally active as an anticancer agent
in vivo.
We measured an IC50 o f ca. 0.5 pM, which is consistent with the original report.
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133
This small molecule is superior as an inhibitor o f p53-MDM2 binding relative to all P*
peptides described here.
3.4 Conclusions
We have shown that improvements in the solid-phase synthesis o f P-peptides using
microwave irradiation and a salt additive can be extended to synthesis on PS macrobeads.
Relative to conventional methods, the microwave approach significantly reduces synthesis time
and required amounts of reagents, which until now have been major limitations on the use o f PS
macrobeads to generate one-bead-one-stock solution combinatorial libraries.
Microwave
irradiation allowed rapid synthesis o f a high-purity P-peptide library via a split-and-mix
approach. This library was screened for inhibitors o f the p53-MDM2 interaction. Hits from this
library provided marginal improvement relative to a previously reported P-peptide sequence.
Our failure to find a more potent inhibitor in this library may reflect the small size o f our library
(insufficient diversity), or this result may indicate that the 14-helix is not well suited to mimic
the a-helical segment o f p53. The relative insensitivity o f the sequence to various substitutions
indicates that these p-peptides are only weakly interacting with the p53-binding site. Schepartz
et al. found that the largest increase in binding affinity was achieved via incorporation o f more
hydrophobic p -hlle residues on the “non-interacting” helix face originally composed o f p hVal.29 In a search for inhibitors of a different a-helix/cleft interaction, the binding o f a BH3
domain to Bcl-xL, we have found P-peptide helices to be unproductive scaffolds, but alternative
foldamers containing both a - and p-amino acid residues lead to effective inhibitors.34
Ultimately, P-peptides and other foldamers may be best suited to disruption o f protein-protein
interactions that involve flatter surfaces on each partner than are found in the p53-MDM2 pair.
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134
Flat interface architectures appear to be particularly challenging for low molecular weight
antagonists.35
The methodological developments reported here will facilitate combinatorial
exploration o f (3-peptides and other foldamers as scaffolds for discovery o f protein-protein
interaction inhibitors.
3.5 Experimental Methods
3.5.1 General Procedures
Fmoc-(1S,,<S)-ACHC-OH and Fmoc-(5',5)-APiC(Boc)-OFI were prepared by the method o f
Schinnerl et al. 3 6
Fmoc-(33 -amino acids were prepared from their corresponding Fmoc-L-a-
amino acids (Novabiochem) as described previously.73 1-Methyl-2-pyrrolidinone (NMP) was
purchased from Advanced ChemTech. Methanol, CH 2 CI2 and acetonitrile were purchased from
Burdick & Jackson.
1-Methylimidazole, piperidine, 1-hydroxybenzotriazole hydrate (FIOBt),
z'P^EtN (DIEA), trifluoroacetic acid (TFA), triethylsilane (TES) and DMSO were purchased
from Aldrich. l-(2-M esitylenesulfonyl)-3-nitro-l,2,4-triazole (MSNT) and O-benzotriazol-l-ylN, N ,N ’,N ’-tetramethyluron ium hexaflurorophosphate (F1BTU), PS Wang resin (100-200 mesh)
and NovaSyn TGR resin were purchased from Novabiochem. Polystyrene A PHB resin (500560 pm “macrobeads”) was purchased from Rapp Polymere. DMF (biotech grade solvent, 99.9+
%) was purchased from Aldrich and stored over Dowex ion exchange resin.
2,4,6-
Trinitrobenzenesulfonic acid (TNBS, 1% solution in DMF) was purchased from Fluka. Nutlin-3
((±)-4-[4,5-6/.v-(4-chlorophenyl)-2-(2-isopropanoxy-4-methoxy-phenyl)-4,5-dihydro-imidazolel-carbonyl]-piperazin-2-one) was purchased from Cayman Chemical. Dry CFI2 CI2 and /P^EtN
were distilled from calcium hydride.
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135
3.5.2 First Residue Loading
First residue loading was accomplished as described with some modifications.6c’37 Fmoc(S)-|33-hGlu(dBu)-OH (0.824 g) was activated with 1-methylimidazole (112 pL) and MSNT (556
mg) in dry CH 2 CI2 (5.6 mL) and added to DMF-swollen Polystyrene A PFIB macrobeads (500
mg, 500-560 pm resin, 0.75 mmol/g initial loading, Rapp Polymere) in a polypropylene solidphase extraction (SPE) tube (25 mL, Alltech). The tube was capped and placed on a wrist-action
shaker (Labquake, Bamstead/Thermolyne). After reaction for 12 hr at room temperature, the
resin was washed (5 x CH2C12, 5 x DMF, 5 x CH 2 CI2 and 5 x MeOH) using a vacuum manifold
(Vac-Man, Promega) connected to a water aspirator and then dried under a stream o f N 2 until
free-flowing. The yield was estimated by UV-quantification of the dibenzofulvene-piperidine
adduct at 290 nm as previously described (0.48 mmol/g, 64%). 38
3.5.3 P-Peptide Synthesis on PS Macrobeads
3.5.3.1 Microwave [3-Peptide Synthesis on PS Macrobeads Using Ramp/Cool Cycles
Loaded PS macrobeads (10 pmol, 21 mg) were placed in a modified polypropylene SPE
tube (4 mL, Alltech, top rim removed with a razor blade) and swelled with DMF for
approximately 10 min.
The resin was washed (5 x DMF, 5 x CH 2 CI2 and 5 x DMF).
Deprotection solution (750 pL o f 20% piperidine in DMF (v/v)) was added to the resin, and the
tube was placed inside a glass 10 mL microwave reaction vessel containing ~ 2 mL o f DMF
(Figure 16). A N 2 line was inserted for agitation, and the vessel was placed in the microwave
reactor (CEM Discover) and irradiated (50 W maximum power, 60°C, ramp 2 min). The sample
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136
pasteur pipette
for N2 agitation
^ 1 0 mL glass vial
^--4 mL polypropylene
SPE tube
^ DMF or 0,8 m
LiCl in NMP
coupling solution
*T
*• « . -H
K g
iS&Q h
^ resin
^
polyethylene frit
-- luer-lock cap
IRsensor
Figure 16. Left) Experimental set-up for small-scale microwave SPPS of P-peptides (SPE = solid-phase extraction)
and Right) CEM Discover microwave reactor (http://www.cem.com/synthesis/discover_s.asp).
was then cooled with a stream o f compressed air for 10 min. This ramp/cool cycle was repeated
a total o f 3 times for a deprotection. The tube was removed from the microwave reactor, and the
resin was washed as before. In a separate vial, Fmoc-P-amino acid (30 pmol) was activated by
adding HBTU (60 pL o f 0.5 M solution in DMF), DMF (440 pL), FIOBt (60 pL o f 0.5 M
solution in DMF), and /P^EtN (60 pL o f 1.0 M solution in DMF). The mixture was vortexed
and added to the resin. The sample was irradiated in the microwave reactor (50 W maximum
power, 50°C, ramp 2 min). The sample was then cooled for 10 min; this ramp/cool cycle was
repeated a total of 6 times per coupling step. Alternatively, the coupling o f Fmoc-(5',5)-ACHCOH was performed by activating with solutions o f FIBTU, HOBt, and /P^EtN in NMP and
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137
adding a solution o f LiCl in NMP for a final concentration of 0.8 M LiCl (620 pL final
volume), and then adding this solution to the resin. The tube was placed in a microwave reaction
vessel containing approximately 2 mL o f 0.8 M LiCl in NMP and irradiated (50 W maximum
power, 45°C, 6 ramp/cool cycles). (The target temperature was set to only 45°C, but the higher
ionic strength o f the 0.8 M LiCl in NMP solution results in a greater efficiency o f energy
transfer, so the final temperature is similar to that observed for a coupling reaction in DMF set at
50°C. Using our reaction vessel with the IR temperature sensor gave reproducible results with
the empirically-derived set temperatures, but the temperature measurements were not accurate.39)
After the coupling reaction, the resin was washed as before. The N-terminal ACHC residue in Ppeptide 3-1 and ACHC-2 and ACHC-5 in p-peptide 3-2 were double-coupled and double­
deprotected.
All other couplings were performed once with DMF as the solvent.
The
deprotection/coupling cycle was repeated in a stepwise manner until reaching the desired
oligomer length.
3.5.3.2 Manual B-Peptide Synthesis on PS Macrobeads
Loaded PS macrobeads (10 pmol, 21 mg) were placed in an SPE tube (4 mL, Alltech)
and swelled with DMF.
The resin was washed (5 x DMF, 5 x CH 2 CI2 and 5 x DMF).
Deprotection solution was added to the resin, and the tube was capped and placed on a shaker for
1 hr at room temperature. The tube was removed from the shaker, and the resin was washed as
before. A solution o f activated Fmoc-p-amino acid was added to the resin, and the tube was
capped and placed on the shaker for 6 hr at room temperature. After the coupling reaction, the
resin was washed as before. The deprotection/coupling cycle was repeated in a stepwise manner
until reaching the desired length o f the hexamer or decamer.
All coupling reactions were
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138
performed in DMF.
The N-terminal ACFIC residue in P-peptide 3-1 and ACFIC-2 and
ACHC-5 in P-peptide 3-2 were double-coupled and double-deprotected.
3.5.3.3 Oil Bath p-Peptide Synthesis on PS Macrobeads
Loaded PS macrobeads (10 pmol, 21 mg) were placed in a SPE tube (4 mL, Alltech) and
swelled with DMF. The resin was washed (5 x DMF, 5 x CH 2 CI2 and 5 x DMF). Deprotection
solution was added to the resin, a N 2 line was inserted for agitation, and the tube placed in an oil
bath at 65 °C for 1 hr. The tube was removed from the oil bath, and the resin was washed as
before. A solution of activated Fmoc-P-amino acid was added to the resin, a N 2 line was inserted
for agitation, and the tube placed in an oil bath at 65 °C for 6 hr. After the coupling reaction, the
resin was washed as before. The deprotection/coupling cycle was repeated in a stepwise manner
until reaching the desired length o f the hexamer or decamer. The N-terminal ACFIC residue in
P-peptide 3-1 and ACHC-2 and ACF1C-5 in P-peptide 3-2 were double-coupled in 0.8 M LiCl in
NMP and double-deprotected. All other coupling reactions were performed once in DMF.
3.5.4 p-Peptide Cleavage and HPLC Analysis
After the final residue had been added and deprotected, the resin was washed (5 x DMF,
5 x CH 2 CI2 , 5 x DMF and 5 x CFI2 CI2 ), and the p-peptide was cleaved from the solid support
with simultaneous side chain deprotection (3 mL, 45:45:5:5 TFA:CH 2 Cl2 :TES:water, 2 h, RT,
with rocking). The cleavage solution was drained and concentrated under a stream o f N 2 . The
crude p-peptide mixture was dissolved in 1.0 mL DMSO, diluted with DMSO (1 to 20) and
analyzed by HPLC (10 pL injection, Shimadzu). The compound was eluted from a C 4 -silica
reverse-phase analytical column (5 pm, 4 mm x 250 mm, Vydac) with a gradient o f acetonitrile
in water (10 - 60%, 50 min, 0.1% TFA in each) at a flow rate of 1 mL/min. Product purity was
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139
determined as peak area percent by integration of the UV absorbance at 220 nm. Integration
was performed over the 15 - 50 min time interval to exclude the large absorbance o f DMSO that
elutes from 5 - 1 5 min. The lower threshold o f integration was set to exclude minor peaks
whose areas were < 1% o f the peak area o f the major species. (3-Peptide masses were measured
by MALDI-TOF-MS (Bruker Reflex II, a-cyano-4-hydroxycinnamic acid matrix).
3.5.5 p-Peptide Characterization Data
p -P eptide 3*1
ACHC1-Addition
^48^68^8^9
C 5 5 H 7 9 N 9 O -1 0
Exact M ass: 1025.59
Exact M ass: 900.51
Fm ocH N '
ACHC1-Deietion
Fm oc-p-Peptide 3-1
C 4 1H 57N 7O 8
C 63 H 78N 80 -n
Exact M ass: 775.43
Exact M ass: 1122.58
Figure 17. Structures, formulas, and calculated masses of P-peptide 3-1 and major side products.
Table 4. Characterization data for P-peptide 3-1 and major impurities.
Com pound
Form u la
C a lc u la ted M a s s
M A L D I-T O F M S
O b s e rve d M ass
R P -H P L C
R eten tio n T im e
[M + H ]+
[M + N a f
(m in )
(3-Peptide 3-1
C48H68N80g
9 0 0 .5 1
9 0 1 .4
9 2 3 .4
3 3 .0
A C H C -1 -D e le tio n
C 41H 57N 7O 8
C 55H 7 9 N 9O 1
7 7 5 .4 3
7 7 6 .2
7 9 8 .2
2 4 .5
0
1 0 2 5 .5 9
1 0 2 6 .3
1 0 4 8 .3
3 6 .0
1 1 2 2 .5 8
1 1 2 3 .5
1 1 4 5 .5
4 5 .5
A C H C -1 -Addition
^63^78^801
F m o c -p -P e p tid e 3-1
1
Table5. Data for Figure 18.
Notebook Reaction Conditions p-Peptide 3-1 ACHC1-Deletion ACHC1-Addition Fmoc-p-Peptide 3-1
JKM V 253
Manual
69
12
2
5
JKMV185
Microwave A
69
8
6
0
JKM V 243
Microwave B
70
5
9
0
JKM VI 069
Microwave C
81
8
5
0
JKM V 255
Oil Bath A
87
0
2
0
JKM VI 225
Oil Bath B
75
5
0
0
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140
HPLC
Peak Area
Percent
■ 3-1
□ ACHC1-Deletion
50
□ ACHC1-Addition
□ Fmoc-3-1
n.
B
Manual
C
A
B
Oil Bath
Microwave
Reaction Conditions
Figure 18. Amount of P-peptide 3-1 and major impurities (peak area percent, from analytical reverse-phase (RP)
HPLC monitored via U V absorbance at 220 nm) from synthesis on PS macrobeads under different conditions
described in Figure 5.
C 0 2H
h 2n
(3-Peptide 3-2
C 74H 113N 13O 15
Exact M ass: 1 4 2 3 .8 5
\
co
2h
P3-hO rn1-Deletion
C 6 8 H 1 01N 1 l O l 4
E xact M ass: 1 2 9 5 .7 5
9 ° 2H
0
"9
NH
0
x ' 1- 1
X
i
AC H C 2-Deletion
C 67H 102N 1 2 O l4
Exact M ass: 1 2 9 8 .7 6
^NH
NH2
Jl
S
0
(
u
/NH;
0
p3-hG lu4-Deletion
C 68H 104N 1 2 ° 1 2
Exact M ass: 1 2 8 0 .7 9
Figure 19. Structures, formulas, and calculated masses of P-peptide 3-2 and major side products.
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141
Table 6. Characterization data for P-peptide 3-2 and major impurities.
MALDI-TOF MS
Observed Mass
[M +Hf [M+Naf
RP-HPLC
Retention Time
(min)
Compound
Formula
Calculated
Mass
P-Peptide 3-2
p3-hOrn1Deletion
ACHC-2Deietion
p3-hGlu4Deletion
C 7 4 H 1 1 3 N 1 3 O 15
1423.85
1425.0
1447.0
41.5
C6 8 H 1 0 1 N 1 1 O 14
1295.75
1296.2
1318.2
44.5
C 6 7 H 1 0 2 N 1 2 O 14
1298.76
1299.8
1321.7
33.5
C6 8 H 1 0 4 N 1 2 O 12
1280.79
1282.1
1304.1
31.5
■ 3-2
50
HPLC
Peak Area 40
Percent
30
■I Orn1 -Deletion
□ ACHC2-Deletion
■ Glu4-Deletion
A
Manual
B
C
A
Microwave
B
Oil Bath
Reaction Conditions
Figure 20. Amount of P-peptide 3-2 and major impurities (peak area percent, from analytical reverse-phase (RP)
HPLC monitored via UV absorbance at 220 nm) from synthesis on PS macrobeads under different conditions
described in Figure 5.
Table 7. Data for Figure 20.
Notebook Reaction Conditions P-Peptide 3-2 p3-Orn1-Deletion ACHC2-Deletion p3-Glu4-Deletion
JKM V 285
56
0
4
0
Manual
JKM V 241
Microwave A
34
0
8
4
54
11
0
JKM V 263
Microwave B
5
JKM VI 079
Microwave C
70
8
0
0
4
JKM V 283
Oil Bath A
69
0
0
JKM VI 227
Oil Bath B
59
5
0
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6
142
3.5.6 Calculation of P-Peptide Yield from a Single M acrobead
Yield from the synthesis o f P-peptide 3-3 on PS macrobeads was quantified by the
method o f Yan et a l 40 P-Peptide 3-3 was synthesized on PS macrobeads on a 10 pmol scale
with microwave irradiation as described above.
Material was cleaved from a portion o f the
beads and analyzed by HPLC as above. The product had a purity o f 64%. This sample o f crude
P-peptide 3-3 was purified by C4 -silica preparative reverse-phase HPLC. The compound was
eluted from the column (10 pm, 22 mm x 250 mm, Vydac) with a gradient o f acetonitrile in
water (24-54%, 30 min, 0.1% TFA in each) at a flow rate o f 15 mL/min. After lyophilization, a
small sample (~1 mg) o f purified P-peptide 3-3 was dissolved in 1.0 mL o f DMSO to make a
0.53 mM stock solution, the concentration being determined by p -hTrp absorbance in 6 M
guanidine hydrochloride 41 The stock solution was then diluted to make a series o f calibration
solutions containing a fixed concentration o f Fmoc-L-Phe-OH (retention time = 40 min) as an
external standard to compensate for instrumental fluctuation and other systematic errors. These
solutions were analyzed by analytical RP-HPLC (C 4 -silica reverse-phase analytical column (5
pm, 4 mm x 250 mm, Vydac) eluted with a gradient o f acetonitrile in water, 10 - 60%, 50 min,
0.1% TFA in each, at a flow rate of 1 mL/min) and monitored by UV absorbance at 220 nm.
The peak area o f P-peptide 3-3 was divided by the peak area o f the external standard to give the
peak area ratio [peak area ratio = (peak area)p_peptidc 3 / (peak area)cxtcrnai standard]- A plot o f peak
area ratio versus concentration yielded a calibration curve (see Supporting Information). The
curve was fit by linear regression [peak area ratio = 10.56 (concentration) - 0.0449] with a
correlation coefficient (R2) o f 0.9921.
The calibration curve was validated by preparing a
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143
solution of [3-peptide 3-3 o f a known concentration and analyzing by HPLC with UV
detection at 220 nm. The concentration determined based on the calibration curve was accurate
within 5%.
To determine the yield from a single macrobead, the crude P-peptide 3-3 mixture from a
single bead was dissolved in 30 pL o f DMSO. External standard was added, the solution was
diluted with DMSO to a total volume o f 100 pL, and 30 pL of the sample was analyzed by
HPLC.
The peak area ratio was measured, and the concentration was determined using the
calibration curve. The amount o f P-peptide 3-3 was calculated [concentration (mM) x volume
(100 pL) = nmol o f P-peptide 3-3], The percent yield was calculated based on the theoretical
yield [(0.48 mmol/g loading) / (8,150 macrobeads/g) = 59 nmol/macrobead]. The average yield
from a single macrobead was 8 nmol, 13%. This low yield was general among microwave, oil
bath, and manual syntheses.
A prior screening o f cleavage conditions (time: 2 or 6 hr;
temperature: RT or 50°C in an incubator; and DCM concentration: 0 - 90% in 10% increments)
identified the reported conditions that gave this yield.
3-3
600-
400Abs
(mV)
200
Sj '-J
10
20
30
Time (min)
40
Figure 21. Analytical RP-HPLC trace of [3-peptide 3-3 synthesized on PS macrobeads (64% purity).
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50
Table 8. Yield calculation for P-peptide 3-3 from a single PS macrobead via correlation of analytical RPHPLC peak area (UV absorbance at 220 nm) with concentration.
Bead 1
Bead 2
Bead 3
Concentration
Peak Area
Peak Area Yield Yield
p-Peptide 3-3 External Standard
(mM)
Ratio
(nmol) (%)
0
0
7874856
0
907477
0.013
9575715
0.0948
1745948
0.026
8964923
0.1948
0.040
3151600
0.3268
9643423
0.053
4818891
9716272
0.4960
0.066
4820827
0.5866
8218128
0.8304
0.079
7078130
8523919
0.092
7857560
7964874
0.9865
0.105
7935660
7678799
1.0335
0.119
8498363
1.1427
7437232
9892579
0.132
7020420
1.4091
0.145
10992294
6573118
1.6723
11800632
1.6217
0.158
7276754
0.171
12810594
7383930
1.7349
0.184
13995017
7481700
1.8706
0.198
14807753
2.0088
7371266
0.211
15811378
7355307
2.1497
5396474
0.076
7157438
0.7540
8
13
0.083
5755582
0.8271
6958365
8
14
0.068
5077679
7580967
0.6698
7
11
Average
8
13
2.5
y = 10.56x-0.0449
R2 = 0.9921
Peak
Area
Ratio
0.5
-0.5
0
0.05
0.1
0.15
0.2
0.25
Concentration (mM)
Figure 22. Calibration curve correlating analytical RP-HPLC peak area (UV absorbance at 220 nm) with
concentration for yield calculation of P-peptide 3-3 from a single PS macrobead.
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145
3.5.7 P-Peptide Library Synthesis on PS Macrobeads with Microwave Irradiation
Loaded PS macrobeads (62 mg, 29.8 pmol, ~ 500 beads) were partitioned into five equal
aliquots by weight, placed in modified SPE tubes (4 mL, Alltech), and swelled with DMF for
approximately 10 min.
The resin was washed (5 x DMF, 5 x CFI2 CI2 and 5 x DMF).
Deprotection solution (750 pL o f 20% piperidine in DMF (v/v)) was added to the resin, and the
tube was placed inside a glass 10 mL microwave reaction vessel containing ~ 2 mL o f DMF. A
N 2 line was inserted for agitation, and the vessel was placed in the microwave reactor (CEM
Discover) and irradiated (50 W maximum power, 60°C, ramp 2 min). The sample was removed
from the microwave reactor and cooled at room temperature for 10 min while the other four
samples were each irradiated in turn. This ramp/cool cycle was repeated a total o f 3 times per
deprotection for each sample.
The resin was then washed as before.
Fmoc-p3-hPhe-OH
(36.2mg, 90 pmol) was activated by adding HBTU (180 pL o f 0.5 M solution in DMF), DMF
(1.32 mL), HOBt (180 pL of 0.5 M solution in DMF), and /Pr2 EtN (180 pL o f 1.0 M solution in
DMF). The mixture was vortexed and 372 pL o f the coupling solution was added to each aliquot
of resin. The first sample was irradiated in the microwave reactor (50 W maximum power, 50°C,
ramp 2 min), removed from the reactor, and cooled for 10 min at room temperature while the
other four samples were each irradiated in turn. This ramp/cool cycle was repeated a total o f 6
times per coupling step for each sample. A few beads were removed from the first sample and
washed as described. The beads were suspended in ~ 1 mL o f DMF, and /Pr2EtN (30 pL o f 1.0
M solution in DMF) and TNBS (30 pL o f a 1% solution in DMF) were added. After 5 min, the
center of the beads was stained red, indicating the presence o f free amino groups and an
incomplete coupling reaction. The five samples were irradiated for an additional 3 ramp/cool
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146
cycles, after which the TNBS test was negative (< 5% o f amino groups are unreacted).
Washing and deprotection were performed as described. Then 18 pmol of Fmoc-p3hVal-OH
(6.4 mg), Fmoc-phGly-OH (5.6 mg), Fmoc-p3hAla-OH (5.9 mg), Fmoc-|33hLeu-OH (6.6 mg),
and Fmoc-(5VS')-ACHC-OH (6.6 mg) were each activated with HBTU (36 pL o f 0.5 M solution
in DMF), DMF (264 pL), HOBt (36 pL o f 0.5 M solution in DMF), and zPr2EtN (36 pL o f 1.0 M
solution in DMF). After vortexing, each coupling mixture was added to a different aliquot o f
resin, and 6 cycles o f microwave ramp/cool cycles were performed for each sample. A TNBS
test o f each aliquot o f resin at the end o f the reaction was negative.
After washing and
deprotection, the resin was combined, suspended in DMF, and thoroughly mixed. The resin was
partitioned into four approximately equal aliquots. Then 22.5 pmol o f Fmoc-p3hOm(Boc)-OH
(10.6 mg), Fmoc-p3hGlu(tBu)-OH (9.9 mg), Fmoc-p3hSer(/Bu)-OH (9.0 mg), and Fmoc-(AA)APiC(Boc)-OH (10.5 mg) were each activated with HBTU (45 pL of 0.5 M solution in DMF),
DMF (330 pL), HOBt (45 pL of 0.5 M solution in DMF), and /Pr2EtN (45 pL o f 1.0 M solution
in DMF). After vortexing, each coupling mixture was added to a different aliquot o f resin, and 6
cycles of microwave ramp/cool cycles were performed for each sample. Fmoc-(S',5)-APiC(Boc)OH was double-coupled to its respective resin aliquot. A TNBS test o f each aliquot o f resin at
the end o f the coupling reaction was negative. The resin was combined, suspended in DMF, and
thoroughly mixed. The resin was partitioned into five approximately equal aliquots. Following
washing and deprotection, Fmoc-p3hTrp(Boc)-OH (48.7 mg) was coupled using the same
procedure as described for Fmoc-P -hPhe-OH. A TNBS test o f each aliquot o f resin at the end
o f the reaction was negative. Following deprotection and washing, Fmoc-p3hVal-OH, FmocphGly-OH, Fmoc-p3hAla-OH, and Fmoc-p3hLeu-OH were coupled as before.
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Fmoc-(1S,,5)-
147
ACHC-OH was coupled to the fifth aliquot in 0.8 M LiCl in NMP. Since this was the sixth
residue from the C-terminus, each residue was double-coupled in its respective solvent.
A
TNBS test of each aliquot o f resin at the end o f the second coupling reaction was negative.
Washing and double-deprotection was performed, followed by coupling o f Fmoc-p3hGlu(/Bu)OH (39.6 mg).
Deprotection, washing, and coupling o f Fmoc-p3hLeu-OH (33.1 mg) was
followed by a final deprotection. The resin was combined, washed (5 x DMF, 5 x CH 2 CI2 , 5 x
DMF, 5 x CH 2 CI2 and 5 x MeOH), and dried under a stream of N 2 until free-flowing. The
macrobeads were arrayed (1 bead per well) into 5 polypropylene V-bottom 96-well plates
(Greiner) using tweezers. The p-peptides were cleaved from the solid support with simultaneous
side chain deprotection (110 pL, 50:50:5:5 TFA:CH2Cl2 :TES:water, 2 h, RT, with orbital
shaking; plate was sealed with a polyolefin mat cover from Fisher Scientific). At the end o f the
reaction, the covered plate was centrifuged (1250 rpm, 1 min) to remove resin and cleavage
solution from the cover.
The cover was then removed and the cleavage solution was
concentrated by rotary evaporation (RT, 1 hr, SpeedVac, Thermo Savant). The crude P-peptide
mixtures were dissolved in 3 pL of DMSO; 2 pL of this stock solution were used in for the
ELISA screening, while 1 pL was reserved for compound identification.
3.5.8 Analytical Characterization of Representative Library Members
The crude P-peptide mixtures from 50 beads were dissolved in 30 pL o f DMSO for
HPLC analysis (Shimadzu); 20 pL was injected on a Gt-silica reverse-phase analytical column
(5 pm, 4 mm x 250 mm, Vydac) and eluted with a gradient o f acetonitrile in water (10 - 60%, 50
min, 0.1% TFA in each) at a flow rate o f 1 mL/min. The product purity was determined as peak
area percent by integration o f the UV absorbance at 220 nm. Integration was performed over the
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
148
1 5 - 5 0 min time interval to exclude the large absorbance o f DMSO that elutes from 5 - 1 5
min. The lower threshold o f integration was set to exclude minor peaks whose areas were < 1%
o f the peak area of the major species. The major peak in each HPLC run was collected and (3peptide
masses
were
measured
by
MALDI-TOF-MS
(Bruker Reflex
II,
a-cyano-4-
hydroxycinnamic acid matrix).
Table 9. Sequences of expected P-peptide library members (1-50).
Com pound #
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
MW
1189.68
1187.66
1187.66
1185.64
1175.67
1175.67
1174.71
1173.65
1173.65
1172.70
1172.69
1172.69
1170.68
1170.68
1170.67
1168.66
1161.66
1160.70
1160.70
1158.69
1158.69
1158.68
1158.68
1156.67
1156.67
1147.67
1147.64
1147.64
1146.69
1145.65
1145.65
1145.62
1145.62
1144.68
1143.63
1133.66
1133.66
1133.63
1133.63
1133.62
1133.62
1132.67
1132.67
1131.64
1131.64
1131.60
1131.60
1130.66
1130.66
1 1 3 0.65
N -te rm in u s I
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
L
ACHC
L
ACHC
V
L
L
ACHC
V
L
ACHC
L
ACHC
L
ACHC
ACHC
V
V
L
V
L
ACHC
V
ACHC
V
L
A
L
V
ACHC
L
ACHC
A
V
ACHC
V
L
A
V
G
L
A
L
ACHC
V
ACHC
G
A
L
ACHC
Sequence
W
E
W
E
w
E
w
E
w
E
w
E
w
o
w
E
w
E
w
APiC
w
O
w
O
w
APiC
w
APiC
w
O
w
APiC
w
E
w
O
w
O
w
APiC
w
APiC
w
O
w
O
w
APiC
w
APiC
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
s
E
E
O
s
s
E
E
APiC
s
S
S
E
E
E
E
O
O
s
s
E
E
APiC
APiC
O
L
L
ACHC
ACHC
L
V
L
V
ACHC
L
L
ACHC
L
ACHC
ACHC
ACHC
V
L
V
L
V
V
ACHC
V
ACHC
L
L
A
V
L
ACHC
A
ACHC
V
ACHC
L
V
V
A
L
G
L
A
V
ACHC
G
ACHC
L
A
A
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
E
E
E
E
E
E
E
E
■E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
IC -te rm in u s
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
149
Table 10. Sequences o f expected P-peptide library members (51-100).
B ead N um ber
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
MW
1130.65
1128.64
1128.64
1119.65
1119.61
1119.61
1 1 1 8.66
1118.66
1118.65
1 1 1 8.65
1 1 1 6 .6 5
1 1 1 6.65
1116.64
1116.64
1 1 1 6.63
1116.63
1114.62
1114.62
1105.63
1105.63
1105.60
1104.64
1104.64
1103.61
1103.61
S equence
N -te rm in u s |
1102.63
1102.63
1091.62
1091.62
1091.61
1091.61
1091.58
1091.58
1090.63
1089.59
1089.59
1088.62
1077.60
1077.60
1077.56
1076.61
1076.61
1074.60
1074.60
1063.59
1062.59
1060.58
1049.57
1049.57
1035.55
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
' E
E
E
E
E
E
E
E
E
E
E
E
E
E
A
ACHC
A
V
G
V
A
V
G
L
A
V
G
L
ACHC
G
ACHC
G
A
L
A
G
V
ACHC
A
G
V
W
W
w
w
w
' w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
A
w
V
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
w
G
L
A
G
A
ACHC
G
A
G
V
G
A
G
A
G
A
G
G
A
G
G
IC -te rm in u s
o
APiC
APiC
s
E
E
0
o
o
o
APiC
APiC
APiC
APiC
O
O
APiC
APiC
s
S
E
O
O
s
s
APiC
APiC
S
s
s
s
E
E
O
s
s
APiC
S
S
E
O
O
APiC
APiC
s
O
APiC
S
S
S
ACHC
A
ACHC
V
V
G
V
A
L
G
V
A
L
G
G
ACHC
G
ACHC
L
A
A
V
G
A
ACHC
G
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
V
F
A
L
G
G
A
A
G
ACHC
A
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
V
V
G
G
G
A
G
A
A
G
G
G
A
G
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
F
E
F
F
F
E
E
E
L = (S )-p3-hom oleucine
E = ( S )-p 3-hom oglutam ic acid
G = p 3-hom oglycine
V = (S )-p3-hom ovaline
A = (S )-p 3-h o m o alan in e
F = (S )-p3-ho m o p h en y lalan in e
W = (S )-p3-hom otryptophan
O = (S )-p 3-hom oornithine
S = (S )-p3-h o m o se rin e
A CHC = (S ,S )-2 -am in o cy clo h ex an ecarb o x y lic acid
APiC = (S ,S )-4 -am in o p ip erid in e-3 -carb o x y lic acid
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
Table 11. HPLC and MS characterization o f material from 50 beads from the library.
Bead #
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
Average
Purity(%) Major lmpurity(%)
10
75
68
9
12
58
9
68
59
17
54
13
64
8
4
72
66
7
14
68
72
6
7
63
11
61
46
13
11
56
54
11
71
9
62
10
72
7
67
7
70
6
12
40
77
6
12
48
70
6
72
10
7
69
64
14
73
8
71
7
12
60
7
62
73
13
71
8
58
9
70
9
67
9
8
66
73
6
58
9
75
6
52
22
47
33
78
6
73
• 16
69
5
70
8
71
8
48
25
67
8
10
65
[M+H]+
1119.4
1105.5
1134.3
1169.4
1106.5
1090.3
1175.9
1171.8
1146.2
1186.3
1146.7
1171.7
1131.0
1132.2
1188.4
1188.4
1190.6
1146.2
1148.3
1063.4
1145.8
1106.4
1174.3
1104.3
1131.4
1131.5
1148.5
1169.2
1171.4
1075.5
1090.5
1146.7
1119.3
1131.4
1119.9
1132.9
1103.7
1117.2
1132.0
1161.3
1157.3
1078.9
1050.5
1077.5
1132.4
1117.4
1171.6
1188.6
1079.2
1104.6
Retention Time (min)
26.3
25.7
36.0
36.0
37.2
21.1
36.6
28.9
34.0
37.1
24.1
27.8
21.5
26.0
35.9
35.9
38.9
23.8
33.5
19.9
34.2
32.1
37.6
33.3
31.9
30.9
33.6
36.0
36.3
23.3
29.2
34.0
26.4
32.6
30.8
30.9
28.0
31.2
30.4
34.4
35.3
27.1
22.9
21.9
36.9
28.9
37.2
38.5
27.1
31.1
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
151
100
90
80
70
HPLC 60
Peak
50
Area
Percent 40
E3 Impurity
■ Product
30
20
10
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49
Bead Num ber
Figure 23. Amount of P-peptide library member and largest impurity from peak area percent of analytical RJPHPLC (UV absorbance at 220 nm).
3.5.9 HPLC ESI-MS/MS Analysis of P-Peptides from Library
P-Peptide sequencing was performed by Dr. Mark Scalf in the laboratory o f Professor
Lloyd M. Smith at the University o f Wisconsin. The pLC-MS/MS system consisted o f an HPLC
connected to an ESI ion trap mass spectrometer (Surveyor HPLC and LCQ deca XPplus,
ThermoFinnigan, San Jose, CA). A ffitless 100 x 365 pm fused-silica capillary microcolumn was
prepared by pulling the tip o f the capillary to approximately 2 pm with a P-2000 laser puller
(Sutter Instruments Co.) and packing with 10 cm o f Ci8-silica beads (5 pm diameter, Western
Analytical Products, Inc, Murrieta, CA).
The capillary column was connected to the HPLC
through a PEEK microcross with a platinum wire inserted into the flow-through to supply a
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
spray voltage o f 1.8 kV. The remaining 10 pL o f P-peptide DMSO stock solution from 10 o f
the randomly selected macrobeads was diluted 1:1 with 95% H 2 O, 0.1% formic acid: 5%
acetonitrile, 0.1% formic acid (95% buffer A, 5% buffer B). Alternatively, the remaining 1 pL
o f P-peptide DMSO stock solution from the ELISA screening hits was diluted with 20 pL of
95% buffer A, 5% buffer B. A total o f 10 pL o f either P-peptide solution was loaded onto the
fused-silica capillary microcolumn at a flow-rate o f 1 pL/min (95% buffer A, 5% buffer B) for
twenty minutes. A gradient from 5% buffer B to 80% buffer B was run over 74 minutes at a
flow-rate of 300 nL/min to elute the mixture.
The ion trap mass spectrometer was run in
“biggest 3” mode, which consists o f a full-mass scan (400 - 2000 m/z), followed by an MS/MS
scan o f each o f the three highest-intensity parent ions with a normalized collision energy o f 45%.
O
NH
Exact Mass: 85.05
Exact Mass: 101.05
(S)-p3-homoglutamic acid
Exact Mass: 143.06
(5)-p3-homovalme
Exact Mass: 113.08
(5)-p3-homoleucine
(6)-p3-homoorni thine
(iS)-p3-homophenylalanine
Exact Mass: 161.08
(.S')-p3-homotryptophan
Exact Mass: 127.10
Exact Mass: 128.09
o
(Syp-homoglycine
Exact Mass: 71.04
(5)-p3-homoalanine
(S)-p3-homoserine
Exact Mass: 200.09
O
NH
NH
(iS',.S')-2-aminocyclohexanecarboxylic acid
Exact Mass: 125.08
(.S',.S')-4-amino-3-piperidine carboxylic acid
Exact Mass: 126.08
h
v ’ '
Figure 24. p-Peptide fragments for sequencing by LC-MS/MS.
R e p ro d u c e d with perm ission of the copyright owner. Further reproduction prohibited without permission.
153
Table 12. Sequencing o f P-peptides by pLC-MS/MS (1-6).
Bead
Number
7
Compound Theoretical
Number
Mass
7
1174.71
904.54
777.44
577.35
449.25
322.15
853.56
726.46
42
49
45
22
44
89
89
44
69
91
Observed Mass
[M+H]+ [M+2H]”
1175.7
905.4
778.4
578.4
450.4
323.3
853.4
726.5
589.1
Full Length Sequence
and Fragments
H
L
E
L
L
|
W
W
w
H
H
L
L
E
E
L
L
w
w
o
o
o
Lo
o
0 I
1077.57
807.42
694.33
494.24
393.19
685.39
1078.9
808.2
695.1
495.2
394.1
H
L
E
V
v
w
w
w
s
s
s
s
6 8 6 .2
H
L
E
V
w
s
1077.57
807.42
694.33
494.24
393.19
685.39
1079.2
808.2
695.1
495.1
394.1
686.2
H
L
E
V
v
w
w
w
s
s
s
s
H
L
E
V
w
s
1131.59
860.43
736.35
536.25
435.25
697.39
1132.7
862.3
737.2
537.2
436.3
697.3
H
L
E
ACHC
| ACHC
W
W
|_ W
W
1105.61
835.45
750.4
550.3
449.25
784.46
657.36
1107.0
836.2
751.2
551.2
450.2
784.2
657.2
1076.59
806.43
721.38
521.29
393.19
322.15
684.41
271.17
1077.7
807.2
722.2
522.3
394.1
324.3
684.4
271.0
567.7
H
L
E
ACHC
H
L
E
A
A
540.3
H
H
L
L
E
E
A
A
H
L
E
A
A
I
S
S
s
s
s
L
L
L
L
L
L
L
E
E
A
G
G
G
G
G
OH
OH
OH
OH
OH
OH
F
F
F
F
F
E
E
E
E
E
OH
OH
OH
OH
OH
F
F
F
F
F
E
E
E
E
E
OH
OH
OH
OH
OH
F
F
F
F
F
E
E
E
E
E
OH
OH
OH
OH
OH
F
F
F
F
F
E
E
E
E
E
OH
OH
OH
OH
OH
F
F
F
F
F
F
E
E
E
E
E
E
OH
OH
OH
OH
OH
OH
G ■
G
G
G
G
G
G
V
V
V
V
V
s
s
s
I s
""I..
w
s
w
S...I
W
o
G
W
o
G
W
0
G
0
G
W
W
W
W
E
E
E
E
E
E
L
G
H
H
F
F
F
F
F
F
O
R e p ro d u c e d with perm ission of th e copyright owner. Further reproduction prohibited without permission.
154
Table 13. Sequencing o f P-peptides by pLC-M S/M S (7-10).
Bead
Com pound
Theoretical
N um ber
39
N um ber
40
M ass
1133.60
863.45
792.41
592.31
449.25
322.15
[M +H ]+
1133.0
862.2
791.2
591.1
448.1
323.2
[M + 2 H ]~
1168.65
898.5
773.41
573.32
447.24
322.15
847.51
722.42
1169.8
899.3
774.3
574.3
448.2
323.1
847.3
722.3
586.2
1173.63
903.48
790.39
590.3
447.24
322.15
852.49
727.4
584.34
1175.0
904.2
791.2
591.2
448.2
323.1
852.3
727.2
584.3
588.3
1049.54
779.39
708.35
508.26
407.21
643.35
1051.0
780.2
709.1
509.1
408.1
643.1
4
23
43
16
9
99
O bserved M ass
Full Length Sequence
H
L
E
G
G
and Fragm ents
E
W
E
W
| W
E
E
L
L
L
L
I
L
L
H
L
E
ACHC
| ACHC
w
w
APiC
APiC
APiC
| APiC
APiC ACHC
I APiC |
W
I
H
H
L
L
E
E
ACHC
ACHC
w
w
H
L
E
V
w
w
v
w
I
E
E
E
E
H
H
H
L
L
L
E
E
E
V
V
V
w
w
H
L
E
G
G
W
W
w
s
s
s
s
G
w
s
H
L
E
W
E
E
ACHC
ACHC
ACHC
ACHC
| ACHC
ACHC
ACHC
ACHC
ACHC
| ACHC
F
F
F
F
F
F
E
E
E
E
.E
E
OH
OH
OH
OH
OH
OH
F
F
F
F
F
F
E
E
E
E
E
E
OH
OH
OH
OH
OH
OH
F
F
F
F
F
F
E
E
E
E
E
E
OH
OH
OH
OH
OH
OH
F
F
F
F
F
E
E
E
E
E
OH
OH
OH
OH
OH
ACHC
J
i
A
A
A
A
A
L = (S )-p 3-homoleucine
E = (S )-p3-homoglutamic acid
G = p3-homoglycine
V = (S )-p3-homovaline
A = (S )-p3-homoalanine
F = (S )-p3-homophenylalanine
W = (S )-p3-homotryptophan
O = (S )-p3-homoornithine
S = (S )-p3-homoserine
ACHC = (S,S)-2-aminocyclohexanecarboxylic acid
APiC = (S,S)-4-aminopiperidine-3-carboxylic acid
R e p ro d u c e d with perm ission of the copyright owner. Further reproduction prohibited without permission.
155
3.5.10 ELISA P rocedure
The ELISA was performed in the laboratory o f Dr. Jiandong Chen at the University of
South Florida.
GST-MDM2 fusion protein containing full-length human MDM2, and His6-
tagged human p53 were expressed in E. coli and affinity purified by binding to glutathione agarose and N i2+-NTA beads under non-denaturing conditions using standard protocols. ELISA
plates were incubated with 2.5 pg/ml His6-p53 in phosphate buffered saline (PBS) for 16 hrs.
After washing with PBS + 0.1% Tween 20 (PBST), the plates were blocked with PBS + 5% non­
fat dry milk + 0.1% Tween 20 (PBSMT) for 0.5 hr. Compounds were dissolved in DMSO. GSTMDM2 (5 pg/ml) was mixed with test compounds in PBSMT + 10% glycerol + 10 mM DTT
and added to the wells. The plates were washed with PBST after incubating for 1 hr at room
temperature, then incubated with MDM2-specific monoclonal antibody 5B10 hybridoma
supernatant diluted 1:10 in PBSMT for 1 hr, followed by washing and incubation with HRPrabbit-anti-mouse Ig antibody for 1 hr. The 5B10 antibody recognizes a C-terminal epitope on
MDM2,42 thus ensuring that the assay detects full-length MDM2 binding to p53. The plates
were developed by incubation with TMB peroxidase substrate (KPL) and measured by
absorbance at 450 nm. P-Peptide hits from the initial library screening were sequenced by LCMS/MS, individually re-synthesized using microwave irradiation,12 purified by preparative RPHPLC, and retested for validation.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
156
Table 14. Raw O.D. data from ELISA screen o f octa-p-peptide library.
1
2
3
4
5
6
7
8
9
10
11
12
Plate 1
A
B
C
D
E
F
G
H
1.134
1.138
1.08
1.248
1.875
0.957
1.251
1.461
1.906
0.927
1.438
2.15
1.764
1.469
1.429
1.368
1.474
0.685
1.289
1.508
1.351
1.426
1.396
1.075
0.642
1.971
1.256
1.15
1.369
2.079
1.344
1.141
1.28
1.141
1.209
1.408
1.486
1.904
1.513
1.125
1.712
1.277
1.396
2.19
1.059
1.199
1.423
1.665
1.332
1.545
1.583
1.759
1.473
1.342
1.865
1.367
0.784
0.808
1.318
0.952
2.078
0.891
1.333
1.416
1.071
1.317
1.246
1.007
1.933
1.289
1.498
1.285
1.601
1.377
1.396
1.412
1.59
1.432
1.77
1.59
2.106
1.501
1.098
0.88
1.268
1.491
1.261
1.245
1.238
1.349
1.584
2.186
1.529
1.129
1.027
1.206
2.801
1.87
1.277
1.504
2.655
1.413
2.093
1.71
1.471
1.559
2.156
3.258
2.163
1.634
1.386
2.6
0.725
2.984
3.123
3.117
2.314
1.298
1.185
2.244
2.027
1.512
2.704
2.808
2.743
2.56
1.858
2.354
1.851
2.405
1.981
1.08
0.745
0.877
1.096
3.132
2.904
1.027
2.06
2.136
2.734
2.968
2.039
2.522
2.111
0.707
0.799
1.531
2.396
2.426
1.229
2.26
3.216
2.485
1.159
2.865
1.506
3.048
2.254
1.585
0.705
1.333
1.934
1.005
2.668
1.045
1.973
1.196
2.245
2.465
2.736
2.761
0.794
1.098
1.234
2.047
1.411
0.757
1.171
1.461
2.866
1.094
0.974
0.936
1.239
0.863
0.803
0.639
1.134
1.299
1.142
1.144
0.717
0.773
1.007
1.169
1.494
1.033
0.798
1.025
1.275
0.697
0.827
1.17
1.227
0.732
1.165
1.243
0.672
1.197
0.987
1
1.114
1.249
1.203
1.15
1.274
1.11
0.776
1.208
1.485
0.663
1.209
1.133
0.995
0.875
0.798
1.266
1.189
0.508
1.062
1.225
1.194
1.096
1.051
1.107
0.682
1.193
1.058
1.063
1.618
0.894
1.03
1.482
1.411
0.926
0.814
1.284
1.368
1.456
1.247
1.209
1.153
1.328
0.723
0.736
1.013
1.392
1.606
1.417
1.403
1.606
1.421
1.251
1.354
1.469
2.144
1.974
1.346
1.6
1.261
0.848
0.809
1.413
1.392
1.322
0.801
1.348
1.229
0.865
1.401
1.707
1.291
1.096
1.044
1.368
1.69
1.027
1.155
1.544
1.784
1.098
2.052
1.66
0.759
0.948
0.783
0.973
1.285
1.19
1.62
0.84
2.196
1.913
2.19
2.343
1.361
1.526
1.257
1.298
1.184
1.048
1.317
1.078
1.374
1.13
1.861
2.068
1.497
0.704
1.223
1.379
2.954
0.818
1.121
1.143
1.505
1.287
2.009
2.661
1.124
1.118
1.098
0.911
1.328
1.571
1.153
0.762
1.444
1.339
1.755
1.114
2.264
1.251
1.2
0.851
1.191
0.784
1.766
1.021
1.161
0.864
0.92
0.989
1.022
1.718
0.869
1.46
1.178
1.496
1.583
1.611
1.043
2.241
1.507
1.51
1.363
1.394
1.756
1.658
1.167
1.193
1.351
1.522
1.572
1.35
1.266
1.524
3.048
1.717
2.561
1.411
2.596
2.367
2.537
1.543
1.383
1.198
1.59
1.161
1.598
0.944
1.587
1.445
1.001 1.018
2.426 1.695
2.669 1.176
1.036 2.575
1.312 2.908
1.201 1.272
1.389 1.464
1.601 . 1.783
2.922
1.646
2.138
2.166
0.953
1.615
1.324
1.392
0.852
2.486
2.423
1.722
1.665
1.074
1.197
1.234
1.149
1.573
1.025
3.159
3.196
1.542
1.189
1.122
1.043
1.133
1.584
1.281
1.368
1.282
1.076
1.562
Plate 2
A
B
C
D
E
F
G
H
1.062
1.974
1.399
1.903
1.244
1.162
2.227
2.224
0.517
0.51
0.37
Plate 3
A
B
C
D
E
F
G
H
1.262
0.897
1.046
1.106
0.994
0.995
1.045
1.173
0.577
0.42
Plate 4
A
B
C
D
E
F
G
H
0.997
1.167
0.671
1.104
1.383
0.944
0.98
1.895
0.541
Plate 5
A
B
C
D
E
F
G
H
1.754
1.567
1.994
1.149
1.584
1.098
1.417
1.598
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
157
Table 15. Sequencing o f P-peptide “hits” by LC-MS/MS.
B ead
C om pound
T h e o r e tic a l
L o c a tio n
N u m b er
26
M a ss
[M + H f
[M +2H]++
1147.65
877.49
750.39
550.29
449.24
322.15
1149.2
878.1
751.0
551.0
450.0
323.0
575.1
1091.56
821.42
708.34
508.25
407.20
322.15
1093.0
822.1
709.0
509.0
408.0
323.2
547.1
1189.66
919.50
792.40
592.30
449.24
322.15
1191.2
920.1
793.1
593.1
450.1
323.0
596.3
1143.62
873.46
748.37
548.28
447.23
322.15
1145.2
874.1
749.1
549.1
448.1
323.1
573.2
1104.62
834.46
721.37
521.28
393.18
322.15
1105.9
835.1
722.1
522.2
394.1
323.1
554.1
1185.63
915.47
790.38
590.29
447.23
322.15
1187.3
916.1
791.1
591.1
448.1
323.0
594.2
1187.65
917.48
792.40
592.30
449.24
322.15
1189.2
918.2
793.1
593.1
450.1
323.1
595.0
2 -F 1 0
3-C9
3-H2
2-H6
2-B5
4-E5
3-F7
79
1
35
73
4
2
O b s e r v e d M a ss
Full L en g th S e q u e n c e
a n d F r a g m e n ts
H
L
L
L
E
W
W
| w
H
H
H
L
L
L
L
L
L
L
L
F
F
F
F
F
F
E
OH
OH
OH
OH
OH
OH
A
A
A
A •
A
F
F
F
F
F
F
E
E
E
E
E
E
OH
OH
OH
OH
OH
OH
I
V
v
W
W
w
I
L
L
W
W
w
E
E
E
E
L
L
L
L
L
F
F
F
F
F
F
E
E
E
E
E
E
OH
OH
OH
OH
OH
OH
w
w
[ w
s
s
s
s
ACHC
ACHC
ACHC
ACHC
| ACHC
F
F
F
F
F
F
E
E
E
E
E
E
OH
OH
OH
OH
OH
OH
E
ACHC
| ACHC
S
s
s
s
I
H
L
E
V
I
IH
L
E
v
ACHC
| ACHC
........ [
H
L
E
E
E
E
E
E
I
E
E
L
S
S
S
S
ACHC
I ACHC
L
w
w
w
o
o
0
o
G
G
G
G
G
F
F
F
F
F
F
E
E
E
E
E
E
OH
OH
OH
OH
OH
OH
w
w
w
E
E
E
E
ACHC
ACHC
ACHC
ACHC
I ACHC
F
F
F
F
E
E
E
E
E
E
OH
OH
OH
OH
OH
OH
E
E
OH
OH
OH
OH
OH
OH
W
w
w
I
F
I
F
|
F
F
F
F
F
F
E
E
E
E
IZ
L = ( S ) - p 3-h o m o le u c in e
E = ( S )-p 3-h o m o g lu ta m ic a cid
G = p 3-h o m o g ly cin e
V = ( S ) - p 3-h o m o v a lin e
A = ( S ) - p 3-h o m o a la n in e
F = (S )-p 3-h o m o p h e n y la la n in e
W = ( S )-p 3-h o m o try p to p h a n
O = ( S )-p 3-h o m o o rn ith in e
S = ( S ) - p 3-h o m o s e rin e
A C H C = (S ,S )-2 -a m in o c y c lo h e x a n e c a rb o x y !ic a cid
R e p ro d u c e d with perm ission of th e copyright owner. Further reproduction prohibited without permission.
E
E
E
E
158
3.5.11 Synthesis of Peptides for ELISA Using Microwave Irradiation
P-Peptide hits were resynthesized using microwave irradiation.10 p3-hGlu-Loaded PS Wang
resin (100-200 mesh, 10 pmol, 14.9 mg) was placed in a modified polypropylene SPE tube (4
mL, Alltech, top rim removed with a razor blade) and swelled with DMF for ~ 10 min. The resin
was washed (5 x DMF, 5 x CH 2 CI2 and 5 x DMF). Deprotection solution (750 pL o f 20%
piperidine in DMF (v/v)) was added to the resin, and the tube was placed inside a glass 10 mL
microwave reaction vessel containing ~ 2 mL o f DMF (See Figure 6). A N 2 line was inserted for
agitation, and the vessel was placed in the microwave reactor (CEM Discover) and irradiated (50
W maximum power, 60°C, ramp 2 min, hold 2 min).
All microwave experiments were
conducted at atmospheric pressure; the temperature was measured via an IR sensor at the base o f
the outer reaction vessel; the temperature was controlled by modulation o f power; and the
sample was cooled with compressed air during the hold time. The tube was removed from the
microwave reactor, and the resin was washed as before. In a separate vial, Fmoc-p-amino acid
(30
pmol)
was
activated
by
adding
0-benzotriazol-l-yl-A(A (/O V ’-tetramethyluronium
hexaflurorophosphate (HBTU, 60 pL o f 0.5 M solution in DMF), DMF (440 pL), 1hydroxybenzotriazole hydrate (HOBt, 60 pL o f 0.5 M solution in DMF), and zP^EtN (60 pL o f
1.0 M solution in DMF). The mixture was vortexed and added to the resin. The sample was
irradiated in the microwave reactor (50 W maximum power, 50°C, ramp 2 min, hold 4 min).
After the coupling reaction, the resin was washed as before. Alternatively, the coupling reaction
ofFm oc-(£5>A C H C at position 3 was performed by activating with solutions o f HBTU, HOBt,
and /P^EtN in NMP and adding a solution o f LiCl in NMP for a final concentration o f 0.8 M
LiCl (620 pL final volume), and then adding this solution to the resin. The tube was placed in a
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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Table 16. Characterization data for assayed peptides.
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
160
microwave reaction vessel containing ~ 2 mL o f 0.8 M LiCl in NMP and irradiated (50 W
maximum power, 45°C, ramp 2 min, hold 4 min). After the coupling reaction, the resin was
washed as before. All other residues at position 3 were double-coupled in DMF. All residues at
position 3 were double-deprotected. The deprotection/coupling cycle was repeated in a stepwise
manner until reaching the desired octamer. The products were cleaved and purified as described
for P-peptide 3-3 except that they were eluted from the preparative HPLC column with different
gradients of acetonitrile in water ((rt-10)-(rt+20)%, 30 min, 0.1% TFA in each, 15 mL/min,
where rt is their retention time from a prior analytical HPLC analysis). The a-peptide p53 15-31
was prepared on NovaSyn TGR resin (Novabiochem) using microwave irradiation.
The N-
terminus was acetylated by adding a solution o f 1.4 mL CH 2 CI2 /O.I mL Et3N/0.5 mL AC2 O and
shaking for 30 min.
P-Peptide 3-5 was synthesized using microwave irradiation; p3-hVal at
position 5 was double-coupled in DMF and double-deprotected.
3.5.12 A dditional ELISA D ata
Table 17. Data for binding curves from competition ELISA (Absorbance at 450 nm vs. concentration, as shown in
Figure 11).
Concentration (pM)
400
200
100
50
25
12.5
6
3
1.5
0.7
0.3
0.1
0.05
0.025
0
Nutlin-3
0.029
0.178
0.39
0.621
0.815
0.937
1.013
1.069
p53 15-31
0
0.017
0.034
0.175
0.40
0.675
0.854
3-P eptide 3-4
0.285
0.599
0.775
0.899
0.919
1.065
1.072
8-P eptide 3-3
0.623
0.728
0.856
0.96
1.159
0.985
1.061
D M SO
0.985
1.133
0.954
1.015
1.063
1.032
1.063
1.034
0.967
1.075
1.00
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 18. ELISA data for Figure 11 fit to a sigmoidal curve with the bottom constrained to 0.0.
■ p53 15-31
▼ p-Peptide
•
p-Peptide
log [competitor], pWl
i i
p 5 '3 15. O f
S -t
P a p t i d i ! 5-S
<1 ir iI.iv h r e s p o n s e o j w o W * s l d s « i
. tin
h i 4 i it* s
BOTTOM
TOP
0.C
II : 4 : /
0 .0
0 .0
; c iv ,
1 J ’l
H IL L S L Q P E
J & 03
T’l . . *;
ecsa
•S t d .
0 .0
1 .0 6 1
i: :; :
2735
- 3 .3 2 6 9
- 0 .6 8 6 7
- 1 .2 6 2
- 1 .3 7 5
0 .4 7 1 1
t
TOP
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H IL L S L C P l
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6 .9 4 5 4 6
0 ,0 7 2 5 7
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0 141 1
0 02535
O O J32J
C -.2479
0 .4 2 6 3
0 .1 0 3 9
0 f : s 2 ? l a 1 .3 5 7
0 . 9 9 5 4 la 1 .1 2 7
2 3 5 3 1'* 3 .1 7 7
-7 D /U t c O , 2 W
-1
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0 3 9 3 3 Lu 0 5 6 3 4
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2 5 :■! tt.' 2 * I r
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4
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0 .9 & 9 2
C -.3750
0 .6 7 3 7
0 .9 9 6 2
A b s o lu te S u m or S q u a re s
0 .S T W J4 7
D. c r t &a
D .0 I 13 4
0 .0 5 4 4 1
0 "2 -j J
0 .0 3 0 -3 5
o m * t *1
o .o s a a i
B O T t O M * 0 .0
HOT
Sy*
£ -.9 2 9 0 la t u
l
2 535
.1 &7D ( r
to 3 3 4 . 9
v
Alu
1502
-■3 4 0 4 6 ’0 - 0 2 4 3 1
£»!•£.-f. -1 U?Ti
C s tr s ! t a u 'i ! >
BOTTOM
DaPi
N u u ! Hf &!.;< valutft'fe
N u ti 1 f f K|
Y
T o t a l n u m b e r o= v a l . i
7
■■
riuu N -rcl nii5»5!*!f| V.T MU’:
1
I
7
7
0
0
TOM * 0 l>
B O tT C M * 7 0
H id r t t i - o o
7
1
1
7
e
0
7
16
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
162
Table 19. ELISA data for library members fit to a sigmoidal curve with the bottom constrained to 0.0 and the
top constrained to <0.81.
p53 15-31
▼
p-Peptide 3-5
fi-Peptide 3-4
♦
26
□
A
35
73
79
V
[3-Peptide 3-3
A
log [competitor], pM
Table 20. Data for Table 19.
C o n cen tratio n (pM)
1000 pM
5 0 0 pM
2 5 0 pM
125 pM
6 2 .5 |JM
31 pM
15 pM
0 |j M
p 5 3 15-31
0 .0 1 7
0 .0 0 9
0 .0 2 2
0.031
0.071
0 .1 7 3
0 .3 9 4
0 .8 0 3
p -P e p tid e 3-5
p -P ep tid e 3-4
26
0.0 6 2
0 .0 6 6
0.1 7 6
0 .3 1 6
0 .4 4 5
0 .5 4 9
0 .6 6 2
0 .6 8 4
0 .0 9
0 .1 8 8
0.3 8 8
0 .5 3 8
0.6 2 7
0.6 4 9
0.7 9 3
0.731
0.1 6 2
0.4 2 3
0.5 9 7
0.62
0.6 1 4
0.72
0.7 3 4
0.6 9 8
35
0 .2 0 4
0.321
0 .5 1 2
0 .5 7 9
0 .5 6 2
0.671
0 .6 7 6
0 .6 9
73
0 .2 9
0 .4 6 2
0 .6 5 7
0.671
0 .6 6 2
0 .6 6 2
0.851
0 .8 0 9
79
p -P e p tid e 3-3
0.306
0.5 7 6
0.69
0.7 6 4
0.6 7 9
0.691
0.8 3 5
0.728
0.2
0 .3 9
0 .5 5 7
0 .6 1 2
0 .6 2 9
0 .6 3 4
0 .6 7 6
0 .7 7 5
3.6 References
1 (a) Arkin, M. R.; Wells, J. A. Nat. Rev. Drug Discovery 2004, 3, 301. (b) Cochran, A. G.
Chem. Biol. 2000, 7, R85. (c) Gadek, T. R.; Nicholas, J. B. Biochem. Pharmacol. 2003, 65, 1.
(d) Berg, T. Angew. Chem. Int. Ed. 2003, 42, 2462. (e) Peczuh, M. W.; Hamilton, A. D. Chem
Rev. 2 000,100, 2479.
2 (a) Fletcher, S.; Hamilton, A. D. Curr. Opin. Chem. Biol. 2005, 9, 632. (b) Yin, H.; Hamilton,
A. D. Angew. Chem. Int. Ed. 2005, 44, 4130.
3 (a) Knight, S. M. G.; Umezawa, N.; Lee, H.-S.; Gellman, S. H.; Kay, B. K. Anal. Biochem.
2002, 3 0 0 ,230. (b) Lee, H.-S.; Umezawa, N.; Gellman, S. H. Unpublished results.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
163
4 Kritzer, J. A.; Lear, J. D.; Hodson, M. E.; Schepartz, A. J. Am. Chem. Soc. 2 0 0 4 ,126, 9468.
5 Kritzer, J. A.; Hodsdon, M. E.; Schepartz, A. J. Am. Chem. Soc. 2 0 0 5 ,127, 4118.
6 Raguse, T. L.; Porter, E. A.; Weisblum, B.; Gellman, S. El. J. Am. Chem. Soc. 2002, 124,
12774.
7 (a) Guichard, G.; Abele, S.; Seebach, D. Helv. Chim. Acta 1998, 81, 187. (b) Arvidsson, P. I.;
Rueping, M.; Seebach, D. Chem. Commun. 2001, 649. (c) Arvidsson, P. I.; Frackenpohl, J.;
Seebach, D. Helv. Chim. Acta 2003, 86, 1522.
8 (a) Merrifield, R. B. J. Am. Chem. Soc., 1963, 85, 2149. (b) Albericio, F. Curr. Op. Chem. Biol.
2004, 8, 211.
9 Sebestyen, F.; Dibo, G.; Kovacs, A; Furka, A. Bioorg. Med. Chem. Lett. 1993, 3, 413.
10 (a) Seneci, P. J. o f Receptor & Signal Transduction Research. 2001, 21, 377. (b) Seneci, P. J.
o f Receptor & Signal Transduction Research, 2001, 21, 409.
11 (a) Gallop, M. A.; Barrett, R. W.; Dower, W. J.; Fodor, S. P. A.; Gordon, E. M. J. Med. Chem.
1994, 37, 1233-1251. (b) Gordon, E. M.; Barrett, R. W.; Dower, W. J.; Fodor, S. P. A.; Gallop,
M. A. J. Med. Chem. 1994, 37, 1386-.
12 Murray, J. K.; Gellman, S.H. Org. Lett. 2005, 7, 1517.
13 (a) Yu, H.-M.; Chen, S.-T.; Wang, K.-T. J. Org. Chem. 1992, 57, 4781. (b) Erdelyi, M.;
Gogoll, A. Synthesis 2002, 1592. (c) Ferguson, J. D. Mol. Div. 2003, 7, 281. (d) Matsushita, T.;
Hinou, H.; Kurogochi, M.; Shimizu, H.; Nishimura, S.-I. Org. Lett, 2005, 7, 877.
14 Tam, J. P.; Lu, Y. A. J. Am. Chem. Soc. 1 9 9 5 ,117, 12058.
15 (a) Thaler, A.; Seebach, D.; Cardinaux, F. Helv. Chim. Acta 1991, 74, 628. (b) Seebach, D.;
Beck, A. K.; Studer, A. M odem Synthetic Methods 1995, 7, 1. (c) Stewart, J. M.; Klis, W. A.
Innovation and Perspective in Solid Phase Synthesis: Peptides, Polypeptides and
Oligonucleotides', Epton, R., Ed.; SPCC: Birmingham, UK, 1990; pp 1-9. (d) Flendrix, J. C.;
Halverson, K. J.; Jarrett, J. T.; Lansbury, P. T. J. Org. Chem. 1990, 55, 4517.
16 Bayer, E. Angew. Chem. Int. Ed. 1991, 30, 113.
17 Blackwell, H. E.; Perez, L.; Stavenger, R. A.; Tallarico, J. A.; Eatough, E. C.; Foley, M. A.;
Schreiber, S. L. Chem. Biol. 2001, 8, 1167.
18 Alluri, P. G.; Reddy, M. M.; Bachhawat-Sikder, K.; Olivos, H. J.; Kodadek, T. J. Am. Chem.
Soc. 2 0 0 3 ,125, 13995.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
164
19 (a) Li, W.; Xiao, X.; Czamik, A. W. J. Comb. Chem. 1 9 9 9 ,1, 127. (b) Groth, T.; Grotli, M.;
Meldal, M. J. Comb. Chem. 2001, 3, 461. (c) Yan, B.; Tang, Q. Ind. Eng. Chem. Res. 2003, 42,
5964.
20 Stavenger, R. A.; Schreiber, S. L. Angew. Chem. Int. Ed. 2001, 40, 3417.
21 Blackwell, H. E. Org. Biomol. Chem. 2 0 0 3 ,1, 1251.
22 Kuhnert, N. Angew. Chem. Int. Ed. 2002, 41, 1863.
23 Stadler, A.; Kappe, C. O. Eur. J. Org. Chem. 2001, 919.
24 Gabriel, C.; Gabriel, S.; Grant, E. H.; Halstead, B. S. J.; Mingos, D. M. P. Chem. Rev. Soc.
1998,27,213.
25 Kussie, P. H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau, J.; Levine, A. J.; Pavletich, N.
P. Science 1996, 274, 948.
26 Dolle, R. E.; Guo, J.; O ’Brien, L.; Jin, Y.; Piznik, M.; Bowman, K. J.; Li, W.; Egan, W. J.;
Cavallaro, C. L.; Roughton, A. L.; Zhao, Q.; Reader, J. C.; Orlowski, M.; Jacob-Samuel, B.;
Carroll, C. D. J. Comb. Chem. 2000, 2, 716.
27 Schreiber, J. V.; Quadroni, M.; Seebach, D. Chimia 1999, 53, 621.
28 Kappe, C. O. Angew. Chem. Int. Ed. 2004, 43, 6250.
29 Kritzer, J. A.; Luedtke, N. W.; Harker, E. A.; Schepartz, A. J. Am. Chem. Soc. 2 0 0 5 ,127,
14584.
30 Burgess, K.; Liaw, A. I.; Wang, N. J. Med. Chem. 1994, 37, 2985.
31 Raguse, T. L.; Lai, J. R.; Gellman, S. H. Helv. Chim. Acta 2002, 85, 4154.
32 (a) Garcia-Echeverria, C.; Chene, P.; Blommers, M. J. J.; Furet, P. J. Med. Chem. 2000, 43,
3205. (b) Sakurai, K.; Chung, H. S.; Kahne, D. J. Am. Chem. Soc. 2 0 0 4 ,126, 16288.
33 Vassilev, L. T.; Vu. B. T.; Graves, B.; Carvajal, D.; Podlaski, F.; Filipovic, Z.; Kong, N.;
Kammlott, U.; Lukas, C.; Klein, C.; Fotouhi, N.; Liu, E. A. Science 2004, 303, 844.
34 Sadowsky, J. D.; Schmidt, M. A.; Lee, H.-S.; Umezawa, N.; Wang, S.; Tomita, Y.; Gellman,
S. H. J. Am. Chem. Soc. 2 0 0 5 ,127, 11966.
35 Arkin, M. R.; Wells, J. A. Nat. Rev. Drug Discovery 2004, 3, 301.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
165
36 Schinnerl, M.; Murray, J. K.; Langenhan, J. M.; Gellman, S. H. Eur. J. Org. Chem. 2003, 721.
37 Blankmeyer-Menge, B.; Nimitz, M.; Frank, R. Tetrahedron Lett. 1990, 31, 1701.
38 Gude, M.; Ryf, J.; White, P. D. Lett. Pept. Sci. 2003, 9, 203.
39 Niichter, M.; Ondruschka, B.; Bonrath, W.; Gum, A. Green Chem. 2004, 6, 128.
40 Yan, B.; Fang, L.; Irving, M.; Zhang, S.; Boldi, A. M.; Woolard, F.; Johnson, C. R.;
Kshirsagar, T.; Figliozzi, G. M.; Krueger, C. A.; Collins, N. J. Comb. Chem. 2003, 5, 547.
41 Edelhoch, H. Biochemistry 1967, 6, 1948.
42 Chen, J.; Marechal, V.; Levine, A. J. Mol. Cell. Biol. 1993, 13,4107.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
166
Chapter 4
Microwave-Assisted Parallel Synthesis
of a 14-Helical P-Peptide Library
Parallel
Synthesis
HP1.C
Peak
Area
Percent
Multimode Microwave Reactor
Portions of this chapter have been published as:
Murray, J. K.; Gellman, S. H. “Microwave-Assisted Parallel Synthesis of a 14Helical P-Peptide Library,” Journal o f Combinatorial Chemistry 2006, 8 (1), 5865.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
167
4.0 Brief Summary of Chapter
To facilitate the preparation o f P-peptide libraries in parallel, we have adapted reaction
conditions for the solid-phase synthesis o f 14-helical P-peptides for use in a multimode
microwave reactor. The low temperature/pressure requirements of microwave-assisted P-peptide
synthesis were found to be compatible with multi-well filter plates composed o f polypropylene.
Microwave heating o f the 96-well plate was sufficiently homogenous to allow the rapid
preparation o f a P-peptide library with acceptable purity.
4.1 Background
4.1.1 Microwave-Assisted Combinatorial Chemistry
Microwave irradiation has been successfully applied to an ever-increasing number o f
organic reactions with a resulting reduction in synthesis time and/or improvement in yield.1 We
determined that the solid-phase synthesis o f P-peptides (oligomers o f P-amino acids) could be
enhanced by microwave irradiation (Chapter 2).2,3 Although microwave irradiation is attractive
for accelerating the discovery o f bioactive molecules,4 harnessing this method o f rapid heating
for the preparation of combinatorial libraries is technically challenging.5
4.1.1.1 Automated Sequential Synthesis
The first challenge in microwave-assisted combinatorial chemistry is to apply the
necessary amount o f microwave irradiation for each reaction step.
In parallel synthesis, the
reaction mixture components differ from one vessel to the next (in our case, the vessels are wells
in a plate). Simultaneous exposure o f the entire set o f reaction vessels to microwave irradiation,
with power control based on the temperature o f a single reference vessel, can lead to quite varied
synthetic results across the library. Therefore, microwave-assisted reactions are often carried out
in an automated sequential manner in order to allow control o f experimental conditions (i.e.,
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
168
temperature).6 The time-saving aspect o f microwave synthesis is diminished by having to
irradiate each reaction mixture individually, one after another, even if each sample is irradiated
for only a short time. We describe a blend o f parallel and sequential microwave irradiation steps
in a multimode reactor, which combines the best aspects o f both methods, for rapid preparation
of a (3-peptide library in inexpensive 96-well polypropylene filter plates.
4.1.1.2 Reaction Vessel for Microwave-Assisted Parallel Synthesis
A major problem with microwave-assisted parallel library synthesis in the past has been
identifying a suitable reaction vessel.7 Early reports described both the possibility o f and the
problems (i.e., inhomogeneous heating and poor mechanical stability at elevated temperatures
and pressures) associated with using polypropylene well plates for microwave-assisted
synthesis.8 In response, reaction blocks composed o f microwave-absorbent material have been
developed for accurate temperature measurement and uniform heating.9
However, such
experimental set-ups (including a rotating drum for agitation) are expensive and cumbersome to
assemble. Furthermore, the glass inserts used with the reaction blocks are fritless, precluding the
rapid washing via bottom-filtration that is such an advantageous feature o f solid-phase synthesis.
Automated liquid handlers capable o f washing the solid support are expensive and slow. These
drawbacks threaten to negate any time-savings achieved by using microwave irradiation,
especially during the synthesis o f oligomeric molecules that require many sequential reactions.
The experimental conditions that we have developed for the solid-phase synthesis o f ppeptides with microwave irradiation employ relatively low temperatures and high-boiling
solvents in open vessels.2,7 Multi-well polypropylene filter plates are sufficiently heat-stable for
these conditions, and these plates are inexpensive and allow bottom-filtration o f the solid
support. We investigated the remaining issues o f accurate temperature measurement with small
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
169
reaction volumes, agitation of the reaction mixtures, and homogeneity o f microwave heating
throughout the plate in the multimode reactor.
We then synthesized a 96-member P-peptide
library in parallel, ■demonstrating the use o f inexpensive polypropylene filter plates and
microwave irradiation in a multimode reactor as a simple and effective method for the rapid
preparation o f peptide libraries on solid support in acceptable purities.
4.1.2 p-Peptide Synthesis
Our desire to discover biologically active P-peptides10 has led us to pursue their
combinatorial
synthesis.
P-Peptides may be
synthesized using
standard
solid-phase
methodology developed for a-peptides,11 but the resulting products are often o f low purity,
which prevents direct evaluation o f their biological properties12 and has limited efforts to the
preparation of small sets o f P-peptides followed by HPLC purification and screening.10
Difficulties with both removal o f the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group and
amide bond formation often arise during the synthesis o f 14-helical P-peptides, usually starting
with the sixth residue from the C-terminus.12 These problems were resolved by application o f
microwave irradiation (Chapter 2), which both increased the purity o f initial P-peptide products
and reduced synthesis time by 10-fold,2 enabling the rapid production o f individual P-peptides in
sufficient purity that they may be reliably screened without HPLC purification.
4.1.3 Microwave-Assisted Synthesis of a Split-and-Mix p-Peptide Library
We combined microwave-assisted p-peptide synthesis with split-and-mix techniques13
and reported the first one-bead-one-compound p-peptide combinatorial library (Chapter 3).14,15
Exposing the polystyrene macrobeads to multiple cycles o f microwave irradiation for each
reaction provided the products in good purity and reduced time relative to room temperature and
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
170
oil bath synthesis.
This advance has allowed the rapid preparation o f large p-peptide
libraries (1,000 members, Chapter 5). The necessity o f high-throughput screening, LC-MS/MS
sequencing, and re-synthesis for hit validation makes this approach useful for the initial
discovery o f biologically active compounds but not for subsequent refinement efforts (Figure 1).
Lead optimization is best accomplished via smaller, spatially addressable libraries, which allow
rapid elucidation o f structure-activity relationships (since the identity o f all library members is
known).16 If such parallel libraries can be synthesized in sufficient purity, then initial screening
can be performed without time-consuming purification o f library members. If library members
are synthesized on a large enough scale, then purification and validation o f compounds identified
as active by initial screening can proceed directly, i.e., without re-synthesis.
Therefore, to
increase the throughput o f P-peptide preparation and evaluation, we have now expanded
microwave-assisted solid-phase P-peptide synthesis methodology to the parallel synthesis o f a
library in a 96-well polypropylene filter plate.
Target
Re-Synthesis
LC-MS/MS
Sequencing
Split-and-Mix
Synthesis
Scaffold
Structure-Based
Design
'Parallel
Synthesis
Combinatorial
p-Peptide
Synthesis
Biochemical
Assay
HPLC Purification
Figure 1. Cycle for development of P-peptide protein-protein interaction inhibitors using either split-and-mix or
parallel combinatorial synthesis.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
171
4.2 Microwave-Assisted Parallel Synthesis of a B-Peptide Library
4.2.1 Synthetic Optimization in Multimode Microwave Reactor
In order to produce a P-peptide combinatorial library in parallel using microwave
irradiation, we needed to adapt the solid-phase P-peptide synthesis reaction conditions that we
previously developed with a monomode microwave reactor (CEM Discover) for use in a
multimode reactor (CEM MARS). P-Peptide 4-1 was selected for synthetic optimization as this
target exemplifies the difficulties of coupling and Fmoc-deprotection in the incorporation o f the
N-terminal ACHC residue (ACHC1, according to standard peptide numbering).2,120
Our
previous study (Chapter 2) showed that manual synthesis produced 4-1 in only 53% purity
(Figure 2 and Figure 3A), even though the penta-p-peptide precursor was 95% pure. Pleating the
reactions either in a monomode microwave reactor or in an oil bath improved the purity o f 4-1 to
80%. The advantage o f microwave irradiation was an overall 10-fold reduction in reaction time,
from 1.5 hr to 6 min for coupling the P-amino acid and from 15 min to 4 min for Fmocdeprotection.
R H N £M
„
H
n.
H
4-1
4-2
4-3
h
N
H
OH
R=H
R = Fmo1'
O
R = H2N
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172
100
C H I p-Peptide 4-1
n ~mmACHC1-Deletion
p-Peptide 4-2
kyy-4 p-Peptide 4-3
80
HPLC 60 ‘
Peak
Area
Percent 40 .
20
I
DMF
NMP
LiCI
Manual
DMF
NMP
LiCI
Monomode
Microwave
■BIN
DMF
DMF
NMP
LiCI
Multimode
Microwave
NMP
LiCI
Oil Bath
Reaction Conditions
Figure 2. Amount of P-peptide 4-1 and major impurities (peak area percent, from analytical reverse-phase (RP)
HPLC monitored via UV absorbance at 220 nm) resulting from different synthetic conditions. All coupling and
deprotection reactions in the synthesis o f the hexamer were conducted under the given reaction condition, i.e.,
manual, monomode or multimode microwave, or oil bath, as described below. The given solvent refers only to the
coupling of ACHC1; all other coupling reactions were performed in DMF. ACHC1 was double-coupled and
double-deprotected in all cases. Manual: 15 min deprotection, 1.5 hr coupling, RT; Monomode Microwave: 4 min
deprotection at 60°C; all couplings were 6 min at 50°C in DMF, except for 6 min at 45°C in 0.8 M LiCI in NMP for
ACHC1 where noted; Multimode Microwave: 4 min deprotection at 75°C; all couplings were 6 min at 70°C in
DMF, except for 0.8 M LiCI in NMP for ACHC1 where noted; Oil Bath: 15 min deprotection, 1.5 hr coupling,
60°C. Results from the manual, monomode microwave, and oil bath syntheses were reported previously (ref. 2).
Table 1. Data for Figure 2.
H P L C P e a k A rea Percent
Com pound
Reaction Conditions
M onom ode M icrowave
Multim ode M icrowave
Manual
DMF
NMP
DMF
LiCI
NMP
LiCI
DMF
JKM V 291
JKM V 199
JK M V 083
JK M V I 0 0 3
55
80
5
62
33
43
31
94
A C H C 1-D eletio n
(3-Peptide 4-2
8
0
21
0
0
6
2
0
0
15
14
4-3
Oil Bath
DMF
LiCI
JK M V I 143 JKM V I 147 JK M V I 0 6 7
Notebook
[1-Peptide 4-1
P-Peptide
NMP
0
NMP
LiCI
JK M V I 0 37
92
4
80
85
7
4
2
0
5
0
12
0
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173
ACHCD eletion
6000
4-1
4-3________________ 4-2
Tv
/V
A) Manual-DMF
A B) Manual-NMP/LiCl
5000
C) M pnom ode Microwave-DMF
I\
4000
D) M o n o m o d e M icrow ave-D M F, H igh T em p
■ E) Monom ode Microwave-NMP/LiCl
iv
F) M ultim ode Microwave-DMF
l\.
G) M ultim ode Microwave-DMF, Fligh Terpp
(V
H) M ultim ode Microwave-NMP/LiCl
fv
{mV)
2000
ft
1000
I) Oil Bath-DMF
0
10
II
J) Oil Bath-NMP/LiCl
20
30
Time (min)
40
50
Figure 3. Analytical RP-HPLC chromatograms (UV absorbance at 220 nm) of P-peptide 4-1 prepared under
different synthetic conditions. All coupling and deprotection reactions in the synthesis o f hexamer 1 were
conducted under the given reaction condition, i.e., manual, monomode microwave, multimode microwave, or oil
bath, as described below. The given solvent refers only to the coupling of ACHC1; all other coupling reactions
were performed in DMF. ACHC1 was double-coupled and double-deprotected in all cases.
A) Manual-DMF: All couplings for 1.5 hr in DMF at RT; 15 min deprotection at RT.
B) Manual-NMP/LiCl: All couplings for 1.5 hr in DMF at RT, except for double-coupling ACHC1 in 0.8 M LiCI in
NMP at RT; 15 min deprotection at RT.
C) Monomode Microwave-DMF: All couplings for 6 min in the monomode microwave at 50°C in DMF; 4 min
deprotection in the microwave at 60°C.
D) Monomode Microwave-DMF, High Temp (JKM VI 219): All couplings for 6 min in the monomode microwave
at 60°C in DMF; 4 min deprotection in the microwave at 60°C.
E) Monomode Microwave-NMP/LiCl: All couplings for 6 min in the monomode microwave at 50°C in DMF,
except for double-coupling ACHC1 in 0.8 M LiCI in NMP at 45°C in the microwave; 4 min deprotection in the
microwave at 60°C.
F) Multimode Microwave-DMF: All couplings for 6 min in the multimode microwave at 70°C in DMF; 4 min
deprotection in the microwave at 75°C.
G) Multimode Microwave-DMF, High Temp (JKM VI 151): All couplings for 6 min in the multimode microwave
at 80°C in DMF; 4 min deprotection in the microwave at 90°C.
H) Multimode Microwave-NMP/LiCl: All couplings for 6 min in the multimode microwave at 70°C in DMF, except
for double-coupling ACHC1 in 0.8 M LiCI in NMP at 70°C in the microwave; 4 min deprotection in the microwave
at 75°C.
I) Oil Bath-DMF: All couplings for 1.5 hr in DMF at 60°C in the oil bath, except for double-coupling ACHC1 in 15
min; deprotection at 60°C in the oil bath.
J) Oil Bath-NMP/LiCl: All couplings for 1.5 hr in DMF at 60°C in the oil bath, except for double-coupling ACHC1
in 0.8 M LiCI in NMP at 60°C in the oil bath; 15 min deprotection at 60°C.
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174
We previously described the difficulties associated with accurate temperature
measurement during microwave irradiation (Chapter 2).2 Target temperatures o f 50°C for amide
bond formation and 60°C for Fmoc-removal had given reproducible results (50 W maximum
power), but because of our reaction vessel configuration and the limitations o f the built-in IR
sensor o f the monomode microwave reactor,17 these set temperatures turned out not to be
accurate. Direct measurement indicated the final temperature of the reaction mixtures to be 6164°C for couplings and 70-75°C for deprotections. Upon transition to the multimode microwave
reactor, we began using a fiber optic probe, which is directly inserted into the reaction mixture
and gives accurate temperature measurements during the course o f the reaction.
We then
synthesized P-peptide 4-1 with magnetic stirring in the multimode microwave instrument
employing 70°C for coupling and 75°C for Fmoc-deprotection to reflect our previouslyoptimized conditions (600 W maximum power).
However, neither coupling nor Fmoc-
deprotection o f the N-terminal ACHC went to completion when DMF was used as solvent, and
P-peptide 4-1 was generated in only 62% purity.
Performing the coupling o f ACHC1 in 1-
methyl-2-pyrrolidinone (NMP) containing 0.8 M LiCI gave P-peptide 4-1 in much improved
2 18
92% purity and 76% yield (Figure 3H). ’ Yield o f P-peptide 4-1 was quantified by correlation
o f peak area in analytical RP-HPLC (UV absorbance at 220 nm) to concentration via a
calibration curve with an external standard.19 The HPLC comparison o f the crude products from
the standard and multimode microwave-enhanced solid-phase syntheses o f 4-1 reveals the extent
o f improvement in the latter case (Figure 4).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 4. HPLC chromatograms (UV absorbance at 220 nm) of (3-peptide 4-1 prepared under reactions conditions
described in Figure 2. A) Manual-DMF; B) Multimode Microwave-NMP/LiCl.
The significant difference between the initial purity of P-peptide 4-1 prepared in the
monomode (80%) and multimode (62%) microwave reactors was investigated. By raising the
temperature to 80°C for coupling and 90°C for Fmoc-deprotection we obtained P-peptide 4-1 in
89% purity using the multimode microwave reactor (JKM V I 151, Figure 3G), similar to our best
results with the monomode reactor.2 The higher temperatures for synthesis in the multimode
instrument were surprising, as earlier attempts to increase reaction temperature in the monomode
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176
instrument (60°C set temperature for coupling) led to the formation of a side-product, Ppeptide 4-3, via premature Fmoc-deprotection and addition o f a second monomer unit during the
double-coupling o f the N-terminal ACHC (JKM VI 219, Figure 3D). While higher temperatures
may be beneficial for the difficult reaction steps, we found through subsequent work that the
polypropylene filter plates from Millipore were not stable to these conditions.
The higher
temperatures also produced other, less difficult a/p-peptide sequences in lower purity than using
70°C for coupling and 80°C for Fmoc-deprotection in the multimode microwave reactor
(Chapter 5). The discrepancy in optimized temperatures between the two different reactors may
be explained by the following factors. In the monomode reactor, the combination o f continuous
cooling o f the sample,20 the experimental set up,2 and the built-in IR temperature sensor, which
measures the external temperature o f the glass reaction vessel,17 result in an observed
temperature that is much lower than the internal temperature of the reaction mixture measured
more accurately with the fiber optic probe o f the multimode instrument.
4.2.2 Microwave-Assisted Synthesis in 96-Well Filter Plate
Having optimized our reaction conditions in the multimode microwave instrument, we
prepared small sets of P-peptides in parallel in individual reaction vessels (4.0 mL polypropylene
solid-phase extraction tubes) using a 52-position turntable available from CEM (data not shown).
However, our ultimate desire was to transition from using many separate vessels to a 96-well
polypropylene filter plate. We evaluated the homogeneity o f microwave heating and its effect on
product purity by synthesizing p-peptide 4-1 in 26 different wells scattered across a plate. Wells
not containing resin were filled with fresh DMF before each reaction.
The fiber optic
temperature probe was placed in well D6 as a reference for the entire plate (Table 2). The plate
and temperature probe were held in place by a microtiter plate turntable (Figure 5). We were
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Ill
pleased to observe that the fiber optic probe could accurately measure the temperature o f the
small volumes (150 pL per well) associated with a 2.5 pmol scale synthesis. Washing o f the
solid support between reaction steps was rapidly accomplished with a vacuum filtration
manifold.
Figure 5. Expermental set-up for microwave-assisted solid-phase p-peptide synthesis.
(3-Peptide 4-1 was synthesized in an acceptable 69% average purity, but some regions of
the plate gave low purities (Table 2).
In particular the products from rows G and H were
synthesized in an average purity o f only 54%, significantly lower than the rest o f the plate. We
believe that this discrepancy resulted from poor agitation rather than uneven heating, since the
impurities resulted from deletion o f a residue at position 2, 3, 4, or 5; heating problems would
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178
have been manifested in the coupling and/or deprotection o f ACHC1. The rows containing
lower purity products (rows G and H) were located on the outer edge o f the turntable, furthest
from the center o f the cavity and the magnetic stirrer.
The stir bars in these wells were
occasionally motionless during the course o f the synthesis.
This problem was resolved by
switching to a smaller stir bar (7 mm in length), which rotates freely within the well, providing
good stirring even at the outer edge of the plate.
The benefit o f this modification was
demonstrated through the subsequent synthesis o f a P-peptide combinatorial library.
Table 2. Initial purity of (3-peptide 4-1 (peak area percent from analytical reverse-phase (RP) HPLC monitored via
UV absorbance at 220 nm) synthesized in various locations on a 96-well plate.
1 2 3 4
A 73 73
B
C
71
75
D
71
72
E
F
70
G
63
H 49 51
5
6
7
8
9
10 11 12
68 71
76
75
74
73
77 76
78 80
79 78
51 64
51 51
4.2.3 Design of 96-Membered P-Peptide Combinatorial Library
We designed a hexa-p-peptide library based on the sequence o f P-peptide 4-1 (Figure 6).
The incorporation o f cyclically constrained residues at positions 1 and 4 ensure that the library
members will have a high 14-helical propensity. The overall design is amphipathic, displaying a
hydrophobic face on 1/3 to 2/3 o f the helix depending upon whether ACHC or APiC is
incorporated at positions 1 and 4. Thus, each P-peptide in the folded state should present a
hydrophobic face for interaction with biomolecular surfaces while also containing charged
3
3
groups to promote water solubility. The inclusion o f p -Val at position 3 and P -Ser at position 2
provides variation in the hydrophobic/hydrophilic pattern.
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179
A)r
N
OH
‘NH
NH
Ps-Phe I p3-Trp I |}*-leu
p3-Phe I p’-Trp I p3-teu / p3-Ser
p3-Val I fP-Om
Figure 6. A) Hexa-P-peptide library ( 2 x 2 ^ 2 x 4 x 3 = 96 members). B) 14-Helical wheel diagram of hexa-Ppeptide library.
4.2.4 Parallel Synthesis of a P-Peptide Combinatorial Library with Microwave Irradiation
Library
synthesis
was performed with
microwave
irradiation using
optimized
power/temperature settings and a blend o f parallel and sequential reactions. Fmoc-P -Glu(tBu)loaded polystyrene Wang resin (100-200 mesh) was distributed in each well o f the 96-well filter
plate using the method o f Lebl et al. with slight modifications.21 Fmoc-deprotection o f all
library members was performed simultaneously. However, we found it necessary to couple one
P-amino acid at a time (in parallel), as the variable microwave absorption properties o f coupling
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180
solutions containing different P-amino acids lead to varied reaction temperatures under
equivalent microwave irradiation conditions.
The N-terminal residues were double-coupled
(ACHC in 0.8 M LiCI in NMP, and APiC in DMF) and double-deprotected. At the end o f the
synthesis, cleavage from the solid support and global side chain deprotection was accomplished
for each resin-bound sample by shaking for 2 hr at room temperature with TFA/CH 2 CI2 , after
which the cleavage solutions were transferred to a solid-bottom polypropylene plate and
concentrated using a rotary evaporator.
The crude p-peptide product mixtures were each
dissolved in DMSO and analyzed by reverse-phase HPLC (UV absorbance at 220 nm). The
major peak in each chromatogram was collected and analyzed by MALDI-TOF MS. In 97% o f
the cases (93 o f 96), the observed mass for the major HPLC peak corresponded to the expected
P-peptide library member. In the other three samples, the second largest peak in the trace was
the desired product. The major peak in these three cases corresponded to the Fmoc-protected
hexa-p-peptide product. The area percent o f the peak corresponding to the desired product in
each chromatogram was determined by integration (Figure 7), revealing that library members
were synthesized in an acceptable 61% average purity (Figure 8) or an average o f 95% for each
o f the 10 reaction steps. P-Peptide 4-1 was synthesized in well D4 in 74% purity (Figure 9),19
comparable to the results obtained in the earlier microwave-assisted parallel synthesis (Table 2).
The reported compound purity as judged by HPLC (UV absorbance at 220 nm) was dependent
upon the conditions employed, and the lower values associated with the steeper elution gradient
and shorter run time (10-60% B solvent over 25 min) have been reported.
The library was
deposited at the NIH Chemical Genomics Center for screening against a panel o f enzymatic
targets.
To date, the library has been screened against five different enzymes.
Five library
members showed activity at concentrations between 54 and 14 pM in an assay for cytochrome
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181
P450 inhibition, but were not pursued because many other compounds in the NIH collection
were active at 100-fold lower concentrations. The lack o f hits coming from the P-peptide library
suggests that these molecules may not be appropriate for enzyme inhibition.
HPLC
Peak
A re a
Percent
Figure 7. Initial purity o f (3-peptide products (peak area percent from analytical RP-HPLC monitored via UV
absorbance at 220 nm) synthesized in parallel with microwave irradiation in a polypropylene 96-well filter plate.
See Figure 10 for complementary presentations of the data.
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182
35
30
25
on
Number
of
Compounds 15
10
5
0
30 - 39
40 - 49
50 - 59
60 - 69
70 - 79
80 - 89
HPLC Peak Area Percent
Figure 8. Product purity of hexa-p-peptide library members, determined as the area percent of the major peak in the
analytical RP-HPLC chromatogram (UV absorbance at 220 nm).
500
74% Purity
400
300
Abs (mV)
200
100
0
10
~A-
J C jV A .
JV ...
20
30
40
50
T im e (m in )
Figure 9. HPLC chromatogram (UV absorbance at 220 nm) of p-peptide 4-1 prepared as a member of the library.
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183
4.2.5 Homogeneity of H eating during L ib rary Synthesis
We sought to determine whether the variation in product purity was due to uneven
heating of the 96-well plate. The three lowest purity members (wells A2, B2, and H I2) were
located at or near the comers o f the plate, but their neighbors were synthesized in higher purities.
We averaged the product purities for a number o f different regions within the plate (Figure 10).
A
B
C
D
E
F
G
H
Av.
B)
C)
A
B
C
D
E
F
G
H
A
B
C
D
E
1
67
50
69
61
73
53
55
49
60
2
35
32
81
75
86
58
61
53
60
3
60
57
76
69
68
57
74
48
64
4
40
53
82
74
88
56
80
44
65
5
63
54
61
43
58
52
67
52
56
6
75
58
68
62
77
71
81
63
69
7
64
57
44
51
62
58
57
64
57
8
62
44
55
45
73
55
63
66
58
9
65
51
64
59
67
55
65
70
62
10
63
54
82
74
81
68
68
62
69
11
62
51
69
64
70
49
69
55
61
50
57
57
69
62
66
57
57
12
51
58
72
57
62
45
58
30
54
Av.
59
52
69
61
72
56
67
55
61
58
62
65
G
H
Figure 10. Positional averages of p-peptide product purity. A) By row and column; B) By region; C) By distance
from the center of the plate.
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184
While products from the edges and/or comers o f the plate may have been synthesized in
slightly lower purity than products in the center, the differences in average purity were typically
< 10%. Thus, the variation in product purity seems to be as much dependent on the sequence as
on the location within the plate.
4.2.6 Characterization of Side Products from Library Synthesis
While product purity was relatively uniform throughout the plate, we wondered whether
investigation o f the side products would provide additional insight on local differences in
reaction temperature. Our previous synthetic optimization o f P-peptide 4-1 had demonstrated
that differences o f ± 10°C in reaction temperature resulted in different types o f impurities.2
Hence, the minor products collected during HPLC analysis o f the library were analyzed by mass
spectroscopy (Figure 11). We found that the identity o f the impurities tended to differ depending
on the location o f the well. Fmoc-protected side products and penta-P-peptide deletion products
were generally identified at the comers and edges o f the plate, possibly indicating slightly lower
reaction temperatures in these areas.
Hepta-P-peptides, resulting from premature Fmoc-
deprotection and coupling o f a second monomer unit during a single reaction cycle, were
typically found closer to the center o f the plate, a signal that reaction temperatures may be
slightly higher in this region.
Without exhaustive identification and quantification o f all
impurities and improved methods o f temperature measurement, however, these speculations
must be regarded as tentative.
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Figure 11. Location of identified impurities from (J-peptide library synthesis. Gray = Fmoc-protected and penta-(3peptide deletion products. Black = Hcpta-p-peptide addition impurities.
4.3 Conclusions
Microwave irradiation has been used to improve the initial purity of P-peptides and
reduce synthesis time.
Use o f a multimode microwave reactor for solid-phase P-peptide
synthesis has facilitated the preparation o f a hexa-p-peptide library using a combination of
parallel and sequential irradiation techniques.
This library is sufficiently pure for initial
screening without HPLC purification, although further improvement in synthetic efficiency
would be desirable.
The use o f inexpensive polypropylene multi-well filter plates for
microwave-assisted parallel library solid-phase synthesis is a simple alternative to more complex
and expensive equipment for the rapid generation o f peptide libraries. These techniques will
facilitate exploration of potential biomedical applications for foldamers.
4.4 Experimental Methods
4.4.1 General Procedures
Fmoc-OS^-ACHC and Fmoc (.S,,5,)-APiC(Boc)-OH were prepared by the method of
Schinnerl et al.22 Fmoc-(S)-p3-Glu(/Bu)-OH, Fmoc-(5)-p3-Phe-OH, Fmoc-(S)-p3-Om(Boc)-OH,
Fmoc-0S)-p3-Trp(Boc)-OH, Fmoc-(5)-p3-Leu-OH, Fmoc-(5)-p3-Ser(fBu)-OH and Fmoc-(5)-p3-
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186
Val-OH were prepared from their corresponding Fmoc-L-a-amino acids (Novabiochem) as
described previously.23
l-Methyl-2-pyrrolidinone (NMP) was purchased from Advanced
ChemTech. Methanol, CH 2 CI2 and acetonitrile were purchased from Burdick & Jackson.
1-
Methylimidazole, piperidine, 1-hydroxybenzotriazole hydrate (HOBt), /P^EtN , trifluoroacetic
acid (TFA), triethylsilane, triisopropylsilane and DMSO were purchased from Aldrich.
Mesitylenesulfonyl)-3-nitro-l,2,4-triazole (MSNT),
l-(2-
polystyrene Wang resin (100-200 mesh)
and O-benzotriazol- l-yl-AW./VV/V’-tetramethyluronium hexaflurorophosphate (FIBTU) were
purchased from Novabiochem.
DMF (biotech grade solvent, 99.9+ %) was purchased from
Aldrich and stored over Dowex ion exchange resin. Dry CH 2 CI2 and z'Pr2EtN were distilled from
calcium hydride.
4.4.2 F irst Residue Loading
First residue loading was accomplished as described.
24
3
Fmoc-(<S)-|3 -hGlu(/Bu)-OH (1.06
g) was activated with 1-methylimidazole (144 pL) and l-(2-mesitylenesulfonyl)-3-nitro-1,2,4triazole (710 mg) in dry CH 2 CI2 (7.5 mL) and added to swollen polystyrene (PS) Wang resin
(500 mg, 100-200 mesh, initial loading: 0.96 mmol/g) in a polypropylene solid phase extraction
(SPE) tube (25 mL, Alltech).
The tube was capped and placed on a wrist-action shaker
(Labquake, Bamstead/Thermolyne). After reaction for 12 hr at room temperature, the resin was
washed (5 x CH 2 CI2 , 5 x DMF, 5 x CFI2 CI2 and 5 x MeOH) using a vacuum manifold (VacMan, Promega) connected to a water aspirator and then dried under a stream o f N 2 until freeflowing. The yield was estimated by UV-quantification o f the dibenzofulvene-piperidine adduct
at 290 nm as previously described (0.67 mmol/g, 70%).
25
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187
4.4.3 M ultim ode M icrowave (3-Peptide Synthesis
Loaded PS Wang resin (10 pmol, 14.9 mg) was placed in a polypropylene SPE tube (4
mL, Alltech) and swelled with DMF for ~ 10 min. The resin was washed (5 x DMF, 5 x CH 2 CI2
and 5 x DMF). Deprotection solution (750 pL of 20% piperidine in DMF (v/v)) was added to
the resin, and a magnetic stir bar (8 mm, VWR) was placed inside the tube. The vessel was
placed inside an empty polypropylene 50 mL centrifuge tube, and placed in one slot o f a 52position turntable inside the multimode microwave reactor (CEM MARS).
The fiber optic
temperature sensor was suspended in the reaction mixture above the stir bar by pressing it
through a small hole (made with a needle) in the plastic top cap o f the SPE tube and placing the
cap loosely on the reaction vessel. The sample was irradiated (600 W maximum power, 75°C,
ramp 2 min, hold 2 min). All microwave experiments were conducted at atmospheric pressure.
The tube was removed from the microwave reactor, and the resin was washed as before. In a
separate vial, Fmoc-P-amino acid (30 pmol) was activated by adding HBTU (60 pL o f 0.5 M
solution in DMF), DMF (440 pL), HOBt (60 pL o f 0.5 M solution in DMF), and /Pr2EtN (60 pL
o f 1.0 M solution in DMF). The mixture was vortexed and added to the resin. The sample was
irradiated in the microwave reactor (600 W maximum power, 70°C, ramp 2 min, hold 4 min).
Alternatively, the coupling reaction o f Fmoc-(S,.S')-ACIIC was performed by activating with
solutions of HBTU, HOBt, and z'P^EtN in NMP and adding a solution o f LiCl in NMP for a final
concentration o f 0.8 M LiCl (620 pL final volume), and then adding this solution to the resin.
The tube was placed in a microwave reactor and irradiated as before (600 W maximum power,
70°C, ramp 2 min, hold 4 min). The accurate temperature measurement o f the fiber optic probe
makes modification o f the target temperature unnecessary. After the coupling reaction, the resin
was washed as before.
The N-terminal ACHC residue in (3-peptide 4-1 (standard peptide
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188
numbering, starting from the N-terminus o f the full sequence) was double-coupled and
double-deprotected in all syntheses. All other couplings were performed once with DMF as the
solvent. The deprotection/coupling cycle was repeated in a stepwise manner until reaching the
desired length o f the hexamer. Please refer to Chapter 2 for experimental procedures for (3peptide synthesis in the monomode microwave reactor or under manual and oil bath conditions.
4,4.4 P-Peptide Cleavage, Work-Up and HPLC
After the final residue had been added and deprotected, the resin was washed (5 x DMF,
5 x CH 2 CI2 , 5 x DMF and 5 x CH 2 CI2 ), and the (3-peptide was cleaved from the solid support
with
simultaneous
side
chain
deprotection
(3
mL,
45:45:5:5
trifluoroacetic
acid
(TFA):CH 2 Cl2 :triethylsilane:water, 2 h, RT, with rocking). The cleavage solution was drained
and concentrated under a stream o f N 2. The crude (3-peptide mixture was dissolved in 1.0 mL
DMSO, diluted (1 to 20) and analyzed by HPLC (10 pL injection, Shimadzu). The C4 -silica
reverse-phase analytical column (5 pm, 4 mm x 250 mm, Vydac) was eluted with a gradient of
acetonitrile in water (10 - 60%, 50 min, 0.1% TFA in each) at a flow rate o f 1 mL/min. Product
purity was determined as peak area percent by integration of the UV absorbance at 220 nm.
Integration was performed over the 1 5 - 5 0 min time interval to exclude the large absorbance o f
DMSO that elutes from 5 - 1 5 min. The lower threshold o f integration was set to exclude minor
peaks whose areas were < 1% o f the peak area o f the major species. (3-Peptide masses were
measured by MALDI-TOF-MS (Bruker Reflex II, a-cyano-4-hydroxycinnamic acid matrix).
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4.4.5 P-Peptide C haracterization D ata
nh2
NH
COoH
h 2n
p-P eptide
4-1
C 48H 68N 80 g
E xact M ass: 900.51
NH
NH.
C 0 2H
OH
A C H C 1 -D e le tio n
C4iH57N70 8
E xact M ass: 7 7 5 .4 3
Fm ocH N
P-Peptide
4-2
C63H78N8Oii
E xact M ass: 1 1 2 2 .5 8
h 2n
OH
4-3
C55H7gNgO10
p -P ep tid e
E xact M ass: 1 0 2 5 ,5 9
Figure 12. Structures, formulas, and calculated masses for p-peptide 4-1 and side products.
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190
Table 3. Characterization data P-peptide 4-1 and side products.
Compound
Formula
Calculated
Mass
MALDI-TOF MS
Observed Mass
RP-HPLC
Retention Time
TM+Hf
rM+Naf
(min)
3-Peptide 4-1
ACHC1Deletion
C48H68N80 9
900.51
901.4
923.4
34.5
C4 1 H5 7 N7 O 8
775.43
776.2
798.2
26.0
3-Peptide 4-2
C6 3 H7 8 N8 O 11
1122.58
1123.5
1145.5
46.5
3-Peptide 4-3
CssfbgNgO-io
1025.59
1026.3
1048.3
37.0
4.4.6 Microwave-Assisted Parallel Synthesis of P-Peptide 4-1 in a 96-Well Filter Plate
Fmoc-(33-Glu(/B u)-loaded polystyrene (PS) Wang resin (100-200mesh, 100 mg) was
distributed in 26 wells o f a 96-well filter plate (MultiScreen Solvinert, 0.45 pm hydrophobic
PTFE membrane from Millipore) by adding a constant volume
o f a homogeneous
resin/DMF/glycerol suspension to each well with a pipette. The resin was washed (5 x CH 2 CI2 ,
5 x DMF). Deprotection solution (250 pL o f 20% piperidine in DMF (v/v)) was added to every
well using a 12-channel multipipette, and a magnetic stir bar (8 mm, VWR) was placed inside
each well. The plate was placed on top o f an empty solid-bottom polypropylene 96-well plate
(250 pL well volume, Greiner) and then slid into a microtiter plate turntable inside the
multimode microwave cavity (CEM MARS). [Note: For best results, we now use a 2 mL deep
well polypropylene filter plate with polyethylene frits and long drip spouts in combination with a
bottom sealing mat (Artie White) instead of the Millipore filter plate mentioned above.] The
fiber optic temperature probe was positioned in well D6 using the arm attached to the turntable,
and the sample was irradiated (600 W maximum power, 75°C, ramp 2 min, hold 2 min, cool-off
5 min). All microwave experiments were conducted at atmospheric pressure. The plate was
removed from the microwave reactor, the resin was washed (5 x DMF, 5 x CH 2 CI2 , 5 x DMF),
and the drip plate was emptied by shaking. In a separate vial, Fmoc-p3-Phe (78.4 mg, 195 pmol)
was activated by adding HBTU (390 pL o f 0.5 M solution in DMF), DMF (2.86 mL), HOBt
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
191
(390 pL of 0.5 M solution in DMF), and /Pr2EtN (390 pL o f 1.0 M solution in DMF). The
mixture was vortexed and 150 pL was added to each resin-containing well using a single­
channel pipette. The rest o f the wells were filled with DMF. The temperature probe was placed
in well D6, and the sample was irradiated (600 W maximum power, 70°C, ramp 2 min, hold 4
min, cool-off 5 min). This stepwise Fmoc-deprotection/monomer coupling cycle was repeated
until the coupling o f residue 1 (standard peptide numbering, starting from the N-terminus o f the
full' hexa-p-peptide sequence) was complete.
The double-coupling o f Fmoc-ACFIC was
performed by activating with solutions o f HBTU, HOBt, and zPr2EtN in NMP and adding a
solution of LiCl in NMP for a final concentration o f 0.8 M LiCl, and then adding this solution to
the resin.
The plate was placed in the microwave reactor and irradiated as before (600 W
maximum power, 70°C, ramp 2 min, hold 4 min, cool-off 5 min). The accurate temperature
measurement o f the fiber optic probe makes modification o f the target temperature unnecessary.2
The N-terminal residue was then double-deprotected. The resin was washed (5 x DMF, 5 x
CH 2 CI2 ). Cleavage from the solid support with global side chain deprotection was accomplished
by adding triisopropylsilane (10 pL), water (10 pL), trifluoroacetic acid (100 pL), and CH 2 CI2
(100 pL) to each well. The plate was wrapped tightly in aluminum foil and shaken for 2 hr at
room temperature on a mini-orbital shaker (Lab-Line Instruments).
The foil covering was
removed, and the cleavage solutions were transferred to a solid-bottom 96-well plate with
vacuum filtration and concentrated using a rotary evaporator (SpeedVac, Thermo Savant). The
crude P-peptide mixtures were dissolved in 250 pL DMSO.
Each sample was analyzed by
HPLC 15 pL injection, Shimadzu). The C 4 -silica reverse-phase analytical column (5 pm, 4 mm
x 250 mm, Vydac) was eluted with a gradient o f acetonitrile in water (10 - 60%, 25 min, 0.1%
TFA in each, followed by a 5 min flush with 95% acetonitrile and 5 min equilibration at the
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
192
starting concentration) at a flow rate o f 1 mL/min. Product purity was determined as peak
area percent by integration o f the UV absorbance at 220 nm. Integration was performed over the
1 0 - 3 0 min time interval to exclude the large absorbance o f DMSO that elutes from 5 - 1 0 min.
The lower threshold o f integration was set to exclude minor peaks whose areas were < 1% o f the
peak area of the major species. (3-Peptide masses were measured by MALDI-TOF-MS (Bruker
Reflex II, a-cyano-4-hydroxycinnamic acid matrix).
4.4.7 Microwave-Assisted Parallel (3-Peptide Library Synthesis
Fmoc-p3-Glu(tBu)-loaded PS Wang resin (240 pmol, 375 mg) was swelled with DMF for
~ 10 min in a polypropylene SPE tube (15 mL, Alltech). The mixture was poured into the center
o f a 96-well polypropylene filter plate (MultiScreen Solvinert, 0.45 pm hydrophobic PTFE
membrane from Millipore).
[Note:
For best results, we now use a 2 mL deep well
polypropylene filter plate with polyethylene frits and long drip spouts in combination with a
bottom sealing mat (Artie White) instead o f the Millipore filter plate mentioned above.] The rest
o f the wells were filled with DMF. A top box (polypropylene cover from a rack o f 0-300 pL
Redi-Tip pipet tips from Fisher Scientific) was placed over the plate.
While being pressed
together, the box and plate were inverted, shaken, and turned right-side-up again, filling each
well with an equal volume o f the homogeneous resin/DMF mixture. The cover was removed,
and the DMF was drained using a vacuum manifold (Millipore) attached to a vacuum pump.
[Note:
For best results, we now suspend the resin in 50 mL o f a 3:2 mixture of
dichloromethane/DMF. The slurry is stirred while 500 pL aliquots are dispensed into each well
using a pipette.] The resin was washed (5 x DMF). Deprotection solution (250 pL o f 20%
piperidine in DMF (v/v)) was added to the resin in each well using a 12-channel multipipette,
and a magnetic stir bar (7 mm, VWR) was placed inside each well. The plate was placed on top
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
193
o f an empty solid-bottom polypropylene 96-well plate (250 pL well volume, Greiner) and
then slid into a microtiter plate turntable inside the multimode microwave cavity (CEM MARS).
The fiber optic temperature probe was positioned in well D6 using the arm attached to the
turntable, and the sample was irradiated (600 W maximum power, 75°C, ramp 2 min, hold 2 min,
cool-off 5 min). All microwave irradiations were conducted at atmospheric pressure. The plate
was removed from the microwave reactor, the resin was washed (5 x DMF), and the drip plate
was emptied by shaking. In a separate vial, Fmoc-p3-hPhe (96.4 mg, 240 pmol) was activated
by adding HBTU (480 pF o f 0.5 M solution in DMF), DMF (3.52 mL), HOBt (480 pF o f 0.5 M
solution in DMF), and /P^EtN (480 pF o f 1.0 M solution in DMF). The mixture was vortexed,
and 150 pF was added to each o f a subset o f the wells using a multipipette (Figure 13). The
temperature probe was placed in the center o f this region o f the plate, and the sample was
irradiated (600 W maximum power, 70°C, ramp 2 min, hold 4 min, cool-off 5 min).
After
washing, Fmoc-|33-hTrp(Boc) was activated and coupled to resin in a different section o f the
plate, followed by Fmoc-p3-hLeu. This constitutes the reaction sequence employed to couple all
residues at position 5 (standard peptide numbering, starting from the N-terminus o f the full hexap-peptide sequence) in the library.
The material in all wells was simultaneously Fmoc-
deprotected as before. The two different residues at position 4 were coupled sequentially. This
stepwise, parallel Fmoc-deprotection/sequential coupling cycle was repeated until the coupling
o f the residues at position 1. Fmoc-APiC(Boc) at position 1 was double-coupled in DMF. The
double-coupling o f Fmoc-ACHC at position 1 was performed by activating with solutions of
HBTU, HOBt, and /P^EtN in NMP and adding a solution o f LiCl in NMP for a final
concentration o f 0.8 M LiCl, and then adding this solution to the resin. The plate was placed in
the microwave reactor and irradiated as before (600 W maximum power, 70°C, ramp 2 min, hold
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
194
4 min, cool-off 5 min). The fiber optic probe provided accurate temperature measurement
even with the highly microwave-absorbent solvent mixture o f 0.8 M LiCl in NMP.2 The Nterminal residues were then double-deprotected. The resin was washed (5 x DMF, 5 x CH 2 CI2 ).
Cleavage from the solid support with global side chain deprotection was accomplished by adding
triisopropylsilane (10 pL), water (10 pL), trifluoroacetic acid (100 pL), and CH 2 CI2 (100 pL) to
each well.
The plate was wrapped tightly in aluminum foil and shaken for 2 hr at room
temperature on a mini-orbital shaker (Lab-Line Instruments). The foil covering was removed,
and the cleavage solutions were transferred to a solid-bottom 96-well plate with vacuum
filtration and concentrated using a rotary evaporator (SpeedVac with well-plate adapter, Thermo
Savant). The crude P-peptide mixtures were dissolved in 250 pL DMSO. Each sample was
analyzed by HPLC (15 pL injection, Shimadzu). The C 4 -silica reverse-phase analytical column
(5 pm, 4 mm x 250 mm, Vydac) was eluted with a gradient o f acetonitrile in water (10 - 60% or
0 - 50%, 25 min, 0.1% TFA in each, followed by a 5 min flush with 95% acetonitrile and 5 min
equilibration at the starting concentration) at a flow rate o f 1 mL/min.
Product purity was
determined as peak area percent by integration o f the UV absorbance at 220 nm. Integration was
performed over the 10 - 30 min time interval to exclude the large absorbance o f DMSO that
elutes from 5 - 1 0 min. The lower threshold o f integration was set to exclude peaks with areas <
10% o f the peak area o f the major species. P-Peptide masses were measured by MALDI-TOFMS (Bruker Reflex II, a-cyano-4-hydroxycinnamic acid matrix).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
195
Position 5
2
3
4
5
6
7
8
9
10
11
12
2
3
4
5
6
7
8
9
10
11
12
2
3
4
5
6
7
8
9
10
11
12
2
3
4
5
6
7
8
9
10
11
12
Position 4
Position 3
Position 2
Position 1
Figure 13. Coupling protocols for step-wise synthesis of spatially defined (3-peptide library.
approach streamlines reagent delivery via a multichannel pipette.
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
This “sector”
Table 4. Sequences o f p-peptide library members 1-48.
C o m p o u n d # W ell
1
A
1
N - T e r m in u s
Sequence
H2N- A P iC -
p 3h P h e -
C - T e r m in u s
p 3hV al-
A PiC -
p 3h P h e - p 3h G lu- OH
A P iC -
p T iP h e - p 3h G lu- OH
2
A
2
H2N- A P iC -
p 3h P h e - p 3h O rn -
3
A
3
H2N-
p dh P h e -
4
A
4
H2N- A P iC -
p 3h P h e - p 3h O rn - A C H C - p 3h P h e - p Jh G lu- OH
5
A
5
H2N- A PiC -
p Jh P h e -
p 3hV al-
A P iC -
p Jh T rp - p ah G lu - OH
6
A
6
H2N- A PiC -
p Jh P h e - p 3h O rn -
A P iC -
p Jh T rp - p Jh G lu - OH
7
A
7
H2N-
p Jh P h e -
p 3hV al- A C H C -
p T iT rp - p Jh G lu - OH
8
A
8
H2N- A P iC -
P ^ h P h e - p 3h O rn - A C H C -
p 3h T rp - p Jh G lu - OH
9
H2N-
p 3h P h e -
A PiC -
A P iC -
A P iC -
p JhV al- A C H C - p 3h P h e - p Jh G lu- OH
9
A
p 3hV al-
A P iC -
p 3ht_eu- p 3h G lu - OH
10
A 10
H2N- A PiC -
p 3h P h e - p 3h O rn -
A P iC -
P3hL e u - |i 3h G lu - OH
11
A 11
H2N-
fi ’h P h e -
12
A 12
H2N- A P iC -
13
B
1
H2N- A C H C - p T iP h e -
p 3hV al-
A P iC -
p 3h P h e - p 3h G lu - OH
14
B
2
H2N- A C H C - P ^ h P h e - p 3h O rn -
A P iC -
p 3h P h e - p 3h G lu - OH
A PiC -
p 3hV al- A C H C - p 3hl_eu- p Jh G lu - OH
p 3h P h e - [ f h O r n - A C H C - p Jh L e u - fl3h G lu - OH
15
B
3
H2N- A C H C - p Jh P h e -
16
B
4
H2N- A C H C - (V’h P h e - p Jh O rn - A C H C - p 3h P h e - fVJh G lu - OH
(l3hV al- A C H C - [i3h P h e - p 3h G lu - OH
17
B
5
H2N- A C H C - P ‘!h P h e -
p ahV al-
A P iC -
p Jh T rp - p Jh G lu - OH
18
B
6
H2N- A C H C - P ’h P h e - |S3h O rn -
A P iC -
p 3h T rp - p ;lh G lu- OH
p 3hV al- A C H C -
p Jh T rp - p ’h G lu- OH
19
B
7
H2N- A C H C - p Jh P h e -
20
B
8
H2N- A C H C - ( f h P h e - p Jh O rn - A C H C -
P Jh T rp - [f h G lu - OH
21
B
9
H2N- A C H C - p Jh P h e -
(SJhV al-
A P iC -
P ’h L e u - p 3h G lu- OH
A P iC -
P ’h L e u - (f h G lu - OH
22
B 10
H2N- A C H C - ( f h P h e - (l3h O rn -
23
B 11
H2N- A C H C - P ’h P h e -
24
B 12
H2N- A C H C - p 3h P h e - p 3h O rn - A C H C - p Jh L e u - p Jh G lu- OH
25
C
1
H2N- A P iC -
P Jh T rp -
p 3hV al-
A P iC -
p ’h P h e - p 3h G lu - OH
26
C
2
H2N- A P iC -
p Jh T rp -
p 3h O rn -
A P iC -
p 3h P h e - p Jh G lu - OH
p 3hV al- A C H C - p 3h L e u - p Jh G lu - OH
27
C
3
H2N- A P iC -
P Jh T rp -
(J3hV al- A C H C - p 3h P h e - (f h G lu - OH
28
C
4
H2N- A P iC -
P 'b T rp -
p 3h O rn - A C H C - p 3h P h e - p Jh G lu - OH
29
C
5
H2N- A P iC -
P Jh T rp -
(f h V a l-
A P iC -
P Jh T rp - (f h G lu - OH
6
H2N- A P iC -
p Jh T rp - p 3h O rn -
A P iC -
p Jh T rp - (f h G lu - O H
7
H2N- A P iC -
| f h T rp -
(f h V a l- A C H C -
8
H2N- A P iC -
p Jh T rp -
(fh O rn - A CHC-
P JhT rp- (f h G lu - OH
9
H2N- A P iC -
P Jh T rp -
ff h V a l-
A P iC -
p Jhl_eu- (f h G lu - O H
A P iC -
p 3hl_eu- p ah G lu - O H
30
31
32
C
C
C
P Jh T rp - p 3h G lu - OH
33
C
34
C 10
H2N- A P iC -
( f h T rp -
(fh O rn -
35
C 11
H2N- A P iC -
P Jh T rp -
p JhV al- A C H C - P3h L e u - [f h G lu - OH
36
C 12
H2N- A P iC -
p Jh T rp -
( f h O r n - A C H C - P ’h L e u - [ f h G lu - OH
D
1
H2N- A C H C -
| f h T rp -
[f h V a l-
A P iC -
p 3h P h e - ( f h G lu - OH
[fh O rn -
A P iC -
p 3h P h e - p 3hG lu - OH
37
38
D
2
H2N- A C H C - P Jh T rp -
39
D
3
H2N- A C H C - p dh T rp -
p 3hV al- A C H C - p 'h P h e - p 3hG lu - OH
( f h T rp -
p 3h O rn - A C H C - p 3h P h e - p Jh G lu - OH
40
D
4
H2N- A C H C -
41
D
5
H2N- A C H C -
p Jh T rp -
p 3hV al-
A P iC -
p 3h T rp - (f h G lu - OH
6
H2N- A C H C -
[ f h T rp -
p 3h O rn -
A P iC -
(fh T rp -
P3h T rp -
p 3hV al- A C H C - p Jh T rp - (f h G lu - OH
42
D
[f h G lu - OH
43
D
7
H2N- A C H C -
44
D
8
H2N- A C H C -
(f h T r p -
(fh O rn - A CHC-
p 3h T rp - fi3h G lu - OH
9
H2N - A C H C -
P Jh T rp -
p 3hV al-
A P iC -
p 3h L e u - p 3h G lu - OH
p 3h O rn -
A P iC -
p 3h L e u - p 3h G lu - OH
45
D
46
D 10
H2N- A C H C -
P 3h T rp -
47
D 11
H2N - A C H C -
p 3h T rp -
p 3hV al- A C H C - P Jh L e u - p 3h G lu - OH
48
D 12
H2N - A C H C -
p Jh T rp -
p 3h O rn - A C H C - p Jh L e u - p 3h G lu - OH
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
Table 5. Sequences o f P-peptide library members 49-96.
