| Peroxyl and other radical oxidants react with stable cyclic nitroxides, such as the piperidinyl and pyrrolidinyl nitroxides to form initially the one electron oxidation product, the oxoammonium cation. For most of the nitroxides studied thus far, the oxoammonium cation can in part be regenerated to the nitroxide through reduction by solution constituents. The reaction mechanisms, however, remain a matter of debate. Further, the highly-sensitive, quantitative determination of peroxyl and other radical oxidants has yet to be achieved, posing a major hurdle to a further understanding of the impact of peroxyl radicals in many biological and environmental processes.;A unique, amino-pyrrolidinyl nitroxide, 3-amino-2,2,5,5,-tetramethyl-1-pyrrolidinyloxy (3-ap) is shown to undergo an irreversible reaction with peroxyl radicals and other radical oxidants to generate a diamagnetic product. When a fluorophore, fluorescamine is covalently coupled to the nitroxide, the resulting compound (3-apf, or I) has very low fluorescence quantum yield. Upon reaction with peroxyl and other radical oxidants, the quantum yield of the product increases dramatically (∼100 fold), thus 3-ap or 3-apf can be used as a highly sensitive and versatile probe to determine oxidant production optically, either by monitoring the changes in fluorescence intensity using a spectrofluorometer, by HPLC analysis with fluorescence detection, or by a combination of both approaches.;By changing the [O2]/[nitroxide] ratio, it is shown that peroxyl radicals can be detected and quantified preferentially in the presence of other radical oxidants, such as ·NO2 and CO3 ·-. When decreasing the [O2]/[nitroxide] ratio, the oxidation product decreases, with a concomitant increase of the alkoxylamine product resulting from reaction of 3-ap (3-apf) with alkyl radicals.;High resolution mass spectrometry and NMR studies indicate that 3-ap is oxidized to form a cyclic peroxide structure, while 3-apf is oxidized to form a cyclic - NH-O- structure, with this difference resulting possibly from the presence of the fluorescamine moiety in 3-apf.;Detection of photochemically produced peroxyl radicals is achieved by employing 3-ap alone, followed by derivatization with fluorescamine, while detection of thermally-generated peroxyl radicals employs 3-apf. Preliminary applications include the detection of peroxyl radicals generated thermally in soybean phosphatidylcholine liposomes by 3-apf and produced photochemically in tap water by 3-ap. |
