Prolinoamino acids as tools to build bifunctionalized, stable β-turns in water

Article abstract of DOI:10.1002/cbic.200900572

(Figure Presented) β-Turn it on: Peptides incorporating cis-3-prolinoamino acids (prolinoleucine or prolinohomotryptophane) and N-methylamino acids (NMePhe/Arg/Lys) have been synthesized to mimic stable β-turns in water (see figure). These 3-substituted p

Full text of DOI:10.1002/cbic.200900572

DOI: 10.1002/cbic.200900572
Prolinoamino Acids as Tools to Build Bifunctionalized, Stable b-Turns in
Water
Cꢀline Mothes,[a, b] Maud Larregola,[a] Jean Quancard,[a] Nicole Goasdouꢀ,[a] Solange Lavielle,[a]
Gꢀrard Chassaing,[a] Olivier Lequin,*[a] and Philippe Karoyan*[a]
The design of peptidomimetics able to mimic the structural
and binding motifs of proteins is a major challenge in the de-
velopment of new pharmaceutical tools. The b-turn motif is
one of the major secondary structure elements that plays an
important role in the folding of globular proteins and is often
implicated as a recognition element in receptor–ligand inter-
actions.[1] Considerable effort has been devoted to the devel-
opment of synthetic templates as reverse turn mimics.[2] One
strategy consists in restricting the conformational space of the
central positions by means of bi- or tricyclic rings. A second
approach is based on stabilizing the N- and C-terminal residues
by covalent bridging or folding through noncovalent cation–p
interactions.[3]
Leu in the i+1 position adopts a stable b-turn structure in or-
ganic solvents and in water.[7]
In this study, we have validated the use of prolinoamino
acids in Piv-d-Pc3Xaa-l-NMeYaa-NHMe sequences (Scheme 1) to
stabilize short b-turns, in water, that possess the two side
Scheme 1. Chemical structures of pseudotetrapeptides Piv-d-Pc3Xaa-l-
NMeYaa-NHMe (4a–c): Piv-d-Pc3Leu-l-NMePhe-NHMe (4a), Piv-d-Pc3HTrp-l-
NMeArg-NHMe (4b) and Piv-d-Pc3HTrp-l-NMeLys-NHMe (4c).
Another strategy to stabilize b-turn conformations is the in-
corporation of proline residues that are known to have a high
b-turn propensity.[4] Heterochiral d-Pro-l-Pro or l-Pro-d-Pro se-
quences adopt types II’ or II b-turn conformations, respective-
ly.[5] The replacement of one proline residue by an N-methyl
amino acid (d-Pro-l-NMeXaa or l-Pro-d-NMeXaa sequences)
further stabilizes the b-turn conformation and enables the in-
troduction of a side chain functionality in the i+2 position of
the b-turn.[6] We have explored the possibility of recovering
the side chain functionality in the i+1 position using cis-3-sub-
stituted prolinoamino acids (Pc3Xaa).[7] These prolinoamino
acids, which are easily accessible by chemical synthesis[8] when
not commercially available, can be considered as chimeras be-
tween proline and a proteinogenic amino acid. The conforma-
tional constraint of the pyrrolidine template limits the confor-
mational space around the f and c1 torsion angles. Prolino-
amino acids can have applications as conformational tools for
probing the bioactive conformations of peptides and for phar-
maceutical use.[9,10] We have previously introduced a cis-3-proli-
noleucine (Pc3Leu) in the Piv-d-Pc3Leu-l-Pro-NHMe sequence
and shown that this short peptide bearing the side chain of
chain functionalities in the i+1 and i+2 positions. We have ex-
amined different types of side chains able to mediate van der
Waals (Leu/Phe, peptide 4a) or cation–p interactions (HTrp/Arg
and HTrp/Lys, peptides 4b and 4c) to investigate the influence
of side chains on b-turn folding and characterize the conforma-
tional space explored by side chains. The sequences of pep-
tides 4b and 4c correspond to turn sequences found in tenda-
mistat[11] and somatostatin,[12] respectively.
The Piv-d-Pc3Xaa-l-NMeYaa-NHMe compounds 4a–c were
obtained by solution phase peptide synthesis (Scheme 2). After
conversion of N-methylamino acids into N-methylcarboxamide
derivatives, couplings of N-protected prolinoamino acids[8]
were realized by using PyBrop or PyAOP/HOAt as coupling
agents with satisfactory yields. After removal of the protecting
group P with TFA (P=Boc) or piperidine (P=Fmoc), the pivalo-
yl group was introduced on the nitrogen of the pyrrolidine
ring. Finally, removal of P1 and P2 side chain protecting groups
when required led to pseudotetrapetides 4a–c.
The conformation of peptides 4a–c was first analyzed by CD
spectroscopy in both methanol and aqueous solutions
(Figure 1). The three peptides show similar CD patterns, with a
broad minimum at around 210–223 nm. This signature was
previously observed for Piv-d-Pc3Leu-l-Pro-NHMe peptide and
other peptide sequences and was assigned to a type II’ b-turn
structure.[7] Interestingly, the CD signature is observed in both
methanol and aqueous solutions; this indicates that the b-turn
conformational propensity of peptides 4a–c is retained in
water. The variation of the minimum molar ellipticity in the
[a] C. Mothes,+ M. Larregola,+ Dr. J. Quancard, Dr. N. Goasdouꢀ,
Prof. S. Lavielle, Dr. G. Chassaing, Prof. O. Lequin, Prof. P. Karoyan
UPMC Universitꢀ de Paris 06
UMR 7203 CNRS-UPMC-ENS and FR2769
Laboratoire des Biomolꢀcules, UPMC
4 Place Jussieu, 75252, Paris Cedex 05 (France)
Fax: (+33)1-44273843
E-mail: olivier.lequin@upmc.fr
philippe.karoyan@upmc.fr
[b] C. Mothes+
Genzyme Pharmaceuticals
4410 Listal (Switzerland)
[+] These authors contributed equally to this work.
210–223 nm region (from À33000 to À16000 degcm2 molÀ1
)
between peptides 4a and 4b/4c might reflect different stabili-
zation of b-turn conformers or different contributions of the
phenyl or indole side chain chromophores to the CD signal.[13]
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.200900572.
ChemBioChem 2010, 11, 55 – 58
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
55

