Improved Aromatic Substitution–Rearrangement-Based Ratiometric Fluorescent Cysteine-Specific Probe and Its Application of Real-Time Imaging under Oxidative Stress in Living Zebrafish

Biothiols, including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), play a crucial role in many physiological processes. Cys production and metabolism is closely connected with Hcy and GSH; meanwhile, the dynamic antioxidant defenses network by Cys is independent of the GSH system, and Cys can serve as a more effective biomarker of oxidative stress. Hence, it is significant and urgent to develop an efficient method for specific detection of Cys over other biothiols (Hcy/GSH). However, most of the present Cys-specific fluorescent probes distinguished Cys from Hcy through response time, which would suffer from an unavoidable interference from Hcy in long-time detection. In this work, in order to improve the selectivity, we employed an improved aromatic substitution–rearrangement strategy to develop a ratiometric Cys-specific fluorescent probe (Cou–SBD-Cl) based on a new fluorescence resonance energy transfer (FRET) coumarin–sulfonyl benzoxadiazole (Cou–SBD) platform for discrimination of Hcy and GSH. Response of Cou–SBD-Cl to Cys would switch FRET on and generate a new yellow fluorescence emission with a 56.1-fold enhancement of ratio signal and a 99 nm emission shift. The desirable dual-color ratiometric imaging was achieved in living cells and normal zebrafish. In addition, probe Cou–SBD-Cl was also applied to real-time monitor Cys fluctuation in lipopolysaccharide-mediated oxidative stress in zebrafish