Stable and Rapid Thiol Bioconjugation by Light‐Triggered Thiomaleimide Ring Hydrolysis

Maleimide‐mediated thiol‐specific derivatization of biomolecules is one of the most efficacious bioconjugation approaches currently available. Alarmingly, however, recent work demonstrates that the resulting thiomaleimide conjugates are susceptible to breakdown via thiol exchange reactions. Herein, we report a new class of maleimides, namely o‐CH2NHiPr phenyl maleimides, that undergo unprecedentedly rapid ring hydrolysis after thiol conjugation to form stable thiol exchange‐resistant conjugates. Furthermore, we overcome the problem of low shelf lives of maleimide reagents owing to their propensity to undergo ring hydrolysis prior to bioconjugation by developing a photocaged version of this scaffold that resists ring hydrolysis. UV irradiation of thiol bioconjugates formed with this photocaged maleimide unleashes rapid thiomaleimide ring hydrolysis to yield the desired stable conjugates within 1 h under gentle, ice‐cold conditions. Light and water go together! A photoactivable reagent for stable thiol bioconjugation under ice‐cold conditions has been rationally designed. The reagent first forms a covalent adduct with the biomolecule of interest, and then undergoes extremely rapid ring hydrolysis upon UV irradiation via intramolecular catalysis orchestrated by a protonated amino group to yield the stable bioconjugate.