Electrochemical and Mechanistic Study of Superoxide Scavenging by Pyrogallol in N , N -Dimethylformamide through Proton-Coupled Electron Transfer

: Scavenging of electrogenerated superoxide radical anion (O 2 •− ) by pyrogallol (PyH 3 ) was investigated on the basis of cyclic voltammetry and in situ electrolytic electron spin resonance spectrum in N , N -dimethylformamide with the aid of density functional theory (DFT) calculations. Quasi-reversible dioxygen/O 2 •− redox coupe was modified by the presence of PyH 3 , suggesting that O 2 •− was scavenged by PyH 3 through proton-coupled electron transfer (PCET) involving two proton transfer and one electron transfer. DFT calculation suggested that the prereactive formation of a hydrogen-bond (HB) complex and the subsequent concerted-two-proton coupled electron transfer characterized by catechol moiety in PyH 3 is plausible mechanism that embodies the superior kinetics of the O 2 •− scavenging by PyH 3 as shown in the electrochemical results. Furthermore, it was clarified that the three hydroxyl groups of PyH 3 promote the formation of HB complex, in comparative analyses using related compounds, resulting in the promotion of the O 2 •− scavenging.

Simultaneously, it is well recognized that the autoxidation is promoted in an alkali solution, showing that the primary deprotonation occurs forming corresponding anion (PyH2 − ) followed by the autoxidation reaction [2,3]. Furthermore, it is considered that forming intermediate peroxiradical (ROS) such as hydroperoxiradical (HO2 • ) followed by a free radical chain reaction is a main mechanism for the net autoxidation. O2 is a moderately good electrophile that can accept electrons from PyH3, rather than O2 •− . However, HO2 • formed after protonation of O2 •− as a Brønsted base is a strong oxidant. Thus, the deprotonation and subsequent oxidation, i.e., PT and ET between PyH3 and oxygen species involving O2 and ROS, are closely related. Most of the reported papers on the antioxidant activity of PyH3 are in aqueous solvents [2,3,6,11,12], and even under pH control, many protons are present. Among these papers, antioxidant reactions of PyH3 involving the autoxidation and ROS scavenging were detected and measured in aqueous media, by increases in O2 consumption or absorbance changes of the solution. Hence, the antioxidant reactions cannot be estimated separately, because several reactant species, i.e., PyH3, PyH2 − , O2, O2 •− , and HO2 • coexist in the experimental solution. Thus, there are still some uncertain issues to be clarified regarding the antioxidant mechanism with the relationship between PT and ET, although the plausible main mechanism for O2 •− scavenging by different reactants, PyH3/PyH2 − , are simply denoted in Scheme 1, forming pyrogallol-orthoquinone radical anion (PyH •− )/radical dianion (Py •2− ) and H2O2. Nasr et al. reported the electrochemical oxidation of PyH3 in acidic aqueous solution [12]. In their pioneering work, the voltammetric results showed that PyH3 oxidation occurs in the same potential region as that of phenol (PhOH). Conversely, the initial deprotonation of PyH3 forming PyH2 − increases electron density in the benzene ring and consequently increases its reactivity to electrophilic attack. Considering the above, the deprotonation first occurs in an aqueous buffer media, then the oxidation processes occur either directly on the electrode surface or can be mediated by O2 (autoxidation) and other oxygen species electrogenerated at the anode surface, implying that it is the main pathway for the oxidation of PyH3 by oxygen species in natural environments such as living body. Several reaction mechanisms for ROS scavenging by phenolic antioxidants such as PyH3 are known, including the superoxide-facilitated oxidation (SFO) [13][14][15], hydrogen atom transfer (HAT) involving PCET [16][17][18][19][20], and sequential proton-loss electron transfer (SPLET) [21]. In the SFO mechanism, the initial PT from the substrate to O2 •− to give HO2 • Nasr et al. reported the electrochemical oxidation of PyH 3 in acidic aqueous solution [12]. In their pioneering work, the voltammetric results showed that PyH 3 oxidation occurs in the same potential region as that of phenol (PhOH). Conversely, the initial deprotonation of PyH 3 forming PyH 2 − increases electron density in the benzene ring and consequently increases its reactivity to electrophilic attack. Considering the above, the deprotonation first occurs in an aqueous buffer media, then the oxidation processes occur either directly on the electrode surface or can be mediated by O 2 (autoxidation) and other oxygen species electrogenerated at the anode surface, implying that it is the main pathway for the oxidation of PyH 3 by oxygen species in natural environments such as living body. Several reaction mechanisms for ROS scavenging by phenolic antioxidants such as PyH 3 are known, including the superoxide-facilitated oxidation (SFO) [13][14][15], hydrogen atom transfer (HAT) involving PCET [16][17][18][19][20], and sequential proton-loss electron transfer (SPLET) [21]. In the SFO mechanism, the initial PT from the substrate to O 2 •− to give HO 2 • is followed by rapid dismutation to give H 2 O 2 and O 2 . Then, the substrate anion is oxidized by the O 2 formed in the dismutation process [15]. Conversely, the other two mechanisms involve direct oxidation by O 2 •− /HO 2 • . Considering the relationship between the structure of PyH 3 and the mechanism of O 2 •− /HO 2 • scavenging, quinone-hydroquinone π-conjugation is inferred to play a role in a PCET mechanism. In our previous studies, it has been reported that O 2 •− /HO 2 • is scavenged by polyphenols [20], diphenols (hydroquinone [22] and CatH 2 [23]), and monophenols [24,25], through a PCET mechanism. In these studies, a concerted twoproton-coupled electron transfer (2PCET) involving two PTs and one ET is a plausible reaction pathway for CatH 2 moiety based on the energetics and kinetics for successful O 2 •− scavenging. Therefore, CatH 2 moiety comprised in PyH 3 is expected to play through the concerted 2PCET, although the third OH group (3OH) gives a different chemical mechanism of PyH 3 from CatH 2 .