The NMR analysis also pro-
vides information about side
chain conformations and interac-
tions. The pyrrolidine ring of the
prolinoamino acid residue in the
three peptides 4a–c adopts a
Cb-exo puckering, as shown by a
strong NOE correlation between
Hb and HdS protons together
with the 3J coupling constant
analysis. This puckering prefer-
ence restricts the c1 torsion
Scheme 2. Syntheses of pseudotetrapeptides 4a–c. Reaction conditions: a) PyBroP, DIEA (3a); b) PyAOP, HOAt,
DIEA (3b, 3c); c) TFA/CH2Cl2 (1:1; 4a); d) piperidine/CH2Cl2 (1:4; 4b, 4c); e) TFA/thioanisole/water (95:2.5:2.5; 4b);
f) TFA/TIS/water (95:2.5:2.5; 4c); P=Boc (1a, 3a) or Fmoc (1b, 1c, 3b, 3c); P1 =Boc (1b, 1c, 3b, 3c); P2 =Mtr
(2b, 3b) or Boc (2c, 3c).
To address this point, the conformation of peptides 4a–c
was further investigated by NMR spectroscopy. Peptides 4b
and 4c were examined in both methanol and aqueous sol-
vents while peptide 4a was studied in methanol only, owing
to its low solubility at mm concentrations in water.
The NOESY or ROESY spectra of the three peptides 4a–c in
methanol revealed NOE correlations characteristic of a b-turn
structure, in particular between the methyl protons of Piv N-
terminal residue and the amide and methyl protons of the C-
terminal NHMe group (Scheme 3). The observed NOE between
the Ha proton of the prolinoamino acid residue and the amide
proton of the NHMe group also supports b-turn folding. In ad-
dition, the amide proton exhibits a small temperature depend-
ence of its chemical shift (DdNH/DT of ca. À3 ppb8CÀ1); this in-
dicates that it is involved in a hydrogen bond. The backbone
sequential NOEs are compatible with (f, y) values found in a
type II’ b-turn conformation. The peptide bond between proli-
noamino acid and N-methylated residues was found to be
trans, with no sign of cis/trans isomerism. Importantly, the
NMR spectroscopy data obtained in water for peptides 4b and
4c were very similar to those in methanol and strongly sup-
port b-turn formation in water, as already suggested by CD
data.
Scheme 3. Characteristic NOE correlations observed for peptides 4a–c in
methanol (2 mm, 258C) or H2O (1 mm, 208C). The selected NOE correlations
were observed in both methanol and H2O solvents for peptides 4b and 4c,
except for peptide 4a (studied in methanol only).
angle of the Leu or HTrp side chains to the trans conformer.
The c2 angle of the Leu side chain in Pc3Leu (peptide 4a) is
3
also trans, as shown by the large value of the JHbÀHg coupling
constant (9.6 Hz). In the case of Pc3HTrp residue in peptides 4b
3
and 4c, the J coupling constant values and the observed NOE
values suggest that the CH2-Ind substituent does not adopt a
unique conformation around its c2 and c3 torsion angles. Re-
Figure 1. CD spectra of peptides 4a–c (20–50 mm, 208C) in: A) methanol, and B) H2O.
56
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ChemBioChem 2010, 11, 55 – 58

