Synthesis of C3-Substituted N1-tert-Butyl 1,2,4-Triazinium Salts via the Liebeskind-Srogl Reaction for Fluorogenic Labeling of Live Cells.

Abstract

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 C3-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 C3 with a C5-(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.