Unveiling the Structure‐Fluorogenic Property Relationship of Seoul‐Fluor‐Derived Bioorthogonal Tetrazine Probes

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Tetrazine (Tz)‐embedded fluorescent probes, known for their fluorogenicity following bioorthogonal inverse electron‐demand Diels–Alder (iEDDA) reactions, are extensively used in bioimaging. Despite extensive research on fluorogenic Tz probes, there has been limited systematic exploration of their fluorogenic responses with various dienophiles. In this study, we elucidate the structure‐fluorogenic property relationship of bioorthogonal Tz probes. We synthesized a series of Seoul‐Fluor‐Tz (SFTz) probes designed to exhibit differentiated turn‐on fluorescence upon iEDDA reactions with three dienophiles: trans‐cyclooctene (TCO), bicyclo[6.1.0]nonyne (BCN), and spiro[2.3]hex‐1‐ene (Sph). Our findings revealed that the fluorogenic properties of the SFTz probes are highly dependent on the structures of Tz‐dienophile adducts. By systematically modifying the electronic properties and employing quantum chemical calculations, we developed a series of SFTz probes with optimal dienophile‐dependent fluorescence. These probes enabled simultaneous dual‐color imaging of different cellular targets using a single probe, providing a robust approach for advanced bioimaging applications that require precise and efficient multicolor labeling strategies.