Stabilisation of a short α-helical VIP fragment by side chain to side chain cyclisation: A comparison of common cyclisation motifs by circular dichroism

Article abstract of DOI:10.1002/psc.2515

A model octapeptide segment derived from vasoactive intestinal peptide (VIP) was utilised to investigate the effect of several conventional cyclisation methods on the α-helical conformation in short peptide fragments. Three of the classical macrocyclisation techniques (i.e. lactamisation, ring-closing metathesis and Huisgen cycloaddition) were applied, and the conformations of the resulting cyclic peptides, as well as their linear precursors, were compared by CD analysis. The visibly higher folding propensity of the triazole-tethered peptide after azide-alkyne CuAAC macrocyclisation illustrates that the secondary structure of a short peptide fragment can differ significantly depending on the chemical strategy used to covalently cross-link side chain residues in a 'helical' fragment. Copyright 2013 European Peptide Society and John Wiley & Sons, Ltd. The effect of three classical macrocyclisation techniques (i.e. lactamisation, ring-closing metathesis and Huisgen cycloaddition) on inducing an alfa-helical conformation in short peptide fragments was investigated using a model octapeptide segment derived from vasoactive intestinal peptide (VIP).The conformations of the resulting cyclic peptides were compared by CD analysis. Based on this analysis, the triazole-tethered peptide after azide-alkyne CuAAC macrocyclisation shows a higher folding propensity in comparison with the two other cyclization methods.

Full text of DOI:10.1002/psc.2515

Research Article
Received: 12 February 2013
Revised: 15 March 2013
Accepted: 8 April 2013
Published online in Wiley Online Library: 27 May 2013
(wileyonlinelibrary.com) DOI 10.1002/psc.2515
Stabilisation of a short a-helical VIP fragment
by side chain to side chain cyclisation: a
comparison of common cyclisation motifs
by circular dichroism
Lukasz Frankiewicz,a{ Cecilia Betti,a{ Karel Guillemyn,a Dirk Tourwé,a
Yves Jacquotb* and Steven Balleta*
A model octapeptide segment derived from vasoactive intestinal peptide (VIP) was utilised to investigate the effect of several con-
ventional cyclisation methods on the a-helical conformation in short peptide fragments. Three of the classical macrocyclisation
techniques (i.e. lactamisation, ring-closing metathesis and Huisgen cycloaddition) were applied, and the conformations of the
resulting cyclic peptides, as well as their linear precursors, were compared by CD analysis. The visibly higher folding propensity
of the triazole-tethered peptide after azide-alkyne CuAAC macrocyclisation illustrates that the secondary structure of a short
peptide fragment can differ significantly depending on the chemical strategy used to covalently cross-link side chain residues
in a ‘helical’ fragment. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: Helix stabilisation; circular dichroism; cyclisation motifs; macrolactamisation; ring-closing metathesis; Huisgen cycloaddition
adopt little, if any, a-helical structure [7]. For this reason, synthetic
techniques or methodologies that stabilise or mimic helical
Introduction
Because the recognition of peptide ligands by their respective
receptor proteins often occurs at the level of well-structured
secondary helical or turn domains [1], all synthetic methodolo-
gies that enable peptides to adopt a specific secondary structure
are of interest for peptide-based drug development. Classically,
after the identification of the so-called hot spot contacts and
the establishment of the pharmacophore, medicinal chemists
attempt to stabilise the appropriate ‘bioactive’ conformation. To
realise this, an important design feature consists of stabilising
the key amino acid side chains in the suited topographical orien-
tation for interaction with the protein, as they contribute signifi-
cantly to the binding energy [2].
The revival of synthetic peptides, especially cyclic ones, as
potential therapeutic candidates [3–5], is a consequence of
the combination of different, related, aspects as follows: (i)
cyclopeptides show a superior resistance to endopeptidase
and exopeptidase, as compared with linear peptides, (ii) a
reduction in molecular flexibility generally yields more potent
and target-selective peptide ligands, and (iii) recent advances
in the synthesis, delivery and formulation of peptides have
been made [3].
It is now also documented that cyclic peptides are suitable for
extended interactions, such as the ones needed for previously
believed ‘undruggable’ protein–protein interactions (PPIs) [6].
Because of the large interaction surfaces that are characteristic
of these PPIs, it is cumbersome to address these difficult targets
with low-molecular weight compounds.
More specifically, a-helical subdomains of proteins often serve as
recognition motifs for PPIs, but when taken out of their tertiary
structure, the peptide fragments that constitute these subdomains
conformations have received considerable attention over the
past decade [8,9]. Next to the development of promising non-
peptide mimetics and modified peptide backbones that mimic
the properties of a-helices, examples of side-chain cross-linked
peptides with remarkable biological activities have been reported.
The ‘stapled peptide’ technology, which is based on RCM reactions
of modified, olefin-containing residues, has resulted in unprece-
dented results in, for example, stabilised a-helical peptides from
the human B-cell lymphoma-2 (Bcl-2) family of proteins and their
therapeutic use for controlling cell death events [10]. The increased
*
Correspondence to: Steven Ballet, Organic Chemistry Department, Vrije Universiteit
Brussel, Pleinlaan 2, B-1050 Brussels, Belgium. E-mail: sballet@vub.ac.be
Yves Jacquot, Department of Chemistry, Ecole Normale Supérieure, 24, rue
Lhomond, 75231 Paris Cedex 05, France. E-mail: yves.jacquot@upmc.fr
{
Authors contributed equally to this work.
a Department of Organic Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, B-1050,
Brussels, Belgium
b Laboratory of the BioMolécules (LBM), Department of Chemistry, CNRS - UMR
7203, Ecole Normale Supérieure/Université Pierre et Marie Curie Paris 6, 24, rue
Lhomond, 75231, Paris Cedex 05, France
Abbreviations: Aib, 2-amino-2-methylpropanoic acid; All, allyl; Alloc,
allyloxycarbonyl; CD, circular dichroism; DIPEA, N,N-diisopropylethylamine;
DMF, N,N-dimethylformamide; HCTU, 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-
tetramethylaminium hexafluorophosphate; MRE, maximum mean residue
ellipticity; Nle, norleucine; Pra, propargylglycine; RCM, ring-closing metathesis;
SPPS, solid-phase peptide synthesis; TBTU, O-(benzotriazol-1-yl)-N,N,N0,N0-
tetramethyluronium tetrafluoroborate; TFE, 2,2,2-trifluoroethanol; VIP, vasoactive
intestinal peptide.
