Solvent Dependence of the Kinetic Isotope Effect in the Reaction of

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J. Phys. Chem. A 2010, 114, 3423–3430

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Solvent Dependence of the Kinetic Isotope Effect in the Reaction of Ascorbate with the 2,2,6,6-Tetramethylpiperidine-1-oxyl Radical: Tunnelling in a Small Molecule Reaction Ivana Sajenko, Viktor Pilepic´, Cvijeta Jakobusˇic´ Brala, and Stanko Ursˇic´* Faculty of Pharmacy and Biochemistry, UniVersity of Zagreb, A. KoVacˇic´a 1. Zagreb, Croatia ReceiVed: July 24, 2009; ReVised Manuscript ReceiVed: January 26, 2010

The oxidation of ascorbate with the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) radical in water and water-dioxane mixed solvent has been demonstrated to be a proton-coupled electron transfer (PCET) process, involving hydrogen tunnelling at room temperature. The magnitude of the kinetic isotope effect (KIE) kH/kD in the reaction increases with decrease of the solvent polarity. The evidence comprise: (a) the spectroscopic and kinetic evidence for the interaction of ascorbate and TEMPO; (b) the observation of KIEs kH/kD of 24.2(0.6) in water and 31.1(1.1) in 1:1 v/v water-diox. (diox ) dioxane), at 298 K; (c) the observation of isotope effect on the Arrhenius prefactor, AH/AD of 0.6(0.2) in the reaction in water and 1.2(0.2) in 1:1 v/v water-diox solvent; (d) the observation of isotope differences in the enthalpies of activation in water and D2O, ∆rH‡ (in H2O) ) 31.0(0.4) kJ/mol, ∆rH‡ (in D2O) ) 40.0 (0.5) kJ/mol; in 1:1 v/v water-diox and 1:1 v/v D2O-diox, ∆rH‡ (in H2O/diox) ) 23.9(0.2) kJ/mol, ∆rH‡ (in D2O/diox) ) 32.1(0.3) kJ/mol; (e) the temperature dependence of the KIEs in water and 1:1 v/v water-dioxane; these KIEs range from 27.3 at 285.4 K to 19.1 at 317.4 K in water and from 34.3 to 24.6 at the corresponding temperatures in 1:1 v/v water-diox, respectively; (f) the observation of an increase of the KIE in 10-40% v/v dioxane-water solvents relative to the KIE in water alone. There is a weak solvent dependence of the rate constant on going from water to 1:1 v/v water-diox. solvent, from 2.20(0.03) mol-1 dm3 s-1 to 5.50(0.14) mol-1 dm3 s-1, respectively, which originates from the mutual compensation of the enthalpy and entropy of activation. Introduction Coupled transfer of a proton and an electron, commonly denoted as the proton-coupled electron transfer (PCET), is essential for many important biological and chemical processes.1-24 These include, among others, photochemical oxidation of water to dioxygen,1-5 respiratory oxygen reduction and numerous enzyme reactions,6-12 chemical13-19 and electrochemical20-22 reactions, involving metal complexes and small molecule systems as well as processes at semiconductor interfaces23 and artificial membranes.24 Most frequently, the reactions that involve the simultaneous transfer of an electron and a proton are termed as proton-coupled electron transfer (PCET)13,14 reactions, but to maintain generality, reactions that involve sequential transfer processes (first the proton transfer then the electron transfer or vice versa, i.e., PT/ET or ET/PT) can be viewed in some circumstances together with the former within the unified framework of PCET reactions.25 This framework also includes hydrogen atom transfer (HAT) reactions in which the electron and proton transfer simultaneously between the same donor and acceptor.13,14,25 These formal HAT reactions can differ in that whether the proton is transferred together with one of its bonding electrons in which case the reaction would be classified to be the simple hydrogen atom transfer (HAT) or the proton and electron are transferred between different sets of orbitals (PCET reaction) but still between the same donor and acceptor site.13,14,25,27 However, it has been pointed out that these distinctions are not rigorous from the theoretical viewpoint because the electron and the proton are not localized to a single point at any given time as a consequence of its quantum mechanical behavior.25 It is important to recall however that * To whom correspondence should be addressed. Phone: +385-14818306. Fax: +385-1-6394400. E-mail: [email protected].

the simultaneous process, usually termed as concerted transfer of a proton and an electron, constitutes a single chemical reaction step, and the direct coupling of the electron and the proton in the transfer is the elementary characteristic of the process. The phenomenon has been greatly studied theoretically25-33 to elucidate and predict fundamental dynamic and structural aspects of the PCET reactions. Important insights into fundamental features of the PCET reactions can be obtained from the kinetic isotope effect (KIE) measured in the reactions. One of the questions related to interpretation of KIEs observed in PCET reactions is whether the magnitude of these KIEs depends on the solvent polarity. Theoretical analyses of KIEs in model PCET reactions have predicted such a possibility.34 Hence, measurements of KIEs in a PCET reaction would also enable these theoretical predictions to be tested experimentally. To gain some insight related to the above question, we have studied the dependence of the KIE on the solvent polarity in the reaction of ascorbate with the 2,2,6,6-tetramethylpiperidine1-oxyl (TEMPO) radical in water and water-1,4-dioxane35 (dioxane henceforth) mixed solvents. The study has been also motivated by some dearth of experimental evidence on the role of solvent in PCET reactions.14 We have chosen the solvent mixture of water with dioxane because the low polarity (r ) 2.21) of dioxane enables the desired variation of mixed solvent polarity. The optical transparency and good miscibility of dioxane with water are additional advantages of the choice. On the other hand, the solvent mixture of water with 1,4-dioxane is representative for many aqueous-organic solvent mixtures.39,40 There are some important studies of PCET reactions in mixed solvents, for instance studies of reaction between an osmium(VI)-nitrido complex and diethylphosphane,41 osmium(IV)-

