Constrained α/γ-peptides: A new stable extended structure in solution without any hydrogen bond and characterized by a four-fold symmetry

Article abstract of DOI:10.1039/c2cc16852a

Small α/γ-peptides alternating α-aminoisobutyric acid and cyclic γ-amino acid residues are described. NMR studies together with restrained simulated annealing revealed that an extended backbone conformation largely dominates in solution for as short as 4-residues long oligomers. This new fold type is devoid of any hydrogen bond and characterized by a four-fold symmetry.

Full text of DOI:10.1039/c2cc16852a

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Constrained a/c-peptides: a new stable extended structure in solution
without any hydrogen bond and characterized by a four-fold symmetryw
a
b
a
a
a
´
Francelin Bouillere, Debby Feytens, Didier Gori, Regis Guillot, Cyrille Kouklovsky,
b
a
´
Emeric Miclet* and Valerie Alezra*
Received 4th November 2011, Accepted 12th December 2011
DOI: 10.1039/c2cc16852a
Small a/c-peptides alternating a-aminoisobutyric acid and cyclic
c-amino acid residues are described. NMR studies together with
restrained simulated annealing revealed that an extended back-
bone conformation largely dominates in solution for as short as
4-residues long oligomers. This new fold type is devoid of any
hydrogen bond and characterized by a four-fold symmetry.
sufficiently rigid units are able to adopt an extended structure
without any hydrogen bond. This extended structure shows a
four-fold symmetry, a fold that, to our knowledge, is unique.
Over the past few years we have developed an asymmetric
synthesis of cyclic or acyclic b,g-diamino acids starting from
natural a-amino acids.9 These compounds are promising sub-
strates for elaboration of a large number of original peptidic
oligomers, the second nitrogen providing a source of molecular
diversity or a hydrophilic substituent.10 Among the accessible
b,g-diamino acids, the cyclic compound 1 (Fig. 1), obtained
from L-aspartic acid, appeared to be a valuable candidate for
the preparation of extended structures because of its intrinsic
rigidity. To further increase this rigidity, and because we are
also very much interested in quaternary a-amino acids,11 we
decided to alternate this lactam 1 (LAC) and a-aminoisobutyric
acid (AIB). Using standard solid phase synthesis (on Rink
amide resin), we thus synthesized several hetero-oligomers
and we embarked on detailed structural studies in DMSO-d6
of tetramer 2, hexamer 3 and octamer 4, which were isolated as
TFA salts12 (Fig. 1).
Construction of unnatural new peptidic architectures that
mimic a-peptides with increased stability is an important
challenge for organic chemists.1 Among these peptidomimetic
compounds, many b-peptides,2 and more recently g-peptides3
or hybrid peptides4 have been proved to efficiently mimic the
various H-bond-stabilized secondary structures found in proteins
(helices, turns and linear strands). Non hydrogen-bonded
secondary structures also play important roles in proteins like
extended PPII domains which are involved in protein–protein
recognition processes.5 However, obtaining conformationally
stable foldamers is more challenging when no hydrogen bond
is present. Within the b-peptide family, Gellman et al. have
studied by circular dichroism or by restrained simulated
annealing several oligomers based on pyrrolidine-3-carboxylic
acid or nipecotic acids. Folded and unfolded conformations in
equilibrium were observed for these oligomers lacking amide
protons.6 Independently, a few groups described oligomers
incorporating constrained b-amino acids which form secondary
structures without any hydrogen bond.7 Within the g-peptide
family, only one example was reported where, according to CD
spectra, rigid bicyclic residue oligomers adopt partially ordered
structures without hydrogen bonding.8 Here we report that
hybrid a/g oligomers of four residues or longer alternating
Oligomers 2–4 are soluble in water, methanol and DMSO.
We decided to perform 1H NMR in DMSO-d6 because the
larger spectral dispersion observed in the NH region compared to
other solvents was interpreted as a probable structure stabili-
zation. Extensive NMR measurements (TOCSY, ROESY,
HSQC, CH2-TROSY,13 and HMBC) allowed complete proton
and carbon resonance assignments as well as homonuclear and
heteronuclear coupling constants determination.12 Chemical shift
dependence on concentration was monitored for compound 3.
