Aluminium ion-promoted radical-scavenging reaction of methylated hydroquinone derivatives

Abstract

The effect of the aluminium ion (Al³⁺) on the scavenging reaction of a 2,2-diphenyl-1-picrylhydrazyl radical (DPPH˙), as a reactivity model of reactive oxygen species, with hydroquinone (QH₂) and its methylated derivatives (Me_nQH₂, n = 1–4) was investigated using stopped-flow and electrochemical techniques in a hydroalcoholic medium. The second-order rate constants (k) for the DPPH˙-scavenging reaction of the hydroquinones increased with the increasing number of methyl substituents. Upon addition of Al³⁺, the k values significantly increased depending on the concentration of Al³⁺. Such an accelerating effect of Al³⁺ on the DPPH˙-scavenging rates of the hydroquinones results from the remarkable positive shift of the one-electron reduction potential (E_red) of DPPH˙ in the presence of Al³⁺. These results demonstrate that Al³⁺, a strong Lewis acid, can act as a radical-scavenging promoter by stabilising the one-electron reduced species of the radical, although Al³⁺ is reported not only to act as a pro-oxidant but also to strongly interact with biomolecules, showing toxicities.

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Introduction

Metal ions are known to play important roles in controlling biological redox reactions.¹ It is also well known that metal ions accelerate electron-transfer reactions by binding to electron acceptors.² Furthermore, they are reported to form transient complexes with the superoxide anion (O₂˙⁻), which is one of the reactive oxygen species (ROS).^3,4 Among them, a complex of Al³⁺ with O₂˙⁻ contributes to promote the Fenton reaction by reducing Fe³⁺ to Fe²⁺ to produce hydroxyl radicals (˙OH), the strongest ROS.⁵ In addition, Al³⁺ as a strong Lewis acid forms a variety of stable complexes with proteins and biomolecules, such as citrate, glutamate and α-ketoglutarate.⁶ Al³⁺ also interacts with molecules containing phosphate groups, such as AMP, ATP, RNA and DNA.⁷ These complexes disrupt the cell metabolic processes, leading to neurological diseases.⁸

Ubiquinol (UQH₂), the reduced form of coenzyme Q₁₀, has a hydroquinone (QH₂) structure as an active centre, showing an efficient scavenging activity against ROS.⁹ In human beings, ROS are generated as side products during the energy production by mitochondrial electron-transport systems or the elimination of virus and bacteria in macrophages and neutrophils. However, excessive ROS cause many diseases, such as cancer, diabetes, cardiovascular disease, Alzheimer's and Parkinson's disease by oxidising proteins, lipids and DNA.^10,11 Thus, antioxidants are very useful to protect against oxidative stress and may lead to prevention of these diseases. UQH₂ is one of the lipid-soluble antioxidants and efficiently scavenges lipid peroxyl radicals.^12,13 Furthermore, UQH₂ reacts with the α-tocopheroxyl radical to regenerate α-tocopherol inside the biomembrane.^14–16 On the other hand, QH₂ is a major benzene metabolite and is widely applied in human and industrial activities. QH₂ is used as an antioxidant in the rubber and food industries due to its antioxidative activity.¹⁷ QH₂ also has a long history of use in pigmentary disorders and is applied in the commercially available cosmetic skin lightning formulations.^18–20

The radical-scavenging reactions of phenolic antioxidants, such as flavonoids and α-tocopherol, are affected by redox-inactive metal ions, such as Mg²⁺, Al³⁺ and Sc³⁺.^21–23 However, little is known about the effects of metal ions on the radical-scavenging reaction of QH₂ and its derivatives. Particularly, whether Al³⁺ promotes the radical-scavenging reactions of antioxidants or acts as a pro-oxidant is yet to be clarified.

