The Combination of Prolinoamino Acids and Cyclopropylamino Acids Leads to Fully Functionalized, Stable β-Turns in Water

Article abstract of DOI:10.1002/cbic.201000707

Tight turns: Peptide mimics based on combinations of cis-3-prolinoamino acids (prolinohomotryptophan) and (Z)-2,3-methanoamino acids (c₃Arg or c₃Lys) form stable β-turns in water. This combination of constraints allows one to mimic t

Full text of DOI:10.1002/cbic.201000707

DOI: 10.1002/cbic.201000707
The Combination of Prolinoamino Acids and Cyclopropylamino Acids Leads
to Fully Functionalized, Stable b-Turns in Water
Karine Guitot,[a, b, c] Maud Larregola,[a, b, c] Tarun K. Pradhan,[d] Jean-Luc Vasse,[d] Solange Lavielle,[a, b, c]
Philippe Bertus,[e] Jan Szymoniak,[d] Olivier Lequin,*[a, b, c] and Philippe Karoyan*[a, b, c]
The conformational freedom of peptides is often considered a
major drawback in their use as suitable therapeutic agents.[1]
Indeed, flexibility both of backbone and of amino acid side
chains is associated with low selectivity, together with poor
metabolic stability. However, this flexibility is also responsible
for the high affinities of peptides for their biological targets,
allowing them to interact in their ideal conformations upon
binding.
structure.[9,11] We have recently validated the use of cis-3-sub-
stituted prolinoamino acids (denoted Pc3Xaa) in Piv-d-Pc3Xaa-l-
NMeYaa-NHMe sequences to stabilize short b-turns (in water)
that retain the side-chain functionalities in both the i+1 and
the i+2 positions of the turn.[9d] These short peptides incorpo-
rate three motifs—a heterochiral sequence, a proline scaffold,
and a N-methyl group—that are known to promote strong b-
turn propensity.[12] The prolinoamino acid allows the canonical
staggered rotamers of the side chains in the i+1 position to
be mimicked with minimal deviation. In contrast, the N-methyl-
ation restricts the conformational space of the side chain in
the i+2 position to gauche- (c1ꢀÀ608) and trans (c1ꢀ1808)
orientations around the c1 angle, the gauche+ rotamer (c1 ꢀ
+608) being destabilized by unfavorable interaction with the
N-methyl group. Here we have studied the replacement of N-
methylamino acids by cyclopropyl amino acids (or 2,3-metha-
noamino acids) in order to characterize the three-dimensional
space available for the side chains of these constrained amino
acids when combined with proline chimeras and to analyze
the influence of side chain interactions on b-turn folding. Piv-
d-Pc3Xaa-d-c3Yaa-NHMe and Piv-d-Pc3Xaa-l-c3Yaa-NHMe se-
quences incorporating the side chains of aromatic hTrp and
cationic Lys or Arg amino acids were prepared (Scheme 1).
The restriction of peptide conformational space is a major
approach to improving both receptor selectivity and metabolic
stability.[2] However, achievement of high binding affinities with
this strategy requires a range of tools allowing fine-tuning in
the control of the three-dimensional space of the peptide
backbone while keeping all the crucial information contributed
by amino acids’ side chains, in terms of functional groups and
spatial orientation.
