Characterization of Free Radicals in γ-irradiated Polycrystalline Uridine 5′-monophosphate: A Study Combining ESR, Spin-trapping and HPLC

Summary

Free radicals generated in γ-irradiated polycrystalline uridine 5′-monophosphate (5′-UMP) were studied by ESR, spin-trapping and high-performance liquid chromatograph (HPLC). After γ-irradiation at 0°C (70 kGy), poly-crystalline 5′-UMP was dissolved in an anaerobic aqueous solution of 2-methyl-2-nitrosopropane as a spin trap at room temperature. Since an ESR spectrum consisting of several components was observed immediately after irradiation, these components were separated with reverse-phase HPLC in the ion-suppression mode and again analysed by ESR spectrometry. Although HPLC ultimately gave four spin-adducts, one component that was originally present disappeared during HPLC. Spin adducts due to two types of C6 radicals were identified. One of these was thought to be formed by electron addition and subsequent protonation at the C6 position, and the other was presumed to be produced by electron addition and subsequent protonation at the O₄ position. The spin adducts derived from the C5 and C5′ radicals were also identified. The spin adduct that disappeared during HPLC was thought to correspond to the C4′-centred radical. Computer simulation of ESR spectra was carried out to estimate the hyperfine splitting constants.