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Inhibited autoxidations-monitored either by O2 consumption or hydroperoxide formation-are the most reliable way to obtain kinetic and stoichiometric information on the activity of radical-trapping antioxidants (RTAs). While many comparatively simple "antioxidant assays" (e.g., the DPPH assay) have supplanted these in popularity, they are generally very poor substitutes since they often do not employ peroxyl radicals as the oxidant and do not account for both the kinetics and stoichiometry of the radical-trapping reaction(s). In an effort to make inhibited autoxidations as simple as the most popular "antioxidant assays", we have developed a spectrophotometric approach for monitoring reaction progress in inhibited autoxidations. The approach employs easily prepared 1-phenylbutadiene-conjugated or styrene-conjugated BODIPY chromophores (PBD-BODIPY or STY-BODIPY, respectively) as signal carriers that co-autoxidize along with a hydrocarbon substrate. We show that inhibition rate constants (kinh) are accurately determined for a range of phenolic and diarylamine RTAs using this approach and that mechanistic experiments, such as kinetic isotope effects and kinetic solvent effects, are equally easily carried out. Moreover, synergistic interactions between RTAs, as well as the unconventional activity of diarylamine RTAs, are captured using this methodology. Lastly, we show that the approach can be employed for monitoring reactions in aqueous solution.

Highlights d FENIX enables screening or counter-screening of lipid peroxidation inhibitors d Equally simple to the DPPH assay, but relays information on kinetics and stoichiometry d FENIX in liposomes predicts the anti-ferroptotic potency of antioxidants in cells d H-bonding to the phospholipid head group attenuates RTA activity in lipid bilayers

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 ≈ 10 to >10 M s) 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.

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