To this end, for the past few years we have been exploring
synthetic methodologies that are easy to scale up, in order to
prepare natural amino acid surrogates such as proline chime-
ras[3] and cyclopropyl[4] and b-amino acid derivatives.[5] These
tools have known applications for pharmaceutical use,[6] for
probing bioactive conformations of peptides,[7] and for stabiliz-
ing peptide secondary structure elements such as PPII helix[8]
or b-turns.[1,9]
The b-turn motif, a recognition element often involved in re-
ceptor–ligand interactions,[10] is a major subject of investigation
in the development of synthetic mimics of peptide secondary
[a] Dr. K. Guitot, Dr. M. Larregola, Prof. S. Lavielle, Prof. O. Lequin,
Prof. P. Karoyan
UPMC Universitꢀ Paris 06, Laboratoire des BioMolꢀcules
UMR 7203 and FR2769, 4
Place Jussieu, 75005 Paris (France)
Fax: (+33)1-44322402
E-mail: olivier.lequin@upmc.fr
philippe.karoyan@upmc.fr
[b] Dr. K. Guitot, Dr. M. Larregola, Prof. S. Lavielle, Prof. O. Lequin,
Prof. P. Karoyan
Ecole Normale Superieure, Dꢀpartement de Chimie
24, rue Lhomond, 75005 Paris (France)
[c] Dr. K. Guitot, Dr. M. Larregola, Prof. S. Lavielle, Prof. O. Lequin,
Prof. P. Karoyan
UMR 7203 CNRS-UPMC-ENS
[d] Dr. T. K. Pradhan, Dr. J.-L. Vasse, Prof. J. Szymoniak
Universitꢀ de Reims Champagne-Ardenne, UMR-CNRS 6229
Institut de Chimie Molꢀculaire de Reims
BP 1039, 51687, Reims Cedex 02 (France)
[e] Prof. P. Bertus
Universitꢀ du Maine, UMR-CNRS 6011
Scheme 1. Chemical structures of the pseudotetrapeptides Piv-d-Pc3Xaa-d-
c3Yaa-NHMe (1a, 1b) and Piv-d-Pc3Xaa-l-c3Yaa-NHMe (2a, 2b): Piv-d-Pc3hTrp-
(2R,3S)-c3Lys-NHMe (1a), Piv-d-Pc3hTrp-(2R,3S)-c3Arg-NHMe (1b), Piv-d-
Pc3hTrp-(2S,3R)-c3Lys-NHMe (2a), and Piv-d-Pc3hTrp-(2S,3R)-c3Arg-NHMe (2b).
Unitꢀ de Chimie Organique Molꢀculaire et Macromolꢀculaire
Avenue Olivier Messiaen, 72085 Le Mans Cedex 9 (France)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201000707.
ChemBioChem 2011, 12, 1039 – 1042
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1039

These pseudotetrapeptides correspond to turn sequences
found in somatostatin[6] and tendamistat,[13] respectively.
stant analysis with the Ha proton, showing that the cis cou-
pling (~9.5 Hz) was larger than the trans coupling (~7.5 Hz), as
typically observed in three-membered rings. Interestingly, the
analysis of NOE correlations in each pair of diastereoisomers
showed different patterns for the NOE correlations involving
the methylenic Hb’ proton of the cyclopropyl ring in a cis rela-
tionship with the amide proton. In particular, for each 1a/2a
and 1b/2b diastereomeric pair, one isomer showed a strong
NOE correlation between the cyclopropyl Hb’ and HN protons,
whereas in the other diastereoisomer the Hb’ proton exhibited
medium-range NOE correlations with the cyclopropyl amide
proton and the methylenic protons of the hTrp side chain
(Figure 1). Structure calculations were carried out with a set of
The N-methyl carboxamide derivatives 3a and 3b were pre-
pared as racemates from the N-Boc-(Z)-2,3-methanoamino acid
analogues of lysine and arginine[4a] (see the Supporting Infor-
mation). The Piv-d-Pc3hTrp-c3Xaa-NHMe compounds 1a, 1b,
2a, and 2b were obtained by solution-phase peptide synthesis
as shown in Scheme 2. Couplings of 3a and 3b with the enan-
Figure 1. Characteristic NOE correlations used for the determination of con-
figurations in the diastereoisomers 1a/1b and 2a/2b. The corresponding in-
terproton distances are indicated in ꢁ (for the distance involving the methyl-
enic protons of hTrp, the minimum distance is indicated).
Scheme 2. Syntheses of the pseudotetrapeptides 1a, 1b, 2a, and 2b. Reac-
tion conditions: a) HCTU, iPr2NEt in CH2Cl2; b) 1-chloro-N,N,2-trimethylprope-
nylamine, then iPr2NEt in CH2Cl2; c) TFA; d) PivCl, Et3N; e) piperidine/CH2Cl2
(1:5);f) Na, NH3 in THF. R=(CH2)3-NH-Fmoc (3a, 5a, 6a) or (CH2)2-NH-C(NTs)-
NH2 (3b, 5b, 6b).
distance restraints involving backbone and pyrrolidine protons
only (Figure S2). In these structures, the two sets of NOEs
could only be satisfied in the (2R,3S) and (2S,3R) configurations,
respectively.
