Original β,γ-diamino acid as an inducer of a γ-turn mimic in short peptides

Article abstract of DOI:10.1039/c2ob26828k

Original αγα tripeptides containing one β,γ-diamino acid have been synthesized and their conformation determined by extensive NMR and molecular dynamic studies. These studies revealed the presence of a C₉ hydrogen bonded turn around the β,γ-diamino acid which was stabilized by bulky side chains of the preceding residue. This turn can be considered as a mimic of the well-known γ-turn. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob26828k

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Organic &
Biomolecular
Chemistry
PAPER
Original β,γ-diamino acid as an inducer of a γ-turn
mimic in short peptides†
Cite this: Org. Biomol. Chem., 2012, 10,
9660
Sophie Thétiot-Laurent,a Francelin Bouillère,a Jean-Pierre Baltaze,a François Brisset,a
Debby Feytens,b,c Cyrille Kouklovsky,a Emeric Miclet*b and Valérie Alezra*a
Received 18th September 2012,
Accepted 22nd October 2012
Original αγα tripeptides containing one β,γ-diamino acid have been synthesized and their conformation
determined by extensive NMR and molecular dynamic studies. These studies revealed the presence of a
C9 hydrogen bonded turn around the β,γ-diamino acid which was stabilized by bulky side chains of the
preceding residue. This turn can be considered as a mimic of the well-known γ-turn.
DOI: 10.1039/c2ob26828k
www.rsc.org/obc
been described, showing the presence of C7 and C9 hydrogen
Introduction
bonded turns around the γ-amino acid.9 A C12 hydrogen
During the past two decades, the study of non-natural oligo- bond has also been observed in solution but not fully
mers that fold into a specific regular structure, that closely characterized.10
resembles the secondary structures adopted by proteins, has
For a few years we have been interested in the asymmetric
gained unprecedented attention. This research field is now synthesis of β,γ-diamino acids starting from natural α-amino
commonly referred to as foldamers.1 A foldamer has been acids11 and in their use as building blocks for γ-peptide syn-
defined by S. Gellman in 1996 as “any polymer with strong ten- thesis, the second nitrogen being a source of molecular diver-
dency to adopt a specific compact conformation”.2 A predomi- sity or a hydrophilic substituent.12 We have already shown that
nant research area in this field concerns the peptidomimetic cyclic β,γ-diamino acids could be used to build α,γ-peptides
foldamers3 that are considered as potential metabolically long- that adopt a stable helical extended conformation devoid of
lived mimetics of bioactive alpha-polypeptides.4 In this field, any hydrogen bond.13 We were then interested in the elabor-
β-peptides have been extensively studied and have shown their ation and the conformational studies of peptidic oligomers
ability to fold and to adopt a secondary structure such as containing an acyclic β,γ-diamino acid. Here we describe αγα
helices, β-sheets, turns and β-hairpins.5 In contrast to the tripeptides containing an original β,γ-diamino acid that are
intensive research on β-peptides, γ-peptides have received far able to form in solution a stable C9 hydrogen bond.
less attention.6 It has been discovered that γ-peptides form
helical secondary structures in solution, detectable by NMR
spectroscopy, with chain lengths as short as four residues.7
Recently, hybrid peptides made of alternate α and γ-amino
Results and discussion
1. Synthesis of peptides
acids, or β and γ-amino acids have emerged and have proven
to adopt also interesting conformations.8 In this field, small
peptides have been often neglected, as they are probably con-
sidered unable to adopt a defined secondary structure. Never-
theless, several crystallographic structures of di- or tripeptides,
containing one γ-amino acid and two α-amino acids, have
The αγα tripeptide sequences were based on compound 1,
which is readily obtained from L-valine.11 Compound 1 was
synthesized in 11 steps (starting from 10 g of L-valine) in 68%
overall yield.14 Peptide synthesis was then achieved under stan-
dard peptide coupling conditions with EDCI, HOBt, DIPEA in
DMF to get three different tripeptides (Scheme 1).
aLaboratoire de Chimie des Procédés et Substances Naturelles, ICMMO, UMR 8182,
Univ Paris-Sud, CNRS, Bât 410, Orsay F-91405, France.