10.1021/jp911086n  2010 American Chemical Society Published on Web 02/12/2010

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hydrazido, osmium(IV)-sulfilimido, and osmium(IV)-phosphoraniminato complexes and benzoquinone42 in 1:1 (v/v) wateracetonitrile, but these do not include a systematic study of change of solvent composition on rates and isotope effects in the reactions. Here we have reported that the reaction of ascorbate with TEMPO radical in water as well as in water-dioxane is a PCET reaction, exhibiting large deuterium KIEs both in water (kH/kD ) 24.2) and in the water-dioxane solvent system (kH/kD ) 31.1 in 1:1 v/v water-dioxane) at room temperature. The observed KIEs in the water-dioxane mixtures increase approximately linearly with the mole fraction of the added dioxane. Thus, the observed KIE increases when the solvent polarity decreases on going from water to water-dioxane mixed solvents, but the observed change of the KIE is moderate, although it does occur in the theoretically predicted direction.34 The observed KIEs kH/kD ) 31.1 in 1:1 v/v water-dioxane and kH/kD ) 24.2 in water are beyond the maximum expected semiclassical value of 7.9 for the dissociation of O-H bond,43 and the isotopic differences in the activation energies of 9 kJ in water and 8.2 kJ in 1:1 (v/v) water-dioxane are beyond the difference in zero-point energies43,44 of 5.1 kJmol-1, which implies that quantum mechanical tunneling can play a role in the reaction. Experimental Methods Materials. The TEMPO radical (99% sublimed) was from Aldrich. Ascorbic acid was from GEHE Pharma (Germany). Sodium acetate, dioxane, glacial acetic acid, and heavy water (99.9% D) were analytical grade (Aldrich or Merck). Heavy water was twice distilled prior the use. Double-distilled water was used throughout. Methods. Determination of pKas of Ascorbic and Acetic Acid. pH measurements were carried out with Mettler Toledo MP 230 pH-meter and combined Mettler Toledo InLab SemiMicro pH electrode designed to give accurate measurement within a temperature range from 0-100 °C. Dioxane (purity of 99.5%) and acetic acid (glacial, pa) were purchased from Merck. Combined electrode was calibrated with commercial aqueous standard buffer solutions pH ) 7.00 and pH ) 4.00 also purchased from Merck. All the solutions were prepared by mixing doubly distilled, freshly boiled, and then chilled water and dioxane, bubbled previously with 99.999% N2 to remove oxygen and carbon dioxide. The titrations were carried out under a nitrogen atmosphere. The solutions 0.1 M of ascorbic and acetic acid in range from 0 to 50% v/v dioxane in water and heavy water were well thermostatted at temperatures from 12 to 45 °C ( 0.1 °C before titration. pH electrode was soaked for 15-20 min in appropriate dioxane-water mixture before the use. The NaOH solution for titration was prepared from pellets of sodium hydroxide (pa, Merck), which were washed three times with doubly distilled, freshly boiled and chilled water, bubbled with nitrogen before dissolution. The obtained NaOH solution was standardized volumetrically with standard 1.000 M HCl (Merck) and diluted with dioxane and water to achieve 0.5 M sodium hydroxide in an appropriate dioxane-water mixture, which was used for titrations. The pKa values45 given in Tables 1-4 in the Supporting Information are the average of at least three titrations. The pKas in dioxane-water mixtures were corrected with UH factors determined by Van Uitert and Haas for dioxane-water solutions.45 All pKa values are expressed in terms of pH and referred to an ionic strength of 0.05. Kinetic Measurements. Pseudo-first-order rate constants for the reaction of ascorbate with TEMPO radical have been determined spectrophotometrically by monitoring the decrease

Sajenko et al. of absorbance of TEMPO at 422 nm in water and at 433 nm in water-dioxane solvents. An HP 8453 UV-vis spectrophotometer was used throughout to collect spectral and absorbance time data. At least in principle the growth of the product dehydroascorbic acid could also be used to measure the kinetics. However, this would imply a very arduous and much less precise high-performance liquid chromatography measurements. Kinetic measurements were performed under carefully maintained constant temperature conditions, i.e., the temperature was always accurate to (0.1K. In the experiments in D2O the deuterium oxide content was 99.5%, and in the experiments in D2O-dioxane 1:1 the deuterium oxide content was 99.1%. The pseudo-first-order conditions were maintained by taking the total ascorbic acid concentration to be at least 25-fold excess over the TEMPO. Typically, total ascorbic acid was 0.100 M and TEMPO 0.004 M, and the pH of the reaction solution usually was in the range 3.2-3.3 in water and 4.5-4.9 in water-dioxane solvent (these figures can be obtained by the below described calculation, and there was no need to measure pH in the kinetics; the pH values in water-dioxane solvent were higher due to the shift in the pKa values on going from water to the mixed solvent, cf., Table 1 in Supporting Information). The appropriate concentration of ascorbate was kept constant during the particular kinetics using the appropriate buffered system, adjusted with sodium acetate. The equilibrium concentration of ascorbate (HAsc-) in this particular experiment was calculated using the appropriate pKa values of ascorbic acid (H2Asc) and acetic acid (HAc) at the given temperature45 by solving the equations

Ka(H2Asc) ) Ka(HAc) )

([HAsc-] - [HAc])[HAsc-] [H2Asc]tot - [HAsc-]

([HAsc-] - [HAc])([Ac-]tot - [HAc]) [HAc]

