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Small Peptide LibrariesCombinatorial Split-Mix Synthesis followed by Combinatorial Amino Acid Analysis of Selected Variants.

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Weinberger, Lichte, Griesinger, and Kutscher
Small Peptide Libraries: Combinatorial Split-Mix Synthesis followed
by Combinatorial Amino Acid Analysis of Selected Variants
Heinz Weinbergera)'')*, Ellen Lichtea),Christian Griesingera),and Bernhard Kutscherb)
Goethe-University of Frankfurt, FrankfurtMain, Germany
Institute of Organic Chemistry, J.W.
ASTA Medica AG, Department of Medicinal Chemistry, Frankfurt/Main, Germany
Key Words: Amino acid analysis; combinatorial peptide library; split-mix synthesis; TentaGel-resin
Peptides from small combinatorial libraries, covalently attached to
polymeric TentaGel beads, can be directly sequenced using amino
acid analysis. For libraries with restricted diversity, generated by
the split-mix synthesis method, the amino acids on a selected single
bead identified by pre-column derivatization with o-phthaldialdehyde (OPA) correlatedirectly with the sequenceof a given peptide.
This is shown on a tripeptide (343 different compounds) and a
tetrapeptide (4096 different compounds) library. This method
allows for rapid peptide sequence determination without relying
on complex encoding strategies.
With the ongoing development of combinatorial chemistry
[11,rapid and readily available analytical tools for structural
determinationof active compounds covalent1 attached to the
bead are now of considerable importance
For peptide
libraries, several strategies have been devised to address this
problem. Edman microsequence analysis has been widely
used for this purpose [4,51, but this expensive method works
only with natural amino acids and is time-consuming. Recently, Youngquist et al.L6] introduced a method for sequencing peptides by partial capping of the growing molecule in
each reaction step and finally analyzing the different peptide
fragments after cleavage from the resin by matrix assisted
laser desorption ionization mass spectrometry. However, this
strategy is compromised by the presence of additional peptides on each single bead which may also interact with the
soluble receptor.
Herein, we describe a method without relying on encoding
strategies that enables rapid determination of the sequence of
peptides from support-bound combinatorial libraries using
amino acid analysis.
Results and Discussion
During the course of our work in searching for high affinity
ligands to cytokine receptors, we constructed several uncoded peptide libraries by standard Fmoc/fB~-chemistry[~]
TentaGel as solid support, following the split-mix synthesis
method developed by Furka [*,'I. After the corresponding
"Present address: ASTA Medica AG, Department of Medicinal Chemistry,
Weismiillerstralk 45, D-603 14 FrankfudMain, Germany
Arch. Phann. Phann. Med. Chem.
bioassay, the identification of immobilized receptor binding
ligands was achieved by bead staining techniques using fluorescein isothiocyanate (FITC) labeled antibodies. Individual
staining beads were revealed by visual inspection and the
determination of the peptide sequence attached to a single
resin bead was accomplished by amino acid analysis using
pre-column derivatization with OPA-reagent [Io1. Although
yielding only information about the amino acid content of the
peptide, in combination with split-mix synthesis amino acid
analysis enables peptide sequencing. The repetition of the
splittinghixing operations namely has an important consequence on the composition of the peptide. As shown in
Figure 1, the 'one-bead, one-peptide' approach["] ensures
that each of the resin beads carries a single peptide sequence.
Moreover, every support bound peptide consists of only one
amino acid of each reaction step, e.g. tripeptides which contain amino acid A cannot hold amino acid B or C at the same
time. The same applied to the amino acids D, E, and F or G,
H, and K.
Thus, if amino acid A is identified by amino acid analysis
it takes position three in the corresponding tripeptide (starting
from the N-terminus).
We now show that for libraries with a restricted diversity it
is possible to derive the absolute position in the peptide from
the result of the amino acid sequencing, resulting in the
definite peptide sequence. To demonstrate the utility of this
method, we have prepared a tripeptide library with 19 different L-amino acids arranged in a 7 by 3 matrix (Figure 2).
