Should I Stay or Should I Go—Leveraging an Overlooked Feature of Click‐to‐Release Chemistry for Affinity Labeling

Click-to-release (C2R) chemistry is widely used for controlled payload liberation, yet the reactivity of its transient intermediates has remained largely unexplored. Here we show that an intermediate formed during the reaction of trans-cyclooctene (TCO)-appended protein-targeting ligands with release tetrazines (rTzs), previously regarded as a byproduct rapidly quenched by water, can be exploited as an electrophile for ligand-directed covalent protein labeling. The bioorthogonally generated dihydropyridazine methide (DHP-methide) enables affinity-directed protein labeling, resulting primarily in capture at surface-exposed histidine residues proximal to the site of electrophile generation and revealing a histidine-biased covalent modification pathway. Incorporation of functional handles into the rTz platform enables modular reporter installation via an orthogonal conjugation step. A fluorogenic leaving-group design further allows direct optical reporting of the electrophile-generating elimination event and real-time monitoring of DHP-methide formation. Selective labeling is retained in cell lysate and can be translated to a cellular context, as demonstrated by confocal imaging of surface-expressed targets. These findings establish a bioorthogonally gated strategy for affinity labeling and reveal a previously unrecognized functional dimension of rTz-TCO click-to-release chemistry.