Energy Transfer and Electron Transfer of Poly(ethylene glycol)-Linked

Decolorisation of azo dyes with hydrogen peroxide catalysed by manganese protoporphyrins. J Tokuda , R Oura , T Iwasaki , Y Takeuchi , A Kashiwada , M...
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Langmuir 1996, 12, 450-458

Energy Transfer and Electron Transfer of Poly(ethylene glycol)-Linked Fluorinated Porphyrin Derivatives in Lipid Bilayers Kouji Iida,† Mamoru Nango,*,† Mitsutaka Matsuura,† Masashi Yamaguchi,† Kiyohito Sato,† Kazumasa Tanaka,† Kyoko Akimoto,† Keiji Yamashita,† Kazuichi Tsuda,† and Yukihisa Kurono‡ Department of Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466, Japan, and Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467, Japan Received November 14, 1994. In Final Form: September 15, 1995X Poly(ethylene glycol) [PEG]-linked porphyrin derivatives separated by spacer methylene groups (Cn), PEG-Cn-MPFPP (M ) H2, Mn; n ) 0, 5, 11), PEG-C11-MTTP (M ) H2, Mn), and PEG-C0-MPFPPBr8 (M ) H2, Mn) (Scheme 1) were synthesized. The porphyrin portion of the poly(ethylene glycol) [PEG]linked fluorinated porphyrin derivative has been anchored onto a lipid bilayer. PEG-linked fluorinated porphyrins easily associated with phospholipid bilayers and are chemically stable against oxidants such as H2O2. An efficient energy transfer from phospholipid-linked zinc porphyrin, PE-C0-ZnPFPP (Scheme 1), to externally added PEG-Cn-H2PFPP (n ) 0, 5, 11) in the lipid bilayer was observed, depending on the length of Cn and the porphyrin structure. Ground state transmembrane electron transfer catalyzed by PEG-Cn-MnPFPP (n ) 0, 5, 11) and PEG-C11-MnTTP revealed that the porphyrin causes a significant accelerated electron transfer especially when n ) 11. Comparison of PEG-C11-MnPFPP- and PEGC11-MnTTP-catalyzed electron transfer is made. The electron transfer rate was controlled not only by the separated spacer methylene groups between the porphyrin and PEG moieties but also by the structures of porphyrins.

Introduction Synthetic porphyrin model compounds can be very helpful in providing an insight into possible reactions of these porphyrin complexes such as occur in photosynthesis and mitochondria.1-3 Porphyrin derivatives play a crucial role in these electron transfer reaction systems.4-7 To provide a model for the electron transfer system, the * Address correspondence to this author. Telephone or Fax: 8152-735-5226. E-mail: [email protected]. † Nagoya Institute of Technology. ‡ Nagoya City University. X Abstract published in Advance ACS Abstracts, November 15, 1995. (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) 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. (f) Iida, K.; Nango, M.; Hikita, M.; Hattori, A.; Yamashita, K.; Yamauchi, K.; Tsuda, K. Chem. Lett. 1994, 753-756. (g) Iida, K.; Nango, M.; Okada, K.; Hikita, M.; Matsuura, M.; Kurihara, T.; Tajima, T.; Hattori, A.; Ishikawa, S.; Yamashita, K.; Tsuda, K.; Kurono, Y. Bull. Chem. Soc. Jpn. 1995, 68, 1959-1968. (h) Nango, M.; Iida, K.; Yamaguchi, M.; Yamashita, K.; Tsuda, K.; Mizusawa, A.; Miyake, T.; Yoshinaga, J. Submitted for publication. (2) (a) Dewa, T.; Satoh, M.; Komiyama, J.; Nango, M.; Tsuda, K. Macromol. Chem. Phys. 1994, 195, 2917-2929. (b) Dewa, T.; Satoh, M.; 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. (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. (8) (a) Wasielewski, M. R. Chem. Rev. 1992, 92, 435-461. (b) Crossley, M.; Burn, P. L. J. Chem. Soc., Chem. Commun. 1991, 15691571.

0743-7463/96/2412-0450$12.00/0

preparation of porphyrin derivatives that are capable of light-induced intra- or intermolecular electron transfer was reported.8-17 However, there has been little study of ground state electron transfer between 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 lipid bilayers.1,3,18-21 In the previous papers,3 Loach and we synthesized and characterized poly(ethylenimine)-linked porphyrin derivatives (PEI-Cn-MnTTP and PEI-CnMnPFPP) (Scheme 1) which should allow a more accurate description of the path of electron transfer from one site to another in biological membrane systems. The resulting porphyrin complexes were of considerable interest in model studies as the porphyrin portion readily and easily inserts into lipid bilayers and also the control of the length of this spacer group in liposome membranes permits systematic (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) 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. (13) Gust, D.; Moore, T. A.; Moore, A. L. Acc. Chem. Res. 1993, 26, 198-205. (14) 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. (15) Osuka, A.; Maruyama, K.; Mataga, N.; Asahi, T.; Yamazaki, I.; Tamai, N. J. Am. Chem. Soc. 1990, 112, 4958-4959. (16) Helms, A.; Heiler, D.; McLendon, G. J. Am. Chem. Soc. 1992, 114, 6227-6238. (17) Groves, J. T.; Fate, G. D.; Lahiri, J. Am. Chem. Soc. 1994, 116, 5477-5478. (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.