(where [H2Asc]tot and [Ac-]tot are the total/added concentrations of ascorbic acid and sodium acetate, respectively). The Mathematica 5.0 program was used for these particular calculations. In a typical kinetic run, at least 200 pairs of absorbance-time data were collected and fitted to the common least-squares algorithm. Very good pseudo-first-order kinetics were obtained throughout. At least four to five observed pseudo-first-rate constants were always used to calculate the corresponding rate parameters under the specified reaction conditions. The observed values of the KIEs were corrected with regard to the corresponding deuterium content in the reaction mixture.45 Results The process we have investigated is the interaction of ascorbate with TEMPO radical (Scheme 1). In water as well as in water containing mixed solvents, ascorbate reduces the TEMPO radical,37 giving the corresponding N-hydroxy compound (TEMPOH) and dehydroascorbic acid. The spectroscopic and product analyses clearly suggest a 1:2 (ascorbate/TEMPO) overall stoichiometry under the conditions employed, in accordance with previous findings.37 Ascorbyl radical anion formed in the relatively slow first step (Scheme 1) disproportionates in the fast subsequent step to ascorbate and dehydroascorbic acid. Since the pKa of TEMPOH is 7.5 (for the protonation of TEMPOH nitrogen),46 the product TEMPOH undergoes fast subsequent protonation (not relevant for the

Reaction of Ascorbate with TEMPO SCHEME 1

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kobs ) 2kHAsc-Ka[H2Asc]0

1 [H ] + Ka +

The rate parameters for the reaction of ascorbate with TEMPO radical (Scheme 1) have been obtained from the pseudo-firstorder rate constants determined spectrophotometrically. An example of the obtained pseudo-first-order kinetics for the reaction is presented by Figure 1. As expected, the obtained kinetic evidence is consistent with Scheme 1, and the fact that under the conditions employed the subsequent disproportionation of ascorbyl radical in water and water-rich mixed solvents proceeds at least for 3-5 orders of magnitude faster48,49 than the first, a relatively slow process of the reduction of the TEMPO radical by ascorbate monoanion. This is further coroborrated by the obtained dependence of the observed rate constants on the reciprocal of H+ ion concentration in water (Figure 2) and 1:1 v/v water-dioxane (not shown here). Hence, when under the pseudo-first-order conditions (with respect to the effectively constant ascorbate concentration in the buffer system) disappearance of TEMPO is monitored, the corresponding empirical rate law should be

with kHAsc-, the second-order rate parameter for the reaction of ascorbate with TEMPO radical, and this parameter is related to the experimentally obtained as 2kHAsc- ) kTEMPO, the observed second-order parameter for the disappearance of TEMPO. Here, Ka is the (first) dissociation constant of ascorbic acid H2Asc, and HAsc- is ascorbate (monoanion). The obtained rate parameters for the reaction in water and in water-dioxane mixtures are listed in Table 1. The obtained second-order rate constant for the reaction of ascorbate with the TEMPO radical in water at 298 K is 2.20 dm3 mol-1 s-1. The corresponding rate parameter in the 1:1 v/v water-dioxane solvent (5.50 mol-1 dm3 s-1) is only 2.5-fold greater than the above rate constant in water. The corresponding rate ratios for water-dioxane solvents containing 10-40% of dioxane are expectedly smaller. The relatively small rate increase could be somewhat surprising with regard to the substantial change of the solvent polarity on going from water to the 1:1 v/v water-dioxane solvent (see also discussion below). The temperature dependence of the reaction rate constant for the reaction in water and in the 1:1 v/v water-dioxane is presented in Figure 3. The activation parameters for the reaction in water and heavy water and in the water-dioxane 1:1 v/v and in the heavy water-dioxane 1:1 v/v are listed in Table 2 along with the corresponding obtained Arrhenius A preexponential factors. Here, the most intriguing result is the observation of the isotopic differences in the enthalpies of activation where ∆∆rH‡ between D2O and H2O is 9.0 and 8.2 kJ/mol in water and 1:1 v/v water-dioxane, respectively. The observed temperature dependence of the KIE both in water and in the 1:1 v/v water-dioxane fits with the normal temperature dependence of isotope effects.50 The observed isotopic ratio of the Arrhenius pre-exponential factors, AH/AD ) 0.6(0.2) in water (Table 2), could be beyond the semiclassical limits43,51-53 of 0.7-1.4 possibly suggesting a tunneling in the PCET process, while the corresponding AH/AD ) 1.2 (0.2) in the reaction in 1:1 v/v water-dioxane can still be fitted within the semiclassical limits for the ratio of isotopic Arrhenius prefactors in a hydrogen transfer process. However, this is not without further qualification as will be discussed below. The observed KIEs in the reaction, both in water and in the water-dioxane solvent systems containing 10-50% v/v of dioxane, are listed in Table 1. The KIEs obtained in water and in the 1:1 v/v water-dioxane in the temperature range from 285.4 to 317.4 K are listed in Table 3. Large KIEs in the reaction were observed in all the cases examined (cf. Table 1 and 3). The values of the KIEs kH/kD ) 31.1 in 1:1 v/v water-dioxane and kH/kD ) 24.2 in water at room temperature are well beyond the maximum semiclassical values calculated to be kH/kD ≈ 10.3 for a hydrogen transfer from oxygen54,55 or even kH/kD ≈ 13 with allowance for bending vibrations.43 A moderate increase of the KIE in the reaction on going from water to water-dioxane solvent systems has been observed. As can be seen from Table 1 and Figure 4, the KIE increases approximately linearly (r ) 0.97) with the increase in mole fraction of dioxane from the value of 24.2 in water to the value of 31.1 in 1:1 v/v water-dioxane, at room temperature.

rate ) -d[TEMPO]/dt ) 2kHAsc-[HAsc-][TEMPO]

Discussion A. PCET Reaction. The obtained kinetic evidence requires that the reaction proceeds according to the Scheme 1, i.e., the

kinetic measurements under the conditions employed in our experiments).47 We have determined (a) rate constants for the reaction of ascorbate with TEMPO radical (Scheme 1), in water and in five water-dioxane solvent systems ranging from 10 to 50% v/v of dioxane; (b) the activation parameters for the reaction in water; (c) the corresponding activation parameters in 1:1 v/v water dioxane; (d) the deuterium KIEs in the process in water and all the mixed solvents used; (e) the temperature dependencies of KIEs in the temperature range of 385.4 K to 317.4 K in water as well as in the water-dioxane 1:1 v/v solvent system. In addition, we have determined the pKa values of ascorbic acid (pKa1, for the dissociation of the first proton) in water and all the mixed solvents used in this study and the temperature dependence of these pKas in water and the 1:1 v/v water-dioxane as well as the corresponding pKas and the temperature dependence of these pKas for acetic acid in the mixed solvents used in this study. These data were used to determine the rate parameters in the corresponding buffered systems. It could be noted here that we have observed an excellent linear dependence of the obtained pKas on the mole fraction of dioxane in water both in the case of ascorbic and acetic acid (see the Experimental section and Supporting Information for details).

