Manganese Porphyrin-Mediated Electron Transfer ... - ACS Publications

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Langmuir 1998, 14, 407-416

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Manganese Porphyrin-Mediated Electron Transfer across a Liposomal Membrane and on an Electrode Modified with a Lipid Bilayer Membrane Mamoru Nango,*,† Takami Hikita,† Takanori Nakano,† Taku Yamada,† Morio Nagata,† Yukihisa Kurono,‡ and Toshiaki Ohtsuka† Department of Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466, Japan, and Department of Pharmaceutics, Faculty of Pharmaceutical Science, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467, Japan Received May 5, 1997. In Final Form: August 27, 1997X Phospholipid-linked mesoporphyrins separated by spacer methylene groups (Cn), PE-Cn-MnMPMME (n ) 0, 5, 11) (Chart 1) were synthesized. Manganese porphyrin-mediated electron transfers across a liposomal membrane and on a glassy carbon (GC) electrode covered with a phospholipid bilayer membrane were examined to provide insight into the effect of the structure of porphyrins on these electron transfers between porphyrins in lipid bilayers. Enhanced transmembrane electron transfers catalyzed by phospholipid-linked manganese porphyrin derivatives separated by spacer methylene groups (Cn)(n ) 0, 5, 11) (Chart 1) were observed in a liposomal membrane only above the phase temperature of the membrane, depending on the length of the Cn and on the structure of the porphyrins. Furthermore, cyclic voltammetry on a SnO2 glass electrode or on a GC electrode modified with a DPPC membrane containing manganese porphyrins showed that one set of waves was clearly visible corresponding to the consecutive monoelectronic reduction of the manganese porphyrin unit (Mn(III)/Mn(II)) only above the phase temperature of the membrane. Phospholipid-linked manganese porphyrins on the electrode modified with a lipid bilayer membrane also caused enhanced electron transfers, depending on the length of the Cn but not on the structure of the porphyrins. Comparison of manganese porphyrin-mediated electron transfers between porphyrins in a liposomal membrane and porphyrins on a GC electrode modified with a lipid bilayer membrane is made.

Introduction Synthetic porphyrin models can be very helpful in studying the effect of porphyrin structure in electron transfer reactions of biological processes.1-14 Porphyrin pigments play a key role in these electron transfers.15-18 * Address correspondence to this author. Telephone and Fax: 052-735-5226. E-mail: [email protected]. † Nagoya Institute of Technology. ‡ Nagoya City University. X Abstract published in Advance ACS Abstracts, December 15, 1997. (1) (a) Nango, M.; Kryu, H.; Loach, P. J. Chem. Soc., Chem. Commun. 1988, 697-698. (b) Nango, M.; Higuchi, M.; Gondo, H.; Hara, M. J. Chem. Soc., Chem. Commun. 1989, 1550-1553. (c) Nango, M.; Mizusawa, A.; Miyake, T.; Yoshinaga, J. J. Am. Chem. Soc. 1990, 112, 1640-1642. (d) Nango, M.; Iida, K.; Kawakita, T.; Matsuura, M.; Harada, Y.; Yamashita, K.; Tsuda, K.; Kimura, Y. J. Chem. Soc., Chem. Commun. 1992, 545-547. (e) Nango, M. MEMBRANE 1992, 17, 105-114. (f) Iida, K.; Nango, M.; Hikita, M.; Tajima, T.; Kurihara, T.; Yamashita, K.; Tsuda, K.; Dewa, T.; Komiyama, J.; Nakata, M.; Ohtsuka, Y. Chem. Lett. 1994, 1157-1160. (g) Iida, K.; Nango, M.; Hikita, M.; Hattori, A.; Yamashita, K.; Yamauchi, K.; Tsuda, K. Chem. Lett. 1994, 753-756. (h) Iida, K.; Nango, M.; Okada, K.; Hikita, M.; Kurihara, T.; Tajima, T.; Hattori, A.; Ishikawa, S.; Yamashita, K.; Tsuda, K.; Kurono, Y. Bull. Chem. Soc. Jpn. 1995, 68, 1959-1968. (2) (a) Dewa, T.; Satoh, M.; Komiyama, J.; Nango, M.; Tsuda, K. Macromol. Chem. Phys. 1994, 195, 2917-2929. (b) Dewa, T.; Mitsuru, S.; Komiyama, J.; Nango, M.; Tsuda, K. Macromol. Chem. Phys. 1994, 195, 1031-1041. (3) (a) Nango, M.; Dannhauser, T.; Huang, D.; Spears, K.; Morrison, L.; Loach, P. A. Macromolecules 1984, 17, 1898-1902. (b) Dannhauser, T.; Nango, M.; Oku, N.; Anzai, K.; Loach, P. J. Am. Chem. Soc. 1986, 108, 5865-5871. (c) Nango, M.; Iida, K.; Yamaguchi, M.; Yamashita, K.; Tsuda, K.; Mizusawa, A.; Miyake, T.; Masuda, A.; Yoshinaga, J. Langmuir 1996, 12, 1981-1988. (d) Iida, K.; Nango, M.; Matsuura, M.; Yamaduchi, M.; Sato, K; Tanaka, K.; Akimoto, K.; Yamashita, K.; Tsuda, K.; Kurono, Y. Langmuir 1996, 12, 450-458. (4) Wasielewski, M. R. Chem. Rev. 1992, 92, 435-461. (5) Photoinduced Electron Transfer; Fox, M. A., Chanon, M., Eds.; Elsevier: Amsterdam, 1988.

There has been little study of ground-state electron transfer between porphyrin complexes to provide an insight into the effect of the structure of porphyrins on the electron transfer, so that a vectorial electron-transfer system between porphyrins may be constructed in the membrane.3,8 Groves et al. reported that vectorial electron transfer and selective, oxidative catalysis have been observed, in which membrane-binding metalloporphyrins were designed and developed in phospholipid arrays to assemble mulicomponent constructs.8 In our previous papers, we synthesized and characterized polymer-linked halogenated tetraphenylporphyrin derivatives and covalently linked porphyrin dimers, in which transmem(6) Sakata, Y.; Nakashima, S.; Goto, Y.; Tatemitsu, H.; Misumi, S.; Asahi, T.; Hagihara, M.; Nishikawa, S.; Okada, T.; Mataga, N. J. Am. Chem. Soc. 1989, 111, 8979-8981. (7) Cusanovich, M. A. Photochem. Photobiol. 1991, 53, 845-857. (8) Groves, J. T.; Fate, G. D.; Lahiri, S. J. Am. Chem. Soc. 1994, 116, 5477-5478. (9) Gust, D.; Moore, T. A.; Moore, A. L.; Leggett, L.; Lin, S.; Degraziano, J. M.; Hermant, R. M.; Nicodem, D.; Craig, P.; Seely, G. R.; Nieman, R. A. J. Phys. Chem. 1993, 97, 7926-7931. (10) Gust, D.; Moore, T. A.; Moore, A. L. Acc. Chem. Res. 1993, 26, 198-205. (11) Stein-berg, G.; Liddell, P. A.; Hung, S-H.; Moore, T. A.; Gust, D.; Moore, A. L. Nature 1997, 385, 239-241. (12) Johnson, D. G.; Niemczyk, M. P.; Minsek, D. W.; Wiederrecht, G. P.; Svec, W. A.; Gaines, G. L., III; Wasielewski, M. R. J. Am. Chem. Soc. 1993, 115, 5692-5701. (13) Osuka, A.; Maruyama, K.; Mataga, N.; Asahi, T.; Yamazaki, I.; Tamai, N. J. Am. Chem. Soc. 1990, 112, 4958-4959. (14) Helms, A.; Heiler, D.; McLendon, G. J. Am. Chem. Soc. 1992, 114, 6227-6238. (15) The Photosynthetic Bacteria; Clayton, R. K., Sistrom, W. R., Eds.; Elsevier/North-Holland Biochemical Press: New York, 1978. (16) Topics in Photosynthesis; Barber, J., Ed.; Elsevier/North-Holland Biochemical Press: New York, 1979. (17) Photosynthesis; Govinjee, Ed.; Academic Press: New York, 1982. (18) Chlorophylls; Sheer, H., Ed.; CRC Press: Boca Raton, FL, 1991.

