An unnatural amino acid that induces β-sheet folding and interaction in peptides

Article abstract of DOI:10.1021/ja025699i

This paper introduces a unique amino acid that can readily be incorporated into peptides to make them fold into β-sheetlike structures that dimerize through β-sheet interactions. This new amino acid, Orn(i-PrCO-Hao), consists of an ornithine residue with

Full text of DOI:10.1021/ja025699i

Published on Web 04/17/2002
An Unnatural Amino Acid that Induces â-Sheet Folding and Interaction in
Peptides
James S. Nowick,*,† Kit S. Lam,‡ Tatyana V. Khasanova,† William E. Kemnitzer,† Santanu Maitra,†
Hao T. Mee,† and Ruiwu Liu‡
Department of Chemistry, UniVersity of California IrVine, IrVine, California 92697-2025, and Department of
Internal Medicine, UniVersity of California DaVis Cancer Center, Sacramento, California 95817
Received January 24, 2002
Peptides generally do not participate in the sort of strong and
specific â-sheet interactions that occur widely among proteins.1
While â-sheet formation between proteins is often involved in
exquisite protein-protein interactions, â-sheet formation between
peptides is generally associated with the formation of insoluble
aggregates of ill-defined structure. Although a variety of elegant
strategies to coax peptides to fold into well-defined â-sheetlike
structures have now been developed, few strategies for achieving
strong and specific â-sheet interactions between peptides have been
reported.2,3 Our (J.S.N.) research group has recently established that
compounds that mimic the structures of protein â-sheets can
dimerize in a highly specific fashion and has shown the facility
with which amino acid building blocks that mimic the hydrogen-
bonding functionality of a peptide â-strand can be used to generate
peptides that dimerize by â-sheet formation.4,5 Here, we introduce
a unique amino acid that can readily be incorporated into peptides
to make them fold into â-sheetlike structures that dimerize through
â-sheet interactions.
This new amino acid, Orn(i-PrCO-Hao), consists of an ornithine
residue with the â-strand-mimicking amino acid Hao5 attached to
its side chain. When Orn(i-PrCO-Hao) is incorporated into a peptide,
or appended to its N-terminus, the Hao group hydrogen bonds to
the three subsequent residues to form a â-sheetlike structure. The
CH2CH2CH2NH side-chain of the ornithine residue allows the Hao
oxalamide carbonyl group to form a hydrogen-bonded ten-
membered ring with the amino group of the subsequent residue,
like a â-turn in a â-hairpin. Chart 1 illustrates the structure of
Orn(i-PrCO-Hao) and that of a peptide containing it.
Fmoc-Orn(i-PrCO-Hao)-OH behaves like a regular amino acid
in peptide synthesis and was uneventfully incorporated into the
peptide o-anisoyl-Val-Orn(i-PrCO-Hao)-Phe-Ile-Leu-NHMe (4)
through standard automated Fmoc solid-phase peptide synthesis
using PS-PEG-indole-NHMe7 resin, with DIC and HOAt8 as the
coupling agent for Fmoc-Orn(i-PrCO-Hao)-OH and o-anisic acid.9
A second synthetic strategy was developed to facilitate the
preparation of peptides with N-terminal Orn(i-PrCO-Hao) residues,
which avoids the need for the preparation of Fmoc-Orn(i-PrCO-
Hao)-OH. In this strategy, Boc-Orn(Fmoc)-OH is used as the
penultimate amino acid in the peptide synthesis, and i-PrCO-Hao-
OH (2) is used as the final amino acid. N-Terminal Orn(i-PrCO-
Hao) peptide H-Orn(i-PrCO-Hao)-Phe-Ile-Leu-NHMe‚TFA (5) was
prepared in a fashion similar to that of 4, using DIC and HOAt8 as
the coupling agent for i-PrCO-Hao-OH.
Chart 1
The amino acid Orn(i-PrCO-Hao) is readily used in peptide
synthesis as its Fmoc derivative, which is conveniently prepared
from hydrazide 1.6 Condensation of hydrazide 1 with ethyl oxalyl
chloride, followed by hydrolysis of the ethyl ester group with NaOH
and passage of the reaction mixture through acidic ion-exchange
resin, yields i-PrCO-Hao-OH (2). Coupling of i-PrCO-Hao-OH and
Boc-Orn-O-t-Bu with EDC and HOBt, followed by cleavage of
the Boc and tert-butyl ester groups with TFA and Fmoc protection
with Fmoc-OSu, yields Fmoc-Orn(i-PrCO-Hao)-OH (3).