In this study, we analyzed the reaction between PyH 3 and electrogenerated O 2 •− comparatively using some related compounds ( Figure 1) in dehydrated N,N-dimethylformamide (DMF) by using electrochemistry and density functional theory (DFT) calculation. Accordingly, herein we present a mechanistic insight into PCET for the O 2 •− scavenging reaction by PyH 3 that constitute some highly reactive antioxidants.
Electrochem 2022, 3, FOR PEER REVIEW 3 is followed by rapid dismutation to give H2O2 and O2. Then, the substrate anion is oxidized by the O2 formed in the dismutation process [15]. Conversely, the other two mechanisms involve direct oxidation by O2 •− /HO2 • . Considering the relationship between the structure of PyH3 and the mechanism of O2 •− /HO2 • scavenging, quinone-hydroquinone π-conjugation is inferred to play a role in a PCET mechanism. In our previous studies, it has been reported that O2 •− /HO2 • is scavenged by polyphenols [20], diphenols (hydroquinone [22] and CatH2 [23]), and monophenols [24,25], through a PCET mechanism. In these studies, a concerted two-proton-coupled electron transfer (2PCET) involving two PTs and one ET is a plausible reaction pathway for CatH2 moiety based on the energetics and kinetics for successful O2 •− scavenging. Therefore, CatH2 moiety comprised in PyH3 is expected to play through the concerted 2PCET, although the third OH group (3OH) gives a different chemical mechanism of PyH3 from CatH2.
In this study, we analyzed the reaction between PyH3 and electrogenerated O2 •− comparatively using some related compounds ( Figure 1) in dehydrated N,N-dimethylformamide (DMF) by using electrochemistry and density functional theory (DFT) calculation. Accordingly, herein we present a mechanistic insight into PCET for the O2 •− scavenging reaction by PyH3 that constitute some highly reactive antioxidants.

Electrochemical and In Situ Electrolytic ESR/UV-vis Spectrum Measurements
Cyclic voltammetry was performed using a three-electrode system comprising a 1.0 mm-diameter glassy carbon (GC) working electrode, a coiled platinum (Pt) counter electrode, and a silver/silver nitrate (Ag/AgNO3) reference electrode (containing acetonitrile solution of 0.1 mol dm −3 tetrabutylammonium perchlorate and 0.01 mol dm −3 AgNO3; BAS RE-5) at 25 °C using BAS 100B electrochemical workstation, coupled to BAS electrochemical software to record data (Supplementary Materials, Table S1). In situ electrolytic ESR/UV-vis spectra were measured using a JEOL JES-FA200 X-band spectrometer/an

Electrochemical and In Situ Electrolytic ESR/UV-vis Spectrum Measurements
Cyclic voltammetry was performed using a three-electrode system comprising a 1.0 mm-diameter glassy carbon (GC) working electrode, a coiled platinum (Pt) counter electrode, and a silver/silver nitrate (Ag/AgNO 3 ) reference electrode (containing acetonitrile solution of 0.1 mol dm −3 tetrabutylammonium perchlorate and 0.01 mol dm −3 AgNO 3 ; BAS RE-5) at 25 • C using BAS 100B electrochemical workstation, coupled to BAS electrochemical software to record data (Supplementary Materials, Table S1). In situ electrolytic ESR/UV-vis spectra were measured using a JEOL JES-FA200 X-band spectrometer/an OCEAN HDX spectrometer (OptoSirius Co., Ltd.). The controlled-potential electrolysis was performed at room temperature in an electrochemical ESR cell using a 0.5 mm-diameter straight Pt wire sealed in a glass capillary as a working electrode/an optically transparent thin-layer electrochemical (OTTLE) cell (path length: 1.0 mm) using a Pt mesh working electrode (Supplementary Materials, Figure S1). Samples were prepared in a glove box completely filled with N 2 gas to prevent contamination by moisture. The DMF solution containing 0.1 mol dm −3 TPAP as a supporting electrolyte was saturated with O 2 by airbubbling the gas for ca. 2-3 min and the gas was passed over the solutions during the electrochemical and spectral measurements to maintain the concentration of O 2 at a constant level. The equilibrium concentration of O 2 was calculated as 4.8 × 10 −3 mol dm −3 .