The conformations of these four pseudotetrapeptides were
investigated by NMR spectroscopy both in methanol and in
aqueous solution (Scheme 3). The peptide bonds between pro-
linoamino acid and methanoamino acids were found to be
trans, with no sign of cis/trans isomerism. The NOESY or ROESY
spectra of the four peptides in
tiomerically pure N-protected prolinohomotryptophan deriva-
tive 4[3d] were performed either with HCTU as coupling agent
(method a) or after conversion of the carboxylic function into
an acyl chloride (method b),[14] leading to 5a and 5b, respec-
tively, each as a mixture of diastereoisomers. After removal of
the Boc protecting group with TFA, the pivaloyl group was
introduced on the nitrogen of the pyrrolidine ring. Finally, re-
moval of side chain protecting groups followed by HPLC sepa-
ration of the diastereoisomers led to the enantiomerically pure
pseudotetrapeptides 1a, 1b, 2a, and 2b.
methanol revealed dipolar correla-
tions characteristic of the b-turn
structure, in particular between
the pivaloyl group methyl protons
and the C-terminal NHMe group
amide and methyl protons. The b-
The absolute configurations of the a and b carbons in the
(Z)-2,3-methanoamino acid residues could be determined by
combining NMR analyses and computational studies of 1a and
2a and of 1b and 2b. Firstly, the stereochemical relationships
between protons in the pyrrolidine and cyclopropyl rings were
deduced from vicinal coupling constants and intraresidual
NOEs. Comparison of the NOE intensities of the amide proton
and of the methylenic Hb’ protons of the cyclopropyl ring in
each case showed that the more strongly upfield-shifted
Hb’ proton has a cis relationship with respect to the amide
proton. This was also confirmed by the vicinal coupling con-
turn folding is also supported by
NOE correlations between the
Ha protons of the prolinoamino
acid residues and the amide pro-
tons of the NHMe groups
Scheme 3. Characteristic NOE
correlations observed for the
(Scheme 3). In addition, these
amide protons exhibit small tem- pseudotetrapeptides 1a, 1b,
2a, and 2b in methanol (0.46
to 2.4 mm, 15 to 308C) or H2O
(0.46 to 2.4 mm, 158C). The
selected NOEs were observed
perature dependences in their
chemical shifts (DdNH/DT between
À4 and À3 ppb8CÀ1), in contrast
with the amide protons of cyclo- both in methanol and in H2O.
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Figure 2. NMR structures of the four pseudotetrapeptides 1a, 1b, 2a, and 2b obtained from NMR spectroscopy data collected in methanol.
propylamino acids (DdNH/DT between À8 and À7 ppb8CÀ1), in-
dicating that they are involved in hydrogen bonds. The back-
bone sequential Ha–HN and HN–HN NOEs are compatible with
(f, y) values found in type II’ b-turn conformations.[15]
of diastereotopic methylenic protons of the cationic side chain,
which can be ascribed to p–cation interactions.
To investigate whether the interactions between the indole
group and the c3Lys/c3Arg side chains play a major role in b-
turn stabilization, we synthesized the peptide Piv-l-Pro-Acc-
NHMe (10, Acc standing for 1-aminocyclopropanecarboxylic
acid), lacking both side chains (Scheme S1), and examined its
conformation. The NMR parameters indicate that the pep-
tide 10 adopts a type II b-turn conformation in methanol solu-
tion (Figure S1), as already observed in chlorinated solvents[1]
and in the crystal state for the related peptide Boc-l-Pro-Acc-
Gly-NH2.[18] Other analogues bearing a single side chain (phenyl
or alkyl) on the cyclopropyl moiety were shown to adopt b-
turns.[1,19] It can be concluded that the proline-cyclopropyl
motif, per se, promotes strong b-turn stabilization.
The peptides 1a and 2a and the peptides 1b and 2b were
found to have similar b-turn propensities, indicating little
effect of the inversion of configurations in the (Z)-2,3-metha-
noamino acids. Noticeably, the NMR spectroscopy data ob-
tained in water for the four peptides 1a, 1b, 2a, and 2b were
very similar to those observed in methanol and indicate the
strong stability of the b-turn conformation in aqueous solution.
