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Effects of 1-Propanol Addition on Quinone Radical Formations in Micellar Solutions Kouichi Nakagawa*,† and Kazuo Tajima‡ Radio Isotope Research Center, Fukushima Medical College, 1 Hikarigaoka, Fukushima-shi 960-1295, and Department of Chemistry, Faculty of Engineering, Kanagawa University, 3-27-1 Rokkakubashi, Kanagawa-ku, Yokohama 221-8686, Japan Received May 1, 1998. In Final Form: August 13, 1998
We have studied the photogenerated free radicals of idebenone (IDB) and a related compound (2,3dimethoxy-5-methyl-1,4-benzoquinone, CoQ0) in micellar solutions of sodium alkyl sulfates with 1-propanol (1-PrOH) addition. These radicals were also studied in 1-PrOH. UV irradiation of IDB with the long side chain in 1-PrOH produced the neutral radical, whereas in the presence of an electron donor (1,4-diazabicyclo[2.2.2]octane, DABCO) the anion radical was formed. On the other hand, UV irradiation of CoQ0 without the side chain produced the anion radical both in the presence and in the absence of DABCO. In micellar solutions, both compounds produced anion radicals, but the EPR (electron paramagnetic resonance) intensity of the IDB radical was very weak, even in the presence of DABCO. When 1-PrOH was added to the micellar solution, the signal intensity of the IDB radical increased approximately 7-fold. The effective electron transfer to IDB to form the radical results in a decrease of the micellar charge density. However, for CoQ0, a significant dilution effect caused by the addition of 1-PrOH was observed. Therefore, the results obtained suggest that the benzoquinone nucleus of IDB can locate in the polar region of the micelle.
Introduction It is known that quinones play an important role in biological electron transport such as redox processes. Relatively stable free radicals are formed by one-electron reduction of quinones.1 We have been investigating the transient free radical of a quinone type antioxidant, called idebenone (IDB). The general chemical and physical properties as well as pharmacology and pharmacokinetics of IDB were reviewed by Zs.-Nagy.2 They also studied spin trapping and chemically generated IDB radicals.3,4 Detailed knowledge of the antioxidative reaction intermediate is the key to understanding the role of the drug. Moreover, the electronic structures of the radicals are an important factor in estimating their adequacy as radical interceptors.5 The relatively stable radicals are measured by EPR (electron paramagnetic resonance) spectroscopy. Photochemistry combined with EPR technique will provide specific information about the quinone intermediates generated during the various electron-transfer. Recently, the hydrated electron (eaq-) ejected from phenothiazine (PTH) in sodium dodecyl sulfate (SDS) has been studied by van Willigen and co-workers using FT-EPR.6 We have observed the eaq- quenching process by IDB in * To whom correspondence should be addressed. E-mail:
[email protected]. Fax: [81]+(24)548-1715. Phone: [81]+ (24)548-2111 ext. 2822. † Fukushima Medical College. ‡ Kanagawa University. (1) Pedersen, J. A., Ed. Handbook of EPR Spectra from Quinones and Quinols; CRC Press: Boca Raton: FL, 1985. (2) Zs.-Nagy, I. Arch. Gerontol. Geriatr. 1990, 11, 177. (3) Zs.-Nagy, I.; Floyd, R. A. Arch. Gerontol. Geriatr. 1990, 11, 215. (4) Murakami, M.; Zs.-Nagy, I. Arch. Gerontol. Geriatr. 1990, 11, 199. (5) Nakagawa, K.; Tero-Kubota, S.; Tsuchihashi, N.; Ikegami, Y. Photochem. Photobiol. 1994, 60, 199. (6) Turro, N. J.; Khudyakov, V.; van Willigen, H. J. Am. Chem. Soc. 1995, 117, 12273.
