Recent Insights on Hydrogen Atom Transfer in the Inhibition of

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Recent Insights on Hydrogen Atom Transfer in the Inhibition of Hydrocarbon Autoxidation Published as part of the Accounts of Chemical Research special issue “Hydrogen Atom Transfer”. Jia-Fei Poon and Derek A. Pratt*

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Department of Chemistry and Biomolecular Sciences, University of Ottawa, 10 Marie Curie Pvt. Ottawa, ON K1N 6N5, Canada CONSPECTUS: Autoxidation, the free radical chain reaction that nominally inserts O2 into hydrocarbons to give peroxides, is primarily responsible for the degradation of all organic materials. Peroxyl radicals propagate autoxidation mainly by abstraction of labile H-atoms from the hydrocarbons, whereas radical-trapping antioxidants (RTAs) inhibit autoxidation by donating an Hatom to the peroxyl radical to give a nonpropagating radical. As such, a detailed understanding of the kinetics and thermodynamics of H-atom transfer (HAT) reactions to peroxyl radicals, and the effects of sterics, electronics, and medium thereupon, is key to understanding the mechanisms and products of autoxidation and the ability of RTAs to inhibit it. Due to their relatively weak O−H and N−H bonds, phenols and aromatic amines have long been utilized as RTAs, but only phenols have been extensively optimized to maximize their reactivity. Amines offer greater structural variability owing to their trivalent central nitrogen atom. Simply linking the two aromatic rings of a diarylamine to afford a phenoxazine offers profound differences in HAT reactivity: 1000-times greater than diphenylamine and 10-fold more reactive than α-tocopherol, Nature’s optimized phenolic RTA. Thus, phenoxazines are an exciting scaffold for RTA development. Indeed, we have recently shown that ring substitution of phenoxazine or 2,4diazaphenoxazine can yield compounds that undergo barrierless HAT reactions with peroxyl radicals. Amines also have the distinct advantage that they can react with peroxyl radicals to yield nitroxides, which can inhibit autoxidation in a catalytic manner utilizing the substrate itself as the stoichiometric reductant. Herein we provide an account of our recent efforts to understand how they manage this feat, which have revealed at least four mechanisms depending on the specific reaction conditions (i.e., saturated hydrocarbons at elevated temperatures, unsaturated hydrocarbons, acidic media, aqueous media/lipid dispersions). We also reiterate how their impressive RTA activity translates from solution to mammalian cell culture, wherein we have demonstrated that diarylamines and their derived nitroxides are potent inhibitors of ferroptosis, a recently characterized form of cell death associated with lipid peroxidation (autoxidation). In addition to phenols and amines, organosulfur compounds have long been used as antioxidants. The prevailing view has been that they undergo ionic reactions with product peroxides, preventing the initiation of further chain reactions. In recent years, we have found that many organosulfur compounds exhibit very good RTA activity. In particular, sulfenic acids (RSOH) and hydropersulfides (RSSH) are found to be among the best HAT agents known, particularly to peroxyl radicals where secondary orbital interactions are found to play a significant role. Consequently, oxidation of the sulfenic acid to a sulfinic acid greatly diminishes its HAT reactivity to peroxyls. Polysulfides and their oxides also undergo direct reactions with peroxyl radicals, thereby inhibiting autoxidation, but do so by homolytic substitution reactions. These insights suggest that the RTA activity of organosulfur compounds may be as important to the inhibition of hydrocarbon autoxidation, if not more so, than their ionic reactions.

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INTRODUCTION Autoxidation, the free radical chain reaction by which organic substrates (RH) react spontaneously with O2, is primarily responsible for the degradation of all hydrocarbon-based products, such as fuels, lubricants, rubbers and plastics. Furthermore, the onset and pathogenesis of a vast array of diseases, including cancer, neurodegeneration and atherosclerosis, have been linked to lipid peroxidation, the autoxidation of biological lipids. As the archetype radical chain reaction, autoxidation involves initiation (eqs 1 and 2), propagation (eqs 3−5), and © XXXX American Chemical Society

termination (eqs 6−8) steps (Figure 1A). The inhibition of hydrocarbon autoxidation can be achieved by the addition of compounds that interfere with either chain-initiation (preventive antioxidants) or chain-propagation (chain-breaking or radical-trapping antioxidants, RTAs).1,2 RTAs generally possess a labile H-atom and interrupt autoxidation by HAT to chain-carrying peroxyl radicals (eq 9). Since the rate constant with which RTAs react with peroxyl radicals (the Received: May 31, 2018