2. Conformational studies of peptides
The conformations of the tripeptides were then explored. The
IR spectra of these peptides in solution (5 mM in CH2Cl2) were
recorded. In the NH-stretching region two absorption maxima
were observed, corresponding to H-bonded and free amide
E-mail: valerie.alezra@u-psud.fr; Fax: (+33) 1 69 15 46 79; Tel: (+33) 1 69 15 76 50
bLaboratoire des BioMolécules, UMR 7203 CNRS-UPMC-ENS, UPMC Univ Paris 06, 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
cOrganic Chemistry Department, Vrije Universiteit Brussel, Pleinlaan 2, B-1050
(for 4a at 3419 and 3340 cm−1, for 4b at 3415 and 3339 cm−1
,
Brussels, Belgium
for 4c at 3417 and 3339 cm−1). Extensive NMR studies (TOCSY,
†Electronic supplementary information (ESI)
available. See DOI:
10.1039/c2ob26828k
NOESY, HSQC, HMBC) were then undertaken as a 5 mM
9660 | Org. Biomol. Chem., 2012, 10, 9660–9663
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Fig. 1 Inter-residue NOE correlations (brown = strong, red = medium, orange =
weak) and hydrogen bonds obtained from the simulated annealing protocol
(green = C7 and blue = C9).
Scheme 1 Synthesis of tripeptides.
(from 6.2 Hz to 7.9 Hz).16 This probably reflects a restricted
flexibility for peptides 4b and 4c compared with peptide 4a.
We therefore wanted to get further insights into the stability
of the NMR structures at 300 K and undertook molecular
dynamics calculations in CHCl3 solvent boxes. Two crucial dis-
tance restraints observed in the NOESY spectra were kept
during 50 ns simulations. The results obtained were highly
dependent on the tripeptide.16 For peptide 4a, the C9 turn was
never observed at 300 K, the C7 turn being present 40% of the
total time.17 In the corresponding conformation, the valine
NH was close to the Cbz carbonyl group (2.20 Å), which could
explain both its high δNH value and the small ΔδNH measured
during the titrations. For peptide 4b, the C9 turn was only
rarely observed in contrast with the C7 turn which largely
dominated (88% of the total time). Actually, the careful exam-
ination of the trajectories predicted for both peptides 4a and
4b indicated that the β,γ residue rapidly exchanged between
several conformational states, which are, on average, consist-
ent with the NOE correlations.18 In contrast, the C9 turn was
present 97% of the time for peptide 4c, sometimes along with
a C7 turn (24% of total time), as observed in 4a and 4b. This
stable C9 turn was in very good agreement with the long-range
NOE correlations observed for this peptide. As displayed on
the dynamics trajectories (Fig. 3),16 the conformational space
appeared much more restricted for this peptide compared
with peptide 4a and, to a lesser extent, with peptide 4b. There-
fore in peptide 4c, the first Phe residue provides sufficient
steric hindrance to induce a stable C9 hydrogen bonded
turn.19 As a γ-turn is defined by the existence of a hydrogen
bond between the CO group of the ith residue and the NH
group of the (i + 2)th residue, this C9 hydrogen bonded turn
can be viewed as a mimic of a γ-turn with a homologated
residue.
solution in CDCl3 for all tripeptides and allowed complete
proton and carbon resonance assignments. The 1H NMR
spectra showed downfield chemical shifts for the valine NH
with δ > 7 ppm (7.60 for 4a, 7.78 for 4b and 7.46 for 4c),
suggesting possible involvement in hydrogen bonds. Moreover
solvent titration studies confirmed this hypothesis since low
Δδ values (<0.20 ppm) were observed for the valine NH in the
three tripeptides.15 NOESY spectra were characterized by
numerous cross peaks, which have been classified into weak,
medium and strong correlations and accordingly converted
into distance restraints. Among the inter-residues correlations,
some are supporting a close proximity between the N- and the
C-termini (Fig. 1). For instance, several NOE correlations imply
the valine NH and either some protons of the first amino acid
or the remote protons of the β,γ-diamino acid.
For each tripeptide, 200 structures have been calculated
starting from extended folds. Vicinal coupling constants and
NOE distance restraints were used in the simulated annealing
protocol. Superimposition of the 10 lowest energy structures
showed the presence of hydrogen bonds, forming a C9 turn
around the β,γ-diamino acid in the three peptides and also a
standard C7 turn around the first α-amino acid in 4a and 4b
(Fig. 1 and 2). These results are in accordance with the
observed NOE correlations. It is noteworthy that although they
all present the same C9 turn, these structures are slightly
different regarding the backbone spatial arrangement. This
means that the starting α-amino acid should play a significant
role in the conformational space explored by the β,γ-diamino
acid within the three peptides. Interestingly, the NH–Hα
vicinal coupling constant of the α-amino acid residues
increased with the steric hindrance of the first amino acid
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Fig. 3 Trajectories obtained for peptide 4c in the time course of the MD simu-
lation. NH⋯OvC distances revealing the formation of (a) a 7-membered ring
hydrogen bond or (b) a 9-membered ring hydrogen bond.
