DBCO/Azide-Appended Thioesters Enable Proximal-Dependent Cysteine to Lysine Labeling through S → N Acyl Shift

Abstract Thioester bonds are unique reactive functional groups with distinct reactivity profiles toward thiol and amine nucleophiles in aqueous solution. Here, we designed and synthesized thioester-appended clickable handles, including DBCO and azide tags, and investigated their reactivity toward biologically relevant thiol and amine nucleophiles. Reactivity studies with free amino acids and free cysteine-containing peptides confirmed thiol labeling through thiol−thioester exchange. Notably, protein-labeling studies revealed that the thioesters preferentially react with free cysteine thiols, thereby facilitating the subsequent labeling of lysine side-chain amino groups through an S-to-N acyl transfer. Furthermore, protein labeling was reduced in the presence of Michael acceptors, which are known to block free thiols. Overall, our findings suggest that thioester-driven, proximity-dependent, site-specific protein labeling may provide new opportunities for the development of antibody−drug conjugates and protein-based fluorescent reporters for therapeutic and biotechnological applications.