Fletcher, SA; (2015) Modification of peptides by disulfide bridging: a biochemical and analytical investigation. Doctoral thesis , UCL (University College London).

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Abstract

The use of chemical reagents for the modification of peptides has broad applications and, as a result, is a rapidly expanding area of research. Such methods serve to improve the pharmacokinetic properties of the peptide or add functionality for a desired application. Chemical modification of peptides has facilitated the development of a variety of bioconjugates for use as analytical probes, diagnostic agents and therapeutics. This project focuses on the modification of peptides by targeting disulfide bonds. Many peptides contain disulfide bonds which serve a crucial role in retaining their structure, function and stability. Reduction of these disulfides affords two reactive cysteine thiolates whose nucleophilicity can be exploited in peptide modification. To this end, a family of 3,4-disubstituted maleimide reagents were synthesised, designed to efficiently re-bridge a reduced, accessible disulfide bond. The bridging reagents vary in reactivity, properties and functionality but all serve to maintain the structural integrity conferred by a disulfide bond. With these reagents in hand, the scope of their utility was tested on peptides of biological and medicinal interest. One such peptide was tertiapin Q, a neurotoxin derived from the venom of the honey bee and consisting of two disulfide bonds in close proximity. Both singly and doubly bridged variants of the peptide were synthesised and isolated. Biological activity of the modified peptide was analysed by whole-cell patch clamp experiments on cells expressing the target of the peptide toxin, the G-coupled inwardly rectifying K+ (GIRK) channel, which tertiapin Q is known to inhibit. Loss of biological activity was observed upon modification, which led into a full structural characterisation study to determine the explanation for this intriguing result. A second peptide target, octreotide, a stabilised analogue of the hormone somatostatin with a single disulfide bond, was modified with relative ease. Biological activity was examined by whole-cell patch clamp on cells expressing the target of octreotide, the somatostatin receptor (SSTR) subtype 2. Pleasingly, nanomolar activity of the modified peptide was observed. A novel candidate for the next generation of diagnostics for SSTR positive tumours was subsequently developed, characterised and tested by confocal microscopy.

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