key: cord-0778280-8cjdeje3
authors: Kohlmann, Tim; Goez, Martin
title: Laser Access to Quercetin Radicals and Their Repair by Co‐antioxidants
date: 2020-11-20
journal: Chemistry
DOI: 10.1002/chem.202001956
sha: fe3ba5254db15b7aafc7dab17ff115fb526b39f6
doc_id: 778280
cord_uid: 8cjdeje3

We have demonstrated the feasibility and ease of producing quercetin radicals by photoionization with a pulsed 355 nm laser. A conversion efficiency into radicals of 0.4 is routinely achieved throughout the pH range investigated (pH 2–9), and the radical generation is completed within a few ns. No precursor other than the parent compound is needed, and the ionization by‐products do not interfere with the further fate of the radicals. With this generation method, we have characterized the quercetin radicals and studied the kinetics of their repairs by co‐antioxidants such as ascorbate and 4‐aminophenol. Bell‐shaped pH dependences of the observed rate constants reflect opposite trends in the availability of the reacting protonation forms of radical and co‐antioxidant and even at their maxima mask the much higher true rate constants. Kinetic isotope effects identify the repairs as proton‐coupled electron transfers. An examination of which co‐antioxidants are capable of repairing the quercetin radicals and which are not confines the bond dissociation energies of quercetin and its monoanion experimentally to 75–77 kcal mol(−1) and 72–75 kcal mol(−1), a much narrower interval in the case of the former than previously estimated by theoretical calculations.

Quercetin(for the structural formula see Scheme2)i so ne of the mosta bundant representatives of the flavonoid antioxidants, asubgroupo fthe polyphenols, and boasts of an activity four times as high as that of a-tocopherol (vitamin E) or ascorbate (vitaminC). [1] An impressive collection of other health benefits has been reported, including antihypertensive, [2] cardioprotective, [3] antithrombotic as wella sa nti-inflammatory, [4] antitumor, [5] and antiviral effects, the latteri np articular against RNA viruses such as SARS-CoV, [6] MERS-CoV, [7] Ebola-CoV, [8] Zika-CoV, [9] and possibly SARS-CoV-2. [10] The oral bioavailability of quercetin is severelyl imited by its low solubility in water,h ence carriers [6] or glucosylation [7] [8] [9] have been testedt oo vercome that limitation. An alternative strategy-although the link between the antioxidative and the other health-protecting properties of quercetin is not clearly proveny et-is provided by redoxc ycling with the aid of ah ydrophilic co-antioxidant exhibiting higherp lasma levels such as ascorbate, which repairst he oxidized quercetin radicals.

There is indeed evidencet hat the antioxidant activity of quercetin is substantially improvedb y, [11] andt hat its antitumor and antiviral potency even relies cruciallyo n, [12, 13] the presence of ascorbate. Yet, and despite nearly 80 000 publications on quercetin listed by SciFinder to date, very little is known about the repairo fi ts radicals by ascorbatea nd related co-antioxidants. Practically all such investigations werec arriedo ut through incubation experiments on long timescales, [14] which inherently can only yield indirect information on the fast processes and are prone to interpretational ambiguities because the quercetin oxidation products mayt hemselves functiona ss econdary antioxidants. [15] [16] [17] To our knowledge,t here have only been two kinetic studies on short timescales; [18, 19] and both used pulse radiolysis with its innate problems of delayedq uercetin radical generation through scavenging, as wella sac onsiderable number of transients and reactions interacting simultaneously.

Herein, we explore an alternative approach, whichw eh ave already successfully applied to the antioxidant resveratrol, [20] namely,d irect generation of the quercetin radicals by laserinduced photoionization. As we will show,t his access to the radicalsi sn ot only completely selectiveo ver aw ide pH range but also quasi-instantaneous comparedt ot he subsequent reactions of the radicals, such that the repair kinetics can be observedi ni solation, their complicatedp Hd ependence can be unravelled, and the bond dissociatione nergy of quercetin can be bracketed experimentally with as ignificantly lower uncertainty compared to reported quantum-mechanical estimates.

! 99.0 %; ascorbic acid, ! 99.7 %; 4-aminophenol, > 98 %; Trolox, 97 %; hydroquinone, > 98 %; NaOH, 99 %; HCl, 99 %). The solvent was ultrapure Millipore Milli-Q water (specific resistance, 18.2 MW cm À1 )o r-for measuring the H/D kinetic isotope effects-D 2 O(99.9 %deuteration, DEUTERO).

