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Aggregation of Amphiphilic Pyranines in Water: Facile Micelle Formation in the Presence of Methylviologen Ryo Sasaki and Shigeru Murata* Department of Basic Science, Graduate School of Arts and Sciences, The UniVersity of Tokyo, Meguro-ku, Tokyo 153-8902, Japan ReceiVed September 13, 2007. In Final Form: December 10, 2007 Four amphiphilic pyranines in which the acidic hydrogen of pyranine was replaced by an octyl, dodecyl, hexadecyl, and eicosyl group, POCn (n ) 8, 12, 16, and 20), were prepared, and their spectroscopic properties and aggregation behaviors in water were investigated. The critical micelle concentration (cmc) of the amphiphilic pyranines was found to be relatively large even in POC20 having a long hydrophobic alkyl chain (ca. 3 × 10-3 M). 1H NMR studies revealed that POC16 and POC20 exist in the compact structure with the pyranine nucleus wrapped with a long methylene chain in water. As in the case of parent pyranine, the addition of methylviologen (MV2+) to an aqueous solution of POCn resulted in the absorption spectral change and efficient quenching of POCn fluorescence. In the case of POC8 and POC12, these spectral changes induced by the MV2+ addition were thoroughly explained in terms of the formation of the electrostatic complex POCn/MV2+ with a complexation constant of ∼3 × 104 M-1. On the other hand, an unexpectedly large dependence of the absorption spectral change, as well as fluorescence quenching, on the total concentration of MV2+ was observed in POC16 and POC20. The Stern-Volmer plot for quenching of the fluorescence of POC16 and POC20 gave a curve deviating largely upward from a straight line. The plot was successfully analyzed by the equation induced by assuming the aggregate formation of the complex POCn/MV2+, which revealed the considerably small cmc values of the complexes, 3.6 × 10-7 and 3.6 × 10-8 M for POC16/MV2+ and POC20/MV2+, respectively. Experimental evidence in support of the aggregate formation was obtained by 1H NMR and dynamic light scattering studies.
Introduction In natural photosynthetic systems, solar energy is collected by light-harvesting complexes and transferred to a reaction center that carries out charge separation and electron transport.1,2 In the light-harvesting complexes, a large number of chromophores having large extinction coefficients are aggregated and arranged in an orderly fashion to achieve the efficient collection of sunlight. In the past decades, a number of artificial chromophore assemblies have been presented toward mimicking of natural light-harvesting complexes.3 One approach to the construction of artificial lightharvesting systems is the synthesis of giant molecules composed of a number of chromophores connected with covalent bonds. Among them are a wide variety of porphyrin arrays4 and various dendritic macromolecules having numerous peripheral lightcollecting chromophores,5,6 in which an efficient intramolecular energy transfer is demonstrated. A supramolecular approach in which molecules are self-assembled with noncovalent interactions is more attractive for aggregating a large number of chromophores. Kobuke and his co-workers reported the synthesis of a variety of supramolecular porphyrin arrays using the complementary coordination of imidazolylporphyrinatozinc(II) derivatives.7 * Corresponding author. Tel: +81 3 5454 6596. Fax: +81 3 5454 6998. E-mail:
[email protected]. (1) Whitmarch, J.; Govindjee In Concepts in Photobiology; Singhal, G. S., Renger, G., Sopory, S. K., Irrgang, K.-D., Govingjee, Eds.; Kluwer Academic Publishers: Dordrecht, 1999; p 11. (2) Ke, B. Photosynthesis; Kluwer Academic Publishers: Doudrecht, 2001, Chapter 1. (3) Ghiggino, K. P.; Smith, T. A. In Energy HarVesting Materials; Andrew, D. L., Ed.; World Scientific: Singapore, 2005; p 219. (4) Nakamura, Y.; Hwang, I.-W.; Aratani, N.; Ahn, T. K.; Ko, D. M.; Takagi, A.; Kawai, T.; Matsumoto, T.; Kim, D.; Osuka, A. J. Am. Chem. Soc. 2005, 127, 236, and references therein. (5) Adronov, A.; Fre´chet, J. M. J. Chem. Commun. 2000, 1701. (6) D’Ambruoso, G. D.; McGrath, D. V. In Energy HarVesting Materials; Andrew, D. L., Ed.; World Scientific: Singapore, 2005; p 281.
Micelles and bilayer membranes are supramolecular aggregates formed through hydrophobic interaction of amphiphilic molecules in water and are one of the most fascinating systems for the construction of artificial light-harvesting assemblies. In 1985, Kunitake and his colleagues reported that double-chain ammonium amphiphiles containing a carbazole or naphthalene moiety as a chromophore aggregated in water to form spherical bilayer membranes (vesicles).8,9 Moreover, they demonstrated efficient energy transfer from the photoexcited chromophoric unit to the acceptors added to the outer aqueous solution. Sisido and his co-workers reported an efficient photoenergy transfer from a naphthyl group to an anthryl group in vesicles composed of amino acid amphiphiles covalently carrying these chromophoric units.10 Although these precedent works reveal the usefulness of bilayer membranes for the field of excitation energy transfer, further studies are required to achieve the construction of artificial light-harvesting membranes in which light energy is collected and transferred to a reaction center with high efficiency. In the course of our studies on artificial photosynthesis using pyrene derivatives as chromophores,11,12 we have investigated the aggregation behaviors of amphiphilic pyrenes in water and their usefulness for artificial light-harvesting systems. Pyrenes have several merits as chromophores for studies on artificial photosynthesis: large extinction coefficients in the near-ultraviolet region, relatively long lifetime of their singlet excited state, intense fluorescence that is sensitive to their surroundings,13 and ease (7) Kuramochi, Y.; Satake, A.; Kobuke, Y. J. Am. Chem. Soc. 2004, 126, 8668, and references therein. (8) Kunitake, T.; Shimomura, M.; Hashiguchi, Y.; Kawanaka, T. J. Chem. Soc. Chem. Commun. 1985, 833. (9) Kakashima, N.; Kimizuka, N.; Kunitake, T. Chem. Lett. 1985, 1817. (10) Sasaki, H.; Sisido, M.; Imanishi, Y. Langmuir 1991, 7, 1944. (11) Yoshida, A.; Harada, A.; Mizushima, T.; Murata, S. Chem. Lett. 2003, 32, 68. (12) Mizushima, T.; Yoshida, A.; Harada, A.; Yoneda, Y.; Minatani, T.; Murata, S. Org. Biomol. Chem. 2006, 4, 4336.