We report herein the effects of Al³⁺ on the scavenging reaction of a 2,2-diphenyl-1-picrylhydrazyl radical (DPPH˙) (Fig. 1) with QH₂ and its methylated derivatives, which are methylhydroquinone (MeQH₂), 2,6-dimethylhydroquinone (Me₂QH₂), trimethylhydroquinone (Me₃QH₂) and tetramethylhydroquinone (Me₄QH₂) (Fig. 2).²⁴ DPPH˙ is frequently used as a reactivity model of peroxyl radicals, which are relatively less reactive ROS compared to hydroxyl and alkoxyl radicals.^25–30 In addition, we investigated the effects of the number of methyl substituents of the hydroquinones on the DPPH˙-scavenging activity. Furthermore, the electron-donating ability of the hydroquinones and the electron-accepting ability of DPPH˙ were also evaluated by cyclic voltammetry (CV) and second-harmonic alternating current voltammetry (SHACV) in the absence and presence of Al³⁺.^31–35 The strong Lewis acidity of Al³⁺ enhanced the electron-accepting ability of DPPH˙, leading to an acceleration of the DPPH˙-scavenging reaction by the hydroquinones.

Fig. 1 DPPH˙ as a model of ROS.

Fig. 2 Structures of UQH₂, QH₂ and its methylated derivatives.

Experimental

Materials

QH₂ and its methylated derivatives were purchased from Tokyo Chemical Industry Co., Ltd, Japan. DPPH˙ was purchased from Wako Pure Chemical industries, Ltd. Tetra-n-butylammonium perchlorate (Bu₄NClO₄), used as a supporting electrolyte for the electrochemical measurements, was purchased from Tokyo Chemical Industry Co., Ltd, Japan. Bu₄NClO₄ was recrystallised from ethanol (EtOH) and dried under vacuum at 313 K. Aluminium perchlorate nonahydrate [Al(ClO₄)₃·9H₂O] was purchased from Wako Pure Chemical Ind. Ltd, Japan. EtOH (spectral grade) used as a solvent was purchased from Nacalai Tesque, Inc., Japan.

Spectral and kinetic measurements

To avoid the effect of molecular oxygen (O₂), the reactions were carried out under strictly deaerated conditions. A continuous flow of argon gas was bubbled through an EtOH–H₂O (9 : 1 v/v) solution (3.0 mL) containing DPPH˙ (5.0–8.9 × 10⁻⁵ M) or the hydroquinones (1.1–6.6 × 10⁻³ M) in a reservoir of the spectrophotometer for 7 min. The rates of the DPPH˙-scavenging reactions of the hydroquinones were determined by monitoring the absorbance change at 517 nm due to DPPH˙ using a stopped-flow technique on a UNISOKU RSP-1000-02NM spectrophotometer. The pseudo-first-order rate constants (k_obs) were determined by least-squares curve fit using an Apple MacBook Pro personal computer. The first-order plots of ln(A − A_∞) vs. time (A and A_∞ denoted as the absorbance at the reaction time and the final absorbance, respectively) were linear until three or more half-lives with the correlation coefficient ρ > 0.999. The reaction of hydroquinones with DPPH˙ in the presence of various concentrations of Al(ClO₄)₃ was carried out in the same manner.

Electrochemical measurements

The cyclic voltammetry (CV) and second-harmonic alternating current voltammetry (SHACV)³⁶ measurements were performed on an ALS-630A electrochemical analyser in deaerated EtOH–H₂O (9 : 1 v/v) containing 0.1 M Bu₄NClO₄ as a supporting electrolyte. The glassy carbon-working electrode (BAS) was polished with BAS polishing alumina suspension and rinsed with methanol before use. The counter electrode was a platinum wire. The measured potentials were recorded with respect to an Ag/AgNO₃ (0.01 M) reference electrode. One-electron oxidation potentials (E_ox) of hydroquinones and one-electron reduction potentials (E_red) of DPPH˙ (vs. Ag/AgNO₃) were converted to those vs. SCE by adding 0.29 V.³⁷ All electrochemical measurements were carried out at room temperature under 1 atm Ar.

pH measurements

The pH values of the solutions of hydroquinones in the presence or absence of the metal ion in EtOH–H₂O (9 : 1 v/v) were measured by using a METTLER TOLEDO SevenEasy pH meter.

