Transmembrane Electron Transfer As Catalyzed by Phospholipid

José Almeida , André M. N. Silva , Susana L. H. Rebelo , Luís Cunha-Silva , Maria Rangel , Baltazar de Castro , Andreia Leite , Ana M. G. Silva. New J...
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Langmuir 1996, 12, 1981-1988

1981

Transmembrane Electron Transfer As Catalyzed by Phospholipid-Linked Manganese Porphyrins Mamoru Nango,* Kouji Iida, Masashi Yamaguchi, Keiji Yamashita, Kazuichi Tsuda, Atsushi Mizusawa,† Takenori Miyake,† Akihiro Masuda,§ and Junji Yoshinaga‡ Department of Applied Chemistry, College of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466, Japan, Department of Applied Chemistry, College of Engineering, University of Osaka Prefecture, Sakai, Osaka 591, Japan, Toray Research Center, Sonoyama 3 cho-me 2-1, Ohtsu 520, Japan, and Sawai Pharmaceutical Co. Ltd., Asahi-ku, Osaka 535, Japan Received September 15, 1995. In Final Form: January 3, 1996X Phospholipid-linked porphyrins separated by spacer methylene groups (Cn), PE-Cn-MTTP and PECn-MPFPP (n ) 0, 5, 11; M ) H2, Mn) (Chart 1) were synthesized. The phospholipid-linked porphyrin complexes easily associated with phospholipid bilayers in a manner that allows the porphyrin portion to penetrate the membrane. Ground state transmembrane electron transfer catalyzed by the manganese complex of the phospholipid-linked porphyrins in egg PC and DPPC liposomes revealed that the porphyrin causes a significant accelerated electron transfer especially when n ) 11 and that the electron transfer rate was controlled not only by the separated spacer methylene groups between the porphyrin and the phospholipid but also by the phase transition temperature of the lipid bilayers. Comparison of PE-CnMnTTP- and PE-Cn-MnPFPP-catalyzed electron transfer is made. Furthermore, the electron transfer was enhanced with an increase of imidazole concentration, implying that imidazole plays an important role in the electron transfer.

Introduction We have been involved in the use of a chemical model to mimic possible reactions of porphyrin complexes in biological membranes such as occur in photosynthesis and mitochondria membranes.1,2 Synthetic porphyrin models can be very helpful for investigating the effect of distance and orientation in electron transfer reactions of many biological processes. To provide a model for the electron transfer system, where porphyrin pigments play the key role, the preparation of porphyrin derivatives that are capable of light-induced intra- or intermolecular electron transfer has been reported.4-16 However, there has been little study of ground state electron transfer between * Address correspondence to this author. Telephone or Fax: 8152-735-5226. E-mail: [email protected]. † University of Osaka Prefecture. § Toray Research Center. ‡ Sawai Pharmaceutical Co. Ltd. X Abstract published in Advance ACS Abstracts, March 15, 1996. (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. Maku 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. (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.; Matsuura, M.; Yamaduchi, M.; Sato, K.; Tanaka, K.; Akimoto, K.; Yamashita, K.; Tsuda, K. Langmuir 1996, 12, 450-458. (4) The Photosynthetic Bacteria; Clayton, R. K. Sistrom, W. R., Eds.; Elsevier/North-Holland Biochemical Press: New York, 1978. (5) Topics in Photosynthesis; Barber, J., Ed.; Elsevier/North-Holland Biochemical Press: New York, 1979. (6) Photosynthesis; Govinjee, Ed.; Academic Press: New York, 1982. (7) Chlorophylls; Sheer, H., Ed.; CRC Press: Boca Raton, FL, 1991.

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porphyrin complexes to provide an insight into the effect of distance and orientation in the electron transfer so that a vectorial electron transfer system may be constructed in the biological membrane.1c-f,11,17-23 In the previous papers,1d,f,3 Loach and we synthesized and characterized poly(ethylenimine) (PEI)- and poly(ethylene glycol) (PEG)linked porphyrin derivatives with various spacer methylene groups between PEI or PEG and the porphyrin derivative, in which the control of the length of this spacer group and the incorporation of these complexes in liposome membranes permit systematic variations of the minimum distances separating two such derivatives incorporated from opposite sides of the membrane bilayer.3 However, (8) (a) Wasielewski, M. R. Chem. Rev. 1992, 92, 435-461. (b) Crossley, M.; Burn, P. L. J. Chem. Soc., Chem. Commun. 1991, 15691571. (9) Photoinduced Electron Transfer; Fox, M. A., Chanon, M., Eds.; Elsevier: Amsterdam, 1988. (10) 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. (11) Cusanovich, M. A. Photochem. Photobiol. 1991, 53, 845-857. (12) Groves, J. T.; Fate, G. D.; Lahiri, J. J. Am. Chem. Soc. 1994, 116, 5477-5478. (13) 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. (14) Gust, D.; Moore, T. A.; Moore, A. L. Acc. Chem. Res. 1993, 26, 198-205. (15) Johnson, D. G.; Niemczyk, M. P.; Minsek, D. W.; Wiederrecht, G. P.; Svec, W. A.; Gaines, I.; Geoge, L.; Wasielewski, R. J. Am. Chem. Soc. 1993, 115, 5692-5701. (16) Osuka, A.; Maruyama, K.; Mataga, N.; Asahi, T.; Yamazaki, I.; Tamai, N. J. Am. Chem. Soc. 1990, 112, 4958-4959. (17) Helms, A.; Heiler, D.; McLendon, G. J. Am. Chem. Soc. 1992, 114, 6227-6238. (18) Lymar, S. V.; Hurst, J. K. J. Am. Chem. Soc. 1992, 114, 94989503. (19) Lymar, S. V.; Hurst, J. K. J. Phys. Chem. 1994, 98, 989-996. (20) Robinson, J. N.; Cole-Hamilton, D. J. Chem. Soc. Rev. 1991, 20, 49-94. (21) Runquist, J. A.; Loach, P. A. Biochim. Biophys. Acta 1981, 637, 231-244. (22) Introduction to Biological Membranes; Jain, M. K., Wagner, R. C., Eds.; John Wiley and Sons: New York, 1980. (23) Membrane Mimetic Chemistry; Fendler, J. H., Ed.; John Wiley and Sons: New York, 1982.

