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

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.


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 Mg 2+ [10] and Al 3+ [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 oneelectron reduction and oxidation to produce DPPH − and the corresponding cation (DPPH + ), 2 of 8 respectively, in organic solvents ( Figure 1A) [19][20][21][22]. We have also reported that an electrontransfer disproportionation of DPPH • to produce DPPH + and DPPHoccurs upon the addition of scandium triflate [Sc(OTf) 3 (OTf = OSO 2 CF 3 )] 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 Sc 3+ with a formation constant of 2.3 × 10 3 M -1 . Recently, Denzo et al. have reported the reactivity of DPPH • in the presence of metal cations (Cu 2+ and Zn 2+ ) and acids (HClO 4 and HNO 3 ) in MeCN [24]. A strong Brønsted acid, such as HClO 4 , 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 DPPHto regenerate DPPH • , demonstrating the reversibility of the Sc 3+ -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.

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 DPPHare suggested to be stabilized by one Sc 3+ , as shown in Figure 1B, although the [(DPPH) 2 Sc] + complex has yet to be detected.

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 DPPHare suggested to be stabilized by one Sc 3+ , as shown in Figure 1B, although the [(DPPH)2Sc] + complex has yet to be detected.  Figure 4 shows the overlapped absorption spectra at 1500 s after the addition of varying amounts of H 2 O. 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. 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.    Figure 5A). Upon addition of Sc(OTf)3 to the DPPH • , the signal intensity was significantly decreased, as shown in F tion of H2O to this reaction system resulted in the regeneration of DPP firmed by the increase in the EPR signal intensity due to DPPH • (Figu The regeneration of DPPH • upon the addition of H 2 O 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 H 2 O 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  6 3+ , has been reported for the Sc 3+ 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-firstorder rate constants (k obs ) vs. [H 2 O]. The slope of this plot (dashed line in Figure 6) Figure 7A. Then, the reaction B (Figure 7) occurs to produce two molecules of DPPH • , DPPH − , and [Sc(H 2 O) 3 ] as the rate-determining step followed by a rapid reaction between DPPH − and DPPH + to produce two molecules of DPPH • ( Figure 7C).   [2] and increased with increasing [H2O] to reach a constant value. It is suggested complex formation of Sc 3+ with H2O may weaken the interaction between DPPH − leading to the electron-transfer comproportionation to produce DPPH • . In fac aaqua complex, Sc(H2O)6 3+ , has been reported for the Sc 3+ hydration in aqueous rate solution [25].
The rise of the absorbance at 519 nm due to DPPH • shown in Figure 3A,B pseudo-first-order kinetics. Figure 6 shows a double logarithmic plot of the pseu order rate constants (kobs) vs. [H2O]. The slope of this plot (dashed line in Figure 6 for the kobs value at 9.3 × 10 −1 M H2O, is about three, suggesting that a triaqua c [(DPPH)2Sc(H2O)3] + , may be formed by the addition of H2O to [Sc(DPPH)2] + as s Figure 7A. Then, the reaction B (Figure 7) occurs to produce two molecules of DPPH − , and [Sc(H2O)3] as the rate-determining step followed by a rapid reaction DPPH − and DPPH + to produce two molecules of DPPH • ( Figure 7C).

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).

Spectral Measurements
Typically, a 10 µL aliquot of Sc(OTf) 3 3 were 7.1 × 10 −5 M and 2.0 × 10 −5 M, respectively, in 3 mL MeCN-H 2 O. UVvis 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 ([H 2 O] = 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 ([H 2 O] = 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.

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 H 2 O (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 Mn 2+ marker. In all cases, solutions were normally equilibrated with air.

Conclusions
The addition of H 2 O to the MeCN solution containing DPPH + and [(DPPH) 2 Sc] + resulted in the regeneration of DPPH • . It is suggested that the complex formation of Sc 3+ with H 2 O may weaken the interaction between DPPH − and Sc 3+ , leading to the electrontransfer 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) 2 Sc(H 2 O) 3 ] + generated upon the addition of H 2 O to [(DPPH) 2 Sc] + is the rate-determining step.