C11/C9 helices in crystals of αβ hybrid peptides and switching structures between helix types by variation in the α-residue

Article abstract of DOI:10.1021/ol5021866

Close-packed helices with mixed hydrogen bond directionality are unprecedented in the structural chemistry of α-polypeptides. While NMR studies in solution state provide strong evidence for the occurrence of mixed helices in (ββ)_n and (αβ)

Full text of DOI:10.1021/ol5021866

Letter
pubs.acs.org/OrgLett
C11/C9 Helices in Crystals of αβ Hybrid Peptides and Switching
Structures between Helix Types by Variation in the α‑Residue
Krishnayan Basuroy, Vasantham Karuppiah, and Padmanabhan Balaram*
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India
S
* Supporting Information
ABSTRACT: Close-packed helices with mixed hydrogen bond directionality are unprecedented in
the structural chemistry of α-polypeptides. While NMR studies in solution state provide strong
evidence for the occurrence of mixed helices in (ββ)n and (αβ)n sequences, limited information is
currently available in crystals. The peptide structures presented show the occurrence of C11/C9
helices in (αβ)n peptides. Transitions between C11 and C11/C9 helices are observed upon varying
the α-amino acid residue.
anonical helical structures, such as the 310 and α-helices
recognized that mixed helices are preferred in sequences with
Cfound in proteins and polypeptides composed of α amino alternating chirality of the β-residues.8b In considering the
“chirality” of β-residues derived by backbone homologation of L-
(S)-α-amino acids, it must be noted that the configuration
specified at the asymmetric center is R.4b Molecular dynamics
simulations have been used to compute the enthalpy difference
between right-handed C12/C10 helix and left-handed C14 helix for
an oligo-β-peptide sequence.9 Kessler and co-workers estab-
lished the C12/C10/C12 helix in an oligo-β-peptide, containing an
alternating sequence of the unsubstituted residue β-homoglycine
(β-hGly) and a sugar amino acid in which torsional freedom
about the Cβ−Cα (θ) bond was constrained by a furanoid sugar
ring.10 In an extensive series of investigations Sharma, Kunwar,
and co-workers have characterized, by NMR, the C12/C10 mixed
helix in oligo-β-peptides containing a sugar amino acid, lacking
covalent backbone constraints, in sequences with alternating
residue chirality.11 These authors demonstrated the occurrence
of C11/C9 mixed helices in hybrid αβ sequences, containing C-
linked carbo-β3-amino acids.12 An ab initio MO study provides
an indication of the intrinsic potential for formation of C11/C9
mixed helices in hybrid αβ sequences.13 The stereochemical
patterning approach has been advanced for the design of novel
helices in β-peptides and hybrid αβ sequences.14
acids, are characterized by intramolecular hydrogen bonds that
run in the same direction.1 The two major helix types have
hydrogen bonds of the type COi···HNi+n (n = 3 for the 310
helix and n = 4 for the α-helix), which require orientation of
peptide units in the same direction, resulting in the development
of a macroscopic dipole moment, with a positive end at the N-
terminus and negative end at the C-terminus.2 Close-packed
helical structures with mixed hydrogen bond directionality in
which alternating CO···HN hydrogen bonds run in opposite
directions are unprecedented in the structural chemistry of α-
polypeptides. The π(L,D) helix proposed for the gramicidin A
