Fluorescence Quenching Properties and Bioimaging Applications of Readily Accessible Blue to Far-Red Fluorogenic Triazinium Salts

Fluorogenic probes, which become fluorescent only upon specific activation, enable no-wash imaging with an excel-lent signal-to-noise ratio. Despite significant progress in the development of such probes, challenges remain in achiev-ing efficient quenching and substantial fluorescence enhancement across the whole visible spectrum while maintain-ing good synthetic accessibility. In this work, we introduce a new class of bioorthogonally activatable fluorogenic probes based on triazinium salts (Trz+), which act as highly efficient fluorescence quenchers. These Trz+-fluorophore conjugates are easily synthesized from common precursors and commercially available fluorophores, covering a broad spectral range from blue to far-red wavelengths. Computational studies provide molecular insights into the unique combined quenching mechanism, attributed to the charged Trz+ core, which triggers substantial fluorescence turn-on upon bioorthogonal reaction with a strained dienophile. The versatility and ease of synthesis of these probes, along with their noteworthy photophysical properties, make them highly valuable for a wide range of bioimaging applica-tions, including the visualization of intracellular organelles, drug molecules, HaloTag fusion proteins, genetically en-coded intra- and extracellular proteins, cell surface antigens, and metabolically labeled glycoconjugates.