A

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Figure 1. Autoxidation and its inhibition by radical-trapping antioxidants. (A) Key reaction steps in hydrocarbon autoxidation. (B) Inhibition of hydrocarbon autoxidation by radical-trapping antioxidants, exemplified by phenols, for which some representative structures and rate-controlling kinetic and thermochemical parameters are given.

inhibition rate constant, kinh) far exceeds the rate constant of the rate-determining step of propagation (kp), only small amounts of RTA are required to protect comparatively large amounts of substrate from autoxidation. Importantly, the radical formed following HAT from the RTA does not propagate the autoxidation chain reaction, meaning that it is unreactive to both the substrate (hydrocarbon) and O2, and often intercepts another peroxyl radical to form (a) nonradical product(s) (eq 10). Phenols are the quintessential RTA. Phenol has a relatively weak O−H bond (87 kcal/mol)3 arising from the highly effective delocalization of the unpaired electron in the phenoxyl radical, which is worth ∼17 kcal/mol when comparing to O−H BDEs in aliphatic alcohols.4 This leads to reactions with peroxyl radicals that are effectively

thermoneutral, as a hydroperoxide has an almost identical O−H BDE.4 The reactions can be rendered exergonic by substitution of phenol with electron-donating groups (EDGs). EDGs stabilize the electron-poor phenoxyl radical and, to a lesser extent, destabilize the electron-rich phenol, leading to decreases in the O−H BDE.5 Conversely, electron-withdrawing groups (EWGs) destabilize the phenoxyl and stabilize the phenol, leading to stronger phenolic O−H bonds.5 The two most recognizable phenolic RTAs, 2,6-di-tert-buyl-4methylphenol (BHT) and α-tocopherol (α-TOH), are adorned with EDGs, which weaken the phenolic O−H BDE by 7 and 10 kcal/mol, respectively (Figure 1B).6 Consistent with the Evans−Polanyi principle, the increased exergonicity of the reaction leads to faster HAT reactions with peroxyl radicals. For example, α-TOH reacts with peroxyl radicals with B

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Figure 2. Relevant data for some interrelated aminic compounds as RTAs (A) Rate-controlling kinetic and thermochemical parameters for some representative diarylamine RTAs. (B) HAT transition state structures for reactions of diphenylamine and phenoxazine with (methyl) peroxyl radicals. (C) Isomeric diazaphenoxazines and diazaphenothiazines have very different reactivities; the synthesis of the more reactive 2,4-diaza isomers must avoid the Smiles rearrangement which arises in previously reported alleged preparations.

kinh = 3.2 × 106 M−1 s−1 at 30 °C, roughly 1000-fold faster than phenol.7 Over the years, phenols have been studied exhaustively as RTAs and their structures have been optimized for use in a wide variety of contexts. For example, as we relayed in a 2015 Account, the incorporation of nitrogen atoms into the phenolic ring enables fine-tuning of the reactivity of phenols, as pyridinols and pyrimidinols (Figure 1B), such that an ideal balance between HAT reactivity and stability to autoxidation can be struck.8 Since then we have demonstrated that aza analogs of α-TOH (so-called tetrahydronaphthyridinols, THNs, Figure 1B) are among the most potent inhibitors of cellular lipid peroxidation ever reported9 and are currently being explored in animal models of degenerative diseases that have been linked to aberrant production of lipid hydroperoxides. The focus of this Account will be on our recent efforts to better understand the reactivity of the two other common, but lesser studied, classes of RTA: amines and organosulfur compounds.

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by today’s standards (N−H BDE ∼ 82 kcal/mol and kinh ∼ 2 × 105 M−1 s−1 for 1),10 but are truly impressive RTAs at elevated temperaturesmore on this later. Similarly to phenols, diphenylamines transfer their aminic H-atom to peroxyl radicals via a syn-TS that enables the interaction of the π-HOMO of the diphenylamine and the π*SOMO of the peroxyl (Figure 2B).10 Maximizing the HAT reactivity of aromatic amines is more challenging than for phenols. Since the starting aromatic amine is more electronrich than phenol and the corresponding aminyl radical is less electron-poor than phenoxyl, smaller interactions arise with ring substituents.5 Although the N−H bond is weakened−and HAT accelerated−by substitution with EDGs, the effect is smaller than in phenols5 and is associated with a precipitous drop in oxidation potential. Incorporation of nitrogen atoms in the aryl rings enables a greater range of substitution,10 and permits the design of air-stable diarylamines with very weak N−H bonds and high reactivity to peroxyl radicals (e.g., compound 2 in Figure 2A)also subject matter discussed in our 2015 Account.8 Fusion of the two aromatic rings of diphenylamines at the ortho positions to yield tricyclic structures enables further manipulation of the HAT activity−particularly when the linkage is a heteroatom. Phenothiazines (sulfur-linked) and phenoxazines (oxygen-linked) are of most interest, as their N− H bonds are 6.5 and 8.6 kcal/mol weaker than the N−H bond in diphenylamine,11 but their oxidation potential is less affected (∼200 mV) because the inductive effect of the heteroatom destabilizes the radical cation.12 We recently synthesized a