Calculation
All solution phase calculations were performed at the DFT level with the Becke three-parameter Lee-Yang-Parr (B3LYP) hybrid functional as implemented in Gaussian 16 Program package [27]. This functional was chosen because it has been shown to give good geometries of the reactants, products and transition states (TS) in PCET reactions between phenolic compounds and free radicals [28]. Geometry optimization, vibrational frequency calculations, the intrinsic reaction coordinate (IRC) calculations, and population analysis of each compound was performed by employing the standard split-valence triple ζ basis sets augmented by the polarization 3df,2p and diffusion orbitals 6-311+G(3df,2p). The solvent contribution of DMF to the standard Gibbs free energies was computed employing the polarized continuum model (PCM) at the default settings of the Gaussian 16, which is widely employed in the description of the thermodynamic characteristics of solvation. The zero-point energies and thermal correction, together with entropy, were used to convert the internal energies to standard Gibbs energy at 298.15 K. The natural bond orbital (NBO) technique was used for electron and spin calculations in population analysis [29].

Cyclic Voltammetry and ESR Analysis of O 2 /O 2
•− in the Presence of PyH 3 In Figure 2, cyclic voltammograms (CV) of saturated O 2 (4.8 × 10 −3 mol dm −3 ) in the presence of PyH 3 and related compounds (Figure 1a-d) in DMF, and ESR spectra of the CV solutions (b) obtained via in situ electrolytic ESR system are demonstrated. CV and ESR in the presence of (c) CatH 2 and CV in the presence of (d) PhOH, were already reported in our previous paper, though are shown here for comparison [22,24]. In aprotic solvents such as DMF, O 2 shows quasi-reversible redox at −1.284 V vs. the ferrocenium ion/ferrocene (Fc + /Fc) couple (Equation (1)) corresponding to generation of O 2 •− in the initial cathodic scan and reoxidation to the starting materials (O 2 ), in the returned anodic scan (1c/1a, bold lines in Figure 2), where O 2 •− is not particularly reactive toward aprotic DMF. The reversible CVs investigated here were all modified to irreversible one by the presence of any compounds (a-d) with concentration dependency (0 to 3.0, 5.0 × 10 −3 mol dm −3 ), supported that CVs of bubbled N 2 showed no peak over the potential range. The reactivity of PyH 3 estimated from a loss of reversibility of the CV is higher than the others. Thus, the loss of reversibility in the CVs of O 2 /O 2 •− is caused by the acid-base reaction; the initial PT from the compounds to O 2 •− as a Brønsted base forming HO 2 • (Equation (2)). With the generation of HO2 • , bielectronic CVs were observed derives from the reduction of HO2 • to hydroperoxyl anion (HO2 − ) (Equation (3)) as shown in Figure 2d, cathodic current 2c. Conversely, in the presence of (a) PyH3, (b) MoCatH2, and (c) MoCatH2, the bielectronic CVs do not appear due to the scavenging of HO2 • by the subsequent ET (Equation (4)) from the deprotonated anion forming its radical (PyH2 − /PyH2 • , , where a cathodic prepeak appeared. In our previous study, the ET involved in the PCET mechanism for successful O2 •− scavenging required two structural characteristics; (1) the quinone-hydroquinone π-conjugated structure characterized by ortho/para-diphenol, and (2) the OH proton for the second PT [20,[22][23][24]30].
