Cyclic peptide disulfides as conformational models; synthesis and spectroscopic studies

Abstract

The study of small peptides has been motivated both by the high biological activity manifested by many peptides and their potential use as model systems for understanding polypeptide and protein structure. The development of structure–function relationships has necessarily required the elucidation of the preferred conformations in stereochemically constrained peptides. Covalent crosslinks formed by disulfide bridges involving Cys side chains serve to generate conformationally well?defined peptide loops. While a large amount of experimental work has been reported on 20?membered cyclic disulfides found in oxytocin and vasopressin, relatively little information is available in the literature on smaller?ring peptide disulfides. The research work presented in this thesis is aimed at developing small?ring peptide disulfides as conformational models. A major part of this investigation is devoted to the synthesis of 14?membered cyclic peptide disulfides and their conformational analysis by spectroscopic methods. The impetus for studying 14?membered disulfide loops—formed when only two spacer amino?acid residues separate the two linked Cys residues—comes from the presence of such a disulfide loop at the active site of the important redox protein thioredoxin. Furthermore, 14?membered peptide disulfides may be used to generate cyclic models for ??turn structures. The conformational analysis of these synthetic peptides has been largely carried out using 270 MHz ¹H NMR. The thesis is divided into 9 chapters, outlined below.

Chapter I The parameters that determine peptide conformation are briefly explained, followed by an account of the methods used for the determination of peptide conformations in solution.

Chapter II This chapter describes the solution?phase synthetic procedures followed to obtain the 14?membered cyclic disulfides: Boc?Cys?Pro?X?Cys?NHMe (X = Gly, L?Ala, D?Ala, Aib, L?Leu) and their characterisation.

Chapter III The stereochemically constrained tetrapeptide disulfide Boc?Cys?Pro?Aib?Cys?NHMe has been investigated both in the solid state and in solution. The results establish a disulfide?bridged consecutive Type III ??turn structure (incipient 3???helix) for the peptide in both states.

Chapter IV A consolidated account of the conformational analysis of the five Pro–X peptide disulfides (X = Gly, L?Ala, D?Ala, Aib, L?Leu) is presented. All five disulfide peptides favour Pro–X ??turn conformations with a transannular 4?1 hydrogen bond in the 14?membered disulfide ring. A comparative study with the corresponding acyclic precursor peptides is also summarized.

Chapter V This chapter describes the synthesis and solution conformational studies of Boc?Cys?Gly?Pro?Cys?NHMe, which mimics the active?site disulfide loop of thioredoxin. To gain insight into the structure of 14?membered cyclic disulfide systems containing ‘X–Pro’ sequences, two more synthetic analogues were studied, replacing Gly with D?Ala and Aib. Spectroscopic methods (NMR, IR, CD) established a consecutive ??turn structure, involving the NH groups of Cys(4) and the terminal methylamide. Comparison of the cyclic disulfide with its acyclic precursor reveals significant structural differences, likely corresponding to conformational changes that occur at the thioredoxin active site during the redox reaction.

Chapter VI A novel emission band arising from the S–S chromophore was observed in some synthetic disulfides. The luminescence characteristics of these peptide disulfides are summarised.

Chapter VII To explore the effect of ring size enlargement, two 17?membered ring disulfides were synthesised and investigated spectroscopically. These studies reveal folded structures and clearly demonstrate the existence of X–Pro cis peptide units and solvent?dependent conformational changes.

Chapter VIII Attempts to synthesise smaller?ring disulfides Boc?Cys?X?Cys?NHMe (X = Gly, L?Leu), containing 11 atoms in the loop, resulted instead in cyclic dimers incorporating two S–S bridges in a 22?membered ring. Spectroscopic investigations, primarily NMR, support the dimeric structure.

Chapter IX The main results and conclusions of this thesis are summarised.