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Expeditious Synthesis of a Novel C3-Symmetric Receptor that Binds Peptides Sequence-Selectively.

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Expeditious Synthesis of a
Novel C,-Symmetric Receptor that
Binds Peptides Sequence-Selectively**
Seung So0 Yoon and W. Clark Still*
One of the most exciting recent advances in molecular recognition is the development of synthetic receptors or host molecules that bind biopolymers sequence-selectively.['] Among the
most selective of these is the C,-symmetric receptor 1.12]It binds
a variety of biooligomers, including glycosides and both N- and
C-protected peptides. with high stereochemical and functionalgroup selectivity. With simple peptides, for example, 1 binds L
peptide residues with enantioselectivities as high as 3 kcalmol( > 99 % ee). Though 1 is available by an optimized synthesis,[2b1
we sought related receptors that could be prepared by even
simpler methods and thus might find practical application to
problems of separation. In this regard, 2 and its derivatives are
Our synthesis of 2 (Scheme 1) began with the coupling of
three equivalents of pentafluorophenyl dimethyl trimesate to
one equivalent of 1,3.S-tri(aminomethyl)benzene to provide 3a
(78 % yield). Saponification of all six methyl ester groups and
activation as the hexakis(pentafluoropheny1) ester then gave 3 b
(30 % yield). This material underwent threefold coupling with a
( 3 R,4 R)-3,4-diaminopyrrolidinelinked to the dye Disperse Red
1 through a succinyl spacer;[31three 21-membered rings were
formed simultaneously, and the brilliant red receptor 2 was
obtained in 26 % yield. We should add that a variety of related
receptors can be prepared with other 1,2-diamines. For example, reaction of commercially available (1 R,2 R)-I ,2-diaminocyclohexane with 3 b yielded an analogous receptor (31 %
yield for the final tricyclization step) in which the pyrrolidine
rings are replaced by simple cyclohexanes.
C02Me
+
H2NH2C
H
.
N
W
Meo2CY
c02c6F5
o
'R
R ' 0 2 C CONH
I
v c 0 2 R '
R'02cp+.CONH
&
H2Nt)N
N
OA(CH2)2C02-Dye
- 2
HNOCp,CozR'
la,R = H
1 b, R = OCH2CH=CH2
1c, R = 0-Dye
C02R'
R
CO2R'
3a, R ' = Me
3b, R'=CeFs
Scheme 1. Synthesis of receptor 2.
To establish the binding properties of our new receptor 2, we
treated a dye-labeled receptor with a large collection or library
of potential substrates.[2d1The substrate library was synthesized
on 50-80 pm Merrifield polystyrene beads in such a way that
each bead carried a single library member. The binding experiment simply involved mixing red 2 with the bead-supported
library and selecting those beads that turned red. Thus binding
of 2 to the entire library (here ca. 50000 acylated tripeptides)
could be surveyed in a single experiment.
We used the polymer-supported substrate library[2d1of general structure 5 . We prepared the library using the split
R
2,R = CO(CH2)2C02-Dye
attractive candidates. Receptors 1 and 2 are closely related in
that their cup-shaped binding cavities have both similar dimensions and analogous patterns on their peripheries of unassociated hydrogen-bond donors and acceptors for substrate binding.
In this communication, we describe a simple synthesis of 2 and
its binding properties as determined by complexation with an
encoded combinatorial library['] of around 50 000 acylated
tripeptide substrates. As we will show, 2 is a remarkably selective receptor for peptides.
[*I
Prof. W. C. Still, S. S . Yoon
Department of Chemistry, Columbia University
New York. N Y 10027 (USA)
Telefax: Int. code (212)932-1289
We acknowledge the support of the National Science Foundation (CHE92
08254).