Results and discussion

Kinetics of the DPPH˙-scavenging reaction of the hydroquinones in the presence or absence of Al³⁺

The rate of the DPPH˙-scavenging reaction of the hydroquinones was measured by monitoring the absorbance change at 517 nm due to DPPH˙ using a stopped-flow technique in deaerated EtOH–H₂O (9 : 1 v/v) at 298 K. The decay of the absorbance at 517 nm obeyed pseudo-first-order kinetics, when the concentration of the hydroquinones ([Me_nQH₂]) (n = 0–4) was maintained at a more than 10-fold excess of the DPPH˙ concentration. The pseudo-first-order rate constants (k_obs) increase with increasing [Me_nQH₂], exhibiting the first order dependence on [Me_nQH₂] (Fig. 3). From the slope of the linear plots of k_obsvs. [Me_nQH₂], the second-order rate constants (k) were determined for the DPPH˙-scavenging reaction (Table 1). The k values of the hydroquinones increased with the increasing number of methyl substituents in the absence of metal ions. Upon addition of Al(ClO₄)₃ (0.05–0.50 M) to the hydroquinone–DPPH˙ system, the k values for all the hydroquinones significantly increased with the increasing Al(ClO₄)₃ concentration (Fig. 4, Table 1).

Fig. 3 Plots of k_obsvs. [Me_nQH₂] (n = 0–4) for the reaction of QH₂ (closed circles), MeQH₂ (open circles), Me₂QH₂ (closed squares), Me₃QH₂ (open squares) and Me₄QH₄ (closed triangles) with DPPH˙ in deaerated EtOH–H₂O (9 : 1 v/v) at 298 K.

Fig. 4 Plots of k vs. [Al(ClO₄)₃] for the reaction of QH₂ (closed circles), MeQH₂ (open circles), Me₂QH₂ (closed squares), Me₃QH₂ (open squares) and Me₄QH₄ (closed triangles) with DPPH˙ in deaerated EtOH–H₂O (9 : 1 v/v) at 298 K.

Table 1 Second-order rate constants (k) for the DPPH˙-scavenging reaction of the hydroquinones in the presence or absence of Al(ClO₄)₃ in deaerated EtOH–H₂O (9 : 1 v/v) at 298 K

	k/M^−1 s^−1
[Al(ClO4)3]/M	QH2	MeQH2	Me2QH2	Me3QH2	Me4QH2
0	1.3 × 10^2	2.0 × 10^2	3.4 × 10^2	1.1 × 10^3	1.4 × 10^3
0.05	3.5 × 10^2	7.0 × 10^2	1.0 × 10^3	2.7 × 10^3	3.0 × 10^3
0.1	3.9 × 10^2	9.3 × 10^2	1.7 × 10^3	3.4 × 10^3	3.5 × 10^3
0.2	3.4 × 10^2	1.1 × 10^3	2.8 × 10^3	5.3 × 10^3	5.4 × 10^3
0.3	5.8 × 10^2	1.3 × 10^3	3.2 × 10^3	6.3 × 10^3	5.8 × 10^3
0.5	5.2 × 10^2	1.1 × 10^3	4.4 × 10^3	7.5 × 10^3	8.2 × 10^3

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Effect of Al³⁺ on the redox behaviour of the hydroquinones and DPPH˙

The one-electron oxidation potentials (E_ox) of the hydroquinones were determined by cyclic voltammetry (CV) and second-harmonic alternating current voltammetry (SHACV)³⁶ measurements in the presence or absence of Al³⁺ in EtOH–H₂O (9 : 1 v/v) to investigate the detailed effects of Al³⁺ on the DPPH˙-scavenging reaction of the hydroquinones. The E_ox values of the hydroquinones were determined from the intersection of SHACV waves (Fig. 5). In the absence of the metal ion, the E_ox values of the hydroquinones were shifted to the negative direction with the increasing number of methyl substituents (Table 2). The electron-donating methyl substituents contribute to an increase in the electron density, leading to an enhancement of the electron-donating ability of the hydroquinones.