AMINIC ANTIOXIDANTS

Maximizing the HAT Reactivity of Aminic RTAs

Diphenylamines and hindered amine light stabilizers are the two types of aminic RTAs commonly added to petroleumderived products. Alkylated diphenylamines (e.g., 1 in Figure 2A) have been extensively used as additives in lubricants and other heavy hydrocarbons since the 1950s. They are considered to be moderate RTAs at ambient temperatures C

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Figure 3. Various catalytic mechanisms by which nitroxides inhibit autoxidation. (A) Korcek cycle. (B) Two mechanisms for rate-determining regeneration of the diarylamine in the Korcek cycle. (C) Nitroxide-catalyzed cross-dismutation of alkylperoxyl radicals and hydroperoxyl radicals arising in unsaturated hydrocarbon autoxidation. (D) Computed syn-TS structures for key HAT steps in the nitroxide-catalyzed cross-dismutation mechanism. (E) Acid-mediated reaction of nitroxides with peroxyl radicals. Acid arises in the autoxidation of saturated hydrocarbons. (F) Computed proton-coupled electron transfer TS structure for the reaction of a (model) protonated nitroxide and peroxyl radical. (G) Catalytic reaction of nitroxides with peroxyl radical in aqueous solution and lipid bilayers. (H) Computed TS structure for the key regeneration reaction.

TOH, the former is characterized by E° = 1.38 V, roughly 0.5 V higher than in α-TOH and consistent with its indefinite stability in air. The latter, with log k ∼ log A (∼8.5), undergoes effectively barrierless reactions with peroxyl radicals (i.e., Ea ∼ 0). Nitrogen incorporation into the aromatic rings of phenoxazines and phenothiazines again enables the design and synthesis of a wide variety of air-stable compounds of remarkable reactivity. Our efforts to demonstrate this point were protracted; although the synthesis of diazaphenothiazines had some precedent in the literature, we discovered that they

library of substituted phenoxazines and systematically evaluated their RTA activity.12 Substitution in the para positions relative to the aminic N−H yielded the expected trends: EDGs weakened the N−H bond and accelerated HAT whereas EWGs strengthened the bond and slowed HAT. The data could be fit to excellent linear free energy relationships bounded by 3-cyano-7-nitrophenoxazine (BDE = 77.5 kcal/ mol and kinh = 4.5 × 106 M−1 s−1) and 3,7-dimethoxyphenoxazine (BDE = 70.7 kcal/mol and kinh = 6.6 × 108 M−1 s−1), the fastest RTA reported to date (compound 3 in Figure 2A).12 Interestingly, despite having BDE and kinh values similar to αD

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Accounts of Chemical Research were incorrectly identified as reaction products.13 For example, 2,4-diazaphenothiazines (see Figure 2C, X = S) that had been allegedly synthesized in 2008 were found instead to be disulfides of the Smiles rearrangement products of the starting material. Optimization of the reaction conditions lead to cyclization to the 1,3-diazaphenothiazine, but inversion of the polarity of the cyclization by switching the positions of the amine and bromide substituents on the pyrimidyl and phenyl rings was necessary to access the desired 2,4-diazaphenothiazines (and 2,4-diazaphenoxazines). As we anticipated on the basis of computational predictions, the 2,4-diaza isomers were orders of magnitude more reactive to peroxyl radicals than the corresponding 1,3-diaza isomers (Figure 2C). In fact, the kinh determined for 2,4-diazaphenoxazine 4 (5.2 × 108 M−1 s−1) is within experimental error of that of 3,7-dimethoxyphenoxazine.13 Given these compounds possess log kinh ∼ log A (∼8.5), it appears that we have reached the limit for optimizing the HAT reactivity of diarylamines.