J. Pept. Sci. 2013; 19: 423–432
Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

FRANKIEWICZ ET AL.
cell permeability of stapled peptides has also made them interest-
ing compounds for targeting intracellular drug targets [11]. A sec-
ond reported strategy to achieve the same goal, that is, obtaining
enhanced helical structure, consists of linking the i and i + 4 side
chains in a peptide via a Cu-catalysed intramolecular Huisgen
azide–alkyne cycloaddition reaction (CuAAC) [12,13]. From the
work of Scrima et al., it was concluded that the number of methy-
lene units present in the side chain bridge, which determine the
length of the cross-link, are important for helicity. Whereas ‘long’
(seven CH2 groups) and ‘short’ (four CH2 groups) linkers did not
stabilise the helical structure, bridged peptides in which the triazole
was flanked by five or six methylene units (corresponding to an
8-atom or 9-atom tether, respectively) nicely accommodated the
desired helix. Triazole-stapling has recently been used for the
stabilisation of B-cell CLL/lymphoma 9 (BCL9) a-helical peptides
[14]. This study, comprising single and double triazole-bridged
analogues, aimed at improved BCL9 – b-catenin binding for
blockade of the transcriptional activity of b-catenin, to suppress
the tumorigenesis of several types of human cancer. When using
triazole bridges, Kawamoto and coworkers showed that a 7-atom
linker was not suited for optimal binding recognition, confirming
short triazole linkers to be inadequate for their purpose. In con-
trast, this linker length was reported to be suited for stabilising
constrained nociceptin derivatives by i ꢀ i + 4 amide bond for-
mation (‘lactamisation’) using the side chain functionalities of
Asp at position i and Lys at i + 4 [15].
The idea of stabilising helices through cross-linkage of a single
turn of the helical peptide domain by RCM was launched and
realised by Grubbs [16]. Through the use of O-allyl serine resi-
dues, located on adjacent helical turns, a tether was installed
between the i and i + 4 residue side chains in a helical model
heptapeptide after RCM conditions were applied.
In a later stage, Verdine and coworkers determined and fine-
tuned the requirements that were important for both iꢀ i + 4 and
i ꢀ i + 7 tethers [17]. When using olefin-containing all-hydrocarbon
(but a,a-disubstituted) (S)-amino acids, they observed that linkers
of 7 or 8 atoms were required.
macrocyclisation techniques (i.e. amide bond formation or
lactamisation, RCM and triazole ‘click’) were employed, taking
into account the aforementioned criteria related to appropriate
linker length for i ꢀ i + 4 bridging. The propensity of each macro-
cyclic linkage to stabilise the helical secondary structure (Figure 1)
was investigated by circular dichroism.
Materials and Methods
General. Mass spectrometry was recorded on a Micromass Q-TOF
spectrometer using electrospray ionisation (positive or negative
ion mode). Data collection was performed with Masslynx soft-
ware. Analytical RP-HPLC was performed using an Agilent 1100
Series system (Waldbronn, Germany) with a Supelco Discovery
BIO Wide PoreW (Bellefonte, PA, USA) RP C-18 column (25 cm
4.6 mm, 5 mm) using UV detection at 215 nm. The mobile phase
(water/acetonitrile) contained 0.1% TFA. The standard gradient
consisted of a 20-min run from 3% to 97% acetonitrile at a flow
rate of 1 ml minꢀ1. Preparative HPLC was performed on a Gilson
apparatus (Gilson, Inc., WI, USA) and controlled with the software
package Unipoint. The reversed phase C18-column (Discovery
BIO Wide Pore 25 cm ꢁ 21.2 mm, 10 mm) was used under the
same conditions as the analytical RP-HPLC but with a flow rate
of 20 ml minꢀ1. A purity of more than 95% was determined
for all compounds by analytical RP-HPLC using the conditions
described in the succeeding text.
Peptide Synthesis
A. Linear Peptides
The linear peptides 2, 3, 4, 5, 9 (Table 2) were synthesised via
standard solid-phase peptide synthesis using Rink amide resin
as the solid support (169 mg, 0.1 mmol, 0.59 mmol gꢀ1). Na-Fmoc
chemistry was applied, and the coupling reactions were
performed with 4 eq of amino acid, 4 eq of HCTU and N-
methylmorpholine in DMF. Removal of the Fmoc protecting
group was realised by means of 20% 4-methylpiperidine in
DMF (5 min, then again for 15 min). Once the sequence was
completed, the resin was treated with a mixture of TFA/H2O/
anisole 95 : 2.5 : 2.5 v/v for 3 h in order to cleave the peptide from
the resin and to remove the side chain protecting groups. After
precipitation in cold diethylether and drying, the crude peptides
were purified by preparative RP-HPLC.