© 1996 American Chemical Society

1982 Langmuir, Vol. 12, No. 8, 1996 Chart 1. Phospholipid-Linked Porphyrin Derivatives

the PEI- or PEG-linked porphyrins could easily associate with the phospholipid bilayer of the liposome, but the porphyrin portion could not be immersed more into the interior phaseof the liposome because of the size effect of the polymer moiety. In this paper, we report the syntheses and characterization of phospholipid-linked fluorinated porphyrins with separated methylene groups (PE-CnMTTP and PE-Cn-MPFPP) (PE ) dipalmitoylphosphatidylethanolamine; Cn ) spacer methylene groups; TTP ) tri-p-tolylporphyrin; PFPP ) tris(pentafluorophenyl)porphyrin; n ) 0, 5, 11; M ) H2, Mn) (Chart 1) which can be easily immersed into the lipid bilayer. Furthermore, the results of transmembrane electron transfer catalyzed by PE-Cn-MnTTP1c and PE-Cn-MnPFPP are reported. Comparisons of PE-Cn-MnTTP- and PE-CnMnPFPP-catalyzed electron transfer and the effect of the imidazole derivative on the rate are 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, Varian Gemini 300, and Jeol JNM-GX-400 instruments with tetramethylsilane as an internal standard for CDCl3 and DMSO-d6. The UV absorption and fluorescence spectra were recorded on Hitachi 124, Hitachi-Perkin Elmer MPF-4, 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. Cyclic voltammetry data were measured by 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-n-butylammonium perchlolate (TBAP). The solutions were degassed by argon bubbling. Electron micrographs were taken with Hitachi H7100FA. The vesicles were stained with 2% phosphotungstic acid. High-purity egg yolk phosphatidylcholine (egg PC), dipalmitoylphosphatidylcholin (DPPC), and dipalmitoylphosphatidylethanolamine (PE) were obtained from Nippon Fine Chemical Co. LTD, Takasago City, Hyogo, Japan. Synthetic Procedure. 5-(4-Carboxyphenyl)-10,15,20-tri-ptolylporphyrin, H2TTPCOOH, 5-[4-(((5-carboxypentyl)amino)carbonyl)phenyl]-10,15,20-tri-p-tolylporphyrin, H2TTPCONH(CH2)5COOH, and 5-[4-(((5-carboxyundecyl)amino)carbonyl)phenyl]-10,15,20-tri-p-tolylporphyrin, H2TTPCONH(CH2)11COOH,

Nango et al. and their manganese complexes, MnTTP(CH2)nCOOH (n ) 0, 5, 11) were prepared as described in our previous paper.3a NDocanoyl-L-histidine methyl ester (Dec-His-OMe) was prepared as described previously.24 5-(4-Carbomethoxyphenyl)-10,15,20-tris(pentafluorophenyl)porphyrin, H2PFPPCOOMe, was prepared by a literature method.25 Pyrrole (1.40 mL, 20 mmol) with a mixture of 4-carbomethoxybenzaldehyde (656 mg, 4 mmol) and pentafluorobenzaldehyde (1.92 mL, 16 mmol) was refluxed in chloroform (800 mL) containing BF3 under a nitrogen atmosphere for 60 h, followed by oxidation with p-chloranil (3.70 g, 15 mmol) for 3 h. The yield was 44% (1.7 g). 1H NMR (CDCl3) δ: -2.92 (2H, s, pyrrole-NH), 4.2 (3H, s, ester-Me), 8.3 (2H, d, Ar 3-H and 5-H), 8.5 (2H, d, Ar 2-H and 6-H), 8.8 (2H, s, β pyrrole), 8.9 (6H, s, β pyrrole). UV/vis (CH2Cl2-10% EtOH) λmax: 412.5 nm ( 327 mM-1 cm-1) (M ) mol dm-3), 506.5 (27.1), 584 (9.83). MS (FAB) m/z: 943 (MH+). 5-((((Carbomethoxypentyl)amino)carbonyl)phenyl)-10,15,20-tris(pentafluorophenyl)porphyrin, H2PFPPCONH(CH2)5COOMe. This compound was prepared in a similar way as that described for H2PFPPC11COOMe. The acid chloride was reacted with 6-aminocaproic acid methyl ester. 1H NMR (CDCl3) δ: -2.8 (2H, s, pyrrole-NH), 1.6-2.0 (6H, m, CCH2C), 2.4-2.6 (2H, m, NCH2C), 3.6-3.8 (5H, m, ester-Me and CCH2COO), 6.5 (1H, bs, amide-NH), 8.2 (2H, d, Ar 3-H and 5-H), 8.3 (2H, d, Ar 2-H and 6-H), 8.8 (2H, s, β pyrrole), 8.9 (6H, s, β pyrrole). MS(FAB) m/z: 1056 (MH+). 5-((((Carbomethoxyundecyl)amino)carbonyl)phenyl)10,15,20-tris(pentafluorophenyl)porphyrin, H2PFPPCONH(CH2)11COOMe. H2PFPPCOOH (1.095 g, 1.18 mmol) was dissolved in benzene (200 mL). Thionyl chloride (60 mL, 840 mmol) was added, and the solution was brought to reflux for 3 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 12-aminododecanoic acid methyl ester (627 mg) in chloroform (150 mL) containing a few drops of triethylamine was added dropwise. The solution was brought to reflux for overnight and 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 (chloroform). The yield was 80% (1.077 g). 1H NMR (CDCl3) δ: -2.92 (2H, s, pyrrole-NH), 1.6-2.0 (18H, m, CCH2C), 2.4-2.6 (2H, m, NCH2C), 3.6 (5H, bs, ester-Me and CCH2COO), 6.4 (1H, bs, amide-NH), 8.2 (2H, d, Ar 3-H and 5-H), 8.3 (2H, d, Ar 2-H and 6-H), 8.8 (2H, s, β pyrrole 3-H and 7H), 8.9 (6H, s, β pyrrole 2-H, 8-H, 12-H, 13-H, 17-H, and 18-H). MS(FAB) m/z: 1140 (MH+). 5-(4-Carboxyphenyl)-10,15,20-tris(pentafluorophenyl)porphyrin, H2PFPPCOOH. H2PFPPCOOMe (1.0 g, 1.07 mmol) was dissolved in THF (60 mL), and 2 N KOH in H2O (60 mL) was added. The mixture was stirred at 40 °C overnight in the dark. The cooled solution was then acidified with 2 N HCl to pH 4, and 0.25 N aqueous ammonium 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 twice with water and dried over MgSO4, and the solvent was removed under reduced pressure. The sample was purified by silica gel chromatography with chloroform acetone ) 4:1 as eluent. The yield of PFPPCOOH was 67% (665 mg). 1H NMR (CDCl3) δ: -2.9 (2H, s, pyrrole-NH), 8.3 (2H, s, Ar 3-H and 5-H), 8.5 (2H, s, Ar 2-H and 6-H), 8.8 (2H, s, β pyrrole), 8.9 (6H, s, β pyrrole). MS(FAB) m/z: 929 (MH+). 5-((((Carboxypentyl)amino)carbonyl)phenyl)-10,15,20tris(pentafluorophenyl)porphyrin, H2PFPPCONH(CH2)5COOH. This compound was prepared in a similar way as that described for H2PFPPCOOH. 1H NMR (CDCl3) δ: -2.8 (2H, s, (24) Ihara, Y.; Kimura, Y.; Nango, M.; Kuroki, N. J. Polym. Sci., Polym. Chem. Ed. 1983, 21, 1535-1542. (25) Iida, K.; Nango, M.; Okada, K.; Hikita, M.; Matsuura, M.; Kurihara, T.; Tajima, T.; Tattori, A.; Ishikawa, S.; Yamashita, K.; Tsuda, K.; Kurono, Y. Bull. Chem. Soc. Jpn. 1995, 68, 1959-1968. (26) Imidazole and Derivatives; Hoffmann, K., Ed.; Interscience Publishers, Inc.: New York, 1953; Part I.