The absence of a significant change between 0.8 mM and 20 mM
excludes aggregation. When applied to peptides, the use of two
separate ranges of temperature coefficients Dd/DT to discriminate
a
´
´
Univ Paris-Sud, Laboratoire de Chimie des Procedes et Substances
Naturelles, ICMMO, UMR 8182, CNRS, Baˆt 410, Orsay, F-91405,
France. E-mail: valerie.alezra@u-psud.fr; Fax: +33 1 69 15 46 79;
Tel: +33 1 69 15 76 50
b
´
Univ Paris 06, Laboratoire des BioMolecules,
UMR 7203 CNRS-UPMC-ENS, 4 Place Jussieu, Paris F-75005,
France. E-mail: emeric.miclet@upmc.fr; Fax: +33 1 44 27 71 50;
Tel: +33 1 44 27 31 15
w Electronic supplementary information (ESI) available: Crystallographic
data of a derivative of 1, experimental details and characterization of all
new monomeric compounds; general procedures for peptide synthesis,
1H NMR, 13C NMR, ROE constrains, coupling constant values,
temperature and concentration studies of oligomers 2, 3 and 4. CCDC
798420. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c2cc16852a
Fig. 1 Structures and nomenclature of lactam 1 used as a monomer
and hybrid a/g oligomers.
c
1982 Chem. Commun., 2012, 48, 1982–1984
This journal is The Royal Society of Chemistry 2012

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0
Table 1 –Dd/DT (ppb/K) of amide protons HN and HN in tetramer 2,
hexamer 3 and octamer 4 (DMSO-d6 between 289 K and 317 K)
Residue
Proton (comp.) LAC2 AIB3 LAC4 AIB5 LAC6 AIB7 LAC8
HN0 (2)
HN (2)
HN0 (3)
HN (3)
HN0 (4)
HN (4)
3.7
5.8
3.7
6.2
3.8
6.0
4.9
—
5.0
—
4.8
—
3.3
6.4
3.5
5.6
3.5
5.6
5.0
—
4.8
—
3.5
6.4
3.2
5.3
4.7
—
3.2
6.2
between H-bonded exchange-protected and solvent-exposed
NH is hazardous.14 However, we noticed that for 0a given amide
proton type (backbone LAC HN, cyclic LAC HN or backbone
AIB HN), temperature coefficients Dd/DT were almost identical
whatever the length of the oligomer or the position of the
amino acid in the sequence, suggesting that no hydrogen bond
was present (Table 1). NMR spectra recorded on compounds
2, 3 and 4 displayed similarities in terms of chemical shifts,
J couplings and ROE correlations. Markedly, within each
residue type, chemical shifts of backbone amide protons were
found to decrease from the N-terminus to the C-terminus
Fig. 2 The CH2-TROSY spectrum of CH2 groups in hexamer 3
(500 MHz, 20 1C). Pure doublets obtained are labelled with corres-
ponding coupling values in Hertz. In this experiment, chemical shifts
1
of methylene groups are downfield-shifted by JCH in the 13C dimen-
sion and upfield-shifted by (1JCH
+
2JHH)/2 in the 1H dimension.13
Lorentz–Gauss window functions were applied in both indirect and
direct detected dimensions with extensive zero-filling prior to Fourier
transformation to yield high digital resolution.
(dHN > dHNi+2). The same behavior was observed for the
i
0
LAC cyclic amide HN . Furthermore, chemical shift differ-
ences between diastereotopic Ha protons in LAC residues, or
b
diastereotopic CH3 methyl in AIB residues grew larger when
approaching the carboxamide terminus. These general features
3
together with observations of non-averaged backbone JHaHb
and (i) ꢀ (i + 2) ROE correlations were indicative of a
common fold for compounds 2, 3 and 4.
In g-amino acids, the number of possible conformers is
increased as a direct consequence of the addition of two
tetrahedral carbon atoms. However, it was shown that the
conformations obtained in g-peptides are inherently more
stable than those in a-peptides.15 In compound 1, the dihedral
angle about the Cb–Cg bond is restricted to about 1201 by the
cyclic lactam as observed in the crystallographic structure of
Fig. 3 Amide network observed in the HN–HN region of the ROESY
spectrum in octamer 4 (500 MHz, 20 1C).