1H NMR spectroscopic studies establish that peptide 4 forms a
dimeric â-sheetlike structure in CDCl3 solution. Most notably, H
1
NMR transverse-ROESY (Tr-ROESY)10 studies in CDCl3 solution
(3.9 mM, 298 K) show five prominent NOEs between the i-PrCO-
Hao unit and the Phe, Ile, Leu, and NHMe groups of the peptide
strand indicative of a folded â-sheetlike structure and three
prominent NOEs between the N- and C-terminal groups of the
peptide (o-anisoyl-Val and Leu-NHMe) consistent with a dimeric
structure. Chart 2 illustrates the structure of the dimer and shows
these NOEs. Particularly compelling among these data is a strong
* To whom correspondence should be addressed. E-mail: jsnowick@uci.edu.
† University of California, Irvine.
‡ University of California Davis Cancer Center.
9
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J. AM. CHEM. SOC. 2002, 124, 4972-4973
10.1021/ja025699i CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Chart 2
Hao and the Ile R-proton, indicating folding. NOEs between the
Phe and Leu R-protons, which would be expected in the antiparallel
â-sheet dimer are absent. NOEs between the i-PrCO and MeN
protons cannot be detected, due to overlap of the i-PrCO methine
and MeN resonances, but are detected in 1:1 CD3OD-CDCl3, in
which these resonances do not overlap. Consistent with proximity
between the Ile side-chain and the aromatic ring of the Hao unit,
the Ile δ- and γ-methyl groups appear upfield at 0.70 and 0.74
ppm, respectively, and one of the Ile γ-methylene protons appears
upfield at 0.85 ppm, while the other appears at 1.37 ppm. That
these NOEs are weaker than those of 4 and the upfield shifting is
more modest suggests that the extent of folding of 5 in CD3OD is
somewhat less than that of 4 in CDCl3. Consistent with a less well-
defined ornithine turn structure, the diastereotopic δ-protons of 5
in CD3OD are less separated than those of 4 in CDCl3, appearing
at 3.64 and 3.22 ppm.
Chart 3
Collectively, these synthetic and spectroscopic studies establish
that the amino acid Orn(i-PrCO-Hao) induces â-sheet structure and
interactions in peptides in suitable organic solvents. Unlike our Hao
amino acid, which acts as a prosthetic to replace three residues of
the peptide strand, the Orn(i-PrCO-Hao) amino acid acts as a splint
that helps enforce a â-sheetlike structure without replacing the
residues and their side chains. This feature of Orn(i-PrCO-Hao) is
important, because it allows the creation of â-sheet structure with
minimal perturbation of the peptide sequence. Another important
feature, which has allowed us to screen and analyze one-bead-one-
compound combinatorial libraries of peptides containing Orn-
(i-PrCO-Hao) for biological activity, is that Orn(i-PrCO-Hao)
behaves like a regular R-amino acid in Edman sequencing.15 We
will describe this work in due course.
interstrand NOE between the proton at the 6-position of the aromatic
group of Hao and the Ile R-proton and a strong intersheet NOE
between the Val and Leu R-protons.
The 3JHNR coupling constants of the peptide strand of peptide 4
are large (8.8-9.8 Hz), and the interresidue NOEs between the
NH and R-protons of the amino acids are strong, reflecting a
â-sheetlike conformation of the peptide strand.11-13 Also consistent
with â-sheetlike structure, the chemical shifts of the R-protons of
the amino acids in 4 are shifted 0.71-1.54 ppm downfield of values
typical of random coil conformations.14 Consistent with the
hydrogen bonding associated with folding and dimerization in
chloroform solution, the NH groups of the peptide strand appear
at 8.3-9.4 ppm, which is substantially downfield of the charac-
teristic chemical shift of peptide NH groups that are not hydrogen-
bonded (ca. 6 ppm). Also consistent with a folded structure in which
the Ile side-chain of the peptide is close to the aromatic ring of the
Hao unit, the Ile δ- and γ-methyl groups appear unusually upfield
at 0.54 and 0.68 ppm, and one of the Ile γ-methylene protons
appears unusually upfield at 0.75 ppm, while the other appears at
1.23 ppm.
