Design, Synthesis, and Characterization of Bis(7-(N-(2-morpholinoethyl)sulfamoyl)benzo[c][1,2,5]oxadiazol-5-yl)sulfane for Nonprotein Thiol Imaging in Lysosomes in Live Cells

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.

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“…Measurement of Lysosome. Lysosome function [18] was determined by using LysoTracker Red (Beyotime, cat: C1046), which was a fluorescent acidotropic probe for labeling and tracking acidic organelle lysosomes. LysoTracker Red probes, consisting of a fluorophore linked to a weak base, are freely permeant to cell membranes and typically concentrate in spherical organelles, thus forming the LysoTracker Red puncta.…”

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“…Based on the thiol specific thiol-sulfide exchange reaction reported from our group [42], we developed a thiol specific and lysosome-selective fluorogenic agent (BISMORX, Probe 77, Figure 69) [206] in 2019 by introducing a lysosome targeting structure morpholine symmetrically to the symmetric benzofurazan sulfide. BSMORX uses a thioether moiety to serve as the thiol recognition units.…”

Fluorescent Probes for Live Cell Thiol Detection

“…Later, Lyso-O-NBD was successfully applied to detect and discriminate Cys/Hcy and GSH in lysosomes in HeLa cells by confocal laser scanning microscopy. Based on the thiol specific thiol-sulfide exchange reaction reported from our group [42], we developed a thiol specific and lysosome-selective fluorogenic agent (BISMORX, Probe 77, Figure 69) [206] in 2019 by introducing a lysosome targeting structure morpholine symmetrically to the symmetric benzofurazan sulfide. BSMORX uses a thioether moiety to serve as the thiol recognition units.…”