Water-Induced Regeneration of a 2,2-Diphenyl-1-picrylhydrazyl Radical after Its Scandium Ion-Promoted Electron-Transfer Disproportionation in an Aprotic Medium

Abstract

A neutral, stable radical, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH^•), has been frequently used to estimate the activity of antioxidants for more than 60 years. However, the number of reports about the effect of metal ions on the reactivity of DPPH^• is quite limited. We have recently reported a unique electron-transfer disproportionation of DPPH^• to produce the DPPH cations (DPPH⁺) and anions (DPPH⁻) upon the addition of scandium triflate [Sc(OTf)₃ (OTf = OSO₂CF₃)] to an acetonitrile (MeCN) solution of DPPH^•. The driving force of this reaction is suggested to be an interaction between DPPH^– and Sc³⁺. In this study, it is demonstrated that the addition of H₂O to the DPPH^•–Sc(OTf)₃ system in MeCN resulted in an increase in the absorption band at 519 nm due to DPPH^•. This indicated that an electron-transfer comproportionation occurred to regenerate DPPH^•. The regeneration of DPPH^• was also confirmed by electron paramagnetic resonance (EPR) spectroscopy. The amount of DPPH^• increased with an increasing amount of added H₂O to reach a constant value. The detailed mechanism of regeneration of DPPH^• was proposed based on the detailed spectroscopic and kinetic analyses, in which the reaction of DPPH⁺ with [(DPPH)₂Sc(H₂O)₃]⁺ generated upon the addition of H₂O to [(DPPH)₂Sc]⁺ is the rate-determining step.

1. Introduction

2,2-Diphenyl-1-picrylhydrazyl radical (DPPH^•) is a neutral, stable radical that has been frequently used to estimate the activity of antioxidants for more than 60 years [1,2,3,4]. It is known that the radical-scavenging reactivity of antioxidants is significantly affected by the reaction environments, such as solvents [5,6], pH [7,8], the presence of metal ions [9,10,11,12,13,14,15,16,17,18], and so on. However, the number of reports about the reactivity of DPPH^• in the presence of metal ions is quite limited. We have demonstrated that the DPPH^•-scavenging reactivity of phenolic compounds, such as a vitamin E model, flavonoids, and hydroquinones, is significantly enhanced in the presence of redox-inactive metal ions with a moderate Lewis acidity, such as Mg²⁺ [10] and Al³⁺ [9]. The coordination of the metal ion to the one-electron reduced species of DPPH^• (DPPH^–) may stabilize the product, resulting in the acceleration of the electron transfer. On the other hand, DPPH^• is known to undergo reversible one-electron reduction and oxidation to produce DPPH⁻ and the corresponding cation (DPPH⁺), respectively, in organic solvents (Figure 1A) [19,20,21,22]. We have also reported that an electron-transfer disproportionation of DPPH^• to produce DPPH⁺ and DPPH^– occurs upon the addition of scandium triflate [Sc(OTf)₃ (OTf = OSO₂CF₃)] to an acetonitrile (MeCN) solution of DPPH^• [23]. Since there is no proton sauce in this reaction system, DPPH⁻ does not undergo protonation to produce DPPH-H. Then, DPPH⁻ may significantly be stabilized by the strong Lewis acidity of Sc³⁺ with a formation constant of 2.3 × 10³ M^–1. Recently, Denzo et al. have reported the reactivity of DPPH^• in the presence of metal cations (Cu²⁺ and Zn²⁺) and acids (HClO₄ and HNO₃) in MeCN [24]. A strong Brønsted acid, such as HClO₄, is required for the disproportionation of DPPH^• to occur. We report herein that the addition of water to the MeCN solution containing DPPH⁺, DPPH⁻ and Sc(OTf)₃ resulted in the electron-transfer comproportionation between DPPH⁺ and DPPH^– to regenerate DPPH^•, demonstrating the reversibility of the Sc³⁺-catalyzed electron-transfer disproportionation of DPPH^•. The reversible redox reactivity of DPPH^• in the presence of the redox-inactive metal ion with strong Lewis acidity shows a unique electron-transfer redox reaction of radical species in aprotic media.