S0743-7463(97)00463-0 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/20/1998

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Chart 1. Manganese Porphyrins and Phospholipid-Linked Manganese Porphyrins

brane electron transfer between porphyrins catalyzed by manganese porphyrins was observed, depending on the structure of the porphyrin derivative.1f,2,3a,b,d Moreover, transmembrane electron transfer catalyzed by phospholipid-linked manganese tetraphenylporphyrin derivatives with separated methylene groups (PE-Cn-MnTTP and PECn-MnPFPP) (n ) 0, 5, 11) (Chart 1) which can be easily immersed into the lipid bilayer is reported.1c,3c The phospholipid-linked manganese porphyrins caused an enhanced electron transfer, depending upon the length of the spacer methylene group (Cn) and also the structure of manganese porphyrins. Alternatively, a large number of chemists have identified that electroactive species such as porphyrins, myoglobin, flavins, viologens, ferrocenes, and quinones can be immobilized on electrode surfaces.1g,19-24 However, there has been little study of electron transfer between porphyrin complexes on an electrode modified with lipid bilayers.1g In the previous paper,1g,2 enhanced electron transfer between porphyrins in polypeptide membranes cast on a galssy carbon electrode or in those membranes was observed especially in hydrophilic polypeptide membranes and also when mesoporphyrin derivatives were used. The result indicated that differences in membrane solubility or mobility of porphyrin and imidazole derivatives are as important as the differences in electron transfer rates among the various porphyrins. Thus, by selection of the proper membrane component and by appropriate choices of the porphyrins, a specific electron transfer at the membrane surface and in lipid bilayers can be studied. These characteristics are of considerable interest to mimic the vectorial electron channel controlled in biological membranes and also to construct some selective electron (19) Molecular Design of Electrode Surfaces; Murray, R.W., Ed.; Techniques of Chemistry Series Vol. 22; Wiley-Interscience: New York, 1992. (20) Subramanian, M.; Mandal, S.-K.; Bhattacharya, S. Langmuir 1997, 13, 153-160. (21) Rojas, M. T.; Han, M.; Kaifer, A. E. Langmuir 1992, 8, 1627. (22) Nakashima, N.; Wake, S.; Nishino, T.; Kunitake, M.; Manabe, O. J. Electroanal. Chem. 1992, 333, 345-351. (23) (a) Rusling, J. F.; Nassar, A.-E. F. J. Am. Chem. Soc. 1993, 115, 11891-11897. (b) Nassar, A.-E. F.; Narikiyo, Y.; Sagara, T.; Nakashima, N. J. Chem. Soc., Faraday Trans. 1995, 91 (12), 1775-1782. (24) Ohtsuka, T.; Hikita, T.; Nango, M. J. Electroanal. Chem. in press.

transfer systems in lipid bilayers. In this paper, we now present the synthesis of phospholipid-linked mesoporphyrins separated by spacer methylene groups (Cn), PECn-MPMME (Chart 1), and the study of electron transfer of the manganese complex across a liposomal membrane and on an electrode modified with a lipid bilayer membrane to provide an insight into the effect of the structure of porphyrins on the electron transfer. We reasoned that mesoporphyrin derivatives can be more embedded into the lipid bilayers than tetraphenylporphyrin derivatives.1g Furthermore, manganese mesoporphyrin derivatives showed an enhanced electron transfer on electrodes modified with keratin and lipid bilayer membranes in comparison to manganese tetraphenylporphyrin derivatives.1g,2 Thus, it is expected that the mesoporphyrin complex is being utilized to systematically examine electron transfer between porphyrins in these lipid-bilayer systems. Our goal is to provide insight into the effect of the structure of porphyrins on porphyrin-mediated electron transfer between porphyrins in a lipid-bilayer membrane as well as to construct vectorial electrontransfer systems of porphyrin derivatives in lipid bilayers. Comparison of the structures of porphyrin derivatives on these electron transfers is a liposomal membrane or on an electrode modified with lipid bilayers is made. Experimental Section General Methods. All reactions and chromatographic separations were carried out in minimum room light. Benzene, chloroform, dichloromethane, pyridine, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), and methanol were distilled and stored over molecular sieves. Other solvents used were spectral grade or better quality. The silica gel used for dry column chromatography was Woelem Silica TSC, activity III/30 mm, obtained from ICN Pharmaceuticals. Merck silica gel GF Uniplates of 1000- and 250-µm thickness were used for preparative or analytical thin layer chromatography. 1H NMR spectra were taken with Jeol JNM-GX-270 and Jeol JNM-GX-400 instruments with tetramethylsilane as an internal standard for CDCl3 and DMSO-d6. The UV-visible absorption and fluorescence spectra were recorded on Hitachi-124 and Jeol FP-777 spectrophotometers, respectively. Mass spectra (MS) were obtained in the FAB mode, in a 3-nitrobenzyl alcohol or thioglycerol matrix, with a JMS-DX300 Jeol instrument. Highpurity egg yolk phosphatidylcholine (egg PC), dimyristoylphos-