C o m p o u n d # W ell
N - T e r m in u s
Sequence
C - T e r m in u s
E
1
H2N-
A P iC -
p 3h L e u -
p shV al-
A PiC -
p T iP h e - p ’ h G lu- OH
50
E
2
H2N-
A P iC -
P ’ h L e u - p ’ h O rn -
A P iC -
P ’ h P h e - p ’ h G lu - OH
51
E
3
H2N-
A P iC -
P Jh L e u -
52
E
4
H2N- A P iC -
P’ h L e u - p Jh O rn - A C H C - p T iP h e - p ’ h G lu - OH
53
E
5
H2N- A P iC -
P Jh L e u -
p ’ hV al-
A P iC -
P’ h T rp - p ’ h G lu - OH
54
E
6
H2N- A P iC -
P^L eu-
p ah O rn -
A P iC -
P’ h T rp - p ’ h G lu - OH
P’ h L e u -
49
A PiC -
p ’ hV al- A C H C - p T iP h e - p 3hG lu- OH
55
E
7
H2N-
p JhV al- A C H C -
P’ h T rp - p ’ h G lu - OH
56
E
8
H2N- A P iC -
P ’ h L e u - p ’ h O rn - A C H C -
p ’ h T rp - p ’ h G lu - O H
57
E
9
H2N- A P iC -
P’hL eu-
p ’ hV al-
A P iC -
P’ h L e u - p 3h G lu - O H
P ’ h L e u - p ’ h O rn -
A P iC -
P’ h L e u - p ’ h G lu - OH
58
E 10
H2N - A P iC -
59
E 11
H2N-
A P iC -
P’hL eu-
60
E 12
H2N-
A P iC -
P ’ h L e u - p ’ h O rn - A C H C - P’ h L e u - p ’ hG lu- OH
F
1
H2N- A C H C - P ’ h L e u -
p ’ hV al-
A P iC -
P ’ h P h e - p ’ hG lu- OH
A P iC -
P 3h P h e - p ’ h G lu - OH
61
p ’ hV al- A C H C - P’ h L e u - p ’ h G lu - OH
62
F
2
H2N - A C H C - P ’h L e u - p ’ h O rn -
63
F
3
H2N- A C H C - P’ h L e u -
64
F
4
H2N- A C H C - P Jh L e u - p Jh O rn - A C H C - P J h P h e - p Jh G lu - OH
65
F
5
H2N- A C H C - p Jh L e u -
p JhV al-
A P iC -
6
H2N- A C H C - p Jh L e u - p Jh O rn -
A P iC -
66
F
p ’ hV al- A C H C - P ’ h P h e - p ’ h G lu - OH
p JhV al- A C H C -
P’ h T rp - p ’ h G lu - OH
P Jh T rp - p ’ h G lu - OH
67
F
7
H2N- A C H C - p Jh L e u -
P JhT rp- P ’ h G lu - O H
68
F
8
H2N- A C H C - p Jh L e u - p Jh O rn - A C H C - p Jh T rp - p ’h G lu - OH
69
F
9
H2N- A C H C - P Jh L e u -
p JhV al-
A P iC -
p Jh L e u - p Jh G lu - OH
A P iC -
p 3h L e u - p Jh G lu- OH
70
F 10
H2N- A C H C - p Jh L e u - p Jh O rn -
71
F 11
H2N- A C H C - P ^ L e u -
72
F 12
H2N- A C H C - P Jh L e u - p ah O rn - A C H C - p Jh L e u - p Jh G lu- OH
73
G
1
H2N-
A PiC -
p Jh S e r -
p JhV al-
A PiC -
p 3h P h e - p^hG lu- OH
2
H2N - A P iC -
(i’h S e r -
(i’h O rn -
A P iC -
P ’h P h e - p ’h G lu - OH
p ’ h S e r-
p ’hV al- A C H C - p Jh P h e - p ’h G lu - O H
p ’ h O rn - A C H C - p 3h P h e - p sh G lu - OH
74
G
p ahV al- A C H C - P Jh L e u - p-’h G lu - OH
75
G
3
H2N - A P iC -
76
G
4
H2N - A P iC -
p ’h S e r-
5
H2N - A P iC -
(i’h S e r -
p ’ hV al-
A P iC -
p ’ h T rp - p sh G lu - OH
(i’ h S e r -
p ’ h O rn -
A P iC -
P JhT rp- p ’ h G lu - O H
p ’h S e r-
p shV al- A C H C - p 3h T rp - p 3h G lu - OH
p ’ h O rn - A C H C - P ’ h T rp - p ’ h G lu - OH
77
G
78
G
6
H 2N- A P iC -
79
G
7
H 2N- A P iC -
80
G
8
H 2N- A P iC -
P’h S e r-
81
G
9
H 2N- A P iC -
P’h S e r-
p ’ hV al-
A P iC -
P’ h L e u - p ’ h G lu - OH
82
G 10
H 2N- A P iC -
P’h S e r-
p ’ h O rn -
A P iC -
P’ h L e u - p ’ h G lu- OH
83
G 11
H 2N- A P iC -
P ’ h S e r-
p ’ hV al- A C H C - P’ h L e u - p ’ h G lu- OH
P’ h S e r-
p ’ h O rn - A C H C - P’ h L e u - p ’ h G lu- OH
84
G 12
H 2N - A P iC -
85
H
1
H 2N- A C H C - P ^ h S e r-
p 3hV al-
A P iC -
P ’ h P h e - p ’ h G lu- OH
86
H
2
H2N- A C H C - p ’ h S e r -
p ’ h O rn -
A P iC -
P ’ h P h e - p ’ h G lu- OH
87
H
3
H2N- A C H C -
P’ h S e r -
p ’ hV al- A C H C - P ’ h P h e - p ’ h G lu- OH
88
H
4
H2N - A C H C - p ’ h S e r-
p ’ h O m - A C H C - P ’ h P h e - p ’ h G lu - OH
89
H
5
H2N - A C H C - P’ h S e r -
p ’ hV al-
A P iC -
P ’ h T rp - p ’ h G lu - OH
90
H
6
H2N - A C H C - P’ h S e r -
p ’ h O rn -
A P iC -
P ’ h T rp - p ’ h G lu - OH
91
H
7
H2N - A C H C - P’ h S e r -
p ’ hV al- A C H C -
92
H
8
H2N - A C H C - P’ h S e r -
p ’ h O rn - A C H C -
P’ h T rp - p ’ h G lu - O H
93
H
9
H2N- A C H C - P ’ h S e r -
p 3hV al-
A P iC -
P’ h L e u - p ’ h G lu - OH
94
H 10
H2N- A C H C - p ’ h S e r -
p ’ h O rn -
A P iC -
P’ h L e u - p ’ h G lu - OH
95
H 11
H2N- A C H C - P ’ h S e r-
p ’ hV al- A C H C - P Jh L e u - p ah G lu- OH
96
H 12
H2N - A C H C - P ’ h S e r-
p ’ h O rn - A C H C - P’ h L e u - p ’ h G lu- OH
P’ h T rp - p ’ h G lu - OH
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 14. P-Peptide library members 1-8.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
199
2h
co
°
h2n^
(
o
O
L nA - A n
N
H
H
H
H ^ N
H
OH
H
NH2
2h
co
O
(
o
O
h2n Z ^ ^ n ^ ^ ' n
N
H
H
n£^N
H
10
O
H ^ N
H
OH
H
2h
co
O
O
h2n £ ^ ^ n '
H
H
v
11
£ s/
N
H
n
OH
NH2
2h
9 0
0
h2n£
( 9
^ n' v
H
H
O
o
O
12
r
o
'N
OH
H
co
0
h2n£
(
0
0
^ n
/
O
h^
O
2h
f'
13
OH
n
h
H
NH2
( _ /
0
h2n£
(
0
o
^ n
o
(
n£ ^ " n-
H ^ N
H
co
O
O
h2n£
^
(
N'
H
0
v
0
'N
H
o
n£
14
OH
H
co
0
2h
I
O
2h
f
15
^ n
OH
NH2
co
0
(
H2n £ ^ 7 > ' ^
H
F ig u r e
o
0
'N
H
( £ ^ n
2h
J o
16
OH
15. P-Peptide library members 9-16.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
// \
o
h2n£
i
NH
c o
19
Q
^ n
OH
N H.
( y
o
h
2h
\
NH
20
9
2n
OH
c o
o
h2n£
(
o
O
^ n'
o
o
n£
^ N
H ^ N
H
2h
o
21
OH
H
NH2
C 0 2H
O
*
H2n£ ^ V
0
^
°
'N
O
n£^"n
h
^
n
O
22
OH
h
H
<f ^
o
h2n£
/
9
O
^ n'
o
23
OH
K 7
h
C 0 2H
(
?
NH,
C 0 2H
24
O
2n
OH
Figure 16. P-Peptide library members 17-24.
R e p ro d u c e d with perm ission of the copyright owner. F urther reproduction prohibited without perm ission.
Figure 17. (3-Peptide library members 25-32.
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
202
co
o
(
h2n £ ^ n '
N
H
H
2h
33
o
^
'N
H
H
'N
H
34
N
H
H
°
N
j
H
O
H
o
N
35
H
36
/ o
H2 n £ ^ 7 ^ N '
N
H
H
v
'N
H
NH
co
O
h 2 n;
N'
NH
^
'N
H
2h
37
j O
^
OH
nh2
c o 2h
o
N'
^
'N '
H
NH
38
J o
v
'O H
C 0 2H
39
0 ( ^ 0
h 2 n;
OH
NH
nh2
co
0
h 2 n;
Figure 18.
2h
J o
40
OH
p -P e p tid e lib ra ry m em bers 3 3 -4 0 .
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
43
O i 9
H2n £ ^ N ' ^ "N
0
(
44
9
^
O
f
H2n £ ^ N ' ^
"N
O
45
o
'N
i- t
^
n
h
H
46
H
’N
H
47
48
Figure 19. P-Peptide library members 41-48.
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
Figure 20. P-Peptide library members 49-56.
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
205
C °2H
o
h2n£
O
^ " n
N
H
H ^N
H
OH
H
NH2
^ ^ n
N
c o 2h
O
O
h2n£
57
O
O
H
58
H ^N
H
OH
H
c o 2h
O
O
o
59
^
N
H
H2N 2 ^ r* N
N H
H
^
NH2
O
h2n£
c o 2h
Q
60
f
o
^ ^ n
N' v
H
H
H
_
O
O
h2n£ ^ t
"n
o
n£^N
H
H
N
H
/
h2n£
0
O
o
f
N
H
H
^
61
OH
c o 2h
O
H ^N
H
l
o
f
o
62
N' ^
H
H
_
o
'OH
c o 2h
NH2
o
^ n
OH
I
'OH
c o 2h
o
i'
63
HzN ^ / ' N
nh 2
h2n£
O
^ n
o
c o 2h
O
/
O
f
o
64
Figure 21. P-Peptide library members 57-64.
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
H ^N
H
N ^
H
N ^
H
OH
/NH2
\
o
(
r
O
f
y
O
O
{
_
c o
O
f
2h
70
O
H
\
H
H
H ''*
H
C02H
H
NH2
o
f
O
r
O
c o 2h
o
N /^ 7
H ^*
72
N' v
H
N ^
H
OH
Figure 22. P-Peptide library members 65-72.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
207
2h
c o
73
o
h 2n £
^
N
H
n
'
v
H
'N
H
9
h^
^
n
9 N
h
OH
h
H
NHo
c o
2h
OH
0
N
H
( 0
H
O
Q
n£
h^
H
^
O
V
n
'
v
h
O
74
OH
n
h
H
c o
?
9
A
^
N
H
o
2h
'N
H
H
75
o
9
OH
NH 2
co
2h
OH
0
(
0
H
9
0
n£
H
9
Xaoh
^ n
76
H
NH
co
*
9
A
H2N £ ^ f " N '
N
H
H
^
O
{
H ^ N
H
'N
H
77
o
9
n£^N ' ^
'N
H
2h
OH
H
NH2
co
2h
OH
0
h2n£
^ N
(
O
0
^ n^ < ^ n
H
^
H
H
H
78
i^rrrr v 'n
< JL
N
’
9
H
79
N
H
H
H
NH2
NH
c o
o
N
H
9
A
H
O
H
?
2h
80
o
OH
Figure 23. P-Peptide library members 73-80.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
H2NZ -C ^ N
N H
H
^
n£
N
H
^ n
H ^N
H
OH
H
NH2
C 02H
OH
O
'
O
o
H2n£ ^
82
O
H ^N
H
OH
H
c o 2h
OH
O
O
H2N £ ^ f
83
o
OH
NH2
\
OH
O
I
N
H
H
84
0
H2N ^ 7 ^ N ' ^
C02h
H
°JUUU
0
N
H
c o 2h
OH
O
O
h2nZ-^7
'N' v
o
/
tz A 'H
N'
'n
H
O
^
H ^N
H
nh 2
h2n£
\ 0
o
0
^ n
n^
f
'N '
I
0
OH
H
c o 2h
o
O
o
/
O
i'
N
/ 'n
c o 2h
OH
h2n^
/ 'n
1
o
87
OH
H
NH2
O
86
f
OH
h2n^
85
c o 2h
/ 'n
H ^N
H
O
'OH
_
OH
Q
OH
O
t
o
H ^ /
H
o
88
f
OH
Figure 24. P-Peptide library members 81-
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
^
i
rr
i
n
OH
NH
co
2h
OH
90
OH
NH
OH
91
OH
NH
NH
co
2h
,OH
92
OH
co
A
o
0
N
n£
H
O
O
^ N
H ^ N
H
2h
S
93
OH
H
NH2
co
2h
OH
O
H2N ^ k N
°
O
n £ ^ n
h^
n
94
OH
h
H
co
I
a
o
p
H
nh
O
N ZiZ-N
95
OH
2
co
OH
0
(
9
P
2h
96
o
h 2n ;
F ig u r e
2h
OH
25. (3-Peptide library members 89-96.
R e p ro d u c e d with perm ission of th e copyright owner. Further reproduction prohibited without permission.
4.4.8 A nalytical C haracterization of P-Peptide L ib rary
Table 6. Characterization of P-peptide library members by MALDI-TOF MS and HPLC retention time.
Com pound Numbers
1
A
1
B
13
C
25
D
37
E
49
F
61
G
73
H
85
2
2
14
26
38
50
62
74
86
3
3
15
27
39
51
63
75
87
4
4
16
28
40
52
64
76
88
5
5
17
29
41
53
65
77
89
6
6
18
30
42
54
66
78
90
7
7
19
31
43
55
67
79
91
8
8
20
32
44
56
68
80
92
9
9
21
33
45
57
69
81
93
10
10
22
34
46
58
70
82
94
11
11
23
35
47
59
71
83
95
12
12
24
36
48
60
72
84
96
2
863.48
862.48
902.49
901.49
829.50
828.50
803.45
802.45
3
847.47
846.47
886.48
885.48
813.49
812.49
787.44
786.44
4
862.48
861.48
901.49
900.49
828.50
827.50
802.45
801.45
5
887.48
886.48
926.49
925.49
853.50
852.50
827.45
826.45
6
902.49
901.49
941.50
940.50
868.51
867.51
842.46
841.46
7
886.48
885.48
925.49
924.49
852.50
851.50
826.45
825.45
8
901.49
900.49
940.50
939.50
867.51
866.51
841.46
840.46
9
814.49
813.49
853.50
852.50
780.51
779.51
754.46
753.46
10
829.50
828.50
868.51
867.51
795.52
794.52
769.47
768.47
11
813.49
812.49
852.50
851.50
779.51
778.51
753.46
752.46
12
828.50
827.50
867.51
866.51
794.52
793.52
768.47
767.47
O bserved M ass [M+H]*
1
2
A
849.22
864.29
B
848.22
863.19
C
903.40
888.36
D
887.26
902.28
E
815.38
830.25
F
814.20
829.33
804.22
G
789.06
H
788.06
803.11
3
848.25
847.65
887.10
886.10
814.33
813.49
788.54
787.07
4
863.37
862.11
902.12
901.27
829.28
828.59
803.59
802.24
5
888.46
887.25
927.26
926.36
854.19
853.43
828.47
827.20
6
903.18
902.35
942.40
941.23
869.18
868.14
843.11
842.27
7
887.20
886.22
926.27
925.44
853.19
852.14
827.07
826.15
8
902.08
901.39
941.19
940.16
868.16
867.28
842.04
841.23
9
815.38
814.16
854.29
853.17
781.18
780.81
755.16
754.32
10
830.47
829.27
869.40
868.14
796.51
795.46
770.34
769.33
11
814.08
813.23
853.35
852.18
780.24
779.10
754.18
753.22
12
829.02
828.23
868.29
867.08
795.21
794.11
769.15
768.16
3
16
23
16
22
16
23
12
17
4
14
21
14
20
14
20
10
16
5
14
17
13
17
13
16
8
11
6
11
15
12
15
11
15
11 ■
V.;14
7
16
23
16
22
16
22
13
17
8
14
20
14
20
13
20
10
16
10
10
14
10
14
'15
11
15
23
16
22
20
23
16
22
12
13
21
14
20
18.
21
14
20
Calculated M ass
1
A
848.47
B
847.47
C
887.48
D
886.48
E
814.49
F
813.49
G
788.44
H
787.44
R etention Time (min
1
A
14
B
17
C
13
D
17
E
13
F
17
G
■ 13
H
11
2
11
15
11
15
11
15
14
9
12
16
13
16
. 17
" 21
■' .'12:'.'
■
1:9 1
.
:;15 A ; .f'-dS ;:'::'
Eluted with a gradient of acetonitrile in w ater (10 - 60%, 25 min, 0.1% TFA in each) a t a flow rate of 1 mL/min.
Eluted with a g r^diehtpf ddetdhitnie;4n:w ater (0 - 50%, 25 Hiin, 0.1 % TFA iii each ) a t a flow rate of 1 mL/min.
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
Table 7. Characterization o f impurities in P-peptide library product mixtures 1-45.
C o m p o u n d # R e te n tio n T im e (m in) O b s e r v e d [M+H]+
8 4 9 .2 2
1
14
9 6 2 .3 4
15
7 3 8 .2 0
2
10
8 6 4 .2 9
11
1 0 8 6 .3 5
17
8 4 8 .2 5
3
16
19
7 2 2 .1 6
8 6 3 .3 7
4
14
20
1 0 8 5 .7 0
14
8 8 8 .4 6
5
11
9 0 3 .1 8
6
8 8 7 .2 0
7
16
14
9 0 2 .0 8
8
12
8 1 5 .3 8
9
8 3 0 .4 7
10
10
8 1 4 .0 8
11
15
8 2 9 .0 2
12
13
13
14
7 2 3 .0 9
17
8 4 8 .2 2
100 9 .6
22
14
10
7 3 8 .1 8
8 6 3 .1 9
15
21
1 0 8 5 .1 3
15
19
7 2 2 .4 0
23
8 4 7 .6 5
21
862.11
16
7 6 6 .0 0
17
14
17
8 8 7 .2 5
11
7 0 2 .1 8
18
15
9 0 2 .3 5
23
8 8 6 .2 2
19
20
9 0 1 .3 9
20
21
14
6 8 9 .0 9
16
8 1 4 .1 6
22
14
8 2 9 .2 7
6 8 6 .0 6
23
18
23
8 1 3 .2 3
24
14
6 6 7 .1 3
21
8 2 8 .2 3
27
1 0 5 0 .4 5
13
8 8 8 .3 6
25
9 0 3 .4 0
26
11
27
16
8 8 7 .1 0
9 0 2 .1 2
28
14
29
13
9 2 7 .2 6
12
9 4 2 .4 0
30
9 2 6 .2 7
31
16
32
14
9 4 1 .1 9
8 5 4 .2 9
33
13
9 6 7 .5 0
15
34
10
8 6 9 .4 0
35
16
8 5 3 .3 5
14
8 6 8 .2 9
36
17
8 8 7 .2 6
37
38
15
9 0 2 .2 8
39
22
8 8 6 .1 0
9 0 1 .2 7
40
20
41
17
9 2 6 .3 6
42
15
9 4 1 .2 3
43
22
9 2 5 .4 4
9 4 0 .1 6
44
20
16
8 5 3 .1 7
45
18
9 7 0 .3 0
Identity
M
M+Val
M-APiC
M
M +Fm oc
M
M-APiC
M
M +Fm oc
M
M
M
M
M
M
M
M
M-APiC
M
M +Phe
M-ACHC
M
M +Fm oc
M-ACHC
M
M
M-ACHC
M
M-Trp
M
M
M
M-ACHC
M
M
M -Leu
M
M -P he
M
M +Fm oc
M
M
M
M
M
M
M
M
M
M+Val
M
M
M
M
M
M
M
M
M
M
M
M
M+Val
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
Table 8. Characterization o f impurities in P-peptide library product mixtures 46-96.
C o m p o u n d # R e te n tio n T im e (m in ) O b s e r v e d [M+H]*
14
8 6 8 .1 4
46
8 5 2 .1 8
47
22
8 6 7 .0 8
48
20
1 0 9 3 .3 4
26
49
13
8 1 5 .3 8
9 2 8 .3 5
15
11
8 3 0 .2 5
50
8 1 4 .3 3
51
16
22
9 2 7 .5 3
14
8 2 9 .2 8
52
8 5 4 .1 9
53
13
9 6 7 .2 3
15
54
11
8 6 9 .1 8
55
16
8 5 3 .1 9
9 6 6 .1 9
20
56
13
8 6 8 .1 6
57
17
7 8 1 .1 8
17
79 6 .5 1
58
7 8 0 .2 4
20
59
79 5 .2 1
60
18
17
8 1 4 .2 0
61
15
8 2 9 .3 3
62
8 1 3 .4 9
63
23
8 2 8 .5 9
64
20
16
8 5 3 .4 3
65
8 6 8 .1 4
66
15
22
8 5 2 .1 4
67
20 '
8 6 7 .2 8
68
21
780.81
69
7 9 5 .4 6
70
19
7 7 9 .1 5 /7 7 9 .1 0
71
2 8 /2 3
7 9 4 .1 4 /7 9 4 .1 1
72
26/21
73
7 /1 3
7 8 9 .5 4 /7 8 9 .0 6
74
8 0 4 .2 2
5 /10
75
12
7 8 8 .5 4
76
10
8 0 3 .5 9
8 2 8 .4 7
77
8
78
5/11
843.11
8 2 7 .0 7
79
13
8 4 2 .0 4
80
10
12
81
7 5 5 .1 6
17
8 6 8 .1 8
82
7 7 0 .3 4
10
83
16
7 5 4 .1 8
84
14
7 6 9 .1 5
11
7 8 8 .0 6
85
86
12
6 7 7 .9 5
8/14
803.11
1 0 2 5 .2 3
27
87
17
7 8 7 .0 7
8 0 2 .2 4
88
16
24
1 0 2 4 .1 8
11
8 2 7 .2 0
89
90
9 /1 4
8 4 2 .1 9 /8 4 2 .2 7
17
8 2 6 .1 5
91
8 4 1 .2 3
92
16
629.11
93
14
7 5 4 .3 2
15
94
13
7 6 9 .3 3
95
22
7 5 3 .2 2
20
7 6 8 .1 6
96
990.11
28
Id e n tity
M
M
M
M +Fm oc
M
M+Val
M
M
M+Val
M
M
M+Val
M
M
M+Val
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M+Val
M
M
M
M
M -ACHC
M
M +Fm oc
M
M
M + F m oc
M
M
M
M
M -ACHC
M
M
M
M
M +Fm oc
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
213
Table 9. Characterization o f impurities in P-peptide library product mixtures by location.
A
B
C
D
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M. M+Val
M, M-APiG, M+Fmoc M, M-APiC M, M+Fmoc
M
M
M, M-APiC. M+Phe M. M-ACHC, M+Fmoc M, M-ACHC
M
M, M-ACHC M, M-Trp
M
M
M
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M
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M
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M, M+Val
M
M, M+Val
M
M, M+Val
M
M
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M
M
M
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M
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M
M, M+Fmoc
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8
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M, M+Va!
M, M+Val
M
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M, M-ACHC
10
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11
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M
M
M
M, M+Fmoc
M
M
M
M
M
M
M
M, M+Fmoc
SJ|0/\UJ
Figure 26. Analytical RP-HPLC chromatograms (UV absorbance at 220 nm) o f p-peptide library members [1-8].
* - p3-Trp-containing sequence with absorbance scaled to 0.3.
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
214
S 1 |0 A U J
Figure 27. Analytical RP-HPLC chromatograms (UV absorbance at 220 nm) of P-peptide library members [9-16].
* - Offset by +1 min.
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
215
CO
fo
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o
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Figure 28. Analytical RP-HPLC chromatograms (UV absorbance at 220 nm) o f P-peptide library members [1724], * - p3-Trp-containing sequence with absorbance scaled to 0.3.
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
216
c-i
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CN
CN
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sijOAUJ
Figure 29. Analytical RP-HPLC chromatograms (UV absorbance at 220 nm) of P-peptide library members [2536]. * - Offset by +1 min.
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
217
r, n
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Figure 30. Analytical RP-HPLC chromatograms (UV absorbance at 220 nm) o f p-peptide library members [3748].
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
218
CN
CO
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CN
00
CHi
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tnj uo
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Figure 31. Analytical RP-HPLC chromatograms (UV absorbance at 220 nm) o f P-peptide library members [4960], * - p3-Trp-containing sequence with absorbance scaled to 0.3.
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
219
CO
CN
00
c-i
CN
CD
CN
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Figure 32. Analytical RP-HPLC chromatograms (UV absorbance at 220 nm) of p-peptide library members [6172]. * - p3-Trp-containing sequence with absorbance scaled to 0.3.
R e p ro d u c e d with perm ission of the copyright owner. F urther reproduction prohibited without perm ission.
220
no
|
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(N *—
0 0 CO
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Figure 33. Analytical RP-HPLC chromatograms (UV absorbance at 220 nm) of P-peptide library members [7384], * - p3-Trp-containing sequence with absorbance scaled to 0.3; ** - Offset by +1 min; *** - Offset by +2 min.
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
221
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Figure 34. Analytical RP-HPLC chromatograms (UV absorbance at 220 nm) of P-peptide library members [8596]. * - p3-Trp-containing sequence with absorbance scaled to 0.3.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
222
4.4.9 Yield Calculation of P-Peptide 4-1
Yields from P-peptide syntheses were quantified by the method o f Yan et al.19 A sample
o f crude P-peptide 4-1 was purified by C4 -silica preparative reverse-phase HPLC (10 pm, 22 mm
x 250 mm, Vydac). The column was eluted with a gradient o f acetonitrile in water (25-55%, 30
min., 0.1% TFA in each) at a flow rate of 15 mL/min. After lyophilization, a small sample (1.0
mg) of purified P-peptide 4-1 was dissolved in 1.0 mL o f DMSO to make a 1.11 mM stock
solution. The stock solution was then diluted to make a series o f calibration solutions containing
a fixed concentration o f Fmoc-L-phenylalanine-OH (retention time = 40 min) as an external
standard to compensate for instrumental fluctuation and other systematic errors. These solutions
were analyzed by analytical RP-HPLC (C 4 -silica reverse-phase analytical column (5 pm, 4 mm x
250 mm, Vydac) eluted with a gradient of acetonitrile in water, 10 - 60%, 50 min, 0.1% TFA in
each, at a flow rate o f 1 mL/min) and monitored by UV absorbance at 220 nm. The peak area o f
p-peptide 4-1 was divided by the peak area o f the external standard to give the peak area ratio
(peak area ratio = (peak area)p_pcptide l / (peak area)cxternai standard)- A plot o f peak area ratio versus
concentration yielded a calibration curve. The curve was fit by linear regression ((peak area
ratio) = 2.6467 (concentration) + 0.0347) with a correlation coefficient (R2) o f 0.9996.
The
calibration curve was validated by preparing a solution of P-peptide 4-1 o f a known
concentration and analyzing by HPLC with LTV detection at 220 nm.
The determined
concentration from the calibration curve was accurate within 5%. Overall, the yields are slightly
lower than the reported purities but follow the same trend. The low yield o f the library synthesis
most likely is due to the small volume o f TFA used for the cleavage reaction. This has been
resolved by using the deep well filter plate mentioned previously.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Concentration
S
E
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00
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JKM VI 239
JKM VI 067
JKM VI 037
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1.751
2.254
1 .2 1 0
1.195
0.734
1.809
1.767
2.270
1.471
2.188
2.048
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0.814
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15301127
15500656
15819171
15278951
16076856
15791007
15987458
14894444
15916048
15855629
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I Error (%) |
j
4.547
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| Cone, (calc.) | Yield (% )|
44
|
26
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11226660
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34680131
23643983
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| 18028510
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15127390
15377421
15710418
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15730859
15680944
15814147
15837856
15513999
Peak Area Ratio |
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Reaction Conditions
I
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9761545
14464317
19229723
24058664
28416845
32884452
37372570
Peak Area
Fmoc-Phe
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20
25
30
35
40
45
Validation
48.5
43.5
38.5
33.5
28.5
23.5
18.5
13.5
8.5
3.5
3
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Vol. DMSO
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CL
0.333
0.444
0.555
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Table 10. Data for calibration curve and yield calculation o f P-peptide 4-1
CO CD
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R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
224
Figure 35. Calibration curve for yield calculation o f [3-peptide 4-1.
y = 2.6467x + 0.0347
R? = 0.9996
2.5
0.5
0.2
0.8
0.6
0.4
Concentration (mM)
Figure 36. Yields of |3-peptide 4-1 under different synthetic conditions.
Manual
Monomode Microwave
Multimode Microwave
Reaction Conditions
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
225
4.4.10 Step-by-Step M icrowave-Assisted P-Peptide Synthesis Protocol
For 25 pmol peptide on NovaSyn TGR resin:
1. Weigh 25 pmol of resin (calculated from resin loading, i.e. 100 mg is 0.25 mrhol/g) into a
4.0 mL Alltech SPE tube.
2. Add 2 mL o f DMF and let swell for 5 min.
3. Transfer tube to wash station and wash 5 x DMF (attach aspirator to wash station, close
stopcock to fill tube with solvent, open to drain.)
4. Remove tube from wash station. Fix bottom cap on tube. Add a flea-sized ( 7 x 2 mm)
stir bar.
5. Weigh amino acid for coupling (3 equiv., 75 pmol) into a 1-dram vial. Add 150 pL o f
0.5 M solution of HBTU in DMF. Add 1.1 mL o f DMF and vortex until the amino acid
is dissolved.
6. Add 150 pL o f 0.5 M solution o f HOBt in DMF. Add 150 pL o f 1.0 M solution o f DIEA
in DMF. Vortex and allow to stand for 60 sec.
7. Add activated amino acid solution to resin.
8. Place vessel in cavity, insert fiber optic temperature probe, and microwave
(“COUPLE70” method: 600W max, 70°C, ramp 2:00 min., hold 4:00 min.)
9. Remove fiber optic probe and wipe clean. Remove vessel from microwave. Remove
bottom cap and quickly transfer tube to wash station.
10. Wash 5 x DMF.
11. Remove tube from wash station. Fix bottom cap on tube.
12. Add 1.5 mL deprotection solution (20% piperidine in DMF (v/v)).
13. Place vessel in cavity, insert fiber optic temperature probe, and microwave
(“DEPRO80” method: 600W max, 80°C, ramp 2:00 min., hold 2:00 min.)
14. Remove fiber optic probe and wipe clean. Remove vessel from microwave. Remove
bottom cap and quickly transfer tube to wash station.
15. Wash 5 x DMF. Remove tube from wash station. Fix bottom cap on tube.
16. Repeat steps 5 through 15 as necessary.
17. Wash the resin (5 x DMF and 5 x DCM).
18. Cleave the P-peptide by adding (3 mL, 90:5:5 TFA:DCM:triethylsilane:H20, 2 h., RT,
with rocking).
19. Drain the cleavage solution and concentrate under a stream o f N 2 .
20. Dissolve the crude P-peptide in DMSO (2.5 mL) using the vortexer and/or sonication.
21. Analyze by C4 -silica reverse-phase analytical FIPLC (5 pm, 4 mm x 250 mm, Vydac),
eluteing the column with a gradient o f acetonitrile in water (10-60%, 50 min., 0.1% TFA
in each) at a flow rate of 1 mL/min.
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
226
4.4.11 Screening D ata for P-Peptide L ib rary
The P-peptide library was assayed against a panel o f enzymatic targets (grefc,
cytochrome P450 1A2, prx2, sOGT, and thal) at the NIH Chemical Genomics Center by Dr.
James Inglese and Dr. Anton Simeonov. The full details o f the assays have not been released,
but beta thalassemia (thal) is a splicing cell assay, prx 2 is a coupled enzyme assay that uses
Schistoma mansoni peroxyredoxin and thioredoxin-glutathione reductase, sOGT is O-linked
glycosyltransferase, and grefc is another pathway cell assay. The columns o f most interest in the
tables below are labeled “qAC50.” Qualified AC50 is the concentration at 50% activity, similar
to IC 5 0 values. The units are in molar concentration. These values are derived from a curvefitting procedure, and numbers greater than approximately 1 x 10‘4 M arc greatly extrapolated
and should be considered inactive. The total change in activity is reported as “dS.” “R ”
measures the goodness o f the fit. “Hill coefficient” is the slope of the Hill equation used to fit
the inhibition data, assuming a single binding-site isotherm. “ActMaxConc” is the activity
measured at the highest compound concentration in the prx 2 assay, giving a single concentration
activity slice. The prx2 “ratio lntercept” is an assay-specific value used to look for fluoresecent
artifacts. “Act_15 pM ” is the value o f activity at that concentration in the sOGT assay. The
“maxact” and “minacf ’ columns show the bracket o f activity in the thal assay, regardless o f
compound concentration. The “object intensity” and “object no” columns pertain to raw data
values for the thal assay. The “cho counter flag” is a thal assay-specific entry from a manual
re-test to see if compounds were providing fluorescent signal. A value o f “ 1” indicates that the
compound is potentially an artifact. The library will continue to be tested over the next couple of
years as assays are added to the panel.
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
227
T able 11. Assay data for compounds 1-48 against grefc and cytochrome P450 1A2.
g re fc
NIH S u b s ta n c e ID s u p p lie r c o m p o u n d id
NCGC00074655-01
uw isc-betapep-01
NCGC00074656-01
uw isc-betapep-02
NCGC00074657-01
uw isc-betapep-03
NCGC00074658-01
uw isc-betapep-04
NCGC00074659-01
uw isc-betapep-05
NCGC00074660-01
uw isc-beta pep-06
NCGC00074661-01
uw isc-betapep-07
NCGC00074662-01
uw isc-betapep-08
NCGC00074663-01
uw isc-beta pep-09
NCGC00074664-01
uw isc-betapep-10
NCGC00074665-01
uw isc-betapep-11
NCGC00074747-01
uw isc-betapep-12
NCGC00074666-01
uw isc-betapep-13
NCGC00074667-01
uw isc-betapep-14
NCGC00074668-01
uw isc-betapep-15
NCGC00074669-01
uw isc-betapep-16
NCGC00074670-01
uw isc-betapep-17
NCGC00074671-01
uw isc-betapep-18
NCGC00074672-01
uw isc-betapep-19
NCGC00074673-01
uw isc-betapep-20
NCGC00074674-01
uw isc-betapep-21
NCGC00074675-01
uw isc-betapep-22
NCGC00074676-01
uw isc-betapep-23
NCGC00074677-01
uw isc-betapep-24
NCGC00074678-01
uw isc-beta pep-25
NCGC00074679-01
uw isc-betapep-26
NCGC00074680-01
uw isc-betapep-27
NCGC00074681 -01
uw isc-betapep-28
NCGC00074682-01
uw isc-betapep-29
NCGC00074683-01
uw isc-betapep-30
NCGC00074684-01
uw isc-betapep-31
NCGC00074685-01
uw isc-betapep-32
NCGC00074686-01
uw isc-betapep-33
NCGC00074687-01
uw isc-betapep-34
NCGC00074688-01
uw isc-betapep-35
NCGC00074689-01
uw isc-beta pep-36
NCGC00074690-01
uw isc-betapep-37
NCGC00074748-01
uw isc-betapep-38
NCGC00074749-01
uw isc-betapep-39
NCGC00074750-01
uw isc-betapep-40
N CGC00074691 -01
uw isc-betapep-41
NCGC00074692-01
uw isc-betapep-42
NCGC00074693-01
uw isc-betapep-43
NCGC00074694-01
uw isc-betapep-44
NCGC00074695-01
uw isc-betapep-45
NCGC00074696-01
uw isc-betapep-46
NCGC00074697-01
uw isc-betapep-47
NCGC00074698-01
uw isc-betapep-48
R2
dS
hill C o efficien t qA C 50(M )
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00206
0.00461
0.00461
0.00461
0.00461
0.00461
C y to c h ro m e P450 1A2
R2
0.7973
0.885
0.8043
0.5828 .
0.8512
0.8403
hill C o efficient
0.9564
1.002
0.424
0.9988
0.8053
0.7897
0.6317
0.8708
0.8737
1.033
0.7573
0.9617
0.6942
0.8415
0.554
0.738
0.6246
1.2
0.5815
0.3395
1.618
1.821
0.6378
0.6081
0.7377
1.749
0.6207
1.248
0.732
1.374
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
qA C50 (M)
0.000211
0.000241
0.002874
0.00258
0.001076
0.002874
0.002874
0.000434
0.002267
0.000859
0.002874
0.002874
0.002874
0.000317
0.001909
0.002874
5.42E-05
0.000106
0.002874
0.002874
0.002874
1.43E-05
0.002874
0.002874
4.66E-05
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.000256
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
228
Table 12. Assay data for compounds 49-96 against grefc and cytochrome P450 1A2.
C ytochrom e P450 1A2
grefc
R2
NIH S u b sta n c e ID supplier c o m p o u n d id
uw isc-betapep-49
NCGC00074699-01
uw isc-betapep-50
NCGC00074700-01
NCGC00074701 -01
uw isc-betapep-51
NCGC00074702-01
uw isc-beta pep-52
NCGC00074703-01
uw isc-betapep-53
uw isc-betapep-54
NCGC00074704-01
NCGC00074705-01
uw isc-betapep-55
uw isc-betapep-56
NCGC00074706-01
uw isc-betapep-57
NCGC00074707-01
uw isc-betapep-58
NCGC00074708-01
NCGC00074709-01
uw isc-betapep-59
N C G C 00074710-01
uw isc-betapep-60
N C G C 00074711-01
uw isc-betapep-61
N C G C 00074712-01
uw isc-betapep-62
uw isc-betapep-63
NCGC00074713-01
uw isc-betapep-64
NCGC00074714-01
uw isc-betapep-65
NCGC00074715-01
uw isc-betapep-66
NCGC00074716-01
NCGC00074717-01
uw isc-beta pep-67
NCGC00074718-01
uw isc-beta pep-68
NCGC00074719-01
uw isc-betapep-69
NCGC00074720-01
uw isc-betapep-70
uw isc-beta pep-71
NCGC00074721-01
uw isc-betapep-72
NCGC00074722-01
NCGC00074723-01
uw isc-betapep-73
NCGC00074724-01
uw isc-betapep-74
uw isc-betapep-75
NCGC00074725-01
NCGC00074726-01
uw isc-betapep-76
uw isc-betapep-77
NCGC00074727-01
NCGC00074728-01
uw isc-betapep-78
uw isc-betapep-79
NCGC00074729-01
NCGC00074730-01
uw isc-betapep-80
uw isc-betapep-81
NCGC00074731-01
uw isc-betapep-82
NCGC00074732-01
NCGC00074733-01
uw isc-beta pep-83
uw isc-betapep-84
NCGC00074734-01
NCGC00074735-01
uw isc-betapep-85
NCGC00074736-01
uw isc-betapep-86
0.8989
uw isc-betapep-87
NCGC00074737-01
uw isc-betapep-88
NCGC00074738-01
uw isc-betapep-89
NCGC00074739-01
NCGC00074740-01
uw isc-betapep-90
uw isc-betapep-91
NCGC00074741-01
NCGC00074742-01
uw isc-betapep-92
uw isc-betapep-93
NCGC00074743-01
uw isc-betapep-94
NCGC00074744-01
NCGC00074745-01
uw isc-betapep-95
NCGC00074746-01
uw isc-betapep-96
dS
hill C oefficient qAC50 (M)
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00206
0.00461
0.00461
0.00461
0.00461
0.00461
0.00206
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
0.00461
2.544
0.000104
339.2
0.00461
0.00461
0.00206
0.00461
0.00461
0.00461
0.00461
0.00461
0.00206
0.00461
R2
0.5612
0.5741
0.5976
hill C oefficient
11.74
16.6
0.417
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
qAC50(M )
1.37E-05
2.95E-05
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.001285
0.001285
0.002874
0.002874
0.001285
0.002874
0.001285
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.001285
0.002874
0.002874
0.002874
0.001285
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.002874
0.001285
0.002874
0.002874
0.002874
0.002874
0.002874
229
Table 13. Assay data for compounds 1-48 against prx2 and sOGT.
sOGT
prx2
NIH Substance ID supplier_compound_id ActMaxConc
NC G C 00074655-01
NC G C 00074656-01
uwisc-beta pep-01
uwisc-betapep-02
N C G C 00074657-01
N C G C 00074658-01
uwisc-betapep-03
uwisc-betapep-04
NC G C 00074659-01
uwisc-betapep-05
uwisc-betapep-06
NC G C 00074660-01
N C G C 00074661-01
N C G C 00074662-01
N C G C 00074663-01
N C G C 00074664-01
uwisc-betapep-07
uwisc-betapep-08
uwisc-betapep-09
uwisc-betapep-10
R2
-7.41
qAC50 (M) ratio intercept Act_15 pM qAC50 (M)
0.0 05 7 1 4
0.0 05 7 1 4
-2.67
-1.53
0.01722
0.01856
0.0 20 7 5
0.0 05 7 1 4
0.0 05 7 1 4
0.0 05 7 1 4
0.0 21 2 4
0.0 18 0 2
0.0 18 5 6
1.725
0.0704
0 .0 07 6 9 2
0 .0 07 6 9 2
-0.2211
-0.8 1 1 7
0 .0 07 6 9 2
0 .0 07 6 9 2
0.02044
0 .0 17 7
-0.5 5 3 5
0.09448
0.0 14 5 4
0.0 16 0 6
0.0 28 8 8
0.0 21 4 2
0.0161
0.0 18 0 9
1.693
-1.42
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
-2.53
-0.31
-4.6
-11.81
-3.2
0.0 02 5 5 6
0.0 05 7 1 4
0 .0 05 7 1 4
0 .0 05 7 1 4
0.0 05 7 1 4
0.005714
0.0 05 7 1 4
0.005714
0.0 05 7 1 4
0.8861
-0.6692
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
-16.18
0 .0 05 7 1 4
0.0 11 6
0.0 12 8 7
0.1354
0.3544
-0.5 1 3 3
-0.7 8 5 6
0 .0 07 6 9 2
0 .0 07 6 9 2
0.0 16 3
0.0 16 0 3
-0.0 1 4 7 4
0.07707
0 .0 07 6 9 2
0 .0 0 7 6 9 2
-0.2 3 4
0.3697
0 .0 07 6 9 2
0 .0 0 7 6 9 2
0.6537
1.181
0 .0 07 6 9 2
0.0 07 6 9 2
0.0 07 6 9 2
0 .0 0 7 6 9 2
-6.62
-6.99
-9.06
3.77
-13.04
-8.4 7
uwisc-betapep-11
uwisc-betapep-12
uwisc-betapep-13
uwisc-betapep-14
N C G C 00074670-01
N C G C 0 0 0 7 4 6 7 1-01
uwisc-betapep-17
uwisc-betapep-18
uwisc-betapep-19
-8.38
-3.4 3
-4.8 8
0.0 05 7 1 4
0.0 05 7 1 4
0 .0 05 7 1 4
0.01582
uwisc-betapep-20
uwisc-betapep-21
-7.2 5
-7.6 2
uwisc-betapep-22
uwisc-betapep-23
uwisc-betapep-24
uwisc-betapep-25
uwisc-betapep-26
uwisc-betapep-27
uwisc-betapep-28
uwisc-betapep-29
-5.1
-1.1 4
-0.05
5.04
-11 .3 6
3.62
-7.2 8
-13 .3 5
uwisc-betapep-30
uwisc-betapep-31
uwisc-betapep-32
-5.3 9
8.16
1.52
0 .0 05 7 1 4
0.0 05 7 1 4
0.005714
0.0 05 7 1 4
0.005714
0.0 05 7 1 4
0.0 05 7 1 4
0.0 05 7 1 4
0.0 05 7 1 4
0 .0 05 7 1 4
0 .0 05 7 1 4
NC G C 00074686-01
NC G C 00074687-01
uwisc-betapep-33
uwisc-betapep-34
-3.2 3
-5.1 8
NC G C 00074688-01
NC G C 00074689-01
NC G C 00074690-01
uwisc-betapep-35
uwisc-betapep-36
uwisc-betapep-37
-3.5
-4.9 8
-7.1 5
0.0 17 2 8
0.0 13 3 8
0.0 13 6 9
0.0 12 6 7
0.0 22 4 5
0.0 17 1 2
0.0 16 5
0.02171
0.0 21 8 4
0.0 18 0 5
0 .0 27 7 8
0.0 30 5 5
0.0 28 9 9
0.0 17 5 8
0.02041
0 .0 19 6 5
0.0 18 6
N C G C 00074748-01
N C G C 00074749-01
N C G C 00074750-01
uwisc-betapep-38
uwisc-betapep-39
uwisc-betapep-40
uwisc-betapep-41
uwisc-betapep-42
uwisc- beta pep-43
uwisc-betapep-44
uwisc-betapep-45
uwisc-betapep-46
-5 .6 4
-7.11
-6.7
uwisc-betapep-47
uwisc-betapep-48
-6.6 3
-10 .7 4
N C G C 00074672-01
N C G C 00074673-01
N C G C 00074674-01
N C G C 00074675-01
N C G C 00074676-01
N C G C 00074677-01
N C G C 00074678-01
N C G C 00074679-01
N C G C 00074680-01
N C G C 00074681 -01
N C G C 00074682-01
N C G C 00074683-01
N C G C 00074684-01
N C G C 00074685-01
NC G C 00074691-01
NC G C 00074692-01
NC G C 00074693-01
N C G C 00074694-01
N C G C 00074695-01
N C G C 00074696-01
N C G C 00074697-01
N C G C 00074698-01
0.0 07 6 9 2
0.0 07 6 9 2
0.005714
-3.79
N C G C 00074665-01
N C G C 00074747-01
N C G C 00074666-01
N C G C 00074667-01
N C G C 00074668-01
N C G C 00074669-01
uwisc-betapep-15
uwisc-betapep-16
-1.5 3 5
0.8782
-9 .9 5
-7 .3 5
-4 .7 4
6.19
-6.91
-7 .2 8
0.7791
0.871
0.0 05 7 1 4
0.0 05 7 1 4
0 .0 05 7 1 4
0.0 05 7 1 4
0.0 05 7 1 4
0 .0 05 7 1 4
0.0 05 7 1 4
0 .0 05 7 1 4
0 .0 05 7 1 4
0.0 05 7 1 4
0.8 42 3
0 .9 5 8 6
0 .9 4 6 2
0.0 02 5 5 6
0.0 05 7 1 4
0.0 05 7 1 4
0.0 05 7 1 4
0.0 02 5 5 6
0.0 05 7 1 4
0.0 05 7 1 4
0.0 05 7 1 4
0.012
0.0 15 6 3
0.01863
0.0 14 6 5
0.0 18 4 8
0.0 20 5 9
0.0 34 7 3
0.0 51 1 6
0.01421
0.0 19 4 5
0.01871
0.01896
-0.3163
-0.4782
-0.6863
0.9721
0.5585
-1.0 6 5
0.5608
1.992
-1.0 7 7
-1.523
2.782
0.3542
-2.6 2 4
0.1106
-1.4 7 9
-0.2 1 8 6
0.8285
-2.2 2 6
1.654
1.823
0.8169
-1.075
1.204
-1.261
1.682
-0.02143
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
0.0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 0 7 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0.0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0.0 07 6 9 2
0.0 07 6 9 2
0 .0 07 6 9 2
0.0 07 6 9 2
0 .0 07 6 9 2
230
Table 14. Assay data for compounds 49-96 against prx2 and sOGT.
sOGT
qAC50 (M) ratio intercept Act_15 pM qAC50 (M)
prx2
NIH Substance ID supplier_compound_id ActMaxConc
N C G C 00074699-01
N C G C 00074700-01
N C G C 00074701-01
N C G C 00074702-01
N C G C 00074703-01
N C G C 0 0 0 7 47 04-01
N C G C 00074705-01
N C G C 00074706-01
N C G C 00074707-01
N C G C 00074708-01
N C G C 00074709-01
N C G C 0 0 0 7 4 7 10-01
N C G C 0 0 0 7 4 7 11-01
N C G C 0 0 0 7 4 7 12-01
N C G C 00074713-01
N C G C 0 0 0 7 4 7 14-01
N C G C 00074715-01
N C G C 00074716-01
N C G C 00074717-01
N C G C 0 0 0 7 4 7 18-01
N C G C 00074719-01
N C G C 00074720-01
N C G C 00074721-01
N C G C 00074722-01
N C G C 00074723-01
N C G C 00074724-01
N C G C 00074725-01
N C G C 00074726-01
N C G C 00074727-01
N C G C 00074728-01
N C G C 00074729-01
N C G C 00074730-01
N C G C 00074731-01
N C G C 00074732-01
N C G C 00074733-01
N C G C 00074734-01
N C G C 00074735-01
N C G C 00074736-01
N C G C 00074737-01
N C G C 00074738-01
N C G C 00074739-01
N C G C 00074740-01
N C G C 00074741 -01
N C G C 00074742-01
N C G C 00074743-01
N C G C 00074744-01
N C G C 00074745-01
N C G C 00074746-01
uwisc-betapep-49
uwisc-betapep-50
uwisc-betapep-51
uwisc-betapep-52
uwisc-betapep-53
uwisc-betapep-54
uwisc-betapep-55
uwisc-betapep-56
uwisc-betapep-57
uwisc-betapep-58
uwisc-betapep-59
uwisc-betapep-60
uwisc-betapep-61
uwisc-betapep-62
uwisc-betapep-63
uwisc-betapep-64
uwisc-betapep-65
uwisc-betapep-66
uwisc-betapep-67
uwisc-betapep-68
uwisc-betapep-69
uwisc-betapep-70
uwisc-betapep-71
uwisc-betapep-72
uwisc-betapep-73
uwisc-betapep-74
uwisc-betapep-75
uwisc-betapep-76
uwisc-betapep-77
uwisc-betapep-78
uwisc-betapep-79
uwisc-betapep-80
uwisc-betapep-81
uwisc-betapep-82
uwisc-betapep-83
uwisc-betapep-84
uwisc-betapep-85
uwisc-betapep-86
uwisc-betapep-87
uwisc-betapep-88
uwisc-betapep-89
uwisc-betapep-90
uwisc-betapep-91
uwisc-betapep-92
uwisc-betapep-93
uwisc-betapep-94
uwisc-betapep-95
uwisc-betapep-96
-7.41
-13 .4 9
-0.8 3
-7.1 5
-12 .8 4
-8.5 3
-5.79
-8.33
-10 .7 9
-3.9 7
-2.5 7
-12.1
-8.58
-9.5
-1.7 6
-8.47
-8.7
-11 .8 5
-9
-12 .0 2
-6.65
-10.02
-4.96
-8.18
-10 .5
-0.8 5
-6.45
-6.0 5
-4.87
-4.14
-12.33
-6.29
-14.31
-7.06
-4.8 2
-4.0 2
-6.62
2.85
-2.2 4
1.19
1.18
-2.4 5
-13.42
-5.73
-8.74
-4.9
-7.3 9
4.4 8
R2
0 .0 05 7 1 4
0.0 05 7 1 4
0.0 05 7 1 4
0.0 05 7 1 4
0 .0 05 7 1 4
0 .0 05 7 1 4
0.0 05 7 1 4
0 .0 05 7 1 4
0 .0 05 7 1 4
0 .0 05 7 1 4
0.01716
0 .4 45
0.01527
0.01745
0.02219
0.01399
0.01886
0.01743
0.01525
0.01819
0.0159
0.7329
-0.8 7 6
2 .2 79
0 .0 05 7 1 4
0 .0 05 7 1 4
0.02121
0 .0 05 7 1 4
0 .0 05 7 1 4
0.0 05 7 1 4
0.0 05 7 1 4
0.0 05 7 1 4
0.0 05 7 1 4
0.0 05 7 1 4
0 .0 05 7 1 4
0 .0 05 7 1 4
0 .0 05 7 1 4
0.0 05 7 1 4
0.0 05 7 1 4
0.0 05 7 1 4
0.0 05 7 1 4
0 .0 05 7 1 4
0 .0 05 7 1 4
0 .0 05 7 1 4
0 .0 05 7 1 4
0 .0 05 7 1 4
0 .0 05 7 1 4
0 .0 05 7 1 4
0 .0 05 7 1 4
0.0 05 7 1 4
0.0 05 7 1 4
0.0 05 7 1 4
0.0 05 7 1 4
0.0 05 7 1 4
0 .0 05 7 1 4
0 .0 05 7 1 4
0 .0 05 7 1 4
0 .0 05 7 1 4
0 .0 05 7 1 4
0 .0 05 7 1 4
0.0 05 7 1 4
0.0 02 5 5 6
0.0 05 7 1 4
0.01959
0 .01402
0.02071
0.02451
0.02138
0.01599
0.01356
0.01339
0.01748
0.01585
0.01441
0.02342
0.0204
0.01654
0.0 17 9 6
0.02298
0.02047
0.01493
0.01382
-0.7 9 3 5
1.655
-0.6 7 4
-1.1 0 7
1.287
1.659
1.191
-0 .6 7 3 8
-1.3 9 5
2.591
-2.0 0 7
0.7 20 2
0.5 77 8
1.302
-0.8 2 2 9
0.2456
1.853
-0.1 1 4 6
-0.7 7 8 6
0.173
0.3027
-0.3538
-2.5 4 5
0.1621
-0.1984
1.881
0.01442
0.01984
0.01787
0.02066
0.01609
0.01996
0.01741
0.01483
0.01997
0.02027
-0.6 0 8 6
-0.404
0.01573
0.0158
0.01422
0.2046
1.578
-1.1 1 7
0.01728
0.272
1.13
0.01619
0.02195
0.01729
0.01968
1.001
0.4341
0 .2 26 9
0.7811
-0.0 8 5 8
-2.181
-0.4 8 7 2
-1.6 9
1.07
-0.02952
-0 .3 7 3 5
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 0 7 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 0 7 6 9 2
0 .0 07 6 9 2
0 .0 0 7 6 9 2
0 .0 0 7 6 9 2
0 .0 0 7 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 0 7 6 9 2
0 .0 0 7 6 9 2
0.0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 0 7 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0 .0 07 6 9 2
0.0 07 6 9 2
0 .0 07 6 9 2
2 31
Table 15. Assay data for compounds 1-48 against thal.
thal
R2
NIH S u b s t a n c e ID s u p p lie r c o m p o u n d id
N C G C 00074655-01
u w isc-b eta p e p -0 1
N C G C 00074656-01
u w isc-b eta p e p -0 2
N C G C 00074657-01
u w is c -b e ta p e p -0 3
N C G C 00074658-01
u w isc-b eta p e p -0 4
u w is c -b e ta p e p -0 5
N C G C 0 0 0 7 4 6 5 9 -0 1
N C G C 00074660-01
u w is c -b e ta p e p -0 6
u w is c -b e ta p e p -0 7
N C G C 00074661 -01
N C G C 00074662-01
u w isc-b eta p e p -0 8
N C G C 00074663-01
u w isc-b eta p e p -0 9
N C G C 00074664-01
u w is c -b e ta p e p -1 0
0 .7 0 4 6
N C G C 00074665-01
u w is c -b e ta p e p -1 1
N C G C 00074747-01
u w is c -b e ta p e p -1 2
N C G C 00074666-01
u w is c -b e ta p e p -1 3
N C G C 0 0 0 7 4 6 6 7 -0 1
u w is c -b e ta p e p -1 4
0 .5 5 5 5
N C G C 00074668-01
u w is c -b e ta p e p -1 5
N C G C 0 0 0 7 4 6 6 9 -0 1
u w is c -b e ta p e p -1 6
0 .3 6 8 4
N C G C 00074670-01
u w is c -b e ta p e p -1 7
N C G C 0 0 0 7 4 6 7 1-01
u w is c -b e ta p e p -1 8
0 .7 5 0 9
N C G C 00074672-01
u w is c -b e ta p e p -1 9
N C G C 0 0 0 7 4 6 7 3 -0 1
u w is c -b e ta p e p -2 0
0 .6 8 8 6
N C G C 00074674-01
u w isc-b eta p e p -2 1
N C G C 00074675-01
u w is c -b e ta p e p -2 2
N C G C 00074676-01
u w is c -b e ta p e p -2 3
N C G C 00074677-01
u w is c -b e ta p e p -2 4
N C G C 00074678-01
u w is c -b e ta p e p -2 5
0 .6 1 5 9
N C G C 00074679-01
u w is c -b e ta p e p -2 6
N C G C 00074680-01
u w is c -b e ta p e p -2 7
N C G C 00074681 -01
u w isc-b eta p e p -2 8
N C G C 00074682-01
u w is c -b e ta p e p -2 9
N C G C 00074683-01
u w is c -b e ta p e p -3 0
N C G C 00074684-01
u w isc-b eta p e p -3 1
N C G C 00074685-01
u w is c -b e ta p e p -3 2
N C G C 0 0 0 7 4 6 8 6 -0 1
u w isc-b eta p e p -3 3
N C G C 0 0 0 7 4 6 8 7 -0 1
u w isc-b eta p e p -3 4
N C G C 0 0 0 7 4 6 8 8 -0 1
u w is c -b e ta p e p -3 5
N C G C 0 0 0 7 4 6 8 9 -0 1
u w is c -b e ta p e p -3 6
N C G C 0 0 0 7 4 6 9 0 -0 1
u w is c -b e ta p e p -3 7
N C G C 0 0 0 7 4 7 4 8 -0 1
u w isc-b eta p e p -3 8
N C G C 0 0 0 7 4 7 4 9 -0 1
u w is c -b e ta p e p -3 9
N C G C 0 0 0 7 4 7 5 0 -0 1
u w is c -b e ta p e p -4 0
u w isc-b eta p e p -4 1
N C G C 0 0 0 7 4 6 9 1 -0 1
N C G C 0 0 0 7 4 6 9 2 -0 1
u w isc -b e ta p e p -4 2
N C G C 0 0 0 7 4 6 9 3 -0 1
u w is c -b e ta p e p -4 3
N C G C 0 0 0 7 4 6 9 4 -0 1
u w is c -b e ta p e p -4 4
0 .7 2 5 7
N C G C 00074695-01
u w isc-b eta p e p -4 5
N C G C 0 0 0 7 4 6 9 6 -0 1
u w is c -b e ta p e p -4 6
N C G C 0 0 0 7 4 6 9 7 -0 1
u w is c -b e ta p e p -4 7
N C G C 00074698-01
u w is c -b e ta p e p -4 8
c h o c o u n te r flag d S
10
1
-1
6
1
-2
3
1
1
1
1
1
0
1
m axact
8 .4 8 8
9 .5 7 7
7 .6 6 5
6.211
9 .7 8 2
10.3
1.378
4 .4 6 8
2 .9 7 4
10.19
8 .6 6 2
3.4 6 8
2 .4 4 6
3.8 6 3
5.3 8 7
9 .1 4 6
0 .8 2 4 5
1.683
6 .6 6 2
7.6 3 4
2 .7 2 2
0 .3 9 6 2
11.93
13.95
10.84
7 .8 5 6
4 .1 0 8
3 .9 6 4
6.421
14.92
7 .9 0 2
1.35
8 .8 0 4
6.451
5.621
3.0 9 9
14.82
12.45
2.79
6 .9 3 4
3.44
3.0 9 5
5 .9 5 8
2 .9 4 2
7 .4 4 4
7 .3 2 5
6 .4 5 2
2 .9 4 3
m in act
-2.289
-8.004
-6.599
-4.39
-10.7
-4.337
-7.483
-7.989
-9.05
-8.331
-1.956
-6.73
-6.29
-14.51
-6.436
-8.424
-5.196
-17.38
-5.47
-9.348
-8 .8 2 7
-9 .1 5 5
-9 .4 8 5
-4.303
-9.984
-2.895
-3.698
-9.735
-3.951
-2.923
-3.695
-11.45
-3.578
-5.729
-6.88
-5.693
2 .4 3 8
-1.841
-7.723
-4.778
-12.54
-5.513
-7.544
-12.44
-8.269
-4.131
-7.571
-8 .3 2 5
o b je c t in te n sity o b j e c t n o qA C 50 (M)
4240
47
5057
47
4024
54
5232
40
4954
36
3855
31
3194
39
4235
39
4495
38
4282
40
-8 .4
3552
47
5981
48
4606
38
5195
42
-9.2
57
4909
54
4906
-5 .5
3872
33
5355
42
-9.1
4806
37
5151
38
-8 .9
3640
38
4282
38
6563
43
4375
33
3544
29
-9 .2
5061
40
3176
43
4245
48
5575
42
4712
31
2033
38
3799
39
5952
45
3160
28
2927
39
3771
39
4364
25
4057
27
3969
41
5644
37
27
3533
3872
24
3950
45
37
2916
-9 .2
5794
31
3910
32
3804
39
3222
42
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
232
Table 16. Assay data for compounds 49-96 against thal.
thal
R2
c h o _ c o u n te r _ fla g d S m a x a c t
NIH S u b s t a n c e ID s u p p lie r c o m p o u n d id
1
4.941
N C G C 00074699-01
u w is c -b e ta p e p -4 9
u w is c -b e ta p e p -5 0
-0.8 3 3 2
N C G C 0 0 0 7 4 7 0 0 -0 1
u w isc-b eta p e p -5 1
6 .1 1 7
N C G C 00074701-01
N C G C 00074702-01
u w is c -b e ta p e p -5 2
12.05
N C G C 0 0 0 7 4 7 0 3 -0 1
u w is c -b e ta p e p -5 3
10.58
5 .0 0 9
N C G C 0 0 0 7 4 7 0 4 -0 1
u w is c -b e ta p e p -5 4
u w is c -b e ta p e p -5 5
7 .5 2 5
N C G C 0 0 0 7 4 7 0 5 -0 1
N C G C 0 0 0 7 4 7 0 6 -0 1
u w is c -b e ta p e p -5 6
2.081
u w is c -b e ta p e p -5 7
10.16
N C G C 0 0 0 7 4 7 0 7 -0 1
5 .2 0 7
N C G C 0 0 0 7 4 7 0 8 -0 1
u w is c -b e ta p e p -5 8
0 .6 5 1 7
6 .2 7 4
N C G C 0 0 0 7 4 7 0 9 -0 1
u w isc-b eta p e p -5 9
9
N C G C 0 0 0 7 4 7 10-01
1.356
u w is c -b e ta p e p -6 0
N C G C 0 0 0 7 4 7 1 1-01
u w isc-b eta p e p -6 1
21 .0 8
u w is c -b e ta p e p -6 2
9.671
N C G C 0 0 0 7 4 7 12-01
3 .2 1 7
N C G C 0 0 0 7 4 7 13-01
u w is c -b e ta p e p -6 3
u w isc-b eta p e p -6 4
15.05
N C G C 0 0 0 7 4 7 1 4 -0 1
N C G C 0 0 0 7 4 7 15-01
u w is c -b e ta p e p -6 5
7 .2 0 5
N C G C 0 0 0 7 4 7 16-01
u w is c -b e ta p e p -6 6
0 .5 2 0 5
8
7 .3 3 9
N C G C 0 0 0 7 4 7 17-01
u w is c -b e ta p e p -6 7
7 .5 6 6
N C G C 0 0 0 7 4 7 18-01
u w is c -b e ta p e p -6 8
3 .4 6 9
N C G C 0 0 0 7 4 7 19-01
u w is c -b e ta p e p -6 9
5 .4 0 6
N C G C 00 0 7 4 7 2 0 -0 1
u w is c -b e ta p e p -7 0
14.08
u w isc-b eta p e p -7 1
10.63
N C G C 00074721 -01
14
3 1 .8 4
u w is c -b e ta p e p -7 2
0 .6 7 5 4
N C G C 00 0 7 4 7 2 2 -0 1
N C G C 00 0 7 4 7 2 3 -0 1
u w is c -b e ta p e p -7 3
0 .4 2 8 7
1
9
7 .8 6 9
N C G C 00 0 7 4 7 2 4 -0 1
u w is c -b e ta p e p -7 4
0 .7 8 4 9
19
11.5
14
N C G C 00 0 7 4 7 2 5 -0 1
u w is c -b e ta p e p -7 5
0 .4 9 6 3
5 .8 7 3
0 .6 9 5 4
N C G C 00 0 7 4 7 2 6 -0 1
u w is c -b e ta p e p -7 6
6
7 .4 1 9
u w is c -b e ta p e p -7 7
2 .9 6 4
N C G C 00 0 7 4 7 2 7 -0 1
N C G C 00 0 7 4 7 2 8 -0 1
u w isc-b eta p e p -7 8
0 .6 7 4 8
15
8 .4 5 8
u w isc -b e ta p e p -7 9
0.6151
12
7 .3 6 9
N C G C 0 0 0 7 4 7 2 9 -0 1
N C G C 0 0 0 7 4 7 3 0 -0 1
u w is c -b e ta p e p -8 0
0 .8 5 1 6
11
9.6 5 8
N C G C 00 0 7 4 7 3 1 -0 1
u w isc-b eta p e p -8 1
0 .5 6 2 5
8
0.11
N C G C 00 0 7 4 7 3 2 -0 1
u w is c -b e ta p e p -8 2
0 .5 2 3 3
24
14 .0 3
N C G C 0 0 0 7 4 7 3 3 -0 1
u w is c -b e ta p e p -8 3
0 .4 6 2 2
9
9 .7 5 5
u w is c -b e ta p e p -8 4
24
13.72
N C G C 00 0 7 4 7 3 4 -0 1
0 .7 6 1 9
N C G C 00 0 7 4 7 3 5 -0 1
u w is c -b e ta p e p -85
0 .4 2 8 9
9
0 .8 7 1 5
N C G C 00 0 7 4 7 3 6 -0 1
u w is c -b e ta p e p -8 6
0 .6 5 7 6
11
10.85
N C G C 0 0 0 7 4 7 3 7 -0 1
u w is c -b e ta p e p -8 7
0 .8 0 7 7
4
2 .5 2 3
N C G C 00 0 7 4 7 3 8 -0 1
u w isc -b e ta p e p -8 8
2 .3 5 3
N C G C 00 0 7 4 7 3 9 -0 1
u w is c -b e ta p e p -8 9
0 .6 4 5 6
6
8.1 7 2
N C G C 00 0 7 4 7 4 0 -0 1
u w isc-b eta p e p -9 0
0 .5 5 1 9
6
9 .0 0 3
N C G C 00 0 7 4 7 4 1 -0 1
u w isc-b eta p e p -9 1
14.65
u w isc -b e ta p e p -9 2
0.8321
19
16.3
N C G C 0 0 0 7 4 7 4 2 -0 1
14
8.741
N C G C 0 0 0 7 4 7 4 3 -0 1
u w isc -b e ta p e p -9 3
0 .6 5 4 8
u w is c -b e ta p e p -9 4
3.061
N C G C 00 0 7 4 7 4 4 -0 1
u w is c -b e ta p e p -9 5
0 .6 2 7 8
19
25.61
N C G C 0 0 0 7 4 7 4 5 -0 1
N C G C 00 0 7 4 7 4 6 -0 1
u w is c -b e ta p e p -9 6
0.861
22
3 4 .6 9
m in act o b j e c t j n t e n s it y o b je c t n o qA C 50 (M)
-7.282
5896
44
2912
41
-9.992
54
-6.119
2565
-0.5072
3821
45
-7.904
3217
28
-5.844
3841
30
-7.623
3433
48
-12.19
5092
44
-7.591
3747
33
-6.446
4218
41
-10.68
4882
56
-5
-9.689
4821
58
-2.956
4280
40
-1.104
3780
45
4082
51
-9.868
3705
72
-0.8 2 3 8
-7.114
3306
43
-8.684
3357
41
-4.6
4695
44
-6.768
-7.579
4067
45
31
-5.031
3989
-4.068
3380
32
-1.845
2782
57
-8.941
5204
-4.7
58
-11.58
5823
53
-4 .8
-10.4
-4.4
4192
59
62
-6 .8
-13.18
3197
71
-8.056
1996
-4.8
-11.58
4228
41
-10.42
2433
54
-5
-8.137
2375
54
-5
-12.23
3629
70
-4.8
3149
-16.29
47
-8.3
-4.519
2704
64
-4
-9.449
4145
60
-4.9
-6.633
72
-4 .5
1835
-14.97
3160
60
-6 .2
-9.434
3013
71
-4.6
-14.56
2545
72
-4.7
3807
-13.32
75
57
-11.56
2969
-4 .9
-9.369
2829
61
-4 .9
-4.135
2300
68
-15.42
1917
67
-4 .6
-15.1
3281
63
-6.1
-13.14
2708
61
-6.792
3044
63
-4 .5
-6.379
3844
89
-4 .7
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233
4.5 References
1 Kappe, C. O. Angew. Chem. Int. Ed. 2004, 43, 6250.
2 Murray, J. K.; Gellman, S.H. Org. Lett. 2005, 7, 1517.
3 (a) Gellman, S. H. Acc. Chem. Res. 1998, 31, 173. (b) Cheng, R. P.; Gellman, S. H.; DeGrado,
W. F. Chem. Rev. 2001, 101, 3219.
4 Blackwell, H. E. Org. Biomol. Chem. 2003, 1, 1251.
5 Kappe, C. O. Curr. Op. Chem. Biol. 2002, 6, 314 and references therein.
6 Stadler, A.; Kappe, C. O. J. Comb. Chem. 2001, 3, 624.
7 Coleman, C. M.; MacElroy, J. M. D.; Gallagher, J. F.; O ’Shea, D. F. J. Comb. Chem. 2002, 4,
87.
8 (a) Cotterill, I. C.; Usyatinsky, A. Y.; Arnold, J. M.; Clark, D. S.; Dordick, J. S.; Michels, P. C.;
Khmelnitsky, Y. L. Tetrahedron Lett. 1998, 39, 1117. (b) Glass, B. M.; Combs, A. P. Rapid
Parallel Synthesis Utilizing Microwave Irradiation. In High-Throughput Synthesis', Sucholeiki,
I., Ed.; Marcel Dekker: New York, 2001; pp. 123-128. (c) Kappe, C. O.; Stadler, A. MicrowaveAssisted Combinatorial Chemistry. In Microwaves in Organic Synthesis', Loupy, A. Ed.; WileyVCH: Weinheim, 2002; pp. 405-433.
9 Selected examples employing the CombiCHEM module for the Milestone MicroSYNTH
Labstation: (a) Alcazar, J. J. Comb. Chem. 2005, 7, 353. (b) Ntichter, M.; Ondruschka, B. Mol.
Diversity 2003, 7, 253. (c) Campiglia, P.; Gomez-Monterrey, I.; Longobardo, L.; Lama, T.;
Novellino, E.; Grieco, P. Tetrahedron Lett. 2004, 45, 1453. (d) Grieco, P.; Campiglia, P.;
Gomez-Monterrey, I.; Lama, T.; Novellino, E. Synlett 2003, 2216. (e) Martinez-Teipel, B.;
Green R. C.; Dolle, R. E. QSAR Comb. Sci. 2004, 23, 854.
10 (a) Werder, M.; Hauser, H.; Abele, S.; Seebach, D. Helv. Chim. Acta 1999, 82, 1774. (b)
Hamuro, Y.; Schneider, J. P.; DeGrado, W. F. J. Am. Chem. Soc. 1999, 121, 12200. (c) Porter, E.
A.; Wang, X.; Lee, H.-S.; Weisblum, B.; Gellman, S. H. Nature, 2000, 404, 565. (d) Gademann,
K.; Seebach, D. Helv. Chim. Acta 2001, 84, 2924. (e) Seebach, D.; Rueping, M.; Arvidsson, P. I.;
Kimmerlin, T.; Micuch, P.; Noti, C.; Langnegger, D.; Hoyer, D. Helv. Chim. Acta 2001, 84,
3503. (f) Liu, D.; DeGrado, W. F. J. Am. Chem. Soc. 2001, 123, 7553. (g) Raguse, T. L.; Porter,
E. A.; Weisblum, B.; Gellman, S. H. J. Am. Chem. Soc. 2002, 124, 12774. (h) Gelman, M. A.;
Richter, S.; Cao, H.; Umezawa, N.; Gellman, S. H.; Rana, T. M. Org. Lett. 2003, 5, 3563. (i)
Kritzer, J. A.; Lear, J. D.; Hodson, M. E.; Schepartz, A. J. Am. Chem. Soc. 2004, 126, 9468. (j)
Potocky, T. B.; Menon, A. K.; Gellman, S. H. J. Am. Chem. Soc. 2005, 127, 3686. (k) Stephens,
O. M.; Kim, S.; Welch, B. D.; Hodsdon, M. E.; Kay, M. S.; Schepartz, A. J. Am. Chem. Soc.
2005, 1 2 7 ,13126.
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234
11 Albericio, F. Curr. Op. Chem. Biol. 2004, 8, 211.
12 (a) Guichard, G.; Abele, S.; Seebach, D. Helv. Chim. Acta 1998, 81, 187. (b) Arvidsson, P. I.;
Rueping, M.; Seebach, D. Chem. Commun. 2001, 649. (c) Arvidsson, P. I.; Frackenpohl, J.;
Seebach, D. Helv. Chim. Acta 2003, 86, 1522.
13 Sebestyen, F.; Dibo, G.; Kovacs, A; Furka, A. Bioorg. Med. Chem. Lett. 1993, 3, 413.
14 Murray, J. K.; Farooqi, B.; Sadowsky, J. D.; Scalf, M.; Freund, W. A.; Smith, L. M.; Chen, J.;
Gellman, S. H. J. Am. Chem. Soc. 2 0 0 5 ,127, 13271.
15 Kritzer, J. A.; Luedtke, N. W.; Flarker, E. A.; Schepartz, A. J. Am. Chem. Soc. 2005, 127,
14584.
16 Porter, E. A.; Weisblum, B.; Gellman, S. H. J. Am. Chem. Soc. 2 0 0 5 ,127, 11516.
17 Nuchter, M.; Ondruschka, B.; Bonrath, W.; Gum, A. Green Chem. 2004, 6, 128.
18 (a) Thaler, A.; Seebach, D.; Cardinaux, F. Helv. Chim. Acta 1991, 74, 628. (b) Seebach, D.;
Beck, A. K.; Studer, A. Modern Synthetic Methods 1995, 7, 1. (c) Stewart, J. M.; Klis, W. A.
Innovation and Perspective in Solid Phase Synthesis: Peptides, Polypeptides and
Oligonucleotides', Epton, R., Ed.; SPCC: Birmingham, UK, 1990; pp 1-9. (d) Hendrix, J. C.;
Halverson, K. J.; Jarrett, J. T.; Lansbury, P. T. J. Org. Chem. 1990, 55, 4517. (e) Leadbeater, N.
E.; Torenius, H. M. J. Org. Chem. 2002, 67, 3145.
19 Yan, B.; Fang, L.; Irving, M.; Zhang, S.; Boldi, A. M.; Woolard, F.; Johnson, C. R.;
Kshirsagar, T.; Figliozzi, G. M.; Krueger, C. A.; Collins, N. J. Comb. Chem. 2003, 5, 547.
20 Leadbeater, N. E.; Pillsbury, S. J.; Shanahan, E.; Williams, V. A. Tetrahedron 2005, 61, 3565.
21 Lebl, M .; Pokomy, V.; Krchnak, V. J. Combi. Chem. 2005, 7, 42.
22 Schinnerl, M.; Murray, J. K.; Langenhan, J. M.; Gellman, S. H. Eur. J. Org. Chem. 2003, 721.
23 Guichard, G.; Abele, S.; Seebach, D. Helv. Chim. Acta 1998, 81, 187.
24 (a) Blankmeyer-Menge, B.; Nimitz, M.; Frank, R. Tetrahedron Lett. 1990, 31, 1701.
Arvidsson, P. I.; Frackenpohl, J.; Seebach, D. Helv. Chim. Acta 2003, 86, 1522.
25 Gude, M.; Ryf, J.; White, P. D. Lett. Pept. Sci. 2003, 9, 203.
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(b)
235
Chapter 5
Optimization of Chimeric (a/p + a)-Peptide
Ligands for Bcl-xLvia Microwave-Assisted
Combinatorial Synthesis
Microwave
Irradiation
Split-and-Mix
vs.
Parallel Synthesis
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236
5.0 Brief Summary of C hapter
We have optimized a chimeric (a /p + a)-peptide ligand for the BH3-recognition cleft o f
B
c 1- x l
to achieve proteolytic stability and good binding affinity through application of
microwave-assisted combinatorial synthesis.
varying the side chains that interact with
B
A one-bead-one-compound library o f oligomers
c 1- x l
in the bound state was found to contain five
analogues with binding affinities similar to the lead compound. Through substitution o f P-amino
acids into the a-peptidic C-terminal segment o f the foldamer ligand, a p-scan approach, we
found that the activity o f the hybrid (a/p + a)-peptide scaffold was extremely sensitive to
backbone modification. Split-and-mix techniques were compared with parallel synthesis for lead
optimization. A fluorescence polarization (FP) assay for the Bcl-Xi/Bak interaction was used to
reliably screen crude peptide products. These synthesis and screening methods facilitated the
rapid evaluation of potential foldamer antagonists and represent important steps toward the
development o f a general approach for identifying foldameric inhibitors o f protein-protein
interactions.
5.1 Background
Protein-protein interactions are involved in many aspects o f cell signaling and growth.
Deregulation o f these pathways is often associated with disease.
Although protein-protein
interactions have emerged as an important new class o f therapeutic targets, a general approach to
the development o f suitable inhibitors has not yet been identified. Protein and antibody therapies
are expensive and limited to extracellular targets.
Antisense and small-interfering RNA
approaches suffer from problems associated with intracellular delivery.
Small molecule
1
2
strategies have proven less effective, with a few notable exceptions. Peptide inhibitors are
often discovered but are not developed because o f poor pharmacokinetic properties, such as
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
237
proteolytic instability and cellular impermeability.
The challenges associated with protein-
protein interaction inhibition have created the opportunity to explore a new class o f molecules
designed to mimic the display o f important amino acid side chains from a protein- structure,
termed proteomimetics, as potential antagonists o f protein-protein interactions.
The Bcl-xi/Bak protein-protein interaction is an important therapeutic target because of
its association with cancer. The anti-apoptotic protein Bcl-xL is often overproduced in tumor
cells . 4 Ligands that bind to
B c 1 -x l
and prevent its complexation with pro-apoptotic Bcl-2 protein
family members, including Bak and Bid, may sensitize malignant cells to cytotoxic signals. An
NMR solution structure o f the Bcl-XL/Bak BH3 peptide complex revealed a focused interfacial
interaction with the a-helical Bak BH3 peptide binding in the hydrophobic BH3-recognition
cleft on
B c 1 - x l.5
Different strategies have been pursued in an effort to discover inhibitors for the
Bcl-xL/Bak interaction . 6 Fesik et al. explored the structure/activity relationships (SAR) o f Bcl-2
family BH3 peptides . 5 ,7
Using their “SAR by NM R” technique , 8 these Abbott researchers
generated a small molecule inhibitor with nanomolar affinity for Bcl-XL.2a,b
Verdine and
coworkers stabilized the a-helical conformation o f the Bid BH3 peptide via cyclization o f the
side chains of residues at the / and i+4 positions o f the helix by olefin methathesis, resulting in an
analogue with greater proteolytic stability and activity than the natural peptide . 3 3 Hamilton and
coworkers have reported proteomimetic terphenyl, terephalamide, and tris-pyridyl amide
scaffolds capable o f binding to
B c 1 - x l.9
Despite progress, development of potent and selective
inhibitors for Bcl-xi/Bak and other protein-protein interactions remains a challenging research
goal.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
238
5 .1 .1
Chimeric (a/p + a)-Peptide Ligands for
B c 1 -x l
We and others are investigating “foldamers,” oligomers with discrete folding
propensities , 10 as proteomimetic scaffolds for the inhibition of protein-protein interactions . 11
The discovery o f chimeric (a/p + a)-peptide 5-1 as a potent ligand for the BH3-recognition
i
domain o f Bcl-xL was recently reported.
The N-terminal segment o f 5-1 is composed of
O
q
h 2n.
<
■)
> -
alternating
> -
oh
y— (
/
oh
\
r
P-
and
a-am ino
acids.
o
H
Incorporation o f the five-membered ring
n
\ s
n
ACPC
constrained
APC
p-amino
acids,
trans-2-
p3-amino acid
aminocyclopentane carboxylic acid (ACPC) and /r<ms-3-aminopyrrolidine-4-carboxylic acid
(APC), promotes the a/p-peptide 14/15-helical conformation , 13 which was observed for the
Ala2-^Lys, Lys 8 -Mle analogue o f 5-1 by 2D NM R spectroscopy in CD 3 OD . 12 The foldamer
scaffold was used to mimic the three-dimensional display o f the hydrophobic residues o f the ahelical Bak BH3 peptide in its binding to the BH3-recognition cleft o f Bcl-x L . 5 A p3-amino acid
residue (p 3 -hNle) was incorporated into the sequence at position 9 to provide the side chain
functionality necessary for mimicking the conserved leucine in the Bak BH3 peptide. However,
neither a “pure” a/p - nor a “pure” P-peptide scaffold was able to effectively replace the Cterminal a-peptide segment o f the Bak peptide . 12 Chimeric (a /p + a)-foldam er 5-1, an oligomer
14/15-Helical a/p-Peptide
i................. :...—
a-Peptide
>..................................... - - ------
............................ -........-......... -......
>
h 2n
5 .1
H
U
H
ooII
II
oO
i
\\
IC 5 0 = 0 .0 6 0 pM
H
Is
U
nh
H2N
H
o0II
00II
I
H
\\
^
A
NH
H
II
oo \
I
H
^=0
H0
II
o \
i
H
Va
W
N h2
Figure 1. Structure of chimeric (a/p + a)-peptide ligand for the BH3-binding domain o f Bcl-xL (ref. 12).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
\
nlh
H2N ^ N H
239
with an a/p-peptide N- terminus and an a-peptide C-terminus, was a high-affinity ligand for
the BH3-recognition cleft on Bcl-xL, having an IC 5 0 o f 0.060 pM in a Bcl-xL/Bak fluorescence
polarization (FP) assay.
One o f the advantages o f foldamers for biomedical applications is their exceptional
proteolytic 14 and metabolic 15 stability; however, the C-terminal a-peptide portion o f molecule 51
is susceptible to proteolysis , 12 limiting the utility o f the (a /p + a ) chimeric foldamer class in
biological applications.
We have explored the SAR o f (a/p + a)-ligand 5-1 and related
analogues through the tandem application o f microwave-assisted combinatorial chemistry and a
high-throughput Bcl-XL/Bak FP assay in an effort to transform the chimeric (a /p + a ) lead
compound into a completely unnatural oligomer with high binding affinity and improved
proteolytic stability.
5.1.2 Microwave-Assisted Synthetic Methods for Rapid Lead Optimization
Ours and other’s attempts to discover and optimize foldamer inhibitors o f protein-protein
interactions have inspired the concurrent development o f improved synthetic techniques for the
rapid generation o f combinatorial libraries. Peptide foldamers may be prepared using the same
solid-phase peptide synthesis (SPPS) procedures developed for the preparation a-peptides , 16 but
foldamers prepared using standard SPPS protocols are usually not o f sufficient purity for direct
evaluation in a biological assay. The necessity o f HPLC purification prior to screening had
limited synthetic efforts to small sets o f oligomers . 17
Parallel solid-phase synthesis o f < 48
oligomers per library led to potent chimeric (a/p + a)-peptide 5-1.
Each peptide was
synthesized at room temperature in an individual reaction vessel and purified by HPLC, resulting
in an increasingly time- and labor-intensive process as the size o f the library increased. Other
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
240
protein-protein interactions have been targeted by [3-peptides with lower potencies using
similar synthesis techniques.11 Progress toward the biological application o f [3-peptides could be
more rapidly accomplished through the synthesis and screening o f combinatorial libraries
without HPLC purification (Figure 2).
Target:
Scaffold:
Bcl-x, /Bak
Advance to
in vivo
Studies
Proteomimetic
Design
High-Throughput
Bcl-xL/Bak
FP Assay
Microwave-Assisted
Combinatorial
Synthesis
HPLC Purification
Figure 2. Paradigm for the discovery and development of foldamer anatagonists for the Bcl-xL/Bak protein-protein
interaction.
The difficult preparation o f 14-helical [3-peptides18 and our need to employ this scaffold
have focused previous synthetic efforts on this class o f foldamers.
We accomplished the
synthetic optimization o f a particularly difficult [3-peptide decamer using microwave irradiation,
resulting in a large increase in initial product purity and a 10-fold reduction in synthesis time
(Chapters 2-4).19,20 Microwave irradiation has been successfully applied to a large number of
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241
organic reactions with impressive results,21 but it has been a challenge to harness this method
for the preparation o f libraries and acceleration o f the discovery process.22 We adapted our
microwave reaction conditions for (3-peptide synthesis on polystyrene (PS) macrobeads and
rapidly prepared, via split-and-mix techniques, a small P-peptide library of potential inhibitors
23 24
for another therapeutically important protein-protein interaction, p53-MDM2 (Chapter 3). ’
We further expanded the utility o f microwave heating by developing methods for the efficient
preparation of P-peptide libraries in parallel using 96-well polypropylene filter plates in
combination with a multimode microwave reactor (Chapter 4).25
a/p-Peptides do not suffer from the synthetic difficulties associated with 14-helical ppeptides and may be readily prepared at room temperature using the traditional SPPS methods.16
However, we accelerated the optimization of (a /p + a)-peptide 5-1 by interfacing the
microwave-assisted synthesis o f combinatorial libraries and our high-throughput FP assay to
further investigate the SAR o f chimeric (a/p + a)-peptide ligands for Bcl-xL and produce a direct
comparison between split-and-mix and parallel synthesis for lead optimization.
synthetic techniques for peptides containing a-am ino acids were developed.
identified a shorter analogue o f 5-1 that displays both high affinity for
B
Improved
We rapidly
c 1- x l
and high
proteolytic stability.
5.2 Optimization of Chimeric (q/B + al-Peptide Ligands for Bcl-xi
5.2.1 One-Bead-One-Compound L ibrary for Side Chain Optimization
The binding affinity o f peptide 5-1 was optimized in collaboration with Jack D.
Sadowsky through combinatorial variation o f the residues that interact directly with Bcl-xL in the
bound state. Mutational studies o f (a /p + a)-peptide ligands identified ACPC3, Leu6, P -hNle9,
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242
and Phel3 (standard peptide numbering, beginning at the N-terminus) as the hydrophobic
residues being most important for the ligand’s interaction with the BH3-binding domain o f the
protein.26 (Arg4 and A s p ll are the most important charged residues.) BH3-domains o f the Bcl2 family of proteins display a large diversity o f hydrophobic residues in their binding epitopes;
therefore, we designed a 1,000-membered library that varied the nature (i.e., aliphatic or
aromatic) and size o f the hydrophobic side chain at each o f these positions (residues 3, 6, 9, and
13 in 5-1, Figure 3). ACPC was included at position 9 to increase the helical propensity o f the
a/p-portion o f the oligomer near the junction between the two segments o f the chimera.
Glutamic acid was incorporated at position 11 to potentially compensate for disturbances o f the
electrostatic interaction o f A spl 1 with the Bcl-xL surface caused by changes to neighboring parts
o f the sequence.
Figure 3. (a/p + a)-Peptide library produced via split-and-pool synthesis on PS macrobeads with microwave
irradiation. Four or five different residues were incorporated at positions 3, 6, 9, and 13; two different residues were
installed at position 11 (5 x 5 x 4 x 2 x 5 = 1,000 members).
The library was constructed in a one-bead-one compound format in order to attain a
higher level o f side chain diversity through a larger library size than was feasible via parallel
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
243
synthesis. Library preparation was accomplished via split-and-pool synthesis in only three
days in a multimode microwave reactor under conditions originally developed for P-peptide
synthesis.25 Multiple cycles of microwave irradiation were used to ensure complete reaction on
the PS macrobeads during both the removal o f the 9-fluorenylmethoxycarbonyl (Fmoc)
protecting group and coupling of the activated amino acids. 23
Reaction temperature was
controlled by modulation of power (600 W maximum; couplings were 6 x (2 min ramp, 10 min
cool) at 80°C; and Fmoc-deprotections were 3 x (2 min ramp, 10 min cool) at 90°C).
Approximately 4,800 PS macrobeads were used; 4.8 beads per compound provided good
statistical coverage o f the library members.27 At the end o f the synthesis, beads were arrayed
(one bead per well) into fifteen 384-well polypropylene plates. Treatment with trifluoroacetic
acid cleaved the peptide product mixture from the resin with simultaneous global side chain
deprotection. After concentration by rotary evaporation, the product mixtures were dissolved in
DMSO.
Material from 50 o f the beads was analyzed by reversed-phase (RP) HPLC.
The
products were not exceptionally pure (Figure 4), but most contained a major peak with a mass
corresponding to an expected library member, as determined by MALDI-TOF MS. The lower
product purity relative to the library in Chapter 3 is due to the increased length o f the oligomer; a
95% yield on each coupling/deprotection reaction cycle results in ~ 40% purity (0.9516 = 0.44).
We wanted to ensure that crude peptide mixtures could be reliably screened with the
competition FP assay developed in our lab for analysis o f ligand binding to the BH3-binding
cleft o f
B
c I- x l
before screening the entire library. To the author’s knowledge, interfacing one-
bead-one-compound peptide libraries with FP screening has not been reported. Peptide 5-2, a
Lys8Ile analogue o f 5-1 with an IC 5 0 o f 150 nM,26 was prepared separately on PS macrobeads as
a test case for oligomer synthesis and the screening o f unpurified peptides. The crude product
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244
DMSO
1000 -
Ph,CH
Abs
(mV)
500-
0
5
10
15
20
25
30
35
Time (min)
Figure 4. Analytical RP-HPLC trace (UV detection at 220 nm) of a representative library member. “M” indicates
the identification by MALDI-TOF MS of an oligomer with a molecular weight corresponding to a predicted library
member.
cleaved from individual beads containing peptide 5-2 was serially diluted and tested directly in
the Bcl-XL/Bak FP assay to generate a dose-response curve (Jack D. Sadowsky, Figure 5). Beadto-bead results showed only slight variability. We found that the interaction was not inhibited at
the concentration resulting from a 1000-fold dilution o f the initial DMSO stock solution o f crude
product 5-2 from single macrobeads.
Bead 1
Bead 2
""A
Bead 3
Bead 4
Bead 5
0.0001
0.001
0.01
0.1
Dilution
Figure 5. Dose-response of crude peptide 5-2 from six individual PS macrobeads. Figure adapted from Jack D.
Sadowsky.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
245
We screened the entire one-bead-one-compound library at the concentration resulting
from a 1:1000 dilution to identify those compounds that inhibit the Bcl-xL interaction with
greater potency than peptide 5-2. A cut-off o f millipolarization units (mP) < 3 6 was chosen after
screening the crude peptide mixtures to separate “hits” from the rest o f the library members
(0.5% hit rate). The compounds from the 26 wells meeting this criterion were sequenced by
pLC-MS/MS (Dr. Mark Scalf), as described previously for |3-peptides. 2 3 ,2 8
This process
identified 16 unique compounds, six o f which were redundantly identified.
Re-synthesis
followed by HPLC purification and testing in the FP assay gave the results listed in Table 1.
Table 1. (a/ft + a)-Peptide hits from library screening and sequencing as validated by re-synthesis, purification,
and re-testing. * - Two beads found in one well.
No.
S eq u en ce
5-1
N
1
2
Ac A PC Ala
3
ACPC
4
5
Arg A C PC
6
Leu
7
8
9
10 11 12 13
14 15 C
A C PC Lys p3-hN le Gly A sp Ala P h e A sn Arg NH2
5-2
Ac A PC Ala
ACPC
Arg A C PC
Leu
A C PC
5-3
Ac APC Ala
ACPC
5-4
Ac APC Ala
ACPC
5-5
Ac APC Ala
5-6
A c A PC Ala
5-7
Ac A PC Ala
5-8
5-9
Ac A PC Ala
Av. m P ICso (pM)
P o ten cy
T im es
(rel. to 1) Identified
N.D.
0.06
1
0
N.A.
0.15
3
N.A.
Arg A C PC xL eu A C PC Lys p 3-h P h e Gly A sp Ala P h e A sn Arg NH2
24.9
0.048
1
3
Arg A C PC xL eu A C PC Lys pJ-hN le Gly A sp Ala P h e A sn Arg NH2
26.9
0.05
1
1*
ACPC
Arg A C PC
Cha A C PC Lys p 3-h P h e Gly A sp Ala P h e A sn Arg n h 2
28.1
0.053
1
1
ACPC
Arg A C PC
Leu
A C PC Lys p3-h P h e Gly A sp Ala P h e A sn Arg NH2
27.7
0.056
1
2
ACPC
Arg A C PC
Phe
A C PC Lys p 3-h P h e Gly A sp Ala P h e A sn Arg |slH2
25.5
0.089
1
5
Ac A PC Ala |}3-hLeu Arg A C PC xL eu A C PC Lys p3-h P h e Gly A sp Ala P h e A sn Arg n h 2
21.2
0.12
2
2
4
ACPC
Arg A C PC
Trp
lie
p J-hN le Gly A sp Ala P h e A sn Arg NH2
A C PC Lys p 3-h P h e Gly A sp Ala P h e A sn Arg NH2
21
0.22
4
5-10 A c A PC Ala p3-hLeu Arg A C PC xL eu A C PC Lys p3-hN le Gly A sp Ala P h e A sn Arg NH2
21.7
0.24
4
1
5-11 Ac A PC Ala
A C PC Lys p3-hN le Gly A sp Ala P h e A sn Arg NH2
29.2
0.28
5
2
5-12 A c A PC Ala p 3-hLeu Arg A C PC xL eu A C PC Lys p 3-h P h e Gly A sp Ala Tyr A sn Arg NH2
27.1
0.29
5
1*
5-13 A c A PC Ala
ACPC
Arg A C PC xL eu A C PC Lys p 3-h P h e Gly A sp Ala Tyr A sn Arg NH2
35.6
0.53
9
1
5-14 Ac A PC Ala
ACPC
Arg A C PC
A C PC Lys p^-hPhe Gly A sp Ala Tyr A sn Arg NH2
33.1
1.1
18
1
5-15 A c A PC Ala
ACPC
Arg A C PC xL eu A C PC Lys p3-hTrp Gly A sp Ala Trp A sn Arg NH2
ACPC
Arg A C PC
Trp
Trp
27.1
1.2
20
1*
5-16 A c A PC Ala p3-hC ha Arg A C PC
Trp
Gly A sp Ala C ha A sn Arg NH2
26.9
26
433
1*
5-17 Ac A PC Ala
P h e A C PC Lys p3-h P h e Gly Glu Ala Trp A sn Arg NH2
23.5
31
517
1
5-18 Ac A PC Ala p 3-h P h e Arg A C PC xL eu A C PC Lys pJ-hN le Gly Glu Ala C ha A sn Arg NH2
32.1
76
1267
1
ACPC
Arg A C PC
A C PC Lys
ACPC
Four (a /p + a ) peptides, 5-3, 5-4, 5-5, and 5-6, were found to have IC 5 0 values of
approximately 50 nM, slightly more active than lead sequence 5-1. All. differences in IC 5 0 values
among these compounds were very small ( < 1 5 nM); their sequences differed at one or two
positions. Lead sequence 5-1 was synthesized as a member o f the library but was not identified
as a hit during the screen, indicating a false negative result.
The other hits had IC50 values
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246
ranging from 0.089 to 76 gilM and were considered false positives. We wondered whether
some amino acid-deletion side product was contributing to the activity of crude products 5-7 to
5-18, causing the activity o f the re-synthesized and purified products to be much lower.
29
Examination of the LC-MS/MS sequencing data for 5-11 showed that the Arg4-deletion product
was present in a significant amount. However, synthesis and testing o f a set o f Arg4-deletion
products revealed that these products were not active (JKM VI 249).
Overall, the synthetic
variability (i.e., purity and yield) o f the crude products lowered the sensitivity o f the assay to the
point that subtle changes in binding affinity resulting from side chain substitutions were almost
completely obscured in the initial screen.
Useful SAR information was gleaned from comparison o f the activity o f the resynthesized hits (Table 2). Among the four most active oligomers (5-3 to 5-6), ACPC3, A s p l l ,
and Phe 13 were retained from lead compound 5-1. Substitution of P -hLeu for ACPC at position
3 resulted in a 2- to 4-fold reduction in potency (5-8 and 5-10). A Phel3->T yr substitution
caused a 2- to 11-fold loss o f inhibitory activity (5-12 and 5-13). Only two oligomers (5-17 and
5-18) contained a glutamic acid residue at position 11. There was considerable variation o f the
residue at position 6 among the hits from the library. The most active compounds contained
leucine, homoleucine (xLeu), or cyclohexylalanine (Cha). A phenylalanine residue at position 6
slightly reduced binding affinity (5-7, IC50 = 0.09 pM), but having a tryptophan at this position
•5
^
reduced the activity by 5-fold (5-9 and 5-11). Substitution o f p -hPhe for p -hNle at position 9
had a negligible effect on the potency o f the oligomer.
Overall, the N-terminal a/p-peptide
segment of the oligomer is much less sensitive than the N-terminus o f the Bak BH3 peptide to
side chain modifications. A variety o f different residues that can be incorporated at positions 6
and 9 without significantly affecting the activity o f the (a /p + a)-peptide oligomer, suggesting
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that the foldamer does not binding in exactly the same manner as the Bak BH3 peptide that it
is intended to mimic. However, minor modifications in the side chains o f the C-terminal a peptide segment of the (a/p + a)-peptide greatly reduced the activity o f the oligomer, and we
concluded that this portion o f the chimeric foldamer is binding to Bcl-xL in a similar manner to
the Bak BH3 peptide.
Table 2. Effects of substitution of |33-Leu for ACPC at position 3.
No.
3
Sequence
9
6
13
IC50 (pM)
Relative
Potency
5-3
ACPC
xLeu
p3-hPhe
Phe
0.048
1
5-8
p3-hLeu
xLeu
p3-hPhe
Phe
0.12
3
5-4
5-10
ACPC
ff-hLeu
xLeu
xLeu
p3-hNle
p3-hPhe
Phe
Phe
0.05
0.24
1
5
5-1
ACPC
Leu
p3-hNle
Phe
0.06
1
5-4
ACPC
xLeu
p3-hNle
Phe
0.05
1
5-11
ACPC
Trp
p3-hNle
Phe
0.28
5
5-3
ACPC
xLeu
p^-hPhe
Phe
0.048
1
5-5
ACPC
Cha
p3-hPhe
Phe
0.053
1
5-6
ACPC
Leu
p3-hPhe
Phe
0.056
1
ACPC
ACPC
p3-hPhe
pJ-hPhe
Phe
Phe
0.089
0.22
2
5-9
Phe
Trp
5-6
ACPC
Leu
p3-hPhe
Phe
0.056
1
5-1
ACPC
Leu
p3-hNle
Phe
0.06
1
5-9
ACPC
Trp
p^-hPhe
Phe
0.22
1
5-11
ACPC
Trp
p3-hNle
Phe
0.28
1
5-3
ACPC
xLeu
p3-hPhe
Phe
0.048
1
5-4
ACPC
xLeu
p3-hNle
Phe
0.05
1
5-8
p3-hLeu
xLeu
p3-hPhe
Phe
0.12
1
5-10
p3-hLeu
xLeu
p3-hNIe
Phe
0.24
2
5-3
ACPC
xLeu
pJ-hPhe
Phe
0.048
1
5-13
ACPC
xLeu
p3-hPhe
Tyr
0.53
11
5-8
p3-hLeu
xLeu
p3-hPhe
Phe
0.12
1
5-12
p3-hLeu
xLeu
p3-hPhe
Tyr
0.29
2
5-9
ACPC
Trp
p3-hPhe
Phe
0.22
1
Trp
p3-hPhe
Tyr
1.1
5
5-7
5-14
ACPC
5
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248
5.2.2 O ne-Bead-O ne-C om pound L ib rary for B ackbone M odification
We decided to pursue modifications o f the backbone since the binding affinity of
compound 5-1 was not enhanced by variation o f the side chains. Our desire to study the activity
o f 5-1 in cells, and eventually in vivo, requires that the oligomer be stable under biological
conditions. Treatment o f 5-1 with a mixture o f pro teases in vitro results in a rapid cleavage of
the C-terminal a-peptide segment.12 Incorporation o f unnatural amino acid residue(s) within this
portion of the sequence was expected to increase its proteolytic stability.
However, such
modifications could not come at the expense o f substantially decreased binding affinity.
Concurrent optimization o f these two properties, binding affinity and proteolytic stability,
required careful examination o f the structure/activity relationship.
Previous studies indicated that use o f alternate oligomer backbones caused large changes
in binding affinity relative to side chain modifications, perhaps o f a sufficient magnitude to be
reliably detected in a one-bead-one-compound library format.12 A library was designed to
combinatorially incorporate either the natural a-am ino acid or its p3-amino acid analogue at
positions 10 through 14.
3
Single a -> p -amino acid replacements (a p-scan) is precedented.
30
Additionally, we decided to probe the effect o f inserting a residue at the junction between the
a /p - and a-peptide segments of the molecule to allow for side chain repositioning. A library
encompassing all combinations o f the described substitutions (Figure 6) was synthesized using
split-and-mix techniques in combination with multiple cycles o f irradiation in a monomode
microwave reactor.
The combinatorial p-scan library is a departure from our general approach for developing
foldamer inhibitors o f protein-protein interactions, primarily because it does not take into
account the available structural information for either the target or the lead compound. The C-
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
H2N ^ O
H
■ M
H
H
Figure 6. (a/p + a)-Peptide library synthesized with microwave irradiation both via split-and-mix techniques and
in parallel. Two different residues (either the natural a-amino acid or its p3-amino acid analogue) were incorporated
at positions 10 through 14; Gly or P-hGly was optionally installed between p3-hNle9 and GlylO (3 x 2 x 2 * 2 x 2 x
2 = 96 members).
terminal portion o f the oligomers in the combinatorial P-scan library was not rationally designed
to mimic the Bak BH3 peptide’s C-terminus. Instead, we approached the development o f Bak
mimetics via combinatorial modification o f the a-portion o f the (a /p + a ) lead sequence in an
effort to identify a non-natural scaffold that binds to Bcl-xL. Substituting P-amino acids one or
more at a time via a P-scan affects the peptide’s local backbone conformation and side chain
placement and has been shown to confer proteolytic stability.30 P-Scanning may be considered a
tool in the peptide chemist’s toolbox (like N-methylation) for conformational restriction o f the
backbone or a tool for biologists to increase the proteolytic stability o f peptides.31 Incorporating
P-amino acids into the backbone gives chemists an option for developing ligands not commonly
observed in Nature.
Screening the one-bead-one-compound P-scan library in the Bcl-XL/Bak FP assay
revealed that the activity o f 5-1 is extremely sensitive to backbone modification within the C-
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
250
terminal a-peptide segment. Material from sixteen beads showed > 30% inhibition (Figure
7) at the concentration used for testing (approximately 200 nM, 2.8% hit rate). Analysis o f the
crude product mixtures by MALDI-TOF MS showed that the five most active samples all
corresponded to the original sequence, peptide 5-1.
Peptide 5-2, used previously for
investigation o f oligomer synthesis on PS macrobeads and compatibility o f the FP assay with
crude peptide products, was only slightly less active than 5-1 in the initial screen, even though its
IC5o value is 2.5-fold weaker than that o f 5-1, indicating slightly reduced assay sensitivity for
screening crude peptide products.26 Compounds in the second tier o f activity included the
peptides with a single a -> [3-amino acid substitution in the C-terminal segment and the Gly and
P-hGly linker insertion sequences. Several hits were re-synthesized, F1PLC purified, and assayed
for validation (Table 3).
(We did not perform LC-MS/MS sequencing, but instead we
synthesized all of the oligomers containing a single a ->[3 substitution.) The excellent correlation
between the relative order o f activities observed in the crude screen and the IC 5 0 values o f the
purified compounds demonstrates that these synthesis techniques can be used in combination
with a FP assay to reliably identify the active members o f a compound library. These results also
demonstrate the potential usefulness o f the split-and-mix library approach during the initial
discovery phase when one is searching for a single compound that is significantly more active
than all the other library members. However, the disadvantages o f this method are the reduced
assay sensitivity (relative to compounds synthesized in parallel as discussed below) and the
significant time and effort required for LC-MS/MS sequencing and hit validation. We concluded
that the one-bead-one-compound library approach is useful for lead discovery but is less suited to
lead optimization, so we pursued a complementary parallel library synthesis approach for SAR
elucidation.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
251
-J17. 78%
F6, 63%
Row
I
2 0 21 2 2 2 3 2 4
17 18 19
g 10 11 12
80
13 14 15 16
Column
r
Sequence
5-1 All a C-terminus
0.060
0.15
5-19to22: Single a~»B substitution 0.99-2.0
5-23; Gly linker insertion
46
5-24: pGly linker insertion
540
co 50
Well
Figure 7. Screening o f the one-bead-one compound “P-scan” library. Top) Plate 1 with a 30% inhibition lower
limit. Bottom) Ranked hits as identified by MALDI-TOF MS.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 3. Binding affinity o f most active compounds from the library described in Figure 6.
Sequence
No.
1
IC50 (JIM)
0.060
° H0v» ■ vX=J \
"‘I
0.15
° HOV» ° \K) ° \
NH
h2n'^,nh
h2n^nh
/o
5-19
NH
HaN'^'NH
o
c/ H
\
>=o 0 \\y-\
_) H2Nh0
o \
HO
0.99
0
h2n^nh
r
5-20
„
P
0T
y°
HO
/"
0 ty=o o
„
1.6
H2N
h2n^nh
/
/"
^ r V 0r v\ "0 A
V0
■ V0 A
\
W
o
^
=
0
HO
H2N
P
5-21
5.5
h^n^ nh
/
5-22
/
/
>
V
P
v
°
P
P
o 1
v
/
P
0 \
h2nx °
»
”
V
-
0
2.0
h2n^nh
5-23
5-22
/
^
>
V
A
o
W
p
0 «1
" ° HO
P
V
/ ”
O yt \
H,N
V
o
)=°
°
( ) °
P
\
46
570
NH
X
h2n'^'nh
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253
5.2.3 Parallel Synthesis of the C om binatorial P-Scan L ib rary
We prepared the combinatorial (3-scan library described above (Figure 6) using
microwave-assisted solid-phase synthesis in 96-well polypropylene filter plates,
25
affording a
direct comparison between split-and-mix and parallel synthetic methods for SAR elucidation.
We synthesized the library in less than 2 days with 6 min coupling reactions at 80°C and
deprotection reactions for 4 min at 90°C.25 HPLC and MS analysis o f the library members
showed that a major species in the product mixture was usually the desired compound, but there
were other significant side products.
The compounds were screened initially without
purification. Testing at three successive 10-fold dilutions showed that lead sequence 5-1 was
again much more active than any other library member (Figure 8). The next most active group
of compounds contained either a single a - to p-amino acid substitution or a Gly or p-Gly residue
insertion, but all o f these were > 10-fold less active than 5-1 (Table 3). Sequences with more
than one substitution were completely inactive (< 20% inhibition at 1 pM).
A similar SAR
among the hits had been observed in the one-bead-one-compound library, but a tremendous timesavings was afforded by having the sequence o f the hits from the parallel library immediately
available simply by knowing their locations within the plate, rather than having to perform LCMS/MS sequencing as with a one-bead-one-compound library. The higher purity and uniform
yield o f peptide products appeared to increase the sensitivity o f the FP assay toward the library
synthesized in parallel. The SAR for members o f the parallel p-scan library was immediately
available and easily discernible, in contrast to the information from the one-bead-one-compound
library o f the same design.
Each member o f the library was subjected to HPLC purification and re-screened to
determine whether there existed significant differences in activity between crude and pure
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
254
Crude
Dilution
% Inhibition
33
1
35
4
49
75
—78------------------------------------------------
lll|» il
■
l III i ikullll J j j |
, ll.lllll
% Inhibition
, . . , 1
II
.
11 l|(| 1 llllj
1:100
4
■
■
■
■
■
% Inhibition
1:10
05
42
41
B1
1 ...M
llilllll.
9 5? § s a 8
I-Lllll
1
llilllll
1:1000
5 -1
. ___ - ■ -*>........ Iplnlll
I i | ......... .................... li.iiil............ .Iililil
? 5 ®S H
i
W e ll
Figure 8. Screening of parallel P-scan library in crude form.
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255
Purified
Dilution
% Inhibition
41
I
.
34
1:10
43
j
,
35________
1
1
l l l r f l
— M
il
J
»
l l
M
l U
ll
y
B
I
■.
l
100
% Inhibition
90
70
60
1:100
50
40
30
20
10
% Inhibition
0
5=1
08925
8245882089248
Figure 9. Screening of parallel p-scan library in purified form.
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
1:1000
256
compounds.
The desired product was collected during the elution o f each mixture. The
collected fractions were re-formatted into two 96-well deep well plates and re-tested in the FP
assay (Figure 9). A considerable advantage o f the parallel library relative to the one-bead-onecompound format is that compounds can be synthesized on a larger scale so that hits can be
purified directly without having to be re-synthesized. All purified library members showed a
slight reduction in activity due to some sample loss during purification or removal o f
contaminants that contributed to the overall activity o f the sample. Some compounds that had
appeared to be weakly active at the highest concentration during screening o f the crude products
were inactive after purification. In general, there was a good correlation between activity o f a
compound in its crude and purified forms (Figure 10). To our knowledge, this is the first report
5-1: Most Active
*
# 5-19 to 22: Single
a —»p substitution
inactive
— 5 - 2 3 .5 -2 4
False positives
100
% Inhibition Crude
Figure 10. Correlation of the percent inhibition of library members from Figure 6 screened before and after HPLC
purification. The line is a manual fit to the data for 5-1.
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257
of a FP assay being used to reliably screen crude peptide products.
Two important
exceptions were the sequences containing the Gly and fl-hGly insertions between residues p3hNle9 and Gly 10, which were much more active before purification than afterwards.
We
attribute this to the fact that the side product from the deletion o f this inserted residue results in a
small amount o f the highly active compound 5-1.
Removal o f this “contaminant” during
purification greatly reduces the activity o f the product.
5.2.4 Second Generation Parallel Library
Flaving observed the extreme sensitivity o f binding affinity to even a single P-amino acid
substitution within the C-terminal a-peptide segment o f 5-1, we wondered whether
combinatorial variation o f only the central residues o f the a-segm ent (Aspl 1, Ala 12, and Phe 13)
with a greater diversity o f side chains might produce a new, tightly binding sequence.
Substitution o f an unnatural amino acid residue at just one o f these positions was hypothesized to
endow sufficient proteolytic stability for future cell-based studies.
At position 12, we
incorporated amino acid residues that would alter the backbone conformation (ACPC, ACHC,
proline, P-hGly, p3-hAla, D-Ala, or a null mutation). A variety o f phenylalanine derivatives
were incorporated at position 13 to compensate for the alteration o f the backbone by re­
positioning the benzyl side chain for interaction with the Bcl-xL surface (Figure 11).
The second generation parallel library was synthesized under the same conditions as the
parallel combinatorial p-scan library, with a few important modifications.
We noted for the
parallel combinatorial p-scan library that the compounds located at the center o f the plate were
synthesized in lower purity than those near the edges o f the plate, which was opposite our results
for the synthesis o f P-peptide combinatorial libraries.25 Also, reaction temperatures near the
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 11. Second generation parallel (a/p + a)-peptide library focused on the central residues of the C-terminal a peptide segment with a greater diversity of side chains (2 x 8 x 6 = 96 members).
center of the plate were higher than at the edges under microwave irradiation conditions,32 which
suggested that reaction temperatures were too high near the center, leading to undesired side
reactions. Reducing the reaction temperatures from 80°C to 70°C for coupling reactions and
from 90°C to 80°C for deprotection reactions increased the purity o f peptide products (Kevin T.
Beier, data not shown). We applied these conditions to the synthesis o f the library described in
Figure 11 and obtained products in higher purity than in the previous parallel combinatorial Pscan library (Figure 12).
The crude (a /p + a ) peptide products were screened at three different concentrations in
the FP assay. The original sequence 5-1 had the highest activity, while compounds with a single
substitution were approximately 10-fold less active (Figure 13), and library members with two or
more substitutions had no detectable activity.
This result is consistent with our previous
findings, indicating that the side chain spacing along the backbone o f this class o f inhibitors is
critical for binding to the B H 3 -recognition cleft o f B c1-x l .
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259
3000
2000
Abs
(mV)
1000
Time (min)
Figure 12. HPLC traces (220 nm) o f A) a representative library member and B) and C) the two lowest purity
members from the second generation parallel library (Figure 11). “M” indicates the identification by MALDI-TOF
MS of an oligomer with a molecular weight corresponding to the expected library member.
90
80
70
60
1: 5-1, all a C-terminus
2: A12-»pGly
^NH 0
4: A12—>ACHC X l j
5: A12-ACPC
(J T
7: A12—*(D)-Ala
ACHC
9; 5-21, D11—# -A sp
17: 5-19, F 1 3 -p 3-Phe
•§ 50
2
2
e
Z 40
30
20
26072267374447^8603428083739^2036461
10
v - ’» * r * - o r o t o a > t N i r > c o
M
N
M
rt
n
f-»
rt
©
t
CO
to
C&
CM
«n
to
Well
Figure 13. Initial screening results for the second generation parallel library (Figure 11) in the Bcl-xL/Bak FP assay
at a 1:1000 dilution.
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260
5.2.5 Structure-Based Design
Concurrent with our combinatorial efforts, we were also working to further understand
the structure/activity relationship of compound 5-1 through traditional techniques. We prepared
and tested a series o f N-terminally truncated peptides and found that APC1 and Ala2 did not
significantly contribute to the binding affinity (Figure 14). We adopted peptide 5 -I 3 . 15, or 5-25,
as the lead compound for further optimization.
log [competitor], pM
•
5 - 1 1 1 5 0 .0 4 2 |iM
▼
■
5 - 1 2 1 5 0 .0 7 4
♦
- I 3 - 1 5 0 .0 4 2
•
A
5
5- 14,s
5- 15-15
5 - 1 6-i 5
3 .8 p M
24
-2 4 0
Figure 14. Dose-response of truncated (a/p + a ) peptides in the Bcl-xL/Bak FP assay. Figure adapted from Jack D.