3
a derivative of compound 1.12 Extraction of precise JHaHb
indicative of trans peptide bond conformations. ROESY
spectra were characterized by numerous cross peaks that were
converted into 47, 70 and 84 distance constraints for tetramer 2,
hexamer 3 and octamer 4, respectively. Almost half of these
correlations corresponds to inter-residue correlations (21, 33,
and 43 for tetramer 2, hexamer 3 and octamer 4, respectively),
which efficiently constrained the structures.17 In particular,
networks of amide protons were identified, involving AIB and
two adjacent residues (Fig. 3).
1
from the H homodecoupled CH2-TROSY spectra13 made it
possible to further constrain the Ca–Cb bond (Fig. 2).
Indeed, 3JHa1 was found stable along the oligomer sequences
Hb
3
with an average value of 8.2 Hz whereas JHa2 was lower at
Hb
4.2 Hz. Using proper Karplus parameters,16 rotameric popu-
lations can be estimated, provided that ideal staggered
conformations are adopted. This would lead to an excess of
the anti rotamer of B70%. Interestingly, among the three
oligomers, the first LAC2 residues were characterized by more
dynamics, since values of 7.6 Hz and 5.4 Hz were measured for
3JHa1 and 3JHa2 , respectively. In the same manner, inspec-
For each oligomer, 200 structures have been calculated
starting from extended folds.18 Complete sets of NMR data
including both homonuclear and heteronuclear vicinal coupling
constants and ROE distance restraints were used in the simulated
annealing protocol.19 Superimposition of the 10 lowest energy
structures of tetramer 2, hexamer 3 and octamer 4 revealed a
common extended fold (Fig. 4). In the three studied oligomers,
AIB1 and LAC2 experienced more dynamics than the following
residues. In contrast, C-terminal tails appeared as stable as the
central region of the oligomers. The dihedral angle observed for
non-starting AIB residues was on average f = ꢀ168 ꢁ 51 and
c = +27 ꢁ 31.20 To our knowledge, although these values
fall in a favoured region of the AIB Ramachandran map,21
Hb
Hb
d
tion of 3JHgH couplings revealed that the LAC2 Cb–Cg–C –C00
d
dihedral angles were experiencing fast exchange between the
two possible values within the cycle (ꢁ151) whereas other LAC
residues were blocked with a +151 puckering.
In the a/g oligomers, the observation of high 3JHN
(ca. 5.8 Hz) in the lactam ring was consistent with the cis
d values
C
0
conformation of the cyclic amide bond. In contrast, the
absence of correlations between all backbone HN and pre-
(i)
ceding Ca
resonances in the HMBC spectra revealed that
(iꢀ1)
the corresponding 3JHN couplings were very weak which was
Ca
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 1982–1984 1983

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R. M. Ortuno, Amino Acids, 2011, 41, 673; (c) G. V. M. Sharma,
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N. Pendem, E. Miclet, C. Didierjean, E. Ennifar and G. Guichard,
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Fig. 4 (a) Overlay of the 10 lowest energy structures of tetramer 2
(blue), hexamer 3 (red) and octamer 4 (green) using the backbone
atoms of residues 2 to 4 for superposition. (b,c) Orthogonal views of
the lowest energy structure of the octamer 4.
5 (a) A. Rath, A. R. Davidson and C. M. Deber, Biopolym. Pept.
Sci., 2005, 80, 179; (b) S. S.-C. Li, Biochem. J., 2005, 390, 641.
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7 (a) S. Abele, K. Vogtli and D. Seebach, Helv. Chim. Acta, 1999,
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no such geometry has been yet described for AIB residues
within a-peptides.
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and C. Tomasini, Org. Biomol. Chem., 2004, 2, 2181; (c) Y. Otani,
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Carroll, M. D. Shoulders and R. T. Raines, Org. Lett., 2010,
12, 5438.
Structures obtained revealed thus a new stable extended fold
characterized by a four-fold symmetry.22 It is noteworthy that
since oligomers as short as 4-residues adopt this extended
conformation, the folding process is probably governed by
local steric interactions, no hydrogen bond being required.
