Benzylic Trifluoromethyl Accelerates 1,6-Elimination Toward Rapid Probe Activation

ABSTRACT Activity-based detection of hydrogen sulfide in live cells can expand our understanding of its reactivity and complex physiological effects. We have discovered a highly efficient method for fluorescent probe activation, which is driven by H 2 S-triggered 1,6-elimination of an α-CF 3 -benzyl to release resorufin. In detecting intracellular H 2 S, 4-azido-(α-CF 3 )-benzyl resorufin offers significantly faster signal generation and improved sensitivity compared to 4-azidobenzyl resorufin. Computed free energy profiles for the 1,6-elimination process support the hypothesis that a benzylic CF 3 group can reduce the activation energy barrier toward probe activation. This novel probe design allows for near-real-time detection of H 2 S in HeLa cells under stimulation conditions.