"IEDDA"-activated fluorescent sensing for protein cysteine redox modifications profiling.

BACKGROUND Protein cysteine sulfenylation is a pivotal reversible modification in redox signaling and disease pathology. However, comprehensive monitoring of these events remains challenging, as most current "always-on" fluorescent probes suffer from high background signals and lack the capability for dynamic, real-time tracking in living systems. RESULTS Here, we developed a background-free "turn-on" sensing strategy based on the inverse electron-demand Diels-Alder (IEDDA) reaction. We designed selective probes, BCN-SH and BCN-SOH, to tag protein thiols and sulfenic acids, respectively, by installing a bicyclo[6.1.0]non-4-yne (BCN) handle. Subsequent reaction with a tetrazine-based fluorophore BODIPY-tetrazine (BTZ) triggers a 248-fold fluorescence enhancement, enabling high-contrast detection. These probes demonstrated excellent selectivity and stability, successfully visualizing endogenous cysteine redox dynamics in live HeLa cells under oxidative stress. Furthermore, we extended this strategy to fluorescence imaging in Caenorhabditis elegans. SIGNIFICANCE Integrating selective cysteine chemistry with fluorogenic bioorthogonal activation, this platform enables background-free, temporally resolved visualization of dynamic redox events. This strategy expands the chemical toolbox for redox biology, facilitating mechanistic studies of oxidative signaling.