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The Catalytic Reaction of Nitroxides with Peroxyl Radicals and its Relevance to their Cytoprotective Properties Markus Griesser, Ron Shah, Antonius T. Van Kessel, Omkar Zilka, Evan A Haidasz, and Derek A. Pratt J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b00998 • Publication Date (Web): 16 Feb 2018 Downloaded from http://pubs.acs.org on February 16, 2018

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Journal of the American Chemical Society

The Catalytic Reaction of Nitroxides with Peroxyl Radicals and its Relevance to their Cytoprotective Properties Markus Griesser, Ron Shah, Antonius T. Van Kessel, Omkar Zilka, Evan A. Haidasz & Derek A. Pratt* Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada ABSTRACT: 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 a 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 to 0.95 V vs. NHE) and that this activity is catalytic in nitroxide. Regeneration of the nitroxide occurs by a twostep process involving initial 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 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.

INTRODUCTION Nitroxides have demonstrated exciting biological activities linked to their ability to mitigate the effects of oxidative stress. Their protective effects have been documented in a staggering array of cell and animal models of disease, including neurodegeneration,1 Alzheimer’s,2 Huntington’s,3 Parkinson’s,4 multiple sclerosis,5 diabetes,6 ischemia-reperfusion injury,7 obesity and hyperlipidemina.8 Furthermore, nitroxide administration has demonstrated significant increase in lifespan in a variety of organisms, including fruit flies,9 freshwater annelids,10 and mice.11 Very recently, mitochondria-targeted nitroxides have been reported to be potent inhibitors of ferroptosis,12 a form of regulated necrosis associated with the accumulation of lipid hydroperoxides,13-15 perhaps hinting that the mechanism that underlies their cytoprotective effects involves the inhibition of lipid peroxidation. The ability of nitroxides to mitigate oxidative stress is often ascribed to their reactivity as superoxide dismutase (SOD) mimics. This reactivity, established largely by the efforts of Samuni, Krishna, Goldstein and co-workers, 16-19 is believed to result from the sequence of reactions illustrated in Scheme 1A for the archetypal nitroxide, 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO). Although there is no doubt that nitroxides undergo this chemistry, it is unclear if this mechanism is physiologically relevant, since cells generally have a comparable or higher level of SOD enzymes;20 the Cu/Zn-SOD to protect the cytoplasm21 and MnSOD specifically to protect mitochondria.22

Scheme 1. Catalytic trapping of peroxyl radicals by nitroxides. TEMPO catalytically reduces hydroperoxyl in aqueous solution (see ref. 19), generally referred to as superoxide dismutation (A), and in the presence of acid (see ref. 22), TEMPO catalytically reduces alkylperoxyl radicals in organic solution (B).

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Another mechanism often invoked to account for the biological activity of nitroxides involves the scavenging of alkyl radicals – a reaction that is purportedly the basis for many newly-developed nitroxide-containing redox probes.23-25 Despite being the quintessential reaction of nitroxides, this reaction is too slow (k ~ 1-3 ´ 108 M-1 s-1)26-27 to compete with the formation of peroxyl radicals from the combination of alkyl radicals and O2 (k ~ 3 ´ 109 M-1 s-1)28 – a competition made more unlikely since O2 is generally present in much higher concentration than the nitroxide (mM vs. µM to nM). Although nitroxides have long been demonstrated to be unreactive to peroxyl radicals in organic solution,29 Goldstein and Samuni provided compelling evidence using pulse radiolysis that nitroxides react readily with peroxyl radicals in water (i.e. k = 310 ´ 107 M-1 s-1 for TEMPO and a variety of alkylperoxyl radicals).30 The mechanism was believed to be an electron transfer – analogous to the first step of the SOD mimicking mechanism shown in Scheme 1A – with concomitant formation of the alkylperoxide anion and the oxoammonium ion derived from TEMPO (2,2,6,6-tetramethylpiperidinium-N-oxide, hereafter TEMPO+). In collaboration with the Valgimigli group, we recently reported that nitroxides undergo acid-promoted reactions with peroxyl radicals,31 and further, that the nitroxide can be recycled from the resultant oxoammonium ion by reduction with a substrate-derived radical (Scheme 1B).32 Thus, nitroxides are catalytic radical-trapping antioxidants (RTAs), and the substrate-de-

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rived radicals are themselves the stoichiometric reductants. Evidence for this mechanism was obtained at ambient temperatures in organic solution to which acids were added31-32 as well as at elevated temperatures in hydrocarbons where acids are formed in situ.32 We wondered whether this mechanism could contribute to the antioxidant activity of nitroxides in aqueous solution, and more importantly, in lipid bilayers where lipid peroxyl radicals propagate the peroxidation of lipids. The results of our investigations are reported here. RESULTS

I. Nitroxides Catalytically Trap Peroxyl Radicals in Aqueous THF. TEMPO was initially investigated for its ability to inhibit the free radical chain oxidation (autoxidation) of THF. THF is a convenient substrate for determination of both the kinetics and stoichiometry of peroxyl radical reactions as it is both miscible with aqueous buffers and sufficiently reactive to participate in a chain reaction with well-defined kinetics.33 Reactions were initiated with the water-soluble azo initiator AAPH and monitored by the consumption of STY-BODIPY, a highly absorbing and oxidizable co-substrate (Figure 1D).34 Representative results obtained at pH 4, 7, and 10 are shown in Figures 1A, B, and C, respectively. Plotted alongside the data are the results of THF/STY-BODIPY co-autoxidations inhibited by Trolox, a water-soluble analog of atocopherol (a-TOH), the most biologically active form of Vitamin E, and an important benchmark of RTA activity.35

Figure 1. Co-autoxidation of THF (3.1 M) and STY-BODIPY (10 µM) initiated by AAPH (1 mM) in either acetate or phosphate buffer (100 mM) of pH 4 (A), 7 (B) and 10 (C) at 37 °C (black) inhibited by 2 µM of TEMPO (red), TEMPO+BF4- (green) or Trolox (blue). Reaction progress was monitored by loss of STY-BODIPY (D) absorbance at 562 nm (ε = 132,261 M-1 cm-1) and enabled the determination of rate constants (kinh) and reaction stoichiometries (n) for reactions of inhibitors with chain-carrying peroxyl radicals (E). Transient concentrations of TEMPO monitored in parallel by EPR at pH 4 (black), 7 (red) and 10 (blue) (F).