Considering these results, we rationalized that O2 •− formation after the primary electrode process associated with PT from the OH group leads to the irreversible overall reduction of O2 to H2O2, which is driven by the exergonic reduction of the resulting HO2 • /HO2 − . Therefore, the CV traces for O2/O2 •− in the presence of phenolic compounds are divided into two typical curves: type A, an irreversible two-electron process observed in electro-chemical-electro reactions (Equations (1)-(3)), and type B, an irreversible oneelectron process (Equations (1), (2), (4), and (5)) leading to O2 •− scavenging. Figure 3 shows the plausible electrochemical mechanism for O2/O2 •− in the presence of (a) PyH3 and (b) PhOH, summarizing Equations (1)-(5). With the generation of HO 2 • , bielectronic CVs were observed derives from the reduction of HO 2 • to hydroperoxyl anion (HO 2 − ) (Equation (3)) as shown in Figure 2d, cathodic current 2c. Conversely, in the presence of (a) PyH 3 , (b) MoCatH 2 , and (c) MoCatH 2 , the bielectronic CVs do not appear due to the scavenging of HO 2 • by the subsequent ET (Equation (4)) from the deprotonated anion forming its radical ( where a cathodic prepeak appeared. In our previous study, the ET involved in the PCET mechanism for successful O 2 •− scavenging required two structural characteristics; (1) the quinone-hydroquinone π-conjugated structure characterized by ortho/para-diphenol, and (2) the OH proton for the second PT [20,[22][23][24]30].
Considering these results, we rationalized that O 2 •− formation after the primary electrode process associated with PT from the OH group leads to the irreversible overall reduction of O 2 to H 2 O 2 , which is driven by the exergonic reduction of the resulting HO 2 • /HO 2 − . Therefore, the CV traces for O 2 /O 2 •− in the presence of phenolic compounds are divided into two typical curves: type A, an irreversible two-electron process observed in electro-chemical-electro reactions (Equations (1)-(3)), and type B, an irreversible oneelectron process (Equations (1), (2), (4), and (5)) leading to O 2 •− scavenging. Figure 3 shows the plausible electrochemical mechanism for O 2 /O 2 •− in the presence of (a) PyH 3 and (b) PhOH, summarizing Equations (1)- (5).
In this scenario, the CV results recorded in the presence of (d) PhOH demonstrate type A (O 2 •− is not scavenged) showing the appearance of a cathodic current ascribed Next, in situ electrolytic UV-vis spectra for the CV solution containing PyH3 (1.0 × 10 -3 mol dm -3 ) using the OTTLE cell (Supporting Information, Figure S2) were measured in the absence of O2 under purging N2 and in the presence of O2 (Figure 4). The spectrum of PyH3 alone has a characteristic absorption band centered at 272 nm. Under the applied potential at 1.0 to −2.0 V vs. Fc + /Fc without O2, the spectrum did not change where any potential was applied (data not shown), demonstrating that PyH3 is not electrolyzed without deprotonation. Conversely, the spectrum has changed in the presence of CH3ONa (5.0 × 10 -3 mol dm -3 ) without applying a potential (red line). Since PyH3 is deprotonated by CH3ONa as a Brønsted base, the spectrum will be attributed to PyH2 -or PyH 2-. On the other side, the spectrum of PyH3 in the presence of saturated O2 (4.8 × 10 -3 mol dm -3 ) has changed, showing the appearance of an absorption band centered at 292 nm at applied cathodic potentials over −1.3 V corresponding electrogeneration of O2 •-. Next, in situ electrolytic UV-vis spectra for the CV solution containing PyH 3 (1.0 × 10 −3 mol dm −3 ) using the OTTLE cell (Supporting Information, Figure S2) were measured in the absence of O 2 under purging N 2 and in the presence of O 2 (Figure 4). The spectrum of PyH 3 alone has a characteristic absorption band centered at 272 nm. Under the applied potential at 1.0 to −2.0 V vs. Fc + /Fc without O 2 , the spectrum did not change where any potential was applied (data not shown), demonstrating that PyH 3 is not electrolyzed without deprotonation. Conversely, the spectrum has changed in the presence of CH 3 ONa (5.0 × 10 −3 mol dm −3 ) without applying a potential (red line). Since PyH 3 is deprotonated by CH 3 ONa as a Brønsted base, the spectrum will be attributed to  These spectral changes have demonstrated that the product of homogeneous rea between PyH3 and O2 •-is PyH •-, and the observed spectrum derives from PyH 2-vi ther 1-electron reduction of PyH •-at the electrode. By analogy with the CV result initial PT (Equation (1)) and the following reactions including ET between HO2 • and P (Equation (4)) rapidly undergo base-catalyzed oxidation. Since PyH •-would be ele lyzed to PyH2 -at the electrode, the radical product was undetectable using the in electrolytic ESR system.
