Catalytic radical reduction in aqueous solution via oxidation of biologically-relevant alcohols
Metalloenzymes that normally perform catalytic antioxidant or radical-degrading functions, as well as small-molecule complexes that mimic them, can also exert pro-oxidant or radical-forming effects depending on the identity of the terminal reductant. Because nitroxyl radicals function as redox active cocatalysts in the aerobic oxidation of alcohols, we hypothesized that catalytic radical reduction could be achieved via the oxidation of biologically-relevant alcohols. Herein we report an organoruthenium complex (Ru1) that catalyzed reduction of 2,2 0 -azino-bis(3-ethylbenzo-thiazoline-6-sulfonate) radical monoanion (ABTSc À ) to ABTS 2À in phosphate buffered saline (pH 7.4) using MeOH, EtOH, i-PrOH, serine, threonine, glucose, arabinose, methyl lactate or dimethyl malate as the terminal reductant. Replacing either the C-H or O-H groups of a -CHOHmoiety resulted in the loss of ABTSc À reducing ability. Moreover, in conjunction with an alcohol terminal reductant, Ru1 was able to inhibit the oxidation of ABTS 2À by H 2 O 2 and horseradish peroxidase, even after multiple successive challenges with excess H 2 O 2 or ABTSc À . Collectively, these results demonstrate that Ru1 inhibits the oxidative formation of and catalyzes the reduction of radicals in aqueous solution via oxidation of biologically-relevant alcohols.
Introduction<!>À<!>Approach and rationale<!>Catalytic radical reduction in aqueous buffer<!>Biologically-relevant terminal reductants<!>Inhibition of oxidative radical formation<!>Conclusions
<p>Reactive oxygen species (ROS) such as superoxide (O 2</p><!><p>) and hydrogen peroxide (H 2 O 2 ) can alter the structures and functions of biomolecules 1,2 via DNA base lesion formation, 3 transition metal ion release from proteins, 4 and membrane phospholipid chain degradation. 5 Antioxidants can prevent or attenuate this damage by reacting with ROS to yield less oxidizing, more stable radical or non-radical products. Superoxide dismutase (SOD) and catalase, for example, are metalloenzymes that protect cells by catalyzing the disproportionation of O 2</p><p>À and H 2 O 2 , respectively. 6 Synthetic Mn-salen and Mn-porphyrin complexes have demonstrated both SOD and catalase activity, 7 and some have entered phase 1 clinical trials as therapeutics for neurodegenerative and pulmonary diseases. 8 Recent studies have shown that these classes of synthetic Mn complexes can also exert pro-oxidant effects at low ROS concentrations, 9,10 dichotomous behavior that is consistent with the enzymes they mimic. Catalase converts 2H 2 O 2 into O 2 + 2H 2 O (Scheme 1-i), 11 wherein the rst equivalent of H 2 O 2 is reduced to H 2 O and generates an Fe(IV)]Oc + intermediate, which then oxidizes the second equivalent of H 2 O 2 to O 2 and releases H 2 O. 12 At low H 2 O 2 concentrations, this Fe(IV)]Oc + intermediate can abstract Hc from other molecules (i.e., A-H) and thus exhibit peroxidase-like reactivity (Scheme 1-ii). 13 Conversely, peroxidase can exhibit catalase-like reactivity at low A-H/high H 2 O 2 concentrations. 14 Because the Mn-salen and Mn-porphyrin complexes proceed through high-valent Mn oxo/ hydroxo intermediates [15][16][17][18] with oxidizing power comparable to Fe(IV)]Oc + , their reactivity will mimic catalase or peroxidase based on the availability of H 2 O 2 . Therefore, the function of catalase and its biomimetic Mn complexes as either Scheme 1 Catalase normally catalyzes the disproportionation of 2H 2 O 2 / O 2 + 2H 2 O (i), but at low H 2 O 2 concentrations it can abstract Hc from other molecules (A-H) and produce radicals (Ac) in a manner similar to peroxidase (ii). Copper or ruthenium complexes can catalyze the oxidation of R 1 -CHOH-R 2 to R 1 -C(]O)-R 2 using a nitroxyl radical (e.g., TEMPO, n ¼ 1 for Cu, n ¼ 2 for Ru) as the Hc abstracting reagent (iii) and O 2 as the terminal oxidant (iv). By a similar mechanism, a complex could catalyze radical reduction using a biologically relevant non-tertiary alcohol as the terminal reductant (v).