© 1996 American Chemical Society

Poly(ethylene glycol)-Linked Porphyrin Derivatives

Langmuir, Vol. 12, No. 2, 1996 451

Scheme 1. Polymer on Phospholipid-Linked Fluorinated Porphyrin

variations of the minimum distances separating two such derivatives incorporated from opposite sides of the membrane bilayer. However, the fluorinated porphyrin, PEICn-MnPFPP, could easily associate with phospholipid bilayers of liposome in comparison to PEI-Cn-MnTTP but the manganese complexes occasionally became insoluble. In the current investigation, we report the preparation of poly(ethylene glycol) [PEG]-linked porphyrins (Scheme 1) as well as that of PEI-Cn-MPFPP (n ) 0, 11; M ) H2, Mn) and characterization of their interactions with lipid bilayers to further systematically study electron transfer model systems in lipid bilayers. We reasoned that the fluorinated porphyrin in the compounds may be easy to insert into the lipid bilayers because of the miscibility of the fluorine compound with the hydrophobic lipid and that it must be chemically stable because of the steric or electrodeficient effect of the fluorine portions on the porphyrin ring as reported preliminarily.1d Furthermore, the PEG moiety on the compound is chemically stable against oxidation in comparison to PEI and also would confer water solubility, while the hydrophobic porphyrin portion should insert into the lipid bilayer as well as the PEI derivative. That is, the porphyrin group in the compounds can be anchored into the lipid bilayer so that a vectorial electron transfer between porphyrin centers may be constructed in biological membranes as a model for electron transfer across biological membranes.1,22,23 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, (22) Introduction to Biological membranes; Jain, M. K., Wagner, R. C., Eds.; John Wiley and Sons: New York, 1980. (23) Fendler, J. H., Ed. Membrane Mimetic Chemistry; John Wiley and Sons: New York, 1982.

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 spectroptometers, respectively. Elemental analyses were performed on a Yanagimoto analytical instrument. Mass spectra (MS) were obtained in the FAB mode, in a 3-nitrobenzyl alcohol or thioglycerol matrix, with JMS-DX300 Jeol instrument. Cyclic voltammetry data was measured on a Yanako P-900. A standard calomel electrode (SCE) and Ag/AgCl served as reference electrodes, glassy carbon was used 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 perchlorate (TBAP). The solutions were degassed by argon bubbling. Fluorescence decay was taken with a Horiba NAES 550 recording spectrophotometer at 25 °C. A solution of zinc 5-(4-(methoxycarbonyl)phenyl)-10,15,20-tris((pentafluorophenyl)porphyrin, ZnPFPPCOOMe, was prepared in CH2Cl2-10% EtOH, and the absorbance of the sample solution was set at 0.2. High purity egg yolk phosphatidylcholine (PC), dipalmitoylphosphatidylcholine (DPPC), and dipalmitoylphosphatidylethanolamine (PE) were obtained from Nippon Fine Chemical Co. LTD, Takasago city, Japan. Poly(ethylene glycol) having one primary amine group (PEG-NH2) with an average molecular weight of 6000 was obtained from Nippon Oil Co. LTD, Tokyo, Japan. The primary amine content was determined by the TNBS (trinitrobenzenesulfonic acid) method.24 PEI with an average molecular weight of about 1800 was a gift from Dr. Irving M. Klotz, Northwestern University (originally purchased from Dow Chemical Co.). These polymers were purified by ultafiltration in an Amicon Diaflo ultrafiltration apparatus. Synthetic Procedure. 5-(4-Carboxyphenyl)-10,15,20-tri-ptolylporphyrin, H2TTPCOOH, 5-(4-carboxyphenyl)-10,15,20-tris(pentafluorophenyl)porphyrin, H2PFPPCOOH, 5-[4-(((5-carboxypentyl)amino)carbonyl)phenyl]-10,15,20-tri-p-tolylporphyrin, H2TTPCONH(CH2)5COOH, 5-[4-(((5-carboxypentyl)amino)carbonyl)phenyl]-10,15,20-tris(pentafluorophenyl)porphyrin, PFPPCONH(CH2)5COOH, 5-[4-(((11-carboxyundecyl)amino)carbonyl)phenyl]-10,15,20-tri-p-tolylporphyrin, H2TTPCONH(CH2)11(24) Johnson, T.; Klotz, I. M. Macromolecules 1974, 7, 149.

