Thiol Specific Fluorogenic Agents for Laser Scanning Confocal Microscopy

描述(由申请人提供)：氧化应激已被发现与各种疾病状态有关。一种可用于监测完整细胞中细胞和亚细胞氧化应激的分析工具将是了解细胞如何对氧化应激刺激做出反应的宝贵工具，并有助于开发氧化应激相关疾病的治疗方法。将合适的荧光或荧光剂与激光扫描共聚焦显微镜(LSCM)相结合，已被广泛用于成像目标分子在完整细胞中不同亚细胞结构中的分布，并将成为监测不同亚细胞位置氧化应激的一种选择方法。硫醇(-SH)由于其高的氧化敏感性和高的丰度，在确定细胞氧化应激状态方面起着重要作用。硫醇密度随着氧化应激的增加而降低，其状态已被用作反映细胞氧化应激的参数。合适的硫醇特异性荧光或荧光剂与激光共聚焦显微镜结合可用于成像完整细胞中的硫醇密度和分布，进而反映亚细胞水平的氧化还原状态。许多荧光剂和荧光剂已被开发用于硫醇分析。然而，这些试剂中的大多数都不是硫醇专一性的。它们在硫醇分析中的使用需要与分离技术相结合，如高效液相色谱(HPLC)或液相色谱/质谱仪(LC/MS)，而不适合于LSCM。因此，它们的使用仅限于组织匀浆的硫醇分析。用激光扫描共聚焦显微镜检测完整细胞中的硫醇只有几种荧光试剂可用，存在着各种缺陷。鉴于氧化应激在各种细胞功能、疾病状态中的重要作用，以及从硫醇分布的完整细胞研究中可以获得的有价值的信息，开发可用于激光扫描共聚焦显微镜的硫醇特异性荧光剂是非常必要的。这一应用的目的是开发硫醇特异性荧光剂，该荧光剂可以用激光扫描共聚焦显微镜对完整细胞内和细胞表面的硫醇密度进行成像。这些试剂的设计是基于硫醇特有的反应：硫醇-二硫键(-S-S-)交换反应。这一反应是最广泛使用的硫醇特定试剂Ellman试剂的基础，Ellman试剂能够通过可见波长测量硫醇。这个项目是利用硫醇专属反应，将二硫键(-S-S-)整合到一个研究得很好并得到广泛应用的苯并呋喃氮类荧光结构中。具体地说，将合成合理设计的硫醇特异性荧光剂及其硫醇加合物。它们的荧光特性将得到充分的表征。通过中国仓鼠卵巢(CHO)细胞在不同程度的硫醇氧化应激下的激光扫描共聚焦显微镜(LSCM)，验证了它们在确定完整细胞内硫醇分布和细胞表面硫醇密度方面的应用。 公共卫生相关性：氧化应激与各种健康问题有关。一种能够检测完整细胞中氧化应激状态的方法将有助于了解氧化应激在各种疾病中的作用，并有助于开发氧化应激相关疾病的治疗方法。这项拟议的应用旨在开发一种可用于检测完整细胞中氧化应激状态的方法。

DESCRIPTION (provided by applicant): Oxidative stress has been found to be associated with various disease states. An analytical tool which can be used to monitor cellular and subcellular oxidative stress in intact cells will be a valuable tool in understanding how cells respond to oxidative stress stimuli and help develop treatments to oxidative stress- associated diseases. A combination of a proper fluorogenic or fluorescent agent with laser scanning confocal microscopy (LSCM) has been widely used to image the distribution of the molecule of interest in different subcellular structures in intact cells and will be a method of choice in monitoring oxidative stress at different subcellular locations. Thiol (-SH) status plays a significant role in determining cellular oxidative stress status due to its high susceptibility to oxidation and high abundance. Thiol density decreases as oxidative stress increases and its status has been used as a parameter to reflect cellular oxidative stress. A proper thiol specific fluorogenic or fluorescent agent in combination with LSCM can be used to image thiol density and distribution in intact cells, which in turns reflects redox state at subcellular levels. Numerous fluorogenic and fluorescent agents have been developed for thiol assays. However, most of these agents are not thiol specific. Their use for thiol assays needs to be coupled with a separation technique, such as high performance liquid chromatography (HPLC) or liquid chromatography/mass spectrometry (LC/MS), and is not suitable for LSCM. Consequently, their use is limited to thiol assays of tissue homogenates. Only a few fluorogenic agents are available for thiol assays in intact cells by LSCM with various drawbacks. In view of the significant role oxidative stress plays in various cellular functions, disease states, and valuable information which can be gained from an intact cell study of thiol distribution, it is highly desirable to develop thiol specific fluorogenic agents which can be used for LSCM. The aim of this proposed application is to develop thiol specific fluorogenic agents which can be used to image thiol density intracellularly and on the cell surface in intact cells with LSCM. The design of these agents is based on a thiol specific reaction: thiol-disulfide (-S-S-) exchange reaction. This reaction is the base of the most widely used thiol specific reagent, Ellman's reagent which enables measurement of thiols through a visible wavelength. This proposed project is to take advantage of the thiol specific reaction and integrate the disulfide bond (-S-S-) into a well studied and widely used benzofurazan fluorogenic structure. Specifically, rationally designed thiol specific fluorogenic agents and their thiol adducts will be synthesized. Their fluorescence properties will be fully characterized. Their application in determining intracellular thiol distribution and thiol density on the cell surface in intact cells will be validated by LSCM with Chinese hamster ovary (CHO) cells under different extents of thiol oxidative stress. PUBLIC HEALTH RELEVANCE: Oxidative stress has been associated with various health issues. A method which can detect the status of oxidative stress in intact cells will help understand the role of oxidative stress in various diseases and facilitate development of treatments to oxidative stress-associated diseases. This proposed application is aimed to develop a method which can be used to detect the status of oxidative stress in intact cells.