Notably, the CV and spectral results demonstrated that (a) PyH3 with its two groups can scavenge O2 •− through the PCET involving two PTs and one ET, wherea role of the 3OH group is unclear. These results imply that the reaction mechanism PyH3 is similar to that of (c) CatH2 (Scheme 1), however their reactivities are differen

Change in HOMO-LUMO Energies upon PCET between PyH3 and O2 •− in DFT Anal
DFT calculations with the frontier molecular orbital analysis were performed t the mechanistic analysis of O2 •− scavenging by PyH3 in DMF. Figure 5 shows HO LUMO changes upon PCET between PyH3/PyH2 − and O2 •− . After the initial PT, som actant species, i.e., PyH3, PyH2 − , O2 •− , and HO2 • , coexist in the solution. The singly pied molecular orbital (SOMO) energy (Hartree) for HO2 • (−0.3142) is much lower HOMO energies of PyH3 and PyH2 − . Thus, the electron acceptor will be HO2 • , not Considering that CV in DMF revealed that HO2 • formed after the initial PT is scave (Figure 2a), the electron donor will be PyH2 − , for which the downhill energy relation is indicated by the bold red line. Thus, this change in HOMO-LUMO (SOMO) ene upon PT between PyH3 and O2 •− forming PyH2 − and HO2 • is reasonable for subseq ET. Next, the HOMO-LUMO relationship between the products after ET (i.e., PyH2 • HO2 − ) is reversed, which is rational for orbital energies in the reverse ET (red dotted However, the HOMO (−0.2754) of the PT-forming H2O2 is lower than HOMO (−0.164 These spectral changes have demonstrated that the product of homogeneous reaction between PyH 3 and O 2 •− is PyH •− , and the observed spectrum derives from PyH 2− via further 1-electron reduction of PyH •− at the electrode. By analogy with the CV results, the initial PT (Equation (1)) and the following reactions including ET between HO 2 • and PyH 2 − (Equation (4)) rapidly undergo base-catalyzed oxidation. Since PyH •− would be electrolyzed to PyH 2 − at the electrode, the radical product was undetectable using the in situ electrolytic ESR system.
Notably, the CV and spectral results demonstrated that (a) PyH 3 with its two OH groups can scavenge O 2 •− through the PCET involving two PTs and one ET, whereas the role of the 3OH group is unclear. These results imply that the reaction mechanism of (a) PyH 3 is similar to that of (c) CatH 2 (Scheme 1), however their reactivities are different.

Change in HOMO-LUMO Energies upon PCET between PyH 3 and O 2 •− in DFT Analyses
DFT calculations with the frontier molecular orbital analysis were performed to aid the mechanistic analysis of O 2 •− scavenging by PyH 3 in DMF. Figure 5 shows HOMO-LUMO changes upon PCET between PyH 3 /PyH 2 − and O 2 •− . After the initial PT, some reactant species, i.e., PyH 3 , PyH 2 − , O 2 •− , and HO 2 • , coexist in the solution. The singly occupied molecular orbital (SOMO) energy (Hartree) for HO 2 • (−0.3142) is much lower than HOMO energies of PyH 3 and PyH 2 − . Thus, the electron acceptor will be HO 2 • , not O 2 •− . Considering that CV in DMF revealed that HO 2 • formed after the initial PT is scavenged (Figure 2a), the electron donor will be PyH 2 − , for which the downhill energy relationship is indicated by the bold red line. Thus, this change in HOMO-LUMO (SOMO) energies upon PT between PyH 3 and O 2 •− forming PyH 2 − and HO 2 • is reasonable for subsequent ET. Next, the HOMO-LUMO relationship between the products after ET (i.e., PyH 2 • , and HO 2 − ) is reversed, which is rational for orbital energies in the reverse ET (red dotted line). However, the HOMO (−0.2754) of the PT-forming H 2 O 2 is lower than HOMO (−0.1648) of HO 2 − , making the reverse ET improbably. Thus, the subsequent PT is dominant in determining the ET direction.  Alternatively, the HOMO-LUMO relationship for O2 •− scavenging by PyH2 − preformed by the initial deprotonation indicates that the similar PCET involving two PTs and one ET occurs: the downhill ET (blue bold line) and reverse ET (blue dotted line). As reported previously, 2PCET occurs between CatH2 moiety and O2 •− after the formation of the pre-reactive hydrogen-bond (HB) complex from the free reactants (FR), and the proton and electron are concertedly transferred in one kinetic step via a TS forming the product complex (PC) [20,23]. Therefore, it is expected that the 2PCET between PyH3 and O2 •− occurs in a similar concerted manner through the HB formed between two OH groups of PyH3 and O2 •− .