Sadowsky.
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261
We hypothesized that substitution o f the alanine residue in the a-peptide segment
with an unnatural a-am ino acid residue could confer the desired proteolytic stability
without
the large reduction in binding affinity caused by incorporation of a p-amino acid residue. We
incorporated the a-helix promoting residue aminoisbutyric acid (Aib), since the peptide ligand is
believed to bind in an a-helical conformation. The compound containing Aib, 5-26, had an IC50
o f 120 nM, only about 2-fold higher than lead compound 5-25 (Table 4).
Table 4. Binding affinity and proteolytic stability o f selected foldamer sequences.
Sequence
No.
5-25
O
0
\ NH
0
HjN^NH
°^s
\^
0
\
0
HjN^NH
5-27
O
5-28
0
\N
H
h!(X „
0=\ / ^
\ .
°\
*°
HO
NHj
,
5-26
% Intact
ti/2 (min)
Peptide
1^50
(nM) 36 hr, Pronase Chymotrypsin
50% FBS
°\
y~\
\
NHZ
59
20
< 10
80
140
92
120
>21 hr
10
180
H2N'^NH
/
0 V,
\ NH
HjN
°\ °\
«r°
O
° \
HjNJss,NH
0
OVN
I^
nh=
° \
° \
0 \0
r°
)~\
\ NH
HO
^J
150
73
0
°\,
\
° \
>•
590
94
° \
b
OV.O
\
>21 hr >21 hr
An A rg->p3-hArg mutation at the C-terminus was expected to confer additional
proteolytic stability with only a minor impact on the binding affinity.
This hypothesis was
verified through the synthesis and testing o f compound 5-27, which was 4-fold less active than 525. The Ala->Aib and A rg->p3-hArg modications were combined in oligomer 5-28. However,
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262
the changes proved to be too dramatic as the IC50 o f 5-28 was 590 nM, 10-fold less potent
than 5-25.
2000
1500
Abs
(mV) 1000
500
Oligomer 5-25
Time = 0 hr
0
5
10
15
a
25
FBS
30
40
35
45
50
55
60
Time <min)
100
o
ts
5-25
-4^- 5-25
5-26
5-27
-mir- 5-28
c
0
5
10
15
20
25
30
35
Time (hr)
Figure 15. Top) HPLC trace (220 nm) o f oligomer 5-25 in 50% FBS at time 0 hr. Bottom) Time course of
oligomer degradation in 50% FBS.
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40
263
5.2.6 Proteolytic Stability
Compound 5-25 and a small set o f oligomers (5-26 to 28) having reasonable binding
affinities and containing unnatural amino acid substitutions in the C-terminal a-peptide segment
were assayed for stability to treatment with a variety o f proteases.3’14 Compound 5-25 was
incubated with 50% fetal bovine serum (FBS) in PBS buffer at 37°C, and its degradation over
time was monitored by RP-HPLC analysis (UV detection at 220 nm) o f the sample mixture.
Only 20% of the original sample remained intact after 36 hr (Table 4, Figure 15). MS analysis o f
new peaks in the chromatogram showed the transient appearance o f a fragment corresponding to
residues 1-9, indicating proteolytic cleavage between Asp9 and AlalO.
Fortunately, the
modification o f AlalO to Aib conferred nearly complete stability at this cleavage site, as 5-26
was still 92% intact after 36 hr. The A rg->p3-hArg modification at the C-terminus increased the
stability o f peptide 5-27 (73% intact) relative to 5-25, but not to the same degree as the Aib
substitution. Combining both modifications did not make peptide 5-28 significantly more stable
than compound 5-26.
A) Pronase
|!1 0 0
f 80
I 60
i «■
|
20
B) Chymotrypsin
■
Substrate
(
5
Cleavage Product
Cleavage Product
■
ti * < 1 ° m,n
10
15
20
Substrate
25
FBS
C) Cleavage Points
--
H
-N , / , ' ' k V N v . - A
Chymotrypsin
)
H
N
v y y :- ... . A
o
,.
▼
jvj
i
I
»
74,
H
N
6
o
,,
▼
a
o
4f
’
>
H
N
HO
NH
H3N ^ N H
25
h2n
O fd
O
20
15
T.m e(h)
Time (h)
H
80 mln
'
10
NHj
o
m2
NH
Pronase
"NH
Figure 16. Time course of proteolysis o f peptide 5-25 by A) Pronase and B) chymotrypsin with cleavage points
indicated in C). Figure adapted from Jack D. Sadowsky.
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264
A similar trend in relative proteolytic stability was observed when peptides 5-25
through 5-28 were treated with Pronase or chymotrypsin.
Compound 5-25, with the all a -
peptide C-terminus, was completely degraded by Pronase in less than 10 minutes. Substitution
of p3-hArg at position 13 increased the half life o f 5-27 to only 10 minutes, but the Ala 10Aib
substitution made peptide 5-26 significantly more stable (fi/2 = 2 hr). Peptide 5-28, with both
sequence modifications, was less than 50% degraded even after 21 hr. Pronase was found to
cleave the amide bond joining residues AlalO and P h e ll. Chymotrypsin cleaved the peptides
between Phel 1 and A snl2, as expected. Treatment with chymotrypsin degraded half o f peptide
5-25 in 80 minutes and half o f compound 5-27 in 180 minutes. The half lives o f peptides 5-26
and 5-28 when treated with chymotrypsin were both greater than 21 hr. We concluded that
incorporating Aib at position 10 in the a-peptidic C-terminus o f the chimeric ligand achieved the
best compromise between increasing proteolytic stability and maintaining binding affinity.
5.3 Conclusions
The optimization of chimeric (a /p + a)-peptide 5-1 for both binding affinity to the BH3recognition domain o f Bcl-xL and proteolytic stability was achieved through application of
microwave-assisted combinatorial synthesis, a high-throughput FP assay, and structure-based
design. The resulting sequence (5-26) was truncated by two residues at the N-terminus and had
an Ala->Aib substitution in the a-peptidic C-terminal segment.
The IC50 value o f this
compound was 140 nM, 2.5-fold higher than lead sequence 5-1, but 5-26 was much more stable
to treatment with FBS, Pronase, and chymotrypsin.
We found that the binding affinity o f the hybrid (a/p + a ) scaffold was extremely
sensitive to side chain and backbone modifications within the a-segment. Substitution o f even a
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265
single P-amino acid within the six C-tenninal residues reduced binding affinity by 2- to 90fold. However, combinatorial variation o f the hydrophobic side chains in peptide 5-1 believed to
interact with
B c 1 -x l
in the bound state produced a number o f compounds that were slightly more
active.
Both split-and-mix and parallel synthetic techniques in conjunction with a highthroughput FP assay were used for lead optimization, and the strengths and weaknesses o f each
synthetic format were revealed during the process. Either method provided rapid access to large
numbers of oligomers in acceptable purities through application o f microwave irradiation,
though the one-bead-one-compound format is recommended if the library size exceeds more than
a few hundred members.
The Bcl-xi/Bak FP assay could be used to reliably screen crude
products in a high-throughput manner. In the one-bead-one-compound format, the FP assay was
capable of distinguishing a hit that was approximately 10-fold more active than the other
members of the library (i.e., compound 5-1 in the combinatorial p-scan library), as is typically
required in the initial discovery phases o f a project. However, if the oligomers were synthesized
in parallel, then compounds with activities differing by 10-fold were much more readily
distinguished.
The assay’s sensitivity was somewhat reduced when screening material from
single beads, perhaps due to the lower purity o f the products relative to those prepared in
parallel, as judged by HPLC analysis.
We found that parallel synthesis, which avoids LS-
MS/MS sequencing and re-synthesis before purification and validation o f active compounds, is
more time-efficient for elucidation o f the SAR o f a lead compound.
We improved our
microwave-assisted 96-well plate parallel synthesis procedures by reducing the reaction
temperatures by 10°C. Although the combinatorial chemistry described herein did not result in a
more active compound, it allowed us extensively test our hypotheses relative to side chain and
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266
backbone modification through investigation o f a large number o f analogues and obtain
results more quickly, so that we knew to explore other avenues.
Application o f these
combinatorial synthetic techniques in combination with the “chimeric foldamer” and (3-scan
design principles may result in a general approach for the inhibition o f therapeutically important
protein-protein interactions.
5.4 Experimental Methods
5.4.1 General Procedures
Fmoc-(5',5)-ACPC and Fmoc-(i?,5)-APC(Boc)-OH were prepared by the method o f Lee
et al.34 Frnoc-(.SVS')-ACIIC was prepared by the method o f Schinnerl et al.35 Fmoc-(iS)-p3-hNleOH, Fmoc-(S)-p3-hPhe-OH, Fmoc-(S)-|33-hTrp(Boc)-OH, Fmoc-(S)-p3-hLeu-OH, Fmoc-0S)-p3hCha-OH (Cha = cyclohexylalanine), Fmoc-(S)-p3-hAsp(fBu)-OFI, Fmoc-(5)-p3-hAla-OH,
Fmoc-(5)-p3-hGln(Trt)-OH, Fmoc-(S)-p3-hArg(Pbf)-OH, and Fmoc-(5)-p3-hxPhe-OH (hxPhe =
backbone homologated, side chain extended phenylalanine) were prepared from their
corresponding Fmoc-L-a-amino acids (Novabiochem and SynPep) as described previously36 or
were purchased from Peptech. Fmoc-(6)-p2-hPhe-OH, Fmoc-(5)-p2-hxPhe-OH, and Fmoc-(R)p2-hPhe-OH were prepared by the method o f Lee et al.37 Methanol, CH 2 CI2 and acetonitrile
were purchased from Burdick & Jackson. Piperidine, 1-hydroxybenzotriazole hydrate (HOBt),
/Pr2 EtN, trifluoroacetic acid (TFA), triethylsilane, triisopropylsilane, and DMSO were purchased
from Aldrich. NovaSyn TGR resin (0.25 mmol/g loading), Fmoc-p-Gly-OH, O-benzotriazol-1yl-A A, N A ’-tetramethyluron ium hexaflurorophosphate (HBTU), and Fmoc-a-amino acids were
purchased from Novabiochem or SynPep. Fmoc-a-aminoisobutyric acid was purchased from
Chem Impex. Polystyrene A RAM macrobeads (500-560 pm diameter, 0.55 mmol/g loading)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
267
were purchased from Rapp Polymere. DMF (biotech grade solvent, 99.9+ %) was purchased
from Aldrich and stored over Dowex ion exchange resin. Dry CH 2 CI2 and z'P^EtN were distilled
from calcium hydride.
5.4.2 Split-and-Mix Library Synthesis on PS Macrobeads with Microwave Irradiation
The side chain optimization library (Figure 3, JKM VI 199) was synthesized using splitand-mix techniques on PS macrobeads using microwave irradiation in a multimode microwave
reactor.
PS A RAM macrobeads (661 mg, 364 pmol, ~ 4,800 beads) were placed in a
polypropylene solid phase extraction (SPE) tube (25 mL, Alltech), and swelled with DMF for
approximately 10 min.
The resin was washed (5 x DMF, 5 x CH 2 CI2 and 5 x DMF).
Deprotection solution (20 mL o f 20% piperidine in DMF (v/v)) was added to the resin, and the
tube was capped and placed on a shaker for 2 hr. The resin was washed as before. In a separate
vial, Fmoc-Arg(Pbf)-OH (708.5 mg, 1092 pmol) was activated by adding HBTU (2184 pL o f
0.5 M solution in DMF), DMF (16 mL), HOBt (2184 pL of 0.5 M solution in DMF), and
/P^EtN (2184 pL of 1.0 M solution in DMF). The mixture was vortexed and allowed to stand
for 1 min before being added to the resin.
The tube was capped and placed on a shaker
overnight. The resin was washed, and deprotection solution was added (20 mL). The vessel was
placed in one slot o f a 52-position turntable inside the multimode microwave reactor (CEM
MARS). The fiber optic temperature sensor was suspended in the reaction mixture by pressing it
through a small hole (made with a needle) in the plastic top cap of the SPE tube and placing the
cap loosely on the reaction vessel. The sample was subjected to three cycles o f irradiation in the
microwave reactor (600 W maximum power, 90°C, ramp 2 min, cool-down 10 min).
All
microwave irradiations were conducted at atmospheric pressure. The tube was removed from the
microwave reactor, and the resin was washed. Fmoc-Asn(Trt)-OH was activated as before and
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
268
added to the resin. The sample was subjected to six cycles of irradiation in the microwave
reactor (600 W maximum power, 80°C, ramp 2 min, cool-down 10 min). The tube was removed
from the microwave reactor, and the resin was washed and Fmoc-deprotected with microwave
irradiation as before. After washing, the resin was partitioned into five aliquots o f approximately
equal volume using a spatula with care not to crush the swollen beads. Each resin sample was
placed in a 15 mL SPE tube (Alltech). Fmoc-Phe-OH, Fmoc-Cha-OH, Fmoc-Leu-OH, FmocTrp(Boc)-OH, and Fmoc-Tyr(?Bu)-OH (218 pmol o f each) were each activated in separate vials
by adding HBTU (437 pL o f 0.5 M solution in DMF), DMF (3.2 mL), HOBt (437 pL o f 0.5 M
solution in DMF), and /Pr2EtN (437 pL o f 1.0 M solution in DMF) and vortexing. One coupling
solution was added to each aliquot o f resin. The samples were evenly distributed around the
turntable within the multimode microwave reactor, the fiber optic probe was inserted into one
sample, and the samples were irradiated (6 cycles, 600 W maximum power, 80°C, ramp 2 min,
cool-down 10 min). (We found later that heating coupling solutions o f different amino acids
simultaneously in the multimode microwave reactor while monitoring the temperature o f one
sample led to differences in reaction temperatures among the different reaction vessels. This
artifact was avoided in the future by performing sequential couplings.) The resin was washed,
combined in the 25 mL SPE tube, suspended in DMF, and thoroughly mixed.
Fmoc-
deprotection and coupling o f Fmoc-Ala-OH were performed. The resin was split into two equal
portions.
Fmoc-Asp(/Bu)-OH (546 pmol ) was coupled to one aliquot o f resin, and Fmoc-
Glu(fBu)-OH was coupled to the other aliquot. The resin was combined, Fmoc-deprotected, and
Fmoc-Gly-OH was coupled. The resin was split into four equal portions. Fmoc-(5)-|3 -hNleOH, Fmoc-(S)-p3-hPhe-OH, Fmoc-(S)-p3-hTrp(Boc)-OH, or Fmoc-ACPC-OH (273 pmol) were
each coupled to a different aliquot o f resin. The resin was recombined and subjected to two
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269
deprotection/coupling cycles to add Fmoc-Lys(Boc)-OH and Fmoc-ACPC-OH. After Fmocdeprotection, the resin was split into five equal portions. Fmoc-Leu-OH, Fmoc-Phe-OH, FmocTrp(Boc)-OH, Fmoc-xLeu-OFI, or Fmoc-Cha-OFI (218 pmol) were each coupled to a different
aliquot o f resin. The resin was recombined and subjected to two deprotection/coupling cycles to
add Fmoc-ACPC-OFl and Fmoc-Arg(Pbf)-OH. After Fmoc-deprotection, the resin was split into
five equal portions.
Fmoc-ACPC-OFI, Fmoc-(S)-p3-hPhe-OFI, Fmoc-(S)-p3-hTrp(Boc)-OFl,
Fmoc-fS’)-p3-Cha-OH, and Fmoc-(5)-p3-Leu-OFI were each coupled to a different aliquot of
resin.
The resin was recombined and subjected to two deprotection/coupling cycles to add
Fmoc-Ala-OFI, and Fmoc-APC(Boc)-OFI. After Fmoc-deprotection and washing (5 x DMF, 5 x
CH2C12, 5 x DMF, and 5 x CH 2 CI2 ), the peptides were N-terminally acetylated by adding 20 mL
o f a 14:5:1 solution o f CFfCL/acetic anhydride/triethylamine and shaking for 30 min. After
washing (5 x CH 2 CI2 and 5 x MeOH) and drying under a stream o f N2, the resin was arrayed
(one bead per well) into 15 384-well polypropylene plates (Costar) using tweezers and a bead
arrayer. The bead arrayer (Figure 17) had a pinhole at each position o f a 384-well plate and was
connected to both a vacuum aspirator and a N 2 line. A sample o f beads was poured into the
trough with a vacuum being pulled on the device. The resin was maneuvered until one bead was
positioned at each pinhole and held there with the vacuum. Excess resin was removed. A 384well plate was inverted and placed in the trough over the beads.
While being held firmly
together, the bead arrayer and plate were inverted, and the valve was switched from the vacuum
source to the N 2 line. The arrayer and plate were tapped smartly on the benchtop to transfer the
beads from the arrayer to the plate.
The vacuum was reapplied, and the bead arrayer was
removed. Any beads remaining in the arrayer were transferred with tweezers. The 384-well
plate was scanned to ensure that each well contained only one bead.
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270
The material on each bead was cleaved from the solid support with simultaneous side chain
deprotection (80 pL, 50:50:5:5 TFA:CH2Cl2 :triethylsilane:water, 2 h, RT, with orbital shaking;
plates were covered with aluminum foil. At the end o f the reaction, the cleavage solutions were
concentrated by rotary evaporation (RT, 4 hr, SpeedVac, Thermo Savant). The crude peptide
mixtures were dissolved in 80 pL o f DMSO; 10 pL o f this stock solution was used for the FP
assay, while the remaining solution was reserved for analytical characterization and compound
(hit) identification.
The crude oligomer mixtures from 50 beads were analyzed by HPLC
(Shimadzu); 30 pL was injected on a C4-silica reversed-phase analytical column (5 pm, 4 mm x
250 mm, Vydac) and eluted with a gradient o f acetonitrile in water (10 - 60%, 25 min, 0.1% TFA
in each) at a flow rate o f 1 mL/min. The chromatograms typically showed a major peak riding
on a mound o f impurities (Figure 4). The major peak in each HPLC run was collected, and
oligomer masses were measured by MALDI-TOF-MS
(Bruker Reflex II, a-cyano-4-
hydroxycinnamic acid matrix). The library was screened in the Bcl-xi7Bak FP assay.
Figure 17. Bead arrayer, courtesy of Harvard Department of Chemistry and Chemical Biology.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
271
Table 5. Sequences of side chain optimization library (Figure 3) members 1-200.
Sequence
ompoun
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
63
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
APC
APC
APC
APC
APC
APC
APC
APC
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
APC Ala
APC Ala
APC Ala
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
ACPC
P’-Leu
Ji '-Cha
(T’-Phe
(C-Trp
ACPC
Jl'-Leu
p'-Cha
Jl’-Phe
|Tl-Trp
Arg ACPC
ACPC
Arg ACPC
Arq ACPC
Arg ACPC
Arq ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
ACPC
Ala jiJ-Leu
ACPC
Ala p'-Cha Arg ACPC
Ala p ’-Phe Arg ACPC
Ala (l!-Trp Arg ACPC
Ala ACPC Arq ACPC
Ala p ’-Leu Arg ACPC
Ala p'-Cha Arg ACPC
Ala Jl’-Phe Arg ACPC
Ala Pj-Trp Arg ACPC
Ala ACPC Arg ACPC
Ala pJ-Leu Arg ACPC
Ala p’-Cha Arg ACPC
Als Jl'-Phe Arg ACPC
Ala p’-Trp Arg ACPC
Ala ACPC Arg ACPC
Ala p'-Leu Arg ACPC
Ala p'-Cha Arg ACPC
Ala p’-Phe Arq ACPC
Ala p’-Trp Arq ACPC
Ala ACPC Arg ACPC
Ala p'-Leu Arg ACPC
Ala p’-Cha Arg ACPC
Ala p’-Phe Arg ACPC
Ala pJ-Trp Arg ACPC
Ala ACPC Arg ACPC
Ala p’-Leu Arg ACPC
Ala p’-Cha Arg ACPC
Ala p ’-Phe Arg ACPC
Ala P'-Trp Arg ACPC
Ala ACPC Arg ACPC
Ala p'-Leu Arq ACPC
Ala P'-Cha Arg ACPC
Ala p ’-Phe Arg ACPC
Ala Pj-Trp Arg ACPC
Ala ACPC Arg ACPC
Ala Jl’-Leu Arg ACPC
Ala p’-Cha Arq ACPC
Ala p ’-Phe Arg ACPC
Ala P’-Trp Arg ACPC
Ala ACPC Arg ACPC
Ala p ’-Leu Arg ACPC
Ala jr’-Cha Arg ACPC
Ala p'-Phe Arg ACPC
Ala P’-Trp Arg ACPC
Ala ACPC Arg ACPC
Ala p’-Leu Arg ACPC
Ala P'-Cha Mg ACPC
Ala p’-Phe Mg ACPC
Ala p’-Trp Mg ACPC
Ala ACPC Arq ACPC
Ala P’-Leu Arg ACPC
Ala p’-Cha Mg ACPC
Ala P’-Phe Mg ACPC
Ala p’-Trp Arg ACPC
Ala ACPC Mq ACPC
Ala p’-Leu Arg ACPC
Ala (i’-Cha Arg ACPC
Ala p’-Phe Arq ACPC
Ala p’-Trp Arg ACPC
Ala ACPC Mg ACPC
Ala p'-Leu Arq ACPC
Ala p’-Cha Arg ACPC
Ala p’-Phe Arq ACPC
Ala P’-Trp Arg ACPC
Ala ACPC Arg ACPC
Ala p'-Leu Mg ACPC
Ala P’-Cha Arg ACPC
Ala P’-Phe Mg ACPC
Ala p’-Trp Arg ACPC
Ala ACPC Arg ACPC
Ala P’-Leu Mg ACPC
Ala p’-Cha Arg ACPC
Ala p'-Phe Mg ACPC
Ala P’-Trp Arg ACPC
Ala ACPC Mg ACPC
Ala p'-Leu Arg ACPC
Ala p'-Cha Mg ACPC
Ala p’-Phe Mg ACPC
Ala P’-Trp Arg ACPC
Ala ACPC Arg ACPC
Ala p’-Leu Mg ACPC
Ala p'-Cha Mq ACPC
Ala p’-Phe Mg ACPC
Ala P’-Trp Mg ACPC
Ala ACPC Arg ACPC
Ala p’-Leu Mg ACPC
Als P’-Cha Mg ACPC
Ala p’-Phe Mg ACPC
Ala pJ-Trp Mg ACPC
Leu
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Trp
Trp
Trp
Trp
Trp
xleu
xLeu
xLeu
xLeu
xLeu
Cha
Cha
Cha
Cha
Cha
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Trp
Trp
Trp
Trp
Trp
xLeu
xLeu
xLeu
xLeu
Cha
Cha
Cha
Cha
Cha
Leu
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Trp
Trp
Trp
Trp
Trp
xLeu
xLeu
xLeu
xLeu
xleu
Cha
Cha
Cha
Cha
Cha
Leu
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Tip
Trp
Trp
Trp
Trp
xLeu
xLeu
xLeu
xLeu
xLeu
Cha
Cha
Cha
Cha
Cha
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
9
Lys Jl’-NIe
Lvs Jl'-NIe
Lys Jl’-NIe
Lvs Jl'-NIe
Lys |V-Nle
Lvs Jl’-NIe
Lvs Jl’-NIe
Lvs (T’-Nle
Lys
Lvs
Lys
IJ'-NIe
Jl’-NIe
p'-NIe
LVS Jl’-NIe
Lvs Jl’-NIe
Lvs Jl’-NIe
Lvs p'-NIe
LVS Jt’-NIe
Lvs [T’-Nle
Lvs JJ’-Nle
Lvs fi'-NIe
Lys Jl’-NIe
Lvs Jl'-NIe
LVs Jl’-NIe
Lys Jl'-NIe
Lvs Jl'-NIe
Lys
Lvs
Lys
Lvs
Lvs
Lvs
Lvs
Lvs
Lys
Lvs
Lys
Lvs
Lys
Lys
Lys
Lys
Jl'-NIe
Jl’-Phe
Jl'-Phe
Jl'-Phe
p'-Phe
Jl’-Phe
P’-Phe
Jl'-Phe
Jl’-Phe
Jl’-Phe
Jl'-Phe
Jl’-Phe
Jl’-Phe
Jl'-Phe
Jl'-Phe
Jl'-Phe
Jl'-Phe
Jl'-Phe
Lvs
Lvs
Lys Jl’-Phe
Lvs |i'-Phe
Lys Jl’-Phe
Lvs p'-Phe
Lys Jl’-Phe
Lys Jl’-Phe
Lvs Jl’-Phe
Lvs Jl’-Phe
Lvs p ’-Trp
Lvs p ’-Trp
Lys p’-Trp
Lys Jl’-Trp
Lvs P’-Trp
Lys p'-Trp
Lys P'-Trp
Lvs P’-Trp
Lys P’-Trp
Lys Jl'-Trp
Lys p'-Trp
Lvs P’-Trp
Lvs p’-Trp
Lys p'-Trp
Lys P'-Trp
Lys pJ-Trp
Lvs p'-Trp
Lvs pJ-Trp
Lys P’-Trp
Lys p’-Trp
Lys P’-Trp
Lvs P’-Trp
Lvs p’-Trp
Lys Jl'-Trp
Lys P’-Trp
Lys ACPC
Lvs ACPC
Lys ACPC
Lvs
Lys
Lys
Lys
Lvs
Lys
Lvs
LVS
Lys
Lvs
Lvs
Lvs
LVS
Lys
Lys
Lys
LVS
Lys
Lys
Lys
Lvs
Lys
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
10
Gly
Glv
Gly
Gly
Gly
Gly
Gly
11
Asp
Asp
Asp
Asp
Asp
Asp
Asp
12
Ala
Ala
Ala
Ala
Ala
Ala
Ala
13
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Asn
Asn
Asn
Asn
Asn
Gly
Gly
Gly
Gly
Asp
Asp
Asp
Asp
Ala
Ala
Ala
Ala
Phe
Phe
Phe
Phe
Asn
Asn
Asn
Asn
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Gly
Gly
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
AJa
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe.
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Glv
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gty
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Glv
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Glv
Gty
Gly
Gly
Glv
Gly
Gly
Gly
Gly
14
Asn
Compound
N
Ac
Ac
Ac
Ac
Ac
Ac
107
Ac APC Ala
108
109
110
111
Ac
Ac
Ac
Ac
C
NH,
NH,
NH,
nh 2
NH,
nh 2
NH,
Arg
Arg
Arg
NH,
NH,
NH2
NH,
NH,
NH,
NH,
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Mg
Arg
Arg
Arg
Mg
Mg
Mg
Mg
Mg
Mg
Mq
Mq
Arg
Mg
Arg
Mg
Mg
Arg
Mg
Mg
Mg
Mg
Mg
Arg
Arg
Arg
Arg
Mg
Arg
Mg
Arg
Arg
Arg
Arq
Arg
Mg
Mg
Mq
Mg
Arq
Arg
Mq
Mg
Mg
Arg
Mg
Arg
Mg
Mg
Mg
Mg
Mq
Mg
Mg
Mg
Mq
Mg
Mg
Mg
Mg
Mg
Mg
Mg
Arg
Arg
Arg
Arg
Mg
Arg
Arg
Mq
Mg
Arg
Arg
Mg
Mg
Mg
Mg
Mg
Mq
Mg
Mg
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
nh2
NH,
NH,
NH,
NH,
nh 2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
nh 2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
nh2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
Sequence
101
102
103
104
105
106
15
Arg
Arg
Arg
Arg
Arg
Arg
Arq
199
200
APC Ala ACPC
APC Ala p'-Leu
APC Ala fl'-Cha
APC Ala Jl'-Phe
APC Ala Jl'-Trp
APC Ala
APC
APC
APC
APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
AC APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Mq
Arg
Arq
Arg
Arg
ACPC Arg
Jl’-Leu Arg
Ji'-eha Arq
p’-Phe Arg
P'-Trp Arq
Ala
Ala
Ala
Ala ACPC
Ala Jl’-Leu
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
Arg ACPC
Arq ACPC
Jl'-Cha Arg ACPC
p'-Phe Arq ACPC
p ’-Trp Arg ACPC
ACPC Arg ACPC
p’-Leu Arg ACPC
Jl’-Cha Arg ACPC
P’-Phe Arq ACPC
P’-Trp Arg ACPC
ACPC Arq ACPC
p’-Leu Arg ACPC
p’-Cha Arg ACPC
p'-Phe Arq ACPC
p’-Trp Arg ACPC
ACPC Arq ACPC
p’-Leu Arg ACPC
p ’-Cha Arg ACPC
P'-Phe Arg ACPC
p’-Trp Arg ACPC
ACPC Arg ACPC
p ’-Leu Arg ACPC
p’-Cha Arg ACPC
p’-Phe Arg ACPC
p’-Trp Arg ACPC
ACPC Arq ACPC
P’-Leo Arg ACPC
p’-Cha Arg ACPC
p’-Phe Arq ACPC
p’-Trp Arg ACPC
ACPC Arg ACPC
p’-Leu Arg ACPC
p'-Cha Arq ACPC
p’-Phe Arq ACPC
pJ-Trp Arg ACPC
ACPC Arq ACPC
p’-Leu Arg ACPC
(lJ-Cha Arq ACPC
p’-Phe Arg ACPC
p’-Trp Arg ACPC
ACPC Arg ACPC
p’-Leu Arq ACPC
p’-Cha Arg ACPC
pJ-Phe Arg ACPC
p'-Trp Arq ACPC
ACPC Arg ACPC
p’-Leu Arq ACPC
p’-Cha Arq ACPC
P’-Phe Arq ACPC
P’-Trp Arq ACPC
ACPC Arq ACPC
p’-Leu Arq ACPC
p’-Cha Arq ACPC
p’-Phe Arg ACPC
P’-Trp Arq ACPC
ACPC Arq ACPC
p'-Leu Arq ACPC
p’-Cha Arq ACPC
p’-Phe Arg ACPC
p'-Trp Arg ACPC
ACPC Arq ACPC
P’-Leu Arq ACPC
pJ-Cha Arq ACPC
p ’-Phe Arg ACPC
P’-Trp Arq ACPC
ACPC Arg ACPC
pJ-Leu Arq ACPC
P’-Cha Arg ACPC
APC
pJ-Phe Arq ACPC
APC
p’-Trp Arg ACPC
APC
APC
ACPC Arq ACPC
p’-Leu Arg ACPC
APC
P’-Cha Arq ACPC
APC
P’-Phe Arq ACPC
APC
P'-Trp Arq ACPC
APC
APC
ACPC Arq ACPC
p’-Leu Arg ACPC
APC
p’-Cha Arg ACPC
APC
p’-Phe Arq ACPC
APC
p’-Trp Arq ACPC
APC
APC
ACPC Arq ACPC
pJ-Leu Arq ACPC
APC
p’-Cha Arq ACPC
APC
p’-Phe Arq ACPC
APC
P’-Trp Arg ACPC
APC
APC
ACPC Arq ACPC
P’-Leu Arq ACPC
APC
p’-Cha Arg ACPC
APC
APC Ala p’-Phe Arq ACPC
APC Ala P’-Trp Arg ACPC
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Leu
Leu
9
Jl'-NIe
p'-NIe
Jl'-NIe
Leu
Leu
Phe
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
Lys
Lys
Lvs
Lvs
Lvs
Lys
Phe
Phe
Phe
Phe
Trp
ACPC
ACPC
ACPC
ACPC
ACPC
Trp
Trp
Trp
Trp
xLeu
xLeu
xLeu
xLeu
xLeu
Cha
Cha
Cha
Cha
Cha
Leu
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Trp
Trp
Trp
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
Lvs Jl’-NIe
Lvs Jl'-NIe
Lys Jl’-NIe
Lvs Jl'-NIe
Lvs Jl’-NIe
Lvs Jl’-NIe
Lys Jl’-NIe
Lys Jl’-NIe
Lys Jl’-NIe
Lvs p’-NIe
Lys p ’-NIe
Lvs Jl’-NIe
Lys Jl’-NIe
Lvs p'-NIe
Lvs p’-NIe
Lvs p'-NIe
LVS p’-NIe
Lys p'-NIe
Lvs P’-NIe
Lvs pJ-Phe
Lvs Jl’-Phe
Lys p’-Phe
Lvs p’-Phe
Lvs p’-Phe
Lys p’-Phe
Lvs p’-Phe
Lvs p’-Phe
Lvs p’-Phe
Lys p’-Phe
Lys P’-Phe
Lvs Jl’-Phe
Lvs p’-Phe
Lvs p'-Phe
LVs p’-Phe
Lvs p ’-Phe
Lvs p'-Phe
LVS p'-Phe
Lvs p’-Phe
Lvs Jl’-Phe
Lvs p’-Phe
Lys P’-Phe
Lvs p’-Phe
Lvs (i’-Phe
Lvs P’-Phe
Lvs P’-Trp
Lys p’-Trp
Lvs P'-Trp
Lys p'-Trp
Lvs P'-Trp
Lys P’-Trp
Lys pJ-Trp
Lvs p’-Trp
LVS p’-Trp
Lvs P’-Trp
Lvs P'-Trp
Lys P’-Trp
Lys P’-Trp
Lvs P’-Trp
Lys p’-Trp
Lvs P’-Trp
Lys P’-Trp
Trp
Trp
xLeu
xLeu
xLeu
xLeu
xLeu
Cha
Cha
Cha
Cha
Cha
Leu
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Trp
Trp
Trp
Trp
Trp
xLeu
xLeu
xLeu
xLeu
xLeu
Cha
Cha
Cha
Cha
Cha
Leu
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Trp
Trp
Trp
Trp
Trp
xLeu
xLeu
xLeu
xLeu
xLeu
Cha
Cha
Cha
Cha
Cha
Lys
Lys
Lys
Lys
Lys
Lvs
Lys
Lys
Lys
Lys
Lys
Lvs
Lys
Lvs
Lys
Lvs
Lys
Lys
LVS
Lys
Lvs
Lys
Lvs
Lvs
Lvs
Lys
Lvs
Lys
Lvs
Lys
LVS
Lys
Lys
Jl’-NIe
Jl’-NIe
Jl'-NIe
P’-Trp
P’-Trp
p’-Trp
P’-Trp
p’-Trp
P’-Trp
P’-Trp
P’-Trp
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
10
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Glv
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Glv
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Glv
Glv
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Glv
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Glv
Gly
Glv
Gly
Glv
Gly
Gly
Gly
Glv
Glv
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
11
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
12
Ala
Ala
Ala
Ala
Ala
Ala
13
Cha
Cha
Cha
Cha
Cha
Cha
14
Asn
Asn
15
Arg
Arg
Asn
Asn
Asn
Asn
Arg
Arg
Arg
Arg
Ala
Ala
Ala
Ala
Ala
Cha
Cha
Cha
Cha
Cha
Asn
Asn
Asn
Asn
Asn
Arg
Arg
Arg
Arg
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ms
Ala
Ala
Ala
Ala
Ala
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Mg
Arg
Arg
Arg
Arg
Arg
Arq
Arq
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arq
Arg
Arg
Arg
Arq
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arq
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arq
Arq
Arg
Arg
Arq
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
AJa
AJa
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
AlaAla
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ale
Ala
Ala
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Ala
Ala
Ala
Ala
Ala
Ala
AJa
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Arg
C
NHj
NH,
NH,
NH,
NH,
NH,
NH;
NH2
NH;
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH2
NH,
NH,
NH,
NH,
NH,
NH,
nh2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
nh 2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
nh2
NH,
NH2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
nh2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
Arq NH,
Arg NH,
Arg NH,
Arq NH,
Arg NH,
Arg NH,
Arg NH?
Arg NH,
Arg NH,
Arg NH,
Arg NH,
272
Table 6. Sequences of side chain optimization library (Figure 3) members 201-400.
Compound
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
. 261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
Seauence
N
Ac APC Ala ACPC Arg ACPC
Ac APC Ala | 'J-Leu Arg ACPC
Ac APC Ala Jl’-Cha Arg ACPC
Ac APC Ala |l ’-Phe Arg ACPC
Ac APC Ala I' Trp Arg ACPC
Ac APC Ala ACPC Arg ACPC
Ac APC Ala Jl’-Leu Arg ACPC
Ac APC Ala [l '-Cha Arg ACPC
Ac APC Ala Jl’-Phe Arq ACPC
Ac APC Ala I*'-Trp Arg ACPC
Ac APC Ala ACPC Arg ACPC
Ac APC Ala |l l-Leu Arg ACPC
Ac APC Ala Jl’-Cha Arg ACPC
Ac APC Ala Jl’-Phe Arq ACPC
Ac APC Ala jv'-Trp Arg ACPC
Ac APC Ala ACPC Arq ACPC
Ac APC Ala [F-teu Arg ACPC
Ac APC Ala Jl'-Cha Arg ACPC
Ac APC Ala Jl’-Phe Arg ACPC
Ac APC Ala |1J-Trp Arg ACPC
Ac APC Al3 ACPC Arg ACPC
Ac APC Ala Jl'-Leu Arg ACPC
Ac APC Ala Jl'-Cha Arg ACPC
Ac APC Ala p'-Phe Arg ACPC
Ac APC Ala Jl’-Trp Arq ACPC
Ac APC Ala ACPC Arg ACPC
Ac APC Ala Jl’-Leu Arg ACPC
Ac APC Ala Jl’-Cha Arg ACPC
Ac APC Ala P ’-Phe Arg ACPC
Ac APC Ala p’-Trp Arg ACPC
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ha
Ala
ACPC
Jl’-Leu
Jl’-Cha
Jl'-Phe
Jl’-Trp
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ha
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
AJa
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
AJa
Ha
ACPC
P’-Leu
p’-Cha
p'-Phe
p’-Trp
ACPC
|t’ ;Leu
Jl’-Cha
J) '-Phe
Jl'-Trp
ACPC
Jl’-Leu
Jl'-Cha
p’-Phe
P’-Trp
ACPC
Jl’-Leu
p’-Cha
Jl’-Phe
Jl'-Trp
ACPC
Jl’-Leu
p ’-Cha
p'-Phe
p’-Trp
ACPC
p ’-Leu
Jl’-Cha
Jl’-Phe
p’-Trp
ACPC
P’-Leu
p’-Cha
p'-Phe
P’-Trp
ACPC
P’-Leu
p’-Cha
p’-Phe
p’-Trp
ACPC
p’-Leu
p’-Cha
p’-Phe
p3-Trp
ACPC
P’-Leu
Arq
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arq
Arg
Arg
Arg
Arq
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arq
Arg
Arg
Arg
Arg
Arg
Arg
Arq
Arg
Arg
Arg
Arg
Arg
p'-Cha Arg
p’-Phe Arg
p’-Trp Arg
ACPC Arg
p’-Leu Arg
p’-Cha Arg
P’-Phe Arg
P'-Trp Arg
ACPC Arg
p ’-Leu Arg
p’-Cha Arg
p’-Phe Arg
p’-Trp Arg
ACPC Arg
P’-Leu Arg
p’-Cha Arg
P’-Phe Arg
P’-Trp Arg
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Trp
Trp
Trp
Trp
Trp
xLeu
xLeu
xLeu
xLeu
xleu
Cha
Cha
Cha
Cha
Cha
Leu
ACPC Lvs
ACPC Lys
ACPC Lvs
ACPC Lys
ACPC Lvs
ACPC Lvs
ACPC Lvs
ACPC Lvs
ACPC Lvs
ACPC lys
ACPC Lvs
ACPC Lvs
ACPC Lys
ACPC Lvs
ACPC Lys
ACPC Lvs
ACPC Lys
ACPC Lys
ACPC Lys
ACPC Lys
ACPC Lys
Trp
Trp
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
Trp
xleu
xLeu
xleu
xleu
xLeu
Cha
Cha
Cha
Cha
Cha
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
Leu
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Trp
Trp
Trp
Trp
Trp
xLeu
xleu
x leu
xLeu
xLeu
Cha
Cha
Cha
Cha
Cha
Leu
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Trp
Trp
Tip
Trp
Tip
xLeu
x leu
xleu
xLeu
Cha
Cha
Cha
Cha
Cha
Leu
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Trp
Trp
Lvs
Lys
LVS
Lvs
Lvs
Lys
Lvs
Lys
LVS
Lvs
Lys
Lys
Lvs
Lvs
LVS
Lys
Lvs
Lvs
Lvs
Lvs
Lvs
Lys
Lvs
Lys
Lvs
Lvs
Lys
LVS
Lys
Lvs
Lvs
Lvs
Lvs
LVS
Lvs
LVS
Lvs
Lys
Lys
Lvp
Lys
L\s
Lys
Lvs
Lvs
Lys
Lvs
Lys
Lys
Lvs
Lys
Lys
Lys
Lvs
Lys
Lys
Lys
Lvs
Lvs
Lvs
Lys
Lys
Lys
Lvs
Lys
Lys
Lys
Jl’-NIe
Jl’-NIe
Jl’-NIe
Jl'-NIe
p ’-NIe
p'-NIe
Jl'-NIe
Jl'-NIe
Jl-NIe
Jl'-NIe
Jl’-NIe
Jl’-NIe
Jl’-NIe
Jl'-NIe
Jl’-NIe
Jl’-NIe
Jl’-NIe
Jl’-NIe
Jl'-NIe
|L’-Nle
p'-NIe
Jl’-NIe
p ’-NIe
P ’-NIe
P’-NIe
Jl’-Phe
P ’-Phe
p’-Phe
P ’-Phe
p’-Phe
p’-Phe
Jl'-Phe
p'-Phe
p’-Phe
p’-Phe
p'-Phe
p'-Phe
p ’-Phe
P ’-Phe
Jl’-Phe
p ’-Phe
p ’-Phe
p'-Phe
Jl’-Phe
Jl’-Phe
p’-Phe
Jl’-Phe
Jl’-Phe
P’-Phe
p’-Phe
Jl’-Trp
p ’-Trp
p’-Trp
p’-Trp
PJ-Trp
p’-Trp
Jl'-Trp
P’-Trp
p ’-Trp
p ’-Trp
p'-Trp
p’-Trp
p’-Trp
Jl’-Trp
p’-Trp
p’-Trp
ji^Trp
P’-Trp
Jl’-Trp
p'-Trp
P’-Trp
Jl'-Trp
p’-Trp
P'-Trp
p'-Trp
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
Lvs ACPC
Lys ACPC
Lys ACPC
Lvs ACPC
Lys ACPC
Lvs ACPC
Lys ACPC
Lvs ACPC
Lvs ACPC
Lvs ACPC
Lys ACPC
Lvs ACPC
10
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
11
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
12
Ala
AJa
Ala
AJa
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Asp
Asp
Gly
Gly
Gty
Gly
Gly
Gly
Gly
Glv
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
AJa
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
AJa
Ala
Ala
Ala
Ala
Ha
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
AJa
Ala
Ala
Aia
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gty
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Glv
Gly
Asp
Asp
Asp
Asp
Asp
Asp
13
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
14
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
15
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arq
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arq
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
C
nh ,
Compound
301
302
303
NH,
nh ,
nh 2
nh 2
NH2
nh2
nh 2
nh2
nh 2
nh2
nh2
NH,
304
305
306
307
308
309
310
311
312
313
nh 2
314
nh 2
315
316
317
318
319
320
321
nh 2
NH,
NH,
NH,
nh 2
NH,
NH,
NH2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH2
NH,
NH,
NH,
NH,
NH,
nh 2
NH,
NH,
NH,’
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
nh 2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
nh2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
3S1
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
Seauence
N
Ac APC Ala ACPC
Ac APC Ala Jl'-Leu
Ac APC Ala Jl’-Cha
Ac APC Ala Jl’-Phe
Ac APC Ala Jl’-Trp
Ac
■Ac
Ac
Ac
Ac
Ac
Ac
Ac
APC
APC
APC
APC
APC
APC
APC
APC
Ala
Ala
Ala
Ala
Aia
Ala
Ala
Ala
ACPC
Jl'-Leu
Jl’-Cha
|t’-Phe
Jl’-Trp
ACPC
Jl'-Leu
Jl’-Cha
Arq ACPC
Arg ACPC
Arg ACPC
Arq ACPC
Arg ACPC
Arg ACPC
Arq ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Ac APC Ala Jl’-Phe Arg ACPC
Ac APC Ala p’-Trp Arq ACPC
Ac APC Ala ACPC Arg ACPC
ACPC
Ac APC Ala Jl’-Leu
Ac APC Ala Jl’-Cha Arg ACPC
Ac APC Ala Jl’-Phe Arq ACPC
Ac APC Ala Jl’-Trp Arg ACPC
Ac APC Ala ACPC Arq ACPC
Ac APC Ala Jl'-Leu Arq ACPC
Ac APC Ala p’-Cha Arq ACPC
Ac APC Ala Jl'-Phe Arq ACPC
Ac APC Ala P’-Trp Arg ACPC
Ac APC Ala ACPC Arg ACPC
Ac APC Ala p’-Leu Arq ACPC
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
Ala P’-Cha Arg ACPC
Ala p'-Phe Arq ACPC
Ala p’-Trp Arq ACPC
Ala ACPC Arg ACPC
Ala p’-Leu Arg ACPC
Ala p'-Cha Arq ACPC
Ala Jl’-Phe Arg ACPC
Ala Jl’-Trp Arg ACPC
Ala ACPC Arq ACPC
Ala Jl’-Leu Arg ACPC
Ala Jl’-Cha Arg ACPC
Ala Jl’-Phe Arg ACPC
Ala p’-Trp Arg ACPC
Ala ACPC Arg ACPC
Ala Jl'-Leu Arg ACPC
Ala p ’-Cha Arq ACPC
Aia p’-Phe Arg ACPC
Ala p’-Trp Arg ACPC
Ala ACPC Arg ACPC
Ala p’-Leu Arg ACPC
Ala p'-Cha Arq ACPC
Ala p’-Phe Arg ACPC
Ala P’-Trp Arg ACPC
AJa ACPC Arg ACPC
Ala Jl’-Leu Arq ACPC
Ala p’-Cha Arq ACPC
Ala p'-Phe Arg ACPC
Ala p’-Trp Arq ACPC
Ala ACPC Arg ACPC
Ala p'-Leu Arg ACPC
Ala p’-Cha Arq ACPC
Ala p’-Phe Arq ACPC
Ala p’-Trp Arg ACPC
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Aia
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
ACPC
P’-Leu
p’-Cha
p’-Phe
Jl’-Trp
Arg
Arg
Arg
Arg
Arg
ACPC
P’-Leu
Jl’-Cha
p’-Phe
p'-Trp
Arq
Arq
Arq
Arg
Arg
Arq
Arq
Arg
Arq
Arq
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
ACPC
P’-Leu
p’-Cha
p'-Phe
p’-Trp
ACPC
Jl'-Leu
p'-Cha
p’-Phe
Jl’-Trp
ACPC
p’-Leu
p’-Cha
p’-Phe
P’-Trp
ACPC
p’-Leu
P’-Cha
p’-Phe
p’-Trp
ACPC
P’-Leu
p’-Cha
p ’-Phe
p’-Trp
ACPC
p’-Leu
p’-Cha
p’-Phe
p’-Trp
Arg
Arg
Arq
Arq
Arg
Arg
Arq
Arg
Arq
Arg
Arq
Arg
Arg
Arg
Arg
Arg
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
Leu
Leu
Leu
ACPC Lys
ACPC Lys
ACPC Lys
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Trp
Trp
Trp
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
Trp
Lys
t-vs
Lvs
Lys
Lvs
Lvs
Lvs
Lvs
Lys
Lys
ACPC Lvs
Trp ACPC Lys
xLeu ACPC Lvs
ACPC LVS
xLeu ACPC Lvs
xleu ACPC Lvs
xleu ACPC Lys
Cha ACPC Lys
Cha ACPC Lvs
Cha ACPC Lys
Cha ACPC Lvs
Cha ACPC Lvs
Leu ACPC Lvs
Leu ACPC LVS
Leu ACPC Lys
Leu ACPC Lvs
Leu ACPC Lys
Phe ACPC Lys
Phe ACPC Lys
Phe ACPC Lys
Phe ACPC Lys
Phe ACPC Lys
Trp ACPC Lys
Trp ACPC Lvs
Trp ACPC Lvs
Trp ACPC Lys
Trp ACPC Lvs
xLeu ACPC Lvs
xleu ACPC Lvs
xLeu ACPC Lys
xLeu ACPC Lvs
xLeu ACPC Lys
Cha ACPC Lys
Cha ACPC Lys
Cha ACPC Lvs
Cha ACPC Lvs
Cha ACPC LVS
Leu ACPC Lys
Leu ACPC Lvs
Leu ACPC Lys
Leu ACPC LVS
Leu ACPC Lys
Phe ACPC Lys
Phe ACPC LVS
Phe ACPC Lys
Phe ACPC Lvs
Phe ACPC Lys
Trp ACPC LVS
Trp ACPC Lys
Trp ACPC Lvs
Trp ACPC LVS
Trp ACPC Lvs
xLeu ACPC Lvs
xleu ACPC Lys
xLeu ACPC Lys
ACPC Lys
xLeu ACPC Lys
Cha ACPC Lvs
Cha ACPC Lvs
Cha ACPC LvS
Cha ACPC Lvs
Cha ACPC Lvs
Leu ACPC Lvs
Leu ACPC Lys
Leu ACPC Lvs
Leu ACPC Lvs
Leu ACPC Lys
Phe ACPC Lvs
Phe ACPC Lys
Phe ACPC Lys
Phe ACPC Lys
Phe ACPC Lvs
Trp ACPC Lys
Trp ACPC Lys
Trp ACPC Lys
Trp ACPC Lys
Trp ACPC Lvs
xLeu ACPC Lys
xleu ACPC Lvs
xleu ACPC Lvs
xLeu ACPC Lvs
xLeu ACPC Lvs
Cha ACPC Lvs
Cha ACPC Lys
Cha ACPC Lvs
Cha ACPC Lvs
Cha ACPC Lys
Jl'-NIe
Jl’-NIe
Jl’-NIe
Jl'-NIe
Jl’-NIe
Ji'-NIe
Jl’-NIe
Jl’-NIe
Jl’-NIe
Jl’-NIe
Jl’-Nte
Ji’-Nte
Jl’-NIe
Ji’-Nte
Jl’-NIe
P’-NIe
p’-NIe
Jl’-NIe
p’-NIe
p'-NIe
p’-NIe
p’-NIe
P’-NIe
p’-NIe
p’-NIe
p’-Phe
p’-Phe
p’-Phe
P’-Phe
p’-Phe
p'-Phe
p’-Phe
P’-Phe
p’-Phe
P’-Phe
p’-Phe
Jl’-Phe
p’-Phe
Jl’-Phe
p'-Phe
p'-Phe
p’-Phe
P’-Phe
Jl’-Phe
p’-Phe
p’-Phe
Jl’-Phe
p’-Phe
p’-Phe
p’-Phe
Jl’-Trp
P’-Trp
P’-Trp
P’-Trp
P’-Trp
p’-Trp
P’-Trp
Jl’-Trp
P’-Trp
p’-Trp
p’-Trp
Jl’-Trp
P’-Trp
p'-Trp
p’-Trp
P’-Trp
p’-Trp
p’-Trp
P’-Trp
p’-Trp
p’-Trp
p ’-Trp
P’-Trp
P’-Trp
p’-Trp
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
10
Gly
Gly
Glv
Glv
Glv
Gly
Gly
Glv
Gly
Glv
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Glv
Glv
Gly
Glv
Gly
Gly
Glv
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Glv
Glv
Glv
GJy
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Glv
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Glv
Glv
Gly
Glv
Glv
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
11
Asp
Asp
12
Ala
Ala
13
Trp
Trp
14
Asn
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Trp
Asn
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Trp
Trp
Trp
Arg
Arg
Arg
Arg
Arg
Arg
Trp
Trp
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Aia
Aia
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Trp
Trp
Trp
Asn
Asn
Asn
Arg
Arg
Arg
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Arq
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
Trp
15
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arq
Arg
Arg
Arq
Arg
Arg
Arg
Arg
Arg
Arg
Arq
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
C
NH,
NH,
NH,
NH,
nh2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
nh2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arq NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arq NH,
Arg NH,
Arq NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arq NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arq NH,
Arg NH,
Arq NH,
Arg NH,
Arg NH2
Arg NH,
Arq NH,
Arg NH,
Arg NH,
Arg NH,
273
Table 7. Sequences o f side chain optimization library (Figure 3) members 401-600.
Sequence
Compound
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Aa
Aa
Ala
Ala
Ala
Aa
Aa
Aa
Ala
Ala
Aa
Aa
Aa
Aa
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
Ala ACPC.
Aa Jl’-Leu
Aa p'-Cha
Aa p ’-Phe
Aa p’-Trp
Aa
Aa
Aa
Aa
Ala
Ala
Ala
Ala
Aa
Ala
Ala
Ala
Aa
As
Aa
Ala
Ala
Aa
Aia
Ala
Ala
Aa
Aa
Aa
Aa
Ala
Aa
Aa
Aa
Aa
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
Ala
Ala
Ala
Ala
Aa
Ala
Aa
Aa
Aa
Ala
Ala
Aa
Aa
Ala
Aa
Aa
Ala
Aa
Ala
Aa
Ala
Ala
Aa
Aa
Aa
Ala
Ala
Aa
Aa
Ala
Ala
Ac
Ac
Ac
Ac
APC
APC
APC
APC
Aa
Aa
Ala
Aa
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
ACPC
Jl'-Leu
Jil-Cha
p ’-Phe
p ’-Trp
ACPC
p ’-Leu
jy'-Cha
Jl’-Phe
Jl’-Trp
ACPC
p ’-Leu
p’-Cha
p’-Phe
p’-Trp
ACPC
p’-Leu
p ’-Cha
(I’-Phe
p'-Trp
ACPC
p’-Leu
p ’-Cha
p ’-Phe
Jl’-Trp
ACPC
Jl’-Leu
p’-Cha
p'-Phe
P’-Trp
ACPC
p ’-Leu
Jl’-Cha
p’-Phe
Jl’-Trp
ACPC
P ’-Leu
p’-Cha
Jl’-Phe
p'-Trp
ACPC
P’-Leu
p'-Cha
Jl’-Phe
P'-Trp
ACPC
p'-Leu
p’-Cha
Jl’-Phe
Jl'-Trp
ACPC
p’-Leu
P’-Cha
p’-Phe
P’-Trp
ACPC
p’-Leu
p'-Cha
p'-Phe
p’-Trp
ACPC
p’-Leu
p’-Cha
p’-Phe
p’-Trp
ACPC
P’-Leu
p'-Cha
p’-Phe
p’-Trp
ACPC
P’-Leu
p'-Cha
p'-Phe
P’-Trp
ACPC
p'-Leu
P’-Cha
p’-Phe
p’-Trp
ACPC
p’-Leu
p'-Cha
P’-Phe
P’-Trp
ACPC
p’-Leu
P’-Cha
P’-Phe
P’-Trp
ACPC
p’-Leu
P’-Cha
P’-Phe
p’-Trp
Arg ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arg ACPC
Arq ACPC
Arg ACPC
Arq ACPC
Arq ACPC
Arg ACPC
Arq ACPC
Arg ACPC
Arq ACPC
Arg ACPC
Arq ACPC
Arq ACPC
Arg ACPC
Arg ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arg ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arg ACPC
Arg ACPC
Arq ACPC
Arg ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arg ACPC
Arq ACPC
Arg ACPC
ArQ ACPC
Arq ACPC
Arq ACPC
Arg ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arg ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arg ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arg ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arg ACPC
Arq ACPC
Arg ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arg ACPC
Arq ACPC
Arg ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arq ACPC
Arg ACPC
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Trp
Trp
Trp
Trp
Trp
xLeu
xleu
xLeu
xleu
xleu
Cha
Cha
Cha
Cha
Cha
Leu
Leu
Leu
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
Leu ACPC
Phe ACPC
Phe ACPC
Phe ACPC
Phe ACPC
Phe ACPC
Trp ACPC
Trp ACPC
Trp ACPC
Trp ACPC
Trp ACPC
xLeu ACPC
xLeu ACPC
xLeu ACPC
xleu ACPC
xLeu ACPC
Cha ACPC
Cha ACPC
Cha ACPC
Cha ACPC
Cha ACPC
Leu ACPC
Leu ACPC
Leu ACPC
Leu ACPC
Leu ACPC
Phe ACPC
Phe ACPC
Phe ACPC
Phe ACPC
Phe ACPC
Trp ACPC
Trp ACPC
Trp ACPC
Trp ACPC
Trp ACPC
xLeu ACPC
xleu ACPC
xleu ACPC
ACPC
xleu ACPC
Cha ACPC
Cha ACPC
Cha ACPC
Cha ACPC
Cha ACPC
Leu ACPC
Leu ACPC
Leu ACPC
Leu ACPC
Leu ACPC
Phe ACPC
Phe ACPC
Phe ACPC
Phe ACPC
Phe ACPC
Trp ACPC
Trp ACPC
Tip ACPC
Trp ACPC
Tip ACPC
xLeu ACPC
xLeu ACPC
xLeu ACPC
xLeu ACPC
xLeu ACPC
Cha ACPC
Cha ACPC
Cha ACPC
Cha ACPC
Cha ACPC
Lvs ji'-Nle
Lys Jl’-NIe
Lys Jl'-NIe
Lys Jl’-NIe
Lvs Jl'-NIe
Lys Jl'-NIe
Lys Jl’-NIe
iw Jl’-NIe
Lys Jl'-NIe
Lys Jl’-NIe
Lys Jl’-NIe
Lvs Jl’-NIe
Lys Jl’-NIe
Lys Jl'-NIe
Lvs Jl’-NIe
Lys Jl’-NIe
Lvs Ji’-Nte
Lys Jl’-NIe
Lys Jl’-NIe
LVS Jl’-NIe
Lys Jl’-NIe
Lys Jl’-NIe
Lys Ji’-Nle
Lys p’-NIe
Lvs p'-NIe
Lys p'-Phe
Lys Jl’-Phe
p’-Phe
p'-Phe
Jl’-Phe
[I’-Phe
Lys p'-Phe
Lys Jl’-Phe
Lys p'-Phe
Lys p'-Phe
Lys Jl’-Phe
Lys Jl'-Phe
Lys p’-Phe
Lys P’-Phe
Lys p’-Phe
Lys p’-Phe
Lvs p ’-Phe
Lys (i’-Phe
Lys p'-Phe
Lvs Jl’-Phe
Lys p’-Phe
Lys p ’-Phe
Lys p ’-Phe
Lvs Jl’-Phe
Lvs p’-Phe
Lys Jl’-Trp
Lys Jl’-Trp
Lys P’-Trp
Lys Jl’-Trp
Lys p’-Trp
Lys P’-Trp
Lys p’-Trp
Lys P'-Trp
Lys Jl’-Trp
Lvs P’-Trp
Lys p’-Trp
Lys p’-Trp
Lys p’-Trp
Lys P’-Trp
Lvs
Lvs
Lvs
Lvs
Lys P’-Trp
Lys p’-Trp
Lys p'-Trp
Lys p'-Trp
Lvs p’-Trp
Lys Jl’-Trp
Lys p’-Trp
Lvs p’-Trp
Lys p’-Trp
Lys p’-Trp
Lys p ’-Trp
Lys ACPC
Lvs ACPC
Lvs ACPC
Lvs ACPC
Lvs ACPC
Lvs ACPC
Lvs ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lvs ACPC
Lys ACPC
Lys ACPC
Lvs ACPC
Lvs ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lvs ACPC
Lys ACPC
Lys ACPC
Lvs ACPC
Lys ACPC
Lys ACPC
10
Gly
Gly
Gly
Glv
Gly
Gly
11
Asp
Asp
Asp
Asp
Asp
Asp
12
Ala
Ala
Ala
Aia
Ala
Ala
13
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
14
Asn
Asn
Asn
Asn
Asn
Asn
15
Arg
Arg
Arg
Arg
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Asp
Asp
Asp
Asp
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Asn
NH,
NH,
NH,
NH,
NH,
NH,
NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg n h 2
Arg NH,
Arg NHj
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH;
Arg NH,
Arg n h 2
Arg NH,
Arq NH;
Arg NH,
Arg NH,
Arg NH,
Arq NH,
Arg NH;
Arg NH,
Arg NH2
Arq NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arq NH,
Arg NH,
Arq NH,
Arg NH,
Arg NH,
Arg n h 2
Arq NH,
Arg NH,
Arq NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NB2
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gty
Gly
Glv
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gty
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
AI3
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
AJa
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Als
Ala
Ala
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tvr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tvr
Tyr
Tvr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tvr
Tyr
Tyr
Tyr
Tyr
Tyr
Tvr
Tvr
Tyr
Tvr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tvr
Tvr
Tyr
Tvr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tvr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tvr
Tyr
Tyr
Tvr
Tyr
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Arg
C
NH;
NH;
NH,
NH,
NH,
NH,
Arg
Arq
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arq
Arg
Arg
Arg
Arg
Arg
Arq
Arg
Arg
Arg
Arg
Arg
Arq
Arg
Arg
Arg
Arq
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arq
Arg
Arg
Arg
NH,
NH,
NH,
NH,
NH,
NH,
nh2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH2
NH,
p
501
502
503
504
505
506
507
508
509
510
511
Sequence
N
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
600
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
Ala
Ala
Ala
Ala
Ala
Ala
Ala
AJa
Ala
Ala
APC Ala
APC Ala
ACPC Arq ACPC
ft '-Leu Arg ACPC
Jl’-Cha Arq ACPC
Jl’-Phe Arg ACPC
Jl'-Trp Arq ACPC
ACPC Arg ACPC
(I'-Leu Arq ACPC
Jl'-Cha Arg ACPC
Jl’-Phe Arg ACPC
Jl'-Trp Arg ACPC
Arg ACPC
Trp
Jl’-Leu Arg ACPC
(i’-Cha Arq ACPC
Jl'-Phe Arg ACPC
(V'-Trp Arg ACPC
ACPC Arq ACPC
Jl’-Leu Arg ACPC
JT’-Cha Arq ACPC
p ’-Phe Arg ACPC
p'-Trp Arg ACPC
ACPC Arg ACPC
p'-Leu Arq ACPC
Jl'-Cha Arg ACPC
Jl’-Phe Arq ACPC
fl’-Trp Arg ACPC
ACPC Arq ACPC
p’-Leu Arg ACPC
p’-Cha Arg ACPC
p’-Phe Arg ACPC
Jl’-Trp Arq ACPC
ACPC Arg ACPC
P’-Leu Arg ACPC
APC Ala p’-Cha Arq ACPC
APC Ala p’-Phe Arq ACPC
APC Ala p’-Trp Arg ACPC
APC Ala ACPC Arg ACPC
APC Ala p'-Leu Arq ACPC
APC Ala |r'-Cha Arq ACPC
APC Ala p ’-Phe Arg ACPC
APC Ala P'-Trp Arg ACPC
APC Ala ACPC Arg ACPC
APC Ala p’-Leu Arg ACPC
APC Ala P’-Cha Arg ACPC
APC Ala p’-Phe Arg ACPC
APC Ala P’-Trp Arq ACPC
APC Ala ACPC Arg ACPC
APC Ala p’-Leu Arq ACPC
APC Ala P’-Cha Arg ACPC
APC Ala P’-Phe Arg ACPC
APC Ala p'-Trp Arq ACPC
APC Ala ACPC Arg ACPC
APC Ala p’-Leu Arq ACPC
APC Ala p ’-Cha Arg ACPC
APC Ala p’-Phe Arg ACPC
APC Ala p'-Trp Arg ACPC
APC Ala ACPC Arg ACPC
APC Ala p ’-Leu Arq ACPC
APC Ala p’-Cha Arg ACPC
APC Ala p’-Phe Arq ACPC
APC Ala p’-Trp Arg ACPC
APC Ala ACPC Arg ACPC
APC Ala P’-Leu Arq ACPC
APC Ala p’-Cha Arq ACPC
APC Ala p’-Phe Arg ACPC
APC Ala p’-Trp Arq ACPC
APC Ala ACPC Arg ACPC
APC Ala P’-Leu Arq ACPC
APC Ala p’-Cha Arg ACPC
APC Ala p'-Phe Arq ACPC
APC Ala p'-Trp Arq ACPC
APC Ala ACPC Arq ACPC
APC Ala p’-Leu Arg ACPC
APC Ala p'-Cha Arq ACPC
APC Ala p’-Phe Arq ACPC
APC Ala p’-Trp Arq ACPC
APC Ala ACPC Arq ACPC
APC Ala p'-Leu Arq ACPC
APC Ala P’-Cha Arq ACPC
APC Ala p’-Phe Arq ACPC
APC Ala P’-Trp Arq ACPC
APC Ala ACPC Arq ACPC
APC Ala pJ-Leu Arg ACPC
APC Ala p’-Cha Arg ACPC
APC Ala P’-Phe Arq ACPC
APC Ala P’-Trp Arq ACPC
APC Ala ACPC Arq ACPC
APC Ala p’-Leu Arq ACPC
APC Ala p'-Cha Arg ACPC
APC Ala p’-Phe Arq ACPC
APC Ala p'-Trp Arq ACPC
APC Ala ACPC Arq ACPC
APC Ala P’-Leu Arg ACPC
APC Ala p’-Cha Arg ACPC
APC Ala p ’-Phe Arg ACPC
APC Ala p’-Trp Arq ACPC
APC Ala ACPC Arq ACPC
APC Ala pJ-L'eu Arq ACPC
APC Ala p’-Cha Arq ACPC
APC Ala pJ-Phe Arq ACPC
APC Ala p’-Trp Arg ACPC
Trp
Trp
Trp
Trp
xleu
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
ACPC
Leu
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Jl’-NIe
p ’-NIe
p’-Nie
p’-NIe
p’-NIe
P’-NIe
ACPC Lys P’-NIe
ACPC Lys p’-Nie
ACPC Lys P’-Nie
ACPC Lys Jl’-NIe
ACPC Lys p’-NIe
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
Lys
Lvs
Lys
Lys
Lys
Lys
ACPC Lys
ACPC
ACPC
ACPC
ACPC
xLeu ACPC
xleu ACPC
xleu ACPC
xleu ACPC
Cha ACPC
Cha ACPC
Cha ACPC
Cha ACPC
Cha ACPC
Leu ACPC
Leu ACPC
Leu ACPC
Leu ACPC
Leu ACPC
Phe ACPC
Phe ACPC
Phe ACPC
Phe ACPC
Phe ACPC
Trp ACPC
Trp ACPC
Trp ACPC
Trp ACPC
Trp ACPC
xLeu ACPC
xLeu ACPC
xLeu ACPC
xLeu ACPC
xleu ACPC
Cha ACPC
Cha ACPC
Cha ACPC
Cha ACPC
Cha ACPC
Leu ACPC
Leu ACPC
Leu ACPC
Leu ACPC
Leu ACPC
Phe ACPC
Phe ACPC
Phe ACPC
Phe ACPC
Phe ACPC
Trp ACPC
Trp ACPC
Trp ACPC
Trp ACPC
Trp ACPC
Lvs
Lvs
Lvs
Lys
Lvs
Lys
Lys
Lys
Lys
Lys
Lvs
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lvs
Lys
Lvs
Lvs
Lvs
Lys
Lys
Lys
Lys
Lys
Lys
Lvs
Lvs
Lys
Lvs
Lys
Lvs
Lys
Lys
Lys
Lys
Lys
Lvs
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lvs
Lys
xLeu
xLeu
xLeu
xLeu
xLeu
Cha
Cha
Cha
Cha
Cha
Leu
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Phe
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lvs
Lys
Lvs
Lys
Lvs
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Phe
Trp
Trp
Trp
Trp
Trp
xLeu
xleu
xleu
xLsu
xLeu
Cha
Cha
Cha
Cha
Cha
ACPC
ACPC
ACPC
ACPC
ACPC
Lys
Lys
Lys
Lys
Lys
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
Lys
Lvs
Lys
Lys
L)®
Lys
Lys
Lys
Lys
Lys
Lys
p ’-NIe
p’-NIe
P’-NIe
p’-NIe
P ’-NIe
p ’-NIe
p’-NIe
Jl’-NIe
p’-Nie
p’-NIe
p’-NIe
P’-NIe
P’-NIe
pJ-Nle
p’-Phe
p'-Phe
P’-Phe
P’-Phe
P’-Phe
P’-Phe
P’-Phe
p'-Phe
p ’-Phe
P’-Phe
P’-Phe
p’-Phe
P’-Phe
p’-Phe
p’-Phe
P’-Phe
p’-Phe
p'-Phe
P’-Phe
p’-Phe
p’-Phe
P’-Phe
pJ-Phe
Jl’-Phe
p ’-Phe
p'-Trp
Jl'-Trp
p’-Trp
pJ-Trp
p’-Trp
p’-Trp
p’-Trp
P’-Trp
p'-Trp
p3-Trp
p’-Trp
P’-Trp
P ’-Trp
P’-Trp
Jl'-Trp
pJ-Trp
P’-Trp
P’-Trp
P’-Trp
p'-Trp
P ’-Trp
PJ-Trp
p’-Trp
P’-Trp
P’-Trp
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
10
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
11
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Gly
Glu
Ala
Phe
Asn
Asn
Asn
Gly
Gly
Gly
Gfy
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Aia
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
AJa
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
12
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
13
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
14
Asn
Asn
Asn
Asn
Asn
Asn
Asn
15
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arq
Arg
Arg
Arg
Arg
Arg
Arq
Arg
Arq
Arg
Arg
Arg
Arg
Arg
Arp
Arg
Arg
Arg
Arg
Arg
Arg
C
nh2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH;
NH,
NH,
NH;
NH,
NH,
NH,
nh2
nh2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH;
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arq NH,
Arg NH,
Arg n h ,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arq
Arg
Arg
Arg
Arq
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arq
Arq
Arq
Arg
Arg
Arg
Arq
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arq
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
274
Table 8. Sequences of side chain optimization library members (Figure 3) 601-800.
Compound
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
Sequence
N
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac APC
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Aia
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Aia
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Als
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Als
Ala
Ala
Als
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ale
Ala
Ala
Ala
Ala
Ala
Ala
AJa
AJa
ACPC
|i'-Leu
((’-Cha
Jl’-Phe
ji'-Trp
ACPC
Jl’-Leu
[(’-Cha
jv’-Phe
((’-Trp
ACPC
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
((’-Leu
ACPC
p’-Cha Arg ACPC
p ’-Phe Arg ACPC
((’-Trp Arg ACPC
ACPC Arq ACPC
p’-Leu Arg ACPC
p’-Cha Arg ACPC
|l’-Phe Arg ACPC
p’-Trp Arg ACPC
ACPC Arg ACPC
p ’-Leu Arg ACPC
p'-Cha Arq ACPC
p’-Phe Arg ACPC
((’-Trp Arg ACPC
ACPC Arg ACPC
p’-Leu Arg ACPC
p’-Cha Arg ACPC
p'-Phe Arg ACPC
(lJ-Trp Arg ACPC
ACPC Arg ACPC
p’-Leu Arg ACPC
P’-Cha Arg ACPC
ft’-Phe Arg ACPC
P’-Trp Arg ACPC
ACPC Arg ACPC
p’-Leu Arg ACPC
p ’-Cha Arg ACPC
[(’-Phe Arg ACPC
p’-Trp Arg ACPC
ACPC Arg ACPC
p’-Leu Arg ACPC
p ’-Cha Arg ACPC
p’-Phe Arg ACPC
p’-Trp Arg ACPC
ACPC Arg ACPC
p’-Leu Arg ACPC
P’-Cha Arg ACPC
p ’-Phe Arg ACPC
p'-Trp Arg ACPC
ACPC Arg ACPC
Jl’-Leu Arg ACPC
p’-Cha Arg ACPC
jl’-Phe Arg ACPC
p'-Trp Arg ACPC
ACPC Arg ACPC
P’-Leu Arg ACRC
p’-Cha Arg ACPC
(('-Phe Arg ACPC
p’-Trp Arg ACPC
ACPC Arg ACPC
p’-Leu Arg ACPC
p’-Cha Arg ACPC
p’-Phe Arg ACPC
p'-Trp Arg ACPC
ACPC Arg ACPC
P’-Leu Arg ACPC
p’-Cha Arg ACPC
p'-Phe Arg ACPC
P’-Trp Arg ACPC
ACPC Arg ACPC
p'-Leu Arg ACPC
p'-Cha Arg ACPC
p’-Phe Arg ACPC
p'-Trp Arg ACPC
ACPC Arg ACPC
P’-Leu Arq ACPC
p’-Cha Arq ACPC
p’-Phe Arg ACPC
P’-Trp Arg ACPC
ACPC Arg ACPC
p'-Leu A/g ACPC
p’-Cha Arg ACPC
P ’-Phe Arg ACPC
p’-Trp Arg ACPC
ACPC Arg ACPC
p’-Leu Arg ACPC
p'-Cha Arg ACPC
p’-Phe Arg ACPC
p’-Trp Arg ACPC
ACPC Arg ACPC
P’-Leu Arq ACPC
p’-Cha Arq ACPC
p’-Phe Arg ACPC
p’-Trp Arg ACPC
ACPC Arg ACPC
P’-Leu Arg ACPC
p’-Cha Arg ACPC
p’-Phe Arg ACPC
p'-Trp Arg ACPC
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Trp
Trp
Trp
Trp
Trp
xLeu
xleu
xLeu
xLeu
xLeu
Cha
Cha
Cha
Cha
Cha
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Trp
Trp
Trp
Trp
Trp
xLeu
xleu
xleu
xLeo
xleu
Cha
Cha
Cha
Cha
Cha
Leu
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Tip
Trp
Trp
Trp
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
Lvs
lys
Lvs
Lvs
Lys
Lys
Lys
Lvs
Lys
Lys
Lvs
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
Lvs
Lvs
Lvs
Lys
Lvs
Lys
Lvs
Lys
Lys
Lys
Lys
Lys
Lys
Lvs
Lys
Lvs
Lvs
Lvs
Lvs
Lvs
Lvs
LVS
Lys
ji’-NIe
Jl'-NIe
js’-NIe
((’-Nle
((’-Nle
((’-Nle
((’•Nle
P’-NIe
p'-NIe
((’-Nle
jf’-Nfe
(I’-Nle
p'-NIe
p’-NIe
p'-NIe
[(‘-Nle
p’-NIe
p ’-NIe
p'-NIe
((’-Nle
p’-NIe
p’-NIe
p'-NIe
p ’-NIe
p'-NIe
p ’-Phe
p'-Phe
p’-Phe
p'-Phe
p’-Phe
P’-Phe
p ’-Phe
p’-Phe
Jl’-Phe
p’-Phe
Lvs
Lys |('-Phe
Lys p'-Phe
Lys p’-Phe
Lvs p’-Phe
Lys p’-Phe
Lys p'-Phe
Lys p ’-Phe
Lvs p ’-Phe
Lvs p ’-Phe
Lvs p'-Phe
Lvs p'-Phe
LVS p’-Phe
Lvs P ’-Phe
Lvs p’-Phe
Lys p ’-Phe
Lys I^-Trp
Lys P ’-Trp
Lvs p’-Trp
Lys Jl'-Trp
Lys p’-Trp
Lys P’-Trp
Lys P’-Trp
Lys p ’-Trp
Lys P’-Trp
Lvs p’-Trp
Lys p ’-Trp
Lvs P’-Trp
Lys P’-Trp
Lvs p ’-Trp
Lys P’-Trp
Lvs p’-Trp
Lys P’-Trp
Lys P ’-Trp
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gty
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Gly
Gly
Gly
Gly
Glu
Glu
Glu
Gki
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Gly
Gly
Gly
Gly
Gly
Gly
Gty
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gty
Gly
Gty
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gty
Gly
Gty
Gly
Gly
Gly
Gly
Gty.
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
Gly
Gly
Gly
Glv
Gly
Gly
Glv
Lvs ACPC
Lvs ACPC
Lvs ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glu
Glu
Glu
Glu
Glu
Glu
Glu
ACPC Lys
ACPC Lvs
Trp ACPC Lys
Trp ACPC Lys
xLeu ACPC Lvs
xLeu ACPC Lvs
xleu ACPC Lys
xLeu
xleu
Cha
Cha
Cha
Cha
Cha
11
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Trp
xLeu
xLeu
xLeu
xleu
Lys p’-Trp
xLeu
Lys P’-Trp
Cha
Lys P’-Trp
Cha
Lys p’-Trp
Cha
Lys P'-Trp
Cha ACPC Lys p’-Trp
Cha ACPC Lys p’-Trp
Leu ACPC Lys ACPC
Leu ACPC Lys ACPC
Leu ACPC Lys ACPC
ACPC Lys ACPC
Leu ACPC Lvs ACPC
Phe ACPC Lvs ACPC
Phe ACPC Lys ACPC
Phe ACPC Lvs ACPC
Phe ACPC Lys ACPC
Phe ACPC Lys ACPC
Trp ACPC Lys ACPC
Trp
Trp
10
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gty
Gly
Gty
Gly
12
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
13
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
14
Asn
Asn
Asn
Asn
Asn
Asn
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
AJa
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ata
Ata
Ala
Aia
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Aia
Ala
Ala
AJa
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Cha
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
15
Arg
Arg
Arq
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
C
NH,
NH,
NH;
NH,
NH,
NH,
NH,
NH,
NH,
NH;
NH,
NH,
NH2
NH,
NHj
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NHj
NH,
NHj
NH,
NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NHj
Arg NH2
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
A/g NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NHj
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
po
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
Sequence
N
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
AJa
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
AJa
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
ACPC
|('-Leu
((’-Cha
[I’-Phe
(j'-Trp
Arg
Arq
Arg
Arg
Arg
Arq
Arq
Arg
Arg
Arg
Arq
Arg
Arq
Arg
Arq
Arg
Arg
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
|(’-Leu
ACPC
[j’-Cha
ACPC
(('-Phe
ACPC
Jl’-Trp
ACPC
ACPC
ACPC
P’-Leu
ACPC
(Jj-Cha
ACPC
[('-Phe
ACPC
[T’-Trp
ACPC
ACPC
ACPC
p ’-Leu
ACPC
p’-Cha Arq ACPC
p’-Phe Arq ACPC
Jl'-Trp Arg ACPC
Leu
Leu
ACPC
ACPC
ACPC
ACPC
Leu ACPC
Phe ACPC
Phe ACPC
Phe ACPC
Phe ACPC
Phe ACPC
Trp ACPC
Trp ACPC
Trp ACPC
Trp ACPC
Trp ACPC
xLeu ACPC
xleu ACPC
xLeu ACPC
xleu ACPC
xLeu ACPC
ACPC Arq ACPC Cha ACPC
p ’-Leu Arq ACPC Cha ACPC
[}'-Cha Arg ACPC Cha ACPC
(('-Phe Arq ACPC Cha ACPC
P'-Trp Arg ACPC Cha ACPC
ACPC Arg ACPC Leu ACPC
P ’-Leu Arq ACPC Leu ACPC
p ’-Cha Arg ACPC Leu ACPC
p’-Phe Arq ACPC Leu ACPC
P’-Trp Arg ACPC Leu ACPC
ACPC Arq ACPC Phe ACPC
P’-Leu Arg ACPC Phe ACPC
pJ-Cha Arq ACPC Phe ACPC
p’-Phe Arq ACPC Phe ACPC
(i'-Trp Arq ACPC Phe ACPC
ACPC Arq ACPC Trp ACPC
p ’-Leu Arq ACPC Trp ACPC
p'-Cha Arg ACPC Trp ACPC
p’-Phe Arq ACPC Trp ACPC
p ’-Trp Arg ACPC Trp ACPC
ACPC Arq ACPC xleu ACPC
P’-Leu Arg ACPC xleu ACPC
p’-Cha Arq ACPC xLeu ACPC
p’-Phe Arq ACPC xleu ACPC
P’-Trp Arg ACPC xleu ACPC
ACPC Arg ACPC Cha ACPC
p’-Leu Arq ACPC Cha ACPC
p’-Cha Arq ACPC Cha ACPC
p’-Phe Arg ACPC Cha ACPC
p'-Trp Arg ACPC Cha ACPC
ACPC Arg ACPC Leu ACPC
p’-Leu Arq ACPC Leu ACPC
p’-Cha Arg ACPC Leu ACPC
p’-Phe Arq ACPC Leu ACPC
p'-Trp Arg ACPC Leu ACPC
ACPC Arq ACPC Phe ACPC
p’-Leu Arg ACPC Phe ACPC
p’-Cha Arq ACPC Phe ACPC
p ’-Phe Arg ACPC Phe ACPC
p'-Trp Arg ACPC Phe ACPC
ACPC Arq ACPC Trp ACPC
p'-Leu Arg ACPC Trp ACPC
p ’-Cha Arg ACPC Trp ACPC
p’-Phe Arg ACPC Trp ACPC
P’-Trp Arq ACPC Trp ACPC
ACPC Arq ACPC xLeu ACPC
p’-Leu Arq ACPC xLeu ACPC
P’-Cha Arg ACPC xLeu ACPC
p ’-Phe Arg ACPC xLeu ACPC
p’-Trp Arq ACPC xLeu ACPC
ACPC Arg ACPC Cha ACPC
(('-Leu Arg ACPC Cha ACPC
P’-Cha Arq ACPC Cha ACPC
p’-Phe Arq ACPC Cha ACPC
p’-Trp Arq ACPC Cha ACPC
ACPC Arq ACPC Leu ACPC
(i’-Leu Arej ACPC Leu ACPC
p’-Cha Arq ACPC Leu ACPC
p’-Phe Arg ACPC Leu ACPC
p’-Trp Arq ACPC Leu ACPC
ACPC Arg ACPC Phe ACPC
p’-Leu Arg ACPC Phe ACPC
p’-Cha Arq ACPC Phe ACPC
p ’-Phe Arg ACPC Phe ACPC
P’-Trp Arg ACPC Phe ACPC
ACPC Arg ACPC Trp ACPC
P'-Leu Arq ACPC Trp ACPC
p’-Cha Arq ACPC Trp ACPC
p’-Phe Arg ACPC Trp ACPC
P’-Trp Arq ACPC Trp ACPC
ACPC Arq ACPC xLeu ACPC
p’-Leu Arq ACPC xLeu ACPC
p’-Cha Arg ACPC xLeu ACPC
p’-Phe Arg ACPC xleu ACPC
p’-Trp Arg ACPC xLeu ACPC
ACPC Arq ACPC Cha ACPC
P’-Leu Arq ACPC Cha ACPC
p'-Cha Arg ACPC Cha ACPC
p’-Phe Arq ACPC Cha ACPC
P’-Trp Arg ACPC Cha ACPC
Lvs
Lvs
Lvs
Lvs
Lys
[I’-Nle
[(’-Nle
(I’-Nte
(I '-Nle
p'-NIe
[I’-NIe
(i ’ Nle
Jl’-NIe
|i ‘-Nle
Lvs js’-NIe
LVS p’-NIe
Lvs ('(’•Nle
Lys p ’-Nle
Lys (('-Nle
Lys p'-NIe
Lys p’-NIe
Lys p’-NIe
Lys p’-NIe
LVS
Lvs
Lvs
Lys
Lys
Lvs
Lys
Lys
Lys
Lys
Lvs
Lys
Lys
Lys
Lys
Lys
Lys
Lw
Lvs
Lys
Lys
Lys
Lys
Lys
Lvs
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lvs
Lys
Lys
Lvs
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
P’-NIe
p’-NIe
p’-NIe
P’-NIe
p’-NIe
p'-NIe
p’-Nfe
P’-Phe
P’-Phe
P’-Phe
p ’-Phe
p ’-Phe
P'-Phe
p ’-Phe
P’-Phe
p’-Phe
p’-Phe
p'-Phe
((’-Phe
p’-Phe
p'-Phe
P’-Phe
p'-Phe
p’-Phe
p’-Phe
(l‘-Phe
p ’-Phe
p’-Phe
p'-Phe
P’-Phe
p’-Phe
P’-Phe
p’-Trp
((’-Trp
P’-Trp
p’-Trp
P’-Trp
P’-Trp
P’-Trp
P”-Trp
p'-Trp
p’-Trp
P’-Trp
P’-Trp
P’-Trp
P’-Trp
P’-Trp
Lys P’-Trp
Lys P’-Trp
Lys P’-Trp
Lvs P’-Trp
Lys P’-Trp
Lys P ’-Trp
Lys P’-Trp
Lys P’-Trp
Lvs P’-Trp
Lys P'-Trp
Lvs ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lvs ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
10
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
11
Glu
Glu
Glu
Glu
Glu
Glu
12
Ala
Ala
Ala
Ala
Ala
Ala
13
Leu
Leu
Leu
Leu
Leu
Leu
Glu
Glu
Glu
Glu
Gfu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Aia
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
14
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
15
Arg
Arg
C
NH,
NH,
NH,
nh.
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
NH,
NHj
NHj
NHj
NHj
NHj
NHj
Arq
Arg
Arg
NHj
NHj
nh2
NH,
NH,
nh ,
NH,
nh .
NH,
NH;
NH,
NHj
NH;
nh2
NHj
NHj
NHj
NHj
NHj
NHj
NHj
NHj
NHj
NHj
NH,
Arg NH,
Arg n h 2
Arg NHj
Arg NHj
Arg NH,
Arg NH,
Arg NHj
Arg NH,
Arg NHj
Arg NH,
Arg NH,
Arg NH,
Arg NHj
Arg- NHj
Arg NHj
Arg NHj
Arg NHj
Arg NH,
Arq NH,
Arg NH,
Arg NH,
Arg NH,
Arg NHj
Arg NH,
Arg NH,
Arg NHj
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arq NH,
Arg NH,
Arg NH,
Arq NH,
Arg NH,
Arq NH,
Arg NH,
Arq NH,
Arg NHj
Arg NH,
Arg NH,
Arg n h 2
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arg NH,
Arq
Arg
Arg
Arq
275
Table 9. Sequences o f side chain optimization library members (Figure 3) 801-1000.
Sequence
p
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859
860
861
862
863
864
865
866
867
668
869
870
671
872
873
874
875
676
877
878
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
897
898
899
900
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Aia
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
AJa
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ata
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ata
AJa
AJa
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
AJa
Ala
Ala
AJa
Ala
Ms
AJa
Ala
Ala
Ala
Ala
Ala
Ala
Ms
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
ACPC
ll'-ieu
iv1-Cha
(l’-Phe
If’-Trp
ACPC
p’-Leu
p'-Cha
p'-Phe
p'-Trp
ACPC
P'-Leu
p ’-Cha
p'-Phe
p’-Trp
ACPC
p'-Leu
p ’-Cha
(I*-Phe
p ’-Trp
ACPC
(l’-Leu
p ’-Cha
(i’-Phe
(I’-Trp
ACPC
pJ-Leu
p’-Cha
p’-Phe
p’-Trp
ACPC
p’-Leu
p'-Cha
p’-Phe
p'-Trp
ACPC
p'-Leu
p ’-Cha
p'-Phe
P’-T.p
ACPC
p'-Leu
p'-Cha
p’-Phe
Pt-T rp
ACPC
p ’-Leu
p ’-Cha
p'-Phe
P’-Trp
ACPC
p ’-Leu
p ’-Cha
p ’-Phe
p’-Trp
ACPC
p'-Leu
pJ-Cha
p’-Phe
p ’-Trp
ACPC
p’-Leu
p’-Cha
P’-Phe
P’-Trp
ACPC
p'-Leu
p ’-Cha
(I’-Phe
P’-Trp
ACPC
p’-Leu
p’-Cha
p ’-Phe
P’-Trp
ACPC
P’-Leu
p’-Cha
p’-Phe
p’-Trp
ACPC
p’-Leu
p’-Cha
p’-Phe
p’-Trp
ACPC
p’-Leu
P’-Cha
p’-Phe
p’-Trp
ACPC
p’-Leu
P’-Cha
p’-Phe
P*-Trp
ACPC
p’-Leu
p’-Cha
p’-Phe
P’-Trp
Arg ACPC
Arg ACPC
Arg ACPC
ACPC
Arg ACPC
Arq ACPC
Arg ACPC
Arg ACPC
Arq ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Mg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Mg ACPC
Mg ACPC
Mg ACPC
Mg ACPC
Mg ACPC
Arq ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Arg ACPC
Mg ACPC
Mq ACPC
Mg ACPC
Arq ACPC
Mg ACPC
Mg ACPC
Mg ACPC
Arg ACPC
Mg ACPC
Mq ACPC
Arg ACPC
Arg ACPC
Mq ACPC
Mg ACPC
Mg ACPC
Mg ACPC
Mg ACPC
Arg ACPC
Arg ACPC
Mg ACPC
Mg ACPC
Mg ACPC
Mq ACPC
Mg ACPC
Mg ACPC
Mg ACPC
Mg ACPC
Mg ACPC
Mg ACPC
Mg ACPC
Leu
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Trp
Trp
Trp
Trp
Trp
xLeu
xLeu
xLeu
xLeu
xLeu
Cha
Cha
Cha
Cha
Cha
Leu
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Trp
Trp
Trp
Trp
Trp
xLeu
xleu
xleu
xleu
Cha
Cha
Cha
Cha
Cha
Leu
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Trp
Trp
Trp
Trp
Trp
xLeu
xleu
xleu
xleu
xleu
Cha
Cha
Cha
Cha
Cha
Leu
Leu
Leu
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Trp
Trp
Trp
Trp
Trp
xleu
xleu
xLeu
xLeu
xLeu
Cha
Cha
Cha
Cha
Cha
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
Lys
Lvs
Lys
Lvs
Lys
1* -Nle
(I -Nle
fl’-NIe
|l ’-Nle
(l '-Nle
Lvs (l’-Nle
Lvs (i'-NIe
Lvs (i’-NIe
Lys (l'-Nle
Lys Jl’-NIe
Lys (1’-Nle
Lys Jl’-NIe
Lys p'-NIe
Lys p’-Nie
Lvs p’-NIe
Lys p’-NIe
Lys p'-NIe
Lys p’-NIe
Lys p’-NIe
Lvs pJ-Nle
Lys p ’-NIe
Lvs p’-NIe
Lvs p’-NIe
Lys p ’-NIe
Lvs p’-NIe
Lys P’-Phe
Lys P’-Phe
LVS pJ-Phe
LVS p’-Phe
Lys p’-Phe
Lvs p’-Phe
Lvs p'-Phe
Lvs p'-Phe
Lvs p'-Phe
Lys p’-Phe
Lvs p'-Phe
Lvs p'-Phe
Lvs p’-Phe
Lys P’-Phe
Lys p’-Phe
Lvs (I’-Phe
Lvs p’-Phe
Lys p'-Phe
Lys P’-Phe
Lys p’-Phe
Lvs p'-Phe
Lys p’-Phe
Lvs p'-Phe
Lvs P’-Phe
Lvs p ’-Phe
Lys p'-Trp
Lvs P’-Trp
Lys p ’-Trp
Lys p’-Trp
LVS p’-Trp
Lvs p’-Trp
LVS (lJ-Trp
Lys p’-Trp
Lys p’-Trp
Lvs P’-Trp
Lys p’-Trp
Lys p’-Trp
Lys P’-Trp
Lys p’-Trp
Lys P’-Trp
Lvs P’-Trp
Lys p’-Trp
Lys P’-Trp
Lvs p’-Trp
Lys p’-Trp
lv s p’-Trp
Lys P’-Trp
Lys P’-Trp
Lvs P’-Trp
Lys P’-Trp
Lvs ACPC
Lys ACPC
Lys ACPC
Lvs ACPC
Lvs ACPC
LVS ACPC
Lvs ACPC
Lvs ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lvs ACPC
Lys ACPC
Lys
Lvs
Lvs
Lys
Lvs
Lvs
Lys
Lvs
Lys
Lvs
Lys
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
10
Gly
Gly
Gly
Gly
Gty
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gty
Gly
Gly
Gly
Gly
Gly
Glv
Glv
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gty
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Glv
Gly
Glv
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gty
Gly
Gly
Gly
Gly
Gty
Gly
Gly
Gly
Glv
Gly
Gly
Glv
Gly
Gly
Gty
Gly
Gly
Gly
Gly
Gly
Gly
11
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
12
13
Ala Trp
Ala Trp
Aia Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Trp
Ala
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
AJa
Trp
Trp
Ala
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Aia Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala ’ Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
Ala Trp
14
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
15
Arg
Arg
Arg
Arq
Mg
Arq
Arq
Mg
Mg
Mg
Mg
Arg
Mg
Arg
Mg
Mg
Arg
Mg
Mg
Arg
Mg
Mg
Mg
Mg
Mg
Mg
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Mg
Mg
Mg
Mg
Mg
Mg
Arg
Mg
Arg
Mg
Arg
Arg
Arg
Arg
Arg
Mg
Arg
Mg
Arg
Mg
Arg
Mg
Mg
Mg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Mg
Mg
Arg
Arg
Mg
Mg
Mg
Mg
Mg
Mg
Arg
Mg
Mg
Mg
Mg
Arg
Mg
Mg
Mg
Arg
Arg
Mg
Mg
Mg
Arg
Mg
Mg
Mg
Mg
Mg
Mg
Mg
Mg
Mq
Mg
Arg
Mg
Arg
Arg
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Mg
Arg
Arg
Arg
C
NHj
NHj
NH,
NH,
NH,
nh2
NH,
NH,
NH,
nh2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
nh2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
nh 2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
n h2
NH,
NH,
NH,
NH,
NHj
NH,
NH,
NH,
NH:
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
P
Sequence
901
902
903
904
905
906
N
Ac
Ac
Ac
Ac
Ac
Ac
907
908
909
910
911
912
913
914
915
916
917
918
919
920
921
922
923
924
925
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
926
927
928
929
930
931
932
933
934
935
936
937
938
939
940
941
942
943
944
945
946
947
948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
969
970
971
972
973
974
975
976
977
978
979
980
981
982
983
984
985
986
987
988
989
990
991
992
993
994
995
996
997
998
999
1000
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
APC
APC
APC
APC
APC
APC
Ala ACPC Arg ACPC
Ala fl’-Leu Arg ACPC
Ala ji’-Cha Arq ACPC
Ala (i’-Phe Arg ACPC
Ala (i'-Trp Arg ACPC
Ala ACPC
ACPC
Ala (I'-Leu Arq ACPC
Ala (S’-Cha Arg ACPC
Ala (i‘-Phe Arq ACPC
Ata fi’-Trp Arg ACPC
Leu
Leu
ACPC Lvs
ACPC Lys
ACPC Lys
ACPC
ACPC
ACPC
APC
ACPC
APC
ACPC
APC
ACPC
APC
ACPC
APC Ala ACPC Arq ACPC
ACPC
APC Ala p’-Leu Arg ACPC
ACPC
APC Ala (i'-Cha Arg ACPC
ACPC
APC Ala p’-Phe Arq ACPC
ACPC
APC Ala p’-Trp Mg ACPC
ACPC
APC Ma ACPC Arq ACPC
ACPC
APC Ala p’-Leu Arg ACPC xleu ACPC
APC Ala (i'-Cha Arg ACPC xLeu ACPC
APC Ala [t’-Phe Arg ACPC xLeu ACPC
APC Ala p3-Trp Arg ACPC xLeu ACPC
APC Ala ACPC Mq ACPC Cha ACPC
APC Ala p ’-Leu Mg ACPC Cha ACPC
APC Ala p’-Cha Mq ACPC Cha ACPC
APC Ala Jl’-Phe Mg ACPC Cha ACPC
APC Ala p ’-Trp Mg ACPC Cha ACPC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
APC
Ala
Ala
Ala
Ala
Ala
Ma
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Aia
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ma
Ala
Ala
Ala
Ala
Ma
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ma
Ala
Ala
Ala
Ala
Ala
Ala
Ala
ACPC
(i’-Leu
p’-Cha
p’-Phe
p’-Trp
ACPC
p’-Leu
p ’-Cha
P’-Phe
P’-Trp
ACPC
P’-Leu
p’-Cha
p’-Phe
p’-Trp
ACPC
p ’-Leu
p’-Cha
p’-Phe
P'-Trp
ACPC
p’-Leu
p’-Cha
p’-Phe
(lJ-Trp
ACPC
(I’-Leu
p’-Cha
P’-Phe
p’-Trp
ACPC
p’-Leu
p’-Cha
p’-Phe
P’-Trp
Arg
Arq
Arg
Arq
Arq
Arq
Arg
Arg
Arg
Arq
Mg
Arq
Arg
Arq
Arq
Mg
Arq
Arg
Arq
Arg
Mg
Mq
Mg
Mg
Mq
Mq
Arq
Mg
Mq
Mg
Mq
Mq
Arq
Mq
Mg
Mq
Mg
ACPC
p’-Leu
p’-Cha Arq
p’-Phe Arg
p’-Trp Arq
ACPC Mq
PJ-Leu Arq
p’-Cha Arq
P’-Phe Arg
P’-Trp Mg
ACPC Mg
p’-Leu Mg
p’-Cha Mq
pJ-Phe Mq
p’-Trp Mg
ACPC Mg
p’-Leu Mq
p’-Cha Mq
p’-Phe Mq
p’-Trp Mq
ACPC Mg
p’-Leu Mg
p’-Cha Mq
jF-Phe Mg
p’-Trp Mq
ACPC Mq
p’-Leu Mg
pJ-Cha Mg
p’-Phe Mq
p'-Trp Mq
ACPC Mq
P’-Leu Arq
PJ-Cha Mq
P’-Phe Mg
P’-Trp Arq
ACPC Mq
p’-Leu Mq
p’-Cha Arq
P’-Phe Mg
APC Ala pJ-Trp Mg
ACPC
ACPC
ACPC
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Trp
Trp
Trp
Trp
Trp
Leu
Leu
Leu
Leu
Leu
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC Phe
ACPC Phe
ACPC Phe
ACPC Phe
ACPC Phe
ACPC Trp
ACPC Trp
ACPC Trp
ACPC Trp
ACPC Trp
ACPC xLeu
ACPC xLeu
ACPC xLeu
ACPC
ACPC xLeu
ACPC Cha
ACPC Cha
ACPC Cha
ACPC Cha
ACPC Cha
ACPC Leu
ACPC Leu
ACPC Leu
ACPC Leu
ACPC Leu
ACPC Phe
ACPC Phe
ACPC Phe
ACPC Phe
ACPC Phe
ACPC Trp
ACPC Trp
ACPC Trp
ACPC Trp
ACPC Trp
ACPC xLeu
ACPC xleu
ACPC xLeu
ACPC xleu
ACPC xleu
ACPC Cha
ACPC Cha
ACPC Cha
ACPC Cha
ACPC Cha
ACPC Leu
ACPC Leu
ACPC Leu
ACPC Leu
ACPC Leu
ACPC Phe
ACPC Phe
ACPC Phe
ACPC Phe
ACPC Phe
ACPC Trp
ACPC Trp
ACPC Trp
ACPC Trp
ACPC Trp
ACPC xLeu
ACPC xLeu
ACPC xleu
ACPC xLeu
ACPC xleu
ACPC Cha
ACPC Cha
ACPC Cha
ACPC Cha
ACPC Cha
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
LVS
Lys
Lys
jl’-NIe
Jl’-NIe
Jl'-NIe
Jl'-NIe
(l’-Nle
Jl'-NIe
(l’-Nle
(l’-Nle
p’-NIe
(l'-Nle
Lvs
Lvs
Lvs
LVS
Lys (l’-Nle
Lys p’-NIe
LVS fl’-NIe
Lvs p’-NIe
Lys fl’-NIe
Lvs p'-NIe
Lvs (i’-NIe
Lvs P’-NIe
LVS p’-NIe
Lys P’-NIe
Lvs (l’-Nle
Lvs P’-NIe
Lys P’-NIe
Lys P'-NIe
Lvs p ’-NIe
Lvs (T’-Phe
Lys p’-Phe
Lys p’-Phe
Lvs p’-Phe
Lvs p’-Phe
Lvs p’-Phe
Lys p’-Phe
Lys (i’-Phe
Lys p’-Phe
Lys p’-Phe
Lvs p’-Phe
Lvs p’-Phe
Lys p’-Phe
Lvs p’-Phe
Lvs p’-Phe
Lys p’-Phe
Lys p’-Phe
Lys p ’-Phe
Lys P’-Phe
LVS p ’-Phe
Lys [I’-Phe
Lys P’-Phe
Lvs p’-Phe
Lys p’-Phe
Lvs p’-Phe
Lys P'-Trp
Lvs p’-Trp
Lvs p’-Trp
Lys Pj-Trp
Lys p ’-Trp
Lvs p’-Trp
Lys p’-Trp
LVS p’-Trp
Lys p’-Trp
Lys pJ-Trp
Lys p’-Trp
Lys p’-Trp
Lvs p’-Trp
Lvs p’-Trp
Lys p’-Trp
Lvs p’-Trp
Lvs p’-Trp
Lys P’-Trp
Lys P’-Trp
Lys p’-Trp
Lvs P’-Trp
Lys P’-Trp
Lys p’-Trp
Lys p'-Trp
Lys p’-Trp
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lvs ACPC
Lys ACPC
Lys ACPC
Lvs ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lvs ACPC
Lys ACPC
Lys ACPC
Lvs ACPC
Lys ACPC
Lvs ACPC
Lvs ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lys ACPC
Lvs ACPC
Lys ACPC
10
Gly
Gly
Gly
Gly
Gly
11
Glu
Glu
Glu
Glu
Glu
12
Ala
Ma
Ma
Ma
Ala
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glu
Gtu
Glu
Glu
Gtu
Glu
Glu
Glu
Ala
Ma
Ala
Ala
Ala
Ma
Ma
Ma
Ma
Ala
Ma
Ala
Ma
Ala
Ala
Ma
Ma
Ma
Ma
Ma
Gly
Gly
Gly
Gly
Glu
Glu
Glu
Glu
Glu
Gly
Gly
Gly
Gly
Gly
Gly
Glu
Gki
Glu
Glu
Glu
Glu
Gly
Gly
Glv
Gty
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Gtu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glv
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glv
Gly
Gly
Gly
Gly
Gly
Gty
Gly
Gly
Gly
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Ma
Ala
Ala
Ala
Ala
Ma
Ala
Ala
Ala
Ma
Ala
Ma
Ma
Ala
Ma
Ma
Ala
Ma
Ma
Ala
Ala
Ala
Ma
Ma
Ma
Ma
Ala
Ma
Ma
Ma
Ma
Ma
Ma
AJa
Ma
Ma
AJa
Ma
Ata
Ma
Ala
Ala
Ma
Ma
Ma
Ala
Ma
Ma
Ma
Ma
Ala
Ma
Ma
Ma
Ma
Ala
Ma
Ma
Ma
Ma
Ma
Ala
Ma
Ma
Ma
Ma
Ala
Ma
Als
Ala
Ma
Ma
Ma
Ma
Ma
13
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tvr
Tyr
Tyr
Tyr
Tyr
Tvr
Tyr
Tvr
Tyr
Tyr
Tyr
Tyr
Tyr
Tvr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tvt
Tyr
Tvr
Tyr
Tyr
Tyr
Tyr
Tyr
Tvr
Tvr
Tyr
Tyr
Tvr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tvr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tvr
Tyr
Tyr
Tyr
Tyr
Tyr
Tvr
Tyr
Tyr
Tyr
Tvr
Tvr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
14
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
Asn
15
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arq
Arq
Arg
Arg
Arg
Arg
C
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
nh 2
NH,
NH,
NH,
NH,
nh2
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
NH,
Mg NH,
Mg NH,
Mq NH,
Mg NH,
Mg NH,
Mg NH,
Mg NH,
Mg NH,
Mg NH,
Mg NH,
Mg NH,
Mg NH,
Mg NH,
Mg NH,
Mg NH,
Mg NH,
Arg NH,
Mg NH,
Mg NH,
Arg NH,
Mg NH,
Mg NH,
Mg NH,
Mg NH,
Mg NH,
Mq NH,
Mq NH,
Mq NH,
Mq NH,
Mg NH,
Mg NH,
Mg NH,
Mq NH,
Mg NH,
Arg NH,
Mq NH,
Mg NH,
Mg NH,
Mq NH,
Arg NH,
Mq NH,
Mg n h 2
Mq NH,
Mg NH,
Mg NH,
Mq NH,
Mg NH,
Mg NH,
Mg NH,
Mq NH,
Mg NH,
Arg NH,
Arg NH,
Mg NH,
Arq NH,
Mg NH,
Mg NH,
Mg NH,
Mg NH,
Mg NH,
Mq NH,
Mg NH,
Mq NH,
Mg NH,
Mg NH,
Mg NH,
Arg NH,
Arq
Arg
Arg
Arg
Arg
Arq
Arq
Mg
Mg
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 10. Raw FP assay data (mP) for plate 1 without compound to provide a reference for 0% inhibition of fluorescently labeled Bak BH3 peptide probe
binding to the B c 1-x l protein.
A
B
C
D
E
F
G
H
1
J
K
L
M
N
0
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
50.34852
53.53989
47.47326
53.92729
57.29403
52.32822
54.82437
56.70989
53.49774
52.78996
52.80851
55.42595
56.33717
52.35049
56.24529
51.12472
55.83631
55.05027
50.34774
54.34627
59.87647
57.97273
55.14841
58.82859
55.95542
60.69475
58.59779
59.05889
57.83128
56.91818
59.76626
58.3217
54.80415
51.20695
54.90554
51.65845
52.72616
51.11069
52.88816
55.53966
54.17389
54.3461
56.50264
57.78052
57.51182
53.32309
52.38589
56.09934
53.97768
54.65912
57.90082
52.90516
52.84938
54.00393
56.21205
55.50981
54.53931
59.39664
53.28512
61.55466
60.68266
56.82787
59.42921
58.78618
54.83835
51.78392
51.81222
49.60656
50.78425
52.08676
51.85996
54.11128
53.75177
54.4019
56.15411
52.485
54.03559
53.23084
51.72741
51.36431
53.12995
57.11695
52.97047
51.95632
51.30629
51.31124
58.21054
55.83988
56.74506
64.32011
60.21133
58.7749
60.49486
56.63003
60.49547
55.16818
52.46589
53.31861
53.31357
60.14494
54.68824
51.50411
57.23014
53.54437
54.47409
54.11783
58.1078
53.92122
59.31615
52.93135
53.50082
52.80535
50.17522
57.58748
57.7803
60.06327
57.69357
57.84949
58.03284
57.34251
56.10114
59.23172
62.19651
61.29785
61.09781
59.84216
58.73101
64.6185
50.65017
56.7419
52.68838
51.17538
53.4639
50.8246
52.64517
54.31703
51.4082
57.11799
55.55111
57.22396
58.42852
51.54355
57.80255
58.58192
52.74984
58.92329
59.56196
53.17228
60.0172
56.3881
54.19871
58.35857
58.24522
63.6549
57.70089
61.57353
61.20768
58.08342
64.39599
64.69893
51.69595
56.02124
57.88476
51.71998
57.13077
51.70776
50.68508
51.32831
52.12874
49.5918
56.2967
54.08997
54.8219
56.89725
57.25187
49.4499
56.30172
55.7458
58.41596
58.25573
53.54843
49.70103
51.66631
59.88614
58.52501
58.36074
58.03901
57.87132
56.70539
65.81823
61.80783
62.12765
50.52214
53.05718
55.27503
57.17894
49.07925
49.53111
51.92994
55.55334
55.96044
56.08507
51.7439
55.14544
54.72439
57.17854
56.40201
50.66463
48.07618
52.76143
55.71011
55.57643
52.07666
57.8714
55.37443
56.02152
56.12041
60.81256
57.77288
60.33237
61.90803
56.55953
59.96425
61.03538
49.3517
51.63233
51.85757
58.20057
55.07036
55.08022
48.4736
54.22629
53.66597
55.01291
55.92506
55.45164
53.18105
54.33374
55.24547
60.36493
47.50358
53.86846
57.50137
53.64987
55.5425
60.6548
54.56276
55.04935
59.60099
61.4213
56.92766
63.47572
58.41146
59.80204
61.35219
62.66763
51.68227
53.99762
55.66758
51.88778
51.21266
57.66056
54.68643
53.66445
63.44752
56.28084
58.87221
50.71847
59.42329
55.20036
60.42994
51.90887
52.59575
54.05007
53.03123
52.33602
54.63962
56.22379
54.76816
58.32532
63.33867
57.51095
63.52952
62.7198
62.04399
62.01787
65.94056
59.57667
52.74815
49.87046
49.98151
49.796
53.03318
49.56039
62.62658
52.3516
53.1449
53.23581
56.96043
54.23088
58.5258
53.96706
56.58013
52.46383
54.96483
51.04252
51.41729
53.32227
56.85867
55.43421
61.09842
54.14952
54.98858
60.02282
65.30818
56.46836
57.76874
58.34702
59.9126
58.60422
53.61379
53.167
52.49861
50.46466
47.60359
48.10199
53.95737
52.44432
52.43133
54.75137
60.35371
51.43805
54.09361
54.91382
51.51112
53.97406
53.29394
57.91864
55.43037
56.43697
56.45354
55.97325
57.81768
56.18646
54.66845
57.28679
63.2508
59.8835
57.82808
56.46032
57.96565
55.27734
54.63792
54.23835
50.73128
53.06078
53.21259
51.2399
53.87357
54.27122
54.88168
55.47388
58.74354
55.2729
55.77454
60.34034
61.04741
55.52575
55.35081
56.24766
50.8418
56.78873
50.86797
51.21543
48.97996
54.60104
56.28697
51.94626
54.75684
57.73193
49.04026
54.96351
52.2747
58.60591
Table 11. Raw FP assay data (mP) for plate 1 with compounds from one-bead-one-compound library (Figure 3) after approximately 3 hr. The lower the mP
value, the greater the extent to which the Bcl-xi/Bak interaction is being inhibited.
A
B
C
D
E
F
G
H
t
J
K
L
M
N
O
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
51.5299
52.14654
48.73242
29.05932
38.56114
50.1729
50.82118
52.50463
48.50177
51.06633
50.99616
47.81408
51.70392
51.2353
51.80723
53.51758
51.90554
51.23676
48.38253
47.12708
52.13133
53.97136
44.33876
55.82008
54.73738
57.72523
40.89055
55.00931
54.68691
55.58986
56.5662
52.72317
54.37455
49.43328
44.81068
33.88329
48.73359
50.1982
48.4644
50.34852
55.61251
47.77299
54.01742
44.35626
54.15162
46.06169
49.85616
61.27319
48.62296
47.34384
47.12265
50.15748
52.98734
53.62631
49.89153
49.03695
53.99249
50.82509
54.82129
58.18709
55.95741
56.67098
45.32972
55.44724
45.65259
45.80871
46.67199
51.6298
52.14243
49.45106
50.8639
50.33254
49.99767
51.59716
53.12047
47.47977
50.24202
50.16508
50.34473
48.66436
47.27615
50.79279
50.0347
55.14386
49.50677
40.99931
52.10943
51.93334
54.59389
57.1391
55.84265
58.07056
49.56498
55.23485
52.17211
54.96958
50.17781
49.66326
49.71481
52.8932
47.07526
54.08195
50.79657
51.6046
53.42932
54.10543
52.7068
50.73917
53.24836
50.85795
49.05782
45.46256
46.85619
52.6724
39.57611
56.7557
51.02491
56.80736
58.84791
55.85411
56.59266
58.14086
58.9672
54.68519
54.93929
57.52776
56.20211
54.14724
48.30156
46.9121
44.51242
50.5598
48.46733
52.63166
49.97167
51.77897
56.4531
53.57166
54.25983
51.50931
55.7764
53.25223
50.69948
54.58807
48.18665
48.76344
49.03796
52.84869
50.34402
57.25853
55.05008
52.79801
57.45846
60.50767
54.80868
55.36018
57.99867
56.13809
55.44636
59.28725
48.37018
47.11229
43.35566
52.76553
41.1962
52.52868
48.51416
50.07893
51.03044
50.98055
52.911
50.44439
47.54205
52.71087
46.35243
46.69039
48.58751
48.60033
47.98042
53.81941
45.65445
53.07433
52.66712
56.88333
56.546
56.81392
53.16364
47.92297
54.33788
54.1724
58.40357
58.34048
52.99222
46.59607
47.72138
56.14401
48.38347
48.00222
50.85787
51.17985
47.09877
50.73235
48.28912
49.06516
55.27036
52.13873
55.70027
49.27917
50.18436
49.79722
49.05479
52.83296
49.97495
54.98786
52.81286
55.62057
51.63939
55.0063
52.83633
54.31729
60.25361
59.99096
38.50427
59.37833
45.16602
48.4614
46.57424
52.97922
46.81952
52.57344
53.1094
54.26338
49.58897
49.82106
52.93753
52.24419
52.61173
51.02205
44.27457
52.61249
49.26272
50.41646
52.37567
53.07172
56.27999
57.33605
54.14057
56.593
51.02354
56.91947
49.20596
54.46465
56.27676
55.35711
53.68993
56.74987
46.74643
48.18363
50.73421
50.84372
54.43468
55.75584
53.20706
56.14723
53.4027
53.68703
54.17698
44.40704
45.2634
51.12403
46.52259
52.42389
50.72191
48.58345
44.03115
53.36682
52.09361
59.7976
54.40466
53.22536
53.21353
58.77745
57.55506
56.65567
59.42514
59.35519
59.67659
58.2709
51.51129
50.51395
44.6168748.46701
49.62985
48.02432
49.31064
53.6444
51.52104
50.55947
51.24944
53.80698
55.7736
52.79434
51.11111
50.1927
48.73759
46.34698
46.79477
49.71942
51.47511
56.98368
57.75043
49.5379
53.58794
56.11333
56.25758
59.48202
58.47354
56.00297
52.49135
50.92206
51.14465
44.65466
46.37006
51.73592
46.27789
39.88443
53.35652
•48.25841
53.50684
49.22419
50.4331
55.19407
50.59977
49.12663
50.96206
46.11793
48.14124
47.50311
47.87434
54.69931
48.12515
55.35568
52.01959
46.36149
52.38573
53.33648
51.45967
57.91043
54.10634
52.45608
58.42737
45.06771
45.85813
42.97623
51.39806
48.77707
51.9078
50.82434
49.98958
46.32478
54.19663
50.91534
48.52256
51.7872
24.05462
59.17569
59.52283
59.65779
44.85149
39.20585
47.46632
43.23383
54.64278
49.59056
51.97395
46.81649
51.58584
47.20902
45.77937
45.80521
47.9475
44.77174
52.38593
61.17903
K>
-u
o\
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Plate 1
10
♦ No Compound
■ + Compound (~3 hours)
1
17
33
49
65
81
97
113 129 145 161 177 193 209 225 241 257 273 289 305 321 337 353 369
Well
277
Figure 18. FP assay data for plate 1 with and without compounds from one-bead-one-compound library (Figure 3).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 12. Raw FP assay data (mP) for plate 2 without compound.
A
B
C
D
E
F
G
H
1
J
K
L
M
N
O
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
53.95441
57.10382
55.21006
53.98756
51.73847
. 51.584
54.75997
53.51348
58.70347
55.56907
58.69308
52.32285
60.16933
55.43507
56.6611
54.42381
58.21541
60.06011
60.31451
62.34284
53.42889
54.40262
58.45399
62.87235
61.75218
63.45495
65.26639
57.61496
64.50907
61.83943
60.14158
57.07545
53.15589
59.54982
63.3056
58.9353
58.48305
60.13194
53.78868
59.56286
57.66945
63.99129
60.29693
59.48444
58.32263
57.09828
53.44453
55.50582
55.99953
63.60317
63.15814
60.24087
56.91081
61.8238
61.90797
60.0842
53.76152
67.26043
63.91417
65.00601
60.77176
59.21371
64.44841
58.98223
55.43407
54.65548
55.81719
55.51131
54.06727
56.19723
59.34987
57.00778
52.29234
62.24524
61.61866
60.08825
57.43356
54.54462
64.42114
55.28795
58.1641
55.19986
58.7848
50.73646
55.64377
57.12776
58.90202
63.03173
61.56129
66.24594
66.86725
57.41295
62.60632
58.20304
59.79613
56.79369
47.53306
51.65074
55.8902
56.75889
57.26988
55.87649
52.27439
57.12989
56.12328
64.36476
64.55247
59.00108
56.48882
56.19281
54.80094
55.95869
51.13245
55.67163
60.59641
61.33893
59.90505
60.03163
55.43283
62.41443
58.51833
67.06532
64.32206
61.4541
64.16079
67.85023
59.15997
64.99444
49.99207
53.59441
55.78242
58.8135
55.37093
54.00051
52.42563
59.20891
55.17848
62.36285
60.2471
59.02926
61.21153
59.86884
58.79321
58.02831
54.06751
58.62959
60.96912
58.44422
59.70895
55.80777
55.44902
60.65623
60.59818
68.64761
57.63138
62.74237
66.84963
66.87785
60.41811
56.74674
53.15826
58.25306
56.60822
55.74154
56.83523
59.16198
51.1292
55.5105
59.50807
60.0261
54.62298
58.35168
61.11802
61.45921
59.83081
50.27057
57.03082
55.53983
58.01318
56.93748
55.87339
61.08521
52.93477
62.62318
62.18825
63.52732
59.51271
58.05296
63.78354
65.44955
59.0864
60.45498
50.73585
54.19303
53.52344
58.07392
57.73764
52.20476
51.02648
56.28717
56.68477
54.4356
53.55873
57.83169
56.51982
59.39267
52.45502
55.49838
54.80329
59.89136
59.71135
57.38068
60.85622
58.16792
57.31158
56.75467
61.84867
60.89065
59.69644
62.4107
64.47112
63.04562
58.83574
56.51369
54.12593
56.09562
53.59744
56.12303
55.98111
58.39872
55.13011
56.56197
63.38053
61.38543
58.49615
60.20412
61.49154
62.68038
57.57723
59.24784
54.75641
55.88194
65.25418
60.43103
59.132
56.6148
56.1563
60.76275
63.42959
64.97604
63.0897
65.51482
61,13642
63.82246
62.33879
63.70433
53.48301
55.69015
58.75094
60.63879
61.00721
59.35519
58.89456
57.98429
67.52369
63.46586
64.70808
65.04555
58.14051
59.13381
60.16334
58.48874
•54.3697
58.73252
55.12225
57.71465
58.21587
58.18825
61.47595
61.72871
62.73406
61.63215
62.05077
61.77946
61.82799
64.74465
58.92085
61.05475
55.53978
58.05923
60.16562
57.57665
57.58599
56.55105
60.06827
56.8349
59.28798
58.55745
59.70183
61.5953
59.46882
60.62877
54.65985
57.44074
54.58096
59.70755
60.49542
59.13573
59.73687
56.65631
57.77153
57.60092
61.06965
60.76922
63.14084
62,1959
61.0004
60.09096
56.52348
59.04124
49.08075
53.32044
60.26108
50.26968
56.80637
55.28161
55.33649
59.55491
62.46178
58.25262
63.97643
60.16238
56.7188
55.15033
56.78074
57.82304
55.68959
57.5348
65.1088
54.45799
58.82429
60.05664
58.95798
60.99348
58.76358
62.45273
62.69611
67.15735
60.92146
60.0325
60.33645
57.53128
60.83785
55.72642
57.68046
55.35336
59.08212
55.28173
53.88
55.55251
63.18838
60.18647
63.70647
57.34019
56.64656
54.93972
63.25112
61.25355
60.54314
56.3183
52.21168
55.53698
56.39344
52.4878
50.961
60.96574
61.05406
63.50665
59.347
53.85329
51.18934
55.73629
59.759
59.63426
Table 13. Raw FP assay data (mP) for plate 2 with compounds from one-bead-one-compound library (Figure 3) after approximately 3 hr.
