Fluorescence "off" and "on" signalling of esculetin in the presence of copper and thiol: a possible implication in cellular thiol sensingElectronic supplementary information (ESI) available: Other supporting experimental data. See DOI: 10.1039/c8pp00157j

The interaction of the cupric ion with esculetin, a dihydroxy coumarin derivative, was studied by absorption and fluorescence spectroscopic methods in aqueous medium. Esculetin formed a complex in the presence of the cupric ion which was characterised by the shift in its absorption band from 350 nm to 389 nm and the quenching of its fluorescence intensity at 466 nm. From Job's plot and fluorescence quenching studies, the stoichiometry of the copper ion and esculetin in the complex was estimated to be 1 : 2 respectively. Interestingly, the incubation of the Cu( ii )-esculetin complex with a thiol peptide, glutathione (GSH), showed restoration of the fluorescence intensity as well as absorption maxima to that of pure esculetin. Incubation with other common thiol and non-sulphur amino acids did not show a similar restoration of the photophysical properties of the complex except in the case of cysteine. Mechanistically, it was evident that two molecules of GSH were consumed in reducing the Cu( ii )-esculetin complex, which subsequently split into the copper ion and esculetin. In this process GSH was converted into oxidised GSH (GSSG) as evident from the mass spectroscopy and HPLC studies. The above florescence regeneration behaviour of the copper-esculetin system in the presence of GSH was also observed in the cellular system using Chinese hamster ovary (CHO) as model cells. In conclusion, these studies may find application in developing sensors for detecting the cellular thiol level. The fluorescence intensity of esculetin was drastically reduced in the presence of a copper ion, which was regenerated in the presence of GSH. The copper-esculetin system was able to detect GSH in a cellular model.