Manganese Porphyrin-Mediated Electron Transfer phatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) and dipalmitoylphosphatidylethanolamine (PE) were kindly provided by Nippon Fine Chemical Co. LTD., Takasago City, Hyogo, Japan. Protoporphyrin-IX dimethyl esters were kindly provided by Hamari Pharmaceutical Co. LTD., Osaka, Japan. Synthetic Procedure. 5,10,15,20-tetra-p-tolylporphyrin, H2TTP, 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin, H2PFPP, and their manganese complexes, MnTTP and MnPFPP, were prepared as described in our previous paper.3 Phospholipidlinked manganese porphyrins with separated methylene groups (PE-Cn-MnTTP and PE-Cn-MnPFPP) (n ) 0, 5, 11) (Chart 1) were also prepared as described previously.3c,d Mesoporphyrin-IX Dimethyl Esters, H2MPDME.25 Protoporphyrin-IX dimethyl esters (2.0 g, 3.39 mmol) were dissolved in formic acid (200 mL) containing methyl methacrylate (2.00 mL) and palladium carbon (2.16 g). The mixture was stirred at 50 °C for 85 min under hydrogen gas, and then 250 mL of diethylether was added. After separation of precipitated palladium, 700 mL of 1% hydrochloric acid was added and then diethyl ether was removed. The acidic solution was neutrized with 10 N NaOH, and then the mesoporphyrin dimethyl esters were extracted into chloroform. The combined extracts were washed several times with water and dried over Na2SO4, and the solvent was removed under reduced pressure. The sample was purified by silica gel chromatography with chloroform containing 0.1% triethylamine as eluent. The yield was 78.8% (1.587 g). 1H NMR (CDCl3): δ -4.0 (2H, s, pyrrole-NH), 1.8 (6H, t, Ar-CH2CH3), 3.2 (4H, m, Ar-CH2CH2COOCH3), 3.5 (12H, m, Ar-CH3), 3.6 (6H, m, Ar-CH2CH2COOCH3), 4.0 (4H, m, Ar-CH2CH3), 4.4 (4H, m, Ar-CH2CH2COOCH3), 10.0 (4H, d, mesoCHdC). UV/vis (CH2Cl2-10% EtOH): λmax 397.5 nm ( 169 mM-1 cm-1) (M ) mol dm-3), 497.0 (14.0), 531.0 (11.0), 567.0 (7.7), 619.0 (4.6). MS(FAB): m/z 595 (MH+). Mesoporphyrin-IX Monomethyl Ester, H2MPMME.26 H2MPDME (1.5 g, 2.5 mmol) was dissolved in 4 N HCl (157 mL), and the mixture was stirred at room temperature for 40 min. The solution was acidified with 2 N HCl to pH 4, and a 0.25 N aqueous ammonium solution was added to pH 8. Chloroform (200 mL) and distilled water (300 mL) were added, and a purple precipitate appeared and was extracted into chloroform. The combined extracts were washed several times with water and dried over Na2SO4, and the solvent was removed under reduced pressure. The sample was purified by silica gel chromatography with chloroform or chloroform-methanol ) 20:1 (v/v) as eluent. The yield of H2MPMME was 45.4% (0.655 g). 1H NMR (CDCl3): δ -4.1 (2H, s, pyrrole-NH), 1.8 (6H, t, Ar-CH2CH3), 3.2 (4H, m, Ar-CH2CH2COOCH3), 3.5-3.6 (15H, m, Ar-CH3 and Ar-CH2CH2COOCH3), 4.0 (4H, m, Ar-CH2CH3), 4.2 (4H, m, Ar-CH2CH2COOCH3), 9.9 (4H, m, meso CHdC). MS(FAB): m/z 585 (MH+). H2MPMME-CONH(CH2)5COOH. H2MPMME (150 mg, 0.258 mmol) was dissolved in dichloromethane (25 mL). NHydroxysuccinic imide (29.7 mg, 0.258 mmol) and dicyclohexylcarbodiimide (DCC, 55.9 mg, 0.271 mmol) were added, and the solution was stirred at 0 °C for 1 h. The solvent was removed under reduced pressure. The residue was redissolved in benzene (10 mL) and once again taken to dryness under reduced pressure to remove traces of thionyl chloride. The acid chloride was dissolved in chloroform (150 mL), and 6-aminocaproic acid (627 mg) in chloroform (150 mL) containing a few drops of triethylamine was added dropwise. The solution was brought to reflux for overnight, and the reaction was quenched by addition of water (80 mL). The chloroform layer was then separated and dried over magnesium sulfate. The chloroform was removed under reduced pressure, and the sample was purified by silica gel chromatography with chloroform or chloroform-methanol ) 20:1 (v/v) as eluent. The yield was 76% (90.6 mg). 1H NMR (CDCl3): δ -3.8 (2H, s, pyrrole-NH), 1.0-1.3 (6H, m, spacer-CH2), 1.8 (6H, t, Ar-CH2CH3), 3.1 (4H, m, Ar-CH2CH2COOCH3), 3.3-3.5 (15H, m, Ar-CH3 and Ar-CH2CH2COOCH3), 3.9 (4H, m, Ar-CH2CH3), 4.2 (4H, m, Ar-CH2CH2COOCH3), 6.2 (1H, s, NHCOPh), 9.9 (4H, m, meso CHdC). MS(FAB): m/z 694 (MH+). (25) Muir, H.; Neuberger, A. Biochem. J. 1949, 45, 163-169. (26) Traylor, T.G.; Chang, C. K.; Geibel, J.; Berzinis, A.; Mincey, T.; Cannon, J. J. Am. Chem. Soc. 1979, 101, 6716-6731.