A
B
C
D
E
F
G
H
1
J
K
L
M
N
O
P
1
2
3
4
5
6
7
8
9
10
48,98425
48.36551
53.24277
50.71273
51.39486
49.5868
54.77935
52.28832
49.41863
54.20191
46.58339
51.42047
45.97948
54.96706
51.63253
52.72513
55.05587
43.57928
49.21705
49.79851
54.10994
48.65241
49.24437
49.94348
49.82876
49.94309
49.46591
50.16699
53.84512
59.30485
47.88117
47.83948
53.61106
52.37513
55.83503
50.22084
53.7274
55.23564
58.25803
58.82916
58.85382
44.89322
60.03378
50.12716
49.05257
56.03948
52.91087
50.15742
55.07624
48.20494
49.22659
46.68269
55.44639
50.07735
53.94464
52.46313
54.98221
54.8379
54.65737
53.37061
51.7888
39.81975
51.57783
52.77646
56.18718
52.31754
56.24541
52.45839
50.23498
48.42562
49.48453
50.61537
53.24042
54.16138
52.91457
53.66234
50.00127
58.12848
49.67597
51.82763
52.85949
59.04883
54.57302
57.24352
51.88729
52.19747
50.69574
51.3275
51.75655
52.3612
57.61239
54.10987
45.98705
51.13776
54.63366
52.83364
55.22788
52.11565
53.79286
50.53325
47.6839
46.9334
53.41303
47.90549
55.19337
52.7744
55.83721
54.68395
48.5243
53.61582
52.73174
52.83278 57.12015 50.38523
50.27558
52.45342
54.18364
51.99118
54.26401
51.92058
53.89633
56.6928
58.8432
56.16752
57.88608
45.92539
54.96721
52.5309
53.36078
53.06891
52.41258
52.28534
57.15647 52.62231
50.62121
52.36583
56.10888
59.60778
55.94799
55.06759
53.4604
58.05499
56.11727
57.2888
53.51816
52.32242
50.90593
52.07466
53.70188
52.63599
47.52026
51.1228
41.89951
11
50.34428
47.57052
53.55984
56.40708
52.90138
52.63588
49.7231
46.77079
52.16614
58.91402 51.27751
55.9102
53.7557
62.24674
53.82555
51.34918
57.53198
51.0842
57.18116
50.08192
56.70409
48.07071
43.50285
12
13
14
15
16
17
18
56.18582
50.71263
55.52674
57.00038
48.04365
50.79279
51.79241
55.10128
54.83419
56.59006
54.86964
53.43854
51.34228
61.52183
51.34939
53.5044
52.90243
50.49399
51.20443
54.02566
49.73896
48.25216
39.59099
47.23437
50.90695
49.85246
50.88927
55.32739
49.24098
52.41885
49.13457
53.3308
51.60967
53.8895
56.26944
51.21116
51.80258
53.65418
49.19887
54.77717
49.6321
56.86935
50.19768
53.41425
58.54485
52.4398
54.07549
56.64698
46.09871
51.48965
50.113
49.88545
46.62633
55.00741
47.63548
47.563
44.36935
52.683 98
41.43973
53.84113
50.8433
53.5379
54.50856
50.44315
50.40108
42.49696
51.65491
54.68015
55.16811
53.68562
51.95192
58.00105
53.69461
54.72732
51.61053
56.40445
54.46708
52.85989
51.62056
55.51417
50.68887
46.48515
48.25205
52.49571
52.52686
51.73649
56.37135
57.2981
56.7536
57.24249
56.42294
55.14522
56.29532
52.13383
49.6815
50.02489
52.61239
53.23291
56.37608
46.85611
56.46935
53.88217
49.94809
55.49509
55.62719
53.81519
58.49114
56.00947
60.63831
56.34876
54.00631
32.06792
19
51.74035
53.38699
51.77372
53.04775
51.75613
56.34111
50.60257
54.89509
46.8329
50.22867
56.22064
56.51101
54.47062
47.32352
46.62078
46.21514
20
21
22
23
24
51.7875
50.00565
51.94039
54.08459
48.3739
54.06177
51.05695
55.87994
53.68222
52.65874
61.4109
58.72086
59.38104
53.59589
53.93463
52.37117
51.61348
52.12904
48.41623
49.85932
50.33274
47.31436
49.37251
51.68243
53.03233
53.98976
53.57386
56.45152
56.35703
41.37155
49.811
48.66487
57.08282
55.55802
55.00877
56.07683
50.09129
51.53175
51.37466
58.88331
55.38407
56.36791
56.27647
59.54142
52.99314
55.30004
48.96367
41.71069
55.71013
48.80652
51.92601
53.21381
53.01777
49.90822
48.13029
44.00882
57.01504
54.72351
52.10512
52.99774
50.42054
51.13311
33.96859
56.23716
52.12619
51.08652
52.82133
53.78496
51.19571
54.02917
46.53721
42.33898
54.4066
50.93996
53.44236
47.30337
46.59475
35.65241
42.47505
43.9079
-J
oo
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Plate 2
20
10
♦ No Compound
■ + Compound (~3 hours)
1
17
33
49
65
81
97
113
129
145 161
177
193 209 225 241
257 273 289
305 321
337 353
369
Well
279
Figure 19. FP assay data for plate 2 with and without compounds from one-bead-one-compound library (Figure 3).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 14. Raw FP assay data (mP) for plate 3 without compound.
A
B
C
D
E
F
G
H
I
J
K
L
M
N
0
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
57.62222
57.62615
64.84953
57.59647
61.11542
57.35709
59.3072
64.99666
59.60881
62.53221
68.68149
54.86621
57.65721
58.2214
56.62804
59.54092
59.09108
61.58745
56.83857
56.58331
59.61722
54.50167
55.60556
56.66198
55.70214
55.86903
56.06781
56.12436
55.71499
61.0923
61.69441
59.31512
58.93885
62.67807
61.89515
60.23354
67.44759
60.57006
63.63018
64.86979
63.9289
66.49349
68.07458
56.64491
55.15591
61.27679
57.54681
59.13788
54.02432
51.91019
57.15519
56.74795
56.79453
57.19489
61.23285
61.58267
56.45553
54.47176
55.40211
54.20767
60.12214
56.0689
58.38745
56.29255
60.96742
57.99019
55.72519
56.40597
59.71361
58.57478
63.19718
62.71226
59.16886
61.34159
62.66894
53.94987
53.65433
56.15636
53.88408
56.41397
55.20566
53.98873
53.77641
55.95774
57.49668
55.06087
55.91347
55.94172
57.11034
61.60417
54.01285
59.69538
54.90816
60.02349
59.41777
57.10419
59.11432
58.06571
62.92971
66.19378
63.19001
59.98988
60.90082
63.04264
62.53299
65.92781
66.41244
58.86713
56.7641
56.84079
58.5815
58.07852
60.20946
55.58671
54.62346
56.54379
56.72108
56.46003
56.09742
57.09227
58.95781
60.28469
61.28041
62.22392
61.53953
62.28806
59.29177
60.687
58.67627
63.27911
59.05263
58.87096
57.56665
61.11216
61.58388
. 60.59345
58.69776
71.59887
67.40451
60.15759
58.12813
60.57622
58.74609
53.87125
57.14859
60.08898
62.53692
63.13662
59.69926
53.34192
64.87664
57.3566
63.16525
65.89182
54.49858
62.38758
61.45238
57.7329
61.13304
55.23782
59.94702
59.61619
62.80344
64.92449
63.50864
59.02393
63.67219
59.89726
66.28937
65.41896
64.23406
58.94368
64.42972
50.3319
57.38855
55.65176
52.45241
55.08637
55.08822
52.37666
60.61192
50.50102
56.65956
61.51432
56.37163
60.90757
53.07326
55.62797
59.49979
52.3016
61.27004
57.91916
57.19172
61.91932
59.34324
60.31503
67.71302
54.39484
59.29677
66.50221
60.97705
66.22143
64.54244
55.81902
57.37894
54.71097
61.23562
55.45657
57.09443
57.76711
61.36285
58.46179
58.41608
55.09689
56.43274
57.57178
55.97811
53.91524
56.26504
57.63063
62.51768
64.9752
61.20612
57.62575
56.68964
63.19957
65.00416
62.66659
60.37283
61.87156
63.09069
60.2544
58.06881
58.39214
64.86149
61.28683
58.89828
65.71787
62.09765
60.97985
50.73636
58.76909
60.37794
59.78274
53.20264
58.7848
57.70327
51.39328
52.55831
59.40456
58.97367
60.12893
61.65384
58.09769
66.23692
59.98032
55.76248
65.0129
63.36746
61.37817
59.40274
63.41558
60.38863
64.64272
64.6838
68.80527
68.55359
58.05727
64.48765
59.69547
62.1415
60.20485
48.25777
57.30895
53.16933
57.81064
53.81486
65.29471
59.04706
58.40383
59.75695
63.56734
58.8312
58.03141
60.54488
61.39365
60.43149
59.64505
58.83745
63.50158
61.67279
63.19251
56.07508
65.11108
66.34457
61.05512
61.00761
65.64329
69.01566
54.14618
52.05311
58.76889
55.16195
53.56586
52.2455
53.76827
55.95175
57.12235
60.52837
56.39774
53.31032
54.2717
63.2682
52.41055
54.46797
59.0114
59.60039
59.47582
58.3949
56.23361
57.21699
58.1636
59.10565
62.48135
63.2407
58.66457
61.02757
59.16495
66.30064
64.45256
67.74568
57.42112
51.16958
59.41987
53.62404
51.63158
51.86773
55.01845
53.98192
56.5892
58.99677
56.67406
55.56016
54.67335
57.03069
48.2238
55.13802
54.94156
56.91589
56.73991
54.82301
54.93461
58.55196
60.69625
63.99454
64.82334
62.2606
65.80552
55.15747
64.46893
65.33122
62.09868
70,42617
65.03633
54.64227
58.41474
49.72551
52.39176
53.01667
55.8384
50.14298
56.52722
55.27429
53.95429
56.08599
55.18772
57.26901
56.64886
52.89099
60.80388
47.67487
51.77159
56.41053
49.30975
Table 15. Raw FP assay data (mP) for plate 3 with compounds from one-bead-one-compound library (Figure 3) after approximately 3 hr.
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
56.24328
54.2618
47.57639
54.56677
55.1242
55.31127
58.75227
56:31687
62.04463
61.9471
49.99412
47.14792
39.43905
49.91785
54,2924
52.94501
52.80862
53.15798
50.00503
49.1153
52.26966
50.16661
47.86748
51.13236
54.44397
55.79799
53.80884
47.78167
53.50179
54.65007
43.45797
49.80225
49.18964
48.80771
49.32044
48.50172
49.37852
53.20994
52.79779
53.52711
58.67365
54.70778
55.76578
54.05284
50.37458
49.89052
51.55976
56.51549
52.69546
50.65846
51.37642
55.85353
54.75139
50.70541
49.60033
51.19039
50.3732
53.21553
50.93179
48.90321
53.94833
57.42839
55.80044
51.75088
57.16219
55.41041
56.11646
48.03872
52.57502
54.93321
53.95807
55.89584
52.44758
52.61731
47.18016
56.28714
60.59164
56.21475
56.37328
53.68286
55.70055
58.79301
60.08615
56.79078
64.5111
50.3356
53.07627
48.17344
45.78824
50.0327
45.82448
51.49206
55.7448
53.551
54.57752
43.31285
47.4418
50.80083
53.95498
52.64349
48.97363
51.91778
58.34414
47.1503
49.28261
54.47897
54.15672
59.95245
56.9734
55.78997
53.36267
54.52218
52.99429
54.16387
58.61416
55.31862
59.64885
49.6975
51.28328
49.34356
48.84423
50.34236
48.52124
52.47508
55.20284
49.59466
49.92752
47.23985
50.49166
51.6407
52.62839
55.8278
58.04533
54.0381
50.35628
53.12157
58.0343
55.60642
58.66502
61.00725
47.48685
49.24414
49.25677
49.3487
49.94513
48.20957
52.27579
54.47762
54.08995
52.68431
51.21125
51.58994
53.42595
46.97269
51.27647
53.05826
53.46239
49.98138
54.80505
52.91164
43.83108
54.50356
59.26628
54.93378
54.30936
57.94079
53.57806
59.8845
52.49909
46.4899
60.13031
58.97697
40.71005
50.54346
52.40435
49.17238
55.51616
47.2517
48.39889
52.84569
51.24383
47.53059
51.46941
54.1875
5150925
53.60959
57.79281
57.30899
49.45141
55.1592
50.82375
52.67165
50.54922
52.91416
54.90282
52.76409
55.81659
54.30161
57.49878
57.73834
59.07891
58.45783
58.08999
66.52945
52.27918
55.9667
52.92859
35.55071
49.34572
49.12083
51.15465
51.70777
50.92576
47.90422
54.81086
53.34698
51.36917
56.57286
49.16201
54.79595
55.7408
57.19069
57.33438
55.46445
53.58348
51.2379
55.23312
54.82707
52.6912
54.56346
54.68302
55.06115
58.55227
58.0676
54.8369
66.42969
50.58573
47.07074
52.26713
49.97478
48.10699
47.32648
41.42661
51.58233
49.6913
47.185
49.07098
53.28231
51.21827
53.46403
45.31187
46.11205
51.56066
51.18238
52.8389
53.05159
49.40299
53.18473
58.89205
59.78592
52.34435
53.01003
51.55771
58.40157
53.50502
54.397
62.73637
58.10645
48.50745
46.59121
48.98042
50.55732
45.06909
48.66938
52.07908
48.58752
48.61788
53.44819
53.19491
52.93849
52.57944
50.05652
49.90296
50.43951
47.22584
55.21406
44.89944
54.6993
48.75474
54,89024
55.21758
53.06799
52.39629
53.80445
57.37893
58.26719
58.30142
55.02877
56.57845
61.45622
47.45171
48.37694
48.96278
49.47495
43.78171
46.47993
48.94663
47.90437
48.20721
49.59222
49.19948
49.8457
49.56281
49.66061
46.41738
42.71827
45.17369
42.63714
46.21669
53.39269
47.66028
55.35075 52.01018 52.25357
58.04812
49.79919
47.96882
51.44269
53.09478
51.46308
50.68909
55.19126
60.0188
54.70413
61.73976
50.6634
53.86289
48.21063
45.51986
52.58104
54.72427
53.13779
51.90853
53.36244
52.54908
53.76899
51.27502
51.21976
60.1726
58.75086
55.76804
63.52105
50.77436
45.8924
52.8933
48.08963
47.87898
59.9808 56.55023
57.33159
56.96304
63.83281
53.79073
49.14185
51.00826
48.40572
45.67448
56.0206
51.21809
54.06425
38.2427
48.65996
50.20569
49.36302
52.29892
54.17902
53.97756
42.55361
56.291
50.31066
50.51169
50.11108
51.23364
60.16051
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 16. Raw FP assay data (mP) for plate 4 without compound.
A
B
C
D
E
F
G
H
1
J
K
L
M
N
O
P
1
2
3
4
5
6
7
8
9
10
58.73956
50.79867
52.8821
52.70279
54.90501
50.02299
60.16584
53.97445
58.95104
60.93698
56.1225
54.06165
53.76772
59.46615
48.07049
57.41545
58.62704
56.42874
56.37658
53.59544
54.63109
56.89221
61.07658
59.04776
60.19337
66.34285
58.02128
54.08515
51.60925
62.07383
53.45225
53.99919
49.75001
58.10754
51.40077
56.2699
59.73216
56.43417
55.69868
55.87097
56.0881
59.82172
53.43574
57.09937
52.26099
57.98333
55.92686
53.41317
53.03651
57.53951
51.70666
57.50389
60.55721
59.71506
61.03373
57.91791
59.72615
65.92459
55.4796
62.72297
58.71444
57.9477
62.26337
52.36218
53.6691
52.69978
57.29718
51.23405
61.05253
58.6361
59.53174
58.11791
59.7219
60.23307
57.84544
59.54326
57.3761
57.82704
56.69178
51.67407
57.44425
55.78265
59.73156
50.26459
63.72929
62.72515
62.82469
60.74124
59.60739
65.68083
64.50055
55.70224
55.9666
56.42097
54.10977
54.68141
54.54631
54.42859
56.04763
60.89594
61.70718
58.46458
63.04227
59.5021
56.72579
63.22026
61.67409
57.62344
58.37466
54.26227
54.42779
54.46733
55.13484
57.17627
54.88557
56.80603
61.9697
63.34911
64.33647
64.46339
64.71621
69.73871
63.2396
55.44093
63.08667
55.65111
56.99079
51.81899
53.45837
59.34969
54.14503
53.91645
55.16223
56.73154
55.62262
52.82526
57.13936
61.53673
62.62829
55.10998
64.56041
54.05759
59.72379
49.27132
55.66309
67.42866
58.93456
55.04609
61.063
60.47417
59.11574
54.90837
60.62824
64.91276
59.46247
63.33117
66.96653
57.344
58.41847
51.73594
11
48.89784
56.40419
53.27907
57.70858
60.13174
57.36252
49.8771
50.76507
61.72847
50.82503
56.16794
57.22496
61.08074
58.50738
58.22435
56.08276
12
13
14
15
16
17
53.91169
58.78501
55.52344
56.80486
61.52223
55.40999
59.31
63.33395
68.93076
54.96845
55.96752
54.37172
60.77099
55.40898
55.7884
57.20137
51.59065
52.53189
59.59145
58.21001
57.24105
57.43264
53.01422
56.506
59.6073
47.17977
53.87927
59.69943
54.9497
55.1005
54.35286
55.45276
52.65707
57.15776
63.07686
64.23443
60.92911
63.13104
57.88894
56.10724
65.75526
60.43486
63.64916
56.6852
60.82307
58.7172
54.5662
52.02165
53.89362
53.84024
61.94453
61.31904
59.08607
56.08073
56.14484
50.78877
57.27849
60.98591
56.4249
54.59548
62.39369
58.29663
60.61338
55.38914
51.52782
59.01397
57.29045
59.77862
63.08842
55.29268
57.50767
58.372
63.79648
67.20416
53.57077
57.0662
57.9603
56.3758
59.40375
52.34102
48.74308
56.27094
58.86917
60.79617
62.519
53.22292
56.46302
57.91614
64.97612
60.1572
62.76109
60.51088
62.03726
54.49359
57.60259
55.24111
18
52.79254
53.31052
52.31585
61.74168
58.93651
62.58333
61.49621
61.85072
69.73541
60.3599
59.69346
60.66651
57.93887
59.82772
57.42231
52.33247
19
20
21
22
23
24
58.29143
53.1179
49.5465
64,58868
55.47341
60.75689
54.84238
55.98623
59.1165
57.10458
57.87557
64.95241
56.06193
59.45515
55.06161
56.03204
60.25729
58.7945
54.40694
55.33126
60.4925
65.16
59.66002
64.61342
66.40183
63.2111
54.93814
54.36087
55.52657
59.19167
55.32879
56.05415
51.93354
54.04932
54.27689
48.08097
58.22078
54.41302
53.63532
53.87748
59.67845
51.21052
56.27781
55.6951
54.28328
54.61128
53.50467
49.14887
52.78392
57.23817
59.50833
55.2767
59.40311
58.98017
56.43446
50.94233
64.96954
57.76245
58.23869
54.51346
55.97335
55.93495
58.56824
51.96419
55.90658
58.16712
60.05106
57.0354
53.80168
65.01278
52.90296
51.6377
59.16885
50.57134
59.27472
55.45254
58.25388
54.25133
57.97326
58.44622
51.21672
54.77521
58.72316
59.1104
49.39022
60.78648
56.86957
58.20152
62.96438
56.10424
57.87582
51.737
54.00026
46.06312
54.76228
52.52939
Table 17. Raw FP assay data (mP) for plate 4 with compounds from one-bead-one-compound library (Figure 3) after approximately 3 hr.
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
56.42049
51.69671
50.53776
51.22239
45.73457
46.75976
49.63345
49.6423
54.2512
49.66637
52.59577
55.50803
50.91166
48.81035
54.32976
54.89304
55.25405
50.00473
49.77276
49.51701
44.39928
49.762
51.45195
49.16684
51.76036
54.04683
49.69497
51.19818
49.48293
52.29228
52.16372
44.99902
53.59927
46.21702
45.98967
50.3445
52.21326
48.91081
52.57012
46.3081
49.69552
51.58202
48.59195
50.70187
45.67352
49.95966
50.66032
53.24295
47.87221
46.56889
50.44201
50.95583
49.31818
45.11876
52.55159
53.2327
55.90771
57.44609
52.35687
52.35716
50.82853
33.66277
54.6258
51.78414
46.0267
46.16695
48.52863
47.26679
48.61227
50.49018
54.9866
50.81935
51.42603
48.79456
53.19985
46.51291
54.19582
42.24133
52.04192
56.60692
51.08862
50.09411
47.34392
49.78167
51.39304
54.31182
55.97095
53.55221
55.95304
53.37868
54.68643
49.58767
55.99645
38.00127
54.65535
55.63351
52.05593
48.94593
45.78541
47.31773
46.08458
49.21782
51.28071
53.92283
47.00952
52.91862
56.81223
50.51179
54.31061
55.08164
59.5541
57.1568
52.1515
54.52223
51.82506
43.39092
55.27148
61.26098
53.13882
61.90252
58.43571
62.46172
51.07591
51.62213
54.31993
56.65456
49.18598
52.16354
46.62516
53.09688
48.14974
48.22044
53.54339
51.58365
54.77667
50.4073
49.0548
54.98743
49.77101
52.09085
60.37244
55.33262
50.31478
47.35385
49.60378
53.04699
55.41951
50.52596
56.11262
53.76749
55.80428
54.10628
56.12992
59.63736
50.83133
50.97887
55.1889
52.38571
54.16515
54.35504
52.10256
48.35101
54.07064
49.71773
54.54041
53.25823
51.44113
49.95375
50.39802
51.38701
48.51509
53.10324
50.44412
54.32647
49.22889
51.01396
51.03198
51.23838
52.46491
50.30992
50.39744
50.58266
52.45536
54.81872
54.03592
56.51701
46.56143
33.05166
41.48567
53.368
56.36753
51.47118
46.35453
49.76059
51.7834
52.48957
50.53946
47.00524
53.89066
52.03742
49.0254
51.15653
48.43166
56.02108
53.48196
49.56459
56.38672
51.30164
51.31765
54.49918
55.03571
48.47775
57.72712
48.99865
53.10566
57.98344
56.42467
53.94446
54.07643
52.47506
57.22084
50.52456
54.47019
46.50481
51.39942
50.27092
48.60426
49.56625
51.49069
49.42017
51.8943
52.53693
52.70032
49.61194
52.02557
49.30357
52.42412
56.48873
49.9026
51.46662
50.10598
52.28662
52.34971
46.84454
51.81092
54.24631
56.5645
58.53755
57.39368
60.09637
50.54614
48.31596
53.67775
56.61863
49.18161
54.78472
41.28002
47.20938
50.15459
48.45799
51.06039
50.06534
53.54833
54.35424
52.69037
53.07161
53.6385
52.31459
57.33063
54.14153
54.53655
44.14732
48.17432
52,42944
46.65107
50.8527
53.49203
56.76185
55.84455
57.04992
56.46006
59.82296
41.31062
52.16185
56.70207
47.26989
50.25911
47.04634
46.78031
51.28525
49.0267
50.02187
47.35796
49.11857
52.35125
50.25336
44.97268
49.12008
54.81636
52.4173
47.60438
48.24732
51.99363
51.45216
52.0454
51.52104
50.9599
47.3064
53.01276
52.79983
56.34018
60.44134
53.70827
58,33746
41.22791
50.31963
51.98364
47.57884
53.30259
55.29482
50.72401
50.3336
46.2909
48.45837
48.93374
51.5924
49.46353
50.87698
50.82786
48.01249
53.92982
51.56099
55.71442
46.39325
45.13532
57.37034
51.77772
53.13013
49.75515
57.53701
47.20122
51.43993
52.73479
51.02101
56.13982
52.70758
45.83166
50.84306
54.99582
53.75509
46.93223
50.48201
46.93224
51.1101
42.77967
52.55728
46.5509
46.56867
43.68753
.41,03763
50.94623
49.19706
45.63715
54.27822
49.25409
46.11864
48,43731
46.6588
47.75054
51.05368
51.89323
47.60435
47.39639
50.26629
49.10389
52.0817
59.92996
55.18971
47.24525
48.23099
46.70833
47,95056
46.88593
49.27855
ro
oo
to
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Plate 4
60
50
40
Q.
E
30
20
10
♦ No compound
■ + Compound (~3 hours)
0
1
17
33
49
65
81
97
113 129 145 161 177 193 209 225 241 257 273 289 305 321 337 353 369
Well
283
Figure 21. FP assay data for plate 4 with and without compounds from one-bead-one-compound library (Figure 3).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 18. Raw FP assay data (mP) for plate 5 without compound.
A
B
C
D
E
F
G
H
1
J
K
L _
M
N
0
P
137.1505
54.67073
56.17329
53.45438
56.69341
56.31989
60.26165
60.17675
57.70598
52.40353
55.62285
53.65868
58.36973
53.14769
55.72101
53.33178
2
3
4
5
6
7
8
9
10
11
12
13
49.50823
58.1066
56.81259
55.98652
63.31828
60.24009
61.69672
60.92211
62.97323
60.95901
59.30733
56.18346
62.29674
56.24444
58.62436
56.54751
46.97065
53.99416
56.59764
57.10496
57.08046
55.12738
54.05663
57.24565
59.97035
60.06408
55.03086
53.78573
52.30961
55.34035
53.53516
53.81367
55.13154
53.34723
50.67364
57.78454
58.31393
52.64356
57.35775
57.57243
55.82803
58.40955
56.98594
58.348
57.83415
54.73391
54.8426
53.87555
51.5518
57.64909
57.01457
54.32299
59.61379
52.73902
53.07017
54.61844
54.95218
58.4339
52.22313
57.79135
54:96545
52.953
53.83127
52.01235
51.77269
55.90968
62.61479
55.21684
61.67125
57.02394
58.0914
57.63396
58.83533
61.16054
56.30206
57.3561
56.20008
55.27176
50.99989
48.9691
58.22674
58.05019
54.97489
56.88531
60.66159
58.76987
58.77895
58.34039
57.17931
65.0123
58.06043
59.92064
55.27223
61.46255
51.27848
49.06953
57.59087
58.97518
57.10209
55.34608
61.38412
57.4804
58.11745
58.53425
59.8282
69.23611
59.07427
60.03681
57.97838
64.53141
53.91432
54.48559
47.85917
57.17896
56.88589
55.65734
59.25982
53.04915
55.79384
55.72309
58.16376
57.14921
54.91397
57.6167
62.33958
63.10526
52.1594
53.48447
52.97542
53.54837
57.68982
59.12005
66.38613
59.06876
61.38201
57.49725
60.23678
60.94375
59.4052
58.14865
66.31264
68.85344
58.72209
56.92521
54.36495
58.40896
55.15606
57.12013
57.97131
57.45171
53.07751
53.73873
59.19797
57.97709
53.61899
57.48162
54.93952
59.9812
54.51346
54.40239
57.6217
58.92103
57.22726
53.81173
58.04303
57.06534
57.2641
57.23181
56.19401
59.71159
57.1968
56.6407
59.26749
57.64515
53.71021
50.88441
53.19422
56.1203
56.47067
54.75726
57.7134
52.77818
55.67821
57.13321
57.05548
53.19346
56.11001
58.85442
55.01681
14
51.76533
60.40245
55.15149
60.97804
60.25512
57.81964
59.87743
57.75665
60.0332
58.88875
58.85288
62.57556
57.4221
57.67822 59.23084
53.94994 57.22154
52.17268 53.55896
15
16
17
55.10814
57.31386
59.30957
62.06685
58.48506
58.31139
54.15319
52.39615
55.86612
64.89834
57.42409
58.6312
55.88925
57.12045
52.17799
54.0514
58.45247
56.10308
59.85421
62.15587
56.86316
62.48347
58.16634
54.05784
60.48239
67.0922
57.0313
61.50997
64.64404
56.43676
53.79575
53.41605
52.71289
60.39824
57.57883
57.032
63.26103
56.45051
59.32245
57.6098
62.65796
60.97199
60.86316
63.15527
57.69254
18
52.76836
56.10567
53.89201
55.15587
62.78383
57.85388
59.26731
56.55193
62.78405
60.65239
58.20639
58.44665
62.57252
57.21957 59.47402
56.52933 53.86422
59.34369 60.65099
19
20
21
49.72682
57.05123
55.3815
54.98894
58.99201
56.74557
49.73528
52.91092
55.28481
53.11238
58.50219
56.89187
55.69379
59.02276
55.65035
55.33893
48.30641
61.602
54.77873
57.35134
59.52015
55.35234
59.4686
53.18623
56.49266
56.55602
64.91022
63.08125
58.63057
56.15824
57.68857
54.38867
48.55473
59.04499
55.87563
53.84617
53.95569
52.2953
57.74478
56.0027
62.48467
58.0334
55.35689
60.13417
51.33199
22
23
24
54.49134 50.85517 53.53362
63.53215 54.56148 52.91765
51.12471 56.25316 53.56075
53.92817 51.89572 49.7842
55.33235 56.98976 55.01108
53.2206 49.84372 52.47452
59.87982 53.10982 61.07096
54.75251 50.22638 55.03818
55.94897 57.18504 54.27018
57.57322 58.23199 59.76651
58.3431 53.99939 56.26979
56.08585 55.41338 52.88604
58.81482 52.77271 56.2776
55.29586 54.78655 54.07495 55 .18644
54.47028 56.84858 57.50854 56.23594
50.87043 52.51694 52.39726 52.64153
Table 19. Raw FP assay data (mP) for plate 5 with compounds from one-bead-one-compound library (Figure 3) after approximately 3 hr.
1
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
2
3
4
5
6
7
46.69319 44.92648 42.12965 47.01111 48.07954 47.84927 49.28718
49.98185 48.74636 49.33072 40.70761 48.34231 50.81651 48.87847
49.94596 49.87872 49.96815 53.81037 53.73138 50.77136
49.741
49.09127 52.55503 52.0268 50.88316 49.61388 40.47032 48.84801
45.88561 41.36285 52.97154 53.00827 45.72706 49.36972 51.72009
47.5576 47.40385 49.08631 39.32864 47.31587 51.45406 49.53792
48.84353 54.59956 52.28722 53.4197 45.22612 56.28128 52.76617
50.9023 49.00732 50.69504 44.99973 49.74643
47.137
31.192
51.57931 51.4679 52.97605 51.94487 50.02684 51.80972 45.01767
52.21492 56.35411 50.86219 52.34036 46.75753 52.05453 52.09643
49.29779 54.36161 51.65515 55.66275 51.74563 49.7318 49.55246
48.8519 47.09048 47.78487 54.06334 42.84776 53.11117 47.41575
48.9943 44.74294 53.15485 53.09328 51.78469 43.16154 52.73486
51.39137 54.44264 52.69849 51.85018 50.51887 48.450041 49.98596
53.849 48.26243 47.62016 51.42237 71.60782 42.85857 51.71611
53.9716 50.97329 52.86605 50.39888 49.80645 40.60023 51.57443
8
9
10
51.52458 50.20972 48.73514
52.68595 51.32867 50.39594
48.781 53.77978 51.56709
51.15914 55.09847 53.00759
55.46597 49.89945 55.19144
'54.301 51.63905 52.81872
59.08919 49.36598 51.23969
44.50977 46.98918 44.67398
47.37209 49.72952 48.97155
52.30393 49.12992 53.71082
55.67909 58.33596 57.23897
56.72437 48.62677 47.81095
53.10638 53.22814 53.50157
57.17638 51.49761 43.88358
52.72555 46.33076 49.77634
57.61464 52.96952 42.7402
11
12
13
52.87887 53.46865 51.71453
50.78098 51.75073 50,59142
49.25029 49.24506 48.65934
51.46242 49.67238 51.89861
48.30817 53.02251 50,15824
49.99705 52.79364 49,32051
48.11511 50.76249
41.484
47.94028 51.96994 50.51044
51.35587 49.22659 41.78198
47.90423 53.44937 47.57554
47.61117 53.67632 35.8789
49.43794 53.58354 49.0223
50.04577 48.72712 47.4556
48.69895 53.84905 51.41848
47.90123 54.10772 51.04824
50.84843 51.3843 51.36496
14
51.15026
54.27136
47.74016
51.14886
50.78282
53.0404
47.70428
53.04906
51.27593
50.45726
51.1601
56.15755
54.22867
56.06396
52.31888
50.72048
15
16
17
48.7841 52.87262 50.6286
52.92622 49.16185 49.67273
45.8491 51.13387 47.0926
48.23133 52.60916 52.76199
49.25682 50.28475 46.52354
49.4547 51.1534 53.04158
49.53296 53.59106 48.47616
54.97093 54.60753 54.59598
47.13728 49.27088 48.49962
53.66252 53.69716 53.19247
51.62202 52.24703 55.53302
46.95596 57.26032 58.83226
48.94245 50.64991 48.28797
49.85992 51.95243 56.25268
48.84043 38.28056 50.50451
44.05537 50.57774 45.18164
18
19
20
21
22
23
24
50.26684
50.79141
46.55558
53.08246
48.85985
48.79119
48.60968
42.16895
56.49028
54.40007
59.04072
58.7206
57.60564
56.5034
52.59028
51.79209
47.34795
52.56051
47.84253
35.44053
50.85731
46.19479
45.3437
52.84912
53.22382
47.04516
52.53919
51.91683
51.40624
43.58819
52.86944
50.51824
47.27324
50.68998
50.07076
48.0422
45.61698
39.2013
49.94344
50.07292
51.63732
49.22685
55.51084
53.40864
43.26104
43.09969
49.91819
54.01368
48.55882
39.06228
46.44694
43.05835
46.98131
41.23422
38.76413
44.20156
50.57877
46.22796
55.07661
50.10523
42.51157
47.29728
45.48065
48.99735
51.33718
51.92645
47.16387
46.43978
52.08413
46.50335
45.7619
50.31099
50.1974
48.67545
55.66343
49.63907
51.58448
52.17561
42.06152
50.08953
46.15845
48.33416
49.38634
50.69203
54.51987
36.74738
46.55319
48.34865
54.14999
48.63327
50.95274
49.52026
44.81337
52.94305
49.81738
52.23434
48.13872
48.56522
44.92254
47.10162
56.03255
45.77204
50.65749
50.09574
46.95253
49.63146
35.2863
48.86198
51.17708
41.84285
49.47311
47.49544
oo
4*.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Plate 5
♦
♦
60
50
40
♦
I
♦
♦ ♦
♦♦♦ ♦
4* ♦5♦>♦<
►
^
A
Sj
% •Ti Pi a
t, £ m
f•m
■■. ^, 1
■VS
■
1
■
■■ ■
r* ■ ■1 I■ ■■*11 ■ ■li
♦
-
♦
Q.
E
■11
♦ ♦♦♦ ♦♦ ♦ ►
♦
♦
Hu W
u k♦ ♦J ►V▲
v.
*
■1#^M
f .I
*i
1 i1 1p1 ■
■■ h ■■■■y
■
■ m
m
■ ■■ ■
I
■
m
m
♦
♦
♦
e
■
-
.
)4
30
20
10
♦
■+-- 1—I-- 1-0 ---1
No Compound
Compound (~3 hours)
1
17
33
49
65
81
97
113 129 145 161 177 193 209 225 241 257 273 289 305 321 337 353 369
Well
285
Figure 22. FP assay data for plate 5 with and without compounds from one-bead-one-compound library (Figure 3).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 20. Raw FP assay data (mP) for plate 6 without compound.
A
B
C
D
E
F
G
H
1
J
K
L
M
N
0
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
51.51667
52.59972
57.77241
52.70827
54.16226
50.99937
49.28468
54.14685
56.13592
57.82512
56.58613
54.66116
57.61682
52.35713
50.63258
51.06102
52.70585
59.01514
59.40178
54.76681
57.91783
57.87033
55.98218
59.14592
59.59303
60.46212
61.7403
56.73831
62.35497
58.82132
55.05833
57.73796
53.46125
56.35028
62.75399
51.9411
57.33134
59.85801
58.17697
61.69673
60.53177
58.17005
60.15318
52.83752
52.77423
54.27285
52.56777
55.2182
54.89408
58.06522
62.41923
56.51674
53.62242
59.20375
59.17255
60.00673
61.68994
60.01307
61.01269
59.51876
58.6736
56.16445
55.99704
55.42806
50.42262
55.64155
53.28268
56.67085
53.68511
56.02534
52.19563
60.35809
65.38931
57.91835
59.74004
56.36653
55.64367
56.46017
54.50252
55.56847
56.05809
57.45744
57.83506
56.90611
59.0703
60.76855
58.77355
64.19244
62.13066
62.33838
65.54503
56.99081
59.8233
61.69218
57.28957
56.93431
55.36768
51.65861
53.74109
55.58415
57.09837
61.47386
55.24989
58.99279
54.29182
54.40397
60.15277
58.66205
56.05671
57.60206
55.59043
54.25319
54.08387
56.60008
54.92677
58.98244
61.84659
63.74583
57.43302
59.19297
58.4036
57.88522
60.51924
59.0367
59.5283
59.86216
56.94326
59.91136
55.41334
53.08982
56.1514
55.20774
58.72379
59.88031
58.26232
55.28402
57.55491
61.52674
60.22114
55.65795
63.93936
56.1416
59.46871
54.42239
61.68972
58.26246
56.27353
59.17229
61.76533
57.92374
63.41245
55.27639
59.15869
59.75691
61.60239
60.72248
62.089
57.50179
58.869
55.48529
52.90669
51.80026
57.76401
53.68988
58.03309
55.04326
52.90317
57.18654
54.6475
51.46113
59.21708
58.6846
51.86231
60.03208
57.68368
57.46784
57.2458
54.40099
60.96178
57.08255
61.04151
52.68825
55.24194
62.64887
54.98364
55,50496
56.25936
56.51798
56.65727
59.87101
58.4975
60.40549
53.55359
54.79492
59.95883
58.88543
57.40052
47.62499
54.6063
57.35718
52.44831
51.51225
53.57994
50.52508
57.69036
52.46631
57.09582
51.89079
53.27999
57.39752
59.23302
60.98587
57.69357
57.10002
56.97629
55.0348
56.71201
56.30226
57.11566
61.76329
58.20238
57.92838
60.64531
58.34113
54.57006
54.8164
56.36453
57.03673
56.67091
59.95993
59.30838
55.71509
53.61855
59.97615
57.81991
54.86642
56.92658
60.12315
54.12
53.90658
54.40471
54.22064
59.73043
51.37052
58.16527
64.55828
59.28101
55.05414
57.76969
62.35494
59.21434
58.12324
59.06945
59.20132
56.92015
53.00838
54.85659
52.75859
58.23147
54.22678
54.64509
59.29792
56.019
57.22442
64.64965
60.33138
62.78332
54.27928
56.4508
62.50322
60.14302
51.01209
57.17722
53.91002
53.39507
51.48482
53.35856
57.00083
57.15595
54.37805
63.27205
58.10455
57.65035
58.58914
56.72443
62.33901
62.1639
58.16536
50.59965
57.0512
59.57612
48.77372
53.11581
50.91561
53.25443
55.15546
56.83332
52.9926
51.93053
52.83625
51.51477
56.04775
56.0351
55.49523
50.88007
55.45076
55.80794
52.23461
54.02808
56.36972
55.60523
54.58389
64.0574
55.61538
58.19669
56.79571
59.28934
56.33514
55.1229
61.49846
57.30114
51.91146
55.95062
54.88499
53.17628
55.46278
.59.80356
58.63251
57.72329
54.11613
55.01634
59.79261
54.89059
61.68572
54.85781
49.47098
55.4479
54.11584
59.328
55.13772
52.66208
59.09554
57.39177
58.75191
58.66526
55.04327
57.18059
62.20483
59.51916
57.97614
60.32257
53.45526
55.77834
54.41905
52.47679
50.5524
56.54439
51.99438
55.37969
51.03773
60.77914
55.44796
55.56378
52.29366
54.18122
52.1304
53.34274
53.35309
53.74742
53.41047
51.24506
51.63146
54.27943
48.33069
54.50564
49.87397
58.60952
58.20154
52.71806
52.65749
57.42251
52.92921
59.14274
53.91472
Table 21. Raw FP assay data (mP) for plate 6 with compounds from one-bead-one-compound library (Figure 3) after approximately 3 hr.
A
B
C
D
E
F
G
H
1
J
K
L
M
N
O
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
48.63544
47.44631
51.61439
48.49066
54.0523
48.1811
46.42288
43.45664
50.47284
48.2143
52.19145
44.79845
48.33681
45.19813
46.01347
46.90111
50.8013
52.3072
50.67989
42.22087
52.66417
48.79732
46.35869
48.89375
53.80329
49.92624
53.13012
52.25483
48.00269
49.62497
47.08933
51.20686
50.51848
48.36938
54.59769
40.69132
51.97658
51.26352
50.0591
46.5024
48.25972
49.02149
44.77754
51.42578
53.64153
50.08243
48.11423
53.30099
48.29823
49.98183
52.20898
47.12707
51.47574
52.82332
50.75447
51.44869
49.73058
49.9 6478
48.73164
53.20637
56.70527
43.80917
48.32795
50.73787
48.45157
48.93087
40.13251
48.08648
47.83899
47.87907
47.34854
48.27694
51.28143
47.79305
47.39792
54.11512
49.26445
48.99167
45.64438
47.59542
53.1979
50.64648
45.51807
49.39346
47.0843
50.33607
51.23278
50.03723
51.29563
47.71553
48.75402
47.14438
49.28905
45.72794
46.62377
47.72238
49.3112
53.18495
50.15225
52.86157
46.58833
50.32312
49.82556
52.36591
53.63547
51.49079
53.13516
54.88734
55.81824
52.39246
49.9108
53.17441
52.55603
54.77903
39.04292
46.94007
49.71896
47.72296
51.5746
50.08624
51.93279
50.28877
48.97473
53.77405
48.00525
50.06481
52.7511
48.60713
47.04275
49.11976
50.55783
51.93576
50.88875
50.84288
54.49868
48.38344
51.82873
51.47091
49.6542
47.51091
47.5745
49.91306
43.75735
49.41791
49.68682
48.34136
50.20618
48.39055
48.61765
49.16567
48.7586
48.04183
51.09406
49.99094
52.18021
50.88554
49.31725
49.74395
45.61137
47.02555
47.53393
51.33467
48.56209
50.64111
46.94234
41.72498
43.60734
44.2647
49.57348
47.30186
46.09156
43.38902
45.77013
51.73894
50.55.133
48.75252
50.04713
50.21918
51.55135
36.51343
48.82039
46.714
48.65471
47.48395
53.04326
54.09308
50.67515
44.0822
48.8122
51.51731
49.26869
54.44857
52.12417
49.91106
54.33925
50.91864
45.4809
50.13238
50.03829
46.47057
48.73941
45.71298
49.47276
47.81212
49.29011
53.00841
52.04924
51.36079
49.5368
52.10254
53.51431
50.78779
49.78793
50.27045
47.73324
50.87051
48.75485
53.08129
51.96294
52.34543
52.28973
50.23372
47.44259
47.38342
48.05977
54.14301
52.71838
50.92344
49.56933
43.19296
49.72123
50.51411
51.55149
54.18371
48.82933
51.766
50.75296
41.50129
52.7969
45.95187
51.23817
53.14937
52.68727
52.57312
50.36148
49.41623
51.48271
48.93089
55.28932
59.2055
54.13079
52.1723
54.04599
43.03373
51.81626
48.54802
51.23434
56.94508
50.41122
53.34233
48.81584
53.23544
46.93417
51.18545
41.15382
47.79607
54.90607
49.19841
43.10673
47.17119
49.66376
48.55404
48.42344
52.31296
49.83564
48.80955
50.17733
53.41144
47.06735
52.22586
51,61471
24.43652
52.89313
52.06152
51.937
47.97527
49.33848
46.80273
23.54045
47.5142
42.73129
44.62368
48.7593
46.98994
49.48546
49.25573
46.50626
46.26708
46.37186
45.23498
49.63073
48.58048
52.66684
44.88842
48.90067
44.11482
48.9773
39.16419
48.18993
50.96081
53.46483
49.73392
52.86509
46.11052
48.95423
48.4485
54.38822
47.03254
48.03771
42.55212
47.39859
49.38152
49.71126
50.35957
46.59295
50.4085
48.88095
44.22827
46.61741
53.19313
50.63409
48.52904
50.6669
47.10651
48.38701
36.98997
51.54762
51.40881
50.01816
45.60166
40.28116
52.33148
49.11749
48.60125
52.17125
50.08739
50.69316
51.28767
51.52252
37.60597 52.16195
51.57761
53.52396
50.42457
51.77367
50.20048
52.93369
51.06922
46.79342
46.34102
40.1329
51.05683
50.00278
55.34604
53.9846
47.8327
50.4963
55.50475
50.05412
55.8753
52.18519
49.98987
50.42757
46.97138
53.72963
51.94567
49.66391
47.72313
49.65133
47.86772
48.46573
47.81656
43.53717
46.81772
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Figure 23. FP assay data for plate 6 with and without compounds from one-bead-one-compound library (Figure 3).
in
o
co
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 22. Raw FP assay data (mP) for plate 7 without compound.
A
B
C
D
E
F
G
H
1
J
K
L
M
N
0
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
55.89361
54.04288
53.13308
54.58443
57.07855
54.34363
53.07036
55.89522
59.27313
56.46199
55.41129
57.95038
56.88102
53.11048
55.21301
50.68853
58.31438
58.25981
55.89092
58.29015
59.14751
58.40526
53.76234
59.01896
60.7632
58.55051
59.83653
57.7342
53.64721
52.63957
56.2027
54.63554
55.11995
53.15368
53.52569
54.04364
52.38562
54.91675
53.40345
55.59712
53.71217
55.87258
59.33093
56.04722
59.90227
54.33464
57.22681
59.06032
59.40628
50.18992
54.91714
54.76095
57.63107
57.36087
57.74939
57.67287
55.75225
58.57753
57.23717
55.11046
62.3493
58.35259
55.7058
58.31402
51.02017
50.48498
58.97032
54.23464
60.41694
56.51301
49.01538
53.73439
57.72014
56.3744
54.58236
52.29676
55.40227
59.42579
55.27861
53.18253
50.96679
53.33614
61.49886
57.4057
60.52936
61.94643
58.0539
57.05969
56.62245
55.31713
60.14607
57.28535
55.17233
63.26958
55.66157
56.46099
52.21043
54.84951
60.12878
55.193
60.63182
59.00744
54.91953
61.464
57.96455
62.26116
59.57937
66.04462
57.5238
58.85872
55.33054
60.88412
49.95421
57.75536
63.56791
57.87995
61.84694
62.28523
57.95238
62.14477
56.28523
58.8167
59.21094
61.2117
58.1681
57.13107
54.68445
57.18747
49.88202
53.64561
59.13882
60.66917
53.64752
54.48336
56.41588
56.33462
56.50656
52.94958
56.65199
53.53113
60.31555
60.74064
59.97255
49.3475
52.5396
51.13303
61.90528
60.4608
57.33784
59.07278
60.95963
57.32061
55.11403
58.85514
58.24998
56.74428
58.30651
59.79535
61.65139
53.10918
50.19089
51.13766
52.10833
55.03265
52.46379
58.93967
55.31996
56.61071
56.82444
56.72591
56.6755
56.38904
58.23581
58.1514
61.86932
57.93294
53.03981
52.43546
55.67719
57.24351
57.37341
63.99827
59.46021
57.54629
57.48403
56.52749
54.28359
53.46614
60.62931
60.36945
58.61195
61.30432
56.69902
55.77196
53.57326
55.5225
60.4366
52.17801
51.13327
56.88801
54.44416
54.30286
61.72845
60.66201
65.45278
60.3672
61.82997
61.05451
56.62707
59.80772
55.78624
56.1242
61.81489
57.66476
56.67402
51.82069
51.87562
59.60089
59.74323
55.89833
67.88581
57.89634
61.31463
57.63984
51.5202
54.99177
56.12625
54.34051
55.10897
57.93099
54.078
54.24145
52.98442
60.64502
62.86273
55.95862
56.58085
56.60931
59.50378
57.14287
54.7253
55.06077
53.90221
53.49167
59.9178
59.8142
60.1593
55.7462
52.30195
61.12548
62.69861
57.39392
57.32074
56.34631
61.57769
54.83458
50.78341
56.00492
53.39184
53.78313
56.70846
58.05412
60.16586
58.83201
59.31166
59.25267
62.27021
63.14518
59.50498
57.22808
61.25542
57.44757
53.0954
51.20685
58.09956
58.37256
58.59092
58.07704
57.56622
54.58344
53.81042
56.67948
63.19253
56.91638
56.82757
56.93418
64.22162
52.9843
49.49581
51.68495
55.75384
57.78283
55.50267
50.21572
54.83039
57.37767
62.5584
57.27977
57.76185
54.47135
55.40084
56.14454
61.84828
54.60688
51.94633
52.86593
59.54135
57.96491
56.02209
59.97614
60.94195
58.4008
61.51786
55.79163
55.35465
54.75106
55.56506
48.94041
60.96286
56.64031
53.60912
52.33028
54.27319
53.53996
54.98748
53.2437
57.30925
56.43353
58.10223
59.21112
56.50408
58.56115
57.79622
54.25037
55.4255
50.42111
56.95545
57.29729
59.09301
57.15989
55.43515
57.66788
62.94425
51.60681
54.64842
55.03703
58.39567
52.57404
56,3446
57.02439
52.48948
52.90812
54.05157
55.38459
54.9834
56.06215
57.96862
59.69705
53.65913
54.39224
64.23
52.77684
56.51022
54.034
61.17581
59.31776
52.26698
57.62511
52.15391
54.43866
53.54775
55.01766
55.42802
56.68837
54.73145
51.79833
55.32695
56.04744
56.02924
55.71401
51.7164
54.44094
53.87476
40.25672
Table 23. Raw FP assay data (mP) for plate 7 with compounds from one-bead-one-compound library (Figure 3) after approximately 3 hr.
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
48.06623
48.84021
52.83721
47.24674
50.76735
45.91425
50.86849
49.52617
48.59701
52.41486
49.07581
50.50828
50.83337
49.96136
49.93501
47.11578
48.17675
45.59821
44.04179
53.37017
52.31107
49.7685
57.52473
51.04348
50.18745
51.66669
39.70549
55.05051
54.20442
52.22603
48.08927
45.54878
45.53932
47.03602
45.12263
47.40448
42.30611
47.31448
55.23165
42.01307
48.82783
48.11496
49.61856
36.92389
54.01884
49.85427
49.25129
46.80238
47.39128
45.7969
46.65123
50.61888
48.33419
52.17201
54.49236
46.94849
46.08983
46.57282
52.82893
51.29261
53.57102
50.80014
44.67539
49.00568
51.13493
47.75882
46.45437
50.75219
46.79389
50.98138
49.56754
44.55986
49.51021
50.93794
53.02363
52.9808
52.48136
49.42575
51.85457
46.50912
50.02065
48.80654
48.23676
50.09884
52.00473
50.3273
52.15613
46.34116
50.65908
52.67504
55.38294
53.73813
53.18378
48.38571
52.43633
47.13763
44.24357
46.6769
47.88173
48.95587
51.16607
45.53357
51.25954
50.37504
49.30657
55.15314
51.4341
48.99414
51.24472
51.26189
51.39806
49.79232
45.80815
47.6767
52.40629
50.10847
53.38318
54.06884
55.78137
49.69993
46.98092
54.23962
54.46875
47.97774
47.95414
48.92882
48.20285
47.59761
45.06646
49.65058
48.91089
50.89034
46.63912
52.84623
50.7751
47.42813
48.34605
54.01253
53.04339
49.34592
52.66307
53.20126
53.86574
44.3153
51.85391
42.88849
51.32804
51.16928
54.31908
52.9531
54.03871
47.07196
45.97682
53.25897
51.64412
51.97191
54.42529
53.27205
53.47559
47.85751
48.44147
47.1197
48.87393
47.71326
39.38723
46.49033
50.15944
47.06558
49.70796
48.78005
50.66615
51.28417
49.79249
51.6411
52.17127
34.08336
46.25953
51.08141
49.04878
52.27741
50.39034
52.86308
48.06624
53.85996
46.08663
49.41145
50.62409
49.32976
54.66424
54.88088
53.46566
44.02725
46.94668
46.98637
43.33134
51.2213
54.62377
50.61228
46.62371
54.41515
45.39862
48.38827
50.24811
50.58756
51.74206
55.37782
50.34642
49.42147
44.50911
44.82661
48.57451
53.36707
58.16659
55.9153
49.69639
48.01099
38.9022
44.4561
53.83815
45.40579
54.8721
51.68507
28.97919
50.32135
43.65598
46.93407
46.97795
52.98121
55.24505
50.49179
44.57111
50.11397
48.49158
53.94108
53.91518
54.3404
52.16861
50.13718
46.35468
48.23185
50.74586
44.96799
51.56215
49.77541
55.24743
51.04523
49.43268
46.68689
48.12249
51.58157
55.68646
53.71598
51.68453
51.8568
50.85453
47.6479
49.64497
41.3305
45.66809
48.94457
51.41229
49.33863
50.24459
38.15029
54.08371
51.83551
52.32874
53.17149
53.33557
54.83457
48.35386
48.88929
46.57701
46.10734
48.53241
50.34801
52.16564
56.40832
54.22565
45.64687
49.5259
51.58861
52.19747
50.82177
52.97536
53.32872
51.05053
50.22078
44.2985
48.15895
45.26495
48.70601
53.54569
49.22817
45.044
43.86061
46.64663
51.90975
46.94254
49.22149
51.51734
54.56507
48.33552
41.8528
49.7926
46.16821
45.4892
41.74458
51.66432
54.28112
51.76092
45.1187
48.31475
50.9678
50.46443
49.65732
47.90309
45.62246
50.07199
48.49005
43.17141
48.62063
42.83558
48.70538
45.96501
48.37578
45.16631
35.66022
53.48929
50.19188
49.58649
53.67255
48.12839
47.42079
40.92439
49.11102
47.4519
50.37221
46.78073
56.09725
47.43536
46.71011
48.03748
41.92105
49.82923
44.50767
48.1459
49.15578
47.32498
47.11218
48.93525
44.10113
43.87479
43.44846
44.58443
55.78495
51.36897
47.75393
41.98384
41.72793
50.24278
51.41711
46.50249
45.74945
49.89644
48.00003
45.59203
44.24982
46.55118
45.15133
48.24158
53.42355
48.32539
49.35814
47.52958
43.54667
49.9355
49.18496
46.41054
46.29099
48.39288
47.42025
47.2632
40.43793
ro
oo
oo
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Plate 7
♦ No Compound
■ + Compound (~3 hours)
---------------------------------------- J
0 I
|
i — 4 -1— q
1
17
33
49
65
81
97
113 129 145 161
-------
J-------1-------------- --------------------- -----------------
177 193 209 225 241 257 273 289 305 321 337 353 369
Well
289
Figure 24. FP assay data for plate 7 with and without compounds from one-bead-one-compound library (Figure 3).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 24. Raw FP assay data (mP) for plate 8 without compound.
A
B
C
D
E
F
G
H
1
J
K
L
M
N
O
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
50.38587
51.13429
50.15778
51.88566
52.00314
50.72112
50.26139
54.02431
53.38226
53.61535
54.82795
53.14559
56.30278
54.91588
55.52693
53.11326
56.31181
53.97628
54.44605
55.11057
55.34919
55.99444
55.77153
56.71134
57.1387
56.69334
57.84164
57.30994
60.20357
57.0644
57.08351
53.54698
52.86115
52.7049
58.03958
54.00465
53.2059
59.4313
53.17371
54.3382
56.07074
55.33384
54.28141
57.7363
55.97095
58.75424
59.556
51.19482
49.2502
51.3984
59.38136
57.38818
54.02004
59.47787
55.56683
56.28261
54.46465
55.63561
55.98905
62.01199
58.93172
60.47989
63.20195
54.97317
47.63234
52.49686
56.50694
50.8595
54.34369
52:67485
53.4535
50.10166
52.49726
56.20002
58.49777
51.23738
53.88479
54,56494
53.76124
51.0429
51.35046
53.95252
56.26325
55.33766
52.67478
59.08995
54.82968
56.55181
55.00439
57.26726
61.54388
57.39994
55.56938
57.52023
55.35036
54.30906
51.09582
53.22766
48.486
55.68913
52.24547
53.4479
56.77369
58.15577
58.05082
57.55879
56.10663
54.46352
58.51524
55.42369
56.86472
53.89487
53.91848
52.63068
55.07852
55.79282
54.51507
58.36537
59.59306
58.1469
61.21184
55.88076
59.28557
56.88963
57.00506
58.05129
51.99981
58.19657
48.72248
61.02258
51.70657
53.1657
53.51709
54.86101
55.43227
55.52919
55.00272
54.8777
55,64149
56.19558
54.60745
60.62488
56.27089
56.09795
51.5438
58.4748
53.37368
55.90457
54.89151
55.81014
55.95259
57.50894
52.18557
57.6013
57.20914
59.38996
56.13065
64.2394
59.94992
57.07576
49.43877
54.03433
51.96224
53.58817
55.56487
52.40122
51.94945
50.13307
52.73968
52.34817
54.88556
56.06898
57.44754
58.87799
56.46976
50.73152
49.79464
51.52808
56.75157
56.61572
56.39867
54.12592
54.80938
57.27533
53.63295
52.26072
56.53843
57.36637
60.63812
58.41561
61.13513
54.10771
49.43392
58.42726
52.52037
61.20301
55.99049
52.06768
49.12096
57.22809
51.96214
52.17538
53.73523
58.90097
52.35674
57.66543
58.77225
52.98568
47.6035
59.97154
58.7809
60.35475
49.80732
56.69576
53.51464
55.62988
52.37874
54.87614
54.35002
64.18799
55.64682
58.4583
56.48796
52.88418
49.25168
55.94742
55.21308
49.7902
54.23318
54.49467
52.23882
53.63746
51.34066
55.55515
54.31785
57.64599
59.56555
56.07436
58.17371
53.831
53.58538
55.98124
53.51687
55.20346
56.70619
56.09107
54.875
56.39972
53.24082
55.62394
57.28954
60.34621
60.48458
59.91929
55.72546
55.32831
49.67169
54.82764
54.65082
52.13681
53.4837
56.19963
53.96219
54.91789
55.1287
56.16373
57.20346
54.95011
59.77328
55.68558
54.94501
52.37449
50.17273
50.01549
54.05646
52.06433
55.10813
55.26457
57.61505
51.31724
56.69776
54.5363
56.35301
59.41493
60.60199
58.97804
57.89439
53.9346
49.08371
54.80815
52.34074
55.04913
53.90072
51.6878
55.12965
55.13676
59.67426
56.30277
56.56182
54.0421
56.24611
57.55571
54.29755
50.80528
48.35566
54.54957
52.93412
48.754
51.73864
54.27304
61.63733
59.22831
60.3547
56.55642
55.80334
59.06486
57.52591
58.58867
53.51777
55.453
48.45688
52.40008
55.46412
49.31155
54.41477
50.51903
52.43225
52.96498
53.80975
57.98325
54.67038
64.1388
53.33289
53.32318
59.13783
47.5792
50.22647
53.69486
52.08419
51.58023
52.64458
52.95406
56.24881
51.9621
52.97754
54.23698
59.33486
62.90053
56.44516
59.70055
57.18632
53.1684
48.61415
51.50848
50.29496
50.53108
50.48072
51.80344
53.22538
48.02871
57.15667
55.50787
53,6338
51.87461
56.68934
52.25548
55.74339
51.72918
47.47218
49.6569
51.22887
49.69796
52.37575
49.20318
53.07628
52.51922
56.73388
57.21792
54.28012
56.8143
58.29886
51.14333
52.84133
51.82536
Table 25. Raw FP assay data (mP) for plate 8 with compounds from one-bead-one-compound library (Figure 3) after approximately 3 hr.
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
46.43844
44.41381
46.14623
47.44806
46.05299
45.31255
46.9522
44.69841
42.03931
50.16505
46.88239
32.20045
48.84539
42.49456
47.43215
44.76955
50.79202
44.85837
45.52697
50.10841
46.11774
45.72254
42.83438
48.57521
48.36079
50.89604
46.54906
47.33704
45.9274
43.10521
50.69862
46.96857
47.85984
46.87023
46.84799
43.1746
44.87981
47.7117
45.42418
42.32567
46.8314
50.17242
48.23158
44.78054
44.73058
43.46029
50.61844
45.25734
47.29887
47.12258
42.91688
37.6737
45.38266
54.34786
39.17092
47.14175
42.17966
50.4678
48.34983
42.685
49.32646
46.79437
49.1766
47.41783
46.52419
46.64829
42.22126
45.84691
41.75893
52.05826
42.81437
45.71809
43.96279
47.75676
45.7034
41.22723
46.94612
44.74362
42.1708
44.81181
47.52136
42.02089
44.35937
47.09225
42.60944
49.06158
36.55467
50.19013
43.02503
48.44671
48.38424
43.5189
47.39371
41.58016
45.72197
48.37791
40.29316
44.88613
44.16495
44.82689
43.99055
48.77091
46.69656
45.2629
46.27626
37.00031
45.5069
40.8531
48.94915
48.69359
44.37767
40.56639
42.87578
49.59022
47.80576
47.51369
44.69276
51.50923
49.29371
47.72798
47.53727
45.96194
51.74364
48.31401
54.10111
53.48156
48.54869
45.54801
41.00787
50.72908
43.4692
40.75778
42.51635
47.31868
44.58218
45.1445
42.81629
47.90666
44.3944
36.39908
48.39779
51.99649
26.89681
45.31971
42.99508
51.43103
45.22954
48.14037
45.35143
47.5063
44.98993
47.49216
44.53756
52.11996
54.17125
44.75799
54.47636
50.44014
51.88974
47.43139
47.4207
51.17415
44.552
44.99798
40.99847
45.15562
44.14599
29.43595
46.49764
48.18764
50.95174
44.60713
49.67515
46.44968
35.28702
43.83313
49.92308
47.63936
50.18338
47.30481
45.73236
42.60501
48.36553
50.39533
49.18483
48.63685
48.8586
46.06502
52.44853
47.45073
47.72897
50.62431
41.77692
44.88499
47.92067
45.36311
47.25959
41.91658
40.72947
45.91213
44.34314
47.54158
44.71016
49.75998
50.72642
41.12205
47.8415
47,48583
46.30838
43.49487
50.40218
50.10677
42.22659
49.33148
40.15707
49.74515
47.74566
43.62876
50.8035
47.41882
51.52586
46.46325
54.08494
50.80032
38.10249
47.88991
47.06127
46.12982
44.57011
41.89069
46.24247
47.85222
45.86417
46.88589
45.93741
43.5851
46.02342
50.14719
48.81041
48.13244
46.32097
35.84248
47.30934
46.62392
50.20928
45.09785
46.31142
55.034
46.45201
47.86678
40.21403
49.02835
48.09111
48.20044
49.83729
53.37244
44.67318
45.73919
42.12294
44.79304
45.16877
50.31304
40.90532
49.89119
48.62035
48.02133
52.60491
48.05414
45.37131
43.25993
48.3847
45.61295
42.41492
32.02213
43.94312
46.99718
45.69806
50.29464
48,03255
47.75135
49.32225
52.1253
53.09219
54.60794
49.08389
46.9331
49.84187
35.89487
43.60205
41.12336
40.61341
44.3642
43.18365
42.29217
42.48691
42.84656
46.01605
49.44903
51.192
48.41241
45.8047
43.49869
50.85353
42.04465
42.71269
42.86404
47.40706
46.962
42.16329
45.61474
47.10146
47.50295
48.44971
46.73067
51.83763
48.96168
50.21472
48.8618
51.7684
37.69829
51.36533
43.47402
43.67357
42.22364
43.2183
45.26908
46.89321
39,85989
45.17207
43.83907
48.29542
44.05795
40.66797
48.71925
41.86386
46.01162
50.23489
44.42908
37.1627
43.36548
45.98867
46.62833
46.72603
47.06504
41.15044
46.36204
48.4412
53.82612
45.80253
51.54385
44.23266
50.12037
37.71367
47.41194
43.04665
42.36873
42.65717
42.93581
33.31658
43.8459
41.66269
46.14316
44.34435
34.37882
45.56502
42.21127
42.47426
44.18768
46.08369
47.22958
43.40649
43.98946
43.04161
45.64302
33.19719
44.29739
46.38484
46.12763
48.09973
48.73027
44.659.16
45.96275
41.27359
40.91568
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R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
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Figure 25. FP assay data for plate 8 with and without compounds from one-bead-one-compound library (Figure 3).
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 26. Raw FP assay data (mP) for plate 9 without compound.
A
B
C
D
E
F
G
H
1
J
K
L
M
N
0
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
53.02223
51.92351
56.67725
53.6263
51.234
52.72414
54.26827
56.68167
53.95284
53.26256
51.77857
51.59403
53.41372
52.93032
50.13184
52.28784
52.85354
55.15625
54.18418
58.1695
55.10856
55.07589
55.3857
55.35405
57.5686
53.44307
57.28826
56.06193
56.64544
57.4746
54.94368
55.40145
52.74395
55.05486
57.22099
54.36853
61.96429
57.87881
59.61838
55.66196
60.1363
57.49613
56.4026
54.12553
52.80682
53.05125
54.17157
53.95476
50.83995
54.61467
56.24694
53.47061
57.19375
55.19579
56.42273
54.76507
59.16783
55.32747
59.59463
55.86591
55.49908
53.96922
55.93215
54.57515
52.4396
53.58812
56.63404
55.7069
54.70577
54.73937
53.30349
53.24658
58.0437
63.26834
61.71637
55.88746
54.47681
52.83304
52.86356
56.56373
49.80052
51.37084
55.67036
55.77378
52.05436
55.02255
54.97551
53.57887
57.33516
61.60167
63.56525
59.37282
57.91957
58.04445
57.01121
57.50769
51.87298
58.22205
54.82525
58.49397
54.81098
56.10778
64.19964
58.49437
56.34591
60.41652
54.96555
55.84456
58.68776
60.81715
53.93499
53.26962
53.56922
59.81969
54.08704
56.55027
59.3197
55.35862
63.75351
56.52939
59.69641
54.66009
59.20294
57.61191
62.7107
64.78005
53.43028
52.87718
49.68065
56.68925
56.43025
59.60749
56.61472
58.34383
55.7678
58.5787
61.99436
66.09849
56.18264
60.21077
56.62541
55.5143
57.72257
52.33035
49.99175
53.40416
53.43079
57.05819
55.23558
56.25416
55.08997
58.7372
59.88447
62.27116
58.49489
62.43756
58.77016
59.73216
59.25892
55.80094
54.25364
53.98449
58.12142
63.13448
56.90882
59.81703
54.2341
54.29715
55.50953
53.93107
61.01512
56.72045
56.00142
52.59095
58.06294
53.06412
53.78019
54.98267
55.65609
61.11502
53.81184
57.23468
53.40178
56.1051
55.94768
53.58181
60.22311
57.12479
57.47873
56.07975
59.94566
57.57305
54.49537
58.47892
64.31393
59.08659
55.893
53.71897
56.54444
64.16003
52.30089
56.00794
50.14461
55.81159
56.82044
56.78817
54.29274
48.43159
53.63007
56.24948
58.78996
56.56752
56.50837
56.84433
53.07917
61.03277
56.06045
57.17168
55.91998
57.09428
56.63054
59.58274
52.86008
54.47419
50.33497
54.85823
56.15614
62.42758
58.97908
57.70085
58.77894
64.25047
53.02895
58.75761
57.69831
56.85563
53.57446
56.42435
51.03713
51.33794
51.96357
51.76336
55.60013
58.92416
56.80605
57.94138
55.58749
63.12269
54.72834
55.40286
60.3699
59.52738
56.79279
55.31038
54.01393
53.22134
52.54323
51.82125
64.93663
56.97336
59.16314
59.64767
61.10429
58.23831
60.46662
61.25961
57.72648
59.52362
61.37497
57.45282
54.55284
53.64189
50.51076
50.2325
55.60186
55.30729
56.32553
58.93881
56.09785
58.47356
57.72059
54.36929
59.32377
60.26701
58.14695
65.40019
53.87421
55.82087
19
21
22
23
52.55008
53.73258
59.58348
56.70817
57.70426
57.05286
56.31394
57.88516
61.61839
64.65929
52.40665
55.95051
55.32419
50.61611
52.09604
53.48294
54.1145
54.82983
57.73911
55.39837
58.63024
56.26768
63.01217
56.89924
57.82789
52.48781
54.1814
51.40846
53.77929
56.28246
58.44163
62.648
58.48243
56.86751
57.0732
55.01916
57.90156
53.53278
54.12913
20
56.59056 58.40684
51.81683 51.02263
55.46698 52.46548
58.85497 53.24246
55.73733 56.28431
56.14017 r 56.793
60.25843 58.09809
56.78473 54.78006
55.08124 58.53921
66.33784 60.64573
54.11535 55.70165
55.69881 54.90915
59.97319 62.51376
51.46892 '55.23191
52.22644 50.89432
51.35849 52.35945
24
50.76828
50.64201
49.37252
51.59386
52.7947
57.92082
60.82662
58.30975
56.19318
53.29397
57.13908
52.52242
54.0287
52.34202 57.82544 52.48373 51.48699
49.93528 40.20917 52.04241 48.8228
54.08177 53.41464 48.37013 50.22101
Table 27. Raw FP assay data (mP) for plate 9 with compounds from one-bead-one-compound library (Figure 3) after approximately 3 hr.
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
55.59061
54.21936
45.22366
43.96496
46.88892
45.16255
50.25366
47.89638
39.7537
48.83475
43.26512
51.21541
47.13151
48.75139
42.66564
45.75024
2
3
53.0647
49.46012
40.43803
50.0422
47.86466
46.76745
45.10102
47.88548
41.6619
43.02026
46.2646
47.77638
56.43949
50.84454
45.6081
50.46864
48.76311
41.42163
39.29918
47.05594
42.43059
51.24065
47.46053
46.19983
46.60825
50.28317
43.96142
32.3288
52.43833
49.75389
42.2705
49.14513
4
5
46.76865 52.8698
42.84283 47.02725
40.10161 44.28811
48.74886 52.78835
44.26761 42.48664
48.80983 48.61486
44.70009 45.9709
50.76833 50.59558
47.92366 48.70345
48.78971 49.44298
52.5578 44.8295
44.13329 46.66588
43.69815 51.32401
49.21121 47.28344
48.61586 43.41304
48.73033 45.67739
6
7
8
9
10
11
12
13
14
15
16
17
18
54.66547
41.9551
48.32079
51.16566
41.38766
43.79911
45.02392
51.01309
45.93448
45.30435
46.02937
50.34597
54.1742
47.70346
46.42792
45.16102
30.63872
42.61615
46.61413
47.688
44.36549
49.24687
45.70731
51.88687
49.18992
50.79401
50.86307
50.68151
47.41826
33.95934
37.60957
46.25841
46.13028
43.47713
43.8708
48.08493
49.11579
50.43323
50.87916
48.19515
31.63275
48.87978
49.68302
55.61484
55.08525
49.16449
45.02636
52.69407
47.04001
44.31756
44.67903
48.48858
47.04796
48.48247
52.29235
45.18042
45.24602
50.2707
45.16911
48.01907
49.93186
46.54013
49.02626
52.09182
46.34286
42.7303
41.32505
45.82253
47.70433
42.6818
51.073
50.53981
45.61614
52.24958
45.3178
48.7336
45.67352
52.63028
49.04284
47.17313
44.62561
46.53296
46.30593
51.35231
46.75987
41.3462
49.8822
47.51694
46.22102
49.97172
42.9223
50.3455
50.96967
43.2554
49.94409
46.80406
46.58153
45.50543
44.06156
49.38294
42.75337
52.20318
45.24797
44.21457
48.17614
43.63278
47.17166
48.61272
51.90251
49.61869
50.6995
53.40145
47.5941
28.14343
40.61411
51.77831
46.71507
44.99635
44.06736
45.05498
42.55025
48.27934
45.24744
48.38183
48.59193
46.09356
46.06248
48.59782
48.88423
42.34632
45.275
50.44729
49.76115
47.03112
49.46173
48.41889
46.83291
47.85877
48.58398
50.64736
50.66424
51.17087
49.08813
54.14858
44.28938
46.15514
46.03525
50.97316
57.1111
44.5457
48.27295
49.55473
43.26503
50.29068
41.2921
49.14313
41.72063
46.91326
40.77862
49.48645
45.36252
42.37259
45.05548
44.78485
53.26171
36.60924
50.59162
48.59625
45.57581
45.58016
52.3597
51.08371
49.17769
47.46621
54.40266
48.35287
50.83943
45.0691
46.80354
52.04379
48.67659
49.59341
54.00407
49.07526
51.55823
49.86737
54.25886
49.62623
50.10119
45.38166
53.25469
45.08043
48.45406
45.68633
41.59998
47.28973
47.20738
49.94073
42.82684
48.03954
52.27655
49.80416
55.47129
50.22677
55.30238
50.65887
52.81285
52.34048
19
20
39.99864 46.8416
44.86184 46.37116
44.97741 ^48.20075
47.75105 43.70057
39.87896 44.22846
42.11423 44.85947
47.27932 52.295,19
48.55186 50.93205
45.64689 47.29608
50.46765 33.50868
47.59927 47.58415
32.20825 49.31295
49.03484 50.59896
46.68243 43.77643
43.05684 44.64473
46.99417 47.75369
21
22
23
24
48.90545
44.37435
45.97665
47.35814
44.75024
38.95345
46.404
50.42946
50.16307
51.65837
44.28926
51.36506
46.09432
47.54474
27.12326
45.71767
34.53127
40.78999
45.91633
47.9413
48.43115
43.14604
43.63718
47.08377
47.60144
47.29491
48.37093
42.75724
49.67512
48.18685
38.50344
47.43684
51.79287
41.83901
43.57102
48.73945
46.14512
44.16588
39.06384
43.19605
45.03841
43.48354
41.65603
46.79006
48.18484
41.84212
42.81813
46.70528
49.5094
43.93199
41.2715
46.13304
39.85115
48.13746
41.44883
44.74798
45.8227
44.91468
50.58125
49.36174
50.8227
47.91078
33.16189
50.41619
K>
VO
ro
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Plate 9
♦
♦
♦
♦
♦
.
♦ :
♦♦
♦♦
♦
♦
♦♦ ♦
♦
t
♦
i
' >■ _ £
i ~
% i
■
K
■
^
*
■ .....
■ ■■
*<
F *
-
if
i :wm
>
♦ *
i
♦
♦
a
▲A
♦ <
97
♦
♦
H r r *
■
■
&
5f
■ ■
K
■
♦
*
i 2
-.«
■ 4F
. *
I I I S
> 1
$
£
S
■
■
m
■
■
■
1 194
♦ No compound
■ + Compound (~3 hours)
--------1-------1-------1-------1
1
17 33 49 65 81
♦
♦
*
S A
■
■i
♦
♦
s
■
■
♦
♦
♦
♦
A
> ■ y
1
♦
♦
113 129 145 161
■
ii
II
■ 335
177 193 209 225 241 257 273 289 305 321 337 353 369
Well
293
F igure 26. FP assay data for plate 9 with and without compounds from one-bead-one-compound library (Figure 3).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 28. Raw FP assay data (mP) for plate 10 without compound.
A
B
C
D
E
F
Q
H
!
J
K
L
M
N
0
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
51.6173
51.1027
53.88094
52.19895
52.83151
58.46605
57.14892
54.87552
52.80274
50.86961
. 55.73408
53.17352
50.33233
52.40573
53.5291
50.71071
54.29712
55.39871
51.24861
56.97144
52.58399
62.1598
57.3062
54.62502
56.42956
54.67077
58.41378
56.64376
54.26826
57.81858
57.08133
50.92876
49.21002
52.88931
50.16091
52.33583
55.83395
54.58145
55.03218
49.6325
55.48315
57.27836
58.56976
56.41729
54.99618
54.246
51.73676
50.64853
54.04596
53.57233
52.85714
53.90346
52:86472
53.55
54.04205
56.77206
55.51328
58.8983
61.16271
55.71232
55.82184
55.73858
56.60661
52.91154
53.07425
52.32056
48.68665
53.99522
55.55915
58.58291
53.3042
58.83377
54.74687
54.33009
56.32279
53.77099
53.00492
54.47697
54.99205
52.35674
52.55979
54.37994
50.73668
58.93166
56.02966
56.25292
55.95744
59.7764
56.24724
56.38876
56.68623
57.25437
56.72185
56.70666
59.02972
49.65372
52.2713
52.76808
53.00957
52.91574
53.20297
54.54332
56.5927
55.74607
54.20475
57.23235
57.51142
59.11914
57.50831
55.22049
55.20272
53.38222
54.14532
54.30814
56.08815
58.03167
55.98516
58.05568
58.57553
57.09767
55.63399
56.03812
60.19407
56.84092
58.97021
56.88806
57.7204
53.33361
52.20414
54.04566
56.87011
54.29066
56.28011
57.42935
55.5746
54.34185
53.60602
54.0013
56.47779
54.96919
56.61869
56.36836
58.91383
53.31398
36.18001
55.37712
61.29274
55.49082
57.51265
57.34662
54.72748
54.75658
54.2155
59.80451
56.81159
60.08149
61.08893
60.70253
60.30977
50.61826
50.46653
53.93194
51.3844
56.24951
58.19977
53.56619
51.6205
54.74135
52.69975
56.99476
54.86473
54.57548
60.64601
54.68771
55.39361
51.64177
50.97785
55.67971
51.54887
58.0472
58.05782
50.54816
56.66015
59.32381
55.43863
58.17211
53.97834
55.34409
63.53623
57.86703
59.36432
56.20572
54.06906
51.14783
52.82051
56.05051
54.23643
51.51601
52.09741
58.49874
52.40326
51.82053
54.35633
55.43438
55.23942
54.82269
56.19329
50.43414
54.55183
53.42273
55.05419
61.38067
55.59869
55.40523
54.76023
56.54128
48.73127
56.55196
56.43606
57.62025
60.63318
60.93703
58.01146
53.96734
54.46899
50.1149
51.4778
57.27853
59.15666
53.99477
54.03875
54.25892
54.44759
53.86729
55.16937
55.2267
58.74132
56.76056
55.12529
54.79063
56.7109
53.02685
52.36694
56.86386
59.22573
57.84954
56.38757
56.07128
55.42094
58.00759
58.01882
60.32534
63.03777
58.92509
56.81763
52.53975
53.46757
56.39033
54.73575
54.11419
51.48543
58.53119
57.09691
57.24168
58.38486
56.11664
58.48499
58.29598
59.49255
59.68902
57.06186
52.32849
48.99835
51.7041
54.06894
55.04013
54.95556
57.11579
59.83945
57.8684
61.43725
57.69238
59.39814
57.38014
61.22053
63.89946
59.2447
52.82125
52.9172
49.53552
54.32847
53.91666
54.87583
55.23685
56.29842
54.81439
53.0143
55.56326
54.09546
55.99501
53.45067
55.03503
55.8441
56.15164
51.60314
52.63611
52.68102
55.13879
53.64554
56.60176
56.78334
55.1699
57.55709
55.88658
56.08806
55.75201
58.79738
56.8852
57.85252
58.00967
51.06698
56.10007
49.54405
52.37615
50.08203
51.99087
53.54302
55.31285
56.73099
54.49919
60.94418
53.26488
51.59525
55.0274
51.01265
48.84471
50.84007
56.42181
51.21221
53.52201
51.4233
54.38446
56.26056
57.82312
55.35871
56.27282
63.56825
57.24271
59.59981
58.9036
55.44745
51.41828
52.28048
52.73286
59.74459
54.08987
50.99032
54.43426
52.47518
55.19499
54.61717
53.0061
56.813
51.86651
55.71424
52.3607
51.78434
53.46945
48.9995
52.22567
56.00734
51.62992
50.04031
52.02515
58.69267
54.32117
56.25583
54.46427
52.63157
52.21616
54.73851
54.93306
51.82991
51.60018
Table 29. Raw FP assay data (mP) for plate 10 with compounds from one-bead-one-compound library (Figure 3) after approximately 3 hr.
A
B
C
D
E
F
G
H
1
J
K
L
M
N
O
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
56.97848
54.53539
51.58345
45.68647
51.58186
47.74165
52.522
52.56736
56.52584
55.06225
48.44491
54.77424
46.16757
42.89729
44.9684
48.7751
56.01937
50.28456
50.33036
51.39489
48.45484
49.92989
56.04093
53.87221
53.58068
40.59725
47.7584
54.51315
37.8357
47.34914
55.57819
52.21992
52.91447
48.70226
46.97731
50.11031
51.58661
51.73942
54.70906
45.01372
46.59924
51.64474
50.04812
46.49344
49.84763
46.26187
51.73471
52.42651
49.63847
53.29986
48.51829
49.59389
47.65314
50.88807
54.99158
51.18905
51.07562
48.64731
48.33301
56.59156
51.95573
50.93684
58.17305
53.57772
49.17599
50.89091
51.95225
53.28009
50.66899
48.46864
53.02474
52.54532
52.06978
47.54629
43.25273
51.72784
50.3645
43.06725
53.05849
49.58203
48.93046
57.86452
50.10597
55.38912
53.19453
49.33254
56.22854
51.1387
52.55566
51.40367
52.42259
55.07962
50.15371
50.77661
51.08307
47.95345
47.51771
48.22851
47.46484
50.88209
48.27191
52.49613
53.49159
48.23092
48.74326
56.81414
54.84442
53.12549
51.32616
47.88213
46.21496
53.64857
49.69911
42.993
53.44261
52.27404
52.4906
51.89653
55.09261
59.90194
52.54896
55.30067
55.69064
57.40721
56.57825
49.34892
51.31226
55.90717
50.40424
46.4179
45.99211
49.75618
51.73469
49.75442
52.04058
53.96671
51.2359
48.59341
53.90921
52.50518
53.50127
41.98176
49.25807
50.16187
45.13078
46.01749
39.51713
49.96986
49.3935
51.6639
51.72037
52.98204
47.23004
51.83408
47.09894
50.40604
55.25255
53.59862
58.84833
46.16471
48.06517
47.84571
52.55043
51.56868
39.43331
51.6434
49.06882
51.9194
35.8943
45.03274
44.35326
50.34145
53.83928
50.74895
52.01145
46.17002
46.43553
52.47307
48.03804
51.8659
52.82374
46.65343
53.586
53.89067
51.79478
50.79655
45.75061
51.62465
54.90533
53.60812
48.12441
46.38659
43.74391
49.06071
47.32701
50.33509
47.65671
48.94747
49.14148
51.78862
45.98962
49.96505
48.25326
51.13308
50.91325
52.17599
50.58329
43.71089
49.6326
44.35822
48.96624
52.65597
44.26676
53.19654
48.77026
55.5803
50.75526
55.34705
59.56359
55.69951
49.38827
56.01405
51.39178
51.26919
44.09532
51.94304
39.95732
52.24077
48.29147
48.36243
51.71461
50.19794
52.39676
51.52653
53.36879
51.50761
50.49426
43.42228
51.56297
51.64554
37.27066
55.38807
47.3993
53.80966
50.89946
38.55129
53.73258
44.37281
55.07109
54.8196
53.3112
57.65156
53.88848
57.47991
55.69295
49.12718
50.8483
48.47309
45.43975
49.78121
52.88088
45.66493
56.2428
47.12812
51.51731
55.24165
56.96266
57.01331
55.67565
50.73662
48.99593
48.08031
53.17775
48.69076
52.3073
51.66347
51.48477
58.10426
47.0424
49.91742
53.40507
51.39776
61.26649
58.29961
53.6964
44.42876
54.84065
45.63501
51.28359
48.34382
53.67715
51.31209
48.09708
48.08134
47.0795
47.75113
51.43878
47.88941
53.79957
49.28229
48.44712
42.40474
49.56991
47.30107
48.76684
47.75709
54.96448
49.21967
49.69943
50.17414
52.08013
49.5986
54.06546
51.69859
57.26559
50.64424
54.42606
56.23908
57.15306
48.79698
47.48202
49.19348
49.2423
47.81253
49.66227
44.887
47.31607
49.97292
51.78862
45.91739
54.77685
47.68703
48.20207
50.5192
47.67747
44.38903
48.55917
55.18797
50.01385
52.9272
55.86378
45.7566
50.20531
53.81042
50.64654
53.23502
56.38821
50.74278
52.99945
49.28636
57.00532
48.46187
48.19663
48.13516
52.14549
49.0241
46.28519
49.26173
47.84363
45.81382
42.79613
47.86046
49.5966
47.89098
45,27378
45.50847
50
45.99728
48.81942
52.20982
50.30183
53.37681
48.54221
53.59443
45.44879
46.66103
53.31777
54.12223
50.33153
45.27594
49.45164
48.13183
49.70785
44.95073
to
'O
4^
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Plate 10
70
40
69
a.
E
30
20
10
♦ No Compound
■ + Compound (~3 hours)
I
I
I
i
I— — — — — — — — — — — — — — — — — — L
1
17 33 49 65 81 97 113 129 145 161 177 193 209 225 241 257 273 289 305 321 337 353 369
Well
295
Figure 27. FP assay data for plate 10 with and without compounds from one-bead-one-compound library (Figure 3).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 30. Raw FP assay data (mP) for plate 11 without compound.
A
B
C
D
E
F
G
H
1
J
K
L
M
N
0
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
50.04875
51.05841
59.8686
51.60794
49.92309
51,03355
54.36986
52.6405
53.41191
53.47204
54.31941
52.56809
53.71297
47,33149
50.8312
50.39663
53.2526
55.69644
62.14795
54.84907
62.02205
53.01326
61.31799
55.59427
57.85831
55.08086
57.49978
56.26848
58.21547
53.5905
53.46158
53.51547
52.77268
51.74383
53.90233
54.76786
54.85941
51.47876
56.20441
62.94533
56.04401
55.65996
54.52881
54.52731
54.43525
52.72585
52.02304
50.76216
56.45712
54.0046
53.20035
53.67013
55.46656
54.43145
56.6233
59.1537
59.0171
55.48914
58.60708
58.30615
54.59146
56.12043
56.21955
50.13892
51.26234
57.89996
55.77453
54.25067
53.8003
52.74919
53.89897
53.27609
54.075
56.68784
53.01921
57.15343
54.49453
59.44228
53.75061
52.33389
53.21073
54.90682
55.43651
54.70705
56.23966
57.30461
55.49453
54.97934
57.92253
57.05327
57.41939
57.06842
58.72824
61.04743
55.00036
54.55697
56.19454
53.70194
56.62603
55.25867
58.0644
56.61033
62.83552
55.65643
58.54679
57.79758
55.47796
55.34264
57.77993
54.36518
54.98293
54.95974
51.31334
52.78797
55.88845
57.06796
61.65675
58.61834
63.19977
56.95341
59.02802
56.90632
59.28901
57.18353
55.43574
59.25801
55.90131
55.69938
52.43629
53.85543
59.19663
55.09914
55.85882
56.84466
57.4689
56.3753
53.98894
55.73596
56.06633
61.19446
55.60916
58.30903
55.17154
50.33181
54.6166
53.09854
58.4164
56.83344
58.20885
54.99947
58.0269
57.18798
56.94482
57.7905
58.83327
63.16795
59.74512
58.93426
57.24217
54.55036
56.41527
59.70073
55.60546
55.65647
54.28947
59.77362
53.92911
54.6848
57.48342
57.04016
54.80635
56.64244
57.41381
53.81297
56.15132
50.6625
57.40041
60.00015
55.95564
54.69388
54.57421
59.80276
55.63752
56.61063
55.78913
60.17204
50.14081
57.48999
55.90786
55.04422
57.27128
55.6733
53.11423
53.55642
55.21808
53.22381
60.16849
53.38686
61.98286
54.97012
52.29033
53.62399
60.10691
53.90424
54.24978
56.93421
54.1297
52.59642
49.92318
53.6514
54.31081
57.07507
64.04587
57.44179
53.90817
41.36111
54.19823
52.47544
61.89759
57.52863
56.13218
58.32967
59.1411
53.23922
51.92159
56.50335
56.66557
57.52765
59.23028
57.54582
53.75724
55.74358
52.02732
55.5063
54.41165
59.80564
59.0108
55.69398
57.63658
53.36204
52.66535
58.26084
56.42932
57.34387
61.20303
56.40486
60.29846
56.37521
55.93393
57.5457
57.01877
60.38185
58.58411
57.92743
58.45056
54.99839
52.92175
55.84472
57.99102
55.81119
57.02106
57.75787
58.43399
56.01028
56.88084
58.88027
61.63715
58.30969
56.23961
59.5124
53.27792
51.45996
48.40544
51.42924
54.9137
54.72267
55.39464
57.64562
53.43217
58.7867
56.68242
56.54812
64.04734
56.87251
57.41221
64.0738
54.54111
54.68577
50.69797
58.89273
52.05773
54.08376
54.68526
54.78991
55.65332
55.96356
53.73155
55.41848
54.69745
53.55622
57.15173
57.81488
54.22352
51.41602
51.62803
53.33144
53.50834
53.1756
54.09861
54.82051
58.09635
57.40278
56.81489
55.68901
58.78294
57.14715
60.53012
60.03573
57.9618
56.27049
57.57715
51.49403
59.75114
53.84329
54.02671
57.20667
52.13829
54.26327
55.21754
57.87192
54.94063
56.08285
55.62686
53.00083
54.07334
49.05104
55.666
52.01164
55.7443
57.24775
53.55028
56.71922
54.35512
53.74776
52.94117
55.99146
56.77725
54.50803
54.79258
57.68607
54.78033
53.27138
53.69056
53.80482
54.63856
54.45553
53.13206
57.8498
53.4753
51.86194
55.24438
51.05504
53.68854
55.36948
51.29685
53.86549
53.46586
51.12617
48.61763
50.44424
51.33278
52.03093
53.04719
52.85736
49.3046
53.41743
54.05592
55.3927
50.75859
53.90182
52.2543
52.19656
50.90782
48.51356
Table 31. Raw FP assay data (mP) for plate 11 with compounds from one-bead-one-compound library (Figure 3) after approximately 3 hr.
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
49.25734
48.59732
46.06991
44.1204
40.60993
42.83067
48.27663
43,40095
44.38442
51.40737
41.83944
54.15365
50.48715
40.22136
51.26935
48.57941
2
3
49.45835
46.14367
49.37196
44.27607
48.2128
41.42533
45.31357
43.38838
41.88245
50.05256
45.79225
48.04112
52.35034
51.37823
53.16445
52.73034
49.39975
52.56216
48.29586
44.52217
48.61293
47.76121
50.37679
43.77427
45.15733
41.81963
43.94215
44.26216
50.00686
46.09253
50.32401
47.42292
4
5
6
47.15864 45.94413 48.44578
47.2847 46.25694 48.91012
29.6719 46.79693 46.18584
48.32304 45.00397 41.63713
45.5743 46.70419 50.90753
47.92884 22.76524 41.33923
54.07915 37.88877 43.88106
43.77815 43.34977 46.45182
43.95377 43.72516 51.17246
49.69666 47.15476 44.55961
47.92782 45.69454 50.00469
46.89522 45.89754 50.10917
47.73336 49.30136 55.03124
53.59415 51.93042 53.49045
54.3611 49.39711 51.71614
43.33882 40.07938 46.34945
7
8
9
10
11
12
13
14
47.86912
46.14898
45.38038
44.84292
47.94301
45.23597
47.19164
48.44745
51.1154
48.72518
50.26968
47.66936
54.33428
50.01981
54.05956
51.52979
44.17121
43.72008
40.57628
48.29816
50.33481
51.40595
51.96319
46.70037
48.41731
48.97509
53.19138
50.38809
53.89056
59.36366
54.68582
51.9858
43.32161
46.7083
47.29685
44.82072
49.10012
52.66236
49.19044
46.54561
46.15113
48.3431
54.08751
52.01099
50.32824
28.5372
49.94427
46.47249
46.51931
44.76082
45.91335
47.22931
51.94819
49.77579
47.0021
49.40468
49.49585
54.63656
52.09012
50.80292
54.11121
53.48597
47.91632
50.19
45.06835
46.67055
49.42744
48.01535
44.61098
44.72083
42.57419
51.13502
45.73086
47.9869
41.6329
44.89083
49.39164
54.18379
49.52214
51.80337
48.00878
46.72526
48.67316
45.51266
46.66224
44.67092
48.81629
48.17899
50.82587
53.27659
39.7277
48.27433
51.781
54.58172
55.0788
54.01097
45.13422
44,02295
49.65584
43.74417
42.85439
46.5996
47.39117
45.21034
44.49683
48.85404
45.58487
44.74239
52.02837
51.59749
51.88763
50.54656
48.03017
41.89241
48.23031
44.90936
55.72196
51.9406
45.63087
34.07874
46.19176
47.61062
47.26034
48.91299
51.24004
53.4032
15
43.56195
43.01505
42.82838
44.40908
52.38242
40.44808
45.84942
48.22239
41.98124
47.22903
39.79517
50.24669
48.44996
48.72617
52.87708
51.5514
53.4037 47.26355
16
17
45.62017
42.60666
46.51443
44.84256
46.48752
46.46702
43.61544
49.0272
44.38681
48.0165
43.11863
50.59118
50.84084
52.74803
53.1113
52.3971
41.03709
45.95891
47.0424
47.9309
50.65456
49.92993
49.62636
48.11626
45.12007
51.25404
48.61479
51.90811
49.55568
47.1395
49.2996
47.21961
18
19
20
44.7166
41.59324
41.52419
44.95531
50.43673
43.13355
50.29429
46.87947
45.17394
45.27221
46.99393
46.93143
45.92835
55.57312
49.78326 47.38252 48.58722
49,73327 44.22879 45.62537
44.26182
44.93254
45.60119
46.30281
50.5993
49.81731
47.36978
48.24866
48.17663
51.10468
49.22431
52.81136
51.88753
52.46978
47.81106
46.98962
42.95043
38.97625
44.17694
42.64235
42.37679
46.12983
39.14089
43.87393
41.79708
42.50821
47.51619
50.57812
21
22
23
24
39.91996
41.94521
46.76284
43.62365
48.55507
40.29691
44.6022
45.20311
45.05647
42.98475
42.37963
46.92556
43.36503
46.49341
52.6131
43.26569
43.47788
42.72445
46.72162
44.72196
44.68024
40.71764
43.19097
44.44527
47.19071
47,06736
44.05314
46.02498
47.77392
47.07031
52.38502
47.35728
44.60923
44.69315
47.22414
43.10908
46.86183
45.96468
47.15315
41.33732
49.33159
44.18786
43.23122
42.11948
43.61807
49.14322
50.94713
40.83769
41.65658
44.85121
40.02631
47.38412
48.72833
45.30815
44.94449
40.88112
49.90277
45.94616
41.41144
48.91794
48.52553
49.80454
53.53411
47.97486
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Table 32. Raw FP assay data (mP) for plate 12 without compound.
A
B
C
D
E
F
G
H
1
J
K
L
M
N
0
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
51.89901
50.27885
54.145
56.82574
56.59042
52.77881
53.16897
53.45421
53.03071
59.94535
56.78626
57.07519
54.32345
52.7009
53.92486
49.34668
52.86749
53.11997
55.27629
59.18569
57.81583
55.69181
54.76867
57.47285
56.66793
63.0408
58.89745
66.42885
56.86061
56.39257
55.30779
57.0886
50.09879
57.89405
54.07684
56.42337
59.68623
54.98702
53.80615
57.39071
53.82103
56.67247
57.00486
57.12751
57.97253
52.26571
52.509
52.48736
53.36624
55.90731
55.56529
56.88522
57.08385
56.48703
54.62989
57.00871
55.83376
58.12404
58.01247
59.7229
57.99498
56.58902
56.16953
53.58301
52.38916
59.50154
60.37211
55.46557
58.63469
54.47079
56.96202
54.81009
57.07947
55.54844
58.66873
57.5846
58.42951
55.53501
59.3749
53.43141
52.49721
59.0096
59.66523
55.83471
58.78189
55.20962
57.97159
57.15617
56.09606
54.60487
60.64156
62.30865
60.24517
55.63346
64.0881
54.29977
50.28311
55.46233
55.30049
57.11591
60.04343
55.51835
56.86245
54.96177
55.51456
60.5438
58.45037
56.42962
56.32479
55.21462
56.26135
53.00213
53.54882
58.80354
57.47804
57.59777
62.50579
59.84592
58.65827
58.39404
58.46099
63.15224
60.08177
60.8637
57.43449
58.31044
57.02955
54.66295
49.80031
54.68825
59.73121
59.01073
57.31679
60.44205
57.54682
55.25801
54.53445
59.42546
61.41593
59.55829
61.41312
55.51263
53.65441
52.21699
50.764
52.16926
58.48493
57.68145
60.14718
61.31445
56.46225
54.5631
54.23915
62.49261
61.05529
63.26291
63.52371
58.41287
55.79577
54.63528
50.22186
57.36812
57.84197
56.11762
52.35763
52.05876
61.14119
54.54668
54.2546
52.43169
53.41191
59.61013
56.22335
61.81446
58.98345
51.74275
49.66877
56.45536
55.44912
58.27643
53.99517
53.0675
61.55672
60.28225
55.32543
53.04809
53.33481
60.7289
55.08838
64.31589
61.54859
55.05011
50.34732
54.97003
57.98438
58.0663
56.20597
56.96611
54.35009
56.97482
52.59251
56.73243
56.65729
59.03549
55.3179
58.27682
57.9803
54.55271
50.21888
55.29878
58.60659
56.95906
58.43193
58.04032
56.14479
56.58281
51.7455
60.5477
59.11326
64,97613
55.58419
60.13009
63.65207
55.92911
49.04979
52.42456
54.70775
56.00058
61.22794
53.42281
55.21103
56.42663
51.0096
56.30769
55.09726
59.89462
. 56.347
57.57455
53.37915
54.42807
51.50235
53.70805
54.15923
57.77524
57.64203
56.74456
56.94115
57.17941
55.2964
59.63659
59.42146
57.42082
55.54305
59.41933
55.17841
55.84735
51.52113
56.09057
57.25586
55.50319
57.21044
58.51559
58.43429
56.98817
57.9979
58.89894
61.46342
57.22963
58.46397
58.53656
54.36927
52.56885
49.85918
52.47605
51.51209
54.34972
54.52415
56.40062
59.85664
56.65306
56.70086
59.23652
58.84819
58.89735
55.15511
58.12939
57.76762
56.20155
53.2784
51.77391
56.93333
55.08593
55.81687
61.27291
59.46724
55.34164
53.88026
59.37925
58.80661
57.39369
59.7352
54.08722
52.21158
52.91316
50.99649
51.81758
56.42245
57.90428
51.69073
58.59337
61.6418
55.2097
56.32571
60.36076
59.34051
58.83901
57.90624
55.39932
56.79009
55.44317
52.96127
55.5033
55.00474
56.08185
60.82353
53.3457
63.01281
55.2605
55.80917
54.42288
56.64438
57.41814
57.75107
55.17789
54.3386
50.59065
51.76107
52.61591
56.67497
54.53335
58.05745
54.49238
59.64788
54.58269
54.49445
59.18742
58.51929
54.74116
59.43186
57.07637
52.89988
54.74152
50.10937
52.21709
53.22748
55.003
58.69583
54.7282
51.15227
52.14721
59.20197
57.48388
58.78975
51.26441
54.50257
56.18531
49.81038
49.97671
48.55867
48.23889
51.56488
51.55645
52.59869
53.20968
51.86766
51.57487
57.52068
55.19144
55.35754
54.0134
52.21538
53.11373
49.15047
50.96199
Table 33. Raw FP assay data (mP) for plate 12 with compounds from one-bead-one-compound library (Figure 3) after approximately 3 hr.
A
B
C
D
E
F
G
H
I
J
K
L
M
N
0
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
46.87039
44.51446
50.90981
43.67697
48.23305
45.6503
47.69066
51.75597
46.3815
50.85027
47.05289
48.97747
45.52083
48.12043
47.23479
52.33089
49.33529
41.20086
47.70772
52.09441
45.10625
50.31464
50.96021
50.62652
43.9543
48.3031
52.224
47.87393
47.06163
42.123
51.76973
41.61098
48.91763
47.79707
49.95969
48.36474
45.81154
49.63526
52.3444
49.98682
50.2445
49.18934
49.34577
45.01921
46.16518
52.26406
51.67973
48.29728
49.46609
52.00159
46.87747
48.96558
48.97744
44.33599
47.24352
52.37858
48.18655
44.86066
49.64065
51.27492
41.93694
46.75383
51.2194
44.35793
43.92082
42.51477
42.35396
50.38854
46.96071
49.38743
49.14514
46.14937
49.16953
42.29469
49.68067
49.23803
43.33163
51.86911
49.13611
46.52187
50.30089
48.93483
44.75263
50.0609
43.85385
47.68169
40.21233
46.36349
48.84834
38.46493
52.53857
51.84939
49.47796
51.31954
53.42346
53.80704
54.28404
50.19694
50.1993
51.94718
38.00241
46.64283
50.09664
49.14378
46.97708
41.57856
45.65519
50.18034
44.92955
40.85006
51.14381
54.45696
50.15921
52.23806
48.9986
51.5201
49.43691
47.19886
47.47986
50.65848
51.08913
47.0564
47.6593
51.3481
34.91107
46.92005
'51.23847
53.49326
45.02614
50.99715
43.23196
46.94074
47.37518
51.17795
45.90796
51.19037
50.35916
46.75846
45.3425
46.16428
44.41068
49.04631
50.00706
54.61581
47.95865
51.92985
46.0188
38.93126
44.39734
52.83824
47.39271
46.59498
47.7492
48.39249
48.05355
42.27737
37.29136
46.50265
54.95555
47.25596
51.29581
50.11887
46.75596
51.77314
47.15405
55.66519
51.89117
49.73222
46.43187
47.87296
46.59677
49.89012
52.74531
57.05343
50.52304
41.71607
45.2852
50.5558
54.00573
48.83058
52.58218
36.3703
52.02023
45.89514
46.00606
51.82447
46.01372
51.91976
50.22233
56.26029
51.17339
48.14863
46.31859
56.76133
54.16893
49.88363
52.26249
49.77743
55.05258
53.18091
48.2325
52.01612
48.40437
45.39342
52.06681
50.80819
51.96895
50.96842
44.84403
47.08306
50.24396
46.6675
46.86573
50.66078
49.51763
51.16479
43.57716
43.23107
45.23191
53.6292
48.68152
53.34328
49.63782
49.07055
48.95607
47.97024
49.62665
39.70118
51.40226
52.33952
51.0059
49.24033
35.86809
50.55615
43.19668
48.9646
50.72198
46.79773
48.57509
42.68317
48.59782
50.66806
34.50509
48.6792
51.56976
46.09785
48.18543
41.66829
38.79449
13.34543
44.71364
41.70475
45.37158
47.04919
47.78367
43.50122
43.88256
39,89386
51.77286
51.11089
46.15623
44.16224
48.39776
42.51338
45.33446
50.49261
48.84644
45.35219
52.24817
52.25404
50.11201
43.85215
40.61861
49.53924
45.56783
48.34043
47.18492
44.62208
46.09691
44.12307
46.01378
44.22485
46.10244
45.53386
48.69009
51.08301
48.72971
43.55605
42.58992
48.99924
47.75985
53.9996
46.45144
50.96986
47.65235
51.15895
45.36469
45.18995
45.04517
48.65855
44.05798
50.39008
52.41052
37.06837
46.16934
47.63744
53.31337
49.64322
41.4811
43.90077
46.65586
46.21212
49.61843
45.79316
46.47472
48.67072
44.20963
46.60319
47.07431
43.30963
46.81065
52.92697
52.43792
42.19447
46.72903
47.90382
45.75444
47.07486
47.46074
47.77478
50.35175
53.9832
45.78917
40.79965
43.70939
48.98385
48.57244
45.89416
48.12153
44.28864 47.73001
38.19574
44.09049
39.62578
48.69019
50.43102
52.70198
51.49287
45.92667
51.16836
42.72922
40.65652
45.13531
41.45434
48.60927
47.56453
42.73979
46.18013
44.12303
36.24023
45.74595
48.46674
50.51117
47.35651
42.96162
44.76754
49.28698
43.88643
45.57432
49.61319
53.19652
48.79054
51.64675
44.25161
54.18703
47.89088
39.78239
48.55035
50.03617
52.24474
52.07889
47.08564
52.29373
49.87733
oo
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Plate 12
20
304
10
♦ No Compound
■ + Compound (~3 hours)
1
17
33
49
65
81
97
113 129 145 161 177 193 209 225 241 257 273 289 305 321 337 353 369
Well
299
F ig u re 29. FP assay data for plate 12 with and without compounds from one-bead-one-compound library (Figure 3).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 34. Raw FP assay data (mP) for plate 13 without compound.
A
B
C
D
E
F
G
H
1
J
K
L
M
N
0
P
52.13421
50.51234
53.61484
53.74665
55.29864
52.22308
52.31129
59.18125
57.66654
61.3221
53.06296
52.20137
54.75544
54.34986
57.20933
56.23131
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
54.254
49.86611
54.07436
54.35876
57.32556
54.31535
55.2547
61.96541
58.25549
62.88571
57.82374
58.33091
57.8196
55.3106
60.69821
43.64611
53.07724
48.0324
60.80517
53.15032
58.17486
53.97211
59.34295
56.17305
56.62834
60.16494
59.51065
59.11792
57.23849
56.98607
58.65885
53.32023
52.29294
57.41713
57.71245
47.01484
57.59203
55.63813
61.0124
55.98427
55.15549
59.08303
58.77328
57.34587
57.50758
56.66439
61.93528
53.77023
51.6412
56.99403
53.70433
52.76536
55.80524
54.46944
59.46716
59.95649
56.54521
58.45772
60.00949
55.87625
57.52858
58.38843
54.44201
52.22228
50.41309
57.74534
53.18438
55.21673
56.70146
55.80538
64.06624
58.5183
56.07445
55.7647
60.68079
58.11968
60.10556
60.42961
55.28035
53.5866
54.91522
54.33789
56.87807
55.43853
54.60264
56.94034
57.77664
56.00429
55.80883
57.98268
58.58478
53.64496
59.72399
56.74227
54.88656
56.97881
54.05601
55.1064
57.98556
56.85748
55.46901
58.65265
56.00939
56.85324
57.87675
58.00596
59.02485
56.65347
60.5753
56.34008
58.01238
57.25481
50.92872
54.27189
54.79205
59.79411
58.49705
57.13107
54.61643
54.47867
60.50364
67.1247
57.44905
60.8977
56.36514
55.31089
56.54001
54.85947
51.46339
53.73797
56.33168
59.85713
54.36543
57.5301
56.45883
53.48855
59.72393
62.56899
56.23924
62.05708
58.4235
58.01399
57.23094
57.58747
52.33273
53.01902
54.89912
57.57762
56.10494
51.28575
53.26062
54.06657
62.82258
54.66935
60.2946
54.46344
58.16383
58.23296
56.72206
55.73068
52.33843
51.462
54.45111
54.68513
52.91555
54.51287
53.37436
54.38367
61.8183.1
53.59742
57.57024
54.81784
58.64796
58.36598
57.60547
57.91932
51.89123
54.13219
54.33478
56.81883
62.52832
54.70289
55.22565
56.00976
55.46636
57.11418
56.61323
59.41257
58.12033
56.71653
56.68012
49.14658
50.58889
57.56718
42.22314
54.01252
58.79328
52.7756
52.65634
50.92171
51.42722
58.42081
53.42514
60.70792
58.59831
53.32739
60.36792
53.86315
53.48478
52.54343
55.00586
58.86728
60.11196
52.72114
55.25281
53.52811
55.0122
60.34346
59.15597
55.57557
56.24945
55.46882
57.95159
52.1634
50.53633
54.15174
53.8778
59.29791
57.79831
55.52768
55.90989
55.2393
53.88982
59.33843
55.358
55.872
56.0289
59.87611
59.61329
52.37393
52.93703
55.70609
57.02288
57.63403
58.36943
57.5212
59.00172
59.01036
56.25563
60.14144
63.71037
57.23365
56.41856
56.33193
56.31714
53.26847
52.22721
54.80937
54.09954
52.73504
51.42506
54.5377
54.03385
56.16892
55.69958
53.51717
58.54558
56.52828
54.9331
57.51785
56.8045
54.54347
57.88112
52.74046
54.79454
57.04948
54.55163
56.65933
55.13181
54.96769
54.58265
60.0988
62.49844
54.30159
61.67073
56.7166
55.34973
55.51684
56.6995
50.20759
54.31895
56.49065
52.02344
54.38429
53.61051
53.98363
54.47766
55.97652
56.53978
57.97328
63.58695
56.41701
57.14727
53.25532
52.49536
55.13691
54.47715
54.59904
59.28284
55.2083
58.87372
58.10657
58.80136
59.47075
59.09152
58.3328
59.67977
54.76125
49.97827
53.00034
48.60248
55.64537
52.30486
53.04446
51.83345
52.53511
55.74407
52.75517
54.14435
55.76443
54.58693
56.26229
55.04164
56.85037
51.14452
54.81965
50.47387
57.29885
55.07824
53.5411
52.40664
56.63554
54.08892
55.30775
55.68189
56.40447
61.65811
53.57246
52.40469
52.29497
51.71913
50.55338
47.69083
55 .0968
52.49333
52.12498
53.00806
52.59545
50.22061
55.0753
52.08224
55.79297
54.53837
55.47622
53.76625
52.13228
52.88196
50.66274
23
24
Table 35. Raw FP assay data (mP) for plate 13 with compounds from one-bead-one-compound library (Figure 3) after approximately 3 hr.
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
1
2
47.20785
54.05803
51.18999
44.54289
31.53271
46.24817
48.40473
47.92405
48.69003
46.6182
45.14062
51.97474
46.7169
44.43919
48.60229
51.09396
48.65798
55.83879
53.17879
47.26762
45.86961
52.31986
50.6845
48.91092
53.21684
46.12427
46.56058
48.84896
45.68069
32.33778
49.95707
53.07982
3
4
5
51.28562 47.69707 48.31821
52.59264 52.79705 49.99381
51.48061
49.6058 52.00243
48.71797 38.80723 35.36607
49.23633 44.86242 45.83131
46.49368 45.87101 44.64726
48.42733 30.83366 46.55039
50.19336 47.14579 48.77429
50.5229 44.91635 47.52458
49.62887 47.86554 51.58802
45.65763 43.73409 43.34809
46.1623 50.86664 43.65804
43.695 45.34514 49.78406
44.44123 47.2962 41.46761
44.52011 47.59829 45.27512
45.56707 48.01591 45.38193
6
7
8
9
10
11
12
13
14
15
16
17
18
19
48.8214
52.3236
53.8027
46.413
46.09225
49.04204
47.28442
48.64368
40.26989
54.17376
21.73699
47.40232
47.81506
45.92116
49.87096
46.86806
46.77317
50.00423
47.39055
52.25042
48.05931
46.56474
50.6934
47.31791
47.51319
51.10645
54.41191
46.61604
44.72435
46.77973
53.16901
48.10976
46.00253
51.44329
44.75054
49.86171
49.88645
42.05139
52.70704
47.7823
49.49174
49.43118
51.17715
52.31791
50.40216
52.61763
55.73197
50.55301
49.64024
49.13006
44.22983
49.77739
42.95128
44.71905
51.4416
48.49275
56.65501
46.14794
46.05843
50.32017
53.20684
44.47239
47.63675
53.83287
47.72857
47.8039
50.35219
47.12109
48.43255
44.0598
47.17041
50.91949
52.35144
42.36215
41.73043
51.96624
53.53775
48.64057
50.38959
46.72721
51.77888
50.15671
48.74622
50.15442
44.72266
45.69565
49.57336
47.17102
50.90418
42.52356
51.78293
52.01782
52.47347
50.42214
45.31183
48.60576
53.88037
47.94349
46.8413
49.23948
44.85343
46.64912
48.08714
50.32775
44.6118
46.18057
50.57745
57.3101
54.66753
49.28292
52.04175
49.96694
51.42009
40.62732
42.71395
53.65358
44.04719
54.81996
44.3784
49.79495
48.50644
41.65369
48.6285
47.20237
52.51269
47.34938
47.49454
49.85626
51.71358
52.7161
46.62979
50.23743
49.00769
50.09957
45.03335
48.47365
45.51435
43.57537
50.76985
50.80708
49.57666
50.00953
48.24315
53.43524
46.69287
47.41135
47.05504
50.27541
57.12524
41.70993
42.85707
47.25604
44.36226
52.90152
48.52615
46.23429
50.25222
49.86064
51.17074
48.32158
51.63541
50.29198
48.23281
44.42603
54.37465
49.26691
33.26342
46.22162
43.71005
48.48573
45.46925
44.92156
51.25298
52.48799
53.77709
49.1509
49.87606
55.88097
51.79002
43.57941
48.34088
46.75408
49.98413
48.31028
48.29848
48.67077
47.66558
53.7408
55.71697
52.16912
48.01706
48.64244
49.79583
51.33719
49.72917
42.99703
43.91932
46.85672
47.87157
47.94525
49.04602
52.32411
46.03603
57.74509
56.65161
51.29563
49.23985
54.93027
46.61345
41.69007
53.51359
45.99861
46.85634
43.04903
44.73962
39.06253
48.72584
54.56286
40.99027
53.75515
38.70281
43.48942
46.81188
54.54851
20
21
47.50519
48.95895
40.99753
42.32563
50.7246
42.11948
39.90443
47.74502
49.41846
53.96313
44.15699
50.1156
52.7711
51.64753
49.43028 50.32732
53.17756 47.22752
42.89551
43.69546
47.63026
46.42118
45.23987
47.68072
45.52417
48.29101
49.61114
51.55106
45.04748
49.40351
47.57525
47.81229
22
51.33214
50.2704
46.38591
38.32139
48.88207
44.40674
44.05112
46.85646
43.58281
47.52699
44.60278
48.71141
48.52259
51.44632
48.73671
49.46923
45.52551
46.72989
42.62801
45.75588
42.86869
44.01881
49.85552
45.87763
45.73602
40.35498
49.32273
47.33347
48.69165
48.93808
43.28924 44.29931
45.40161 46.60086
47.39913
47.48956
44.58112
47.3994
46.00869
46.41381
30.53852
45.12579
46.95414
47.04893
40.09921
45.83901
41.34508
41.31783
OJ
o
o
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Plate 13
60
50
f t
i?
40
0l
E
I 55
30
I 3J 9
01
20
10
♦ No Compound
■ + Compound (-3 hours)
0
1
17
33
49
65
81
97
113 129 145 161
177 193 209 225 241 257 273 289 305 321 337 353 369
Well
Figure 30. FP assay data for plate 13 with and without compounds from one-bead-one-compound library (Figure 3).
w
o
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 36. Raw FP assay data (mP) for plate 14 without compound.
