Hybrid αγ polypeptides: Structural characterization of a C 12/C10 helix with alternating hydrogen-bond polarity

Article abstract of DOI:10.1002/anie.200801665

A different twist: A mixed C₁₂/C₁₀ helix with alternating hydrogen-bond directionality has been observed in the crystal structure of the hybrid αγ peptide Boc-Leu-Gpn-Leu-Aib-OMe (see structure; Boc: tert-butoxycarbonyl; Leu: leucine; Gpn: 1-(aminomethyl) cyclohexaneacetic acid (gabapentin); Aib: aminoisobutyric acid). The structure may be generalized to a regular mixed helix. (Chemical Equation Presented)

Full text of DOI:10.1002/anie.200801665

Communications
DOI: 10.1002/anie.200801665
Helical Structures
Hybrid ag Polypeptides: Structural Characterization of a C12/C10 Helix
with Alternating Hydrogen-Bond Polarity**
Prema G. Vasudev, Sunanda Chatterjee, Kuppanna Ananda, Narayanaswamy Shamala,* and
Padmanabhan Balaram*
The insertion of the higher homologues of the a-amino acids,
specifically b, g, and d residues, into a peptide sequences
results in hybrid structures with novel hydrogen-bonding
possibilities.[1] Research in this area of hybrid polypeptides
has been stimulated by the observation of novel helical
structures in b oligopeptides in which the hydrogen-bond
polarities are reversed. For example, the 12 helix (2.512 or
2.51-P) in a (b)n sequence maintains the same directionality
Figure 1 shows the molecular conformation of tetrapep-
tide 1 in the crystal. The folded conformation is stabilized by
two intramolecular hydrogen bonds. A C12 interaction is
=
(C O(i)···NH(i+3)) as the canonical 310 helixin an ( a)n
sequence[2] and may be formally considered as an expanded
version of the latter. By contrast, the 14 helix(3 14 or 31-M) in a
(b)n sequence possesses a reversed hydrogen-bond polarity
[3]
=
(NH(i)···C O(i+2)). Initial crystallographic results with
hybrid ab and ag sequences suggest that the expanded
analogues of the 310 helix, namely, the C11 (ab) and C12 (ag)
helices, can indeed be observed in short peptides.[4] Calcu-
lations suggest that helices with alternating hydrogen-bond
polarities may indeed be stable structures in hybrid sequen-
ces.[1c,d] In this Communication, we report the C12/C10 mixed
hydrogen-bonding pattern in the crystal structure of the
tetrapeptide Boc-Leu-Gpn-Leu-Aib-OMe (1; Boc: tert-
butoxycarbonyl; Gpn: 1-(aminomethyl)cyclohexaneacetic
acid (gabapentin); Aib: aminoisobutyric acid), which was
serendipitously obtained during the synthesis of longer hybrid
sequences. This novel helical conformation can be general-
ized as an (ag)n sequence. The sequence contains the
stereochemically constrained g residue gabapentin, which is
an achiral b,b-disubstituted g-amino acid. The presence of
gem-dialkyl substituents on the Cb atom limits the torsion
Figure 1. a) Molecular conformation of 1 in crystals. The Aib4 side
chain, along with the CO(OMe) group, is disordered over two
positions, with occupancies of 0.58 and 0.42. Only the major confor-
mer is shown for clarity. The backbone torsion angles are: Leu1:
f=ꢀ71.98, y=121.18; Gpn2: f=87.88, q1 =37.78, q2 =45.18,
y=ꢀ129.38; Leu3: f=ꢀ64.88, y=143.38; Aib4: f=ꢀ56.88,
y=ꢀ30.78 (f=33.48, y=ꢀ154.58 for the minor conformer). The C12
hydrogen-bond parameters are N···O=2.93 Š, H···O=2.17 Š, and
N-H-O=146.78. The C10 hydrogen-bond parameters are N···O=2.91 Š,
H···O=2.06 Š, and N-H-O=166.78. b) The backbone of the
Leu1-Gpn2-Leu3 segment showing the C12/C10 hydrogen bonds with
opposite hydrogen-bond directionalities. c) The C12/C10 mixed helix
generated by extending the conformation observed in crystals.
ꢀ
ꢀ
angles about the Cg Cb (q1) and Cb Ca (q2) bonds to
approximately ꢁ 608.[5]
[*] P. G. Vasudev, Prof. N. Shamala
Department of Physics, Indian Institute of Science
Bangalore, 560 012 (India)
Fax: (+91)80-2360-2602
E-mail: shamala@physics.iisc.ernet.in
S. Chatterjee, K. Ananda, Prof. P. Balaram
Molecular Biophysics Unit, Indian Institute of Science
Bangalore, 560 012 (India)
=
observed between the Boc C O and Leu3 NH groups, while a
C
10 hydrogen bond with inverted polarity is observed between
=
Fax: (+91)80-2360-0535
the Gpn2 NH and Leu3 C O groups. In this structure, both of
the a residues, Leu1 and Leu3, adopt the semiextended
polyproline (PII) conformation (f~ ꢀ608, y ~ +1208). This
mixed C12/C10 unit can readily be extended into a regular
12/10 helixwith alternating hydrogen-bond directionalities, as