sequence, where amino acid chirality alternates, has C16 and C14
hydrogen bonds in opposite directions but has a channel running
through the body of the helix.3
The discovery of new hydrogen bonding patterns in
polypeptide helices followed rapidly after the observation that
helices with both hydrogen bond types COi···HNi+n and
NHi···COi+n are possible in poly-β-peptides, which have an
additional atom inserted into the backbone.4 The novel C14 helix
in which successive hydrogen bonds of type NHi···COi+2 are
formed was the first member of this class to be structurally
characterized in solution and the solid state.5 Seebach and co-
workers provided the first NMR evidence for helices with mixed
hydrogen bonding patterns of the type C12/C10/C12 (12/10/12
mixed helix) in their study of oligo-β-peptides with alternating
substitution patterns (β2 and β3).5c,6 Theoretical calculations by
the groups of Hofmann7 and Wu8 provided a comprehensive
evaluation of the energetics of helical structures in sequences
containing β residues, permitting an assessment of relative
stabilities of helices stabilized by unidirectional and mixed
hydrogen bonding patterns. Most importantly, Wu and Wang
While studies in solution provide strong evidence for the
occurrence of mixed helices in oligo-β-peptides and hybrid αβ
sequences, limited information is currently available in the
crystalline state. Reported peptide crystal structures have been
largely confined to short sequences of 3−4 residues, permitting
characterization of isolated C11/C9 structures in αβ peptides15
and two examples of C12/C10 structure in αγαα and αγαγ
Received: July 24, 2014
Published: August 21, 2014
© 2014 American Chemical Society
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sequences16. Recently, Lee et al. have described mixed C11/C9
helices in peptides with the alternating D-Ala−cis-ACHC
sequences [in the cis-ACHC residue the Cα−Cβ torsion angle
θ is constrained by covalent backbone cyclization].17 During the
course of studies of hybrid peptides containing the β2,2-
disubstituted residue β2,2Ac6c (1-aminomethylcyclohexanecar-
boxylic acid), we observed formation of the mixed C11/C9
structure in the tripeptide Boc-Aib-β2,2Ac6c-Aib-OMe.15d Inter-
estingly, in this example both of the 310/α-helix promoting Aib
(α-aminoisobutyric acid) residues adopted the rarely observed
semiextended polyproline (PII, ϕ ≈ −60°, ψ ≈ 120°; PII′, ϕ ≈
60°, ψ ≈ −120°)-like conformations.18 Extension of this
sequence to 4 (αβαβ) and 5 (αβαβα) residue sequences yielded
incipient C11 helices with conventional hydrogen bond
directionality.15d In order to facilitate mixed helix formation by
promoting PII conformation at the α residues, we turned to (αβ)n
sequences containing alternating chiral α-amino acid and
β2,2Ac6c residues.
We describe in this report the crystallographic characterization
of two tripeptides, Boc-Leu-β2,2Ac6c-Aib-OMe (1) and Boc-Leu-
β2,2Ac6c-Leu-OMe (2); one tetrapeptide, Boc-[Leu-β2,2Ac6c]2-
OMe (3); and one hexapeptide, Boc-[Leu-β2,2Ac6c]3-OMe (4)
(Figure 1). The tripeptides Boc-Leu-β2,2Ac6c-Aib-OMe (1) and
Figure 2. Molecular conformations of peptides in crystals. (a) Boc-Leu-
β2,2Ac6c-Aib-OMe (1). (b) Boc-Leu-β2,2Ac6c-Leu-OMe (2). (c) Boc-
[Leu-β2,2Ac6c]2-OMe (3). (d) Boc-[Aib-β2,2Ac6c]2-OMe.15d
Figure 1. Structures of peptides. (a) Boc-Leu-β2,2Ac6c-Aib-OMe 1. (b)
Boc-Leu-β2,2Ac6c-Leu-OMe 2. (c) Boc-[Leu-β2,2Ac6c]2-OMe 3 and
Boc-[Leu-β2,2Ac6c]3-OMe 4.