A
B
C
D
E
F
G
H
1
J
K
L
M
N
0
P
48.55565
51.38529
46.34446
50.01068
50.85268
49.67072
54.53355
49.86992
50.33276
51.09972
50.90699
51.03562
59.55609
50.82937
50.86848
49.88204
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
47.54213
51.80711
53.19146
50.6323
59.60231
51.84187
58.05201
56.30263
54.63836
56.23128
54.51853
55.80299
54.30845
54.64815
53.51727
47.34648
47.87548
51.5058
51.10074
52.08844
51,19827
53.75554
54.07926
53.98908
53.31721
53.41979
49.57944
52.67704
52.2087
52.83184
55.66129
54.68537
54.47809
54.18016
56.58053
54.62498
56.43118
55.62903
58.55346
55.00125
56.57437
53.55268
45.3372
51.22581
55.447
56.3331
54.20609
56.37405
54.7519
54.41222
53.913
53.22555
53 .71266
53.12197
53.37745
53.22329
54.43129
52.06212
51.01732
51.48723
50.86092
60.26895
58.63461
57.76676
55.79655
54.95294
57.37446
57.55161
55.25351
57.1171
55.55918
57.3053
53.45711
51.48557
49.6971
55.46939
53.60731
54.80729
51.95944
56.12795
53.23287
53.45589
54.75015
57.38896
59.74109
56.90197
56.49044
56.19859
54.40581
53.16853
51.82256
55.2694
52.32472
56.57188
59.68058
56.5847
58.35149
55.36232
60.39436
57.31173
50.64122
52.55127
52.54588
55.35288
55.67478
56.39845
61.20022
54.60731
55.75292
56.99544
56.12483
56.1697
58.92525
55.33281
57.98764
49.66415
48.93026
54.54225
53.58932
56.77679
59.11986
56.27854
61.2858
54.92206
42.49631
56.05919
49.15759
53.84923
56.64874
53.21815
55.65376
54.68235
51.68039
51.35778
54.92333
53.31611
53 .96038
59.75068
54.74741
59.30439
59.73877
51.08829
51.03487
50.01033
55.96751
57.63887
56.62563
55.65803
53.1679
56.92162
57.3084
45.95879
48.44726
50,92087
54.95227
55.75578
54.7208
53.38203
53.01577
60.4779
54.12817
52.7604
59.14966
57.87691
55.78472
56.59938
56.27129
51.73668
46.47505
52.31448
56.77194
56.09433
59.10256
58.21258
58.39817
61.98976
55.01495
55.44979
50.66419
51.59954
53.76114
56.61978
57.7429
55.82816
55.05629
54.52385
52.65494
57.50578
51.6146
55.24067
54.60557
56.59792
59.68775
56.84196
58.7653
57.79335
55.61258
57.36515
64.17914
58.92632
56.33554
60.97845
56.11367
52.55932
47.96934
54.40478
55.26439
58.83597
56.77675
58.86649
56.48271
58.0373
56.19162
58.22922
57.6469
57.52491
59.93113
56.35836
56.51131
53.44948
52.61755
49.05402
53.15988
57.57034
58.66665
57.85487
56.09898
58.28128
58.15449
58.1593
62.12849
59.75452
59.37986
57.07497
55.40936
52.36178
50.65988
51.80239
54.99799
55.21631
55.08234
54.83016
56.7593
55.92354
53.46629
59.19381
47.70268
51.93939
54.99151
53.81048
57.99175
56.40552
58.84898
59.43006
57.48357
58.3154
51.09556
53.53749
55.23518
52.89942
57.5542
57.35505
54.90668
55.88765
54.61255
62.07519
51.11345
54.4767
53.44531
55.43822
57.3793
56.03909
54.15952
56.84748
55.40978
61.63412
57.81763
58.68658
57.86679
59.26407
55.18254
52.03288
50.1494
56.01209
57.75813
53.86965
53.10261
50.88228
63.91723
55.72964
59.42408
55.02522
55.85074
51.25316
55.37426
60.3955
55.9191
57.03147
58.62321
52.56454
53.03393
58.85616
56.10034
62.50638
56.41223
54.36954
60.7726 60.93249
57.40678
57.98037
57.2635
55.97295
51.44665
56.5358
58.29963
59.94733
58.42803
54.00012
56.29215 57.20362 56.55912
58.03368
59.2614
54.70679
52.4519
52.40002
56.45606
57.11696
54.12931
53.88202
50.75404
56.64734
61.27802
56.26056
55.2398
51.44767
23
55.23289
53.40356
52.50755
57.35003
55.20454
54.4939
51.93151
51.87928
55.36448
57.58327
54.49577
58.11949
51.16922
52.80117
59.608
53.95388
24
54.54618
49.59468
51.6554
51.93352
56.54109
56.09866
52.97667
55.63305
52.44846
55.30156
53.94953
52.45348
53.03648
49.57216
54.64572
51.84282
Table 37. Raw FP assay data (mP) for plate 14 with compounds from one-bead-one-compound library (Figure 3) after approximately 3 hr.
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
55.39546
54.51938
52.98829
52.69884
51.21442
45.99751
52.71531
51.96138
50.14806
52.6584
49.63511
47.36546
56.28251
46.0831
53.50316
50.06373
50.66714
49.61586
56.15955
48.44751
53.17665
50.82908
52.56484
54.27729
52.67988
52.94008
51.82019
51.04396
51.6282
49.36384
54.2714
47.94176
46.69493
50.79498
53.97282
53.51937
51.45511
50.21126
51.92356
53.84515
50.79902
49.22731
51.79335
47.01384
47.10081
51.29548
45.18238
48.29199
48.39626
48.93443
45.01557
51.85801
55.40551
49.14004
50.79272
51.16544
54.28816
52.00769
50.67945
47.44115
48.54262
54.457
52.674
50.9383
50.73881
47.76347
51.14906
47.26748
50.11665
41.49894
41.2141
51.71967
40.62111
52.22766
47.15975
52.09552
46.42635
36.84964
47.41003
52.39877
49.14856
41.51817
49.13051
39.88494
54.27934
50.25547
45.91197
51.94919
51.0033
55.84937
46.13555
55.71628
49.36835
44.56999
51.33297
52.54667
50.07291
51.8099
47.68548
47.68898
46.12839
53.56832
52.17892
48.42652
50.06878
53.45214
42.97898
51.68147
50.76217
51.73071
52.74647
54.08885
51.95114
51.13522
49.48446
50.69901
51.74345
58.71692
55.89544
47.70489
52.50637
52.58686
48.96771
49.57694
53.04987
51.26468
54.21834
47.71559
47.41688
49.15674
52.23206
53.51536
50.71127
55.03154
49.69511
44.55738
54.99053
45.4243
46.53102
50.66396
54.05085
48.81538
50.77789
46.21762
50.13612
50.00083
52.99538
55.94981
52.61719
54.34579
54.88307
53.54428
54.95654
55.67248
50.58493
53.29499
52.06325
50.72332
51.82629
50.96399
50.45778
53.18799
47.54267
47.19242
54.48137
51.42051
46.65982
5 1.973
47.89586
48.42015
50.99591
44.13607
50.11844
49.17546
45.27006
47.67652
49.53475
52.19231
48.8207
40.8367
55.0116
53.1722
44.44453
53.06911
49.05268
42.2336
51.46993
49.51405
49.33944
49.65248
47.95021
47.58362
49.26094
53.34293
52.12882
54.07744
48.83318
53.78547
43.19746
53.94048
47.80511
46.72648
50.38235
49.18915
43.01838
43.32944
56.42354
51.23521
51.01654
55.24973
49.71359
58.07554
52.94301
56.6906
50.70169
53.02185
52.67935
50.91711
52.32879
52.526
51.41137
56.22861
52.65561
50.30367
52.50077
55.03901
45.99061
51.93883
49.98443
51.65111
47.72386
53.05518
36.39363
53.37571
52.8445
50.13985
51.55184
52.09718
46.9938
48.14536
54.21209
54.16565
49.44703
51.19894
56.91274
53.30584
55.00997
53.96477
54.52639
56.4118
52.54164
53.60851
53.41482
54.31921
53.58305
37.02919
49.88173
48.93043
54.76178
54.92459
55.86721
49.32488
50.4942
51.72457
49.34897
51.51656
52.99806
57.49233
50.56544
52.35686
51.5946
53.55639
54.89397
52.42082
54.32262
48.40106
52.76472
48.86446
53.68818
51.36075
30.06081
49.75033
47.79215
53.65787
49.30073
51.00756
49.62079
44.68285
54.48949
52.90914
47.50511
48.44085
49.8771
44.19495
48.22753
48.85771
48.9081
51.01525
49.01965
.52.73201
50.65234
50.52479
55.56654
51.05475
54.04441
52.23829
50.22896
53.83296
51.12453
45.3772
50.16086
47.43617
48.92977
50.50877
53.44176
44.90267
47.73286
44.64191
48.48582
51.46706
48.48901
50.75453
52.08326
50.9316
48.07939
48.41735
45.54596
51.38567
50.98565
51.69351
49.23921
48.64361
47.85604
46.68777
51.42923
52.07843
48.28892
50.55441
54.06145
53.8266
49.56205
51.03693
49.48603
46.28049
49.37696
48.8946
51.25084
54.10476
50.77812
44.04284
47.19871
49.58215
50.60315
54.61481
55.44864
49.69162
41.73711
51.73066
52.73634
50.4552
47.65338
49.0209
41.78012
31.80003
50.06492
47.6708
49.87531
53.27937
54.68354
52.69757
55.61136
50.78052
51.74455
46.37315
49.54691
48.32814
47.95224
45.85047
46.55037
51.70541
44.3881
43.31664
49.68699
50.64109
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Plate 14
a.
E
1290
30
20
10
♦ No compound
■ + Compound (-3 hours)
1
17
33
49
65
81
97
113 129 145 161
177 193 209 225 241 257 273 289 305 321 337 353 369
Well
F ig ure 31. FP assay data for plate 14 with and without compounds from one-bead-one-compound library (Figure 3).
382
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 38. Raw FP assay data (mP) for plate 15 without compound.
A
B
C
D
E
F
G
H
1
J
K
L
M
N
O
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
51.95606
55.38026
54.67194
55.31165
48.81659
53.58706
53.05392
48.59096
50.54198
51.68504
50.64129
52.68597
53.46949
50.67898
48.86636
48.07486
50.44642
54.47094
51.18538
54.34011
48.02779
52.53281
52.57444
54.26072
51.12074
51.36741
54.51923
53.22877
55.06592
54.68638
51.78354
49.2894
57.17403
53.62482
64.01804
56.49825
53.91592
52.97397
51.53249
52.15254
53.52271
56.00138
44.76797
53.4268
54.96707
51.15553
51.78229
49.04052
48.45946
48.25828
45.31815
47.15622
47.9406
54.6553
39.47595
53.32506
54.57748
56.9154
52.10163
52.21852
55.59604
52.58299
54.31834
50.63689
49.44416
53.21893
48.21275
51.64136
54.58537
50.73423
53.70685
54.90346
52.67707
52.62545
52.34111
51.81896
52.56811
53.11852
51.56217
51.62701
45.30074
47.33379
46.44266
51.27189
55.05972
52.44439
49.08265
62.69896
55.43279
51.40424
50.67383
51.3222
55.86518
52.13677
54.38321
49.90307
47.72271
50.26585
49.15442
52.34251
51.02655
51.43944
49.5151
55.90801
53.50477
52.97937
55.74945
56.99191
56.155
56.27277
51.88946
49.81749
21.65186
34.42029
48.38242
48.76305
51.79142
51.99173
54.62113
53.75422
53.36868
49.76923
55.77721
57.81133
53.93935
59.55899
54.08113
52.18396
41.21518
47.38477
53.61854
50.76344
52.8392
51.76597
49.49395
52.83921
52.72139
53.52673
52.37107
54.8395
51.88056
54.03217
52.01325
53.92641
37.72681
48.4921
48.28845
49.63147
50.48344
52.01649
51.08415
54.09689
49.14234
52.67385
50.41585
55.52835
55.18772
53.4375
54.02878
54.23479
51.09396
52.33526
50.64386
47.50531
48.51248
51.18075
51.17037
52.34045
43.46815
51.21708
54.95133
52.64466
54.31468
56.26806
51.12582
49.59246
46.34413
49.69862
51.45147
48.66519
49.97084
49.82396
39.34953
54.24758
50.17611
36.16366
54.45412
51.15467
55.2078
55.34292
52.50059
51.70345
49.93644
51.67099
52.49526
52.28316
52.11049
48.36387
48.30601
53.65838
50.91827
47.01627
53.69924
52.88647
60.7659
55.90966
54.5242
49.87883
45.50193
44.22411
52.70931
50.29478
51.2655
50.00591
32.63994
54.3254
47.00031
49.99478
53.61729
53.63358
56.56957
53.20497
54.94547
52.58152
46.90962
50.25614
51.7814
50.26375
51.61703
49.96997
58.03115
52.41418
48.59026
50.29483
50.94013
55.80005
51.24112
53.23949
50.94849
49.43539
50.63939
48.80475
49.54271
47.69886
49.75758
53.40021
53.79459
55.59582
49.92973
51.4365
55.08595
55.73556
53.59885
53.56915
53.56756
51.95172
52.88155
55.53264
55.01688
53.33877
53.47305
54.70862
53.76394
53.15081
52.34053
51.39583
53.30281
57.8899
54.53432
55.09076
52.54761
51.74138
48.16518
49.95927
46.45564
48.48571
52.80936
50.27686
48.65482
54.59021
52.6744
52.06752
54.35972
56.70587
52.28545
51.72546
54.24061
53.36331
51.77052
56.81013
55.51225
53.94407
56.2133
50.85438
51.45546
52.83289
51.69484
50.47725
55.46898
55.02311
52.88404
53.86047
51.61221
53.97582
48.10881
50.83646
49.6394
45.82765
48.80137
51.69603
51.85081
58.82375
51.99947
49.01711
55.20258
54.01535
54.82508
54.02399
51.95941
49.66725
54.77344
55.38387
52.85278
54.8212
56.09813
63.01281
52.2814
51.68629
51.16469
55.93127
55.14184
54.82152
53.25495
55.85869
55.82786
56.88506
47.76107
54.13031
50.1588
50.70493
54.24176
53.54164
51.96783
52.57463
49.10496
56,02701
55.57334
51.49751
49.70874
51.46537
49.79408
51.5693
53.09851
53.46123
56.74582
57.56526
54.93401
54.23815
55.30159
49.61954
55.05472
52.36318
51.86111
53.25603
49.63374
57.12605
63.69804
57.63344
47.5959
52.7041
49.95202
50.1009
.47.83809
51.31483
44.57283
49.1915
52.24824
51.98475
53.46629
50.98932
49.64487
52.13369
52.97042
46.35064
Table 39. Raw FP assay data (mP) for plate 15 with compounds from one-bead-one-compound library (Figure 3) after approximately 3 hr.
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
52.03339
36.15941
43.04296
48.82484
50.49253
58.3211
45.89049
45.63429
47.44391
47.67998
47.33858
52.30971
46.32341
42.17773
36.31504
40.47841
52.96151
49.26115
48.76139
21.6775
50.0889
50.93488
52.80463
47.39354
47.4256
46.79747
45.77377
54.50164
53.51693
61.29922
55.33397
55.10165
50.18615
42.68349
53.25224
47.31954
47.40062
40.73633
43.93168
48.24934
46.62845
42.92734
36.40894
48.27214
43.00008
46.03093
41.54286
45.58142
45.08485
45.31969
55.7946
45.98855
45.19996
49.45081
47.17877
49.33441
44.84664
38.66157
46.38742
52.97427
53.12667
48.61046
52.26308
51.11328
45.15885
43.81582
43.66308
44.12013
44.43725
46.17182
51.7647
34.1855
21.19244
32.12515
48.30265
47.74925
49.23333
47.0925
45.58684
47.45262
45.5181
48.86163
50.33106
51.26478
47.20163
46.40239
48.19688
45.66042
31.5788
49.1023
41.18111
48.83739
47.02835
47.06621
49.52692
50.64202
49.01736
42.18853
44.11997
48.48702
49.23339
50.81287
51.05841
49.65459
50.26621
48.67922
51.51453
49.47478
46.22746
46.90945
51.68251
46.97604
58.18158
46.63109
46.57575
48.22413
52.57456
35.52237
40.47875
48.93093
46.76288
47.23939
46.8523
41.67532
44.43845
54.41037
49.39255
51.59407
52.27567
47.99316
46.68641
52.28746
49.52484
50.22002
41.23844
50.48664
49.9475
49.99029
49.26182
42.11331
52.221
53.7509
55.65122
48.97545
48.18177
44.52228
43.4722
48.47135
47.47575
43.82479
44.34341
39.25328
49.78113
49.50627
45.35371
40.93277
53.0739
54.07252
54.0501
47.279
46.85118
47.13323
46.60224
49.67494
53.0267
43.0894
47.88528
47.49078
48.33993
50.29474
49.57856
46.90267
34.6396
45.75351
47.87721
50.26676
47.12808
44.5432
46.8678
46.23325
46.97757
45.42902
44.03401
48.7135
45.7792
47.45222
42.87786
43.20299
41.40884
44.63887
49.02174
48.26881
50.36851
48.26613
41.61295
50.17238
45.23787
47.67081
47.4448
49.61895
50.52542
49.27222
54.42257
42.78874
43.41798
45.61321
44.0703
47.72463
48.18189
45.22905
47.19983
46.43507
45.60299
47.36551
22.84576
45.92251
48.71832
35.855
43.68549
43.53423
46.44501
45.50952
47.77059
46.06563
48.76233
45.73707
52.30329
50.31834
50.67651
47.11776
49.72014
45.44759
51.39381
48.71932
47.27615
46.94358
48.80875
52.04395
57.33255
43.33695
50.34102
48.15664
44.22102
47.21799
49.79484
50.96603
42.17371
51.02565
45.82912
46.33401
45.82634
43.21673
44.8217
43.43828
35.77303
46.52201
48.56379
48.43458
41.36204
45.91577
49.20912
52.08565
49.5839
48.09511
50.73551
44.61526
39.95417
43.34765
53.03971
53.83801
59.0363
54.39524
47.85783
53.98072
48.36967
45.1442
50.28353
11.58934
48.84665
46.68933
48.06657
47.70247
45.29054
47.17789
47.74111
49.80973
43.72955
42.76282
41.91239
54.08332
57.05882
52.22992
52.86733
50.54924
46.84346
47.47034
51.68422
49.75848
41.90834
44.35829
52.47705
54.90725
49.94246
46.84472
53.39849
52.52305
45.16143
45.39165
48.19914
44.74028
45.37206
46.30581
46.07486
36.29801
56.90537
48.57918
50.04995
49.3787
43.31046
51.84067
52.91311
45.1259
45.53082
47.55982
49.56566
44.75584
51.83611
44.68481
23.95208
44.75979
40.24322
48.57481
39.58653
50.76675
47.1009
54.89536
53.40383
52.89376
51.15433
48.53078
41.2011
41.42273
44.57675
46.12131
43.52004
50.28507
44.15646
59.25442
43.22568
37.11541
43.68557
50.01285
41.69955
46.09047
47.26122
27.2263
43.41861
47.55862
47.83025
46.79017
43.84403
38.14443
19.50243
43.46891
50.83052
43.7547
49.36559
44.80843
46.36427
48.04913
44.81323
45.72347
40.84087
40.10348
47.35239 45.57348
45.83379
49.69868
47.4417
50.83507
48.03731
52.16515
52.89849
48.98245
u>
o
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Plate 15
70
0L
E
■ 235
383
♦ No Compound
■ + Compound (~3 hours)
4 — I — I------ 1—
1
17 33 49 65 81
97
113 129 145 161 177 193 209 225 241 257 273 289 305 321 337 353 369
Well
305
F ig u re 32. FP assay data for plate 15 with and without compounds from one-bead-one-compound library (Figure 3).
306
A cut-off o f mP < 36 was chosen after screening the crude peptide mixtures to
separate hits from the rest o f the library members (0.5% hit rate). The compounds from the 26
wells meeting this criterion were sequenced by pLC-MS/MS (Dr. Mark Scalf), as described
previously for (3-peptides.23 This process identified 16 unique compounds, 6 o f which were
identified more than once. The pLC-MS/MS system consisted o f an HPLC connected to an ESI
ion trap mass spectrometer (Surveyor HPLC and LCQ deca XPplus, ThermoFinnigan, San Jose,
CA). A fritless 100 x 365 pm fused-silica capillary microcolumn was prepared by pulling the tip
of the capillary to approximately 2 pm with a P-2000 laser puller (Sutter Instruments Co.) and
packing with 10 cm o f Ci 8-silica beads (5 pm diameter, Western Analytical Products, Inc,
Murrieta, CA). The capillary column was connected to the HPLC through a PEEK microcross
with a platinum wire inserted into the flow-through to supply a spray voltage o f 1.8 kV. The
peptide DMSO stock solution (10 pL) was diluted 1:1 with 95% H20 , 0.1% formic acid: 5%
acetonitrile, 0.1% formic acid (95% buffer A, 5% buffer B). A total o f 10 pL o f peptide solution
was loaded onto the fused-silica capillary microcolumn at a flow-rate o f 1 pL/min (95% buffer
A, 5% buffer B) for twenty minutes. A gradient from 5% buffer B to 80% buffer B was run over
74 minutes at a flow-rate o f 300 nL/min to elute the mixture. The ion trap mass spectrometer
was set to run in “biggest 3” mode, which consists o f a full-mass scan between 400 - 2000 m/z,
followed by an MS/MS scan o f each o f the three highest-intensity parent ions with a nofmalized
collision energy o f 45%.
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
307
Table 40. Conversion from well location to compound number for correlation of hits to location.
1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
Table 41. Predicted (based on possible oligomers with a matching overall MW) and observed (highlighted) masses
of N-terminal fragments from pLC-MS/MS sequencing of hits from library (JKM VI 199, Figure 3). Some samples
were redundantly identified. Nomenclature is “plate.column row,” i.e., 8.11H = plate 8, column 11, row H.
%
X.
°v
X?
v?,
«
1
\
\
\
\
c' y
'V
Compound v
- '7
"%
~ 'V'9
"%
“«U
%
%
\
° \p
* *
^
%
V>.
\
%
^
X
^
-9
" too
V>.
'Vr
>^
\
' <?s
*7
V
\
'V7&T
<?
>**
8 .1 1 H
1 5 5 .0 8
2 2 6 .1 2
3 3 7 .1 9
4 9 3 .2 9
6 Q 4 .3 6
7 1 7 .4 4
8 2 8 .5 1
9 5 6 .6
0 6 7 .7
1 1 2 4 .7
1 2 3 9 .7
1 3 1 0 .8
1 4 2 3 .8 : 1 5 3 7 .9 ,
1694
1710
8 .9 0
1 5 5 .0 8
2 2 6 .1 2
3 3 7 .1 9
493 29
. 6 0 4 .3 6
7 3 1 .4 6
8 4 2 .5 3
9 7 0 .6 2
0 9 7 .7
1 1 5 4 ./
1 2 6 9 .fr :
1 3 4 0 .8
1 4 8 7 .9 : 1 6 0 1 .9
1758
1774
1 3 .6 K
1 5 5 .0 8
2 2 6 .1 2
3 5 3 .2 2
5 0 9 .3 2 ; n 6 2 0 .3 9
; 7 4 7 .4 9
8 5 8 .5 6
9 8 6 .6 5
1 1 3 .8
1 1 7 0 .8
1 7 8 5 .8
1 3 5 6 .8
1 5 0 3 .9
1 7 7 4 .1
1 7 9 0 .1
1 1 .5 F
08
226. 12
3 3 7 .1 9
4 9 3 .2 9
7 1 7 ,4
8 2 8 .5 1
9 5 6 .6
1 1 7 .7
1 3 6 0 .?
1 5 0 7 .9 ; 1 6 2 1 .9
1778
1794
1 4 .1 9 B
.0 8
226. 12
3 3 7 .1 9
4 9 3 .2 9 : 6 0 4 .3 6
7 1 7 .4 4 :
8 2 8 .5 1
9 5 6 .6
1 1 7 .7
1 1 7 4 .7
1 1 7 4 .7
1 3 6 0 .?
1 5 0 7 .9
1778
1794
1 3 6 0 .?
1 5 0 7 .9 i 1 6 2 1 .9
6 0 4 .3 6
1 4 .1 9 B
08
226. 12
3 3 7 .1 9
4 9 3 .2 9
6 0 4 .3 6
7 1 7 .4 4
8 2 8 .5 1
9 5 6 .6
1 1 7 .7
1 1 7 4 .7
1 5 .6 C
08
226. 12
3 3 7 .1 9
4 9 3 .2 9
6 0 4 .3 6
7 3 1 .4 6
8 4 2 .5 3
9 7 0 .6 2
1 3 1 .7
1 1 8 8 .7
6.20M
08
226. 12
3 3 7 .1 9
4 9 3 .2 9 :
6 0 4 .3 6
7 3 1 .4 6
8 4 2 .5 3
9 7 0 .6 2
1 3 1 .7
1 .1 D
08
226. 12
3 3 7 .1 9
4 9 3 .2 9
6 0 4 .3 6
7 3 1 -4 6
8 4 2 -5 3
9 7 0 .6 2
1 3 1 .7
3 .1 7 0
1 5 5 .0 8
2 2 6 .1 2
3 3 7 .1 9
498 2 9 . 60 4 ^6
7 3 1 .4 6
8 4 2 .5 3
9 7 0 .6 2
1 5 .2 4 0
155. 0 8
2 2 6 . 1 2 i f r 5 8 2 2 ; 5 0 9 .3 2
6 2 0 .3 9
7 4 7 .4 9
8 5 8 .5 6 : 9 8 6 , 6 5 :
1 5 .1 5 K
■mm
9.7,
)7
17
1618
1 6 2 1 .9 ;
1778
1794
1.W3 8
1 3 7 4 .8 : 1 5 2 1 .9
1 6 3 5 .9
1792
1808
1 1 8 8 .7
1 3 0 3 .8
1 3 7 4 .8 : 1 5 2 1 .9 : 1 6 3 5 .9
1792
1808
1 1 8 8 .7
1 3 0 3 .8
1 3 7 4 .8
1 5 2 1 .9 : 1 6 3 5 .9 :
1792
1808
1 3 1 .7
1 1 8 8 .7
1303 8
1 3 7 4 .8
1 5 3 7 .9
1 6 5 1 .9
1808
1824
1 4 7 .7
1 2 0 4 .8
1 J 1 9 .8
1 3 9 0 .8
1 5 3 7 .9 ; 1 6 5 1 .9
1808
1 8 2 4 .1
155. 0 8
'2 2 6 . 1 2
3 5 3 .2 2
6 2 0 .3 9
1 2 0 4 .8
1 3 1 9 .6
1 3 9 0 .8 ; 1 5 3 7 .9 : 1 6 5 1 .9
1808
1 8 2 4 .1
08
226. 12
3 3 7 .1 9
6 0 4 fr€ T
W«.
1 4 7 .7
15.21
862fr
99059
1 5 1 .7
1 2 0 8 .7
13 2 3 7 : 1 3 9 4 .8 ; 1 5 4 1 .8 ! 1 6 5 5 .9
1812
1828
1 5 .2 2 M
08
226. 12
3 3 7 .1 9
6 0 4 .3 6
7 M M ' - : 8 6 2 .5
9 9 0 .5 9
1 5 1 .7
1 2 0 8 .7
1323 7
1 3 9 4 .8
1 5 4 1 .8 • 1 6 5 5 .9 ;
1812
1828
I .2 3 M
08
2 26. 12
3 3 7 .1 9
7 5 1 .4 3
8625
9 9 0 .5 9
1 5 1 .7
1 2 0 8 .7
1 3 2 3 .7
1 3 9 4 .8
1 5 4 1 .8 : 1 6 5 5 .9 ;
1812
1828
I I .9N
0 8 . 226. 12
3 3 7 .1 9
4 9 3 .2 9
6 0 4 .3 6
7 5 1 .4 3
8 6 2 .5
9 9 0 .5 9
1 5 1 .7
1 2 0 8 .7
1 3 2 3 .7
1 3 9 4 .8
1 5 4 1 .8
1 6 5 5 .9
1812
1828
7 .1 4 0
08
226. 12'
3 3 7 .1 9
4 9 3 .2 9
6 0 4 .3 6
751.43
8 6 2 .5
1 5 1 .7
1 2 0 8 .7
1323 7
1 3 9 4 .8
1 5 4 1 .8
1 6 5 5 .9
1812
1828
1 5 .2 4 H
08
226. 12
3 3 7 .1 9
4 9 3 .2 9
6 0 4 .3 6
7 9 0 .4 4
9 0 1 .5 1
1 0 2 9 .6
1 5 6 .7
1 2 1 3 .7
1 3 2 8 .8
1 3 9 9 .8
1 5 4 6 .9
1 6 6 0 .9
1817
1833
5 .7 H
08
7 9 0 .4 4
9 0 1 .5 1
10296
1 5 6 .7
1 2 1 3 .7
1 3 2 8 .8
1 3 9 9 .8 i 1 5 4 6 .9 : 1 6 6 0 .9
1817
1833
U 3B
1 5 7 .1 3
»
1 5 9 .8
12188
1 3 3 1 .8
1 4 0 2 .9 ■ 1 5 4 9 .9
1 8 2 0 .1
1 8 3 6 .1
1824
1 8 4 0 .1
4 9 3 .2 9
■if: 3 3 7 .1 9 M i ;
:2MM
4*
226.
« » .4 1
7 5 9 .5 3
j >
1664
9 .2 1 0
1 5 5 .0 8
2 2 & 1 2 ': 3 5 3 .2 2 V 5 Q 9 .3 2 v : 6 2 0 f r 9
1 4 7 .7
1 2 0 4 .8
1 3 1 9 .8
1 3 9 0 .8 ; 1 5 5 3 .9 : 1 6 6 7 .9 ;
2 .1 8 P
1 5 5 .0 8
2 2 6 .1 2
3 8 7 .2 1
5 4 3 fri
6 5 4 :3 8
1 4 7 .7
1 2 0 4 .8
1 3 3 3 .8
1 4 0 4 .8
1 5 .1 9 J
08
226. 12
3 3 7 .1 9
1 2 4 7 .7
1 3 6 2 .7
1 4 3 3 .8 : 1 5 8 0 .9
1 6 9 4 .9 !
1851
1867
08
226. 12
3 3 7 .1 9
6 0 4 .3 6
RT\A .’iias,
1 9 0 .7
1 2 .1 9 P
4 9 3 .2 9
A ci'z hQ
1 9 0 .7
1 2 4 7 .7
1 3 6 2 .7
1 4 3 3 .8
1 5 8 0 .9
1 6 9 4 .9 !
1851
1867
8 . 1 1H
0 8 ^ ^ 2 6 . 12
3 3 7 .1 9
4 9 3 .2 9
6 0 4 .3 6
1 9 0 .7
1 2 4 7 .7
1 3 6 2 .7
1 4 3 3 .8
1 5 8 0 .9
1 6 9 4 .9 ,
1851
1867
1 1 .4 C
08
226. 12
3 3 7 .1 9
1 9 0 .7
1 2 4 7 .7
1362 7
1 4 3 3 .8 : 1 5 8 0 .9
1 6 9 4 .9 I
1851
S .9 0
1 5 5 .0 8
2 2 6 .1 2
3 9 3 .2 5
1 2 5 3 .8
1 3 6 8 .8 V 1 4 3 9 .8 ! 1 5 9 2 .9
6 .2 1 D
1 5 5 .0 8
mM
1 9 6 .7
3 3 7 .1 9
4 .1 2 L
1 5 5 .0 8
2 2 6 ) 1 2 :: 3 3 7 .1 9
9 .2 1 0
1 5 5 .0 8
901.51
w . 4 3 .1.,604- 30:
5 4 9 ,3 5
9 0 1 .5 1
7 9 0 .4 4
9 0 1 .5 1
6 6 0 .4 2
4 9 3 .2 9
5 0 9 .3 2
1 0 2 9 .6
6 2 0 .3 9
8 0 6 .4 7
1558
:
1672
1707
; 1 8 2 8 .1
; 1 8 6 3 .1
1 8 4 4 .1
1867
1 8 7 9 .1
1 5 1 .7
1 2 0 8 .7
1 3 3 7 .7
1 4 0 8 .?
15 9 4 .S
1 7 0 8 .9
1865
1881
9 0 1 .5 1
1 0 2 9 .6
1 9 0 .7
1 2 4 7 .7
1 3 6 2 7-
1 4 3 3 .8
1 5 9 6 .8
1 7 1 0 .9 :
1867
1883
9 1 7 .5 4
1 0 4 5 .6
1 5 6 .7
1 2 1 3 .7
1 3 4 2 .8
1 4 1 3 .8
1 5 9 9 .9
1 7 1 3 .9
1870
1886
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
308
Table 42. Predicted and observed (highlighted) masses of C-terminal fragments from pLC-MS/MS sequencing
of hits from library (JKM VI 199, Figure 3).
Jo
A'
Of
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rv '
rS*
A
yP
C om pound
s>'
_?>
. £>'
of
<v'
A'
'W
•
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.$>
A
, <b
<3
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O
^
.$>
A
^
<o'
<D'
*
a*'
<b
y. ^
^
o>
«M
^
^
k/
^
.Vf
tv
O'
V-*
O^v
s^
J>
O
O'
y
^
A*
<*
VN
N*
^
^
O'
N>
n>
O'
O'
O
-V o'.-O'
V'
oO
O'
O~N
*'
5^
A.'
O
\
O'
^
V
O '
k> '
1710
1 6 6 6 .9 8
1 5 5 4 .9 2
1 4 8 3 .8 8
1 3 7 2 .8 1
1 2 1 6 .7 1
1 1 0 5 .6 4
9 9 2 .5 6
8 8 1 .4 9
7 5 3 .4
6 4 2 .3 3
5 8 5 .3 1
8 .9 0
1 7 7 4 .0 4
1 7 3 1 .0 2
1 6 1 8 .9 6
1 5 4 7 .9 2
1 4 3 6 .8 5
1 2 8 0 .7 5
1 1 6 9 .6 8
9 3 1 .5 1
8 0 3 .4 2
4 7 0 .2 8
5 0 4 .2 7
1 7 9 0 .0 7
1 7 4 7 .0 5
1 6 3 4 .9 9
1 5 6 3 .9 5
9 3 1 .5 1
8 0 3 .4 2
1 1 .5 F
1 7 9 4 .0 1
1 7 5 0 .9 9
1 6 3 8 .9 3
1 0 7 6 .5 7
9 6 5 .5
8 3 7 ,4 1
6 7 6 .3 2
6 1 9 .3
6 1 9 .3
5 0 4 .2 7
5 0 4 .2 7
1 7 9 4 .0 1
1 7 5 0 .9 9
1 6 3 8 .9 3
1 6 3 8 .9 3
1 5 6 7 .8 9
1 5 6 7 .8 9
1 1 6 9 .6 8
1 1 8 9 .6 5
1 4 .1 9 B
1 4 3 6 .8 5 1 2 8 0 .7 5
1 4 5 6 .8 2 1 3 0 0 .7 2
1 4 5 6 .8 2 1 3 0 0 .7 2
6 7 6 .3 2
6 7 6 .3 2
6 1 9 .3
1 3 .6 K
1 0 4 2 .5 8
1 0 4 2 .5 8
1 0 7 6 .5 7
1 1 8 9 .6 5 1 0 7 6 .5 7
9 6 5 .5
.8 3 7 .4 1 83741;
6 7 6 .3 2
6 7 6 .3 2
6 1 9 .3
5 0 4 .2 7
1 4 .1 9 B
1 7 9 4 .0 1
1 7 5 0 .9 9
1 5 .6 C
6 .2 0 M
1 8 0 8 .0 3
1 8 0 8 .0 3
1 7 6 5 .0 1
1 7 6 5 .0 1
1 .1 D
3 .1 7 0
1 5 .2 4 0
1 5 .1 5 K
4 3 3 .2 3
2 8 6 .1 6
1 7 2 .1 2
4 3 3 .2 3
4 3 3 .2 3
2 8 6 .1 6
2 8 6 .1 6
1 7 2 .1 2
1 0 7 6 .5 7
8 3 7 .4 1
6 7 6 .3 2
5 0 4 .2 7
5 0 4 .2 7
1 0 9 2 .5 6
9 8 1 .4 9
6 9 2 .3 1
6 7 6 .3 2
6 3 5 .2 9
4 4 9 .2 2
4 3 3 .2 3
1 7 2 .1 2
83741:
83741
6 7 6 .3 2
6 7 6 .3 2
6 1 9 .3
6 1 9 .3
6 1 9 .3
5 2 0 .2 6
5 0 4 .2 7
2 8 6 .1 6
2 8 6 .1 6
9 6 5 .5
8 5 3 .4
8 3 7 .4 1
5 0 4 .2 7
5 0 4 .2 7
4 3 3 .2 3
6 7 6 .3 2
6 1 9 .3
5 0 4 .2 7
6 7 6 .3 2
6 1 9 .3
9 6 5 .5
83741
83741
8 3 7 .4 1
6 1 9 .3
9 6 5 .5
83741
6 7 6 .3 2
6 7 6 .3 2
5 0 4 .2 7
5 0 4 .2 7
6 1 9 .3
9 3 1 .5 1
80342
6 7 6 .3 2
6 1 9 .3
1 :3 1 4 .7 4
1 3 3 0 .7 3
1 3 1 4 ,7 4
1 2 0 3 .6 7
1 2 1 9 .6 6
1 5 9 7 .9 4
1 4 7 0 .8 4
15.21
1 7 8 4 .9 8
1 6 7 2 .9 2
1 6 0 1 .8 8
1 4 9 0 .8 1
1 2 0 3 .6 7
1 3 1 4 J 4 : 1 2 0 3 .6 7
■1334.71: 1 2 2 3 .6 4
1 0 7 6 .5 7
9 6 5 .5
9 6 5 .5
1 5 .2 2 M
1828
1 7 8 4 .9 8
1 6 7 2 .9 2
1 6 0 1 .8 8
1 4 9 0 .8 1
1 3 3 4 .7 1
1 2 2 3 : 6 4 1 0 7 6 .5 7
9 6 5 .5
1 .2 3 M
1828
1 6 7 2 .9 2
1 6 0 1 .8 8
1 2 2 3 .6 4
9 6 5 .5 :
1 6 7 2 .9 2
1 6 0 1 .8 8
7 .1 4 0
1828
1828
1 4 9 0 .8 1
1 4 9 0 .8 1
1 3 3 4 .7 1
1 1 .9N
1 7 8 4 .9 8
1 7 8 4 .9 8
1 7 8 4 .9 8
1 6 7 2 .9 2
1 6 0 1 .8 8
1 4 9 0 .8 1
1 3 3 4 .7 1
1 5 .2 4 H
1 8 3 3 .0 2
1790
1 6 7 7 .9 4
1 6 0 6 .9
1 4 9 5 .8 3
1 3 3 9 .7 3
1 3 3 9 .7 3
5 .7 H
1 8 3 3 .0 2
1 8 3 6 .1
9 .2 1 0
1 8 4 0 .0 5
1 8 4 4 .1 2
2 .1 8 P
1 5 .1 9 J
1 6 7 7 .9 4
1 6 0 6 .9
1 4 9 5 .8 3
1 6 8 1 .0 2
1 6 0 9 .9 8
1 4 9 8 .9 1
1 7 9 7 .0 3
1 6 8 4 .9 7
1 6 1 3 .9 3
1618
1 4 8 6 .8 3
1 3 3 0 .7 3
1 6 8 9 .0 4
1 4 5 6 .9 1
1 3 0 0 .8 1
1 3 7 3 .7 2
1 3 7 3 .7 2
1 3 7 3 .7 2
1790
1 7 9 3 .0 8
1 3 4 2 .8 1
9 3 1 .5 1
5 6 7 .5 5 ,
9 8 1 .4 9
1 0 6 2 .6 4
1 2 6 2 .6 5
1 0 7 6 .5 7
1 0 7 6 .5 7
9 5 1 .5 7 :
9 6 5 .5
1 8 6 7 .0 1
1 7 1 1 .9 3
1 6 4 0 .8 9
1 5 2 9 .8 2
1 8 6 7 .0 1
1 8 2 3 .9 9
1 7 1 1 .9 3
1 6 4 0 .8 9
8 .1 1 H
1 8 6 7 .0 1
1 8 6 7 .0 1
1 8 2 3 .9 9
1 8 2 3 .9 9
1 6 4 0 .8 9
1 6 4 0 .8 9
1 5 2 9 .8 2
1 3 7 3 .7 2
1 2 6 2 .6 5
1 8 7 9 .1
1 8 3 6 .0 8
1 7 1 1 .9 3
1 7 1 1 .9 3
1 7 2 4 .0 2
1 5 2 9 .8 2
1 5 2 9 .8 2
1 6 5 2 .9 8
1 4 8 5 .8 5
1 3 2 9 .7 5
6 .2 1 D
1 8 8 1 .0 2
1838
1 7 2 5 .9 4
1 6 5 4 .9
1 5 4 3 .8 3
1 3 8 7 .7 3
1 2 1 8 .6 8
1 2 7 6 .6 6
4 .1 2 L
1883
1 8 8 6 .0 4
1 8 3 9 .9 8
1 8 4 3 .0 2
1 7 2 7 .9 2
1 6 5 6 .8 8
1 6 5 9 .9 2
1 5 4 5 .8 1
1 5 3 2 .8 2
1 3 8 9 .7 1
1 3 7 6 .7 2
1 7 3 0 .9 6
1 0 4 2 .5 8
1 0 7 8 .6 2
1 0 9 2 .5 6
1 2 .1 9 P
9 .2 1 0
1 0 4 2 .5 8
1 2 1 9 .6 6
1 1 8 9 .7 4
1 8 0 1 .1
1 8 2 3 .9 9
1 1 .4 C
8 .9 0
1 0 7 6 .5 7
1 0 7 6 .5 7
1 0 7 6 .5 7 .
1 2 2 3 .6 4 ; 1 0 7 6 .5 7
1 2 2 3 .6 4 1 0 7 6 .5 7
1 2 2 8 .6 6
1 2 3 1 .7 4
1 2 6 2 .6 5
1 2 6 2 .6 5
1 7 2 .1 2
1 7 2 .1 2
5 0 4 .2 7
9 6 5 .5
1 8 2 4 .0 6
1828
9 .1 3 B
2 8 6 .1 6
5 0 4 .2 7
1 4 7 0 .8 4
1 2 2 8 .6 6
4 3 3 .2 3
4 3 3 .2 3
6 1 9 .3
1 4 8 6 .8 3
1 4 7 0 .8 4
1 3 3 4 .7 1
1 7 2 .1 2
6 1 9 .3
6 1 9 .3
1 5 8 1 .9 1
1 5 9 7 .9
1 5 9 7 .9 4
1 6 6 8 .9 8
1 6 6 8 .9 8
1 7 2 .1 2
. 2 8 6 .1 6
2 8 6 .1 6
6 1 9 .3
1 0 7 6 .5 7
1 0 7 6 .5 7
1 7 8 1 .0 4
1 7 8 1 .0 4
1 7 2 .1 2
2 8 6 .1 6
6 7 6 .3 2
1 2 0 3 .6 7
1 8 2 4 .0 6
2 8 6 .1 6
4 3 3 .2 3
4 3 3 .2 3
6 7 6 .3 2
1 2 0 3 .6 7
1 3 1 4 .7 4
1 6 5 2 .9 5
3 9 9 .2 4
8 3 7 .4 1 :
1 3 1 4 .7 4
1 4 7 0 .8 4
1 6 6 8 .9 4
or
8 3 7 .4 1 :
1 4 7 0 .8 4
1 5 8 1 .9 1
1781
# 'v
o'
9 6 5 .5
1 4 5 6 .8 2
1 5 8 1 .9 1
1 6 5 2 .9 5
1 7 6 5 .0 1
^
9 6 5 .5
9 6 5 .5
1 5 6 7 .8 9
1 6 5 2 .9 5
1 8 0 8 .0 3
1 8 2 4 .0 2
1 3 0 0 .7 2
V
O'
^
8 .1 1 H
1 1 8 9 .6 5
&^
1 0 7 6 .5 7
1 0 7 6 .5 7
1 0 3 2 .6
1 2 7 8 .6 4
1 1 2 9 .5 9
1 0 9 2 .5 6
1 2 6 5 .6 5
1 0 7 9 .5 7
9 6 5 .5
9 6 5 .5
9 6 5 .5
9 2 1 .5 3
1 0 1 8 .5 2
9 8 1 ,4 9
9 6 8 ,5
4 3 3 .2 3
2 8 6 .1 6
2 8 6 .1 6
2 8 6 .1 6
4 3 3 .2 3
4 3 3 .2 3
4 3 3 .2 3
1 7 2 .1 2
5 0 4 .2 7
4 3 3 .2 3
2 8 6 .1 6
1 7 2 .1 2
4 3 3 .2 3
2 8 6 .1 6
6 1 9 .3
5 0 4 .2 7
5 0 4 .2 7
4 3 3 .2 3
2 8 6 .1 6
1 7 2 .1 2
6 2 1 .3 5
5 0 6 .3 2
4 3 5 .2 8
2 8 8 .2 1
2 8 6 .1 6
1 7 4 .1 7
1 7 2 .1 2
6 9 2 .3 1
6 9 6 .3 8
6 3 5 .2 9
5 2 0 .2 6
4 4 9 .2 2
6 3 9 .3 6
4 3 9 .2 8
8 3 7 .4 1
8 3 7 .4 1
6 7 6 .3 2
6 7 6 .3 2 -
6 1 9 .3
5 1 0 .3 2
5 0 4 .2 7
4 3 3 .2 3
2 8 6 .1 6
2 8 6 .1 6
5 0 4 .2 7
5 0 4 .2 7
4 3 3 .2 3
4 3 3 .2 3
2 8 6 .1 6
2 8 6 .1 6
8 4 0 .4 1
1 7 2 .1 2
1 7 2 .1 2
8 5 3 .4
82348
. 6 1 9 .3
6 7 6 .3 2 . : 6 1 9 .3
6 7 6 .3 2
6 1 9 .3
6 8 2 :3 7
6 2 5 .3 5
7 2 9 .3 4
1 7 2 .1 2
1 7 2 .1 2
2 8 6 .1 6
2 8 6 .1 6
4 3 3 .2 3
6 7 6 .3 2
6 7 8 .3 7
7 9 3 .4 4
8 9 0 .4 3 ,
8 5 3 .4
1 7 2 .1 2
2 8 6 .1 6
8 0 3 .4 2
63946
83741
8 3 7 .4 1
1 7 2 .1 2
6 7 2 .3 2
6 9 2 .3 1
6 3 5 .2 9
7 2 9 ,3 4 -. 6 7 2 .3 2
1 7 2 .1 2
1 7 2 .1 2
1 7 2 .1 2
1 7 2 .1 2
1 7 2 .1 2
1 7 2 .1 2
1 7 2 .1 2
5 0 4 .2 7
4 3 3 .2 3
2 8 6 .1 6
5 1 0 .3 2
5 4 3 .2 8
4 3 9 .2 8
4 7 2 .2 4
2 8 6 .1 6
2 8 6 .1 6
1 7 2 .1 2
5 2 0 .2 6 1 4 4 9 .2 2
4 7 2 .2 4
5 4 3 .2 8
2 8 6 .1 6
1 7 2 .1 2
1 7 2 .1 2
2 8 6 .1 6
1 7 2 .1 2
Table 43. Sequences of hits from side chain optimization library (JKM V I 199, Figure 3).
S eq u en ce
Plate. Well
W e ll#
8 . 11H
168
14 3
91
70
290
290
83
317
4
271
383
235
25
349
365
14 2
223
376
10 4
19 4
335
288
298
304
8 .9 0
13.6K
1 1 . 5F
1 4 .1 9 B
1 4 .1 9 B
1 5 .6 C
6.20M
1.1D
3 .1 7 0
1 5 .2 4 0
15 .1 5 K
15.21
15.22M
I.2 3 M
I I . 9N
7 .1 4 0
1 5 .2 4 H
5.7H
9 .1 3 B
9 .2 1 0
2 .1 8 P
1 5 .1 9 J
1 2 .1 9 P
8 .1 1 H
1 1 .4 C
8 .9 0
6 .2 1 D
4 .1 2 L
9 .2 1 0
16 8
51
14 3
324
188
33 5
[M+H]+ N
4
5
6
7
8
11
12
15
C
Leu
xLeu
xLeu
ACPC
ACPC
ACPC
Lys
Lys
Lys
Gly
Gly
Gly
A sp
A sp
A sp
Ala
Ala
Ala
13
L eu
Phe
Phe
14
ACPC
ACPC
ACPC
9
ACPC
B3Nle
B3Nle
10
A rq
A rg
A rq
A sn
A sn
A sn
Arq
Arq
Arq
NH2
NH2
NH2
1710
1 7 7 4 .0 4
1 7 9 0 .0 7
Ac
Ac
Ac
APC
APC
APC
A la
A la
A la
3
ACPC
ACPC
B3L eu
1 7 9 4 .0 1
Ac
APC
Ala
ACPC
A rg
ACPC
Leu
ACPC
Lys
B3Phe
Gly
A sp
Ala
Phe
A sn
Arg
NH2
1 8 0 8 .0 3
Ac
APC
Ala
ACPC
A rg
ACPC
x L eu
ACPC
Lys
B3Phe
Gly
A sp
A la
Phe
A sn
Arg
NH2
1 8 2 4 .0 2
Ac
APC
Ala
ACPC
A rg
ACPC
x L eu
ACPC
Lys
B3Phe
Gly
A sp
Ala
Tyr
A sn
A rg
NH2
1 8 2 4 .0 6
Ac
APC
Ala
B 3L eu
A rg
ACPC
x L eu
ACPC
Lys
B3Phe
Gly
A sp
A la
Phe
A sn
A rg
NH2
1828
Ac
APC
Ala
ACPC
A rg
ACPC
Phe
ACPC
Lys
B 3Phe
Gly
A sp
Ala
Phe
A sn
A rg
NH2
2
1 8 3 3 .0 2
Ac
APC
Ala
ACPC
A rg
ACPC
T rp
ACPC
Lys
B3Nle
Gly
A sp
A la
Phe
A sn
A rg
NH2
1 8 36.1
1 8 4 0 .0 5
1 8 4 4 .1 2
Ac
Ac
Ac
APC
APC
APC
A la
A la
A la
ACPC
B3L eu
B3Phe
A rq
A rq
A rg
ACPC
ACPC
ACPC
C ha
x L eu
x L eu
ACPC
ACPC
ACPC
Lys
Lys
Lys
B3Phe
B3Phe
B3Nle
Gly
Gly
Gly
A sp
A sp
Glu
A la
Ala
A la
Phe
Tyr
Cha
A sn
A sn
A sn
A rq
A rq
A rg
NH2
NH2
NH2
1 8 6 7.01
Ac
APC
Ala
ACPC
A rg
ACPC
T rp
ACPC
Lys
B3Phe
Gly
A sp
Ala
Phe
A sn
A rg
NH2
1 879.1
1 8 8 1 .0 2
1883
1 8 8 6 .0 4
Ac
Ac
Ac
Ac
APC
A PC
APC
APC
A la
A la
A la
Ala
B 3C ha
ACPC
ACPC
ACPC
A rg
Arg
Arg
Arg
ACPC
ACPC
ACPC
ACPC
T rp
Phe
T rp
x L eu
A CPC
ACPC
ACPC
ACPC
Lys
Lys
Lys
Lys
ACPC
B 3Phe
B 3Phe
B3Trp
Gly
Gly
Gly
Gly
A sp
G lu
A sp
A sp
Ala
Ala
Ala
Ala
Cha
T rp
T yr
T rp
A sn
A sn
A sn
A sn
A rq
A rq
A rg
A rq
NH2
NH2
NH2
NH2
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without permission.
mP
2 9 .4
2 6 .9
2 1 .7
2 2 .8
30.1
30.1
2 1 .2
2 4 .4
29.1
3 5 .6
1 9 .5
2 2 .8
2 1 .7
24
24.1
2 8 .5
29
2 7 .2
3 1 .2
28.1
27.1
32.1
1 1 .6
1 3 .3
2 9 .4
2 9 .7
2 6 .9 .
2 3 .5
33.1
27.1
309
Table 44. Activity of re-synthesized, purified peptide hits from library (JKM VI 223). mP, Av. mP, and # of
Occurrences are from initial screening of data.
Sequence
Peptide
RH1415-a-34 #2
JKM-VI-199 # t
Ac
Ac
APC
APC
ACPC
Ala
ACPC
Ala
Arg
Arg
ACPC
ACPC
Leu
Phe
ACPC
ACPC
Lys
Lys
JKM-VI-199 #2
Ac
APC
Ala
ACPC
Arg
ACPC
Trp
ACPC
Lys
JKM-VI-199 #3
Ac
APC
Ala
ACPC
Arg
ACPC
xLeu
ACPC
JKM-VI-199 #4
Ac
APC
Ala
ACPC
Arg
ACPC
Trp
JKM-VI-199 #5
Ac
APC
Ala
ACPC
Arg
ACPC
JKM-VI-199 #6
Ac
APC
Ala
03Leu
Arg
ACPC
Ac
APC
APC
APC
APC
APC
APC
APC
APC
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
ACPC
ACPC
ACPC
ACPC
ACPC
03Leu
Arg
ACPC
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Arg
Arg
ACPC
ACPC
Arg
Arg
Ac
Ac
Ac
APC
APC
APC
Ala
Ala
Ala
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
ACPC
Cha
Trp
xLeu
Phe
xLeu
xLeu
xLeu
Trp
xLeu
xLeu
Leu
JKM-VI-199 #7
JKM-VI-199 #8
JKM-VI-199 #9
JKM-VI-199 #10
JKM-VI-199 #11
JKM-VI-199 #12
JKM-VI-199 #13
JKM-VI-199 #14
JKM-VI-199 #15
JKM-VI-199 #16
B3Leu
Arg
Arg
B3Cha
B3Phe
ACPC
ACPC
Arg
Arg
Arg
Arq
mP
B3Nle
03Phe
Gly
Gly
Asp
Asp
Ala
Ala
Phe
Phe
Asn
Asn
Arg
Arg
NH2
p3Phe
Gly
Asp
Ala
Phe
Asn
Arg
NH2
Lys
p3Phe
Gly
Asp
Ala
Phe
Asn
Arg
NH2
ACPC
Lys
p3Nle
Gly
Asp
Ala
Phe
Asn
Arg
NH2
Leu
ACPC
Lys
p3Phe
Gly
Asp
Ala
Phe
Asn
Arg
NH2
xLeu
ACPC
Lys
p3Phe
Gly
Asp
Ala
Phe
Asn
Arg
NH2
B3Phe
03Phe
B3Nte
B3Phe
B ^ rp
B3Phe
8 3Nle
ACPC
B3Nle
B3Phe
ACPC
Gly
Asp
Asp
Asp
Glu
Asp
Asp
Asp
Asp
Glu
Asp
Asp
Ala
Ala
Phe
Tyr
Phe
Trp
Trp
Tyr
Phe
Cha
Cha
Tyr
Leu
Asn
Asn
Arg
NH2
NH2
ACPC
Lys
ACPC
ACPC
Lys
Lys
ACPC
ACPC
ACPC
ACPC
ACPC
Lys
Lys
Lys
ACPC
ACPC
ACPC
Lys
Lys
Lys
Lys
Lys
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Asn
Asn
Asn
Asn
Arg
Arg
Arg
Arg
Arg
Av. mP (St. Dev.) IC50 (pM) # of O ccurrences
NH2
NH2
NH2
NH2
NH2
Asn
Asn
Arg
Arg
NH2
NH2
Asn
Asn
Asn
Arg
Arg
Arq
NH2
NH2
NH2
0.061
21.7
24
24.1
28.5
29
11.6
13.3
29.4
29.7
21.2
24.4
29.1
27.2
31.2
22.8
30.1
19.5
22.8
28.1
25.5 (3.2)
0.089
5
0.22
4
21.0(9.9)
24.9 (4.0)
29.2 (2.8)
27.7 (4.2)
21.2(2.3)
33.1
26.9
23.5
27.1
27.1
21.7
26.9
32.1
35.6
29.4
0.048
3
0.28
2
0.056
2
0.12
0.053
1.1
2
0.05
31
1.2
0.29
. 0.24
26
76
0.53
b ' - 1-r. :*
f
t
A 0f
■d- /.'A
J V J \-i\9
1 ;: ; 3 :
>'
r‘4
13 I'.:';
&.}
;p:
Hd f ; v :;.
'17 > i
4A
--'I
*6
TlfJif •[.'T-ii:)
Figure 33. LC-MS/MS sequencing oligomer 5-11 with side products noted.
5.4.3 Split-and-Mix Synthesis of the P-Scan Library Using Microwave Irradiation
The P-scan library (JKM VII 121, Figure 6) was synthesized using split-and-mix
techniques on PS macrobeads using microwave irradiation in a monomode microwave reactor.
PS A RAM macrobeads (70 mg, 40 jamol, ~ 500 beads) were placed in a polypropylene solidphase extraction (SPE) tube (4 mL, Alltech), and swelled with DMF for 10 min. The resin was
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
310
washed (5 x DMF, 5 x CH2C12 and 5 x DMF).
Deprotection solution (2 mL o f 20%
piperidine in DMF (v/v)) was added to the resin, and the tube was capped and placed on a shaker
for 2 hr. The resin was washed as before. In a separate vial, Fmoc-Arg(Pbf)-OFl (120 pmol)
was activated by adding HBTU (240 pL of 0.5 M solution in DMF), DMF (1.76 mL), HOBt
(240 pL of 0.5 M solution in DMF), and /Pr2EtN (240 pL o f 1.0 M solution in DMF). The
mixture was vortexed and allowed to stand for 1 min before being added to the resin. The tube
was capped and placed on a shaker overnight. The resin was washed, and deprotection solution
was added (2 mL).
The tube was placed inside a glass 10 mL microwave reaction vessel
containing ~ 2 mL of DMF. A N2 line was inserted for agitation, and the vessel was placed in
the monomode microwave reactor (CEM Discover) and irradiated (50 W maximum power, 60°C,
ramp 2 min).
The monomode reactor was used instead o f the multimode reactor to avoid
simultaneously irradiating samples containing different coupling solutions and the resulting
differences in reaction temperature. The sample was removed from the microwave reactor and
cooled at room temperature for 10 min. This ramp/cool cycle was repeated 3 times. The tube
was removed from the microwave reaction vessel, and the resin was washed as before. The resin
was partitioned into two aliquots of approximately equal volume using a spatula with care not to
crush the swollen beads.
Fmoc-Asn(Trt)-OH (60 pmol) and Fmoc-|33-Gln(Trt)-OH were
activated in separate vials by adding HBTU (120 pL o f 0.5 M solution in DMF), DMF (880 pL),
HOBt (120 pL o f 0.5 M solution in DMF), and z'Pr2EtN (120 pL o f 1.0 M solution in DMF) to
each. The mixtures were vortexed. The Fmoc-Asn(Trt)-OH-containing coupling solution was
-j
added to one aliquot o f resin, and the activated Fmoc-P -Gln(Trt)-OH was added to the other
aliquot. The first sample was irradiated in the microwave reactor (50 W maximum power, 50°C,
ramp 2 min), removed from the reactor, and cooled for 10 min at room temperature while the
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
311
second sample was irradiated. This ramp/cool cycle o f microwave irradiation was repeated a
total o f 6 times for each sample. The resin was washed, combined, and suspended in DMF, and
thoroughly mixed. After Fmoc-deprotection, the resin was again divided into two equal portions.
Fmoc-Phe-OFI was coupled to one aliquot o f the resin, and Fmoc-(33-Phe-OH was coupled to the
other aliquot in separate reactions. The resin was washed, combined, mixed, Fmoc-deprotected,
and split into two equal portions. Fmoc-Ala-OH was coupled to one aliquot o f the resin, and
Fmoc-p3-Ala-OH was coupled to the other aliquot in separate reactions. The resin was washed,
combined, mixed, Fmoc-deprotected, and split into two equal portions. Fmoc-Asp(7Bu)-OH was
coupled to one aliquot o f the resin, and Fmoc-p3-Asp(7Bu)-OH was coupled to the other aliquot
in separate reactions. The resin was washed, combined, mixed, Fmoc-deprotected, and split into
two equal portions. Fmoc-Gly-OH was coupled to one aliquot of the resin, and Fmoc-P-Gly-OFI
was coupled to the other aliquot in separate reactions. The resin was washed, combined, mixed,
Fmoc-deprotected, and split into three equal portions. Fmoc-Gly-OFI was coupled to one aliquot
o f the resin, and Fmoc-P-Gly-OH was coupled to the second aliquot in separate reactions.
Flowever, no monomer unit was coupled to the third resin aliquot (a null substitution). The resin
was washed, combined, mixed, and Fmoc-deprotected. The coupling/deprotection reaction cycle
was repeated with Fmoc-p3-Nle-OFI, Fmoc-Lys(Boc)-OFI, Fmoc-ACPC-OFI, Fmoc-Leu-OH,
Fmoc-ACPC-OH, Fmoc-Arg(Pbf)-OH, Fmoc-ACPC-OH, Fmoc-Ala-OFI, and Fmoc-APC(Boc)OH. After washing (5 x DMF, 5 x CH 2 CI2 , 5 x DMF, and 5 x CH 2 CI2 ), the peptides were Nterminally
acetylated
by
adding
2
mL
of
a
14:5:1
solution
of
CFkCVacetic
anhydride/triethylamine and shaking for 30 min. After washing (5 x CH 2 CI2 and 5 x MeOH)
and drying under a stream o f N2, the resin was arrayed (one bead per well) into 2 384-well
polypropylene plates (Costar) using tweezers and a bead arrayer as described above.
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
The
312
oligomers were cleaved from the solid support with simultaneous side chain deprotection (80
pL, 50:50:5:5 TFA:CH2Cl2:triethylsilane:water, 2 h, RT, with orbital shaking; plates were
covered with aluminum foil. At the end o f the reaction, the cleavage solution was concentrated
by rotary evaporation (RT, 4 hr, SpeedVac, Thermo Savant). The crude oligomer mixtures were
dissolved in 80 pL o f DMSO; 10 pL o f this stock solution was used for the FP assay, while the
remaining
solution was
reserved
for
analytical
characterization
and
compound
(hit)
identification. The crude oligomer mixtures from 50 beads were analyzed by HPLC (Shimadzu);
30 pL was injected on a C 4 -silica reversed-phase analytical column (5 pm, 4 mm x 250 mm,
Vydac) and eluted with a gradient o f acetonitrile in water (10 - 60%, 25 min, 0.1% TFA in each)
at a flow rate o f 1 mL/min. The major peak in each HPLC run was collected, and peptide masses
were measured by MALDI-TOF-MS (Bruker Reflex II, a-cyano-4-hydroxycinnamic acid
matrix). The library was screened in the Bcl-xi/Bak FP assay.
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 45. Screening data (time = 0 hr) for plate 1 of the split-and-mix P-scan library (Figure 6). Percent inhibition = 100 * (l-((mP - 39.25)/(av. mP - 39.25))).
2
A
B
C
D
E
F
G
H
1
J
K
L
M
N
0
P
162.72498
155.00956
164.1519
165.38089
159.71816
161.06095
161.50261
165.09047
169.3136
168.965
159.26429
165.89081
161.00183
164.76537
148.09325
165.16716
155.80573
168.43289
166.06971
172.27304
167.49822
167.73227
152.86734
168.06173
171.07569
170.37426
173.82814
166.04446
129.19036
126.84694
172.27789
168.78933
3
158.2519
166.1481
164.4967
166.3912
161.4691
167.8432
153.5417
167.7758
168.962
172.9319
145.8146
165.6494
128.4696
171.9206
165.8373
172.7568
4
156.1325
167.4261
168.1795
167.3734
172.0682
172.9949
170.497
174.8994
174.1248
167.2236
172.6341
171.6908
168.2179
132.7332
166.9408
144.4093
5
160.5346
159.4637
167.128
161.7907
174.4128
170.6515
166.647
177.4516
177.0961
167.6226
169.7208
161.8623
174.5845
119.6966
170.3471
. 170.3601
162.1556
160.8385
170.1314
169.4761
172.1901
94.2231
167.7695
172.2686
167.2304
171.7632
174.1412
172.8741
175.6274
166.154
-171.9055
167.5329
4
7.7
-1.3
-1.9
- 1.2
-4.9
-5.7
-3.7
-7.2
-6.6
- 1.1
-5.4
-4.6
-1.9
26.1
-0.9
16.9
5
4.2
5.0
- 1.0
3.2
-6.8
-3.8
-0.6
-9.2
-8.9
-1.4
-3.1
3.1
-6.9
36.4
-3.6
-3.6
2.9
3.9
-3.4
-2.9
-5.0
56.6
-1.5
-5.1
- 1.1
-4.7
-6.6
-5.6
-7.7
-0.3
-4.8
-1.3
6
7
157.7771
160.5497
171.4399
168.1241
170.9236
171.6729
173.5811
176.6388
167.186
169.4403
177.2637
177.9342
174.7603
180.7219
173.9173
154.9908
8
164.7262
168.8838
170.1502
174.6968
174.3078
170.6721
161.3077
172.6716
169.0142
173.0696
172.7217
169.2473
159.3216
174.034
175.5346
168.8327
9
162.8702
162.7642
170.0805
166.4537
174.1595
167.4002
172.847
168.776
174.5746
162.4832
166.6337
174.1285
153.8763
154.7634
169.9715
144.4369
10
162.7604
172.3012
171.4391
122.8303
176.4811
174.23
178.1638
173.7235
170.9147
181.9385
178.294
177.8811
168.5621
171.6142
175.81
167.6831
11
12
174.0268
167.6665
168.4483
169.1588
175.3724
171.1042
166.9383
166.5568
168.9761
123.1394
167.2183
174.2677
164.9554
166.9611
173.6581
171.0378
169.7579
164.3028
165.3027
169.8593
161.9772
170.8216
156.5844
166.4334
168.9688
169.8144
170.7264
173.6731
176.9938
168.9193
172.7738
166.5885
13
164.7571
164.5673
164.346
164.4111
167.4782
175.2207
170.4371
147.5398
176.8905
163.218
169.1925
165.7284
166.6487
146.6886
178.4711
175.819
14
156.6898
109.0491
166.1417
163.7378
157.2758
159.3096
170.2389
173.406
172.2113
167.9154
169.8061
172.6223
166.5109
166.4102
174.6445
167.0416
15
159.7122
167.3538
171.5754
159.8316
170.2664
160.5757
165.2325
167.3517
169.5772
176.9725
172.9186
170.2958
150.4928
173.1343
175.399
166.0127
16
169.116
160.5057
180.2108
167.9371
169.4189
171.6857
172.0286
167.854
170.2714
170.8097
177.1062
178.8044
170.7241
169.7872
172.6344
151.6906
17
162.8749
151.8874
172.5589
165.4053
174.6821
166.0977
169.1693
167.5808
181.2762
68.19155
161.9351
156.0337
173.0181
106.8379
176.0881
181.9418
18
159.1452
162.4651
167.3255
164.3614
173.9983
151.5802
171.3314
169.8546
170.6531
168.3507
170.8858
171.7944
167.5573
164.57
175.4917
135.7968
14
7.2
44.9
-0.2
1:7
15
4.8
- 1.2
-4.5
4.7
-3.5
4.1
0.5
- 1.2
-3.0
-8.8
-5.6
-3.5
16
-2.6
4.2
-11.4
-1.7
-2.8
-4.6
-4.9
- 1.6
-3.5
-3.9
-8.9
-10.3
-3.9
-3.1
-5.4
17
2.3
18
5.3
2.7
- 1.2
19
162.2913
161.6412
170.3953
173.5405
166.8959
172.8401
167.7285
170.262
180.6012
177.8535
170.4461
159.5084
159.6729
168.7341
168.3846
175.4331
20
21
22
162.2877
171.0452
159.5636
172.4422
168.7888
171.3268
169.9834
93.23531
173.5175
173.1488
179.2481
175.5352
173.2226
173.7351
165.4729
167.3673
162.5551
166.4262
168.9665
164.7413
169.8004
169.0568
167.2379
174.2238
171.5356
168.8542
163.5301
164.5499.
164.7648
159.7763
167.8198
149.1391
165.7482
161.8253
164.5907
161.3
168.4264
162.279
168.1282
175.1837
177.9946
173.346
171.3133
183.3176
167.928
167.7057
169.8714
153.0446
2.8
20
2.8
21
2.6
22
0.1
3.3
-3.6
-6.1
-0.8
-5.5
-1.5
-3.5
-11.7
-9.5
-3.6
5.0
4.9
-2.3
-2.0
-7.6
-4.1
4.9
-5.2
-2.3
-4.3
-3.3
57.4
-6.1
-5.8
- 10.6
-7.7
-5.8
-6.2
0.3
- 1.2
-0.5
-2.5
0.9
-3.1
- 2.6
- 1.1
-6.6
-4.5
-2.4
3.2
23
169.5293
160.8572
167.0694
163.7186
163.1963
163.1581
141.4118
167.2208
167.9863
158.061
165.3157
154.5505
147.3021
148.1126
152.5687
154.9703
24
166.6824
159.6869
166.7468
158.0841
164.9001
166.6404
171.1039
171.1011
173.4209
167.05
167.297
183.0959
164.3446
166.0832
163.9931
163.3373
23
-2.9
3.9
- 1.0
1.7
24
-0.7
4.9
-0.7
2.1
2.1
0.7
-0.6
-4.2
-4.2
-6.0
- 1.0
- 1.2
-13.6
cent Inhibition
A
B
C
D
E
F
G
H
I
J
K
L
M
N
0
P
1
2
3
2.5
8.5
1.3
0.4
4.8
3.8
3.4
7.9
-2.1
-0.2
-5.1
-1.3
-1.5
6.0
0.6
-2.8
-2.5
5.2
0.0
3.8
0.8
14.0
0.5
10.2
- 1.8
-4.1
-3.6
-6.3
-0.2
28.9
30.8
-5.1
-2.3
-0.3
1.1
-0.4
3.4
- 1.6
9.7
-1,5
-2.5
-5.6
15.8
0.1
29.5
-4.8
0.0
-5.5
6
7
6.4
4.2
-4.4
- 1.8
-4.0
-4.6
-6.1
-8.5
- 1.1
-2.9
-9.0
-9.6
-7.1
- 11.8
-6.4
8.6
8
0.9
-2.4
-3.4
-7.0
-6.7
-3.8
3.6
-5.4
-2.5
-5.7
-5.4
-2.7
5.1
-6.5
-7.7
-2.4
9
2.3
2.4
-3.4
-0.5
-6.6
-1.2
-5.5
-2.3
-6.9
2.6
-0.6
-6.6
9.4
8.7
-3.3
16.9
10
11
12
13
2.4
-5.1
-4.4
34.0
-8.4
-6.6
-9.7
-6.2
-4.0
-12.7
-9.8
-9.5
-2.2
-4.6
-7.9
-1.5
-6.5
-1.5
- 2.1
-2.6
-7.5
-4.2
-0.9
-0.6
-2.5
33.7
- 1.1
-6.7
0.7
-0.9
-6.2
-4.1
-3.1
0.8
1.0
1.2
1.1
1.2
0.4
-3.2
3.0
-3.9
7.3
-0.5
-2.5
-3.1
-3.9
-6.2
-8.8
-2.4
-5.5
-0.6
-1.3
-7.4
-3.6
14.4
. -8.7
2.1
-2.7
0.1
-0.6
15.1
- 10.0
-7.9
6.8
5.1
-3.5
- 6.0
-5.0
- 1.6
-3.1
-5.4
-0.5
-0.5
-7.0
- 1.0
12.1
-5.8
-7.6
-0.1
11.2
11.0
-5.3
0.3
-7.0
-0.2
-2.6
-1.4
- 12.2
77.1
3.1
7.7
-5.7
46.6
-8.1
-12.7
1.2
-6.5
11.3
-4.3
-3.2
-3.8
-2.0
-4.0
-4.7
-1.4
1.0
-7.6
23.7
19
1.8
1.0
0.8
4.8
- 1.6
. 13.2
1.0
3.6
- 2.1
2.8
- 1.8
-7.4
-9.6
-5.9
-4.3
-13.8
-1.7
-1.5
-3.2
10.1
19.3
- 1.1
-1.7
6.1
0.4
8.9
14.6
14.0
10.5
8.6
6.1
1.2
-0.2
1.4
2.0
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 46. Screening data (time = 4 hr) for plate 1 of the split-and-mix |3-scan library (Figure 6). Percent inhibition = 100 * (l-((mP - 39.25)/(av. mP - 39.25))).
Data taken at a later time point to ensure that the system has reached equilibrium.
2
A
B
C
D
e
F
G
H
1
J
K
L
M
N
0
P
167.7471
157.9375
167.0207
172.0217
168.0494
167.6651
168.7807
172.8161
167.1492
169.5179
163.8773
169.5931
167.6115
173.8606
144.696
178.8393
169.4224
173.1209
174.0188
177.893
178.5984
179.5442
162.807
177.9333
182.5934
172.017
179.929
183.74
148.3396
119.5745
179.8298
187.8949
3
171.20909
164.1753
165.86746
159.65346
171.02809
169.07681
153.24096
170.01376
170.30701
163.90495
141.57014
163.72838
143.29089
174.44159
167.12569
173.61533
4
175.3356
169.322
178.7331
170.9175
180.4656
175.9376
179.6811
172.8463
179.5932
175.0772
181.5813
175.0986
180.3779
101.4351
184.8901
145.0525
5
171.7401
162.8632
156.0207
157.8067
163.4035
168.23
162.2953
173.1664
170.4686
176.3137
168.1512
161.0234
157.1282
110.8429
167.7634
174.1578
3
-0.4
4.9
3.6
8.4
-0.3
4
-3.6
5
-0.8
5.9
6
172.1113
174.2396
167.0919
174.57
171.0439
87.93347
179.3119
173.5698
182.3265
185.3306
194.255
181.3796
191.1706
176.2828
186.2602
182.2047
7
162.9857
160.2607
175.0715
168.1912
167.1576
169.6729
173.8442
180.3768
167.5689
162.9945
179.8231
155.8383
159.3662
159.0862
147.089
177.6494
8
175.2603
172.8905
178.2649
172.5169
176.0354
171.7085
172.2365
186.2779
173.8386
174.8622
199.7067
204.427
179.6284
190.7603
176.7873
183.7447
9
168.7235
171.0973
167.3819
171.3651
171.5919
166.7555
165.0051
173.5146
175.245
167.2451
173.3267
175.2662
146.835
153.4314
165.2068
167.7836
10
11
12
170.7592
177.0373
171.0515
142.9774
176.523
173.3059
179.8863
178.1359
198.5203
175.4296
179.4372
183.1008
178.3345
183.6561
192.2171
176.655
169.5928
166.8363
167.5057
172.1017
171.6143
174.5547
160.1044
170.1329
173.5481
113.6003
168.9698
166.0412
155.094
158.37
167.217
172.6838
168.3067
166.233
172.8835
180.4526
169.5565
177.859
173.7725
182.8787
174.1325
174.4729
169.6916
175.6756
187.2507
190.1981
179.3293
176.5099
13
170.5995
161.3406
172.4207
164.6721
172.5611
174.9353
159.384
128.9872
167.747
148.2984
158.1963
164.1756
180.6381
159.3074
174.3558
178.9003
14
172.1424
113.3021
176.0753
182.3686
166.6926
183.8637
178.0371
187.6834
183.0417
170.2185
190.2862
175.1788
194.8748
187.5434
173.4196
171.7543
15
163.0054
166.0217
165.6721
165.5171
170.7778
159.5894
167.08
165.7698
170.264
171.0056
169.7146
158.0627
181.7517
178.6114
166.7526
169.9629
16
178.205
169.8947
177.1335
174.9503
174.3528
190.7074
180.2158
187.8193
177.9468
178.4703
179.7916
177.4867
184.6591
181.2695
176.3196
176.7434
17
165.0015
149.6689
172.0603
168.0347
167.9565
174.0144
163.7586
173.9017
169.3709
68.67982
157.95
159.716
160.3642
125.1022
161.9202
181.1837
10
11
0.8
12
1.8
13
0.0
3.4
-1.7
-7.5
15
5.8
3.5
3.8
3.9
-0.1
8.4
2.7
3.7
0.3
-0.3
0.7
9.6
-8.4
-6.1
3.0
0.5
16
-5.7
2.9
2.4
- 1.1
-0.7
-3.0
14
-1.1
43.6
-4.1
-8.9
3.0
- 10.1
-5.6
-13.0
-9.4
0.3
-14.9
-3.4
-18.4
-12.9
-2.1
-0.8
17
4.3
16.0
- 1.1
18
170.7335
170.3929
165.9415
181.9747
169.8033
182.8584
175.9725
189.9157
198.6395
166.2259
180.4022
176.1952
179.4773
185.9237
177.1592
167.2636
19
169.0117
173.8608
171.3543
168.0056
172.7825
169.0083
164,1925
172.2249
170.3937
160.5166
158.0802
156.2106
152.5251
165.3572
153.8203
162.618
20
21
172.4604
174.9052
168.6248
183.0003
172.5826
187.6316
203.2023
89.37959
189.349
193.1201
191.5768
182.0368
179.5621
191.6169
174.7399
195.5917
164.726
171.3561
170.2252
162.1513
172.1765
169.6368
163.7783
173.6113
170.997
164.7659
152.2802
167.7297
153.2933
157.0538
160.526
159.6425
22
171.7652
170.9987
179.5641
176.0773
174.1348
174.0312
173.038
184.622
186.0517
175.1728
180.1908
195.1358
179.5391
172.4423
173.0893
179.8516
23
168.0936
163.3525
176.0743
165.9103
164.768
167.4315
145.7869
176.8382
152.5525
160.2715
171.3194
164.9436
168.8245
164.8281
175.0038
154.5417
24
170.123
166.6974
178.565
162.1881
171.0943
186.2535
191.2353
194.8096
194.8943
192.9426
181.8601
187.5797
188.4463
181.7937
179.3368
165.5292
24
0.4
3.0
-6.0
6.4
-0.3
-11.9
-15.7
-18.4
-18.4
-17.0
-8.5
-12.9
-13.5
-8.5
-6.6
3.9
cent Inhibition
A
B
C
D
E
F
G
H
I
J
K
L
M
N
0
P
1
2.2
9.7
2.8
- 1.0
2.0
2.3
1.4
- 1.6
2.7
0.9
5.2
0.8
2.3
-2.4
19.8
-6.2
2
0.9
-1.9
-2.6
-5.5
-6.0
-6.8
6.0
-5.5
-9.1
- 1.0
' -7.1
- 10.0
17.0
38.9
-7.0
-13.1
1.2
13.3
0.5
0.3
5.1
22.1
5.3
20.8
-2.9
2.7
-2.3
1.0
-6.1
-0.2
-7.5
-4.0
-6.9
-1.7
-6.8
-3.4
-8.3
-3.4
-7.4
52.7
- 10.8
19.5
11.1
9.8
5.5
1.8
6.4
-1.9
0.1
-4.3
1.9
7.3
10.3
45.5
2.2
-2.7
6
- 1.1
-2.7
2.7
-3.0
-0.3
63.0
-6.6
-2.2
-8.9
- 11.2
-18.0
-8.2
-15.6
-4.3
-11.9
-8.8
7
5.8
7.9
-3.4
1.9
2.7
0.7
-2.4
-7.4
2.4
5.8
-7.0
11.3
8.6
8.8
17.9
-5.3
8
-3.5
-1.7
-5.8
-1.4
-4.1
-0.8
- 1.2
-11.9
-2.4
-3.2
-22.1
-25.7
-6.8
-15.3
-4.7
- 10.0
9
1.5
-0.3
2.5
-0.5
-0.7
3.0
4.3
-2.2
-3.5
2.6
-2.0
-3.5
18.1
13.1
4.1
2.2
-0.1
-4.9
-0.3
21.1
-4.5
-2.0
-7.0
-5.7
-21.2
-3.6
-6.7
-9.5
-5.8
-9.9
-16.4
-4.6
8.0
0.4
-2.2
43.4
1.3
3.5
11.8
9.4
2.6
-1.5
0.8
-5.5
-2.4
-9.3
-2.6
-2.9
0.7
-3.8
- 12.6
-14.9
-6.6
-4.5
7.1
-1.3
4.6
-1.4
-3.3
8.6
31.7
2.2
17.0
9.5
4.9
-7.6
8.6
-2.8
-6.3
0.6
-4.9
-3.3
-2.8
-15.3
-7.3
-13.1
-5.5
-5.9
-7.0
-5.2
• -10.7
- 8.1
-4.3
-4.6
2.0
2.1
-2.6
18
-0.1
0.2
3.6
-8.6
0.6
6.6
-9.3
-4.0
-14.7
-21.3
3.4
-7.4
-4.2
-6.7
- 11.6
-4.9
-8.0
2.6
5.2
-2.5
1.0
77.6
9.7
8.3
7.8
34.7
19
1.3
-2.4
-0.5
2.0
- 1.6
1.3
4.9
- 1.2
0.2
7.7
9.6
11.0
13.8
4.0
12.8
6.1
20
21
22
23
-1.4
-3.2
1.5.
-9.4
-1.5
-12.9
-24.8
61.9
-14.2
-17.1
-15.9
-8.7
-6.8
-16.0
-3.1
-19.0
4.5
-0.5
0.3
6.5
- 1.2
-0.8
-0.3
-6.8
-4.1
-2.6
-2.6
-1.8
- 10.6
-11.7
-3.4
-7.3
-18.6
-6.8
-1.4
-1.9
-7.0
2.0
0.8
5.2
-2,2
-0.3
4.5
14.0
2.2
13.2
10.4
7.7
8.4
5.6
-4.1
3.6
4.5
2.5
18.9
-4.7
13.8
7.9
-0.5
4.3
1.4
4.4
-3.3
12.3
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 47. Screening data at time = 0 hr (top), and time = 4 hr (bottom) for plate 2 of the split-and-mix P-scan library (Figure 6). Percent inhibition = 100 * (1((mP - 39.25)/(av. mP - 39.25))). Data taken at a later time point (bottom) to ensure that the system has reached equilibrium.
2
A
B
C
D
E
F
G
H
87.78329
141.8384
157.0371
170.3275
157.5872
158.2525
157.416
149.467
95.38878
157.3421
157.7651
169.4454
160.9292
162.5184
158.6017
150.8174
3
78.3099
151.9884
163.8303
161.0427
157.9034
163.9553
157.6769
157.9852
4
93.19124
146.6213
161.4179
160.3323
165.0972
170.2741
163.0525
154.3813
5
145.4284
159.9203
161.4732
164.9449
164.7032
171.3761
176.2263
171.8642
3
67.9
7.2
-2.5
-0.2
2.3
- 2.6
2.5
2.3
4
55.6
5
6
7
12.6
3.6
-0.5
0.3
-3.6
-7.8
-1.9
5.2
0.7
-0.6
-3.4
-3.2
-8.7
-12.7
-9.1
3.2
0.3
-3.2
-4.6
-4.8
-3.4
0.0
0.0
-6.0
3
93.58385
166.218
167.6759
164.3543
169.1272
164.3396
171.7154
155.3818
4
122.6397
160.6209
162.2648
169.4499
171.1674
185.7374
181.5445
155.2411
5
175.7844
172.5953
171.9882
164.5103
170.2595
173.6567
174.4604
174.7055
208.2809
169.6801
174.6868
168.6316
166.6768
176.6283
176.804
165.6587
3
58.6
3.2
4
36.4
7.5
2.1
6.2
0.8
5
-4.1
- 1.6
- 1.2
4.5
6
156.3553
158.1544
156.8625
160.3501
164.6825
166.3147
166.6304
164.863
7
160.7555
160.7203
168.0527
167.3582
173.1545
166.5702
169.5144
169.1375
8
159.5004
156.1811
155.8967
162.7153
168.2084
169.5831
146.6388
166.5438
9
157.8816
163.9777
159.9475
171.5126
168.6531
167.7432
169.8884
170.5447
10
11
12
159.5453
158.5921
154.2002
169.605
165.2478
164.1556
134.4517
170.9099
160.3589
163.1698
161.9096
171.5863
165.7751
162.8038
168.1972
171.934
150.5797
159.6867
158.865
166.7359
164.4134
165.8632
169.9893
170.5846
13
156.8881
92.24532
164.725
164.3572
172.559
178.0708
166.8461
168.8111
14
160.8693
160.1316
151.7811
167.3299
169.1528
150.8084
162.1842
165.5423
15
166.428
165.4031
161.1071
171.7784
164.5107
78.00423
168.937
169.5385
16
166.8207
158.3189
157.1611
166.6641
162.936
166.9373
165.3272
167.623
17
164.2076
163.1798
164.9929
163.5906
165.1649
169.9425
172.9403
178.6695
18
161.7057
160.8399
148.5064
160.7147
162.6264
166.9178
172.7641
169.9467
19
163.0791
164.4818
163.0396
165.9299
167.9887
142.6888
170.6859
169.9864
13
3.2
56.4
-3.3
-3.0
-9.7
-14.3
-5.0
-6.6
14
-0.1
0.5
7.4
-5.4
-6.9
15
-4.7
-3.8
-0.3
-9.1
-3.1
16
-5.0
18
-0.8
-0.1
8.2
- 1.2
68.1
3.0
-4.9
- 1.8
-5.1
-3.8
-5.7
17
-2.8
-2.0
-3.5
-2.3
-3.6
-7.6
- 10.0
-14.7
-1.5
-5.1
-9.9
-7.6
19
-1.9
-3.1
-1.9
-4.3
-6.0
14.9
-8.2
-7.6
-5.8
-2.0
-6.2
-2.3
13
175.4942
90.76106
169.1325
171.4667
172.5745
160.8201
172.5729
174.0543
14
175.5559
173.095
164.5595
176.6712
1.69.1376
168.6117
177.8804
172,5783
15
178.2378
171.538
170.7869
177.2428
175.8319
85.20012
169.8266
175.8476
16
171.8256
164.9396
168.2031
174.6362
180.6767
187.4702
170.9191
171.0011
17
172.7522
169.899
177.2312
161.5796
172.9409
157.5431
176.9598
178.6222
18
168.8852
165.3485
171.0316
166.9438
170.322
174.2185
174.9303
169.384.1
19
176.3196
170.1173
175.8886
176.5189
178.7128
159.1873
177.3783
183.1404
20
21
22
170.2416
175.8485
174.186
181.3495
178.6625
173.4715
172.7646
179.2133
180.8519
177.574
176.054
169.2719
135.4513
171.1341
178.1436
178.3106
171.6456
135.575
168.718
165.2153
174.7156
189.6559
171.1532
168.7153
13
-3.8
60.7
14
-3.9
-2.0
4.5
-4.7
15
-5.9
-0.8
-0.3
-5.2
-4.1
65.0
0.5
-4.1
16
- 1.1
4.2
1.7
-3.2
-7.8
-13.0
-0.4
-0.4
17
- 1.8
0.4
-5.2
18
19
-4.5
0.3
-4.1
-4.6
-6.3
20
21
22
162.1617
159.6633
160.436
159.9856
167.7618
163.1693
168.3436
163.5577
168.3103
165.1321
165.5434
171.4138
130.0438
168.0415
173.3029
170.4503
161.6062
115.0994
157.9664
165.12
160.7003
167.8065
164.7932
160.9142
23
159.0934
163.8476
167.0764
168.3271
167.4361
171.642
178.9037
167.3608
24
157.6073
158.1142
163.4534
164.7824
162.0197
167.7408
175.8355
166.5019
cent Inhibition
A
B
C
1
2
60.1
15.6
3.1
-7.9
53.8
2.8
E
F
G
H
2.6
2.1
2.5
-7.2
-0.1
-1.5
2.7
9.3
1.8
8.2
A
B
C
D
E
F
G
H
93.61811
151.5232
171.274
173.0455
162.1513
160.4243
171.1718
152.6901
113.48
169.7617
174.9719
173.0133
171.2068
170.5693
174.3933
149.4467
0
2
11.6
2.1
6
-5.4
- 10.2
-4.8
-7.2
-6.9
7
179.6358
176.2914
169.4679
174.2751
171.7775
170.3607
184.186
174.2071
8
1.0
3.8
4.0
- 1.6
-6.1
-7.3
11.6
-4.8
8
224.8909
171.5759
163.1881
169.2324
171.8077
180.6561
175.9971
175.725
9
2.4
-2.7
0.7
-8.9
-6.5
-5.8
-7.5
- 8.1
10
1.0
1.8
5.4
-7.3
-3.7
-2.8
21.6
-8.4
11
12
0.3
-2.0
- 1.0
-8.9
-4.1
-1.7
-6.1
-9.2
8.4
0.9
1.6
-4.9
-3.0
-4.2
-7.6
-8.1
9
221.9637
175.2292
168.9641
162.316
169.8133
173.0243
180.0652
178.4546
10
11
12
226.9203
175.6371
167.7459
167.3122
171.1455
183.5839
156.6404
173.5114
227.4178
171.1614
167.4124
177.5169
174.1142
162.3693
169.7546
176.4046
178.6568
171.4384
162.6426
176.128
174.8412
176.2795
171.1332
174.1135
9
-39.3
-3.6
10
11
12
-43.0
-4.0
-43.4
-0.5
2.3
-5.4
- 2.8
-6.3
-0.8
5.9
-4.3
-3.3
-4.4
-0.5
-2.8
-3.9
-6 .7 __|
-7.2
2.0
10.1
0.0
20
21
22
- 1.2
0.9
0.3
- 6.2
-3.6
-3.9
-8.8
25.3
-6.0
-10.3
-8.0
-0.7
37.6
2.3
-3.6
0.6
0.0
-5.8
-3.3
-0.1
23
1.4
-2.5
-5.2
-6.2
-5.5
-9.0
-14.9
-5.4
24
-3.3
- 1.0
-5.7
-12.4
-4.7
23
174.7065
167.88
175.259
160.3015
172.7192
156.0782
183.4753
178.7763
24
168.1315
168.7732
167.8815
168.3357
169.2166
186.3618
174.0487
175.3456
2.6
2.2
- 2.2
cent Inhibition
A
B
C
0
E
F
G
H
1
2
58.6
14.4
-0.6
-2.0
6.3
7.6
-0.6
13.5
43.4
0.5
-3.4
-2.0
-0.6
-0.1
-3.0
16.0
4.6
1.0
4.7
- 1.0
11.5
-0.5
-11.7
-8.5
11.6
0.1
-2.4
-3.1
-3.2
6
-28.8
0.6
-3.2
1.4
2.9
-4.7
-4.8
3.7
7
-7.0
-4.5
0.7
-2.9
- 1.0
0.1
-10.5
-2.9
8
-41.5
-0.9
5.5
0.9
- 1.0
-7.8
-4.2
-4.0
1.1
6.2
0.5
-2.0
-7.3
- 6.1
2.1
2.4
-0.5
- 10.0
10.5
-2.3
6.2
0.5
-4.5
1.0
-0.8
- 1.6
7.3
- 1.6
-.2.7
1.0
1.4
-5.7
- 1.6
6.8
-1.9
9.8
-5.0
-6.2
1.2
3.9
-0.4
2.7
0.1
-2.9
-3.4
0.8
8.6
-5.3
-9.7
20
0.2
-4.1
-2.8
-8.3
-6.3
-2.3
- 1.8
-6.7
21
22
-7.9
-5.4
-4.3
0.9
26.7
-0.5
-5.9
-6.0
-0.9
26.6
1.3
.4.0
-3.3
-14.6
-0.5
1.3
24
23
-3.2
1.8
2.0
1.3
-3.7
7.7
-1.7
0.9
11.0
-9.9
-6.3
2.0
1.6
- 12.1
-2.7
-3.7
u i
%lnhibition
316
Column
Figure 34. Percent inhibition by compounds in plate 2 o f the split-and-mix combinatorial P-scan library (Figure 6).
See Figure 7 for results from Plate 1.
Table 48. Hits from screening of split-and-mix P-scan library (Figure 6) identified by MALDI-TOF MS.
Plate-Well Percent Inhibition MALDI Identification
1-J17
7 7 .6
5-1
5-1
65 .0
2 -F 1 5
5-1
1-F6
63.0
1-H 20
61.9
5-1
60.7
5-1
2-B 13
5-2 + deletion peptides
58.6
2-A 3
5-2
2-A1
58.6
5-19,21,22, or 23
1-N 4
52.7
5-24
1-N 5
4 5 .5
5-1
1-B14
4 3 .6
5-2 + deletion peptides
4 3 .4
2-A2
5-19, 21,22, or 23
4 3 .4
1-J11
5-1, 5-1 + Leu
1-N2
38.9
5-2 + deletion peptides
2-A 4
36.4
5-19,21,22, or 23
1-N 17
34.7
5-19,21,22, or 23
31.7
1-H 13
5-19,21,22, or 23
26.7
2-E21
5-19,21,22, or 23
2-B 22
26.6
5-19, 21,22, or 23
21.1
1-D 10
5-19, 21,22, or 23
1-M 3
20.8
5-19, 21,22, or 23
19.8
1 -0 1
5-19, 21,22, or 23
1-P4
19.5
1-G 23
18.9
5-23
R e p ro d u c e d with permission of the copyright owner. Further reproduction prohibited without perm ission.
317
5.4.4 Microwave-Assisted Parallel Synthesis of the p-Scan L ibrary (Figure 6)
For the parallel p-scan library (JKM VI 241, Figure 6), NovaSyn TGR resin (250 pmol, 1
g) was suspended in 50 mL of a 3:2 mixture o f dichloromethane/DMF.
The slurry was stirred
while 500 pL aliquots were dispensed using a pipette into each well o f a 2 mL deep well
polypropylene filter plate with polyethylene frits and long drip spouts sealed with a bottom mat
(Artie White). The resin was washed (5 x DMF). A magnetic stir bar (7 mm, VWR) was placed
inside each well. In a separate vial, Fmoc-Arg(Pbf)-OH (720 pmol) was activated by adding
HBTU (1440 pL o f 0.5 M solution in DMF), DMF (10.56 mL), HOBt (1440 pL o f 0.5 M
solution in DMF), and /Pr2EtN (1440 pL o f 1.0 M solution in DMF). The mixture was vortexed,
and 150 pL was added to each well using a using a 12-channel multipipette. The plate was
placed on a microtiter plate turntable inside the multimode microwave cavity (CEM MARS).
The fiber optic temperature probe was positioned in well D6 using the arm attached to the
turntable, and the sample was irradiated (600 W maximum power, 80°C, ramp 2 min, hold 4
min).
All microwave irradiations were conducted at atmospheric pressure.
The plate was
removed from the microwave reactor, and the resin was washed (5 x DMF). The bottom sealing
mat was reaffixed, and the coupling procedure was repeated to double couple Fmoc-Arg(Pbf)OH. After washing, deprotection solution (250 pL o f 20% piperidine in DMF (v/v)) was added
to the resin in each well.
The temperature probe was placed in the center o f this region o f the
plate, and the sample was irradiated (600 W maximum power, 90°C, ramp 2 min, hold 2 min).
After washing, a solution o f activated Fmoc-Asn(Trt)-OH (360 pmol, 720 pL o f 0.5 M FIBTU in
DMF, 5.28 mL DMF, 720 pL of 0.5 M HOBt in DMF, and 720 pL o f 1.0 M zPr2EtN in DMF,
150 pL per well) was added to half o f the wells in the plate (rows A-D, columns 1-12, Figure 35)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
318
using a 12-channel multipipette, and DMF (150 pL) was added to the remaining wells (rows
E-H, columns 1-12). The plate was placed in the multimode microwave reactor, and the fiber
optic probe was positioned in the center o f the Fmoc-Asn(Trt)-OH-containing section o f the
■y
plate (well C6). The sample was irradiated, and the resin was washed. Activated Fmoc-|3 Gln(Trt)-OH (360 pmol, 150 pL per well) was added to the second half o f the wells in the plate
(rows E-H, columns 1-12, Figure 35) using a 12-channel multipipette, and DMF (150 pL) was
added to the remaining wells (rows A-D, columns 1-12). The plate was placed in the multimode
microwave reactor, and the fiber optic probe was positioned in the center o f the Fmoc-(33-Gln
(Trt)-OH-containing section o f the plate (well F6). The sample was irradiated, and the resin was
washed.
The two-step process described for coupling Fmoc-Asn(Trt)-OH and Fmoc-(33-Gln
(Trt)-OH constitutes the sequential coupling o f the two residues at position 15. The Fmoc■y
deprotection/sequential coupling steps were repeated to incorporate Fmoc-Phe-OH and Fmoc-(3 Phe-OH at position 14, Fmoc-Ala-OH and Fmoc-p3-Ala-OH at position 13, Fmoc-Asp(fBu)-OH
and Fmoc-(33-Asp(?Bu)-OH at position 12, and Fmoc-Gly-OH and Fmoc-p-Gly-OH at position
11 (Figure 35). At position 10, Fmoc-Gly-OH was coupled to one-third o f the library members,
Fmoc-p-Gly-OH was coupled to the second third o f the library members, and the final one-third
o f the library was not coupled to any monomer unit (a null substitution).
The Fmoc-y
deprotection/coupling reactions were repeated with all library members to incorporate Fmoc-P Nle-OH, Fmoc-Lys(Boc)-OH, Fmoc-ACPC-OH, Fmoc-Leu-OH, Fmoc-ACPC-OH, FmocArg(Pbf)-OH, Fmoc-ACPC-OH, Fmoc-Ala-OH, and Fmoc-APC(Boc)-OH. Following the final
Fmoc-deprotection, the resin was washed (5 x DMF,. 5 x CH 2 CI2 ), and the peptides were
acetylated for 15 min at room temperature with stirring (150 pL o f 14:1:5 CH 2 CI2 /TEA/AC2 O).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
319
After washing (5 x CH2C12), cleavage from the solid support with global side chain
deprotection was accomplished by adding triisopropylsilane (50 pL), water (50 pL), and
trifluoroacetic acid (1.0 mL) to each well. The plate was wrapped tightly in aluminum foil and
stirred for 2 hr at room temperature on a stir plate. The foil covering was removed, and the
cleavage solutions were transferred to a solid-bottom deep well 96-well plate by gravity filtration
and concentrated using a rotary evaporator (SpeedVac with well-plate adapter, Thermo Savant).
The crude peptide mixtures were dissolved in 250 pL DMSO. All 96 samples were analyzed by
HPLC (15 pL injection, Shimadzu). The C 4 -silica reversed-phase analytical column (5 pm, 4
mm x 250 mm, Vydac) was eluted with a gradient o f acetonitrile in water (10 —60% B solvent,
25 min, 0.1% TFA in each, followed by a 5 min flush with 95% acetonitrile and 5 min
equilibration at the starting concentration) at a flow rate o f 1 mL/min. The major peaks from the
HPLC chromatogram were collected, and peptide masses were measured by MALDI-TOF-MS
(Bruker Reflex II, a-cyano-4-hydroxycinnamic acid matrix).
The library was diluted (1:10,
1:100, and 1:000) and screened in the Bcl-xL/Bak FP assay. A portion o f each library member
was purified by HPLC (200 pL injection).
The C4 -silica reversed-phase semi-preparative
column (5 pm, 10 mm x 250 mm, Vydac) was eluted with a gradient o f acetonitrile in water (10
- 60% B solvent, 25 min, 0.1% TFA in each, followed by a 5 min flush with 95% acetonitrile
and 5 min equilibration at the starting concentration) at a flow rate o f 3 mL/min. The fractions
o f the eluent corresponding to the major peaks from the HPLC chromatogram were transferred to
two deep well 96-well plates and concentrated via rotary evaporation (SpeedVac). The purified
peptides were dissolved in 200 pL o f DMSO, diluted (1:10, 1:100, and 1:000), and screened in
the Bcl-xL/Bak FP assay.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Posisiton 16: a-Arg
Position 15:
A
B
C
D
E
F
G
H
Position 14:
12
A
B
C
D
E
F
G
H
a-Phe
a-Phe
Position 13:
3
6
4
7
8
9
10
11
12
7
8
9
10
11
12
a-Ala
A
B
C
D
E
F
G
H
BJ-Ala
a-Ala
|i -Ala
a-Ala
a-Ala
a*
Position 12:
2
1
A
B
C
D
a-Asp
E
F
G
H
3
4
P3-Asp
5
6
a-Asp
u-Asp
P3-Asp
P’-Asp
Position 11:
7
8
9
10
11
12
1
2
3
4
5
6
A
B
C
D
a-Gly P-Gly a-Gly P-Gly a-Gly P-Gly a-Gly P-Gly a-Gly P-Gly a-Gly P-Gly
E
F
G
H
Position 10:
1
2
3
4
5
6
7
8
9
10
11
12
Position 9: P -Nle
Position 8: a-Lys
Position 7: ACPC
Position 6: a-Leu
Position 5: ACPC
Position 4: a-Arg
Position 3: ACPC
Position 2: a-Ala
Position 1: ACPC
Figure 35. Spatial addressing for parallel synthesis o f (3-scan library (JKM V I 241, Figure 6). This “sector”
approach streamlines reagent delivery via a multichannel pipette.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
321
T able 49. Sequences o f (3-scan library members 1-48 (JKM VI 241 and JKM VII 121, Figure 6).
C o m p o u n d Well
N |
1 I 2 I
Leu
S equence
8
9 I 10
Gly
A CPC Lys p’-NIe
A CPC
Leu
A CPC
Lys
p’-NIe
Arg
ACPC
Leu
ACPC
Lys
ACPC
Arg
ACPC
Leu
ACPC
Lys
A CPC
Arg
A CPC
Leu
A CPC
3
|
4
1
A 1 Ac
APC
Ala
ACPC
Arg
5
ACPC
2
B 1 Ac
APC
Ala
ACPC
Arg
3
c
1 Ac
APC
Ala
ACPC
4
D 1 Ac
APC
Ala
Ala
I
6
7
|
I 11 I 12 |
14
15
Gly
Asp
Ala
P he
Asn
Gly
Gly
Asp
P’-Ala
P he
Asn
Arg n h 2
p’-NIe
Gly
Gly
Asp
Ala
p ’-P he
Asn
Arg n h 2
p’-NIe
Gly
Gly
Asp
A sn
Arg n h 2
Lys p ’-NIe
Gly
Gly
Asp
Ala
13
|
fs-Ala p’-P he
5
E
1 Ac
APC
6
F
1 Ac
APC
Ala
A CPC
Arg
ACPC
Leu
A CPC
Lys
p-’-NIe
Gly
Gly
Asp
p’-Ala
7
G
1 Ac
APC
Ala
A CPC
Arg
A CPC
Leu
ACPC
Lys
p’-NIe
Gly
Gly
Asp
Ala
8
H 1 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
ACPC
Lys
Pj -Nle
Gly
Gly
Asp
Arg
A CPC
Leu
ACPC
Lys
p-’-NIe
Gly
p-hGly
Asp
| 16| C
Arg n h 2
P he
p’-GIn Arg n h 2
P he
P’-GIn Arg n h 2
p ’-P he p’-GIn Arg n h 2
p -A la p ’-P h e p’-GIn Arg n h 2
Arg n h 2
9
A 2 Ac
APC
Ala
A CPC
Ala
Phe
Asn
10
B 2 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
ACPC
Lys
p’-NIe
Gly
p-hGly
Asp
p’-Ala
P he
Asn
Arg n h 2
11
C 2
Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
p’-NIe
Gly
P-hGly
Asp
Asn
Arg n h 2
12
D 2 Ac
APC
Ala
ACPC
Arg
ACPC
Leu
A CPC
Lys
p’-NIe
Gly
P-hGly
Asp
Ala p’-P he
P’-Ala p’-P he
Arg
A CPC
Leu
A CPC
Lys
p’-NIe
Gly
P-hGly
A sp
Ala
E 2 Ac
APC
Ala
ACPC
14
F 2 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
p’-NIe
Gly
P-hGly
A sp
P’-Ala
15
G 2 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A C PC
Lys
P’-NIe
Gly
p-hGly
A sp
Ala
13
P he
Asn Arg n h 2
p’-GIn Arg n h 2
P he
p’-GIn Arg n h 2
p - P h e p -G in Arg n h 2
p’-Ala p’-P h e p’-GIn Arg n h 2
16
H 2 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
p’-NIe
Gly
P-hGly
A sp
17
A 3 Ac
APC
Ala
ACPC
Arg
ACPC
Leu
A C PC
Lys p-’-NIe
Gly
Gly
P’-Asp
18
B 3 Ac
APC
Ala
A CPC
Arg
ACPC
Leu
A C PC
Lys p’-NIe
Gly
Gly
P’-A sp p’-Ala
19
C 3 Ac
APC
Ala
A CPC
Arg
ACPC
Leu
ACPC
Lys
pJ-Nle
Gly
Gly
P’-Asp
20
D 3 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
AC PC
Lys p ’-NIe
Gly
Gly
p’-A sp p’-Ala p’-P he
21
E 3 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
p’-NIe
Gly
Gly
p’-Asp
22
F 3 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
p’-NIe
Gly
Gly
P’-A sp p’-Ala
23
G 3 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A C PC
Lys
p’-NIe
Gly
Gly
p’-Asp
24
H 3 Ac
APC
Ala
ACPC
Arg
ACPC
Leu
A CPC
Lys
p-’-NIe
Gly
Gly
P’-A sp p’-Ala p’-P h e p’-GIn Arg n h 2
ACPC
Arg
A C PC
Leu
A CPC
Lys
p’ -NIe
Gly
p-hGly p’-Asp
P-hGly p’-Asp p’-Ala
Ala
Asn
Asn
Arg n h 2
p’-P he
Asn
Arg n h 2
Asn
Arg n h 2
Ala
Ala
Arg n h 2
Phe
Phe
Ala
Phe
p’-GIn Arg n h 2
Phe
p’-GIn Arg n h 2
p - P h e p -G in Arg n h 2
Ala
Phe
Asn
Arg n h 2
P he
Asn
Arg n h 2
25
A 4 Ac
APC
Ala
26
B 4 Ac
APC
Ala
A CPC
Arg
A CPC
Leu
A CPC
Lys p ’-NIe
Gly
27
C 4
APC
Ala
A CPC
Arg
A CPC
Leu
A CPC
Lys
p-’-NIe
Gly
p-hGly p’-Asp
p’-P he
Asn
Arg n h 2
Gly
P-hGly P’-A sp P’-Ala p’-P he
A sn
Arg n h 2
Ac
Ala
28
D 4 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
p’-NIe
29
E 4 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
p’-NIe
Gly
P-hGly p ’-Asp
30
F 4 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
p’-NIe
Gly
P-hGly p ’-A sp p’-Ala
31
G 4 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
p’-NIe
Gly
P-hGly p’-Asp
A CPC
Lys p-’-NIe
Gly
Ala
Phe
p’-GIn Arg n h 2
P he
p’-GIn Arg n h 2
Ala p’-P h e p’-GIn Arg n h 2
P-hGly p’-A sp p’-Ala P’-P h e p’-GIn Arg n h 2
32
H 4
Ac
APC
Ala
ACPC
Arg
A CPC
Leu
33
A 5 Ac
APC
Ala
A CPC
Arg
A CPC
Leu
A CPC
Lys
p’-NIe
Gly
Asp
Ala
Phe
Asn
Arg n h 2
34
B 5 Ac
APC
Ala
A CPC
Arg
ACPC
Leu
A CPC
Lys
p’-NIe
Gly
Asp
p’-Ala
P he
Asn
Arg n h 2
35
C 5 Ac
APC
Ala
A CPC
Arg
A CPC
Leu
A CPC
Lys
p’-NIe
Gly
Asp
Ala
p’-P he
Asn
Arg n h 2
36
D 5 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
p’-NIe
Gly
A sp
p’ -Ala p ’-P he
Phe
A sn Arg n h 2
p ’-GIn Arg n h 2
P he
p’-GIn Arg n h 2
37
E 5 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
p’-NIe
Gly
A sp
Ala
38
F 5 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A C PC
Gly
Asp
p’-Ala
39
G 5 Ac
APC
Ala
A CPC
Arg
A C PC
Leu
A C PC
Lys p’-NIe
Lys p’-NIe
Gly
A sp
Ala
40
H 5 Ac
APC
Ala
A CPC
Arg
ACPC
Leu
A CPC
Lys p ’-NIe
Gly
A sp
41
A 6 Ac
APC
Ala
A CPC
Arg
ACPC
Leu
A CPC
Lys p ’-NIe
p-hGly
A sp
Ala
P he
A sn
42
B 6 Ac
APC
Ala
A CPC
Arg
A CPC
Leu
ACPC
Lys p ’-NIe
P-hGly
Asp
p’-Ala
P he
A sn
Arg n h 2
43
C 6 Ac
APC
Ala
A CPC
Arg
A CPC
Leu
ACPC
Lys
p’-NIe
P-hGly
Asp
Ala
p ’-P he
Asn
Arg n h 2
44
D 6 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
p’-NIe
p-hGly
A sp
Asn
Arg n h 2
45
E 6 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys p’-NIe
p-hGly
A sp
Ala
46
F 6 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
p’-NIe
P-hGly
A sp
p’-Ala
47
G 6 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A C PC
Lys
p ’-NIe
P-hGly
A sp
Ala
48
H 6 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
p’-NIe
P-hGly
A sp
p’-P h e p -G in Arg n h 2
P’-Ala p’-P h e p’-GIn Arg n h 2
p’-Ala p’-P he
Arg n h 2
Phe
p’-GIn Arg n h 2
P he
p’-GIn Arg n h 2
p’-P he p’-GIn Arg n h 2
p’-Ala p’-P h e p’-GIn Arg n h 2
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
322
T able 50. Sequences o f p-scan library members 49-96 (JKM VI 241 and JKM VII 121, Figure 6).
C o m p o u n d Well
49
N|
1 I
2
I
3
|
4
5
|
6
Arg
A CPC
Leu
S equence
9 |
7 | 8
ACPC Lys pJ-Nle
I
10
|
11
i1 2
pJ-Asp
I 13
14
15
Phe
Asn
Arg n h 2
Phe
Asn
Arg n h 2
pJ-P he
Asn
Arg n h 2
Asn
Arg n h 2
]
ACPC
APC
Ala
ACPC
Arg
ACPC
Leu
ACPC
Lys
p'-N Ie
Gly
Ala
pJ-Asp P^-Ala
APC
Ala
ACPC
Arg
ACPC
Leu
A CPC
Lys
pJ-Nle
Gly
pJ-Asp
D 7 Ac
APC
Ala
ACPC
Arg
ACPC
Leu
A CPC
Lys
pJ-Nle
Gly
pJ-A sp pJ-Ala pJ-P he
E 7 Ac
APC
Ala
ACPC
Arg
ACPC
Leu
A CPC
Lys
pJ-Nle
Gly
pJ-Asp
APC
50
B 7 Ac
51
c
7 Ac
52
53
Gly
|
Ala
A 7 Ac
Ala
Phe
|1 6 |
C
pJ-Gln Arg n h 2
p-'-GIn Arg n h 2
54
F
7 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
pJ-Nle
Gly
Ala
pJ-Asp pJ-Ala
55
G 7 Ac
A PC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys p J-Nle
Gly
pJ-Asp
56
H 7 Ac
APC
Ala
ACPC
Arg
ACPC
Leu
A CPC
pVA sp p^-Ala p - P h e p -G in Arg n h 2
A 8 Ac
APC
Ala
ACPC
Arg
ACPC
Leu
ACPC
Lys p-’-NIe
Lys pJ-Nle
Gly
57
58
B 8 Ac
APC
Ala
ACPC
Arg
ACPC
Leu
ACPC
Lys
pJ-Nle
P-hGly pJ-A sp pJ-Ala
59
C 8 Ac
APC
Ala
ACPC
Arg
ACPC
Leu
A CPC
Lys
pJ-Nle
P-hGly pJ-Asp
60
D 8 Ac
APC
Ala
A CPC
Arg
A CPC
Leu
A CPC
Lys
61
E 8 Ac
APC
Ala
A CPC
Arg
A CPC
Leu
A CPC
Lys
62
F 8 Ac
APC
Ala
ACPC
Arg
ACPC
Leu
ACPC
Lys pJ-Nle
P-hGly pJ-A sp pJ-Ala
63
G 8 Ac
A PC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys p J-Nle
p-hGly p-’-Asp
64
H 8 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys p J-Nle
P-hGly pJ-A sp pJ-Ala pJ-P h e pJ-Gln Arg n h 2
65
A 9 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
pJ-Nle p-hGly
Gly
Asp
Ala
P he
Asn
Arg n h 2
66
B 9 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
pJ-Nle p-hGly
Gly
A sp
pJ-Ala
P he
Asn
Arg n h 2
67
C 9 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
pJ-Nle p-hGly
Gly
Asp
Ala
p^-Phe
Asn
Arg n h 2
Arg
A CPC
Leu
A CPC
Lys p J-Nle p-hGly
Gly
Asp
p-*-Ala p^-Phe
Asn
Arg n h 2
A CPC
Leu
A CPC
Lys pL-NIe p-hGly
Gly
Asp
Ala
P-hGly p^-Asp
Ala
P he
pJ-P he p J-Gln Arg n h 2
Phe
Asn
Arg n h 2
Phe
A sn
Arg n h 2
P^-Phe
Asn
Arg n h 2
pJ-Nle
P-hGly pJ-Asp pJ-Ala pJ-P he
Asn
Arg n h 2
p3-Nle
P-hGly pJ-Asp
Ala
Ala
Ala
Ala
P he
p J-Gln Arg n h 2
P he
p'-G In Arg n h 2
p J-P he pJ-Gln Arg n h 2
68
D 9 Ac
A PC
Ala
ACPC
69
E 9 Ac
APC
Ala
ACPC
Arg
70
F 9 Ac
APC
Ala
ACPC
Arg
ACPC
Leu
A CPC
Lys
Pa-Nle p-hGly
Gly
Asp
pJ-Ala
71
G 9 Ac
APC
Ala
ACPC
Arg
ACPC
Leu
A CPC
Lys
pJ-Nle p-hGly
Gly
A sp
Ala
72
H 9 Ac
APC
Ala
A CPC
Arg
A CPC
Leu
A CPC
Lys
pa-Nle p-hGly
Gly
Asp
73
A 10 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
pJ-Nle p-hGly P-hGly
Asp
Ala
P he
Asn
74
B 10 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys Ps-Nle p-hGly p-hGly
Asp
pJ-Ala
P he
A sn
Arg n h 2
75
C 10 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
p-’-NIe p-hGly p-hGly
Asp
Ala
p J-P he
Asn
Arg n h 2
76
D 10 Ac
APC
Ala
A CPC
Arg
A CPC
Leu
ACPC
Lys
pJ-Nle p-hGly p-hGly
A sp
77
E 10 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
pJ-Nle p-hGly p-hGly
Asp
Ala
P he
p J-Gln Arg n h 2
Phe
pJ-Gln Arg n h 2
pJ-P he pJ-Gln Arg n h 2
pJ-Ala p^-Phe p -G in Arg n h 2
pJ-Ala pJ-P he
Phe
Arg n h 2
Asn Arg n h 2
p J-Gln Arg n h 2
p^-GIn Arg n h 2
78
F 10 Ac
A PC
Ala
ACPC
Arg
A CPC
Leu
ACPC
Lys pJ-Nle p-hGly p-hGly
Asp
pJ-Ala
79
G 10 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
AC PC
Lys
pJ-Nle p-hGly p-hGly
Asp
Ala
80
H 10 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
ACPC
Lys
pJ-Nle p-hGly p-hGly
81
A 11 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
ACPC
Lys p J-Nle p-hGly
Gly
pJ-Asp
82
B 11 Ac
APC
Ala
ACPC
Arg
ACPC
Leu
ACPC
Lys
pJ-Nle p-hGly
Gly
pJ-A sp pJ-Ala
83
C 11 Ac
APC
Ala
A CPC
Arg
A CPC
Leu
A CPC
Lys
pJ-Nle p-hGly
Gly
pJ-Asp
84
D 11 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
AC PC
Lys
p3-Nle p-hGly
Gly
pJ-Asp p3-Ala p3-P he
85
E 11 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A C PC
Lys
pJ-Nle p-hGly
Gly
p^-Asp
86
F 11 Ac
A PC
Ala
A CPC
Arg
A CPC
Leu
ACPC
Gly
pJ-A sp pJ-Ala
Gly
pJ-Asp
Gly
pJ-A sp pJ-Ala pJ-P h e pJ-Gln Arg n h 2
A sp
Phe
p^-Phe pJ-Gln Arg n h 2
pJ-Ala pJ-P he pa-Gln Arg n h 2
Phe
A sn
Arg n h 2
Phe
Asn
Arg n h 2
pJ-P he
Asn
Arg n h 2
Ala
Ala
Ala
87
G 11 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys p-’-NIe p-hGly
Lys pJ-Nle p-hGly
88
H 11 Ac
APC
Ala
A CPC
Arg
A CPC
Leu
A CPC
Lys
pJ-Nle p-hGly
89
A 12 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
pJ-Nle p-hGly p-hGly p^-Asp
90
B 12 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A C PC
Lys
p3-Nle p-hGly P-hGly P^-Asp pJ-Ala
91
C 12 Ac
APC
Ala
A C PC
Arg
A CPC
Leu
A CPC
Lys
pL-NIe p-hGly p-hGly pJ-Asp
92
D 12 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
93
E 12 Ac
APC
Ala
A CPC
Arg
A CPC
Leu
ACPC
Lys
Ala
P he
A sn Arg n h 2
pJ-Gln Arg n h 2
Phe
pJ-Gln Arg n h 2
pJ-P h e p J-Gln Arg n h 2
P he
A sn
Arg n h 2
P he
A sn
Arg n h 2
p-'-Phe
Asn
Arg n h 2
Lys pJ-Nle p-hGly p-hGly pJ-A sp pJ-Ala pJ-P he
Asn
Arg n h 2
pJ-Nle p-hGly p-hGly pJ-Asp
Ala
Ala
Ala
P he
p J-Gln Arg n h 2
pJ-Gln Arg n h 2
94
F 12 Ac
APC
Ala
A CPC
Arg
ACPC
Leu
A CPC
Lys
pJ-Nle p-hGly p-hGly pJ-A sp pJ-Ala
95
G 12 Ac
APC
Ala
ACPC
Arg
A CPC
Leu
A CPC
Lys
P'-N Ie p-hGly p-hGly pJ-A sp
96
H 12 Ac
APC
Ala
A CPC
Arg
A C PC
Leu
A CPC
Lys
pJ-Nle p-hGly p-hGly pJ-A sp pJ-Ala pJ-P he p-’-GIn Arg n h 2
Ala
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Phe
pJ-P h e pJ-Gln Arg n h 2
323
Table 51. Characterization data for parallel P-scan library members (JKM V I 241, Figure 6).
