Design and development of a fluorescent probe for monitoring hydrogen peroxide using photoinduced electron transfer.

A novel fluorescent probe, 7-hydroxy-2-oxo-N-(2-(diphenylphosphino)ethyl)-2H-chromene-3-carboxamide (DPPEA-HC) was developed for use in monitoring hydrogen peroxide (H2O2) production. DPPEA-HC, which consists of a diphenylphosphine moiety and a 7-hydroxycoumarin moiety, reacts with H2O2 to form DPPEA-HC oxide, which is analogous to the reaction of triphenylphosphine with hydroperoxides such as H2O2 to form triphenylphosphine oxide. Photoinduced electron transfer (PET) was applied in the design of DPPEA-HC. Since the diphenylphosphine moiety and the 7-hydroxycoumarin moiety would act as the PET donor and the acceptor, respectively, it would be expected that DPPEA-HC would rationally cancel the PET process via the formation of DPPEA-HC oxide, based on the calculated energy levels of the donor and the acceptor moieties using the B3LYP/6-31G*//AM1 method. The fluorescence intensity of DPPEA-HC increased on the addition of a H2O2 solution in 100 mM sodium phosphate buffer (pH7.4), as predicted from the energy level calculation and a good correlation between increase in the fluorescence of DPPEA-HC and the concentration of H2O2 was observed. DPPEA-HC was also fluoresced by H2O2, which was enzymatically produced in xanthine/xanthine oxidase/superoxide dismutase (XA/XOD/SOD) system. The increase in the fluorescence of DPPEA-HC in the presence of H2O2 immediately ceased on the addition of catalase (CAT), which catalyzes the disproportionation of H2O2. In addition, DPPEA-HC was found to have a much higher selectivity for H2O2 and a greater resistance to autoxidation than 2',7'-dichlorodihydrofluoresein (DCFH). Time-resolved fluorescence measurements of DPPEA-HC and DPPEA-HC oxide confirmed that the fluorescence off/on switching mechanism of DPPEA-HC is based on the PET on/off control.