</p><p>antioxidants or pro-oxidants will be determined by the nature of the terminal reductant.</p><p>A catalytic oxidation reaction important in synthetic chemistry is the aerobic oxidation of R 1 -CHOH-R 2 to R 1 -C(]O)-R 2 that does not cause any (1) over-oxidation to R-COOH or (2) undesired reactions at other functional groups. [19][20][21][22][23] One strategy employs a copper or ruthenium catalyst in conjunction with a nitroxyl radical, such as 2,2,6,6-tetramethylpiperidinyl-N-oxyl (TEMPO), to oxidize R 1 -CHOH-R 2 to R 1 -C(]O)-R 2 with concomitant formation of TEMPO-H (Scheme 1-iii). Subsequent regeneration of the TEMPO radical occurs with the oxidation of TEMPO-H by O 2 , which functions as the terminal oxidant for this reaction (Scheme 1-iv). Because the TEMPO radical is reduced by R 1 -CHOH-R 2 in the aerobic alcohol oxidation catalytic cycle, we hypothesized that the catalytic reduction of other radicals could also be achieved via the oxidation (i.e., dehydrogenation) of non-tertiary alcohols (Scheme 1-v). Herein we report the validation of this hypothesis with an organoruthenium complex that catalyzes the reduction and inhibits the formation of radicals in buffered aqueous solutions via the oxidation of biologically-relevant non-tertiary alcohols. Given the successes of group 8 transfer hydrogenation catalysts as chemotherapeutics 24,25 in particular and the burgeoning scope of medicinal applications for transition metalbased catalytic systems [26][27][28] in general, we anticipate that the reduction of radicals via the Ru-catalyzed oxidation of alcohols will lead to new strategies for protecting against ROS in vivo.</p><!><p>We recently reported a Ru complex supported by a chelating N-heterocyclic carbene-carboxylate ligand (Ru1, Scheme 2A) that catalyzed the hydrogenation of C]O, C]N and C]C bonds, in which the H 2 transferred was obtained from the oxidation of i-PrOH to acetone. 29 Based on this catalytic chemical reactivity observed with Ru1 and previous reports of other Ru complexes with chelating carboxylate ligands exerting antioxidant effects in cells, which derived from irreversible stoichiometric reactions with nitric oxide, 30,31 we hypothesized that Ru1 would function as a catalytic antioxidant, reducing radicals using non-tertiary alcohols as terminal reductants. Using methods previously reported by our group, 29,32 the N-heterocyclic carbene (NHC) ligand precursor [1H 2 ][Br] was treated with Ag 2 O to afford the Ag-NHC complex [Ag(1)] n (2). The NHC ligand was subsequently transferred to Ru via the reaction of 2 with [{RuCl(h 6 -cymene)} 2 (m-Cl) 2 ], which yielded the Ru-NHC complex [RuCl(1)(h 6 -cymene)] (Ru1).</p><p>As a radical substrate to probe antioxidant activity, 2,2 0azino-bis(3-ethylbenzothiazoline-6-sulfonate) radical monoanion (ABTSc À , Scheme 2B) was selected on the basis of its large extinction coefficient (3) at long wavelengths (3 750 ¼ 1.6 Â 10 4 M À1 cm À1 in EtOH and 3 734 ¼ 1.5 Â 10 4 M À1 cm À1 in aqueous buffer), 33 its reversible one-electron reduction to ABTS 2À (E 1/2 ¼ 0.68 V vs. NHE) falling within the range of potentials accessible in biological systems (À0.4 to +0.8 V vs. NHE), 34 and its stability in aerobic, protic media. The longer absorption wavelength and greater extinction coefficient for ABTSc À enables spectroscopic analysis of radical-degrading and -forming reactions (1) with less interference from other optically absorbing species present in biological systems and (2) at lower concentrations ‡ more relevant to oxidative stress than would be possible with direct measurements of O 2 c À (3 250 ¼ 1.9 Â 10 3 M À1 cm À1 ) or H 2 O 2 (3 240 ¼ 43.6 M À1 cm À1 ). 