Phospholipid-Linked Mn Porphyrins as Catalysts pyrrole-NH), 1.6-2.0 (6H, m, CCH2C), 2.4-2.6 (2H, m, NCH2C), 3.7 (2H, bs, CCH2COO), 6.4 (1H, bs, amide-NH), 8.2 (2H, d, Ar 3-H and 5-H), 8.3 (2H, d, Ar 2-H and 6-H), 8.8 (2H, s, β pyrrole 3-H and 7H), 8.9 (6H, s, β pyrrole 2-H, 8-H, 12-H, 13-H, 17-H, and 18-H). MS(FAB) m/z: 1042 (MH+). 5-((((Carboxyundecyl)amino)carbonyl)phenyl)-10,15,20tris(pentafluorophenyl)porphyrin, H2PFPPCONH(CH2)11COOH. This compound was prepared in a similar way as that described for H2PFPPCOOH. The yield was 84%. 1H NMR (CDCl3) δ: -2.8 (2H, s, pyrrole-NH), 1.6-2.0 (18H, m, CCH2C), 2.4-2.6 (2H, m, NCH2C), 3.66 (2H, bs, CCH2COO), 6.4 (1H, bs, amide-NH), 8.2 (2H, d, Ar 3-H and 5-H), 8.3 (2H, d, Ar 2-H and 6-H), 8.8 (2H, s, β pyrrole 3-H and 7H), 8.9 (6H, s, β pyrrole 2-H, 8-H, 12-H, 13-H, 17-H, and 18-H). MS(FAB) m/z: 1126 (MH+). Manganese Fluorinated Porphyrin Complexes. MnPFPPCOOH, MnPFPPCONH(CH2)5COOH, MnPFPPCONH(CH2)11COOH. As an example, the synthesis of MnPFPPCONH(CH2)5COOH will be described. H2PFPPCONH(CH2)5COOH (200 mg, 0.192 mmol) was dissolved in 1,3-dimethyl-2imidazolidinone (DMI) (19 mL), and Mn(C5H7O2)2 (243 mg, 0.960 mmol) was added. The solution was brought to 150 °C for 30 min. The DMI was removed under reduced pressure. Distilled water was added to the residue, and the precipitate was filtered by membrane filter. The precipitate was dissolved in chloroform and dried over magnesium sulfate. The chloroform was removed under reduced pressure, and the sample was purified by silica gel chromatography (chloroform-10% MeOH). The yield was 79% (210 mg). UV/vis (CHCl3) nm: 468, 569.5. MS(FAB) m/z: 1094 (M). MnPFPPCOOH. UV/vis (CHCl3) nm: 468.5, 569.5. MS(FAB) (M): 981. MnPFPPCONH(CH2)11COOH. UV/vis (CHCl3) nm: 468, 569.5. MS(FAB) (M): 1178. PE-C0-H2TTP. H2TTPCOOH (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 2.4% methanol in chloroform as eluent, giving 172 mg (65%). 1H NMR (CDCl ) δ: -2.85 (2H, s, pyrrole NH), 0.82 (6H, bs, 3 CCH3), 1.2 (56H, m, PE-CH2), 3.95 (6H, bs, PE-CH2OPO2OCH2 and PE-CCCH2OCO), 4.4 (1H, bs, PE-CHOCO), 5.2 (1H, bs, PENHCO), 7.5 (6H, bs, tolyl-3,5), 8.1-8.3 (10H, m, carbomethoxyphenyl-2,3,5,6 and tolyl-2,6), 8.7-8.9 (8H, m, β pyrrole). UV/ vis (CHCl3) 418, 515, 550, 590, 647. MS(FAB) (MH+): 1431. Mp: 55-58. PE-C5-H2TTP and PE-C11-H2TTP. These PE-linked porphyrins were prepared in a similar way as that described for PE-C0-H2TTP, giving 66% and 70% yield, respectively. PE-C5-H2TTP. 1H NMR (CDCl3) δ: -2.85 (2H, s, pyrrole NH), 0.82 (6H, bs, CCH3), 1.2 (56H, m, PE-CH2), 1.5 (6H, bs, spacer-CH2), 3.95 (6H, bs, PE-CH2OPO2OCH2 and PE-CCCH2OCO), 4.4 (1H, bs, PE-CHOCO), 2.2 (4H, bs, PE-COCH2), 2.4 (2H, bs, CCH2NHCOPh), 3.5 (bs, PENCH2CH2OP), 3.95 (6H, bs, PE-CH2OPO2OCH2 and PECH2OCO), 5.2 (1H, bs, PE-NHCO), 7.5 (6H, bs, tolyl-3,5), 8.1-8.3 (10H, m, carbomethoxyphenyl2,3,5,6 and tolyl-2,6), 8.7-8.9 (8H, m, β pyrrole). UV/vis (CHCl3) nm: 418, 515.5, 550, 590.5, 647. MS(FAB) (MH+): 1516. PE-C11-H2TTP. 1H NMR (CDCl3) δ: 2.85 (2H, s, pyrroleNH), 0.82 (6H, bs, CCH3), 1.2 (56H, m, PE-CH2), 1.5 (18H, bs, spacer-CH2), 3.95 (6H, bs, PE-CH2OPO2OCH2 and Pe-CCCH2OCO), 4.4 (1H, bs, PE-CHOCO), 2.2 (4H, bs, PE-COCH2), 2.4 (2H, bs, CCH2NHCOPh), 3.5 (bs, PE-NCH2CH2OP), 3.95 (6H, bs, PE-CH2OPO2OCH2 and PE-CH2OCO), 5.2 (1H, bs, PENHCO), 7.5 (6H, bs, tolyl-3,5), 8.1-8.3 (10H, m, carbomethoxyphenyl-2,3,5,6 and tolyl-2,6), 8.7-8.9 (8H, m, β pyrrole). UV/ vis (CHCl3) nm: 419, 515, 550, 590.5, 647. MS(FAB) (MH+): 1629. PE-C0-H2PFPP. H2PFPPCOOH (50 mg) was dissolved in a mixture of CHCl5 (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 (75 mg) dissolved in CHCl3 (5 mL) for 24 h at room temperature. The chloroform was removed under reduced pressure, and the sample was purified by silica gel preparative thin layer chromatography with 50% methanol in chloroform as eluent, giving 41 mg (48% yield). 1H NMR (CDCl3) δ: -2.85 (2H, bs, pyrrole-NH), 0.82