The solutions were deoxygenated with argon, or with N 2 Ow hen the hydrated electron had to be blanked out as explained in the main text, for at least 30 minutes. Both gases were of purity 5.0 and obtained from AirLiquide. The quercetin weight-in concentration was 5 mm throughout, which kept the solutions optically thin and thus avoided filter effects caused by nonlinear absorption. The desired pH values were adjusted under pH meter control by the addition of NaOH or HCl. All measurements were carried out at room temperature. Mechanistic and kinetic experiments studies were performed on a home-made laser flash photolysis setup described elsewhere. [21] Its main characteristics pertinent to this investigation are excitation with af requency-tripled (355 nm) pulsed Nd:YAG laser (Continuum Surelite-III;p ulse width, ca. 5ns);h omogeneous illumination of the observed volume (window,2 4mm; optical path length, 4mm) in as uprasil flow cell;d etection at right angles to the excitation beam and with atime resolution down to 1ns. The steady-state absorption and the fluorescence spectra were recorded on aS himadzu UV-1800 spectrophotometer and an Edinburgh Instruments FS5 TCSPC spectrometer.

Access to the quercetin radicals Figure 1f ocuseso nt he photophysicso fq uercetin as far as is essential fort his work. The antioxidante xists in five protonation states with typical separations between pK a values by less than 2u nits, [22] which is comparablet ot he spread of the reported individual values (e.g.,b etween 5.50 and 7.65 for the lowest pK a ). [22, 23] Herein, only the fully protonated and singly deprotonated forms H 2 Qa nd HQ À (for the structures see the summarizing Scheme2)p layarole because we observed sample deterioration above pH 9o nt he timescale of 1-2 h, which restricted the pH range accessible to our experiments.

The complexity of the system is reflected by the pH-dependent absorption spectra of Figure1a, which exhibit no welldefinedi sosbestic points;h owever,3 76 nm providesagood approximationo fs uch ap oint up to pH%8.5, i.e.,e xcept for the highest pH in Figure 1 . Excitation at 376 nm thus allowed recording the fluorescences pectra ( Figure 1b )w ith no, or only am inor ( % 7%)c orrection. At the wavelength of our ionizing laser,3 55 nm, the profile displayed as the inset of Figure1a can be fitted with at itration curve with ap K a of 7.5, although no plateau value is reached at the high-pHe nd of the region. The observed ground-state extinction coefficient e GS decreases by about one third when going from pH 4t op H9.T his reduces the rate of excitation by our laser in proportion but does not cause sensitivityp roblems because e GS is still quite high in the basic medium.

The absorption-correctedf luorescences pectra of Figure 1b clearly show aw eakly emitting speciesa tl ower pH and a much more strongly emitting species at higher pH, with emission maximaa t5 21 nm and 554 nm. Ap rofile at the longer wavelength (see, inset of the Figure) is represented well by a titration curve with ap K a of 7.3. On account of the distinct and complete curve shape, we regard this pK a -which hardly differs from the above one-asr eliable;a nd we interpret the data of Figure 1b ya bsorption of, and emissionf rom, the same protonation state, the concentration of which is determined by the ground-state equilibrium between H 2 Qa nd HQ À ,w ith pertaining pK a of 7.3. Phenols are more acidic in the excited S 1 state compared to the ground state; [24] hence,t he obvious absence of proton transfer in the excited state points to av ery short S 1 lifetime.

Laser flash photolysis at 355 nm affords transients pectra that can be decomposed into the spectrals ignatures displayed in Figure 2a .The occurrence of the hydrated electron eC À aq is evidenced by its characteristic strong and broad absorption with maximum at about7 20 nm, and by the fact that this spectral feature is absent when the solutionh as been saturated with the specific eC À aq scavenger N 2 Oo rw hen the pH lies below about 2. Both N 2 Oa nd H + are known to reactd iffusion-con- trolled with eC À aq to give the nonabsorbing radicals HOC and HC; [25] and under the described conditions the life of eC À aq is shortened so much (to af ew ns, which is the duration of our laser pulses)a st or ender it undetectable. These observations thus identify the laser-inducedp rocess as ap hotoionization.

