Schumacher, FF; (2013) Functional bridging of protein disulfide bonds with maleimides. Doctoral thesis (PhD), UCL (University College London).

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Abstract

The application of chemical methods to biological systems has led to great advances in all life sciences and the discovery of novel approaches for therapy and diagnosis. Pivotal amongst these methods is the ability to chemically modify proteins to enhance their biophysical properties or add new functionality. Despite the success, the chemical toolbox of efficient and widely applicable protocols is relatively limited. In the work presented in this thesis the idea of protein modification via the targeting of solvent accessible disulfide bonds is explored. These are fairly common in secreted proteins and their reduction affords two cysteine residues, which are highly reactive nucleophiles. However, to preserve their native function – stabilisation and maintenance of the protein structure – ideally bis-reactive compounds are used that react simultaneously with both thiols and keep the covalent connection of the disulfide bond intact. To this end a selection of maleimides substituted with good leaving groups in the 3 and 4 positions as well as their N-functionalised versions were synthesised and tested for their reactivity. The findings were transferred to the small peptide hormone somatostatin, which served as a model system to explore kinetics and feasibility of the proposed “functionalisation by bridging”. Changing the leaving groups from halides to thiols enabled the development of in situ protocols where the bridging reagent could be employed in tandem with the reducing agent, greatly decreasing the reaction time and unwanted side reactions such as dimerisation or unfolding. The developed methods were then utilised to bridge the cystines of insulin as well as a selection of full length antibodies and antibody fragments. PEGylated, biotinylated, fluorescent or spin labelled analogues of these proteins were also synthesised. The biological activity, stability and functionality of the conjugates were assessed in biochemical and biophysical assays. Overall the functionalisation of disulfide bonds with maleimides was found to be site-specific, fast, high yielding and the inserted bridge was stable under various conditions. The modification was well tolerated and all prepared analogues exhibited the desired functionality. The scope and potential of present and future applications of the method are discussed.

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