Non-protein thiol imaging and quantification in live cells with a novel benzofurazan sulfide triphenylphosphonium fluorogenic compound.
Non-protein thiol imaging and quantification in live cells with a novel benzofurazan sulfide triphenylphosphonium fluorogenic compound.
复制标题
使用新型苯并呋喃硫化物三苯基磷荧光化合物对活细胞中的非蛋白质硫醇成像和定量。
DOI：
10.1007/s00216-017-0285-y
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发表时间：
2017
影响因子：
4.3
通讯作者：
Guan,Xiangming
中科院分区：
文献类型：
--
作者：
Yang,Yang;Guan,Xiangming
硫醇 (-SH) 在生物系统中发挥着多种作用。它们分为蛋白质硫醇（PSH）和非蛋白质硫醇（NPSH）。由于硫醇在各种生理/病理功能中发挥重要作用，因此已开发出许多用于硫醇测定的分析方法。这些方法大多数是针对谷胱甘肽（NPSH 的主要形式）而开发的。这些方法中的大多数需要在分析前进行组织/细胞均质化。由于缺乏有效的硫醇特异性荧光/荧光试剂，活细胞中硫醇的成像和定量方法受到限制。活细胞中分析物的测定可以揭示细胞匀浆分析无法揭示的信息。之前，我们报道了硫醇特异性硫醇-硫化物交换反应。基于该反应，开发了苯并呋喃硫醚硫醇特异性荧光试剂。该试剂能够通过荧光显微镜有效地对活细胞中的总硫醇 (PSH+NPSH) 进行成像和定量。该试剂后来被命名为GUALY试剂。在这里，我们想通过合成一种新型苯并呋喃硫醚三苯基鏻衍生物来报告该工作的扩展[(((7,7′-thiobis(苯并[c][1,2,5]恶二唑-4,4′-磺酰基))双(甲基氮杂二基))双(丁烷-4,1-二基))双(三苯基鏻)(TBOP)]。与 GUALY 试剂一样，TBOP 是一种硫醇特异性荧光剂，它不发出荧光，但以硫醇特异性方式形成荧光硫醇加合物。与 GUALY 试剂不同，TBOP 只与 NPSH 发生反应，而不与 PSH 发生反应。 TBOP 可通过荧光显微镜有效地对活细胞中的 NPSH 进行成像和定量。 TBOP 是 GUALY 试剂的补充试剂，通过荧光显微镜测定活细胞中 PSH、NPSH 和总硫醇在硫醇相关生理/病理功能中的作用。图解摘要 TBOP 的活细胞成像和非蛋白硫醇定量
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 AbstractLive cell imaging and quantification of non-protein thiols by TBOP