When eC À aq is blanked out by N 2 Os aturation, the transient spectra of the accompanying quercetin-derived radicals are obtained in pure form because any subsequenta ttack of HOC on residual HQ À or H 2 Qa ffords the same radical as the photoionization does. [27] By the two independent procedures described below,wec alibrated the spectra and corrected them for depletion of the startingq uercetin ( Figure 2a ). In accordance with a pulse-radiolytic study and at heoretical investigation, which concluded that the radical cation initially resulting from H 2 Qi s deprotonated quasi-instantaneously, [19, 28] the limiting spectra at the low and high ends of our pH range are assignedt ot he neutralr adical HQC and the radical anion QC À (see Scheme2). Compared to the previously reported experimental results, [19] we find as imilar pK a of HQC (4.5, i.e.,h igher by 0.3 units) but much highere xtinction cooefficients at maximum (19 750 m À1 cm À1 and 9700 m À1 cm À1 for QC À and HQC,i .e., higher by factorsofa bout 2a nd 4). These observations strongly suggest the spectralc alibration as the origin of the discrepancies because ac alibration error will additionally distort the weakera nd hypsochromica bsorption of HQC (maximum at 510 nm, i.e.,i nt he outer wing of the longest-wavelength bands in Figure 1a )t hrough depletion of the startingq uercetin, whereas that effect is absent fort he stronger and bathochromic absorption of QC À (maximum at 560 nm);a nd calibration errors cancel to first order in the pK a determinationa tt he maximum of the QC À band. Figure 2b addresses the post-generation fate of the transients. Both HQC and QC À are seen to be intrinsically long-lived intermediates that decay on the timescale of af ew hundreds of mst og ive other absorbings pecies.Asuccessful global fit of a first-order kinetic modeli ndicates that the rate constantso f these transformations are independento fp H, and that the ex-tinctionc oefficients of the products are similart o, but slightly smallert han, those of QC À and HQC,r espectively.T he products are most likelyd ifferentp rotonation states of the structure displayed at the lower right in Scheme 2, [29] but further characterization was not warrantedb ecause these conversions fall outside the temporal window of the repair reactions by our coantioxidants.

In contrast to the quercetin-derived radicals, the natural decay of eC À aq occurs within af ew msi nb asic medium( see, right inset of the Figure) and increasingly more rapidlyw hen the pH is lowered. This has two implications. On one hand, an attack of eC À aq on H 2 Qo rH Q À plays no role in this work because the mm concentrationsr ender these reactions noncompetitive;a nd the same holds true for HOC when N 2 Os aturation is employed, or for HC when eC À aq is generated in acidic medium. On the other hand, down to about pH 3t he life of eC À aq is long enough for precise determinationso ft he initial post-flashc oncentrations, which in turn provides the basis for our first calibration procedure:O nt he premise that the ejectiono fe C À aq is the only pathway to the quercetin radicals, stoichiometric equivalence demands ap roportionality between the ratio of the extinctions and that of the extinction coefficients. The possibility of blanking out eC À aq makes this at rivial task. First, the superposition of the absorptions of eC À aq plus HQC and/or QC À is recorded in argon-saturated solution;t hen, N 2 Os aturation is used to recordo nly the absorptions of the quercetin radicals;a nd a difference of the two measurements yields the pure eC À aq absorption. The concentrations of the transientsa re varied most conveniently through the laser intensity. The left inset of Figure 2b illustrates the proportionality for the spectral maximum of QC À (560 nm) and ac onvenient wavelength for eC À aq (not the 720 nm maximum but 824 nm, entirely for technical reasons). [30] Obviously,t he validity of this calibration approachh ingeso n the absence of homolytic photodissociation of the OÀHb ond as am ajor pathway to the quercetin radicals that bypasses photoionization. However,t his assumption is very reasonable Figure 2 . Characterization of the intermediates (radical anion QC À ,blue;neutral radicalHQC,red;hydrated electroneC À aq , [26] violet) followingq uercetin photoionizationi nhomogeneousaqueous solution.G raph (a): mainplot, calibrated absorption spectra corrected for absorption of the starting quercetin;inset, pH dependence of the apparent extinction coefficient of the quercetinradicals at 560 nm (pK a = 4.5, from the dotted best-fit titrationc urve). Graph (b): mainplot, slow decays of the relative absorptionsa t5 60 nm of QC À (pH 8.2) and HQC (pH 2.3) superimposed on monoexponential fits (grayc urves)w ith global rate constanto f1 /(320 ms) and local end valuesof0 .76 and 0.57;right inset,f ast eC À aq decay(pH 8.2) from as tarting concentration c 0 of about 2.2 mm;left inset, illustration of the spectral calibration of QC À at pH 8.2 by comparing the initial extinctions E of QC À at 560 nm and of eC À aq at 824 nm. For furtherexplanation,see the text.

