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04
J . Org. Chem. 1998, 63, 904-905
Communications
Ch em ica l Evolu tion : A Mod el System Th a t
Selects a n d Am p lifies a Recep tor for th e
Tr ip ep tid e (D)P r o(L)Va l(D)Va l
The experiments we carried out involved first finding a
disulfide-linked small molecule receptor (X-SS-X) that binds
a particular peptide. The question we then asked was:
given an equilibrating mixture of disulfides that includes
X-SS-X (i.e. X-SS-X, X-SS-Y, Y-SS-Y, Z-SS-Z...), can the
position of the equilibrium be significantly shifted to favor
X-SS-X by introduction of the peptide it binds? Or more
generally, can a system of equilibrating disulfides evolve in
the presence of a given substate to significantly increase the
amount of (i.e. to amplify) the particular disulfide that best
binds the substrate?
Hideaki Hioki and W. Clark Still*
Department of Chemistry, Columbia University,
New York, New York 10027
Received September 25, 1997
One of the most remarkable advances in recent years is
the development of in vitro selection systems that evolve to
enrich mixtures of chemical compounds in those components
having selected properties. Indeed, mixtures of RNA aptam-
ers can evolve to yield highly effective receptors or catalysts
by an in vitro process involving selection and amplification.1
Using such an approach more generally to effect evolution
in simple chemical systems, however, is difficult because few
other classes of molecules can be efficiently copied to effect
the amplification step (which in the case of RNA aptamers
is accomplished enzymatically using reverse transcription
to DNA, PCR amplification, and retranscription to RNA).2
In recent years, an alternative approach to chemical evolu-
tion has emerged in which a reversible equilibrium between
different chemical compounds is set up and the desired
compounds are somehow selected and removed from the
equilibrating mixture.3 In such an approach, enrichment
in the selected compound (i.e. amplification) results from the
equilibration process that carries out a preferential destruc-
tion and recycling of unselected compounds rather than from
an explicit copying of selected compounds as in the aptamer
system. Several simple systems have been described in
recent years that employ such equilibrium shifting to effect
chemical evolution.4 Some of the first examples operated
by equilibrating a mixture of stereoisomeric molecules in the
To find a suitable disulfide-linked receptor, we drew on
previous work indicating that many molecules containing
linked oligomers of isophthalic acids and trans-1,2-diamines
(
e.g. the two-armed receptor below) are highly sequence-
5
,6
selective receptors for peptides.
Although most previous studies of such receptors em-
ployed 1,2-diamine linkers, considerable variation in the
linker structure is tolerated by the receptors and we thought
it likely that an analogous receptor employing a disulfide
linker might also have the properties we sought. Thus we
prepared the dansyl-labeled, fluorescent mercaptan A-SH
and oxidized (I ) it to the receptor-like disulfide A-SS-A.
2
Using fluorescence microscopy to detect binding to an
4
a
presence of a cationic substrate (barium ion, methylguani-
dinium ion4d) to select and amplify those molecules that bind
the substrate most tightly. Others equilibrated systems of
imine-linked synthons to evolve DNA-binding DNA
analogues4b and small molecule ligands for a protein. In
this communication, we describe a simple study that shows
how equilibrium shifting can be used to select and amplify
a hostlike receptor for a complex substrate, here the trip-
eptide sequence (D)Pro(L)Val(D)Val. In this work, the recep-
tor molecule is formed by a disulfide exchange reaction, and
amplification is driven thermodynamically by binding of the
selected receptor to the cognate tripeptidic substrate on a
solid support.
encoded combinatorial library of 3375 different N-acetyl
tripeptides on polystyrene (PS) beads,7 we found that
A-SS-A does indeed selectively bind peptides. In particular,
A-SS-A in CHCl preferentially bound the following poly-
3
4e,f
styrene-supported tripeptide sequences: Ac(D)Pro(L)Val(D)-
Val-PS, Ac(L)Asn(L)Pro(D)Xxx-PS, and Ac(L)Pro(L)Pro(D)Xxx-
PS. Though we were unable to accurately measure the
binding constants for these peptides, the 8 µM equilibrium
concentration of A-SS-A from which the beads were picked
4
5
implies binding constants on the order of 10 -10 .
(
1) Review: Szostak, J . W. Acc. Chem. Res. 1996, 29, 103.
(2) For progress in molecular amplification by replication, see: Orgel,
L. E. Acc. Chem. Res. 1995, 28, 109. Wintner, E. A.; Conn, M. M.; Rebek, J .
Acc. Chem. Res. 1994, 27, 198.
(3) The approach has precedents in classical synthetic organic chemistry.
For example, in his total synthesis of steroids, Woodward was able to set
up an equilibrium between the cis and trans Diels-Alder adducts of 1,3-
butadiene and 2-methoxy-5-methyl-p-quinone and drive that equilibrium
to the less stable trans adduct in 90% yield by crystallization. Woodward,
R. B.; Sondheimer, F.; Taub, D.; Heusler, K.; McLamore, W. M. J . Am. Chem.
Soc. 1952, 74, 4223.
(5) Review of sequence-selective peptide-binding host molecules: Still,
W. C. Acc. Chem. Res. 1996, 29, 155.
(
4) (a) Still, W. C., Hauck, P.; Kempf, D. Tetrahedron Lett. 1987, 28, 2817.
b) Goodwin, J . T.; Lynn, D. G. J . Am. Chem. Soc. 1992, 114, 9197. (c)
Kramer, R.; Lehn, J .-M.; Marquis-Rigault, A. Proc. Natl. Acad. Sci. U.S.A.
(6) (a) Wennemers, H.; Yoon, S. S.; Still, W. C. J . Org. Chem. 1995, 60,
1108. (b) Shao, Y.; Still, W. C. J . Org. Chem. 1996, 61, 6086. (c) Pan, Z.;
Still, W. C. Tetrahedron Lett. 1996, 37, 8699. (d) Iorio, E. J .; Still, W. C.
Bioorg. Med. Chem. Lett. 1996, 2673. (e) Torniero, M.; Still, W. C.
Tetrahedron 1997, 53, 8739.
(
1
1
2
993, 90, 5394. (d) Eliseev, A. V.; Nelen, M. I. J . Am. Chem. Soc. 1997,
19, 1147. (e) Huc, I.; Lehn, J .-M. Proc. Natl. Acad. Sci. U.S.A. 1997, 94,
106. (f) Swann, P. G.; Casanova, R. A.; Desai, A.; Frauenhoff, M. M.;
3
(7) The library had 3375 (15 ) members having the structure Ac-AA1-
Urbancic, M.; Slomczynska, U.; Hopfinger, A. J .; Le Breton, G. C.; Venton,
D. L. Biopolym. 1996, 40, 617.
AA2-AA3-polystyrene where AAn ) Gly, (D and L) Ala, Ser, Val, Pro, Asn,
Gln, Lys. See ref 5 for details.
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Published on Web 01/22/1998