Conditional Relay Activation of Theranostic Prodrug by Pretargeting Bioorthogonal Trigger and Fluorescence‐Guided Visible Light Irradiation

Abstract Bioorthogonalized light‐responsive click‐and‐uncage platform has enabled precise cell surface engineering and timed payload release, but most of such photoactivatable prodrugs have “always‐on” photoactivity leading to the dark toxicity. On the other hand, the conditionally activatable photocage is limited to the application of fluorogenic probe/photosensitizer liberation. Herein, we devise a conditionally activatable theranostic platform based on the tetrazine (Tz)‐boron‐dipyrromethene (BODIPY) construct, in which tetrazine serves as a quencher motif to disable both the fluorescence and photoresponsivity of BODIPY. The uncaged BODIPY upon the bioorthogonal transformation of the tetrazine reinstates fluorescence on‐target to guide the subsequent visible light irradiation thereby releasing the therapeutic payload in situ . The proof of concept for such “light where is lit up”‐activated theranostics was demonstrated by the tetrazine‐BODIPY conjugated SN‐38 ( 1 ) with masked fluorescence, up to 70 times‐reduced cytotoxicity, and good photostability. Upon an inverse electron‐demand Diels–Alder (IEDDA) reaction between tetrazine unit on 1 and a biotinylated trans ‐cyclooctene (biotin‐TCO) as a pretargeted trigger on tumor cell surface, the green fluorescence from BODIPY was fully restored for cell‐selective imaging, which guided the green light irradiation (500–520 nm) to activate the caged drug SN‐38 exerting potent cytotoxicity against tumor cells, offering high spatiotemporal precision of disease diagnosis and therapy.