These features recall the polyproline II helix properties, stable
without hydrogen bonding, and showing a three-fold symmetry
for the pyrrolidine ring.23
8 F. Machetti, A. Ferrali, G. Menchi, E. G. Occhiato and
A. Guarna, Org. Lett., 2000, 2, 3987.
9 (a) C. T. Hoang, V. Alezra, R. Guillot and C. Kouklovsky, Org. Lett.,
2007, 9, 2521; (b) C. T. Hoang, F. Bouillere, S. Johannesen, A. Zulauf,
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12 See ESIw.
13 G. Guichard, A. Violette, G. Chassaing and E. Miclet, Magn.
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14 N. H. Andersen, J. W. Neidigh, S. M. Harrix, G. M. Lee, Z. Liu
and H. Tong, J. Am. Chem. Soc., 1997, 119, 8547.
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17 See ESIw, Tables S11–S13.
18 We used crystallographic data of a derivative of 1 to determine
some distances within the pyrrolidine ring and to incorporate them
into the protocol.
In conclusion, we have shown that oligomers alternating
AIB and cyclic g-amino acid 1 are able to adopt an extended
structure in solution, without any hydrogen bond, regardless of
the peptide length. Concerning the AIB residue, the dihedral f
and c angles observed in our structure are close to a local
minimum in the Ramachandran map but have not been found
in other structures. We have thus highlighted a new extended
helix showing a four-fold symmetry. To our knowledge, there is
no precedent for this type of extended structure in the b-peptide
or g-peptide families. Additional peptides with a functionalized
nitrogen lactam or without the AIB residue are under investiga-
tion as well as potential biological applications of these com-
pounds, such as inhibition of protein–protein interactions.
This research was supported by the Ministere de l’Enseignement
Superieur et de la Recherche (doctoral grant to F.B.) and by
´
ANR (Agence Nationale de la Recherche; ANR grant no
ANR-08-JCJC0099). D. F. was a research assistant of the Fund
for Scientific Research Flanders (FWO-Vlaanderen).
19 F. Delaglio, Dynamo NMR Molecular Structure Engine, version
2.1. http://spin.niddk.nih.gov/NMRPipe/dynamo/.
20 This is consistent with the following observations: for compounds
23 and 4, the AIB intraresidue ROE correlations between HN
Notes and references
b
amide protons and each CH3 methyl group were of the same
magnitude and in the medium range which is incompatible with a
1 Reviews: (a) T. A. Martinek and F. Fulop, Chem. Soc. Rev., DOI:
¨
¨
10.1039/c1cs15097a; (b) G. Guichard and I. Huc, Chem. Commun.,
single f–dihedral angle at f = +601 or at f = ꢀ601. Further-
hetero-
C
2011, 47, 5933; (c) D. Seebach, D. F. Hook and A. Glattli,
more, the relatively high value of the measured 3JH
N
¨
Biopolym. Pept. Sci., 2006, 84, 23; (d) C. M. Goodman, S. Choi,
S. Shandler and W. F. DeGrado, Nat. Chem. Biol., 2007, 3, 252.
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Chem. Rev., 2001, 101, 3219; (b) D. Seebach and J. Gardiner,
Acc. Chem. Res., 2008, 41, 1366.
nuclear couplings (3.0 ꢁ 0.1 Hz) precludes rapid exchange between
the two f = ꢁ 601 dihedral angles, both being characterized by a
very weak 3JH
C (ca. 0.5 Hz), see P. E. Hansen, Prog. Nucl. Magn.
N
Reson. Spectrosc., 1980, 14, 175.
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22 Vicinal scalar couplings were constant in the 293–321 K tempera-
ture range which indicates that the extended conformation is not
disrupted when increasing the temperature.
3 Review on g-peptides: F. Bouillere, S. Thetiot-Laurent,
´
C. Kouklovsky and V. Alezra, Amino Acids, 2011, 41, 687. Recent
examples: (a) L. Guo, W. Zhang, A. G. Reidenbach, M. W.
Giuliano, I. A. Guzei, L. C. Spencer and S. H. Gellman, Angew.
Chem., Int. Ed., 2011, 50, 5843; (b) R. Gutierrez-Abad, D. Carbajo,
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23 T. P. Creamer, Proteins, 1998, 33, 218.
c
1984 Chem. Commun., 2012, 48, 1982–1984
This journal is The Royal Society of Chemistry 2012