Synthesis of C 3 -Substituted N 1- tert -Butyl 1,2,4-Triazinium Salts via the Liebeskind-Srogl Reaction for Fluorogenic Labeling of Live Cells.

We recently described the development and application of a new bioorthogonal conjugation, the triazinium ligation. To explore the wider application of this reaction, in this work, we introduce a general method for synthesizing C 3 -substituted triazinium salts based on the Liebeskind-Srogl cross-coupling reaction and catalytic thioether reduction. These methods enabled the synthesis of triazinium derivatives for investigating the effect of different substituents on the ligation kinetics and stability of the compounds under biologically relevant conditions. Finally, we demonstrate that the combination of a coumarin fluorophore attached to position C 3 with a C 5 -(4-methoxyphenyl) substituent yields a fluorogenic triazinium probe suitable for no-wash, live-cell labeling. The developed methodology represents a promising synthetic approach to the late-stage modification of triazinium salts, potentially widening their applications in bioorthogonal reactions.