PTH/SDS.7 In the process, the knowledge of the location of IDB in the SDS micelle is necessary for further understanding of the quenching reaction to form the IDB radical. To extract information regarding the location of solute quinones, we perturbed micellar solutions with alcohol. Addition of 1-propanol (1-PrOH) can change the motility of solute quinone in the micelle. Location and motility of the quinone are directly related to photochemical generation of the quinone radical. The solubilization of polar molecules like alcohols in aqueous surfactant system is quite complex.8 However, it is known that there are two major effects.9,10 With regard to aqueous micellar phase, alcohols are generally found to be solubilized in the micelles with the polar group anchored in the surface group region. If the hydrocarbon chain of the alcohol is sufficiently long, it will presumably penetrate the micellar core. The longer aliphatic alcohol effectively perturbs the interface SDS micellar solutions by intercalation of the alcohol into the head-group region.9 Then, higher polar solute ratio results in the molecular disorder of the interface. Second, the direct constant at the micellar interface decreases.11 Also, the entropy of the mixing due to the penetration of alcohol increases. We first examined the effect of 1-PrOH addition to SDS and SOS (sodium octyl sulfate) using various spin labels. The spin probe technique can provide information regarding dynamics and location in the micelle with 1-PrOH addition. The effects of 1-PrOH addition was significant for small molecular motion of the spin probe but less (7) Nakagawa, K.; Katsuki, A.: Tero-Kubota, S: Tsuchihashi, N.: Fujita, T. J. Am. Chem. Soc. 1996, 118, 5778. (8) Hqiland, H.; Blokhus, A. M. In Handbook of Surface and Collide Chemistry; Birdi, K. S., Ed.; CRC Press: Boca Raton: New York, 1997; Chapter 8. (9) Baglioni, P.; Kevan, L. J. Phys. Chem. 1987, 91, 1516. (10) Shinoda, K.; Nakagawa, T.; Tamamushi, B.; Isemura, T. Colloidal Surfactants: Some Physicochemical Properties; Academic Press: New York, 1963; p 72. (11) Zana, R.; Yiv, S.; Strazielle, C.; Lianos, P. J. Colloid Interface Sci. 1981, 80, 208.
10.1021/la980511n CCC: $15.00 © 1998 American Chemical Society Published on Web 10/02/1998
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Figure 1. Chemical structures of various compounds; the surfactants SDS and SOS; the spin labels 5DS, 7DS, 12DS, and TEMPOL; and the quinones, IDB with the side chain and CoQ0 without the chain.
effective for the various doxylstearic acids studied. Then, to have detailed information about IDB location in the micelle, we studied the photogenerated IDB radical with 1-PrOH addition. Comparison was also made with the related quinone compound. The photochemical reaction of IDB in micellar solution was quite different from that in 1-PrOH. Our results demonstrate that the reaction microenvironment and the long side chain of IDB affected the radical formation in micellar solutions. Experimental Section Materials. Idebenone [(6-(10-hydroxydecyl)-2,3-dimethoxy5-methyl-1,4-benzoquinone, IDB] was donated by Takeda Chemical Industries, Ltd. 2,3-Dimethoxy-5-methyl-1,4-benzoquinone was purchased from Tokyo Chemical Co. and used as received.12 The electron donor 1,4-diazabicyclo[2.2.2]octane was purchased from Aldrich. The concentration was kept at 2 mM. Sodium dodecyl sulfate and sodium octyl sulfate were obtained from Aldrich Chemical Co. They were recrystallized from ethanol. The concentrations of SDS and SOS were 0.15 and 0.18 M, respectively. All other reagents were of the highest quality and used as received. The spin labels 5-((4,4-dimethyl-3-oxazolidinyl)oxy)octadecanoic acid, known as 5-doxylstearic acid (5DS), and the related compounds 7-doxylstearic acid (7DS), 12-doxylstearic acid (12DS), and the water soluble 4-hydroxy-2,2,6,6-tretramethylpiperidine 1-oxy (TEMPOL) were obtained from Aldrich Chemical Co. and used as received. Concentrations of spin labels were kept at 0.1-0.5 mM in the solution. Diagrams of the surfactants and solutes are presented in Figure 1. Measurements. EPR signals were measured by a JEOL FE1X X-band EPR spectrometer. This spectrometer is equipped with a cylindrical TE011 mode cavity. Quinone/DABCO/micellar solutions were illuminated within the EPR cavity with an xenon short arch lamp (Ushio model UXL 500D, 500W). An interference filter (290 nm maximal transmission) was placed in front of the EPR cavity. Typical EPR conditions were as follows: microwave frequency, 9.35 GHz; microwave power, 10 mW; modulation amplitude, 0.32 G; time constant, 1 s; scan rate, 3.125 G/min. All spectra were recorded at 22 ( 1 °C. Prior to EPR measurements, argon gas was bubbled through the quinone solutions to achieve deoxygenation. For measurements of quinone/micellar solutions, a 0.3 mm path length flat cell with a 60 µL sample volume was used. Sample solutions were not flowing during the measurements unless otherwise noted. (12) IDB is soluble in water and in methanol at ∼0.1 and 1 g/mL, respectively. The values were provided by Takeda Chemical Industries, Ltd. Also, a general description of IDB is given in: The Merck Index, 12th ed.; Merck & Co., Inc.: Rahway, NJ, 1996; p 840. CoQ0 is soluble in methanol at 20 mg/mL. The information was provided by Sigma technical service.