10.1021/la702849c CCC: $40.75 © 2008 American Chemical Society Published on Web 02/09/2008
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of preparation of a wide variety of their derivatives. There are two approaches to the molecular design of amphiphilic pyrenes: introduction of a hydrophilic substitution group into a hydrophobic pyrenyl ring14 and introduction of a hydrophobic long alkyl chain into a water-soluble pyrene derivative. The experimental results described in this paper are concerned with amphiphilic pyrenes designed by the latter strategy. Pyranine (trisodium 8-hydroxypyrene-1,3,6-trisulfonate, POH) is a commercially available water-soluble pyrene derivative that has been employed as a pH probe because of its highly pHdependent fluorescence.15 The substitution of a hydrophobic long alkyl chain for the acidic hydrogen of its hydroxyl group leads to amphiphilic pyrene derivatives. Although Koller and his coworkers demonstrated that the pyranines having a decyl and octadecyl alkyl chain acted as excellent fluorescent probes for determination of critical micelle concentration (cmc) of nonionic detergents,16 no detailed studies on pyranines having a long alkyl chain have been reported so far. In this paper, we report the spectroscopic properties and aggregation behaviors of pyranines having a long alkyl chain.17 Since the cmcs of these amphiphilic pyranines were relatively large, they unfortunately appear to be unsuitable for the component chromophores to construct artificial light-harvesting systems. However, we found and successfully analyzed a novel complexation-induced micelle formation, where the cmc of the amphiphilic pyranine having a C20 alkyl chain decreased dramatically by a factor of 105 in the presence of an electron acceptor, methylviologen (MV2+). Experimental Section General Methods. Trisodium 8-hydroxypyrene-1,3,6-trisulfonate (pyranine) and 1,1′-dimethyl-4,4′-bipyridinium dichloride (methylviologen dichloride) were purchased from Tokyo Chemical Industries Co., Ltd. Methanol (MeOH) was purchased from Kanto Chemical Co., Inc. Distilled water used for spectroscopic experiments was purchased from Wako Pure Chemical Industries Co., Ltd. N,NDiisopropylethylamine was distilled from calcium hydride. Quinine sulfate employed as a standard for fluorescence quantum yield determination was recrystallized from water. Analytical thin-layer chromatography (TLC) was performed on E. Merck silica gel 60 F254 precoated plates (0.25-mm thickness). Preparative thin-layer chromatography (PLC) was performed on E. Merck silica gel 60 F254 precoated plates (2-mm thickness). Melting points (mp) were recorded on a YAZAWA BY-2. 1H and 13C NMR spectra were recorded on a JEOL JNM-A500 or ECA-500 spectrometer. Chemical shifts are reported in ppm from tetramethylsilane with reference to a signal due to the solvent [CHD2OD (δ 3.30) for 1H NMR and CD3OD (δ 49.0) for 13C NMR]. The following abbreviations are used to designate the signal multiplicities: s ) singlet, d ) doublet, t ) triplet, m ) multiplet, br ) broad peak. High-resolution mass spectra (HRMS) were recorded on a JEOL JMS-700 mass spectrometer under fast atom bombardment (FAB) conditions using m-nitrobenzyl alcohol as a matrix. UV-visible absorption spectra of the samples were measured on a JASCO V-560 spectrometer with a 400 nm/min scanning speed and a 2.0 nm bandwidth. Fluorescence spectra of the samples were measured on a JASCO FP-777 spectrofluorometer with a 200 nm/min scanning speed and a 1.5 nm bandwidth. (13) It is known that the vibronic fine structure of pyrene fluorescence is very sensitive to the solvent environment: Dong, D. C.; Winnik, M. A. Can. J. Chem. 1984, 62, 2560. (14) Research on amphiphilic pyrenes designed by this strategy is ongoing in our laboratory. A preliminary oral presentation has been given: Mizushima, T.; Murata, S. Proceedings of the Annual Meeting on Photochemistry 2002; Kyoto, 2002; p 315. (15) Winschel, C. A.; Kalidindi, A.; Zgani, I., Magruder, J. L.; Sidorov, V. J. Am. Chem. Soc. 2005, 127, 14704, and references therein. (16) Kriechbaum, M.; Wolfbeis, O. S.; Koller, E. Chem. Phys. Lipids 1987, 44, 19. (17) For a preliminary report, see: Sasaki, R.; Murata, S. Chem. Lett. 2007, 36, 364.
Sasaki and Murata Synthesis of Amphiphilic Pyranines. POC8. Trisodium 8-octyloxypyrene-1,3,6-trisulfonate (POC8) was synthesized as follows: To a refluxing solution of trisodium 8-hydroxypyrene-1,3,6trisulfonate (596.5 mg, 1.14 mmol) in MeOH was added 1-bromooctane (0.7 mL, 4.02 mmol) and N,N-diisopropylethylamine (0.5 mL, 2.94 mmol), and the resulting mixture was refluxed with stirring for 6 days. The solution was cooled to room temperature and then filtered and concentrated under reduced pressure. Purification by PLC on silica gel (CHCl3:MeOH:water ) 7:3:0.5) afforded POC8 (212.0 mg, 0.333 mmol, 28%) as a yellow solid: mp 335-338 °C (dec); 1H NMR (500 MHz, CD3OD) δ 9.38 (s, 1H), 9.25 (d, J ) 9.8 Hz, 1H), 9.19 (d, J ) 9.6 Hz, 1H), 9.11 (d, J ) 9.9 Hz, 1H), 8.66 (d, J ) 9.7 Hz, 1H), 8.40 (s, 1H), 4.45 (t, J ) 6.2 Hz, 2H), 2.08-2.03 (m, 2H), 1.70-1.64 (m, 2H), 1.52-1.46 (m, 2H), 1.411.38 (m, 2H), 1.32 (br, 4H), 0.91 (t, J ) 6.7 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ 154.4, 142.1, 138.1, 137.9, 131.3, 130.8, 128.5, 127.2, 126.6, 126.3, 124.6, 123.6, 122.5, 122.2, 110.5, 70.4, 33.0, 30.5, 30.4, 27.4, 23.7, 14.4; HRMS (FAB) calcd for C24H24Na3O10S3 [(M + H)+] 637.0225, found 637.0253. POC12. Trisodium 8-dodecyloxypyrene-1,3,6-trisulfonate (POC12) was prepared in 37% yield by a similar procedure to that described for POC8 using 1-bromododecane: yellow solid; mp 340 °C (dec); 1H NMR (500 MHz, CD OD) δ 9.39 (s, 1H), 9.24 (d, J ) 9.6 Hz, 3 1H), 9.18 (d, J ) 9.6 Hz, 1H), 9.11 (d, J ) 9.9 Hz, 1H), 8.65 (d, J ) 9.6 Hz, 1H), 8.39 (s, 1H), 4.45 (t, J ) 6.4 Hz, 2H), 2.09-2.03 (m, 2H), 1.71-1.65 (m, 2H), 1.49-1.46 (m, 2H), 1.28 (br, 22H), 0.88 (t, J ) 6.9 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ 154.4, 142.1, 138.0, 137.8, 130.8, 