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Figure 1. Change of the absorbance at 422 nm (points) of the reaction mixture of TEMPO (0.004 M) and ascorbic acid (0.100 M) in presence of sodium acetate (0.010 M) in water at 25 °C. The line is the fit of the first-order kinetics obtained from these data points.

Figure 2. Dependence of the rate constants for the reaction of ascorbic acid with TEMPO radical in water on the reciprocal proton concentration (r ) 0.999) at 25 °C. Total ascorbic acid concentration was 0.1 M throughout.

TABLE 1: Dependence of Rate Constants and KIEs for the Reaction of Ascorbate with TEMPO Radical on Solvent Compositiona,b v/v dioxane/% 0.0 10.0 20.0 30.0 40.0 50.0

kHAsc- (H2O-dioxane)/ 10×kHAsc- (D2O-dioxane)/ M-1s-1 M-1s-1 2.20 (0.03) 2.65 (0.02) 3.20 (0.01) 3.82 (0.01) 4.67 (0.03) 5.50 (0.14)

0.91 (0.02) 1.05 (0.01) 1.23 (0.00) 1.44 (0.01) 1.68 (0.01) 1.77 (0.04)

KIE 24.2 (0.6) 25.2 (0.3) 26.0 (0.1) 26.5 (0.2) 27.8 (0.2) 31.1 (1.1)

a At 25 °C. b Rate constants for the reaction were determined as described in the Experimental section (see also Supporting Information) and are an average of 4-5 runs.

proton of the product N-hydroxyamine (TEMPOH) came from the ascorbate. Furthermore, the consideration of obtained experimental evidence in comparison with the thermochemical analysis of the corresponding consecutive (PT/ET or ET/PT) reactions (i.e., usual consideration using the “square scheme”)13 clearly show that the interaction of ascorbate with TEMPO radical (Scheme 1) should be a concerted, PCET process, both in water and in the 1:1 v/v water-dioxane solvent system. Thus, if there would be a sequential PT/ET, the initial proton transfer should be endergonic by no less than 96.4 kJ/mol, i.e., 65.0

kJ/mol would be needed for the dissociation of the 2-OH of ascorbate taking into account the pKa value of 11.3 of the ascorbate 2-OH group,57 and 31.4 kJ/mol for the protonation of TEMPO, taking the pKa of TEMPO to be58 -5.5 in accord to the well-known thermochemical equation ∆rG° ) 2.303RTpKa). This barrier would be greater by at least 25 kJ/ mol than the experimentally obtained activation energy ∆rG‡ of 71 kJ/mol (Table 2), and consequently this process should be ruled out. The initial electron transfer (in assumed sequential ET/PT) appears to be energetically highly unfavorable with respect to the observed activation free energy (71 kJ/mol) in the reaction. E° for ascorbate monoanion is57 0.720 V; therefore the initial ET from ascorbate requires ca. 69.4 kJ/mol, which is about 1.6 kJ/mol below the experimentally obtained activation energy ∆rG‡ of 71 kJ/mol. However, the electron transfer to TEMPO should be endergonic by more than 22 kJ/mol as E°(TEMPO•/ TEMPO-) is -0.228 V.45,46,59 Collectively, the free energy barrier for the initial ET would amount at least 91 kJ/mol which is 20 kJ/mol more endergonic than experimentally obtained ∆rG‡ and should be highly unlikely to occur. It could be added that if the initial ET from ascorbate occurred and if TEMPO anion and protonated ascorbyl radical would be formed, taking into regard the kinetic evidence, this process must be followed by a

Reaction of Ascorbate with TEMPO

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Figure 3. Eyring plots for the reaction of ascorbate with TEMPO in water (b), D2O (O), water-dioxane (9) and D2O-dioxane mixture (0). Correlation coefficients of the straight lines are always 0.999 or better.

TABLE 2: Thermodynamic Parameters for the Reaction of Ascorbate with TEMPO Radical in Water, D2O, 1:1 v/v Water-Dioxane and 1:1 v/v D2O-Dioxane

solvent

∆H‡/ kJ mol-1

∆S‡/ J K-1 mol-1

∆G‡/ kJmol-1 (298.15 K)

H2 O D2O H2O-dioxane D2O-dioxane

31.0 (0.4) 40.0 (0.5) 23.9 (0.2) 32.1 (0.3)

-134 (2) -130 (2) -151 (1) -152 (1)

71.0 (0.7) 78.8 (0.8) 68.9 (0.4) 77.4 (0.4)

AH/AD 0.6 (0.2) 1.2 (0.2)

TABLE 3: Temperature Dependence of the KIEs kH/kD for the Reaction of Ascorbate with the TEMPO Radical in Water and 1:1 v/v Water-Dioxanea T/°C

KIE in water

KIE in water-dioxane

12.4 16.6 21.3 25.0 29.6 34.4 37.9 44.4

27.3 (0.2) 27.1 (0.5) 26.2 (0.6) 24.2 (0.6) 22.8 (0.4) 22.2 (0.9) 20.3 (0.4) 19.1 (0.7)

34.3 (0.6) 32.9 (0.6) 31.5 (1.3) 31.1 (1.1) 28.9 (0.4) 26.6 (0.8) 25.9 (0.3) 24.6 (0.3)

a The corresponding observed rate constants are listed in Supporting Information.

