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.