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Visual comparison of the reaction progress data reveals that TEMPO is both significantly more reactive to peroxyl radicals (lower initial rate) and has a greater radical-trapping capacity (longer inhibited period) than Trolox. The rate constants derived from the initial rates of the inhibited autoxidations (Eq. 1, Figure 1E), indicate that the reactivity of TEMPO towards peroxyl radicals is essentially pH-independent, viz. kinh = (2.8 ± 0.4) ´ 106, (4.6 ± 0.2) ´ 106, and (3.7 ± 0.2) ´ 106 M-1 s-1 at pH 4, 7, and 10, respectively. These data can be compared with those obtained for Trolox (kinh = 2.1 ´ 105, 4.1 ´ 105, and 8.7 ´ 105 M-1 s-1, respectively), which increase ever so slightly with pH due to the increasing contribution of a sequential proton-loss electron-transfer mechanism.33 The rate constants determined for the reactions of TEMPO are roughly one order of magnitude lower than those determined by Goldstein et al. by pulse radiolysis, consistent with the depressed polarity of the current medium.30 The benefits of carrying out inhibited autoxidations in lieu of pulse radiolysis experiments to assess the reactivity of the nitroxides to peroxyl radicals are not limited to the ‘cleanliness’ of the reaction,36 but include the ability to directly determine the stoichiometry of peroxyl radical trapping, which is derived from the length of the inhibited period (tinh) as in Eq. 2 (Figure 1E). While this is easily done for Trolox, yielding n ~ 2 at each pH, this is possible for TEMPO only at pH 4, since the autoxidations at pH 7 and 10 remain retarded well beyond the initial inhibited period. The duration of the inhibited period at pH 4 indicates that TEMPO traps roughly 3-fold more radicals than Trolox (i.e. n ~ 6). Thus, as in organic solution, TEMPO appears to catalytically trap radicals in aqueous solution – but in the absence of acid. THF/STY-BODIPY co-autoxidations were also carried out in the presence of TEMPO+. The reaction progress curves are also included in Figure 1. Interestingly, the data are practically superimposable on those obtained with TEMPO, suggesting that TEMPO+ can be reduced in situ to yield TEMPO. To corroborate this result, corresponding experiments were carried out in the cavity of an EPR spectrometer. The results are shown in Figure 1D, from which it is clear that the temporal evolution of the TEMPO signal at each pH is consistent with the extent to which each of the autoxidations are inhibited. That is, while TEMPO is consumed relatively rapidly at pH 4, it is consumed progressively more slowly with increasing pH. Since the autoxidation remains inhibited and TEMPO is not consumed, it must be reformed in situ. Moreover, since no external reductant is supplied, the substrate must serve as the stoichiometric reductant – perhaps via THF-derived alkyl radical as in Scheme 1B.

II. The Substrate is the Stoichiometric Reductant in NitroxideCatalyzed Inhibited Autoxidations of THF. During efforts to monitor the foregoing THF/STY-BODIPY co-autoxidations by EPR it became apparent that TEMPO+ was not stable under the reaction conditions, yielding significant amounts of TEMPO at t = 0, even in the absence of STY-BODIPY and AAPH. Since THF and phosphate buffer were the only other components of the reaction mixture, their interaction with TEMPO+ was investigated by monitoring the loss of its absorbance at 300 nm (where TEMPO+ and TEMPO have a maximum difference in their spectra, see Figure 2A inset). Representative results are shown as a function of THF concentration in Figure 2A, from which the rate constant for the reaction was determined to be (1.4 ± 0.3) ´ 10-4 M-1 s-1.

The observation that TEMPO+ can oxidize THF was somewhat surprising, since there are few examples of ether oxidation by oxoammonium ions in the literature and they are limited to activated (benzylic) ethers. Alkyl ethers are generally described as non-reactive.37,38,39

Figure 2. TEMPO+ (1 mM) in pH 7 phosphate buffer reacts with THF as monitored by loss of its absorbance at 300 nm Inset: Difference in absorbance of TEMPO+ and TEMPO (A). CBS-QB3calculated transition state structure for the reaction of TEMPO+ with THF (B).

The oxidation of alcohols by oxoammonium ions has been the subject of considerable study,40 and is believed to occur by formal hydride transfer from the Ca-H to the oxygen atom of the oxoammonium cation.41 The results of CBS-QB342 calculations (including a continuum solvation model) suggest that a similar mechanism is at play for the reaction of THF and TEMPO+, as illustrated in the transition state structure shown in Figure 2B. The calculated free energy barrier for the reaction of DG‡ = 26.2 kcal mol-1 predicts a second order rate constant of 5.3 ´ 10-5 M-1 s-1 upon application of transition state theory, which is in excellent agreement with the experimental data. Additional support for this mechanism was obtained by carrying out kinetic experiments in d8-THF, which yielded a kinetic isotope effect of kH/kD = 3.5 on the oxidation of THF by TEMPO+ (see Supporting Information for the data). To assess the relevance of each of the TEMPO+/THF and the TEMPO+/R• reactions on the turnover of TEMPO in the autoxidation of THF, we constructed a kinetic model of the autoxidation using the simulation software COPASI.43 An abbreviated scheme (omitting the reactions of STY-BODIPY for clarity) and the corresponding rate constants are given below, and the results of the simulation are visualized in Figure 3. The complete scheme and associated rate constants can be found in the Supporting Information.



