Surprisingly Long-Lived Ascorbyl Radicals in Acetonitrile: Concerted Proton-Electron Transfer Reactions and Thermochemistry

Abstract

Proton-coupled electron transfer (PCET) reactions and thermochemistry of 5,6-isopropylidene ascorbate (iAscH⁻) have been examined in acetonitrile solvent.iAscH⁻ is oxidized by 2,4,6-^tBu₃C₆H₂O^• and by excess TEMPO^• to give the corresponding 5,6-isopropylidene ascorbyl radical anion (iAsc^•−), which persists for hours at 298 K in dry MeCN solution. The stability of iAsc^•− is surprising in light of the transience of the ascorbyl radical in aqueous solutions, and is due to the lack of the protons needed for radical disproportionation. A concerted proton-electron transfer (CPET) mechanism is indicated for the reactions of iAscH⁻. Redox potential, pK_a and equilibrium measurements define the thermochemical landscape for 5,6-isopropylidene ascorbic acid and its derivatives in MeCN. These measurements give an O–H bond dissociation free energy (BDFE) for iAscH⁻of 65.4 ± 1.5 kcal mol⁻¹ in MeCN. Similar studies on underivatized ascorbate indicate a BDFE of 67.8 ± 1.2 kcal mol⁻¹. These values are much lower than the aqueous BDFE for ascorbate of 74.0 ± 1.5 kcal mol⁻¹ derived from reported data.

Ascorbic acid (vitamin C) is involved in a wide range of biochemical processes.¹ It is primarily a redox cofactor, being oxidized to the ascorbyl radical and then to dehydroascorbate. Studies of the ascorbyl radical in aqueous media are complicated by its transient nature.^1c Here we report that in ‘dry’ acetonitrile, the soluble 5,6-isopropylidene ascorbyl radical (iAsc^{• −}, eq 1) is surprisingly long-lived, as is the parent ascorbyl radical.² These long lifetimes have facilitated detailed reactivity and thermo-chemical studies. While ascorbate reactivity is often described as electron transfer,¹ at pH 7 one-electron oxidation to the ascorbyl radical occurs with concomitant loss of a proton (a proton-coupled electron transfer (PCET) process).³ Ascorbate often reacts by concerted transfer of e⁻ and H⁺, including reactions with cytochrome b₅₆₁ nitrosobenzenes, quinones and the reduction of tocopheroxyl radicals to tocopherols (vitamin E).^4,5 These were historically called hydrogen atom transfer reactions, but they are probably better termed PCET or (our preference) concerted proton-electron transfer (CPET), because the ascorbate proton is in the molecular plane while the electron is removed from a π-orbital.⁶ Described here are a number of CPET reactions of ascorbates in MeCN, which reveal an unusual solvent dependence of the O–H bond dissociation free energy.

5,6-Isopropylidene semidehydroascorbyl radical (iAsc^•−) is conveniently generated by reaction of 5,6-isopropylidene ascorbate (iAscH⁻) with the 2,4,6-tri-tert-butyl phenoxyl radical (^tBu₃PhO^•)⁷ in MeCN (eq 1). Rapid and quantitative reduction of ^tBu₃PhO^• to

(1)

^tBu₃PhOH is observed by UV-Vis and¹H NMR spectroscopies. 5,6-Isopropylidene ascorbic acid (iAscH₂) is an organic soluble ascorbate analog,⁸ available from Sigma-Aldrich. iAscH⁻ can be isolated as ⁿBu₄N⁺iAscH⁻, a low melting solid, or generated in situ by addition of one equivalent of the strong base DBU to iAscH₂ in MeCN (DBU = l,8-diazabicyclo[5.4.0]undec-7-ene).⁹

The ascorbyl radical product of reaction 1 (iAsc^•−) has been characterized by its UV-visible, EPR, and ESI-mass spectra. By stopped flow spectrophotometry, the disappearance of ^tBu₃PhO^• (λ_max = 382, 401, 634 nm) is accompanied by growth of a band at 377 nm (Figure 1a). The λ_max and ε₃₇₇ (3900 ± 200 M⁻¹ cm⁻¹) of this species are similar to those reported for the ascorbyl radical anion in water (360 nm, 3300 M⁻¹ cm⁻¹).^1b,10 ESI-mass spectra of iAscH⁻ + ^tBu₃PhO^• reaction mixtures⁹ show a negative ion at m/z = 214, as well as major peaks at m/z = 213 and 215 due to iAscH⁻ and dehydroascorbate (iAsc)¹¹ (from loss of H⁺ from the 4-position¹²). The m/z = 214 ion is the ascorbyl radical anion.

Figure 1.

