Tetrazine‐Isonitrile Bioorthogonal Fluorogenic Reactions Enable Multiplex Labeling and Wash‐Free Bioimaging of Live Cells

Abstract Developing fluorogenic probes for simultaneous live cell labeling of multiple targets is crucial for understanding complex cellular events. The emerging [4+1] cycloaddition between tetrazine and isonitriles holds promise as a bioorthogonal tool, yet existing tetrazine probes lack reactivity and fluorogenicity. Here, we present the development of a series of tetrazine‐functionalized bioorthogonal probes. By incorporating pyrazole adducts into the fluorophore scaffolds, the post‐reacted probes displayed remarkable fluorescence turn‐on ratios, up to 3184‐fold. Moreover, these modifications are generalizable to various fluorophores, enabling a broad emission range from 473 to 659 nm. Quantum chemical calculations further elucidate the turn‐on mechanisms. These probes enable the simultaneous labeling of multiple targets in live cells, without the need for a washing step. Consequently, our findings pave the way for advanced multiplex imaging and detection techniques for cellular studies.