Langmuir, Vol. 12, No. 8, 1996 1983 (6H, s, CCH3), 1.2 (56H, m, PE-CH2), 2.2 (4H, bs, PE-COCH2), 3.5 (bs, NCH2CH2OP), 3.95 (6H, bs, PE-CH2OPO2OCH2 and PECH2OCO), 4.4 (1H, bs, PE-CHOCO), 5.2 (1H, bs, PE-NH), 7.5 (1H, bs, NHCOPh), 8.3 (4H, m, Ar), 8.9 (8H, m, β pyrrole). UV/ vis (CH2Cl2-10% EtOH) nm: 414, 507, 583. MS(FAB) (MH+): 1659. PE-C5-H2PFPP and PE-C11-H2PFPP. PE-C5-H2PFPP and PE-C11-H2PFPP were prepared in a similar way as that described for PE-PFPP, giving 60% and 80% yield, respectively. PE-C5-H2PFPP. 1H NMR (CDCl3) δ: -2.85 (2H, bs, pyrrole NH), 0.82 (6H, s, CCH3), 1.2 (56H, m, PE-CH2), 1.5 (6H, bs, spacerCH2), 2.2 (4H, bs, PE-COCH2), 2.4 (2H, bs, CCH2NHCOPh), 3.5 (bs, PENCH2CH2OP), 3.95 (6H, bs, PE-CH2OPO2OCH2 and PECH2OCO), 4.4 (1H, bs, PE-CHOCO), 5.2 (1H, bs, PE-NH), 7.5 (1H, bs, NHCOPh), 8.3 (4H, m, Ar), 8.9 (8H, m, β pyrrole). UV/ vis (CH2Cl2-10% EtOH) nm: 413, 509, 584. MS(FAB) (MH+): 1772. PE-C11-H2PFPP. 1H NMR (CDCl3) δ: -2.85 (2H, s, pyrroleNH), 0.82 (6H, bs, CCH3), 1.2 (56H, m, PE-CH2), 1.5-1.6 (18H, bs, spacer-CH2), 2.2 (4H, bs, PE-COCH2), 2.4 (2H, bs, CCH2NHCOPh), 3.5 (bs, PENCH2CH2OP), 3.95 (6H, bs, PE-CH2OPO2OCH2 and PE-CH2OCO), 4.4 (1H, bs, PE-CHOCO), 5.2 (1H, bs, PE-NH), 7.5 (1H, bs, NHCOPh), 8.3 (4H, m, Ar), 8.9 (8H, m, β-pyrrole). UV/vis (CH2Cl2-10% EtOH) nm: 416, 508, 583. MS(FAB) (MH+): 1856. Manganese Complexes of PE-Linked Porphyrins: PEC0-MnTPP, PE-C5-MnTTP, and PE-C11-MnTTP. For an example, the synthesis of PE-C0-MnTTP will be described as shown in (a) and (b). (a) MnTTPCOOH (30 mg) was dissolved in THF (5 mL) containing triethylamine (0.1 mL) and ECC (0.04 mL) at room temperature. The solution was stirred for 3 h and treated with PE (27.5 mg) dissolved in THF (1 mL) for 3 h at room temperature. The THF was removed under reduced pressure, and the sample was purified by silica gel preparative thin layer chromatography with 10% methanol in chloroform as eluent, giving 12.5 mg (23% yield, mp 136-142 °C). (b) A saturated solution of tris(pentane2,4-dionate)manganese in pyridine was added with stirring to a CHCl3 solution of PE-C0-H2TTP. The solution was refluxed for 24 h. Progress of conversion to the manganese complex was followed by visible spectroscopy. The manganese complex was purified by the same method described for PE-C0-H2TTP. The compounds obtained from methods (a) and (b) were identical, determined by thin layer chromatography. UV/vis (CHCl3) nm: 478, 580, 617. MS(FAB) (M): 1483. PE-C5-MnTTP. UV/vis (CHCl3) nm: 477, 579, 616. MS(FAB) (M): 1568. PE-C11MnTTP. UV/vis (CHCl3) nm: 477.5, 580, 617. MS(FAB) (M): 1680. Manganese Complexes of PE-Linked Fluorinated Porphyrins: PE-C0-MnPFPP, PE-C5-MnPFPP, and PEC11-MnPFPP. For an example, the synthesis of PE-C5MnPFPP will be described as follows. MnPFPPCONH(CH2)5COOH (50 mg) was dissolved in CHCl3 (15 mL) containing triethylamine (0.1 mL) and ECC (0.04 mL) at room temperature. The solution was stirred for 3 h and treated with PE (189 mg) for 1.5 h at room temperature. The CHCl3 was removed under reduced pressure, and the sample was purified by silica gel preparative thin layer chromatography with 25% methanol in chloroform as eluent, giving 230 mg. UV/vis (CHCl3) nm: 472, 570. MS(FAB) (M): 1824. PE-C0-MnPFPP and PE-C11MnPFPP were prepared in a similar way as described for PEC5-MnPFPP, giving 61% yield and 70% yield, respectively. PEC0-MnPFPP. UV/vis (CHCl3) nm: 473, 572. MS(FAB) (M): 1711. PE-C11-MnPFPP. UV/vis (CHCl3) nm: 472.5, 572. MS(FAB) (M): 1907. Electron Transfer Assay. 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 or 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 published3b for preparation of small unilamellar vesicles incorporating PE-linked porphyrin complexes. Liposome-containing PE-linked porphyrins were prepared by previously reported methods for incorporating metalloporphyrins into

1984 Langmuir, Vol. 12, No. 8, 1996

Nango et al.

egg PC vesicles. For an example, the preparation will be described. 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 Tomy Seiko Model U-200 or 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 the same day.