Between the three coupling steps, the resin was pooled, mixed
and subsequently divided in 7 reaction vessels, thus resulting
in 73 = 343 trimers. Following the corresponding bioassay,
the brightest beads were physically picked out under a fluorescence microscope with a glass syringe. After removal of
the receptor complex with 5 % sodium n-dodecyl sulfate
(SDS), individual stained beads were hydrolysed in 6 N HCl.
The resulting mixture was derivatized with OPA-reagent and
analyzed by HPLC. As shown in Figure 2, the amino acids
alanine, tyrosine, isoleucine, and the linker e-aminocaproic
acid (Aca) were identified by single-bead amino acid analysis.
In the 7 by 3 amino acid matrix Ile is contained only in the
subset for AAl and therefore is AA1. Only the subset for A A 2
and AA3 contains Ala and Tyr, respectively, assigning these
amino acids to AA2 and AA3 unambiguously.Therefore, the
peptide sequence of the trimer results in H-Tyr-Ala-Ile-OH.
Our studies established that the amount of the peptide
present on any given single bead (250 pmol maximum) is
more than sufficient since the detection limit in OPA-based
0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1997
0365-6233/97/0404-0109$17.50 +.50/0
Combinatorial Amino Acid Analysis of Selected Variants
Figure 1. The split-mix synthesis method of preparing a combinatorial library. In this illustration only three synthetic cycles with nine different amino acids
(A-K) are used, giving a combinatorial library of 27 trimeric peptides.
Figure 2. Amino acid analysis of a single-bead bound tripeptide after the
bioassay (PEG = polyethylene glycol). The amino acids found are highlighted in the 7 by 3 matrix on top.
amino acid analysis is 20-50 pmol. For generating larger
libraries with greater diversity, it is inevitable to use amino
acids several times. In this case, the above demonstrated
principle of exclusion will lead to an intense reduction in the
number of sequence possibilities up to the unique peptide
sequence. Therefore, another tetrapeptide library attached to
the above linked resin was prepared consisting of only 16
amino acids arranged in a 8 by 4 matrix resulting in 84 = 4096
different compounds (Figure 3). After the bioassay, isolation
and hydrolysis, a single-bead bound tetrapeptide was analyzed. Therein, glutaminic acid, tyrosine, valine, lysine, and
the linker Aca were identified by amino acid analysis. The
remarkably low signal intensity of the isoindole derivative of
lysine was found for all lysine containing peptide hydrolyzates.
The amino acid sequence can be derived from the amino
acid analysis in the below way. Val is contained only in subset
AA3. Therefore, Lys, which is contained both in subset AA3
and AAI can only be AAi. By the same taken, Tyr being in
subset AAI as well as in A A 2 must represent AA2. A A 4
remains for either glutamine or glutaminic acid, because Gln
is hydrolyzed to Glu by treating with 6 N HCl. Consequently,
amino acid analysis reduces the number of tetrapeptide sequences to the following 2 out of 4096 possibilities: H-GluVal-Tyr-Lys-OH or H-Gln-Val-Tyr-Lys-OH.