128.4, 127.1, 126.5, 126.3, 124.6, 123.6, 110.4, 70.3, 33.0, 30.7, 30.5, 30.4, 27.4, 23.7, 14.4; HRMS (FAB) calcd for C28H32Na3O10S3 [(M + H)+] 693.0851, found 693.0838. POC16. Trisodium 8-hexadecyloxypyrene-1,3,6-trisulfonate (POC16) was prepared in 22% yield by a similar procedure to that described for POC8 using 1-bromohexadecane: yellow solid; mp 300 °C (dec); 1H NMR (500 MHz, CD3OD) δ 9.38 (s, 1H), 9.24 (d, J ) 9.9 Hz, 1H), 9.19 (d, J ) 9.6 Hz, 1H), 9.11 (d, J ) 9.9 Hz, 1H), 8.66 (d, J ) 9.6 Hz, 1H), 8.40 (s, 1H), 4.45 (t, J ) 6.4 Hz, 2H), 2.09-2.03 (m, 2H), 1.70-1.64 (m, 2H), 1.51-1.46 (m, 2H), 1.27 (br, 22H), 0.88 (t, J ) 6.9 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ 154.4, 138.2, 137.9, 137.1, 131.3, 130.9, 128.5, 127.2, 126.6, 126.4, 124.7, 123.6, 122.5, 122.2, 70.4, 33.1, 30.8, 30.6, 30.5, 27.4, 23.7, 14.4; HRMS (FAB) calcd for C32H39Na4O10S3 [(M + Na)+] 771.1296, found 771.1298. POC20. Trisodium 8-eicosyloxypyrene-1,3,6-trisulfonate (POC20) was prepared in 11% yield by a similar procedure to that described for POC8 using 1-bromoeicosane: yellow solid; mp 302 °C (dec); 1H NMR (500 MHz, CD OD) δ 9.39 (s, 1H), 9.24 (d, J ) 9.8 Hz, 3 1H), 9.18 (d, J ) 9.6 Hz, 1H), 9.11 (d, J ) 9.9 Hz, 1H), 8.65 (d, J ) 9.6 Hz, 1H), 8.39 (s, 1H), 4.44 (t, J ) 6.5 Hz, 2H), 2.05 (m, 2H), 1.67 (m, 2H), 1.49 (m, 2H), 1.40-1.27 (br, 30H), 0.88 (t, J ) 6.9 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ 154.4, 142.1, 138.1, 137.9, 131.3, 130.9, 128.5, 127.2, 126.6, 126.3, 124.6, 123.6, 122.5, 122.2, 110.4, 70.4, 33.1, 30.8, 30.6, 30.5, 27.4, 23.7, 14.4; HRMS (FAB) calcd for C36H48Na3O10S3 [(M + H)+] 805.2103, found 805.2095. Fluorescence Quantum Yield Determination. Measurements of fluorescence quantum yields were carried out for 1 × 10-5 M aqueous solutions of POCn (n ) 8, 10, 12, and 20) in a quartz cell (10 mm × 10 mm) under air. All fluorescene spectra were recorded on excitation at 366 nm, and emission and excitation bandwidths were set at 1.5 nm. Quantum yields were determined by using the following equation
()
FAx Absst Ist nx Φx ) Φst FAst Absx Ix nst
2
where Φ is the fluorescence quantum yield, FA is the emission peak area, Abs is the optical density at the excitation wavelength, n is the refractive index of the solvent, I is the light intensity at the excitation wavelength, and both x and st are subscripts denoting an unknown sample (POCn) and a standard, respectively. A solution of quinine sulfate in 1 N H2SO4 was used as a standard (Φst ) 0.55).
Aggregation of Amphiphilic Pyranines in Water Determination of Critical Micelle Concentrations (cmcs). A solution of Nile Red in ethanol was placed in the vials and concentrated under reduced pressure. The samples were dried in vacuo overnight. An aqueous solution of POCn was added into the vials, and then the vials were sonicated for 10 min. The clear supernatant solution of the resulting mixture was collected, and the fluorescence spectra of the solution were recorded on excitation at 520 nm. The cmcs were evaluated on the basis of the fluorescence intensity of Nile Red. Absorption Spectra Measurements and Job Plot Experiments. An aqueous solution of POCn (1 × 10-5 M, 3 mL) was placed in a quartz cell (10 mm × 10 mm). The absorption spectral changes induced by the addition of MV2+ were recorded by repetition of the following cycles: to the POCn solution was added an aqueous solution of MV‚Cl2 (1 × 10-3 M, 20 µL), and after the mixture was stirred for 3 min, the absorption spectra were recorded. Job plot experiments were carried out for POC8/MV2+ and POC16/MV2+. Aqueous solutions of POCn containing various amounts of MV‚Cl2 were prepared with their total concentration kept constant, and the absorption spectra were measured. The difference of absorbance (∆Abs) was determined by subtracting the observed absorbance from that calculated by assuming the absence of the complex. Fluorescence Spectra Measurements and Quenching Studies. Fluorescence spectra were measured for an aqueous solution of POCn (1 × 10-5 M, 3 mL) in a quartz cell (10 mm × 10 mm) on excitation at 350 nm under air. The fluorescence quenching by the addition of MV2+ was recorded by repetition of the following cycles: to the POCn solution was added an aqueous solution of MV‚Cl2 (1 × 10-3 M, 20 µL), and after the mixture was stirred for 3 min, the fluorescence spectra were recorded. Relative fluorescence intensities (I0/I) were determined by measuring the peak of heights for the maxima. 1H NMR and 1H-1H COSY Spectra Measurement in Water. A solution of POCn in deuterium oxide (D2O) at a 1 × 10-2 M concentration was prepared by dissolving appropriate amounts of each compound in D2O. Solutions at 1 × 10-3 and 1 × 10-4 M concentrations were prepared by diluting each sample with D2O in sequence. Similarly, an aqueous solution of POCn at a 1 × 10-2 M concentration containing equimolar MV2+ was prepared by dissolving POCn and MV‚Cl2 in D2O, and solutions at 1 × 10-3 and 1 × 10-4 M concentrations were prepared by diluting each sample with D2O in sequence. 1H NMR was recorded on a JEOL ECA-500 spectrometer with a 5-mm indirect detection triple-axis gradient probe (IDT3G5005VJ). A deuterium lock (D2O) was used, and chemical shifts were reported in ppm from tetramethylsilane with reference to the signal of HDO (δ 4.75). Dynamic Light Scattering Studies. Dynamic light scattering experiments were performed on a Protein Solutions DynaPro-99D-50 dynamic scattering instrument (Charlottesville, VA). The measurement range for dynamic light scattering was set from 0.1 to 1000 nm. The average hydrodynamic radius was determined by using DYNAMICS operating software. All samples were measured at 20 °C. Calculations. The lengths of the POC16 and POC20 molecules were evaluated on the basis of the structures optimized by semiempirical molecular orbital calculations, which were carried out by the WinMOPAC (ver. 3.0, Professional) program package provided by Fujitsu. The geometrical optimizations were performed at the PM3 level of theory.