2. Results and Discussion

When Sc(OTf)₃ was added to an MeCN solution of DPPH^•, a decrease in the absorption band of DPPH^• at 519 nm was observed, accompanied by an increase in the absorption band at 380 nm due to the electron-transfer disproportionation [23]. The band at 380 nm is characteristic of DPPH⁺. The spectral titration conducted in this study shows the Sc(OTf)₃/DPPH^• molar ratio being 1:4 (Figure 2). Thus, two molecules of DPPH^– are suggested to be stabilized by one Sc³⁺, as shown in Figure 1B, although the [(DPPH)₂Sc]⁺ complex has yet to be detected.

Upon the addition of H₂O to this solution, the absorption band at 519 nm due to DPPH^• increased. The time course changes in the absorbance at 519 nm after the addition of several amounts of H₂O are shown in Figure 3A,B. At all the concentrations of H₂O, the reaction has completed after 1500 s. Figure 4 shows the overlapped absorption spectra at 1500 s after the addition of varying amounts of H₂O. The absorption band at 380 nm due to DPPH⁺ decreased, accompanied by an increase in the absorption band at 519 nm due to DPPH^• with clear isosbestic points at 344 and 449 nm.

The regeneration of DPPH^• upon the addition of H₂O to the DPPH^•–Sc(OTf)₃ system in MeCN was also confirmed by the electron paramagnetic resonance (EPR) spectroscopy. The well-resolved five lines having a g value of 2.0036 were observed in the EPR spectrum of DPPH^• in MeCN (Figure 5A). Upon addition of Sc(OTf)₃ to the MeCN solution of DPPH^•, the signal intensity was significantly decreased, as shown in Figure 5B. The addition of H₂O to this reaction system resulted in the regeneration of DPPH^•, which was confirmed by the increase in the EPR signal intensity due to DPPH^• (Figure 5C).

Figure 3C shows [DPPH^•] vs. [H₂O] at 1500 s after the addition of H₂O to the 3 mL MeCN solution containing DPPH^• (7.1 × 10⁻⁵ M) and Sc(OTf)₃ (2.0 × 10⁻⁵ M). The [DPPH^•] values were calculated using the extinction coefficient (ε) of 1.2 × 10⁴ M⁻¹ cm⁻¹ at 519 nm [2] and increased with increasing [H₂O] to reach a constant value. It is suggested that the complex formation of Sc³⁺ with H₂O may weaken the interaction between DPPH⁻ and Sc³⁺, leading to the electron-transfer comproportionation to produce DPPH^•. In fact, a hexaaqua complex, Sc(H₂O)₆³⁺, has been reported for the Sc³⁺ hydration in aqueous perchlorate solution [25].

The rise of the absorbance at 519 nm due to DPPH^• shown in Figure 3A,B obeyed pseudo-first-order kinetics. Figure 6 shows a double logarithmic plot of the pseudo-first-order rate constants (k_obs) vs. [H₂O]. The slope of this plot (dashed line in Figure 6), except for the k_obs value at 9.3 × 10⁻¹ M H₂O, is about three, suggesting that a triaqua complex, [(DPPH)₂Sc(H₂O)₃]⁺, may be formed by the addition of H₂O to [Sc(DPPH)₂]⁺ as shown in Figure 7A. Then, the reaction B (Figure 7) occurs to produce two molecules of DPPH^•, DPPH⁻, and [Sc(H₂O)₃] as the rate-determining step followed by a rapid reaction between DPPH⁻ and DPPH⁺ to produce two molecules of DPPH^• (Figure 7C).

3. Materials and Methods

3.1. Materials

DPPH^• was commercially obtained from Tokyo Chemical Industry Co., Ltd., Tokyo, Japan. Sc(OTf)₃ was purchased from Sigma–Aldrich, St. Louis, MO, USA. MeCN (spectral grade) used as a solvent was commercially obtained from Nacalai Tesque, Inc., Kyoto, Japan, and used as received. The water used in this study was freshly prepared with a Milli-Q system (Millipore Direct-Q UV3) (Merck Millipore, Burlington, MA, USA).