because for phenols in water the quantum yields of such homolyses are known to be practically zero. [31] This is corroborated by juxtaposing the photolysesa tp H5.8 and pH 8.2. These start out near quantitatively from H 2 Qa nd from HQ À (including their S 1 states as discussed above), and despite significantly different bond dissociation energies (according to the literature: 77.2 AE 5.9 kcal mol À1 for H 2 Q; [28, [32] [33] [34] [35] and 73.0 AE 2.1 kcal mol À1 for HQ À ) [28, 32, 35] yield practically the same calibrated extinction coefficient of QC À (with HQC accounting for lesst han 5% in both experiments). It is also vindicated by our second calibration procedure (see next section). Figure 3f inally deals with the efficiency of the photoionization access to the quercetin radicals.T he intensity dependences of the eC À aq concentrations (main plot) show that more than 40 %o ft he starting quercetin can be converted into radicals by as ingle laser pulse, meaning that radicalc oncentrations of 2 mm are routinelya ttainable in situ and within 5ns. Surprisingly,t he protonation state of the quercetin plays only an insignificant role:a tg iven laser intensity,H 2 Q( at pH 5) and HQ À (above pH 8) afford eC À aq concentrations that are so similara st o fall nearly within the margin of experimental error.

Phenol(ate) ionizations are known to be biphotonic. [31] In apparento pposition, no dependence of the eC À aq yield on the square of the laser intensity is discernible, but it is well understood that unfavourable combinationso fp arameters often preventaclear manifestation of this limiting relationship. [30, 36] As the best-fit curve to ab iphotonic ionization model in the main plot of Figure 3s hows, the validity of the quadratic approximation is restricted to an almost imperceptibly small range near the origin. More importantly,t he fit converges on complete ionization at infinite intensity,i no ther words, suggests that homolytic dissociation of the phenolateO ÀHb ond is absent under our conditions, whichf urthers upports our above calibration of the extinction coefficients of the quercetin radicals.

The unexpectedly negligible influence on the eC À aq yield of the decrease of the ground-state extinction coefficient by 30 % when going from HQ À to H 2 Qi sr evealed most clearly by the absence of ap Hd ependence at constantl aser intensity in the inset of the Figure. The reasonm ust be an accidental cancellation by different extinction coefficients of, lifetimes of, and photodetachment efficiencies from,t he excited states of H 2 Q and HQ À .A ll these parameters are inaccessible to our experimentso nn st imescales, but in the context of this work, their knowledge is also irrelevant. It is sufficent that photoionization of quercetin,regardless of whether it is present as H 2 Qo rH Q À , with a3 55 nm laser provides ad irect access to useable concentrationso fq uercetin radicals, as the experiments of this section have demonstrated and Scheme 2a tt he end of this "Results and Discussion" sectionsums up.

Whereas transformations of HQC and/or QC À on their own are hardlyn oticeable during the first 20 msa fter the generating laser flash, the additiono ft he archetypal co-antioxidant ascorbate changes the situation. Figure 4a epitomizes the effects, on which our second procedure for calibrating the extinction coefficientso ft he quercetin radicals is based.

The experimentalp Ho f6 .5 ensures as ingle protonation state for the quercetin radicals and fort he ascorbate, namely, QC À and the monoanion HAsc À (compare Scheme1;H Asc À is completely transparent above 320 nm). Thes ame condition is fulfilled for the ascorbyl radical AscC À (Scheme1), which hasa n absorption maximum at 360 nm with an extinction coefficient of 4500 m À1 cm À1 . [26] Best suited for observation at this pH are thus 560 nm (maximum of the QC À absorption) and 360 nm. The former wavelength responds to QC À only whereas the latter capturesQ C À ,depletion of quercetin, and AscC À .