observation that, at low rates of initiation, nitroxides are excellent RTAs, but only in the autoxidation of unsaturated hydrocarbons (e.g., styrene, hexadecene). Not only did TEMPO and bis(4-tert-butylphenyl)nitroxide apparently react very rapidly with peroxyl radicals (yielding kinh = 2.1 × 106 and 7.9 × 106 M−1 s−1, respectively), but they gave rise to lengthy inhibited periods consistent with the trapping of multiple radicals. Extensive studies converged on a mechanism whereby the nitroxide catalyzes the cross-dismutation of hydroperoxyl and alkylperoxyl radicals (Figure 3C); the former being formed from a tunneling-enhanced 1,4-HAT in an intermediate αalkoxylalkylperoxyl radical followed by elimination. Hydroperoxyl radical (O−H BDE ∼ 55 kcal/mol) was predicted to react with TEMPO via a syn HAT TS with k = 2 × 1010 M−1 s−1 (calculated), giving rise to the corresponding hydroxylamine, TEMPOH (O−H BDE ∼ 70 kcal/mol), which could then react with an alkylperoxyl via a syn TS with k = 3 × 106 M−1 s−1 (experimental) (Figure 3D). This mechanism bypasses the need for nitroxide to compete with O2 for alkyl radicals as well as the need for elevated temperatures to convert the alkoxyamine back to amine via the retro-carbonylene reaction or homolysis/in-cage disproportionation (HAT). The autoxidation of saturated hydrocarbons can also be inhibited directly by nitroxides, but only in the presence of acids. In collaboration with the Valgimigli group at the University of Bologna, we discovered that the addition of acid to autoxidations of styrene or cumene enables a very rapid formal HAT reaction from protonated TEMPO to peroxyl radicals, e.g., kinh ∼ 1 × 108 M−1 s−1.19 We characterized this reaction as a proton-coupled electron transfer (PCET) because the proton and electron are moving between two pairs of nominally orthogonal orbitals. Remarkably, both styrene and cumene autoxidations were perpetually inhibited by TEMPO when they were carried out in the presence of weak acids (carboxylic acids). Subsequent mechanistic studies revealed that the oxoammonium ion derived from the nitroxide can be reduced by chain-carrying alkyl radicals by single electron transfer (k = 1−3 × 1010 M−1 s−1) that is competitive with the reaction of alkyl radicals with O2 (k = 3 × 109 M−1 s−1).20 The complete mechanism is shown in Figure 3E alongside the computed TS for the key PCET reaction in Figure 3F. Because carboxylic acids are known to form in the autoxidation of saturated hydrocarbons, we proposed that the in situ formation of acids contributes to the mechanism of nitroxides derived from hindered amine light stabilizers typically added to plastics and other polymeric products. Indeed, the addition of an otherwise nonparticipating base (2,4,6-tri-t-butylpyridine) to autoxidations of a representative saturated hydrocarbon (nhexadecane) at 160 °C abrogated the inhibitory activity of a nonvolatile TEMPO derivative. Electron transfer reactions are generally enabled by polar solvents, and indeed, pulse radiolysis (PR) studies have shown that nitroxides react directly with peroxyl radicals by single electron transfer in aqueous solutions.21 We recently expanded on the PR experiments by carrying out inhibited autoxidations of THF in phosphate buffer, and confirmed that no acid is required to enable this reaction.22 In fact, acid inhibits the reaction. The results, from which kinh = 5.4 × 106 and 1.3 × 106 M−1 s−1 were derived for TEMPO and bis(4-tert-butylphenyl)nitroxide, not only corroborated the PR data, but interestingly, they also showed that the nitroxides were catalytic inhibitors. Mechanistic studies revealed that the substrate (THF) was able to reduce the nitroxide-derived oxoammonium ion by hydride