Figure 2. UV irradiation of 5 mM IDB in 1-propanol: (A) experimental EPR spectrum and (B) the calculated spectrum. The g value of the radical is indicated. Analyses of EPR Spectra. Rotational correlation times (τR) for spin labels were calculated by the equation13
τR ) 5.47 × 10-10 [(h0/h1)0.5 + (h0/h-1)0.5 - 2] ∆Hpp0 (1) where h0, h1, and h-1 are EPR hyperfine amplitudes of mI ) +1, 0, and -1 transitions, respectively. ∆Hpp0 is the limiting peakto-peak line width of the central hyperfine comportment. In this study, we obtained ∆Hpp0 from 1-PrOH since we investigated the effects of 1-PrOH addition to micellar solution. For photoinduced quinone radicals the strongest line intensity was used for the analyses.
Results and Discussion Spin Labels in 1-Propanol Solution. We studied spin labels in 1-propanol solution in two respects. First, 1-PrOH was added to examine the motive effects of spin labels in the micelles. Second, to calculate τR, the peakto-peak width of the center line was used to obtain ∆Hpp0 in eq 1. The τR values obtained for DSs were similar. However, the value for TEMPOL was 1 order of magnitude smaller than that for DSs. It is expected that the smaller molecule TEMPOL tumbles faster. Quinones in 1-Propanol Solution. Figure 2A shows the EPR spectrum measured during UV irradiation of IDB in 1-propanol. When the light was turned off, the signal disappeared over a period of seconds. The calculated spectrum is presented in Figure 2B. The hyperfine coupling values (units in Gauss) obtained are given in (13) Poole, C. P., Jr.; Farach, H. A. Theory of Magnetic Resonance, 2nd ed.; John Wiley & Sons: New York, 1987; Chapter 18, p 319.
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Table 1. Hyperfine Coupling Constants (G) of Benzoquinone Radicals
Figure 4. UV irradiation of 5 mM CoQ0 in 1-propanol in the flow experiment: (A) experimental spectrum and (B) simulated spectrum.
a Solvent: P, 1-propanol; S, SDS; P+ and S+, in the presence of DABCO.
Figure 5. Rotational correlation times (τR) of various spin labels in SDS with 1-propanol addition (vol %). The right-hand in the figure (1-propanol 100 vol %) is the τR obtained for various spin labels in 1-propanol. Open diamond, filled circle, and open square represent 5DS, 7DS, and 12DS, respectively.
Figure 3. (A) EPR spectrum obtained during UV irradiation of 10 mM IDB in the presence of 10 mM DABCO and (B) the simulated spectrum.
Table 1 together with the g value and indicate that the electron spin mostly localizes at the 3- and 5-positions of the benzene ring. Thus, on the basis of the spectrum, we determined the radical as the neutral semiquinone radical. The neutral radical can be produced as a result of hydrogen abstraction from the solvent. By changing the solvent from 1-propanol to 2-propanol the signal amplitude of the radical was slightly increased. The hydrogen abstraction from 2-propanol is well-known. UV irradiation of IDB in the presence of electron donor agent DABCO generated a nine-line spectrum, as shown in Figure 3A. The calculated spectrum is shown in Figure 3B. The hyperfine values are given in Table 1. The hyperfine values obtained suggest that the radical is an anion radical in which most of the electron spin localizes in the 4-position of the oxygen. The photochemically generated IDB anion radical was observed at 22 ( 1 °C for the first time. This spectrum is identical to the one produced by potassium metal.4 In the case of CoQ0 under the same experimental conditions, the anion radical was observed in the flow experiments as shown in Figure 4A. The calculated EPR spectrum is given in Figure 4B. The hyperfine coupling
values obtained are listed in Table 1 and indicate that the unpaired electron spin localizes throughout the benzene ring. In the presence of DABCO, the identical anion radical was observed, but the signal intensity was about 10 times stronger. The results obtained suggest that CoQ0 is more reactive than IDB. Therefore, we have observed the clear differences in the photochemically generated radicals for IDB and CoQ0, even though they have the same benzoquinone nucleus. Spin Labels in Micellar Solution. To further clarify the differences in the photochemical reaction properties between IDB and CoQ0 in homogeneous solution, we performed UV photolysis of the quinone samples in micellar solution. At first, we studied the dynamics and location of spin labels in micellar solution with 1-PrOH addition. Then, on the basis of the information regarding spin labels in micelle, we were able to adequately interpret photogenerated quinone radicals. Figure 5 shows the rotational correlation times (τR) of various spin labels in SDS. The right-hand of the figure indicates τR for the spin labels in 1-PrOH solution as a reference. Values of τR are similar for the DS spin labels. The tumbling motion of TEMPOL without the long chain is much faster than that of other spin labels. TEMPOL tumbles 1 order of magnitude faster than DSs. In addition, τR for 12DS was a little faster than that for 5DS and 7DS in the micellar solution. The results imply that the nitroxyl radical of 12DS is located in a liquid-like region of the SDS micelle core.
