NEW FLUORESCENT THIOL PROBES FOR FLOW CYTOMETRY AND PCR

我们建议合成新的探针用于测定细胞内的 谷胱甘肽和其他细胞硫醇。在第一阶段，我们合成了五种新的 优于现有硫醇染料CPM的硫醇反应性荧光团 关于量子产率和斯托克斯位移和灵敏度的辅助DACM 用于硫醇定量。至少有一种染料比现有的更好 细胞内硫醇染料对于 谷胱甘肽在其他细胞内硫醇的存在下。在第二阶段，我们 计划制备具有相同基本分子结构的其他染料 但具有将赋予较长波长吸收的取代基， 发射和在水介质中更好的溶解性。合成的探针 将在体外和细胞培养物中测试吸收和释放 当与谷胱甘肽和其他蛋白质硫醇结合时的性质。细胞 用于流式细胞术的染料的毒性和适用性也将被 测定新的染料也将与现有的染料进行评估， 荧光标记物在其他应用中，例如HPLC检测 硫醇、酶测定和PCR技术。

We propose to synthesize new probes for the determination of intracellular glutathione and other cellular thiols. In Phase I we synthesized five new thiol reactive fluorophores which are superior to existing thiol dyes CPM aid DACM with respect to quantum yield and Stokes shifts and sensitivity for thiol quantitation. At least one of the dyes was better than existing intracellular thiol dyes with respect to selectivity and sensitivity for glutathione in the presence of other intracellular thiols. In Phase II we plan to prepare additional dyes with the same basic molecular structure but with substituents which will impart longer wavelength absorption and emission and better solubility in aqueous media. The synthesized probes will be tested in vitro and in cell cultures for absorption and emission properties when bound to glutathione and other protein thiols. Cell toxicity and suitability of the dyes for flow cytometry will also be determined. The new dyes will also be evaluated against existing dyes as fluorescent markers in other applications such as HPLC detection of thiols, enzyme assays, and in PCR technology.