Catalytic Mechanisms of Aminic RTA Activity

Diphenylamines have long been known to possess catalytic RTA activity at elevated temperature; that is, the diphenylamine is regenerated in situ, leading to the trapping of far more than the nominal two radicals accounted for by eqs 9 and 10 in Figure 1B. Although estimates of the number of radicals trapped per molecule of diphenylamine ranging from dozens to hundreds can be found in the literature, precise quantitation is challenging since homolysis of the product peroxides (formed by eq 4 and/or 5) occurs at elevated temperatures, leading to a progressive increase in the rate of initiation. In 1995, Korcek and co-workers suggested a mechanism that accounts for the catalytic activity of diphenylamines and the requirement for elevated temperatures (Figure 3A).14 Briefly, following HAT from the diphenylamine to a peroxyl radical, the diphenylaminyl radical reacts with a second equivalent of ROO• leading to a transient peroxyamine that undergoes O− O homolysis to produce a diphenylnitroxide. The diphenylnitroxide is proposed to react with an alkyl radical to form an alkoxylamine that decomposes via rate-limiting N−O homolysis and in-cage disproportionation (HAT) to yield the starting diphenylamine and a carbonyl compound. Enabled by the synthesis of authentic N,N-diarylalkoxyamines, we recently showed that the regeneration step in the Korcek cycle proceeds by a pericyclic retro-carbonyl-ene (RCE) reaction for unsaturated substrates (Figure 3B). This is supported by the larger entropy cost for the process than expected for a simple (N−O) bond dissociation (log A ∼ 13 vs 16) and a significant kinetic isotope effect observed when the in-flight hydrogen atom is replaced with deuterium.15 While the Korcek mechanism accounts for the catalytic activities of diphenylamine RTAs at elevated temperatures, it does so only under conditions when the nitroxide can accumulate to levels in excess of O2. This is simply because the reaction of alkyl radicals with O2 is faster than the reaction of alkyl radicals with nitroxides (compare k ∼ 3 × 109 M−1 s−1 for the former16 vs k ∼ 1−3 × 108 M−1 s−1 for the latter17 at 25 °C). As the temperature is raised, this difference will decrease, but as long as O2 levels are comparable to−or exceed−those of the nitroxide, alkoxyamine formation and subsequent turnover of the catalytic cycle will not be competitive. An alternative mechanism was uncovered in our recent revisitation of the reaction (or purported lackthereof) between nitroxides and peroxyl radicals.18 We had made the surprising E

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Figure 4. Some cytoprotective amines and their corresponding nitroxides. (A) Inhibiton rate constants in solution and lipid bilayers as well as EC50 values for inhibiton of RSL3-induced ferroptosis for some representative amine RTAs. (B) The HAT reactivity of amines is less sensitive to medium (i.e., solvent, S) effects due to less favorable H-bonding interactions than phenols. (C) Inhibiton rate constants in lipid bilayers and EC50 values for inhibiton of RSL3-induced ferroptosis for some representative nitroxide RTAs.

significantly more reactive in lipid bilayers−consistent with their greater potency as inhibitors of ferroptosis (e.g., 4.6 × 104 M−1 s−1 and 1.2 × 104 M−1 s−1 for Fer-1 and Lip-1 vs 4.7 × 103 M−1 s−1 for α-TOH).9 Medium effects account for these differences−primarily H-bonding−which retards the HAT reactions of phenols to a far greater extent than amines (Figure 4B). We subsequently showed that there is not anything particularly special about the structures of Fer-1 and Lip-1; similarly reactive aminic RTAs, such as the industrial standard bis(4-alkylphenyl)amine 1, are also potent ferroptosis inhibitors.24 Moreover, amines with greater RTA activity, e.g. phenoxazine, were even more potent.24 Interestingly, we were also able to show that the nitroxides that derive from aromatic amines (e.g., phenoxazine N-oxyl), as well as hindered aliphatic amines related to TEMPO, of appropriate lipid-solubility (e.g., lipo-TEMPO) are also potent inhibitors of ferroptosis.22 The ability of good RTAs to inhibit cell death underscores the central role of lipid autoxidation in the execution of ferroptosis.

transfer to produce a hydroxylamine in situ (Figures 3G and H). This reaction is analogous to the common synthetic transformation which employs TEMPO as a catalyst in the hypochlorite-mediated oxidation of an alcohol to a carbonyl compound. The hydroxylamine, a potent HAT agent (vide supra), then traps another peroxyl radical to reform the nitroxide. We extended our investigations from THF to lipid bilayers, where we showed that nitroxides retained high reactivity to peroxyl radicals, and the oxoammonium ions derived therefrom could be reduced by NAD(P)H, Nature’s hydride donor. These results provide a mechanistic basis for the well-known cytoprotective properties of nitroxide-based drugs. Aminic and Nitroxide RTAs: Emerging Cytoprotectants