Results and Discussion Synthesis of Phospholipid-Linked Porphyrins. The synthetic sequence leading to the compound PECn-MTTO or PE-Cn-MPFPP (M ) H2, Mn) followed these steps. The porphyrins 5-(4-carboxyphenyl)-10,15,20-tritolylporphyrin, TTPCONH(CH2)nCOOH (n ) 0, 5, 11), and 5-(4-carboxyphenyl)-10,15,20-tris(pentafluorophenyl)porphyrin, PFPPCONH(CH2)nCOOH (n ) 5, 11), and their manganese porphyrin complexes were prepared as described previously.1d,3 TTPCONH(CH2)nCOOH and PFPPCONH(CH2)nCOOH and their manganese complexes were treated with ethyl chloroformate in chloroform at low temperature and reacted with dipalmitoylphosphatidylethanolamine (PE) to give the phospholipid-linked porphyrins PE-Cn-MTTP and PE-Cn-PFPP (n ) 0, 5, 11; M ) H2, Mn), followed by chromatographic separation (silica gel 10% methanol in chloroform). The 1H NMR and mass spectra of PE-Cn-MTTP or PE-Cn-MPFPP (n ) 0, 5, 11; M ) H2, Mn) support unambiguously the assigned structure as described in the Experimental Section. The UV/vis absorption spectra of PE-Cn-MTTP or PE-Cn-MPFPP (n ) 0, 5, 11; M ) H2, Mn) in chloroform and egg yolk phosphatidylcholine (egg PC) vesicles 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 chloroform and in the phospholipid vesicles. No change of these absorption spectra of PE-Cn-MTTP and PE-Cn-MPFPP in phospholipid vesicles was observed due to the change of their concentrations from 2.5 to 50 (nmol/10 mg of lipid) (data not shown). The fluorescence spectra of PE-C11-H2PFPP in CH2Cl2-10% EtOH and egg PC vesicles as a typical example are shown in Figure 1. 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 egg PC vesicles at 25 °C, similar to the fluorescence intensity in CH2Cl2-10% EtOH. The data, therefore, imply that in the vesicle systems the porphyrin moiety is immersed within the hydrophobic interior of the membrane. Similar results were obtained for other phospholipid-linked fluorinated porphyrins and PE-Cn-H2TTP (n ) 0, 5, 11), as summarized in Table 1, where the reason why the intensity of the fluorescence yield in CH2Cl2-10% EtOH goes down in PE-Cn-H2PFPP as the linker increases is not clear. Gel Filtration. To further examine the interaction of the phospholipid-linked porphyrin complexes with the lipid bilayer, we attempted to remove the phospholipidlinked porphyrin complexes from the external vesicle surface. Proof of insertion into the bilayer was noted by the sharpening and increased intensity of the porphyrin Soret band and an increase in fluorescence yield. A total of 2-3 mL of porphyrin embedded vesicles was then applied to a Sephadex G-50 Column (45 cm × 1.0 cm i.d.), eluted with 10 mM Bis-Tris buffer, pH 7.0, and the vesicle

Figure 1. Fluorescence emission spectra of PE-C11-H2PFPP in CH2Cl2-10% EtOH (s) and egg PC vesicles (0.01 M Bis-Tris buffer, pH 7.0) (- - -) at 25 °C. The solution of the porphyrin derivative was adjusted to have an equal absorbance of 0.20 at the Soret band λmax. The vesicles contained phospholipid-linked porphyrin derivatives in the molar ratio 3000 lipids to 1 porphyrin. The spectra was corrected by Rhodamine B.

fraction was collected. The visible spectra of the vesicles containing porphyrins were measured before and after gel filtration, indicating that the porphyrin portion of PEC0-MnPFPP was almost completely immersed in either the egg PC vesicle or the DPPC vesicles. Similar results were obtained with other lipid-linked porphyrins, PECn-MnPFPP (n ) 5, 11) and PE-Cn-MnTTP (n ) 0, 5, 11). Furthermore, the data of electron micrographs for PE-C0-MnPFPP and PE-C0-MnTTP indicated the formation of the vesicle (data not shown). Release of Calcein. Calcein, a charged water-soluble fluorescent dye was used for the determination of the permeability properties of liposomes containing Pe-CnMnPFPP (n ) 0, 5, 11). Calcein (75 mM) loaded in liposomes showed a quite weak fluorescence due to selfquenching. Any calcein released from the liposomes exhibits an intense fluorescence, providing a sensitive test of the liposome integrity. The fluorescence of the calcein was continuously monitored at 520 nm, excited at 488 nm. One hundred percent efflux of calcein was determined after addition of octyl-β-D-glucoside. Figure 2 shows plots of release of calcein form the liposomes containing PECn-MnPFPP (50 nM/10 mg of egg PC) at 0.4 M imidazole buffer, pH 7.0, at 25 °C as a function of time. The data illustrate that none of the phospholipid-linked manganese porphyrins caused significant calcein efflux from the liposomes under these conditions in spite of the various carbon lengths. Similar results were obtained for all PECn-MnTTP (n ) 0, 5, 11) (data not shown). Effects of Various Carbon Lengths (Cn) in PECn-MnPFPP and PE-Cn-MnTTP on Transmembrane Electron Transfer. 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 lipsome (either egg PC or DPPC) was measured anaerobically at 0.4 M imidazole buffer or 0.1 M phosphate buffer, as mediated by a catalyst of manganese porphyrin derivatives incorporated in the vesicle bilayer, as described in the previous papers.1c,3b,c 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 electron transfer with minimal spectroscopic interference from the other components of the model system. PECn-MnTTP- and PE-Cn-MnPFPP-catalyzed electron transfer was examined at pH ) 7.0. The initial electron