Langmuir, Vol. 14, No. 2, 1998 409 H2MPMME-CONH(CH2)11COOH. This compound was prepared in a similar way as that described for H2MPC5COOMe. The acid chloride was reacted with 12-aminododecanoic acid. The yield was 68% (91.3 mg). 1H NMR (CDCl3): δ -4.0 (2H, s, pyrrole-NH), 1.0-1.3 (6H, m, spacer-CH2), 1.9 (6H, t, Ar-CH2CH3), 3.2 (4H, m, Ar-CH2CH2COOCH3), 3.5-3.6 (15H, m, Ar-CH3 and Ar-CH2CH2COOCH3), 4.0 (4H, m, Ar-CH2CH3), 4.3 (4H, m, ArCH2CH2COOCH3), 6.1 (1H, s, NHCOPh), 10.0 (4H, m, meso CHdC). MS(FAB): m/z 778 (MH+). PE-C0-H2MPMME. H2MPMME (134 mg) was dissolved in a mixture of CHCl3 (5 mL) containing triethylamine (0.06 mL) and ethyl chloroformate (ECC, 0.04 mL) at room temperature. The solution was stirred for 2.5 h and treated with PE (259 mg) dissolved in CHCl3 (5 mL) for 6 h at room temperature. The chloroform was removed under reduced pressure, and the sample was purified by silica gel preparative thin layer chromatography with chloroform containing 2.4% methanol as eluent, giving 188 mg (87%). 1H NMR (CDCl3): δ -3.9 (2H, s, pyrrole-NH), 0.9 (6H, s, CCH3), 1.0-1.3 (48H, m, DPPE-CH2), 1.4 (4H, s, DPPECOCH2CH2), 1.8 (6H, t, Ar-CH2CH3), 2.2 (4H, m, DPPE-COCH2CH2), 3.3 (4H, m, Ar-CH2CH2COOCH3), 3.6 (15H, m, Ar-CH3 and Ar-CH2CH2COOCH3), 4.1 (4H, m, Ar-CH2CH3), 4.4 (4H, m, Ar-CH2CH2COOCH3), 7.5 (1H, s, PE-NH), 10.1-10.2 (4H, m, meso CHdC). MS(FAB): m/z 778 (MH+). UV/vis (CH2Cl2-10% EtOH): λmax 397.0 nm, 496.5, 531.0, 566.6, 618.0. FABMS (MH+): 1254. PE-C5-H2MPMME and PE-C11-H2MPMME. These PElinked mesoporphyrins were prepared in a similar way as described for PE-C0-H2MPMME, giving 83% and 82% yields, respectively. PE-C5-H2MPMME. 1H NMR (CDCl3): δ -3.8 (2H, s, pyrroleNH), 0.8 (6H, s, CCH3), 1.0-1.2 (54H, m, DPPE-CH2 and spacerCH2), 1.3 (4H, s, DPPE-COCH2CH2), 1.8 (6H, t, Ar-CH2CH3), 2.1 (4H, m, DPPE-COCH2CH2), 3.2 (4H, m, Ar-CH2CH2COOCH3), 3.6 (15H, m, Ar-CH3 and Ar-CH2CH2COOCH3), 4.1 (4H, m, ArCH2CH3), 4.4 (4H, m, Ar-CH2CH2COOCH3), 6.3 (1H, s, NHCOPh), 7.8 (1H, s, DPPE-NH), 10.1-10.2 (4H, m, meso CHdC). UV/vis (CH2Cl2-10% EtOH): λmax 396.0 nm, 497.5, 531.5, 565.5, 618.0. FABMS (MH+): 1367. PE-C11-H2MPMME. 1H NMR (CDCl3): δ -3.8 (2H, s, pyrroleNH), 0.8 (6H, s, CCH3), 1.1-1.3 (66H, m, DPPE-CH2 and spacerCH2), 1.6 (4H, m, DPPE-COCH2CH2), 1.8 (6H, t, Ar-CH2CH3), 2.3 (4H, m, DPPE-COCH2CH2), 3.2 (4H, m, Ar-CH2CH2COOCH3), 3.4 (15H, m, Ar-CH3 and Ar-CH2CH2COOCH3), 4.1 (4H, m, ArCH2CH3), 4.4 (4H, m, Ar-CH2CH2COOCH3), 6.2 (1H, s, NHCOPh), 7.1 (1H, s, DPPE-NH), 10.1-10.2 (4H, m, meso CHdC). UV/vis (CH2Cl2-10% EtOH): λmax 396.5 nm, 496.5, 531.0, 565.5, 618.0. FABMS (MH+): 1451. Manganese Porphyrins, MnMPMME, PE-C0-MnMPMME, PE-C5-MnMPMME, and PE-C11-MnMPMME. For example, the synthesis of PE-C5-MnMPMME will be described as follows.3c A saturated solution of tris(pentane-2,4-dionate)manganese in pyridine was added with stirring to the CHCl3 solution of PE-C5-H2MPMME. The solution was refluxed for 2 h at 40 °C. Progress of conversion to the manganese complex was followed by visible spectroscopy. The manganese complex was purified by the same method as described for PE-C0-H2TTP. MnMPDME, MnMPMME, PE-C0-MnMPMME, and PE-C11MnMPMME were prepared in a similar way as described for PE-C5-MnMPMME, giving 66.4%, 88.6%, 52.5%, and 72.9% yield, respectively. MnMPDME. UV/vis (CH2Cl2-10% EtOH): 457.5 nm, 543, 571.5. FABMS (M): 648. MnMPMME. UV/vis (CH2Cl2-10% EtOH): 458 nm, 550.5, 582.0. FABMS (M): 633. PE-C0MnMPMME. UV/vis (CH2Cl2-10% EtOH): 458.5 nm, 550.5, 582.0. FABMS (M): 1307. PE-C5-MnMPMME. UV/vis (CH2Cl2-10% EtOH): 457.5 nm, 551.0, 584.0. FABMS (M): 1420. PE-C11-MnMPMME. UV/vis (CH2Cl2-10% EtOH): 458.0 nm, 550.0, 583.5. FABMS (M): 1504. Electron Transfer Assay.3c,d,27 Transmembrane electron transfer from an external reductant, indigotetrasulfonic acid (ITSAH2, 1.0 × 10-5 M) reduced by Na2S2O4 (1.0 × 10-4 M), to potassium ferricyanide (0.1 M) trapped within an egg PC liposome (27) Runquist, J. A.; Loach, P. A. Biochim. Biophys. Acta 1981, 637, 231-244.

410 Langmuir, Vol. 14, No. 2, 1998 or a DPPC liposome as mediated by a catalyst (e.g., a manganese porphyrin) incorporated in the lipid bilayer was studied by monitoring the appearance of oxidized indigotetrasulfonic acid (ITSA, λmax 605 nm). Conditions for this assay were nearly identical with those previously published.3c,d Liposome containing PE-linked porphyrins was prepared by previously reported methods for incorporating metalloporphyrins into egg PC vesicles.3c,d For example, egg PC or DPPC (100 mg) was dissolved in a few milliliters of CHCl3. An aliquot of the desired PE-linked porphyrin or cholesterol in CHCl3-CH3OH (1:1) was added and mixed, and the solvent was removed on a rotary evaporator. The lipid film was suspended in 3.0 mL of 0.1 M K3Fe(CN)6 in 0.4 M imidazole buffer, pH 7.0, by gentle swirling. The suspension was sonicated at 0 °C for egg PC or 55 °C for DPPC by using a Branson 250 sonifier. The vesicle samples were then applied to a Sephadex G-25-80 gel filtration column using 0.15 M KCl in 0.4 M imidazole buffer, pH 7.0, as the eluting buffer. Oxygen was removed from the vesicles by passing argon gas over and through the vesicle solution for about 1 h. The vesicles were then stored under inert gas until needed for the electron assay, which was performed on the same day. Cyclic Voltammogram Measurement. Cyclic voltammetry data were measured with a Yanako P-900. A standard calomel electrode (SCE) and Ag/AgCl served as reference electrodes, glassy carbon served as the working electrode, and platinum functioned as the counter electrode. Solutions containing porphyrins were prepared in DMSO containing 0.1 M tetra-nbutylammonium percholate (TBAP). The solutions were degassed by argon bubbling. The modified electrode was prepared by a cast method: 0.05 mL of CHCl3 solution into which manganese porphyrin was dissolved with dipalmitoylphosphatidylcholine (DPPC) was dropped on a polished glassy carbon (GC) ((45-60) × 10-4 W/cm, Tokai Carbon Co.) or transparent SnO2 electrode (9.9 W/cm, gifted from Nippon Itagalasu Co.) (surface area 2 cm2) at room temperature, immediately dried at about 60 °C, and then cooled at room temperature. This level of coverage results in the DPPC membrane having a thickness of approximately 8-10 µm, measured with an Elecont micrometer (Mitsutoyo Co.), and the thin membrane of DPPC contains 150 mmol of Mn-porphyrins per 1 g of the DPPC membrane. The cyclic voltammogram (CV) of the modified electrode was measured in a pH 7.0 aqueous phosphate buffer containing 0.1 mol dm-3 phosphate and 0.2 mol dm-3 sulfate ions. Impedance Measurement. The impedance measurement was done by using a frequency response analyzer, NF Circuit Design 5020 with an AC amplitude of 10 mV rpm at frequencies from 20 kHz to 10 mHz superimposed on DC bias in a pH 7.0 aqueous phosphate buffer containing 0.1 mol dm-3 phosphate and 0.2 mol dm-3 sulfate ions.24 The absorption change of the cast membrane with potential was measured in a spectrophotometer, JASCO V-520, where the membrane-covered transparent electrode was set in a small glass cell with an Ag/AgCl reference and a Pt plate counter electrode. The potential was referenced to with an Ag/AgCl electrode in a 0.1 mol dm-3 KCl solution. The experimental temperature was thermostatically controlled within 0.2 °C in a range from 25 to 50 °C.