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COOH, 5-[4-(((11-carboxyundecyl)amino)carbonyl)phenyl]10,15,20-tris(pentafluorophenyl)porphyrin, H2PFPPCONH(CH2)11COOH, and their manganese complexes, MnTTP(CH2)nCOOH and MnPFFFP(CH2)nCOOH (n ) 0, 5, 11) were prepared as described in our previous paper.1h,3a Zinc Fluorinated Porphyrin Complex: ZnPFPPCONH(CH2)11COOH. The zinc porphyrin derivative was prepared as described previously. H2PFPPCONH(CH2)11COOH (202 mg, 0.180 mmol) was dissolved in chloroform (18 mL). Zinc acetate dihydrate (892 mg, 1.79 mmol) dissolved in methanol (5 mL) was added dropwise. The reaction mixture was stirred at 61 °C for 30 min after which 50 mL of water was added. The reaction products were extracted with 20 mL of chloroform, washed three times with water, and dried over MgSO4. After evaporation of the solvent, the resulting solid was purified by silica gel column chromatography (chloroform-methanol, 9:1). The yield of ZnPFPPCONH(CH2)11COOH was 190 mg (89.2%). UV/vis: λmax 429 nm, 554. MS(FAB): m/z 1187 (M). 5-(4-(Carbomethoxy)phenyl)-10,15,20-tris(2,3,4,5,6-pentafluorophenyl)-2,3,7,8,12,13,17,18-octabromoporphyrin: H2PFPPBr8COOH. ZnPFPPCOOH (800 mg, 0.807 mmol) and N-bromosuccinimide 2.87 g (16.1 mmol) were dissolved in carbon tetrachloride (80.7 mL) in a 300-mL 3-neck bottle. Trifluoroacetic acid (2.47 mL, 32.3 mmol) was added to the solution, and the resulting solution was brought to reflux for 5 h and then kept at room temperature. Sodium sulfate was added, neutralized by sodium bicarbonate, and washed with distilled water. Zinc acetate dihydrate (886 mg, 4.04 mmol) dissolved in methanol (20 mL) was added, and the mixture was stirred for 10 min. The resulting solution was washed with distilled water and dried over MgSO4, and the solvent was removed under reduced pressure. After evaporation of the solvent, the resulting solid was purified by silica gel column chromatography using chloroform. The yield of ZnPFPPBr8COOH was 521 mg (39.8%). UV/vis (CHCl3): 466 nm, 600. ZnPFPPBr8COOH (500 mg, 0.308 mmol) was dissolved in chloroform (20 mL) in a 100-mL 2-neck bottle. Trifluoroacetic acid (2.36 mL, 30.8 mmol) was added to the solution, and the resulting solution was stirred for 1 h at room temperature. Distilled water (10 mL) was added to the solution in an ice bath, and this mixture was brought to room temperature and sat for 3 h. The organic phase was washed with distilled water and neutralized by sodium bicarbonate. The organic phase was again washed with distilled water and dried over MgSO4. The solvent was removed under reduced pressure. After evaporation of the solvent, the resulting solid was purified by silica gel column chromatography using chloroform. The yield of PFPPBr8COOH was 251 mg (52.3%). 1H NMR (CDCl3): δ -1.5 (2H, s, pyrrole NH), 8.3 (2H, d, Ar 3-H and 5-H), 8.5 (2H, d, Ar 2-H and 6-H). MS(FAB): m/z 1560 (MH+). UV/vis (CH2Cl2-10% EtOH): λmax 462.5 nm ( 107 mM-1 cm-1), 558.0 (10.5), 604.0 (7.09). Manganese Fluorinated Porphyrin Complexes: MnPFPPCOOH, MnPFPPCONH(CH2)5COOH, MnPFPPCONH(CH2)11COOH, and MnPFPPBr8COOH. As an example, the synthesis of MnPFPPCONH(CH2)5COOH will be described.1g,h H2PFPPCONH(CH2)5COOH (200 mg, 0.192 mmol) was dissolved in 1,3-dimethyl-2-imidazolidinone (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 through a 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 of MnPFPPCONH(CH2)5COOH was 79% (210 mg). UV/vis: λmax 468 nm, 569.5. MS(FAB): m/z 1094 (M). MnPFPPCOOH.1h UV/vis (CHCl3): λmax 468.5 nm, 569.5. MS(FAB): m/z 981 (M). MnPFPPCONH(CH2)11COOH.1h UV/vis: λmax 468 nm, 569.5. MS(FAB): m/z 1178 (M). MnPFPPBr8COOH. UV/vis: λmax 473.5 nm, 585.5. MS(FAB): m/z 1613 (MH+). PEI-C0-H2PFPP. H2PFPPCOOH (0.12 g) was dissolved in chloroform (15 mL) containing 2 drops of triethylamine; ethyl chloro carbonate (6 drops) was added to the solution, and the solution was stirred at room temperature. PEI (0.24 g) dissolved