The RTA activity of the diarylamine and nitroxides discussed above has recently been demonstrated to translate to cell culture. Specifically, some of these compounds have been shown to be potent inhibitors of ferroptosis, a recently characterized form of nonapoptotic cell death driven by lipid peroxidation.23 Ferroptosis has been the subject of considerable attention in recent years owing to its potential involvement in a number of degenerative conditions. It is readily induced upon inhibition of the (phospho)lipid hydroperoxide detoxifying enzyme glutathione peroxidase 4 (Gpx4), deletion of the gene encoding it, or starving Gpx4 of its reducing cosubstrate, glutathione. The two prototype inhibitors of ferroptosis, the arylamines ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1) (Figure 4A), were identified from high-throughput screening of compound libraries in cell models of ferroptosis. Given their structures, we surmised that Fer-1 and Lip-1 would be effective RTAs. Indeed, we found that they undergo HAT to peroxyl radicals with kinh = 3.5 × 105 M−1 s−1 (Fer-1) and 2.4 × 105 M−1 s−1 (Lip-1).9 Although their reactivity is inferior to α-TOH (Nature’s premier RTA) in homogeneous organic solution, they were

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ORGANOSULFUR ANTIOXIDANTS

Thiols, Sulfenic, Sulfinic, and Sulfonic Acids

Organosulfur compounds have long been employed as antioxidant additives to various hydrocarbon-based products, but are generally believed to act as “preventive antioxidants” rather than RTAs. Preventive antioxidants are generally ascribed ionic (two-electron) peroxide decomposing activities; precluding their accumulation and decomposition to autoxidation chain-initiating species (i.e., alkoxyl and/or hydroxyl radicals, i.e. eq 1). This is based on sulfur’s relatively high nucleophilicity and oxidizability to acids that are capable of dehydrating and/or cleaving peroxides in Hock-like processes. Although thiols have relatively weak S−H bonds (87 kcal/ mol)4 and are highly useful HAT reagents in organic synthesis (mainly to reduce alkyl radicals, k = 1 × 107 M−1 s−1),25 they F

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Figure 5. Relevant data for some interrelated organosulfur compounds as RTAs. (A) The persistent 9-triptycenesulfenic acid enabled kinetic and thermochemical studies of the RTA (and other HAT) reactivity of sulfenic acids. Sulfenic acids are predicted to react with peroxyl radicals via a syn TS. (B) Activated thiosulfinates undergo Cope-type elimination to form sulfenic acids, which trap peroxyl radicals. (C) Unactivated thiosulfinates react with thiols to produce sulfenic acids. (D) Trisulfide-1-oxides react with peroxyl radicals by homolytic substitution. (E) Hydropersulfides are predicted to react with peroxyl radicals via a syn TS.

from the preferred syn TS geometry for the HAT reaction (Figure 5A).28,32 Experimental characterization of sulfenic acid HAT chemistry was enabled by the synthesis and study of 9triptycenesulfenic acid;29,31 without a bulky substituent, sulfenic acids rapidly self-condense to thiosulfinates, precluding quantitative studies.33 Further oxidation of sulfenic acids to sulfinic acids raises their O−H BDE by ca. 6−8 kcal/mol.34 With an O−H BDE of ∼78 kcal/mol (essentially identical to that in α-tocopherol), it may be expected that sulfinic acids are excellent RTAs. Interestingly, we were unable to characterize them as such. Despite extensive efforts utilizing both indirect (inhibited autoxidations) and direct (laser flash photolysis) methods, we were unable to measure a rate constant for their reaction with peroxyl radicals. CBS-QB3 calculations suggest an insignificant kinh ∼ 1 M−1 s−1!34 It is apparent that the secondary orbital interaction between the sulfur lone pair and the peroxyl π* is

are comparatively poor H-atom donors to peroxyl radicals, and the propensity of the product thiyl radicals to dimerize and react with O2 generally limits the stoichiometry of their RTA reactions to (at most) a single peroxyl radical.26 While studying the antioxidant mechanism of allicin, the characteristic odorous thiosulfinate from garlic (vide infra), we uncovered the impressive HAT chemistry of sulfenic acids (RSOH). Sulfenic acids arise from the two-electron oxidation of thiols,27 which reduces the strength of the bond to the labile H-atom from ∼87 kcal/mol4 to ∼70 kcal/mol28,29 (Figure 5). Consistent with the greater driving force, the reactivity of sulfenic acids with peroxyl radicals is roughly 10 000-fold greater than with thiols.28,30,31 Computations again suggest that this reaction benefits from a secondary orbital interaction−in this case between the nominally nonbonded (lone) pair on the sulfur atom and the contribution of the inner oxygen atom of the peroxyl radical π* orbital−evident G

