A Click-Triggered Bioorthogonal Strategy for Dual Activation of Covalent Warheads and Reporter Payloads

Abstract Michael acceptors are indispensable electrophilic motifs in chemical biology and covalent drug discovery, yet their intrinsic “always-on” reactivity frequently causes off-target modification and limits therapeutic precision. Here we report a bioorthogonal strategy for externally controlling Michael acceptor reactivity through hydroxylamine masking and cyclooctyne-triggered activation. Specifically, hydroxylamine masks the electrophilic site, and a subsequent bioorthogonal click reaction initiates a rapid Cope elimination, regenerating the active electrophile under mild conditions. This platform accommodates a broad range of Michael acceptors─including α,β-unsaturated carbonyl, cyano-, sulfonyl-, phosphonate-, and maleimide-based warheads, with activation kinetics tunable through electronic and steric modulation. Moreover, incorporation of appropriately functionalized cyclooctynes couples electrophile activation with the release of imaging or functional payloads, thereby affording concurrent signal readout that directly reports on the activation event. Application of this strategy to covalent inhibitors demonstrates controlled activation in cellular settings, establishing a general framework for precision regulation of covalent reactivity in complex biological environments.