shown in Figure 1c. The observed backbone torsion angles
are very close to those computed for theoretical models.[1c]
The structure of peptide 1 illustrates some important
conformational features of hydrogen-bonded turns in hybrid
E-mail: pb@mbu.iisc.ernet.in
[**] We thank Dr. K. Nagarajan, Hikal R&D Centre, Bangalore, for gifts of
1-(aminomethyl)cyclohexaneacetic acid (Gpn) and for his contin-
ued interest. P.G.V. and S.C. thank the Council of Scientific and
Industrial Research (CSIR), India, for the award of Senior Research
Fellowships. Research grant support was from the CSIR, India, and
from the Department of Biotechnology, India. The CCD facility is
funded under the IRHPA program of the Department of Science and
Technology, India.
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Angewandte
Chemie
ag sequences. Figure 2 compares the conventional type I
(type III) and type II b-turn conformations of aa segments[6]
with the C12 turns observed in the ag peptides. It is evident
Figure 2. a) Type I/III (left) and type II (right) b turns in a polypep-
tides. b) C12 turns characterized in ag hybrid peptides of Gpn: average
conformation of ag helical C12 turn (left) and the C12 turn observed in
the Leu1–Gpn2 segment of peptide 1 (right).
Figure 3. 500 MHz 1H NMR spectrum of peptide 1 at 300 K (lower
panel) and 278 K (middle panel). The top panel shows an expanded
view of the amide NH resonances of peptide 1 in CDCl3 at 278 K. A–C
refer to the three distinct conformational species.
that ag turns can also be classified into two categories. In the
“helical turns”, the
g residues adopt a conformation
(f=ꢀ1358, q1 = 548, q2 = 608, y = ꢀ1088)[5b] that will permit
the formation of a successive ga turn. This structure is an
expanded version of the helical type III b turn, which is
repeated to generate a 310 helixin all- a polypeptides.[2,7]
Interestingly, the C12 ag turn observed in tetrapeptide 1 is
formally analogous to the type II b turn in an aa segment. The
two types of ag C12 turns illustrated in Figure 2 are formally
related by an approximately 1808 flip of the central peptide
unit, a feature that also relates the type I and type II b turns in
a peptides.[8] The structure in Figure 1c illustrates the fact that
the ag analogue of the type II b turn can be accommodated
into the hybrid C12/C10 (ag)n helix. By using internal hydro-
gen-bonded turns as the repeating structure in helices, the ag
C12 helixwould have a two-residue repeating unit, while the
C12/C10 helixwould have a three-residue repeating unit. If
torsion angles were used as a descriptor, both types of ag
helices would be considered as (ag)n repeat units.
peaks observed in two-dimensional ROESY experiments
confirm that multiple resonances arise from slowly intercon-
verting conformational states of the peptide.
Figure 4 summarizes the conformational regions that are
likely to be populated in these ag sequences. The Gpn residue
is predominantly restricted to gauche conformations about
ꢀ
ꢀ
the Cg Cb (q1) and Cb Ca (q2) bonds, so the residue
conformation may be represented in a two-dimensional f–y
plane. The conformational diagrams in Figure 4 illustrate the
The aga segment in tetrapeptide 1 could, in principle,
adopt two distinct conformations, both of which are stabilized
by two intramolecular hydrogen bonds. These are the C12/C10
structure described above and the C12/C12 structure, which has
been previously characterized in the tetrapeptide Boc-Aib-
Gpn-Aib-Gpn-OMe.[4c] Distinct conformational states may
indeed be characterized in solution and possibly in poly-
1
morphic crystal forms. The amide H NMR resonances of
peptide 1 in CDCl3 at 300 K are broad (Figure 3), in contrast
1
to the sharp well-resolved H NMR resonances observed for
the side-chain protons in the region between 0 and 4 ppm (not
shown). Cooling of the sample to 278 K results in the
appearance of multiple resonances, with as many as three
distinct species being observed. The estimated populations
are 66% of A, 22% of B, and 11% of C. Exchange cross-
Figure 4. Representation of individual residue conformations in two-
dimensional f–y planes for the a and g residues. For a residues, the
normal Ramachandran space is shown. For g residues, the values of q1
and q2 are both gauche and have the same sign.
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6431