Results and Discussion Preparation of Amphiphilic Pyranines. In order to investigate the effect of hydrophobic alkyl chain length on photophysical properties and aggregation behaviors, four amphiphilic pyranines, in which the acidic hydrogen of POH was replaced by an octyl, dodecyl, hexadecyl, and eicosyl group, were synthesized. They are referred to in this paper as POCn (n ) 8, 12, 16, and 20), respectively (Figure 1). We were able to obtain all POCns by the direct Williamson alkylation of POH with the corresponding alkyl bromide. After many attempts to optimize reaction conditions, we found that the reaction of POH with a large excess
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Figure 1. Structures of pyranine POH and amphiphilic pyranines POCns. Table 1. Absorption and Fluorescence Spectral Data of Amphiphilic Pyranines in Water compound POC8 POC12 POC16 POC20
absorptiona λmax/nm (/M-1 cm-1)
fluorescencea,b λmax/nm
Φfc
290 (25 300), 374 (19 000), 405 (24 300) 290 (21 100), 375 (16 400), 405 (20 700) 290 (25 600), 375 (19 300), 405 (24 900) 290 (26 100), 375 (19 600), 405 (24 600)
436
0.856
434
0.863
433
0.849
432
0.860
[POCn] ) (1.2-1.3) × 10-5 M. The absorption and fluorescence spectra of POC20 are shown in Figure S1 (Supporting Information.) b λex ) 350 nm. c Fluorescence quantum yield (λex ) 366 nm). a
of the bromide in the presence of diisopropylethylamine as a base in refluxing methanol18 afforded the desired compounds in moderate yields. After purification using preparative thin-layer chromatography, all POCns were obtained as yellow granules, which were fully characterized by the use of 1H NMR spectra recorded in CD3OD. Photophysical Properties and Aggregation Behaviors of Amphiphilic Pyranines in Water. (1) Absorption and Fluorescence Spectra. All amphiphilic pyranines synthesized (POCn) were soluble in water. The absorption and fluorescence spectra of all POCns in water are practically identical at concentrations up to 10-4 M, indicating the absence of aggregation, even in the amphiphile having an extremely long alkyl chain (n ) 20). In spite of the presence of a long alkyl chain, the flexible dynamic motion of which could induce an additional nonradiative deactivation of the photoexcited state, all POCns exhibited relatively high fluorescence quantum yields of 0.85-0.86, which are slightly greater than that reported for parent POH19 but smaller than that in acidic media.20 The absorption and fluorescence spectral data obtained in an aqueous solution of POCns are summarized in Table 1. (2) Estimation of cmcs. As mentioned in the previous section, all POCns exhibited practically the same fluorescence spectra at concentrations up to 10-4 M. However, in aqueous solutions of POC16 and POC20 at a 10-3 M concentration, the fluorescence maximum appeared at around 445 nm. Since the red shift of the fluorescence maximum with an increase in concentration appeared to be a sign of aggregation of the chromophores, estimation of the cmcs of POCns was conducted by means of incorporating a hydrophobic fluorescent dye, Nile Red. An aqueous POC20 solution (1.3 × 10-4 M) prepared by sonication in the presence of Nile Red gave no fluorescence due to the dye, while in the solution of POC20 at 2.6 × 10-3 M, Nile Red fluorescence appeared at 646 nm on excitation of 520 nm. Moreover, in the solution at 1.4 × 10-2 M the intensity of Nile Red fluorescence was considerably enhanced and the fluorescence maximum was (18) Whitaker, J. E.; Hauglnad, R. P.; Moore, P. L.; Hewitt, P. C.; Reese, M.; Haugland, R. P. Anal. Biochem. 1991, 198, 119. (19) de Borba, E. B.; Amaral, C. L. C.; Politi, M. J.; Villalobos, R.; Baptista, M. S. Langmuir 2000, 16, 5900. (20) Tran-Thi, T.-H.; Prayer, C.; Millie´, Ph.; Uznanski, P.; Hymes. J. T. J. Phys. Chem. A 2002, 106, 2244.
