Overcoming Spectral Dependence: A General Strategy for Developing Far‐Red and Near‐Infrared Ultra‐Fluorogenic Tetrazine Bioorthogonal Probes

Bioorthogonal fluorogenic dyes are indispensable tools in wash-free bioimaging of specific biological targets. However, the fluorogenicity of existing tetrazine-based bioorthogonal probes deteriorates as the emission wavelength shifts towards the NIR window, greatly limiting their applications in live cells and tissues. Herein, we report a generalizable molecular design strategy to construct ultra-fluorogenic dyes via a simple substitution at the meso-positions of various far-red and NIR fluorophores. Our probes demonstrate significant fluorescence turn-on ratios (10² -10³ -fold) in the range 586-806 nm. These results will greatly expand the applications of bioorthogonal chemistry in NIR bioimaging and biosensing.