title: Non-protein thiol imaging and quantification in live cells with a novel benzofurazan sulfide triphenylphosphonium fluorogenic compound.
abstract: Thiols (-SH) play various roles in biological systems. They are divided into protein thiols (PSH) and non-protein thiols (NPSH). Due to the significant roles thiols play in various physiological/pathological functions, numerous analytical methods have been developed for thiol assays. Most of these methods are developed for glutathione, the major form of NPSH. Majority of these methods require tissue/cell homogenization before analysis. Due to a lack of effective thiol-specific fluorescent/fluorogenic reagents, methods for imaging and quantifying thiols in live cells are limited. Determination of an analyte in live cells can reveal information that cannot be revealed by analysis of cell homogenates. Previously, we reported a thiol-specific thiol-sulfide exchange reaction. Based on this reaction, a benzofurazan sulfide thiol-specific fluorogenic reagent was developed. The reagent was able to effectively image and quantify total thiols (PSH+NPSH) in live cells through fluorescence microscopy. The reagent was later named as GUALY's reagent. Here we would like to report an extension of the work by synthesizing a novel benzofurazan sulfide triphenylphosphonium derivative [(((7,7'-thiobis(benzo[c][1,2,5]oxadiazole-4,4'-sulfonyl))bis(methylazanediyl))bis(butane-4,1-diyl))bis(triphenylphosphonium) (TBOP)]. Like GUALY's reagent, TBOP is a thiol-specific fluorogenic agent that is non-fluorescent but forms fluorescent thiol adducts in a thiol-specific fashion. Different than GUALY's reagent, TBOP reacts only with NPSH but not with PSH. TBOP was effectively used to image and quantify NPSH in live cells using fluorescence microscopy. TBOP is a complementary reagent to GUALY's reagent in determining the roles of PSH, NPSH, and total thiols in thiol-related physiological/pathological functions in live cells through fluorescence microscopy. Graphical Abstract Live cell imaging and quantification of non-protein thiols by TBOP.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Profession, South Dakota State University, 1055 Campanile Ave, Box 2202C, Brookings, SD, 57007, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Profession, South Dakota State University, 1055 Campanile Ave, Box 2202C, Brookings, SD, 57007, USA. Xiangming.Guan@sdstate.edu.
authors.forename: Xiangming
authors.initials: X
authors.lastname: Guan
mesh_terms: D001583:Benzoxazoles; D001681:Biological Assay; D045744:Cell Line, Tumor; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008175:Lung Neoplasms; D008856:Microscopy, Fluorescence; D015394:Molecular Structure; D009943:Organophosphorus Compounds / Q000737:chemistry*; D015203:Reproducibility of Results; D013438:Sulfhydryl Compounds
chemical_list: D001583:Benzoxazoles; D005456:Fluorescent Dyes; D009943:Organophosphorus Compounds; D013438:Sulfhydryl Compounds
keywords: Fluorescence microscopy; Live cell thiol imaging; Non-protein thiol; Thiol-specific fluorogenic agent