Difluoroboronated tetrazine probes for rapid bioorthogonal fluorescence activation, no-wash STED imaging, and triggered drug release

A unique class of bioorthogonal fluorogenic probes, based on difluoroboronated tetrazine (<b>Tz-BFs</b>), has been developed. These probes are the smallest tetrazine-based fluorescent structures and exhibit a high fluorescence turn-on ratio (up to 582-fold) following the inverse electron-demand Diels-Alder reaction. By engineering π-conjugated systems and using push-pull groups, their fluorescence emission can be tuned across blue to yellow (440 to 570 nanometers). They offer no-wash fluorogenic behavior, excellent cell permeability, rapid reaction kinetics, and high photostability, ideal for real-time biomolecule tracking, suborganelle bioorthogonal imaging, and metabolic labeling. In addition, they enable stimulated emission depletion (STED) microscopy for subdiffraction resolution imaging and have click-to-release functionality for simultaneous fluorescence activation and therapeutic agent release, such as TCO (<i>trans</i>-cyclooctene)-DOX (doxorubicin). These probes provide broad potential for advanced imaging and drug delivery applications.