AAPH à 2 R•

(3)

R• + O2 à ROO•

(4)

ROO• + THF à ROOH + THF•

(5)

THF• + O2 à THF-OO•

(6)

THF-OO• + THF à THF-OOH + THF•

(7) = kp

2 ROO• / THF-OO• à non-radical products

(8) = kt

ROO• / THF-OO• + TEMPO à ROO- / THF-OO- + TEMPO+ TEMPO+ + R• / THF• à TEMPO + R+ / THF+ TEMPO+ + THF à TEMPOH + THF+

(9) = kinh

ROO• / THF-OO• + TEMPOH à ROOH / THF-OOH + TEMPO

(12) = kinh2

TEMPO+ + TEMPOH à 2 TEMPO

(13)

TEMPO + R• / THF• à TEMPO-R / TEMPO-THF

(14)

(10) = kreg1 (11) = kreg2

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Simulations of inhibited autoxidations carried out without inclusion of the TEMPO+/THF reaction (Eq. 11) or subsequent reactions of TEMPOH (Eqs. 12 and 13) in the kinetic model displayed neither the protracted inhibited period nor the subsequent retardation of the autoxidation observed experimentally (Figure 3A). This suggests that the steady state concentration of alkyl radicals that propagate the autoxidation is simply too low to sustain enough TEMPO to effectively inhibit the reaction. Therefore, once TEMPO is initially consumed (corresponding to n = 1), the autoxidation proceeds at the uninhibited rate. Inclusion of the TEMPO+/THF reaction in the kinetic model, as well as the subsequent reaction of TEMPOH with peroxyl radicals (Eq. 12, reported kinh = 2.4 x 106 M-1 s-1),31 affords simulated data that is strikingly consistent with experiment. The known comproprotionation of TEMPO+ and TEMPOH to yield TEMPO was also included (Eq 13), but given the known rate constant44 of k = 50 M-1 s-1, this is not expected to be a major pathway as it requires simultaneous accumulation of both species, while TEMPOH will react readily with ROO•. The loss of TEMPO via its combination with alkyl radicals (Eq. 14) limits the turnover of the cycle; omission of this reaction in the kinetic scheme leads to perpetual retardation of the autoxidation.

Figure 3. Simulation of the reaction progress curves for the TEMPO-inhibited autoxidation of THF according to variations in the (abbreviated) kinetic model defined by Eqs. 3-8 (dotted lines), Eqs. 3-10 (dashed lines) and Eqs. 3-13 (solid lines) (A). Numerical fits of experimental traces of THF/STY-BODIPY co-autoxidations inhibited by TEMPO at pH 7 and 10, as well as the best possible fits without including Eq. 11 (B). Experimental data at pH 4 inhibited by TEMPO (red) as well as TEMPOH (green) in contrast to the theoretical fit without Eq. 11 (blue, dashed) (C).


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The data sets obtained at both pH 7 and pH 10 could be readily fit to this kinetic model, but the data sets at pH 4 could not. Attempts to do so yielded rate constants that exceeded diffusion for Eq. 14 (k ~ 2 ´ 1011 M-1 s-1), suggesting that at least one other off-cycle reaction limits radical-trapping activity. Since the pKa of TEMPOH2+ has been reported to be ca. 7.5,44 we surmised that at pH 4 TEMPOH is largely protonated, and a much poorer Hatom donor. In fact, the O-H BDE of TEMPOH2+ is calculated to be 116 kcal mol-1 (CBS-QB3). Consistent with this expectation, when autoxidations were carried out at pH 4 in the presence of TEMPOH, little inhibition was observed (see Figure 3C).

III. Peroxyl Radical-Trapping Antioxidant Activity Extends to Other Relevant Nitroxides. Since arylnitroxides are intermediates in the catalytic inhibition of hydrocarbon autoxidation by diarylamine RTAs45-47 and may contribute to the anti-ferroptotic activity of liproxstatin (and other monoarylamines),48 we expanded the foregoing studies to include a model diarylnitroxide (bis(4-tert-butylphenyl)nitroxide, TBPNO) and a monoarylnitroxide (the dihydroquinoline-N-oxyl, DHQNO) derived from a liproxstatin model compound (shown in Figure 4A). The results of THF/STY-BODIPY co-autoxidations in phosphate buffer of pH 7 inhibited by these compounds are shown in Figure 4B (corresponding results obtained at pH 4 and 10 are included in the Supporting Information). Introduction of the aryl substituents change the reactivity of the nitroxide considerably. Although they remain more reactive than Trolox, the profiles differ from both TEMPO and each other. The reactivity of the monoaryl nitroxide DHQNO to peroxyl radicals is initially indistinguishable from TEMPO (kinh = (5.4 ± 0.4) ´ 106 M-1 s-1), whereas the diarylnitroxide TBPNO is noticeably less reactive (kinh = (1.3 ± 0.4) ´ 106 M-1 s-1), but only initially so (more on this later). However, while DHQNO traps fewer radicals than TEMPO (similarly to Trolox, for which n ~ 2), TBPNO traps more.

To rationalize these observations, and based on the evidence which suggests that the (initial) reaction between the nitroxide and peroxyl radical is an electron transfer, the standard potentials of the three nitroxides were determined by cyclic voltammetry. The results are shown in Figure 4C, where it is clear that the trend in the potentials matches kinh; DHQNO is very slightly more oxidizable than TEMPO (E° = 0.85 and 0.88 V vs. NHE, respectively), and both are more oxidizable than TBPNO (E° = 0.99 V). The corresponding relative instability of the diaryloxoammonium ion (hereafter TBPNO+) suggests that it will be the most easily reduced by the substrate, providing a rationale for its greater catalytic activity. Indeed, the CBS-QB3-calculated rate constant for the reaction of TBPNO+ with THF is 1.6 M-1 s-1 (recall for TEMPO+ it was 5.3 ´ 105 M-1 s-1). While some of the predicted rate acceleration must be based on the differing electronics, the lesser steric hindrance surrounding the key N-O moiety of TBPNO+ as compared to TEMPO+ must also contribute. The relative stability of the monoaryloxoammonium ion (hereafter DHQNO+) implies that its reaction with THF will be slower than that of TEMPO+, but due to the reduced steric hindrance, it is predicted to be 6-fold faster (3.2 ´ 10-4 M-1 s-1). The poor turnover of the monoaryl compound may therefore be attributed to its decomposition by other reactions. The (highly reproducible) ‘kink’ in the initial inhibited region of the TBPNO-inhibited autoxidation (see inset, Figure 4B) deserves further comment. Since the conversion of TBPNO+ to its hydroxylamine (TBPNOH) via reaction with THF is predicted to be relatively facile, we wondered if the inhibition improves with time because TBPNOH is a better peroxyl radical-trapping agent than TBPNO. Hence, we independently synthesized TBPNOH and carried out the same inhibited autoxidations. Indeed, the data obtained were essentially superimposable on those shown in Figure 4B after the initial decay in STY-BODIPY between 0 and ca. 500 s (kinh = (4.7 ± 0.5) ´ 106 M-1 s-1, see Supporting Information).

