Differential Chemoselectivity of Homobifunctional and Monofunctional Maleimide Reagents for Bioconjugation

Maleimides are widely utilized in bioconjugation because they efficiently and selectively react with thiols. Owing to their favorable reaction kinetics, maleimides have found broad applications in bioconjugate chemistry, including the synthesis of antibody-drug conjugates for cancer therapy, fluorescence labeling of proteins, or functionalization of materials. As increasingly complex platforms are developed, multivalent linkers are being incorporated more frequently into synthetic strategies. Although the chemistry of monovalent maleimides is well understood, the chemoselectivity of multivalent maleimide linkers has not been fully established. To investigate differences in the chemoselectivity of mono- and bivalent maleimides, we performed bioconjugation of mal-PEG11-mal or mPEG12-mal with the physalis mottle virus-like particle. Through a series of chemical assays and characterization studies, we demonstrate that while monovalent maleimides are highly selective for cysteine modifications, bivalent maleimides exhibit cross-reactivity toward both lysine and cysteine residues. Overall, this study highlights how linker valency can influence the chemoselectivity of maleimide-based conjugation chemistry.