Precisely controlling the activation of covalent inhibitors through the caging and decaging of their reactive warheads is pivotal, yet this strategy is rarely pursued due to its formidable technical challenges. In this contribution, we report a novel tandem bioorthogonal retro-Cope and Cope elimination designed for efficient and selective activation of the covalent inhibitors bearing an acrylamide or vinylsulfonamide warhead in live cells. Notably, this strategy can be simultaneously tailored to coactivate both the covalent inhibitor and a fluorescent reporter, enabling real-time monitoring of prodrug activation. We successfully demonstrate the proof of concept through on-demand activation of two distinct EGFR covalent inhibitors and a BRD4-targeting molecular glue in live cells. This approach allows precise control over antiproliferative activity or induced protein degradation exclusively upon triggering via the tandem bioorthogonal reaction. We anticipate that this methodology will open new avenues for the selective delivery and controlled activation of covalent inhibitors, with broad potential in chemical biology and targeted therapy.
Huang et al. (2026) studied this question.