Phospholipid-Linked Mn Porphyrins as Catalysts

Langmuir, Vol. 12, No. 8, 1996 1985

Table 1. Fluorescence Emission Wavelength (nm) and Relative Fluorescence Yieldsa for PE-Linked Fluorinated Porphyrin Derivatives CH2Cl2-10% EtOH porphyrin PE-C0-H2TTP PE-C5-H2TTP PE-C11-H2TTP PE-C0-H2PFPP PE-C5-H2PFPP PE-C11-H2PFPP

λmax wavelength (nm) 653.5 651 651 642.5 642.5 643

egg PC vesicle (0.01 M Bis-Tris, pH 7.0) yield (%)

720 721 721 707 707 706

59 74 82 86 74 66

λmax wavelength (nm) 655 653 653 644.5 644.5 645.5

720 722 722 707.5 708 707

yield (%) 100 100 100 100 100 100

a Solutions of the porphyrin derivatives were adjusted to have equal absorbances of 0.20 at the Soret band λ max. The ratio of porphyrin derivative/lipids was 1/3000. The spectra were corrected using rhodamine B. Values are normalized to the highest yields found for the vesicle samples, which were set at 100.

Figure 2. Plots of the release of calcein (%) vs time (h): (4) PE-C0-MnPFPP; (0) PE-C5-MnPFPP; (O) PE-C11-MnPFPP at 25 °C. Calcein (75 mM) was incorporated in the egg PC vesicle (lipid 4.4 mM) inner phase. The excitation wavelength was 488 nm, and the emission intensity was monitored at 520 nm. 100% release of calcein was determined by the addition of octyl-β-D-glycoside (OG) to 0.4%. The buffer was 0.01 M Bis-Tris (pH 7.0). The vesicles contained phospholipidlinked porphyrin derivatives in the molar ratio 3000 lipids to 1 porphyrin.

Figure 3. Rate (V0) of transmembrane electron transfer catalyzed by PE-Cn-MnPFPP (n ) 0, 5, 11) and PE-C11MnTTP as a function of porphyrin concentration in the egg PC vesicle, 0.4 M imidazole buffer, pH 7.0 at 25 °C: (b) PE-C11MnPFPP; (() PE-C5-MnPFPP; (9) PE-C0-MnPFPP; (O) PEC11-MnTTP.

transfer rate (V0) was determined from the initial slope of the change of the absorbance band at 600 nm. For an example, Figure 3 illustrates the rate of electron transport across egg PC liposomes (V0) at 25 °C. As is apparent from Figure 3, PE-C0-MnPFPP shows little or not catalytic activity by itself, consistent with the rate of

control. However, PE-C5-MnPFPP and PE-C11-MnPFPP catalysis increased transmembrane electron transport with increased porphyrin concentration in the liposome, in which an enhanced electron transfer is observed for PE-C11-MnPFPP in comparison to PEC5-MnPFPP. Furthermore, PE-C5-H2PFPP or PEC11-H2PFPP showed little or no catalytic activitgy by itself (data not shown). These results are similar to those observed for PE-Cn-MnTTP (n ) 0, 5, 11), which catalyzed the transmembrane electron transfer especially when the spacer methylene group is n ) 11, as shown in Figure 3 and reported preliminarily.1c A similar result was observed for PEI- or PEG-linked manganese porphyrins with various spacer methylene groups (Cn) (PEIor PEG-Cn-MnTTP, n ) 0, 5, 11), in which an enhanced electron transfer was observed when n ) 11.3b,c At the relatively low concentration of reduced indigotetrasulfonic acid (1.0 × 10-5 M) employed under these experimental conditions, [K3Fe(CN)6] . [ITSAH2] > [PE-Cn-MnTTP or PE-Cn-MnPFPP] (see the 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.3b,c,21 Separate experiments showed that the rate of catalysis was independent of the potassium ferricyanide concentration under these conditions (data not shown). Thus, the rate-limiting step for electron transport by either catalyst when n ) 5 or 11 is the oxidation of ITSAH2,3b,21 in which the mechanism of electron transfer is presumed to follow a pathway as illustrated in Figure 4. The similarity between the phospholipid-linked porphyrins, PE-Cn-MnTTP and PE-Cn-MnPFPP, is not surprising, in which the polar head group of the PE-linked porphyrins is estimated to be at the level of the choline head group moiety of the lipid bilayers. By varying the lengths of the various carbon bonds, the position of the membrane-bound phospholipidlinked porphyrins in the lipid bilayers can be systematically changed, and consequently, the minimum distance separating two such porphyrins inserted from opposite sides of the membrane can also be varied. 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, as illustrated in Figure 4. The reduced manganese porphyrin is then viewed as changing to an 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 ferricyanide 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. According to the data of Figure 3 and the presumed

1986 Langmuir, Vol. 12, No. 8, 1996

Figure 4. Schematic representation of transmembrane electron transfer as catalyzed by phospholipid-linked manganese porphyrins. The reduced form ITSAH2 was oxidized to the oxidized form ITSA by a two-electrons release. The porphyrins and the head group of the phospholipid (PE) are represented by squares and circles, respectively. See text for the detailed discussion.

Figure 5. Schematic diagram of the minimum distances separating phospholipid-linked manganese porphyrins inserted from opposite sides of egg PC lipid bilayers. Each phospholipidlinked porphyrin is represented in its extended conformation. The distance from the PE(head group)/spacer amide alkylcarbonyl to the far edge of the porphyrin pyrrole ring is estimated to be the maximum depth the porphyrin can penetrate in the bilayer. See text for further discussion.

mechanism of Figure 4, the rate-limiting step for the PECn-MnTTP/PE-Cn-MnTTP or PE-Cn-MnPFPP/PECn-MnPFPP (n ) 5 or 11) system was predominantly the oxidation of ITSAH2 under these conditions, as described above. As the number of carbons connecting MnTTP or MnPFPP to PE is decreased, the rate-limiting step is assumed to shift to k3 and thus reflect the increasing distance between MnPFPP or MnTTP molecules anchored to their PE constituents at opposite aqueous interfaces. To allow a more detailed evaluation of our results, we have constructed molecular models of PE-linked porphyrins and from these estimated the positions of the porphyrins in the bilayers. The minimum distance separating two PE-linked porphyrins inserted from opposite sides of the membrane can be calculated by substracting the distance these PE-linked porphyrins penetrate into the bilayer from the width of the lipid membrane, as shown in Figure 5. The anhydrous bilayer thickness of egg PC has been measured and found to be 37 Å at room temperature.23 We have assumed the polar head group of the PE-linked porphyrins is estimated to be at the level of the choline head group moiety of the lipid builayers. The estimated distances penetrated into the bilayer by PE-linked porphyrins are estimated by using

Nango et al.