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shifted to 634 nm (Figure S2, Supporting Information). Taking into account the fact that the fluorescence maximum of Nile Red is shifted to a shorter wavelength as the polarity of the environment of the dye decreases,21 this observation indicates that POC20 molecules start to aggregate in water with a cmc of ca. 3 × 10-3 M to form a hydrophobic domain in which the hydrophobic dye is incorporated22 and that the size of the domain increases in the solution at a 10-2 M concentration. Judging from the maximal wavelength of Nile Red emission of 634 nm, the interior of POC20 aggregates is slightly more hydrophobic compared with that of the micelles composed of sodium dodecyl sulfate (SDS).23 In this manner, the cmcs of POCns were estimated to be >20 mM for POC8, 20 mM for POC12, 10 mM for POC16, and 3 mM for POC20 (Figure S3, Supporting Information). Thus, it is found that, contrary to our expectation, all POCns exhibit relatively large cmc values. In particular, although it is expected that a C20 alkyl chain is long enough to induce an effective hydrophobic interaction, the cmc of POC20 is of the same order as that of SDS.24 These results are thought to be attributed to an intense electrostatic repulsion among the pyranine rings of POCns having triple negative charges, which prevents aggregation by the hydrophobic interaction. Unfortunately, these observations suggest that POCns are unsuitable as molecules to construct the aggregates of chromophores in which light energy is harvested effectively. (3) Conformation of Amphiphilic Pyranines in Water Determined by 1H NMR Studies. In the course of 1H NMR characterization of the synthesized POCns, we found an interesting solvent effect of signal positions. In the case of POC8, practically identical 1H NMR spectra were obtained in CD3OD and D2O, in which the triplet signal assigned to a terminal methyl group appeared in the highest magnetic region of δ 0.6-0.8 ppm. However, the 1H NMR spectrum of POC16, as well as POC20, recorded in D2O was dramatically different from that recorded in CD3OD, as shown in Figure 2. It should be pointed out that the multiplet signals appeared in a higher magnetic field region than the methyl triplet signal in the spectrum recorded in D2O, the intensity of which corresponded to 8 and 10 protons for POC16 and POC20, respectively. The concentration of POCn in D2O had little influence on the 1H NMR signal positions. However, a considerable broadening of signals was observed in the 1H NMR spectra of POC16 recorded at a 10-2 M concentration, while in the case of POC20 the broadening of 1H NMR signals started to be observed from a 10-3 M concentration. These observations indicate that the shift of 1H NMR signals to the higher magnetic field region recorded in D2O originates from not an intermolecular interaction among POCn molecules induced by aggregation, but a conformational change of an individual POCn molecule. With the aid of the 1H-1H COSY spectrum (Figure S5, Supporting Information), we concluded that, in the case of POC16, methylene protons connected to carbons from the 9 to 13 position of the 1-hexadecyl group could be responsible for the signals being shifted upfield, although a detailed assignment of the signals could not be achieved. It is reasonable to assume that the extreme upfield shift of the methylene signals is due to the ring current effect of the pyranine ring. Thus, it is proposed that POCns having a (21) Sackett, D. L.; Wolff, J. Anal. Biochem. 1987, 167, 228. (22) It is possible that molecules incorporated in micelle pseudophase affect the cmc of surfactants, as reported by Baptista and his co-workers: Junqueira, H. C.; Severino, D.; Dias, L. G.; Gugliotti, M. S.; Baptista, M. S. Phys. Chem. Chem. Phys. 2002, 4, 2320. Thus, Nile Red may affect the aggregation behavior in water of POCn, but we suppose that the effect of neutral and hydrophobic Nile Red on the cmc of POCn is not appreciably large. (23) Nizri, G.; Mgdassi, S. J. Colloid Interface Sci. 2005, 291, 169. (24) Helenius, A.; Simons, K. Biochim. Biophys. Acta 1975, 415, 29.
Sasaki and Murata
Figure 2. 1H NMR spectra of POC16 recorded in (a) CD3OD and (b) D2O (1.0 × 10-3 M, 500 MHz). The intense peaks around δ 5 in both spectra are due to water. 1H NMR spectra of other POCns recorded in D2O are illustrated in Figure S4 (Supporting Information).
Figure 3. Illustration of a plausible structure of POC16 in water estimated from its 1H NMR spectral data.
longer alkyl group (n ) 16 and 20) exist in a conformation where the pyranine nucleus is wrapped with a long methylene chain in water, and the methylene protons near a terminal methyl group are located in the shielding zone of the pyranine ring, as illustrated in Figure 3. This compact structure reduces the surface area of the molecule to minimize unfavorable interactions with water. However, this structure is entropically unstable, so in less polar methanol, the long alkyl chain of POCns is unwrapped from the pyranine nucleus to adopt a conformation having a flexible methylene chain. It seems that this compact conformation of POC16 and POC20 in water, which is unfavorable for an intermolecular hydrophobic interaction among long alkyl chains, is partly responsible for their relatively large cmc.
Aggregation of Amphiphilic Pyranines in Water
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Figure 5. Dependence of the absorbance for absorption maximum of POCn in water on the total concentration of MV2+ (Cq): POC8 (b), POC12 (O), POC16 (2). Broken straight lines are drawn by fitting the experimental data to the Benesi-Hildebrand equation (eq 1).
maximum was slightly displaced to a longer wavelength, and very weak absorption at around 440 nm appeared with an isosbestic point at 417 nm. A Job plot using absorption spectra was carried out to confirm that POC8 and MV2+ formed a complex having a 1:1 stoichiometry (Figure S6a, Supporting Information). According to the Benesi-Hildebrand model, when POC8 and MV2+ are in equilibrium with the 1:1 complex POC8/MV2+, the absorbance for the POC8 absorption maximum (A) correlates with a total concentration of MV2+ (Cq) by eq 1 Figure 4. Change in absorption spectrum of an aqueous solution of (a) POC8 (1.32 × 10-5 M) and (b) POC20 (1.32 × 10-5 M) with increasing concentration of MV2+. The corresponding data of POC12 and POC 16 are given in Figure S7 (Supporting Information).
Photophysical Properties and Aggregation Behaviors of Amphiphilic Pyranines in the Presence of MV2+. It is known that the fluorescence of pyranine POH is effectively quenched with MV2+ in water by the formation of an electrostatic complex. Baptista and his colleagues reported the photophysical and photochemical properties of POH/MV2+ complexes in aqueous and in micellar solutions,19 and the dynamics of the chargetransfer state formed through the photoexcitation of the complexes was investigated by femtosecond transient absorption experiments by Tran-Thi’s group.25 Moreover, the efficient quenching of POH fluorescence with MV2+ derivatives has been applied to sensors of biologically important molecules including glucose26 and guanosine triphosphate.27 Thus, in order to reveal the effect of a hydrophobic alkyl chain on POH/MV2+ complexation, the interaction of POCn with MV2+ in water was examined. (1) Absorption Spectral Changes with the Addition of MV2+. An addition of MV‚Cl2 to an aqueous solution of POCn (∼1 × 10-5 M) caused a decrease in the intensity of absorption due to POCn. In the typical case of POC8 (Figure 4a), as the concentration of MV2+ was increased, the intensity of the absorption due to POC8 gradually decreased, the absorption (25) Prayer, C.; Tran-Thi, T.-H.; Pommeret, S.; d’Oliveira, P.; Meynadier, P. Chem. Phys. Lett. 2000, 323, 467. (26) Gamsey, S.; Miller, A.; Olmstead, M. M.; Beavers, C. M.; Hirayama, L. C.; Pradhan, S.; Wessling, R. A.; Singaram, B. J. Am. Chem. Soc. 2007, 129, 1278, and references therein. (27) Neelakandan, P. P.; Hariharan, M.; Ramaiah, D. J. Am. Chem. Soc. 2006, 128, 11334.