In this study, a model octapeptide segment, derived from vaso-
active intestinal peptide (VIP), was utilised to verify the effect of
several conventional cyclisation methods on the a-helical confor-
mation in short peptide fragments. This helical conformation is
characterised for more than half of the complete 28-amino acid
long VIP sequence 1 [18]. It was reported that a C-terminal
truncation of the VIP peptide results in a loss of helicity and a
concomitant drop in bioactivity [18]. Because this feature is
common in a-helical peptides, that is, a decreased helicity upon
truncation and taking into account that one of the classic steps
in the peptide-to-drug process consists of identifying the shortest
fragment with retained activity, we have taken the central VIP
(15–22) (fragment 2 in Table 1) and submitted it to different
side chain to side chain macrocyclisations. Three of the classical
Table 1. Peptide sequences of VIP 1 and model peptide 2 [18]
Compound
Peptide sequence*
VIP 1
H-His1-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-
Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-
Leu-Asn-Ser-Ile-Leu-Asn28-NH2
Fragment 2
H-Lys15-Gln-Met-Ala-Val-Lys-Lys-Tyr22-NH2
Figure 1. Schematic representation of a full helical sequence (left) and
stabilisation of helical conformation in a fragment (blue) by Huisgen
azide–alkyne cycloaddition, RCM and amide bond formation (right).
* The underlined amino acids are part of an a-helical region.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 423–432

STABILISATION OF A SHORT ALFA-HELICAL VIP FRAGMENT
Table 2. Peptide sequences and compound numbers
phenylsilane (24 eq) and tetrakis(triphenylphosphine)-palladium
(0) (0.2 eq) in dry DCM (2 times 15 min, r.t.). The cyclisation
was performed overnight on resin by use of 4 eq HCTU and
8 eq of DIPEA in DMF. Completion of the cyclisation was verified
by the Kaiser test. Removal of the terminal Fmoc protecting
group, cleavage of the peptide from the resin and concomitant
side chain protecting group removal were followed by final
Sequence
Compound
number
H-Lys1-Gln-Met-Ala-Val-Lys-Lys-Tyr8-NH2
H-Lys1-Gln-Nle-Aib-Val-Lys-Lys-Tyr8-NH2
H-Lys-Gln-Nle-Aib-Val-Lys-Lys-NH2
2
3
4
5
6
H-Lys-Gln-Nle-Aib-Val-Lys-Lys-Ala-NH2
purification as described earlier. HPLC (standard gradient): tret
=
9.21 min.; ESI-HRMS [M + H+]: m/z = 973.5944 (calculated for
C
47H80H+N12O10: 973.6120).
Synthesis of compounds 7 and 8
Fmoc-Lys(Boc)-Ser(All)-Nle-Aib-Val-Ser(All)-Lys(Boc)-Tyr(tBu)-NH2
was synthesised according to the general SPPS protocol
(0.1 mmol scale). The peptide-resin was subsequently dried for
3 h and swollen in 3 ml of degassed 1,2-dichloroethane under
argon atmosphere for 30 min in a round-bottom flask. Next,
the Hoveyda–Grubbs catalyst (20 mg in 3 ml of degassed 1,2-
dichloroethane) was added, and the mixture was stirred
overnight at 50 ꢂC. Intermediate small scale cleavage indicated
incomplete conversion and hence, a second portion of catalyst
was added, and the mixture was stirred for an additional 2 h.
Fmoc removal, cleavage from the resin and purification yielded
the pure CH2CHCHCH2-tethered peptide 7. Finally, in order to
obtain the saturated linker in 8, compound 7 was reduced in
MeOH solution by means of 10% Pd/C (10% (w/w)) under H2
atmosphere (20 psi, r.t.) for 3 h. The crude peptide was again
purified as described in the general method.
7
8
H-Lys-Pra-Nle-Aib-Val-eN3Lys-Lys-Tyr-NH2
9
10
H-Lys-c[Ser(CH2CH=)-Nle-Aib-Val-Ser(CH2CH=)]-Lys-Tyr-NH2 7.
HPLC (standard gradient): tret = 10.71 min.; ESI-HRMS [M + H+]:
m/z = 961.1655 (calculated for C46H77H+N11O11: 961.1707)
H-Lys-c[Ser(CH2CH2-)-Nle-Aib-Val-Ser(CH2CH2-)]-Lys-Tyr-NH2
8. HPLC (standard gradient): tret = 10.31 min.; ESI-HRMS [M + H+]:
m/z = 963.1855 (calculated for C46H79H+N11O11: 963.1866).
Synthesis of compound 10
Peptide characterisation. H-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-
NH2 2 (VIP 15–22): HPLC (standard gradient): tret = 8.00 min. ESI-
HRMS [M + H+] : m/z =994.5612 (calculated for C45H79H+N13O10S:
994.5794)
H-Lys-Gln-Nle-Aib-Val-Lys-Lys-Tyr-NH2 3: HPLC: tret = 7.99 min.
ESI-HRMS [M+ H+]: m/z =990.6300 (calculated for C47H83H+N13O10:
990.6386)
In solution: Compound 9 (0.008 mmol, 10 mg) was dissolved in
10 ml tBuOH/H2O (1:1 v/v) and 6 eq of sodium ascorbate (6 eq,
0.048 mmol, 9 mg) and 4 eq of CuSO.45H2O (4 eq, 0.032 mmol,
8 mg) were added. The reaction mixture was stirred overnight
at room temperature. After full conversion (HPLC monitoring),
the crude cyclised peptide was purified as described in the gen-
eral method.
