α/γ4-Hybrid peptide helices: Synthesis, crystal conformations and analogy with the α-helix

Article abstract of DOI:10.1039/c2cc32911e

Synthesis, crystal conformations of α/γ⁴-hybrid peptide helices containing proteinogenic amino acid side-chains, and the analogy with the α-helix are reported. Results suggest that α/ γ⁴-hybrid peptides adopted helical conformations

Full text of DOI:10.1039/c2cc32911e

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a/c4-Hybrid peptide helices: synthesis, crystal conformations and analogy
with the a-helixw
Anupam Bandyopadhyay, Sandip V. Jadhav and Hosahudya N. Gopi*
Received 24th April 2012, Accepted 27th May 2012
DOI: 10.1039/c2cc32911e
Synthesis, crystal conformations of a/c4-hybrid peptide helices
containing proteinogenic amino acid side-chains, and the
analogy with the a-helix are reported. Results suggest that
a/c4-hybrid peptides adopted helical conformations with
12-membered H-bond pseudocycles in single crystals.
Scheme 1 Direct transformation of a/E-vinylogous hybrid peptides
into a/g4-hybrid peptides.
was synthesized in solution-phase using Boc-chemistry. The
Designing synthetic protein structures from non-natural amino
E-vinylogous amino acid dgF was synthesized using the Wittig
acids has immense importance not only to understand the
reaction.7 The pure peptide was directly transformed to its
saturated a/g4-hybrid peptide analogue (P1) by catalytic
hydrogenation in the presence of 20% Pd/C in ethanol. The
protein folding but also from the perspective of medicinal
chemistry. Significant progress has been achieved in this regard
using the oligomers of b- and mixed sequences containing
a/b hybrid peptides.1,2 However, the research in the area of
reaction was monitored by MALDI-TOF and HPLC. The
g4-peptides containing proteinogenic amino acid side-chains
complete conversion of peptide D1 to P1 was achieved within
six hours and the pure a/g4-hybrid peptide was isolated in 96%
yield. The atomic resolution structures are very important in
understanding the conformational properties of peptides;
however, peptides containing g4-amino acids showed poor
record of X-ray structures in the literature. Keeping this in
mind, we attempted to grow the single crystals of a/g4-hybrid
peptides in various solvent combinations. Single crystals of
a/g4-hybrid peptide P1 obtained from the slow evaporation of
methanol solution yield the structure shown in Fig. 1A. Two
molecules of P1 were observed in the asymmetric unit with
slight variation in the torsional values. Instructively, the
tetrapeptide adopted a right-handed 12-helix [12-atom ring
H-bonds of CQO (i)Á Á ÁH–N(i + 3), C12] conformation in the
crystalline state. Recently, Hofmann and co-workers have
predicted the formation of the 12-helical conformation in
1 : 1 alternating a- and unsubstituted g-amino acids using
theoretical calculations.8 In addition, Balaram and colleagues9
and Gellman et al.10 have shown the formation of a 12-helix in
the a/g-hybrid peptides containing 3,3-dialkyl g-amino acids
and cyclic g-amino acids, respectively. However, mixed 10/12
helices have also been reported from the hybrid peptides
containing carbo-g-amino acids as well as 3,3-dialkyl g-amino
acids.11,12 The crystal structure analysis of P1 reveals that the
two continuous twelve membered backward (1 ’ 4) H-bonds,
CQO(Boc, i)ÁÁÁNH(3, i + 3) [CQOÁÁÁH–N dist. 2.10 A, OÁÁÁN
dist. 2.95 A and +OÁÁÁH–N 1611] and CQO(1)ÁÁÁNH(4)
[CQOÁÁÁH–N dist. 2.07 A, OÁÁÁN dist. 2.90 A and +OÁÁÁH–N
1561], stabilize the helical conformation (Fig. 1A). The conforma-
tional analysis of g-residues reveals that g4Phe2 adopted the
gauche+, gauche+ (g+, g+, y1 E y2 E 601) local conformation
about the Cb–Cg and Ca–Cb bonds, while the C-terminal g4Phe4
(g4-amino acids, double homologated a-amino acids) is lagging
behind that of b- and hybrid b-peptides, this is in part probably
due to the difficulty of obtaining stereochemically pure
g4-amino acids. In addition, very little is known about the
heterooligomers containing a- and g4-amino acids. The advantage
of the hybrid peptides containing a- and backbone homologated
amino acids is that a new class of helical structures can be
generated by varying the amino acid sequence patterns.3 None-
theless, Seebach and colleagues4 and Hanessian et al.5 in their
pioneering work recognized the 14-helical conformations from the
oligomers of g4-amino acids. Herein, we report the synthesis, single
crystal conformations of a/g4-hybrid peptides Boc-Aib-g4Phe-Leu-
g4Phe-OEt (P1), Boc-Aib-g4Phe-Leu-g4Phe-Aib-dgF-OEt (P2)
and Boc-Aib-g4Phe-Leu-g4Phe-Aib-g4Phe-OEt (P3), and the
structural analogy with the a-helix.
Recently, we reported the utility of E-vinylogous amino
acids in the construction of a stable and functionalizable
hybrid b-hairpin and its direct transformation to the g-peptide
analogue.6 We adopted a similar strategy for the synthesis of
a/g4-hybrid peptides (Scheme 1). To understand the conforma-
tional properties of hybrid peptides with 1 : 1 alternating a- and
g4-amino acids, the a/E-vinylogous hybrid peptide Boc-Aib-dgF-
Leu-dgF-OEt (D1) (dgF = a,b-unsaturated g-phenyl alanine)
Department of Chemistry, Indian Institute of Science Education and
Research, Dr Homi Bhabha Road, Pashan, Pune-411021, India.
E-mail: hn.gopi@iiserpune.ac.in; Fax: +91-20-2589-9790;
Tel: +91-20-2590-8075
w Electronic supplementary information (ESI) available: Experimental
details, NMR spectra, mass spectra and X-ray crystallography (for
peptides P1–P3). CCDC 878071–878073. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/c2cc32911e
c
7170 Chem. Commun., 2012, 48, 7170–7172
This journal is The Royal Society of Chemistry 2012