S0 1 1 1 ) + A0 - A ∆l ∆lKs Cq
(1)
where S0 and A0 are the initial concentration of POC8 and the absorbance in the absence of MV2+, respectively, and l is the cell length. Moreover, ∆ and Ks represent the difference in extinction coefficient for the absorption maximum between POC8 and POC8/MV2+ and the complexation constant, respectively. As displayed in Figure 5, the plot of S0/(A0 - A) against 1/Cq gives a straight line, indicating that the change in the absorption spectrum of POC8 observed with the addition of MV2+ is reasonably explained in terms of the 1:1 complex formation of POC8 with MV2+. By the least-square analysis of the plot, we obtain Ks and ∆ as 2.5 × 104 M-1 and 3800 M-1 cm-1, respectively. The Ks value is comparable with that reported for POH in acidic media.19 In the same manner, the absorption spectral change induced by the addition of MV2+ was analyzed for the other three POCns. As shown in Figure 5, the plot for POC12 also gives a straight line, from which Ks and ∆ are evaluated to be 3.4 × 104 M-1 and 3800 M-1 cm-1, respectively. On the other hand, in the case of POC16 and POC20, a decrease in the intensity of the absorption caused by the addition of MV2+ was considerably larger compared with the case of POC8 and POC12. When the MV2+ concentration reached around 3 × 10-5 M, little further spectral change was observed (Figures 4b and S7b, Supporting Information). The Job plot indicates the 1:1 complex formation of POC16 and MV2+ (Figure S6b, Supporting Information), and the same Benesi-Hildebrand treatment according to eq 1 affords Ks > 105 M-1 for POC16. However, since the complexation of POCn with MV2+ is basically derived from an electrostatic interaction, a reasonable explanation cannot be given for the result that the
2392 Langmuir, Vol. 24, No. 6, 2008
Sasaki and Murata
Figure 6. Change in fluorescence spectrum of an aqueous solution of (a) POC8 (1.32 × 10-5 M) and (b) POC20 (1.32 × 10-5 M) with increasing concentration of MV2+ (λex ) 350 nm). The corresponding data of POC12 and POC16 are given in Figure S8 (Supporting Information).
complexation constant jumps considerably with an increase in the alkyl chain length from C12 to C16. Thus, these observations strongly suggest that the mechanism of the complexation of MV2+ with POC16 and POC20, which caused the absorption spectral change, is different from that with POC8 and POC12. To gain further information on the complexation of POCn with MV2+, we examined the fluorescence spectra in detail, which possess advantages over the absorption spectra in detecting small spectral changes with accuracy. (2) Quenching of Fluorescence by MV2+. In analogy with parent POH,19 the fluorescence of POCn (∼1 × 10-5 M in water, λex ) 350 nm) was quenched by the addition of MV2+. As suggested in the absorption studies, the quenching efficiency was considerably dependent on the alkyl chain length of the POCns. Figure 6a displays a decrease in the fluorescence intensity of POC8 with the addition of MV2+. As shown in Figure 7a, the quenching data of POC8, as well as those of POC12, obey the Stern-Volmer equation (eq 2)
I0 ) 1 + KqCq I
(2)
where I0 and I are the fluorescence intensity in the absence and presence of quencher, respectively, and Kq is the quenching constant. By least-square analysis of the plots, Kq is obtained as 2.6 × 104 M-1 for POC8 and 2.9 × 104 M-1 for POC12. These Kq values are in fair agreement with the Ks values obtained by the Benesi-Hildebrand plots of the change in the absorption spectra, indicating that the fluorescence quenching occurs by a
Figure 7. Dependence of the intensity of POCn fluorescence in water on the total concentration of MV2+: POC8 (b), POC12 (O), POC16 (2), POC20 (4). (a) Difference in the quenching efficiency with the length of alkyl chain. Broken straight lines for POC8 and POC12 are drawn by fitting the experimental data to the SternVolmer equation (eq 2). (b) Detailed quenching data for POC16 and POC20. Broken lines represent the curves fitted with the experimental data by using the nonlinear least-squares analysis according to eq 5.
static mechanism. Thus, the fluorescence quenching, as well as the change in the absorption spectra, of POCn having a shorter alkyl chain (n ) 8 and 12) observed with the addition of MV2+ is thoroughly explained by assuming that POCn and MV2+ form a complex having a 1:1 stoichiometry of POCn/MV2+ with a complexation constant of ∼3 × 104 M-1 in water. As shown in Figures 6b and S8b, however, the fluorescence quenching of POCn having a longer alkyl chain (n ) 16 and 20) occurred much more effectively compared with that of POC8 and POC12. Furthermore, it should be pointed out that the plot of I0/I against Cq (Figure 7a,b) gave a curve deviating strongly upward from the straight line predicted by the Stern-Volmer equation, and the deviation for POC20 is much larger than that for POC16. Baptista and his co-workers reported Stern-Volmer plots with upward curvature in quenching of POH with MV2+ in acidic and basic media and nicely analyzed the plots by eq 3, which considers the coexistence of static and dynamic quenching processes19
( )
I0 -1 I ) Kqd + Kqs + KqdKqsCq Cq
(3)
Aggregation of Amphiphilic Pyranines in Water
Langmuir, Vol. 24, No. 6, 2008 2393
Scheme 1. Aggregate Formation of POCn (n ) 16 and 20) in the Presence of MV2+
where Kqd and Kqs are the quenching constants for the dynamic process and the static process, respectively. However, it is confirmed that neither the plot of I0/I against Cq for POC16 nor that for POC20 follows this equation. Again, the plot of I0/I deviated strongly upward from the line predicted by eq 3. It is apparent that the efficiency of the fluorescence quenching of POCns by MV2+ is highly dependent on the alkyl chain length, suggesting that the aggregation of POCn molecules by a hydrophobic interaction participates in the quenching process. Thus, we propose the following mechanism for the observed effective quenching of the fluorescence of POCn having a longer alkyl chain (n ) 16 and 20) by MV2+. Upon addition of MV2+ to a solution of POCn, the complexation of POCn with MV2+ is caused by π-stacking and electrostatic interactions to form the complex POCn/MV2+ with a complexation constant of Ks. In the case of POCns having a shorter alkyl chain (n ) 8 and 12), their fluorescence quenching with MV2+ is completely explained by this scenario, while for POCns having a longer alkyl chain (n ) 16 and 20), an additional intermolecular interaction acts among POCns molecules. In the complex POCn/MV2+, the long alkyl chain is unwrapped from the pyranine nucleus and the electrostatic repulsion among polar pyranine moieties is considerably reduced compared with POCn because the triple negative charges of POCn are partially neutralized by the complexation with MV2+, having double positive charges. Therefore, a hydrophobic interaction among long alkyl chains of the complex POCn/MV2+ operates to induce the formation of aggregates (POCn/MV2+)agg, which shifts the equilibrium to the direction of the complexation of POCn with MV2+ (Scheme 1). It is reasonable to think that the only POCn molecules free from complexation fluoresce because the POCn excited-state is deactivated predominantly by a rapid charge transfer to MV2+ in the complex POCn/MV2+ and its aggregate (POCn/MV2+)agg. Moreover, it can be assumed that once the aggregates (POCn/ MV2+)agg are formed in the solution, the concentration of the free complex POCn/MV2+ is kept constant at its cmc (Sc) with an increase in the total concentration of MV2+. Therefore, the ratio of the fluorescence intensity in the absence of MV2+ to that in the presence of MV2+ (I0/I), which is equal to the ratio of the concentration of POCn free from the complexation in the absence of MV2+ to that in the presence of MV2+ (S0/S), correlates with the total concentration of MV2+ (Cq) by eq 4. (Derivation of eq 4 is given in the Supporting Information.)