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acids (∼70 kcal/mol), and our experimental investigations confirm that they are potent HAT agents. Predicted once again to be enabled by secondary orbital interactions, hydropersulfides react quickly with peroxyl radicals with kinh = 2 × 106 M−1 s−1 (Figure 5E). Most importantly, because hydropersulfides (like thiols) are very weak H-bond donors, their HAT kinetics are not significantly diminished in strong H-bond accepting solvents/media. Consistent with their weak S−H bond, hydropersulfides were also found to undergo very rapid HAT reactions with alkyl (∼5 × 108 M−1 s−1), alkoxyl (∼1 × 109 M−1 s−1), and thiyl (>1 × 1010 M−1 s−1) radicals, acting as “superthiols” of sorts.40 Interest in hydropersulfides has surged in recent years owing to their presence at significant levels in mammalian tissues. It has been suggested that they serve merely as a reservoir for H2S, but also that due to their enhanced nucleophilicity, they may be important for sensing and/or detoxifying oxidants (e.g., H2O2). Our work prompts the question of whether their HAT reactivity could be relevant. The very weak H-bond donating character of the hydropersulfides make them excellent H-atom donors in good H-bonding media, such as water and its interfacial region with lipid bilayers, areas where the reactivity of phenols, such as α-TOH, is significantly suppressed. Indeed, the RTA reactivity of hydropersulfides translates well from organic solutions to aqueous media.40 Interestingly, we found that their RTA activity is pH dependent, with kinh reaching a maximum of ∼5 × 106 M−1 s−1 above pH ∼ 8, coincident with their pKa, suggesting a so-called SPLET (sequential proton-loss electron transfer) mechanism.40 The efficacy of the hydropersulfides as RTAs in aqueous solution is limited by their stability; the stoichiometry of the radical-trapping reaction is significantly less than 1 and decreases with increasing pH owing to competing decomposition of the hydropersulfide to non-RTA products.40 Transient formation of hydropersulfides where they are stable (i.e., the lipid bilayer) may minimize their decomposition and maximize their efficacy.

significantly diminished upon oxidation, driving up the barrier despite the favorable thermodynamics. Further oxidation of the sulfinic acid to a sulfonic acid raises the O−H BDE further to 107 kcal/mol,34 precluding any HAT activity (particularly to peroxyl radicals). (Poly)sulfide Oxides

Allicin (diallyl disulfide S-oxide) and petivericin (dibenzyl disulfide S-oxide) represent two of the most well-known thiosulfinates. The former is widely is widely believed to account for account for garlic’s health benefits while the latter is the active compound in Petiveria alliacae, another medicinal plant from the same family. It had been demonstrated by others that both thiosulfinates possess RTA activity,35 but their suggested mechanism of HAT from the allylic/benzylic positions to peroxyl radicals was unlikely to be correct. Based on an earlier proposal that di-tert-butylsulfoxide was an effective RTA at elevated temperatures because it underwent Cope elimination in situ to form tert-butylsulfenic acid,36 we suggested and then demonstrated that an analogous mechanism is more likely to operate for allicin and petrivercin, giving rise to radical-trapping allyl and benzyl sulfenic acids, respectively (Figure 5B).28,30,31 Although thiosulfinates must be activated to undergo efficient Cope-type elimination at ambient temperatures and produce sulfenic acids at a rate sufficient to compete with propagation of autoxidation in solution, we demonstrated that an ionic mechanism is also available to unactivated thiosulfinates in biphasic systems, such as lipid bilayers (Figure 5C).37 Thiols, such as N-acetylcysteine (NAC), react with the unoxidized sulfur of thiosulfinates to liberate a sulfenic acid (and disulfide). This may be relevant in biological contexts where thiols abound. The chemistry is facilitated by phase separation of the sulfenic acid and the activating thiol, which minimizes competing disulfide formation, which is likely what renders allicin and petivericin useless in cellular contexts.38 In contrast, a hexylated petivericin derivative was highly effective in preventing lipid peroxidation in the presence of NAC; significantly bettering α-TOH and demonstrating catalytic activity in the presence of excess NAC presumably due to the recycling of the sulfenic acid from its sulfinyl radical.37 Moreover, in contrast to allicin and petivericin, it retained some of its reactivity in cell culture.38 Insertion of a third sulfur atom into the thiosulfinate functionality (now a trisulfide-1-oxide) activates them to direct reactions with peroxyl radicals. The reaction is surprisingly fast (kinh = 1.5 × 104 M−1 s−1 at 37 °C),39 essentially the same as that for BHT, but it must occur by a completely different (nonHAT) mechanism. Our experiments and computations point to a homolytic substitution mechanism, whereby the peroxyl radical attacks the sulfinyl sulfur atom and liberates a perthiyl radical (Figure 5D), which is persistent, unreactive to O2, and simply dimerizes (k = 6.0 × 109 M−1 s−1) to yield an innocuous tetrasulfide.39 The homolytic substitution reaction is equally facile for tetrasulfide-1-oxides (and presumably higher polysulfide-1-oxides) owing to the similar radical stabilities of RSnS•.39