+
[**I
2458
:(>
VCH Ver.lugsgesellschuft mhH, 0-69451 W<,inheirn.I994
RCO-AA 3-AA2-AA 1 -NH(CH,),CONHCH,-polystyrene
5
AAn (n = 1-3) = Gly , D-Ala, L-Ah, 03-IBu-o-Ser, 03-fBu-~-Ser,D-Val,
L-Val, o-Pro, L-Pro, N4-trityl-o-Asn, N'-trityl-L-Asn, NS-trityl-D-Gln, N 5 tntyl-L-Gln , N'-Boc-~-Lys, N6-Boc-~-Lys
R = Me, Et, iPr, tBu, iBu, neopentyl (mope), trifluoromethyl (TFM),
methoxymethyl (MOM), cyclopropyl (cPr), cyclobutyl (cBu), cyclopentyl
(rPe), acetoxymethyl (AcOM), Ph, Me,N, morpholino (Mor)
synthesis.[41 Thus, the total possible number of different substrates in the library was 154 (50625). To allow for determination of the structure of the substrate on any bead that bound 2,
we employed the binary encoding method that uses electroncapture gas chromatography (ECGC) to analyze tag-encoded
structural i n f o r m a t i ~ n . [ ~ ]
O57O-OX33/9412323-245~S 1 O . O O t .2SiU
Angew. Chem. I n t . Ed. Engl. 1994, 33. No. 23/24
COMMUNICATIONS
In the binding assay, a sample of the above peptide library
(10 mg. ca. 10’ beads) was equilibrated with a 50m > M solution
of 2 in CHCI, (ca. 0.3 mL). After 24 h of agitation, approximately 1 % of the beads had developed deep red-orange colorations. These beads carried peptides that bound red 2 most
tightly.r61By measuring the concentration of 2 in solution over
the beads at equilibrium, we established that the assay allowed
selection of beads having association constants K, 2 3 000. Fifty
of these most deeply colored beads were picked under a microscope (4 x magnification), and their tag-encoded structural information was decoded by ECGC. The residue types found in
the peptides on these beads are listed in Table 1 .
Table 1. Residues R and AA 1-AA 3 in acylated, protected peptide substrates that
are selectively bound by 2 (no. of occurrences from 50 decoded beads in parentheses).
R
MOM (31)
Me,N (13 )
Et (4)
Me (2)
AA3
NS-trityl-u-Gln (31)
h’4-trityl-D-Asn ( 7 )
Nb-BOc-U-LyS (5)
N*-trityl-r-Asn (4)
u-Ala (2)
GlY (1)
AA2
AAI
Gly (15)
N4-trityl-~-Asn(8)
NS-trityI-D-Gh (5)
D-Aka (4)
NS-trityl-L-Gh ( 3 )
N ~ - B O C - U - L(3)
~S
L-Val (3)
N ~ - B O C - L - L(2)
~S
N4-trityl-1.-Asn (2)
O-vdl (2)
L - A (1)
~
@-tBu-~-Ser (1)
O3-IBu-u-Ser (1)
L-Pro (11)
U-Akd (lo)
D-Pro (9)
Gly ( 7 )
NS-trityl-o-Gln (3)
NS-trityl-L-Gln (2)
L-Ala (2)
N4-trityl-~-Asn(2)
o-Val (2)
03-tBu-r-Ser (2)
In many ways, the binding selectivities of 2 are similar to
those recently reported for l.‘Zdl
Thus, both receptors show the
highest selectivity for residues at the terminus of the peptide
chain: they selectively bind certain R groups having three nonhydrogen atoms and AA3 = N5-trityl-Gln. However, 1 binds
AA3 = N5-trityl-~-Gln,whereas 2 prefers the D isomer. This
difference in enantioselection is rationalized by the enantiomeric patterns of hydogen bonding between substrate and groups at
the periphery of the binding cavities of 1 and 2 (Scheme 2).
Scheme 2 Schematic representation of receptors 1 and 2. Ar = aromatic ring;
- indicate exposed H-bond donors and acceptors, respectively.
+,
According to molecular modeling, these groups surround the
opening to the binding cavity and are well positioned to bind
substrate through intermolecular hydrogen bonds. Other
donors and acceptors of 1 and 2 are unavailable for substrate
binding because they project away from the opening to the
binding cavity and further are involved in intramolecular hydrogen bonds.
Other selectivity differences between 1 and 2 are found at
AA2 (1 prefers L-Pro, 2 prefers Gly) and AA 1 (1 shows virtually
no selectivity, 2 prefers D-Pro, L-Pro or D-Ala) residues. Interestingly, the remarkable preference of 1 for R = cyclopropyl[2d1is
absent with 2. Neither 1 nor 2 bind peptides having
Angew. Chnn. I n l . Ed. Engl. 1994, 33, No . 23/24
R = isopropyl. These and other selectivities were verified by
independent binding energy measurements with 2 that showed,
inter alia, that changing R = MOM to cyclopropyl or
AA 3 = N’-trityl-D-GIn to N’-trityl-L-Gln diminished binding
by 1.3 kcalmol-’ relative to the preferred substrate MOMCON5-trityl-~-Gln-Gly-~-Pro (AGh,nding= - 4.3 kcalmol- ’).
