Protein modification is a powerful and important tool for the generation of bioconjugates of immense diagnostic and therapeutic utility. Strategies towards achieving efficient, selective, and accessible bioconjugation are widely studied and sought after, particularly for the development of antibody-drug conjugates (ADCs). However, existing approaches towards synthesising ADCs on native antibodies suffer from numerous limitations, resulting in heterogeneous mixtures of unpredictable tolerability, pharmacokinetics, and efficacy. Whilst engineering antibodies can overcome some of these limitations, these technologies are expensive, time-consuming, and complex.

The cysteine-to-lysine transfer (CLT) approach enables an accessible way of synthesising homogeneous ADCs, modified site-selectively at lysine residues to generate stable amide products. This strategy utilises cysteine residues as initial ligating hooks before an acyl transfer enables modification of proximal lysine residues. Whilst CLT enables site-selective lysine conjugation on off-the-shelf antibodies utilising easily accessible alkyl thioesters, currently, it is a time-consuming process, and competing hydrolysis inhibits quantitative product formation.

This work concerns the development of the next generation of CLT reagents. To enable CLT to become a more widely accessible strategy, chemical synthesis is conducted to develop several novel reagents with unique reactivity. Electron-deficient thioesters were developed to improve thioester reactivity, and these represent the fastest currently available reagents for CLT. Of particular interest is a tetrazine thioester that demonstrates rapid reactivity and click chemistry on N-terminal cysteine, with complete modification observed within minutes. Additionally, a plethora of carbonyl-related functional groups were investigated for their potential as Fab and protein modification reagents. These studies represent the first attempt of the exploration of many of these functional groups for stable and controlled antibody modification, and some particularly promising and useful chemistry is demonstrated. Finally, early works into the expansion of thioesters is discussed, involving dual-reactive thioester reagents that offer two sites of protein reactivity.