Figure 4. Dependence of the KIE in the reaction on mole fraction n of dioxane (r ) 0.97).

diffusion-controlled proton transfer between the strong acid (pKa of the protonated ascorbyl radical is60 -0.45) and the strong base, the TEMPO anion having a pKa 13.7 or even more. The obtained experimental evidence shows that the very large KIEs are observed in the reaction (cf. Table 1 and 3) both in water and in water-dioxane solvents which clearly indicates that the proton transfer occurs in the rate-controlling step. If there would

be a diffusion-controlled proton-transfer process, only very small isotope effects could be observed. Taken together, it seems reasonable to conclude that the interaction of ascorbate with TEMPO proceeds neither by sequential PT/ET nor sequential ET/PT but is a concerted PCET process. Similar reasoning applies to the water-dioxane solvent systems taking however into regard the corresponding changes in the pKas and E° values on going from water to the mixed water-dioxane solvents. An instructive example for such a consideration presents the analogous analysis of Mayer and Warren,38 who have demonstrated that the interaction of the isopropylidene derivatized ascorbate with TEMPO in acetonitrile should be a concerted PCET process. B. Solvent Effects. An important question related to the results obtained in this study is whether the hydrogen-bonded complex consisting of ascorbate and the molecule of organic cosolvent is formed under the conditions of the performed experiments. Ingold and co-workers61 have studied reactions of phenols with free radicals in various solvents (not in water or mixed solvents containing water). The very important result there is the elucidation of the role of hydrogen bonding of substrate (a phenol) to a hydrogen bond acceptor (HBA) solvent molecule (in pure HBA solvent) in the reactions. This HBA-HBD interaction leads to the formation of hydrogenbonded complexes and a diminished effective concentration of a phenol substrate. Since the hydrogen-bonded complex is not involved in the reaction of a phenol with a radical, the corresponding kinetic solvent effects result. However, it could be reasonable to expect a very different situation in waterdioxane (mole fraction of water in 1:1 water-dioxane is 0.83) because not only ascorbate but also water should provide a hydrogen bonding (HBD) competition to dioxane and to ascorbate (HBA and HBD) too. A crude calculation, taking into account these interactions (see Supporting Information) gave about 1% or less of the relevant dioxane · · · HAsc- complex (with regard to HAsc-tot.). Collectively, it seems that a potential change of the concentration of “free” ascorbate due the formation of dioxane · · · HAsc- hydrogen-bonded complex can be considered as kinetically insignificant under the conditions of our experiments. It seems that the experimental evidence could be in line with the above consideration. The obtained UV spectroscopic evidence does not suggest a formation of the hydrogen-bonded complex in 1:1 v/v water dioxane. The observed 2 ( 1 nm shift of the 266-nm peak of ascorbate in water on going to the 1:1 water-dioxane does not resemble a solvatochromic shift due

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to ascorbate-dioxane hydrogen-bonded complex and probably comes from medium effects. Also, there is no change in the molar absorption coefficient at λmax of ascorbate. Worth noting in this regard is that we have observed a small 1 ( 1 nm shift of the 266 nm ascorbate peak on going from water to D2O. Taken together with the above consideration, one should not expect the kinetic solvent effects under the reaction conditions employed in this reaction system. Finally, the obtained evidence shows no saturation kinetics over more than 4 orders of magnitude in ascorbate concentration in the reaction. An observation of saturation kinetics could suggest the formation of a hydrogen-bonded complex intermediate in the reaction which might be relevant to the interpretation of the observed KIEs.14,41,42 C. Activation Parameters and Isotope Effects. The observed enthalpy of activation in the reaction decreases from the value of 31 kJ/mol for the reaction in water to 23.9 kJ/mol in the case of the water-dioxane 1:1 v/v reaction (Table 2), while the corresponding figure for the entropy of activation decreases from -134 to -151 J K-1 mol-1. The observed decrease of the activation enthalpy on going from water to water-dioxane likely reflects a change in the ground-state enthalpy of solvation of ascorbate. The ion-dipole interactions between ascorbate anion and water dipoles are much stronger than other interactions with the anion and also much stronger than interactions between uncharged species (which is relevant to the solvation of TEMPO reactant). The (effective) ion-dipole interactions should be weaker in the water-dioxane solvent than in water. Hence, less enthalpy could be required for a (partial) desolvation of the reactants on going from the ground state to the activated complex. Okazaki and Kuwata37 reported a similar enthalpy difference in the reaction of di-tert-butylnitroxide (DTBN) with ascorbate on going from water to the water-methanol (1:1) solvent (∆∆rH‡ ) 5.4 kJ/mol) and the water-acetone (2:1) solvent (∆∆rH‡ ) 8.5 kJ/mol). The great negative entropy of activation observed in the reaction (see Table 2) is expected to principally reflect the loss of translational entropy when two freely diffusing reactants come together to achieve the transition state as the corresponding figure for the reaction in water (-134 J K-1 mol-1) is close to the anticipated value55b of ca. -126 J K-1 mol-1. The origin of the change in the observed entropy of activation on going from water to the water-dioxane solvent system, where the activation entropy is ca. 17 entropy units more negative in the mixed solvent than in water (cf. Table 2), seems to be perhaps less obvious. It could indicate that a difference in the entropy changes during the partial desolvation of reactants in forming the transition state, principally the desolvation of ascorbate anion reactant, on going from water to the waterdioxane solvent, may lead to the difference in the entropies of activation. The ground-state entropy of solvation will reflect also a degree of ordering of solvent around the reactant; the main contribution would come from ion-dipole interactions with ascorbate anion. More ordering of the solvent (water) molecules around the ascorbate anion due to the solvation should be expected in water than in the mixed solvent, i.e., the hydrated anion probably involves more ordering of solvent molecules as compared to the solvated ascorbate anion in the mixed solvent. These differences in the ordering of solvent could be less important in the transition state for the reaction in water and in the mixed solvent, as the charge could be more dispersed in transition state for the reaction as compared with the anion in reactant state. Hence, a partial desolvation of ascorbate anion in water on going from the ground state to the activated complex could generate more entropy than the analogous process of

