Abstract
Thiols are critical to cellular structures and functions. Disturbance of cellular thiols has been found to affect cell functions and cause various diseases. Intracellularly, thiols were found unevenly distributed in subcellular organelles. Probes capable of detecting subcellular thiol density in live cells are valuable tools in determining thiols' roles at the subcellular level. The subcellular organelle lysosome is the place where unwanted macromolecules are removed through degradation by hydrolytic enzymes. The degradation also serves as a regulation of a variety of cellular functions such as autophagy, endocytosis, and phagocytosis to maintain cellular homeostasis. Thiols are found to be involved in the lysosomal degradation process. A probe that can detect lysosomal thiols in live cells will be a valuable tool in unveiling the roles of thiols in lysosomes. We would like to report bis(7-(N-(2-morpholinoethyl)sulfamoyl)benzo[c] [1,2,5]-oxadiazol-5-yl)sulfane (BISMORX) as a thiol specific fluorogenic agent for live cell nonprotein thiol (NPSH) imaging in lysosomes through fluorescence microscopy. BISMORX itself shows no fluorescence and reacts readily with a NPSH to form a fluorescent thiol adduct with excitation and emission wavelengths of 380 and 540 nm, respectively. BISMORX does not react with compounds containing nucleophilic functional groups other than thiols such as -OH, -NH2, and -COOH. No reaction was observed either when BISMORX was mixed with protein thiols. BISMORX was able to image, quantify, and detect the change of NPSH in lysosomes in live cells. A colocalization experiment with LysoTracker Red DND-99 confirmed that the thiols imaged by BISMORX were indeed lysosomal thiols.