Boc-Leu-β2,2Ac6c-Leu-OMe (2) crystallized in the orthorhombic
space group P212121 with one peptide molecule in the
crystallographic asymmetric unit (Supplemental Table S1). In
both cases, mixed C11/C9 conformations are observed (Figure
2a,b). Extension to the tetrapeptide, Boc-[Leu-β2,2Ac6c]2-OMe
(3), revealed the persistence of the mixed C11/C9 hydrogen-
bonded conformation (Figure 2c). This is in sharp contrast to the
analogous tetrapeptide, Boc-[Aib-β2,2Ac6c]2-OMe (3), in which
Leu (1) and Leu (3) are replaced by Aib residues (Figure 2d) and
two consecutive C11 hydrogen bonds are observed.15d The
tetrapeptide 3 crystallized in the orthorhombic space group
P212121 with one peptide molecule and one co-crystallized
solvent molecule (CHCl3) in the crystallographic asymmetric
unit (Supplemental Table S2).
Encouraged by these observations, we turned to an
examination of longer (Leu-β2,2Ac6c)n sequences. Diffraction
quality single crystals in the monoclinic space group C2 were
obtained for the hexapeptide Boc-[Leu-β2,2Ac6c]3-OMe (4)
containing two peptide molecules in the crystallographic
asymmetric unit along with one water molecule (Supplemental
Table S2). Both molecules fold into a right-handed mixed C11/C9
helix containing four intramolecular hydrogen bonds, C11/C9/
C11/C9 (Figure 3). The relevant backbone torsion angles are
listed in Supplemental Table S3. The backbone torsion angles
Figure 3. Molecular conformations of (αβ)n C11/C9 and C11 helices in
the peptide sequences (a) Boc-[Leu-β2,2Ac6c]3-OMe (4) and (b) Boc-
[Aib-β2,2Ac6c]2-Aib-OMe,15d respectively. (c, d) View of the backbone
conformations of the two symmetry independent molecules in the
crystallographic asymmetric unit of the hexapeptide Boc-[Leu-
β2,2Ac6c]3-OMe (4).
reveal that all six Leu residues, in the two independent molecules,
adopt semiextended polyproline-like conformations (hydrogen
bond parameters for all the peptides are listed in Supplemental
Table S4). The structure of the hexapeptide 4 may be compared
with a related αβαβα pentapeptide,15d containing Aib residues,
which forms a C11 helical structure containing three successive
C11 (4 → 1) hydrogen bonds. The crystal structures described in
this report provide definitive experimental support for the
conformational requirement (PII) at the α-residues in mixed
helices. The β-residue necessarily requires a gauche conformation
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Letter
(g+/g−) about the Cβ−Cα (θ) bond, depending on the chirality at
the α-residue, in order to form hydrogen bonds with alternating
directionality.
bonding patterns (Supplemental Tables S3, S5, and S6 provide
the listing of torsion angles, bond lengths, and bond angles used
for making the model decapeptide). Figure 5 summarizes the
Figure 4a provides a distribution of backbone ϕ−ψ values from
both α and β residues observed in the crystal structures of short
Figure 5. Average backbone torsion angles of the two types of hydrogen-
bonded 4 → 1 (αβ) C11 turns and the 1 → 2 (βα) C9 turn. a) C11 turn in
unidirectional C11 helices. b) C11 turn in alternate directionality C11/C9
helices. c) C9 turn in alternate directional C11/C9 helices (Average
backbone torsion angle values are shown).
backbone torsion angles characterizing two distinct C11 turn
types and the C9 hydrogen-bonded turn observed in structurally
characterized (αβ)n hybrid peptides (Supplemental Tables S7
and S8 provide a listing of available structures). The two possible
helix types, the C11 helix and the mixed C11/C9 helix
characterized in (αβ)n sequences containing the β2,2Ac6c residue,
differ only slightly in the observed values of θ (Cβ−Cα). It may be
noted that the θ values in unconstrained β residues lie close to the
ideal gauche value of 60°, while somewhat larger values of 80°−
90° were observed in cyclically constrained residues.19 As seen
from Figure 3a,b, a transition between these two helix types may
be achieved by major changes in the ψ value at the α residues (PII
→ αR, Δψ ≈ 180°) and ϕ values at the β residue (−90° → +90°,
Δϕ ≈ 180°). Transitions between helices of the same
handedness do not involve changes in the sign of θ and can be
achieved by 180° flips of alternate peptide units in αβ segments,
with relatively minor torsion angle change at βα segments.