H-Lys-Gln-Nle-Aib-Val-Lys-Lys-NH2 4: HPLC (standard gradient):
On solid support. The protected peptide Fmoc-Pra-Nle-Aib-
Val-eN3Nle-Lys(Boc)-Tyr(tBu)-NH-Rink was synthesised following
the general procedure (0.05 mmol scale), and the cyclisation
was carried out as follows: the resin was swollen in DMF, and
sodium ascorbate (2 eq, 0.1 mmol, 20 mg), CuI (2 eq, 0.1 mmol,
19 mg) and DIPEA (3 eq, 0.15 mmol, 25 mL) were added. The
mixture was shaken overnight, filtered, and the resin was
washed three times with DMF, iPrOH, DCM and finally five times
(5 ml) with a sodium diethyl dithiocarbamate/DIPEA solution in
DMF (50 mg/25 ml in 25 ml). After Fmoc removal, the final Lys
residue was coupled by addition of Na-Boc-Lys(Boc)-OH.DCHA
(3 eq, 0.15 mmol, 79 mg), TBTU (3 eq, 0.15 mmol, 48 mg) and
DIPEA (9 eq, 0.45 mmol, 74 ml) in DMF. Standard coupling of
90 min was followed by washing steps and peptide cleavage
and purification.
t
ret = 7.86min.; ESI-HRMS [M+ H+]: m/z = 827.5800 (calculated for
C38H74H+N12O8: 827.5753)
H-Lys-Gln-Nle-Aib-Val-Lys-Lys-Ala-NH2 5: HPLC (standard gra-
dient): tret =7.87 min.; ESI-HRMS [M+ H+]: m/z= 898.5920 (calculated
for C41H79H+N13O9: 898.6124)
H-Lys-Pra-Nle-Aib-Val-«N3Nle-Lys-Tyr-NH2 9: HPLC (standard
gradient): tret = 10.71min.; ESI-HRMS [M+ H+]: m/z = 983.6005 (cal-
culated for C47H78H+N14O9: 983.6076).
B. Cyclic Peptides
Synthesis of H-Lys-c[Lys-Nle-Aib-Val-Glu]-Lys-Tyr-NH2 6
The resin-bound linear precursor Fmoc-Lys(Boc)-Lys(Alloc)-Nle-
Aib-Val-Glu(All)-Lys(Boc)-Tyr(tBu)-Rink amide resin was prepared
following the general procedure (0.1 mmol scale). Alloc. . ./All
protecting groups were selectively removed with a solution of
HPLC (standard gradient): tret = 9.58 min.; ESI-HRMS [M + H+]:
m/z = 983.5830 (calculated for C47H78H+N14O9: 983.6076)
J. Pept. Sci. 2013; 19: 423–432 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

FRANKIEWICZ ET AL.
b-forms [23] such as hairpin and turns). The mechanisms by
which TFE induces the structural organisation of peptides are
not entirely resolved. It may occur (i) through direct association
with the peptide of interest [24], (ii) by limiting H2O–peptide
interactions [25], (iii) by favouring the formation of –C¼O---HN–
intramolecular H-bonds [25] and (iv) by increasing the viscosity of
the medium (density of TFE 1.391). The folding kinetics of the
open and cyclised peptides in 50% TFE was studied by record-
ing CD spectra as a function of the percentage of TFE (0, 10,
20, 30, 40, 50 and 60). Likewise, the conformational stability
was investigated as a function of the temperature of the medium
(5, 15, 25, 35, 45 and 55 ꢂC).
Circular Dichroism
Circular dichroism spectra were recorded with a Jobin Yvon CD6
(Jobin Yvon Horiba, Longjumeau, France) apparatus by using a
250 ml (1 mm path length) quartz cell. Each peptide was dissolved
in 18.2 MΩ deionized water (milliQ, Millipore) alone or with various
amounts of 2,2,2-trifluoroethanol (TFE) at a concentration of 50 mM.
Prior to recording, each sample was allowed to equilibrate for
5 min at different temperatures (5, 15, 20, 25, 35, 40, 45 and 55 ꢂC)
or in various water/TFE mixtures (0, 10, 20, 30, 40, 50 or 60% TFE in
water). Absorbance differences were recorded at 1 nm interval (1 s
integration time) from 190 to 260 nm and were averaged over four
scans. After solvent subtraction, smoothing and baseline correction,
absorbance data were treated by using the Dichrograph Software
version 1.2 and converted into mean residue molar circular dichro-
ism Δe (in Mꢀ1 cmꢀ1) following the equation Δe=(ΔA/lc)/n, where l
corresponds to the cell path length (in cm), c corresponds to the
peptide concentration (in M) and n to the number of residues. Then,
Δe values were converted into mean residue molar ellipticity [(θ), in
10ꢀ3 deg cm2 dmolꢀ1] from the equation [θ] = 3.298 Δe.
Linear Peptides
First, we determined the spectroscopic signature of the wild-type
peptide fragment VIP (15–22) (H-Lys1-Gln-Met-Ala-Val-Lys-Lys-
Tyr8-NH2 2, Table 2).
Because of the presence of a phenolic chromophore at the side
chain of the C-terminal Tyr8 in 2, an absorbance band that is
not correlated to the polypeptide backbone conformation may
appear. In fact, this phenomenon occurs when the flexibility of
the aromatic amino acid side chains (tryptophan, tyrosine and
phenylalanine) is restricted around wi dihedral angles, encourag-
ing chirality. The phenol of the tyrosine is usually characterised
by three absorption bands that are associated with pp* transi-
tions: at 190 nm (Ba and Bb transition states), 230 nm (La transition
state) and 280 nm (Lb transition state), following Platt’s nomen-
clature [26–29].
