Should I Stay or Should I Go – Leveraging an Overlooked Feature of Click-to-Release Chemistry in Affinity Labeling

Herein we report a ligand-directed affinity labeling platform that leverages a previously overlooked electrophilic intermediate of a click-to-release reaction. Upon TCO – release tetrazine (rTz) reaction, an electrophilic dihydropyridazine methide is formed transiently, which covalently captures proximal nucleophilic residues on the protein of interest, enabling robust and selective modification. The meth-od showed strict ligand dependence and covalent target modification, confirmed through displacement assays and MS/MS mapping of the modified protein. Installation of further functional handles onto the rTz platform enabled modular reporter ligation via strain-promoted click chemistry. This bioorthogo-nally triggered strategy provides a valuable alternative to light-based electrophile activation-based approaches, particularly in systems where optical activation is impractical.