Sajenko et al. partial desolvation in the mixed solvent which in turn is reflected in the less negative entropy of activation in water system. Again, Okazaki and Kuwata37 reported a similar entropy differences in the reaction of DTBN with ascorbate on going from water to the water-methanol (1:1) solvent (∆∆rS‡ ) 17 J/Kmol) and the water-acetone (2:1) solvent (∆∆rS‡ ) 27 J/Kmol). The interactions between TEMPO and water can be less important in this regard since the attractive forces between uncharged species generally are expected to be much weaker than ion-dipole interactions; therefore, only a relative small differences in the ground state enthalpies and entropies of solvation of TEMPO can be expected on going from water to the mixed solvents in the case. Finally, the absence of the (abovementioned) saturation kinetics do not suggest a formation of hydrogen-bonded intermediate consisting of TEMPO and ascorbate, and following the results of Ingold et al.61 other HB complexes with TEMPO would be generally absent or insignificant in the reaction. It has been noted above that the observed reaction rate constant increases by a factor of only 2.5 on going from water to the water-dioxane 1:1 v/v solvent, although this change of solvent involves a relatively great change in the solvent polarity in the reaction. This finding could be somewhat unexpected as the rates of PCET reactions involving a charge transfer are commonly dependent on the medium polarity (through the reorganization parameter λ).13,14,28 However, the origin of this apparent paradox probably should be traced in the observed decrease of the activation entropy by 17 J K-1 mol-1 in this PCET reaction on going from water to the water-dioxane solvent, as the corresponding observed decrease of the enthalpy of activation of 7.1 kJ/mol is almost entirely compensated by the decrease in the entropy of activation. Without this compensating entropy contribution and taking into account the observed decrease of 7.1 kJ/mol in activation enthalpy, the rate constant would increase 18-fold on going from water to the mixed solvent. This could be an expected rate increase with the decrease of solvent polarity in a reaction involving a charge transfer. KIEs collected in Table 1 revealed an approximately linear (r ) 0.97) increase of KIE (shown also in Figure 4) with the increase in mole fraction of dioxane in the reaction. The increase ranges from the value of 24.2 in water to the value of 31.1 in 1:1 v/v water-dioxane, at room temperature. The observation of an increase in KIE on decreasing the solvent polarity in a PCET reaction could be in line with the theoretical predictions based on a model PCET system.34 According to theoretical predictions based on this model reaction, the KIE of a PCET reaction will increase as the probability of the coupled electron and proton transfer increases and as the localization of and distance between the reactant and product proton vibrational wave function increase. The probability of the coupled electron and proton transfer mechanism increases, among others, if the free-energy barrier for electron transfer increases relative to that of coupled electron and proton transfer as a consequence of decreasing the solvent polarity and altering the reorganization energies.34 Furthermore, all the KIEs determined in this reaction are large and obviously beyond the maximum semiclassical expected values calculated to be kH/kD ≈ 10.3 for a hydrogen transfer from oxygen54,55a or even kH/kD ≈ 13 with allowance for bending vibrations.43,44 Such large isotope effects could signal a hydrogen tunneling in the reaction, providing that they are perhaps not related to some other coupled process.50 Our evidence clearly shows that these isotope effects should be ascribed to the

Reaction of Ascorbate with TEMPO examined reaction exclusively. The isotopic differences in the enthalpies of activation ∆∆rH‡ between D2O and H2O are 9.0 kJ/mol in water and 8.2 kJ/mol in 1:1 v/v water-dioxane, which is greater than the semiclassical value of 5.1 kJ/mol for the difference between zero-point energies E0H - E0D for dissociation of O-H bond indicating a possible hydrogen tunnelling.43,50 Kreevoy and Kim44 also suggested that values of Ea(D) - Ea(H) greater than 5 kJ/mol will usually signal tunnelling for a hydrogen transfer between massive polyatomic donors and acceptors. The isotopic ratio of the Arrhenius prefactors AH/AD ) 1.2 (0.2) in the reaction in water-dioxane can be fitted within the semiclassical limits43,51-53 of 0.7-1.4 for the ratio of isotopic Arrhenius AH/AD prefactors in a hydrogen transfer process. The situation in water seems to be less clear. The corresponding isotopic ratio of the Arrhenius prefactors AH/AD ) 0.6 (0.2) is relatively close to the figure of 0.7, which has been considered as the corresponding semiclasical limit43,51-53 when the ratio AH/AD < 1. Both the ratios of isotopic Arrhenius AH/AD prefactors may be not indicative of a hydrogen tunnelling in the reaction which seemingly contradicts the above evidence (the very large isotope effects and the isotopic differences in the enthalpies of activation ∆∆rH‡ between D2O and H2O) in support of the tunnelling. However, this apparent contradiction can be resolved taking into regard two other lines of evidence. First, Stern and Weston52 and Bell43 have pointed out that in certain circumstances the degree of tunnelling can be large, although AH/AD is close to their semiclassical values, i.e., the observations of values close to the semiclassical ones does not exclude a large degree of tunnelling provided that the observed isotope effect is large. Second, it has been well-known for years that the transfer of anions from H2O to D2O involves an entropy change64 of the order 2-8 J K-1 mol-1. Combining the Arrhenius and Eyring equation for the rate constant can give the expression

AH/AD ) exp(∆rS‡H-∆rS‡D)/R On adding the entropy ∆transferS°D,H for the transfer of ascorbate anion from H2O to D2O to the equation we arrive to the expression