In the case of the peptide 2, no absorption band ascribed to
the phenol of Tyr8 was observed (spectrum recorded from 180
to 380 nm, data not shown). Thus, we assumed that the CD spec-
tra of this peptide and of those explored in the present study will
exclusively reflect the spatial orientation of the backbone. Like-
wise, any potential influences of the modified amino acid side
chains on the overall peptide conformation was verified by
recording the CD spectrum of an initial set of linear peptides
in a large wavelength range (from 180 to 380 nm) in water
alone and at 25 ꢂC. The CD signatures of VIP fragment 2, its
analogue 3 (H-Lys-Gln-Nle-Aib-Val-Lys-Lys-Tyr-NH2) and the
peptides H-Lys-Gln-Nle-Aib-Val-Lys-Lys-NH2 (compound 4) and
H-Lys-Gln-Nle-Aib-Val-Lys-Lys-Ala-NH2, (compound 5), corre-
sponding to fragment 3 devoid of the C-terminal tyrosine or
bearing an alanine instead of a tyrosine, respectively, were
recorded and displayed similar spectroscopic profiles. These
results confirmed the absence of any tyrosine-induced chromo-
phoric effect [Figure 2(A)].
Results and Discussion
Several strategies are available for the stabilisation of the most
abundant protein secondary structure in nature, the a-helix, and
comprise both covalent and non-covalent (e.g. electrostatic and
metal complexation) stabilisation [8]. During the last decade,
the covalent stabilisation by a side chain to side chain tether
has become a common technique to stabilise helical and turn
conformations of promising, bioactive peptide segments. How-
ever, these stabilisation techniques have rarely been applied to
the same peptide in order to verify which technique might be
the most suited for fixing the desired helical form. Different
cyclisation motifs were however compared in a recent study that
aimed at mimicking the b-turn structure of tendamistat [19]. Next
to the conventional disulfide-bridged and backbone-cyclised
lactam cyclopeptide analogues, the triazole-cyclised and linear
b3 and/or b2-amino acid-containing peptides were checked for
their propensity to induce a b-turn conformation. This compara-
tive study demonstrated that the Huisgen cycloaddition (forma-
tion of triazole) and linear b-amino acid-containing peptides
(e.g. with inclusion of b3 homo-amino acids or the turn-inducing
b2-b3 dipeptide motif) [20] were not giving way to the desired
b-turn stabilisation. This is in contrast to the linear and back-
bone ‘lactamised’ and to a lesser extent, the disulfide-bridged
analogues, which seemed to readily adopt the folded structure
in solution [19].
Herein, and along the same line of the aforementioned study,
the propensity of cyclised peptides as well as of their linear pre-
cursors (Table 2) to adopt a helical conformation was investi-
gated using CD, a powerful method for exploring the secondary
structure of peptides.
For the study of the conformational state of a bioactive
compound, which is relevant to the one in its natural environ-
ment, water seemed to be the most appropriate medium. How-
ever, because of its high polarity and its propensity to create
intermolecular hydrogen bonds, it is a poorly structuring solvent.
For this reason, our spectroscopic studies were carried out in
water with 2,2,2-trifluoroethanol (TFE, 0 ! 60% v/v). Similarly to
CHCl3, TFE promotes peptide folding, allowing access to sec-
ondary structures (i.e. mainly helix [21,22] or to a lesser extent
In water, compound 2 displays a CD signature relevant to
a random form with a strong negative maximum at 194 nm
[(θ) = ꢀ27 253 deg cm2 dmolꢀ1
, pp* transition, Figure 2(A)].
Because of medium change, this band is slightly red-shifted
at 199 nm [(θ) = ꢀ9839 deg cm2 dmolꢀ1] in 50% TFE [Figure 2
(B)]. Thus, random populations predominate, even in structur-
ing conditions. In 50% TFE, analogues 4 (without Tyr8) and 5
(Tyr8 ! Ala8) behave differently than the ‘native’ VIP fragment
(but with Met3 ! Nle3 substitution, compound 3), suggesting
that the C-terminal tyrosine participates, even modestly, to
peptide folding [Figure 2(B)].
The peptide analogue which was used as a reference in this
study, that is, compound 3, resulted from the well-established
Met to Nle substitution to avoid an anticipated Met3 side chain
wileyonlinelibrary.com/journal/jpepsci Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 423–432

STABILISATION OF A SHORT ALFA-HELICAL VIP FRAGMENT
a)
b)
Figure 2. CD spectra of the linear peptides 2, 3, 4 and 5 at 50 mM and at 25 ꢂC (a) in water and (b) in 50% TFE. The mean residue ellipticity [θ] values are
expressed in 10ꢀ3 deg cm2 dmolꢀ1
.
oxidation and from the observation that H-Lys-Gln-Nle-Ala-Val-
Lys-Lys-Tyr-NH2 peptide (i.e. [Ala4]-3) did not cyclise efficiently
under the applied RCM conditions (hence, the substitution to
Aib, see later discussion of RCM peptides 7 and 8). Aib is a well-
established inducer of 310 helices [30–32] and is often introduced
to make a sequence prone to folding. Looking more closely at
compound 3 in water alone and at 25 ꢂC, this peptide is
characterised by a negative maximum at 193 nm [(θ) = ꢀ16 898
deg cm2 dmolꢀ1], revealing the presence of random statistical-
coil conformation states [Figure 3(A)]. In a 50% TFE in water
medium [Figure 3(A)], this pp* transition is of lower intensity
and red-shifted to 205 nm [(θ) = ꢀ14 830 deg cm2 dmolꢀ1], an
observation that can be caused by the medium or to some fold-
ing events. Because of the additional positive maximum recorded
at 197 nm [(θ) = 7947 deg cm2 dmolꢀ1] and the local negative
shoulder at ~220 nm [(θ) = ꢀ10 477 deg cm2 dmolꢀ1], helix struc-
ture is likely. The fact that the intensity of the band at ~220 nm
is smaller to the one at 205 nm suggests the formation of a 310
helix, confirming the role of Aib in this context [33,34]. It is note-
worthy that the low intensity of the band at 197 nm is also in
favour of 310 helix [16,35,36]. However, 11 and 14/15-helix (i.e.