These results were obtained by resynthesizing selected substrate
sequences and using UV spectrophotometry to quantitate the
concentrations of free receptor both before and after treatment with a known quantity of the polymer-supported substrate.
We also carried out an analogous survey of 2 with a substrate
library having deprotected peptide side chains (Table 2). Measurement of the concentration of 2 at equilibrium indicated that,
in this case, the color assay allowed selection of substrates having K, 2 5 000.
0 VCH
Table 2. Residues R and AA 1- A A 3 in acylated, deprotected peptide substrates
that are selectively bound by 2 (no. of occurrences from 45 decoded beads in
parentheses).
R
MOM (12)
Me,N (10)
Ph (6)
Mor (6)
Et (4)
cPr (3)
Me (1)
cPen (1)
tBu ( I )
AcOm (1)
AA3
AA2
AA 1
o-Gln (23)
L-Gln (18)
D-Lys (8)
L-LYS(3)
Gly (4)
D-Gh (3)
D-Asn (2)
D-Ala (2)
L - A I ~( 2 )
r-Pro (1)
o-Ser (1)
r-Ser (1)
L-Pro ( 2 3 )
!)-Pro (21)
u-Gln (1)
D-Val(7)
D-LYS(4)
u-Pro (3)
D-Ala (2)
D-Ser (2)
o-Asn (2)
L-Asn (1)
L-Ser (1)
Whereas our previous studies found 1 to have similar selectivities for protected and deprotected peptide substrates, receptor
2 bound a different set of peptides with the deprotected library.
It was most discriminating at the internal AA 1 site where Pro
was strongly preferred. In particular, 2 preferentially bound the
sequences with AA3 = D-Gh, AA2 = D amino acid, and
AAI = L-Pro (40% of beads) or with AA2 = L-Gln and
AA1 = D-Pro (40% of beads). The relationship between the
Pro configuration at the AA 1 position and both the configuration and position of the downstream Gln is particularly interesting. This novel Gln-Pro selectivity may reflect the presence of
hydrogen bonding between the Gln side chain and amides at the
bottom of 2’s binding cavity-a possibility not available to 1 .
The ability of 2 to selectively bind peptide spans with as many
as three residues is remarkable for such a small host molecule.
These studies and others soon to be reported suggest that a
variety of receptors similar in structure to 1 or 2 have significantly sequence-selective peptide binding properties and that
the specific selectivity of such a receptor can be controlled by
varying the details of its structure. While the full range of variations that can be tolerated without loss of binding affinity has
yet to be established, we have found that the full macrotricyclic
framework of 2 is necessary for peptide binding: deletion of the
central aromatic linker obliterates tight peptide binding as detected by our solid-phase color assay. Given the simplicity of 2’s
synthesis, its highly selective peptide-binding properties, and the
availability of many building blocks for the construction of
analogues, it is likely that a variety of C,-symmetric receptors
having a range of peptide sequence selectivies can be prepared
along the lines described here.
Verlugsgesellschafi mbH, D-69451 Weinherm, 1994
0570-0833/94/2323-2459$ 10.0Oi .2S:0
2459
COMMUNICATIONS
Experimental Procedure
Single-Step Six-Electron Transfer in a
Heptanuclear Complex Isolation of Both Redox
Forms**
3a:To a solution ofpentafluorophenyl dimethyl trimesate (0.42 g. 1.04mrnol) and
:
tri(aminomethy1)benzene triHCl salt (86mg, 0.31mmol) in 10 mL of dry N,Ndimethylacetamide (DMA) was added 0.36m L of iPr,NEt. After the mixture had
been stirred for 8 h, it was concentrated at reduced pressure and purified by flash
Jean-Luc Fillaut, Jorge Linares, and Didier Astruc*
chromatography (silica gel. 5 % MeOH in CH,CI,) to give 3a as an amorphous
white solid (0.20 g, 701%). ' H N M R (CDCI,) 6 = 3.90(s, 6 H ) , 4.38(m. 2 H ) , 7.10
Dedicated to Dr. Jacqueline Zarembovitch
(s, 1 H). 7.30(m. 1 H), 8.61 (s, 2 H ) , 8.70(s, 1 H). "C NMR (CDCI,) 6 = 165.74.
