A highly stable short α-helix constrained by a main-chain hydrogen-bond surrogate

Article abstract of DOI:10.1021/ja0466659

Herein we describe a strategy for the preparation of artificial α-helices involving replacement of one of the main-chain hydrogen bonds with a covalent linkage. To mimic the C=O...H-N hydrogen bond as closely as possible, we envisioned a covalent bond of the type C=X-Y-N, where X and Y are two carbon atoms connected through an olefin metathesis reaction. Our results demonstrate that the replacement of a hydrogen bond between the i and i + 4 residues at the N-terminus of a short peptide with a carbon-carbon bond results in a highly stable constrained α-helix at physiological conditions as indicated by CD and NMR spectroscopies. The advantage of this strategy is that it allows access to short α-helices with strict preservation of molecular recognition surfaces required for biomolecular interactions. Copyright

Full text of DOI:10.1021/ja0466659

Published on Web 09/10/2004
A Highly Stable Short r-Helix Constrained by a Main-Chain Hydrogen-Bond
Surrogate
Ross N. Chapman, Gianluca Dimartino, and Paramjit S. Arora*
Department of Chemistry, New York UniVersity, New York, New York 10003
Received June 7, 2004; E-mail: arora@nyu.edu
Analyses of helix-coil transition in peptides emphasize the
energetically demanding organization of three consecutive amino
acids into the helical orientation as the slow step in helix formation.1
Preorganization of these amino acid residues is expected to
overwhelm the intrinsic nucleation propensities and initiate helix
formation.2 In an R-helix, a hydrogen bond between the CdO of
the ith amino acid residue and the NH of the i + 4th amino acid
residue stabilizes and nucleates the helical structure (Figure 1a).
Herein we describe a strategy for the preparation of artificial
R-helices involving replacement of one of the main-chain hydrogen
bonds with a covalent linkage. To mimic the CdO‚‚‚H-N hydrogen
bond as closely as possible, we envisioned a covalent bond of the
type CdX-Y-N, where X and Y would be part of the i and the
i + 4 residues, respectively. The exceptional functional group
tolerance displayed by the olefin metathesis catalysts for the facile
introduction of non-native carbon-carbon constraints in the
preparation of peptidomimetics suggested that X and Y could be
two carbon atoms connected through an olefin metathesis reaction
(Figure 1b).3 This hydrogen bond replacement approach is inspired
by the work of Satterthwait and co-workers, who explored the use
of a hydrazone link to stabilize R-helices.4 The metathesis-based
method affords a stable and irreversible bond as compared to the
hydrazone strategy and should be applicable to a broader range of
peptide sequences.
Figure 1. (a) Strategy for the stabilization of R-helices by replacement of
an i and i + 4 hydrogen bond (CdOsH-N) with a covalent link (CdX-
Y-N). (b) Replacement of the hydrogen bond with a carbon-carbon bond
through an olefin metathesis reaction affords a stabilized R-helix.
Scheme 1. Synthesis of an Internally Constrained R-Helix
The main-chain hydrogen-bond surrogate strategy is attractive
because placement of the cross-link on the inside of the helix does
not block solvent-exposed molecular recognition surfaces of the
molecule. Thus far, helix stabilization methods have relied pre-
dominantly on side-chain constraints,5 capping templates,2,6 or non-
natural amino acid substitutions.7 These methods either block
solvent-exposed surfaces of the target R-helices or remove important
side-chain functionalities. Our strategy allows strict preservation
of the helix surfaces and affords helices with unprecedented
stability.
To test the stabilization properties of the metathesis-derived
internal cross-link, we synthesized an 8-mer constrained peptide 3
bearing a cross-link at the N-terminus (Scheme 1). This particular
peptide was chosen for the initial studies because the control peptide
(AcGEAAAAEA-OMe, 1) does not display any R-helicity, allowing
us to observe an increase in R-helical content due to the modifica-
tion. Two glutamic acid residues were incorporated in this alanine-
rich peptide at different faces of the putative helix to increase the
solubility of the constrained peptide in aqueous buffers. The
constrained peptide 3 was synthesized from precursor peptide 2
via a ring-closing metathesis reaction with Hoveyda-Grubbs
metathesis catalyst8 followed by the removal of the tert-butyl ester
groups. The isolated metathesized peptide 3 contained a trans alkene
as indicated by NMR spectroscopy.9
performed in 30 mM phosphate buffer, pH 7.0, to obtain a
quantitative measure of the helical content (Figure 2). The CD
spectrum of 3 displays a double minimum at 202 and 222 nm,
characteristic of R-helices.10 The constrained peptide 3 does not
show any increase in helicity upon addition of the R-helix inducing
solvent trifluoroethanol (20% TFE in 30 mM phosphate buffer, pH
7.0), as measured by molar ellipticity at 222 nm; TFE does cause