(AH/AD)obs ) exp(∆rS‡H-∆rS‡D + ∆transferSOD,H)/R Obviously, this would change the observed AH/AD (in which ∆transferS°D,H is buried) significantly and could lead to the AH/AD values that are outside the semiclassical limits. Collectively, the large isotope effects and the isotope differences in the enthalpies of activation in this reaction cannot be explained without allowance for a hydrogen tunnelling, and taking into regard the additional evidence described above, a hydrogen tunneling likely occurs in the process. Recently, Wu, Borden, and Mayer67 reported the observation of hydrogen tunnelling in a HAT reactions that involve the pseudo self-exchange reactions of nitroxyl radicals and hydroxylamines, including the reactions of the TEMPO radical. The reaction reported here presents one more example of tunnelling in reactions involving TEMPO. To the best of our knowledge, there is no report about tunnelling in a reaction involving ascorbate, and this report can be the first. Conclusions The oxidation of ascorbate with the TEMPO radical in water and water-dioxane mixed solvents has been demonstrated to

J. Phys. Chem. A, Vol. 114, No. 10, 2010 3429 be a concerted PCET process, exibiting the very large KIEs in all solvent mixtures employed. The reaction likely involves hydrogen tunnelling at room temperature, both in water and in the mixed solvents. The magnitude of the KIE kH/kD in the reaction increases approximately linearly with the decrease of the solvent polarity. This increase of the KIE is moderate. This could be of relevance to the question of influence of solvent polarity on the change of the magnitude of the KIE in PCET reactions. The reaction reported here presents the first report about hydrogen tunnelling in a reaction involving ascorbate. This could perhaps suggest that ascorbate used hydrogen tunneling in reactions in biological systems too. Acknowledgment. We thank the Croatian Ministry of Science and Education for support (Contract No. 0063082-0354). Supporting Information Available: The detailed experimental kinetic and thermodynamic data related to the investigated reaction system. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Tommos, C.; Babcock, G. T. Acc. Chem. Res. 1998, 31, 18. (2) Tommos, C.; Babcock, G. T. Biochim. Biophys. Acta 2000, 1458, 199. (3) McEvoy, J. P.; Brudvig, G. W. Chem. ReV. 2006, 106, 4455. (4) Meyer, T. J.; Huynh, M. H. H.; Thorp, H. H. Angew. Chem., Int. Ed. 2007, 46, 5284. (5) Liu, F.; Concepcion, J. J.; Jurss, J. W.; Cardolaccia, T.; Templeton, J. T.; Meyer, T. J. Inorg. Chem. 2008, 47, 1727. (6) Proshlyakov, D. A.; Pressler, M. A.; Babcock, G. T. Proc. Natl. Acad. Sci., U. S. A. 1998, 95, 8020. (7) Stubbe, J.; Nocera, D. G.; Yee, C. S.; Chang, M. C. Y. Chem. ReV. 2003, 103, 2167. (8) Reece, S. Y.; Nocera, D. G. Annu. ReV. Biochem. 2009, 78, 673. (9) Belevich, I.; Verkhovsky, M. I.; Wikstro¨m, M. Nature 2006, 440, 829. (10) Itoh, S.; Fukuzumi, S. Acc. Chem. Res. 2007, 40, 592. (11) Bollinger, J. M., Jr.; Jiang, W.; Green, M. T.; Krebs, C. Curr. Opin. Struct. Biol 2008, 18, 650. (12) Becker, C. F.; Watmough, N. J.; Elliott, S. J. Biochemistry 2009, 48, 87. (13) Mayer, J. M. Annu. ReV. Phys. Chem. 2004, 55, 363. (14) Huynh, M. H. H.; Meyer, T. J. Chem. ReV. 2007, 107, 5004. (15) Rosenthal, J.; Nocera, D. G. Acc. Chem. Res. 2007, 40, 543. (16) Markle, T. F.; Rhile, I. J.; DiPasquale, A. G.; Mayer, J. M. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 8185. (17) Markle, T. F.; Mayer, J. M. Angew. Chem., Int. Ed. 2008, 47, 738. (18) Herres-Pawlis, S.; Verma, P.; Haase, R.; Kang, P.; Lyons, C. T.; Wasinger, E. C.; Florke, U.; Henkel, G.; Stack, T. D. P. J. Am. Chem. Soc. 2009, 131, 1154. (19) Hammarstro¨m, L.; Styring, S. Phil. Trans. R. Soc. B 2008, 363, 1283. (20) Costentin, C. Chem. ReV. 2008, 108, 2145. (21) Costentin, C.; Robert, M.; Save´ant, J. M.; Teillout, A. L. ChemPhysChem 2009, 10, 191. (22) Abhayawardhana, A. D.; Sutherland, T. C. J. Phys. Chem. C 2009, 113, 4915. (23) Li, B.; Zhao, J.; Onda, K.; Jordan, K. D.; Yang, J.; Petek, H. Science 2006, 311, 1436. (24) Parthasarathy, M.; Gopinath, C. S.; Pillai, V. K. Chem. Mater. 2006, 18, 5244. (25) Hammes-Schiffer, S.; Soudackov, A. V. J. Phys. Chem. B 2008, 112, 14108. (26) Cukier, R. I.; Nocera, D. G. Annu. ReV. Phys. Chem. 1998, 49, 337. (27) Cukier, R. I. J. Phys. Chem. B 2002, 106, 1746. (28) Hammes-Schiffer, S. Acc. Chem. Res. 2001, 34, 273. (29) Hammes-Schiffer, S. Acc. Chem. Res. 2006, 39, 93. (30) Hammes-Schiffer, S.; Hatcher, E.; Ishikita, H.; Skone, S.; Soudackov, J. H. Coord. Chem. ReV. 2008, 252, 384. (31) Presse´, S.; Silbey, R. J. Chem. Phys. 2006, 124, 164504. (32) Marcus, R. A. J. Phys. Chem. B 2007, 111, 6643. (33) Tischcenko, O.; Truhlar, D. G.; Ceulemans, A.; Nguyen, M. T. J. Am. Chem. Soc. 2008, 130, 7000. (34) Decornez, H.; Hammes-Schiffer, S. J. Phys. Chem. A 2000, 104, 9370.

