Styryl Benzoxazolium Salts as Environment-Sensitive Mitochondrial Probes for Imaging Ferroptosis

Abstract

Styryl benzoxazolium salts are promising fluorescent scaffolds, yet systematic studies on their structure–property relationships and biological applications remain limited. To address this gap, we synthesized five novel derivatives (A1–A5) bearing diverse electron-donating groups and evaluated their photophysical behavior, viscosity sensitivity, and bioimaging performance. All derivatives exhibited strong intramolecular charge transfer (ICT) character, high environmental sensitivity, and notable Stokes shifts, with A5 showing near-infrared emission and significant viscosity-dependent fluorescence enhancement (53-fold). Probes A1, A2, A4, and A5 selec-tively targeted mitochondria in live COS-7 cells with high Pearson correlation coefficients (PCC > 0.82), while A5 additionally enabled real-time monitoring of mitophagy and mitochon-drial viscosity changes during ferroptosis. Cytotoxicity assays confirmed their biocompatibility up to 40 μM, and A5’s responsiveness was validated via ferrostatin-1 rescue and malondialde-hyde assays. These findings establish A1–A5 as structurally tuneable, organelle-specific fluor-ophores with diagnostic potential for cellular dynamics and regulated cell death pathways. Our study reports, for the first time, the synthesis, imaging, and biological application of this dye class, laying the groundwork for advanced probe design in cell biology.