Novel approaches for cysteine bioconjugation

This thesis describes and investigates novel strategies for cysteine modification to achieve protein bioconjugation. Chapter 1 provides an introduction to the research project with an overview of protein modification techniques. Chapter 2 describes the development of a site selective dual labelling strategy based on substrate controlled cysteine modification. The application of this strategy to green fluorescent protein is also detailed. Chapter 3 describes the development and evaluation of thiophosphonium as a platform for protein modification. An in-depth discussion on reaction mechanisms, pathways, stability of thiophosphonium and the utility of this platform is also included. Chapter 4 describes the development of bromomaleimide based reversible cysteine modification, with particular focus on development of improved strategies for conversion of thiomaleimides back to free cysteines. The utility of the approach was demonstrated using a proof of concept experiment

A novel approach to the site-selective dual labelling of a protein via chemoselective cysteine modification

Local protein microenvironment is used to control the outcome of reaction between cysteine residues and 2,5-dibromohexanediamide. The differential reactivity is exploited to introduce two orthogonal reactive handles onto the surface of a double cysteine mutant of superfolder green fluorescent protein in a regioselective manner. Subsequent elaboration with commonly used thiol and alkyne containing reagents affects site-selective protein dual labelling

A rapid, site-selective and efficient route to the dual modification of DARPins.

Designed ankyrin repeat proteins (DARPins) are valuable tools in both biochemistry and medicine. Herein we describe a rapid, simple method for the dual modification of DARPins by introduction of cysteine mutations at specific positions that results in a vast difference in their thiol nucleophilicity, allowing for clean sequential modification

Reversible protein affinity-labelling using bromomaleimide-based reagents

Reversible protein biotinylation is readily affected via conjugation with a bromomaleimide-based reagent followed by reductive cleavage. The intermediate biotinylated protein constructs are stable at physiological temperature and pH 8.0. Quantitative reversibility is elegantly delivered under mild conditions of using a stoichiometric amount of a bis-thiol, thus providing an approach that will be of general interest in chemical biology and proteomics

Post-translational site-selective protein backbone α-deuteration

Isotopic replacement has long-proven applications in small molecules. However, applications in proteins are largely limited to biosynthetic strategies or exchangeable (for example, N-H/D) labile sites only. The development of postbiosynthetic, C-1H → C-2H/D replacement in proteins could enable probing of mechanisms, among other uses. Here we describe a chemical method for selective protein α-carbon deuteration (proceeding from Cys to dehydroalanine (Dha) to deutero-Cys) allowing overall 1H→2H/D exchange at a nonexchangeable backbone site. It is used here to probe mechanisms of reactions used in protein bioconjugation. This analysis suggests, together with quantum mechanical calculations, stepwise deprotonations via on-protein carbanions and unexpected sulfonium ylides in the conversion of Cys to Dha, consistent with a 'carba-Swern' mechanism. The ready application on existing, intact protein constructs (without specialized culture or genetic methods) suggests this C-D labeling strategy as a possible tool in protein mechanism, structure, biotechnology and medicine