Fig. 2 Overlay of the 10 lowest energy structures of compounds 4a, 4b and 4c
(hydrogen-bonds are shown in dashed lines; for clarity, only the backbone is
shown).
3. Macroscopic properties
Several attempts have been made in order to get crystals of the
tripeptides. Although this was unsuccessful, slow evaporation
of a solution of compound 4a in a mixture AcOEt–petroleum
ether (1 : 1) led to a white fibrous solid. Only one absorption
maximum at 3321 cm−1 in the NH-stretching region was
observed on the IR spectrum of this solid. This is consistent
with H-bonded amides. In the carbonyl stretching region three
absorption maxima were distinguished at 1739, 1690 and
1650 cm−1, confirming therefore the presence of hydrogen
bonds.20
A small piece of this solid was then observed by SEM
(Fig. 4). Thin strands of 130 nm to 700 nm width and of
several hundreds of microns length were visible. These
strands are probably formed by self-assembly of peptide 4a,
stabilized by a network of hydrogen bonds as shown by the
IR spectrum.
Fig. 4 SEM image of solid thin strands 4a.
Moreover, compound 4c showed also interesting supra-
molecular properties. A solution of compound 4c (15 mM in
CHCl3 or CH2Cl2) forms rapidly a gel, that is probably signifi-
cant of a self-assembly of peptide arising from specific inter-
action between molecules. These macroscopic properties have
to be explored further to get real insight into the spatial
arrangement of molecules.
9662 | Org. Biomol. Chem., 2012, 10, 9660–9663
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8 Reviews: (a) A. Roy, P. Prabhakaran, P. Kumar Baruah and
G. J. Sanjayan, Chem. Commun., 2011, 47, 11593;
(b) W. S. Horne and S. H. Gellman, Acc. Chem. Res., 2008,
Conclusions
We have shown that αγα peptide containing only three resi-
dues, including a β,γ-diamino acid, is able to adopt a defined
stable conformation, as long as the first α-amino acid pos-
sesses the ideal steric hindrance. The propensity of this
β,γ-diamino acid to form a C9 hydrogen bond could be seen as
an opportunity to have a superior homologue of the γ-turn of
α-amino acids or as a good chance to get either a C9 ribbon or
a 9-helix by the repetition of this residue,21 as this latter has
been described as very stable by theoretical calculations.22
Further studies in this field are currently under investigation
in our laboratory.
41, 1399; Recent examples:
(c) L. Guo, W. Zhang,
I. A. Guzei, L. C. Spencer and S. H. Gellman, Tetrahedron,
2012, 68, 4413; (d) L. Guo, W. Zhang, I. A. Guzei,
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(e) B. Dinesh, K. Basuroy, N. Shamal and P. Balaram, Tetra-
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9 (a) S. Aravinda, K. Ananda, N. Shamala and P. Balaram,
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Acknowledgements
This research was supported by the M.E.S.R. (a doctoral grant
to F. B.) and by ANR (ANR grant no. ANR-08-JCJC0099). D. F. 10 G. V. M. Sharma, V. B. Jadhav, K. V. S. Ramakrishna,
was a research assistant at the Fund for Scientific Research
Flanders (FWO-Vlaanderen).
P. Jayaprakash, K. Narsimulu, V. Subash and A. C. Kunwar,
J. Am. Chem. Soc., 2006, 128, 14657.
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Org. Lett., 2007, 9, 2521; (b) C. T. Hoang, F. Bouillère,
S. Johannesen, A. Zulauf, C. Panel, D. Gori, A. Pouilhès,
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Notes and references
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almost no intermediate purification was actually necessary
(see ESI† for more details).
4 C. Rosés, D. Carbajo, G. Sanclimens, J. Farrera-Sinfreu, 15 Solvent titration studies were carried out by sequential
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additions of DMSO-d6 (up to 140 μL) to a 5 mM solution of
peptide in CDCl3 (500 μL). In compound 4b, both NH
groups of the β,γ-diamino acid showed also Δδ < 0.20 ppm.
5 Reviews: (a) D. Seebach and J. Gardiner, Acc. Chem. Res., 16 See ESI† for further information.
2008, 41, 1366; (b) L. K. A. Pilsl and O. Reiser, Amino Acids, 17 Two other conformations were also populated in the MD
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This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 9660–9663 | 9663