Acknowledgment. We thank the NIH and NSF for grant support
(GM-49076 and CHE-9813105).
Supporting Information Available: Synthetic procedures and
spectral data (PDF). This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(1) Maitra, S.; Nowick, J. S. In The Amide Linkage: Structural Significance
in Chemistry, Biochemistry, and Materials Science; Greenberg, A.,
Breneman C. M., Liebman, J. F., Eds.; Wiley: New York, 2000; Chapter
15.
(2) Venkatraman, J.; Shankaramma, S. C.; Balaram, P. Chem. ReV. 2001,
101, 3131-3152.
The 1H NMR studies also suggest that the ornithine unit adopts
a well-defined turn conformation. Particularly prominent in the Tr-
ROESY spectrum of 4 is a Very strong NOE between the R-proton
of Orn and one of its two diastereotopic δ-protons. This proton
appears dramatically downfield of the other diastereotopic δ-proton
(4.39 ppm vs 3.09), indicating that these two protons reside in very
different environments. No NOE is seen between the R-proton of
Orn and the other diastereotopic δ-proton, confirming the difference
in environments. These two protons also exhibit widely different
coupling patterns, with the downfield δ-proton resonance resembling
a quartet or a triplet of doublets with three large (10-12 Hz)
coupling constants, and the upfield δ-proton resembling a broad
doublet with one large coupling constant. Molecular modeling gives
a turn structure consistent with these NOE data and shift data, in
which the pro-S δ-proton is in contact with the Orn R-proton and
is shifted downfield by proximity to the adjacent carbonyl group
of the Hao group and by proximity to the carbonyl of the adjacent
Val residue. Chart 3 illustrates this turn structure.
(3) Phillips, S. T.; Rezac, M.; Abel, Y.; Kossenjans, M.; Bartlett, P. A. J.
Am. Chem. Soc. 2002, 124, 58-66.
(4) Nowick, J. S.; Tsai, J. H.; Bui, Q.-C. D.; Maitra, S. J. Am. Chem. Soc.
1999, 121, 8409-8410.
(5) Nowick, J. S.; Chung, D. M.; Maitra, K.; Maitra, S.; Stigers, K. D.; Sun,
Y. J. Am. Chem. Soc. 2000, 122, 7654-7661.
(6) (a) Nowick, J. S.; Pairish, M.; Lee, I. Q.; Holmes, D. L.; Ziller, J. W. J.
Am. Chem. Soc. 1997, 119, 5413-5424. (b) Holmes, D. L.; Smith, E.
M.; Nowick, J. S. J. Am. Chem. Soc. 1997, 119, 7665-7669.
(7) Estep, K. G.; Neipp, C. E.; Stephens Stramiello, L. M.; Adam, M. D.;
Allen, M. P.; Robinson, S.; Roskamp, E. J. J. Org. Chem. 1998, 63, 5300-
5301.
(8) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397-4398.
(9) The o-anisoyl group enhances the solubility of peptide 4 and minimizes
its aggregation by intramolecularly hydrogen bonding to the valine NH
group.
(10) (a) Hwang, T.-L.; Shaka, A. J. J. Am. Chem. Soc. 1992, 114, 3157-
3159. (b) Hwang, T.-L.; Shaka, A. J. J. Magn. Reson. Series B 1993,
102, 155-165.
(11) Wu¨thrich, K. NMR of Proteins and Nucleic Acids; Wiley: New York,
1986; pp 125-129.
(12) Nowick, J. S.; Smith, E. M.; Pairish, M. Chem. Soc. ReV. 1996, 25, 401-
415.
(13) Wu¨thrich, K. NMR of Proteins and Nucleic Acids; Wiley: New York,
1986; pp 166-168.
Analogous 1H NMR studies of peptide 5 indicate that this TFA
salt folds but does not dimerize in CD3OD solution. Most notably,
Tr-ROESY studies in CD3OD solution (10.1 mM, 298 K) show an
NOE between proton at the 6-position of the aromatic group of
(14) (a) Wishart, D. S.; Sykes, B. D.; Richards, F. M. J. Mol. Biol. 1991, 222,
311-333. (b) Wishart, D. S.; Sykes, B. D.; Richards, F. M. Biochemistry
1992, 31, 1647-1651.
(15) Lam, K. S.; Lebl, M.; Krchna´k, V. Chem. ReV. 1999, 97, 4411-448.
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