Free Energy Calculations of PCET between PyH3 and O2 •−
For a mechanistic analysis of the O2 •− scavenging by PyH3 in DMF, DFT calculations were performed at the (U)B3LYP/PCM/6-311+G(3df,2p) level. In Figure 6, the equilibrium schemes and standard Gibbs free energy changes (ΔG°/kJ mol −1 , 298.15 K) of the six diabatic electronic states for the PCET involving two PTs and one ET between (a) PyH3 and O2 •− , and between (b) O2 •− and PyH2 − formed after the initial deprotonation are shown. The important factors in determining the sequential processes shown in this scheme are the ΔG°s for the individual reactions; the acid-base interaction and redox potentials of the components. In Figure 6a, ET1 (ΔG° = 405.3 kJ mol -1 ) is strongly endergonic, thus, PT1 (17.9) forming PyH2 − and HO2 • must primarily occur, as shown in the CV result. In the following pathway shown in the lower rectangle, both PT3 (357.9) and ET2 (28.2) are uphill endergonic, suggesting that the sequential PCET does not proceed but the concerted PCET (−39.5) is a thermodynamically feasible pathway. Alternatively, one-step one-electron transfer concerted with sequential two-proton transfer after initial formation of the HB complexes between PyH3 and O2 •− without generating high-energy intermediates, which we refer to as concerted 2PCET reactions, is another feasible pathway [23,28]. For a Alternatively, the HOMO-LUMO relationship for O 2 •− scavenging by PyH 2 − preformed by the initial deprotonation indicates that the similar PCET involving two PTs and one ET occurs: the downhill ET (blue bold line) and reverse ET (blue dotted line). As reported previously, 2PCET occurs between CatH 2 moiety and O 2 •− after the formation of the pre-reactive hydrogen-bond (HB) complex from the free reactants (FR), and the proton and electron are concertedly transferred in one kinetic step via a TS forming the product complex (PC) [20,23]. Therefore, it is expected that the 2PCET between PyH 3 and O 2 •− occurs in a similar concerted manner through the HB formed between two OH groups of PyH 3 and O 2 •− .

•−
For a mechanistic analysis of the O 2 •− scavenging by PyH 3 in DMF, DFT calculations were performed at the (U)B3LYP/PCM/6-311+G(3df,2p) level. In Figure 6, the equilibrium schemes and standard Gibbs free energy changes (∆G • /kJ mol −1 , 298.15 K) of the six diabatic electronic states for the PCET involving two PTs and one ET between (a) PyH 3 and O 2 •− , and between (b) O 2 •− and PyH 2 − formed after the initial deprotonation are shown. The important factors in determining the sequential processes shown in this scheme are the ∆G • s for the individual reactions; the acid-base interaction and redox potentials of the components. In Figure 6a, ET1 (∆G • = 405.3 kJ mol -1 ) is strongly endergonic, thus, PT1 (17.9) forming PyH 2 − and HO 2 • must primarily occur, as shown in the CV result. In the following pathway shown in the lower rectangle, both PT3 (357.9) and ET2 (28.2) are uphill endergonic, suggesting that the sequential PCET does not proceed but the concerted PCET (−39.5) is a thermodynamically feasible pathway. Alternatively, one-step one-electron transfer concerted with sequential two-proton transfer after initial formation of the HB complexes between PyH 3 and O 2 •− without generating high-energy intermediates, which we refer to as concerted 2PCET reactions, is another feasible pathway [23,28]. For a successful O 2 •− scavenging in either PCET pathway, the second PT coupled to ET is necessary, as reported in our previous studies [25,31]. On the other side, a PCET reaction between PyH 2 − and O 2 •− shown in Figure 6b is also plausible, in case that the initial deprotonation of PyH 3 will partially occur in an aprotic DMF solution. Although, since both PT1 (88.5) and ET1 (253.5) are uphill, the only feasible pathway is 2PCET forming quinone-radical-dianion (Py •2− ) as a product of the net reaction involving three PTs and one ET from PyH 3 .