Figure 6. Rate (V0) of transmembrane electron transfer catalyzed by PE-C11-MnPFPP (17 nmol/10 mg of egg PC) and PE-C11-MnTTP (55 nmol/10 mg of egg PC) in the egg PC vesicle, 0.4 M imidazole buffer, pH 7.0, at various temperatures: (b) PE-C11-MnPFPP; (O) PE-C11-MnTTP.

the extended conformations rather than the statistical average positions, which are expected to be somewhat shorter due to random kinks.3b According to the data of Figure 3 and the presumed model of Figure 5, the rate of transmembrane electron transfer is likely to decrease with increasing distance separation, and a significant electron transfer occurs only when the edge-to-edge separation of the two porphyrins becomes too small in the lipid bilayers. Similar result was observed for polymer-linked manganese porphyrins with spacer methylene groups (PEIor PEG-Cn-MnTTP) where the polymer is estimated to be at the level of the choline head group of the lipid because the polymer could not penetrate into the membrane.1c,3c Effect of Temperature on the Transmembrane Electron Transfer across the DPPC Liposome. Figure 6 shows the rate of transmembrane electron transfer catalyzed by PE-C11-MnTTP and PE-C11MnPFPP in DPPC liposome at various temperatures, where the result of PE-Cn-MnTTP (n ) 0, 5, 11) was reported preliminarily.1c PE-C11-MnTTP and PE-C11MnPFPP exhibited major transitions in electron transfer rate across the lipid bilayer near 33 and 42 °C, respectively. These temperatures are near the prephase transition and phase transition temperatures of the lipid, respectively.22,23 It is considered that below the phase transition temperature the lipids are arranged in tiled one-dimensional lattices and then at the pretransition temperature twodimensional arrangements of the lipid are formed with periodic undulations.23 Above the main phase transitions lipids revert to one-dimensional lattice arrangements, separated somewhat from each other, and assume mobile liquidlike conformations.23 Thus, near the pretransition and the main phase transition PE-C11-MnTTP and PEC11-MnPFPP begin to associate with the structural transformation of lipid bilayers and can catalyze electron transfer such as that in egg PC liposome described above (see Figure 3). However, PE-C0-MnTTP cannot catalyze the electron transfer because of the shorter spacer length as well as the result in egg PC liposome described above and in DPPC liposome described preliminarily.1c Below the phase transition of the lipid bilayer of DPPC liposome all PE-Cn-MnTTP and PE-Cn-MnPFPP showed little or no catalytic activity, where electron transfer from this manganous porphyrin to a manganic porphyrin tethered to a PE molecule on the opposite side of the bilayer cannot occur because the lipids are arranged in a tiled onedimensional lattice and then the porphyrins are frozen in

Phospholipid-Linked Mn Porphyrins as Catalysts

Langmuir, Vol. 12, No. 8, 1996 1987

Figure 8. Plots of C0/k0 vs S0 in the egg PC vesicle, 0.4 M imidazole buffer, at 25 °C: (b) PE-C11-MnPFPP (20 nmol/10 mg of egg PC); (O) PE-C11-MnTTP (18 nmol/10 mg of egg PC).

Figure 7. Effect of imidazole derivative and pH on the rate of transmembrane electron transfer catalyzed by PE-C11MnPFPP at 25 °C. (a) Rate (V0) of transmembrane electron transfer catalyzed by PE-C11-MnPFPP (23 nmol/10 mg of egg PC) as a function of the concentration of imidazole derivatives: (O) imidazole; (b) Dec-His-OMe. (b) Rate (Vcorr) of transmembrane electron transfer catalyzed by PE-C11-MnPFPP (31 nmol/10 mg of egg PC) in the presence of Dec-His-OMe (350 nmol/10 mg of egg PC) as a function of pH, 0.1 M phosphate buffer.

the lipid bilayers. These results reviewed that the phospholipid-linked manganese porphyrins exhibited electron transfer across the lipid bilayer, depending on the structural change of the lipid bilayer such as the pretransition and main phase transition properties of the lipid bilayer and also on the length of the spacer methylene groups of the phospholipid-linked manganese porphyrins incorporated in the phospholipid vesicles. Effect of Imidazole on Transmembrane Electron Transfer Catalyzed by PE-C11-MnPFPP. Figure 7 shows the rate of electron transfer catalyzed by PE-C11MnPFPP at 0.1 M phosphate buffer at 25 °C, pH 7.0, in egg PC liposome as a function of the concentration of imidazole or decanoyl-L-histidine methyl ester (Dec-HisOMe) (Figure 7a) and of pH (Figure 7b). As is apparent from Figure 7a, the rate was enhanced with increasing concentration of imidazole or its derivative. No or little electron transfer was observed with increasing concentration of Dec-His-OMe in the liposome containing no manganese porphyrin. As is apparent from Figure 7b, the rate increases clearly with increasing pH from 7.0 to 8.0, and the rate Vcorr may be extracted from the observed rates (Vo) after correction for the contribution of the rate catalyzed by the manganese porphyrin in the absence of Dec-His-OMe at each pH. These results imply that imidazole derivatives play an important role in the electron

transfer, since the pKa of imidazole is around 7.27 Thus, it is considered that the electron transfer takes place by the catalytic reaction of Mn(III)/(II) porphyrin coupled with proton transport by imidazole.2,21 Furthermore, imidazole is likely to facilitate the electron transfer by coordinating with the manganese complex, as reported previously.2,21 Effect of the Porphyrin Structure on the Electron Transfer. As is apparent from Figure 3, PE-C11MnTTP and PE-C11-MnPFPP catalyzed transmembrane electron transport in egg PC liposomes with an enhanced rate of electron transfer observed for PE-C11-MnPFPP in comparison to PE-C11-MnTTP. To further examine the effect of the porphyrin structure on the electron transfer rate, kinetic measurements were made with these manganese porphyrins, as described previously.3c According to the mechanism of Figure 4, the rate-limiting step for the PE-C11-MnTTP/PE-C11-MnTTP or PEC11-MnPFPP/PE-C11-MnPFPP system is likely to be the oxidation of ITSAH2 at the outside surface of the membrane under these conditions. Thus the kinetics of the oxidation of reduced indigotetrasulfonic acid, ITSAH2, by manganese porphyrins were analyzed in a format similar to that in enzymatic catalyses, as described previously.3c,27,28 If C represents one catalytic site on the porphyrin and S the substrate, ITSAH2, then the following scheme may be formulated: k1

k2

S+C\ {k } SC 98 product + C

(1)

-1

For certain sets of experiments, the initial concentration of substrate S0 was kept in great excess over that of catalyst C0. Under these conditions, S0 . C0, initial velocities V0 are measured, and the steady-state expression for the scheme in eq 1 is

k0 ) k2C0/(KM + S0)

(2)

where k0 is the initial rate constant, V0/S0, and KM ) (k-1 (27) Suh, J.; Scarpa, I. S.; Klotz, I. M. J. Am. Chem. Soc. 1976, 98, 7060-7064. (28) Nango, M.; Kimura, Y.; Ihara, Y.; Kuroki, N. Macromolecules 1988, 21, 2330-2335.