Figure 4. Investigated nitroxides (A). Co-autoxidation of THF (3.1 M) and STY-BODIPY (10 µM) initiated by AAPH (1 mM) in phosphate buffer (100 mM) of pH 7 at 37 °C (black) and inhibited by 2 µM of TEMPO, TBPNO, DHQNO, and Trolox (B) monitored at 562 nm (ε = 132,261 M-1 cm-1). Cyclic voltammograms of TEMPO, TBPNO, and DHQNO recorded in acetonitrile (E° vs. NHE) (C).

IV. Nitroxides Inhibit the Autoxidation of Phosphatidylcholine Lipid Bilayers. The reactivity of nitroxides as catalytic RTAs in aqueous solution – but not organic solution – prompted our investigation of the same series of compounds in biphasic media

more representative of the biological contexts in which this reactivity may be relevant. Unilamellar liposomes of egg phosphatidylcholine (Egg-PC) supplemented with STY-BODIPY as the signal carrier was used for this purpose, such that the kinetics and



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Figure 5. Co-autoxidations of egg phosphatidylcholine lipids (1 mM) and STY-BODIPY (8 μM, monitored at 565 nm, ε = 123,676 M−1 cm−1) suspended in phosphate-buffered saline (10 mM) at pH 7.4 initiated by MeOAMVN (0.2 mM) at 37 °C and inhibited by 2 μM of nitroxides (A). Co-autoxidations under identical conditions except initiated by 0.68 mM MeOAMVN and using PBD-BODIPY (10 µM, monitored at 584 nm, ε = 118,548 M−1 cm−1) as indicator. In addition, the experiments were performed in an EPR cavity under identical conditions to directly monitor the nitroxide concentrations. The inhibitory effect of 8 µM nitroxide, 8 µM NADPH as well as their combination is depicted for TEMPO (B), lipo-TEMPO (C), TBPNO (D), and DHQNO (E). stoichiometry of radical-trapping could be determined as above for the THF/STY-BODIPY co-autoxidations.48-49 Representative results are included in Figure 5A The reaction progress curves reveal that each of the nitroxides are reactive RTAs in lipid bilayers, and that all are significantly more reactive than a-tocopherol (from the initial rates, kinh = (3.2 ± 0.2) ´ 104, (3.3 ± 0.4) ´ 104, and (5.3 ± 0.3) ´ 104 M-1 s-1 for TEMPO, TBPNO and DHQNO, respectively, versus (4.7 ± 0.4) ´ 103 M-1 s-1 for a-TOH). In this medium, the RTA activity of the nitroxides is not obviously catalytic. In fact, TEMPO appears to trap only a single peroxyl radical, and after the initial inhibited period of ca. 1000 s, the reaction progress is essentially that of the uninhibited reaction. On the other hand, the autoxidations inhibited by TBPNO and DHQNO are retarded beyond the initial inhibited period, but only slightly so. Given the lack of THF or other potential hydride donors to reduce the oxoammonium ions, it is possible that the reductant is a lipid-derived alkyl radical as we first surmised. If this were the case, TEMPO, being the most polar of the three nitroxides, may not turn over at all because TEMPO+ more readily partitions to the aqueous phase away from lipid radicals. The foregoing experiments were also carried out in the presence of NADPH to determine if a hydride donor can facilitate catalytic RTA activity in liposomes as in aqueous THF. The results are shown in Figure 5B-D where, superimposed on the reaction progress determined by PBD-BODIPY50 co-autoxidation, the concentrations of the nitroxides (determined by EPR spectroscopy) are also indicated. In the presence of 1 equivalent of NADPH, the inhibition period corresponding to the reaction of

the nitroxide with chain-carrying peroxyl radicals at least doubled compared to its absence. This result implies that NADPH acts synergistically with the nitroxide – presumably by reduction of the corresponding oxoammonium ion51 – since NADPH alone only retards the oxidation.52 The changes in concentration of the nitroxide over the course of the autoxidations indicate very different dynamics between TEMPO and either TBPNO or DHQNO. In the absence of NADPH, the rate of nitroxide loss (and corresponding formation of oxoammonium ion) is similar for all compounds, with the small differences being consistent with the relative reactivity of each with peroxyl radicals (kinh). When NADPH is present, similar curves are observed for DHQNO and TBPNO wherein the rate of loss of nitroxide is slightly lower. However, the TEMPO profile is strikingly different. While the initial loss of TEMPO is similar, the nitroxide concentration then rebounds at ca. 1500 s (see Figure 5B), peaking at around half the initial concentration at ca. 3500 s and then falling to zero – all while the autoxidation remains retarded. These results can be rationalized on the basis of the greater hydrophilicity of TEMPO+ relative to DHQNO+ and TBPNO+. Thus, upon oxidation of TEMPO with a peroxyl radical, TEMPO+ partitions to the aqueous phase where it is readily reduced to TEMPOH by NADPH. TEMPOH and TEMPO+ can comproportionate to yield TEMPO that accumulates in the aqueous phase, gradually partitioning back to the lipid where it can inhibit the autoxidation. In contrast, DHQNO+ and TBPNO+ remain largely in the lipid and/or interfacial region where, once reduced by NADPH, the hydroxylamines readily undergo reactio-