Results and Discussion Synthesis of Phospholipid-Linked Porphyrins. The synthetic sequence leading to the compound PE-CnMnMPMME followed these steps. The porphyrins H2MPMME and H2MPMMECONH(CH2)nCOOH (n ) 5, 11) and their manganese porphyrin complexes were prepared as described in the Experimental Section.3c H2MPMME and H2MPMMECONH(CH2)nCOOH (n ) 5, 11) and their manganese complexes were treated with hydroxysuccinimide in chloroform at low temperature and reacted with dipalmitoylphosphatidylethanolamine (PE) to give the phospholipid-linked porphyrins PE-Cn-H2MPMME (n ) 0, 5, 11) and their manganese complexes, followed by chromatographic separation (silica gel, 10% methanolchloroform). The lH NMR and mass spectra of PE-CnH2MPMME (n ) 0, 5, 11) and their manganese complexes support unambiguously the assigned structure as de-

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Figure 1. Rate (V0) of transmembrane electron transfer catalyzed by PE-Cn-MnMPMME (n ) 0, 5, 11) as a function of porphyrin concentration in an egg PC vesicle, 0.4 M imidazole buffer, pH 7.0 at 25 °C: (b) PE-C11-MnMPMME; ([) PE-C5MnMPMME; (9) PE-C0-MnMPMME.

scribed in the Experimental Section. PE-Cn-MnPFPP and PE-Cn-MnTTP (n ) 0, 5, 11) were prepared as described in the previous papers.3c The UV-visible absorption spectra of PE-Cn-H2MPMME (n ) 0, 5, 11) and their manganese complexes in CH2Cl2-10% EtOH were identical, and all showed the presence of a normal porphyrin chromophore, as shown in the Experimental Section, indicating no intra- and intermolecular interaction in the solvent. The fluorescence spectra of PE-Cn-H2MPMME (n ) 0, 5, 11) were also recorded in CH2Cl2-10% EtOH and egg PC and DPPC vesicles (data not shown). The excitation of the Soret band of the porphyrin produces a normal porphyrin fluorescence. It can be seen that a higher fluorescence intensity is observed in these vesicles at 25 °C, similar to the fluorescence intensity in CH2Cl2-10%EtOH. These results imply that in the vesicle systems the porphyrin moiety is immersed within the hydrophobic interior of the membrane as described for PE-Cn-H2PFPP and PE-Cn-H2TTP in the previous papers.3c Phospholipid-Linked Manganese Porphyrin-Mediated Electron Transfer across a Liposomal Membrane: Effect of Various Spacer Methylene Groups (Cn). Electron transfer from an external reductant (reduced indigotetrasulfonic acid, ITSAH2, 1.0 × 10-5 M) to potassium ferricyanide (0.1 M) trapped within a phospholipid liposome (either egg PC or DPPC) was measured anaerobically at 0.4 M imidazole buffer as mediated by a catalyst of manganese porphyrin derivatives incorporated in the vesicle bilayer as described in the previous papers.3c,d,27 The oxidized form of the dye, ITSA, has an intense absorbance band at λmax ) 600 nm. The intensity and positions of this band permitted us to measure the rate of the electron transfer with minimal spectroscopic interference from the other components of the model system. PE-Cn-MnMPMME-catalyzed electron transports were examined at pH ) 7.0. The initial electron transfer rate (V0) was determined from the initial slope of the change of the absorbance band at 600 nm. For example, Figures 1 and 2 illustrate the rate of electron transport across egg PC liposomes (V0) at 25 °C. As is apparent from Figure 1, PE-C0-MnMPMME and PEC5-MnMPMME show little or no catalytic activity by themselves, consistent with the rate of control. However, catalyzed PE-C11-MnMPMME increased transmembrane electron transfer with increased porphyrin concentration in the liposomal membrane. At the relatively low concentration of reduced indigotetrasulfonic acid ( 1.0 × 10-5 M) employed under these experimental conditions,

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Scheme 1. Schematic Representation of Transmembrane Electron Transfer as Catalyzed by Phospholipid-Linked Manganese Porphyrins in a Liposomal Membranea

a Reduced form ITSAH was oxidized to the oxidized form ITSA by 2 electron release. The porphyrins and the head groups of 2 phopholipid (PE) were represented by squares and circles, respectively. See text for the detailed discussion.

Figure 2. Rate (V0) of transmembrane electron transfer catalyzed by PE-linked Mn porphyrins as a function of porphyrin concentration in an egg PC vesicle, 0.4 M imidazole buffer, pH 7.0 at 25 °C: (b) PE-C11-MnPFPP; (2) PE-C11-MnTTP; (9) PEC11-MnMPMME.

[K3Fe(CN)6] . [ITSAH2] > [PE-linked manganese porphyrins] (see Experimental Section), the rate-limiting step of catalysis is a bimolecular reaction between the manganese porphyrin and ITSAH2 at the outside surface of the membrane.3c,d,27 The mechanism of electron transfer is presumed to be the pathway shown in Scheme 1.3d Electron transfer occurs from external reduced indigotetrasulfonic acid to an oxidized manganese porphyrin which momentarily assumed a folded position relative to its attached PE group. The semiquinone form of indigotetrasulfonic acid would be expected to disproportionate rapidly in water. The reduced manganese porphyrin is then viewed as changing to its extending configuration relative to the PE group. Electron transfer from this manganous porphyrin to a manganic porphyrin tethered to a PE molecule on the opposite side of the bilayer can then occur if, in their lateral diffusion, they approach each other sufficiently closely. The electron transfer to ferri-

cyanide is then completed by movement of the manganous porphyrin on the inner half of the bilayer to a folded position near the aqueous interface where it can donate an electron to a ferricyanide molecule. As the number of carbons connecting the manganese porphyrins to PE was shortened, the rate-limiting step is assumed to shift to the electron-transfer reaction between the manganese porphyrin molecules and thus reflect the increasing distance between the manganese porphyrin molecules anchored to their PE constituents at opposite aqueous interfaces. Thus, the rate of transmembrane electron transfer is likely to decrease with increasing distance of separation and a significant electron transfer occurs only when the edge-to-edge separation of the two porphyrins becomes small in the lipid bilayers (Scheme 2a). These results were similar to those observed for PE-Cn-MnTTP and PE-Cn-MnPFPP (n ) 0, 5, 11) which catalyzed the transmembrane electron transfer especially when n ) 5 or n ) 11 and above the phase temperature of the lipid membrane.3c MnMPMME also catalyzed electron transfer as described previously, indicating that MnMPMME can move in the membrane so that an electron can be transferred from external ITSAH2 to potassium ferricyanide trapped within a phospholipid liposome from ITSA. Effect of the Structure of Porphyrins. As is apparent from Figure 2, PE-C11-MnMPMME showed the slowest electron-transfer rate in comparison to PE-C11MnTTP and PE-C11-MnPFPP, in which an enhanced electron transfer was observed in the following order, PEC11-MnPFPP > PE-C11-MnTTP > PE-C11-MnMPMME. It is expected that differences in lipid solubility or mobility are important to the differences in electron-transfer rates among the various porphyrins because mesoporphyrins can be more easily immersed into the lipid bilayers than tetraphenylporphyrin derivatives. However, at the relatively low concentration of reduced indigotetrasulfonic acid (1.0 × 10-5 M) employed under these experimental conditions, the rate-limiting step of catalysis is a bimo-