For a comparative study, the ∆G • values of the PCET pathways for CatH 2 and MoCatH 2 were calculated ( Table 1). From a thermodynamic viewpoint, the total values of ∆G • for the net PCET were obtained from the sum of the values for the two PTs and one ET. If the PCET occurs along a pathway involving the unfeasible single PT/ET (PT1, PT3, ET1, and ET2), the total values cannot embody the energetic driving force because the ∆G • for the unfeasible PT/ET has been summed in it. However, since the concerted PCET (ET2-PT4/PT3-ET3) after the initial PT1 is endergonic for both CatH 2 (−55.7) and MoCatH 2 (−79.1), the total values (−36.2 and −32.9) can embody the exergonic driving force through PT1-concerted PCET pathway, similar to PyH 3 (concerted: −39.6, total: −21.6). Notably, both the ∆G • values (concerted and total) for PyH 3 are larger than those for CatH 2 and MoCatH 2 , showing a lower reactivity of PyH 3 . The effect of the substituted group of MoCatH 2 , PyH 3 , and CatH 2 , on the O 2 •− scavenging through the PCET is primarily considered to be due to the electron-donating ability (-OCH 3 > -OH > -H) increasing electron density in the benzene ring, known as the Hammett equation [32]. Additionally, the intramolecular HB formed at the 3OH group strongly stabilizes the negatively charged deprotonated species along the PCET; PyH 2 − , PyH 2− , and its trianion (Py 3− ), consequently suppressing their reactivities to electrophilic attack. Thus, these ∆G • values confirm that the PCET mechanism in Figure 6a alone cannot explain the reason for the higher reactivity of PyH 3 than the others toward electrogenerated O 2 •− shown in the CVs (Figure 2). As a result of the comparative analyses of the ∆G • values, the involvement of three reaction pathways is plausible for efficient O 2 •− scavenging by PyH 3 ; PT-concerted PCET and 2PCET between PyH 3 and O 2 •− , and 2PCET between PyH 2 − and O 2 •− .
Electrochem 2022, 3, FOR PEER REVIEW 9 successful O2 •− scavenging in either PCET pathway, the second PT coupled to ET is necessary, as reported in our previous studies [25,31]. On the other side, a PCET reaction between PyH2 − and O2 •− shown in Figure 6b is also plausible, in case that the initial deprotonation of PyH3 will partially occur in an aprotic DMF solution. Although, since both PT1 (88.5) and ET1 (253.5) are uphill, the only feasible pathway is 2PCET forming quinoneradical-dianion (Py •2− ) as a product of the net reaction involving three PTs and one ET from PyH3. For a comparative study, the ΔG° values of the PCET pathways for CatH2 and MoCatH2 were calculated (Table 1). From a thermodynamic viewpoint, the total values of ΔG° for the net PCET were obtained from the sum of the values for the two PTs and one ET. If the PCET occurs along a pathway involving the unfeasible single PT/ET (PT1, PT3, ET1, and ET2), the total values cannot embody the energetic driving force because the ΔG° for the unfeasible PT/ET has been summed in it. However, since the concerted PCET (ET2-PT4/PT3-ET3) after the initial PT1 is endergonic for both CatH2 (−55.7) and MoCatH2 (−79.1), the total values (−36.2 and −32.9) can embody the exergonic driving force through PT1-concerted PCET pathway, similar to PyH3 (concerted: −39.6, total: −21.6). Notably, both the ΔG° values (concerted and total) for PyH3 are larger than those for CatH2 and MoCatH2, showing a lower reactivity of PyH3. The effect of the substituted group of MoCatH2, PyH3, and CatH2, on the O2 •− scavenging through the PCET is primarily considered to be due to the electron-donating ability (-OCH3 > -OH > -H) increasing electron density in the benzene ring, known as the Hammett equation [32]. Additionally, the intramolecular HB formed at the 3OH group strongly stabilizes the negatively charged deprotonated species along the PCET; PyH2 − , PyH 2− , and its trianion (Py 3− ), consequently suppressing their reactivities to electrophilic attack. Thus, these ΔG° values confirm that the PCET mechanism in Figure 6a alone cannot explain the reason for the higher reactivity of PyH3 than the others toward electrogenerated O2 •− shown in the CVs (Figure 2). As a result of the comparative analyses of the ΔG° values, the involvement of three reaction pathways is plausible for efficient O2 •− scavenging by PyH3; PT-concerted PCET and 2PCET between PyH3 and O2 •− , and 2PCET between PyH2 − and O2 •− .     