1988 Langmuir, Vol. 12, No. 8, 1996

Nango et al.

Table 2. Kinetic Constants for the Transmembrane Electron Transfera and Redox Potentialsb of Porphyrin Derivatives Mn porphyrin

k2 (s-1)

105KM (M)

10-4k2/KM (M-1 s-1)

redox potential (V vs SCE)

PE-C11-MnPFPP PE-C11-MnTTP PEG-C11-MnPFPPc PEG-C11-MnTTPc MnPFPPd MnTTPd

0.33 0.33 1.2 1.2 0.45 0.50

2.9 4.5 5.2 6.2 1.9 3.3

1.1 0.73 2.3 1.9 2.4 1.5

-0.11 -0.22 -0.09 -0.18 -0.08 -0.25

a Experimental conditions of PE-linked porphyrin derivatives were as in Figure 8. The concentrations of MnPFPP and MnTTP were 10.5 and 6.1 nmol/10 mg of PC in the presence of 0.4 M imidazole at pH 7.0 at 25 °C, respectively. b Redox potentials (E1/2) of PE-linked porphyrins were measured by cyclic voltammetry in DMSO-0.1 M TBAP without imidazole derivatives. c Poly(ethylene glycol)-linked MnTTP or MnPFPP with spacer methylene groups (Cn, n ) 11).3c d MnPFPP: manganese 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin. MnTTP: manganese 5,10,15,20-tetrakis(p-tolyl)porphyrin.3c

+ k2)/k1. A linear transform of eq 2 is

C0/k0 ) (KM/k2) + (1/k2)S0

(3)

Fitting experimental data at S0 . C0 to eq 3, one can evaluate k2 and KM. A substantial acceleration is produced by PE-C11-MnTTP or PE-C11-MnPFPP which shows saturation behavior, as is to be expected from eq 1. Figure 8 illustrates the fit of the data for S0 . C0 to the linear eq 3. From the parameters of the lines shown in Figure 8, the kinetic constants given in Table 2 were determined. Comparing catalytic effectiveness between PE-C11MnTTP and PE-C11-MnPFPP, we note that the difference of the second-order rate parameter, k2/KM, between PE-C11-MnTTP and PE-C11-MnPFPP is influenced by the contribution of KM rather than by that of k2. Similar kinetic results were observed for the difference of the electron transfer rate between MnPFPP and MnTTP, as shown in Table 2, indicating that the KM step is responsible for the difference of the electron transfer rate between these fluorinated porphyrins and nonfluorinated porphyrins. Thus, the increased rate observed by fluorinated porphyrins in comparison to nonfluorinated porphyrins is likely to be responsible for the KM step. As is shown in eq 1, we assume that the KM step is responsible for the complex forming between the substrate, ITSAH2, and the manganese porphyrin at the outside surface of the membrane and that the k2 step is responsible for the subsequent electron transfer process from the substrate to the porphyrin derivative and for release of the substrate from the lipid surface. Thus, the kinetic results imply that the complex forming between the substrate, ITSAH2, and the manganese porphyrin at the outside surface of the membrane is responsible for the increased rate observed by fluorinated porphyrins in comparison to nonfluorinated porphyrins under these conditions. However, manganese fluorinated porphyrins which show a high oxidation potential (E1/2 value), determined by cyclic voltammetry in comparison to the MnTTP series (see Table 2), may accelerate the electron transfer rate by the difference of the redox potential, ∆E°, between electron transfer species because the electron transfer rate strongly depended on the difference of ∆E°.29 These results indicated that fluoro portions of the phenyl groups on the (29) Marcus, R. A. Angew. Chem., Int. Ed. Engl. 1993, 32, 11111121.

porphyrin ring contribute to the specific interaction with substrate on the membrane surface, in which the fluoro portions of the porphyrin ring provide an electrodeficient or steric effect on the porphyrin ring. Similar results were observed on the difference of the electron transfer rate between MnPFPP and MnTTP and between the poly(ethylene glycol)-linked manganese porphyrins PEGC11-MnPFPP and PEG-C11-MnTTP (see Table 2).3c It is considered that, by selection of the proper aqueous redox component and by appropriate choices of the porphyrins, a specific reaction at the membrane surface can be studied. Conclusions Phospholipid-linked porphyrin complexes were synthesized. The fluorinated porphyrins can be more easily embedded in the lipid bilayer of liposomes than nonfluorinated porphyrins. The manganese complexes of the phospholipid-linked porphyrins catalyzed a selective transmembrane electron transfer in the presence of imidazole, depending not only on the length of the spacer methylene groups of the compounds but also on the structure of the porphyrin and the lipid bilayers. It is considered that a significant electron transfer occurs only when the edge-to-edge separation of the two porphyrins becomes too small in the lipid bilayers and when imidazole is present. Furthermore, the complex forming between manganese porphyrin complexes and the substrate reduced indigotetrasulfonic acid in the lipid bilayers plays an important role in the electron transfer, in which the fluoro portions of the phenyl groups of the porphyrin ring contribute to the electrodeficient or steric effect on the porphyrin ring. By selection of the proper aqueous redox component and by appropriate choices of the phospholipidlinked porphyrins, a specific reaction at the membrane surface can be studied. These characteristics are of considerable interest to mimic the vectorial electron channel controlled in biological membranes and to construct some selective electron transfer systems in lipid bilayers. Thus, these porphyrin complexes are being utilized to systematically examine electron transport in these membrane systems. Acknowledgment. The present work was partially supported by Grant-in-Aid No. 07241235 from the Ministry of Education, Science and Culture, Japan. LA9507693