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Fig. 2 Circular dichroism spectra of a/g4-hybrid peptides P1, P2, and
P3 in MeOH (200 mM). The CD signature showed similarity with
b-peptide 12-helices.
similar to P2, peptide P3 also adopted 12-helical structure in
single crystals and the structure is stabilized by four consecu-
tive C12 intramolecular 1 ’ 4 H-bonds. In addition, due to the
lack of a H-bond donor (NH) at the C-terminal, a weak 10
membered C–HÁ Á ÁO H-bond between the acidic a-hydrogens
and the CQO of the preceding gPhe residue (1 ’ 3) was
observed in all hybrid peptides. The H-bond parameters of all
hybrid helical peptides (P1–P3) are tabulated in the ESI.w
Circular dichroism spectra of all a/g4-hybrid peptide helices
recorded in methanol solution (200 mM) are shown in Fig. 2.
Recently, Gellman and colleagues demonstrated the CD
signature for 12-helices containing cyclic b-amino acids.13
Instructively, a/g4-hybrid peptide 12-helices P1 and P2
displayed a similar CD pattern to that of b-peptide 12-helices
with CD maxima at B205 nm and weak minima at 220 nm. In
contrast, P2 displayed a little distorted CD spectrum with the
CD maxima at 204 nm and minima at 218 nm, this may be due
to the interference of C-terminal a,b-unsaturated ester.
The crystal structure analysis of the a/g4-hybrid peptides
P1–P3 reveals that the a-residues adopted right handed helical
conformations with average f and c values being À59 Æ 51
and À38 Æ 51, respectively. The dihedral angles of g4-residues
were measured by introducing two additional variables y1
(N–Cg–Cb–Ca) and y2 (Cg–Cb–Ca–C) as shown in Fig. 1A.
The torsional angles of g4-residues in all hybrid peptides
are tabulated in Table 1. The average f and c values of
g4-residues in the helix were found to be À126 Æ 101 and
À118 Æ 101, respectively. Further, the top view of the a/g4-hybrid
peptide helix P3 signifies the projection of amino acid side-chains
at four corners of the helical cylinder (Fig. 1A(i)). In comparison
to the b- and g-peptides, the distinct orientation of the amino
acid side-chains was observed in a/g4-hybrid helical peptides.
The intriguing results of the hybrid peptides P1–P3 encourage
us to assess the hybrid 12-helical structure with respect to the
Fig. 1 (A) X-ray structures of P1, P2 and P3, (i) top view of P3
showing the distinct projections of amino acid side-chains, (ii) helix
favouring the g+, g+ conformation adopted by the g4Phe residue in
12-helices. (B) Continuous 12-membered backward H-bonds observed
in the hybrid peptides P2 and P3.
residue displayed the gauche+ (Cb–Cg), anti (Ca–Cb) conforma-
tion due to the lack of a terminal H-bond donor (NH). To
introduce the additional H-bond at the C-terminal of P1 and
also to understand the behaviour of E-vinylogous amino esters
at the C-terminal, hexapeptide P2 (Boc-Aib-gPhe-Leu-gPhe-
Aib-dgF-OEt) was synthesized from P1 through a 4 + 2
convergent strategy. It is worth mentioning that pure P2 was
crystallized within 30 min. in many solvents including acetone
and ethyl acetate/hexane. Single crystals of P2 obtained from
the ethyl acetate/hexane solution yield the structure shown
in Fig. 1A. The crystal structure analysis demonstrates that
the 12-helical structure of P2 is stabilized by four consecutive
backward 1 ’ 4 hydrogen bonds and g4Phe4 adopted the g+,
g+ conformation. Surprisingly, the C-terminal vinylogous
residue nicely accommodated into the helix through a 10 membered
weak C–HÁÁÁO H-bond between the dgF6 Ca–H and the carbonyl
of gPhe4 (1 ’ 3). The torsional angle of the vinylogous residue is
231 for y1 and the conjugated ester adopted the local s-trans
conformation with the c value of 281 (Fig. 1).7 Further, P2 was
transformed into a completely saturated analogue P3 (Boc-Aib-
g4Phe-Leu-g4Phe-Aib-g4Phe-OEt) by catalytic hydrogenation and
its X-ray structure is shown in Fig. 1A. The analysis revealed that
Table 1 Backbone torsional variables of a/g4-hybrid peptide (P1, P2
and P3) 12-helices
Peptide Residue f
y1
y2
C
P1
P2
gPhe1 À126.5 Æ 3.5 50.1 Æ 2.5 63 Æ 3
gPhe2 À109.5 Æ 7.5 62.5 Æ 0.5 Æ180
gPhe1 À137.5 Æ 0.5 61.5 Æ 0.5 59
À111 Æ 4
—
À115 Æ 1
À120 Æ 3
29.5 Æ 1.5
À114 Æ 1
gPhe2 À123 Æ 2
gPhe3 À113
49 Æ 1
63 Æ 1
24.5 Æ 0.5 173 Æ 1
59 Æ 1 61
P3
gPhe1 À135
gPhe2 À122.5 Æ 2.5 46.5 Æ 2.5 63.5 Æ 2.5 À126.5 Æ 3.5
gPhe3 À116 Æ 3
55 Æ 1
177.5 Æ 0.5 24.5 Æ 0.5
c
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Chem. Commun., 2012, 48, 7170–7172 7171

