MECHANISTIC STUDIES ON THE FORMATION OF AMINOXYL RADICALS FROM 5,5-DIMETHYL-1-PYRROLINE-N-OXIDE IN FENTON SYSTEMS - CHARACTERIZATION OF KEY PRECURSORS GIVING RISE TO BACKGROUND ESR SIGNALS

To assign unidentified ESR signals obtained in a Fenton system with a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), fundamental reactions of DMPO have been thoroughly investigated. When the concentration of Fe2+ in Fenton systems exceeded 1% of that of DMPO, background ESR signals totally distinguishable from 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxy (DMPO-OH) appeared, and simultaneously absorbance at 520 nm increased, which was characteristic of hydroxamic acids complexed with Fe3+. To prove the postulated formation of hydroxamic acid as a key intermediate, DMPO was subjected to Fenton reaction or was treated with Fe3+ in aqueous solution to produce DMPO-OH (Makino et al. Biochem. Biophys. Res. Commun. 172, 1073 (1990)), and a major product was isolated by RPLC. Based on H-1, C-13 NMR and MS measurements, the structure of the major product was assigned to 1-hydroxy-5,5-dimethyl-1-pyrrolid-2-one (HDMPN) having a hydroxamic acid structure. Temperature dependence of NMR spectra and careful analysis of the fragmentation patterns in MS further revealed that HDMPN was present in equilibrium with 2-hydroxy-5,5-dimethyl-1-pyrroline-N-oxide (HDMPO), a tautomer of HDMPN. It has also been shown that the oxidation of DMPO yielding HDMPN and HDMPO occurs via DMPO-OH and is driven by Fe3+. These oxidation products (HDMPN and HDMPO) were readily converted to the corresponding ESR visible aminoxyl radicals by oxidation and .OH addition in aqueous solution, respectively, and superimposition of the ESR signals arising from these radicals accounts for the background signals observed in Fenton systems with DMPO as a spin trap.