(a) UV-Visible spectrum and (b) X-band EPR spectrum of iAsc’“ in CH₃CN at 298K, generated via reaction (1)

The room temperature X-band EPR spectrum of iAsc^•− in MeCN (Fig. 1b), prepared by mixing a slight excess of iAscH⁻ with ^tBu₃PhO^•, is centered at g_MeCN = 2.0059 with hyperfine couplings of 1.91 [H4], 0.05 [H5], and 0.12, 0.16 G [H6].⁹ These are close to the values reported for iAsc^•− in DMSO and in water (e.g., g_dmso =2.00563, g_H2O=2.00520).¹³

iAsc^•− generated via reaction 1, at ca. 0.1 mM concentrations, decays over about two hours at room temperature. This stability is surprising given that the underivatized radical anion disproportionates rapidly in aqueous solutions at pH 7 (k ~ 3 × 10⁶ M⁻¹ s⁻¹; t_1/2 ~3 ms at 0.1 mM).¹⁴ The decay of iAsc^•− in MeCN forms iAscH⁻ and iAsc by¹H NMR and ESI-MS (eq 2), analogous to the

$$\mathbf{2}{\mathit{i}\mathbf{Asc}}^{\cdot -}+{\text{H}}^{+}\rightleftharpoons \mathit{i}\mathbf{Asc}{\mathbf{H}}^{-}+\mathit{i}\mathbf{Asc}$$ (2)

decay of the ascorbyl radical in H₂O.¹⁴ The decay of iAsc^•− in MeCN appears to be highly dependent on the proton activity: drying the MeCN with activated alumina extends the lifetime, and addition of 56 mM H₂O causes complete decay within 30 min. The addition of 0.1 mM CF₃CO₂H results in immediate loss of the absorbance at 377 nm. This indicates that the stability of iAsc^•− arises from effectively “starving” the reaction of protons, since, unlike the decay of most organic radicals, disproportionation stoichiometrically requires H⁺ (eq 2). Analogous preparations of iAsc^•− in “dry” CH₂Cl₂ and DMSO give similarly stable solutions.

Stopped-flow kinetic studies of reaction 1 give k₁, = (3.4±0.5) × 10⁶ M⁻¹ s⁻¹ and k_H/k_D 3.2 ± 0.6 at 289 K.¹⁵ The analogous reaction of 2,6-di-tert-butyl-4-methoxy-phenoxyl forms iAsc^•− and the corresponding phenol, about six times slower: k = (5.3 ± 0.5) × 10⁵ M⁻¹ s⁻¹; k_H/k_d = 3.5 ± 0.6 The reaction of iAscH⁻ and the nitroxyl radical 2,2,6,6-tetramethyl-piperidin-1-oxyl (TEMPO) in MeCN results in production of iAscH^•− and TEMPOH (eq 3) as observed by UV-Vis and ¹H NMR spectroscopies. Aqueous ascorbic acid has long been known to reduce nitroxyl radicals.¹⁶ Kinetic measurements in MeCN using excess TEMPO yielded k₃ = 1720 ± 150 M⁻¹ s⁻¹ at 298 K, and ΔH₃^‡ = 3.4±0.5 kcal mol⁻¹,

(3)

ΔS₃^‡ = −32 ± 2 cal K¹ mol⁻¹. Spectrophotometric titration of iAscH⁻ with limited TEMPO gives the equilibrium constant for eq 3, k₃ = 1.2 ± 0.2 (ΔG°₃ = −0.15 ± 0.10 kcal mol⁻¹).⁹

The stability of iAsc^•− allows the ready determination of the thermochemistry of electron, proton, and hydrogen atom transfers in this system (Scheme 1; all data in MeCN).⁹ Titrations of iAscH₂ with N-methyl morpholine (pk_a = 15.59 in MeCN),¹⁷ and iAscH⁻ with 1,5,7-triazabicyclo[4.4.0]dec-5-ene (pk_a = 26.03),¹⁸ give pk_a(iAscH₂) = 18.3 ± 0.3 and pk_a(iAscH⁻) = 28.8 ± 0.5. Cyclic voltammetry of iAscH⁻ and iAsc²⁻¹⁹ show quasi-reversible waves (ΔE_P ~ 0.2 V) at −0.410 ± 0.015 and −1.30 ± 0.02 V vs. Cp₂Fe^+/0. The pk_a(AscH^•) is calculated from these E_1/2 and pk_a values, using the square in Scheme 1 as a thermochemical cycle: 1.37[pk_a(iAscH⁻)−pk_a(iAscH^•)] − 23.1[E(iAscH⁻)− E(iAsc²⁻)] = 0.

