Temperature-dependence of radical-trapping activity of phenoxazine, phenothiazine and their aza-analogues clarifies the way forward for new antioxidant design.

The prediction and/or rationalization of diarylamine radical-trapping antioxidant (RTA) activity at the elevated temperatures where they are most useful presents a significant challenge, precluding the development of new technology. Whilst structure-activity relationships at ambient temperatures are well established, their predictive capacity at elevated temperatures is poor due to competing reactions. A striking example involves phenoxazine, which is a superior RTA relative to its sulfur analog phenothiazine at ambient temperature (e.g. k = 3.9 × 107 vs. 7.6 × 106 M-1 s-1 at 37 °C, respectively), but is demonstrably inferior at elevated temperatures. Despite being inherently less oxidizable in electrochemical experiments and high-accuracy computations, phenoxazine is more rapidly consumed than phenothiazine in autoxidations at 160 °C - a result which we attribute to a lower reorganization energy barrier to oxidation. Given these observations, we surmised that incorporation of an electronegative N-atom into the phenoxazine ring system would decrease the driving force for oxidation and 'rescue' its activity. Indeed, this was found to be the case for nitrogen incorporation at any position, regardless of the impact on the inherent RTA activity. Analogous experiments were carried out on phenothiazines into which nitrogen atoms were incorporated, revealing little benefit at 160 °C. These results suggest that for highly reactive diarylamines (i.e. those with k > 106 M-1 s-1), further enhancements in reactivity do not materially improve their ability to inhibit hydrocarbon autoxidation at elevated temperatures. Instead, their stability to one-electron oxidation determines their efficacy.

Temperature-Dependent Effects of Alkyl Substitution on Diarylamine Antioxidant Reactivity.

Alkylated diphenylamines are among the most efficacious radical-trapping antioxidants (RTAs) for applications at elevated temperatures since they are able to trap multiple radical equivalents due to catalytic cycles involving persistent diphenylnitroxide and diphenylaminyl radical intermediates. We have previously shown that some heterocyclic diarylamine RTAs possess markedly greater efficacy than typical alkylated diphenylamines, and herein, report on our efforts to identify optimal alkyl substitution of the scaffold, which we had found to be the ideal compromise between reactivity and stability. Interestingly, the structure-activity relationships differ dramatically with temperature: para-alkyl substitution slightly increased reactivity and stoichiometry at 37 and 100 °C due to more favorable (stereo)electronic effects and corresponding diarylaminyl/diarylnitroxide formation, while ortho-alkyl substitution slightly decreased both reactivity and stoichiometry. No such trends were evident at 160 °C; instead, the compounds were segregated into two groups based on the presence/absence of benzylic C-H bonds. Electron spin resonance spectroscopy indicates that increased efficacy was associated with lesser diarylnitroxide formation, and deuterium-labeling suggests that this is due to abstraction of the benzylic H atom, precluding nitroxide formation. Computations predict that this reaction path is competitive with established fates of the diarylaminyl radical, thereby minimizing the formation of off-cycle products and leading to significant gains in high-temperature RTA efficacy.

Inhibition of hydrocarbon autoxidation by nitroxide-catalyzed cross-dismutation of hydroperoxyl and alkylperoxyl radicals.

Nitroxides are putative intermediates in the accepted reaction mechanisms of the diarylamine and hindered amine antioxidants that are universally added to preserve synthetic and natural hydrocarbon-based materials. New methodology which enables monitoring of hydrocarbon autoxidations at low rates of radical generation has revealed that diarylnitroxides and hindered nitroxides are far better inhibitors of unsaturated hydrocarbon autoxidation than their precursor amines, implying intervention of a different mechanism. Experimental and computational investigations suggest that the nitroxides catalyze the cross-dismutation of hydroperoxyl and alkylperoxyl radicals to yield O2 and a hydroperoxide, thereby halting the autoxidation chain reaction. The hydroperoxyl radicals - key players in hydrocarbon combustion, but essentially unknown in autoxidation - are proposed to derive from a tunneling-enhanced intramolecular (1,4-) hydrogen-atom transfer/elimination sequence from oxygenated radical addition intermediates. These insights suggest that nitroxides are preferred additives for the protection of hydrocarbon-based materials from autoxidation since they exhibit catalytic activity under conditions where their precursor amines are less effective and/or inefficiently converted to nitroxides in situ.

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.

Phenoxazine: A Privileged Scaffold for Radical-Trapping Antioxidants.