35,36 Furthermore, the 1e À reduction potential for ABTSc À is lower than or comparable to the standard reduction potentials for several of the oxidizing species associated with oxidative stress in biological systems (e.g., E ¼ 1.78 V for H 2 O 2 , 1.6 V for ROc, 1.0 V for ROOc, 0.92 V for Cys-Sc, 0.70 for O 2 , etc.), 34 therefore ABTSc À is a reasonable substrate for approximating biologically-relevant oxidants.</p><p>As a non-catalytic antioxidant control, Trolox (Scheme 2C) was employed given its use as a benchmark in a variety of radical degradation and antioxidant studies. 37 Trolox can serve as a 1e À or 2e À reductant, whereby the rst 1e À oxidation is accompanied by rapid H + loss to form a phenoxyl radical (TrOc), which can then undergo a second 1e À oxidation to form a phenoxonium cation (TrO + ). However, in methanol (MeOH) or ethanol (EtOH) solutions, these processes converge into a single 2e À oxidation. 38 Subsequent hydrolysis of TrO + can then cleave the tertiary carbon-oxonium bond, which renders the 2e À oxidation of Trolox irreversible.</p><p>Because ROS and other oxidants can cause damage to critical biomolecules, inhibiting the formation of these oxidizing species in biological systems can potentially prevent the onset of harmful pathologies. 2 As a model system, we selected the oxidation of ABTS 2À to ABTSc À by horseradish peroxidase (HRP) and H 2 O 2 (i.e., Scheme 1-ii). 39 However, before studying the ability of Ru1 to inhibit ABTSc À formation, it was rst necessary to explore the fundamental reactivity of Ru1 with ABTSc À under controlled conditions and achieve the catalytic reduction of ABTSc À to ABTS 2À in buffered aqueous solution. was essential for the reduction of ABTSc À to ABTS 2À and that CH 3 CN did not impact ABTSc À stability. No ABTSc À formation was observed in an aerobic solution containing 5 mM Ru1 and 50 mM ABTS 2À , indicating that Ru1 does not oxidize ABTS 2À to ABTSc À under these conditions. In contrast to Ru1, the addition of 5 mM Trolox caused a rapid (within mixing time) 22% decrease in ABTSc À concentration (Fig. 1, blue line), which corresponded to the reduction of 10 mM ABTSc À (2 equiv. vs. Trolox) and was consistent with the ability of Trolox to serve as a non-catalytic 2e À reductant in EtOH solution. 38 To assess its catalytic potential and corresponding regeneration, the reactivity of Ru1 with multiple sequential aliquots of excess ABTSc À was examined. Aer the reduction of 50 mM ABTSc À by 5 mM Ru1 was complete (Fig. 2, red line), 2 additional 50 mM ABTSc À aliquots were introduced § and complete ABTSc À reduction was observed in each instance, indicating that Ru1 remained catalytically competent for the complete reduction of 150 mM ABTSc À (30 turnovers). Unlike Ru1, the addition of successive 10 mM aliquots § of ABTSc À following the initial reduction by 5 mM Trolox (Fig. 2, blue line) only caused the radical absorbance to increase by amounts equivalent to the concentration of ABTSc À added, indicating that the antioxidant capacity of Trolox had been exhausted aer the reduction of only 2 equiv. of ABTSc À . However, subsequent addition of 5 mM Ru1 to the exhausted Trolox solution containing 70 mM ABTSc À resulted in 100% radical reduction within 30 min.</p><p>Addition of 5 mM ascorbate (AscH À ) or 5 mM glutathione (GSH) to a solution of 50 mM ABTSc À in EtOH caused the ABTSc À concentration to decrease by 5% or 60% (Fig. S2 and S3 †), values that were consistent with their ability to function as 1e À or 6e À reductants, respectively, under these experimental conditions. [40][41][42] Similar to the experiments with Trolox, subsequent addition of ABTSc À only afforded increases in radical absorbance equal to the concentration of ABTSc À added, which likewise indicated that the antioxidant capacity of AscH À and GSH had been exhausted. Quantitative radical reduction could nonetheless be achieved via addition of 5 mM Ru1 to the exhausted AscH À or GSH solutions that contained excess ABTSc À . Collectively, these data demonstrate that Ru1 can catalytically reduce ABTSc À and, because it is regenerated aer each reaction cycle, that a catalytic antioxidant can offer signicantly greater protection than traditional stoichiometric antioxidants, such as Trolox, AscH À and GSH.