a slight change in the intensity of the 202-nm band (Figure 2a).
The relative percent helicity of peptides can be estimated by the
mean residue ellipticity at 222 nm, although these estimates are
typically not accurate for short helices.11a We calculate this
constrained peptide to be 100% helical based on Yang’s method11b
and Baldwin’s correction11a for short peptides (see Supporting
Information for details). As expected, the unconstrained peptide 1
does not display any R-helicity in aqueous buffer (Figure 2a) and
0-100% TFE solutions (Supporting Information). The constrained
peptide remains fully helical when heated from 5 to 95 °C,12
indicating that the peptide is structurally robust (Figure 2b). To
further gauge the stability of the constrained peptide, we performed
The structure of constrained peptide 3 was determined using
circular dichroism (CD) and 2D NMR spectroscopies. CD studies
on the constrained peptide 3 and the control peptide 1 were
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C O M M U N I C A T I O N S
strong evidence for R-helical structure, were observed for the entire
sequence as shown in the NOE correlation chart (Figure 3). The
ROESY spectrum also reveals several medium range NOEs, for
example, dRN(i,i + 3), that provide unequivocal evidence for the
helical structure. The fact that we can detect NOEs involving the
last residue (alanine-8) indicates that the helix has not started fraying
at the ends.
In summary, these results demonstrate that the replacement of a
hydrogen bond between the i and i + 4 residues at the N-terminus
of a short peptide with a carbon-carbon bond results in a highly
stable constrained R-helix at physiological conditions as indicated
by CD and NMR spectroscopies. The advantage of this strategy is
that it allows access to short R-helices with strict preservation of
molecular recognition surfaces required for biomolecular interac-
tions.
Acknowledgment. We thank Neville R. Kallenbach and Simon
H. Friedman for helpful insights and John Kulp and Kang Chen
for help with the 2D NMR experiments. P.S.A. thanks NYSTAR
for support of this work in the form of a James D. Watson
Investigator award.
Supporting Information Available: Synthesis, 1H NMR, 13C
NMR, and HRMS of modified amino acids. Synthesis, metathesis, and
characterization of peptides. Description of ROESY, TOCSY, and
circular dichroism studies (PDF). This material is available free of
charge via the Internet at http://pubs.acs.org.
Figure 2. (a) (i) Circular dichroism spectra of R-helix 3 in 30 mM
phosphate buffer (pH 7.0) and (ii) 20% TFE/phosphate buffer. (iii) Spectrum
of unconstrained peptide 1 (AcGEAAAAEA) in phosphate buffer. The
spectra were recorded at 25 °C. Effect of (b) temperature and (c) GnHCl
on the stability of R-helix 3.
References
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(8) Hoveyda, A. H.; Gillingham, D. G.; Van Veldhuizen, J. J.; Kataoka, O.;
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2, 8-23. The ring-closing metathesis reaction with the “second-generation”
Grubbs catalyst afforded significantly lower yields.
(9) Based on the alkene proton coupling constant of 16.1 Hz. Typical ring-
closing metathesis reactions yield a mixture of cis and trans alkene isomers,
favoring trans for large macrocycles (Furstner, A.; Langemann, K.
Synthesis 1997, 792-803); however, we could not detect the cis alkene
isomer in the reaction mixture by HPLC.
Figure 3. ROESY correlation chart for 3. The alanine-4 residue does not
contain an NH. Filled rectangles indicate relative intensity of the NOE cross-
peaks. Empty rectangles indicate NOE that could not be unambiguously
assigned because of overlapping signals.
a guanidinium chloride (GnHCl) titration experiment (Figure 2c).
The intensity of the [θ]222 band for the constrained peptide shows
that the peptide is still 85% helical at a concentration of 4 M GnHCl;
the peptide started to unravel between 6 and 7 M GnHCl. These
GnHCl titration studies illustrate that compound 3 adopts an
exceedingly stable R-helical structure whose stability compares
favorably to a previously reported constrained R-helix with three
side-chain lactam bridges.5d
The R-helix structure of 3 was further confirmed by NMR
spectroscopy. A combination of 2D TOCSY and ROESY spectra
was used to assign 1H NMR resonances for the constrained peptide.
Sequential NN (i and i + 1) ROESY cross-peaks, which provide
(10) In canonical R-helices, the 202-nm band typically appears between 207
and 209 nm.
(11) Chin, D. H.; Woody, R. W.; Rohl, C. A.; Baldwin, R. L. Proc. Natl.
Acad. Sci. U.S.A. 2002, 99, 15416-15421. (b) Chen, Y. H.; Yang, J. T.;
Martinez, H. M. Biochemistry 1972, 11, 4120-4131.
(12) This conclusion is based on the thermal stability of the 222-nm band,
which is typically used to gauge R-helicity; we observe a slight change
in the intensity of the 202-nm band upon heating (Please see Supporting
Information for details on this experiment.)
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