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

Bioconjugate Chemistry

October 09, 2026 doi.org/10.1021/acs.bioconjchem.6c00470

N. Fathima , Anagha S. Nair, A. M. Amrutha,Nagarjun Narayanaswamy

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.