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Scheme 2. Schematic Diagram of Minimum Distances Separating Phospholipid-Linked Manganese Porphyrins Inserted from Opposite Sides of Egg PC or DPPC Lipid Bilayersa

Figure 3. UV-visible absorption spectra of MnTTP (150 µmol/g DPPC) in a DPPC membrane cast on the SnO2 glass electrode. A reduction of Mn(III) to Mn(II) and an oxidation of Mn(II) to Mn(III) from the light absorption measurement in the DPPC membrane cast on the SnO2 glass electrode as a function of potencial.

a Each phospholipid-linked porphyrin is represented in its extended conformation. The distance from the PE(head group)/ spacer amide carbonyl to the far edge of the porphyrin pyrrole ring is estimated to be the maximum depth the porphyrin can penetrate in the bilayer. (a) In liposomal membrane. (b) On glassy carbon electrode modified with DPPC lipid bilayers. See text for further discussion.

lecular reaction between the manganese porphyrin and ITSAH2 at the outside surface of the membrane, as described above.3c,d,27 Thus, the reason for the differences in electron-transfer rates among the various porphyrins is likely to be that the manganese pentafluoroporphyrin derivative which has the highest oxidation potential (E1/2 value) in comparison to other porphyrins (see Table 1) may accelerate the electron-transfer rate by the difference of the redox potential, ∆E°, between the electron transfer species because electron transfer rate strongly depended on the difference of ∆E°, as described previously.3c,d,28 Electron Transfer on an Electrode Modified with a Lipid Bilayer Membrane Containing Manganese Porphyrins. Figure 3 illustrates that UV-visible absorption spectra of MnTTP in a DPPC membrane cast on the SnO2 glass electrode were identical and that all showed the presence of a normal porphyrin chromophore. The cyclic voltammetric response of the glassy carbon electrode or SnO2 glass electrode covered with a DPPC membrane containing manganese porphyrins in an aqueous phosphate buffer solution showed that one set of waves is clearly visible corresponding to the consecutive monoelectronic reduction of the manganese porphyrin unit (Mn(III)/Mn(II)) only above the phase temperature of the membrane. For example, Figure 4 illustrates the CV curves on the glassy carbon electrode modified with a DPPC bilayer membrane containing MnTTP at 42 °C. The current peaks were observed at about -0.27 and -0.15 V, which conformed to a reduction of Mn(III) to Mn(II) and an oxidation of Mn(II) to Mn(III) from the light absorption measurement in a DPPC membrane cast on (28) Marcus, R. A. Angew. Chem., Int. Ed. Engl. 1993, 32, 11111121.

Figure 4. Cyclic voltammograms on the glassy carbon electrode modified with a DPPC membrane containing MnTTP (150 or 200 µmol/g DPPC) in a pH 7.0 aqueous phosphate buffer containing 0.1 mol dm-3 phosphate and 0.2 mol dm-3 sulfate ions at 42 °C. Scan rate: 0.1 V-1. The current peaks were observed at about -0.27 and -0.15 V, which conformed to a reduction of Mn(III) to Mn(II) and an oxidation of Mn(II) to Mn(III) from the light absorption measurement in a DPPC membrane cast on the SnO2 glass electrode, as shown in Figure 3.

the SnO2 glass electrode, as shown in Figure 3. The absorption spectra of an MnTTP-embedded DPPC membrane on the SnO2 glass electrode showed that the absorption peaks at the wavelengths 472 nm (at the potential -0.15 V) and 439 nm (at potentials lower than -0.27 V), respectively, correspond to the Soret bands of Mn(III) and Mn(II) in MnTTP. A similar cyclic voltammetric response of a GC electrode covered with a DPPC membrane containing other manganese porphyrins was also observed. Effect of Temperature. The cathodic peak currents for the reduction of Mn(III) to Mn(II) as a function of temperature which show a sharp break point in the current-temperature behavior reflecting the melting of the multilayer assembly, that is, the gel-to-fluid phase transition, can be observed. For example, Figure 5 showed cathodic peak currents for the reduction of Mn(III) to Mn(II) on the glassy carbon electrode modified with various lipid membranes containing MnMPDME. As is apparent from Figure 5, a sharp break point in the current-

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Figure 5. Cathodic peak currents for the reduction of Mn(III) to Mn(II) on the glassy carbon electrode modified with various lipid membranes containing MnMPDME (150 µmol/g DPPC) as a function of temperature in a pH 7.0 aqueous phosphate buffer containing 0.1 mol dm-3 phosphate and 0.2 mol dm-3 sulfate ions. Scan rate: 0.1 V-1 (b) DMPC; (9) DPPC; (2) DSPC.

temperature behavior is likely to reflect the melting of the multilayer assembly, in which the sharp break points for DMPC, DPPC, and DSPC were observed at 32, 42, and 52 °C, respectively. That is, the gel-to-fluid phase transition can be observed.29-32 These characteristic temperatures correspond to a transient from a gel form to a fluid form of DMPC, DPPC, and DSPC bilayers.1g,3c,21-23,29-32 The free movement and diffusion of Mn porphyrin derivatives may be a crucial factor for the electron transfer from the carbon electrode for Mn(III)/ Mn(II) reduction or between Mn(III) and Mn(II) in manganese porphyrins. Similar results were observed for the cyclic voltammetric response of a GC electrode covered with DMPC, DPPC, and DSPC membranes containing other manganese porphyrins (data not shown). These temperatures are near the phase-transition temperatures of the lipid, respectively.30,31 It is considered that below the phase-transition temperature the lipids are arranged in tiled one-dimensional lattices and that then at pretransition temperature two-dimensional arrangements of the lipid are formed with periodic undulations. Above the main phase transitions lipids revert to one-dimensional lattice arrangements, separated somewhat from each other, and assume mobile liquid-like conformations. Thus, near the main-phase transition manganese porphyrins become associated with this structural transformation of lipid bilayers and can catalyze electron transfer such as that in egg PC liposome, as described above and in the previous paper.1g Below the phase transition of the lipid bilayer membrane all manganese porphyrins showed little or no catalytic activity, where electron transfer from this manganous porphyrin to a manganic porphyrin cannot occur because the lipids are arranged in a tiled one-dimensional lattice and then the porphyrins are frozen in the lipid bilayers. These results again showed that the phospholipid-linked manganese porphyrins exhibited electron transfer in the lipid membranes, depending on the structural changes of the lipid bilayer, such as the main-phase transition properties of the lipid bilayer, and also on the length of the spacer methylene groups of the phospholipid-linked (29) Rusling, J. F.; Zhang, H. Langmuir 1991, 7, 1791-1796. (30) Introduction to Biological Membranes; Jain, M. K., Wagner, R. C., Eds.; John Wiley and Sons: New York, 1980. (31) Membrane Mimetic Chemistry; Fendler, J. H., Ed.; John Wiley and Sons: New York, 1982. (32) Suga, K.; Bradley, M.; Rusling, J. F. Langmuir 1993, 9, 30633066.