A. Calculated M asses
1
A
B
C
D
E
F
G
H
1816
1830
1830
1844
1842
1856
1856
1870
2
3
4
1830
1844
1844
1858
1856
1870
1870
1884
1830
1844
1844
1858
1856
1870
1870
1884
1844
1858
1858
1872
1870
1884
1884
1898
MALDI-TOF O bserved M asses
1
2
3
1817.7
1832.0
183 1 .7
A
1846.3
184 6 .2
B
1831.8
C
1832.3
D
1845.6
1860.1
186 0 .0
E
F
1860.1
1874.1
1817.1
187 4 .5
G
1887.9
H
1874.1
1888.1
HPLC Retention Time (10-60%B over 25
1
2
3
A
17.0
16.0
16.5
B
17.0
16.0
16.0
C
17.0
16.5
D
17.0
16.0
17.0
E
17.0
16.5
16.0
F
16.5
16.0
16.5
16.5
G
17.0
16.5
H
16.0
17.0
17.0
C. HPLC Purity (See Figure 36)
1
2
A
B
C
D
E
F
G
H
_
5
6
7
1759
1773
1773
1787
1785
1799
1799
1813
1773
1787
1787
1801
1799
1813
1813
1827
1773
1787
1787
1801
1799
1813
1813
1827
IT_______ 9________10_______ 11_______ 12
1787
1801
1801
1815
1813
1827
1827
1841
1830
1844
1844
1858
1856
1870
1870
1884
1844
1858
1858
1872
1870
1884
1884
1898
1844
1858
1858
1872
1870
1884
1884
1898
1858
1872
1872
1886
1884
1898
1898
1912
4
5
6
7
8
9
10
11
12
184 6 .0
1860.2
1860.7
1761.3
1775.4
1775.0
1790.0
1775.5
1789.8
1832.0
1847.0
1846.5
1860.2
-
-
-
1789.3
1803.0
1803.7
-
-
-
1788.8
-
1803.1
-
-
-
1860.5
1874.6
1874.5
1888.1
-
-
-
-
-
-
-
1845.7
1860.7
1859.6
1874.2
1874.2
-
1803.2
-
1817.1
1817.5
1830.9
-
-
1888.0
-
-
-
18 74.4
1888.1
-
1889.0
-
-
1902.1
1916.1
-
-
-
190 1 .9
1816.9
1830.9
min)
4
16.0
16.0
16.0
-
16.0
16.0
16.0
16.0
-
-
5
6
7
8
9
10
11
12
18.0
17.0
18.0
17.5
17.0
15.5
17.0
16.5
16.0
16.0
16.0
16.0
-
17.0
16.0
17.0
16.5
16.5
16.5
16.0
16.5
16.5
16.0
16.5
16.5
16.0
16.0
16.5
-
17.5
16.5
18.0
16.5
17.0
16.0
17.5
16.0
15.5
15.5
16.0
16.0
16.0
16.0
16.0
16.0
15.0
16.0
16.0
16.0
16.0
16.0
16.0
16.0
16.0
16.0
16.0
16.0
10
11
12
-
17.0
-
16.0
16.0
16.0
-
Figure 36. Representative
HPLC
traces
(UV
absorbance 220 nm) o f
parallel
P-scan
library
(Figure
6)
members
1500
corresponding to purity
ranking o f “ 1” (top), “2”
(middle), and “3” (bottom)
from Table 51. A ranking
o f “ 1” indicates that the
500
m ajor peak corresponds to
the desired product by
v— MALDI-TOF MS. A “2”
100
126
)
17
6
20
0
22
5
25
0
27 5
30 0
32 5
35 0
0.0
has two major products, one
T im e (m in)
o f which is the desired
product, and a “3” has a three or more major products. “M ” indicates the identification by MALDI-TOF MS o f an
oligomer with a m olecular weight corresponding to a predicted library member.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
324
Table 52. Reformatting o f parallel p-scan library members (Figure 6) after purification. The crude compounds
in the library were numbered according to their position in the plate during synthesis according to the table at the
top. HPLC purification o f each compound in the library resulted in the collection o f one or more fractions for each
sample. Those fractions were transferred to two 96-well deep plates. The compounds from the odd-numbered
columns were placed in Plate A, and the compounds from the even-numbered columns were placed in Plate B.
Using two plates doubled the number o f available wells so that the fractions corresponding to minor products
collected during HPLC purification could be preserved and re-tested in the FP assay along with the major products.
Com pound/Plate Numbering
2
1
A
1
9
B
2
10
11
C
3
D
E
F
4
5
6
G
H
7
8
12
13
14
15
16
te A: Odd-Numbered Colum ns
1
2
A
1
B
2
C
3
D
E
F
G
H
4
5b
6
7
8
5
3
4
5
6
7
8
9
10
11
12
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
3
4
8
17
18
19
20
21
22
23
24
Plate B: Even-Numbered Colum ns
1
2
3
A
9
B
10
C
12
D
E
F
G
H
13b
13
14
15
16
31b
X
5
6
7
3 3b
34
3 5b
3 6b
37
38b
39
40
33
34
35
36
49
50
51b
52b
53
54
55
56
38
4
5
6
25
26
27
4 1b
42b
41
42
43
44
45
46
47
48
X
-
29
30
31
32
X
X
51
52
9
10
11
12
65b
66
67
68
6 9b
70
71
72
65
81b
82b
8 3b
8 4b
8 5b
71b
8 7b
88
81
82
83
84
85
71
87
6 7b
X
69
X
7________ 8_ _______9________10_______ 11_______ 12
61 b
63
64 b
57
58
59
60
61
62
63 b
64
X
X
X
73
74
75
76
89b
90b
X
X
-
7 8b
X
78
79
80
94
89
90
91
92
93
9 4b
95
96
Notes:
in d ic a tes th a t th e m ajo r p e a k in th e H PLC tra c e w a s n o t collected.
"x“ in d ic a tes th a t a s e c o n d p e a k in th e H PLC tra c e w a s c ollected.
"b" in d ic a tes th a t th e fraction co n tain in g th e m a jo r p e a k from th e H PLC tra c e w a s divided b e tw e e n tw o w ells in th e deep-w ell plate.
5000
4000
65__,
A bs
(mV)
3000
2000
Figure 37. HPLC traces (UV absorbance 220 nm) o f hits from
parallel P-scan library. The compound numbers correspond to
those from Table 52 (top). “M ” indicates the identification by
MALDI-TOF MS o f an oligom er with a molecular weight
corresponding to a predicted library member.
1000
12.5
15.0
17.5
Time (min)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
20.0
325
Table 53. Raw assay data for initial screening o f parallel (3-scan library (JKM VI 241, Figure 6).
1:10
Well
mP
Well
mP
Well
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
116.3968
196.1513
186.1838
225.9101
229.0621
229.593
228.5938
216.076
162.6828
214.1119
215.692
209.5269
210.5716
216.3792
220.67
212.6762
214.1174
223.8374
226.56
216.4005
228.9908
233.2378
234.6604
234.3909
214.2865
214.3614
217.3031
213.9584
210.774
214.0321
206.0891
214.0787
105.0433
124.213
124.9119
200.2121
219.7856
228.3332
210.3514
210.8704
122.7156
170.7332
203.8809
207.8498
218.9178
214.8782
195.8459
217.566
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
148.7717
217.7958
220.0034
212.777
227.5344
230.5879
239.0773
211.0229
203.7422
211.9269
207.8279
209.3357
202.0109
207.6696
200.1485
192.384
163.3587
218.8152
215.4357
201.4343
233.7718
211.1043
239.8
227.4008
198.9886
207.1299
182.9876
197.5874
201.5637
186.4421
209.9782
210.2267
219.4129
227.2849
213.3895
213.3212
212.8232
211.1735
221.0736
228.751
202.0642
191.0432
213.0488
199.3905
209.6189
201.1843
216.9176
203.6956
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Dilution
1:100
mP
Well
175.0909
222.4317
220.6454
228.8718
226.6001
223.5968
230.1269
227.9429
210.4351
220.802
216.4727
223.025
214.3611
223.1255
217.5331
219.4138
232.3087
229.8532
229.2645
228.6597
225.8587
238.2385
233.5511
234.0062
213.3718
214.627
216.504
217.0556
210.385
214.5155
216.9625
215.3475
113.8379
179.2413
186.9834
227.5718
226.0977
225.5631
230.8195
228.6776
180.9197
211.6696
220.5711
216.6076
216.4974
217.6174
206.5562
216.8029
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
1:1000
mP
Well
mP
Well
mP
204.3041
229.7964
228.6155
226.93
229.9908
228.1376
227.6654
234.4969
213.0574
215.0773
215.7185
213.72
211.2876
211.097
211.4398
210.4201
216.4359
236.9648
233.1694
230.4473
230.4864
229.2776
240.5404
232.1757
212.277
218.8957
209.4346
216.8851
213.8968
210.688
215.0226
213.5706
227.8833
231.1695
231.7343
225.639
225.1361
225.9331
229.156
223.4705
211.8552
214.3704
216.2873
213.8703
207.429
213.1997
213.8813
209.1929
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
218.2042
223.7954
223.1218
223.6651
225.8857
231.1452
227.6157
226.3835
218.998
225.4825
223.6825
216.9509
219.8323
219.5505
216.5522
214.6367
233.3212
228.0558
223.0877
229.8047
229.122
232.0539
228.3728
235.6746
213.8268
217.0694
211.6538
213.8646
213.6487
211.7897
213.2593
214.1853
163.0975
218.1072
217.5215
229.357
229.8907
231.2857
225.01
225.7747
212.1732
217.1001
217.6363
219.5829
221.0632
211.9556
214.3644
212.0728
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
226.1568
229.2328
230.317
227.055
232.3762
230.5484
230.3013
227.9759
217.0083
212.0165
214.576
216.6824
217.18
210.3533
211.0565
216.4127
232.3035
233.4088
235.4769
235.3533
232.5986
233.808
226.7819
230.7136
213.2754
213.7422
217.4788
214.6102
215.8058
214.9882
210.1064
220.5207
232.081
232.6346
229.3431
232.8128
233.4865
228.4005
230.7644
231.2435
217.3156
212.3073
216.3115
212.5559
215.1044
213.0148
215.0101
210.5449
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
326
T able 54. Raw assay data for screening o f plate A o f purified parallel p-scan library (JKM VI 241, Figure 6).
1:10
Well
mP
Well
mP
Well
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
17.14446
-1.93799
2.60934
2.707139
2.256799
1.859189
3.098617
3.038418
-0.71183
-3.70855
0.990571
1.444268
2.739278
2.869417
2.32773
2.918424
0.150536
-2.56506
1.211089
2.356599
2.182318
2.005117
2.808182
2.83833
-0.16995
-1.48928
1.873494
1.817903
2.494022
2.184022
2.409134
3.644945
77.41611
59.15699
15.46275
1.677442
1.948651
3.515645
3.950412
6.238092
88.68262
47.13312
54.7578
7.377053
2.135553
2.128963
2.641114
4.578466
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
40.61179
0.602894
2.00765
2.539032
1.879282
2.609503
2.327788
4.452801
1.286198
0.32215
2.284853
1.942434
1.779285
1.821825
2.849353
4.06645
5.119966
1.951694
1.991223
1.3348
1.801267
1.75126
2.590619
3.739947
7.222011
0.011639
1.965046
1.396957
0.736848
1.329389
2.15433
3.32609
1.499307
0.79076
1.502817
1.630752
2.676469
1.263159
3.457137
3.859947
1.375969
-0.5855
2.057959
1.295748
1.639387
2.324912
2.949032
3.945653
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Dilution
1:100
mP
Well
2.877129
0.167646
1.649184
0.71062
2.661812
2.667586
2.449999
3.367899
0.249061
0.432216
1.622599
1.5826
1.883003
3.249729
3.466477
4.029575
-0.33266
0.147802
1.021793
1.772202
3.212493
2.563689
2.967167
3.513303
0.139381
0.5158
0.514525
2.314499
3.004281
2.974671
4.134099
4.934485
37.25912
19.61258
3.23101
2.521705
2.855849
3.195134
4.822087
4.305405
76.50257
11.70149
16.80699
3.799799
3.728547
3.756212
4.632831
5.396451
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
1:1000
mP
Well
mP
7.977971
0.260185
1.043696
2.735168
2.664266
3.752173
4.081034
5.56291
1.305647
1.322503
1.310392
2.290755
2.302195
4.564953
3.566165
5.676936
1.009291
0.647941
1.531295
1.784641
4.269357
3.660271
3.996797
4.940775
1.077085
0.914488
3.670721
3.30824
2.537608
4.479294
4.567694
3.582505
0.705088
0.887083
1.772088
2.539998
2.856466
4.533589
4.097984
4.331596
1.419607
1.661211
3.314714
3.0639
3.294483
4.015184
4.13813
5.159422
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
4.53653
4.593475
4.586397
5.480847
6.583587
6.275779
7.256121
7.288777
4.506848
4.404352
5.50514
4.226216
5.727794
7.578229
8.545626
8.051019
3.658995
4.012457
4.921398
4.726571
5.74861
7.393519
8.39331
8.333279
4.305941
4.131961
4.737407
5.604542
6.267194
7.372636
8.993525
9.292087
11.18446
7.086406
5.320604
5.426055
6.191674
7.087418
9.47117
8.460837
37.3513
5.76119
7.664004
6.048185
7.47801
7.178315
8.74998
10.52323
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Well
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
' 88
89
90
91
92
93
94
95
96
mP
4.943417
4.493354
5.642436
5.284742
6.783813
7.262006
9.049362
10.21333
5.397115
4.747771
5.530715
6.395279
7.018308
7.509563
9.207836
10.11731
4.021818
4.662526
5.250116
5.658895
6.046737
7.362966
1.938607
9.184698
4.77887
4.961352
5.851421
6.595442
6.731762
8.007461
8.528938
10.12034
4.82595
5.205876
6.406355
5.70075
7.201204
8.082645
8.133823
8.309964
5.857838
5.353831
6.860857
6.644425
7.087953
8.091233
8.338439
8.845546
Ill
T able 55. Raw assay data for screening o f plate B o f purified parallel p-scan library (JKM VI 241, Figure 6).
1:10
Well
mP
Well
mP
Well
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
0.648584
1.400686
1.468463
3.366835
2.614864
3.662619
1.38784
0.707364
4.156631
1.875417
2.089614
3.428954
2.611182
2.42221
4.151561
3.054818
2.361288
1.929521
2.758116
3.716615
3.705998
3.584127
4.827356
3.423782
2.476844
2.055597
2.377552
2.959566
3.201415
3.999755
4.830871
4.463167
10.41239
4.265844
2.821293
3.638566
3.672185
3.679604
5.29287
4.594398
50.65826
2.796647
10.8858
4.015734
4.074113
4.461714
5.586745
5.60944
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
2.251976
1.795833
3.261459
4.217501
4.007789
5.59538
5.255982
5.7085
4.162568
1.869016
4.535461
3.844261
3.326736
4.606368
5.026358
4.663258
2.227544
2.311028
1.515178
3.711448
3.798266
4.257004
7.541327
4.905413
3.170269
1.2256
4.297267
4.488238
4.157091
4.209531
5.857882
4.738462
1.599175
1.870835
2.369093
4.611666
2.033285
4.378158
4.844967
3.890363
1.942423
2.450393
2.948928
4.788008
3.389218
3.21375
3.80438
2.712083
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Dilution
1:100
mP
Well
4.239951
5.300156
5.982411
6.091518
5.740267
6.26464
7.018883
7.328147
4.630024
4.441716
4.503901
4.94826
4.158761
5.467996
6.739779
6.009735
4.629472
4.54519
5.411912
5.740672
6.607689
6.908915
6.995386
7.517704
3.814433
4.378955
4.745622
5.69305
5.544674
5.978334
7.51703
7.028924
5.073374
4.762955
5.304023
5.018233
6.809635
7.020878
8.077702
8.453496
16.88442
4.818991
5.898241
5.51439
5.72712
5.617216
6.956802
8.572202
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
1:1000
mP
W ell
mP
Well
mP
4.356598
4.84383
5.252325
6.575157
6.187831
6.948035
7.723557
8.280904
4.339154
4.337794
5.063055
5.21552
5.628179
5.985144
7.861682
8.204699
4.804189
4.851131
5.501308
6.331644
6.348569
6.714895
6.114675
8.850357
3.140842
4.950358
5.423646
6.062618
6.268959
7.144465
6.202844
7.460785
4.976205
5.421971
5.936321
5.795328
7.624332
6.359412
7.603031
6.443468
3.992762
4.207433
5.63535
5.685545
7.614153
7.081704
6.625388
6.821086
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
6.026365
7.428905
7.73879
8.593373
9.031633
10.12341
10.51445
10.68182
6.398217
6.655134
7.733467
7.24147
8.545652
10.43346
10.78311
10.28673
6.886119
6.590394
7.532972
8.265397
8.877879
10.91188
11.43833
11.73738
6.549188
6.733831
6.830745
7.6105
9.093054
9.754192
11.99947
11.8458
6.565969
6.41561
8.512294
7.803067
8.933493
10.50171
12.12283
12.06445
7.460266
6.270439
7.667061
7.099205
8.443876
10.22953
11.30688
12.43861
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
6.845066
6.712545
8.301695
8.351107
9.076311
11.19769
11.46632
12.58737
6.356862
6.49092
6.650329
7.681042
9.096186
10.40707
11.08358
11.87119
6.321133
6.713837
8.097876
8.01496
8.406022
10.21293
10.45303
11.32716
6.247244
6.098678
7.924042
6.92495
10.05624
10.45027
9.899386
11.44569
6.517532
6.385047
8.716224
8.345671
9.089111
9.914622
10.9513
11.48537
6.287548
6.756013
8.2325
8.182575
9.626815
9.9843
10.18975
11.00303
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
328
Table 56. Percent inhibition data (calculated from mp data in Table 54 and Table 55) for parallel P-scan library
(Figure 6) screened in both crude and purified forms at three different dilutions (Figure 8 (right) and Figure 9 (left)).
Percent Inhibition = 100 * (l-((m P - blank)/(mP without compound - blank))).
C ru d e
P u rifie d
D ilu tio n
W e ll
1:10 1:100 1 :1 0 0 0
W e ll
D ilu tio n
1:10 1 :1 0 0 1:1 0 0 0
W e ll
D ilu tio n
1:10 1:100 1 :1 0 0 0
W e ll
1:10
D ilu tio n
1 :1 0 0 1:1 0 0 0
1
2
8 4 .9
19.5
36.8
1.5
-3.1
49
5 8 .4
12.9
-2.0
50
1.8
-8 .0
3
2 7 .7
-4 .8
-0.5
-7.3
-2 .6
-3 .0
51
52
0 .0
5.9
-7.1
4
-5 .7
-8 .5
-5 .8
-7 .4
-5 .4
-4 .8
53
-6 .2
-8 .2
-10.1
-7 .9
-2 .9
-9.1
54
-8 .7
-6 .7
-8 .6
-7 .0
-8 .3
-6 .2
55
-15 .6
-6 .3
-8 .4
-6 .5
1
2
17.1
2 .9
4 .5
49
4 0 .6
8 .0
4 .9
-1 .9
0 .2
4 .6
50
0.6
0.3
4 .5
3
4
2 .6
2 .7
1.6
0 .7
4 .6
5 .5
51
52
2.3
1.9
1.3
2.3
5.5
6 .4
5
2.7
1.9
5 .7
53
1.9
2.7
6.8
5
6
1.9
2 .7
6 .3
54
2 .6
3 .8
7 .3
6
7
3.1
2 .4
7 .3
55
2 .3
4.1
9 .0
7
-5 .0
-7 .6
8
3.0
3 .4
7 .3
56
4 .5
5.6
10.2
8
3 .2
-6 .5
-5 .2
56
7.4
-1 1 .9
9
4 .2
4 .6
6 .4
57
4 .2
4 .3
6 .4
9
4 7 .0
7.8
0 .8
57
13.3
5 .7
2.5
10
1.9
4 .4
6 .7
58
1.9
4 .3
6 .5
10
4 .8
-0.7
-4 .5
58
6.6
4 .0
6 .5
59
4 .5
5.1
6.7
11
3 .5
2.9
-3 .0
59
10.0
3 .5
4 .4
12
3 .4
4 .9
7 .2
60
3.8
5.2
7.7
12
8 .6
-2 .5
2 .5
60
8.7
5.1
2 .7
13
2 .6
4 .2
61
3.3
5.6
7 .7
4 .6
0.1
61
14.7
7.1
2 .4
5.5
62
4 .6
6.0
9.1
10.4
13
14
8 .5
10.4
14
3 .0
-2 .6
0 .4
62
10.1
7 .3
2 .3
7.9
15
16
17
4 .2
6.7
10.8
7 .0
10.3
3 .7
2 .8
4 .4
16.3
6 .0
-0 .3
63
64
63
3.1
0.2
2 2 .6
4 6 .4
7 .9
2 .9
-10.1
18
-2 .6
0.1
4 .0
65
66
64
65
66
1.0
-1 3 .9
-1 1 .0
19
1.2
1.0
4 .9
20
2 .4
21
2 .2
1.8
3.2
4 .7
5.7
22
23
2 .0
2.8
2.6
3.0
7 .4
24
2.8
3 .5
25
26
2 .5
27
11
5 .3
7 .7
11.5
15
-0 .5
2.0
4 .7
7 .2
8.2
1.1
11.9
4 .8
16
17
6 .0
4 .8
0.5
-10.1
2.0
0 .6
4 .7
18
-8.1
-1 0 .9
-6 .6
67
2.0
1.5
5 .3
19
-3.1
-5 .4
-7 .6
-2 .5
67
3.7
-1 0 .8
-1 2 .7
68
1.8
20
21
3 .0
-7 .4
-7.1
-4 .8
68
15.2
-7 .5
69
-1 1 .3
-8 .6
-8 .6
-1 2 .6
2 .5
5.7
6.7
-8 .0
69
1.3
0 .7
1.8
2 .6
3 .7
4 .0
7 .4
-1 0 .9
-1 2 .0
-11.1
-9 .9
-6 .9
70
71
7 .3
-1 6 .2
-7 .6
1.9
22
23
-1 4 .9
8 .4
70
71
-1 6 .8
-1 1 .3
-5 .6
8 .3
72
3 .7
4 .9
9 .2
24
-1 1 .8
-11 .5
-1 2 .8
72
-6.1
-1 0 .0
-8 .8
3.8
6 .5
73
3.2
3.1
6 .2
25
4 .7
5.4
5.1
73
17.2
6 .3
5 .5
2.1
4 .4
6 .7
74
1.2
5.0
6.1
26
4 .6
4.4
2 .4
74
10.5
0 .9
5.1
2 .4
4 .7
6 .8
75
4 .3
5 .4
7 .9
27
2 .2
6 .8
75
3 0 .3
8 .7
2.1
76
4 .5
6.1
6 .9
28
5 .0
2.9
2 .4
5.0
76
18.4
2 .6
4 .4
7 .6
4 .9
7.9
4.5
5.2
6 .7
77
10.5
29
30
15.1
2 7 .5
5 .0
7 .6
3 .4
4.1
28
29
3.2
30
31
4 .0
4 .8
32
4 .5
33
8 8 .7
34
47.1
35
5 4 .8
7 .4
16.8
36
37
5.5
6.0
9.1
9 .8
7.5
7.0
77
4 .2
12.0
78
79
5.9
6.2
9 .9
31
11.4
2.5
5 .5
78
79
11.8
80
4 .7
7 .5
11.4
32
4 .9
3.8
4 .8
80
7 6 .5
3 7 .4
81
1.4
1.4
5.9
33
9 4 .2
8 7 .0
4 6 .6
11.7
5.8
7 .7
82
-0.6
1.7
5 .4
34
2.1
3 .3
35
1.3
1.6
3.1
3 .3
6 .9
6 .6
33.4
27.1
1.6
83
84
7 8 .5
7 7 .9
36
37
16.2
-6.2
2.0
-7 .7
83
84
0.2
38
-6 .8
7.9
-5.0
-4 .6
1.9
3.8
2 .9
38
2.1
3 .8
39
40
4 .0
6 .2
4 .8
4 .3
41
5 0 .7
42
2 .8
43
10.9
44
4 .0
6 .0
6 .2
7 .2
85
7.1
7.1
9 .5
86
87
2 .9
8 .5
88
3.9
16.9
7 .5
6 .3
89
90
1.9
4 .8
2 .5
5 .9
7 .7
91
2.9
5 .6
8.2
5 .5
7.1
92
4 .8
5.7
8 .2
4.1
4 .3
8.3
7 .3
2.9
-1 0 .3
8.2
4.1
8.1
8.0
5 .3
-0 .4
81
0.5
-6 .5
-9 .9
82
-6 .0
5.4
-9 .2
-1 0 .4
5 .5
-9 .6
-4 .6
5 .9
7 .2
-4 .2
-4 .9
-8.1
85
86
87
-0 .9
-7 .5
-7 .2
-2 .8
-7 .7
-1 0 .5
-11.1
-6 .9
-8 .9
-9 .3
-4.1
7 .5
-7.1
-4 .7
88
41
7 9 .7
3 2 .0
6 .4
89
14.7
6 .7
2 .2
42
4 0 .4
6.8
2 .4
90
2 3 .7
4 .6
6 .3
43
13.2
-0 .5
1.9
91
5.7
3.0
44
10.0
2.8
0 .3
92
16.9
5 .0
3 .0
6.1
4 .0
8.3
39
40
4 .0
6 .3
4 .2
6 .8
-8 .8
-9 .2
45
4.1
5 .7
8 .4
93
3.4
7 .6
9.6
45
0 .9
2.9
-0 .9
93
8.5
10.3
46
4 .5
5.6
10.2
94
4 .4
6.4
9.9
46
4 .2
2.0
6 .6
94
15.4
5 .6
5.7
47
5.6
5 .6
7 .0
8 .6
11.3
12.4
95
3 .8
2.7
6 .6
6 .8
10.2
11.0
47
48
19.8
2 .0
11.0
2.6
4 .6
6 .5
95
96
2 .5
13.4
5 .0
8 .9
4.1
7 .8
48
96
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
329
5.4.5 Synthesis of the Second Generation Parallel Library Using Microwave
Irradiation (Figure 11)
The second generation parallel library (JKM VI 265, Figure 11) was synthesized using
the same procedure as described for the parallel p-scan library (JKM VI 241) with an important
modification.
Coupling reactions were performed at 70°C, and Fmoc-deprotection reactions
were performed at 80°C.
The reduction in reaction temperature significantly improved the
average purity of the oligomers. The oligomers were synthesized in parallel with the sequential
coupling (one amino acid residue after another in different sections o f the plate (Figure 11), as
described for the synthesis o f the combinatorial p-scan library) o f six residues at position 13
(Fmoc-(S)-Phe-OH, Fmoc-(iS)-p3-Phe-OH, Fmoc-(5)-p2-Phe-OH, Fmoc-(S)-p3-xPhe-OH, Fmoc(5)-p2-xPhe-OH, and Fmoc-(i?)-p2-xPhe-OH), eight residues at position 12 (Fmoc-(5)-Ala-OH,
Fmoc-P-Gly-OH, Fmoc-(5)-Pro-OH, F m o c-^ ^ -A C H C -O H , Fmoc-OS^-ACPC-OH, Fmoc(f?,f?)-ACPC-OH, Fmoc-f/?)-Ala-011, and null), and two residues at position 11 (Fmoc-(5)Asp(fBu)-OH and Fmoc-(5)-p3Asp(tBu)-OH.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
330
Position 15:
Position 14:
Position 13:
A
B
C
D
E
F
G
H
P-xPhe
Position 12:
A
B
C
D
E
F
G
H
ACPC
Position 11:
1
Position
Position
Position
Position
Position
Position
Position
Position
Position
Position
2
3
4
5
6
7
8
9
10
11
10: Gly
9: p3-Nie
8: Lys
7: ACPC
6: Leu
5: ACPC
4: Arg
3: ACPC
2: Ala
1: APC
F ig u re 38. Spatial addressing for parallel synthesis o f follow-up parallel library (JKM V I 265, Figure 11).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
12
331
T able 57. Sequences o f follow-up parallel library members 1-48 (JKM VI 265, Figure 11).
Sequence
9
10
14
15
C
11
12
13
1
Well
A 1
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P J-N le G ly
A sp
A la
Phe
A sn A rg N H 2
2
B
1
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P^-NIe Gly
A sp
p-G ly
Phe
A sn A rg N H 2
3
C
1
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P^-NIe Gly
A sp
P ro
Phe
A sn A rg N H 2
4
D
1
A c A P C A la A C P C A rg A C P C L eu A C P C Lys p J-N le Gly
A sp
ACHC
Phe
A sn A rg N H 2
5
E
1
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys PJ-N le Gly
A sp
A CPC
Phe
A sn A rg N H 2
6
F
1
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys p J-N le Gly
A sp
(R .R )-A C P C
Phe
A sn A rg N H 2
7
G
1
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P^-NIe Gly
A sp
(D )-A la
Phe
A sn A rg N H 2
8
H
1
A c A P C A la A C P C A rg A C P C L eu A C P C Lys p J-N le . Gly
A sp
9
A
2
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P ^ N Ie G ly P^-Asp
C om pound
N
1
2
3
4
5
6
7
8
Phe
A sn A rg N H 2
Ala
Phe
A sn A rg N H 2
A sn A rg N H 2
10
B
2
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys PJ-N le G ly P '-A s p
P-Gly
Phe
11
C
2
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P ’-N Ie G ly p J-A sp
P ro
Phe
A sn A rg N H 2
12
D
2
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys PJ-N le G ly P J-A sp
ACHC
Phe
A sn A rg N H 2
ACPC
13
E
2
A c A P C A la A C P C A rg A C P C L eu A C P C L ys P ’-N Ie G ly P ^ A s p
Phe
A sn A rg N H 2
14
F
2
A c A P C A la A C P C A rg A C P C L eu A C P C L ys p J-N le G ly p J-A sp (R .R )-A C P C
Phe
A sn A rg NH2
15
G
2
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P'!-N le G ly P ’-A sp
Phe
A sn A rg N H 2
16
H
2
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys p 4-N le G ly PJ-A sp
Phe
A sn A rg N H 2
17
A
3
A c A P C A la A C P C A rg A C P C L eu A C P C Lys P'!-N le Gly
A sp
A la
p J- P h e
A sn A rg N H 2
18
B
3
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys PJ-N le G ly
A sp
p-G ly
p J- P h e
A sn A rg N H 2
19
C
3
A c A P C A la A C P C A rg A C P C L eu A C P C Lys PJ-N le Gly
A sp
P ro
Pj - P h e
A sn A rg N H 2
20
D
3
A c A P C A la A C P C A rg A C P C L eu A C P C
A sp
ACHC
Pj-P h e
A s n A rg N H 2
21
E
3
A c A P C A la A C P C A rg A C P C L eu A C P C Lys p J-N le Gly
A sp
ACPC
p J- P h e
A sn A rg N H 2
22
F
3
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P^-NIe Gly
A sp
(R .R )-A C P C
p ^ -P h e
A sn A rg NH2
23
G
3
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P ’-N Ie Gly
A sp
(D )-A la
p J- P h e
A sn A rg NH2
24
H
3
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P 3-N le Gly
A sp
25
A
4
A c A P C A la A C P C A rg A C P C L eu A C P C Lys p J-N le G ly p J-A sp
L ys P ’-N Ie Gly
(D )-A la
P ^ -P h e
A sn A rg NH2
Ala
p ^ -P h e
A sn A rg NH2
26
B
4
A c A P C A la A C P C A rg A C P C L eu A C P C
P-G ly
p J- P h e
A sn A rg N H 2
27
C
4
A c A P C A la A C P C A rg A C P C L eu A C P C Lys p J-N le G ly p J-A sp
P ro
P ^ -P h e
A sn A rg N H 2
Lys p J-N le Gly P J-A sp
28
D
4
A c A P C A la A C P C A rg A C P C L eu A C P C Lys P^-NIe G |y p J-A sp
ACHC
p J- P h e
A sn A rg N H 2
29
E
4
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P '-N Ie G ly [lJ-A sp
ACPC
p J- P h e
A s n A rg N H 2
30
F
4
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P ’-N Ie G ly p J-A sp (R .R )-A C P C
p J- P h e
A s n A rg NH2
31
G
4
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P J-N le G ly p J-A sp
p 3-P h e-
A sn A rg NH2
32
H
4
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P J-N le G ly P J-A sp
p ^ -P h e
A sn A rg N H 2
33
A
5
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P'*-Nle G ly
A sp
A la
P ^ -P h e
A sn A rg N H 2
34
B
5
A c A P C A la A C P C A rg A C P C L eu A C P C Lys P^-NIe Gly
A sp
p-G ly
P ^ -P h e
A sn A rg N H 2
35
C
5
A c A P C A la A C P C A rg A C P C L eu A C P C Lys P^-NIe Gly
A sp
P ro
p ^ -P h e
A sn A rg N H 2
36
D
5
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys p J-N le Gly
A sp
ACHC
p 2- P h e
A sn A rg N H 2
37
E
5
A c A P C A la A C P C A rg A C P C L eu A C P C Lys p J-N le Gly
A sp
A CPC
p ^ -P h e
A sn A rg NH2
(R .R )-A C P C
P ^ -P h e
A sn A rg N H 2
(D )-A la
p ^ -P h e
A s n A rg N H 2
p ^ -P h e
A sn A rg N H 2
38
F
5
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys p J-N le G ly
A sp
(D )-A la
39
G
5
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys p d-N le G ly
A sp
40
H
5
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys p J-N le G ly
A sp
41
A
6
A c A P C A la A C P C A rg A C P C L e u A C P C Lys p J-N le G ly PJ-A sp
A la
p 2- P h e
A sn A rg N H 2
42
B
6
A c A P C A la A C P C A rg A C P C L eu A C P C L ys P '-N Ie G ly p J-A sp
p-G ly
pV phe
A sn A rg N H 2
43
C
6
A c A P C A la A C P C A rg A C P C L eu A C P C Lys p J-N le G ly (iJ-A sp
P ro
p ^ -P h e
A sn A rg N H 2
44
D
6
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P'^N Ie G ly p J-A sp
ACHC
p ^ -P h e
A sn A rg NH2
45
E
6
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P J-N le G ly p J-A sp
A CPC
P ^ -P h e
A sn A rg N H 2
46
F
6
A c A P C A la A C P C A rg A C P C L eu A C P C Lys p d-N le Gly [lJ-A sp (R .R )-A C P C
p ^ -P h e
A sn A rg N H 2
47
G
6
A c A P C A la A C P C A rg A C P C L eu A C P C Lys p J-N le G ly p J-A sp
p ^ -P h e
A sn A rg N H 2
48
H
6
A c A P C A la A C P C A rg A C P C L eu A C P C L ys P^-NIe G ly [)3-A sp
p ^ -P h e
A sn A rg N H 2
(D )-A la
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
332
T able 58. Sequences o f follow-up parallel library members 49-96 (JKM VI 265, Figure 11).
Sequence
2
3
4
5
6
7
8
9
10
Well N 1
C om pound
49
A 7 A c A P C A la A C P C A rg A C P C L eu A C P C Lys P^-NIe Gly
50
51
52
53
54
B
C
D
E
F
7
7
7
7
7
A c A P C A la A C P C A rg A C P C L eu A C P C Lys P^-NIe Gly
A c A P C A la A C P C A rg A C P C L eu A C P C
A c A P C A la A C P C A rg A C P C L eu A C P C
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P '-N Ie Gly
Lys p J-N le Gly
Lys P ’-N Ie Gly
A c A P C A la A C P C A rg A C P C L eu A C P C Lys p J-N le Gly
11
12
13
A sp
Ala
p J-x P h e
A sn A rg N H 2
A sp
p-G ly
P ^ -x P h e
A sn A rg N H 2
P ro
p J-x P h e
A sn A rg NH2
ACHC
P ^ -x P h e
A sn A rg NH2
A CPC
p J-x P h e
A sn A rg N H 2
(R .R )-A C P C
p 3-x P h e
A sn A rg N H 2
(D )-A la
p J-x P h e
A sn A rg N H 2
A sp
A sp
A sp
A sp
14
15
C
G
7
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys PJ-N le Gly
A sp
56
H
7
A c A P C A la A C P C A rg A C P C L eu A C P C Lys PJ-N le G ly
A sp
|f - x P h e
A sn A rg N H 2
57
A
8
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P^-NIe G ly P'5-A sp
Ala
P ^ -x P h e
A s n A rg N H 2
58
B
8
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P 3-N le G ly p J-A sp
p-G ly
|F - x P h e
A sn A rg N H 2
59
C
8
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P J-N le G ly p J-A sp
P ro
P ^-x P h e
A sn A rg N H 2
60
D
8
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P J-N le G ly P ’-A sp
ACHC
p J-x P h e
A sn A rg N H 2
61
E
8
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P^-NIe G ly PJ-A sp
A CPC
p J- x P h e
A sn A rg N H 2
62
F
8
A c A P C A la A C P C A rg A C P C L eu A C P C
L ys P^-Nle G ly p J-A sp (R .R )-A C P C
p J- x P h e
A sn A rg N H 2
63
G
8
A c A P C A la A C P C A rg A C P C L eu A C P C
L ys P J-N le G ly p J-A sp
p J- x P h e
A sn A rg N H 2
64
H
8
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys p J-N le G ly P'’-A sp
p J-x P h e
A sn A rg N H 2
65
A
9
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P ’-N Ie Gly
A sp
Ala
p ^ -x P h e
A sn A rg N H 2
66
B
9
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P^-NIe G ly
A sp
P -G ly
(F -x P h e
A s n A rg NH2
67
C
9
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P J-N le Gly
A sp
P ro
P ^ -x P h e
A sn A rg NH2
68
D
9
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P ’-N Ie Gly
A sp
ACHC
P ^-x P h e
A s n A rg NH2
69
E
9
A c A P C Ala A C P C A rg A C P C L eu A C P C Lys p J-N le Gly
A sp
A CPC
p ^ -x P h e
A s n A rg N H 2
70
F
9
A c A P C A la A C P C A rg A C P C L eu A C P C Lys PJ-N le Gly
A sp
(R .R )-A C P C
P ^ -x P h e
A sn A rg N H 2
71
G
9
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P '-N Ie Gly
A sp
(D )-A la
p z-x P h e
A sn A rg N H 2
A sp
55
(D )-A la
72
H
9
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys p J-N le Gly
P ^ -x P h e
A sn A rg N H 2
73
A
10
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys p J-N le Gly p J-A sp
Ala
p ^ -x P h e
A sn A rg N H 2
Lys P J-N le G ly PJ-A sp
74
B
10
A c A P C A la A C P C A rg A C P C L eu A C P C
p-G ly
p z-x P h e
A sn A rg NH2
75
C
10
A c A P C A la A C P C A rg A C P C L eu A C P C Lys p J-N le G ly PJ-A sp
P ro
p a-x P h e
A s n A rg NH2
76
D
10
A c A P C A la A C P C A rg A C P C L eu A C P C L ys P^-NIe G ly PJ-A sp
ACHC
p z-x P h e
A sn A rg N H 2
77
E
10
A c A P C A la A C P C A rg A C P C L eu A C P C Lys P '-N Ie G ly p J-A sp
ACPC
p z-x P h e
A sn A rg N H 2
78
F
10
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys p J-N le G ly PJ-A sp (R .R )-A C P C
p z-x P h e
A sn A rg N H 2
79
G
10
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P^-NIe G ly p J-A sp
p z-x P h e
A sn A rg N H 2
80
H
10
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys p J-N le G ly p J-A sp
p z-x P h e
A sn A rg N H 2
81
A
11
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys p J-N le Gly
A sp
Ala
(D )-p z-x P h e A sn A rg N H 2
82
B
11
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys PJ-N le Gly
A sp
p-G ly
(D )-pz- x P h e A s n A rg N H 2
83
C
11
A c A P C A la A C P C A rg A C P C L eu A C P C Lys PJ-N le G ly
A sp
P ro
(D )-pz- x P h e A s n A rg N H 2
84
D
11
A c A P C A la A C P C A rg A C P C L eu A C P C Lys P^-NIe Gly
A sp
ACHC
(D )-pz- x P h e A s n A rg N H 2
85
E
11
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P J-N le Gly
A sp
ACPC
(D J-p ^-x P h e A sn A rg N H 2
86
F
11
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys p J-N le G ly
A sp
(R ,R )-A C P C
(D )-pz-x P h e A sn A rg N H 2
87
G
11
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys p J-N le G ly
A sp
(D )-A la
(D )-pz-x P h e A sn A rg N H 2
88
H
11
A c A P C A la A C P C A rg A C P C L eu A C P C Lys P J-N le Gly
A sp
89
A
12
A c A P C A la A C P C A rg A C P C L eu A C P C
90
B
12
A c A P C A la A C P C A rg A C P C L eu A C P C
(D)-Ala
(D )-p z- x P h e A s n A rg N H 2
(D )-p z-x P h e A sn A rg N H 2
Lys P J-N le G ly p J-A sp
Ala
L ys p J-N le G ly p J-A sp
p-G ly
(D )-p z-x P h e A sn A rg N H 2
91
C
12
A c A P C A la A C P C A rg A C P C L eu A C P C L ys P ’-N Ie G ly P^-A sp
P ro
(D )-p z- x P h e A s n A rg N H 2
92
D
12
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P^-NIe G ly P ’-A sp
ACHC
(D )-p z-x P h e A s n A rg N H 2
93
E
12
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys p J-N le G ly P '-A s p
A CPC
(D )-p z-x p h e A sn A rg N H 2
94
F
12
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P '-N Ie G ly p J-A sp (R .R )-A C P C
95
G
12
A c A P C A la A C P C A rg A C P C L eu A C P C
Lys P'5-N le G ly p J-A sp
96
H
12
A c A P C A la A C P C A rg A C P C L eu A C P C Lys P^-NIe G ly PJ-A sp
(D )-A la
(D )-pz- x P h e A sn A rg N H 2
(D )-p - x P h e A s n A rg N H 2
(D )-pz-x P h e A sn A rg N H 2
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
333
Table 59. Characterization data for follow-up parallel library (JKM VII 265, Figure 11). All 96-library members
were analyzed by HPLC, and fractions were collected for 41 samples and characterized by MALDI-TOF MS.
A. Calculated Masses
1
2
A
1759.9 1773.9
1759.9 1773.9
B
1785.9 1799.9
C
D
1813.9 1828.0
E
1799.9 1813.9
1799.9 1813.9
F
1759.9 1773.9
G
H
1688.9 1702.9
3
4
5
6
7
8
9
10
11
12
1773.9
1773.9
1799.9
1828.0
1813.9
1813.9
1773.9
1702.9
1787.9
1787.9
1813.9
1842.0
1828.0
1828.0
1787.9
1716.9
1773.9
1773.9
1799.9
1828.0
1813.9
1813.9
1773.9
1702.9
1787.9
1787.9
1813.9
1842.0
1828.0
1828.0
1787.9
1716.9
1787.9
1787.9
1813.9
1842.0
1828.0
1828.0
1787.9
1716.9
1801.9
1801.9
1828.0
1856.0
1842.0
1842.0
1801.9
1730.9
1787.9
1787.9
1813.9
1842.0
1828.0
1828.0
1787.9
1716.9
1801.9
1801.9
1828.0
1856.0
1842.0
1842.0
1801.9
1730.9
1787.9
1787.9
1813.9
1842.0
1828.0
1828.0
1787.9
1716.9
1801.9
1801.9
1828.0
1856.0
1842.0
1842.0
1801.9
1730.9
7
8
9
10
11
12
MALDI-TOF Observed Masses
1
2
3
4
A
1761.0 1774.9 1775.0 1789.4
B
1761.9 1776.0 1775.7 1789.4
1803.2 1812.0
1789.0
C
1815.3 1829.4 1829.3 1843.2
D
E
F
G
H
5
6
1775.5
1774.9
1803.2
1828.2
1789.3
1786.7
1815.2
1843.3
HPLC Retention Time (10-60%B over
1
2
3
4
18.0
16.5
A
18.0
17.0
16.0
16.0
15.5
16.0
B
17.0
17.0
17.0
17.0
C
17.0
18.5
17.0
19.0
D
16.5
15.0
18.5
15.0
E
16.0
17.0
16.0
F
17.0
16.0
16.5
16.0
16.0
G
15.5
16.0
16.0
16.0
H
25 min)
5
6
16.5
16.0
16.0
18.0
16.0
17.5
16.5
16.0
16.5
15.5
16.0
16.5
16.5
16.0
15.5
15.5
1789.0 1802.9 1788.8 1803.2 1788.6 1804.9
1786.7 1800.6 1784.7 1798.8 1787.6 1803.0
1815.1 1829.1 1815.2 1829.1
1815.1
1843.4
7
8
9
10
11
12
18.0
17.0
17.0
19.5
19.0
17.0
16.5
17.0
18.0
16.0
17.0
18.0
18.0
16.0
16.5
16.5
17.0
16.5
16.5
19.0
18.0
18.0
17.0
17.0
17.0
16.0
16.5
17.0
17.0
16.5
16.0
16.5
16.5
16.0
16.0
18.0
17.0
17.0
16.5
16.5
16.0
16.0
16.0
17.0
15.5
16.0
16.0
16.0
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334
Table 60. Percent inhibition data (calculated from mp data by Jack Sadowsky) from initial screen o f follow-up
parallel library (JKM VII 265, Figure 11).
D ilu tio n
W ell
1 :1 0
% In h ib itio n
W elt
% I n h ib itio n
W ell
1 :1 0 0
% I n h ib itio n
W ell
% I n h ib itio n
W ell
1 :1 0 0 0
% I n h ib itio n
W e ll
% In h ib itio n
1
107
49
48
1
100
49
14
.1
80
49
12
2
98
50
24
2
66
50
9
2
29
50
12
3
20
51
13
3
9
51
9
3
13
51
14
4
81
52
24
4
33
52
8
4
17
52
14
5
103
53
27
5
67
53
9
5
29
53
14
6
24
54
16
6
10
54
10
6
13
54
14
7
101
55
16
7
66
55
11
7
30
55
15
8
23
56
18
8
10
56
11
8
12
56
17
9
88
57
15
9
41
57
6
9
17
57
11
10
23
58
13
10
8
58
6
10
11
58
10
11
20
59
12
11
9
59
8
11
13
59
12
12
15
60
16
12
8
60
8
12
14
60
13
13
28
61
16
13
10
61
7
13
14
61
14
14
15
62
15
14
9
62
10
14
14
62
14
15
45
63
17
15
16
63
10
15
15
63
14
16
15
64
16
16
10
64
9
16
14
64
15
17
102
65
22
17
68
65
7
17
28
65
11
18
45
66
15
18
12
66
7
18
12
66
10
19
12
67
12
19
8
67
8
19
13
67
12
20
25
68
20
20
10
68
7
20
14
68
13
21
54
69
19
21
17
69
8
21
16
69
13
22
17
70
18
22
10
70
9
22
14
70
14
23
38
71
16
23
14
71
8
23
15
71
14
24
16
72
20
24
9
72
11
24
14
72
14
25
29
73
13
25
9
73
6
25
12
73
■ 11
26
12
74
12
26
8
74
6
26
12
74
11
27
12
75
12
27
8
75
8
27
13
75
13
28
15
76
15
28
8
76
6
28
13
76
13
29
17
77
16
29
9
77
10
29
15
77
14
30
15
78
15
30
10
78
10
30
15
78
14
31
16
79
15
31
10
79
10
31
15
79
14
32
15
80
16
32
10
80
9
32
14
80
14
33
18
81
29
33
7
81
9
33
12
81
11
34
17
82
15
34
8
82
7
34
12
82
11
35
11
83
12
35
8
83
8
35
13
83
13
36
21
84
18
36
9
84
9
36
14
84
13
37
19
85
18
37
8
85
10
37
13
85
14
38
18
86
18
38
10
86
10
38
14
86
14
39
16
87
16
39
10
87
11
39
15
87
14
40
17
88
17
40
11
88
10
40
15
88
15
41
11
89
11
41
6
89
7
41
11
89
11
42
12
90
12
42
7
90
6
42
11
90
12
43
12
91
12
43
9
91
8
43
14
91
11
44
17
92
14
44
8
92
8
44
14
92
12
45
37
93
15
45
12
93
9
45
14
93
14
46
15
94
14
46
10
94
9
46
14
94
14
47
14
95
13
47
9
95
9
47
14
15
96
15
48
11
96
11
48
17
95
96
13
48
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
14
335
.2
60
17964298459443
W eil
F ig u re 39. Percent inhibition o f crude library members (JKM VII 265, Figure 11) at 1:10 dilution.
*
50
2353482353233123235353235348234823532353235323532348234823
N
*
^
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p
'
<
8
>
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&
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Figure 40. Percent inhibition o f crude library members (JKM VII 265, Figure 11) at 1:100 dilution.
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Figure 41. Percent inhibition o f crude library members (JKM VII 265, Figure 11) at 1:1000 dilution.
5.4.6 Solid-Phase Oligomer Synthesis Using Microwave Irradiation
Library hits and follow-up compounds (JKM VI 249) and oligomers from our structurebased design efforts were synthesized using microwave irradiation in a multimode reactor.
NovaSyn TGR resin (10 pmol, 40 mg) was placed in a polypropylene SPE tube (4 mL, Alltech)
and swelled with DMF for ~ 10 min. The resin was washed (5 x DMF, 5 x CH 2 CI2 and 5 x
DMF). In a separate vial, Fmoc-amino acid (30 pmol) was activated by adding HBTU (60 pL o f
0.5 M solution in DMF), DMF (440 pL), HOBt (60 pL of 0.5 M solution in DMF), and /Pr2EtN
(60 pL of 1.0 M solution in DMF).
The mixture was vortexed and added to the resin.
A
magnetic stir bar (8 mm, VWR) was placed inside the tube. The vessel was placed inside an
empty polypropylene 50 mL centrifuge tube and placed in one slot o f a 52-position turntable
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337
inside the multimode microwave reactor (CEM MARS). The fiber optic temperature sensor
was suspended in the reaction mixture above the stir bar by pressing it through a small hole
(made with a needle) in the plastic top cap of the SPE tube and placing the cap loosely on the
reaction vessel. The sample was irradiated in the microwave reactor (600 W maximum power,
70°C, ramp 2 min, hold 4 min).
pressure.
All microwave irradiations were conducted at atmospheric
The tube was removed from the microwave reactor, and the resin was washed as
before. Deprotection solution (750 pL o f 20% piperidine in DMF (v/v)) was added to the resin,
and the sample was irradiated (600 W maximum power, 80°C, ramp 2 min, hold 2 min). The
coupling/deprotection cycle was repeated in a stepwise manner until reaching the desired
oligomer length.
After the final residue had been added and deprotected, the resin was washed (5 x DMF,
5 x CH 2 CI2 , 5 x DMF and 5 x CFI2 CI2 ). An acetylation cocktail was prepared containing 1.4 mL
CH 2 CI2 , 0.1 mL TEA, and 0.5 mL acetic anhydride. Acetylation was accomplished by adding
1.0 mL o f the cocktail to the resin and shaking for 15 min. After washing the resin a (5 x
CH 2 CI2 ), the peptide was cleaved from the solid support with simultaneous side chain
deprotection (3 mL, 45:45:5:5 trifluoroacetic acid (TFA):CH 2 Cl2 :triethylsilane:water, 2 h, RT,
with rocking). The cleavage solution was drained and concentrated under a stream o f N 2 . The
crude oligomer was dissolved in 1.0 mL DMSO and purified by HPLC (200 pL injection,
Shimadzu). The C 4 -silica reversed-phase semi-preparative column (5 pm, 10 mm x 250 mm,
Vydac) was eluted with a gradient o f acetonitrile in water (10 - 60%, 50 min, 0.1% TFA in each)
at a flow rate o f 3 mL/min. The major peak in the HPLC chromatogram was collected, and
oligomer masses were measured by MALDI-TOF-MS
(Bruker Reflex II, a-cyano-4-
hydroxycinnamic acid matrix).
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338
Table 61. Oligomer characterization data.
[M + H p
N o teb oo k
S eq uen ce
JKM VI 249 1
1638.9 Ac APC Ala ACPC
-
ACPC
ACPC Lys pS3-Phe Gly Asp
Ala
Phe Asn
Arg
nh2
JKM VI 249 2
1652.9 Ac APC Ala ACPC
-
ACPC xLeu ACPC Lys p3-Phe Gly Asp
Ala
Phe Asn
Arg
nh2
JKM VI 249 3
1672.9 Ac APC Ala ACPC
-
ACPC
Ala
Phe Asn
Arg
nh2
Leu
Phe ACPC Lys p3-Phe Gly Asp
JKM VI 249 4
1711.9 Ac APC Ala ACPC
-
Ala
Phe Asn
Arg
JKM VI 249 5
1669.0 Ac APC Ala P3-Leu
-
ACPC Trp ACPC Lys p3-Phe Gly Asp
ACPC xLeu A C P C Lys [J3-Phe Gly Asp
nh2
Ala
Phe Asn
Arg
nh2
JKM VI 249
1677.9 Ac APC Ala ACPC
-
ACPC
ACPC Lys p3-Nle Gly Asp
Ala
Phe Asn
Arg
nh2
6
Trp
JKM VI 295 1
1761
Ac APC Ala ACPC Arg ACPC
Leu
ACPC Lys p3-Nle Gly Asp
3-Gly
Phe Asn
Arg
nh2
JKM VI 295 2
1775.1
Ac APC Ala ACPC Arg ACPC
Leu
ACPC Lys B3-Nle Gly Asp
Aib
Phe Asn
Arg
nh2
JKM VI 295 3
1815.1
Ac APC Ala ACPC Arg ACPC
Leu
A C P C Lys |?3-N|e Gly Asp
A CHC
Phe Asn
Arg
nh2
JKM VI 2 95 4
1801.1
Ac APC Ala ACPC Arg ACPC
Leu
ACPC Lys 33-Nle Gly Asp
ACPC
Phe Asn
Arg
nh2
JKM VI 295 5
1761.1
Ac APC Ala ACPC Arg ACPC
Leu
ACPC Lys 33-Nle Gly Asp (D )-A la Phe Asn
Arg
nh2
JKM VI 295
1773.2
Ac APC Ala ACPC Arg ACPC
Leu
ACPC Lys p3-Nle Gly Asp
AC3 C
Phe Asn
Arg
nh2
1775.2 Ac APC Ala ACPC Arg ACPC
Leu
ACPC Lys p3-Nle Gly Asp
p-Gly
ACPC Lys p3-Nle Gly Asp
6
8
1789.3 Ac APC Ala ACPC Arg ACPC
Leu
Aib
Phe Asn p3-Arg n h 2
Phe Asn B3-Arg n h 2
JKM VI 295 9
1829.4 Ac APC Ala A C P C Arg ACPC
Leu ACPC Lys 33-Nle Gly Asp
ACHC
Phe Asn 33-Arg n h 2
JKM VI 295 10
1815.4 Ac APC Ala ACPC Arg ACPC
Leu
ACPC Lys 33-Nle Gly Asp
ACPC
Phe Asn B3-Arg n h 2
JKM VI 295 11
1775.2 Ac APC Ala ACPC Arg ACPC
Leu
ACPC Lys 33-Nle Gly Asp (D )-A la Phe Asn p3-Arg n h 2
JKM VI 295 12
1787.2 Ac APC Ala ACPC Arg ACPC
Leu
ACPC Lys 33-Nle Gly Asp
AC3 C
Phe Asn
JKM VII 037 3
1603.8 Ac
-
-
ACPC Arg ACPC
Leu
A C P C Lys p3-Nle Gly Asp
AC3 C
Phe Asn p3-Arg n h 2
JKM VII 037 4
1632.5 Ac
-
-
ACPC Arg ACPC
Leu
ACPC Lys 33-Nle Gly Asp
ACPC
Phe Asn p3 -Ar 9 n h 2
JKM VI 295 7
JKM VI 295
3 3-Arg
NH2
5.4.7 O ligom er Stability in Serum
The stability o f the oligomers to proteolytic degradation in 50% fetal bovine serum (FBS)
was monitored over time by HPLC. Buffer (100 mM phosphate, pH 7.5) was mixed with an
equal volume o f FBS to make a 50% FBS solution. A sample o f oligomer (10 pL o f a 20 mM
DMSO stock solution) was added to the 50% FBS solution (1 mL). A small aliquot (100 pL)
was withdrawn from the solution with a pipette and analyzed by HPLC (Shimadzu).
remaining solution was incubated at 37°C with orbital shaking.
The
Additional aliquots were
withdrawn and analyzed after approximately 6, 20, and 36 hours. The oligomers were injected
onto a C 4 -silica reversed-phase analytical column (5 pm, 4 mm x 250 mm, Vydac) and eluted
with a gradient o f acetonitrile in water (10 - 60%, 50 min, 0.1% TFA in each) followed by a 5
min flush at 95 % B solvent and a 5 min equilibration at 10% B solvent at a flow rate o f 1
mL/min. The retention time o f the oligomers was between 32 and 33 min, just before the bulk o f
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339
the serum proteins began to elute. The peak corresponding to the oligomer (UV absorbance
220 nm) was integrated to determine the relative amount o f oligomer that was still intact (Table
62). Peaks with shorter retention times appearing in the HPLC traces at later time points were
collected and identified by MALDI-TOF MS as corresponding to oligomer degradation products
(Table 63). The column was flushed extensively with 95% acetonitrile after each use, but the
colum n’s performance was sub-optimal with later samples.
Table 62. Peptide degradation in 50% FBS monitored by HPLC (UV absorbance 220 nm).
N oteb ook
JKM Vtl 0 3 7 1
JKM VII 0 3 7 1
JKM VII 0 5 3
JKM VII 0 3 7 5
[M +Hf
1 5 7 7 .4
C om pound Tim e (hr)
Sequence
A c A C P C Arg A C PC Leu A C PC Lys p3-N le Gly A sp
Ala
P h e A sn
Arg
NH2
5-25
A c A C P C Arg A C P C Leu A C PC Lys p3-N le Gly A sp
Ala
P h e A sn
Arg
NH2
5-25
repeat
1 5 9 1 .7 A c A C PC Arg A C PC Leu A C PC Lys p3-N le Gly A sp
Aib
P h e A sn
Arg
NH2
5-26
1 5 9 1 .3
Ala
P h e A sn p3-Arg NH2
5-27
1 5 7 7 .4
A c A C PC Arg A C P C Leu A C PC Lys p3-N le Gly A sp
P eak Area
% Intact
0.0
5951811
6 .8
19 .7
4001556
2143236
1209396
6570344
4180990
100
67
36
3 6 .4
0 .0
7.1
2 1 .0
3 6 .4
0 .0
6 .9
2 0 .4
3 6 .5
0 .0
6 .8
19 .7
3 6 .4
JKM VII 0 3 7 2
1 6 0 6 .0
A c A C PC Arg A C PC Leu AC PC Lys p3-N le Gly A sp
Aib
P h e A sn p3-Arg NH2
5-28
0 .0
6 .7
1 9 .9
3 6 .3
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
2031843
877725
6626401
6352948
6289777
6080326
5611177
5376270
4914949
4095667
20
100
64
31
13
100
96
95
92
100
96
5869969
5546799
88
73
100
94
5420538
5494136
92
94
340
mVolts
200
100
0
18
20
22
24
26
28
30
32
34
28
30
32
34
Minutes
mVolts
200
100 -
18
20
22
26
24
Minutes
mVolts
200
100
18
20
22
24
26
28
30
32
34
Minutes
Figure 42. Degradation o f oligomers 5-25 (top, rt = 32.5 min), 5-27 (middle, rt = 32.5 min), and 5-28 (bottom, rt =
33 min) over time monitored by HPLC.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
18
20
22
24
26
28
30
32
34
28
30
32
34
Minutes
200
100
0
18
20
22
24
26
Minutes
Figure 43. Degradation o f peptides 5-26 (top, rt = 32.5 min) and 5-25 (bottom, repeat, rt = 32.5 min) over time
monitored by HPLC.
Table 63. Characterization o f proteolytic fragments o f 5-25 (JKM V I I 063).
Ac
43
43
43
43
43
43
43
ACPC
111.07
111.07
111.07
111.07
111.07
111.07
111.07
Arg
156.1
156.1
156.1
156.1
156.1
156.1
156.1
ACPC
111.07
111.07
111.07
111.07
111.07
111.07
111.07
Leu
113.08
113.08
113.08
113.08
113.08
113.08
113.08
ACPC
111.07
111.07
111.07
111.07
111.07
111.07
111.07
Sequence
0a-Nle Gly
Lys
128.09 127.1 57.02
128.09 127.1 57.02
128.09 127.1 57.02
128.09 127.1 57.02
128.09 127.1 57.02
128.09 127.1 57.02
128.09 127.1
A sp
115.03
115.03
115.03
115.03
115.03
Ala
71.04
71.04
71.04
71.04
P he
147.07
147.07
147.07
A sn
114.04
114.04
Arg n h 2
156.1 16
16
16
16
16
16
16
MW Calc. [M +H f O bs.
1576.9
1420.8
1306.7
1159.7
1088.6
973.6
916.6
1090.5
975.5
R eten tio n
Tim e (Min)
32.5
27.5
26.5
5.5 References
1 (a) Arkin, M. R.; Wells, J. A. Nat. Rev. D rug Discovery 2004, 3, 301. (b) Cochran, A. G.
Chem. Biol. 2000, 7, R85. (c) Gadek, T. R.; Nicholas, J. B. Biochem. Pharmacol. 2003, 65, 1.
(d) Berg, T. Angew. Chem. Int. Ed. 2003, 42, 2462. (e) Peczuh, M. W.; Hamilton, A. D. Chem
Rev. 2 0 0 0 ,100, 2479.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
342
2 (a) Petros, A. M.; Dinges, J.; Augeri, D. J.; Baumeister, S. A.; Betebenner, D. A.; Bures, M. G.;
Elmore, S. W.; Hajduk, P. J.; Joseph, M. K.; Landis, S. K.; Nettesheim, D. G.; Rosenberg, S. H.;
Wang, S.; Thomas, S.; Wang, X.; Zanze, I.; Zhang, H.; Fesik, S. W. J. Med. Chem. 2006, 49,
656. (b) Oltersdorf, T.; Elmore, S. W.; Shoemaker, A. R.; Armstrong, R. C.; Augeri, D. J.; Belli,
B. A.; Bruncko, M.; Deckwerth, T. L.; Dinges, J.; Hajduk, P. J.; Joseph, M. K.; Kitada, S.;
Korsmeyer, S. J.; Kunzer, A. R.; Letai, A.; Li, C.; Mitten, M. J.; Nettesheim, D. G.; Ng, S. C.;
Nimmer, P. M.; O'Connor, J. M.; Oleksijew, A.; Petros, A. M.; Reed, J. C.; Shen, W.; Tahir, S.
K.; Thompson, C. B.; Tomaselli, K. J.; Wang, B.; Wendt, M. D.; Zhang, H.; Fesik, S. W.;
Rosenberg, S. H. Nature 2005, 435, 677. (c) Arkin, M. R.; Randal, M.; DeLano, W. L.; Hyde, J.;
Luang, T. N.; Oslob, J. D.; Raphael, D. R.; Taylor, L.; Wang, J.; McDowell, R. S.; Wells, J. A.;
Braisted, A. C. Proc. Nat. Acad. Sci. U.S.A. 2 0 0 3 ,100, 1603. (d) Vassilev, L. T.; Vu, B. T.;
Graves, B.; Carvajal, D.; Podlaski, F.; Filipovic, F.; Kong, N.; Kammlott, U.; Lukacs, C.; Klein,
C.; Fotouhi, N.; Liu, E. A. Science 2004, 303, 844. (e) Grasberger, B. L.; Lu, T.; Schubert, C.;
Parks, D. J.; Carver, T. E.; Koblish, H. K.; Cummings, M. D.; LaFrance, L. V.; Milkiewicz, K.
L.; Calvo, R. R.; Maguire, D.; Lattanze, J.; Franks, C. F.; Zhao, S.; Ramachandren, K.; Bylebyl,
G. R.; Zhang, M.; Manthey, C. L.; Petrella, E. C.; Pantoliano, M. W.; Deckman, L C.; Spurlino,
J. C.; Maroney, A. C.; Tomczuk, B. E.; Molloy, C. J.; Bone, R. F. J. Med. Chem. 2005, 48, 909.
3 (a) Walensky, L. D.; Kung, A. L.; Ischer, I.; Malia, T. J.; Barbuto, S.; Wright, R. D.; Wagner,
G.; Verdine, G. L.; Korsmeyer, S. J. Science 2004, 305, 1466. (b) Tugyi, R.; Uray, K.; Ivan, D.;
Fellinger, E.; Perkins, A.; Hudecz, F. Proc. Nat. Acad. Sci. U.S.A. 2 0 0 5 ,102, 413.
4 (a) Adams, J. M.; Cory, S. Science 1998, 281, 1322. (b) Coultas, L.; Strasser, A. Semin.
Cancer Biol. 2 0 0 3 ,13, 115. (c) Letai, A.; Bassik, M. C.; Walensky, L. D.; Sorcinelli, M. D.;
Weiler, S.; Korsmeyer, S. J. Cancer Cell 2002, 2, 183.
5 Sattler, M.; Liang, H.; Nettesheim, D.; Meadows, R. P.; Harlan, J. E.; Eberstadt, M.; Yoon, H.
S.; Shuker, S. B.; Chang, B. S.; Minn, A. J.; Thompson, C. B.; Fesik, S. W. Science 1997, 275,
983.
6 (a) Baell, J. B.; Huang, D. C. S. Biochem. Pharmacol. 2002, 64, 851. (b) O'Neill, J.; Manion,
M.; Schwartz, P.; Hockenbery, D. M. Biochim. Biophys. Acta 2 0 0 4 ,1705, 43.
7 Petros, A. M.; Nettesheim, D. G.; Wang, Y.; Olejniczak, E. T.; Meadows, R. P.; Mack, J.;
Swift, K.; Matayoshi, E. D.; Zhang, H.; Thompson, C. B.; Fesik, S. W. Protein Sci. 2000, 9,
2528.
8 (a) Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Science 1996, 274, 1531. (b)
Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Science 1997, 278, 497.
9 (a) Yin, H.; Lee, G.-i.; Sedey, K. A.; Kutzki, O.; Park, H. S.; Omer, B. P.; Ernst, J. T.; Wang,
H.-G.; Sebti, S. M.; Hamilton, A. D. J. Am. Chem. Soc. 2 0 0 5 ,127, 10191. (b) Yin, H.; Lee, G.i.; Park, H. S.; Payne, G. A.; Rodriguez, J. M.; Sebti, S. M.; Hamilton, A. D. Angew. Chem. Int.
Ed. 2005, 44, 2704. (c) Yin, H.; Lee, G.-i.; Sedey, K. A.; Rodriguez, J. M.; Wang, H.-G.; Sebti,
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343
S. M.; Hamilton, A. D. J. Am. Chem. Soc. 2005, 127, 5463. (d) Yin, H.; Hamilton, A. D. Bioorg.
Med. Chem. Lett. 2 0 0 4 ,14, 1375. (e) Ernst, J. T.; Becerril, J.; Park, H. S.; Yin, H.; Hamilton, A.
D. Angew. Chem. Int. Ed. 2003, 42, 535. (f) Kutzki, O.; Park, H. S.; Ernst, J. T.; Omer, B. P.;
Yin, H.; Hamilton, A. D. J. Am. Chem. Soc. 2 0 0 2 ,124, 11838.
10 (a) Gellman, S. H. Acc. Chem. Res. 1998, 31, 173. (b) Cheng, R. P.; Gellman, S. H.; DeGrado,
W. F. Chem. Rev. 2 0 0 1 ,101, 3219.
11 (a) Werder, M.; Hauser, H.; Abele, S.; Seebach, D. Helv. Chim. Acta 1999, 82, 1774. (b)
Gademann, K.; Seebach, D. Helv. Chim. Acta 2001, 84, 2924. (c) Seebach, D.; Rueping, M.;
Arvidsson, P. I.; Kimmerlin, T.; Micuch, P.; Noti, C.; Langnegger, D.; Hoyer, D. Helv. Chim.
Acta 2001, 84, 3503. (d) Kritzer, J. A.; Lear, J. D.; Hodson, M. E.; Schepartz, A. J. Am. Chem.
Soc. 2 0 0 4 ,126, 9468. (e) Stephens, O. M.; Kim, S.; Welch, B. D.; Hodsdon, M. E.; Kay, M. S.;
Schepartz, A. J. Am. Chem. Soc. 2 0 0 5 ,127, 13126. (f) English, E. P.; Chumanov, R. S.;
Gellman, S. H.; Compton, T. J. Biol. Chem. 2006, 281, 2661.
12 Sadowsky, J. D.; Schmidt, M. A ; Lee, H.-S.; Umezawa, N.; Wang, S.; Tomita, Y.; Gellman,
S. H. J. Am. Chem. Soc. 2 0 0 5 ,127, 11966.
13 Hayen, A.; Schmitt, M. A.; Ngassa, F. N.; Thomasson, K. A.; Gellman, S. H. Angew. Chem.
Int. Ed. 2004, 43, 505.
14 Frackenpohl, J.; Arvidsson, P. I.; Schreiber, J. V.; Seebach, D. ChemBioChem 2001, 2, 445.
15 Wiegand, H.; Wirz, B.; Schweitzer, A.; Camenisch, G. P.; Perez, M. I . R.; Gross, G.;
Woessner, R.; Voges, R.; Arvidsson, P. I.; Frackenpohl, J.; Seebach, D. Biopharm. D rugDisp.
2002,23,251.
16 (a) Merrifield, R. B. J. Am. Chem. Soc., 1963,8 5 , 2149. (b) Albericio, F. Curr. Op. Chem.
Biol. 2004,3, 211.
17 Porter, E. A.; Weisblum, B.; Gellman, S. H. J. Am. Chem. Soc. 2 0 0 5 ,127, 11516.
18 Arvidsson, P. I.; Frackenpohl, J.; Seebach, D. Helv. Chim. Acta 2003, 86, 1522.
19 Murray, J. K.; Gellman, S. H. Org. Lett. 2005, 7, 1517.
20 (a) Yu, H.-M.; Chen, S.-T.; Wang, K.-T. J. Org. Chem. 1992, 57, 4781. (b) Erdelyi, M.;
Gogoll, A. Synthesis 2002, 1592. (c) Ferguson, J. D. Mol. Div. 2003, 7, 281. (d) Matsushita, T.;
Hinou, H.; Kurogochi, M.; Shimizu, H.; Nishimura, S.-I. Org. Lett. 2005, 7, 877.
21 Kappe, C. O. Angew. Chem. Int. Ed. 2004, 43, 6250.
22 (a) Blackwell, H. E. Org. Biomol. Chem. 2 0 0 3 ,1, 1251. (b) Kappe, C. O. Curr. Op. Chem.
Biol. 2002, 6, 314 and references therein.
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23 Murray, J. K.; Farooqi, B.; Sadowsky, J. D.; Scalf, M.; Freund, W. A.; Smith, L. M.; Chen, J.;
Gellman, S. H. J. Am. Chem. Soc. 2 0 0 5 ,127, 13271.
24 A one-bead-one-compound (3-peptide library was prepared on TentaGel macrobeads: Kritzer,
J. A.; Luedtke, N. W.; Flarker, E. A.; Schepartz, A. J. Am. Chem. Soc. 2 0 0 5 ,127, 14584.
25 Murray, J. K.; Gellman, S. H. J. Comb. Chem. 2005, 8, 58.
26 Sadowsky, J. D.; Gellman, S. H. Submitted.
27 Burgess, K.; Liaw, A. I.; Wang, N. J. Med. Chem. 1994, 37, 2985.
28 Schreiber, J. V.; Quadroni, M.; Seebach, D. Chimia 1999, 53, 621.
29 Morken, J. P.; Kapoor, T. M.; Feng, S.; Shirai, F.; Schreiber, S. L. J. Am. Chem. Soc. 1998,
120, 30.
30 (a) Guichard, G.; Zerbib, A.; Le Gal, F.-A.; Hoebeke, J.; Connan, F.; Choppin, J.; Briand, J.P.; Guillet, J.-G. J. Med. Chem. 2000, 43, 3803. (b) Reinelt, S.; Marti, M.; Dedier, S.; Reitinger,
T.; Folkers, G.; Lopez de Castros, J. A.; Rognan, D. J. Biol. Chem. 2001, 276, 24525. (c) Steer,
D.; Lew, R.; Perlmutter, P.; Smith, A. I.; Aguilar, M.-I. Biochemistry 2002, 41, 10819.
31 Adessi, C.; Soto, C. Curr. Med. Chem. 2002, 9, 963.
32 (a) Cotterill, I. C.; Usyatinsky, A. Y.; Arnold, J. M.; Clark, D. S.; Dordick, J. S.; Michels, P.
C.; Khmelnitsky, Y. L. Tetrahedron Lett. 1998, 39, 1117. (b) Glass, B. M.; Combs, A. P. Rapid
Parallel Synthesis Utilizing Microwave Irradiation. In High-Throughput Synthesis', Sucholeiki,
I., Ed.; Marcel Dekker: New York, 2001; pp. 123-128. (e) Kappe, C. O.; Stadler, A. MicrowaveAssisted Combinatorial Chemistry. In Microwaves in Organic Synthesis', Loupy, A. Ed.; WileyVCH: Weinheim, 2002; pp. 405-433.
33 (a) Shrimpton, C. N.; Abbenante, G.; Lew, R. A.; Smith, A. I. Biochem. J. 2000, 345, 351. (b)
Yamaguchi, H.; Kodama, H.; Osada, S.; Kato, F.; Jelokhani-Niaraki, M.; Kondo, M. Biosci.
Biotechnol. Biochem. 2003, 67, 2269.
34 Lee, H.-S.; LePlae, P. R.; Porter, E. A.; Gellman, S. H. J. Org. Chem. 2001, 66, 3597.
35 Schinnerl, M.; Murray, J. K.; Langenhan, J. M.; Gellman, S. H. Eur. J. Org. Chem. 2003, 721.
36 Guichard, G.; Abele, S.; Seebach, D. Helv. Chim. Acta 1998, 81, 187.
37 Lee, H.-S.; Park, J.-S.; Kim, B. M.; Gellman, S. H. J. Org. Chem. 2003, 68, 1575.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
345
Chapter 6
Efforts toward the Discovery of
Proteomimetic Inhibitors for the
Transforming Growth Factor 03/
Type II Receptor Interaction
Disulfide
Bond
Asp32
TGF|33
Phe30
Thr51
Glu55
Leu27
Figure adapted from Hart, P. J.; Deep, S.; Taylor, A. B.; Shu, Z.; Hinck, C. S.; Hinck, A.
P. Nature Struct. Biol. 2002, 9, 203-208, PDB code 1KTZ.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
346
6.0 Brief Summary of Chapter
Attempts were made to inhibit the transforming growth factor P3 (TGFp3)/type II
receptor (TpRII) protein-protein interaction by mimicking the display o f important TpRII
residues from the binding interface on either a P-hairpin a-peptide or 14-helical P-peptide
scaffold. Combinatorial libraries based on each design were synthesized in parallel using
microwave irradiation. Despite numerous apparent hits during initial screening, to date
no compounds have been validated as having significant inhibitory activity.
6.1 Background
The protein-protein interactions targeted by the two previously described efforts
for development o f foldamer inhibitors shared a common structural theme.
The
p53/MDM2 (Chapter 3) and Bcl-xL/BH3-domain (Chapter 5) interactions both involve
the binding of an a-helical protein segment into a complementary hydrophobic cleft. In
each case, the natural a-helix provided a starting point for the structure-based design o f
potential foldamer inhibitors.
However, the foldamer approach to protein-protein
interaction inhibition should not be limited to mimicking an a-helical protein structure.
By displaying various functional groups on a well-defined structural scaffold, foldamers
can potentially be used to inhibit protein-protein interactions with differing binding
epitopes, including P-hairpins,1 polyproline helices,2 and the even relatively flat
interfaces that have proven intractable with small molecule approaches.
This hypothesis
was tested by attempting to develop inhibitors for the TGFP3/TPRII interaction, which
has a P-sheet binding epitope.
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347
6.1.1 TGFp in Breast Cancer
TGFp plays a complex role in cell growth regulation, extracellular matrix
production, and immune modulation in normal cells4 and in malignant cells during the
development of breast cancer.5 In the early stages of the disease, TGFp is a negative
growth factor for both healthy and malignant mammary epithelial cells.6 However,
during tumor progression, this growth inhibitory response is lost or attenuated, and TGFp
becomes a facilitator of metastasis.6 Tumor cells produce higher levels of TGFP, and
TGFp acquires a tumor-promoting effect by facilitating angiogenesis and the
development of stromal tissue (leading to tumor invasion) while inhibiting immune
responses.6 In preclinical studies, inhibition of TGFp signaling through a number of
traditional
approaches,
including
neutralizing
antibodies
to
TGFp,
antisense
oligonucleotides to TGFp mPNA, soluble receptor fusion proteins, and small molecule
inhibitors of the receptor kinase activity, blocks tumor growth and/or metastasis.7 Each
of these therapeutic approaches has its weaknesses (Chapter 1), creating an opening for a
foldamer-based approach.
6.1.2 Interactions and Signaling of TGFP
There are three mammalian TGFp isoforms, TGFpi, TGFP2, and TGFP3, which
bind to a heteromeric complex of transmembrane serine/threonine kinases, the type I and
type II TGFp receptors (TpRI and TpRII).4
These ligands also bind a large
transmembrane proteoglycan referred to as the type III TGFp receptor (also called
betaglycan) the role of which is to present TGFp to TpRII.4 Following ligand binding to
TpRII, TPRI is recruited to the ligand-receptor complex.5 Interaction of TpRII with
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348
Tf3RI allows for the constitutively active TpRII kinase to transphosphorylate and
activate the TpRI kinase, which, in turn, phosphorylates the receptor-regulated Smad2
and Smad3 proteins.5 Smad2 and Smad3 then associate with a common mediator,
Smad4, and translocate to the nucleus where they regulate gene transcription.6
6.1.3 TGFp3/TpRII Assay
A homogenous time-resolved fluorescence assay (HTRF) for the TGFP3/TPRII
interaction was developed in collaboration with Dr. F. Michael Hoffmann (Oncology
Department, University of Wisconsin-Madison Medical School, McArdle Laboratory for
Cancer Research). The proteins used in the assay, His-tagged TpRII-ectodomain (ED)
and TGFp3, were obtained from Dr. Andrew P. Hinck (Department of Biochemistry and
Center for Biomolecular Structure Analysis, University of Texas Health Science Center
at San Antonio). The His-tagged TpRII-ED was labeled indirectly with an antibody to
the His tag that bears a europium chelate.8 TGFp3 was labeled with IC5 to provide the
acceptor fluorophore. When the two proteins bind to one another, fluorescence resonance
energy transfer (FRET) occurs between the excited europium chelate and the IC5
acceptor (Figure 1). Unlabeled TGFpl was used as a positive control to compete with
labeled TGFP3 for binding to TpRII-ED and reproducibly exhibited 60-70% inhibition at
6 nM. The assay could be carried out in 384-well plates with a 30 pL assay volume,
allowing for high-throughput screening of potential inhibitors.
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Eu-labeled
anti-His antibody
Figure 1. Europium chelate FRET assay. Figure adapted from Dr. F. Michael Hoffmann.
In an effort to benchmark the assay, we prepared and assayed a set of a-peptides
reported to inhibit the TGFP3/TPRII interaction.9,10 While the most active compound
(AC-ETWIWDLVWN-NH 2 ) gave results similar to those reported in the literature (~
90% inhibition at 20 pM, ref. 9), its activity dropped to around 30% inhibition at 11 pM.
This peptide exhibited > 90% inhibition of the Smad3/Fox-Hl interaction at 11 pM. We
decided not to investigate this peptide further due to its steep dose-response curve
(characteristic of a critical concentration required for aggregation of the peptide and/or
protein target), strong inhibition of a structurally unrelated protein-protein interaction,
and its low aqueous solubility.
These observations indicated the possibility of non­
specific inhibition by the peptide, a common problem for protein-protein interaction
inhibitors with low pM potencies.11 The lack of other compounds known to inhibit the
TGFp3/TpRII interaction prevented further investigation, and we proceeded with the
assay without additional modification.
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350
6.1.4 S tru ctu re of the TG Fp3/T|3RII Complex
A crystal structure of the extracellular ligand-binding domain of TpRII in
complex with TGFP3 from Hinck and coworkers facilitated the structure-based design of
potential inhibitors.12,13
In contrast to previous a-helix-mediated protein-protein
interaction targets (Chapters 3 and 5), a surface ridge formed by p-strands 1 and 4 of
TpRII binds in a cleft on the surface of the TGFp3 ligand. TpRII residues Ile50, Thr51,
and Ile53 in p-strand 4 and Leu27 and Phe30 in p-strand 1 of the ridge make hydrophobic
contacts with TGFp3 residues Trp32, Tyr90, Tyr91, and Val92 within the cleft. Two
pairs of charged residues are located opposite one another at the periphery of the binding
site. Residues Arg25 and Arg94 on TGFp3 form salt-bridge interactions with TpRII
residues G lu ll9 and Asp32, respectively. TpRII residues Glu55 and Glu75, which are
located near TGFP3 residue Arg94, have also been shown to contribute significantly to
the binding affinity.14 We hypothesized that mimicking the three-dimensional display of
the important interfacial residues on TpRII using an appropriate foldamer scaffold would
produce a new class of inhibitors of the TGFP3/TPRII interaction.
6.2 Attempted Development of B-Hairpin q-Peptide Inhibitors
We decided to explore the interface of the TGFp3/TpRII interaction through the
design of P-hairpin a-peptide inhibitors intended to mimic the TpRII contact surface, in
preparation for the later development of foldamer inhibitors. As mentioned above, all of
the important interacting residues on TpRII, except G lull9 and Glu75, are contained
within P-strand 1 (residues Leu27 to Asp32) and P-strand 4 (residues Cys48 to Glu55).
These strands form a two-strand antiparallel p-sheet with three pairs of cross- strand
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35 1
Figure 2. A) Top view and B) front view of design for (3-hairpin inhibitors. Crystal structure from PDB
code 1KTZ, ref. 12. Solvent-exposed surface of TGF(33 rendered using MOLCAD in Sybyl. Some side
chains have been omitted for clarity.
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352
hydrogen-bonded residues (C=0 of Gln26 to H-N of Cys54, both C =0 and H-N of
Cys28 to H-N and C=0 of Ser52, and H-N of Phe30 to C=0 of Ile50). These strands are
covalently tethered via a disulfide bond between the side chains of Cys31 and Cys48.
We wondered if the N-terminus of p-strand 1 could be synthetically linked to the Cterminus of P-strand 4 via a reverse turn to yield a disulfide-cyclized p-hairpin capable of
inhibiting the TGFp3/TpRII interaction (Figure 2).
6.2.1 P-Hairpin L ibrary Design
We prepared a 96-membered parallel library based on our P-hairpin design for
screening in the TGFp3/TpRII assay (Figure 3). Our primary objective was to identify a
suitable reverse turn for linking the two strands, so four different turn units of varying
length and rigidity were incorporated.15 The residues N- and C-terminal to the turn
segment were also varied. For synthetic ease, residues Cys28 and Cys54 in the natural
TpRII sequence, which are not involved in the disulfide bond between P-strands 1 and 4,
were replaced with serine. Since the hydrophobic pocket formed by TGFp3 residues
Trp32, Tyr90, and Val92 is only partially filled by TpRII residue Ile53,12 phenylalanine
or tryptophan was incorporated in addition to lie at this position in the library. The
geometry of the disulfide bond was varied by omitting Ser49 (i.e., a null mutation) from
half the compounds in the library. The first synthesis of the library was performed at
room temperature (1.5 hr couplings, 15 min deprotections) in a deep well filter plate with
agitation on the mini orbital shaker (JKM VI 303). However, these conditions were not
sufficient, as HPLC and MALDI-TOF MS analyses revealed that the product mixtures
contained very little of the full length products.
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353
Trp
; F m o c H I'
null
w
ou2n j
P he
Lys
A s n -G ly
P ro -A sp -G ly
G ly-G ly
G ly-G ly-G ly
32
30
27
Glu
Figure 3. Parallel library of disulfide-cyclized P-hairpins screened against the TGFp3/TpRII interaction (2
x 3 * 2 x 4 x 2 = 96 compounds, JKM VI 303 and JKM VII 033). Numbers correspond to position in the
natural TpRII sequence.
6.2.2 Synthetic Optimization of p-Hairpin
The library members were produced in much higher purity through a combination
of microwave heating and inclusion of a pseudoproline dipeptide unit.
First, a
representative sequence from the library was selected for synthetic optimization (6- 1,
Figure 4).
Given our success with microwave-enhanced P-peptide synthesis,16 we
prepared the octa-a-peptide corresponding to residues 9 through 16 of 6-1 (Gly-Glu-Leu-
. 0
6-1
o
Ac-Cys-Ser-lle-Thr-Ser-lle-Ser-Gly-Gly-Glu-Leu-Ser-Lys-Phe-Cys-Asp-NH 2
Figure 4. Sequence of a-peptide 6-1 used for synthetic optimization.
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354
Ser-Lys-Phe-Cys-Asp) in the multimode microwave reactor with our optimized
reaction conditions (70 °C couplings, 80 °C deprotections, JKM VII 003). The resulting
product was > 90% pure by HPLC analysis (UV absorbance at 220 nm), in spite of the
potential for epimerization of the activated a-amino acids under microwave conditions.
The synthesis of 6-1 was carried to completion by incorporating residues 1 through 8, and
a small aliquot of resin was withdrawn after each coupling reaction to monitor the purity
of the intermediates (JKM VII 007). When the final product was produced in low purity,
HPLC analysis of the intermediates identified the couplings of Ser2 and Cysl as
proceeding in low yield (Figure 5). Double coupling of Ser2 in 0.8 M LiCl in NMP16
followed by double coupling of Cysl in DMF with microwave irradiation failed to
resolve the problem (JKM VII 009).
We wondered whether incorporating a pseudoproline dipeptide at an appropriate
position within the sequence would alleviate the difficult couplings.17 Position 4 was
selected for incorporation of an Ile-Thr oxazolidine dipeptide since this position is near
the site of the difficult coupling, does not include a position of diversity in the library,
and accomplishes the coupling of sequential P-branched residues (Figure 3). Microwaveassisted coupling of Fmoc-Ile-Thr(\|/M°’Mepro)-OH, followed by double coupling of Ser2
and Cysl yielded the desired product in acceptable purity (JKM VII 027). Dissolving the
product mixture in DMSO and stirring for 48 hr open to air accomplished oxidation of
the cysteine residues to form the disulfide-cyclized product (JKM VII 031). Parallel
synthesis under these optimized conditions produced the library in a much higher average
purity than before (JKM VII 033, Figure 6).
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355
100 0
800
600
Abs
(mV)
400
200
22
24
26
Time (min)
28
30
32
Figure 5. HPLC traces o f Fmoc-protected intermediates in the synthesis of a-peptide 6-1 from Figure 4.
Numbers correspond to the position of the N-terminal residue.
M ox
M red
1500
M ox
1000
A bs
(mV)
500
20
22
24
26
28
30
32
34
38
38
40
Time (min)
Figure 6. HPLC traces o f A) typical and B) high purity representative library members (JKM V II033).
6.2.3 P-Hairpin L ibrary Screening
The library was screened (without purification) for inhibitors of the TGFP3/TPRII
interaction, and several potential hits were identified.
Screening at an approximate
peptide concentration of 333 pM identified 13 compounds with > 90% inhibition (Figure
7). However, a 10-fold dilution of the most active compounds reduced their activity to
around 15% inhibition. These compounds were re-synthesized and purified, but none of
the hits showed any activity when retested (Table 1). In an effort to determine the
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356
reliability of screening crude product mixtures, 14 compounds from the library
synthesis were purified by HPLC. Screening results for the purified compounds were
compared to the previous results obtained for the crude product mixtures, but no
correlation was apparent (Figure 8). However, compound H ll did show 38% inhibition
on two different occasions after this initial purification. The mass spectrum showed that
this sample contained the desired product and a Ser-deletion side product. Compound
HI 1 and the three possible Ser-deletion peptides were re-synthesized and purified (JKM
VII 103), but none was active upon retesting (Table 2).
100
c
o
§
40
C
CM
ID
O
00
00
t-
(O
o
o
•£E "IE
^
t-
^
-
LL
o
O
O)
CM
N
o o o
X3'—X X"
Com pound
Figure 7. Screening data for (3-hairpin library (JKM VII 033) at -333 pM in TGFp3/T(3RII assay.
Table 1. Apparent hits from screening o f the P-hairpin library (JKM V II033).
Compound
B02
D05
F03
D04
E04
C08
F07
D03
D11
D10
D07
D08
E02
% Inhibition
Crude (333 pM) crude (33 pM)
99
99
99
99
98
97
97
96
94
94
93
91
90
12
17
21
13
11
13
16
17
13
19
12
17
16
Sequence
Pure (100 pM)
N
-11
-7
1
4
-8
-8
-7
2
-7
-3
-5
-8
-8
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Cys
Cys
Cys
Cys
Cys
Cys
Cys
Cys
Cys
Cys
Cys
Cys
Cys
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
C
null
Ser
Ser
null
null
null
Ser
Ser
Ser
null
S er
null
null
lie
lie
lie
lie
lie
lie
lie
lie
lie
lie
He
He
He
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Ser
Ser
Ser
Ser
S er
S er
Ser
S er
Ser
Ser
Ser
S er
Ser
Trp
Phe
Trp
Trp
Trp
Phe
Phe
Trp
lie
He
Phe
Phe
Trp
Ser
Ser
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Ser
Lys
Lys
Ser
Gly
Gly
Gly
Gly
Asn
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Asn
Asp
Gly
Asp
Gly
Gly
Gly
Asp
Gly
Gly
Gly
Gly
Gly
Gly
Pro
Gly
Pro
Gly
Glu
Glu
Gin
Glu
Gin
Glu
Gin
Glu
Glu
Glu
Glu
Glu
Gin
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
PhePhe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Cys
Cys
Cys
Cys
Cys
Cys
Cys
Cys
Cys
Cys
Cys
Cys
Cys
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
Pro
Gly
Gly
Gly
Gly
Gly
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
357
100
90
80
70
C
o
60
50
40
1
o
!♦
30
^0
1
♦
-io_
♦
-10
♦
10 .
20>..........
30...
40.
50
Pure % Inhibition
Figure 8. Correlation of percent inhibition obtained by screening p-hairpin library members (JKM VII
033) in both crude and purified forms.
Table 2. Sequence and activity of compound HI 1 and all possible Ser-deletion side products (JKM VII
103).
% Inhibitio n (200 ^M )
7
to
3
-6
S eq u en ce
Ac
Ac
Ac
Ac
C ys
C ys
C ys
C ys
Ser
Ser
S er
-
ile
lie
He
ile
Thr
Thr
Thr
Thr
Ser
-
Ser
Ser
ile
lie
lie
Ile
Lys
Lys
Lys
Lys
G ly
G ly
G ly
G ly
G ly
G ly
G ly
Gly
Gly
G ly
Gly
Gly
G in
G in
G in
G in
L eu
L eu
L eu
L eu
Ser
Ser
Ser
Lys
Lys
Lys
Lys
Phe
Phe
Phe
Phe
C ys
C ys
C ys
C ys
A sp
A sp
A sp
A sp
NH2
NH2
NH2
NH2
6.2.4 Summary of P-Hairpin Designs
Preparation of P-hairpin a-peptide inhibitors for the TGFp3/TpRII interaction
provided useful synthetic insights.
Microwave-assisted a-peptide synthesis proceeds
well under the conditions previously developed for P-peptides.
Incorporation of a
pseudoproline dipeptide unit can be very effective for the synthetic optimization of
difficult a-peptide sequences.
Air oxidation is a simple and effective method for
disulfide bond formation during parallel library synthesis. These synthetic techniques
will be applied where appropriate to future efforts.
In spite of promising initial screening results, no hits from the P-hairpin library
could be validated.
This suggested that further assay development was needed, that
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
358
compounds should be screened at lower concentrations to avoid aggregation, or that
only purified compounds should be assayed.
Adding a detergent (e.g., Pluronic® or
Triton X-100) to the assay buffer had no effect, indicating that the problem with
identification of false positives may not be related an aggregation phenomenon.11
Recently, titration of the TpRII protein showed that its concentration could be greatly
reduced without lowering the intensity of the assay signal (Dr. Seth Home and Dr. Grace
Jurkowski). The concentration of the peptides in the assay can now also be lowered,
while identifying hits with a IQ of ~ 10 pM.
r~ -
't
6-2 Ac-25PQLSK 30F C D V R 35FS T C D 40N Q K SC 45M S N C S 50ITSIS 55EK -N H 2
Figure 9. Sequence of a-peptide 32-mer.
The design of the inhibitors could potentially be improved by using a longer
sequence from TpRII (residues Pro25 to Lys56) that naturally joins p-strands 1 and 4 (62, Figure 9). The conformation of this 32-residue segment of the protein is stabilized by
two disulfide bonds, which may render it a suitable scaffold for inhibitor development
through combinatorial synthesis or phage display (Figure
10).
Peptide 6-2,
corresponding to the natural sequence except for Cys28->Ser and Cys54->Ser mutations
for synthetic convenience, was rapidly synthesized with microwave irradiation, the Ile50Thr51 pseudoproline dipeptide being incorporated as before. The inner disulfide bond
between Cys38 and Cys44 was formed by air oxidation in DMSO after removal of the
trityl protecting groups from the side chain sulfur atoms during standard TFA cleavage.
The Cys31 and Cys48 residues were incorporated as acetimidomethyl (Acm) protected
derivatives, allowing selective formation of the second disulfide bond by treatment with
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359
Figure 10. A) Top view and B) front view of (3-strands 1-4 of TpRII in complex with TGFP3. Crystal
structure from PDB code 1KTZ, ref. 12. Solvent-exposed surface o f TGFP3 rendered using MOLCAD and
in Sybyl. Side chains of TpRII not involved in intramolecular disulfide bonds were omitted for clarity.
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
360
iodine after initial HPLC purification.18 However, this compound did not show any
activity in the TGF|33/TpRII assay.
6.3 Toward the Development of Foldamer Inhibitors
We began to explore the possibility of inhibiting the TGFp3/TpRII interaction
with foldamers shortly after initiating our investigation of the P-hairpin a-peptide designs
described above. The fact that this interaction is modulated by a p-sheet rather than an ahelical protein segment presented a new challenge for the design of helical foldamer
inhibitors. Examination of the crystal structure of the complex reveals that the cleft on
TGFP3 is deep and wide but very short and that the ridge on TpRII has a high density of
interacting residues.
This architecture contrasts sharply with the long, narrow
hydrophobic cleft and well-spaced interaction sites of Bcl-xL19 and other previous targets.
6.3.1 Structure-Based Design of 14-Helical P-Peptide Inhibitors
Our inhibitor design aimed to mimic the receptor, developing foldamers to bind in
the hydrophobic cleft on the surface of TGFP3.
Modeling suggested the use of a
foldamer scaffold with a small rise per turn in order for three turns of the helix to be
accommodated lengthwise within the cleft. Manual docking of a left-handed 14-helical
P-peptide (6-3, Figure 11) into the groove on TGFP3 (Figure 12) suggested that the cleft
was sufficiently wide to accommodate the backbone and sufficiently deep that all three
faces of the helix could contact the protein. Superimposition of P-peptide 6-3 with TpRII
showed that a majority of the interacting residues could be presented from this scaffold.
One face of the 14-helix was aligned with p-strand 4 in the bottom of the cleft,
positioning p -hlle8 and p -hlle2 near the binding sites of TpRII residues Ile53 and Ile50,
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361
respectively. We observed that a (3 -amino acid at position 5 could potentially be
used to explore a pocket on the surface of TGFP3 that is unoccupied by TpRII. The
scaffold uses the two remaining faces of the helix to display p3-hGlul, p3-hPhe4, and p3hLeu9 near the binding sites of Asp32, Phe30, and Leu27 in P-strand 1 of TpRII. A
potential improvement over the previous P-hairpin designs is that TpRII residue G lu ll9
can be mimicked by p3-hGlu6 in 6-3.
However, the possible hydrogen-bonding
interactions of TpRII residues Ile53, Ser52, and Ser49 with TGFP3 residues Tyr91,
Gly93, and His94 in the floor of the groove cannot be mimicked by the 14-helical Ppeptide scaffold.
Cyclic residues APiC3 and ACHC7 were included to promote 14-
helical conformational stability.
TGpll Residues: Asp32
Ile50
I
c o
6-3
o \
H
Phe30
Glu119
Ile53
Leu27
j
2h
o
'Y
H
o
o
H
Figure 11. Left-handed 14-helical P-peptide 6-3, a potential mimic of TpRII.
6.3.2 First Generation 14-Helical P-Peptide L ibrary Design
P-Peptide 6-3 served as the basis for design of a 96-membered parallel library
(Figure 13).
Acyclic residues were incorporated at positions 3 and 7 to vary the
conformational stability of the 14-helix. At positions 4 and 9, the display of the side
chain was varied by including p2-hPhe and p3-hxLeu, respectively. To avoid a potential
steric clash with the floor of the cleft, p-hGly was included at position 5. Hydrophobic
3
3
residues P -hPhe and P -hTrp were included at position 8 to completely fill the binding
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
Figure 12. A) Top and B) front views o f P-peptide 6-3 m anually docked with TFGP3.
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n
363
site of TpRII residue Ile53. The parallel library (JKM VI 293, Figure 13) was rapidly
synthesized with the multimode microwave reactor. The resulting product mixtures were
of acceptable purity (the major peak in the HPLC trace corresponded to the desired
library member by MALDI-TOF MS) but were difficult to dissolve in DMSO, most
likely due to the peptides’ low net charge (-1) and high proportion of hydrophobic
residues. A portion of the DMSO stock solution was transferred from the 96-well plate to
a 384-well plate. Three other 96-membered parallel libraries (JKM VI 239 for the NIH
described in Chapter 4 and JKM VI 241 and JKM VI 265 for B c1-xl from Chapter 5)
were added to the plate, and the resulting foldamer compound collection (JKM VI 297)
was screened in the TGFP3/TPRII assay.
Figure 13. Left-handed 14-helical (3-peptide library design (JKM V I293 ,2
* 2 x 2 x 2 x 3 * 2 = 96).
6.3.3 Screening Results for Foldam er Compound Collection
Compounds from the collection showed three different effects in the
TGFp3/TpRII HTRF assay.
A majority had no effect on TGFp3 binding to TpRII.
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364
Some inhibited the fluorescence of the europium chelate (615 nm), disrupting the
assay readout (665 nm), perhaps due to acidification of the assay mixture. However, at a
100 pM concentration, 65 compounds nearly eliminated the FRET signal (potentially
100% inhibition of TGFp3/T(3RII binding). However, when these “hits” were diluted 1:3
and re-tested, they showed no inhibition. The same compounds were tested in a different
assay (Smad-FAST) and showed strong inhibition. It was concluded that the “hits” were
acting through a non-specific mechanism. About half of the “hits” were from the library
deposited at the NIH (Chapter4), and the other half had come from one of the libraries
designed for Bcl-xL (JKM VI 241, Chapter 5). It is interesting to note that the latter
compounds had been perfectly well-behaved in the FP assay for Bcl-xL/Bak, perhaps
because they had been screened at significantly lower concentrations.
Finally, no
compounds from the 14-helical p-peptide library designed to inhibit the TGFp3/TpRII
interaction showed any activity.
6.3.4 Second Generation 14-Helical P-Peptide L ibrary
A second, more general library of P-peptides was designed and synthesized
(Figure 14). We had learned from previous work on Bcl-xL inhibitors that changing the
nature and size of hydrophobic groups often results in only very small changes in binding
affinity.
However, if a charged residue is buried in a hydrophobic pocket, then the
binding affinity is significantly weakened. To take advantage of this idea, we developed
a strategy for performing a combinatorial “hydrophile scan” via parallel library synthesis.
In the 14-helical conformation, library members should display p3-hlle2 and p3-hlle8 on
one face of the helix, which is designed to bind in the bottom of the cleft on TGFP3 by
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365
mimicking TpRII residues Ile50 and Ile53, respectively. With that hydrophobic face
acting as an anchor, all the other positions in the library were varied between an acidic
residue (p3-hGlu) and a hydrophobic residue (p3-hLeu or p3-hPhe). It was anticipated
that a hit from this library would reveal an appropriate pattern of charged and
hydrophobic residues for binding to TGFP3. The generality of this design did not bias
the library members toward binding in either an N-^C or C->N orientation within the
cleft; the library also included both amphipathic and scrambled sequences. A maximum
number of negatively charged residues were incorporated to improve the solubility of the
compounds relative to previous 14-helical library (Figure 13). The library (JKM VII 035,
Figure 14) was synthesized with microwave irradiation and screened for activity.
nh2
nh2
Figure 14. Second generation library of potential 14-helical P-peptide inhibitors (JKM V II035, 2 x 2 x 3
x 2 x 2 x 2 = 96).
6.3.5 Screening Results for Second Generation 14-Helical p-Peptide L ibrary
Initial screening identified a number of weak hits from the second generation 14helical p-peptide library. The library was tested without purification in the TGFP3/TPRII
assay, and 14 compounds showed > 10% inhibition at a concentration of 160 pM (Figure
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
366
15). Although this is a low level of activity, examination of the sequences of the hits
suggested a possible structure/activity relationship (Table 3). For example, all 14 hits
had p3-hPhe at position 7. Most of the hits differed from one another by substitution at
only one or two positions. The hits were re-synthesized (JKM VII 073 and JKM VII
077), but HPLC purification proved challenging.
40
-..............
35 30 --------------------25 -
-15 J...
Com pound
Figure 15. Screening data for second generation 14-helical P-peptide library (JKM V II035) at
approximately 160 pM in TGFP3/TPRII assay after 4 hr.
Table 3. Sequence and activity of hits from initial screening of second generation 14-helical P-peptide
library (JKM VII 035).
Com pound % Inhibition (160 pM)
D06
F08
H12
H11
D07
D08
H01
D05
D12
B07
H08
H06
H07
B08
33
20
20
20
19
15
15
14
13
12
12
11
11
10
S eq u en ce
N
1
2
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
p^-Glu
pJ-Glu
p’ -Glu
p’ -Glu
P’-Glu
Pj -Glu
P’ -Glu
P’ -Glu
p J-Glu
P’-Glu
p ’-Glu
P’-Glu
P’-Glu
P"-Glu
p J-Leu
p J-Leu
p’ -Leu
3
pJ-lle
p^-lle
pJ-lle
P^-He
pJ-lle
pJ-Leu
P He
pJ-lle
p'-lle
p’ -Leu
pJ-Leu
P’ -Leu
P“-Leu
P -He
pJ-lle
P^-He
pJ-lle
P’-lle
i“-lle
4
p’-Glu
BJ-Leu
B’-Leu
B’-Leu
B’-Leu
BJ-Leu
P’ -Glu
P’ -Glu
B’-Leu
B’-Leu
B’-Leu
pJ-Glu
B’-Leu
r- L e u
5
B’-P h e
B^-Phe
P’ -Leu
p’ -Leu
P’-P h e
j’-P h e
P’-Glu
P’-P h e
P’-Leu
P’-P h e
P’-P h e
P’-P h e
P’-P h e
r-P h e
6
p’-Ala
p J-Ala
S’ -Ala
B’ -Ala
P’ -Ala
P’ -Ala
P’ -Ala
P’ -Ala
p-’-Ala
P°-Ala
p’ -Ala
p’ -Ala
p’ -Ala
r- A la
7
8
p’-P h e
p -P h e
|S -P h e
p’-P h e
p -P h e
I1-P h e
-P h e
1 Phe
p’-P h e
1 -P h e
p’-P h e
J -P h e
I Phe
fl J -P h e
p’-Leu
p J-Glu
; P’-Leu
p ’-Leu
P’-Leu
B’-Leu
p ’-Leu
B’-Leu
p ’-Leu
p ’ -Glu
p ’-Leu
p ’-Leu
P’-Leu
p“-Glu
9
pJ-lle
pJ-lle
pJ-lle
pJ-lle
pJ-lle
pJ-lle
pJ-lle
pJ-lle
pJ-lle
pJ-lle
pJ-lle
pJ-lle
pJ-lle
p -lie
10
P’-Leu
P’-L eu
P’-Leu
p J-L eu
P’ -Leu
p-’-Leu
P’ -Leu
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11
C
P^G Iu
pJ-Glu
p ’-G lu
P’-G lu
P’-G lu
pJ-G lu
p-’-G lu
P’-G lu
P’-G lu
P’-G lu
P’-G lu
p ’-G lu
P’-G lu
P"-Glu
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
367
6.3.6 H PLC Purification of H ydrophobic/A nionic p-Peptides
Modification of the B solvent facilitated HPLC purification of the re-synthesized
hits from the second generation 14-helical P-peptide library. The P-peptides eluted as
very broad peaks at very high concentrations of B solvent with the standard HPLC
buffers (A Solvent: water, B Solvent: acetonitrile, 0.1% TFA in each).
Under these
acidic conditions the side chains of the p3-hGlu residues are protonated, making them
minimally hydrophilic, at best.
Switching from TFA to a triethylammonium acetate
(TEAA) buffer (pH 6.5) converted the p3-hGlu side chains to the carboxylate anion and
greatly reduced the concentration of B solvent required for elution. We identified an
inverse correlation between retention time and the number of p3-hGlu residues in the
sequence (Figure 16). We were able to purify the seven P-peptides containing either
three or four p 3 -hGlu residues. The sequences containing only two p 3 -hGlu residues, one
at the each terminus, were abandoned for being too hydrophobic.
Unfortunately,
lyophilization of TEAA-containing P-peptide mixtures often resulted in a glassy
1200
600
200
0
5
10
15
20
25
30
35
40
45
50
55
60
65
75
80
Tim e (min)
Figure 16. HPLC traces of P-peptides containing 4, 3, and 2 p3-hGlu residues eluted with A Solvent: water
plus 100 mM TEAA (pH 6.5), B Solvent: acetonitrile, 1% per minute gradient, starting at 10% B solvent.
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
368
material rather than a powder, in which case aqueous acetic acid was added to the Ppeptide followed by a second lyophilization before screening for inhibition of
TGFP3/TPRII.
We eventually adopted an HPLC solvent system with a more
hydrophobic B solvent that both reduced retention times and afforded a nicely lyophilized
powder (Solvent A: water, Solvent B: 1/1 acetonitrile/isopropanol, 0.1% TFA in each).
6.3.7 Evaluation of Hits from Second Generation 14-Helical P-Peptide L ibrary
Re-synthesis and testing of the hits from the second generation 14-helical ppeptide library resulted in the identification of a very weak potential lead compound.
Three of the seven re-synthesized and purified hits showed some activity in the
TGFp3/TpRII assay (Table 4).
Based on these results, the active compounds were
further investigated through generation of a dose-response curve (Figure 17). The most
active compound, JKM VII 073 2, was selected for further optimization. Nine different
single substitution analogues were synthesized varying the N- and C-termini,
incorporating P3 -hGlu at the positions that were constant in the library (p 3 -hlle3, P3 3
3
hAla6, and p -hlle9), and increasing the hydrophobicity at positions 5 and 7 with p -hTrp
(JKM VII 105). However, none of these P-peptides showed any activity, including a new
batch of the lead compound (Table 5). The original batches of the P-peptide hits were re­
tested (Table 4), and JKM VII 073 2 dropped to being only 7% active at 200 pM as
opposed to 40% active at 100 pM 6 weeks previously. The second most active hit from
the second generation library JKM VII 077 5 (6-4, Figure 18), which differs from JKM
VII 073 2 only by the deletion of p3-hLeulO, showed similar results in both rounds of
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
369
testing. A new batch of this compound was synthesized and purified; however, its
testing was delayed due to re-optimization of the TGF|33/TpRII assay conditions.
Table 4. Assay results for re-synthesized, purified hits from second generation 14-helical P-peptide library
(JKM VII 035).
Cm pd.
JK M VII
35
% Inhib,
(-1 6 0 pM)
D 06
33
Cm pd. Purification
JK M VII (HPLC/Lyo)
077 1
TEAA
TEAA/AcOH
% Inhib.
(100 pM)
11/23/05
% Inhib.
(200 pM)
1/3/06
-3
4
3
1
A c |53-GIu p 3-L eu p 3-lle p 3-Glu p 3- P h e p 3-Ala p 3- P h e p 3-L eu p 3-lle
N
1
2
3
4
5
Sequence
6
7
8
9
10
11
C
p 3-Glu NH2
F0 8
20
073 2
TFA
40
7
A c PJ-G lu p 3-L eu p 3-lle p J-L eu p J- P h e p 3-Ala p 3- P h e p 3-G lu p 3-lle p J-L eu p J-Glu NH2
H01
15
073 7
TFA
-1 3
-8
A c p 3-G lu
p 3-lle p J-Glu
D 05
14
077 2
TEAA/AcOH
-4
0
A c p 3-G lu
p 3-lie p “-Glu p 3-P h e p 3-Ala p 3- P h e p 3-L eu p 3-lle
p J-Glu N H 2
B07
12
077 3
TEAA/AcOH
-4
3
A c p 3-G lu
P3-Ile p J-L eu p 3- P h e p 3-Ala p 3- P h e p J-Glu p 3-lle
p 3-Glu NH2
H 06
11
077 4
TEAA/AcOH
14
-
A c p J-G lu p J-L eu p 3-lle p J-Glu p 3-P h e p J-A la p J- P h e p J-L eu p 3-lle p 3-L eu p J-G lu NH2
B 08
10
077 5
TEAA
TEAA/AcOH
17
5
23
-
p J-Glu
p 3-Ala p 3- P h e p 3-L eu p 3-lle p 3-L eu p 3-Glu N H 2
A c p 3-GI u p 3-L eu p 3-lle p 3-L eu p 3-P h e p 3-A la p 3- P h e p 3-Glu p 3-lle
p 3-Glu NH2
50
40
30
- ♦ — JKM VII 073 2
.n 20
!c
c
*«ll “ JKM VII 077 5 TEAA
JKM VII 0774
“ iti—
10
0
100
■10
Concentration (pM)
Figure 17. Dose-response curves for weak potential leads from second generation 14-helical P-peptide
library.
Table 5. Assay results for analogues of 14-helical P-peptide lead compound. Note: JKM VII 105 1 is a
newly synthesized batch of JKM V II073 2.
C om pound % Inhibition
(200 pM)
JKM VII 105
1
2
3
4
5
6
7
8
9
10
-1
1
7
-8
-6
-8
5
-8
1
1
N
1
Ac
Ac
Ac
p J-G lu
(V-Glu
p 3-G lu
p 3-G lu
P 3-G lu
pV G Iu
p 3-G lu
p°-G lu
p 3-Glu
p J-Glu
A c-^-G lu
Ac
Ac
Ac
Ac
Ac
H2N
2
3
4
p J-L eu pV lle p 3-L eu
p J-L eu p J-lle p J-L eu
p 3-L eu p N Ie P3-L eu
p 3-L eu p°-lle p 3-L eu
p 3-L eu pV lle p 3-L eu
p J-L eu p 3-lle P3-L eu
p J-Leu p J-lle p J-L eu
p J-L eu pJ-Glu pJ-L eu
p J-Leu pM Ie pJ-L eu
p 3-Leu p J-lle p 3-L eu
5
Sequence
6
7
p J-P h e p J-Ala p 3-P h e
p J-P h e p 3-A la p 3-P h e
p ° -P h e p J-A la p J-P h e
p J-P h e p 3-Ala p J-P h e
pJ-P h e p3-Glu pJ-P h e
p3-Trp fV-Ala p J-P h e
p 3-P h e fi'5-Ala pJ-Trp
p 5-P h e p ’-A la p 3-P h e
p J- P h e pV A Ia p J-P h e
p V P h e p J-A la p 3-P h e
8
9
10
p J-Glu p v ile p 3-L eu
p J-Glu p J-lle p 3-L eu
p 3-Glu p 3-lle pV L eu
p 3-Glu p 3-lle p 3-L eu
fV-Glu P j""e P3-L eu
p 3-Glu P -lie p J-L eu
p J-Glu p 3-lle p 3-L eu
p°-G lu p 3-lle pJ-L eu
p 3-G lu P°-Glu P3-L eu
p 3-G lu p J-lle p 3-L eu
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
11
C
p 3-Glu
nh2
p 3-Glu
OH
p^-Glu 0 3-Glu-NH?
p J-Glu
NH,
p J-Glu
nh2
p 3-Glu
nh2
p 3-Glu
nh2
P3-Glu
nh2
p 3-Glu
nh2
p 3-Glu
nh2
370
6.3.8 Sum m ary of 14-Helical (3-Peptide Designs
The potential for the 14-helical P-peptide scaffold to inhibit the TGFP3/TPRII
interaction has been explored, but no clear success has been achieved to date. TGFp
remains an interesting and challenging therapeutic target. Microwave-assisted parallel
synthetic techniques (Chapter 4) were extremely useful for the rapid preparation of 14helical P-peptide libraries in acceptable purities, and these methods have been adopted by
other members of the research group. Advances were made in the areas of library design
through the concept of combinatorial hydrophile scanning (i.e., incorporating either a
charged or hydrophobic residue at each position of the library). Conditions for HPLC
purification of hydrophobic, anionic peptides were developed.
These efforts have
resulted in the identification of P-peptide 6-4 (JKM VII 073 2, Figure 18) as a potential
weak lead. However, as mentioned previously, the HTRF assay for TGFp3/TpRII needs
additional optimization to improve the reliability of screening crude peptide mixtures.
Figure 18. Structure o f P-peptide 6-4, a potential weak lead for the inhibition o f TGFP3/TPRII.
6.4 Conclusions and Future Directions
A suitable foldamer scaffold for inhibition of the TGFP3/TPRII interaction has
not yet been identified. We have found that selecting the appropriate scaffold is the most
critical step for developing foldamer inhibitors of protein-protein interactions and that
this selection cannot always be made based on molecular modeling.
20
To date, designs
have focused on the P-peptide 14-helix, but investigation of several other helical
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
371
foldamer scaffolds (e.g., the P-peptide 12-helix, a/p-peptide 11- and 14/15 helices,
and a/a/p-peptide helix) for mimicking the p-sheet epitope of the TGFP3/TPRII
interaction are underway. (Of note is a report of the reverse scenario: a p-sheet was
designed that successfully mimicked the function of the a-helical segment of p53 in
binding to MDM2.21)
Combinatorial exploration of the different structural scaffolds
using newly optimized assay conditions represents the best opportunity for identifying an
inhibitor. The reverse turn length and rigidity should be explored further. New designs
for potential inhibitors could be generated by attempting to mimic the display of
important interfacial TGFp3 residues, rather than trying to mimic residues from TpRII as
has been described. Finally, new structural information on the heteromeric complex of
dimeric TGFp3 with TpRII and TpRI could present new surfaces for targeting with
foldamer inhibitors (Hinck et al., unpublished results). TGFp3 is a therapeutically import
target, and a continued effort toward modulation of its activity is justified.
6.5 Experimental Methods
6.5.1 General Procedures
The general procedures used for the attempted development of inhibitors of the
TGFp3/TpRII interaction are the same as described in Chapter 5, with one addition:
Fmoc-Ile-Thr(\|/Me,Mepro)-OH was purchased from Novabiochem.
6.5.2 Multimode Microwave a - and P-Peptide Synthesis
NovaSyn TGR resin (10 pmol, 40 mg) was placed in a polypropylene SPE tube (4
mL, Alltech) and swelled with DMF for ~ 10 min. The resin was washed (5 x DMF, 5 x
CH2C12 and 5 x DMF). In a separate vial, Fmoc-P-amino acid (30 pmol) was activated
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
372
by adding HBTU (60 pL of 0.5 M solution in DMF), DMF (440 pL), HOBt (60 pL of
0.5 M solution in DMF), and /P^EtN (60 giL of 1.0 M solution in DMF). The mixture
was vortexed and added to the resin. A magnetic stir bar (8 mm, VWR) was placed
inside the tube. The vessel was placed inside an empty polypropylene 50 mL centrifuge
tube and placed in one slot of a 52-position turntable inside the multimode microwave
reactor (CEM MARS). The fiber optic temperature sensor was suspended in the reaction
mixture above the stir bar by pressing it through a small hole (made with a needle) in the
plastic top cap of the SPE tube and placing the cap loosely on the reaction vessel. The
sample was irradiated in the microwave reactor (600 W maximum power, 70°C, ramp 2
min, hold 4 min). All microwave irradiations were conducted at atmospheric pressure.
The tube was removed from the microwave reactor, and the resin was washed as before.
Deprotection solution (750 pL of 20% piperidine in DMF (v/v)) was added to the resin,
and the sample was irradiated (600 W maximum power, 80°C, ramp 2 min, hold 2 min).
The coupling/deprotection cycle was repeated in a stepwise manner until reaching the
desired length of the peptide.
6.5.3 a- and |3-Peptide Cleavage, W ork-Up and HPLC
After the final residue had been added and deprotected, the resin was washed (5 x
DMF, 5 x CII 2 CI2 , 5 x DMF and 5 x CH2C12). An acetylation cocktail was prepared
containing 1.4 mL CH2C12, 0.1 mL TEA, and 0.5 mL acetic anhydride. Acetylation was
accomplished by adding 1.0 mL of the cocktail to the resin and shaking for 15 min. After
washing the resin a (5 x CH2C12), the peptide was cleaved from the solid support with
simultaneous
side
chain
deprotection
(3
mL,
45:45:5:5
trifluoroacetic
acid
(TFA):CH2Cl2:triethylsilane:water, 2 h, RT, with rocking). The cleavage solution was
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
373
drained and concentrated under a stream of N2. The crude peptide mixture was
dissolved in 1.0 mL DMSO and analyzed by HPLC (30 pL injection, Shimadzu). The
C4 -silica reverse-phase analytical column (5 pm, 4 mm x 250 mm, Vydac) was eluted
with a gradient of acetonitrile in water (10 - 60%, 50 min, 0.1% TFA in each) at a flow
rate of 1 mL/min. Anionic peptides were eluted from a C4 -column with a gradient of
acetonitrile in water (10 - 60%, 50 min, 100 mM triethylammonium acetate (TEAA) of
pH 6.5 in each).
Alternatively, anionic peptides were eluted with a gradient of 1:1
acetonitrile/isopropanol in water (10 - 60%, 50 min, 0.1% TFA in each). Product purity
was determined as peak area percent by integration of the UV absorbance at 220 nm.