In particular, the four pseudotetrapeptides showed NOEs char-
acteristic of b-turns (Scheme 3). The low temperature coeffi-
cients of the NHMe amide protons also support their involve-
ment in hydrogen bonds. The temperature coefficients of the
cyclopropyl amide protons were surprisingly low in peptides
2a and 2b (+0.2 and +0.6 ppb8CÀ1) in relation to peptides 1a
and 1b (À8.5 and À8.0 ppb8CÀ1). Atypical temperature coeffi-
cients have already been described for peptides in aqueous
solution.[16] This could be due either to sequestration by the
aromatic group of hTrp[17] or to the existence of temperature-
dependent conformational equilibria involving the side chains.
The strong temperature dependency of the chemical shifts of
several side chain protons supports the latter hypothesis.
The NMR analysis also provides information about side chain
conformations and interactions. The pyrrolidine rings of the
cis-3-prolinoamino acid residues in the four peptides each
adopt a Cb-exo puckering, as shown by vicinal coupling con-
stant analysis and NOE correlations between the Hb and HdS
protons. This puckering preference restricts the c1 torsion
angle of the hTrp side chain to the trans conformer. The 3J cou-
pling constant values and the observed NOEs indicate that the
CH2-indole substituents do not adopt unique conformations
around their c2 and c3 torsion angles. Two rotamers around
the c2 torsion angle are populated, corresponding to gauche
and trans conformations. For the i+2 position, the populations
of the different conformers of the cyclopropylamino acid side
chain could not be determined because of the lack of coupling
constants available. Several medium-intensity NOEs are ob-
served between the side chain protons (Hg in particular) of the
cyclopropylamino acid side chain and the methylenic or aro-
matic protons of the hTrp side chain, indicating the presence
of conformers with close proximity of these side chains. This is
further confirmed by the strong chemical shift inequivalence
The three-dimensional structures of the peptides 1a, 1b, 2a,
and 2b (Figure 2) were calculated by restrained molecular dy-
namics and energy minimization with the DISCOVER program
using interproton distance restraints derived from NOEs and
dihedral angle restraints derived from homonuclear vicinal
coupling constants measured in methanol. All low-energy
structures have a single backbone conformation with (f, y)
values of around (608, À1258) and (À758, 08) for the prolinoa-
mino acid and the cyclopropanylamino acid residues, respec-
tively, corresponding to the canonical (f, y) values observed in
type II’ b-turns.[15]
For all isomers, the observed NOEs could not be satisfied by
single conformations of hTrp and Arg or Lys side chains, con-
firming the flexibility of the side chains in both the i+1 and
the i+2 positions. The conformational restriction induced by
the pyrrolidine ring enables partial control of the side chain
conformational space. In the i+2 position, the presence of cy-
clopropyl amino acids with different a and b carbon absolute
configurations allows different side chain orientations to be ex-
plored (Figure 2).
Although the c1 torsion angle of cyclopropylamino acid sig-
nificantly deviates from those of ideal amino acid rotamers be-
cause of its nearly eclipsed value, comparison of the calculated
NMR structures with somatostatin and tendamistat pharmaco-
phores shows good overall superimpositions of some low-
energy structures of the Piv-d-Pc3hTrp-l-c3Yaa-NHMe peptides
2a and 2b (Figure 3). Importantly, the introduction of a (Z)-2,3-
methanoarginine unit in the i+2 position (2b) enables the ob-
tention of a rotamer suitable for mimicking the tendamistat
ChemBioChem 2011, 12, 1039 – 1042
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Figure 3. Superimposition of selected conformers of the pseudotetrapeptides 2a and 2b with b-turn motifs of somatostatin and tendamistat. A), B) Two dif-
ferent views showing the superimposition of 2a and the d-Trp-Lys b-turn of a somatostatin analogue (octreotide, PDB ID: 1SOC). C), D) Two different views
showing the superimposition of 2b with the Trp-Arg b-turn of tendamistat (PDB ID: 1BVN). The carbon atoms of octreotide and tendamistat are colored in
cyan whereas those of peptides 2a and 2b are colored in green. Nitrogen and oxygen atoms are shown in blue and red, respectively. The structures were
superimposed by best fitting of backbone atoms. The rms deviations of backbone N, Ca, and C’ atoms are 0.16 and 0.46 ꢁ for 2a and 2b, respectively. The
rms deviations for all heavy atoms are 1.08 and 1.14 ꢁ for 2a and 2b, respectively.
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Keywords: beta-turns
spectroscopy · peptidomimetics · prolinoamino acid
·
cyclopropylamino acids
·
NMR
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Received: November 23, 2010
Published online on March 29, 2011
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