</p><p>Although ABTSc À degradation has been previously observed with other Ru complexes, [43][44][45] Ru1 is the rst to demonstrate catalytic ABTSc À reduction. It is important to note that the previous studies measured percentage of ABTSc À degradation with respect to Ru complex concentration, for the purpose of determining the dose dependence of radical degradation. However, the reported absorbance vs. time plots displayed signicantly slower ABTSc À degradation for the Ru complexes compared to Trolox (much like Ru1 vs. Trolox, Fig. 1) and 100% ABTSc À degradation at multiple Ru concentrations (demonstrating dose independence). Thus, it is possible that a previously reported Ru complex may have degraded ABTSc À catalytically, but its signicantly slower reactivity compared to Trolox created the appearance that the percent of ABTSc À degraded was dependent on Ru complex concentration and led to the conclusion that the observed radical degradation was non-catalytic.</p><!><p>As a catalyst for the 1e À reduction of ABTSc À to ABTS 2À , Ru1 itself cannot serve as the terminal reductant for this reaction, a role most likely played by the EtOH solvent for the experiments displayed in Fig. 1. To test this hypothesis and identify the chemical features required for terminal reductant function, it was rst necessary to determine experimental conditions under which no ABTSc À degradation occurred in the presence of Ru1 alone. Phosphate-buffered saline (PBS, pH 7.4) was selected as a suitable reaction medium because neither the solvent (H 2 O) nor the buffer components (Na 2 HPO 4 , KH 2 PO 4 , NaCl, KCl) would undergo oxidation to supply the electrons necessary  for the reduction of ABTSc À (i.e., Fig. 3-i). No degradation of ABTSc À in PBS was observed aer treatment with 5 mM Ru1 (Fig. 3-ii), indicating this solution lacked a suitable terminal reductant. Subsequent addition of 50 mM EtOH to this PBS solution containing 5 mM Ru1 and 50 mM ABTSc À caused the ABTSc À concentration to decrease (Fig. 3-iii), evidence that the electrons needed for the ABTSc À reduction observed in Fig. 1 were ultimately supplied by the EtOH solvent.</p><p>If the hypothesis that the oxidation of R 1 -CHOH-R 2 to R 1 -C(]O)-R 2 provides the electrons necessary for the reduction of ABTSc À to ABTS 2À is correct (i.e., Scheme 1-v), then both the O-H and C-H groups in a -CHOHmoiety will be essential for terminal reductant function. To test this hypothesis, PBS solutions containing 5 mM Ru1 and 50 mM ABTSc À were treated with 50 mM MeOH, i-PrOH, t-BuOH, ethylene glycol (EG) or 1,2dimethoxyethane (DME) and the ABTSc À concentration was measured. With MeOH, i-PrOH and EG, the ABTSc À concentration began to decrease immediately following addition of the alcohol, indicating that these alcohols could serve as terminal reductants (Fig. S4-S6 †). In contrast, no ABTSc À reduction was observed with DME or t-BuOH (e.g., Fig. 4-ii). However, when the addition of 50 mM t-BuOH was followed by 50 mM EtOH, ABTSc À reduction did occur (Fig. 4-iii), revealing that the lack of reactivity with t-BuOH was not due to catalyst deactivation but rather the inability of t-BuOH to serve as a terminal reductant. Collectively, these results show that the C-H and O-H groups of a -CHOHmoiety are both essential for terminal reductant function, which is consistent with the hypothesis that oxidation of R 1 -CHOH-R 2 to R 1 -C(]O)-R 2 provides the reducing equivalents necessary to convert ABTSc À to ABTS 2À . Replacing either the C-H group with C-Me or the O-H group with O-Me precludes this oxidation and thus the ability to function as a terminal reductant, which is supported by the lack of ABTSc À reduction observed with t-BuOH and DME.