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ns with peroxyl radicals. The nitroxides never accumulate because they are formed in the lipid bilayer – or at least at the interface – and react readily with peroxyl radicals as they are formed in the lipid. To provide some additional insight on this point, we synthesized a TEMPO derivative of greater lipophilicity (hereafter lipoTEMPO; 4-amino-TEMPO acylated with dodecanoic acid) and examined its ability to inhibit lipid peroxidation in the same liposomes. While this nitroxide is inherently less reactive than TEMPO (due to both the inductive electron withdrawal of the amide group and the reduced mobility in the bilayer due to the C12 sidechain, see Supporting Information for more details), it is still an effective inhibitor (Figure 5E). Moreover, although it appears to act synergistically with NADPH, the nitroxide is not initially consumed and then reformed as is TEMPO – presumably due to its anchoring in the lipid. Instead, the concentration of the nitroxide is largely maintained while the autoxidation is inhibited. Since the nitroxide itself is less reactive than the corresponding hydroxylamine, its concentration does not drop until all the NADPH – and therefore hydroxylamine – is depleted.

V. Nitroxides Can Be Potent Inhibitors of Ferroptosis. To ascertain how the RTA activity of nitroxides in liposomes may translate to cells, their potencies as inhibitors of ferroptosis were determined. Ferroptosis was induced in Pfa1 mouse embryonic fibroblasts53 by inhibition of the lipid hydroperoxide detoxifying enzyme glutathione peroxidase (Gpx4) with (1S,3R)-RSL3.13, 54-55 The results are shown in Figure 6, alongside corresponding data obtained with ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1), the archetype ferroptosis inhibitors.

Figure 6. Dose-response curves obtained from RSL3 (100 nM) induced ferroptosis in Pfa1 cells inhibited by either TEMPO (●), lipo-TEMPO (■ ), TBPNO (▲), DHQNO (♦) compared alongside Fer-1 (▼) and Lip-1 (◄). The reactivity trends of the nitroxides observed in the liposome autoxidations translate well to the potency of the compounds in cell culture. That is, the cytoprotection afforded by nitroxide supplementation follows the same general trend wherein TBPNO ~ DHQNO > lipo-TEMPO > TEMPO. Although TEMPO was a rather poor inhibitor of ferroptosis (EC50 = 5300 nM), simple attachment of a lipophilic sidechain resulted in much greater potency (EC50 = 241 nM). The results are consistent with our hypothesis that TEMPO (and/or TEMPO+) partitions significantly to the aqueous phase, reducing its efficacy at

inhibiting lipid peroxidation occurring in the lipid rich regions of the cell. The monoaryl and diarylnitroxides are both more lipophilic than TEMPO and have preferential reactivity for both the initial electron transfer and the subsequent regeneration steps, leading to EC50 values of 99 and 80 nM, respectively, only ca. 2- fold higher than those of Fer-1 (45 nM) and Lip-1 (35 nM). DISCUSSION The cytoprotective properties of nitroxides have long been attributed to ‘radical-scavenging’ either by combination with alkyl radicals or by catalysis of superoxide dismutation. Unfortunately, these mechanisms are at odds with either the inherent chemistry or physical attributes of the nitroxides that display these properties. That is, the reaction of nitroxides with alkyl radicals must be irrelevant in all but the most hypoxic of tissues since the reaction of alkyl radicals with O2 proceeds with a significantly greater rate constant (~10´)27-28 and O2 is generally present at much higher concentrations than the nitroxide (mM vs. µM or less). Likewise, catalysis of superoxide dismutation is unlikely since most cells express superoxide dismutase enzyme(s) that are greater in both concentration and specific activity than the nitroxides. If one considers these facts, it is likely that another reactivity is key. Insight into an alternative mechanism of cytoprotection comes from Goldstein’s oft-overlooked work that shows a direct reaction between nitroxides and peroxyl radicals,30,56 which are the propagating species in lipid peroxidation. Our results are in good agreement with Goldstein’s. Although the absolute rate constants we have determined from inhibited autoxidations are about one order of magnitude lower (4.6 x 106 vs. 3-5 x 107 M-1 s1 ), this seems reasonable given that our reactions contain a significant amount of THF which will slow electron transfer from the nitroxides to peroxyls to form the ionic oxoammonium and peroxide products. Most importantly, our results clearly show that the oxoammonium ion can be recycled – by THF or other hydride donors in solution (see data obtained for for hydride transfer with dioxane, dimethoxyethane and co-autoxidations with 4ethylbenzoic acid in the Supporting Information). These results enable the proposal of a mechanistic scheme accounting for the catalytic radical-trapping antioxidant activity of nitroxides under ambient conditions in the absence of added acid, which is illustrated with TEMPO in Scheme 2. TEMPO undergoes electron transfer to a peroxyl radical, inhibiting the propagation of autoxidation and generating TEMPO+. The rate of regeneration of TEMPO via reduction of TEMPO+ with an alkyl radical is generally too slow to contribute, therefore requiring the presence of a hydride donor to convert TEMPO+ to TEMPOH. TEMPOH can trap another peroxyl radical via Hatom transfer, thereby regenerating TEMPO. The catalytic cycle is limited by the oft-invoked, but deleterious (in this context, at least), combination of TEMPO with an alkyl radical, which forms a stable alkoxylamine. Moreover, turnover is limited at low pH due to the protonation of TEMPOH, whose conjugate acid is not an H-atom donor. This becomes important at pH < 7 (the pKa of TEMPOH2+). The catalytic radical-trapping antioxidant reactivity of nitroxides extends well beyond aliphatic hindered nitroxides, such as TEMPO and related piperidine-N-oxyls, to include monoarylnitroxides, such as those that may derive from liproxstatins, as well as diarylnitroxides, such as those derived from dialkylated