(
I 0 S0 K s C - S0 + ) I 2 Sc q
x
(Cq - S0)2 +
)
4Sc Ks
(4)
Since S0 is equal to the initial concentration of POCn, if we define parameter R as R ) Ks/Sc, eq 4 is rewritten as eq 5.
(
I 0 S 0R Cq - S0 + ) I 2
x(C - S ) + R4) 2
q
0
(5)
As illustrated with the broken lines in Figure 7b, nonlinear least-square analysis revealed that the plots of I0/I against Cq for POC16 and POC20 were satisfactorily reproduced by eq 5 with R ) 8.4 × 1010 and 8.3 × 1011 M-2, respectively. It is reasonable
to think that complexation constants Ks of POC16 and POC20 are comparable with that of POCn having a shorter alkyl chain (n ) 8 and 12), because the complexation is basically caused by an electrostatic interaction of the pyranine nucleus with MV2+ and the electronic property of the pyranine chromophore of POCn is almost independent of the alkyl chain length. By assuming Ks ) 3.0 × 104 M-1, cmcs of the complex POC16/MV2+ and POC20/ MV2+ (Sc ) Ks/R) are estimated to be 3.6 × 10-7 and 3.6 × 10-8 M, respectively. Thus, it is revealed that the cmc of POC20, which is estimated to be ca. 3 × 10-3 M in the absence of MV2+, is reduced 105 times by the addition of MV2+. It is likely that the reduction of electrostatic repulsion and the conformational change of the long alkyl chain caused by the complexation with MV2+ are responsible for the facile aggregation of the complex POC16/MV2+ and POC20/MV2+. Moreover, it should be pointed out that the POC16/MV2+ cmc value of 3.6 × 10-7 M is significantly smaller compared with those of surfactants having a single negative charge on their polar head and a hydrophobic alkyl chain of a similar length:28 1.05 × 10-3 M for CH3(CH2)15SO3Na, 5.4 × 10-4 M for CH3(CH2)15OSO3Na, and 5.35 × 10-4 M for p-CH3(CH2)15C6H4SO3Na. This observation indicates that the π-stacking of the pyranine and MV2+ aromatic rings including a charge-transfer interaction contributes largely to the facile aggregation of the POCn/MV2+ complexes.29 (3) EVidence for Aggregate Formation. As demonstrated in the previous section, the unusually effective fluorescence quenching of POCn having a long alkyl chain (n ) 16 and 20) by MV2+ is successfully explained by the mechanism involving the aggregate formation of the POCn/MV2+ complex. In order to obtain experimental evidence supporting the aggregate formation, 1H NMR and dynamic light scattering (DLS) experiments were conducted. In the 1H NMR spectra of a D2O solution of a 1:1 mixture of POC8 and MV‚Cl2 at a 1.0 × 10-4 M concentration, all signals were observed but were slightly broadened, and the chemical shifts of pyranine and MV2+ aromatic protons shifted upfield compared with those recorded in separate D2O solutions of POC8 and MV‚Cl2. Even at a 1.0 × 10-3 M concentration, all signals were observed and aromatic protons appeared in a region further upfield (Figure S9, Supporting Information). These observations are consistent with the results obtained by the absorption and fluorescence experiments that POC8 and MV2+ exist in equilibrium with the complex POC8/MV2+. The proportions of the complex are evaluated to be 54 and 82% for a total POC8 concentration of 1.0 × 10-4 and 1.0 × 10-3 M, respectively, on the basis of a complexation constant Ks of 2.5 × 104 M-1. It is reasonable to assume that in the complex POC8/MV2+, the aromatic protons of the POC8 pyranine ring are located in the shielding zone of the MV2+ aromatic ring, and vice versa, to induce upfield shifts of all aromatic protons. Analogous results were obtained in the 1H NMR experiments for POC12, although a large signal broadening was observed at a 1.0 × 10-3 M concentration. On the other hand, in the 1H NMR spectra of a D2O solution of a 1:1 mixture of POCn having a longer alkyl chain (n ) 16 (28) Kikukawa, K. Ed. The Handbook of Oil Chemistry: Lipids and Surfactants; Maruzen; Tokyo, 2001. (29) We examined the effect of other dicationic species on the aggregation behavior of POCn having a longer alkyl chain. However, the 1H NMR spectrum of a D2O solution of a 1:1 mixture of POC16 and CaCl2 at a 1.0 × 10-4 M concentration was identical to that recorded in the absence of Ca2+, although broadening of signals was observed at a 1.0 × 10-3 M concentration. These observations demonstrate that the aggregation of POC16 in water is promoted by the addition of Ca2+, but its effect is significantly smaller compared with MV2+, indicating the large contribution of π-stacking to the facile micelle formation of POC16 in the presence of MV2+.