a/b-mixed peptides) is not totally excluded [37,38].
represent the evolution of the intensity of the negative maximum
at ~205 nm as a function of the percentage of TFE. Such a graph
is informative regarding the propensity of a given peptide to
adopt a regular structure. In this regard, it should be stressed that
for all the studied peptides, the evolution of the intensity of the
negative maximum at ~222 nm (data not shown) correlated to
that recorded at ~205 nm. When recording CD profiles as a
function of an incremental concentration of TFE (v/v), a limited
decrease of the intensity of the negative band at ~205 nm for
compound 2 was observed, confirming not only that the peptide
adopts a more regular structure when TFE concentration increases
but also that the random states participate in the absorbance
recorded at this wavelength [Figure 4(A)]. Note that a red-shift
(from 195 to 220nm) is observed, allowing to consider two confor-
mational populations, depending on the percentage of TFE: the
populations where the band is at 185 nm would be relevant to
random populations, whereas those that share a band at 200nm
are relevant to structured populations.
Cyclised Peptides
In Figure 4(A), the CD spectra of the wild-type peptide 2 in
different concentrations of TFE are shown. The graph insets
The conformational effects resulting from different side chain to side
chain cyclisation strategies were explored and placed in perspective
J. Pept. Sci. 2013; 19: 423–432 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

FRANKIEWICZ ET AL.
a)
b)
c)
d)
e)
Figure 3. CD spectra of the synthesised peptides at a concentration of 50 mM in water alone or in water with 50% TFE, at 25 ꢂC. (a) Peptide 3, (b) peptide 6,
(c) peptide 7, (d) peptide 8 and (e) peptide 10. The mean residue ellipticity [θ] values are expressed in 10ꢀ3 deg cm2 dmolꢀ1
.
with earlier studies, such as those of Scrima [12] and Kawamoto
[14]. Aside from disulphide bridge formation, the most commonly
utilised cyclisation techniques were directly compared in this work
and involved lactamisation, RCM and Cu-catalysed Huisgen cyclo-
addition (CuAAC) reactions.
A macrocyclic amide analogue of reference peptide 3, that is,
H-Lys-c[Lys-Nle-Aib-Val-Glu]-Lys-Tyr-NH2 (peptide 6), was pre-
pared and its CD signature was recorded. The applied cyclisation
conditions (HCTU/DIPEA in DMF for 4 h) did not allow the i to i + 4
side chain to side chain cyclisation for the synthesis of the c[Glu2,
Lys6]-analogue. As a consequence, this latter analogue was not
included in the present study.
at ~ 204 nm fluctuates modestly between ꢀ10000deg cm2 dmolꢀ1
and ꢀ15 000 deg cm2 dmolꢀ1 [inset Figure 4(B)]. It should be noted
that an increase in concentration of TFE has little effect on the
structure. In the same context, a thermal unfolding study
performed at 205 nm [Figure 5(A)] and 222 nm [Figure 5(B)],
between 5 and 55ꢂC, showed marginal changes. Such an observa-
tion is relevant to a stable conformational state. Thus, the amide-
mediated cyclisation of peptide 3 exerts structuring and stabilising
effects in TFE, which are in the favour of a/b populations.
In water, the CD spectrum of the peptide cyclised through RCM
and possessing an unsaturated diether bridge (compound 7) is
typical for a random form [(θ) = ꢀ5908 deg cm2 dmolꢀ1 at 197 nm,
Figure 3(C)].
In the presence of 50% TFE, this peptide exhibits a red-shifted neg-
ative maximum at 204 nm [(θ) = ꢀ6,770 deg cm2 dmol-1] and a weak
negative signal observed at ~223 nm [(θ) = ꢀ2,113 deg cm2 dmol-1,
np* transition, Figure 3(C)].
Recently, Manning and Woody have provided convincing sim-
ulation data concerning the 310-helix CD signature [33]. More pre-
cisely, they showed that 310-helix is characterised by an intense
In peptide 6, the amide bond formation between the residues i
(Lys2) and i + 4 (Glu6) induces a negative absorption band rele-
vant to a random state at 193 nm [(θ) = ꢀ20,255 deg cm2 dmolꢀ1
,
pp* transition], in water at 25 ꢂC [Figure 3(B)]. In the presence
of 50% TFE, this band is strongly red-shifted to 203 nm [(θ) =
12 404 deg cm2 dmolꢀ1], suggesting a/b (a-helix and/or b turn)
populations or more structural organisation [Figure 3(B)].