Fig. 5 Cyclic voltammogram (CV) (dashed line) and second-harmonic alternating current voltammogram (SHACV) (solid line) of QH₂ (2.4 × 10⁻³ M) recorded at the scan rate of 0.1 V s⁻¹ and 4 mV s⁻¹, respectively, on a glassy carbon working electrode in EtOH–H₂O (9 : 1 v/v) (0.1 M Bu₄NClO₄).

Table 2 The E_ox values of hydroquinones and the E_red values of DPPH˙ in EtOH–H₂O (9 : 1 v/v) in the presence or absence of Al(ClO₄)₃

	E ox (vs. SCE)^a/V					E red (vs. SCE)/V
[Al(ClO4)3]/M	QH2	MeQH2	Me2QH2	Me3QH2	Me4QH2	DPPH˙
a Determined by SHACV at the rate of 4 mV s^−1 on a glassy carbon working electrode (0.1 M Bu4NClO4).
0	+0.39	+0.36	+0.29	+0.23	+0.19	+0.23
0.05	+0.48	+0.42	+0.36	+0.29	+0.24	+0.67^a
0.1	+0.54	+0.47	+0.39	+0.33	+0.28	+0.69^a
0.2	+0.55	+0.48	+0.40	+0.34	+0.28	+0.70^a
0.3	+0.56	+0.50	+0.42	+0.36	+0.30	+0.70^a
0.5	+0.56	+0.50	+0.42	+0.36	+0.32	+0.69^a

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Upon addition of Al(ClO₄)₃ (0.05–0.5 M), the E_ox values of hydroquinones were significantly shifted to the positive direction depending on the concentration of Al³⁺ (Table 2). Thus, the one-electron donating ability of the hydroquinones was decreased in the presence of Al³⁺. The pK_a values of the hydroquinones in H₂O and DMSO are listed in Table 3.^38,39 The pH values of the hydroquinone solutions in the presence or absence of Al(ClO₄)₃ in EtOH–H₂O (9 : 1 v/v) are shown in Table 4. The pK_a values of the hydroquinones used in this study in aqueous solution are in the range of 9.9–11.3 (Table 3). Thus, all the hydroquinones do not undergo deprotonation and exist in their neutral forms. The one-electron reduction potential (E_red) of DPPH˙ was also determined using CV in the presence or absence of Al³⁺. In the absence of Al³⁺, two well-defined reversible redox waves were observed as shown in Fig. 6 (dashed line), from which the one-electron reduction potential of DPPH˙ was determined to be +0.23 V vs. SCE. In the presence of 0.1 M Al(ClO₄)₃, however, the cyclic voltammogram of DPPH˙ was remarkably changed and the E_red value of DPPH˙ was significantly shifted to the positive direction (Fig. 6, Table 2). These results indicated that the DPPH anion (DPPH⁻), the one-electron reduced species of DPPH˙, strongly interacted with Al³⁺ to enhance the electron-accepting ability of DPPH˙. The effects of metal ions on the E_red of DPPH˙ differed by the Lewis acidity.⁴⁰

Fig. 6 Cyclic voltammograms of DPPH˙ (2.0 × 10⁻³ M) in the absence (dashed line) and presence (solid line) of 0.1 M Al(ClO₄)₃ recorded at the scan rate 0.1 V s⁻¹ on a glassy carbon working electrode in EtOH–H₂O (9 : 1 v/v) (0.1 M Bu₄NClO₄).

Table 3 The pK_a values of hydroquinones

	QH2	MeQH2	Me2QH2	Me3QH2	Me4QH2
a The pKa values in aqueous solution.^38 b The pKa values in DMSO.^39
pKa	9.9^a	10.1^a	—	—	11.3^a
	19.1^b	18.3^b	19.0^b	17.8^b	20.0^b

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Table 4 The pH values of hydroquinone solutions in EtOH–H₂O (9 : 1 v/v) containing 0.1 M Bu₄NClO₄ in the presence or absence of Al(ClO₄)

[Al(ClO4)3]/M	QH2	MeQH2	Me2QH2	Me3QH2	Me4QH2
0	5.5	4.7	4.7	4.5	4.6
0.05	2.1	2.2	2.1	2.1	2.1
0.1	1.7	1.8	1.6	1.7	1.7
0.2	1.5	1.5	2.0	1.6	1.5
0.3	1.3	1.1	1.2	1.1	1.1
0.5	0.9	0.7	0.8	0.8	0.8