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and 20) and MV‚Cl2 at a 1.0 × 10-4 M concentration, the signals assigned to the POCn and MV2+ protons were hardly detected, owing to an extreme broadening of these signals. At a 1.0 × 10-3 M concentration, considerably broad and fused signals appeared in aromatic and aliphatic regions, together with a broad singlet assigned to the methyl protons of MV2+ at δ 3.8-3.9 (Figure S10, Supporting Information), which shifted further upfield compared with δ 4.2 observed in the solution of a mixture of POCn having a shorter alkyl chain (n ) 8 and 12) and MV‚Cl2. These observations substantiate the assumption that complexes of POCn having a longer alkyl chain (n ) 16 and 20) and MV2+ form aggregates, in which the motions of POCn and MV2+ molecules are enormously restricted and the π-stacking structure of the POCn and MV2+ aromatic rings in the aggregates induces a larger anisotropic magnetic effect on their protons. More reliable evidence of aggregate formation in the complex POCn/MV2+ (n ) 16 and 20) was obtained by DLS experiments (Figure S11, Supporting Information). The measurement of the particle size distribution of an aqueous solution of a l:1 mixture of POC16 and MV‚Cl2 at a 1.2 × 10-3 M concentration using the DLS method revealed the formation of particles with a hydrodynamic diameter of ca. 5.2 nm. By the measurement of POC20 under the same conditions, the slightly larger value of the hydrodynamic diameter (ca. 5.5 nm) was obtained. Thus, the DLS studies unambiguously demonstrate that the complex POCn/ MV2+ (n ) 16 and 20) aggregates to form micelle-size particles in water. Since the length of the POCn molecule having a fully extended conformation of the alkyl chain is estimated to be 3.1 and 3.6 nm for n ) 16 and 20, respectively (Figures S12 and S13, Supporting Information), the hydrodynamic diameters observed are not inconsistent with the size of the micelles composed of the complex.
Conclusions We synthesized four amphiphilic pyranines, POCns, in which the acidic hydrogen of parent pyranine POH was replaced by a C8, C12, C16, and C20 alkyl group and investigated their spectroscopic properties and aggregation behaviors in water. It was disappointing that the cmcs of the amphiphilic pyranines were relatively large, ca. 3 mM, even for POC20 having an extremely long hydrophobic alkyl chain. 1H NMR studies revealed that POCns having a long alkyl chain (n ) 16 and 20) exist in a conformation in which the pyranine nucleus is wrapped with a long methylene chain. It appears that this compact structure, as well as the large electrostatic repulsion among the pyranine rings having triple negative charges, is responsible for their relatively large cmcs. As in the case of parent pyranine, the addition of MV2+ to an aqueous solution of POCn caused the formation of the complex POCn/MV2+, which induced the absorption spectral change and quenching of POCn fluorescence. In the case of POCn having a shorter alkyl chain (n ) 8 and 12), the dependence of the absorption spectral change, as well as fluorescence quenching, on the total concentration of MV2+ was thoroughly explained in terms of the complex formation with a complexation constant of ∼3 × 104 M-1. However, an unexpectedly larger dependence was observed for POCn having a longer alkyl chain (n ) 16 and 20). We proposed that in the complex POCn/MV2+ (n ) 16 and 20), the aggregates of the complexes were formed by the reduction of electrostatic repulsion and the conformational change of the long alkyl chain caused by the complexation, which shifted the equilibrium further in the direction of the complexation of POCn and MV2+. The aggregate formation was confirmed by 1H NMR and DLS studies. On the basis of analysis of the fluorescence quenching data, the cmc of
Sasaki and Murata
the complex POC20/MV2+ was estimated to be 3.6 × 10-8 M, indicating that the cmc of POC20 decreased 105 times by the addition of MV2+. As mentioned in the Introduction, supramolecular assemblies composed of molecules having chromophores are of great interest from the viewpoint of mimicking natural light-harvesting complexes. Unfortunately, it appears that, in spite of the large extinction coefficient in the near-ultraviolet region, the amphiphilic pyranines described in this paper are not suitable molecules with which to construct artificial light-harvesting systems, because the attractive interaction among the molecules is not sufficient to form stable aggregates. To overcome this problem, novel amphiphilic pyranines having double hydrophobic alkyl chains have been designed, and their synthesis is ongoing in our laboratory. On the other hand, it is revealed that the formation of micelles is readily induced by the addition of MV2+ to a solution of POCn having a longer alkyl chain in water. The micelle of the complex POCn/MV2+ provides a new class of supramolecular aggregation systems of chromophores, which are composed of an electron-donating chromophore and an electron acceptor. Upon photoexcitation of POCn in the POCn/ MV2+ micelles, the excitation energy is quenched rapidly through electron transfer to MV2+ to produce the charge separation state [POCn+•/MV+•]. If the charge generated is delocalized in the micelles through interaction with the other POCn/MV2+ complexes, it is expected that the charge separation state will have a long lifetime.30 Work is in progress to construct supramolecular assembly systems based on amphiphilic pyrene derivatives and to elucidate their photophysical and photochemical properties. Acknowledgment. This work was supported by a Grantin-Aid for Scientific Research 16550030 from the Japan Society for the Promotion of Science. This work (R.S.) was also supported by Research Fellowships of 21st century COE program for Frontiers of fundamental chemistry from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Prof. Hiroshi Hirota and Miss Wakana Ohashi in RIKEN for help with DLS measurements. Supporting Information Available: Absorption and fluorescence spectra of POC20 recorded in water, fluorescence spectra recorded in an aqueous solution of POC20 prepared in the presence of Nile Red, the dependence of the intensity of Nile Red fluorescence on the concentration of POCn, 1H NMR spectra of POCn (n ) 8, 12, and 20) in D2O, 1H-1H COSY spectrum of POC16 in D2O, Job plot for the complex formation of MV2+ with POCn (n ) 8 and 16), the change in absorption and fluorescence spectra of POCn (n ) 12 and 16) with the addition of MV2+, 1H NMR spectra of POC8 and POC20 in the presence of equimolar amounts of MV2+ in D2O, DLS data of POC16/MV2+ and POC20/MV2+, geometry of POC16 and POC20 optimized with PM3, and the derivation of eq 4. This information is available free of charge via the Internet at http://pubs.acs.org. LA702849C (30) We performed nanosecond laser flash photolysis (LFP) studies for the detection of MV+• under the irradiation of an aqueous solution containing POC16/ MV2+ micelles by using a pulse Q-switch Nd:YAG laser (SOLAR LF114) as the excitation source (355 nm, 20 ns, 4 mJ pulse-1). However, no transient absorption due to MV+•(605 nm) could be observed. Thus, at the present stage, we have no experimental data indicating that a lifetime of the charge separation state elongates in the micelles. It is known that the charge separation state generated by the irradiation of the complex PO-/MV2+ has a considerably short lifetime. TranThi’s group reported that in LFP studies of the complex PO-/MV2+ the singlet excited state of the complex was detected with a lifetime of 3.7 ps, but the rise of any absorption that could correspond either to PO• or MV+• was not observed, suggesting that the charge separation state [PO•/MV+•] relaxed to the ground state more quickly than its formation (ref 25).