Further studies in solution are necessary to address the question
whether a conformational equilibrium is indeed detectable. In
(αβ)n sequences where both residues are chiral, mixed C11/C9
helices may be anticipated for sequences with alternating chirality
(heterochiral), as demonstrated in earlier NMR studies.12,20
In the case of homochiral sequences, continuous helices (C11
or C14/C15) may be accessible in the absence of local
conformational constraints. Indeed, Gellman and co-workers
have provided NMR evidence for rapid interconversion between
the 11-helix (C11) and 14/15-helix (C14/C15) for a protected
octapeptide composed of alternating sequences of (S,S)-trans-2-
aminocyclopentanecarboxylic acid (ACPC) and L-α-amino
acids.21 It is noteworthy that in the octapeptide Boc-[Aib-
β3(R)Val]4-OMe a right-handed C14/C15 helix, which is an
analogue of the α-peptide α-helix (C13) observed in
crystals.22The backbone conformational parameters differ only
slightly from those of the continuous C11 helix. Some general
features of value in engineering specific helix types in αβ hybrid
peptides emerge from these structural results. In achiral (αβ)n
sequences, as exemplified by (Aib-β2,2Ac6c)n peptides, both
continuous C11 helices and mixed C11/C9 structures are indeed
Figure 4. (a) Scatter plot in ϕ−ψ space for (αβ)n C11 and C11/C9 helices
[θ average of β-residues for C11 and C11/C9 helices are 90.6°(9.0°) and
60.5°(4.1°), respectively]. Supplemental Tables S7 and S8 provide the
listing of the structures used. (b) Schematic representation of
enantiomeric helix types from both C11 and C11/C9 structures.
Transitions between helices of the same handedness do not require
significant changes in the values of θ. The points correspond to the
average values determined from the analysis of the available crystal
structures (see Supplemental Tables S7 and S8). (c) A view of the right-
handed (αβ)n C11/C9 decapeptide helix, generated using the average
backbone geometries (see Supplemental Tables S3, S5, and S6).
peptides containing a mixed C11/C9 hydrogen bonding pattern.
In the case of observations made on peptides lacking chiral α-
residues, two molecules of opposite handedness related by
reflection or inversion symmetry are observed in crystals,
characterized by achiral space groups. In such cases, only one
set of torsion angles that corresponds to those observed for L-α-
amino acid conformations has been chosen, resulting in right-
handed helix types. For the β-residues the mean value of the
torsion angle about the Cβ−Cα (θ) is ∼60°. The ϕ−ψ values for
the β-residues cluster closely around 90°, with opposite signs. An
alternative helical structure that may be considered for (αβ)n
sequences is the continuous C11 helix, with conventional C
Oi···HNi+3 intramolecular hydrogen bonds, which is a backbone
expanded analogue of the α-polypeptide 310 helix. Cluster plots
for observed ϕ−ψ values in (αβ)n C11 helices are also shown in
Figure 4a, to permit direct comparison between two helix types.
Figure 4b shows a representation of the right- and left-handed
C11/C9 and continuous C11 helices on a 3D ϕ−θ−ψ plot. Figure
4c shows a view of a right-handed, hybrid αβ C11/C9 mixed helix
generated for a model decapeptide using the average residue
geometries derived from crystal structures of the peptides
containing a β2,2Ac6c residue, with C11/C9 mixed hydrogen-
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(5) (a) Seebach, D.; Overhand, M.; Kuhnle, F. N. M.; Martinoni, B.;
Oberer, L.; Hommel, U.; Widmer, H. Helv. Chim. Acta 1996, 79, 913.
(b) Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.;
Gellman, S. H. J. Am. Chem. Soc. 1996, 118, 13071. (c) Seebach, D.;
Abele, S.; Gademann, K.; Guichard, G.; Hintermann, T.; Jaun, B.;
Matthews, J. L.; Schreiber, J. V.; Oberer, L.; Hommel, U.; Widmer, H.