garding the i+2 position, the side chain of NMePhe residue in
peptide 4a adopts the gaucheÀ rotamer around its c1 angle,
al constraint driving type II’ b-turn formation. In addition to
type II’ b-turns, type II b-turn peptides could be easily obtained
by inverting the Ca configurations of both amino acids.
The incorporation of prolinoamino acid and N-methylamino
acid enables the introduction of two side chain functionalities
in positions i+1 and i+2 of the b-turn. The pyrrolidine scaffold
leads to minimal deviation from ideal staggered rotamers of
side chains and enables side chain contacts through van der
Waals or p–cation interactions. The c1 torsion angle of proli-
noamino acid side chain is dependent on the puckering of pyr-
rolidine ring. In cis-prolinoamino
3
as shown by JHaÀHb coupling constant analysis. In the case of
peptides 4b and 4c, the same analysis reveals a conformation-
al equilibrium around the c1 angle of NMeArg and NMeLys res-
idues between gaucheÀ and trans rotamers with respective
populations of ~3:1.
The 3D structures of peptides 4a–c (Figure 2) were calculat-
ed by restrained molecular dynamics and energy minimization
with the DISCOVER program by using interproton distance re-
acids, the Cb-exo ring pucker pre-
dominates and causes a unique
c1 trans orientation of the sub-
stituent (c1 ~À1558). However,
other side-chain orientations
might be accessible by using
trans-prolinoamino
acids.
Indeed, we have shown previ-
ously that the energy difference
was smaller between Cb-exo and
Cb-endo ring puckers in the cor-
responding trans-prolinoamino
acids, so that both trans (c1 ~
1558) and gauche+ (c1 ~908)
conformations around the c1
angle might be populated.[9] The
side-chain orientations of the N-
methylamino acid in position
Figure 2. NMR structures of peptides 4a–c obtained from NMR spectroscopy data collected in methanol solvent.
The low energy minima structures corresponding to different side chain conformers were superimposed by best
fitting of the backbone heavy atoms.
straints derived from NOEs and dihedral angle restraints de-
rived from homonuclear vicinal coupling constants measured
in methanol solvent. A unique conformation of the backbone
was found in the three peptides with (f, y) values around
(608, À1258) and (À1258, 558) for prolinoamino acid and N-
methylamino acid residues, respectively. The Leu and Phe side
chains in peptide 4a adopt unique orientations and interact
with each other through van der Waals interactions, in agree-
ment with several NOE values observed between the aliphatic
and aromatic resonances. In peptides 4b and 4c, the observed
NOEs cannot be satisfied by a single conformation of HTrp and
Arg or Lys side chains. Cation–p interactions are observed in
different conformers of the NMR structure family and are ex-
perimentally confirmed by the up-field shifts of Arg and Lys
side chain protons together with weak NOE correlations ob-
served between indolic protons of Pc3HTrp and side chain pro-
tons of the cationic residues.
i+2 are restricted to gaucheÀ and trans orientations around
the c1 angle, the gauche+ rotamer being destabilized by un-
favorable interaction with the N-methyl group. The structures
of peptides 4b and 4c were compared to bioactive conforma-
tions that are found in tendamistat and somatostatin, respec-
tively. For the NMR structure family of peptide 4c, several low
energy conformers fit well with the conformation of a soma-
tostatin analogue (Figure S5 in the Supporting Information). In
the case of peptide 4b, the side-chain positions deviate more
significantly from those in tendamistat because the gauche+
conformer of the Arg residue in position i+2 of the tendami-
stat b-turn was not observed in the NMR structures of 4b.
In conclusion, prolinoamino acids represent valuable pepti-
domimetic tools that can be directly incorporated in peptide
sequences and can mimic most side chains of proteinogenic
amino acids. They can be used to stabilize b-turns in water
that retain the side-chain functionalities on both i+1 and i+2
positions of b-turns. The heterochiral prolinoamino acid/N-
methylamino acid sequence enables the design of short b-turn
mimetics and can be easily incorporated in longer b-hairpin
peptides.
It can be concluded from CD and NMR spectroscopy data
that peptides 4b and 4c adopt a stable type II’ b-turn confor-
mation in both methanol and aqueous solutions. Such a b-turn
structure was also observed by NMR spectroscopy of peptide
4a in methanol and the similarity of the CD signatures in both
solvents strongly supports a similar type II’ b-turn conforma-
tion of peptide 4a in water. Therefore, we have shown that
heterochiral peptide sequences incorporating a prolinoamino
acid and an N-methylamino acid in positions i+1 and i+2
adopt stable b-turn conformations in water. The pyrrolidine
scaffold and the N-methylation provide a strong conformation-
Experimental Section
All experimental details can be found in the Supporting Informa-
tion.
ChemBioChem 2010, 11, 55 – 58
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www.chembiochem.org
57

