Defining peptide structure with metathesis.

Abstract

Understanding protein structure and function is central for the development of therapeutics for the treatment of diseases and also novel biocompatible materials. Herein describes studies on the control of peptide structure and function through synthetic modifications, for the synthesis of novel enzyme inhibitors and biomaterials, primarily using olefin metathesis chemistry. Metathesis is chosen for the manipulation of peptide structure in order to induce conformational constraint in novel macrocyclic peptidomimetic inhibitors and to develop novel hydrogel matrices, which are of importance in the advancement of the pharmaceutical and medical industries. The realization that enzymes bind their substrates in an extended β-stranded conformation has led to the development of inhibitors that mimic this bioactive conformation. The controlled organization of secondary structures in peptides by conformational constraint has been utilized to design two novel series of macrocyclic inhibitors, which are constrained by the P₁ and P₃ residues or the P₂ and P₄ residues using ring closing metathesis (RCM). These inhibitors contain a pyrrole group in the peptide backbone, thereby decreasing the peptidic nature of these inhibitors minimising susceptibility to proteolysis, while maintaining the appropriate geometry for inhibitor binding. The corresponding P₁-P₃ and P₁-P₄ acyclic inhibitors are designed and synthesized to provide an insight into the importance of cyclisation on the potency of inhibition against serine and cysteine protease. The macrocyclic and acyclic inhibitors synthesized are assayed against a series of cysteine (calpain and cathepsin) and serine proteases (α-chymotrypsin, human leukocyte elastase and trypsin). These enzyme assays analyse the efficacy of the inhibitors against the enzymes tested. The potency of the inhibitors against the aforementioned proteases provides an insight into the effect of cyclisation, ring size and introduction of aryl groups into the ring system, as well as trends in selectivity between proteases of the same family (calpain vs. cathepsin and α-chymotrypsin vs. HLE and trypsin) and between the cysteine and serine protease families. The ability to mimic the natural environment of structural proteins in wound healing, has led to the development of biocompatible materials, such as hydrogels, through the manipulation of natural peptide structure. The controlled organization of the tertiary structure of naturally occurring peptides is investigated by aqueous metathesis in the synthesis of biocompatible hydrogels derived from gelatin. Novel gelatin-gels are obtained by reacting methacrylate-functionalized gelatin and norbornene dicarboxylic acid in the presence of a catalyst in aqueous media. Optimisation of the hydrogel formation is investigated by; i) varying catalyst utilised and ii) varying ratios of starting gelatin and norbornene dicarboxylic acid. These polymer gels exhibited physical and chemical properties that might be useful in regenerative medicine. Mechanistic studies using MALDI is also performed to provide an insight into the mode of hydrogel formation.