Abstract:

Bioconjugation strategies that enable precise, site-selective covalent modification of proteins are essential tools for therapeutics, diagnostics, and drug delivery. The histidine (His) sidechain presents an attractive yet underexplored bioconjugation site due to its rarity in protein sequences, surface accessibility, and highly reactive imidazole ring. We leveraged the unique reactivity of electron-deficient azidomaleimides to develop a new protein bioconjugation strategy that achieves selective modification at the C-terminal His6 epitope while leaving native residues untouched. Using maltose binding protein (MBP) as a model system, we systematically evaluated the reactivity of electron-deficient N-phenylazidomaleimide toward histidine. Our results demonstrate an efficient, physiologically compatible, and highly sequence-selective protein conjugation method targeting the His6 epitope, providing a simple and broadly applicable handle for precise protein modification.