[7] J. Quancard, P. Karoyan, O. Lequin, E. Wenger, A. Aubry, S. Lavielle, G.
Chassaing, Tetrahedron Lett. 2004, 45, 623–625.
Acknowledgements
[8] a) P. Karoyan, J. Quancard, J. Vaissermann, G. Chassaing, J. Org. Chem.
2003, 68, 2256–2265; b) J. Quancard, A. Labonne, Y. Jacquot, G. Chas-
saing, S. Lavielle, P. Karoyan, J. Org. Chem. 2004, 69, 7940–7948; c) C.
Mothes, S. Lavielle, P. Karoyan, J. Org. Chem. 2008, 73, 6706–6710.
[9] J. Quancard, P. Karoyan, S. Sagan, O. Convert, S. Lavielle, G. Chassaing,
O. Lequin, Eur. J. Biochem. 2003, 270, 2869–2878.
P.K. thanks Daniel Scheidegger, Benoit Oswald and Thomas
Nsenda from Genzyme Pharmaceuticals for their involvement in
this work. C.M. is grateful to Genzyme Pharmaceuticals for finan-
cial support of her Ph.D. grant.
[10] a) M. W. Holladay, C. W. Lin, C. S. May, D. S. Garvey, D. G. Witte, T. R.
Miller, C. A. W. Wolfram, A. M. Nadzan, J. Med. Chem. 1991, 34, 455–457;
b) S. A. Kolodziej, G. V. Nikiforovich, R. Skeean, M. F. Lignon, J. Martinez,
G. R. Marshall, J. Med. Chem. 1995, 38, 137–149; c) M. Cai, C. Cai, A. V.
Mayorov, C. Xiong, C. M. Cabello, V. A. Soloshonok, J. R. Swift, D. Trivedi,
V. J. Hruby, J. Pept. Res. 2004, 63, 116–131; d) J. A. Tran, F. C. Tucci, M.
Arellano, W. Jiang, C. W. Chen, D. Marinkovic, B. A. Fleck, J. Wen, A. C.
Foster, C. Chen, Bioorg. Med. Chem. Lett. 2008, 18, 1931–1938.
[11] G. Wiegand, O. Epp, R. Huber, J. Mol. Biol. 1995, 247, 99–110.
[12] G. Weckbecker, I. Lewis, R. Albert, H. A. Schmid, D. Hoyer, C. Bruns, Nat.
Rev. Drug Discovery 2003, 2, 999–1017.
Keywords: beta-turn
spectroscopy · peptidomimetics · prolinoamino acid
·
bioorganic chemistry
·
NMR
[1] J. D. Tyndall, B. Pfeiffer, G. Abbenante, D. P. Fairlie, Chem. Rev. 2005, 105,
793–826.
[2] a) J. B. Ball, P. F. Alewood, J. Mol. Recognit. 1990, 3, 55–64; b) K. S. Kee,
S. D. S. Jois, Curr. Pharm. Des. 2003, 9, 1209–1224; c) A. J. Souers, J. A.
Ellman, Tetrahedron 2001, 57, 7431–7448.
[3] D. Barbaras, K. Gademann, ChemBioChem 2008, 9, 2398–2401.
[4] a) C. M. Wilmot, J. M. Thornton, J. Mol. Biol. 1988, 203, 221–232;
b) M. W. MacArthur, J. M. Thornton, J. Mol. Biol. 1991, 218, 397–412.
[5] a) A. Aubry, B. Vitoux, M. Marraud, Biopolymers 1985, 24, 1089–1100;
b) B. Chatterjee, I. Saha, S. Raghothama, S. Aravinda, R. Rai, N. Shamala,
P. Balaram, Chem. Eur. J. 2008, 14, 6192–6204.
[13] a) A. Chakrabartty, T. Kortemme, S. Padmanabhan, R. L. Baldwin, Bio-
chemistry 1993, 32, 5560–5565; b) R. Banerjee, G. Basu, FEBS Lett. 2002,
523, 152–156.
[6] a) D. K. Chalmers, G. M. Marshall, J. Am. Chem. Soc. 1995, 117, 5927–
5937; b) Y. Takeuchi, G. M. Marshall, J. Am. Chem. Soc. 1998, 120, 5363–
5372.
Received: September 11, 2009
Published online on November 24, 2009
58
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