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 excellent signal-to-noise
ratio. Despite significant progress in the development of such probes,
challenges remain in achieving efficient quenching and substantial
fluorescence enhancement across the whole visible spectrum while maintaining
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.
Mechanistic studies provide molecular insights into the 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 applications,
including the visualization of intracellular organelles, drug molecules,
HaloTag fusion proteins, genetically encoded intra- and extracellular
proteins, cell surface antigens, and metabolically labeled glycoconjugates.