A bis(trifluoromethyl)benzenesulfonate-caged red-emissive fluorescent probe for cellular imaging of superoxide anions.

Acetaminophen (APAP) overdose is a leading cause of drug-induced liver injury and acute liver failure, where excessive reactive oxygen species (ROS), particularly superoxide anions (O2˙-), play a key early role in oxidative stress. To enable selective imaging of O2˙- in biological systems, we developed a red-emissive fluorescent probe (TCFCl-SA) based on a bis(trifluoromethyl)benzenesulfonate-caged fluorophore, exhibiting fluorescence emission centered at 620 nm after activation. The electron-withdrawing protecting group effectively suppresses intramolecular charge transfer, resulting in a weakly fluorescent state, while reaction with O2˙- triggers deprotection to release the strongly fluorescent TCFCl. The probe exhibits a linear fluorescence response to O2˙- (0-50 µM, R2 = 0.9991) and high selectivity over various reactive species and biologically relevant ions. In living cells, TCFCl-SA shows low cytotoxicity and enables fluorescence imaging of endogenous and APAP-induced O2˙- in RAW 264.7 and HepG2 cells, with signal suppression by ROS scavengers confirming specificity. These results demonstrate that TCFCl-SA is a useful tool for monitoring intracellular O2˙- dynamics and investigating APAP-induced oxidative stress at the cellular level.