165.36,139.11,134.99,132.84.132.18,130.77,126.35,
52.44,43.71.IR (neat) 3324,
Polymers containing ferrocene units are useful redox catalysts
2815,1730,1674,1573 c m - ' : HR-MS (FAB) for C,,H,,N,O,,: calcd: 827.2537
[ M + Hi],
found: 827.2539.
for the modification of electrodes['] and as biosensors.I*] A new
2:To 3 a (0.1 g, 0.121mmol) in T H F ( 5 mL). MeOH (3 mL), and water (1 mL) was
generation of polynuclear compounds intermediate between
added a 1 h NaOH solution (0.85 mL). After stirring for 5 11, the mixture was
mononuclear and polymeric complexes has recently appearedacidified with 1 N HCI solution and extracted with EtOAc (3 x 50 mL). The extractthe
and ~rganometallic[~]
molecular "trees". These
ed hexaacid was dissolved in T H F (3 mL) and CH,CI, (IOmL), and C,F,OH
well-defined molecules promise to be useful in the construction
(0.1 5 g. 0.84mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiirnide(EDC)
(0.16g, 0.84mmol) were added. Stirring for 5 h, concentration. and flash chroof dendrimers,I5]in molecular electronic devices,[61and in multimatography (silica gel. 20% acetone in CH,CI,) gave 3b as an amorphous white
electron redox catalysis.171FeCp+ can serve as an excellent startsolid (63mg. 30%).
ing core for molecular trees, since polyalkylation, polyallylaA solution of 3b (44mg,0.025 mmol) and 4.2CF3CO2H(63 mg, 0.084mmol) in
tion, and polybenzylatjon of polymethyl arenes leads to various
DMA (IOmL) was added with stirring to a solution of iPr,NEt (0.19mL.
topologies.['] The reaction of [FeCp(C,Me,)]PF, with excess
1.09mmol) in dry T H F (200 rnL) at room temperature over 20 h by syringe pump.
Stirring for an additional 8 h, concentration. and flash chromatography (silica gel.
base and an alkvl halide results in selective hexasubstitution
10% MeOHinCH,CI,)gaveZasanamorphousred solid(15 mg, 26%). ' H N M R
[Eq. (a)].[']
(CD,OD/CDCI, 1;l) 6 = 0.70(t. 3H, J =7.0Hz). 2.45 (m. 4H), 3.01 (q, 2H.
J=7.0Hz),3.36(br.s,ZH),3.50(br.s,2H).3.74(in,lH),3.92(m.1H).4.12(m,
4H).4.58(m,1H),4.68(m,1H),6.64(d,2H,J=9.1Hz),7.72(m,5H),7.98(m,
I H ) , 8.10 (d. 2H. J=Y.OHz). 8.23(m, lH),01.64(m. 1H); I3C N M R ( l /
1 = CD,OD/CDCI,) S =172.82,170.20.169.72,169.61,167.70,138.51,136.06,
Since the introduction of organometallic groups on the
133.53, 131.27. 130.54, 130.46,129.54,126.72. 125.92.125.22,124.37,124.24.
"branches" is sometimes tedious and requires multistep synthe122.31,118.X5.112.22,61.34,55.74,50.54,45.70.
44.42.38.28.28.76,28.27.27.52,
sis,[8a1 we have engaged in research aimed at grafting
27.22,11.94;IR (neat) 3324,2815. 1720,1675,1573 c m - ' ; MS (FAB) mi: 2127
[M H + I .
organometallic complexes directly on the branches in the
+
Received: July 25. 1994 (Z7172IEI
German version: Angew. Chem. 1994,106, 2517
Reviews: H.-J. Schneider, Anger.. Cllrm. 1993. 105, 890; Ange.Pw. Chem.
Inl. Ed. Engl. 1993,32. 848;T. H. Webb, C. S. Wilcox, Chem. Soc. Rev. 1993,
383.
a) J:I. Hong, S. K. Namgoong, A. Bernardi. W C. Still, J. A m . C'heni. SOC.1991,
f 1 3 , 5111;b) S. D.Erickson, J. A. Simon, W. C . Still. J. Org. Chem. 1993,58,
1305;c) R. Liu, W. C. Still, Terrahedron Lert. 1993,34, 2573;d) A. Borchardt,
W. C. Still, J A m . Chem. Soc. 1994,116, 373.
(R,R)-Diaminopyrrolidine preparation: D. R. Reddy. E. R. Thornton,
J. Chem. SOC.Chem. Commun. 1992,172;S . S.Yoon. W. C. Still, Tetruhedron, in
press.
a) A. Furka. M . Sebestyen, M. Asgedom, G. Dibo, Ahstr. 14th Inr. Congr.