Table 2. Notably, there is almost no difference in each Ea value (PyH3: 53.9, CatH2: 52.5, and MoCatH2: 50.7), and either of those is as low as the hydrogenbonding energy. Conversely, there are about 10-20 kJ mol −1 differences between the ΔG° values for forming each of the PRC from the FRs (PyH 3 : 81.2, CatH2: 71.6, and MoCatH2: 61.4). Thus, the ΔG° values for the formation of the PRC from the FRs (step i), rather than the kinetics of the 2PCET reaction to the PC via a TS (step ii), embody the superior O2 •−scavenging ability of PyH3 with a good correlation with the CV results in DMF. Considering the IRC results together with the CV (Figure 2) and the ΔG° results ( Figure 6), the 2PCET between O2 •− and PyH2 − formed after the deprotonation of PyH3 is not feasible. In an aprotic DMF solution, the initial reaction between PyH3 and O2 •− will be the formation of the PRC stabilized at −81.2 kJ mol −1 via two HBs, rather than the initial PT (PT1 in Figure  6a: 17.9 kJ mol −1 ) or a deprotonation (proton loss in the solution). Taken together, these findings indicate that the O2 •− scavenging by PyH3 in DMF is governed by the concerted 2PCET after forming PRC via two HBs, which corresponds to a moving along the red diagonal line of the two rectangles shown in Figure 6a. In Figure   Figure 7.  Table 2. Notably, there is almost no difference in each E a value (PyH 3 : 53.9, CatH 2 : 52.5, and MoCatH 2 : 50.7), and either of those is as low as the hydrogen-bonding energy. Conversely, there are about 10-20 kJ mol −1 differences between the ∆G • values for forming each of the PRC from the FRs (PyH 3 : 81.2, CatH 2 : 71.6, and MoCatH 2 : 61.4). Thus, the ∆G • values for the formation of the PRC from the FRs (step i), rather than the kinetics of the 2PCET reaction to the PC via a TS (step ii), embody the superior O 2 •− -scavenging ability of PyH 3 with a good correlation with the CV results in DMF. Considering the IRC results together with the CV (Figure 2) and the ∆G • results (Figure 6), the 2PCET between O 2 •− and PyH 2 − formed after the deprotonation of PyH 3 is not feasible. In an aprotic DMF solution, the initial reaction between PyH 3 and O 2 •− will be the formation of the PRC stabilized at −81.2 kJ mol −1 via two HBs, rather than the initial PT (PT1 in Figure 6a: 17.9 kJ mol −1 ) or a deprotonation (proton loss in the solution). Taken together, these findings indicate that the O 2 •− scavenging by PyH 3 in DMF is governed by the concerted 2PCET after forming PRC via two HBs, which corresponds to a moving along the red diagonal line of the two rectangles shown in Figure 6a. In Figure 8, the net mechanism of the O 2 •− scavenging by PyH 3 in DMF is shown. In the 2PCET mechanism, ET occurs between oxygen-π-orbitals orthogonal to the molecular framework, then, PT occurs between oxygen-σ-orbitals along the HBs [23]. It is presumed that the higher reactivity of PyH 3 with O 2 •− than that for CatH 2 , is due to the sequential reactions; the initial formation of the PRC, followed by the 2PCET. 8, the net mechanism of the O2 •− scavenging by PyH3 in DMF is shown. In the 2PCET mechanism, ET occurs between oxygen-π-orbitals orthogonal to the molecular framework, then, PT occurs between oxygen-σ-orbitals along the HBs [23]. It is presumed that the higher reactivity of PyH3 with O2 •-than that for CatH2, is due to the sequential reactions; the initial formation of the PRC, followed by the 2PCET.

Conclusions
In conclusion, we have investigated the O2 •− scavenging by PyH3 through the PCET in DMF. As a result, we have clarified;  PyH3 scavenges O2 •− through the 2PCET involving concerted two PTs and one ET, in a similar mechanism for CatH2;  the net mechanism involves the initial formation of PRC followed by concerted 2PCET;  the 3OH group thermodynamically promotes the formation of PRC via two HBs but does not promote the latter 2PCET, resulting in an effective O2 •− scavenging ability of PyH3.
Although the results presented in this manuscript are for a chemical reaction in aprotic DMF solvent, the PCET theory is adaptable to biological processes in such as a lipid bilayer. Therefore, we hope that the findings obtained in this study will provide evidence for the mechanistic actions of O2 •− scavenging by various antioxidants involving PyH3 moiety.

Conclusions
In conclusion, we have investigated the O 2 •− scavenging by PyH 3 through the PCET in DMF. As a result, we have clarified; • PyH 3 scavenges O 2 •− through the 2PCET involving concerted two PTs and one ET, in a similar mechanism for CatH 2 ; • the net mechanism involves the initial formation of PRC followed by concerted 2PCET; • the 3OH group thermodynamically promotes the formation of PRC via two HBs but does not promote the latter 2PCET, resulting in an effective O 2 •− scavenging ability of PyH 3 .
Although the results presented in this manuscript are for a chemical reaction in aprotic DMF solvent, the PCET theory is adaptable to biological processes in such as a lipid bilayer. Therefore, we hope that the findings obtained in this study will provide evidence for the mechanistic actions of O 2 •− scavenging by various antioxidants involving PyH 3 moiety.