Scheme 2. Catalytic cycle of autoxidation inhibition by TEMPO. diphenylamines that are common industrial RTAs. Although the diarylnitroxide undergoes slowest initial reactions with peroxyl radicals (consistent with its higher E°), it displays good catalytic activity, which can be ascribed to the greater ease with which it can be reduced from the oxoammonium ion to the hydroxylamine. The monoarylnitroxide has a faster reaction with peroxyl radicals (consistent with its lower E°), but possesses less efficient catalytic activity, than both the diarylnitroxide and TEMPO. The oxoammonium ion derived from the monoarylnitroxide is predicted to react more slowly with THF than the diaryloxoammonium ion, but faster than piperidinium-N-oxide. Thus, the poorer catalytic activity for the monoarylnitroxide may result from the instability of the oxoammonium ion to water. The results in buffered THF translate well to lipid bilayers, wherein it becomes clear that the differing polarity of the nitroxide and oxoammonium ion have a significant impact on the catalytic turnover of the nitroxide. Even in the absence of additional reductants, the more lipophilic molecules give a better overall stoichiometry. This can be rationalized by their ability to react with lipid derived alkyl radicals (or with HOO• that is reportedly formed in small amounts during the autoxidation of linoleate57). As our model system does not contain any lipophilic hydride donor, the generated oxoammonium ions must access the interface or transition to the aqueous phase to be reduced (e.g. by NADPH). The difference in the dynamics is most visible between TEMPO and lipo-TEMPO. Since the conversion of lipoTEMPO+ to lipo-TEMPOH is the rate determining step of the catalytic cycle and the reaction between lipo-TEMPO and peroxyl radicals is slowed owing to its higher redox potential, lipoTEMPO in the lipid phase. In contrast, in the TEMPO-inhibited autoxidation, TEMPOH accumulates. These results clearly demonstrate the effective recycling of nitroxides by NADPH, whose reaction was first suggested by Goldstein,51 but not demonstrated experimentally. Given that mitochondria produce significant amounts of reactive oxygen species and the mitochondrial dysfunction that can arise as a result is believed to contribute to a number of pathologies, efforts have been made to target nitroxides to the mitochon-

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dria. Two of the more prominent examples are Murphy’s MitoTEMPOL58 and Wipf’s XJB-5-131.3 Some time ago it was acknowledged that rather than operating as superoxide dismutase mimics, the nitroxides were catalytic inhibitors of lipid peroxidation because they are reduced to the corresponding hydroxylamine in situ,59 which in turn can transfer an H-atom to lipid-derived peroxyl radicals to inhibit lipid peroxidation, along with reformation of starting nitroxide.58 The stoichiometric reductant was proposed to be the reduced form of the mitochondrial electron carrier coenzyme Q10 (co-QH2) which has been shown to undergo direct reaction with the nitroxide to yield the hydroxylamine. The foregoing results indicate that co-QH2 is not strictly required for these compounds to display catalytic behaviour. If the nitroxide reacts with a peroxyl radical to yield the corresponding oxoammonium ion in the presence of a suitable hydride donor – such as the NAD(P)H normally used to regenerate co-QH2 from co-Q – this pathway can be bypassed.60 This may explain why non-mitochondria-targeted nitroxides are also cytoprotective. As shown above, each of the lipophilic TEMPO, monoarylnitroxide and diarylnitroxide are potent inhibitors of cellular lipid peroxidation and ferroptotic cell death. The ability of mitochondria-targeted nitroxides (e.g. XJB-5131) to subvert ferroptosis has motivated investigators to consider a role for mitochondrial oxidation in ferroptosis.12, 14, 61-62 Interestingly, despite lacking a mitochondria-targeting element, lipo-TEMPO appears to be similarly effective to XJB-5-131 in subverting ferroptosis. That is, lipo-TEMPO was found to be ca. 5fold less potent than Fer-1 in preventing RSL-3 induced ferroptosis in MEFs; essentially the same difference between XJB-5-131 and Fer-1 (albeit in HT-1080 cells).12 Moreover, the diarylnitroxide and monoarylnitroxide studied here, which also lack mitochondria-targeting elements, were almost as potent as Fer-1. These results suggests that mitochondrial oxidation may play little to no role in ferroptosis – consistent with the observation that a mitochondria-targeted derivative of coenzyme Q10 (a good RTA) is less effective in subverting ferroptosis than is the nontargeted Q10 analog decylubiquinone.69 Moreover, they suggest that alterations in the structure of the nitroxide to increase its lipophilicity and/or enhance the rate of the initial electron transfer may be most useful in the design of more potent nitroxidebased therapeutic and/or chemopreventive compounds – not specific targeting to the mitochondria. As proof of principle, we synthesized phenoxazine-N-oxyl (PHOXNO):

expecting that is would be easier to oxidize than the diarylnitroxide, as is true for the corresponding precursor amines.63 Indeed, PHOXNO was much more reactive to peroxyl radicals in buffered THF than the other nitroxides (kinh = (2.7 ± 0.3) ´ 107 M-1 s1 ), consistent with the lower E° we measured by cyclic voltammetry (0.79 V vs. NHE) – see Supporting Information for the data. The greater inherent reactivity of PHOXNO translated to liposomes, where again it was by far the most reactive of the nitroxides studied (kinh = (2.1 ± 0.2) ´ 105 M-1 s-1). Moreover, PHOXNO was the most potent of the nitroxides as an inhibitor of RSL3induced ferroptosis in mouse embryonic fibroblasts with an EC50


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of only 13 nM – demonstrably better than both Fer-1 and Lip-1. It is interesting to note that the potency of PHOXNO is essentially indistinguishable from that of the parent phenoxazine (with a kinh = (4.5 ± 0.5) ´ 105 M-1 s-1 and EC50 of 9 nM), which we recently described to be the most potent scaffold of ferroptosis inhibitor described to date.49 Since diarylamines are known to be converted to their corresponding diarylnitroxides under autoxidative conditions, these results further highlight the privileged nature of the phenoxazine scaffold as both an RTA and ferroptosis inhibitor: not only is it a potent compound, but so is its oxidation product. EXPERIMENTAL METHODS