From 20 to 60% of TFE, the intensity of the negative band
wileyonlinelibrary.com/journal/jpepsci Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 423–432

STABILISATION OF A SHORT ALFA-HELICAL VIP FRAGMENT
Peptide 2
a)
0
-5
4
2
0
-2
0% TFE
-4
10%TFE
20%TFE
30%TFE
40%TFE
50%TFE
60%TFE
-6
-10
-15
-20
-25
-8
-10
-12
-14
-16
-18
-20
-22
-24
190 200 210 220 230 240 250 260
0
10
20
30
40
50
60
70
Wavelength (nm)
%TFE
Peptide 6
b)
0
-5
2
0
-2
-4
0%TFE
-6
-8
-10
-12
-14
-16
-18
-20
10%TFE
20%TFE
30%TFE
40%TFE
50%TFE
60%TFE
-10
-15
-20
-25
-22
0
10
20
30
40
50
60
70
190 200 210 220 230 240 250 260
Wavelength (nm)
% TFE
c)
Peptide 7
0
-5
4
2
0
-2
-4
-6
-8
-10
-12
-14
-16
0%TFE
10%TFE
20%TFE
30%TFE
40%TFE
50%TFE
60%TFE
-10
-15
-20
-25
190
200
210
220
230
240
250
260
0
10
20
30
40
50
60
70
Wavelength (nm)
% TFE
d)
Peptide 8
0
-5
6
4
2
0%TFE
10%TFE
20%TFE
30%TFE
40%TFE
50%TFE
60%TFE
0
-10
-15
-20
-25
-2
-4
-6
-8
-10
190 200 210 220 230 240 250 260
0
10
20
30
40
50
60
70
Wavelength (nm)
% TFE
e)
Peptide 10
30
0
-5
25
20
15
10
5
0
-5
-10
-15
-20
-25
0%TFE
10%TFE
20%TFE
30%TFE
40%TFE
50%TFE
60%TFE
-10
-15
-20
-25
0
10
20
30
40
50
60
70
190
200
210
220
230
240
250
260
Wavelength (nm
% TFE
Figure 4. Spectra of the cyclized peptides in function of the amount of TFE (from 0 to 60% TFE, v:v) and evolution of the maximum MRE in function of
the percentage of TFE: (a) peptide 2, (b) peptide 6, (c) peptide 7, (d) peptide 8 and (e) peptide 10. The mean residue ellipticity [(θ), MRE] values are
expressed in 10ꢀ3 deg cm2 dmolꢀ1. Inset: intensity of the negative maximum at 205 nm as a function of the percentage of TFE. We represent only
the evolution of the signal for the red-shifted negative maximum, which is relevant to structuring events.
J. Pept. Sci. 2013; 19: 423–432 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

FRANKIEWICZ ET AL.
a)
b)
Figure 5. Unfolding of the secondary structures of the cyclised compounds 6, 7, 8 and 10 monitored at 205 (a) and 222 nm (b). The mean residue
ellipticity [θ] values are expressed in 10ꢀ3 deg cm2 dmolꢀ1
.
208 nm band relative to the 222 nm band, in contrast with an
a-helix [34]. Indeed, the a-helix is usually characterised by a
strong positive maximum at ~ 190 nm and two negative bands
at 208 nm and 222 nm. In 1996, Toniolo et al. proposed to use
the ratio R = [θ]222/[θ]208 (i.e. [θ]n!p*/[θ]p!p*) to detect a 310-helix:
R ≤ 0.4 in the case of 310 helix and R ꢃ 1 in the case of a-helix
[35,34]. More precisely, the values would be 0.20 < R < 0.40 in the
case of 310 helix and 0.85 < R < 0.90 in the case of the a-helix
[39,40]. The ratio [θ]223/[θ]205 value being of ~0.31 in our case, a
could be relevant to a 310 helix [Figure 3(D)]. The study of the con-
formation of the derivative 8 as a function of the percentage of TFE
reveals not only random forms but also helix contribution, as shown
by the shoulder at 225 nm [inset Figure 4(D)]. By referring to the
maximum at 204 nm, modest modifications of the absorbance at
this wavelength were observed [Figure 4(D)]. However, the increase
of the maximum at 191 nm when TFE increases confirms an enrich-
ment of peptide populations adopting a helical form.
Next, we studied the CD signature of cyclopeptide 10, which
was obtained by the Cu-catalysed Huisgen cycloaddition reaction
involving modified amino acid side chains, in this case, (S)-
propargylglycine (Pra) in position 2 and (S)-e-azido-norleucine
(eN3ꢀNle) in position 6. In contrast to the lactam analogue (com-
pound 6), peptide 10 appears to be more structured and this as
early as 0% TFE, as evidenced by the negative maximum at
205 nm [Figure 4(E)].
Remarkably, the behaviour of this peptide is different from
the others. In contrast to the lactam (6) and the RCM derivatives
(7 and 8), it is much more structured into 310 helix as evidenced
by the positive maximum at 192 nm, the negative maximum at
204 nm and the weak minimum at 222 nm [Figures 3(E) and 4(E)].
Accordingly, the ratio [θ]222/[θ]205 is approximately 0.60 and
remains constant during titration [Figure 6(A)] [16, 34–36].
Thus, the triazole motif appears particularly important for pep-
tide folding and stabilisation into 310 helix. In agreement with this
statement, a negative maximum near 205 nm (pp* transition) and
a negative shoulder between 215 and 230 nm (np* transition)
were observed in the absence and in the presence of TFE, giving
310 helix for 7 seems likely [16,35,36]. As previously observed, the
linear decrease of the intensity of the negative maximum at
204 nm with increasing amounts of TFE (from ꢀ15 deg cm2 dmolꢀ1
to ꢀ2 deg cm2 dmolꢀ1) is relevant to a lack of cooperativity, and
therefore, a redistribution of random population [inset Figure 4
(C)]. It is noteworthy that peptide populations are unchanged
between 5 and 55 ꢂC, as stressed by the measure of the ratio
[θ]205 (T ꢂC)/[θ]205 (5 ꢂC) [Figure 5(A)] and the ratio [θ]222 (T ꢂC)/
[θ]222 (5 ꢂC) [Figure 5(B)].