Scheme 1.

Thermochemistry of the AscH₂ system in MeCN

The pk_a and E_1/2 values indicate, using eq 4, that the O-H bond dissociation free energies (BDFEs) for iAscH₂ and iAscH⁻ in MeCN are 70.5 ± 2.0 and 64.4 ± 2.0 kcal mol⁻¹, respectively (C_G = 54.9 kcal mol⁻¹ in MeCN versus Fc^0/+).²⁰ The equilibrium in eq 3

$$\text{BDFE}[\text{X}-\text{H}]=23.06E°+1.37\text{p}{K}_{\text{a}}+C_{\text{G}}(\text{in}\text{kcal}{\text{mol}}^{-1})$$ (4)

(K₃ = 1.2) and the known²¹ BDFE(TEMPO-H) of 66.5 ± 1 kcal mol⁻¹ give an independent measure of BDFE(iAscH⁻), 66.4 ±1.0 kcal mol⁻¹. This value is in good agreement with that obtained from E_1/2 and Pk_a data, and indicates a consensus BDFE for iAsc⁻ of 65.4 ±1.5 kcal mol⁻¹ in MeCN (Scheme 1). This corresponds to a bond dissociation enthalpy of roughly 70 ± 1 kcal mol^−1.20b

With underivatized ascorbate, the tetrabutylammonium salt is sufficiently soluble in MeCN to examine its similar reactivity. Reaction with TEMPO forms the parent ascorbyl radical, based on its characteristic optical spectrum (λ_max = 377 nm), which then decays slowly over ca. 1 hour. This reaction has k_eq = 0.11 ± 0.07 which implies BDFE(ascorbate) = 67.8 ± 1.2 kcal mol⁻¹ in MeCN.⁹ Aqueous thermochemical data for ascorbate²² yield an aqueous O–H BDFE of 74.0 ± 1.5 kcal mol⁻¹ (eq 4 with C_G = 57.5 kcal mol⁻¹ in H₂O versus NHE²⁰).⁹ The BDFE is 6.2 ± 1.8 kcal mol⁻¹ higher in H₂O than in MeCN. Preliminary studies of the aqueous ascorbate + TEMPO reaction (pH 7 phosphate buffer) seem consistent with these conclusions. The difference in BDFE of 6.2 ± 1.8 kcal mol⁻¹ is a very large given that homolytic bond strengths are typically not very sensitive to the solvent.²³

The mechanism of the reaction of iAscH⁻ and TEMPO in MeCN is indicated to be concerted H_+/e⁻ transfer following the analysis used by Njus to implicate this mechanism for ascorbate + tocopheroxyl radicals.^4b Alternative pathways of (i) initial proton transfer (PT) followed by electron transfer (ET) or (ii) initial ET followed by PT are not possible because of the high free energies of the initial steps: ΔG°_PT ≅ 40 kcal mol⁻¹ and ΔG°_ET ≅ 35 kcal mol⁻¹ - from the data in Scheme 1 and the known thermochemical values for TEMPO(H).²⁴ These ground state free energy changes are the minimum activation barriers for these pathways (ΔG₃^‡ ≥ ΔG°) and both are much larger than the observed Eyring barrier, ΔG₃^‡ = 13.0 ± 0.1 kcal mol⁻¹. The same treatment indicates that iAscH⁻ + ^tBu₃PhO^• (eq 1) also likely proceeds via CPET.⁹

In conclusion, the 5,6-isopropylidene ascorbyl radical (iAsc^•−) is readily generated from the corresponding ascorbate by phenoxyl and nitroxyl radicals. Remarkably, this radical is stable for hours in ‘dry’ acetonitrile, likely because protons are required for radical disproportionation (eq 2). Mechanistic and thermo­chemical data indicate that these reactions proceed by concerted H⁺/e⁻ transfer, rather than a stepwise path involving separate electron and proton transfers. The O–H bond dissociation free energy (BDFE) of iAscH⁻ in MeCN is determined to be 65.4 + 1.5 kcal mol⁻¹ from two different approaches, and the BDFE of underivatized ascorbate in MeCN is 67.8 +1.2 kcal mol⁻¹. These values are significantly lower than the BDFE of ascorbate, 74.0 + 1.5 kcal mol⁻¹, derived from aqueous thermochemical measurements. Future studies will explore the origin of this large variation in ascorbate O–H BDFEs, which could reflect an unusual sensitivity to local solvation effects and could be important for enzymatic reactions of ascorbate.

Supplementary Material

SI1

Supporting Information Available: Experimental details for kinetic and thermochemical studies. This material is available free of charge via the Internet at http://pubs.acs.org.