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Figure 6. Rotational correlation times (τR) of various spin labels in SOS with 1-propanol addition. It should be noted that the τR obtained for various spin labels in 1-propanol is plotted at 100 vol % in the figure. Open diamond, filled circle, and open square represent 5DS, 7DS, and 12DS, respectively.
Effective 1-PrOH addition to TEMPOL in SDS solution was observed. TEMPOL showed a prominent change on τR. Addition of 1-PrOH affects τR for DSs with the long chain much less. Furthermore, among TEMPOL and DSs the effect of 1-PrOH addition on τR was distinguishable for the first 10 vol % addition. Further addition showed less change on τR, as presented in Figure 5. We also examined the molecular motion in shorter alkyl sulfate (SOS) solution. The rotational correlation times of TEMPOL are nearly 10 times faster than that in SDS. For DSs values of τR are similar in SDS and in SOS. It is interesting to note that 5DS tumbles a little faster than 7DS and 12DS, as shown in Figure 6. The nitroxyl radical in 5DS is near the COO- group. Also, considering the size of the SOS micelle, the radical in 5DS may be located in the outer sphere of the micelle. The 1-PrOH addition does not make tumbling much faster for all the spin probes studied. Thus, in the case of SOS solution, the contribution of 1-PrOH addition on the rotational motion is less significant than that for SDS. Quinones in Micellar Solutions. To examine the validity of the expectation, we investigated the micellar effect on IDB molecule. The location of IDB in the micelle is the key for the formation of the radical. Changes in micelle with 1-PrOH addition could prove the photochemical reaction processes of IDB. (1) IDB. The EPR spectra observed for UV irradiation of IDB/SDS in the presence of hygroscopic DABCO are presented in Figure 7. The very weak signal of the IDB radical was observed, as shown in Figure 7A. The EPR signal intensity became stronger with the addition of 1-PrOH when the original sample concentrations were diluted (Figure 7B,C). The normalized EPR intensities are plotted against 1-PrOH addition in Figure 8. The signal intensity increased up to ∼23 vol % addition to the original sample volume. The results suggest that the effective electron transfer to IDB produces the IDB anion radical with 1-PrOH addition. After approximately 23 vol % addition, the EPR intensity started to decease. Moreover, similar results were obtained with the addition of ethanol. We observed a noticeable EPR intensity for the IDB radical with no 1-PrOH addition in SOS (Figure 8). The intensity is about 3 times stronger than that in SDS. The production of the radical in SOS is more efficient. Then, a moderate rise of the EPR intensity was observed. The
Nakagawa and Tajima
Figure 7. EPR spectra obtained by UV irradiation of 2 mM IDB in 0.15 M SDS in the presence of 2 mM DABCO: (A) no 1-propanol was added to the solution, (B) 1-propanol (9 vol %) was added, and (C) 1-propanol (23 vol %) was added to the original micellar solution.
Figure 8. Normalized EPR intensity of photochemically generated IDB anion radical in SDS (filled circle) and SOS (open circle) as a function of 1-propanol addition (vol %).
