Benzofurazan sulfides for thiol imaging and quantification in live cells through fluorescence microscopy

Thiol groups play a significant role in various cellular functions. Cellular thiol concentrations can be affected by various physiological or pathological factors. A fluorescence imaging agent that can effectively and specifically image thiols in live cells through fluorescence microscopy is desirable for live cell thiol monitoring. Benzofurazan sulfides 1a-1e were synthesized and found to be thiol specific fluorogenic agents except 1d. They are not fluorescent but form strong fluorescent thiol adducts after reacting with thiols through a sulfide-thiol exchange reaction. On the other hand, they exhibit no reaction with other biologically relevant nucleophilic functional groups such as -NH(2), -OH, or -COOH revealing the specificity for the detection of thiols. Sulfide 1a was selected to confirm its ability to image cellular thiols through fluorescence microscopy. The compound was demonstrated to effectively image and quantify thiol changes in live cells through fluorescence microscopy using 430 and 520 nm as the excitation and emission wavelengths, respectively. The quantification results of total thiol in live cells obtained from fluorescence microscopy were validated by an high-pressure liquid chromatography/ultraviolet (HPLC/UV) total thiol assay method. The reagents and method will be of a great value to thiol redox-related research.