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predominantly existing natural a-helix. The superimposition
of the backbone conformations of P3 with the a-helix of
chicken egg white lysozyme14 (PDB code: 1HEL, sequence:
C-E-L-A-A-A-M-K, 6–13) is shown in Fig. 3. Instructively, the
backbone conformation of hybrid hexapeptide P3 is well
correlated with the eight residue a-helix, except the H-bonding
pattern. However, the internal H-bonding orientation and
the macrodipole of a/g4-hybrid peptides are analogous to the
a-peptide helix. The top view of the superimposed P3 and the
a-helix signifies the projection of the amino acid side-chains
(Fig. 3). The backbone correlation and the side-chain projec-
tions of a/g4-hybrid peptide helices with respect to the a-helix
suggest that these hybrid peptides can be exploited as mimics of
a-peptide helices. Further, with the availability of broad side-
chain diversity in both a-and g4-amino acids, these a/g4-hybrid
peptides stand unique than the other a/g-hybrid peptides.
In conclusion, we presented the facile transformation and
the structural analysis of the a/g4-hybrid peptides containing
backbone homologated g4-amino acids. Though there is a
possibility of conceiving different types of H-bond pseudo-
cycles, the atomic resolution data of a series of a/g4-hybrid
peptides revealed the unprecedented 12-helical conformations.
The solution conformations of these peptides and the analogy
with other helices are under investigation. The conformational
analysis and the unique side-chain projections of a/g4-hybrid
peptide helices presented here may provide fundamental
information for the design of functional foldamers.
This work is supported by Department of Science and
Technology, Govt. of India. A. B and S. V. J are thankful to
CSIR, Govt. of India, for senior research fellowship.
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Notes and references
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c
7172 Chem. Commun., 2012, 48, 7170–7172
This journal is The Royal Society of Chemistry 2012