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.

Chemical Biology Research Group Institute of Organic Chemistry HUN REN Research Centre for Natural Sciences Magyar Tudósok Krt 2 1117 Budapest Hungary

Department of Physical Chemistry University of Chemistry and Technology Prague Technická 5 166 28 Prague Czech Republic

Institute of Organic Chemistry and Biochemistry of the CAS Flemingovo nám 2 16000 Prague Czech Republic

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