Helv. Chim. Acta 1998, 81, 932. (d) Appella, D. H.; Barchi, J. J.; Durell, S.
R.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 2309. (e) Appella, D. H.;
Christianson, L. A.; Karle, I. L.; Powell, D. R.; Gellman, S. H. J. Am.
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stereochemically feasible. The observed switch from the C11/C9
mixed helix to the C11 continuous helix on going from the
tripeptides to the tetrapeptides may be attributed to the energy
penalty that needs to be paid for the Aib residues to adopt the PII
conformation,18 which is a requirement for C11/C9 helix
formation. In principle, at the tetrapeptide level two intra-
molecular hydrogen bonds will be formed in both types of
helices. The balance between these two helix types is then tilted
by the conformational preferences of the Aib residue, which are
determined by intraresidue nonbonded interactions. Hybrid
(αβ)n sequences containing chiral α-residues and achiral β
residues may provide an attractive model system for studying
transitions in peptide helices between structures with unidirec-
tional hydrogen bonds and mixed helices. Designed, model (ββ)n
peptides containing alternating chiral and achiral residues may be
useful in experimentally probing the distribution of C14 helices (1
→ 3, reverse hydrogen bond directionality), C12 helices (4 → 1,
conventional hydrogen bond directionality), and C12/C10
structures (mixed directionality). Notably the analogous C10/
C8 structures in all α-polypeptides may be difficult to realize
because of the requirement of a cis peptide bond to facilitate the
C8 hydrogen bond.23
(6) (a) Seebach, D.; Gademann, K.; Schreiber, J. V.; Matthews, J. L.;
Hintermann, T.; Jaun, B.; Oberer, L.; Hommel, U.; Widmer, H. Helv.
Chim. Acta 1997, 80, 2033. (b) Rueping, M.; Schreiber, J. V.; Lelais, G.;
Jaun, B.; Seebach, D. Helv. Chim. Acta 2002, 85, 2577.
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H.-J. Biopolymers 1999, 50, 167. (b) Baldauf, C.; R. Gunther, R.;
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Hofmann, H.-J. Angew. Chem., Int. Ed. 2004, 43, 1594. (c) Baldauf, C.; R.
Gunther, R.; Hofmann, H.-J. Biopolymers 2005, 80, 675. (d) Schramm,
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P.; Sharma, G. V. M; Hofmann, H.-J. Biopolymers 2010, 94, 279.
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(b) Wu, Y.-D.; Wang, D.-P. J. Am. Chem. Soc. 1999, 121, 9352. (c) Wu,
Y.-D.; Han, W.; Wang, D.-P.; Gao, Y.; Zhao, Y.-L. Acc. Chem. Res. 2008,
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(9) Lin, Z.; Timmerscheidt, T. A.; van Gunsteren, W. F. J. Chem. Phys.
2012, 137, 064108.
The present study emphasizes the utility of the constrained β-
residue β2,2Ac6c in designing specific types of hydrogen-bonded
helical structures in (αβ)n hybrid sequences, by varying the
nature of the α residue.
(10) Gruner, S. A. W.; Truffault, V.; Voll, G.; Locardi, E.; Stockle, M.;
̈
Kessler, H. Chem.Eur. J. 2002, 8, 4365.
(11) (a) Sharma, G. V. M; Reddy, K. R.; Krishna, P. R.; Ravi Sankar, A.;
Narsimulu, K.; Kiran Kumar, S.; Jayaprakash, P.; Jagannadh, B.; Kunwar,
A. C. J. Am. Chem. Soc. 2003, 125, 13670. (b) Sharma, G. V. M; Reddy,
K. R.; Krishna, P. R.; Ravi Sankar, A.; Jayaprakash, P.; Jagannadh, B.;
Kunwar, A. C. Angew. Chem., Int. Ed. 2004, 43, 3961. (c) Sharma, G. V.