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We previously reported that the radical-scavenging mechanism of a vitamin E model is significantly affected by solvents.^22b In a protic solvent, such as methanol, an electron transfer from the vitamin E model to radicals followed by proton transfer is favorable compared to one-step hydrogen-atom transfer. Based on these results, the DPPH˙-scavenging reaction by hydroquinones in the hydroalcoholic medium would proceed via the electron transfer. The free energy change (ΔG_et) of the electron transfer from hydroquinones to DPPH˙ is calculated by using eqn (1),

ΔG_et = F(E_ox − E_red) (1)

where F is the Faraday constant, which is negative (ΔG_et < 0) in the presence of Al³⁺. Al³⁺ enhanced the electron transfer by strongly stabilizing DPPH⁻ and increased the k values of the hydroquinones depending on its concentration, in spite of decreasing the one-electron donor ability of hydroquinones.

Fig. 7 shows the free-energy-change dependence of log k for the radical-scavenging reaction of DPPH˙ with a series of hydroquinones in the absence and presence of Al³⁺ (0.5 M).

Fig. 7 Plots of log k vs. ΔG_et for the DPPH˙-scavenging reaction by the hydroquinone derivatives in the absence of metal ions (closed circles) and the presence of Al³⁺ (0.5 M) (open circles). The lines represent the curve fits to eqn (2) with λ = 1.78 and 2.19 eV for the reaction without and with Al³⁺ (0.5 M), respectively.

The dependence of outer-sphere electron transfer has been well established by Marcus as given by eqn (2),⁴¹

1/k = 1/k_diff + 1/{Zexp[–(λ/4)(1 + ΔG_et/λ)²/k_BT]} (2)

in which k_diff is the diffusion rate constant (10¹⁰ M⁻¹ s⁻¹),^42,43λ is the reorganization energy of electron transfer, k_B is the Boltzmann constant and Z is the collision frequency (1 × 10¹¹ M⁻¹ s⁻¹). The λ value is determined to be 1.78 eV for the radical-scavenging reaction in the absence of metal ions. In the presence of Al³⁺, the rate constants increase with a decrease in the ΔG_et value as shown in Fig. 7. The λ value in the presence of Al³⁺ is 2.19 eV, which is larger than that in the absence of metal ions, suggesting that trivalent Al³⁺ strongly binds to DPPH⁻ as an electron transfer product in electron transfer from the hydroquinones to DPPH˙.^2,44

Conclusions

The DPPH˙-scavenging activity of the hydroquinones increased with the increasing number of methyl substituents in the dearated hydroalcoholic medium. Upon addition of Al(ClO₄)₃ to the hydroquinone–DPPH˙ system, the k values of the hydroquinones were significantly increased depending on the concentration of Al³⁺, in spite of decreasing the electron-donating ability of the hydroquinones. Al³⁺, one of the strong Lewis acids, strongly stabilized the DPPH⁻ generated by the electron transfer from the hydroquinones to DPPH˙. In addition, the electron transfer occurred voluntarily (ΔG_et < 0) in the presence of Al³⁺ according to the electrochemical data. The strong Lewis acids, such as Al³⁺, significantly accelerated the electron-transfer reaction and enhanced the radical-scavenging reaction of phenolic antioxidants. The results obtained in this study demonstrate that Al³⁺, a strong Lewis acid, can act as a radical-scavenging promoter by stabilising the one-electron reduced species of the radical, although Al³⁺ is reported not only to act as a pro-oxidant but also to strongly interact with biomolecules, showing toxicities.

Acknowledgements

This work was partially supported by Grant-in-Aid (No. 26460056 to I. N., 26620154, 266288037 and 16K13964 to K. O.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, Acenet Inc. Foundation (to K. O.) and SENTAN projects from JST (to S. F.). We thank Mr T. Waki (Chiba University) for his help in the measurements of redox potentials.

Notes and references

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