We stress that all three contributionsa re already present directly after the laser flash, i.e.,a t0ms: because the experiment is performed in N 2 Os aturated solution, eC À aq yields HOC within nanoseconds;i nt urn, HOC is scavenged even faster by the high concentration of HAsc À (100 mm)t og ive AscC À ;i ns um, eC À aq is thus quantitativelya nd quasi-instantaneously converted into AscC À .E xperimental proof is provided by comparing the initial absorptiona t3 60 nm, which deceptively lies near zero, with that in ac ontrol experiment withoutH Asc À but under otherwise identical conditions. The negative and persistent transienta bsorption in the control experiment reveals the true contributions of QC À and quercetin depletion. In the experiment with ascorbate,atransients pectrum after 15 msi sc ompletely dominated by the band of AscC À ,w hich identifies the main reaction as the repair of the quercetin radical, in accordance with expectation.

The absorption trace at 560 nm does not decay to zero but to as mall residual value, about 4% of the initial absorption, on account of the competition of the repair with the natural conversion of QC À into an unspecified absorbing product (compare, Figure2b) . However,a t3 60 nm that product and QC À must have practically the same extinction coefficient, as is evident from the constant absorption trace in the control experiment. After the straightforwarda nd very smallc orrections for the described side reaction, the final absorption at 360 nm in the experiment with ascorbate is proportional to twice the extinction Figure 3 . Photoionization process of quercetin in homogeneous aqueouss olution. Main plot, dependence of the eC À aq yields relative to the quercetin weight-in concentration c 0 (5 mm)o nt he laser intensity I 355 .B lue data points, pH 8.1;o rangedata points, pH 5.0. The solid curve is the fit of ab iphotonic model to all data;best-fit limiting eC À aq yield at infinite intensity,100 %; other fit parameters without relevance. Inset, pH dependence of relative eC À aq yield at I 355 = 507 mJ cm À2 .For furtherexplanation, see the text. coefficient of AscC À ;a nd the constant of proportionality is the same as that between the initial absorption andt he extinction coefficient of QC À in the 560 nm trace.

The extinction coefficients obtained by this second calibration procedure (standard, AscC À )a nd by the above-described first one (standard, eC À aq )d iffer by 2% only.T his consistency not only lendss upportt ot he much higher value herein compared to the literature [19] but also, and far more importantly,e stablishes that QC À is exclusively formed through photoionization and not to anys ignificant degree through homolytic photocleavage of the OÀHb ond.

Even though the maximum of the QC À absorption band (560 nm) intuitively appearsb est suited for observation, two considerations were instrumental for our choice of as horter monitoring wavelength for all further experiments of this Section, namely,5 15 nm. First, the sensitivity of our detection system is noticeably highera t5 15 nm;a nd second, the dy-namic range of the detection signal is better equalized at 515 nm when the pH is varied (compare the spectra of HQC and QC À in Figure 2a ). By control experiments, we established that the decay curves at the two wavelengths are linearf unctions to one another.

On the basis of the first-order intrinsic decay (Figure 2b) , the repair is expected to obey Stern-Volmer kinetics. Figure 4b illustratest hat this surmise holds true, and that the pseudo first-order rate constant k obs at constant pH is al inear function of the ascorbatew eight-inc oncentration c Asc .H owever,t he apparentb imolecular rate constant obtained from the slope in the inset is not meaningful per se, on account of ap ronounced andi ntriguing pH dependence, which Figure 5a investigates at constant c Asc .T he relationship is seen to be ab ellshaped curve with an asymmetric tail to the side of higher pH. For improved precision, we measured k obs (pH) in that region with ah igher c Asc (100 mm)a nd recalculated it to c Asc of the rest of the data (5 mm)a si nF igure 4b.

In the pH range from 2t o9 ,t he quercetin radicals can exist as HQC or QC À and ascorbate as the acid H 2 Asc or the monoanion HAsc À ;h ence,f our combinations of radical and co-antioxidant need to be taken into account. However,asignificant involvement of the pair HQC/H 2 Asc is immediatelyr uled out by the evident decrease of k obs (pH) towards zero in acidic medium, where HQC and H 2 Asc are presentp ractically exclusively.

