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多种疾病状态有关。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成为了解细胞如何响应氧化应激刺激和帮助开发氧化应激相关疾病治疗的有价值的工具。适当的荧光剂或荧光剂与激光扫描共聚焦显微镜（LSCM）的组合已被广泛用于成像完整细胞中不同亚细胞结构中感兴趣的分子分布，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）由于其高氧化敏感性和高丰度，在决定细胞氧化应激状态中起着重要作用。硫醇密度随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。适当的硫醇特异性荧光剂或荧光剂结合LSCM可用于成像完整细胞中的硫醇密度和分布，从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开发用于硫醇测定。然而，大多数这些药物不是硫醇特异性的。它们用于硫醇分析需要结合分离技术，如高效液相色谱（HPLC）或液相色谱/质谱（LC/MS），不适合LSCM。因此，它们的使用仅限于组织匀浆的硫醇测定。只有少数荧光剂可用于LSCM对完整细胞进行硫醇测定，并且存在各种缺陷。鉴于氧化应激在各种细胞功能和疾病状态中发挥着重要作用，并且可以从完整的细胞研究中获得有价值的信息，因此开发可用于LSCM的硫醇特异性荧光剂是非常必要的。本应用的目的是开发硫醇特异性荧光剂，可用于用LSCM成像完整细胞内和细胞表面的硫醇密度。这些试剂的设计是基于硫醇特异性反应：硫醇-二硫（- s - s -）交换反应。这个反应是最广泛使用的硫醇特异性试剂的基础，埃尔曼试剂可以通过可见波长测量硫醇。本课题拟利用巯基特异性反应，将二硫键（- s - s -）整合到研究充分、应用广泛的苯并呋喃唑荧光结构中。具体而言，将合成合理设计的硫醇特异性荧光剂及其硫醇加合物。它们的荧光性质将被充分表征。在不同程度硫醇氧化应激的中国仓鼠卵巢（CHO）细胞中，通过LSCM验证它们在测定细胞内硫醇分布和细胞表面硫醇密度方面的应用。

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.