Biochem., Prag, 1988. 5 , 47: b) Abslr. 10th Int. S w ~ p Med.
.
Chem., Budapest.
1988.288;c) Inl. J. P e p . Protein Res. 1991,37.487;
d) K. S. Lam. S. E. Salmon,
E. M. Hersh, V. J. Hruby. W. M. Kazmierski. R . J. Knapp, Nature (LondonJ
1991,354, 82.
M.H. J. Ohlmeyer. R. N.Swanson, L. W. Dillard, J. C. Reader. G. Asouline. R.
Kobayashi, M. Wigler. W. C. Still. Proc. Natl. Acad. Sci. U S A 1993, 90,
10922.
Previous studies have shown that the color assay readily distinguishes substrates
differing in binding energy by as little as 1.0kcalmol-'. Control experiments
established that head coloration reflects binding of the peptide and not the dye:
neither the dye nor the succinylated dye stained any of the library beads under
the assay conditions.
See, for example. P. Eckes, Angew. Chem. 1994, 106, 1649:Angew. Chem. Int.
Ed. €ng/. 1994. 33,1573.
polyalkylation process[sb1and report here our first results. We
have isolated and characterized both the reduced and oxidized
forms of a heptametallic hexaferrocene system.['I The central
iron units in this complex serve as an internal standard in the
determination of the number of ferrocenyl groups on the
branches by Mossbauer spectroscopy and cyclic voltammetry.
The 4-ferrocenylbutyl iodide (1) was synthesized in the form
of orange-red crystals according to Scheme 1. Reaction of a
threefold excess of 1 with KOH and [Fe"Cp(C,Me,)]PF, (2)
gives the PF, salt of the heptanuclear Fe" complex cation 3
(orange-red microcrystals) in 65 % yield. The Mossbauer spectrum of 3 at 200K shows a ferrocene doublet with QS =
2.384 mms-' and IS = 0.487 m m s - ' as well as a shoulder for
the [Fe"Cp(arene)J doublet at QS =1.879 m m s - ' and IS =
0.487 mm s- Photolysis of 3 in CH,CN in the presence of one
equivalent of PPh, using visible light gives [FeCp(PPh,)(NCMe),]PF, and the hexanuclear neutral complex 4 (orange
microcrystals) in 96 YO yield, which results from the selective
decomplexation of the central cationic FeCp+ unit. Oxidation
of 3 with H,SO, followed by ion exchange with aqueous
NH,PF, solution provides the PF, salt of the heptanuclear
hexaferricinium complex 5 as purple-black microcrystals in
98 % yield. The Mossbauer spectrum of 5 at 200 K (as well as at
4 K ) shows a Fe" doublet at QS =1.95 mms-' and IS =
0.5 m m s - l andalargeferriciniumsignalat QS = 0.164mms-'
'.
[*I
[**I
2460
8
VCH VerlagrgesellscAufl mbH. 0-69451 Weinheim, 1994
Prof. D. Astruc, Dr. J.-L. Fillaut
LabOratoire de Chimie Organique et Organometallique
Universite Bordeaux I, URA CNRS N" 35
351,Cows de la Libtration, F-33405Talence Cedex (France)
Telefax: Int. code + 56846646
Dr. J. Linares
Departement de Recherches Physiques
Universite Pierre et Marie Curie, URA CNRS N" 71
Tour 22, 4. place Jussieu. F-75252Paris Cedex (France)
This research was supported by the Centre National de la Recherche Scientifique (CNRS), the Universities Bordeaux 1 and Paris V1, and the Region
Aquitaine. We thank Prof. F. Varret and K. Boukheddaden for help with the
MoSsbaUer spectroscopy. S. Treavett for synthetic collaboration, and Dr. F.
Moulines for discussions.
0570-0833/94/2323-2460$10.00+ .25,'0
Angeic. Chem. Int. Ed. Engi. 1994, 33. No. 23/24
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