Bispidine as a helix inducing scaffold: Examples of helically folded linear peptides

Article abstract of DOI:10.1039/c3cc45649h

We designed and synthesized bispidine-anchored peptides and showed that these peptides as small as B3 (containing four chiral α-amino acid residues) adopt a right handed helical conformation. Bispidine anchored linear peptide B3 adopts a helical conformat

Full text of DOI:10.1039/c3cc45649h

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Bispidine as a helix inducing scaffold: examples of
helically folded linear peptides†
Cite this: Chem. Commun., 2013,
49, 10980
V. Haridas,* Sandhya Sadanandan, M. V. S. Gopalakrishna, M. B. Bijesh,
Ram. P. Verma, Srinivas Chinthalapalli and Ashutosh Shandilya
Received 24th July 2013,
Accepted 8th October 2013
DOI: 10.1039/c3cc45649h
www.rsc.org/chemcomm
We designed and synthesized bispidine-anchored peptides and
showed that these peptides as small as B3 (containing four chiral
a-amino acid residues) adopt a right handed helical conformation.
Bispidine anchored linear peptide B3 adopts a helical conformation
in solution and in the solid state.
Controlling the 3D structure of proteins is the underlying theme
of many recent research activities. The alpha helical shape is one
of the topologies employed by nature to give structural and
functional form to biological molecules. The prevalence of a
helical conformation in many protein–protein interactions
prompted chemists to mimic this conformation via a variety of
methods.1 For example, different peptidomimetic approaches
for mimicking helical conformations have been investigated.
Fairlie et al.2 and Arora et al.3 used cyclization approaches for
restricting the conformation of the peptide to a helix. Their
approaches involved the stapling of side chains using amide
bonds or click reactions. These covalent cross-links stabilized
the helical conformation if the targeted linking residues were
installed at appropriate positions. Hamilton et al.4 used non-
peptide systems, such as biphenyl, to generate a helical topology
and to effectively inhibit protein–protein interactions.
A variety of folding paradigms were investigated by Gellman
and coworkers for specific types of folded molecules (namely,
foldamers), thereby opening a new area of study. Gellman et al.5
and Horne et al.6 used different combinations of a and b amino
acids to synthesize a variety of folded molecules. The continuing
saga of protein folding and the engineering of peptides with
predictable 3D structures still requires a more fundamental
understanding before perfect designs can be obtained.7
Fig. 1 Proline-inspired design of a template for secondary structure nucleation.
structures in peptides were successfully demonstrated by various
groups. b-Aminocyclopentanecarboxylic acid (ACPC),10 b-amino
cyclohexanecarboxylic acid (ACHC),6a aminopiperidine carboxylic
acid,11 1-(aminomethyl)cyclohexaneacetic acid (gabapentin),12
bicyclic systems,13 and diproline14 systems are effective in inducing
secondary structures in peptides (Fig. 1). We conceived the incor-
poration of a cyclic structure into peptide backbones as a strategy to
induce a turn that, if further reinforced by appropriate hydrogen
bonds, might facilitate nucleation of a specific secondary structure.
We envisaged that bispidine – with its two fused piperidine
rings – would provide rigidity and kink in the backbone due to its
bicyclic architecture with co-facially arranged nitrogen atoms. The
two closely spaced nitrogens of bispidine keep the appended
peptide chains in close proximity, which is favorable for intra-
molecular interactions leading to secondary structure formation.15
We envisaged the closely appended nitrogen, decorated with
peptides in an antiparallel way, as an attractive option for
secondary structure nucleation.
Template-facilitated peptide folding is an attractive option for
engineering secondary structures.8 Recently, turns have been
hypothesized to act as helix-nucleating elements in peptides.9
Approaches based on proline-like structures to induce secondary
In order to accomplish this, we converted bispidine into an
imino acid-like structure, by linking one of the nitrogens to
bromoacetic acid. In other words, incorporation of bispidine into
the peptide backbone could be visualized as incorporation of an
unnatural amino acid surrogate, where one side of the bispidine
was functionalized with bromoacetic acid to obtain the imino acid
(highlighted region in Fig. 2). The antiparallel arrangement
of anchored peptide chains on the bispidine provides ample
Department of Chemistry, Indian Institute of Technology Delhi, New Delhi-110016,
India. E-mail: h_haridas@hotmail.com
† Electronic supplementary information (ESI) available: Synthesis procedure and data
of all compounds and NMR, IR, HRMS, X-ray, and CD studies. CCDC 946097. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/c3cc45649h opportunities for intramolecular hydrogen bonding.
c
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concentration of 22% (v/v), indicating non-hydrogen-bonded solvent-
exposed NH. Conversely, Leu NH (Dd = 0.12), Ala NH (Dd = 0.07), and
Phe NH (Dd = 0.03) showed almost no change in the amide protons,
indicative of intramolecular H-bonding (Fig. S5, ESI†). The solution
FT-IR studies of B3 in chloroform showed bands at 3328 cmÀ1 and
3426 cmÀ1, typical for hydrogen-bonded and non-hydrogen-bonded
amide NHs (Fig. S6, ESI†). The amide I band at 1666 cmÀ1 supported
a helical conformation.18
