Lamb, Charles MG; (2024) Electrochemical Desulfurization and its Application to Native Chemical Ligation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Native chemical ligation (NCL), a synthetic technique where two peptide fragments are linked using the thiol chemistry of the cysteine side chain, has rapidly proved itself to be the most significant finding in peptide chemistry since solid phase synthesis. This method allows the production of large and complex targets including cyclic peptides, glycopeptides, and even small proteins. The major downside of the process is the necessity for cysteine in the reaction, an uncommon amino acid in nature. The development of desulfurization methods that can convert the cysteine of the product to alanine has allowed for traceless ligations and the development of readily cleaved ligation auxiliaries that enable ligations at non cysteine junctions. The currently used methods for cysteine desulfurization have key downsides such as a tendency for peptide aggregation, non-selectivity for cysteine and the requirement of noxious and potentially explosive reagents. This work first explores the application of synthetic organic electrochemistry to cysteine desulfurization aiming to produce a milder and more environmentally friendly alternative. Using an amino acid model, a reduced reagent alternative to radical desulfurization was developed with a high isolated yield of 90%. The method was then applied to more complex linear and cyclic peptide targets but demonstrated lower yields due to two side reactions at other amino acid sites. The same desulfurization procedure was used to demonstrate cleavage of the 2-mercapto-2 phenethyl class of ligation auxiliary from a peptide ligation model. Finally, our experiments into auxiliary-mediated glycosylation are outlined and the developed electrochemical procedures applied to the topic. A sugar- auxiliary building block is developed, and peptide models are used to demonstrate the plausibility of the technique using C-terminus and side chain thioesters. Electrochemical reduction led to incomplete cleavage. Glycosylating thioester containing expressed proteins proved challenging likely due to the choice of model protein.

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