Cook, Declan T; (2025) Installing and Identifying Disulfide Bonds in Cystine-bridged Multicyclic Peptides. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Cystine-bridged multicyclic peptides represent a promising class of molecules in the development of novel therapeutics. However, the installation and elucidation of complex disulfide architectures in disulfide-rich peptides (DRPs) is non-trivial. Here, a novel chemical approach exploits pseudoprolines to strongly direct the oxidative folding of a synthetic DRP toward a single positional isomer. A strategic enzymatic digestion method is used to determine the disulfide connectivity in the bridged peptide. This work serves as a foundation for expanding the chemical diversity present in disulfide-directing motifs in DRPs. Conformational analyses and conformer-specific collision activation cyclic ion mobility-tandem mass spectrometry (cIM-MS/MS) experiments suggest that cis-trans interconversions occur at the pseudoproline units to introduce conformational variability in the model cystine-bridged peptide. The oxidative folding pathway of ProTx-II, a triply cystine-bridged DRP, was investigated using an alkylation and reduction approach coupled to cIM-MS. While conformational heterogeneity of given oxidative products complicated analysis, fragmentation data supported existing work that proposes the oxidative folding of ProTx-II initiates at the C-terminus of the peptide. Attempts were made to develop novel methodologies for the determination of the disulfide connectivity within natively folded ProTx-II. The compact nature of ProTx-II prevented the successful development of a workflow that aimed to utilise partial in-source reduction (ISR) to elucidate the peptide’s disulfide connectivity. Chemical strategies to modify and extend cystine linkages into thioacetals were explored, laying the foundation for the development of methodologies that relax highly compact and knotted DRPs. Lastly, the effects of the electron-based fragmentation technique electron-capture dissociation (ECD) on various Cys S-alkylations were explored. The fragmentation profiles of these different S-modifications were analysed to gain insights into likely fragmentation mechanisms for these systems. These data will prove useful for informing data analyses in workflows that rely on Cys S-alkylations.

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