3.2. Spectral Measurements

Typically, a 10 µL aliquot of Sc(OTf)₃ (6.1 × 10⁻³ M) in MeCN was added to a quartz cuvette (10 mm i.d.) which contained DPPH^• in MeCN (2.95, 2.90, 2.85, 2.80, 2.75 or 2.70 mL). This led to an electron-transfer disproportionation of DPPH^• to produce DPPH⁺ and [(DPPH)₂Sc]⁺. After 1 h, water (50, 100, 150, 200, 250, or 300 µL) was added to this MeCN solution (2.95, 2.90, 2.85, 2.80, 2.75, or 2.70 mL, respectively). The molar concentrations of 50, 100, 150, 200, 250, and 300 µL H₂O in 3 mL MeCN–H₂O are 9.3 × 10⁻¹, 1.9, 2.8, 3.7, 4.2, 5.6 M, respectively. The final concentrations of DPPH^• and Sc(OTf)₃ were 7.1 × 10⁻⁵ M and 2.0 × 10⁻⁵ M, respectively, in 3 mL MeCN–H₂O. UV-vis spectral changes associated with the reaction were monitored using an Agilent 8453 photodiode array spectrophotometer thermostated with a Peltier temperature control at 298 K (Agilent Technologies, Santa Clara, CA, USA). The regeneration rates of DPPH^• were followed by monitoring the absorbance change at 519 nm due to DPPH^• on the Agilent 8453 photodiode array spectrophotometer ([H₂O] = 9.3 × 10⁻¹ and 1.9 M) or on a UNISOKU RSP-1000-02NM stopped-flow spectrophotometer (UNISOKU Co., Ltd., Osaka, Japan), which was thermostated with a Thermo Scientific NESLAB RTE-7 Circulating Bath (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 298 K ([H₂O] = 2.8, 3.7, 4.2, and 5.6 M). The k_obs values were obtained by a least-square curve fit using an Apple MacBook Pro personal computer (Apple Inc., Cupertino, CA, USA). The first-order plots of ln(A_∞–A) vs. time (A and A_∞ are the absorbance at the reaction time and the final absorbance, respectively) were linear until three or more half-lives, with a correlation coefficient ρ > 0.999. In each case, it was confirmed that the k_obs values derived from at least three independent measurements agreed within experimental error of ±5%. In all cases, solutions were normally equilibrated with air.

3.3. EPR Measurements

The EPR spectra of DPPH^• (7.1 × 10⁻⁵ M) in the presence or absence of Sc(OTf)₃ (2.0 × 10⁻⁵ M) and/or H₂O (5.6 M) in MeCN were taken using an LLC-04B ESR sample tube (LABOTEC Co., Ltd., Tokyo, Japan) on a JEOL X-band spectrometer (JES-RE1X) (JEOL Ltd., Tokyo, Japan) at room temperature under the following conditions: microwave frequency 9.43 GHz, microwave power 8 mW, center field 338 mT, sweep width 15 mT, sweep rate 3 mT min⁻¹, modulation frequency 100 kHz, modulation amplitude 0.2 mT, and time constant 0.1 s. EPR data acquisition was controlled by the WIN-RAD ESR Sata Analyzer System (Radical Research, Inc., Tokyo, Japan). The g values were calibrated with an Mn²⁺ marker. In all cases, solutions were normally equilibrated with air.

4. Conclusions

The addition of H₂O to the MeCN solution containing DPPH⁺ and [(DPPH)₂Sc]⁺ resulted in the regeneration of DPPH^•. It is suggested that the complex formation of Sc³⁺ with H₂O may weaken the interaction between DPPH⁻ and Sc³⁺, leading to the electron-transfer comproportionation to produce DPPH^•. The detailed mechanism of regeneration of DPPH^• was proposed based on the detailed spectroscopic and kinetic analyses, in which the reaction of DPPH⁺ with [(DPPH)₂Sc(H₂O)₃]⁺ generated upon the addition of H₂O to [(DPPH)₂Sc]⁺ is the rate-determining step.