General. Reagents and solvents were purchased from commercial sources and used without further purification unless otherwise indicated. TEMPO+BF4−,64 TEMPOH,65 TBPNO,66 TBPNOH,67 DHQNO,48 Fer-1,68 Lip-1,48 STY-BODIPY,34 PBDBODIPY,34 and RSL-369 were synthesized according to literature procedures. Autoxidations employed inhibitor-free HPLC grade solvents. Buffered solutions were made in milliQ water at 100 mM phosphate (pH 7 and 10) and 100 mM sodium acetate/acetic acid (pH 4). UV−visible spectra were measured with a Cary 100 spectrophotometer equipped with a thermostatted 6×6 multicell holder. Electron paramagnetic resonance (EPR) spectra were recorded on a Bruker EMXplus (X-band) spectrometer equipped with an ER 4119HS cavity. The radical concentration was determined using the quantitative EPR package of the Bruker Xenon software. Synthetic Procedures. 4-Lauramido-2,2,6,6-tetramethylpiperidine-l-oxyl (lipo-TEMPO). LipoTEMPO was synthesized from its corresponding amine in a manner similar to TBPNO.66 Briefly, 4-lauramido-2,2,6,6-tetramethylpiperidine (1 mmol) was dissolved in methanol (4.5 mL) and heated to reflux. To this, a 30% solution of hydrogen peroxide (0.4 mL) was added dropwise followed by a solution of sodium tungstate decahydrate (0.1 mmol) in water (0.07 mL). The solution was refluxed until the reaction was deemed complete by TLC. The solution was extracted using dichloromethane three times, washed with brine and dried using MgSO4. The solvent was filtered and then evaporated under reduced pressure. The crude product was purified by column chromatography (hexanes → 15% Et2O/hexanes) to yield 22%. EPR (benzene, g = 2.0064, a (G)): 15.50 (N), 0.41 (12H). HRMS (EI, magnetic sector): calcd for C21H41N2O2 353.3168, found 353.3198. Phenoxazine-N-oxyl. Phenoxazine 1.83 g (10 mmol) was dissolved in 30 mL of diethyl ether. Seperately, 3.50 g of 77% mCPBA was dissolved in 14 mL of diethyl ether, and then slowly added to the phenoxazine solution, while swirling. After 2 minutes, the solution turned from colourless to yellow/black. By scratching the walls of the flask with a pipette induced crystallization, which proceeded for ~1 hour. The solvent was decanted, and the crystals were rinsed three times with cold diethyl ether then three times with petroleum ether which afforded 635 mg (32 %) of the phenoxazine-N-oxyl; mp 127-129 oC; EPR (benzene, g = 2.0051, a (G)): 9.03 (N), 2.40 (H1,3,7,9), 0.55 (H2,4,6,8). HRMS (EI, magnetic sector): calcd for C12H8NO2 198.0555, found 198.0571.

Inhibited Autoxidation of THF. A 3.5 mL quartz cuvette was loaded with unstabilized THF (0.625 mL) and the appropriate buffer (1.810 mL). The cuvette was then preheated to 37 °C for approximately 5 min. After blanking, 12.5 μL of 2 mM STYBODIPY in DMSO and 50 μL of 50 mM AAPH in buffer were added and the solution thoroughly mixed. After 8 min, a 10 µL aliquot of antioxidant stock solution in buffer was added, and the loss of absorbance at 562 nm was followed. The inhibition rate constant (kinh) and stoichiometry (n) were determined for each experiment according to the equations in Figure 1E. Kinetics are derived from a miminum of three independent experiments and are reported as the mean ± standard deviation. An analogous procedure was used for EPR monitoring where the measurements was performed in a capillary with 80 µL volume in the EPR cavity. Therefore, the solutions were completely mixed initially, including the antioxidant, and then placed in the cavity at 37 °C. Inhibited Autoxidation of Egg-PC Liposomes. To a 3 mL cuvette, 2.34 mL of 10 mM PBS at pH 7.4 and liposomes (125 μL of 20 mM stock in PBS at pH 7.4) were added and the solution was equilibrated for 5 min at 37 °C. After blanking, 10 μL of 2 mM STY-BODIPY (or PBD-BODIPY) in DMSO and 10 μL of 0.05 M MeOAMVN in acetonitrile were added and the solution was thoroughly mixed. After 5 min, a 10 µL aliquot of antioxidant stock solution in DMSO was added and the loss of absorbance at 565 nm followed. The inhibition rate constant (kinh) and stoichiometry (n) were determined for each experiment according to Figure 1E. Kinetics are derived from a miminum of three independent experiments and are reported as the mean ± standard deviation. Matching EPR experiments were performed as described above. Inhibition of Ferroptosis Induced by Gpx4 Inhibition with (1S,3R)-RSL3. Mouse embryonic fibroblasts (Pfa-1 cells, 3,000 in 100 µL) were seeded in 96-well plates and incubated overnight. The next day the media was removed, the cells were washed twice with PBS and the cells were suspended in new media and treated with different compounds for 30 minutes before addition of (1S, 3R)-RSL3 (100 nM) in a final volume of 100 µl. Cell viability was assessed 6 hours later using the AquaBluer (MultiTarget Pharmaceuticals, LLC) assay according to the manufacturer’s instructions. Cell viability was calculated by normalizing the data to untreated controls. Each experiment was carried out in six analytical replicate per concentration of inhibitor and repeated independently at least three times. Electrochemistry. Standard potentials were measured against Fc/Fc+ by cyclic voltammetry at 25 °C in dry acetonitrile with 0.1 M NBu·PF6 as supporting electrolyte. Experiments were carried out with a potentiostat equipped with a glassy-carbon working electrode, a platinum auxiliary electrode, and a Ag/AgNO3 (5 mM) reference electrode. Calculations. Calculations were carried out using the CBS-QB3 complete basis set method42 as implemented in the Gaussian 16 suite of programs.70 To get accurate energies for the charged species, we employed the SMD71 continuum solvent model for all calculations (except BDE). Rate constants were calculated via transition state theory at 37 °C.



ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional autoxidation traces for THF/buffer, 4-ethylbenzoate, and liposomes, EPR spectra, voltammagrams, extracted kinetics data (inhibition and hydride transfer), collected EC50 values, detailed kinetics model, and optimized geometries and energies for computational results. (PDF)

AUTHOR INFORMATION Corresponding Author * [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada and the Canada Foundation for Innovation and through generous access to the computational resources of the Centre for Advanced Computing (cac.queensu.ca). DAP and RS also acknowledge the support of the Canada Research Chairs and NSERC Post-Graduate Scholarships programs, respectively. The authors thank Luke Farmer for synthesis of the phenoxazine-N-oxyl,

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that lipid-rich regions are the target area, and thus, that their ability to inhibit lipid peroxidation by trapping chain-propagating lipid peroxyl radicals underlies their bioactivity. See: (a) Cighetti, G.; Allevi, P.; Debiasi, S.; Paroni, R., Chem. Phys. Lipids 1997, 88 (2), 97106. (b) Patel, K.; Chen, Y.; Dennehy, K.; Blau, J.; Connors, S.; Mendonca, M.; Tarpey, M.; Krishna, M.; Mitchell, J. B.; Welch, W. J.; Wilcox, C. S., Am. J. Physiol. 2006, 290, R37-R43. (c) Cimato, A. N.; Piehl, L. L.; Facorro, G. B.; Torti, H. B.; Hager, A. A., Free Radical Biol. Med. 2004, 37 (12), 2042-2051. (57) Tikhonov, I. V.; Pliss, E. M.; Borodin, L. I.; Sen, V. D.; Kuznetsova, T. S., Russ. Chem. Bull. 2015, 64 (10), 2438-2443. (58) Trnka, J.; Blaikie, F. H.; Smith, R. A. J.; Murphy, M. P., Free Radical Biol. Med. 2008, 44 (7), 1406-1419. (59) It should be acknowledged that accumulation of nitroxides in the mitochondrial intermembrane space, which has a lower pH than the cytosol, would lead to decreased catalytic activity via protonation of the hydroxylamine. (60) We cannot also rule out that the nitroxide can be regenerated from the oxoammonium ion by reaction with chaincarrying lipid radicals, as in heavy hydrocarbons and shown in Scheme 1B, although as indicated before this would be an overall slow reaction rate. (61) Wang, Y.-Q.; Chang, S.-Y.; Wu, Q.; Gou, Y.-J.; Cui, Y.-M.; Yu, P.; Shi, Z.-H.; Gao, G.; Chang, Y.-Z.; Jia, L.; Wu, W.-S., Front Aging Neurosci 2016, 8, 308. (62) Neitemeier, S.; Jelinek, A.; Laino, V.; Hoffmann, L.; Eisenbach, I.; Eying, R.; Ganjam, G. K.; Dolga, A. M.; Oppermann, S.; Culmsee, C., Redox Biol. 2017, 12, 558-570. (63) Farmer, L. A.; Haidasz, E. A.; Griesser, M.; Pratt, D. A., J. Org. Chem. 2017, 82 (19), 10523-10536. (64) Holan, M.; Jahn, U., Org. Lett. 2014, 16 (1), 58-61. (65) McGrath, A. J.; Garrett, G. E.; Valgimigli, L.; Pratt, D. A., J. Am. Chem. Soc. 2010, 132 (47), 16759-16761. (66) Golubev, V. A.; Sen, V. D., Russ. J. Org. Chem. 2013, 49 (4), 555-558. (67) Golubev, V. A.; Tkachev, V. V.; Sen, V. D., Russ. J. Org. Chem. 2014, 50 (5), 678-684. (68) Skouta, R.; Dixon, S. J.; Wang, J.; Dunn, D. E.; Orman, M.; Shimada, K.; Rosenberg, P. A.; Lo, D. C.; Weinberg, J. M.; Linkermann, A.; Stockwell, B. R., J. Am. Chem. Soc. 2014, 136 (12), 4551-4556. (69) Friedmann Angeli, J. P.; Schneider, M.; Proneth, B.; Tyurina, Y. Y.; Tyurin, V. A.; Hammond, V. J.; Herbach, N.; Aichler, M.; Walch, A.; Eggenhofer, E.; Basavarajappa, D.; Radmark, O.; Kobayashi, S.; Seibt, T.; Beck, H.; Neff, F.; Esposito, I.; Wanke, R.; Foerster, H.; Yefremova, O.; Heinrichmeyer, M.; Bornkamm, G. W.; Geissler, E. K.; Thomas, S. B.; Stockwell, B. R.; O'Donnell, V. B.; Kagan, V. E.; Schick, J. A.; Conrad, M., Nat. Cell Biol. 2014, 16 (12), 1180-1191. (70) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Petersson, G. A.; Nakatsuji, H.; Li, X.; Caricato, M.; Marenich, A. V.; Bloino, J.; Janesko, B. G.; Gomperts, R.; Mennucci, B.; Hratchian, H. P.; Ortiz, J. V.; Izmaylov, A. F.; Sonnenberg, J. L.; Williams; Ding, F.; Lipparini, F.; Egidi, F.; Goings, J.; Peng, B.; Petrone, A.; Henderson, T.; Ranasinghe, D.; Zakrzewski, V. G.; Gao, J.; Rega, N.; Zheng, G.; Liang, W.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Throssell, K.; Montgomery Jr., J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M. J.; Heyd, J. J.; Brothers, E. N.; Kudin, K. N.; Staroverov, V. N.; Keith, T. A.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A. P.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Millam, J. M.; Klene, M.; Adamo, C.; Cammi, R.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Farkas,



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