Integration was performed over the 15 - 50 min time interval to exclude the large
absorbance of DMSO that elutes from 5 - 1 5 min.
The major peaks in the HPLC
chromatogram were collected, and peptide masses were measured by MALDI-TOF-MS
(Bruker Reflex II, a-cyano-4-hydroxycinnamic acid matrix).
R e p r o d u c e d with p e r m i s s io n of t h e co p y rig h t o w n e r. F u r th e r r e p ro d u c tio n prohibited w ith o u t p e rm is s io n .
6.5.4 Peptide C haracterization D ata
Table 6. Characterization data for a-peptide controls.
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375
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Figure 19. Testing of an a-peptide positive control. Percent binding of TGFfl3 to TpRII in blue (data
values at the top of each bar and concentration indicated on x-axis), and Smad3 binding to peptide from
FoxHl transcription factor in grey. Compound 503 is an inhibitor o f Smad3/FoxHl and JKM V II039-1 is
A c-ETWIWDLVVVN-NH2 from ref. 9. The peptide inhibited the TGFp3/TpRII interaction but was more
active against Smad3/FoxHI.
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
Table 7. Raw assay data for a-peptide controls (Figure 19). Percent binding —100 *
(LANCE/LANCE non Inhibitor). LANCE = normalized fluorescence, taking into account the fluorescence
of a blank containing the assay buffer.
C om pound
T ype
LB
LC
M
LH
LH
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
No protein
No inhibitor
No inhibitor
JKM VII 039-1 1:1
JKM VII 039-1 1:2
JKM VII 039-1 1:3
JKM VII 039-1 1:4
JKM VII 039-1 1:9
5uM 503
10uM 503
20uM 503
3nM T G F b l
6nM T G F b l
10nM T G Fbl
1ul JKM V II039-2
1ul JKM VII 039-3
1ul JKM VII 039-4
1ul JKM VII 039-5
1ul JKM VII 045-1C
1ul JKM VII 045 2B
LANCE
High
Count
615
(C o u n ts)
38
1 7 065
15131
14381
14098
11132
14606
13904
1 4440
14775
14122
14089
14095
14775
14603
1 4 839
LANCE
High
Count
665
( C o u n ts)
10
67
66
447
51 9
45
56
32 9
39 5
476
505
468
429
235
153
141
T G F -b e ta
/R e c e p to r
1 2:09
-48
0
5
385
46 7
-3
-3
27 9
332
402
45 2
41 5
376
169
92
78
iip h -o e ta
and
R e c e p to r
3 :4 9 p lu s
10nM IC5
b e ta 3
-51
0
-4
1079
1122
6
29
747
1018
1051
1243
1197
1167
513
254
190
T G F -b e ta
/R e c e p to r
p lu s 66nM
IC5 b e ta 3
4 :5 6
-41
0
-1
1525
1643
11
122
1414
1548
1583
1548
1694
1541
1018
613
481
P e rce n t
B in d in g
100
1
3
68
93
96
113
109
106
47
23
17
Sm ad
S m a d FoxH1
FoxH1 12:12
3:53
-36
-41
0
0
149
128
2182
2059
2171
2041
153
137
195
127
243
222
557
290
1006
471
1659
1617
1296
1385
245
180
2152
2073
2105
1913
2084
1952
1416
1437
7 56
564
1635
1835
A v e ra g e
P e rce n t
B in d in g
2050
100
0
0
•5
8
18
77
65
3
101
93
95
67
68
33
23
78
89
1200
t
24.0
24.5
25.0
25.5
28.5
29.5
tones
Figure 20. HPLC traces o f Fmoc-Gly-Glu-Leu-Ser-Lys-Phe-Cys-Asp-NH2 synthesized with microwave
irradiation (JKM VII 003).
Table 8. Characterization data for synthetic optimization of peptide 6-1 (JKM VII 007, 027, and 031).
C om pound
6-1
[M+H]+
N
1 6 8 5 .5
1 6 8 7 .5
Ac
Ac
1 8 6 7 .7
1 7 6 5 .2
1 6 7 7 .6
1 2 4 1 .3
S eq u en ce
C
N o te s
C y s S e r lie T h r S e r lie S e r G ly G ly G lu L eu S e r Lys P h e C y s A s p N H 2 o x id iz e d
C y s S e r lie T h r S e r lie S e r G ly G ly G lu L eu S e r L ys P h e C y s A sp N H 2 r e d u c e d
F m o c C y s S e r lie T h r S e r lie S e r G ly G ly G lu L e u S e r L ys P h e C y s A sp N H 2
Fm oc
S e r lie T h r S e r lie S e r G ly G ly G lu L e u S e r L ys P h e C y s A sp N H 2
Fm oc
lie T h r S e r lie S e r G ly G ly G lu L eu S e r L ys P h e C y s A sp N H 2
Fm oc
S e r He S e r G ly G ly G lu L e u S e r L ys P h e C y s A sp N H 2
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
377
1000
800:
Mreci
600:
Abs
(mV)
M red
400;
M ox
200 ;
16
18
20
24
22
26
28
30
Time (min)
Figure 21. HPLC trace o f 6-1 after stirring for 48 hr A) open to air and B) in a capped vial (JKM V II 031).
6.5.5 Microwave-Assisted Parallel P-Hairpin Library Synthesis
For the a-peptide P-hairpin library (JKM VII 033, Figure 3 and Figure 4),
NovaSyn TGR resin (250 pmol, 1 g) was suspended in 50 mL o f a 3:2 mixture of
dichloromethane/DMF.
The slurry was stirred while 500 pL aliquots are dispensed into
each well o f a 2 mL deep well polypropylene filter plate with polyethylene frits and long
drip spouts sealed with a bottom mat (Artie White) using a pipette.
The resin was
washed (5 x DMF). A magnetic stir bar (7 mm, VWR) was placed inside each well. In a
separate vial, Fmoc-(5)-Asp(tBu)-OH (296.3 mg, 720 pmol) was activated by adding
HBTU (1440 pL of 0.5 M solution in DMF), DMF (10.56 mL), HOBt (1440 pL o f 0.5 M
solution in DMF), and z'P^EtN (1440 pL o f 1.0 M solution in DMF). The mixture was
vortexed, and 150 pL was added to each well using a using a 12-channel multipipette.
The plate was placed on a microtiter plate turntable inside the multimode microwave
cavity (CEM MARS). The fiber optic temperature probe was positioned in well D6 using
the arm attached to the turntable, and the sample was irradiated (600 W maximum power,
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
378
70°C, ramp 2 min, hold 4 min).
All microwave irradiations were conducted at
atmospheric pressure. The plate was removed from the microwave reactor, and the resin
was washed (5 x DMF).
The bottom sealing mat was re-affixed, and deprotection
solution (250 pL o f 20% piperidine in DMF (v/v)) was added to the resin in each well.
The temperature probe was placed in the center o f this region of the plate, and the sample
was irradiated (600 W maximum power, 80°C, ramp 2 min, hold 2 min). After washing,
the coupling/deprotection cycle was repeated with Fmoc-(S)-Cys(Trt)-OH, Fmoc-(6)Phe-OH, Fmoc-(5)-Lys(Boc)-OH, Fmoc-(5)-Ser(tBu)-OFI, and Fmoc-(5)-Leu-OH. Two
different residues, Fmoc-(5)-Gln(Trt)-OH and Fmoc-(5)-Glu(tBu)-OH, were coupled one
after the other (i.e., sequentially) to different halves o f the library members (Figure 22).
The material in all wells was simultaneously Fmoc-deprotected as before. The different
turn residues were incorporated, followed by Fmoc-(5)-Lys(Boc)-OH or Fmoc-(A)Ser(/Bu)-OH at position 7, Fmoc-(S')-Trp(Boc)-OII, Fmoc-(6)-Phe-OH, or Fmoc-(*S)-IleOFI at position 6, and Fmoc-(£)-Ser(fBu)-OFI at position 5. A pseudoproline dipeptide
(Fmoc-Ile-Thr(vj/Mc,Mcpro)-OH) was then incorporated, followed by double coupling of
Fmoc-(5)-Ser(®u)-OH to half the library members and double coupling o f Fmoc-(S)Cys(Trt)-OFI to all library members.
Following Fmoc-deprotection, the resin was
washed (5 x DMF, 5 x CH 2 CI2 ), and the peptides were acetylated for 15 min at room
temperature with stirring (150 pL o f 14:1:5 CH 2 CI2 /TEA/AC2 O). After washing (5 x
CH 2 CI2 ), cleavage from the solid support with global side chain deprotection was
accomplished by adding triisopropylsilane (50 pL), water (50 pL), and trifluoroacetic
acid (1.0 mL) to each well. The plate was wrapped tightly in aluminum foil and stirred
for 2 hr at room temperature on a stir plate. The foil covering was removed, and the
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
379
cleavage solutions were transferred to a solid-bottom deep well 96-well plate by
gravity filtration and concentrated using a rotary evaporator (SpeedVac with well-plate
adapter, Thermo Savant). The crude peptide mixtures were dissolved in 250 pL DMSO
and stirred open to air for 72 hr to accomplish cyclization via disulfide bond formation.
Several samples were analyzed by HPLC (15 pL injection, Shimadzu).
The C4-silica
reverse-phase analytical column (5 pm, 4 mm x 250 mm, Vydac) was eluted with a
gradient o f acetonitrile in water (10 - 60% or 0 - 50% B solvent, 25 min, 0.1% TFA in
each, followed by a 5 min flush with 95% acetonitrile and 5 min equilibration at the
starting concentration) at a flow rate o f 1 mL/min. The major peaks from the HPLC
chromatogram were collected, and peptide masses were measured by MALDI-TOF-MS
(Bruker Reflex II, a-cyano-4-hydroxycinnamic acid matrix).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
380
Position
Position
Position
Position
Position
Position
Position
17:
16:
15:
14:
13:
12:
11:
Asp
Cys
Phe
Lys
Ser
Leu
1
2
10
11
12
A
B
C
D
E
F
G
H
Position 10
Position 9:
Position 8:
Position 7:
Position 6:
Position 5: Ser
Positions 4 and 3: lie-Thr
Position 2:
Position 1: Cys
F ig u re 22. Spatial addressing for synthesis o f [3-hairpin library (JKM V II 033, Figure 3 and Figure 4).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
381
T ab le 9. Sequence o f P-hairpin library members 1-48 (JKM VII 033, Figure 3 and Figure 4).
Compound Well
1
N
1
2
3
4
5
2
A01
A02
Ac Cys Ser lie Thr Ser
Ac Cys null lie Thr Ser
3
A03
Ac Cys
4
A04
Ac Cys
null lie Thr Ser
5
A05
6
7
8
Sequence
9 10 11
12
13
14
15
16
17
C
Trp
Ser Asn
Trp
Ser Asn
Gly
Gly
Ser lie Thr Ser Trp
Lys Asn
Gly
-
Glu Leu Ser Lys Phe Cys Asp NHp
Glu Leu Ser Lys Phe Cys Asp NHp
Glu Leu Ser Lys Phe Cys Asp NH?
Lys Asn
Gly
-
Glu Leu Ser Lys Phe
Cys Asp NHp
Ac Cys Ser lie Thr Ser Phe Ser Asn
Gly
-
Glu Leu Ser Lys Phe
Cys Asp NH?
Trp
-
6
A06
Ac Cys
null lie Thr Ser Phe
Ser Asn
Gly
-
Glu Leu Ser Lys Phe
Cys Asp NH?
7
A07
Ac Cys Ser lie Thr Ser Phe
Lys Asn
Gly
-
Glu Leu Ser Lys Phe
Cys Asp NHp
8
A08
Ac Cys
Lys Asn
Gly
-
Glu Leu Ser Lys Phe
Cys Asp NH?
9
A09
Ac Cys Ser lie Thr Ser
lie
Ser Asn
Gly
-
Glu Leu Ser Lys Phe
Cys Asp NH?
10
A10
Ac Cys
null lie Thr Ser
lie
Ser Asn
Gly
-
Glu Leu Ser Lys Phe
Cys Asp NH?
11
A11
Ac Cys Ser lie Thr Ser
lie
Lys Asn
Gly
-
Glu Leu Ser Lys Phe
Cys Asp NH?
12
A12
Ac Cys null lie Thr Ser
lie
Lys Asn
Gly
-
Glu Leu Ser Lys Phe
Cys Asp NHp
13
B01
Ac Cys
Ser lie Thr Ser Trp
Ser
Gly
Asp Pro Glu Leu Ser Lys Phe
Cys Asp NH?
14
B02
Ac Cys null lie Thr Ser Trp
Ser
Gly
Asp Pro Glu Leu Ser Lys Phe
Cys Asp NHp
15
B03- Ac Cys Ser lie Thr Ser
Trp
Lys
Gly
Asp Pro Glu Leu Ser Lys Phe
Cys Asp NHp
16
B04
Trp
Lys
Gly
Asp Pro Glu Leu Ser Lys Phe
Cys Asp NHp
17
BOS Ac Cys Ser lie Thr Ser Phe Ser
Giy
Asp Pro Glu Leu Ser Lys Phe
Cys Asp NH?
18
B06
Ac Cys
Gly
Asp Pro Glu Leu Ser Lys Phe
Cys Asp NH?
19
B07
Ac Cys Ser lie Thr Ser Phe
Gly
Asp Pro Glu Leu Ser Lys Phe
Cys Asp NHp
20
B08
Ac Cys
Lys
Gly
Asp Pro Glu Leu Ser Lys Phe
Cys Asp NHp
21
B09
Ac Cys Ser lie Thr Ser
lie
Ser
Gly
Asp Pro Glu Leu Ser Lys Phe
Cys Asp NHp
22
B10
Ac Cys
lie
Ser
Gly
Asp Pro Glu Leu Ser Lys Phe
Cys Asp NH?
23
B11
Ac Cys Ser lie Thr Ser
lie
Lys
Gly
Asp Pro Glu Leu Ser Lys Phe
Cys Asp NHp
24
B12
Ac Cys null lie Thr Ser
lie
Lys
Gly
Asp
Pro Glu Leu Ser Lys Phe
Cys Asp NH?
25
C01
Ac Cys Ser lie Thr Ser
Trp
Ser
Gly
Gly
-
Glu Leu Ser Lys Phe
Cys Asp NHP
26
C02
Ac Cys null lie Thr Ser
Trp
Ser
Gly
Gly
-
Glu Leu Ser Lys Phe
Cys Asp NH?
27
C03
Ac Cys Ser lie Thr Ser
Trp
Lys
Gly
Gly
-
Glu Leu S er Lys Phe
Cys Asp NHp
28
C04
Ac Cys null lie Thr Ser
Trp
Lys
Gly
Gly
-
Glu Leu Ser Lys Phe
Cys Asp NHp
Ac Cys
null lie Thr Ser Phe
null lie Thr Ser
null lie Thr Ser Phe Ser
null lie Thr Ser Phe
null lie Thr Ser
Lys
29
C05
Ac Cys Ser lie Thr Ser Phe Ser
Gly
Gly
-
Glu Leu Ser Lys Phe
Cys Asp NHp
30
C06
Ac Cys null lie Thr Ser Phe
Ser
Gly
Gly
-
Glu Leu Ser Lys Phe
Cys Asp NH?
Cys Asp NHp
31
C07
Ac Cys Ser lie Thr Ser Phe
Lys
Gly
Gly
-
Glu Leu Ser Lys Phe
32
C08
Ac Cys null lie Thr Ser Phe
Lys
Gly
Gly
-
Glu Leu Ser Lys Phe
Cys Asp NHp
33
C09
Ac Cys Ser lie Thr Ser
lie
Ser
Gly
Gly
-
Glu Leu Ser Lys Phe
Cys Asp NHp
Cys Asp NHp
34
C10
Ac Cys null lie Thr Ser
lie
Ser
Gly
Gly
-
Glu Leu Ser Lys Phe
35
C11
Ac Cys Ser lie Thr Ser
lie
Lys
Gly
Gly
-
Glu Leu Ser Lys Phe
Cys Asp NHp
36
C12
Ac Cys null lie Thr Ser
lie
Lys
Gly
Gly
-
Glu Leu Ser Lys Phe
Cys Asp NHp
37
D01
Ac Cys Ser lie Thr Ser
Trp
Ser
Gly
Gly
Gly Glu Leu Ser Lys Phe
Cys Asp NHp
38
D02
Ac Cys null lie Thr Ser
Trp
Ser
Giy
G!y
Gly Glu Leu Ser Lys Phe
Cys Asp NHp
39
D03
Ac Cys Ser lie Thr Ser
Trp
Lys
Gly
Gly
Gly Glu Leu Ser Lys Phe
Cys Asp NH?
40
D04
Ac Cys null lie Thr Ser
Trp
Lys
Gly
Gly
Gly Glu Leu Ser Lys Phe
Cys Asp NHp
41
D05
Ac Cys Ser lie Thr Ser Phe
Ser
Gly
Gly
Gly Glu Leu Ser Lys Phe
Cys Asp NHp
42
D06
Ac Cys null lie Thr Ser Phe Ser
Gly
Gly
Gly Glu Leu Ser Lys Phe
Cys Asp NHp
43
D07
Ac Cys Ser lie Thr Ser Phe
Lys
Gly
Gly
Gly Glu Leu Ser Lys Phe
Cys Asp NHp
44
D08
Ac Cys null lie Thr Ser Phe
Lys
Gly
Gly
Gly Glu Leu Ser Lys Phe
Cys Asp NHp
45
D09
Ac Cys Ser lie Thr Ser
lie
Ser
Gly
Gly
Gly Glu Leu Ser Lys Phe
Cys Asp NHp
46
D10
Ac Cys null lie Thr Ser
lie
Ser
Gly
Giy
Gly Glu Leu Ser Lys
Phe
Cys Asp NHp
47
D11
Ac Cys Ser lie Thr Ser
lie
Lys
Gly
Gly
Gly Glu Leu Ser Lys Phe
Cys Asp NHp
48
D12
Ac Cys null lie Thr Ser
lie
Lys
Gly
Gly
Gly Glu Leu Ser Lys Phe
Cys Asp NH?
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
382
T able 10. Sequence o f p-hairpin library members 49-96 (JKM VII 033, Figure 3 and Figure 4).
Com pound
W ell
49
E01
N
1
2
3
4
5
6
7
8
Ac Cys Ser lie Thr Ser Trp
Ser Asn
S equence
9
10
Gly
-
11
12
13
14
15
16
17
C
Gin Leu Ser Lys Phe
Cys Asp n h 2
Cys Asp NH?
50
E02
Ac Cys null lie Thr Ser Trp
Ser Asn
Gly
-
Gin Leu Ser Lys Phe
51
E03
Ac Cys Ser lie Thr Ser
Trp
Lys Asn
Gly
-
Gin Leu Ser Lys Phe
Cys Asp NHp
52
E04
Ac Cys null lie Thr Ser
Trp
Lys Asn
Gly
-
Gin Leu Ser Lys Phe
Cys Asp NHp
53
E05
Ac Cys Ser lie Thr Ser Phe
Ser Asn
Gly
-
Gin Leu Ser
Cys Asp n h 2
54
E06
Ac Cys
null lie Thr Ser Phe
Ser Asn
Gly
-
Gin Leu Ser Lys Phe Cys Asp NH?
55
Lys Phe
E07
Ac Cys Ser lie Thr Ser Phe
Lys Asn
Gly
-
Gin Leu Ser
Lys Phe Cys Asp NH?
56
E08
Ac Cys null lie Thr Ser Phe
Lys Asn
Gly
-
Gin Leu Ser
Lys Phe Cys Asp NH?
57
E09
Ac Cys Ser lie Thr Ser
He
Ser Asn
Gly
-
Gin Leu Ser Lys Phe Cys Asp NH?
58
E10
Ac Cys null lie Thr Ser
lie
Ser Asn
Gly
-
Gin Leu Ser Lys Phe Cys Asp NH?
59
E11
Ac Cys Ser lie Thr Ser
He
Lys Asn
Gly
-
Gin Leu Ser Lys Phe Cys Asp NH?
60
E12
Ac Cys null lie Thr Ser
He
Lys Asn
Giy
-
Gin Leu Ser Lys Phe Cys Asp n h 2
61
F01
Ac Cys Ser lie Thr Ser Trp
Ser
Gly
Asp
Pro Gin Leu Ser Lys Phe Cys Asp NHp
62
F02
Ac Cys null lie Thr Ser Trp
Ser
Gly
Asp
Pro Gin Leu Ser Lys Phe Cys Asp NH?
63
F03
Ac Cys Ser lie Thr Ser Trp
Lys
Gly
Asp
Pro Gin Leu Ser Lys Phe Cys Asp NH?
64
F04
Ac Cys null lie Thr Ser
Lys
Gly
Asp
Pro Gin Leu Ser Lys Phe Cys Asp NH?
65
F05
Ac Cys Ser lie Thr Ser Phe Ser
Gly
Asp
Pro Gin Leu Ser Lys Phe
Cys Asp NHp
66
F06
Ac Cys null lie Thr Ser Phe
Gly
Asp
Pro Gin Leu Ser Lys Phe
Cys Asp NH?
67
F07
Ac Cys Ser lie Thr Ser Phe
Lys
Giy
Asp
Pro Gin Leu Ser Lys Phe Cys Asp NH2
68
F08
Ac Cys null lie Thr Ser Phe
Lys
Gly
Asp
Pro Gin Leu Ser Lys Phe Cys Asp NH?
69
F09
Ac Cys Ser lie Thr Ser
lie
Ser
Gly
Asp
Pro Gin Leu Ser Lys Phe Cys Asp NHp
70
F10
Ac Cys null lie Thr Ser
He
Ser
Gly
Asp
Pro Gin Leu Ser Lys Phe Cys Asp NHp
71
F11
Ac Cys Ser He Thr Ser
lie
Lys
Gly
Asp
Pro Gin Leu Ser Lys Phe Cys Asp NHp
72
F12
Ac Cys null lie Thr Ser
He
Lys
Gly
Asp
Pro Gin Leu Ser Lys Phe Cys Asp NHp
73
G01
Ac Cys Ser lie Thr Ser
Trp
Ser
Gly
Gly
-
74
G 02 Ac Cys null lie Thr Ser Trp
Ser
Gly
Gly
-
Gin Leu Ser Lys Phe Cys Asp NHp
75
G 03 Ac Cys Ser lie Thr Ser Trp
Lys
Gly
Gly
-
Gin Leu Ser Lys Phe Cys Asp NH?
76
G04 Ac Cys null lie Thr Ser Trp
Lys
Gly
Gly
-
Gin Leu Ser Lys Phe Cys Asp NH?
77
G 05 Ac Cys Ser lie Thr Ser Phe Ser
Gly
Gly
-
Gin Leu Ser Lys Phe Cys Asp NHp
Trp
Ser
Gin Leu Ser Lys Phe Cys Asp n h 2
78
G06 Ac Cys null lie Thr Ser Phe Ser
Gly
Gly
-
Gin Leu Ser Lys Phe
79
G07 Ac Cys Ser lie Thr Ser Phe Lys
Gly
Gly
-
Gin Leu Ser Lys Phe
Cys Asp NHp
80
G08 Ac Cys null lie Thr Ser Phe Lys
Gly
Gly
-
Gin Leu Ser Lys Phe
Cys Asp NHp
81
G09 Ac Cys Ser He Thr Ser
lie
Ser
Gly
Gly
-
Gin Leu Ser Lys Phe
Cys Asp NHp
82
G 10 Ac Cys null He Thr Ser
lie
Ser
Gly
Gly
-
Gin Leu Ser Lys Phe Cys Asp NHp
Cys Asp NHp
83
G11
Ac Cys Ser lie Thr Ser
lie
Lys
Gly
Gly
-
Gin Leu Ser Lys Phe Cys Asp NHp
84
G12
Ac Cys null lie Thr Ser
lie
Lys
Gly
Gly
-
Gin Leu Ser Lys Phe Cys Asp NHp
85
H01
Ac Cys Ser He Thr Ser Trp
Ser
Gly
Gly
86
H02
Ac Cys null He Thr Ser
Trp
Ser
Gly
Gly
Gly Gin Leu Ser Lys Phe Cys Asp NHp
87
H03 Ac Cys Ser He Thr Ser Trp
Lys
Gly
Gly
Gly Gin Leu Ser Lys Phe Cys Asp NHp
Trp
Gly Gin Leu Ser Lys Phe Cys Asp NHp
88
H04
Ac Cys null lie Thr Ser
Lys
Gly
Gly
Gly Gin Leu Ser Lys Phe Cys Asp NHp
89
H05
Ac Cys Ser He Thr Ser Phe Ser
Gly
Gly
Gly
90
H06 Ac Cys null lie Thr Ser Phe Ser
Gly
Gly
Gly Gin Leu Ser Lys Phe Cys Asp NHp
Gin Leu Ser Lys Phe Cys Asp NHp
91
H07 Ac Cys Ser lie Thr Ser Phe Lys
Gly
Gly
Gly
Gin Leu Ser Lys Phe Cys Asp NHp
92
H08 Ac Cys null He Thr Ser Phe Lys
Gly
Gly
Gly
Gin Leu Ser Lys Phe Cys Asp NHp
93
H09 Ac Cys Ser He Thr Ser
Gly Gin Leu Ser Lys Phe Cys Asp NHp
94
95
H11
96
He
Ser
Gly
Gly
H10 Ac Cys null lie Thr Ser
He
Ser
Gly
Giy
Gly
Gin Leu Ser Lys Phe Cys Asp NHp
Ac Cys Ser lie Thr Ser
He
Lys
Gly
Gly
Gly
Gin Leu Ser Lys Phe Cys Asp NHp
H12 Ac Cys null He Thr Ser
lie
Lys
Gly
Gly
Gly Gin Leu Ser Lys Phe
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Cys Asp NH,
383
6.5.6 Characterization of f3-Hairpin Library
T able 11. Calculated masses o f p-hairpin library members (JKM VII 033, Figure 3 and Figure 4).
A
B
C
D
E
F
G
H
1
2
3
4
5
6
7
8
9
10
11
12
1 8 1 4 .5
1 7 2 7 .5
1 8 5 5 .5
1 7 8 2 .5
1 8 2 7 .5
1 6 7 2 .5
1 7 2 9 .5
1 7 2 8 .5
1 8 3 9 .5
1 6 8 4 .5
1 7 4 1 .5
1 7 5 2 .5
1 5 9 7 .5
1 6 5 4 .5
1 8 8 0 .5
1 7 2 5 .5
1 7 8 2 .5
1 6 9 5 .5
1 7 9 3 .5
1 8 1 4 .5
1 8 1 3 .5
1 8 1 6 .5
1 9 1 4 .5
1 7 5 9 .5
1 8 1 6 .5
1 8 1 5 .5
1 6 5 4 .5
1 9 5 3 .5
1 7 9 8 .5
1 8 5 5 .5
1 8 5 4 .5
1 6 8 8 .5
1 7 8 6 .5
1 6 3 1 .5
1 6 8 8 .5
1 6 8 7 .5
1 7 4 1 .5
1 8 2 5 .5
1 6 7 0 .5
1 7 2 7 .5
1 7 2 6 .5
1 7 7 5 .5
1 8 7 3 .5
1 7 1 8 .5
1 7 7 5 .5
1 7 7 4 .5
1 7 2 9 .5
1 9 1 2 .5
1 7 5 7 .5
1 7 6 8 .5
1 8 6 6 .5
1 7 1 1 .5
1 7 6 8 .5
1 7 6 7 .5
1 7 4 0 .5
1 9 1 1 .5
1 7 5 6 .5
1 8 2 4 .5
1 6 6 9 .5
1 7 8 5 .5
1 6 3 0 .5
1 6 8 7 .5
1 9 1 3 .5
1 7 5 8 .5
1 8 1 5 .5
1 8 2 6 .5
1 6 7 1 .5
1 8 3 8 .5
1 6 8 3 .5
1 6 5 3 .5
1 7 5 1 .5
1 7 2 6 .5
1 8 6 5 .5
1 7 1 0 .5
1 7 6 7 .5
1 8 7 2 .5
1 7 1 7 .5
1 8 1 3 .5
1 9 5 2 .5
1 7 9 7 .5
1 8 5 4 .5
1 7 2 8 .5
1 7 4 0 .5
1 7 7 4 .5
1 7 8 1 .5
1 6 3 8 .5
1 6 9 5 .5
1 6 9 4 .5
1 5 9 6 .5
1 8 7 9 .5
1 7 2 4 .5
1 6 3 7 .5
1 6 5 3 .5
1 7 8 1 .5
1 6 9 4 .5
1 7 9 2 .5
T ab le 12. Peptide masses from major peak o f HPLC analysis o f selected members o f P-hairpin library
(JKM VII 033, Figure 3 and Figure 4) measured via MALDI-TOF MS. For highlighted wells, the expected
mass (Table 11) matches the observed mass.
1
1 8 1 6 .2
2
3
4
5
6
7
8
9
1 7 7 6 .8
10
11
12
1 6 5 5 .8
1 8 5 4 .9
1 9 4 9 .3
1 7 6 1 .1
1 6 8 5 .9
1 7 3 0 .6
1 6 3 9 .7
1 7 4 2 .5
1 6 5 4 ,5
1 9 4 8 .2
1 8 5 3 .6
1 7 5 9 .6
1 6 3 8 .5
1 7 2 9 .9
1 7 4 2 .3
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
384
T able 13. Initial screening data for P-hairpin library members A01-D12 (JKM VII 033, Figure 3 and
Figure 4) and controls. Data for Figure 7.
C om pound
LB
LC
M
LH
LH
A01
A02
A 03
LANCE
High
C ount
615
(C ou n ts)
46
13968
13821
10432
9943
9757
9103
10350
LANCE
High
C ount
665
(C oun ts)
13
56
54
2093
2035
2072
1889
2285
C o u n ts
1ul o f
~10mM
into 30ul
-31
0
-1
2000
2040
2118
2068
2205
P ercen t
Inhibition
C ou n ts
1ul o f
~1mM
into 30ul
P ercen t
Inhibition
1816
12
1804
13
17
-2
0
-7
A04
95 3 5
1386
1436
31
A 05
10256
2336
2276
-10
A 06
9852
2231
2262
-9
A 07
10134
2302
2270
-10
A 08
9577
2022
2106
-2
A 09
10463
2254
2151
-4
A 10
10883
2468
2267
-10
A11
9783
2090
2132
-3
A 12
87 5 5
1921
2189
-6
B01
10265
1517
1461
29
B 02
8639
50
12
99
B03
10088
2117
2093
-1
B04
10387
2008
1925
7
B05
10621
1964
1840
11
B06
10474
2194
2090
-1
B 07
10727
1719
1589
23
B08
10559
2353
2226
-8
B09
9393
2035
2162
-4
B10
9323
1935
2069
0
B11
9383
2021
2149
-4
B12
9721
2116
2173
-5
C01
9285
1702
1822
12
C02
9860
1906
1925
7
C 03
10952
2161
1967
5
C 04
10508
1862
1761
15
C 05
10765
2266
2101
-1
C 06
11085
2348
2115
-2
C 07
10726
2220
2065
0
C 08
12648
135
67
97
C 09
10320
1876
1808
13
C10
11000
2217
2010
3
C11
10014
1865
1853
10
C 12
10502
2150
2042
1
D01
10801
1802
1656
20
D02
11023
2053
1854
10
D03
11621
133
74
96
1709
D04
12911
72
15
99
1795
13
D05
13287
73
15
99
1718
17
D06
10928
2347
2144
-4
D07
10905
2 07
150
93
1825
12
D08
11241
246
180
91
1718
17
D09
10199
1242
1196
42
1705
18
D10
11278
184
123
94
1685
19
D11
12094
193
120
94
1801
13
D12
9951
2005
2008
3
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
385
T able 14. Initial screening data for p-hairpin library members E01-H12 (JKM VII 033, Figure 3 and
Figure 4). Data for Figure 7. Compound E04 is highlighted because the counts at 615 nm were low,
indicating potential degradation o f the Eu chelate through acidification o f the assay mixture.
C om pound
LANCE
High
C ount
615
(Counts)
LANCE
High
C ount
665
(Counts)
C ounts
1ul of
~10mM
P ercen t
into 30ul Inhibition
C ounts
1ul of
~1mM
P ercen t
into 30ul Inhibition
E01
11730
2117
1796
13
E 02
11156
282
214
90
1745
E03
10365
1309
1242
40
1754
15
E 04
1843
25
35
98
1841
11
E05
10032
2023
2010
3
E06
9662
2005
2069
0
22
16
E 07
10069
1642
1617
E08
10480
2044
1943
6
E 09
10730
1599
1474
29
1720
17
E 10
11367
1514
1314
37
1 753
15
E11
10997
2192
1987
4
E12
10006
1993
1985
4
1724
17
1645
21
1736
16
1752
15
1671
19
F01
10956
1207
1079
48
F02
11141
2307
2066
0
F03
12613
70
15
99
F04
10435
1547
1466
29
F05
9407
1811
1916
7
F06
9588
1890
1963
5
F07
11458
126
69
97
F 08
9142
1816
1978
4
F09
8761
1558
1765
15
F10
8803
1428
1606
22
F11
9725
1034
1038
50
F12
9277
1928
2072
0
G01
10744
1970
1824
12
G 02
10316
1774
1708
18
G 03
11000
2016
1824
12
G 04
11137
1835
1635
21
G 05
10508
2243
2131
-3
G 06
10059
1895
1875
9
G 07
11180
2331
2081
-1
-1
G 08
10785
2260
2091
G 09
10763
2082
1927
7
G 10
10441
2134
2038
2
G11
10782
2024
1869
10
G 12
11036
2282
2063
0
H01
11540
2225
1921
7
H 02
12348
2360
1905
8
H 03
12227
1561
1258
39
H 04
12935
1806
1381
33
10
H 05
12152
2267
1858
H 06
11597
2031
1741
16
H 07
11253
1704
1500
28
H 08
11058
2291
2067
0
H 09
11508
2130
1843
11
H 10
10772
2052
1897
8
H11
10494
1980
1878
9
H12
10681
2185
2040
1
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
T able 15. Characterization o f re-synthesized “hits” from P-hairpin library (JKM VII 033, Table 1 and
Table 2).
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
387
T able 16. Raw assay data for re-synthesized, purified “hits” from [3-hairpin library (Figure 3 and
Figure 4).
Du plicate
Average
Compound (10G Counts Counts Counts Percent Counts Percent
Percent
(5 min) (1 hr) (2 hr) Inhibition (2 hr) Inhibition Inhibition
(iM)
LANCt
L X rJb t
No T(5RII protein
No Compound
No Compound
T G F -p i (6 nM)
TG F-pi (6 nM)
5
2050
2209
1720
1606
23
2681
2697
1089
1024
17
2546
2622
1050
1003
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
JKM
2183
2334
2096
2183
2326
2180
2308
2200
2204
2162
2297
2137
2127
2112
2223
2829
3014
2706
2646
2930
2886
2869
2704
2917
2899
2899
2872
2689
2860
2860
2519
2953
2447
2512
2766
2758
2784
2726
2751
2716
2790
2810
2635
2727
2793
VII
VII
VII
VII
VII
VII
VII
VII
VII
VII
VII
VII
VII
VII
VII
075
075
075
075
075
075
075
075
075
075
075
075
075
079
079
1
2
3
4
5
6
7
8
9
10
11
12
13
12
13
16
3086
3170
914
980
59%
61%
3%
-14%
5%
3%
-7%
-7%
71%
69%
3159
3384
3049
3103
3412
3429
3334
3245
3414
3446
3323
3265
3198
3172
3292
-8%
-5%
-6%
-5%
-8%
-9%
-2%
-6%
-8%
65%
-1%
-8%
3%
1%
-9%
-10%
-7%
-4%
1%
-1 1 %
4%
2%
-8%
-8%
-7%
-5%
-8%
-8%
-7%
-7%
-2%
-3%
-7%
-9%
-10%
-6%
-4%
-2%
-1%
-5%
T able 17. Peptide characterization data for purified members o f the library and data for Figure 8.
L ocation C rude
B07
23
B08
-8
D05-1
99
D05-2
99
D06
-4
D07-16
93
D08-12
91
D12
3
F08
4
HOt
7
H06-11
16
H06-12
16
H07
28
H08-13
0
H08-14
0
H11
9
H12
1
% Inhibition
P ure P u re R e te s t
[M +H f
m a s s (mg) MW (+TFA) pm ol
-3
1.01
2208.5
0.46
1983.2
21
17
1895.9
0.13
2121
0.06
22
9
1794.7
0.09
1889.5
0.05
-5
1798.7
0.87
1891.5
0.46
7
1690.4
0.16
1802.5
0.09
4
1818.3
0.3
0.15
2043.5
5
1731.6
0.26
1956.5
0.13
16
1696.9
0.01
1922.5
0.01
-1
1895.4
0.81
2120.5
0.38
•1
1814.8
0.58
1927.5
0.30
27
1689.1
0.04
1801.5
0.02
21
12
1602.2
0.11
1714.5
0.06
-9
1816.7
1.07
2042.5
0.52
10
1730.1
0.12
1955.5
0.06
18
1 .
1643.1
0.06
1868.5
0.03
38
38
1782.7, 1695.7
0.07
2008.5
0.03
4
1696.0
0.15
1921.5
0.08
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Cys
C ys
Cys
Cys
Cys
Cys
Cys
Cys
Cys
Cys
Cys
Ser
null
S er
S er
null
S er
null
null
null
Ser
null
lie
lie
lie
He
lie
He
He
lie
He
lie
lie
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Thr
Ser
S er
S er
Ser
Ser
S er
S er
Ser
Ser
S er
Ser
Phe
Phe
Phe
Phe
Phe
Phe
Phe
lie
Phe
Trp
Phe
Lys
Lys
S er
Ser
Ser
Lys
Lys
Lys
Lys
S er
Ser
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
S equence
Asp Pro
Asp Pro
Gly Gly
Gly Gly
Gly Gly
Gly Gly
Gly Gly
Gly Gly
Asp Pro
Gly_ Gly
Gly Gly
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Glu
Gin
Gin
Gin
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
S er
Ser
Ser
Ser
Ser
Ser
Ser
S er
Ser
Ser
Ser
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Cys
C ys
Cys
Cys
Cys
Cys
Cys
Cys
C ys
Cys
Cys
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
Ac Cys S e r lie Thr S er P h e Lys Gly Gly Gly Gin Leu S e r Lys P h e Cys A sp NH2
Ac Cys null lie Thr S e r P h e Lys Gly Gly Gly Gin Leu S er Lys P h e Cys Asp NH2
Ac C ys S er He Thr S er
Ac Cys null lie Thr S er
lie
lie
Lys Gly Gly Gly Gin Leu S e r Lys P h e Cys A sp NH2
Lys Gly Gly Gly Gin Leu S e r Lys P he Cys A sp NH2
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
N o tes
M+65
M+65
M+Na oxidized
M+Na reduced
M
M
M
M
M+65
M
M
M-Ser
M
M
M-Ser
M, M-Ser
M
388
T able 18. Raw assay data for purified P-hairpin library members (Figure 3 and Figure 4).
Re-Tes 1 1/3/06
D uplicate
LANUC
Com pound
Controls
High
Count
615
(Counts)
High
Count
665
(Counts)
LANCE
(Counts)
High
Count
615
(Counts)
High
Count
665
(Counts)
LANCE
(Counts)
Average
LANCE
LANCE
High
High
Count
665
LANCE
Percent
Percent Count 615
Inhibition (Counts) (Counts) (Counts) Inhibition
57
7
-6 7
23290
89
0
18553
3474
3465
19247
3744
3603
22853
1225
941
1269
1006
LB
76
14
-5 6
LC
23429
100
0
M
21233
74
-1 3
No com pound
15222
3212
3156
No co m p o u n d
15359
3350
3265
22237
73%
71%
T G F -b 1 ( 6 nM )
T G F -b 1 (6 nM )
17309
1306
1085
18478
1654
1301
B07
14494
3382
3497
16955
3517
3103
3300
-3 %
B 08
16623
2825
2530
18009
3078
2545
2537
21%
23268
3684
2920
17%
D 05-1
16727
2975
2651
17047
2689
2343
2497
22%
22755
3945
3204
9%
D 0 5 -2
14958
3527
3535
17463
3725
3193
3364
-5 %
20780
3499
3110
12%
. 1193
63%
D 06
15974
3221
3014
18206
3577
2936
2975
7%
D 0 7 -1 6
17270
3681
3191
17932
3576
2981
3086
4%
D 0 8 -1 2
17296
3648
3157
16921
3329
2939
3048
5%
D 12
17695
2862
2405
17102
3399
2970
2688
16%
JKM VII 033 F 0 8
15919
3656
3442
16907
3464
3064
3253
-1 %
H 01
15304
3482
3409
19117
3945
3088
3248
-1 %
H 06 -1 1
17159
2220
1910
21136
3891
2749
2329
27%
H 0 6 -1 2
16809
2792
2471
19859
3457
2595
2533
21%
H 07
16797
4180
3736
19274
4169
3240
3488
-9 %
H 0 8 -1 3
17664
3382
2859
18725
3651
2914
2887
10%
H 0 8 -1 4
16096
2803
2593
19220
3424
2657
2625
18%
20620
3875
3479
1%
H11
17622
2239
1874
21871
3064
2076
1975
38%
23426
2792
2180
38%
H 12
17188
3675
3201
17714
3537
2985
3093
4%
JK M VII 1 0 3 0
19084
3399
3293
7
JK M VII 1 0 3 1
18621
3193
3167
10
JK M VII 1 0 3 2
JK M VII 1 0 3 3
19290
3574
3428
3
19444
3912
3729
-6
6.5.7 First Generation 14-Helical P-Peptide L ibrary Synthesis and Characterization
The first generation 14-helical p-peptide library (JKM VI 293, Figure 13) was
synthesized in parallel with microwave irradiation using the same general methods as
described for the P-hairpin a-peptide library. No residues were double coupled. The
product mixtures were dissolved in 500 pL (per well) and sonicated. A portion o f the
stock solution from each well (20 pL) was transferred to a 384-well plate to make a
compound collection (JKM VI 297). The collection was screened in the TGFP3/TPRII
assay.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
389
P o sition 9:
P o sitio n 8:
A
B
C
D
E
F
G
H
■■
iip,
P o sitio n 7:
P o sitio n 6: p -Glu
P o sitio n 5:
___
P o sitio n 4:
10
11
12
-Sf^3V^
P o sitio n 3
P o sitio n 2: p -lie
P o sitio n 1: p3-Glu
F ig u re 23. Spatial addressing for synthesis o f first generation 14-helical P-peptide library (JKM V I 293,
Figure 13).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
T able 19. Sequences o f first generation 14-helical P-peptide library members 1-48 (JKM VI 293,
Figure 13).
S equence
1
2
3
4
5
6
7
C om pound Well N
1
A01 Ac- P 'G lu - p 'lle - A PiC - p dP h e - pG ly- p JG lu- A C H C -
8
9
C
PJHe-
pJL eu-
NH2
2
A 02
A c-
PJG lu- p 'lle - p JO m - p JP h e -
pG ly- p^G lu- A C H C -
PJHe-
pJL eu- NH2
3
A 03
Ac-
pJG lu- p Jlle- A PiC -
P ^P h e-
pG ly- p JG lu- A C H C -
fflle -
pdLeu-
NH2
4
A 04
A c-
pJG lu- p J lle- p JO rn - p2P h e -
pG ly- p JG lu- A C H C -
p JHe-
pJLeu-
NH2
5
A 05
A c-
pJG lu- p Jlle- A PiC -
p dP h e -
pG ly- p JG lu- A C H C - p dP h e -
pJL eu- NH2
6
A06
A c-
pdG lu- pJ lle- p JO rn - p JP h e -
pG ly- p JG lu- A C H C - p dP h e -
pJL eu- NH2
A07
A c-
pJG lu- p Jlle- A PiC -
P ^P he-
pG ly- p JG lu- A C H C - p dP h e -
paLeu- NH2
A 08
A c-
p dG lu- p Jlle- p JO m - p 'P h e -
pG ly- p 'G lu - A C H C - p dP h e -
pJLeu-
NH2
P^GIu- p dlle- A PiC -
p JP h e -
pG ly- p JG lu- A C H C - PdTrp-
pV eu-
NH2
7
8
9
A 09
A c-
10
A 10
A c-
p dG lu- p Jlle- p aO rn - p JP h e -
pG ly- p JG lu- A C H C - P^Trp-
pV eu-
NH2
11
A11
A c-
PJG lu- p Jlle- A PiC -
P ^P he-
pG ly- p^G lu- A C H C - P 'T rp -
PJLeu-
NH2
12
A12
A c-
pJG lu- p Jlle- P^O rn- p 'F h e -
pG ly- p JG lu- A C H C - p T r p -
pJL eu- NH2
13
B01
A c-
PJG lu- p Jlle- A PiC -
p JP h e - p dA la- p JG lu- A C H C -
PJ He-
pJL eu- NH2
14
B02
A c-
PdG iu- p J lle- p JO rn - p JP h e - p JA la- p JG lu- A C H C -
PJ He-
p \e u -
NH2
15
B 03
A c-
PJG lu- p Jlle- A PiC -
P ^P he- p JA la- p JG lu- A C H C -
P ^ le -
p \e u -
NH2
16
B 04
A c-
p dG lu- p Jlle- p JO rn - p aP h e - p JA la- p^G lu- A C H C -
PJMe-
pdLeu-
NH2
17
B05
A c-
P^GIu- p Jlle- A PiC -
P ^P h e- p JA la- p JG lu- A C H C - PJP h e -
pV eu-
NH2
18
B06
A c-
p JG lu- p dlle- p JO rn - p JP h e - p JA la- p JG lu- A C H C - PJP h e -
pJLeu-
NH2
19
B 07
A c-
pJG lu- p Jlle- A PiC -
pJL eu- NH2
20
B 08
A c-
P j GIu - p Jlle- p JO rn - p aP h e - p^Ala- p JG lu- A C H C - p dP h e -
pJLeu-
p JL eu- NH2
p aP h e - p JA la- p JG lu- A C H C - p JP h e -
21
B 09
A c-
pJG lu- p J lle- A PiC -
22
B 10
A c-
pdG lu- p-’lle- p-’O rn - p JP h e - p JA la- p JG lu- A C H C - p T r p -
paL eu-
NH2
B11
A c-
PJG lu- p Jlle- A PiC -
p ^ P h e - p JA la- p JG lu- A C H C - pJT rp-
pJL eu-
NH2
24
B 12
A c-
P j GIu - p Jlle- p JO rn - p ^ P h e - p JA la- p JG lu- A C H C - PJT rp-
pJLeu-
NH2
25
C 01
Ac-
PdG lu- p i l e -
p alle-
pV eu-
NH2
26
C 02
A c-
p dG lu- p 4 le - pJO rn - p JP h e -
pG ly- p JG lu- p JL eu-
p Jlle-
pJL eu- NH2
27
C 03
A c-
pJG lu- p Jlie- A PiC -
P ^P he-
pG ly- p JG lu- p JL eu-
p J lle-
pSL eu- NH2
28
C 04
A c-
pJG lu- p J lle- p^O rn- p 'P h e -
pG ly- p JG lu- p V e u -
p Jlle-
pJL eu- NH2
29
C05
A c-
pJG lu- p J lle- A PiC -
p dP h e -
pG ly- p JG lu- p JL eu- p aP h e -
pJL eu- NH2
30
C 06
A c-
pJG lu- p Jlle- p JO rn - p JP h e -
pG ly- p JG lu- p JL eu- p JP h e -
pV eu-
NH2
31
C07
A c-
p dG lu- p i l e -
P 'T h e -
pG ly- p JG lu- p JL eu- p dP h e -
p \e u -
NH2
32
C08
A c-
p dG lu- p i l e - p JO rn - p ^ P h e -
pG ly- p JG lu- p JL eu- p JP h e -
pJLeu-
NH2
33
C 09
A c-
p JG lu- p Jlle- A PiC -
pG ly- p-’G lu- p JL eu-
p^Trp-
pJL eu- NH2
34
C 10
A c-
pJG lu- p Jlle- p JO rn - p JP h e -
pG ly- p JG lu- p JL eu-
p^Trp-
pJL eu- NH2
C11
A c-
pJG lu- p J lle- A PiC -
P ^ P h e-
pG ly- p JG lu- pJL eu-
p T rp -
pdLeu- NH2
C 12
A c-
p JG lu- p J lle- p J0 m - paP h e -
pG ly- p JG lu- p JL eu-
pJT rp-
pJL eu- NH2
D01
A c-
p dG lu- p Jlle- A PiC -
p aP h e - p JA la- p-’G lu- p \ e u -
p Jlle-
paL eu-
NH2
38
D02
Ac-
p dG lu- p 4 le - p a0 m - p JP h e - p JA la- p JG lu- p aLeu-
p Jlle-
pJLeu-
NH2
39
D03
A c-
p JG lu- p 4 le - A PiC - p dP h e - p JA la- p^G lu- p JLeu-
p alle-
pJL eu- NH2
40
D04
A c-
p dG lu- p Jlle- p JO rn - p ^ P h e - p-’A la- p JG lu- p JL eu-
p Jlle-
pJLeu-
41
D 05
A c-
pJG lu- p Jlle- A PiC -
p JP h e - p JA la- p JG lu- pJL eu- p ^P h e-
pJL eu- NH2
42
D 06
A c-
PJG lu- p Jlle- p aO rn - p JP h e - p JA la- p JG lu- p JL eu- p aP h e -
pJL eu- NH2
43
D 07
A c-
PJG lu- pJ lle- A PiC -
44
D 08
A c-
pJG lu- p Jlle- p JO rn - p zP h e - p JA la- p JG lu- p JL eu- p JP h e -
pJL eu-
45
D 09
A c-
p dG lu- p Jlle- A PiC -
p dP h e - p JA la- p JG lu- p JL eu-
pJT rp-
pJL eu- NH2
23
35
36
37
A PiC -
A PiC -
P 'P h e - p 'A la - p JG lu- A C H C -
p dP h e -
p JP h e -
pG ly- p^G lu- p JLeu-
p T rp -
NH2
P ^P he- p JA la- p JG lu- p JL eu- p JP h e -
NH2
pJL eu- NH2
NH2
46
D 10
A c-
p dGlu- p Jlle- pJO rn - p JP h e - p JA la- p JG lu- p JL eu-
pJT rp-
pdLeu-
NH2
47
D11
A c-
PJGIu - p i l e - A PiC -
P ^P h e- pJA la- p JG lu- p JL eu-
P',Trp-
p \e u -
NH2
48
D 12
A c-
pJG lu- p-’lle- p JO rn - p ^ P h e - p JA la- p JG lu- p-'Leu-
p^Trp-
pJL eu- NH2
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
T able 20. Sequences o f first generation 14-helical P-peptide library members 49-96 (JKM VI 293,
Figure 13).
Sequence
1
2
3
4
5
6
7
C om pound Well N
E01 A c- P^GIu- p J lle- A PiC - p JP h e - pG ly- p JG lu- A C H C 49
8
9
C
PJ He-
p JxL eu- NH2
p 'x L e u - n h 2
50
E 02
A c-
PJG lu- p Jlle- p J0 m - p 3P h e -
pG ly- p JG lu- A C H C -
p JHe-
51
E 03
A c-
PJG lu- p alle- A PiC -
pG ly- p JG lu- A C H C -
P ^ le -
p JxL eu - NH2
52
E04
A c-
p JG lu- p Jl!e- p aO rn - p^Phe- pG ly- p 3G lu- A C H C -
PSHe-
p Jx L e u - NH2
53
E 05
A c-
pJG lu- p Jlle- A PiC -
54
E 06
A c-
p JG lu- pJ lle- p JO rn- p JP h e -
pG ly- p-’G lu- A C H C - p JP h e - p 'x L e u - NH2
55
E 07
A c-
P^GIu- p Jlle- A PiC -
pG ly- p 3G lu- A C H C - p 3P h e - p JxL eu- n h 2
P ^P he-
p JP h e -
P 'F h e -
pG ly- p 3G lu- A C H C - p 3P h e - p JxL eu - NH2
56
E 08
A c-
P^GIu- p Jlle- p JO m - p ^ P h e -
PGly- p JG lu- A C H C - p JP h e - p JxL eu - NH2
57
E09
A c-
P^GIu- p Jlle- A PiC -
p JP h e -
pG ly- p 3G lu- A C H C - p JT rp- p JxL eu- NH2
58
E10
A c-
PSGIu - p alle- p 3O rn - p JP h e -
pG ly- p3G lu- A C H C - P^Trp- p JxL eu - NH2
59
E11
A c-
PJG lu- p 3lle- A PiC -
P 'F h e -
pG ly- p 3G lu- A C H C - PJT rp- p JxL eu - NH2
60
E12
A c-
p 3G lu- p Jlle- p 3O rn - p3P h e -
PGly- p JG lu- A C H C - PJT rp- p V L e u - NH2
61
F01
A c-
PJG lu- p^lle- A PiC -
p JP h e - p JA la- p JG lu- A C H C -
PJ He-
p JxL eu- NH2
62
F02
A c-
p 3G lu- p 3lle- p JO rn - p 3P h e - p JA la- p JG lu- A C H C -
P 3» e -
p JxL eu- NH2
63
F03
A c-
PJG lu- p 3lle- A PiC -
P ^P he- p JAla- p JG lu- A C H C -
P3He-
p JxL eu- NH2
F04
A c-
pJG lu- p 3lle- p 3O rn - p 3P h e - p-’Ala- p 3G lu- A C H C -
p alle-
p3xL eu - NH2
65
F05
A c-
P^GIu- p 3lle- A PiC -
66
F06
A c-
pJG lu- p-’lie- p JO rn - p JP h e - p JA la- p JG lu- A C H C - p 3P h e - pJx L e u - NH2
67
F 07
A c-
PJG lu- p Jlle- A PiC -
68
F 08
Ac-
p 3G lu- pJ lle- p 3O rn - p ^ P h e - p 3A la- p JG lu- A C H C - P ^P he- pJx L eu- NH2
69
F 09
A c-
pJG lu- p 3lle- A PiC -
70
F 10
A c-
P^GIu- p Jlle- p J0 m - p-’P h e - p JAla- p 3G lu- A C H C -
71
F11
A c-
PJG lu- p 3lle- A PiC -
p JG lu- p Jlle- p JO rn - p 'P h e - p JA la- p JG lu- A C H C - p JT rp- p Jx L e u - NH2
64
p JP h e - p JA la- p 3G lu- A C H C - p JP h e - pJxl_eu- NH2
P ^P he- p JA la- p JG lu- A C H C - p JP h e - pJxL eu- NH2
P ^P h e- p 3Ala- pJG lu- A C H C - PJT rp- p Jx L eu- NH2
PJT rp- pJx L eu- NH2
P ^P he- p^Ala- p JG lu- A C H C - PJT rp- p3xl_eu- NH2
72
F12
A c-
73
G01
A c-
p JG lu- p-’lle- A PiC -
p 3P h e -
pG ly- p JG lu- p 3L eu-
p J lle-
pJx L e u - NH2
74
G 02
A c-
p JG lu- pJlle- p 3O rn - p3P h e -
pG ly- p JG lu- p 3L eu-
p J lle-
pJx L eu- n h 2
75
G 03
A c-
PJG lu- p Jlle- A PiC -
P ^P he-
pG ly- p JG lu- p \ e u -
palle-
p 3x L eu- NH2
76
G 04
A c-
pJG lu- p 3lle- p^O rn- p ^ P h e -
pG ly- p 3G lu- p JL eu-
p Jlle-
p3x L eu- NH2
77
G 05
A c-
PJG lu- p 3lle- A PiC -
pGly- p 3G lu- p aL eu- p JP h e - p3x L eu- NH2
78
G 06
A c-
pJG lu- p 3lle- p JO rn - p JP h e -
pG ly- p JG lu- p JL eu- p JP h e - pJx L eu- NH2
79
G 07
A c-
pJG lu- p Jlle- A PiC -
pG ly- p JG lu- p 3L eu- p 3P h e - pJx L eu- NH2
80
G 08
A c-
PJG lu- p J lle- p aO rn - p3P h e -
pG ly- p JG lu- p \ e u -
81
G 09
A c-
P3G lu- pJ lle- A PiC -
p 3P h e -
pG ly- p JG lu- p JL eu-
p JT rp- pJx L eu- NH2
82
G 10
A c-
p JG lu- p Jlle- p 3O rn - p 3P h e -
pG ly- p JG lu- p JL eu-
p JT rp- p Jx L e u - NH2
83
G11
A c-
pdG lu- p 3lle- A PiC -
P ^P h e-
pGly- p 3G lu- p JL eu-
pJT rp- pJx L eu- NH2
84
G 12
A c-
pJG lu- p 3lle- p JO rn - p 3P h e -
pGly- p 3G lu- p JL eu-
pJT rp- pJx L eu- NH2
85
H01
A c-
pJG lu- p Jlle- A PiC -
p JP h e - p JA la- p JG lu- p JL eu-
p Jlle-
pJxL eu- NH2
86
H02
A c-
p 3G lu- p Jlle- p30 r n - p JP h e - p 3A la- p JG lu- p JL eu-
p J lle-
pJxL eu- NH2
87
H03
A c-
pJG lu- pJ lle- A PiC -
P ^P he- p 3A la- p JG lu- p 3Leu-
p Jlle-
p Jx L eu- NH2
88
H04
A c-
pJG lu- p 3lle- p 30 m - p ^ P h e - p 3Ala- pJG lu- p JL eu-
p 3lle-
pJx L eu- NH2
89
H 05
A c-
PJG lu- p 3lle- A PiC -
90
H06
A c-
p3G lu- p 3lle- p JO rn - p JP h e - p aAla- p 3G lu- p JL eu- p 3P h e - pJx L eu- NH2
91
H07
A c-
PJG lu- p J lle- A PiC -
92
H08
A c-
P JG lu- p Jlle- p JO rn - p 3P h e - p 3A la- p JG lu- p 3L eu- p 3P h e - p Jx L e u - NH2
93
H09
A c-
P3GIu- p3lle- A PiC -
p 3P h e - p JA la- p JG lu- p 3L eu-
p JT rp- pJxL eu- NH2
H10
A c-
P3G lu- p Jlle- p 3O m - p 3P h e - p JA la- p3G lu- p JL eu-
p JT rp- p 3x L eu- NH2
95
H11
A c-
P3G lu- p 3lle- A PiC -
paT rp - pJx L eu- n h 2
96
H12
A c-
PJG lu- p 3lle- p JO rn - p 3P h e - p 3A la- p JG lu- p 3L eu-
94
p aP h e -
P^P he-
p 3P h e - pJxL eu- NH2
p JP h e - p^Ala- p 3G lu- p JL eu- p JP h e - pJx L eu- NH2
P ^P h e- p JA la- p JG lu- p 3L eu- p 3P h e - pJxL eu- NH2
P ^P he- p^Ala- p 3G lu- p JL eu-
p ^ rp -
pJx L eu- NH2
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
392
T ab le 21. Calculated masses o f first generation 14-helical P-peptide library members (JKM VI 293,
Figure 13).
1
2
3
4
5
6
7____8___ 9___ 10___ 11___ 12
A
1 2 1 0 .8
1 2 1 2 .8
1 2 1 0 .8
1 2 1 2 .8
1 2 4 4 .8
1 2 4 6 .8
1 2 4 4 .8
1 2 4 6 .8
1 2 8 3 .8
1 2 8 5 .8
1 2 8 3 .8
1 2 8 5 .8
B
1 2 2 4 .8
1 2 2 4 .8
1 2 6 0 .8
1 2 9 7 .8
1 2 4 8 .8
1 2 6 2 .8
1 2 4 8 .8
1 2 9 7 .8
1 2 8 5 .8
1 2 9 9 .8
1 2 4 6 .8
1 2 5 8 .8
1 2 4 6 .8
1 2 6 0 .8
1 2 1 2 .8
1 2 2 6 .8
1 2 2 6 .8
. 1 2 1 4 .8
1 2 5 8 .8
C
1 2 2 6 .8
1 2 1 4 .8
1 2 8 7 .8
1 2 6 0 .8
1 2 6 2 .8
1 2 9 9 .8
1 3 0 1 .9
1 2 8 5 .8
1 2 9 9 .8
1 2 9 9 .8
1 2 8 7 .8
1 2 6 0 .9
1 2 5 8 .9
1 2 9 7 .9
1 2 9 9 .9
1 2 7 4 .9
1 2 6 0 .9
1 2 7 4 .9
1 3 1 1 .9
1 2 6 2 .9
1 2 9 9 .9
1 3 1 3 .9
1 3 0 1 .9
1 2 7 6 .9
1 3 1 3 .9
1 3 1 5 .9 .
1 2 1 2 .8
1 2 2 8 .9
1 2 2 6 .8
1 2 2 4 .9
1 2 2 8 .9
1 2 6 0 .8
1 2 2 6 .9
1 2 3 8 .9
1 2 4 0 .9
1 2 5 8 .9
1 2 7 2 .9
1 2 2 6 .9
1 2 2 8 .9
1 2 4 2 .9
D
E
1 2 2 4 .9
1 2 2 6 .9
F
1 2 3 8 .9
G
1 2 2 6 .9
1 2 4 0 .9
1 2 2 8 .9
H
1 2 4 0 .9
1 2 4 2 .9
1 2 4 0 .9
1 2 6 0 .9
1 2 6 2 .9
1 2 7 2 .9
1 2 6 0 .9
1 2 7 4 .9
1 2 7 6 .9
1 2 7 4 .9
1 3 0 1 .9
1 2 9 9 .9
1 2 9 7 .9
1 3 1 1 .9
1 3 1 3 .9
1 2 9 9 .9
1 3 0 1 .9
1 3 1 3 .9
1 3 1 5 .9
T ab le 22. M asses from major peak o f HPLC analysis measured via MALDI-TOF MS for selected
members o f the first generation 14-helical p-peptide library (JKM V I 293, Figure 13). For highlighted
wells, the expected mass (Table 21) matches the observed mass.
1
2
3
4
5
6____7___ 8___ 9
1 2 4 4 .6
A
10___ 11___ 12
1 2 8 3 .6
1 2 8 3 .6
1 2 9 7 .7
B
1 2 9 7 .8
c
1 2 9 1 .7
1 3 0 1 .9
D
E
1 2 9 7 .7 .
F
1 3 1 1 .6
1 3 0 1 .9
1 2 9 7 .6
1 3 1 1 .7
1 3 0 5 .9
1 3 0 5 .8
1 3 1 9 .9
G
H
1 3 1 5 .8
T able 23. Spatial addresses o f compounds in 384-well plate collection JKM VI 297 from libraries JKM VI
239, 241, 265, and 293. Each well in 8-well sectors described below contains a 50 pL aliquot o f sample
from the original library, denoted by notebook page, row, and column.
1 2
3
4
A c _ c_
c_
B 7 s 7 s 7 s 7S ;
C < < •« . <
IS) |SJ IS)
D IS)
O) -K O ) K
Ol
Ol
E
F > > is ) >IS)
G X X X X
N ) N)
H
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c_
c_
7s
J
S
£■ 8
K < < lis t <
Is)
IS)
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CO
CD CO
CO
CO
p
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A
lX
isJS)
P "rf..
5
6
7
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s
c_
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IS)
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7s ■
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ISO
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00
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t ;
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9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
X
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# 5
c_
7s
S
c_
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00
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CO 3
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o
e&t/t c_
c_
c_
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7s t ;
7s
7s i i
5 : 7 s ■S‘
2 '3 E ;
< <
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iso
IS) IS) IS) IS) IS) wm 00
CO CO 0 0
CO 0 0
CO
CO CO CO CO CO
>
00
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CO
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CD
>
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9
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CO
X
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c_ c_ c_
t; 7s 7s
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< < <
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s
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IS) IS)
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IS)
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X X
a . -S
IS)
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C_
c_
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7s
2
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< < < <
IS) IS) IS) Is)
tp s 00 CO 00
.:<*)• CO 00 CO
>
m * >s a>
O
c_ c_
o
X K
o
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
>
>s
>
IS)
■X.
IS)
C2ITS
_i.
m>
N> IS)
X X
IS)
- A- u
X
393
Table 24. Raw assay data for screening o f compound collection members A1-D24 (JKM VI 297,
Table 23).
LANCE LANCE
LANCE
LANCt
High
FollowHigh
High
High
FollowCount
Count
Count
Up
Count
Up
665
665
LANCE
(See)
JKM VI
615
JKM VI 297
615
LANCE
(See)
(Counts) (Counts) (Counts) Table 28) 297 Well (Counts) (Counts) (Counts) Table 28)
Well
A01
A02
A03
A04
A05
A06
A07
A08
A09
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
A23
A24
B01
B02
B03
B04
B05
B06
B07
B08
B09
B10
B11
B12
B13
B14
B15
B16
B17
B18
B19
B20
B21
B22
B23
B24
20870
19413
19068
19969
19312
18849
19717
18247
19774
16425
20547
1551
20120
23256
19996
21000
21227
20550
20062
9208
19567
522
19633
224
18889
19344
19860
17519
19272
17881
20312
18697
19235
1225
20715
47
20423
17153
19918
20671
20513
23620
20022
3525
18870
585
17736
42
4384
3329
3877
3766
4188
3379
4037
3282
4154
58
4165
15
4227
141
4090
2145
4336
1290
4385
51
3892
21
3924
18
4058
3331
4305
2878
4209
2167
4259
3129
3986
13
3845
16
4204
94
4197
2065
4298
87
4170
21
3850
19
3775
8
4049
3292
3916
3628
4182
3444
3944
3455
4049
-4
3905
-50
4050
50
3941
1933
3936
1161
4216
22
3830
188
3849
250
4142
3306
4180
3149
4212
2304
4042
3210
3993
-80
3570
570
3967
35
4062
1889
4039
4
4014
-20
3930
95
4102
-3091
X
X
X
X
X
C01
C02
C03
C04
C05
C06
C07
C08
C09
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
C24
D01
D02
D03
D04
D05
D06
D07
D08
D09
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
D20
D21
D22
D23
D24
20119
18157
19686
20494
19478
18609
20356
22761
19921
18865
19907
19392
17982
18609
19303
13028
19383
13143
18703
32
19133
32
19375
8036
19208
16804
19988
18224
19181
637
19485
19879
19043
20284
19524
16901
19259
453
20254
16025
20056
40
18950
45
19535
46
19269
585
4283
2869
4184
3947
4094
2728
4155
132
4246
318
4219
86
3707
588
4101
61
4066
51
4020
14
4036
14
3921
54
3895
2219
3922
1222
3883
19
4140
3798
3853
94
4019
2166
3782
15
3922
83
4005
17
3980
12
3426
13
3449
22
4104
3027
4097
3707
4051
2803
3933
45
4109
259
4086
16
3971
548
4095
14
4043
-2
4144
-363
4066
-363
3898
40
3906
2516
3777
1243
3899
82
4096
3676
3897
21
3966
2440
3780
-35
3727
28
3846
1169
4047
-1146
3368
-696
3439
195
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
X
X
X
X
X
X
X
X
394
T ab le 25. Raw assay data for screening o f compound collection members E1-H24 (JKM VI 297,
Table 23).
LANCE
LANCL
High
High
Follow -
Count
JKM VI 297
Count
615
Up
(See)
W ell
E01
E02
E03
E04
E05
E06
E07
E08
E09
E10
E11
E12
E13
E14
E15
E16
E17
E18
E19
E20
E21
E22
E23
E24
F01
F02
F03
F04
F05
F06
F07
F08
F09
F10
F11
F12
F13
F14
F15
F16
F17
F18
F19
F20
F21
F22
F23
F24
(Counts)
19392
18058
20285
19045
19927
18732
19294
18851
19719
19996
20723
19161
18950
16926
19425
382
18749
12494
17822
559
18371
41
17551
14750
19591
18122
20452
17782
19950
18582
18538
17122
19645
20555
18178
18220
19339
19169
20474
12431
19658
46
19835
37
20748
35
19025
245
665
(Counts)
4019
3336
4451
3230
4067
3378
3950
1378
3991
3523
4158
3434
3851
2610
3456
13
3852
60
3563
12
3788
15
3648
72
4088
3243
4211
3286
4109
2359
3965
1624
4080
151
3795
3090
3944
491
3729
47
4184
6
4113
15
4173
18
3755
16
LANCE
(Counts) Table 28)
3993
3551
4233
3255
3932
3465
3944
1362
3898
3385
3865
3443
3914
2951
3418
-133
3958
15
X
3848
-144
3972
183
4004
20
X
4021
3437
3968
3552
3969
2418
4123
1787
4002
74
X
4022
3254
3928
431
X
3502
-5
X
4103
-3680
3996
209
3874
1905
3799
64
LANCE
LANCE
High
Count
High
Count
JKM VI
615
665
297 W ell
G01
G02
G03
G04
G05
G06
G07
G08
G09
G10
G11
G12
G13
G14
G15
G16
G17
G18
G19
G20
G21
G22
G23
G24
H01
H02
H03
H04
H05
H06
H07
H08
H09
H10
H11
H12
H13
H14
H15
H16
H17
H18
H19
H20
H21
H22
H23
H24
(Counts)
19303
18621
20670
19914
21000
17277
21926
642
19685
19523
20213
44
19335
19489
18487
290
19676
19669
19564
1267
19629
121
19351
21220
19667
3700
19712
18485
19791
18444
18336
17501
19184
17498
19385
18779
18356
17966
19211
231
17942
15078
18901
17348
18212
17754
17435
18483
(Counts)
4011
3359
3823
3632
4464
2880
4497
10
3982
3710
4164
11
4020
98
3802
10
4120
92
4008
17
4192
20
3926
148
4145
30
4165
3395
4123
2835
3808
3391
3993
1798
3921
2998
3780
85
4025
10
3882
69
4044
3148
4029
87
3642
2454
Follow Up
LANCE
(See)
(Counts) Table 28)
4004
3466
3557
3506
4099
3196
3953
-194
3896
3656
3970
-1616
4006
X
28
3961
-361
4036
21
3947
-18
4117
835
3908
67
4062
26
4073
3530
4015
2943
4001
3727
4011
1943
3896
3059
3967
21
4038
-438
4171
15
4124
3486
4266
24
4024
2532
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
X
X
X
X
X
Table 26. Raw assay data for screening o f compound collection members I1-L24 (JKM VI 297, Table
23).
JKM VI 297
W ell
LANCE
High
C ount
615
(C o u n ts)
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
I20
121
I22
I23
I24
J01
J02
J03
J04
J05
J06
J07
J08
J09
J10
J11
J12
J13
J14
J15
J16
J17
J18
J19
J20
J21
J22
J23
J24
20738
19129
20333
19731
20051
17323
18901
20019
20920
18508
20661
16915
20271
18443
19771
18088
20961
17955
20718
19306
20527
19460
19340
19616
19716
20570
21499
17942
19867
19625
19949
16307
19859
17972
20844
16753
16532
18721
20968
19478
19632
16488
20457
19703
20832
19013
18589
19703
TANCE
High
C ount
665
(C o u n ts)
4181
2585
4252
3941
4214
2870
3885
3883
4202
3559
3925
3166
4222
1692
4085
3742
4303
257
3782
3666
4287
3938
4080
3809
4281
4273
4558
2852
4307
1195
4089
84
4380
2786
4150
72
3127
371
4389
327
4304
69
4338
1163
4479
3312
3933
3894
Follow Up
LANCE
(See)
JKM VI
(C o u n ts) T ab le 28) 297 W ell
3884
2579
4031
3846
4051
3176
3959
3733
3869
3699
3655
3597
4014
1727
3981
3985
3956
209
3510
3653
4025
3898
4066
3737
4187
4004
4088
3045
4181
1123
3949
27
4255
2968
3834
11
3636
317
4035
258
4228
9
4088
1087
4146
3345
4078
3805
X
X
X
X
X
X
K01
K02
K03
K04
K05
K06
K07
K08
K09
K10
K11
K12
K13
K14
K15
K16
K17
K18
K19
K20
K21
K22
K23
K24
L01
L02
L03
L04
L05
L06
L07
L08
L09
L10
L11
L12
L13
L14
L15
L16
L17
L18
L19
L20
L21
L22
L23
L24
LANCE
LANCE
High
C ount
615
(C o u n ts)
High
C ount
665
(C ounts)
20897
19093
20952
20493
20665
12516
19489
10590
20264
6212
20905
4469
20579
8625
20285
18868
21027
10748
21442
17678
21147
18790
18403
18859
20445
17671
4248
3899
4191
2789
4273
50
4006
41
3955
39
4129
32
4288
40
4020
164
4243
45
4081
69
4294
3594
20403
18848
20065
13288
19982
11317
19753
13830
19406
16374
19386
17741
19907
13144
18225
3044
20456
19029
19817
12237
19638
16611
3805
3538
3963
3524
4003
2100
4266
195
4007
57
3904
61
3776
85
4186
112
4123
62
3921
21
4178
104
4367
66
3863
3076
Follow Up
LANCE
(See)
(C o u n ts) T ab le 28)
3917
3933
3853
2599
3985
-1
3960
-7
3757
21
3803
21
4016
2
3816
99
3887
-1
3663
4
3912
3680
3983
3608
3731
3838
3777
2114
4099
210
3862
17
3805
10
3745
28
4163
52
3991
15
4147
-14
3935
36
4251
26
3787
3558
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
X
X
X
X
X
X
X
X
X
X
X
X
X
396
T able 27. Raw assay data for screening o f compound collection members M1-P24 (JKM VI 297,
Table 23).
JKM VI 297
W ell
LA N C t
High
C ount
615
(C o u n ts)
LANdl:
H igh
C ount
665
(C o u n ts)
M01
M02
M03
M04
M05
M06
M07
M08
M09
M10
M11
M12
M13
M14
M15
M16
M17
M18
M19
M20
M21
M22
M23
M24
N01
N02
N03
N04
N05
N06
N07
N08
N09
N10
N11
N12
N13
N14
N15
N16
N17
N18
N19
N20
N21
N22
N23
N24
20952
19002
20622
13656
20311
7406
20938
8866
21247
5425
20384
8665
20736
6143
21839
15653
20814
17180
20908
10678
20722
10628
19395
19553
20813
19379
20500
19079
20514
14804
20314
9854
20345
924
21050
10170
20343
9919
19747
5279
19193
2554
19913
14225
20080
13897
18627
17455
4294
3214
4022
64
4197
85
4048
54
4010
28
3844
45
4267
41
4316
78
4192
74
3904
42
4213
47
3861
2345
4154
3596
3968
78
4443
57
4335
46
4068
15
4200
40
4429
53
3966
30
3936
10
4150
50
4216
79
3850
3080
Follow Up
LANCE
(S ee)
JKM VI
(C o u n ts) T ab le 28) 297 Well
3949
3246
3755
15
3982
130
3721
31
3631
-7
3628
13
3965
28
3806
23
3880
12
3592
-6
3917
4
3833
2281
3844
3568
3726
9
4177
0
4115
6
3851
-46
3843
-7
4199
20
3868
1
3950
-91
4016
-7
4047
35
3982
3388
X
X
X
X
X
X
X
X
X
X
X
X
O01
002
003
004
005
006
007
008
009
O10
0 11
012
013
014
015
016
017
018
019
020
021
022
023
024
P01
P02
P03
P04
P05
P06
P07
P08
P09
P10
P11
P12
P13
P14
P15
P16
P17
P18
P19
P20
P21
P22
P23
P24
LANCE
LANc e
High
C ount
615
(C o u n ts)
High
C ount
665
(C o u n ts)
20645
19101
20856
20572
20323
19591
20810
13239
20384
17396
4105
3595
3951
2591
3966
21323
17178
20133
18591
20608
13924
18952
18808
21054
18381
20251
18310
19376
19680
35
34
26987
27088
19490
19730
19505
18345
20397
17297
20936
17186
20398
18267
20208
18059
19853
18813
21182
19268
20002
18938
19686
21406
111
3988
63
3915
70
4015
71
3710
92
3815
57
3939
99
3958
92
4114
434
3851
3506
11
18
95
89
3983
4076
3862
2759
4045
2093
3899
2892
4151
3066
3680
2786
4145
327
4131
3486
4050
3237
3900
967
F ollow -1
Up
LANCE
(See)
(C o u n ts) T ab le 28)
3829
3620
3645
2400
3757
40
3688
16
3696
6
3623
8
3543
26
3560
4
4005
32
3617
26
3914
393
3827
3423
-2017
1962
2
-2
3937
3981
3812
2878
3819
2300
3582
3227
3920
3220
3501
2953
4024
270
3755
3477
3900
3281
3814
816
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
X
X
X
X
X
X
X
X
X
X
397
T able 28. Re-evaluation o f hits from compound collection (JKM VI 297, Table 23) from library JKM
VI 241 at a 1:3 dilution and against Smad/Fast.
* t
i l l
4
e,
•—y as. — ■-
ct H ^
= U o Is-- < S■- u«•.
®
-S (3
5s
> 5*®.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
398
T able 29. Re-evaluation o f hits from compound collection (JKM VI 297, Table 23) from library JKM
VI 239 at a 1:3 dilution and against Smad/Fast.
n o rm a liz e d
% ln h ib itio n
LANCE
JKM VI 297 L ib rary a n d N o rm alize d
In h ib itio n o f o f T G F p3LANCE
O rig in a l
384-W ell
In h ib itio n o f TG Fp3-TpRII
TpRH
P la te
P la te
b in d in g
TGFP3-TPRII 1:3 d ilu tio n
L o c a tio n
L o c a tio n
5025
95%
V I23 9 A9
209
118
VoinniDitton
o f TG Fp3TpRII
b in d in g a t
1:3 d ilu tio n
%".......
In h ib itio n
of Sm adF ast
B in d in g
0%
76%
Sequence
H2N- APiC- P 'h P h e - p 'h V al- APiC-
P 'h L e u - p 'h G lu - OH
5110
99%
-2%
94%
H2N- ACHC- P 'h P h e - p 'h O rn - ACHC- P 'h P h e - P'h G lu - OH
11
5257
100%
-5%
96%
H2N- ACHC- P 'h P h e - p-'hO m - APiC-
317
•5 2 3 7
92%
-5%
91%
H2N- ACHC- P 'h P h e - p 'hV al- ACHC- P 'h T rp - p 'h G lu - OH
5202
93%
-4%
71%
H2N- ACHC- P 'h P h e - p 'h O rn - ACHC- P 'h T rp - P'h G lu - OH
4997
100%
0%
95%
H2N- ACHC- P 'h P h e - p 'h V al-
4864
100%
3%
95%
H2N- APiC-
-7
5020
100%
0%
96%
H2N- A PiC-
P'hT rp- p 'h O rn - ACHC- P 'h P h e - p°hGlu- OH
99
5101
97%
-2%
90%
H2N- APiC-
P 'h T rp - p 'h O rn - ACHC- P 'h T rp - p 'h G lu - OH
-1
4961
100%
1%
96%
H2N- APiC-
P 'h T rp - p 'h V a l-
APiC-
P 'h L e u - p 'h G lu - OH
4
4784
100%
4%
94%
HZN- APiC-
P 'h T rp - P 'h O rn - APiC-
P 'h L eu - p 'h G lu - OH
2 10
4765
95%
5%
86%
H2N- ACHC- P 'h T rp - P'hV al- ACHC- P 'h P h e - p 'h G lu - OH
VI 23 9 D4
17
4805
100%
4%
96%
H2N- ACHC- P 'h T rp - p 'h O rn - ACHC- P 'h P h e - p 'h G lu - OH
VI 23 9 D5
10
4897
100%
2%
95%
H2N- ACHC- P 'h T rp - p 'h V al-
APiC-
P 'h T rp - p 'h G lu - OH
L12
VI 23 9 D6
28
5082
99%
-2%
92%
H2N- ACHC- P'hT rp- p 'h O rn - APiC-
P 'h T rp - P'h G lu - OH
L14
V I 23 9 D7
52
5008
99%
0%
95%
H2N- ACHC- P'hT rp- p 'h V al- ACHC- P 'h T rp - p 'h G lu - OH
L16
V I 2 3 9 D8
15
5086
100%
-2%
90%
H2N- ACHC- P 'h T rp - p 'h O rn - ACHC- P 'h T rp - p 'h G lu - OH
L20
VI 23 9 D10
36
5059
99%
-1%
92%
H2N- ACHC- P 'h T rp - p JhO rn- A PiC-
L22
VI 23 9 D11
26
4636
99%
7%
93%
H2N- ACHC- P 'h T rp - p 'h V al- ACHC- P 'h L e u - p 'h G lu - OH
M04
VI 2 3 9 E2
15
5059
100%
-1%
94%
H2N- APiC-
P 'h L eu - p 'h O rn - APiC-
M16
VI 23 9 E8
23
4870
99%
3%
95%
H2N- A PiC-
P^hLeu- pJhO rn- ACHC- PJPTrp- p JhGlu- OH
M18
VI 23 9 E9
12
4868
100%
3%
94%
H2N- APiC-
p JPLeu- p°hVal-
A PiC-
P°hLeu- p-’hGlu- OH
M20
VI 23 9 E 10
-6
4626
100%
7%
94%
H2N- APiC-
p JhLeu- p-hO rn- APiC-
P°hLeu- p JhGlu- OH
M22
VI 23 9 E11
4
4632
100%
7%
96%
H2N- APiC-
P°hLeu- p JhVal- ACHC- PJhLeu- p°hG lu- OH
N04
VI 2 3 9 F2
9
4816
100%
4%
94%
H2N- ACHC- P°hLeu- p-hO m - APiC-
N06
V I 2 39 F3
0
4966
100%
1%
94%
H2N- ACHC- pJhLeu- p JhVal- ACHC- P^hPhe- p JhGlu- OH
27
J08
VI 2 3 9 B4
J12
VI 2 3 9 B6
J1 4
V I2 3 9 B7
J1 6
V I2 3 9 B8
258
J18
V I 23 9 B9
9
K06
VI 23 9 C3
-1
K08
V I 23 9 C4
K16
V I 23 9 C8
K18
V I 23 9 C9
K20
V I 2 39 C 10
L06
VI 2 3 9 D3
L08
L10
APiC-
P 'h T rp - pJhGlu- OH
P 'h L eu - p 'h G lu - OH
P'hT rp- P'hV al- ACHC- P 'h P h e - p 'h G lu - OH
P 'h L eu - p 'h G lu - OH
P 'h P h e - p-hG lu- OH
P^hPhe- p JhGlu- OH
N08
VI 2 3 9 F4
6
4888
100%
2%
94%
H2N- ACHC- pJhLeu- p JhO rn- ACHC- P°h P h e- P'h G lu - OH
N12
VI 2 3 9 F6
-7
4752
100%
5%
95%
H2N- ACHC- PJhLeu- p JhO rn- A PiC-
N14
VI 2 3 9 F7
20
5076
99%
-2%
94%
H2N- ACHC- P^hLeu- p JhVal- ACHC- p JhTrp- p-'hGlu- OH
P°hTrp- p-'hGlu- OH
N20
VI 23 9 F10
-7
4900
100%
2%
96%
H2N- ACHC- P'’hLeu- p JhO rn- APiC-
N22
VI 23 9 F11
35
4897
99%
2%
94%
H2N- ACHC- P',hLeu- p 'h V al- ACHC- P 'h L e u - p 'h G lu - OH
p JhLeu- pJhGlu- OH
006
VI 23 9 G 3
40
5012
99%
0%
95%
H2N- APiC-
008
VI 23 9 G 4
16
4840
100%
3%
94%
H2N- APiC-
P °hSer- p-'hOm- ACHC- P 'h P h e - p JhGlu- OH
010
VI 2 3 9 G 5
6
4575
100%
8%
93%
H2N- APiC-
P ''h S er- p JhVal-
APiC-
P“hTrp- p JhGlu- OH
012
VI 2 3 9 G 6
8
4710
100%
6%
94%
H2N- APiC-
p-'hSer- p°hO m - APiC-
P 'h T rp - p 'h G lu - OH
014
VI 2 3 9 G 7
26
4843
99%
3%
94%
H2N- APiC-
P 'h S e r- p 'h V al- ACHC- P 'h T rp - p-'hGlu- OH
016
VI 2 3 9 G 8
4
4631
100%
7%
95%
H2N- APiC-
P 'h S e r- p-'hO m - ACHC- P 'h T rp - p 'h G lu - OH
018
VI 2 3 9 G9
32
4798
99%
4%
95%
H2N- APiC-
P 'h S e r- p JhVal-
A PiC-
P 'h L e u - p 'h G lu - OH
020
VI 23 9 G10
26
5022
99%
0%
82%
H2N- APiC-
P 'h S e r- p 'h O rn - APiC-
P 'h L e u - p 'h G lu - OH
022
VI 23 9 G11
39 3
5013
90%
0%
75%
H2N- APiC-
P 'h S e r- p 'hV al- ACHC- P 'h L e u - p 'h G lu - OH
P 18
VI 23 9 H9
270
4878
93%
2%
92%
H2N- ACHC- P 'h S e r- P'hV al-
P 'h S e r- p JhVal- ACHC- p-'hP he- p-’hGlu- OH
APiC-
P 'h L eu - p 'h G lu - OH
6.5.8 Second Generation 14-Helical p-Peptide Library Synthesis and
Characterization
The second generation 14-helical P-peptide library (JKM VII 035, Figure 14) was
synthesized in parallel with microwave irradiation using the same general methods as
described above.
No residues were double coupled. . The product mixtures were
dissolved in 250 pL (per well). The library was screened in the TGFp3/TpRII assay.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
399
P osition 9:
1
2
3
■» ' J.
4
5
6
7
8
9
10
11
12
5
6
7
8
9
10
11
12
W*--?r\
P o sitio n 8: p3-lle
P o sitio n 7:
P osition 6:
P o sitio n 5: p -Ala
P o sitio n 4:
1
2
3
4
P o sitio n 3:
P osition 2: p3-lle
P osition 1
F ig u re 24. Spatial addressing for synthesis o f second generation 14-helical P-peptide library (JKM VII
035, Figure 14).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
400
Table 30. Sequences o f second generation 14-helical (3-peptide library members A1-D12 (JKM VII
035, Figure 14).
Com pound
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Well
A01
A02
A03
A04
A05
A06
A07
A08
A09
A10
A11
A12
B01
B02
B03
B04
B05
B06
B07
B08
B09
B10
B11
B12
C01
C02
C03
C04
C05
C06
C07
C08
C09
C10
C11
C12
D01
D02
D03
D04
D05
D06
D07
D08
D09
D10
D11
D12
N
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
1
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
2
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
3
p3-Glu
p3-Glu
p3-Leu
p3-Leu
p3-Glu
p3-Glu
p3-Leu
p3-Leu
p3-Glu
p3-Glu
p3-Leu
p3-Leu
p3-Glu
p3-Glu
p3-Leu
p3-Leu
p3-Glu
p3-Glu
p3-Leu
p3-Leu
p3-Glu
p3-Glu
p3-Leu
p3-Leu
p3-Glu
p3-Glu
p3-Leu
p3-Leu
p3-Glu
p3-Glu
p3-Leu
p3-Leu
p3-Glu
p3-Glu
p3-Leu
p3-Leu
p3-Glu
p3-Glu
p3-Leu
p3-Leu
p3-Glu
p3-Glu
p3-Leu
p3-Leu
p3-Glu
p3-Glu
p3-Leu
p3-Leu
S eq u en ce
4
5
6
7
p3-Glu p3-Ala p3-Glu
p3-Glu p3-Aia p3-Glu
p3-Glu p3-Ala p3-Glu
p3-Glu p3-Ala p3-Glu
p3-Phe p3-Ala p3-Glu
p3-Phe p3-Ala p3-Glu
p3-Phe p3-Ala p3-Glu
p3-Phe p3-Ala p3-Glu
p3-Leu p3-Ala p3-Glu
p3-Leu p3-Ala p3-Glu
p3-Leu p3-Ala p3-Glu
p3-Leu p3-Ala p3-Glu
p3-Glu p3-Ala p3-Phe
p3-Glu p3-Ala p3-Phe
p3-Glu p3-Ala p3-Phe
p3-Glu p3-Ala p3-Phe
p3-Phe p3-Ala p3-Phe
p3-Phe p3-Ala p3-Phe
p3-Phe p3-Ala p3-Phe
p3-Phe p3-Ala p3-Phe
p3-Leu p3-Ala p3-Phe
p3-Leu p3-Ala p3-Phe
p3-Leu p3-Ala p3-Phe
p3-Leu p3-Ala p3-Phe
p3-Glu p3-Ala p3-Glu
p3-Glu p3-Ala p3-Glu
p3-Glu p3-Ala p3-Glu
p3-Glu p3-Ala p3-Glu
p3-Phe p3-Ala p3-Glu
p3-Phe p3-Ala p3-Glu
p3-Phe p3-Ala p3-Glu
p3-Phe p3-Ala p3-Glu
p3-Leu p3-Ala p3-Glu
p3-Leu p3-Ala p3-Glu
p3-Leu p3-Ala p3-Glu
p3-Leu p3-Ala p3-Glu
p3-Glu p3-Aia p3-Phe
p3-Glu p3-Ala p3-Phe
p3-Glu p3-Ala p3-Phe
p3-Glu p3-Ala p3-Phe
p3-Phe p3-Ala p3-Phe
p3-Phe p3-Ala p3-Phe
p3-Phe p3-Ala p3-Phe
p3-Phe p3-Ala p3-Phe
p3-Leu p3-Ala p3-Phe
p3-Leu p3-Ala p3-Phe
p3-Leu p3-Ala p3-Phe
p3-Leu p3-Ala p3-Phe
8
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
p3-Leu
9
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-IIe
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Giu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
p3-Glu
C
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
401
Table 31. Sequences o f second generation 14-helical (1-peptide library members E1-H12 (JKM V ll
035, Figure 14).
C om pound
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
Well
E01
E02
E03
E04
E05
E06
E07
E08
E09
E10
E11
E12
F01
F02
F03
F04
F05
F06
F07
F08
F09
F10
F11
F12
G01
G02
G03
G04
G05
G06
G07
G08
G09
G10
G11
G12
H01
H02
H03
H04
H05
H06
H07
H08
H09
H10
H11
H12
N
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
1
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
p3-Glu
p3-Glu p3-Leu
2 3
p3-lle p3-Glu
p3-lle p3-Glu
p3-lle p3-Leu
p3-lle p3-Leu
p3-lle p3-Glu
p3-lle p3-Glu
p3-lle p3-Leu
p3-lle p3-Leu
p3-lle p3-Glu
p3-lle p3-Glu
p3-lle p3-Leu
p3-lle p3-Leu
p3-lle p3-Glu
p3-lle p3-Glu
p3-lie p3-Leu
p3-lle p3-Leu
p3-lle p3-Glu
p3-lle p3-Glu
p3-lle p3-Leu
p3-lle p3-Leu
p3-lle p3-Glu
p3-lle p3-Glu
p3-lle p3-Leu
p3-lle p3-Leu
p3-lle p3-Glu
p3-lle p3-Glu
p3-lle p3-Leu
p3-lle p3-Leu
p3-lle p3-Glu
p3-lle p3-Glu
p3-lle p3-Leu
p3-lle p3-Leu
p3-lle p3-Glu
p3-lle p3-Glu
p3-lle p3-Leu
p3-lle p3-Leu
p3-lle p3-Glu
p3-lle p3-Glu
p3-lle p3-Leu
p3-lle p3-Leu
p3-lle p3-Glu
p3-lle p3-Glu
p3-lle p3-Leu
p3-lle p3-Leu
p3-lle p3-Glu
p3-lle p3-Glu
p3-lle p3-Leu
p3-lle p3-Leu
S eq u en ce
4
5
6
7
8
p3-Glu p3-Ala p3-Glu p3-Glu
p3-Glu p3-Ala p3-Glu p3-Glu
p3-Giu p3-Ala p3-Glu p3-Glu
p3-Glu p3-Ala p3-Glu p3-Glu
p3-Phe p3-Ala p3-Glu p3-Glu
p3-Phe p3-Ala p3-Glu p3-Glu
p3-Phe p3-Ala p3-Glu p3-Giu
p3-Phe p3-Ala p3-Giu p3-Glu
p3-Leu p3-Ala p3-Glu p3-Glu
p3-Leu p3-Ala p3-Glu p3-Glu
p3-Leu p3-Ala p3-Glu p3-Glu
p3-Leu p3-Ala p3-Glu p3-Glu
p3-Glu p3-Ala p3-Phe p3-Glu
p3-Glu p3-Ala p3-Phe p3-Glu
p3-Glu p3-Ala p3-Phe p3-Glu
p3-Glu p3-Ala p3-Phe p3-Glu
p3-Phe p3-Ala p3-Phe p3-Glu
p3-Phe p3-Ala p3-Phe p3-Glu
p3-Phe p3-Ala p3-Phe p3-Glu
p3-Phe p3-Ala p3-Phe p3-Glu
p3-Leu p3-Ala p3-Phe p3-Glu
p3-Leu p3-Ala p3-Phe p3-Glu
p3-Leu p3-Ala p3-Phe p3-Glu
p3-Leu p3-Ala p3-Phe p3-Glu
p3-Glu p3-Ala p3-Glu p3-Leu
p3-Glu p3-Ala p3-Glu p3-Leu
p3-Glu p3-Ala p3-Glu p3-Leu
p3-Glu p3-Ala p3-Glu p3-Leu
p3-Phe p3-Ala p3-Glu p3-Leu
p3-Phe p3-Ala p3-Glu p3-Leu
p3-Phe p3-Ala p3-Glu p3-Leu
p3-Phe p3-Ala p3-Glu p3-Leu
p3-Leu p3-Ala p3-Glu p3-Leu
p3-Leu p3-Ala p3-Glu p3-Leu
p3-Leu p3-Ala p3-Glu p3-Leu
p3-Leu p3-Ala p3-Glu p3-Leu
p3-Glu p3-Ala p3-Phe p3-Leu
p3-Glu p3-Ala p3-Phe p3-Leu
p3-Glu p3-Ala p3-Phe p3-Leu
p3-Glu p3-Ala p3-Phe p3-Leu
p3-Phe p3-Ala p3-Phe p3-Leu
p3-Phe p3-Ala p3-Phe p3-Leu
p3-Phe p3-Ala p3-Phe p3-Leu
p3-Phe p3-Ala p3-Phe p3-Leu
p3-Leu p3-Ala p3-Phe p3-Leu
p3-Leu p3-Ala p3-Phe p3-Leu
p3-Leu p3-Ala p3-Phe p3-Leu
p3-Leu p3-Ala p3-Phe p3-Leu
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
p3-lle
9
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Giu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
p3-Leu p3-Glu
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
C
NH2
NH2
NH2
NH2
NH?
NH2
NH2
NH2
NH2
NH2
NH?
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
402
Table 32. Calculated masses o f second generation 14-helical (3-peptide library members (JKM VII
035, Figure 14).
1
2
3
4
5
6
A
1 2 5 7 .6
1 3 8 4 .7
1 2 4 1 .7
1 3 6 8 .8
1 2 7 5 .7
1 4 0 2 .8
1 2 5 9 .7
7_________8_________9________ 10________11________ 12
1 3 8 6 .8
1 2 4 1 .7
1 3 6 8 .8
1 2 2 5 .7
1 3 5 2 .8
B
1 2 7 5 .7
1 4 0 2 .8
1 2 5 9 .7
1 3 8 6 .8
1 2 9 3 .7
1 4 2 0 .8
1 2 7 7 .7
1 4 0 4 .8
1 2 5 9 .7
1 3 8 6 .8
1 2 4 3 .8
C
1 2 4 1 .7
1 3 6 8 .8
1 2 2 5 .7
1 3 5 2 .8
1 2 5 9 .7
1 3 8 6 .8
1 2 4 3 .8
1 3 7 0 .9
1 2 2 5 .7
1 3 5 2 .8
1 2 0 9 .8
1 3 7 0 .9
1 3 3 6 .9
D
1 2 5 9 .7
1 2 4 3 .8
1 3 7 0 .9
1 2 7 7 .7
1 4 0 4 .8
1 2 4 3 .8
1 3 7 0 .9
1 3 8 4 .7
1 4 0 2 .8
1 3 6 8 .8
1 5 2 9 .9
1 5 4 7 .9
1 5 1 3 .9
1 3 6 8 .8
G
1 3 6 8 .8
1 4 9 5 .9
1 4 7 9 .9
1 3 8 6 .8
1 5 1 3 .9
1 3 7 0 .9
1 3 8 6 .8
1 3 5 2 .8
H
1 3 8 6 .8
1 5 1 3 .9
1 3 5 2 .8
1 3 7 0 .9
1 5 3 1 .9
1 4 9 8 .0
1 4 9 5 .9
1 5 1 3 .9
1 4 7 9 .9
1 3 8 6 .8
1 4 0 2 .8
1 4 2 0 .8
1 2 2 7 .8
1 3 5 2 .8
1 5 2 9 .9
1 4 9 5 .9
1 5 1 3 .9
1 2 6 1 .8
1 3 8 6 .8
1 3 8 8 .9
E
F
1 3 8 6 .8
1 5 1 1 .8
1 4 9 8 .0
1 4 0 4 .8
1 5 3 1 .9
1 3 8 8 .9
1 5 1 6 .0
1 3 7 0 .9
1 4 0 4 .8
1 3 5 4 .9
1 3 7 0 .9
1 4 9 8 .0
1 4 7 9 .9
1 3 3 6 .9
1 4 6 4 .0
1 4 9 8 .0
1 3 5 4 .9
1 4 8 2 .0
T able 33. Raw assay data for screening o f second generation 14-helical p-peptide library members A lD12 (JKM VII 035, Figure 14). LANCE = normalized fluorescence, taking into account the fluorescence
o f a blank containing the assay buffer. LANCE LH = normalized fluorescence with no compound added.
Percent inhibition = 100 * (1-(LANCE/LANCE LH)).
LANCE
High
Count
615
Compound (Counts)
LANCE
High
Percent
Percent
Percent
Count
665
LANCE Inhibition LANCE Inhibition LANCE Inhibition
(1 hr)
(5 min) (5 min)
(Counts) (4 hr)
(4 hr)
(1 hr)
LB
67
12
-61
-6 0
LC
23811
90
0
0
-8 5
0
M
25939
94
-2
0
-1
LH
18631
4141
4068
4
4388
1
4438
0
A 01
16714
3979
4361
-3
4503
-1
4604
-4
A 02
16342
4106
4606
-9
4781
-8
4648
-5
A 03
16928
4147
4490
-6
4705
4663
-5
A 04
16087
3927
4473
-6
4738
-6
-7
4667
-5
-4
A 05
18084
4295
4352
-3
4556
-3
4624
A 06
17608
4250
4423
-4
4725
-6
4597
-4
A 07
176 5 1
4455
4629
-9
4555
-3
4522
-2
A 08
181 2 1
4436
4487
-6
4488
-1
4448
0
A 09
17990
4394
4477
-6
4629
-4
4561
-3
-2
A 10
18118
4280
4328
-2
4661
-5
4534
A 11
17975
4196
4276
-1
4645
-5
4357
2
A 12
17832
4183
4297
-1
4593
-3
4765
-7
B 01
16233
3903
4405
-4
4633
-4
4569
-3
B 02
15602
3836
4505
-6
4540
-2
4610
-4
B 03
16304
3960
4450
-5
4763
-7
4388
1
B 04
17204
4079
-3
4530
-2
4503
-1
B 05
17343
4206
4343
4444
-5
4647
-5
4683
-5
B 06
B 07
17727
4307
4453
-5
-5
4688
-6
18876
3842
3719
12
4679
4367
2
4499
-1
B 08
17903
3720
3798
10
4328
3
4590
-3
B 09
18522
4395
4348
-3
4482
-1
4374
1
B 10
186 8 1
4387
4302
-2
4572
-3
4375
1
B 11
17957
4236
4322
-2
4505
-1
4436
0
B 12
19164
4376
4182
1
4517
-2
4364
2
C 01
16296
3965
4458
-5
4687
-6
4515
-2
C02
16369
4466
-5
4554
-3
4642
-5
C 03
17332
3989
4144
4381
-3
4559
-3
4597
-4
-3
4499
-1
C 04
16970
4053
4375
-3
4587
C 05
17428
4246
4465
-5
4574
-3
4695
-6
C 06
17237
4163
4426
-4
4562
-3
.4 4 2 6
0
C 07
17752
4419
4564
-8
4751
-7
4228
5
C 08
18640
4547
4472
-6
4477
-1
4339
2
C 09
18027
4459
4535
-7
4693
-6
4493
-1
C 10
17585
4256
4435
-5
4416
1
4477
-1
C 11
18630
4589
4516
-7
4382
1
4399
1
C 12
18585
4557
4495
-6
4570
-3
4357
2
D01
16414
-1
4420
0
4552
-3
4334
-2
4472
-1
4427
0
D 03
16728
16957
3826
3958
4268
D 02
4133
4467
-5
4512
-2
4439
0
D 04
17259
4109
4362
-3
4544
-2
1 8 241
3658
3663
14
4060
9
4498
4237
-1
D 05
D 06
19603
3059
2835
33
3416
23
4242
4
D 07
17733
3348
3444
19
3804
14
4208
5
D 08
19015
3736
3588
15
4077
8
4281
4
D 09
17743
4007
4134
2
4220
5
4497
-1
D 10
18949
4536
4387
-4
4471
-1
4368
2
D11
19358
4155
3926
7
4332
2
4345
2
D 12
19553
3927
3669
13
3901
12
4258
4
5
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
T ab le 34. Raw assay data for screening o f second generation 14-helical P-peptide library members
E1-H12 (JKM VII 035, Figure 14).
LANCE
LANCE
High
High
Percent
Percent
Percent
Count
Count
LANCE Inhibition LANCE Inhibition LANCE Inhibition
615
665
(5 min)
(5 min)
(4 hr)
(1 hr)
(1 hr)
Compound (Counts) (Counts) (4 hr)
E01
E 02
E 03
E 04
E 05
E 06
E 07
E 08
E 09
E 10
E11
E 12
F01
F02
F03
F 04
F 05
F 06
F07
F08
F09
F10
F11
F12
G01
G 02
G 03
G 04
G 05
G 06
G 07
G 08
G 09
G 10
G11
G 12
H01
H02
H03
H04
H05
H06
H07
H08
H09
H10
H11
H12
16726
16049
16930
16350
17420
1 6668
1 7780
1 8213
18059
17982
18351
17935
16965
16721
16594
16596
1 5812
16040
17266
1 9035
1 8188
18304
18611
17584
17679
16571
1 5 678
1 6 857
17279
16104
1 7720
1 9199
17059
17747
19470
20018
18585
17811
1 7706
18590
17365
18984
19051
19340
19188
19948
19915
20482
3903
3807
4105
3896
4162
3992
4297
4325
4352
4220
4424
4179
4005
3915
3942
3968
3681
3955
3921
3525
4023
4090
4297
4273
4345
4443
4365
4377
4388
3770
4126
4003
3816
4022
4168
4009
4581
4601
4089
4198
4428
4508
3680
4319
4199
4356
3708
3909
3931
3960
4240
4383
3707
3803
3921
4275
4426
4459
4371
4420
4563
4743
4392
4392
4333
4165
4123
3616
4444
4345
4293
3904
3764
3772
3742
4044
4021
3396
3387
4429
4351
4416
4299
4418
4268
4324
4288
4351
4380
4262
4519
4157
3377
4048
4090
4229
-1
-3
-5
-3
-3
-4
-5
-3
-4
-1
-4
-1
-2
-1
-3
-3
-1
-7
2
20
4
3
0
7
-1
-4
-5
-3
-4
-8
-12
-4
-4
-2
2
3
15
-5
-3
-1
8
11
11
12
5
5
20
20
4564
4674
4709
4534
4634
4693
4501
4518
4707
4630
4507
4336
4491
4458
4280
4376
4516
4702
4436
3729
4385
4166
4519
4192
4602
4587
4554
-3
-5
-6
-2
-4
-6
-1
-2
-6
-4
-1
2
-1
0
4
1
-2
-6
0
16
1
6
-2
6
-4
4561
4499
4501
4578
4432
4358
4344
4374
4296
4161
4441
4300
4405
4139
4140
4045
4223
4365
4323
3821
3827
-3
-1
-1
-3
0
2
2
1
3
6
0
3
1
7
7
-3
-3
9
5
2
3
14
14
4568
4528
4446
4556
4529
4650
4425
4488
4505
4376
4383
4341
4571
4503
4561
4396
4447
4517
4313
4116
4329
4155
4443
4332
4492
4430
4417
4398
4445
4355
4497
4642
4285
4228
4300
4314
4086
4499
4401
4398
4408
4272
4346
4398
4382
4341
4078
4279
-3
-2
0
-3
-2
-5
0
-1
-1
1
1
2
-3
-1
-3
1
0
-2
3
7
2
6
0
2
-1
0
1
1
0
2
-1
-5
3
5
3
3
8
-1
1
1
1
4
2
1
1
2
8
4
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 35. Characterization data for re-synthesized hits from second generation P-peptide library
(Figure 14).
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
405
Table 36. Raw assay data for re-synthesized hits from second generation P-peptide library (Figure
14). LANCE = normalized fluorescence, taking into account the fluorescence o f a blank containing the
assay buffer. Percent inhibition = 100 * (1-(LANCE/LANCE No Compound)).
Duplicate
LANCE
P ercen t
LANCE
P ercent
LANCE
LANCE
C oncentration (C ounts, 5 min) (C ounts, 1 hr) (C ounts, 2 hr) Inhibition (C ounts, 2 hr) Inhibition
Com pound
N o TpRII pro tein
No C o m p o u n d
No C om pound
TG F-p1
6 nM
6 nM
TGF-p1
JKM VII 0 7 3 2
JKM VII 0 7 3 7
JKM VII 077TE A A
JKM VII 077TE A A
JKM VII 0 77A cO H
JKM VII 077A cO H
JKM VII 077A cO H
JKM VII 077A cO H
JKM VII 077A cO H
1 00 pM
1 00 pM
1
5
1
2
3
4
5
100
100
1 00
100
1 00
100
1O0
pM
pM
pM
pM
pM
pM
pM
5
2050
2209
1 720
1 606
23
2681
17
2546
1%
16
3086
2697
1089
1024
2622
1050
1003
-1%
59 %
61 %
3170
914
980
1 877
2220
2071
1 903
2187
2183
2175
2076
2147
1679
2829
2720
2076
2730
2738
2771
2497
2477
1554
2914
2649
2156
2470
2681
2697
2214
2445
40%
-1 3 %
-3%
17%
4%
-4%
-4%
14%
5%
2371
3355
3140
2554
3179
3224
3363
2938
3019
71 %
69 %
24 %
-7%
0%
18%
-2%
-3%
-8%
6%
3%
Table 37. Raw assay data for dose-response curve for weak p-peptide lead compounds (Figure 17).
LANCE = normalized fluorescence, taking into account the fluorescence o f a blank containing the assay
buffer. Percent inhibition = 100 * (1-(LANCE/LANCE No Compound)).
C om pound
C oncen tratio n
LANCE
P e rc en t
(C ounts, 2 hr) Inhibition A verage
N o TpR II p ro te in
16
No com pound
3086
No C om pound
T G F - p1
TGF- [11
JKM VII 073 2
6 nM
6 nM
71%
69%
2827
10%
2 5 pM
2791
2816
2637
11%
10%
16%
10
0.6
5 0 pM
2402
23%
18
4 .5
2656
2188
15%
30%
1881
2388
40%
24%
31
8.2
3323
3219
-6 %
-5
1.8
3305
3022
-6 %
3%
0
3.4
20
0 .8
3
4 .4
8
0.9
4
1.2
1 0 0 pM
2 5 pM
JKM VII077TEAA 5
3170
914
980
5 0 pM
1 0 0 pM
2 5 pM
3125
3232
5 0 pM
1 0 0 pM
-3 %
0%
2484
-3%
21%
2532
2506
19%
20%
3087
1%
0%
3118
2866
2849
JKM VII 077AcOH 4
S tan d ard
Deviation
8%
2900
9%
7%
2892
2963
8%
5%
2970
3033
5%
3%
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
406
Table 38. Characterization data for analogues o f 14-helical P-peptide lead compound JKM VII 073.
Set
C om pound [M+NaJ* M ass (mg) MW (w/TFA) pmol pL for 10 mM
1
2
JKM VI1105
1553.9
1554.6
1.31
1.13
1531
1532
0.86
86
S equence
Ac
Ifl-Glu [V-Leu ifl-lle
pJ-Leu |iJ-P he ir'-Ala [t'-Phe (fl-Glu (i-lie |r-L eu ir’-Giu
(i’-lle |fl-Leu p3-Glu
NH,
0 .7 4
74
Ac
|t3-Glu p3-Leu
jfl-lle
|t3-Leu p3-P he [t3-Ala |fl-Phe |t3-Glu
Ac
p3-Glu |s1-Leu
[fl-lle
|t3-Leu p3-P he |v’-Ala |t'-P h e |C-Glu |t3-lle |i3-Leu |t3-G(u |f'-Glu
nh2
c o 2h
3
1697.1
2.33
1674
1.39
139
4
1697.5
2.34
1674
1.40
140
Ac
ps-Glu |l‘-Glu pJ-Leu
jfl-lle
p’-Leu |v!-P h e ir’-Ala |i- P h e |V!-Glu
[v’-lle |fl-Leu [tJ-Glu
nh2
5
1612.5
2.8
1589
1.76
176
Ac
|tJ-Glu pJ-Leu
pJ-lle
(fl-Leu p -P h e {v'-Glu It’-P h e p'-Glu
p5-lle |t5-Leu |t5-Glu
nh2
6
1592.9
0 .74
1570
0.4 7
47
Ac
H’-Glu |v‘-Leu
p3-lle
p -L eu p '-trp
[v'-leu [i‘-Glu
nh2
7
1593.1
2.56
1570
1.63
163
Ac
|lJ-G!u |r-L eu
iv-lle
|i(-Leu p'-P he p-A la pJ-T rp pJ-Glu
iC-Ala p - P h e p’-Glu
(C-Leu l^-Glu
nh2
ivMIe |tJ-Leu (tJ-Glu
nh2
|r-L eu ifl-Phe |i3-Ala pJ-P h e |iJ-Giu H3-Glu (\J-Leu |i3-Glu
p’-Leu |C-Phe |V-Ala (L-Hhe pJ-Glu jfl-lle (L-Leu
nh2
8
1 570.5
2.72
1547
1.76
176
Ac
|i‘-Glu p ’-Leu fiJ-Glu |i!-Leu |fl-Phe p-A la |iJ-P h e p’-Giu
9
1570.6
' 1.06
1547
0.69
69
Ac
10
1512.1
2.32
1633
1.42
142
nh2
|is-Glu li'-Leu
|V‘-Glu |t‘-Leu
p3-lle
ir’-lle
|tJ-lle
nh2
Table 39. Raw assay data for screening o f analogues o f 14-helical P-peptide lead compound JKM VII 073
(JKM VII 105) and re-testing o f re-synthesized hits from second generation 14-helical P-peptide library.
LANCE = normalized fluorescence, taking into account the fluorescence o f a blank containing the assay
buffer. Percent inhibition = 100 * (1-(LANCE/LANCE No Compound)).
LANCE LANCE
High
High
Count
Count
LANCE Percent
615
665
Compounds (200pM) (Counts) (Counts) (Counts) Inhibition
LB
LC
No Compound
No Compound
TGF-|31 (6nM)
TGF-p1 (6nM)
JKM VII 105-1
JKM VII 105-2
JKM VII 105-3
JKM VII 105-4
JKM VII 105-5
JKM VII 105-6
JKM VII 105-7
JKM VII 105-8
JKM VII 105-9
JKM VII 105-10
JKM VII 073-2
JKM VII 073-7
JKM VII 077-1 TEAA
JKM VII 077-1 AcOH
JKM VII 077-5 TEAA
JKM VII 077-2 AcOH
JKM VII 077-3 AcOH
57
23290
18553
19247
22853
22237
17376
17743
20109
17320
18564
18538
18306
20145
17781
18411
18374
17224
18364
18171
19434
18609
18637
7
89
3474
3744
1225
1269
3353
3335
3549
3557
3743
3793
3300
4145
3354
3480
3246
3549
3390
3438
2886
3545
3430
-67
0
3465
3603
941
1006
3573
3478
3263
3807
3737
3793
3333
3816
3491
3499
3265
3820
3415
3502
2733
3527
3405
73
71
-1
1
7
-8
-6
-8
5
-8
1
1
7
-8
3
1
23
0
3
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
407
6.5.9 12-Helical P-Peptide L ib rary Synthesis and C haracterization
Potential inhibitors of the TGFp3/TpRII interaction based on the 12-helical ppeptide scaffold were designed, and a library based thereupon was synthesized (Corbin
Occhino) and screened. An idealized 12-helical p-peptide scaffold was manually docked
in the cleft on the surface of TGFP3, overlaying P-amino acid residues 3 and
8
with
TpRII residues Ile53 and Ile50, respectively. A p3-hAla residue was incorporated at
position 5 to avoid a potential steric clash with the floor of the cleft. For improved
oligomer solubility, p3-hGlu residues were added to both termini and at position 4, which
was predicted to be solvent-exposed in the bound conformation. The other positions in
the sequence were varied between ACPC (for added 12-helical stability) and hydrophobic
residues intended to mimic the side chains displayed by TpRII in complex with TGFP3.
Initial screening of the library produced a number of apparent hits, none of which was
active after a 1:10 or 1:100 dilution (Dr. Grace Jurkowski, data not shown).
Figure 25. Library of 12-helical (3-peptides designed to inhibit the TGF(33/T(3RII interaction (2 x 3 x 2 x 2
x 2 x 2 = 96 members).
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
408
Position 9: p3-Glu
Position 8:
1
2
3
4
5
6
7
8
9
10
11
12
10
11
12
9
10
11
12
9
10
11
12
10
11
12
Position 7:
Position 6:
1
B
c
I;-.'.---'- ■
8
(SsSeSji'
D
E
F
G
h
| .r.;
Position 5:
Position 4: p -Glu
Position 3:
Position 2
Position 1: p3-Glu
Figure 26. Spatial addressing for synthesis of 12-helical p-peptide library (JKM V I I 137, Figure 25).
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
Table 40. Sequences o f 12-helical P-peptide library members 1-48 (JKM VII 137, Figure 25).
A01
2
A02
Ac P°-Glu p^-Glu
pJ-lle
p-’-Glu A C PC A C PC A C PC ACPC P°-Glu NH2
3
A03
Ac P°-Glu ACPC
pJ-Me
P^-Glu p J-Ala A C PC A C PC ACPC P°-Glu NH2
4
A04
Ac P°-Glu p-’-Glu
p J-lle
p^-Glu pJ-Ala A C PC A C PC ACPC P^-Glu NH2
5
A05
Ac P^-Glu A C PC PJ-Glu p-’-Glu A C PC A C PC A C PC ACPC P^-Glu NH2
6
A06
A c P^-Glu p J-Glu p^-Glu p-’-Glu A C PC A C PC A C PC ACPC P^-Glu NH2
7
A07
Ac pJ-Glu A C PC P”-Glu P^-Glu p°-Ala A C PC A C PC ACPC P^-Glu NH2
8
A08
Ac p^-Glu p^-Glu p”-Glu p°-Glu p J-Ala A C PC A C PC ACPC pJ-Glu NH2
9
A09
Ac p^-Glu A C PC P^-Phe pJ-Glu A C PC A C PC A C PC ACPC P^-Glu NH2
W ell
1
3
(F-He
Sequence
4
5
6
7
8
9
C
pJ-Glu A C PC A C PC ACPC ACPC P°-GIu NH2
N
1
2
Ac (V-Glu AC PC
C om pound
10
A10
Ac P°-Glu p ’-Glu p ’-P h e p°-Glu A C PC A C PC A C PC ACPC P'hGlu NH2
11
A11
Ac P°-Glu A C PC P"-Phe p ’-Glu p ’-Ala A C PC A C PC ACPC P°-Glu NH2
12
A12
Ac P^-Glu pJ-Glu pJ-P h e p ’-Glu p ’-Ala A C PC A C PC ACPC P°-Glu NH2
13
B01
A c P°-Glu A C PC
fV-ile
[G-Glu A C PC P^-Phe A C PC ACPC P^-Glu NH2
14
B02
A c P^-Glu p ’-Glu
p J-lle
p^-Glu A C PC P °-P he A C PC ACPC P ’-Glu NH2
15
B03
A c pJ-Glu A C PC
P^-He
P ’-Glu p-’-Ala pJ-P h e A C PC ACPC P ’-Glu NH2
16
B04
A c P '-G lu p '-G lu
p’-lle
pJ-Glu p ’-Ala p ’-P h e A C PC A CPC P"-Glu NH2
17
B05
A c P^-Glu A C PC P ’-Glu p J-Glu A C PC P^-Phe A C PC ACPC P°-Glu NH2
18
B06
A c P^-Glu p ”-Glu pJ-Glu p ”-Glu A C PC P^-Phe A C PC A CPC P°-Glu NH2
19
B07
A c P”-Glu A CPC P ’-Glu p ’-Glu pMMa p J-P h e A C PC A CPC P°-Glu NH2
20
B08
Ac P”-Glu pJ-Glu p '-G lu p ’-Glu p J-Ala pJ- P h e A C PC A CPC P'hGlu NH2
21
B09
Ac P^-Glu A C PC P °-Phe p°-Glu A C PC pJ-P h e A C PC ACPC (I’-Glu
22
B10
Ac P^-Glu p°-Glu p°-P he p ’-Glu A C PC pJ-P h e A C PC A CPC P ’-Glu NH2
23
B11
Ac P°-Glu A C PC P^-Phe p°-Glu p J-Ala pJ-P h e A C PC ACPC p 3-Glu NH2
24
B12
Ac P°-Glu p°-Glu pJ-P h e pJ-Glu p J-Ala p-’-P h e A C PC ACPC P^-Glu NH2
25
C01
Ac pJ-Glu A C PC
P^He
P^-Glu A C PC A C PC p M e u A CPC P°-Glu NH2
C02
Ac pJ-Glu pJ-Glu
p J-lle
pJ-Glu A C PC A C PC P°-Leu ACPC P°-Glu NH2
27
C03
Ac P '-G lu A C PC
PJ-He
p ’-Glu p°-Ala A C PC p M e u A CPC P°-Glu NH2
28
C04
Ac p 3-Glu p ”-Glu
p-’-lle
p ’-Glu p’-Ala A C PC p M e u ACPC P ’-Glu NH2
29
C05
Ac P°-Glu A C PC P°-Glu p^-Glu A C PC A C PC P ’-Leu A CPC P’-Glu NH2
30
C06
A c P°-Glu P ’-Glu pJ-Glu p’-Glu A C PC A C PC P’-Leu A C PC P’-Glu NH2
31
C07
Ac P’-Glu A C PC P j -GI u p ’-Glu p ’-Ala A C PC p J-Leu A C PC P’-Glu NH2
32
C08
Ac P’-Glu p ’-Glu p ’-Glu p’-Glu p ’-Ala AC P C P’-Leu A CPC P’-Glu NH2
33
C09
Ac P’-Glu A C PC P’-P h e p ’-Glu A C PC A C PC P’ -Leu A CPC P’-Glu NH2
34
C10
A c P’-Glu p’-Glu p ’-P h e p ’-Glu A C PC A C PC P’-Leu ACPC P’-Glu NH2
35
C11
Ac P’-Glu A C PC pJ-P h e p ’-Glu p ’-Ala A C PC P’-Leu ACPC P°-Glu NH2
36
C12
A c P°-Glu p-’-Glu p 3-P h e p J-Glu p'-A la A C PC p"'L eu A C PC P '-G lu NH2
37
D01
A c P^-Glu A C PC
P ’-He
p J-Glu A C PC P °-P he [i’-Leu ACPC P°-Glu NH2
38
D02
Ac P '-G lu p°-Glu
p"-lle
p J-Glu A C PC P '-P h e p"-Leu ACPC P"-Glu NH2
D03
Ac P"-Glu ACPC
pM le
p J-Glu pJ-Ala pJ-P h e pJ-Leu A C PC P '-G lu NH2
40
D04
Ac p°-Glu p^-Glu
p"-lle
p°-Glu [V’-Ala (F -P h e p°-Leu A C PC P '-G lu NH2
41
D05
Ac P^-Glu A C PC P'hGlu p°-Glu A C PC P ^ P h e p°-Leu ACPC P°-Glu NH2
42
D06
Ac P°-Glu pJ-Glu p°-Glu p-’-Glu A C PC P^-Phe pJ-Leu ACPC P°-Glu NH2
43
D07
Ac pJ-Glu A C PC P^-Glu pJ-Glu p J-Ala p°-P he p^-Leu A C PC p J-Glu NH2
44
D08
Ac p J-Glu p°-Glu pJ-Glu p a-Glu p J-Ala p °-P h e p°-Leu ACPC P°-Glu NH2
45
D09
Ac P°-Glu A C PC P °-P he P '-G lu A C PC P ’-P h e P°-Leu ACPC P°-Glu NH2
46
D10
Ac p J-Glu p^-Glu pJ-P h e p J-Glu A C PC P^-Phe |f -L e u ACPC P°-Glu NH2
47
D11
Ac p J-Glu A C PC P °-Phe p '-G lu p ’-Ala p °-P h e p J-Leu ACPC P°-Glu NH2
48
D12
Ac P°-Glu p ’-Glu p ’-P h e p ’-Glu p ”-Ala pJ-P h e p ’-Leu ACPC P ’-Glu
26
39
nh2
n H2
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
Table 41. Sequences o f 12-helical p-peptide library members 49-96 (JKM VII 137, Figure 25).