</p><!><p>A wide variety of biomolecules comprise -CHOHgroups, therefore we hypothesized that sugars, amino acids and citric acid cycle metabolites could serve as suitable terminal reductants in Ru1-catalyzed ABTSc À reduction. To test this hypothesis, PBS solutions containing 5 mM Ru1 and 50 mM ABTSc À were treated with 50 mM serine, threonine, glucose, arabinose, malic acid or lactic acid. Reduction of ABTSc À was markedly slower following the addition of the amino acids compared to the sugars, consistent with the greater number of CH-OH groups per molecule in the latter (Fig. 5). No signicant differences in reactivity were observed between serine and threonine or between glucose and arabinose.</p><p>Malic acid and lactic acid are metabolites of the citric acid cycle and each comprises a -CHOHgroup. However, no ABTSc À reduction occurred following the addition of 50 mM malic acid or lactic acid to 5 mM Ru1 and 50 mM ABTSc À in PBS (Fig. 6). Malate and lactate will be the predominant species in pH 7.4 PBS, and these carboxylate anions can function as chelating bidentate ligands, which could potentially disrupt the catalytic activity of Ru1. To prevent catalyst deactivation, the methyl esters of these substrates were used. Addition of 50 mM methyl lactate (Me-lactate) or dimethyl malate (Me 2 -malate) to PBS solutions containing 5 mM Ru1 and 50 mM ABTSc À caused the ABTSc À concentration to decrease rapidly. None of the biologically-relevant alcohols afforded ABTSc À reduction in the</p><!><p>Having obtained unambiguous evidence that Ru1 catalyzes ABTSc À reduction using biologically-relevant alcohols as terminal reductants, we next sought to demonstrate that Ru1 could also inhibit radical formation. As the model system, we utilized the in situ oxidation of ABTS 2À to ABTSc À by horseradish peroxidase (HRP) and H 2 O 2 (i.e., Scheme 1-ii). 39 Addition of 10 mM H 2 O 2 to a PBS solution of 10 nM HRP, 20 mM ABTS 2À , 5 mM Ru1, and 50 mM EtOH caused the ABTSc À concentration to increase immediately. The ABTSc À concentration peaked at 1.0 mM aer 2.3 min, which then gradually declined and complete ABTSc À reduction was observed 13 min aer the peak (Fig. 7A, red line). No ABTSc À formation occurred when 10 mM H 2 O 2 was added to 5 mM Ru1 and 20 mM ABTS 2À in PBS, indicating that Ru1 did not cause any pro-oxidant reactions. Because Ru1 and Trolox were added as 30 mL aliquots from 5.0 mM stock solutions in CH 3 CN, 10 mM H 2 O 2 was added to a PBS containing 10 nM HRP, 20 mM ABTS 2À and 0.19 M CH 3 CN to control for the inuence of this solvent. The radical absorbance immediately began to increase and reached a maximum of 17.9 mM aer 50 min (Fig. 7A, dotted green line and Fig. S7 †). In contrast, the addition of 10 mM H 2 O 2 to a PBS solution of 5 mM Trolox, 10 nM HRP and 20 mM ABTS 2À resulted in no change in ABTSc À concentration for the rst 7.6 min, demonstrating that Trolox completely halted ABTSc À formation (Fig. 7A, blue line). Aer 7.6 min, however, the ABTSc À concentration began to increase and reached a maximum of 9.3 mM at 12 min aer the onset of ABTSc À formation. Notably, the maximum [ABTSc À ] observed in the presence of Trolox was 8.6 mM lower than the control, which was consistent with the ability of Trolox to function as a 2e À reductant (cf., the 22% decrease observed with Trolox in Fig. 1A). For the control and Trolox experiments, the decline in ABTSc À concentration aer peaking was consistent with normal ABTSc À thermal decay.