Figure 6. Effect of the scan-rate depedence on the cathodic peak current for the reduction of manganase porphyrin (150 µmol/g DPPC) on the glassy carbon electrode modified with a DPPC membrane in a pH 7.0 aqueous phosphate buffer containing 0.1 mol dm-3 phosphate and 0.2 mol dm-3 sulfate ions at 42 °C. (a) PE-C5-MnPFPP; (b) MnPFPP.

manganese porphyrins incorporated in the phospholipid membranes. Effect of the Scan-Rate Depedence. Figure 6 illustrates the effect of the scan-rate depedence on the cathodic peak current for the reduction of PE-C5-MnPFPP or MnPFPP on the glassy carbon electrode modified with a DPPC membrane at 42 °C. The cathodic peak currents for the reduction of PE-C5-MnPFPP increase linearly with the first order of the scan rate rather than the square root of the scan rate (Figure 6a). Similar results of the scanrate dependency were observed for PE-C5-MnTTP and PE-C5-MnMPMME. These facts are in accord with ideal, one-electron, reversible thin-layer electrochemistry.1g,19,29 The cathodic peak current for the reduction of MnPFPP increases linearly with the square root of the scan rate, as shown in Figure 6b. Similar results of the scan-rate dependency were observed for MnTTP and MnMPMME. These facts indicate that the electrochemistry of phospholipid-linked manganese porphyrins inside the lipid membrane is under conditions under which diffusion of charge occurs during the voltammetric scan.1g,19, 29 Electrode Impedance. Alternatively, Figure 7 illustrates the impedance diagrams for MnMPMME (Figure 7a) and PE-Cn-MnMPMME (Figure 7b) on the glassy carbon electrode modified with a DPPC membrane at -0.30 V, in which the imaginary part of impedance is plotted against the real part of impedance (i.e., the ColeCole plot), as a function of the frequency parameter. As is apparent from Figure 7, at lower frequencies, the plots

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Figure 8. Redox peak currents of manganese porphyrin derivatives on the glassy carbon electrode modified with a DPPC membrane at 42 °C as a function of the concentration of manganese porphyrin derivatives in a pH 7.0 aqueous phosphate buffer containing 0.1 mol dm-3 phosphate and 0.2 mol dm-3 sulfate ions. Scan rate: 0.1 V-1. (b) MnTTP; (9) MnPFPP; (2) PE-C0-MnPFPP.

Figure 7. Impedance diagram for manganase porphyrin on the glassy carbon electrode modified with a DPPC membrane (150 µmol/g DPPC) at -0.30 V, in which the imaginary part of impedance is plotted against the real part of impedance (i.e., the Cole-Cole plot), as a function of frequency parameter in a pH 7.0 aqueous phosphate buffer containing 0.1 mol dm-3 phosphate and 0.2 mol dm-3 sulfate ions at 42 °C: (a) MnMPMME; (b) PE-C5-MnMPMME.

for MnMPMME showed linearly first order, while that for PE-C5-MnMPMME showed no linearity, and similar results of the impedance diagram were observed for other manganese porphyrins, implying that the electrochemistry of phospholipid-linked manganese porphyrins inside the lipid membrane is not diffusion-controlled but is an electron-hopping system, while that of non-phospholipidlinked manganese porphyrins is diffusion-controlled. More detailed impeadance analysis will be reported elsewhere.24 Effects of Porphyrin Structure. Figure 8 showed the redox peak current of manganese porphyrins on the glassy carbon electrode modified with DPPC bilayers at 42 °C. As is apparent from Figure 8, MnTTP and MnPFPP increased the redox peak current with increasing concentration of porphyrins until 100 µmol of porphyrins/g of DPPC membrane and then largely decreased the peak current with increasing concentration of porphyrins. Large differences of peak currents among these porphyrins were not observed. Interestingly, the redox peak current of PE-Cn-MnTTP, PE-Cn-MnPFPP, and PE-Cn-MnMPMME (n ) 0, 5, 11) increased with increasing concentration of the porphyrins and then exhibited a saturation behavior as shown for an example of PE-C0-MnPFPP in Figure 8 or Figure 9. These results implied that MnTTP and MnPFPP became aggregated in the lipid bilayer membrane above the concentration of 100 µmol of porphyrins/g of DPPC membrane; however, this was not the case of phospholipid-linked manganese porphyrins because a phospholipid-linked porphyrin complex could be easily

Figure 9. Redox peak currents of PE-Cn-MnMPMME (n ) 0, 5, 11) on the glassy carbon electrode modified with a DPPC membrane at 42 °C as a function of the concentration of porphyrin in a pH 7.0 aqueous phosphate buffer containing 0.1 mol dm-3 phosphate and 0.2 mol dm-3 sulfate ions. Scan rate: 0.1 V-1 (9) C0; (b) C5; (2) C11.

immersed into the lipid bilayer.3c Phospholipid-linked manganese porphyrins yield reversible voltammetric waves in nonaqueous solvents, like DMSO and CH2Cl2, that are similar to those in the lipid bilayers, indicating that the porphyrin subunit of PE-linked manganese porphyrin resides in an essentially nonaqueous environment. This, in turn, suggests that a certain level of molecular organization of phospholipid-linked manganese porphyrins exists within the lipid bilayer membrane.22,23,29,32 Effect of Various Spacer Methylene Groups (Cn). As is apparent from Figure 9, PE-C5-MnMPMME and PEC11-MnMPMME showed increased peak currents with increasing concentration of the porphyrins, and an enhanced peak current was observed for PE-C5-MnMPMME in comparison to PE-C11-MnMPMME (Figure 9). PE-C0-MnMPMME showed the slowest current by itself with increased concentration of the porphyrin. These results were similar to those observed for PE-Cn-MnTTP and PE-Cn-MnPFPP (n ) 0, 5, 11). However, large differences of peak currents between the structures of porphyrins were not observed (data not shown). Thus, the peak currents of phospholipid-linked manganese porphyrins on the electrode were controlled by the separated spacer methylene groups between the porphyrin and the phospholipid but not by the structure of porphyrins. The CV and impedance measurements on electrodes

