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. A general tetrazine probe design strategy overcomes the spectral dependence of the energy transfer mechanism, endowing excellent fluorogenicity in the NIR window. The high specificity, signal‐to‐noise ratio, and photostability of the synthesized probes make them highly suited for live‐cell bioimaging and theranostics.