In order to investigate the intramolecularly H-bonded pattern in
B3, we studied the simpler peptides B1 and B2. The solution FT-IR
spectrum of B1 showed a band at 3424 cmÀ1, indicating that the
predominant form in solution was non-bonded NHs (Fig. S6, ESI†).
NMR titration studies of B1 in CDCl3 with DMSO-d6 showed
exposed NH, as expected (Fig. S7, ESI†). FT-IR and DMSO-d6
titration studies of B2 showed that both the NHs were intra-
molecularly hydrogen-bonded (Fig. S6–S8, ESI†). Comparing B1
and B2, the possible interactions could be ascribed to Leu NH
with Ala CO and Ala NH with Leu CO. This was further supported
by molecular dynamics studies of B2 in chloroform. All possible
H-bonded conformations of B2 were modelled and analyzed to
find the most favoured hydrogen bonding. It was found that the
involvement of Leu NH/Ala CO and Ala NH/Leu CO is the most
favoured compared to other possible hydrogen bonds (Table S2,
ESI†). The three-dimensional structures of B3 in chloroform and
methanol were calculated using molecular dynamics simulation
(SANDER, Amber 10). The ten lowest-energy structures showed a
right handed helical structure (Fig. S9, ESI†) and a backbone
RMSD of 0.21–0.59 Å as compared to the crystal structure.
CD spectra of the peptides B3–B7 showed minima at 214 nm and
Fig. 2 Structures of the bispidine anchored peptides B1–B7.
We designed and synthesized bispidine-conjugated peptides 228 nm, which are characteristic of a right handed helical conforma-
B1–B7 (Fig. 2). Selective functionalization16 of the two nitrogen tion (Fig. 3 and Fig. S10b–e, ESI†). The small red shift in the
atoms yielded asymmetrically-appended bispidine with peptides wavelength was attributed to the presence of an unnatural moiety
(Scheme S1, ESI†). One of the two bispidine nitrogen atoms was in the backbone, or to deviations from the typical a-helical protein
functionalized with peptides via amide linkage, and the other structure. Such a shift in the CD spectra is known in many synthetic
nitrogen atom was functionalized by reacting with peptides func- systems.19 B1 and B2 showed no helical characteristics in the CD
tionalized with bromoacetic acid at the terminus. The two different spectrum (Fig. S10a, ESI†). The longer peptides B6 and B7 showed
linkages on bispidine-conjugated peptides offer possibilities for enhanced helicity (Fig. S10d–e, ESI†) compared to the shorter
intramolecular hydrogen bonding because of the proximity of the peptides B3, B4 and B5. A minimum of four residues were required
complementary groups (i.e., amide NHs and COs). Tetrapeptides for helical conformation, as observed from the CD studies. B3 and
B3–B5 and pentapeptides B6 and B7 incorporating a bispidine B4 remained fully helical during CD melting experiments at tem-
moiety were synthesized by solution phase peptide synthesis under peratures from 10–55 1C, indicating that they are structurally robust
DCC/N-hydroxysuccinimide-based coupling conditions (ESI†).
(Fig. S11, ESI†). The addition of trifluoroethanol (TFE) showed only a
The solution conformations of bispidine-anchored peptides were slight increase in the CD signal (Fig. 3 and Fig. S12, ESI†).
studied by NMR, IR and circular dichroism (CD) studies. The
1H NMR spectra of tetrapeptides B3, B4 and B5 in CDCl3 showed
highly dispersed NHs. Careful analysis of the NMR spectra of B1 and
B2 showed exchange peaks of amide NH as a result of the rotation of
the imide carbonyls. The rotamer population decreases with
increased peptide length (Table S1, ESI†). Detailed 2D NMR spectro-
scopic analyses of these compounds were performed using TOCSY,
ROESY, and HSQC (Fig. S1–S4, ESI†). The ROESY spectrum of
compound B3 in CDCl3 showed NOE peaks (Fig. S1 and S2, ESI†)
between PheNH/LeuNH, ValNH/AlaNH, LeuNH/AlaNH, AlaaCH/
ValNH and Leu NH/PheaCH; these are typical for helical conforma-
tions (Fig. S5b, ESI†). To test for the presence of intramolecular
H-bonding, we performed 1H NMR titration of the peptides in CDCl3
D after addition of 50%
Fig. 3 CD spectrum of B4 B in methanol and
with DMSO-d6.17 The Val NH showed a shift of (Dd) 0.332 at a DMSO
trifluoroethanol.
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peptides with no significant preference for a helical conforma-
tion is a noteworthy finding.
We thank the Department of Science and Technology (DST
and DST-FIST) and CSIR, New Delhi, for the financial support.
We thank Prof. Narayanan Kurur (IITD) for discussions regard-
ing NMR experiments. We also thank Prof. Venugopalan Paloth,
Punjab University, for the help in X-ray structure analysis.
Notes and references
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conformation (Fig. S14–19, ESI†).
Compound B3 was crystallized from acetonitrile and
dimethylformamide (Table S3, ESI†). The X-ray crystal structure
of B3 showed topological similarity to a right-handed helical
conformation (Fig. 4) with torsional angles (Table S5, ESI†)
different from a typical a-helix. The two piperidine rings of
bispidine adopted chair conformations, resulting in a chair–chair
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Leu NH (2.072 Å) and Phe NH with Val CO (2.398 Å) (Fig. S20, ESI†).
These hydrogen bonds resulted in 10, 11, and 12-membered rings
(Fig. S20, ESI†). The crystal structure further showed that free Val
NH present in B3 is involved in an intermolecular H-bond with the
Phe CO of another molecule to form a linear chain of helical
molecules (Fig. 4c).
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