By contrast, ar eaction between QC À and HAsc À clearly takes place, as is manifest from the constant nonzero rate constant in the higherp Hr ange where QC À andH Asc À are the only protonation forms. This process adds ac ontribution k'(pH) to k obs ,

where k dep -the true, pH-independent rate constant of the reaction between QC À and HAsc À -constitutes the upper limit of k'(pH),w hich is approached at high pH. Equation 1i ss een to remain invariant when pK a1 and pK a2 are interchanged;i ts plot is virtually indistinguishable from at itration curve characterized by the higherofthe two pK a values as long as j pK a1 ÀpK a2 j is larger than about 1.5;a nd when that absoluted ifference becomes smaller, the mid-point slightly shifts to higher pH, with am aximum deviation of 0.4 pH units being reached for identical pK a values. The two "mixed" reactions, betweeno ne protonated and one deprotonated species, are indistinguishable by their pH dependence:i nt erms of the average M and the half-difference D of the two pK a values [Eq. (2), Eq. (3)]

¼ pK a1 þ pK a2 2 ð2Þ

their rate constant k(pH) can be formulated to give [Eq. (4)]

The invariance of the denominatoro ft he final expression with respect to an interchange of pK a1 and pK a2 is obvious. Equation (4) has been set up for species 1r eactingi ni ts protonated form and species 2i ni ts deprotonated form, but an interchange of the two species, tantamountt oa ni nterchange of the two pK a values, thus merely leads to as calingo ft he numeratort hat cannot be separated from ad ifferent value of k mix .

To determinew hether k mix is the pH-independent rate constant of the reactionb etween HQC and HAsc À or that between QC À and H 2 Asc (or as uperposition of the two) and therebyt o extract its correct numerical value, argumentso utside mathematics have to be invoked. Fortunately,t his is straightforward in our system because HAsc À is af ar better antioxidant than is H 2 Asc (compare the bond dissociation energies BDE in Scheme1). Hence, we assign the mixed process exclusively to the repair of HQC by HAsc À .E ven without knowledge of the BDE, this is entirely consistent with it being much faster than the high-pHr epair of QC À by HAsc À ,m eaning that HQC is more reactive than is QC À and implying that ar eaction between QC À and H 2 Asc would be slower than the already unobservable one betweenHQC and H 2 Asc.

Equations (1) and (4) share the same pK a values, one of which (the pK a of HQC)w as determined herein under exactly the same experimental conditions. Because of considerable spread in the reportedp K a of H 2 Asc (between 3.96 [37] and 4.25 [38] ), we treated that parameter as adjustable, in addition to k dep and k mix .T he resulting three-parameter best fit of the sum of Equations (1) and (4) to k obs (pH) is displayed in Figure 5a .A s emerges from the separated contributions that have also been plotted, the fit is extremelyw ell-conditioned because k dep can practically be read off from the data in the region of high pH.

The fit converged near the lower of the two pK a values of H 2 Asc cited above;a nd dividing k mix and k dep by c Asc finally yielded the true second-order rate constants, 1.6 10 8 m À1 s À1 and 3.0 10 6 m À1 s À1 for the reactions of HAsc À with HQC and with QC À .

Only two kinetici nvestigations on fast timescales were carried out on quercetin/ascorbate systemss of ar,a nd both were performed at as ingle pH only,8 .5 [18] or 10.8. [19] The latter work lists ar ate constant of 2.4 10 5 m À1 s À1 but, according to the authors' conclusions, the quercetin radicals are doubly deprotonated at pH 10.8 (secondp K a ,9 .4);h ence, that rate constant appliest oareaction different from ours. From ah ighly complex kinetic modelling, the former study extracts ar ate constant of 5.0 10 6 m À1 s À1 for what must be the reactionb etween QC À and HAsc À .O ur result is 40 %l ower but we believe it to be more reliable because we obtained it by direct kinetic measurements of the isolated process.

For the reaction between HQC and HAsc À ,n or ate constant seems to have been reported to date. We stress that, in view of the bell-shaped pH dependence (Figure 5a )a nd its underlying formula, measurements at asingle pH would have suggested values that are grossly too low: even if, through lucky coincidence, these experiments had been carriedo ut at the maximum of the bell-shaped curve [Eq. (5)],

the pK a -dependent fraction in Equation (5) would have necessitated ac orrection by af actor of 2.5 with our parameters (pK a1 ÀpK a2 = 0.48) to obtain k mix ;t his would have risen to a factor of 4i fb oth pK a values happened to be equal; and, from Equation (4), to much more if some pH in the outer wings of the curve had accidentally been chosen. The regenerationo ft he quercetin from HQC could be ac oncerted process( proton-coupled electron transferP CET) or as equentialo ne (rate-determining electron transfer followed by proton transfer,S ETPT;o rr ate-determining proton loss followed by electron transfer,S PLET). The pertinent pK a values, 11.74 of HAsc À and À0.45 of HAscC, [38, 39] already disfavor SETPT and SPLET,a nd the H/D kinetic isotope effects (KIEs), which have also been included in Figure5a, provide direct experimental evidence. SETPT could only lead to as mall secondary KIE, and SPLET to as imilarly small thermodynamic isotope effect on the deprotonation equilibrium of HAsc À , [40] whereas PCET is expected to exhibit aprimary KIE.