N
1
2
3
4
S equ en ce
5
6
7
8
9
C
C om pound
W ell
49
E01
A c P’-Glu ACPC
P’-He
p ’-Glu A C PC A CPC A C PC
P’-He p ’-Glu NH2
50
E02
A c P’-Glu p’-Glu
p ’-lle
p ’-Glu ACPC A C PC A C PC
P’-He p ’-Glu NH2
51
E03
A c P’-Glu ACPC
P°-He
p ’-Glu p’-Ala A C PC A C PC
P°-lle P’-Glu NH2
52
E04
Ac P’-Glu pJ-Glu
p ’-lle
p’-Glu p ’-Ala A C PC A C PC
P’-He p J-Glu NH2
53
E05
Ac P’-Glu ACPC P’-Glu p-’-Glu A C PC A C PC A C PC
P’-Me p ’-Glu NH2
54
E06
Ac pJ-Glu p’-Glu p ’-Glu p’-Glu A C PC A C PC A C PC
P’-He p J-Glu NH2
55
E07
A c P’-Glu A C PC P’-Glu p ’-Glu p ’-Ala A C PC A C PC
P’-Me P’-Glu NH2
56
E08
Ac P’-Glu p’-Glu p ’-Glu p ’-Glu p ’-Ala A C PC A C PC
P’-He P’-Glu NH2
57
E09
Ac P’-Glu ACPC P’-P h e p ’-Glu A C PC A C PC A C PC
P’-He P’-Glu NH2
58
E10
Ac P’-Glu p ’-Glu p ’-P h e p’-Glu A C PC A C PC A C PC
P’-He P’-Glu NH2
59
E11
Ac P’-Glu A C PC P’-P h e p’-Glu p ’-Ala A C PC A C PC
PJ-He pJ-Glu NH2
60
E12
Ac P’-Glu p ’-Glu pJ-P h e pJ-Glu p ’-Ala A C PC A C PC
P’-He P’-Glu NH2
61
F01
Ac P’-Glu A C PC
P’-He
p ’-Glu A C PC P’-P h e A C PC
P’-He pJ-Glu NH2
62
F02
A c P’-Glu p ’-Glu
p’-lle
p ’-Glu A C PC p J-P h e A C PC
P’-He P’-Glu NH2
63
F03
Ac P’-Glu A C PC
P’-He
P’-Glu p ’-Ala p’-P h e A C PC
P’-He P’-Glu NH2
64
F04
Ac P’-Glu p ’-Glu
p’-lle
p ’-Glu p’-Ala p ’-P h e A C PC
P’-He p J-Glu NH2
65
F05
Ac p ’-Glu A C PC P’-Glu p ’-Glu A C PC P’-P h e A C PC
P’-He P’-Glu NH2
66
F06
Ac P’-Glu p ’-Glu p ’-Glu p ’-Glu A C PC P’-P h e A C PC
P’-He pJ-Glu NH2
67
F07
A c P’-Glu A C PC pJ-Glu p ’-Glu p’-Ala p’-P h e A C PC
P’-He P’-Glu NH2
68
F08
A c p J-Glu p ’-Glu p’-Glu p ’-Glu p’-Ala p’-P h e A C PC
P’-He p ’-Glu NH2
69
F09
Ac P’-Glu A C PC p ’-P h e p ’-Glu A C PC P’-P h e A C PC
P’-He p ’-Glu NH2
70
F10
Ac P’-Glu p ’-Glu p ’-P h e p J-Glu A C PC P’-P h e A C PC
P’-He p ’-Glu NH2
71
F11
A c p J-Glu A CPC P’-P h e p ’-Glu p ’-Ala p’-P h e A C PC
P’-He p ’-Glu NH2
72
F12
A c p J-Glu p’-Glu p ’-P h e p ’-Glu p ’-Ala p’ -P h e A C PC
P’-Me P’-Glu NH2
73
G01
A c P’-Glu A C PC
P’-Me
p ’-Glu A C PC A C PC p J-Leu
p’-lle p ’-Glu NH2
74
G 02
A c P’-Glu p’-Glu
p’-lle
p ’-Glu A C PC A C PC P’-Leu
p’-lle
75
G 03
A c p J-Glu A C PC
P’-Me
p ’-Glu p ’-Ala A C PC P’-Leu
p’-lle
p ’-Glu NH2
76
G 04
A c P’-Glu p’-Glu
p’-lle
p ’-Glu p ’-Ala A C PC p ’-Leu
pJ-lle
p J-Glu NH2
77
G 05
A c P’-Glu A CPC P’-Glu p ’-Glu A C PC A C PC P’-Leu
p’-lle
p ’-Glu NH2
78
G 06
A c p ’-Glu p’-Glu p J-Glu pJ-Glu A C PC A C PC P’-Leu
p’-lle
p ’-Glu NH2
G 07
A c P’-Glu A C PC P’-Glu p’-Glu p ’-Ala A C PC P’-Leu
p’-lle
p ’-Glu NH2
80
G 08
A c pJ-Glu p’-Glu p ’-Glu p’-Glu p ’-Ala A C PC P’-Leu
p’-lle p ’-Glu NH2
81
G 09
A c PJ-Glu A C PC P’-P h e p’-Glu A C PC A C PC P’-Leu
p’-lle p ’-Glu NH2
82
G 10
A c PJ-Glu p’-Glu p ’-P h e p’-Glu A C PC A C PC P’-Leu
p’-lle
83
G11
Ac P’-Glu A C PC P’-P h e p’-Glu p ’-Ala A C PC P’-Leu
p ’-lle
p’-Glu NH2
84
G 12
Ac P’-Glu p’-Glu p’-P h e p’-Glu p ’-Ala A C PC P’-Leu
p ’-lle
p’-Glu NH2
79
p ’-Glu NH2
p’-Glu NH2
85
H01
Ac P’-Glu A C PC
P^-He
p’-Glu A C PC P’-P h e p’-Leu
p ’-lle
p’-Glu NH2
86
H02
Ac P’-Glu p’-Glu
p ’-lle
p’-Glu A C PC P’-P h e p’-Leu
p’-lle
p’-Glu NH2
87
H03
Ac P’-Glu A C PC
P’-Me
P’-Glu p’-Ala p ’-P h e p’-Leu
p ’-lle
p’-Glu NH2
88
H04
Ac P’-Glu p’-Glu
p ’-lle
p ’-Glu p’-Ala p ’-P h e p’-Leu
p ’-lle
p’-Glu NH2
89
H05
Ac P’-Glu A C PC P’-Glu p’-Glu A C PC P’-P h e p ’-Leu
p’-lle
p’-Glu NH2
90
H06
Ac P’-Glu p ’-Glu p ’-Glu p ’-Glu A C PC P’-P h e p’-Leu
p ’-lle
p ’-Glu NH2
91
H07
Ac P’-Glu A C PC P’-Glu p ’-Glu p’-Ala p’-P h e p ’-Leu
p’-lle
p ’-Glu NH2
H08
Ac p ’-Glu p ’-Glu p’-Glu p ’-Glu p’-Ala p’-P h e p ’-Leu
p’-lle
p ’-Glu NH2
93
H09
Ac P’-Glu A C PC P’-P h e p ’-Glu A C PC P’-P h e p ’-Leu
p’-lle
p ’-Glu NH2
94
H10
Ac p ’-Glu p ’-Glu p ’-P h e p ’-Glu A C PC P’-P h e p ’-Leu
p’-lle
p ’-Glu NH2
95
H11
Ac P’-Glu A C PC P’-P h e p ’-Glu p’-Ala p ’-P h e p ’-Leu
p’-lle p ’-Glu NH2
96
H12
Ac P’-Glu p ’-Glu p ’-P h e p ’-Glu p ’-Ala p’-P h e p ’-Leu
p’-lle
92
p ’-Glu NH2
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
41 1
Table 42. Calculated masses for 12-helical P-peptide library members (JKM VII 137, Figure 25).
8________ 9________ 10_______ 11________12
1
2
3
4
5
6
7
A
1 1 6 4 .6
1 1 9 6 .6
1 1 3 8 .6
1 1 7 0 .6
1 1 8 6 .6
1 2 1 8 .6
1 1 6 0 .6
1 1 9 2 .6
1 2 0 4 .6
1 2 3 6 .6
1 1 7 8 .6
B
1 2 1 4 .6
1 2 4 6 .6
1 1 8 8 .6
1 2 2 0 .6
1 2 3 6 .6
1 2 6 8 .6
1 2 1 0 .6
1 2 4 2 .6
1 2 5 4 .6
1 2 8 6 .6
1 2 2 8 .6
1 2 6 0 .6
C
1 1 8 0 .6
1 2 1 2 .6
1 1 5 4 .6
1 1 8 6 .6
1 2 0 2 .6
1 2 3 4 .6
1 1 7 6 .6
1 2 0 8 .6
1 2 2 0 .7
1 2 5 2 .6
1 1 9 4 .6
1 2 2 6 .6
D
1 2 3 0 .7
1 2 6 2 .6
1 2 0 4 .6
1 2 3 6 .6
1 2 5 2 .6
1 2 8 4 .6
1 2 2 6 .6
1 2 5 8 .6
1 2 7 0 .7
1 3 0 2 .7
1 2 4 4 .7
1 2 7 6 .7
E
1 1 8 0 .6
1 2 1 2 .6
1 1 5 4 .6
1 1 8 6 .6
1 2 0 2 .6
1 2 3 4 .6
1 1 7 6 .6
1 2 0 8 .5
1 2 2 0 .6
1 2 5 2 .6
1 1 9 4 .6
1 2 2 6 .6
F
1 2 3 0 .6
1 2 6 2 .6
1 2 0 4 .6
1 2 3 6 .6
1 2 5 2 .6
1 2 8 4 .6
1 2 2 6 .6
1 2 5 8 .6
1 2 7 0 .6
1 3 0 2 .6
1 2 4 4 .6
1 2 7 6 .6
G
1 1 9 6 .6
1 2 2 8 .6
1 1 7 0 .6
1 2 0 2 .6
1 2 1 8 .6
1 2 5 0 .6
1 1 9 2 .6
1 2 2 4 .6
1 2 3 6 .6
1 2 6 8 .6
1 2 1 0 .6
1 2 4 2 .6
H
1 2 4 6 .6
1 2 7 8 .6
1 2 2 0 .6
1 2 5 2 .6
1 2 6 8 .6
1 3 0 0 .6
1 2 4 2 .6
1 2 7 4 .6
1 2 8 6 .6
1 3 1 8 .6
1 2 6 0 .6
1 2 9 2 .6
1 2 1 0 .6
Table 43. Raw assay data from screening of 12-helical P-peptide library members 1-48 (JKM VII 137,
Figure 25).
Duplicate
Compound Well 615 (Counts) 665 (Counts) LANCE (Counts
615 (Counts) 665 (Counts) LANCE (Counts; Average % Inhibition
1
A 01
2100
28
120
3779
30
46
83
99
2
A 02
21414
123
36
28015
124
8
22
100
3
A 03
22582
128
34
25163
110
3
19
100
4
A 04
24202
129
26
28285
115
-8
9
100
5
A 05
24453
18 1
83
29889
331
301
192
95
6
A 06
24595
141
37
29688
171
67
52
99
7
A 07
20242
115
33
29165
140
25
29
100
8
A 08
24454
222
128
29328
167
64
96
99
9
A 09
29294
4198
3791
31233
2678
3579
3685
38
10
A 10
24023
3504
3859
31311
850
1007
2433
82
11
A 11
23097
3195
3653
28542
1375
1928
2791
66
12
A 12
25175
885
840
26478
146
55
447
99
13
B 01
22291
611
627
30553
3218
4438
2533
23
14
B02
24346
3840
4182
32228
4110
5414
4798
6
15
B03
22601
3060
3572
30251
3326
4641
4107
19
16
B04
23726
3885
4346
31684
4128
5534
4940
3
17
B05
22652
3583
4194
33252
3351
4240
4217
26
18
B06
26960
4908
4847
37507
5323
6047
5447
-6
19
B 07
24360
1997
2117
29886
223
142
1129
98
20
B 08
25462
3576
3712
30156
2854
3969
3840
31
21
B09
25126
4337
4589
30849
.4 3 6 9
6032
5310
-5
22
B 10
26102
4352
4429
34380
5278
6555
5492
-1 4
23
B 11
24062
3611
3974
31231
4511
6155
5064
-7
24
B12
24760
4118
4417
25897
142
53
2235
99
25
C 01
22389
2821
3316
29482
4015
5792
4554
-1
26
C 02
23728
2176
2382
29953
2648
3695
3038
36
27
C 03
23633
2539
2811
28734
2222
3208
3009
44
28
C 04
23405
2369
2641
31493
3845
5175
3908
10
29
C 05
22834
2929
3378
28862
2543
3681
3530
36
30
C 06
21553
3547
4368
29201
4104
5983
5175
-4
31
C 07
22686
3456
4035
29691
2734
3856
3946
33
32
C 08
24564
1703
1772
29329
1137
1516
1644
74
33
C 09
23402
2482
2773
29981
3171
4457
3615
22
34
C 10
25250
4081
4289
31444
4614
6256
5273
-9
35
C 11
25111
4572
4847
30625
4233
5882
5364
-3
36
C 12
25585
557
475
30609
2543
3462
1968
40
37
D 01
22718
150
60
28567
2736
4018
2039
30
38
D 02
26067
3987
4053
30647
4031
5588
4821
3
39
D 03
22189
2970
3530
30868
4055
5581
4556
3
40
D 04
26399
4035
4050
29253
3740
5426
4738
5
41
D 05
26747
3708
3663
26706
3307
5248
4455
8
42
D 06
21104
2542
3164
25966
2190
3514
3339
39
43
D 07
13356
843
1593
25795
150
67
830
99
44
D 08
21134
2791
3481
31285
3358
4527
4004
21
45
D 09
27272
4053
3935
26882
3579
5657
4796
1
46
D 10
23183
4132
4741
24807
3505
6013
5377
-5
47
D 11
24028
3487
3839
35985
4174
4909
4374
14
48
D 12
25097
2054
2113
28683
1793
2558
2336
55
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
412
Table 44. Raw assay data from screening o f 12-helical p-peptide library members 49-96 (JKM VII
137, Figure 25).
Duplicate
Compound Well 615 (Counts) 665 (Counts) LANCE (Counts
615 (Counts) 665 (Counts) LANCE (Counts; Average % Inhibition
49
E 01
21743
3476
4239
28922
4036
5939
5089
-4
50
E 02
24334
298
215
30206
232
152
183
97
12
51
E 03
22036
3249
3901
29774
3530
5019
4460
52
E 04
25304
3875
4058
38674
4917
5399
4728
6
53
E05
19972
1756
2276
27443
2092
3159
2718
45
54
E 06
23647
1521
1635
27208
2258
3456
2545
40
55
E 07
19945
2734
3617
27624
3154
4825
4221
16
56
E08
23304
3738
4255
27445
3187
4910
4583
14
57
E09
27860
2088
1926
32586
2023
2541
2234
56
58
E10
27000
4491
4418
30999
4162
5709
5063
0
59
E 11
24466
4299
4673
31691
4460
5993
5333
-5
60
E12
27774
464
338
33345
443
400
369
93
61
F01
21583
769
851
28181
224
163
507
97
-4
62
F02
23055
3958
4562
29536
4146
5976
5269
63
F03
23096
3957
4553
31691
3973
5318
4936
7
64
F 04
24781
4169
4469
29797
3727
5305
4887
7
65
F05
26446
3796
3796
28342
3096
4609
4203
20
66
F06
17703
3104
4658
27114
3809
5979
5319
-4
67
F07
24694
3768
4043
29968
3691
5221
4632
9
68
F 08
22815
4078
4755
28770
3070
4498
4626
22
69
F09
27718
4810
4615
31375
4606
6259
5437
-9
70
F10
26584
4739
4744
31413
4477
6071
5407
-6
71
F 11
26925
1559
1461
32260
1457
1798
1630
69
72
F12
24162
4105
4514
31178
4303
5873
5194
-2
73
G 01
22628
1488
1673
27788
2302
3450
2561
40
11
74
G 02
23489
3579
4036
29149
3520
5115
4575
75
G 03
23835
4007
4466
29814
4267
6097
5281
-6
76
G 04
24590
4390
4750
31585
4758
6428
5589
-1 2
77
G 05
24865
3897
4155
34273
4373
5417
4786
5
78
G 06
28242
4250
3987
19692
2688
5797
4892
-1
79
G 07
23393
3810
4322
28683
4119
6117
5220
-7
80
G 08
23777
3981
4447
30114
4230
5980
5214
-4
81
G 09
23317
3756
4274
29560
4101
5904
5089
-3
82
G 10
23099
3746
4303
31588
4566
6160
5232
-7
83
G 11
25022
4138
4391
31964
4599
6131
5261
-7
84
G 12
23684
1309
1388
30337
3763
5259
3324
8
85
H 01
23415
3810
4318
29472
3664
5271
4795
8
86
H 02
25484
4384
4573
30997
4531
6231
5402
-9
87
H 03
24022
4435
4916
31809
4967
6670
5793
-1 6
88
H 04
24346
4160
4541
31030
4394
6031
5286
-5
89
H 05
23909
4065
4518
29427
4181
6051
5284
-6
90
H 06
22607
3993
4697
19612
4334
9506
7101
91
H 07
24158
3858
4236
36372
5505
6461
5349
92
H 08
24841
4398
4710
37030
4585
5253
4981
8
93
H 09
24558
3922
4237
38861
5423
5944
5090
-4
94
H 10
23564
4229
4775
37204
4713
5378
5077
6
95
H 11
24771
4224
4532
31636
4361
5866
5199
-2
96
H 12
23507
1986
2185
29369
193
103
1144
98
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-1 3
413
100
40 -
20
-20
Compound
Figure 27. Screening data for 12-helical p-peptide library (JKM VII 137, Figure 25) in TGFP3/TPRII
assay.
6.5.10 Experim ental Procedure for TGFP3/TPRII HTRF Assay
The TGFp3/TpRII HTRF assay was developed and performed by Dr. F. Michael
Hoffmann and Dr. Grace Jurkowski. The assay buffer was prepared by mixing 536 pL
water, 50 pL of 0.5 M Na-HEPES (pH 7.0), 400 pL 0.25 M KC1, 10 pL BSA (100
mg/mL), and 4 pL of TWEEN (25%). Buffer was added (30 pL) to well A01 of 384well microtiter plate for the LANCE blank. (LANCE refers to normalized fluorescence,
which takes into account the fluorescence of a blank containing only assay buffer.) The
Eu-anti-His antibody was added to the buffer (3.34 pL of a 1:10 dilution of a 445 pg/mL
stock). This solution was added to well A02 for the cross-talk control. The TGFP3-IC5
conjugate was added to the buffer (2,25 pL of a 1:10 dilution of an 80 pM stock). The
resulting solution was added to well A03 as a third control. The TGpRII was added to
the buffer (3.65 pL of a ~2 mg/mL stock). This solution was added to well A04 for the
maximum LANCE signal. The final buffer containing antibody, ligand, and receptor was
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414
distributed to the remaining wells in the plate (30 pL per well). Compounds were
added to each well (1 pL of DMSO stock solution), and the plate was read after 5 min, 1
hr, and 2 hr incubation times.
6.6 References
I Gordon, N. C.; Pan, B.; Hymowitz, S. G.; Yin, J.; Kelley, R. F.; Cochran, A. G.; Yan,
M.; Dixit, V. M.; Fairbrother, W. J.; Starovasnik, M. A. Biochemistry 2003, 42, 59775983.
Ball, L. J.; Kuhne, R.; Schneider-Mergener, J.; Oschkinat, H. Angew. Chem. Int. Ed.
2005, 44, 2852-2869.
3
Pan, B.; Li, B.; Russell, S. J.; Tom, J. Y. K.; Cochran, A. G.; Fairbrother, W. J. J. Mol.
Biol. 2002, 316, 769-787.
4
Massague, J. Annu. Rev. Biochem. 1998, 67, 753-91.
5
Benson, J. R. Lancet Oncol. 2004, 5, 229-239.
Muraoka-Cook, R. S.; Dumont, N.; Arteaga, C. L. Clin. Cancer Res. 2 0 0 5 , 11, 937s943s.
6
7
Dumont, N.; Arteaga, C. L. Cancer Cell, 2003, 3, 531-536.
8
Jurkowski, G.; Peters, N.; Floffmann, F. M. In Preparation.
9
Arandjelovic, S.; Freed, T. A.; Gonias, S. L. Biochemistry 2003, 42, 6121-6127.
1 0 (a) Ezquerro, I.-J.; Lasarte, J.-J.; Dotor, J.; Castilla-Cortazar, I.; Bustos, M.; Penuelas,
I.; Blanco, G.; Rodriguez, C.; Lechuga, M. d. C. G.; Greenwel, P.; Rojkind, M.; Prieto, J.;
Borras-Cuesta, F. Cytokine 2003, 22, 12-20. (b) Santiago, B.; Gutierrez-Canas, I.; Dotor,
J.; Palao, G.; Lasarte, J. J.; Ruiz, J.; Prieto, J.; Borras-Cuesta, F.; Pablos, J. L. J. Invest.
Dermatol. 2005, 125, 450-455.
I I (a) Ryan, A. J.; Gray, N. M.; Lowe, P. N.; Chung, C.-W. J. Med. Chem. 2003, 46,
3448-3451. (b) McGovern, S. L.; Helfland, B. T.; Feng, B.; Shoichet, B. K. J. Med.
Chem. 2003, 46, 4265-4272.
Hart, P. J.; Deep, S.; Taylor, A. B.; Shu, Z.; Hinck, C. S.; Hinck, A. P. Nature Struct.
Biol. 2002, 9, 203-208.
12
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415
13 Hinck, A. P.; Archer, S. J.; Qian, S. W.; Roberts, A. B.; Spom, M. B.; Weatherbee, J.
A.; Tsang, M. L.-S.; Lucas, R. ; Zhang, B.-L.; Wenker, J.; Torchita, D. A. B i o c h e m i s t r y
1996,55,8517-8534.
14 De Crescenzo, G.; Hinck, C. S.; Shu, Z.; Zuniga, J.; Yang, J.; Tang, Y.; Baardsnes, J.;
Mendoza, V.; Sun, L.; Lopez-Casillas, F.; O’Connor-McCourt, M.; Hinck, A. P. J . M o l .
B i o l . 2006, 3 5 5 , 47-62.
15
Hutchinson, E. G.; Thornton, J. M. P r o t e i n
16
Murray, J. K.; Gellman, S.H.
O rg . L e tt.
S ci.
1994, 3, 2207-2216,
2005, 7, 1517.
Mutter, M.; Nefzi, A.; Sato, T.; Sun, X.; Wahl, F.; Wuhr, T. J .
153.
17
P e p t. R es.
1995,
8,
145-
Kamber, B.; Hartmann, A.; Eisler, K.; Riniker, B.; Rink, H.; Sieber, P.; Rittel, W. H e l v .
C h i m . A c t a 1980, 6 3 , 899-915.
18
19 Sattler, M.; Liang, H.; Nettesheim, D.; Meadows, R. P.; Harlan, J. E.; Eberstadt, M.;
Yoon, H. S.; Shuker, S. B.; Chang, B. S.; Minn, A. J.; Thompson, C. B.; Fesik, S. W.
S c i e n c e 1997, 2 7 5 , 983-986.
Sadowsky, J. D.; Schmidt, M. A.; Lee, H.-S.; Umezawa, N.; Wang, S.; Tomita, Y.;
Gellman, S. H. J . A m . C h e m . S o c . 2005, 1 2 7 , 11966.
20
(a) Fasan, R.; Dias, R. L. A.; Moehle, K.; Zerbe, O.; Vrijbloed, J. W.; Obrecht, D.;
Robinson, J. A. A n g e w . C h e m . I n t . E d . 2004, 4 3 , 2109. (b) Fasan, R.; Dias, R. L.;
Moehle, K.; Zerbe, O.; Obrecht, D.; Mittl, P. R. E.; Griitter, M. G.; Robinson, J. A.
C h e m B i o C h e m 2006, 7, 515-526.
21
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
416
Chapter 7
Conclusions and Future Directions
Target
Re-Synthesis
LC-MS/MS
Sequencing
Split-and-Mix
Synthesis
Scaffold
Structure-Based
Design
^Parallel
Synthesis
Combinatorial
(3-Peptide
Synthesis
Biochemical
Assay
HPLC Purification
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417
7.1 Conclusions and F u tu re Directions
The rapid preparation of p-peptide combinatorial libraries in high purity is a significant
advancement toward developing biologically active foldamers, but much remains to be done.
Our current approach to the discovery of foldamer inhibitors is hindered by the relatively lowthroughput screening available to us via collaboration with academic biologists. While we have
cultivated many good relationships and experienced success in our cooperative efforts, in
practice this paradigm often results in slow assay turn-around times and limits libraries to a size
that can be easily tested (< 96 members). The protein-protein interaction target with which our
lab has had the most success in identifying potent inhibitors, Bcl-xL/Bak, is also the first
interaction for which our lab developed an assay. Having an in-house, high-throughput assay
facilitated quicker, and over time, more iterations of the discovery cycle, which eventually
resulted in the identification of high-affinity ligands.
Current efforts within the lab toward
protein production and assay development are critical to take full advantage of the combinatorial
synthetic techniques described in this Thesis and to our group’s future success within this area of
research.
We are seeing within the Gellman lab the same sequence of events that has played out on
a much larger scale in the pharmaceutical industry over the past decade. With the development
of combinatorial chemistry, the number of new compounds being synthesized surpassed the
capacity of the screening facilities. The bottleneck in the discovery process was temporarily
shifted downstream but not removed. Biologists and engineers rose to the challenge. Their
combined efforts gave birth to high-throughput screening (HTS) and restored equilibrium with
the synthetic chemists. Now, when a new target is identified, a company’s entire compound
collection can be tested within a few days.
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418
Initial screening rarely produces a hit as targets in the pharmaceutical industry change
from enzymes to protein-protein interactions. As a result, some experts have proclaimed the
failure of combinatorial chemistry to fulfill expectations. While combinatorial chemistry may be
a “failure” in terms of the small number of new drugs produced in exchange for the tremendous
economic investment, the problem may be that combinatorial chemistry’s potential was largely
over-estimated in the first place. We still have not begun to explore all of chemical space, no
matter how large the library of compounds.
The time and effort invested in combinatorial
chemistry did buy an important negative result:
the strategies, techniques, and types of
molecules developed in the past for enzyme inhibitors were not going to be effective against a
new generation of protein-protein interaction targets. Combinatorial chemistry and HTS simply
helped us reach this conclusion much more quickly by allowing us to synthesize and screen
every molecule possible within the current parameters of structure-based design and synthetic
chemistry.
Some researchers understood the implications of the failure of combinatorial
chemistry and began, not to synthesize even more molecules, but to develop new strategies for a
new class of targets. A renaissance in peptide chemistry and the development of fragment-based
drug discovery and proteomimetics occurred through these efforts.
Combinatorial chemistry has positively impacted science. The greatest effect is on the
rapidity with which we can apply the scientific method. We now think of molecules in terms of
sets, rather than individually, in part because we make them that way.
We explore the
structure/function relationship of a new molecule and its analogues by comparing the activities
of members within a library to determine the essential attributes of our molecule, thereby
refining and formulating new hypotheses that lead us to a more optimal compound through a
better understanding of the system.
The challenge is to consistently harness the power of
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419
combinatorial chemistry for information generation by designing libraries that help us learn
something from each experiment rather than just performing a high-speed process of trial-anderror.
In the absence of a lead compound, the underlying question of how to most effectively
explore chemical space is of central importance. Schreiber and co-workers have prepared large
libraries (~
1
million members) with complex, natural product-like architectures in a diversity-
oriented synthesis approach. 1 Workers at Sunesis and Abbott have developed “Tethering” and
“SAR by NMR,” respectively, to screen large collections of small (MW ~ 100) fragments for
low-affinity ligands that are subsequently linked to create a more potent inhibitor. We have
found in developing foldamer inhibitors of protein-protein interactions that the most critical
factor is selection of the appropriate scaffold. Computer modeling has not been a reliable means
for deciding which scaffold to pursue.
Using the parallel synthesis techniques described
previously (Chapter 4), we have searched for new protein ligands by synthesizing a few 96membered libraries based on each of the different foldamer scaffolds (Chapter 6 ), rather than
preparing much larger one-bead-one-compound libraries.
The next great step in the development of foldamers as ligands for important biological
targets may come through the application of
in v i t r o
selection techniques. We covet the power
of phage display (Chapter 1) for the development of natural peptide ligands and have wondered
if selection/amplification techniques could be employed for foldamers via either ribosomal
peptide synthesis (translation) or DNA-display. A system for the efficient incorporation of (3amino acids into the peptide chain via the ribosome must first be developed in order to use
ribosomal peptide synthesis.
Szostak and coworkers have demonstrated the enzymatic
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420
2
3
aminoacylation of tRNA with p - and p -amino acids by aminoacyl-tRNA synthetases.
2
However, P-amino acids have not yet been successfully translated.
We have envisioned DNA-display as a method to potentially synthesize and screen all
a/p-foldamer scaffolds for biological activity. Harbury and co-workers recently reported the
synthesis, selection, and amplification of peptides synthesized on unprotected DNA, or DNAdisplay.
The DNA sequence encodes the attached peptide’s identity by physically guiding the
growing peptide through a split-and-mix synthesis. The beauty of the method is that library
members are generated in minute quantities, but through a few rounds of selection and
subsequent DNA amplification, compounds showing the desired biological activity are readily
identified. This would allow us to do the high-throughput screening in-house and accomplish
discovery and much of the optimization simultaneously. We could apply this method to the
exploration of P-peptides and new foldamer backbones as potential inhibitors for protein-protein
interactions. In initial efforts we synthesized a hexa-P-peptide on DNA . 4
In conclusion, the discovery of bioactive foldamers is a lofty research goal, with
challenges in many areas of chemistry and biology. Development of synthetic methods for the
preparation of foldamer libraries was an important piece of the puzzle, but it was only one piece.
Selection of appropriate protein-protein interaction targets, identification of additional foldamer
backbones, further development of robust high-throughput assays, and methods for the efficient
exploration of chemical space will all contribute to reaching the goal of identifying biologically
active foldamers. My experience in foldamer research has prepared me for an exciting career in
search of novel treatments for human disease.
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7.2 References
1
Schreiber, S. L.
2
Hartman, M. C. T.; Josephson, K.; Szostak, J. W.
3
Halpin, D. R.; Harbury, P.B. P L o S
4
Price, J. L.; Murray, J. K.; Gellman, S. H. Unpublished results.
S cie n c e
2000, 287, 1964
B io l.
P ro c . N a t. A c a d .
S ci. U S A
2006,
2004, 2 , 1022.
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103,
4356.
422
Appendix A
Scalable, Asymmetric Synthesis of
14-Helix-Promoting 0-Amino Acids
O
X = NBn HCI
= CH2
FmocHN
X = NBoc, Fmoc-APiC(Boc)-OH
= CH2, Fmoc-ACHC-OH, 182 g, 17%
Portions of this appendix have been published as:
Schinnerl, M.; Murray, J. K., Langenhan, J. M.; Gellman, S. H. “Asymmetric Synthesis
of a New Helix-Forming j3-Amino Acid: ^ram,-Aminopiperidine-3-Carboxylic Acid,”
European Journal o f Organic Chemistry 2003, 721-726.
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423
A.O B rief Sum m ary of the A ppendix
We report a synthesis of a protected derivative of fnms,-4-aminopiperidine-3-carboxylic
acid (APiC).
The route provides either enantiomer.
All intermediates are purified by
crystallization, and large-scale preparation is therefore possible. An analogous route provides
either enantiomer of fram-2-aminocyclohcxanecarboxylic acid (ACHC), and a large-scale
synthesis is described. We have previously shown that P-peptide oligomers containing ACHC
adopt a helical conformation defined by 14-membered N-Hj->0 =Ci+ 2 hydrogen bonds (“ 14helix”). It was shown that APiC residues could be incorporated into the 14-helix.1 Access to
large quantities of these monomers is important for the discovery and development of
biologically active 14-helical p-peptides.
A .l Background
Unnatural oligomers with a strong tendency to adopt specific and predictable
conformations in solution (“foldamers”) have been the subject of extensive investigation in
recent years . 2 P-Amino acid oligomers (“P-peptides”) have been particularly well studied in this
regard; P-peptides can adopt several distinct folding patterns if residue substitution patterns are
chosen appropriately. 2 Constrained P-amino acid residues, e.g., those in which the Ca-Cp bond
is part of a small ring, lead to extremely stable conformations. Gellman and coworkers have
shown, for example, that P-peptides containing as few as six residues adopt either of two helical
conformations in aqueous solution depending on the type of residue constraint. 3 , 4
Five-
membered ring constraint (as in trara-2-aminocyclopentanecarboxylic acid (ACPC)) gives rise
to the
12
-helix, defined by
•>
1 2
-membered ring C=Oj->H-Ni+ 3 hydrogen bonds, while six-
membered ring constraint (as in /ram-2-aminocyclohexanecarboxylic acid (ACHC)) leads to the
14-helix, defined by 14-membered ring N-H,-> 0 =Ch 2 hydrogen bonds . 4
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In contrast,
424
conventional peptides (a-amino acid residues) do not form helices at such short lengths, nor
do most (3-peptides comprised of unconstrained p-amino acid residues. 5
A short and enantioselective synthesis of tram-4-aminopiperidine-3-carboxylic acid
(APiC), a new P-amino acid with a six-membered ring constraint, was developed. The route
employs a-methylbenzylamine as a chiral auxiliary and nitrogen source, which allows one to
generate either enantiomer of APiC. No chromatography is required; intermediates are purified
by crystallization.
Thus, this method is amenable to large-scale preparations.
A similar
synthetic route provides access to either enantiomer of ACHC, again without chromatography.
The scalability of the route is demonstrated through single-batch preparation of > 180 g of
Fmoc-(S,S)-ACHC-OH. We have show that a hexa-P-peptide containing both ACHC and APiC
forms a 14-helix in aqueous solution . 1 The large quantities of material available through this
synthetic route have facilitated exploration of potential biological applications of 14-helical (3peptides through combinatorial synthesis.
A.2 Preparation of 14-Helix-Promoting B-Amino Acids
A.2.1 Synthesis of APiC
Our synthetic approach (Scheme 1) follows the strategy previously employed by Gellman
et al. for protected P-amino acids with five-membered ring constraints, including ACPC 6 and a
pyrrolidine analogue. 7
This approach was inspired by the asymmetric synthesis of
cis-2 -
o
aminocyclohexanecarboxylic acid reported by Wu et al. The route to Fmoc-APiC(Boc)-OH (A4) starts with commercially available P-ketoester A -l, which bears a benzyl group on the ring
nitrogen.
Hydrogenolytic removal of the benzyl group and reaction with di-tert-butyl
dicarbonate provided the Boc-protected p-ketoester, which was allowed to react with (R ) - ( + ) - a -
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425
O
1) 10% P d/C , H C 0 2N H 4
Ph
2) B oc 20 , N a H C 0 3
^
N - HCI
Ph
X
Plf'N^
!
1) N a B H 4, is o b u ty ric acid
2) Na, a bs. E tO H
NH
COoEt
”
3) HCI
4 ) R e c ry s ta lliz a tio n
p T sO H (5 m o le %)
16%
B oc
71%
A-1
Ph
A-2
1) L iO H - H20
NH
F m o cH N
^C02H
2 ) 10% P d/C , H C 0 2N H 4
3) F m o c-O S u
N
90%
B oc
B oc
A-3
A-4
Scheme 1.
methylbenzylamine9 in refluxing benzene with catalytic p-toluenesulfonic acid to form enamine
A-2. The enamine was then allowed to react with NaBH 4 in isobutyric acid. Proton NMR
analysis indicated that this reduction generated a 4:1 mixture of the two
cis
diastereomers of the
expected P-amino ester. This mixture was treated with sodium ethoxide in ethanol, causing
epimerization to the
tra n s-
P-amino esters.
suggested a 2.5:1 preference for
tra n s
vs.
cis
Proton NMR analysis of the product mixture
diastereomers. A single
tra n s
diastereomer was
isolated from this mixture via a two-stage crystallization protocol. The crude diastereomeric
mixture was dissolved in diethyl ether, and 4 N HCI in dioxane was added slowly. Adding
hexanes and storing this solution at 0°C led to the precipitation of a white crystalline solid.
Recrystallization of the precipitated solid from acetonitrile provided the pure diastereomer A-3
(> 99%
de
according to NMR analysis) in 16% yield from crude A-2.
The absolute
configuration of A-3 prepared using (6 )-(-)-a-methylbenzylamine was established by removal of
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426
the Boc group and conversion to the bis-HCl salt A-5, for which a crystal structure was
obtained . 1 The desired Fmoc-P-amino acid A-4, obtained from A-3 via the final three steps
indicated in Scheme 1, was crystallized to > 99 % e e as demonstrated by chiral HPLC analysis of
the corresponding dinitrophenyl derivative A-6 .
X = NBoc A-6
CHo A-12
A.2.2 Synthesis of ACHC
An analogous route (Scheme 2) provided enantiomerically pure Fmoc-ACHC-OH (A -ll)
from commercially available p-ketoester A-7. Reduction of enamine A- 8 yielded predominantly
the
cis
P-amino esters, and the desired diastereomer A-9 was isolated by crystallization of the
HBr salt, recrystallization from acetonitrile, and a base wash.
provided a diastereomeric mixture with a 4:1
obtained in > 99%
de
tra n s'.c is
o
ratio.
Base-catalyzed epimerization
Pure diastereomer A-10 was
via a two-stage crystallization protocol; the overall yield from crude A- 8
was 25%. The absolute configuration of Fmoc-ACHC-OH (A -ll) obtained from (R)-(+)-amethylbenzylamine was established as
(R ,R )
by comparing the optical rotation with
(R ,R )-
Fmoc-
ACHC-OH obtained via a route that involves an enzymatic desymmetrization. 10 The new route
provides access to either enantiomer of Fmoc-ACHC, since either enantiomer of a methylbenzylamine can be used. The enantiomeric excess of A -ll was shown to be > 99 % via
chiral HPLC analysis of the corresponding dinitrophenyl derivative A-12.
This synthetic
protocol represents a substantial improvement in terms of efficiency relative to earlier routes to
enantiomerically pure ACHC derivatives. 10,11 (After our work was completed, Berkessel et al.
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427
reported an alternative and very practical synthesis of ACHC, in either enantiomeric form.)
The synthesis of (5,5)-Fmoc-ACHC-OH was scaled up to provide 182 g of final product (17%
overall yield) in under three weeks time.
1) NaBH4, isobutyric acid
2) HBr
pTsOH (5 mole %)
3) Recrystallization
4) NaHC03
49%
A -6
A -7
FmocHN
1) Na, abs. EtOH^ P h '^ ^ N H
,C02Et 2) 10% Pd/C, H C02NH4
3) Fmoc-OSu
3) Recrystallization
51%
A -8
77%
A-9
A -10
Scheme 2.
A.3 Conclusions
Cyclically constrained P-amino acids are very important building blocks for the creation
of short P-peptides that adopt defined conformations in aqueous solution. Here we identify a
new preorganized residue that can confer aqueous solubility, APiC, and we provide a short,
practical and scalable synthetic route that leads to either enantiomer.
In addition, we have
extended this route to perform a large-scale synthesis of enantiomerically pure ACHC. These
building blocks have furthered the exploration of 14-helical p-peptides, and they have been
useful for a variety of biological applications.
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428
A.4 Experimental Methods
A.4.1 General Procedures
Melting points were determined on a capillary melting point apparatus and were
uncorrected. Optical rotations were measured using sodium light (D line, 589.3 nm). Benzene
and toluene were distilled from sodium/benzophenone ketyl under N 2 and ethanol from
sodium/di ethyl phthalate. Unless otherwise noted, all other commercially available reagents and
solvents were purchased from Aldrich and used without further purification, except for 4 N HCI
in dioxane, which was purchased from Pierce, and Fmoc-OSu, which was purchased from
Advanced ChemTech. Analytical thin-layer chromatography (TLC) was carried out on Whatman
TLC plates precoated with silica gel 60 (250 /rm layer thickness). Visualization was
accomplished using either a UV lamp or phosphomolybdic acid (PMA) stain (10%
phosphomolybdic acid in ethanol). Column chromatography was performed on EM Science
silica gel 60 (230-400 mesh). Solvent mixtures used for TLC and column chromatography are
reported in v/v ratios. Diastereomeric excesses were determined using 'H NMR. Enantiomeric
excesses were determined using chiral HPLC.
A.4.2 Synthesis of Fmoc-APIC(Boc)-OH
A.4.2.1 Enamine A-2
To a clear solution of ethyl N-benzyl-4-oxo-piperidine-3-carboxylate hydrochloride (15.3
g, 51.4 mmol) in absolute ethanol (480 ml) under a nitrogen atmosphere was added Pd/C (10%,
2.40 g) and ammonium formate (15.7 g, 249 mmol). The mixture was refluxed for 1 h. The
cooled solution was filtered through Celite and washed with ethanol. The filtrate was
concentrated to obtain a white solid (9.85 g, 92%). This solid was taken up in chloroform (85
ml), and a solution of NaHCCE (4.17 g, 49.6 mmol) in water (80 ml) and NaCl (8.33 g, 143
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
429
mmol) were added.
A solution of di-ferf-butyl dicarbonate (10.4 g, 47.5 mmol) in
chloroform (30 ml) was slowly added at room temperature (15 min), and the mixture was then
refluxed for 15 h. The organic layer was separated, and the aqueous phase was extracted with
chloroform (3x50 ml). The combined organic layers were dried over Na2 S0 4 , and the solvent
was evaporated to obtain a yellow solid (R f = 0.29, 7:1 hexane/ethyl acetate). A stirred solution
of this solid, (R)-(+)-a-methylbenzylamine (6.05 g, 49.9 mmol) and a catalytic amount of p toluenesulfonic acid (451 mg, 2.37 mmol, 5 mole %) in 150 ml of dry benzene was refluxed
under a nitrogen atmosphere with continuous removal of water by using a Dean-Stark trap for 7
h. The cooled reaction mixture was washed twice with saturated aqueous NaHC0 3 (2x100 ml).
The organic layer was dried over Na2 SC>4 . After removal of the solvent, the yellow oily residue
was filtered through a pad of silica gel (CH2 CI2 , wash until the filtrate was colorless; a yellow
color remained on the silica gel). The filtrate was concentrated to obtain 13.7 g of enamine A -2
as a pale yellow oil (71% over three steps): R f = 0.31, 6:1 hexane/ethyl acetate; JH NMR (300
MHz, CDCI3 ) 5 9.25 (d, J
J
=
7.3 Hz, 1H), 7.35-7.22 (m, 5H), 4.60 (quin, J
=
6.7 Hz, 1H), 4.19 (q,
= 7.0 Hz, 2H), 4.07 (s, 2H), 3.46-3.38 (m, 1H), 3.33-3.26 (m, 1H), 2.43-2.35 (m, 1H), 2.09-
1.99 (m, 1H), 1.50 (d, J
=
6.7 Hz, 3H), 1.43 (s, 9H), 1.29 (t, J
=
7.0 Hz, 3H); 13C NMR (75 MHz,
CDCI3 ) 5 168.77 (C), 156.91 (C), 154.33 (C), 145.00 (C), 128.28 (CH), 126.85 (CH), 125.62
(CH), 88.35 (C), 79.37 (C), 58.73 (CH2), 52.02 (CH), 41.21 (CH2), 39.67 (CH2), 28.16 (CH3),
26.02 (CH2), 24.98 (CH3), 14.34 (CH3); MS-ESI
m /z
calc, for C2 1 H3 0 N 2 O4 , 374.22 [M]; obs.
375.2 [M + H]+, 397.2 [M + Na]+, 771.4 [2M + Na]+.
A.4.2.2 HCI Salt A -3
To isobutyric acid (60.0 ml, 647 mmol) was added sodium borohydride (3.60 g, 95.2
mmol) portionwise under N 2 at 0 °C. This mixture was further stirred at room temperature for 0.5
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
430
h and then 10 ml absolute toluene were added and the mixture was cooled to 0 °C. A solution
of enamine A -2 (11 . 8 g, 3 1. 6 mmol) in dry toluene (40 ml) was added dropwise under N 2 at 0 °C.
The mixture was stirred at 0 °C for 1 h, and then additional sodium borohydride (0.7 g, 18.5
mmol) was added in four portions over a 4 h period. When the reaction was complete (7 h), 100
ml of water was added carefully, and the reaction mixture was stirred for
10
min at room
temperature. Afterwards the mixture was brought to pH 10 with 3 N NaOH and extracted with
EtOAc (3x150 ml). The combined organic layers were dried over MgS 0 4 and concentrated
under reduced pressure. The resulting yellow oil was applied to a plug of silica gel and washed
with 2:1 hexane/ethyl acetate. The filtrate was concentrated to obtain a colorless oil (11.3 g, 30.1
mmol, 95%; R f 0.35, 2:1 hexane/ethyl acetate). This oil (dried over night under vacuum) was
dissolved in dry ethanol (50 ml) under N2. In a separate flame-dried Schlenk flask was placed
dry ethanol (250 ml), and sodium (2.06 g, 89.6 mmol) was added portionwise under N2. The
mixture was kept under N 2 and vented to remove evolved gases until all of the sodium dissolved.
The clear solution of the carboxylate was then transferred to the NaOEt solution, and the mixture
was stirred at 50 °C under N 2 for 15 h. The solvent was removed in vacuo, and after addition of
brine (150 ml) the mixture was brought to pH 10 with 1 N NaOH and extracted with ethyl
acetate (3x100 ml). The combined organic layers were dried over MgS 0 4 and concentrated
under reduced pressure. The resulting oil was applied to a plug of silica gel and washed with 2:1
hexane/ethyl acetate. The filtrate was concentrated and dried overnight under vacuum to obtain a
pale yellow oil (9.51 g, 25.3 mmol, 84%). This oil was dissolved in diethyl ether (25 ml), and 4
N HCI in dioxane (6.2 ml, 24.8 mmol) was added dropwise. The solution was stirred for 1 h, and
a precipitate formed during this time. The precipitation was completed by adding hexanes (125
ml) and storing the mixture at 0 °C for 1 h. The white solid was isolated by filtration and washed
with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohib ited w ith o u t p e r m is s io n .
431
with hexanes. This crude product was purified by recrystallization from acetonitrile. The
solid was suspended in acetonitrile
(2 0
ml) and heated to reflux until the solid completely
dissolved. The solution was then cooled to 0 °C overnight. The resulting precipitate was isolated
by filtration and washed three times with 5 ml portions of cold acetonitrile. The mother liquor
and the washings were combined and condensed to about half volume and cooled to 0 °C to get a
second crop. The combined crops were further dried under vacuum to give 2.07 g of A-3 as a
white crystalline solid (16% yield from A-2). Characterization of A-3: mp 197-198 °C;
+
[ a ] JR
=
11.7 (c 1.03, MeOH); !H NMR (300 MHz, CDC13) 5 10.20 (br s, 1H), 9.92 (br s, 1H), 7.79-
7.76 (m, 2H), 7.44-7.40 (m, 3H), 4.60 (br s, 1H), 4.27 (q,
J =
7.2 Hz, 2H), 3.87 (br d,
J =
11.8
Hz, 1H), 3.28-3.19 (m, 2H), 3.00-2.78 (m, 1H), 2.54 (br s, 1H), 2.06-1.93 (m, 4H), 1.74-1.60 (m,
1H), 1.44-1.30 (m, 13H); 13C NMR (75 MHz, CDC13) 5 171.30 (C), 153.67 (C), 136.10 (C),
129.24 (CH), 129.02 (CH), 128.34 (CH), 80.05 (C), 61.63 (CH2), 59.78 (CH), 54.86 (CH), 44.74
(CH), 44.12 (CH2), 41.70 (CH2), 28.56 (CH2), 27.99 (CH3), 20.46 (CH3), 13.88 (CH3); MS-ESI
m /z
calc, for C2 lH 3 2 N 2 0 4, 376.24 [M]; obs. 377.2 [M-C1]+, 399.2 [M-HCl+Naf, 775.4 [2M-
HCl+Na]+. 'H NMR of the corresponding free amine indicated the diastereomeric excess to be >
99%; 'H NMR (300 MHz, CDC13) 5 7.34-7.18 (m, 5H), 4.28-4.12 (m, 3H), 4.04-3.88 (br, 1 H),
3.81 (q,
J -
6 .6
Hz, 1H), 2.66
{id , J =
13, 2.4 Hz, 1H), 2.30 (td,
J =
10.3, 3.7, 1H), 1.74 (br d,
1H), 1.5-1.4 (m, 12 H), 1.36-1.24 (m, 7H).
A.4.2.3 (R,RVFmoc-APiC(BocVOH (A-4f
Compound A-3 (1 .8 9 g, 4 .5 8 mmol) was dissolved in THF/EtOH/H20 (2 :1 :1 , 100 ml),
and this clear solution was cooled to 0 °C. LiOH H20 (1 .0 2 g, 24.3 mmol) dissolved in 10 ml
H20 was added. The mixture was stirred at 0 °C for 16 h. The solvent was removed under
reduced pressure to give a white solid (R f 0 .3 0 , 10:1 CH2 Cl2 /MeOH). To a turbid solution of this
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
432
white solid in 200 ml MeOH, Pd/C (10%, 1.1 g) and ammonium formate (2.38 g, 37.7 mmol)
were added under N 2 at room temperature. The mixture was refluxed for 2 h. After the reaction
was complete (disappearance of starting material, as monitored by TLC), the cooled solution was
fdtered through Celite, and the fdtrate was concentrated to obtain a white solid. This solid was
dissolved in acetone/H20 (2:1, 200 ml) and cooled to 0 °C, and Fmoc-OSu (1.53 g, 4.53 mmol)
and NaHCCL (3.60 g, 42.8 mmol) were added. The turbid reaction mixture was stirred at 0 °C for
1 h and was then allowed to stir at room temperature overnight. The acetone was removed under
reduced pressure. The aqueous residue was diluted with H2 O (50 ml), stirred for 1 h at room
temperature with diethyl ether (200 ml), and the layers were separated. The organic phase was
washed with saturated aqueous NaHCCL (3x100 ml). All the aqueous phases were combined,
acidified with 1 N aqueous HCI, and extracted with ethyl acetate (3x100 ml). The combined
organic layers were dried over MgSCL and concentrated to give a white solid. The crude product
was purified by crystallization from refluxing chloroform
(1 0 0
ml) after careful addition of
MeOH (ca. 3 ml) until all solid was dissolved. For complete precipitation, hexanes was added to
the cold reaction mixture until the solution became turbid. After storage at 0 °C overnight 1.92 g
(90%) of A -4 was obtained as a white solid: mp 202-203 °C; R f = 0.32, 8:1 CH 2 Cl2 /MeOH;
[a ]R
J
= - 4.9 (c 0.51, MeOH); *H NMR (300 MHz, CDCI3 /CD 3 OD)
7.61 (d, J
=
8
7.77 (d, J
=
7.4 Hz, 2H),
7.5 Hz, 2H), 7.42-7.29 (m, 4H), 4.36-4.34 (m, 2H), 4.24-4.19 (m, 2H), 4.06-4.02 (m,
1H), 3.92-3.85 (m, 1H), 3.03-2.85 (m, 2H), 2.47-2.41 (m, 1H), 2.01-1.97 (m, 1H), 1.47 (s, 10H);
13C NMR (75 MHz, CDCI3 /CD 3 OD) 5 173.44 (C), 156.04 (C), 154.46 (C), 143.49 (C), 140.95
(C), 127.33 (CH), 126.72 (CH), 124.68 (CH), 119.53 (CH), 80.25 (C), 66.35 (CH2), 49.96 (CH),
47.26 (CH), 46.81 (CH), 44.59 (CH2), 41.87 (CH2), 30.87 (CH2), 27.79 (CH3); MS-ESI
for C2 6 H 3 0 N 2 O6 , 466.21 [M]; obs. 243.1 [M-Fmoc]', 465.2 [M-H]~, 931.3 [2M-H]7
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
m /z
(S ,S )-
calc,
Fmoc-
433
t
~ \R T
a \D
-
+ 5.0 (c 0.51,
MeOH).
A.4.2.4 Chiral HPLC Assay of A-6 and ent-A-6
Compound A-4 was converted to A-6 by esteriflcation, Fmoc-deprotection, and reaction
with 2,4-dinitrofluorobenzene. Similarly, (S',5)-Fmoc-APiC was derivatized to obtain ent-A-6.
Racemic A-6 in 9:1 hexanes/isopropanol (15 pi of a 1.0 mg/ml solution) was injected onto a
CHIRALCEL OD column (4.6 x 250 mm; Daicel Chemical Ind., Ltd.).
Elution with 1:1
hexanes/isopropanol at 0.6 ml/min resulted in separation of the enantiomers (A-6: tr = 19.7 min;
ent-A-6: tr = 32.4 min). Solutions of A-6 and ent-A-6 (1.0 mg/ml) were similarly analyzed, and
integration of peak areas showed > 99%
ee
for both enantiomers.
A.4.3 Synthesis of (^ )-F m o c-A C H C -O H
A.4.3.1 Enamine A-8
A stirred solution of (A)-(+)-a-mcthylbenzylamine (8.01 g, 66.1 mmol), ethyl 2-oxocyclohexanecarboxylate (11.0 g, 64.7 mmol) and a catalytic amount of /?-toluenesulfonic acid
(618 mg, 3.25 mmol, 5 mole %) in 100 ml dry benzene was refluxed under a nitrogen
atmosphere with continuous removal of water by using a Dean-Stark trap for 4 h. The cooled
reaction mixture was washed twice with saturated aqueous NaHCCE (2x50 ml). After drying
over Na2 SC>4 and removal of the solvent, the resulting yellow oily residue was fractionally
distilled to give 15.4 g (87%) of A-8 as a pale yellow oil: bp 150-155°C, 0.6 mm Hg; Rf = 0.31,
20:1 hexane/ethyl acetate; *H NMR (300 MHz, CDC13)
5H), 4.64 (quin, J
=
8
9.42 (d, J
=
7.5 Hz, 1H), 7.36-7.22 (m,
7.2 Hz, 1H), 4.18 (dq, 7 = 1.0 Hz, 7.2 Hz, 2H), 2.30-2.25 (m, 3H), 1.98-1.91
(m, 1H), 1.54-1.47 (m, 7H), 1.31 (t, J
=
7.2 Hz , 3H); l3C NMR (75 MHz, CDCI3 ) 5 170.76 (C),
158.88 (C), 145.65 (C), 128.48 (CH), 126.59 (CH), 125.23 (CH), 90.34 (C), 58.50 (CH2), 51.79
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
434
(CH), 26.43 (CH2), 25.17 (CH3), 23.61 (CH2), 22.39 (CH2), 22.00 (CH2), 14.49 (CH3); MSESI m / z calc, for C l7 H2 3 N 0 2, [M] 273.17; obs. 296.1 [M + Na]+, 569.3 [2M + N af.
A.4.3.2
cis
(3-Amino ester A-9
To isobutyric acid (40.0 ml, 431 mmol) was added sodium borohydride (2.45 g, 64.8
mmol) portionwise under N 2 at 0 °C. This mixture was further stirred at room temperature for 0.5
h and then cooled to 0 °C. A solution of A- 8 (5.89 g, 21.6 mmol) in dry toluene (24.0 ml) was
added dropwise under N 2 at 0 °C. The mixture was stirred at 0 °C for 1 h, and then another
portion of sodium borohydride (0.50 g, 13.2 mmol) was added. The reaction mixture was stirred
at 0 °C for another 2 h. When the reaction was complete, 100 ml of water was added carefully,
and the reaction mixture was stirred for 10 min at room temperature. Afterwards the mixture
was brought to pH 10 with 3 N NaOH and extracted with EtOAc (3x150 ml). The combined
organic layers were dried over MgS 0 4 and concentrated under reduced pressure. The resulting
oil was applied to a plug of silica gel and washed with 1:1 hexane/ethyl acetate. The filtrate was
concentrated to obtain a colorless oil (5.92 g, 21.5 mmol, 99%; Rf 0.41, 4:1 hexane/ethyl
acetate). This oil was dissolved in ethyl acetate (125 ml) and cooled to 0 °C. To this solution
30% (w/w) HBr in propionic acid (5.3 ml, 25.8 mmol) was added dropwise with vigorous
stirring. A voluminous white precipitate formed during the addition. The mixture was stored at
0 °C overnight for complete precipitation. The white solid was isolated by filtration and washed
with three portions of cold ethyl acetate.
The crude product was further purified by
recrystallization from acetonitrile. The solid was suspended in acetonitrile (55 ml) and refluxed
for 1 h. While hot, the solution was filtered through a cotton wool plug, and the filtrate was
stored at 0 °C overnight. The resulting white crystals were isolated by filtration and dried under
vacuum (3.82 g, 10.7 mmol, 50%). This solid was mixed with an excess of saturated aqueous
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
435
NaHCCE (150 ml) then extracted with diethyl ether (3x50 ml).
The combined organic
extracts were dried over MgSCb, concentrated, and dried overnight under vacuum to give clear
oil A-9 (2.91 g, 10.6 mmol, 99%). 'H NMR indicated the diastereomeric excess to be > 99%.
Characterization of A-9: Rf = 0.41, 4:1 hexane/ethyl acetate; 'H NMR (300 MHz, CDCI3 )
7.36-7.17 (m, 5H), 4.17 (q,
1H), 2.73 (dt, J
=
J =
7.1 Hz, 2H), 3.86 (q,
7.2, 3.7 Hz, 1H), 1.86 (dtd,
J =
J =
6 .6
Hz, 1H), 2.82 (dt,
J =
8
7.8, 3.7 Hz,
9.6, 7.8, 3.5 Hz, 1H), 1.75-1.38 (m, 6 H), 1.38-
1.15 (m, 8 H); 13C NMR (75 MHz, CDC13) d 174.62 (C), 146.70 (C), 128.47 (2CH), 126.88 (CH),
126.75 (2CH), 60.09 (CH2), 55.16 (CH), 53.55 (CH), 44.71 (CH), 30.06 (CH2), 25.67 (CH2),
24.79 (CH3), 23.46 (CH2), 22.99 (CH2), 14.53 (CH3); MS-ESI
m /z
calc, for Ci7 H2 5 N 0 2, 275.19
[M]; obs. 276.1 [M + H]+, 298.1 [M + N a f, 573.2 [2M + Na]+.
A.4.3.3 HCI Salt A-fO
Compound A-9 (5.92 g, 21.5 mmol) was dissolved in dry ethanol (50 ml) under N2. In a
separate flame-dried Schlenk flask was placed dry ethanol (200 ml), and sodium (2.47 g, 107
mmol) was added portionwise under N2. The mixture was kept under N 2 and vented to remove
evolved gases until all of the sodium dissolved. The clear solution of A-9 was then transferred to
the NaOEt solution, and the mixture was stirred at 80 °C under N 2 for 15 h. The solvent was
removed under vacuum, and after addition of brine (150 ml) the mixture was brought to pH 10
with 1 N NaOH and extracted with ethyl acetate (4x100 ml). The combined organic layers were
dried over MgS 0 4 and concentrated under reduced pressure. The resulting oil was applied to a
plug of silica gel and washed with 3:1 hexane/ethyl acetate. The filtrate was concentrated to
obtain a pale yellow oil (4.85 g, 17.6 mmol, 82%). This oil was dissolved in ethyl acetate (20
ml), and 4 N HCI in dioxane (5.3 ml, 21.2 mmol) was added dropwise at room temperature. The
resulting solution was cooled to 0 °C and allowed to stand at 0 °C overnight. A precipitate
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
436
formed during this time. The white solid was filtered and washed three times with 20 ml
portions of cold ethyl acetate to provide the desired material. This crude product could be
purified by recrystallization from acetonitrile. The solid was suspended in acetonitrile (50 ml)
and heated to reflux for 1 h. The mixture was then filtered through cotton wool and cooled to 0
°C overnight. The resulting precipitate was isolated by filtration and washed three times with 10
ml portions of cold acetonitrile. The mother liquor and the washings were combined and
condensed to about half volume to get a second crop. The combined crops were further dried
under vacuum to give 3.44 g of A-10 as a white crystalline solid (51% yield from A-9). 'H NMR
of the corresponding free amine indicated the diastereomeric excess to be >99%.
Characterization of A-10: mp 211-212 °C;
[ a f j
=
- 37.5 (c 1.05, MeOH). *H NMR (300 MHz,
CDC13) 8 9.76 (br s, 1H), 7.80-7.77 (m, 2H), 7.47-7.38 (m, 3H), 4.59 (q, J
=
7.0 Hz, 1H), 4.34-
4.18 (m, 2H), 3.19-3.06 (m, 2H), 2.22-2.18 (m, 1H), 1.93-1.60 (m, 8H), 1.34-1.24 (m, 5H), 1.030.90 (m, 1H); ,3C NMR (75 MHz, CDC13) 5 174.10 (C), 136.59 (C), 128.93 (CH), 128.83 (CH),
128.40 (CH), 61.13 (CH2), 59.44 (CH), 56.48 (CH), 45.87 (CH), 29.95 (CH2), 29.25 (CH2),
23.91 (CH2), 23.69 (CH2), 20.48 (CH3), 13.93 (CH3); MS-ESI
m /z
calc, for Ci7H25N 0 2, 275.19
[M]; obs. 276.1 [M-C1]+, 298.2 [M-HCl+Na]+.
A.4.3.4 fRRVFmoc-ACHC-OH fA-111
Compound A-10 (2.80 g, 8.98 mmol) was dissolved in THF/EtOH/H20 (2:1:1, 100 ml),
and the clear solution was cooled to 0 °C. LiOH H20 (1.89 g, 45.1 mmol) dissolved in 10 ml
H20 was added. The mixture was stirred at 0 °C for 36 h. The solvent was removed under
reduced pressure to obtain a white solid (R f 0.31, 8:1 CH2Cl2/MeOH). To a turbid solution of
this white solid in 200 ml MeOH, Pd/C (10%, 2.1 g) and ammonium formate (2.83 g, 44.9
mmol) were added under N2 at room temperature. The mixture was refluxed for 2 h. After the
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
437
reaction was complete (disappearance o f starting material, as monitored by TLC), the cooled
solution was filtered through Celite, and the filtrate was concentrated to obtain a white solid.
This solid was dissolved in acetone/H 2 0 (2:1, 100 ml) and cooled to 0 °C, and Fmoc-OSu (3.03
g, 8.98 mmol) and NaHCCh (7.32 g, 87.1 mmol) were added. The turbid reaction mixture was
stirred at 0 °C for 1 h and was then allowed to stir at room temperature overnight. The acetone
was removed under reduced pressure. The aqueous residue was diluted with H 2 O (50 ml), stirred
for 1 h at room temperature with diethyl ether (200 ml), and the layers were separated. The ether
layer was washed with saturated aqueous NaHCCE (3x100 ml).
The aqueous layers were
combined, acidified with 1 N aqueous HC1 and extracted with ethyl acetate (3x100 ml). The
combined organic layers were dried over MgSC^ and concentrated to give a white solid. This
crude product was purified by crystallization from refluxing chloroform (300 ml) with careful
addition of MeOH (ca. 10 ml) until all solid dissolved. For complete precipitation, hexanes was
added to the cooled solution until the solution became turbid. After storage at 0 °C overnight
2.52 g (77%) A - ll was obtained as a white solid: mp 206-207 °C; Rf = 0.44, 10:1
CH 2 Cl2 /MeOH; [ a f j = - 36.4 (c 0.50, acetone); !H NMR (300 MHz, CDCI3 /CD 3 OD)
8
7.77 (d,
J = 7.3 Hz, 2H), 7.62 (d, J = 7.0 Hz, 2H), 7.42-7.29 (m, 4H), 4.33-4.19 (m, 3H), 3.73-3.67 (m,
1H), 2.35-2.28 (m, 1H), 2.02-1.98 (m, 2H), 1.78-1.73 (m, 2H), 1.63-1.51 (m, 1H), 1.41-1.22 (m,
3H); 13C NMR (75 MHz, CDCI3 /CD 3 OD) 5 180.61 (C), 160.16 (C), 147.69 (C), 147.53 (C),
144.92 (C), 131.26 (CH), 130.67 (CH), 128.69 (CH), 123.47 (CH), 70.23 (CH2), 55.12 (CH),
52.45 (CH), 50.84 (CH), 36.27 (CH2), 32.73 (CH2), 28.35 (CH2), 28.11 (CH2); MS-ESI m/z calc,
for C 2 2 H 2 3 NO 4 [M], 365.16; obs. 388.1 [M + Na]+, 753.2 [2M + Na]+. (A 8 >Fm oc-ACHC was
prepared by starting with (5)-(-)-a-methylbenzylamine: \ccfo = + 36.7 (c 0.52, acetone).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
438
A.4.3.5 Chiral HPLC Assay o f A-12 and ent-A-12
Compound A -ll was converted to A-12 by esterification, Fmoc-deprotection, and
reaction with 2,4-dinitrofluorobenzene. Similarly, (iS’,S)-Fmoc-ACHC was derivatized to obtain
ent-A-12.
Racemic A-12 in 9:1 hexanes/isopropanol (15 pi o f a 1.0 mg/ml solution) was
injected onto a CHIRALCEL OD column (4.6 x 250 mm; Daicel Chemical Ind., Ltd.). Elution
with 1:1 hexanes/isopropanol at 1.0 ml/min resulted in separation o f the enantiomers (A-12: tr =
26.0 min; ent-A-12: tr = 29.3 min). Solutions o f A-12 and ent-A-12 (1.0 mg/ml) were similarly
analyzed, and integration o f peak areas showed > 99% ee for both enantiomers.
A.4.4 Large-Scale Synthesis of (SVS)-Fmoc-ACHC-OH
O
^
Ph^N H 2
C o2Et
1) NaBH4, isobutyric acid
NH
2) HBr
Ph
pTsOH (5 mole %)
^ / A / C 0 2Et
I
1
3^ RecrySta ||ization
4) NaHC03
56% over two steps
A-6
ent-A-7
r
1) Na, abs. EtOH
Ph^NH
^
,\C02Et 2) HCI
3) Recrystallization
Ph^
©Cl
NH
^
FmocHl^
> ^ . C 0 2Et 2) 10% Pd/C, H C 02NH4
|
|
3) Fmoc-OSu
51%
ent-A-8
1)LiOH-H2Q
|
^L/Uorl
|
61%
ent-A-9
ent-A-10
182 g, 17% overall
Scheme 3.
A .4.4.1 Enamine ent-A-7
A stirred solution o f (S)-(-)-a-methylbenzylamine (364 g, 3.00 mol), ethyl 2-oxocyclohexanecarboxylate (500 g, 2.94 mol) and a catalytic amount o f y>-toluenesulfonic acid (28
g, 147 mmol, 5 mol %) in 4.5 L benzene was refluxed in a 3-necked 5 L round bottom flask
using a heating mantle under a nitrogen atmosphere with continuous removal o f water by using a
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439
Dean-Stark trap for 4 h. The cooled reaction mixture was washed twice with saturated
aqueous NaHCCE. After drying over Na 2 SC>4 and removal o f the solvent by rotary evaporation,
the resulting yellow oily residue was fractionally distilled (bp 170-175°C under vacuum, second
fraction) to give ent-A-7 as a pale yellow oil (JKM IV 101, ~ 80% yield). Rf = 0.31, 20:1
hexane/ethyl acetate, stained with PMA. Characterization data matched A-7.
A.4.4.2 cis B-Amino ester ent-A-8
To isobutyric acid (3.0 L, 32.35 mol) was added sodium borohydride (267 g, 7.06 mol)
portionwise under N 2 at 0 °C. This mixture was further stirred at room temperature for 0.5 h and
then cooled to 0 °C. A solution of ent-A-7 (~ 2.35 mol) in toluene (2.6 L) was added dropwise
under N 2 at 0 °C. The mixture was stirred at 0 °C for 1 h, and then another portion o f sodium
borohydride was added. The reaction mixture was stirred at 0 °C for another 2 h. When the
reaction was complete, 1 L o f water was added carefully, and the reaction mixture was stirred for
10 min at room temperature. Afterwards the mixture was brought to pH 10 with 3 N NaOH and
extracted with EtOAc. The combined organic layers were dried over M gS 0 4 and concentrated
under reduced pressure. The resulting oil was applied to a plug o f silica gel and washed with 1:1
hexane/ethyl acetate. The filtrate was concentrated to obtain a colorless oil (JKM IV 107, ~ 99%
yield; Rf 0.41, 4:1 hexane/ethyl acetate). This oil was dissolved in ethyl acetate (9 L) and cooled
to 0 °C. To this solution 30% (w/w) HBr in propionic acid (578 mL) was added portionwise
with vigorous overhead mechanical stirring. A voluminous white precipitate formed during the
addition. The mixture was stored at 0 °C overnight for complete precipitation. The white solid
was isolated by filtration and washed with three portions o f cold ethyl acetate.
The crude
product was further purified by recrystallization from acetonitrile. The solid was suspended in
acetonitrile and refluxed for 1 h and then stored at 0 °C overnight. The resulting white crystals
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440
were isolated by filtration and dried under vacuum (530 g, ~ 99% de). A second crop was
obtained by another round o f recrystallization from acetonitrile (34 g, ~ 90% de). The two crops
were combined (564 g, 1.583 mol, 54% over two steps, JKM IV 111). This solid was mixed
with an excess o f saturated aqueous NaHCCE (3 L) and extracted with diethyl ether.
The
combined organic extracts were dried over MgSC>4 , concentrated, and dried overnight under
vacuum to give clear oil ent-A-8 (JKM IV 113). Characterization data matched A-8.
A.4.4.3 HC1 Salt ent-A-9
Compound ent-A-8 (~ 1.583 mol) was dissolved in dry ethanol (1 L) under N 2 . In a
separate flame-dried flask was placed dry ethanol (4 L), and sodium (135 g) was added
portionwise under N 2 . The mixture was kept under N 2 and vented to remove evolved gases until
all o f the sodium dissolved. The clear solution o f ent-A-8 was then transferred to the NaOEt
solution, and the mixture was stirred at 80 °C under N 2 for 15 h. The solvent was removed under
vacuum, and after addition o f brine (1.5 L) the mixture was brought to pH 10 with 1 N NaOH
and extracted with ethyl acetate. The combined organic layers were dried over MgSC>4 and
concentrated under reduced pressure. The resulting oil was applied to a plug o f silica gel and
washed with 3:1 hexane/ethyl acetate. The filtrate was concentrated to obtain a pale yellow oil
(JKM IV 115). This oil was dissolved in ethyl acetate (1.5 L), and 4 N HC1 in dioxane (390 mL)
was added slowly at room temperature. The resulting solution was cooled to 0 °C and allowed to
stand at 0 °C overnight. A precipitate formed during this time. The white solid was filtered and
washed three times with portions o f cold ethyl acetate to provide the desired material. This crude
product could be purified by recrystallization from acetonitrile. The solid was suspended in
acetonitrile and heated to reflux for 1 h. The mixture was then filtered through cotton wool and
cooled to 0 °C overnight. The resulting precipitate was isolated by filtration and washed three
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44 1
times with 10 ml portions of cold acetonitrile. The mother liquor and the washings were
combined and condensed to about half volume to get a second crop. The combined crops were
further dried under vacuum to give 254 g o f ent-A-9 as a white crystalline solid (51% yield from
ent-A-8, JKM IV 117). 111 NMR o f the corresponding free amine indicated the diastereomeric
excess to be 98%. Other characterization data matched A-9.
A.4.4.4 (S. 5V Fm o c -AC11C-011 (ent-A-10J
Compound ent-A-9 (254 g, 0.815 mol) was dissolved in TIIF/EtOH/fFO (2:1:1, 4.8 L),
and the clear solution was cooled to 0 °C. LiOH-EhO (171.6 g) dissolved in 1 L H 2 O was added.
The mixture was stirred at 0 °C for 36 h. The solvent was removed under reduced pressure to
obtain a white solid (R f 0.31, 8:1 CH 2 Cl2 /MeOH, JKM IV 119). To a turbid solution o f this
white solid in 5 L MeOH, Pd/C (10%, wet, lOOg) and ammonium formate (210 g) were added
under N 2 at room temperature. The mixture was refluxed for 2 h. After the reaction was complete
(disappearance o f starting material, as monitored by TLC), the cooled solution was filtered
through Celite, and the filtrate was concentrated to obtain a white solid (JKM IV 121). This solid
was dissolved in acetone/H^O (2:1, 4.5 L) and cooled to 0 °C, and Fmoc-OSu (275 g) and
NaHCC>3 (664 g) were added. The turbid reaction mixture was stirred at 0 °C for 1 h and was
then allowed to stir at room temperature overnight during which time the reaction mixture turned
into a thick white sludge. The residue was partitioned between sat. NaHCCh solution and diethyl
ether (200 ml). The aqueous phase and remaining solid was acidified with 1 N aqueous HC1 and
extracted with ethyl acetate.
The combined organic layers were dried over MgSC>4 and
concentrated to give a white solid. This crude product was purified by crystallization from
refluxing chloroform with a small amount o f MeOH. The mixture was filtered while hot to
remove the remaining solid.
For complete precipitation, hexanes were added to the cooled
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442
solution until the solution became turbid. After storage at 0 °C overnight 161 g ent-A-10 was
obtained as a white solid. A second crop was obtained from another round o f recrystallization
(21g). The two crops were combined (182 g, 17% overall yield). Characterization data matched
A-10.
A.5 References
1
2
Schinnerl, M.; Murray, J. K., Langenhan, J. M.; Gellman, S. H. Eur. J. Org. Chem. 2003, 721.
(a) Gellman, S. H. Acc. Chem. Res. 1998, 31, 173-180. (b) Hill, D. J.; Mio, M. J.; Prince, R. B.;
Hughes, T. S.; Moore, J. S. Chem. Rev. 2001, 101, 3893-4011. (c) Seebach, D.; Matthews, J. L. J.
Chem. Soc., Chem. Commun. 1997, 2015-2022. (d) Cheng, R. P.; Gellman, S. H.; DeGrado, W. F.
Chem. Rev. 2001, 101, 3219-3232.
(a) Wang, X.; Espinosa, J. F.; Gellman, S. H. J. Am. Chem. Soc. 2000, 122, 4821-4822. (b)
Lee, H.-S.; Syud, F. A.; Wang, X.; Gellman, S. H. J. Am. Chem. Soc. 2001, 123, 7721-7722.
3
4
Appella, D. H.; Barchi, J. J.; Durell, S. R.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 23092310.
5
(a) Abele, S.; Guichard, G.; Seebach, D. Helv. Chim. Acta 1998, 81, 2141-2156. (b) Gung, B.
W.; Zou, D.; Stalcup, A. M.; Cottrell, C. E. J. Org. Chem. 1999, 64, 2176-2177. (c) Cheng, R.
P.; DeGrado, W. F. J. Am. Chem. Soc. 2001, 123, 5162-5163. (d) Arvidsson, P. I.; Rueping,
M.; Seebach, D. J. Chem. Soc., Chem. Commun. 2001, 649-650.
6
Lee, H.-S.; LePlae, P. L.; Porter, E. A.; Gellman, S. H. J. Org. Chem. 2001, 66, 3597-3599.
7
LePlae, P. L.; Umezawa, N.; Lee, H.-S.; Gellman, S. H. J. Org. Chem. 2001, 66, 5629-5632.
8
Wu, D.; Prasad, K.; Repic, O.; Blacklock, T. J. Tetrahedron: Asymmetry 1997, 8, 1445-1451.
9
Juaristi, E.; Leon-Romo, J. L.; Reyes, A.; Escalante, J. Tetrahedron: Asymmetry 1999, 10,
2441-2495.
10
11
12
(a) Appella, D. H.; LePlae, P. R.; Raguse, T. L.; Gellman, S. H. J. Org. Chem. 2000, 65, 47664769. (b) Raguse, T. L.; Porter, E. A.; Weisblum, B.; Gellman, S. H. J. Am. Chem. Soc. 2002,
124, 12774-12785.
Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.; Gellman, S. H. J. Am. Chem.
Soc. 1999, 121, 6206-6212.
Berkessel, A.; Glaubitz, K.; Lex J. Eur. J. Org. Chem. 2002, 17, 2948-2952.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
443
Appendix B
Efforts Toward Stabilization of the y-Peptide
14-Helix via Monomer Cyclic Constraint
j
A
I
H2 N
/
/
CO2 H
(
?
y-AGPC
■ -...i \ )~'i
H2N
# " C 0 2H
/
y-ACHC
f
"X
7-P ep tid e 14-Helix
Portions of this appendix have been published as:
Gellman, S. H.; Woll, M. G.; Lai, J. R.; Murray, J. “Polypeptides containing yamino acids,” U.S. Patent 6,958,384 B2, October 25, 2005.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
444
B.O Brief Summary of the Appendix
New foldamer 1 scaffolds are important for the development o f future biological
applications. Cyclically constrained y-amino acid residues were prepared and incorporated into
y-peptide oligomers, following the strategy previously employed by Gellman et al. for the
stabilization of {3-peptide helices . 2 The propensity o f these y-peptides to adopt a stable helical
secondary structure was investigated; however, no evidence of secondary structure formation
was observed.
B .l Background
B.1.1 Foldamers
Foldamers, unnatural oligomers with a propensity to adopt stable secondary structure
have been the subject o f intense research . 1 |3-Amino acid oligomers, or {3-peptides, are one class
o f foldamers that has been particularly well developed.
•i
These folded, nonbiological polymers
are characterized by each {1-amino acid residue having an additional backbone methylene group
relative to an a-am ino acid residue and represent a small but significant step away from
naturally-occurring a-peptides (Figure 1).
{3-peptides can adopt several distinct secondary
structures depending on appropriate residue substitution patterns . 4
Preorganization o f the 13-
amino acid residues by cyclically constraining the Ca-Cp bond as part o f a small ring leads to
more stable conformations. In fact, p-peptides containing as few as six residues adopt either o f
two helical conformations in aqueous solution according to the size o f the cyclic constraint . 5 ,6
Five-membered ring constraint (as in /ra«v-2-aminocyclopentanecarboxylic acid (ACPC)) leads
to the
1 2
-helix, defined by
1 2
-membered ring C=Oi->H-N,i3 hydrogen bonds , 5 while six-
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445
membered ring constraint (as in /rara-2-aminocyclohexanecarboxylic acid (ACHC)) gives
rise to the 14-helix, defined by 14-membered ring C=Oi-^H-Nj _2 hydrogen bonds . 6
OH
h 2n
OH
a -a m in o acid
p-am ino acid
ACPC
nh2
lC 0 2H
y-am ino acid
ACHC
Figure 1. Structures o f amino acids.
Combining constrained and substituted, acyclic P-amino acid residues allows one to
prepare P-peptides that display specific arrays o f diverse side chains on a stable threedimensional scaffold. The predictable relationship between p-amino acid sequence and folding
raises the prospect o f endowing P-peptides with useful functions. A number o f applications have
n
been reported for P-peptides, including their use as proteomimetics for the inhibition o f proteinprotein interactions.
Proteolytic 8 and metabolic 9 stability and the prospect o f intracellular
delivery7j make P-peptides very attractive from a biomedical perspective. However, Gellman
and coworkers have found through the development o f foldamer inhibitors for a number o f
protein-protein interactions that no scaffold is capable o f mimicking all targeted protein
structures. Consequently, we have begun to investigate new foldamer backbones. Mixing a -
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446
and P-amino acid residues in varying proportions has produced novel helical structures 10 that
have already proven useful as proteomimetics . 11
B.1.2 y-Peptides
y-Peptides have been identified as a class o f foldamers. Formal insertion o f a methylene
unit into the backbone o f each residue in a p-peptide results in the formation o f a y-peptide
(Figure 1). The secondary structures common to a-peptides (i.e., sheets, turns, and helices) have
also been characterized for y-peptides. A sheet architecture was observed by Schreiber et al. for
their “vinylogous peptides . ”
12
Seebach and coworkers reported that y-peptide B-l adopted a
right-handed 14-helix defined by 14-membered ring C = O j - > H - N , n hydrogen bonds with 2.6
residues per turn, a pitch o f 5.0 A, and an N-AC-terminal helix dipole (Figure 2 ) . 13 Hanessian et
al. demonstrated the importance of substitution and stereochemistry at the a-position o f the yamino acid monomers by showing that tetrapeptide B-2 forms a 14-helix14 while peptide B-3
adopts a reverse turn conformation . 1 5 ,1 6
Seebach and coworkers obtained an X-ray crystal
structure of peptide B-4 in a 14-helical conformation . 17 Gellman et al. characterized a parallel
sheet secondary structure for y-peptide B-5, which contains the cyclically constrained y-amino
acid residue trara-3-aminocyclopentanecarboxylic acid.
»o
The preparation and secondary
structure of other y-peptides and a few heteroatomic analogues have also been reported . 19
Reports o f y-peptide 14-helix formation in aqueous solution are lacking , 2 0 despite the fact
that the 14-helix has been observed both in organic solvents and in the solid state with oligomers
only four residues in length 1 2 ,1 4
The propylene unit o f each y-amino acid residue is a
conformationally flexible element, which makes adopting a specific conformation entropically
unfavorable . 16 However, only a few conformations are available to a substituted propylene unit
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447
due to yy«-pentane interactions, which are even more energetically unfavorable than the
alternative entropic cost . 18 We hypothesized that cyclically constraining the backbone o f the yamino acid residues as part of a small (5- or 6 -membered) carbocyclic ring would preorganize
the residue in a 14-helical conformation and that incorporation o f these constrained residues into
a y-peptide oligomer would stabilize the 14-helical secondary structure. Subsequent replacement
of the carbocyclic rings with nitrogen-containing heterocycles would confer additional water
solubility upon the oligomers and allow structural characterization o f the y-peptide 14-helix in
water. The y-peptide 14-helix could then be used as a scaffold for biological applications.
OH
H
N.
■OBn Boc'
OTMSE
Boc'
B-2
B-3
.OBn
Boc'
NH
B-4
PhH2C.
HN’
B-5
Figure 2. Structures o f y-peptides.
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448
B.2 Preparation and Characterization of y-Peptide Oligomers
B.2.1 Synthesis of Ring Constrained y-Amino Acids
The development o f (3-peptides in our lab served as a guide to the synthesis o f y-peptides.
The enantiomerically pure /V-protected (33-amino acid monomers used for solid-phase (3-peptide
synthesis were readily available from commercially available a-am ino acids via the two-step
Amdt-Eistert homologation procedure . 21 We prepared our cyclically constrained y-amino acids
from the corresponding (3-amino acids using the same procedure (Figure 3). Previous attempts
by Seebach et al. to prepare acyclic y-amino acids in this way had proceeded in low yield.
1
3
Scalable synthetic routes to the cyclically constrained P-amino acids ACPC 2 2 and transaminopyrollidine-4-carboxylic
acid
(APC ) 2 3
and
ACHC
and
/ram-4-aminopiperdine-3-
carboxylic acid (APiC ) 2 4 had been developed previously and provided sufficient starting material
to accomplish the transformations.
ACPC, APC, ACHC, and APiC were successfully homologated to their corresponding yamino acids via the Amdt-Eistert procedure (Figure 3).
First the enantiomerically pure N-
protected amino acid was converted to the corresponding diazoketone by reaction o f the mixed
anhydride (formed with isobutyl chloroformate and A-mcthyl morpholine (NMM)) with CH 2 N2.
This reaction was compatible with the necessary 9-fluorenylmethoxycarbonyl (Fmoc) and tertbutyloxycarbonyl (Boc) protecting groups. The diazoketone underwent the W olff rearrangement
under the influence o f a catalytic amount o f silver benzoate with sonication to produce the
corresponding ketene. Acid hydrolysis afforded the C-terminally homologated amino acid in
moderate yield. Fmoc-ACPC-OH was synthesized and subjected to Amdt-Eistert conditions to
give the diazoketone (57%) and Fmoc-y-ACPC-OH (30%). Boc-ACPC-OH (56% overall yield),
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449
Fmoc-APC(Boc)-OH (50%), Boc-ACHC-OH (31%), and Fmoc-APiC(Boc)-OH (16%) were
also homologated to the corresponding y-amino acids in two steps.
o
FmocHN,
C0 2H 1 - i-BuOCOCI, NMM, THF FmocHN,
r \
2. CH2 N2, Et20
Fmoc-ACPC-OH
1 L ^ N2 1 , PhCOOAg, sonicate FmocHN
(~\
H
2. HCI (aq)
57%
BocHN,
Boc-ACPC-OH
FmocHN,
FmocHN,
Fmoc-y-APC(Boc)-OH
BocHN,
C 02H
Boc-ACHC-OH
31%
16%
Fmoc-APiC(Boc)-OH
/ —COzH
Boc-y-ACHC-OH
FmocHN,
.C02H
-NBoc
r-C 02H
N
Boc
50%
Fmoc-APC(Boc)-OH
BocHN,
o
Boc-y-ACPC-OH
FmocHN,
C 02H
N
Boc
^
Fmoc-y-ACPC-OH
r ~ C 02H
BocHN,
56%
C 02H
(~\
30%
C 02H
r~
g
—C 02H
-NBoc
Fmoc-y-APiC(Boc)-OH
Figure 3. Preparation o f cyclically constrained y-amino acids.
B.2.2 Synthesis and Characterization of y-ACPC-Containing Oligomers
Homo-oligomers o f y-ACPC were prepared by standard solution-phase peptide synthesis
(Figure 4) despite the susceptibility o f y-amino acids to y-lactam formation upon activation o f the
carboxylic acid for peptide coupling. The carboxylic acid o f Boc-y-ACPC-OH was protected as
the benzyl ester by reaction with CS2 CO 3 and benzyl bromide in DMF (87%). The Boc group
was removed from Boc-y-ACPC-OBn with 4 N HCI in dioxane and coupled with Boc-y-ACPCOH using EDCI and DMAP, giving the cyclically constrained y-peptide dimer B-6. A portion of
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NH2-y-ACPC-OBnHCI
Boc-y-ACPC-OBn
Boc-y-ACPC-OH
EDCI, DMAP
\
J-N H
O
H2, Pd/C/
jrA
/ - “- N H
O
°-
Boc-(y-ACPC)2-OBn
/
B-6
O
> -N H
r A
V
O
C"+HdN
O 0H
Boc-(y-ACPC)2-OH
NH2 -(y-ACPC)2-OBnHCI
EDCI, DMAP
O
O
" -NH
^ —NH
O
O
r— NH
O
z—
O
^
Boc-(y-ACPC)4 -OBn
B-7
O
O
NH
/ —
°
HCI
O
O
/ —\
NH
6
C r +H^N
/ —
O
NH
'CS
"
/ —
U
Boc-(y-ACPC)2-OH
'
O
NH
C
/ —
O
NH
/ —k
o
>
°
NH2 -(y-ACPC)4-OBnHCI
EDCI, DMAP ^
O
^ —NH
O
/ —
NH
a
O
'
^ NH
A
O
'
NH
O
.—U—NH
O
a
^ NH
O
/ —^
Boc-(y-ACPC)e-OBn
B-8
Figure 4. Solution-phase synthesis o f Boc-(y-ACPC)6-OBn.
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45 1
dipeptide B-6 was subjected to hydrogenation on a Parr apparatus to remove the benzyl
protecting group. Standard solution-phase peptide coupling conditions were again employed to
yield y-tetrapeptide B-7 (53%). This compound was no longer soluble in pure chloroform but
required some methanol as a co-solvent. Boc-(y-ACPC)2-OH and H 2 N-(y-ACPC)4 -OBn were
coupled to give y-hexapeptide B-8. This compound was soluble only in mixtures o f chloroform
with trifluoroethanol (TFE) and was consequently difficult to purify. These homo-oligomers
were synthesized with the intent o f obtaining an X-ray crystal structure. A number o f trials to
this end were performed by dissolving B-7 and B-8 in chloroform, methylene chloride, or 1,2dichloroethane with small amounts o f methanol or TFE and then spurring crystal growth by
solvent evaporation or liquid-liquid or liquid-vapor diffusion with ether or /7 -heptane. To date,
no suitable crystals have been obtained.
Proton NMR analysis o f B-7 in chloroform and
methanol was unsuccessful due to poor signal dispersion (data not shown).
y-Hexapeptide B-9 (Figure 5) was prepared in pursuit of an NMR solution structure o f
the 14-helix. Solid-phase peptide synthesis with alternating couplings o f Fmoc-y-APC(Boc)-OFl
and Fmoc-y-ACPC-OH gave B-9 in good yield. The product was subjected to 2D-NMR analysis
in CD 3 OD, but overlap in the amide and y-proton regions prevented structural assignment.
CF3CO2
B -9
CF3CO2"
CF3CO2"
Figure 5. Structure o f water soluble g-peptide oligomer for 2D NMR analysis.
Only one cyclic y-amino acid monomer was thoroughly investigated, and no conclusive
evidence for y-peptide 14-helix formation was obtained. Continued crystallization efforts o f B-7
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452
and B-8 could prove fruitful. Varying the temperature or adding pyridine may produce the
dispersion necessary for further analysis o f B-7 and B-9 by NMR spectroscopy. Incorporation o f
derivatized, acyclic y-amino acid residues could also lead to better dispersion in the NMR
spectrum. It is possible that future efforts could identify a secondary structure adopted by this
group o f oligomers or that y-ACPC does not promote secondary structure formation.
Investigation of the cyclohexane ring-containing series o f y-peptides was initiated.
Examination o f the NMR solution structure o f the y-peptide 14-helix revealed that the Ca-Cp
bond has an average dihedral angle % o f 63° and the average dihedral angle 0 for the Cp-Cy bond
was 60°, suggesting that a cyclohexyl ring bearing two adjacent substituents in a trans
configuration could be used to enforce the local geometry needed to form the 14-helix about
either bond.
The homodimer, Boc-(y-ACHC) 2 -OBn, was constructed by solution-phase
synthesis but not structurally characterized.
Further investigations were pursued and then
abandoned because o f synthetic difficulty (Soo Hyuk Choi, unpublished results).
Figure 6. Left) Torsion angles in the y-peptide
backbone. Right) Values o f torsion angles for ypeptide B-2 in a 14-helical conformation.
R e sid u e
1
2
3
4
5
6
A v era g e
d>
-132
-119
-157
-131
-135
-103
-130
0
52
66
62
59
63
59
60
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X
60
67
64
65
63
61
63
-163
-152
-131
-154
-135
-157
-149
453
B.3 Conclusions
Stabilization o f a y-peptide in the 14-helical conformation by constraining the Cp-Cy bond
o f the y-amino acid residue backbone as part o f a five-membered ring has been attempted.
Cyclic y-amino acids were prepared from their corresponding P-amino acids via the AmdtEistert homologation. Various y-peptide oligomers were prepared; however, none crystallized or
was suitable for analysis by NMR spectroscopy. Development o f the y-peptide 14-helix as a
scaffold for future biological applications o f foldamers is desirable but will require further
investigation.
B.4 Experimental Methods
B.4.1 Genera] Procedures
THF was distilled from sodium/benzophenone ketyl under N 2.
distilled from calcium hydride.
Triethylamine was
All commercially available reagents and solvents were
purchased from Aldrich and used without further purification, except for 4 N HCI in dioxane,
which was purchased from Pierce and Fmoc-OSu, which was purchased from Advanced
ChemTech.
Analytical thin-layer chromatography (TLC) was carried out on Whatman TLC
plates precoated with silica gel 60 (250 jum layer thickness). Visualization was accomplished
using a UV lamp, phosphomolybdic acid (PMA) stain (10% phosphomolybdic acid in ethanol),
or KMnC>4 stain. Column chromatography was performed on EM Science silica gel 60 (230-400
mesh). Solvent mixtures used for TLC and column chromatography are reported in (v/v) ratios.
Diastereomeric excesses were determined using 1H NMR.
Enantiomeric excesses were
determined using chiral HPLC.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
454
B.4.2 Synthesis of Boc-y-ACPC-OH
According to Scheme 1, (lA2S)-2-Amino-cyclopentanecarboxylic acid 2 2 (NH 2 -ACPCOH, JK M I91, 5.18 g, 40.1 mmol) was dissolved in methanol (409 mL). Triethylamine (11.16
mL, 80.2 mmol) was added via syringe followed by di-/er/-butyl-dicarbonate (8.74 g, 40.1
mmol). The reaction mixture was stirred at room temperature for 2 h. The solvent was removed
by rotary evaporation. The residue was diluted with ethyl acetate (250 mL), washed with 0.5M
NaHS 0 4 (1 x 100) and saturated aqueous NaCl solution (1 x 100). The organic layer was dried
over MgSCL, filtered, and concentrated to give a white solid. The crude product was purified by
column chromatography (7:3:0.3) hexane:ethyl acetate:acetic acid). Acetic acid was removed on
the high vacuum rotary evaporator. Benzene (2 x 100 mL) was added and removed on the rotary
evaporator. The residue was dried under high vacuum overnight to give a white solid (BocACPC-OH, JK M I103, 7.65 g, 83%). ]H NMR (250MHz, C D C I 3 ):
8
= 11.22 (br. s, 1 H), 4.93
(br. s, 1 H), 4.02 (br. s, 1 H ), 2.69 (br. s, 1H), 2.14-1.89 (m, 3 H), 1.68 (quin., J = 6.7 Hz, 2 H),
1.51-1.35 (m, 10 H) ppm.
(17?,25}-2-tert-Butoxycarbonylamino-cyclopentanecarboxylic
acid
(Boc-ACPC-OH,
JK M I103, 6.94 g, 30.3 mmol) was placed in an oven-dried flask with a clear-seal joint
containing an oven-dried stir bar and dissolved in dry THF (61 mL) under a N 2 atmosphere. The
solution was cooled to -14°C with an ice/brine bath. iV-methylmorpholine (3.50 mL, 31.8 mmol)
and isobutylchloroformate (4.13 mL, 31.8 mmol) were added via syringe.
The mixture was
stirred for 1 h and allowed to warm to 0°C. A white precipitate formed during this time. The
flask was then fitted with an oven-dried Diazald apparatus, and the joint was sealed with
parafilm. A solution o f KOH (12 g, 214 mmol) in H20 (20 mL) and 2-methoxyethanol (16 mL)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Boc-ACPC-OH
JKM103
h2n -a c p c -o h
JKMI91
CgH-i -|N02
Boc-ACPC-CHN2
JKMI125
Ci -| H-19NO4
Mol. Wt.: 229.27
Mol. Wt.: 129.16
C 12H 19N 3O 3
Mol. Wt.: 253.30
/?
0
NH
W
O
C I+ H 3 N
f—
OH
Boc-y-ACPC-OBn
JKMI129
Boc-y-ACPC-OH
JKMI127
C-12 H2 1 NO4
C1 9 H2 7 NO4
Mol. Wt.: 243.30
Mol. Wt.: 333.42
NH2 -y-ACPC-OBnHCI
JKMI133
c 14h 20 c i n o 2
Mol. Wt.: 269.77
NH
B-6
Boc-(y-ACPC)2-OBn
JKMI135
C 26H3 8 N 2 0 5
Mol. Wt.: 458.59
o
$ — NH
O
NH
<
NH
OH
Boc-(y-ACPC)2-OH
JKMI139
H2 N-(y-ACPC)2-OBnHCI
JKMI141
c 2 1 h 3 1 c in 2 o 3
Mol. Wt.: 394.94
C-|9 H3 2 N2 0 5
Mol. Wt.: 368.47
o
O
-NH
-NH
O
-H-NH
B-7
o
O
/-A
0
Boc-(y-ACPC)4-OBn
JKMI147
O 40H 60N 4O 7
Mol. Wt.: 708.93
Scheme 1. Synthetic routes to Boe-y-ACPC-OH, Boc-(y-ACPC)2-OBn (B-6), and Boc-(y-ACPC)4-OBn (B-7).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
456
was placed in the well o f the apparatus with a stir bar and heated to 75°C. A saturated
solution of Diazald (19.5 g, 91 mmol) in diethyl ether (100 mL) was decanted into a dropping
funnel attached to the apparatus. The vacuum adapter was fitted with a septum, and the system
was placed under N 2 . The cold finger was cooled to -78°C with a dryice/isopropanol mixture.
The Diazald solution was then dropped into the KOH solution over a period o f 30 min. The
yellow diazomethane distilled over, condensed on the cold finger, and dropped into the reaction
mixture. Once distillation o f the diazomethane was complete, and the reaction mixture had a
persistent yellow color, the Diazald apparatus was removed. The flask was stoppered, placed
under N 2, and stirred for 4 h, being allowed to warm from 0°C to room temperature. Acetic acid
(1 mL) was then added to neutralize any excess diazomethane. The reaction mixture was diluted
with diethyl ether (200 mL), washed with saturated aqueous NaHC 0 3 (2 x 100 mL), aqueous
HCI (IN , 100 mL), and saturated aqueous NaCl solution (100 mL). The organic layer was dried
over MgSCL, filtered, and concentrated.
The crude product was purified by column
chromatography (8:2 hexane:ethyl acetate) to give a pale yellow solid (B 0 C-ACPC-CHN 2 ,
JK M I125, 4.75 g, 62%). 'H NMR (250MHz, CDCI3 ):
6
= 5.58 (br. s, 1 H), 4.70 (br. s, 1 H),
3.99 (quin., J = 6.3 Hz, 1 H), 2.66 (br. s, 1 H), 2.03-1.55 (m, 5 H), 1.49-1.29 (m, 10 H) ppm.
This solid (Boc-ACPC-CHN2, JK M I125, 2.71 g, 10.68 mmol) was dissolved in
water/dioxane (1/5, 480 mL), and the flask was covered in aluminum foil to exclude light. Silver
benzoate (0.245 g, 10 mol%) was added as a catalyst, and the mixture was sonicated at room
temperature under N 2 for lh. At 0°C the solution was acidified to pH 2 with aqueous 0.5M
NaHS 0 4 . The solution was extracted with diethyl ether (4 x 150 mL). The organic layer was
dried over MgSCL, filtered, and concentrated to give a white solid.
The crude product was
purified by column chromatography (6:4:0.3 hexane:ethyl acetate:acetic acid). Acetic acid was
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
457
removed on the high vacuum rotary evaporator.
Benzene (2 x 200 mL) was added and
removed on the rotary evaporator. The residue was dried under high vacuum overnight to give a
yellowish white solid (((li?,25)-2-/ert-Butoxycarbonylamino-cyclopentyl)-acetic acid, Boc-yACPC-OH, JKMI127, 2.36 g, 91%). This compound exists as slowly interconverting rotamers.
'H NMR (300 MHz, CDC13): 5 = 11.63 (br. s, 1 H), 6.53 (br. s, 1 H), 5.04 (d, J = 7.4 Hz, 1 H),
3.59-3.46 (m, 1 H), 2.65-2.55 (m, 1 H), 2.28-2.23 (br. s, 1 H), 2.07-1.93 (M, 3 H), 1.66-1.24 (m,
12 H) ppm. ,3C NMR (62.9MHz, CDC13): 5 = 178.04 (C), 157.84, 156.14 (C), 80.82, 80.75 (C),
58.20, 56.91 (CH), 42.84 (CH), 38.00 (CH2), 32.38 (CH2), 30.03 (CH2), 28.45 (3CH3), 21.51
(CH2) ppm. MS-ESI: m/z = 242.2 [M-H]-, 485.3 [2M-H]'.
B.4.3 Synthesis of Boc-(y-ACPC)2-OBn (B-6)
According to Scheme 1, this solid (Boc-y-ACPC-OH, JKMI127, 1.00 g, 4.12 mmol) and
CSCO3 (1.34 g, 4.12 mmol) were dissolved in THF (41 mL). Benzyl bromide (0.53 mL, 4.44
mmol) was added, and the mixture was stirred at room temperature under N 2 covered in
aluminum foil for 24 h. The mixture was diluted with ethyl acetate (200 mL) and washed with
saturated aqueous N aH C 0 3 (1 x 100 mL) and saturated aqueous NaCl (1 x 100 mL) solutions.
The organic layer was dried over MgS 0 4 , filtered, and concentrated to give a white solid. The
crude product was purified by column chromatography ( 8 : 2 hexane:ethyl acetate) and dried
overnight under high vacuum to give white solid (Boc-y-ACPC-OBn, JKMI129, 0.58 g, 42%).
‘H NMR (300 MHz, CDC13):
6
= 7.40-7.30 (m, 5 H), 5.12 (s, 2 H), 4.59 (br. s, 1 H), 3.57 (br. t, J
= 8.2 Hz, 1 H), 2.65 (dd, J = 15.8, 4.9 Hz, 1 H), 2.32 (dd, J = 15.6,
8 .8
Hz, 1 H), 2.11-1.88 (m, 3
H), 1.70-1.59 (m, 2 H), 1.43-1.21 (m, 11 H) ppm. The combined aqueous layers were acidified
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
458
with acetic acid and extracted with ethyl acetate (3 x
75
mL) to isolate unreacted starting
material (Boc-y-ACPC-OH).
This white solid (Boc-y-ACPC-OBn, JKMI129, 0.839 g, 2.52 mmol) was dissolved in
4N HCI in dioxane (10 mL) and stirred under N 2 for three hours at room temperature. The
solvent was blown off overnight under a stream o f N2. The residue was placed under high
vacuum for one hour and then carried on without further purification (NH 2 -y-ACPC-OBn»HCl,
JKMI133).
Boc-y-ACPC-OH (JKMI127, 0.612 g, 2.52 mmol), NH 2 -y-ACPC-OBn*HCl (JKMI133,
0.679 g, 2.52 mmol), and 4-dimethylaminopyridine (1.076 g, 8.817 mmol) were dissolved in
DMF (15 mL, anhydrous). The solution turned a pale orange color. To the solution was added
l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.726 g, 3.78 mmol), and the
reaction mixture was stirred under N 2 at room temperature overnight. The mixture was diluted
with 100 mL ethyl acetate and washed with 0.5M aqueous NaHSCL (1 x
aqueous N aH C 0 3 (1 x
7 5
mL), and saturated aqueous NaCl (1 x
layer was dried over MgSCL, filtered, and concentrated.
7 5
7 5
mL), saturated
mL) solutions. The organic
The crude product was purified by
column chromatography (1:1 hexane:ethyl acetate) to give a white solid (Boc-(y-ACPC)2 -OBn,
JKMI135, 1.004 g, 87%). 'H NM R (300 MHz, CDC13): 5 = 7.38-7.28 (m, 5 H), 6.77 (d, J = 7.0
Hz, 1 H), 5.10 (s, 2 H), 4.82 (d, J = 7.8 Hz, 1 H), 3.87 (quin., J = 8.1 Hz, 1 H), 3.56 (br. quin., J =
7.8 Hz, 1 H), 2.64 (dd, J = 15.8, 4.9 Hz, 1 H), 2.40-2.29 (m, 2 H), 2.19-1.83 (m, 7 H), 1.72-1.47
(m, 4 H), 1.43-1.21 (m, 13 H) ppm. MS-ESI: m/z = 481.3 [M+Na]+, 939.5 [2M+Na]+.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
459
B.4.4 Synthesis of Boc-(y-ACPC)4-OBn (B-7)
According to Scheme 1, Boc-(y-ACPC)2-OBn (JKMI135, 0.375 g, 0.819 mmol) was
dissolved in methanol (8.2 mL), and the flask was flushed with N2. To the flask was added Pd/C
10% (0.056 g), and the flask was attached to a Parr apparatus and shaken overnight at an H 2
pressure of 44 psi. The reaction mixture was filtered through a syringe filter and concentrated on
the rotovap to give a white solid (Boc-(y-ACPC)2 -OH, JKMI139, 0.289 g, 96%). The crude
product was carried on without further purification.
Boc-(y-ACPC)2-OBn (JKMI135, 0.375 g, 0.819 mmol) was dissolved in 4N HCI in
dioxane (10 mL) and stirred under N 2 at room temperature for 2 h. The solvent was blown off
overnight under a stream of N2. The residue was placed under high vacuum for 1 h. The residue
(NH 2 -(y-ACPC)2 -OBn»HCl, JKMI141) was carried on without further purification.
Boc-(y-ACPC)2-OH (JKMI139, 0.289 g, 0.786 mmol), NH 2 -y-ACPC-y-ACPC-OBn*HCl
(JKMI141), and 4-dimethylaminopyridine (0.350 g, 2.87 mmol) were dissolved in DMF (8.2
mL, anhydrous).
To the solution was added l-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (0.236 g, 1.23 mmol), and the reaction mixture was stirred under N 2 at room
temperature overnight. A precipitate formed, and the solution turned pale orange during this
time. Water (25 mL) was added to the reaction mixture to completely precipitate the product,
which was then isolated by suction filtration. The solid was dissolved in CH 2 C12 (100 mL). The
aqueous layer was extracted with CH 2 C12 (3 x 25 mL). The combined organic extracts were
washed with 0.5M aqueous N aH S 0 4 (1 x 25 mL), saturated aqueous NaHCCL (1 x 25 mL), and
saturated aqueous NaCl (1 x 25 mL) solutions.
filtered, and concentrated.
The organic layer was dried over M gS 04,
The organic layers were pooled, washed with saturated aqueous
NaCl, dried over M gS 04, and concentrated to give a white solid. The crude product was purified
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
460
by column chromatography (CH 2 CI2
10%MeOH in CH 2 CI2 , loaded by adsorption to silica
gel) to give a white solid (Boc-(y-ACPC)4 -OBn, JKMI147, 0.306 g, 53%). *H NMR (300 MHz,
10:1 CDC13 :CD3 0D ): 5 = 7.58 (br. s, 2 H), 7.41-7.29 (m, 5 H), 7.23 (br. s, 2 H), 5.11 (s, 2 H),
3.87-3.74 (m, 3 H), 3.54-3.42 (m, 2 H), 2.63 (dd, J = 15.5, 4.3 Hz, 1 H), 2.37-1.84 (m, 22 H),
1.74-1.52 (m, 7 H), 1.43-1.15 (m, 14 H) ppm. MS-ESI: m/z = 731.5 [M+Na]+.
B.4.5 Synthesis of Boc-(y-ACPC)6-OBn (B-8)
According to Scheme 2, Boc-(y-ACPC)4 -OBn (JKMI147, 0.150 g, 0.212 mmol) was
dissolved in 4N HCI in dioxane (10 mL) and stirred under N 2 at room temperature for 2 h. The
material fully dissolved, then a white precipitate formed. The solvent was blown off overnight
under a stream of N2. The residue was placed under high vacuum for 1 h. The residue (NH 2 -(yACPC)4 -OBn*llCl, JKMI151) was carried on without further purification.
Boc-y-(ACPC)2-OH (JKMI139, 0.078 g, 0.212 mmol), NH 2 -(y-ACPC)4 -OBn*HCl
(JKMI151), and 4-dimethylaminopyridine (0.091 g, 0.742 mmol) were dissolved in DMF (5
mL, anhydrous).
To the solution was added l-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (0.061 g, 0.318 mmol), and the reaction mixture was stirred under N 2 at room
temperature for 48 h. The solvent was then removed under a stream o f N 2 and then under high
vacuum. The residue was diluted with H 2 0 . The insoluble material was isolated by filtration
and dried under vacuum.
This solid was washed with diethyl ether and ethyl acetate then
dissolved in 5:1 CHCl3 :CF3 CH2OH and washed with 0.5 M N aH S 04, saturated aqueous
N aH C 03, and saturated aqueous NaCl solutions.
A solid precipitated upon addition of the
aqueous solutions. This solid was isolated by filtration and dried under vacuum to give a white
solid (Boc-(y-ACPC)6 -OBn, JKMI155, approximately 0.1 g).
'H NMR (300 MHz, 5:1
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
461
CD 2 Cl2 :CF3 CD 2 OD): 5 = 7.37-7.34 (m, 5 H), 6.95-6.82 (br. m, 4 H), 6.65 (d, J = 6.0 Hz, 1
H), 5.15 (s, 1 H), 5.10 (s, 2 H), 3.93-3.89 (m, 2 H), 3.88-3.58 (m, 3 H), 3.59-3.40 (m, 11 H), 2.57
(dd, J = 15.6, 4.9 Hz, 1 H), 2.36-2.26 (m, 3 H), 2.13-1.83 (m, 15 H), 1.70-1.63 (m,
8
H), 1.41-
1.18 (m, 26 H) ppm. MALDI-TOF MS: m/z = 981.5 [M+Na]+.
O
o
o
-U-NH
-NH
B-7
■
0
'
\
w—k
°
Boc-(y-AC PC )4 -OB n
JKMI147
C 40H 60N 4O 7
Mol. Wt.: 708.93
\
O
o
$— NH
"
o
o
-N H
r~A
o
c i- +H 3N,
0H
H2 N-(y-ACPC)4-OBnHCI
JKMI151
Boc-(y-AC PC )2- 0 H
JKMI139
Mol. Wt.: 368.47
NH
NH
NH
NH
NH
B-8
Boc-(y-ACPC)6 -OBn
JKMI155
C54H 82N 6O 9
Mol. Wt.: 959.26
Scheme 2. Synthetic route to Boc-(y-ACPC)(, - 0 Bn (B-8).
B.4.6 Synthesis of Fmoc-y-ACPC-OH
According
to
Scheme
3,
(LS,25,)-2-(9//-fluoren-9-ylmethoxycarbonylamino)-
cyclopentane carboxylic acid (Fmoc-ACPC-OH, JK M I97, 0.56 g, 1.59 mmol) was placed in an
oven-dried flask with a clear-seal joint containing an oven-dried stir bar and dissolved in dry
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
462
THF
(8
mL) under a N 2 atmosphere. The solution was cooled to -14°C with an ice/brine
bath. /V-methylmorpholine (183 pL, 1.67 mmol) and isobutylchloroformate (216 pL, 1.67
mmol) were added via syringe. The mixture was stirred for 1 h and allowed to warm to 0°C. A
white precipitate formed during this time. The flask was then fitted with an oven-dried Diazald
O
%
K qh
°
^-N H
P
NH / — ^
V -/
OH
O
JKMI113
C 2 i H 21NO 4
Fmoc-y-APC(Boc)-OH
JKMII9
JKMII7
O2 5 H2 8 N2 O6
O2 6 H3 0 N2 O6
O2 6 H2 8 N4 O5
Mol. Wt.: 452.50
Mol. Wt.: 466.53
Mol. Wt.: 476.52
O
O
-NH
O
-NH
N
H2+
h
2+
o
o
-NH
N
CF3 CO2
Mol. Wt.: 365.42
Fmoc-APC(Boc)-CHN2
Fmoc-APC(Boc)-OH
JKMI299
NH
C2 2 H2 3 NO4
C2 2 H2 1 N3 O3
Mol. Wt.: 375.42
Mol. Wt.: 351.40
O
Fmoc-y-ACPC-OH
JKMI115
Fmoc-ACPC-CHN2
Fmoc-ACPC-OH
JKMI97
B-9
CF3 CO2
o
W
— ^
'■
f—
<
-NH
- U - n h
n h 2
N
H2+
CF3 CO2 "
Ac-(y-APC-y-ACPC)-N H2 as TFA Salt
JKMII23
C4 7 H7 1 F9 N-10O -13
Mol. Wt.: 1155.11
O4 1 H5 8 N 1 0 O7 (no TFA)
Mol. Wt.: 813.04
Scheme 3. Synthetic routes to Fmoc-y-ACPC-OH and Fmoc-y-APC-OH with Ac-(y-APC- y-ACPC)3-OBn (B-9)
shown at bottom.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
463
apparatus, and the joint was sealed with parafilm. A solution of KOH (4 g, 71.4 mmol) in
H20 (6.7 mL) and 2-methoxyethanol (5.3 mL) was placed in the well o f the apparatus with a stir
bar and heated to 75°C. A saturated solution o f Diazald (1.02 g, 4.76 mmol) in diethyl ether
(lOmL) was decanted into a dropping funnel attached to the apparatus. The vacuum adapter was
fitted with a septum, and the system was placed under N2. The cold finger was cooled to -78°C
with a dryice/isopropanol mixture.
The Diazald solution was then dropped into the KOH
solution over a period o f 30 min. The yellow diazomethane distilled over, condensed on the cold
finger, and dropped into the reaction mixture.
Once distillation o f the diazomethane was
complete, and the reaction mixture had a persistent yellow color, the Diazald apparatus was
removed. The flask was stoppered, placed under N 2, and stirred for 4 h, being allowed to warm
from 0°C to room temperature. Acetic acid (1 mL) was then added to neutralize any excess
diazomethane. An off-white precipitate formed at this time. The reaction mixture was diluted
with CH 2 C12 (200 mL), washed with saturated aqueous NaHC 0 3
(2
x 100 mL), aqueous HC1 (1
N, 100 mL) and saturated aqueous NaCl solution (100 mL). The organic layer was dried over
MgSCL, filtered, and concentrated. The crude product was purified by column chromatography
(1% methanol in CH 2 C12, loaded by adsorption onto silica gel) to give a white solid (FmocACPC-CHN2, JK M I113, 0.42 g, 71%). *H NMR (250MHz, DMSO_d6):
8
= 7.89 (d, J = 7.0 Hz,
2 H), 7.68 (d, J = 7.3 Hz, 2 H), 7.41 (t, j = 7.4 Hz, 2 H), 7.32 (t, J = 7.4 Hz, 2 H), 6.01 (s, 1 H),
4.32-4.17 (m, 2 H), 4.01-3.92 (m, 1H), 3.56-3.26 (m, 2 H), 2.74-2.55 (m, 1 H), 1.99-1.43 (m,
6
H) ppm.
This solid (Fmoc-ACPC-CHN2, JK M I113, 0.145 g, 0.387 mmol) was dissolved in
water/dioxane (1/5, 20 mL), and the flask was covered in aluminum foil to exclude light. Silver
benzoate (9 mg, 10 mol%) was added as a catalyst, and the mixture was sonicated at room
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
464
temperature under N 2 for lh. At 0°C the solution was acidified to pH 2 with aqueous HC1 (1
N). The solution was extracted with diethyl ether (4 x 25 mL). The organic layer was dried over
MgSCL, filtered, and concentrated to give a white solid. The crude product was purified by
column chromatography (1:1:0.3 hexane:ethyl acetate:acetic acid). Acetic acid was removed on
the high vacuum rotary evaporator. Benzene (2 x 100 mL) was added and removed on the rotary
evaporator.
The residue was recrystallized from CH 2 Cl2 /hexane to obtain a white solid
([(li?,25)-2-(9//-Fluoren-9-ylmethoxycarbonylamino)-cyclopentyl]-acetic acid, Fmoc-y-ACPCOH, JK M I115, 0.041 g, 29%). *H NMR (300 MHz, CD 3 OD):
8
= 7.78 (d, J = 7.3 Hz, 2 H),
7.64 (d, J = 7.5