Communications
positions and were refined as riding over the atoms to which they
were bonded. CCDC 662233 contains the supplementary crystallo-
graphic 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.
structural transitions necessary for interconversions between
the C12/C10 and C12 helical structures. In the aga segment, the
a residue must transit from the helical region of f–y space
(aR) to the polyproline region (PII); this requires an approx-
imately 1808 rotation about the dihedral angle y. The
g residue must transit from a f value of ꢀ1308 in the C12
helixto a f value of + 878 in the C12/C10 helical conformation.
Although these conformational transitions require only
rotation about single bonds, appreciable barriers may be
anticipated, which result in the observation of slowly
exchanging species, even at a temperature as high as 278 K.
It is clear that the C12/C10 structure is conformationally fragile
in solution. In this short peptide sequence, nevertheless, the
trapping of this unusual, incipient helixsuggests that the
construction of helices with alternating hydrogen-bond polar-
ity can be readily achieved in longer sequences.
The results described above establish that ag sequences
containing stereochemically constrained residues can serve
not only to characterize new helical folds in polypeptides but
also to provide model systems to investigate folding transi-
tions and conformational interconversions in solution. The
designed structures, with hydrogen-bonding patterns unpre-
cedented in proteins and all-a peptide sequences, provide an
entry to new families of foldamers.[9]
Received: April 9, 2008
Published online: July 10, 2008
Keywords: amino acids · helical structures · hydrogen bonds ·
.
peptides · structure elucidation
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[5] a) K. Ananda, S. Aravinda, P. G. Vasudev, K. M. P. Raja, H.
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[10] a) G. M. Sheldrick, SHELXS-97, a program for automatic
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Experimental Section
Tetrapeptide 1 was prepared by coupling Boc-Leu-Gpn-OH to Leu-
Aib-OMe by using N,N’-dicyclohexylcarbodiimide (DCC)/1-hydrox-
ybenzotriazole (HOBt) as the coupling reagent. The dipeptides were
obtained by conventional procedures. The final peptide was purified
by reversed-phase medium-pressure liquid chromatography (C18, 40–
60m) and high-performance liquid chromatography on a reversed-
phase C18 column (5–10 m, 7.8–250 mm) with methanol/water gra-
dients. The peptide was characterized by electrospray-ionization mass
spectrometry (ESI MS) on an HP-1100 mass spectrometer and by
complete assignment of the 500 MHz 1H NMR spectra. NMR experi-
ments were carried out on a Bruker AV 500 spectrometer. All
processing was done by using BRUKER XWINNMR software.
Single crystals of peptide 1 (C31H56N4O7, Mw = 596; crystal size:
0.3 ” 0.23 ” 0.04 mm3) were obtained by slow evaporation from a
methanol/dioxane mixture. The peptide crystallized in orthorhombic
space group P212121 with a molecule of methanol in the asymmetric
unit. X-ray intensity data were collected at room temperature on a
Bruker AXS SMART APEX CCD diffractometer (MoKa radiation,
l = 0.71073 Š, w scan). The structure was solved by direct methods by
using the SHELXS-97[10a] program and was refined against F2, with
the full-matrixleast-squares methods by using the SHELXL-97
program.[10b] The unit-cell dimensions and refinement parameters
were a = 10.854(1), b = 18.385(1), c = 20.038(1) Š; V= 3998.6(4) Š3;
Z = 4; 1calc = 1.04 gcmꢀ3; m = 0.07 mmꢀ1; F(000) = 1360; 2qmax = 538;
Rint = 0.05; 7564 independent reflections; 4342 unique reflections;
2729 observed reflections (Fo ꢂ 4s j Fo j ); parameters = 443; R1 =
0.0767 and wR2 = 0.1860; GOF = 1.194; residual electron density
D1max = 0.47 eŠꢀ3, D1min = ꢀ0.19 eŠꢀ3. The C-terminal CO(OMe)
group, along with the side chain of the Aib4 residue, is disordered
over two positions, which were refined with an occupancy ratio of
0.58:0.42. Hydrogen atoms were fixed geometrically in idealized
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