5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO, 2), a new spin trap, has been synthesized via a two-step synthetic route, and its ability to spin trap oxy radicals in biological milieu has been addressed. The in vitro spin trapping of hydroxyl and superoxide radicals was investigated in a phosphate buffer 0.1 M, and the hyperfine coupling constants of the spin adducts were determined. The rates of spin trapping of hydroxyl and superoxide radicals with 2 were found to be close to those reported for 5,5-dimethyl-1-pyrroline N-oxide (DMPO). However, the DEPMPO-superoxide spin adduct was shown to be significantly more persistent (15 times at pH 7) than the DMPO--superoxide spin adduct. Using 2 as a spin trap, the production of superoxide has been clearly characterized during the reperfusion of ischemic isolated rat hearts.

Using a novel phosphorylated spin trap, 5-diethoxy-phosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), an analog of the commonly used trap 5,5'-dimethyl-1-pyrroline N-oxide (DMPO), we have investigated the reactions of sulfur-centered radicals produced from the oxidation of thiols and sulfite by peroxynitrite. The predominant species trapped in all cases are the corresponding sulfur-centered radicals, i.e. glutathionyl radical (GS) from glutathione (GSH), N-acetyl-DL-penicillamine thiyl radical (S-NAP) from N-acetyl-DL-penicillamine (NAP) and sulfate anion radical (SO3-) from sulfite. These radicals consume molecular oxygen forming either peroxyl or superoxide anion radicals. GS, S-NAP, and (SO3-)-derived radicals react with ammonium formate to form the carbon dioxide anion radical (CO2-). Further support of spin adduct assignments and radical reactions are obtained from photolysis of S-nitrosoglutathione and S-nitroso-N-acetyl-DL-penicillamine. We conclude that the direct reaction of peroxynitrite with thiols and sulfate forms thiyl and sulfate anion radicals, respectively, by a hydroxyl radical-independent mechanism. Pathological implications of thiyl radical formation and subsequent oxyradical-mediated chain reactions are discussed. Oxygen activation by thiyl radicals formed during peroxynitrite-mediated oxidation of glutathione may limit the effectiveness of GSH against peroxynitrite-mediated toxicity in cellular systems.

5-Diethoxyphosphoryl-5-methyl-1 -pyrroline N-oxide (DEPMPO), a newly synthesized spin trap, is used for the in vitro spin trapping of hydroxyl and superoxide radicals, and though the spin trapping rates are close to those reported for 5,5-dimethyl-1 -pyrroline N-oxide (DMPO), the DEPMPO-superoxide spin adduct is significantly more persistent than its DMPO analogue, a difference which allows the detection of superoxide during the reperfusion of ischaemic isolated rat hearts.

Three analogues of 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO, 1) labelled with two (1-d2), five (1-d5) or seven (1-d7)2H were synthesized and used to trap the tert-butylperoxyl radical. The EPR spectra of 1-d2-OOBu(t) and 1-d7-OOBu(t) spin adducts exhibited more straightforward patterns and better signal to noise ratio than those obtained with 1 or 1-d5. The use of the easily available 1-d2 as spin trap could help significantly the analysis of the EPR signals when the signal of either superoxide or alkylperoxyl spin adduct is superimposed with the signals of other spin adducts.