Manganese Porphyrin-Mediated Electron Transfer

modified with lipid membranes imply that a schematic model for the electron transfer on an electrode modified with a membrane containing manganese porphyrins can be considered, as shown in Scheme 2b.22,23,29,32 The electron transfer from a GC electrode to Mn(III)/Mn(II) may be controlled by a diffusion process of the porphyrins in the DPPC membrane when manganese porphyrins are not linked with the phospholipid. When manganese porphyrin is covalently linked with the phospholipid, electron transfer becomes slow and largely depends on the length of the spacer methylene groups (Cn) between the manganese porphyrin and the phospholipid. The order of the electron transfer rate for the spacer methylene groups (Cn) is C5 > C11 > C0 for all phospholipid-linked manganese porphyrins, indicating that the length of the spacer methylene groups is an important factor for controlling the electron transfer between Mn(III) and Mn(II) of the manganese porphyrin moiety. The manganese porphyrin moieties cannot approach each other to short enough distance to bring about the fast electron transfer both between electrode and porphyrin and also between porphyrins when n ) 0. In contrast, the free movement of manganese porphyrin is greatly limited because of the long methylene length, and thus the electron transfer does not take place at a fast enough rate both between electrode and porphyrin and also between porphyrins when n) 11. Thus, n ) 5 is probably suitable for the electron transfer on the electrode because the manganese moieties can freely move and approach each other enough for electron transfer both between electrode and porphyrin and also between porphyrins. However, as is apparent from Figure 1, an enhanced transmembrane electron transfer catalyzed by PE-Cn-MnMPMME was observed in a liposomal membrane only when n ) 11 at this condition. These results imply that the distance between the phospholipid-linked porphyrins on the electrode becomes sufficiently closely in comparison to that in the liposomal membrane so that electron transfer may occur on the electrode even when n ) 0. The reasons are likely that, in comparison to the liposomal membrane, the structure of the lipid bilayers on the electrode is partially perturbed and also the alkyl chains of the DPPC lipid bilayers on the electrode are shrunken because dehydration in the lipid bilayers on the electrode occurs, such as observed on addition of ethanol into the lipid bilayers.30 According to the data of electron transfer on the electrode, it is likely that the length between the lipid bilayers on the GC electrode is partially perturbed or shrunken about 50 Å to 40 Å, causing electron transfer between the manganese porphyrins even when n ) 0, as shown in Scheme 2b). A.-E.F. Nassar et al. reported that there is an organized monolayer on the surface of electrode.23b However, polished gassy carbon contains a significant fraction of oxygen functionality on its surface, and it is relatively hydrophilic. Thus, it is likely that cast lipid films have head down orientations on hydrophobic surfaces for a variety of head group types, as shown in Scheme 2b.29,32 Effect of the Structure of Porphyrins on Manganese Porphyrin-Mediated Electron Transfer in a Liposomal Membrane or on an Electrode Modified with Lipid Bilayers. Transmembrane electron transfer catalyzed by the manganese complex of the phospholipidlinked porphyrins in egg PC liposome revealed that the porphyrin causes a significantly accelerated electron transfer, depending upon the structure of the porphyrin, following the order PE-C11-MnPFPP > PE-C11-MnTTP > PE-C11-MnMPMME (Figure 2). It is expected that differences in lipid solubility or mobility are important to the differences in electron-transfer rates among the

Langmuir, Vol. 14, No. 2, 1998 415 Table 1. Redox Potentials (V vs Ag/AgCl (0.1 M KCl)) of Manganese Porphyrin Derivatives in DMSO and in DPPC Membranea Mn(II/III) manganese porphyrins

in DMSO

in DPPC membrane

MnPFPP MnTTP MnMPDME MnMPMME PE-C0-MnMPMME PE-C5-MnMPMME PE-C11-MnMPMME PE-C11-MnPFPP PE-C11-MnTTP

-0.12 -0.30 -0.48 -0.52 -0.51 -0.52 -0.51 b b

-0.13 -0.21 -0.41 -0.45 -0.46 -0.43 -0.44 -0.16c -0.27c

a Redox potentials were obtained as the midpoint potential between the anodic and cathoic peak potentials. Scan rate: 0.1 V s-1 b Not measured. c Reference 3c.

various porphyrins. However, at the relatively low concentration of reduced indigotetrasulfonic acid employed under these experimental condition, the rate-limiting step of catalysis is a bimolecular reaction between the manganese porphyrin and ITSAH2 at the outside surface of the membrane described above.3c,d,27 It is considered that manganese fluorinated porphyrins which have the highest oxidation potential (E1/2 value) (see Table 1) may accelerate the electron-transfer rate by the difference of the redox potential, ∆E°, between electron-transfer species.3c,d,28 Thus, by selection of the proper aqueous redox component and by appropriate choices of the phospholipid-linked porphyrins, a specific electron-transfer reaction at the membrane surface can be studied. These characteristics are of considerable interest to mimic the vectorial electron channel controlled in biological membranes. However, electron transfer on an electrode modified with phospholipid membranes containing the manganese complex indicated that large differences of electron-transfer rate among various manganese porphyrins were not observed although manganese mesoporphyrin derivatives show the lowest oxidation potential (E1/2 value) for the manganese porphyrins. The reason for this is not certain, but these results imply that the difference of the redox potential, ∆E°, between electron-transfer species, such as between manganese complexes and between a manganese complex and an electrode, does not play a crucial role on electron transfer on the electrode. It is expected that differences in lipid solubility or mobility are important to the differences in electron-transfer rates among the various porphyrins, as shown in Figure 8. Thus, by selection of the proper membrane component and by appropriate choices of the porphyrins, an efficient electron transfer on an electrode can be studied. These characteristics are of considerable interest to construct an efficient electrontransfer system on an electrode modified with a lipid bilayer membrane. Conclusions. Phospholipid-linked mesoporphyrins separated by spacer methylene groups (Cn), PE-CnMnMPMME (n ) 0, 5, 11) (Scheme 1) were synthesized. Electron transfer across a liposomal membrane and on a glassy carbon (GC) electrode covered with a phospholipid bilayer membrane containing manganese porphyrins in an aqueous phosphate buffer solution was examined. An enhanced transmembrane electron transfer catalyzed by phospholipid-linked manganese porphyrins with spacer methylene groups (Cn) was observed in egg PC or DPPC liposome only when n ) 11 and above the phase temperature of the membrane. By selection of the proper aqueous redox component and by appropriate choices of the phospholipid-linked porphyrins, a specific reaction at

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the membrane surface can be studied. Cyclic voltammetry on a SnO2 glass electrode or on GC electrode modified with a DPPC membrane containing manganese porphyrins showed that one set of waves was clearly visible corresponding to the consecutive monoelectronic reduction of the manganese porphyrin unit (Mn(III)/Mn(II)) only above the phase temperature of the membrane. Phospholipid-linked manganese porphyrins in a liposomal membrane and on the electrode modified with a lipid bilayer membrane caused an enhanced electron transfer. Electron transfer of the manganese porphyrins in a liposomal membrane depended on the length of the spacer methylene group (Cn) and also the structure of porphryins, while the electron transfer on the electrode depended only upon the Cn. It is expected that differences in lipid solubility or mobility are important to the differences in electron transfer rates among the various porphyrins.

Nango et al.

Thus, by selection of the proper membrane component and by appropriate choices of the porphyrins, an efficient electron transfer on an electrode can be studied. These characteristics in a liposomal membrane or on an electrode modified with lipid bilayers are of considerable interest to mimic the vectorial electron transfer controlled in biological membranes and to construct an efficient electrontransfer systems on an electrode. Thus, these porphyrin complexes are being utilized to systematically examine electron transfer in these lipid bilayer systems. Acknowledgment. The present work was partially supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan. M.N. thanks Drs. K. Yamashita and K. Tsuda for some helpful discussions and Mmes S. Kawamoto, K. Ogura, and M. Mogami for some fundamental CV measurements. LA970463R