rise of EPR intensity observed is similar to that in SDS solution. These results together with the DS spin probe experiments in SOS imply that the motility of IDB with the addition may increase slowly. However, the production of the radical is efficient in SOS. The difference in the production between SDS and SOS can be due to the size of the micelle. Also, SOS molecule frequently changes between two phases: the bulk and the micellar phases. There are two major effects of 1-PrOH addition on micellar solutions.9,10 First, the micellar size changes because of 1-PrOH partition. Second, the micellar charge density decreases with 1-PrOH addition. As discussed in the previous section, the motive effects with 1-PrOH addition on DSs are small. Therefore, the motive effect on IDB with the long side chain may likewise not be significant. The increasing intensity of the IDB radical as 1-PrOH is added indicates effective electron transfer from DABCO as a result of deceasing micellar charge density. Thus, the benzoquinone nucleus of IDB may become easier to encounter with the electron donor by 1-PrOH addition. Under such conditions, UV irradiation of IDB can yield more of the corresponding radical species. It is important to note that the dilution effect with 1-PrOH addition is minor. (2) CoQ0. The anion radical was produced by the photolysis of CoQ0 in SDS micellar solution, as shown in Figure 9A. The identical EPR hyperfine structure and a stronger signal intensity than that for 1-PrOH were observed. The calculated spectrum is presented in Figure
Quinone Radical Formations in Micellar Solutions
Figure 9. (A) EPR spectrum obtained during UV irradiation of 2 mM CoQ0 in 0.15 M SDS in the presence of 2 mM DABCO without 1-PrOH addition and (B) the simulated spectrum. It is noted that this signal amplitude for CoQ0 was intense in comparison with that of IDB.
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micellar solution becomes more dominant. This can be the major influence on the decay of EPR signal intensity. The results of CoQ0 in SOS solution were similar to those of CoQ0 in SDS. However, a relatively moderate rise in SOS was observed in comparison with SDS. Thus, CoQ0 showed a rise with a small percent of 1-PrOH addition and then exhibited the effective dilution. In the 1-PrOH-sodium alkyl sulfates system, the photochemical reaction for electron transfer may occur in two phases: one is the bulk phase and the other is the micellar phase. IDB and CoQ0 are distributed in two phases. The distribution of the solutes can be changed by 1-PrOH addition. The change in distribution for both phases could reflect the photochemical production of the radicals. The solubility of IDB in water suggests that IDB is in the micellar phase.12 CoQ0 can be in both phases and estimated by the strong EPR intensities with and without 1-PrOH addition. Furthermore, the motility of CoQ0 should be more significant than that of IDB on the basis of the TEMPOL/surfactant/1-PrOH results. For IDB, the addition of 1-PrOH is less effective than that of CoQ0 in terms of the motility but more effective in generating the IDB radical. IDB showed the continuous rise of the EPR signal with 1-PrOH addition. The results suggest that IDB may locate in the polar region of the micelle. Conclusions
Figure 10. The normalized EPR signal intensities as a function of 1-propanol addition. The CoQ0 anion radicals in SDS (filled circle) and in SOS (open circle) are indicated.
9B. It is noted that the signal-to-noise ratio of CoQ0 radical is approximately 12 times larger than that of IDB in SDS (Figures 7A and 9A). Half of the maximum intensity in SDS was observed with no addition, as shown in Figure 10. This strong intensity may be attributed to either the motility or the solubilization or both. Then, the signal intensity of CoQ0 radical increased when 1-PrOH was added. The sharp rise of the signal with a small percent of 1-PrOH addition is similar to the observation for the spin label TEMPOL. The rise can be due to an increase of the motilities for both CoQ0 and DABCO in the solution. However, it started to decrease after ∼5 vol % addition. The dilution effect of
The 1-PrOH addition makes molecular tumbling faster for TEMPOL but has little effect on DS spin labels. In SOS solution, effects of 1-PrOH addition on all spin labels studied were not notable. UV photolysis of IDB produced neutral and anion radicals in the presence of the electron donor in 1-PrOH. However, in SDS micellar solution, it produced the anion radical with a very weak signal intensity. The present results obtained indicate that IDB with the long side chain in the micellar photoreaction environment can affect the formation of the radical. By the addition of 1-PrOH, IDB interacts with DABCO effectively and yields more of the corresponding radical during UV irradiation. For CoQ0 without the side chain, a significant dilution effect upon the addition of 1-PrOH was observed in SDS solution. These results were also supported by various spin probe experiments. The present results obtained imply that the benzoquinone nucleus of IDB can locate in the polar region of the micelle. Therefore, the long side chain in the IDB molecule in micellar solutions plays an important role in the formation of the radical. Acknowledgment. We thank Takeda Chemical Industries, Ltd. for the gifts of idebenone and its chemical and physical information. K.N. also thanks Prof. H. van Willigen for useful discussion on the results. This research was supported in part by Grant-in-Aid for General Scientific Research (C) (No. 10640562) from the Ministry of Education, Science, Sports, and Culture of Japan (K.N.). LA980511N
