The Catalytic Reaction of Nitroxides with Peroxyl Radicals and Its Relevance to Their Cytoprotective Properties.

Sterically-hindered nitroxides such as 2,2,6,6-tetramethylpiperidin- N-oxyl (TEMPO) have long been ascribed antioxidant activity that is thought to underlie their chemopreventive and anti-aging properties. However, the most commonly invoked reactions in this context-combination with an alkyl radical to give a redox inactive alkoxyamine or catalysis of superoxide dismutation-are unlikely to be relevant under (most) physiological conditions. Herein, we characterize the kinetics and mechanisms of the reactions of TEMPO, as well as an N-arylnitroxide and an N, N-diarylnitroxide, with alkylperoxyl radicals, the propagating species in lipid peroxidation. In each of aqueous solution and lipid bilayers, they are found to be significantly more reactive than Vitamin E, Nature's premier radical-trapping antioxidant (RTA). Inhibited autoxidations of THF in aqueous buffers reveal that nitroxides reduce peroxyl radicals by electron transfer with rate constants ( k ≈ 106 to >107 M-1 s-1 ) that correlate with the standard potentials of the nitroxides ( E° ≈ 0.75-0.95 V vs NHE) and that this activity is catalytic in nitroxide. Regeneration of the nitroxide occurs by a two-step process involving hydride transfer from the substrate to the nitroxide-derived oxoammonium ion followed by H-atom transfer from the resultant hydroxylamine to a peroxyl radical. This reactivity extends from aqueous solution to phosphatidylcholine liposomes, where added NADPH can be used as a hydride donor to promote nitroxide recycling, as well as to cell culture, where the nitroxides are shown to be potent inhibitors of lipid peroxidation-associated cell death (ferroptosis). These insights have enabled the identification of the most potent nitroxide RTA and anti-ferroptotic agent yet described: phenoxazine- N-oxyl.