Conformations of Heterochiral and Homochiral Proline-Pseudoproline ConformationsSegments in of Peptides: Heterochiral Context and Dependent HomochiralCis–Trans Proline-PseudoprolinePeptide SegmentsBond Isomerization in Peptides: Context Dependent Cis–Trans Peptide Bond Isomerization Kantharaju,1 Srinivasarao Raghothama,2 Upadhyayula Surya Raghavender,3 Subrayashastry Aravinda,3 Narayanaswamy Shamala,3 Padmanabhan Balaram1 1 Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka 560012, India 2 NMR Research Centre, Indian Institute of Science, Bangalore, Karnataka 560012, India 3 Department of Physics, Indian Institute of Science, Bangalore, Karnataka 560012, India Received 9 March 2009; accepted 7 April 2009 Published online 16 April 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.21207 ABSTRACT: stabilized by two intramolecular hydrogen bonds. Further truncation of the sequence gives an appreciable rise in the The pseudoproline residue (CPro, L-2,2-dimethyl-1,3- D thiazolidine-4-carboxylic acid) has been introduced into population of cis conformers in the tripeptide Piv- Pro- C heterochiral diproline segments that have been previously Pro-Leu-OMe (6). In the homochiral segment Piv-Pro- C shown to facilitate the formation of b-hairpins, containing Pro-Leu-OMe (7)onlythecis form is observed with the b central two and three residue turns. NMR studies of the NMR evidence strongly supporting a type VIa -turn ? octapeptide Boc-Leu-Phe-Val-DPro-CPro-Leu-Phe-Val- conformation, stabilized by a 4 1 hydrogen bond OMe (1), Boc-Leu-Val-Val-DPro-CPro-Leu-Val-Val-OMe between the Piv (CO) and Leu (3) NH groups. The crystal CH,CH3 (2), and the nonapeptide sequence Boc-Leu-Phe- structure of the analog peptide 7a (Piv-Pro- Pro- Val-DPro-CPro-DAla-Leu-Phe-Val-OMe (3)established Leu-NHMe) confirms the cis peptide bond geometry for CH,CH3 well-registered b-hairpin structures in chloroform the Pro- Pro peptide bond, resulting in a type VIa b # solution, with the almost exclusive population of the trans -turn conformation. 2009 Wiley Periodicals, Inc. conformation for the peptide bond preceding the CPro Biopolymers (Pept Sci) 92: 405–416, 2009. residue. The b-hairpin conformation of 1 is confirmed by Keywords: pseudoproline; peptides; cis-trans isomeriza- single crystal X-ray diffraction. Truncation of the strand tion; NMR; turn length in Boc-Val-DPro-CPro-Leu-OMe (4) results in an increase in the population of the cis conformer, with a cis/ This article was originally published online as an accepted L preprint. The ‘‘Published Online’’ date corresponds to the trans ratio of 3.65. Replacement of CPro in 4 by Pro in 5, preprint version. You can request a copy of the preprint by results in almost exclusive population of the trans form, emailing the Biopolymers editorial office at biopolymers@wiley. resulting in an incipient b-hairpin conformation, com INTRODUCTION he peptide bonds in polypeptide and protein struc- Additional Supporting Information may be found in the online version of this article. Correspondence to: Narayanaswamy Shamala; e-mail: [email protected] tures occur predominantly as the energetically more or Padmanabhan Balaram; e-mail: [email protected] favored trans conformer.1,2 However, a small but sig- Contract grant sponsor: Council of Scientific and Industrial Research, India Contract grant sponsor: Department of Biotechnology, India nificant number of peptide bonds occur in the cis VC 2009 Wiley Periodicals, Inc. T form and are of structural and functional impor- PeptideScience Volume 92 / Number 5 405 406 Kantharaju et al. tance.3–13 An interesting structural feature that is found in Homochiral (L,L) diproline segments and conformationally proteins is the type-VIa b-turn, which possess a central cis constrained, covalently bridged templates have been shown amide linkage and facilitates chain reversal.14–23 The design to be inducers of helical folding in short peptides.48–54 Hetero- and synthesis of conformationally well-defined peptides as chiral (D,L) Pro-Pro sequences a have strong tendency to folding models and as analogs of biologically active peptides form type II0 b-turn structures,55,56 facilitating the nucleation requires strategies for the rational introduction of cis peptide of peptide b-hairpins, in which antiparallel strands are held bonds into polypeptide sequences. Mutter and coworkers together by registered cross-strand hydrogen bonds.57–59 have pioneered the use of pseudoproline (CPro) residues as Peptide hairpins containing a central three residue, DPro- a means of stabilizing cis peptide bonds in linear sequen- Pro-DAla, loop have also been characterized.57 In this article, ces.24–26 In the pseudoprolines (see Figure 1), the presence of we examine the effect of replacement of the LPro residue by d L geminal substituents at the C carbon atom of the five-mem- CPro (CPro, L-2,2-dimethyl-1,3-thiazolidine-4-carboxylic