Arch. P h a m P h a m Med Chem 330,109-111 (1997)
Weinberger, Lichte, Griesinger, and Kutscher
AA, = amino acid
were attached to the resin serving as a linker between the polyethyleneglycol
(PEG) chains and the peptide by using a 4-fold excess of Fmoc-Aca in each
coupling reaction. The peptide libraries were synthesized on the resin by
standardFmoc chemistryfollowingthe split-mix synthesismethod.Coupling
of protected amino acids was carried out with DIC in the presence of HOBt
using a 20-fold excess of Fmoc-amino acid. Reactions were run on a 4.5 pM
(tripeptide) and 5 pM (tetrapeptide) scale. After each coupling and mixing
step, the N-a-Fmoc protecting groups of the building blocks were removed
by 40 % ( v h ) piperidine in DMF. After completion of peptide synthesis, the
libraries were deprotected using 4-6 ml of a mixture of TFA (8.25 ml), Hz0
(0.5ml), thioanisole (0.5ml), phenol (0.5 g) and 1,2-ethanedithiol(O.25ml)
per 1 0 0 mg of peptidyl resin. After 4-6 h the resin was washed five times
with DMF (5 ml),three times with ether (5 ml) and finally dried under high
For amino acid sequencing, a selected single bead was washed thoroughly
with 5 % SDS and placed into a melting point glass tube. 30 pI of aqueous
6 N HCI (containing0.1 % Phenol) was added, the glass tube was sealed and
the hydrolysis were run at 110 "C for 22 h. To the resulting hydrolysate was
added 30 pl of 5 N NaOH and a 30 fl aliquot of that solution was mixed with
20fl each of borate buffer and OPA-reagent, purchased from GROM
(Herrenberg, Germany). After 3 min standing at room temperature,20 pl of
1 N HCL was added and a 60 p1 aliquot of that mixture was placed directly
on a GROM-SIL OPA column (5 pn, 4 x 250 nun) under the following
conditions: eluant A 50 mmol Na acetate pH 7.2, eluant B: acetonitrile; for
tripeptide analysis: gradient from 95% to 40% A and 5% to 60% B over 50
min, flow 1.O d m i n ; for tetrapeptide analysis: gradient from 100% to 0%
A and 0%to 80% B over 50 min, flow 1.O ml/min; fluorescence detection at
340 nm (excitation maximum), 450 nm (emission maximum).
Figure 3. Amino acid analysis of a single-bead bound tetrapeptide after the
bioassay. The four amino acids found are highlighted in the 8 by 4 matrix on
In contrast to the time consuming and expensive Edman
degradation method, we have developed a strategy which is
simple to use and does not require any tagging procedures.
Furthermore, the identification of non-natural amino acids as
well as the distinction between L- and D-aminoacids and the
sequencing of cyclic peptides is also possible. For small
combinatorial peptide libraries, the combination of the splitmix synthesis method and amino acid analysis can be a powerful tool for the rapid discovery of novel biologically-active
lead compounds.
This work has been supported by the Bundesministerium fiir Bildung,
Wissenschaft,Forschung und Technologie(BMBF), BOMunder No. 10792.
The amino acid analyzer was provided by the DFG under Gr 121111-2. We
are indebted to Prof. Dr. A. Kleemann (ASTA Medica AG) and Prof. Dr. G.
Quinkert (University of Frankfurt) for their active interest in establishing and
running the joint postdoctoral research program.
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J. Med. Chem. 1994,37, 1233-1251.
[2] X.Williard, I. Pop, L. Bowel, R. Baudelle, P. Melnyk, B. Dkprez, A.
Tartar,Eur. J. Med. Chem. 1996,31,87-98.
[3] F. Balkenhohl, C. von dem Bussche-Hiinnefeld,A. Lansky, C. Zechel,
Angew. Chem. 1996, 108, 2436-2488; Angew. Chem. Int. Ed. Engl.
[4] K. S. Lam,S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M. Kazmierski,
R. J. Knapp, Nature (London) 1991,354,82-84.
[5] K. S. Lam, V. J. Hruby, M. Lebl, R. J. Knapp, W. M. Kazmierski, E.
M. Hersh, S. E. Salmon, Bioorg. Med. Chem. Lett. 1993,3,419424.
[6] R. S. Youngquist, G. R. Fuentes, M. P. Lacey, T. Keough,J. Am. Chem.
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Experimental Section
TentaGel@S-NH2 resin (0.25 meqlg, 8.9 x lo5 beaddg, 130 pm beads)
was purchased from Rapp Polymere (Tiibingen, Germany). Three Aca units
Arch Phann Pharm Med Chem. 330,1WI11~1997)
[ 1I] M. Lebl, V. Krchnak, N. F. Sepetov, B. Seligmann,P. Strop, S.Felder,
K. S. Lam,Biopolymers (Pept. Sci.) 1995.37. 177-198.
Received: February 20, 1997 [FP191]
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