Because all hydroxylic protons of quercetin and ascorbate are exchangeable and the reagent concentrations are very small, complete deuteration at the relevantp ositions is achieved before the start of the photoionization in D 2 O. Determining KIEs for ar eaction with complex pH dependence faces the problemt hat pK a valuesi nH 2 Oa nd D 2 Om ight be slightly different;a nd the same appliest op Ha nd pD readings. Potential pitfalls are thus comparing data at the maximum of the curve (Figure 5a )i no ne solventw ith off-maximum data in the other solvent; and, independent from the former,o verlooking different trailing fractions in Equation (5) caused by ad iscrepancy of D in the two solvents. To avoid these sources of errors, we carried out al imited pD variation to localize the maximum in D 2 O and to ensure that the width of the bell-shaped dependence, which is only relatedt oD butn ot to M,d oes not change. Figure 5a displays the outcome, which gave the same D in D 2 O and apparently also the same M as in H 2 O. Although the latter might well be due to an equal influence of the deuterated solvent on the potential of the glass electrode, Equation (4) is insensitivet os uch as hift of the horizontal scale as aw hole. Direct comparison of the maximay ields aK IE of 1.9, whose magnitude can only be reconciled with ap rimary KIE and thus the PCET mechanism. [40] Only an estimate can be given for the KIE in basic medium because the reactioni nD 2 Ob ecomes so slow that the correction for the decay without quencher (compare, Figure 2b )e n-tails ar ather large error.S ingle-point measurements at pH 8.0 gave aK IE between 3a nd 5, whichs uggests aP CET mechanism for the repair of QC À by HAsc À as well.

Theoretical investigations report bond dissociation energies (BDEs) in aqueous solution between 71.3 kcal mol À1 and 83.1 kcal mol À1 for H 2 Q, [32, 35] and between 70.9 kcal mol À1 and 75.0 kcal mol À1 for HQ À . [32, 35] Precise experimental values are unavailable, but our results of Figure 5a allow bracketing these key quantities, in particulart hat of H 2 Qw ith am uch lower uncertainty.APCET is thermodynamically feasible when the BDE of the co-antioxidant is smaller than the BDE of the regenerated quercetin, each in its respective protonation state. The combination of no discernible repair of the quercetin radicals by H 2 Asc (BDE, 78.0 kcal mol À1 ) [39] and successful repairsb yH Asc À (BDE, 71.8 kcal mol À1 ) [39] thus puts af irst upper and lower limit on the BDEs of H 2 Qa nd HQ À ;i nf ull accordance with this, we found reduced glutathione (BDE, 87.2 kcal mol À1 ) [39] to be inoperative as ar epairing agent;t he upper boundary is substantiated and even slightly tightened by our observations that neither hydroquinone (BDE, 79.7 kcal mol À1 ) [39] nor aw atersoluble analogue of a-tocopherol( Trolox;B DE, 76.7 kcal mol À1 ) [39] are capable of repairing the quercetin radicals in the pH range of Figure 5a ,w here no deprotonations of the co-antioxidanth ydroxylic groups need to be taken into account; and our following experiments (Figure 5b) , which demonstrate that p-aminophenol pAP (BDE, 75.0 kcal mol À1 ) [39] can repair HQC but not QC À ,b oth raise the lower limit forH 2 Qa nd decrease the upper limit for HQ À still further.

The deprotonation of the phenolic OH-group of pAP falls outside the pH range investigated, but the amino group already becomes protonated in slightly acidic medium( p K a , 5.48), [41] whereby this phenol loses its good antioxidant properties. In line with expectation, no repairi st hus observed at low pH, where only HQC andN -protonated pAP exist. When the pH is raised,t he increase of available co-antioxidant through deprotonation at Ni sc ountered by the transformation of HQC into the less readily repaired QC À ,s uch that againabell-shaped pH dependence of k obs results. On its high-pHs ide, however, no constant floor is detectable;h ence,n or epair of QC À by pAP occurs.