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(35) We have started this study at the time we have reported some preliminary results on the solvent dependence of KIEs in the interaction of ascorbate with nitrosobenzenes.36 An ESR study on the reactivity of nitroxide radicals with ascorbic acid was reported many years ago,37 and during this investigation a study of interaction of 5,6-isopropilidene ascorbate with TEMPO in pure acetonitrile38 appeared. (36) Vuina, D.; Pilepic´, V.; Ljubas, D.; Sankovic´, K.; Sajenko, I.; Ursˇic´, S. Tetrahedron Lett. 2007, 48, 3633. (37) Okazaki, M.; Kuwata, K. J. Phys. Chem. 1985, 89, 4437, and references therein. (38) Warren, J. J.; Mayer, J. M. J. Am. Chem. Soc. 2008, 130, 7546. (39) Ahn-Ercan, G.; Krienke, H.; Schmeer, G. J. Mol. Liq. 2006, 129, 75. (40) Bako´, I.; Pa´linka´s, G.; Dore, J. C.; Fisher, H. E. Chem. Phys. Lett. 1999, 303, 315. (41) Huynh, M. H. H.; Meyer, T. J. Angew. Chem., Int. Ed. 2002, 41, 1395. (42) Huynh, M. H. H.; Meyer, T. J. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 13138. (43) Bell, R. P. The Tunnel Effect in Chemistry; Chapman & Hall: London, 1980; pp 77-101. (44) Kim, Y.; Kreevoy, M. M. J. Am. Chem. Soc. 1992, 114, 7116. (45) Details are given in Supporting Information. (46) Israeli, A.; Patt, M.; Samuni, A.; Kohen, R.; Goldstein, S. Free Radical Biol. Med. 2005, 38, 317. (47) Certainly, a fast protonation of TEMPOH takes place subsequently, after the formation of TEMPOH in reaction 1, i.e., this protonation is not a part of the reaction coordinate for the concerted proton and electron transfer in the reaction. It can be seen, among others, that if TEMPOH(H)+ would be a “product” in reaction 1, then H+ must be a “reactant” and the rate of reaction should not depend on the reciprocal of H+ concentration. Furthermore, if this would be the case, the observed rate constant should be independent on H+; in contrast, the linear dependence on the 1/H+ is what indeed has been observed. The only consequence of that subsequent protonation of TEMPOH is that the concentration of ascorbate in our buffered reaction mixture is more close to a “constant” during the kinetics since TEMPOH consumed a proton liberated in the dissociation of ascorbic acid (ascorbic acid is in excess, see Experimental section) in the buffer system. (48) Bielski, B. H. J.; Allen, O.; Schwarz, H. A. J. Am. Chem. Soc. 1981, 103, 3516.

Sajenko et al. (49) The same kinetic pattern should be obtained if there would be a hypothetical fast subsequent reaction of ascorbate radical anion with TEMPO radical and a proton. The reaction of TEMPO with ascorbate dianion (ET) should be unimportant under the conditions employed in these experiments (equilibrium concentrations of the ascorbate dianion less than of 10-10 mol dm-3), taking also into account the E°(TEMPO•/TEMPO-)45 as well as the corresponding thermochemical considerations. (50) Romesberg, F. E.; Schowen, R. L. AdV. Phys. Org. Chem. 2004, 39, 27. (51) Kwart, H. Acc. Chem. Res. 1982, 15, 401. (52) Stern, M. J.; Weston, R. E. J. J. Chem. Phys. 1974, 60, 2808. (53) Kohen, A.; Klinman, J. P. Acc. Chem. Res. 1998, 31, 397. (54) More O’Ferrall, R. A. Proton-Transfer Reactions; Caldin, E., Gold, V., Eds.; Chapman and Hall: London, 1975, Chapter 8. (55) (a) Espenson, J. H. Chemical Kinetics and Reaction Mechanisms, McGraw-Hill, New York 1995, p 217. (b) p 158. (56) Here, we maintain the term PCET for this reaction in the most usual meaning that the reaction involves a concerted PCET13,14,25 as we do not have the evidence whether the proton is transferred together with one of its bonding electrons in which case the reaction would be classified to be the simple hydrogen atom transfer (HAT) or the proton and electron are transferred between different sets of orbitals (PCET reaction).65,66 (57) Njus, D.; Kelley, P. M. Biochim. Biophys. Acta 1993, 1144, 235. (58) Malatesta, V.; Ingold, K. U. J. Am. Chem. Soc. 1973, 95, 6404. (59) Kato, Y.; Shimizu, Y.; Yijing, L.; Unoura, K.; Utsumi, H.; Ogata, T. Electrochim. Acta 1995, 40, 2799. (60) Laroff, G. P.; Fessenden, R. W.; Schuler, R. H. J. Am. Chem. Soc. 1972, 94, 9062. (61) Litwinienko, G.; Ingold, K. U. Acc. Chem. Res. 2007, 40, 222. (62) Mulder, P.; Korth, H.-G.; Pratt, D. A.; DiLabio, G.; Valgimigli, L.; Pedulli, G. F.; Ingold, K. U. J. Phys. Chem. A 2005, 109, 2647. (63) Nakayama, H.; Shinoda, K. J. Chem. Thermodyn. 1971, 3, 401. (64) Conway B. E. Ionic Hydration in Chemistry and Biophysics; Elsevier Scientific Publishing Co.: Amsterdam, 1981; Chapter 26. (65) Mayer, J. M.; Hrovat, D. A.; Thomas, J. L.; Borden, W. T. J. Am. Chem. Soc. 2002, 124, 11142. (66) DiLabio, G.; Ingold, K. U. J. Am. Chem. Soc. 2005, 127, 6693. (67) Wu, A.; Mader, E. A.; Datta, A.; Hrovat, D. A.; Borden, W. T.; Mayer, J. M. J. Am. Chem. Soc. 2009, 131, 11985.

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