Thiol-specific fluorogenic agent for live cell non-protein thiol imaging in lysosomes

Thiol molecules play a significant role in cellular structures and functions. These molecules are distributed in cells unevenly at the subcellular level. Disturbance of cellular thiols has been associated with various diseases and disorders. Probes that are able to detect subcellular thiol density in live cells are valuable tools in determining thiols' roles at the subcellular level. Lysosomes are a subcellular organelle involved in the degradation of macromolecules through the action of proteolytic enzymes. The degradation not only serves as a way to dispose of unwanted macromolecules but also a way to regulate a variety of cellular functions such as autophagy, endocytosis, and phagocytosis to maintain cell homeostasis. A probe that can detect lysosomal thiols in live cells will be useful in unveiling the roles of thiols in lysosomes. Currently, limited probes are available to detect lysosomal thiols in live cells. We would like to report 4,4'-{[7,7'-thiobis(benzo[c][1,2,5]oxadiazole-4,4'-sulfonyl)]bis(oxy))bis(naphthalene-2,7-disulfonicacid) (TBONES) as a thiol-specific fluorogenic agent for lysosomal thiol imaging in live cells through fluorescence microscopy. TBONES exhibits no fluorescence and readily reacts with non-protein thiols to form fluorescent thiol adducts with λ_ex = 400 nm and λ_em = 540 nm. No reaction was observed when TBONES was mixed with compounds containing nucleophilic functional groups other than thiols such as -OH, -NH₂, and -COOH. No reaction was observed either when TBONES was mixed with protein thiols. When incubated with cells, TBONES selectively and effectively imaged lysosomal thiols in live cells. Imaging of lysosomal thiols was confirmed by a co-localization experiment with LysoTracker™ Blue DND-22.