As the main differencet ot he ascorbate case of Figure5a, D [Eq. (3)] is negative for HQC/pAP.F rom the above discussion it emerges that this does not influence the shape of the pH dependence in any way.H owever,i ts trongly decreases the scale factor given by Equation (5) such that the curve at its maximum amounts to lesst han 6% of k mix only,w hereas that factor was sevent imes higher for HQC/HAsc À .T he resulting lower k obs thus totally maskst hat the true rate constantf or the repair of HQC by pAP,6 .0 10 8 m À1 s À1 ,i sa bout four times larger than that with the co-antioxidant HAsc À .W es tress that this difference is not due to ac hange of mechanism:t he KIE of 2.2 in Figure5b, whichw as obtained by the same procedure as before,c onfirmst hat the reactionr emains aP CET when pAP replaces HAsc À .

Despite the lower thermodynamicd rivingf orce, pAP repairs HQC faster than does HAsc À .W eh ave already observed the same phenomenonf or the repairso ft he resveratrol radical by these two co-antioxidants. [20] Most likely,s teric constraints on the highly ordered PCET transition state provide the underlying reason.

The findings of this work regarding the pathways to the quercetin radicals HQC and QC À ,t he feasibility or infeasibility of their repairs by co-antioxidants,a nd the derived intervals for the BDEs of H 2 Qa nd HQ À have been collected in Scheme 2.

As has emerged, flash photolysis with an inexpensive near-UV pulsed solid-state laser (355 nm) provides an extremely convenient access to quercetin radicals through photoionization. In fact, not as inglea dditive is neededp er se, although as a variant one can blank out the only by-product eC À aq by saturating the solution with N 2 O. Neither eC À aq nor its blanking product HOC interfere with residual quercetin or its radicals for kinetic reasons; the timescale of the radical generationi ss et by the duration of the laser flash, af ew ns, which is tantamount to instantaneous generation when typicals econdary reactions with co-antioxidants on mst imescales are studied;a nd within a wide pH range (between 2-3 and 9), the attainable post-flash radicalconcentrationi su pt oo ne-half the substrate concentration while being easily controlled by the laser power.

Besidesa llowing ac haracterization of the neutrala nd anion radicals HQC and QC À ,i ncluding ar ecalibration of their absorption spectra with very different resultsc ompared to the literature, the photoionization approachp aved the way to ad etailed study of the interactionso fH Q C and QC À with the ascorbate monoanion HAsc À and other co-antioxidants, such as 4-aminophenol. Most conspicuous were the bell-shapedp Hd ependences of the repair kinetics of HQC,i nc onsequence of which the maxima of the observed rate constantsa sf unctions of the pH represent only af raction of the true rate constants for the repair of HQC by the co-antioxidant, such that singlepoint measurements even at the exact maximac ould be grossly misleading. Given that we were able to identify the repair mechanisms as proton-coupled electron transfers, ac omparison of different co-antioxidants with known bond dissociation energies allowedu st os pecify the bond dissociation energies of quercetin and its monoanionw ith much smalleru ncertainties than previously estimated by quantum-mechanical calculations.

On the basis of the Stern-Volmer behavior of the repairs with ascorbate, the 50 times lower reactivity of the quercetin radicala nion compared to the neutral radical, and ap K a value of the latter below 5, it is evident that no complete repair is achievable with reasonableascorbate concentrations at physiological pH in homogeneous solution. However,t hat pessimistic picture shouldn ol ongerb ev alid in organized systems;a nd we envisage that our laser flash photolysis approach, which generates the radicals at the exact locations of their precursors, will prove useful in such situations, as we have already shown in the case of the antioxidant resveratrol and itsr epairs through micelle-water interfaces. [42] 

Antimicrob.Agents Chemother

Anti-Cancer Drugs

Principles of Fluorescence Spectroscopy,3

Vitamin C: Its Chemistrya nd Biochemistry,1

CRC Handbook of Chemistry and Physics

Accepted manuscript online

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www.chemeurj.org 2020 The Authors

Open access fundinge nabled and organized by DEAL.Scheme2.Quercetin-derived species [35] with theira bbreviations;connecting pathways pertinenttot his work, pK a values, and bond dissociation energies BDE, as determined herein.

The authors declare no conflict of interest.Keywords: antioxidants · kinetics · laser chemistry · photoionization