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PERSPECTIVE The chemistry of phenolic and aminic radical-trapping antioxidantsand in particular, their H-atom transfer reactionsis a mature field. The community enjoys a comprehensive understanding of the thermodynamics and kinetics of these reactions, and steric, electronic and medium effects thereupon have now been well-explored. In recent years, computation and guided experimentation have further clarified the origins of these facile HAT reactions, particularly with respect to the contribution of the interactions of orbitals localized on the groups bonded to the atoms between which the H-atom is being transferred (i.e., secondary orbital interactions). In the authors’ opinion, the primary challenge in the field in recent years has been to reconcile the catalytic RTA activities of amines and their corresponding nitroxides at low concentrations of amine/nitroxide (i.e., < 1 mM). The accepted mechanismsthe Korcek and Denisov cycles for diphenylamines and hindered amines, respectivelyrequire nitroxides to outcompete O2 for alkyl radicals; an unlikely scenario at these concentrations under aerobic conditions. The efforts we describe above go some of the way to addressing this issue, but much remains to be learned; in particular, how our insights translate to real-world applications. Given that not only the stoichiometry and kinetics, but the catalytic mechanisms of nitroxides, vary with reaction conditions, it follows that the constituents of (or additives to) hydrocarbon-

Hydropersulfides

The stability of the perthiyl radical formed upon homolytic substitution of peroxyl radicals on trisulfide-1-oxides prompted us to consider the HAT chemistry of hydropersulfides (also known as perthiols, Figure 5E).40 Computations suggest that they have similar RSS-H BDEs to the RSO-H BDEs in sulfenic H

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based materials can have a significant impact on RTA performance. This information will be vital to the design of the next generation of aminic/nitroxide RTAs. The characterization of ferroptosis as a specific form of regulated cell death associated with lipid oxidation has led to a renaissance of sorts in the study of lipid peroxidation (autoxidation) and its inhibition in biological contexts. The development of inhibitors of ferroptosis may prove useful in the prevention and/or treatment of degenerative diseases associated with the accumulation of lipid peroxidation products. Employing the knowledge gleaned from fundamental studies on the reactivity of RTAs is vital to rationalizing the biological activity of existing compounds and enabling the design of new compounds. Relative to phenols and amines, the HAT chemistry of organosulfur compounds remains less well-explored. Our recent efforts to probe the reactivities of sulfenic acids, sulfinic acids and hydropersulfides goes some way to addressing this, providing quantitative assessment of the thermodynamics and kinetics of their HAT reactions relative to their far better studied precursor thiols. The relevance of the reactions of these compounds in both industrial and biological contexts, however, remains unclear. Moreover, since we have now shown that precursor polysulfides and their oxides can also undergo (non-HAT) homolytic substitution reactions with peroxyl radicals, the interplay between both types of reactions and predictions of which is the more important contributor under specific conditions needs to be clarified.

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Article

REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Derek A. Pratt: 0000-0002-7305-745X Notes

The authors declare no competing financial interest. Biographies Jia-Fei Poon received his B.Sc. and M.Sc. degrees (Org. Chem.) in 2011 from Linköpings University and his Ph.D. degree (Org. Chem.) in 2016 from Uppsala University. He is currently a postdoctoral fellow at the University of Ottawa. His research interests include antioxidant chemistry for both industrial and biological applications. Derek Pratt received his B.Sc. (Hon. Chem.) degree from Carleton University in 1999 and his Ph.D. degree from Vanderbilt University in 2003. After completing postdoctoral work at the University of Illinois at Urbana−Champaign in 2005, he returned to Canada as Canada Research Chair in Free Radical Chemistry at Queen’s University. In 2010, he moved to the University of Ottawa, where he is currently Full Professor in the Department of Chemistry and Biomolecular Science.

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ACKNOWLEDGMENTS The authors would like to acknowledge the efforts of Pratt Group researchers whose dedication contributed to many of the insights presented above. They are named explicitly in the corresponding references. The research recounted here was made possible primarily by the continued support of the Discovery Grant program of the Natural Sciences and Engineering Research Council of Canada. I

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