M; Manohar, V.; Dutta, S. K.; Subash, V.; Kunwar, A. C. J. Org. Chem.
2008, 73, 3689.
ASSOCIATED CONTENT
* Supporting Information
■
S
Details of synthetic procedure, X-ray diffraction data, structure
solution, and refinement procedures and supplementary tables.
This material is available free of charge via the Internet at http://
pubs.acs.org. CCDC deposition numbers for the peptides are
988391 (1), 988392 (2), 988393 (3), and 988394 (4), which
contain the supplementary crystallographic data for this paper.
These data can be obtained free of charge from the Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_
request/cif.
(12) Sharma, G. V. M; Nagendar, P.; Jayaprakash, P.; Krishna, P. R.;
Ramakrishna, K. V. S.; Kunwar, A. C. Angew. Chem., Int. Ed. 2005, 44,
5878.
(13) Baldauf, C.; Gunther, R.; Hofmann, H.-J. Biopolymers 2006, 84,
̈
408.
(14) Man
Vass, E.; Fulop, F. Angew. Chem., Int. Ed. 2009, 48, 2171.
́
́ ́
dity, I. M.; Weber, E.; Martinek, T. A.; Olajos, G.; Toth, G. K.;
̈
̈
(15) (a) Tanaka, M.; Oba, M.; Ichiki, T.; Suemune, H. Chem. Pharm.
Bull. 2001, 49, 1178. (b) Saavedra, C.; Hernan
E. J. Org. Chem. 2009, 74, 4655. (c) Andre, C.; Legrand, B.; Deng, C.;
́
́
dez, R.; Boto, A.; Alvarez,
́
AUTHOR INFORMATION
Corresponding Author
■
Didierjean, C.; Pickaert, G.; Martinez, J.; Averlant-Petit, M. C.; Amblard,
M.; Calmes, M. Org. Lett. 2012, 14, 960. (d) Basuroy, K.; Karuppiah, V.;
Shamala, N.; Balaram, P. Helv. Chim. Acta 2012, 95, 2589.
(16) (a) Vasudev, P. G.; Chatterjee, S.; Ananda, K.; Shamala, N.;
Balaram, P. Angew. Chem., Int. Ed. 2008, 47, 6430. (b) Guo, L.; Zhang,
W.; Guzei, I. A.; Spencer, L. C.; Gellman, S. H. Org. Lett. 2012, 14, 2582.
(17) Lee, M.; Shim, J.; Kang, P.; Guzei, I. A.; Choi, S. H. Angew. Chem.,
Int. Ed. 2013, 52, 12564.
(18) Aravinda, S.; Shamala, N.; Balaram, P. Chem. Biodiversity 2008, 5,
1238.
(19) (a) Choi, S. H.; Guzei, I. A.; Gellman, S. H. J. Am. Chem. Soc. 2007,
129, 13780. (b) Choi, S. H.; Guzei, I. A.; Spencer, L. C.; Gellman, S. H. J.
Am. Chem. Soc. 2008, 130, 6544.
(20) Srinivasulu, G.; Kiran Kumar, S.; Sharma, G. V. M; Kunwar, A. C.
J. Org. Chem. 2006, 71, 8395.
(21) Hayen, A.; Schmitt, M. A.; Ngassa, F. N.; Thomasson, K. A.;
Gellman, S. H. Angew. Chem., Int. Ed. 2004, 43, 505.
(22) Basuroy, K.; Dinesh, B.; Shamala, N.; Balaram, P. Angew. Chem.,
Int. Ed. 2012, 51, 8736.
*E-mail: pb@mbu.iisc.ernet.in.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported under the DBT-IISc partnership
program. V.K. was supported by a UGC D. S. Kothari
postdoctoral fellowship. We thank Dr. S. Raghothama for the
13C NMR spectra.
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