In water, the CD spectrum of 8 [Figure 3(D)], the compound in
which the double bond has been reduced shows a CD profile sim-
ilar to the one of its unsaturated precursor (peptide 7), with a strong
negative maximum at 194 nm [(θ) = ꢀ7902 deg cm2 dmolꢀ1]. In
50% TFE and with respect to the absorption bands intensities,
the positive maximum at 192 nm [(θ) = 4391 deg cm2 dmolꢀ1
,
pp* transition polarised perpendicular to the helix axis], the nega-
tive maximum (shoulder at 204 nm, [θ] = ꢀ2165 deg cm2 dmolꢀ1
,
pp* transition polarised parallel to the helix axis) and the negative
shoulder at 225nm [(θ) = ꢀ1296 deg cm2 dmolꢀ1, np* transition],
wileyonlinelibrary.com/journal/jpepsci Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 423–432

STABILISATION OF A SHORT ALFA-HELICAL VIP FRAGMENT
a)
30
25
20
-2
-7
0%TFE
15
10
5
0
-5
10%TFE
20%TFE
30%TFE
40%TFE
50%TFE
60%TFE
205 nm
222 nm
222 nm/205 nm
-12
-17
-22
-10
-15
-20
-25
0
10
20
30
40
50
60
190 200 210 220 230 240 250 260
Wavelength (nm)
% TFE
b)
2
0
8
3
0%TFE
10%TFE
20%TFE
30%TFE
40%TFE
50%TFE
60%TFE
-2
205 nm
222 nm
222 nm / 205 nm
-4
-2
-6
-7
-8
-12
-10
190 200 210 220 230 240 250 260
0
10
20
30
40
50
60
70
Wavelength (nm)
%TFE
Figure 6. Intensity of the [θ] values (in 103 deg cm2 dmolꢀ1) recorded at ~205 and 222 nm for compounds 10 (a) and 9 (b). Inset: the ratio values [θ]
222 nm/[θ]205 nm has no unit.
substantial weight to a superior structural arrangement of the
peptide, even in water [Figure 4(E)].
2–5 in water). In the presence of 50% TFE, random coil and minor
secondary structure are observed. With regard to the cyclised
peptides, one can conclude that the lactam analogue 6 also pre-
fers random coil conformations in water but seems to adopt
structured forms that are in favour of a-helix/b-turn populations
when the measurements were carried out in 50% TFE/water (v/v).
Both analogues resulting from RCM cyclisation (i.e. compounds 7
and 8) again show random conformations in water. The CD
spectra of these compounds are, however, indicative of 310
helix populations at high TFE content (50%). With respect to
the linear precursor of the CuAAC reaction and the triazole-
bridged peptide analogue (9 and 10, respectively), a higher pro-
pensity of folding is observed, as compared with the RCM and
lactam peptides. Whereas the click precursor 9 seems to fold
into a 310 helix at a TFE content equal to 20% and higher, the
triazole-tether stabilises the peptide 10 in that same conforma-
tion, even in the absence of TFE. As a result, we have obtained
the following ranking of the different structural motifs for
helix stabilisation: triazole tether > linear azide/alkyne precursor
amide tether > RCM tethers. In consequence, the results are dif-
ferent from those in an earlier study that compared different
strategies to stabilise the b-turn structure [19]. Although, these
results clearly seem to be in favour of the Huisgen cycloaddition
for helix stabilisation, the observations in this work might be
sequence dependant. The remarkable folding propensity of
the linear azide/alkyne analogue 9 prompts us to investigate
the generality of this observation more in depth.
It is noteworthy that at 0 and 10% TFE 9 is random in con-
trast to the cyclised analogue. Starting from TFE percentages
of 20%, a CD profile similar to 10, with a positive maximum
at ~192 nm, a strong minimum near 205 nm and a negative
band near 222 nm [Figure 6(B)], is exhibited by the linear pre-
cursor of the click cyclisation (i.e. H-Lys-Pra-Nle-Aib-Val-e
N3Nle-Lys-Tyr-NH2, 9). When compared with the CD spectra of
peptide 10 [Figure 6(A)], the intensity of the bands recorded
for 9 is systematically lower, an observation consistent with
the fact that random populations participate in the signal at
205 nm. In view of the absorbance changes of the spectrum
recorded for 2 and 6, the spectral signature associated with 9
and 10 suggests also that the Pra and/or eN3Nle functionalities
aid in peptide folding [15].
Lastly, the ratio value of the mean residue ellipticities recorded
at 205 and 222 nm shows that R = [θ]222/[θ]205 ~ 0.45 and remains
constant during titration, which is indicative of the presence of
3
10 helix [Figure 6(B)] [16,34–36]. Note that cooperativity appears
evident (inset), revealing a well-defined conformation distinct
from random species.
Hence, the triazole-mediated cyclisation is not only associ-
ated with a helix-turn conformation, but it seems that the Pra
and eΝ3Νle side chains are involved in peptide folding in such
a way that a structural organisation is quickly achieved.
Accordingly, the ratio of the mean-residual ellipticity values
recorded at 205 and 222 nm, as a measure of conformational
changes, remains constant during titration with values around
0.60 [Figure 5(A)] [16,35,36].
Acknowledgements
The work of SB, DT and CB was supported by a collaboration con-
vention between the Ministère du Développement économique,
de l’Innovation et de l’Exportation du Québec (MDEIE) and the
Fund for Scientific Research Flanders (FWO Vlaanderen) (FWO
G.A055.10 N and PSR-SIIRI-417). The work of YJ was supported
by the Centre National pour la Recherche Scientifique (CNRS), the
University Pierre et Marie Curie and the Ecole Normale Supérieure.
Conclusions
This study has shown that in general, linear peptide analogues of
a VIP octapeptide segment lose their helical propensity when
taken out of an extended a-helix domain (cf. spectra of peptides
J. Pept. Sci. 2013; 19: 423–432 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

FRANKIEWICZ ET AL.
19 Larregola M, Lequin O, Karoyan P, Guianvarc’h D, Lavielle S. b-Amino
acids containing peptides and click-cyclized peptide as b-turn mimics:
a comparative study with ‘conventional’ lactam- and disulfide-bridged
hexapeptides. J. Pept. Sci. 2011; 17: 632–643. DOI: 10.1002/psc.1382
20 Seebach D, Gardiner J. b-Peptidic peptidomimetics. Acc. Chem. Res.
2008; 41: 1366–1375. DOI: 10.1021/ar700263g
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