bered ring results in nonbonded interactions, which tilt the acid), in both the two residue (DPro-LPro) and three residue conformational equilibrium towards the cis conformer. In an (DPro-LPro-DAla) loop containing hairpins. We establish that elegant series of studies Mutter’s group has established the the CPro containing octapeptides Boc-Leu-Phe-Val-DPro- easy synthetic accessibility of a variety of pseudoprolines, CPro-Leu-Phe-Val-OMe (1), Boc-Leu-Val-Val-DPro-CPro- containing substituents at the Cd position and a heteroatom Leu-Val-Val-OMe (2), and the nonapeptide Boc-Leu-Phe- [O,S] at the c-position.27–31 Pseudoprolines have been incor- Val-DPro-CPro-DAla-Leu-Phe-Val-OMe (3) adopt b-hairpin porated into several model peptides and biologically active conformations with the DPro-CPro bond existing exclusively sequences including cyclosporin C,32,33 analogues of morphi- as the trans conformer. Studies with truncated sequences 34 D D ceptin and endomorphin-2, the gp120 V3 loop of HIV- Boc-Val- Pro-CPro-Leu-OMe (4) and Boc-Val- Pro-Pro- 1,35,36 the peptide hormone oxytocin,37 and a d-conotoxin Leu-OMe (5) establish that folding into b-hairpins and the EVIA peptide analog.38 The incorporation of pseudoprolines resultant cross-strand hydrogen bond formation provides the into peptide backbone is also useful in limiting peptide energetic driving force for tilting the DPro-CPro peptide aggregation and facilitating chain extension in solid-phase bond towards the trans conformation. The effect of the con- peptide synthesis.39–42 The Dumy and Mutter groups have figuration of the preceding Pro residue is established by com- also presented an extensive series of studies which establish paring the diastereomeric sequences Piv-DPro-CPro-Leu- the effect of Cd substitution on the cis-trans equilibrium.43–47 OMe (6) and Piv-Pro-CPro-Leu-OMe (7). Diproline templates have proved valuable in synthetic pep- tide design for the construction of both helices and hairpins. MATERIALS AND METHODS Peptide Synthesis Peptides were synthesized by conventional solution-phase chemis- try, using a racemization free, fragment condensation strategy. The Boc group was used for N-terminal protection, and the C-terminus was protected as a methyl ester. Deprotection was performed by using 98% formic acid and by saponification for the N- and C-ter- minus, respectively. Couplings were mediated by N,N-dicyclohexyl- carbodiimide (DCC) and 1-hydroxy-1H-benzotriazole (HOBT). The intermediate Boc-DPro-Cys(CCH3,CH3Pro)-Leu-Phe-Val-OMe was obtained by the fragment condensation of Boc-DPro- CH3,CH3 Cys(C Pro)-OH and H2N-Leu-Phe-Val-OMe. The dipeptide Boc-DPro-Cys(CCH3,CH3Pro)-OH was synthesized by the coupling D of Boc- Pro-OH and the L-2,2-dimethyl-1,3-thiazolidine-4-carb- oxylic acid (readily prepared by refluxing a mixture of L-cysteine hydrochloride monohydrate (7 g, 0.04 mol), 100 mL of 2,2-di- methoxypropane, and 500 mL of dry acetone for about 2 h. After cooling to room temperature, the resulting white platelets were col- lected by simple filtration), by using O-benzotriazole-N,N,N0,N0-tetra- methyluronium-hexafluorophosphate (HBTU)/HOBT as coupling agents and two equivalents of diisopropylethylamine. The final FIGURE 1 Trans and cis conformers of proline (top) and pseudo- step in the synthesis of the peptides was achieved by the frag- proline (bottom). ment condensation of Boc-Leu-Phe-Val-OH and HN-DPro- Biopolymers (Peptide Science) Hetero and Homochiral Proline-Pseudoproline Segments in Peptides 407 CH3,CH3 Cys(C Pro)-Leu-Phe-Val-OMe. The Boc group was removed 5657 observed reflections with F0 >4r(F0) and 1366 parameters by using 1% TFA in dry dichloromethane for about 1 hr (detailed where the data-to-parameter ratio is 4.1:1.0 and S ¼ 1.063. The larg- procedures are provided as Supporting Information). All of the est difference peak was 0.59 e/A˚ 3 and the largest difference hole was intermediates were characterized by 1H NMR spectroscopy À0.31 e/A˚ 3. For peptide 7a, x scan type was used, with 2h ¼ 53.808, (500 MHz) and thin layer chromatography (TLC) on silica gel and for a total of 2493 unique reflections. The crystal size was 0.45 mm were used after purification using reverse-phase medium pressure 3 0.02 mm 3 0.04 mm. The space group was P31 with a ¼ b ¼ ˚ ˚ ˚ 3 liquid chromatography (C18, 40–60 lm). The final peptides were 14.6323(22) A, c ¼ 10.4359(32) A, V ¼ 1935.0(7) A , Z ¼ 3 for À3 purified by HPLC on a reverse phase C18 column (5–10 lm, 7.8– chemical formula C22H38N4O4S1, qcalc ¼ 1.17 g cm , l ¼ 0.16 250 mm) by using methanol–water gradients. Peptides 1–7 were mmÀ1, and F(000) ¼ 738.0. The structure was solved by direct characterized by electrospray ionization mass spectrometry (ESI-MS) methods using SHELXD.60 This gave a fragment containing 28 on a Bruker Daltonics Esquire-3000 instrument and by complete atoms.