Diphenylamines are widely used to protect petroleum-derived products from autoxidation. Their efficacy as radical-trapping antioxidants (RTAs) relies on a balance of fast H-atom transfer kinetics and stability to one-electron oxidation by peroxidic species. Both H-atom transfer and one-electron oxidation are enhanced by substitution with electron-donating substituents, such as the S-atom in phenothiazines, another important class of RTA. Herein we report the results of our investigations of the RTA activity of the structurally related, but essentially ignored, phenoxazines. We find that the H-atom transfer reactivity of substituted phenoxazines follows an excellent Evans-Polanyi correlation spanning kinh = 4.5 × 106 M-1 s-1 and N-H BDE = 77.4 kcal mol-1 for 3-CN,7-NO2-phenoxazine to kinh = 6.6 × 108 M-1 s-1 and N-H BDE = 71.8 kcal mol-1 for 3,7-(OMe)2-phenoxazine (37 °C). The reactivity of the latter compound is the greatest of any RTA ever reported and is likely to represent a reaction without an enthalpic barrier since log A for this reaction is likely ∼8.5. The very high reactivity of most of the phenoxazines studied required the determination of their kinetic parameters by inhibited autoxidations in the presence of a very strong H-bonding cosolvent (DMSO), which slowed the observed rates by up to 2 orders of magnitude by dynamically reducing the equilibrium concentration of (free) phenoxazine as an H-atom donor. Despite their remarkably high reactivity toward peroxyl radicals, the phenoxazines were found to be comparatively stable to one-electron oxidation relative to diphenylamines and phenothiazines (E° ranging from 0.59 to 1.38 V vs NHE). Thus, phenoxazines with comparable oxidative stability to commonly used diphenylamine and phenothiazine RTAs had significantly greater reactivity (by up to 2 orders of magnitude). Computations suggest that this remarkable balance in H-atom transfer kinetics and stability to one-electron oxidation results from the ability of the bridging oxygen atom in phenoxazine to serve as both a π-electron donor to stabilize the aminyl radical and σ-electron acceptor to destabilize the aminyl radical cation. Perhaps most excitingly, phenoxazines have "non-classical" RTA activity, where they trap >2 peroxyl radicals each, at ambient temperatures.

Diazaphenoxazines and Diazaphenothiazines: Synthesis of the "Correct" Isomers Reveals They Are Highly Reactive Radical-Trapping Antioxidants.

The preparation of 2,4-diazaphenothiazines and 2,4-diazaphenoxazines via a copper-catalyzed intramolecular amination is described. Literature approaches which utilize easily accessed (2'-aminophenyl) 4-pyri(mi)dyl sulfides undergo a Smiles rearrangement that gives rise to the 1,3-diaza derivatives instead, confirmed by X-ray crystallography. Inversion of the polarity of the cyclization avoids the rearrangement and affords the desired products. Preliminary kinetic studies suggest that 2,4-diazaphenothiazines and diazaphenoxazines, but not the 1,3-diaza isomers, are remarkably potent radical-trapping antioxidants.

Acid Is Key to the Radical-Trapping Antioxidant Activity of Nitroxides.

Persistent dialkylnitroxides (e.g., 2,2,6,6-tetramethylpiperidin-1-oxyl, TEMPO) play a central role in the activity of hindered amine light stabilizers (HALS)-additives that inhibit the (photo)oxidative degradation of consumer and industrial products. The accepted mechanism of HALS comprises a catalytic cycle involving the rapid combination of a nitroxide with an alkyl radical to yield an alkoxyamine that subsequently reacts with a peroxyl radical to eventually re-form the nitroxide. Herein, we offer evidence in favor of an alternative reaction mechanism involving the acid-catalyzed reaction of a nitroxide with a peroxyl radical to yield an oxoammonium ion followed by electron transfer from an alkyl radical to the oxoammonium ion to re-form the nitroxide. In preliminary work, we showed that TEMPO reacts with peroxyl radicals at diffusion-controlled rates in the presence of acids. Now, we show that TEMPO can be regenerated from its oxoammonium ion by reaction with alkyl radicals. We have determined that this reaction, which has been proposed to be a key step in TEMPO-catalyzed synthetic transformations, occurs with k ∼ 1-3 × 10(10) M(-1) s(-1), thereby enabling it to compete with O2 for alkyl radicals. The addition of weak acids facilitates this reaction, whereas the addition of strong acids slows it by enabling back electron transfer. The chemistry is shown to occur in hydrocarbon autoxidations at elevated temperatures without added acid due to the in situ formation of carboxylic acids, accounting for the long-known catalytic radical-trapping antioxidant activity of TEMPO that prompted the development of HALS.