</p><p>To determine if Ru1 remained catalytically competent aer the complete reduction of ABTSc À formed by 10 nM HRP and 10 mM H 2 O 2 , two additional 10 mM aliquots of H 2 O 2 were introduced (Fig. 7B, red line). Impressively, the concentration of ABTSc À never exceeded 0.4 mM and decreased back to zero in less than 16 min aer the introduction of the second and third H 2 O 2 aliquots. In contrast, addition of the second 10 mM H 2 O 2 aliquot to the Trolox experiment caused the ABTSc À concentration to gradually increase by 7.9 mM over the course of 38 min (Fig. 7B, blue line), effectively resulting in complete oxidation of ABTS 2À to ABTSc À (96% of the 17.9 mM observed for control). It was therefore unsurprising that the third 10 mM H 2 O 2 aliquot produced no change in radical absorbance. Subsequent addition of 50 mM EtOH and 5 mM Ru1 caused the ABTSc À concentration to decrease rapidly, affording quantitative ABTSc À reduction in less than 30 min.</p><p>To conrm that the results obtained with Ru1 arose from its ability to catalyze ABTSc À reduction, rather than inhibiting HRP or another chemical reaction, two 10 mM aliquots of chemically synthesized ABTSc À were introduced aer the ABTSc À concentration had decreased to zero following the initial addition of 10 mM H 2 O 2 (Fig. 7C, red line). The rst and second ABTSc À aliquots produced immediate increases in ABTSc À concentration corresponding to 6.3 and 6.7 mM, respectively, followed by rapid decreases in radical absorbance that resulted in quantitative radical reduction within 75 s of aliquot addition. With the Trolox experiment, however, the rst and second ABTSc À aliquots caused [ABTSc À ] to increase by 9.9 and 9.7 mM, respectively, with  no observable changes in radical absorbance other than thermal decay (Fig. 7C, blue line). Addition of 50 mM EtOH and 5 mM Ru1 aer the second ABTSc À aliquot afforded complete ABTSc À reduction within 30 min, similar to the experiment with multiple H 2 O 2 aliquots. Both AscH À and GSH inhibited the HRP-induced oxidation of ABTS 2À , albeit for shorter periods of time and less effectively than Trolox. The introduction of additional H 2 O 2 (Fig. S8 and S9 †) or ABTSc À aliquots (Fig. S10 and S11 †) only afforded increases in ABTSc À concentration that mirrored the behaviors observed for Trolox (i.e., Fig. 7B and C). Subsequent addition of 50 mM EtOH and 5 mM Ru1 to the AscH À and GSH solutions containing excess ABTSc À produced quantitative radical reduction, consistent with the Trolox experiments.</p><!><p>An organoruthenium complex (Ru1) that was previously reported to catalyze the hydrogenation of unsaturated organic substrates, using the oxidation of i-PrOH to acetone to supply the needed H 2 , has now been shown to also catalyze the reduction of ABTSc À to ABTS 2À in buffered aqueous solution. By itself, Ru1 was unreactive towards ABTSc À and the presence of a non-tertiary alcohol was essential for ABTSc À reduction to occur. Replacing either the C-H or O-H group in a -CHOHmoiety with C-Me or O-Me, respectively, resulted in the loss of ABTSc À reducing ability. Given the previously reported transfer hydrogenation activity of Ru1 and the fact that Ru1-catalyzed ABTSc À reduction required a reactant comprising a -CHOHmoiety, we deduced that the reducing equivalents necessary for the 1e À reduction of ABTSc À to ABTS 2À were supplied by the oxidation of R 1 -CHOH-R 2 to R 1 -C(]O)-R 2 . Consistent with this deduction, a diverse array of biologically-relevant nontertiary alcohols, including amino acids, sugars and citric acid cycle metabolites, were found to be suitable terminal reductants for the reduction of ABTSc À catalyzed by Ru1. Furthermore, in conjunction with an alcohol terminal reductant, Ru1 was able to inhibit the oxidation of ABTS 2À to ABTSc À by H 2 O 2 and horseradish peroxidase. In contrast, non-catalytic antioxidants (Trolox, ascorbate, glutathione), only offered limited protection that, once exhausted, had no impact on ABTSc À formation or stability. Impressively, no ABTSc À formation or other prooxidant effects by Ru1 were observed under any experimental conditions. The mechanism for the catalytic reduction of ABTSc À by Ru1 and non-tertiary alcohols, along with the biological applications, will be detailed in subsequent reports.</p>