A Continuous Visible Light Spectrophotometric Approach To Accurately Determine the Reactivity of Radical-Trapping Antioxidants.

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.

Polysulfide-1-oxides react with peroxyl radicals as quickly as hindered phenolic antioxidants and do so by a surprising concerted homolytic substitution.

Polysulfides are important additives to a wide variety of industrial and consumer products and figure prominently in the chemistry and biology of garlic and related medicinal plants. Although their antioxidant activity in biological contexts has received only recent attention, they have long been ascribed 'secondary antioxidant' activity in the chemical industry, where they are believed to react with the hydroperoxide products of autoxidation to slow the auto-initiation of new autoxidative chain reactions. Herein we demonstrate that the initial products of trisulfide oxidation, trisulfide-1-oxides, are surprisingly reactive 'primary antioxidants', which slow autoxidation by trapping chain-carrying peroxyl radicals. In fact, they do so with rate constants (k = 1-2 × 104 M-1 s-1 at 37 °C) that are indistinguishable from those of the most common primary antioxidants, i.e. hindered phenols, such as BHT. Experimental and computational studies demonstrate that the reaction occurs by a concerted bimolecular homolytic substitution (SH2), liberating a perthiyl radical - which is ca. 16 kcal mol-1 more stable than a peroxyl radical. Interestingly, the (electrophilic) peroxyl radical nominally reacts as a nucleophile - attacking the of the trisulfide-1-oxide - a role hitherto suspected only for its reactions at metal atoms. The analogous reactions of trisulfides are readily reversible and not kinetically competent to inhibit hydrocarbon autoxidation, consistent with the longstanding view that organosulfur compounds must be oxidized to afford significant antioxidant activity. The reactivity of trisulfides and their oxides are contrasted with what is known of their shorter cousins and predictions are made and tested with regards to the reactivity of higher polysulfides and their 1-oxides - the insights from which may be exploited in the design of future antioxidants.

Unprecedented inhibition of hydrocarbon autoxidation by diarylamine radical-trapping antioxidants.

The reactivities of novel heterocyclic diarylamine radical-trapping antioxidants (RTAs) are profiled in a heavy hydrocarbon at 160 °C, conditions representative of those at which diphenylamine RTAs are used industrially. While carboxylic acids produced during the autoxidation are shown to deactivate these more basic RTAs, the addition of a sacrificial base leads to efficacies that are unprecedented in the decades of academic and industrial research in this area.

The catalytic mechanism of diarylamine radical-trapping antioxidants.

Diarylamine radical-trapping antioxidants are important industrial additives, finding widespread use in petroleum-derived products. They are uniquely effective at elevated temperatures due to their ability to trap multiple radicals per molecule of diarylamine. Herein we report the results of computational and experimental studies designed to elucidate the mechanism of this remarkable activity. We find that the key step in the proposed catalytic cycle-decomposition of the alkoxyamine derived from capture of a substrate-derived alkyl radical with a diarylamine-derived nitroxide-proceeds by different mechanisms depending on the structure of both the substrate and the diarylamine. N,N-Diarylalkoxyamines derived from saturated substrates and diphenylamines decompose by N-O homolysis followed by disproportionation. Alternatively, those derived from unsaturated substrates and diphenylamines, or saturated substrates and N-phenyl-ß-naphthylamine, decompose by an unprecedented concerted retro-carbonyl-ene reaction. The alkoxyamines that decompose by the concerted process inhibit hexadecane autoxidations at 160 °C to the same extent as the corresponding diarylamine, whereas those alkoxyamines that decompose by the N-O homolysis/disproportionation pathway are much less effective. This suggests that the competing cage escape of the alkoxyl radicals following N-O homolysis leads to significantly less effective regeneration of diarylamines and implies that the catalytic efficiency of diarylamine antioxidants is substrate-dependent. The results presented here have significant implications in the future design of antioxidant additives: diarylamines designed to yield intermediate alkoxyamines that undergo the retro-carbonyl-ene reaction are likely to be much more effective than existing compounds and will display catalytic radical-trapping activities at lower temperatures due to lower barriers to regeneration.