Iida et al. in chloroform (20 mL) was added dropwise, and the solution was refluxed for 3 h. EtOH (20 mL) was added to the solution, and chloroform was removed under reduced pressure. Distilled water (50 mL) was added, and the solution was ultrafiltrated for 1 day, using the Amicon Diaflo apparatus with a UM-2 membrane. The solution in the cell was frozen and lyophilized. The yield was 74% on the basis of the weight of PEI-18 starting material. 1H NMR (CDCl3): δ -2.8 (2H, b s, pyrrole NH), 2.65 (155H, b s, C2H4N), 8.25 (4H, b s, Ar), 9.5 (8H, b s, β-pyrrole), indicated PEI(H2PFPP)1.1. UV/vis (CH2Cl2-10% EtOH): λmax 413 nm, 508, 582. UV/vis (H2O): λmax 413 nm, 510, 583. PEI-C11-H2PFPP. 1H NMR (CDCl3): δ -2.8 (2H, b s, pyrrole NH), 1.35 (18H, m, CCH2C), 2.65 (160H, b s, C2H4N), 8.25 (4H, b s, Ar), 9.5 (8H, b s, β-pyrrole), indicated PEI(H2PFPP)1.0. UV/vis (CH2Cl2-10% EtOH): λmax 417 nm, 511, 583. UV/vis (H2O): λmax 416 nm, 511, 583. PEI-C0-MnPFPP. This compound was prepared in a similar manner to that described for PEI-C0-H2PFPP, using MnPFPPCOOH. UV/vis (CH2Cl2-10% EtOH): λmax 463 nm, 566. UV/vis (H2O): λmax 465 nm, 571. PEI-C11-MnPFPP. This compound was prepared in a similar way as that described for PEI-C0-H2PFPP. UV/vis (CH2Cl2-10% EtOH): λmax 465 nm, 567. UV/vis (H2O): λmax 466 nm, 572. PEG-C0-H2PFPP. PFPPCOOH (20 mg, 0.0216 mmol) was dissolved in benzene (2.2 mL), and thionyl chloride (0.16 mL, 2.16 mmol) was added. The mixture solution was brought to reflux for 2 h. The solvent was removed under reduced pressure. The residue was redissolved in benzene (2 mL) and once again taken to dryness under reduced pressure to remove traces of thionyl chloride. The acid chloride was dissolved in chloroform (2.0 mL), and PEG-NH2 (88.2 mg, 0.0173 mmol) in chloroform (1.9 mL) containing a few drops of triethylamine was added. The solution was stirred at room temperature for 1 h, and the solvent was removed under reduced pressure. The sample was purified by Sephadex LH-20 gel chromatography (EtOH). The yield was 91.1% (225 mg). 1H NMR (CDCl3): δ -2.8 (2H, s, pyrrole NH), 3.4-3.8 (425H, m, OCH2CH2O), 7.68 (1H, b s, 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 7-H), 8.9 (6H, s, β-pyrrole 2-H, 8-H, 12-H, 13-H, 17-H, and 18-H). UV/vis (CH2Cl2-10% EtOH): λmax 413 nm ( 383 mM-1 cm-1), 507 (25.7), 584 (10.8). PEG-C5-H2PFPP. 1H NMR (CDCl3): δ -2.8 (2H, s, pyrrole NH), 1.6-2.0 (18H, m, CCH2C), 2.4-2.6 (2H, m, NCH2C), 3.43.8 (425H, m, OCH2CH2O and CCH2COO), 6.3 (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 7-H), 8.9 (6H, s, β-pyrrole 2-H, 8-H, 12-H, 13-H, 17-H, and 18-H). UV/vis (CH2Cl2-10% EtOH): λmax 413 nm ( 369 mM-1 cm-1), 508 (23.9), 584 (7.28). PEG-C11-H2PFPP. 1H NMR (CDCl3): δ -2.8 (2H, s, pyrrole NH), 1.35 (18H, m, CCH2C), 2.4-2.6 (2H, m, NCH2C), 3.4-3.8 (425H, m, OCH2CH2O and CCH2COO), 6.3 (1H, b s, 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 7-H), 8.9 (6H, s, β-pyrrole 2-H, 8-H, 12-H, 13-H, 17-H, and 18-H). UV/vis (CH2Cl2-10% EtOH): λmax 413 nm ( 382 mM-1 cm-1), 508 (26.5), 584 (8.82). PEG-C0-H2PFPPBr8. 1H NMR (CDCl3): δ -1.5 (2H, s, pyrrole NH), 3.4-3.8 (425H, m, OCH2CH2O), 7.68 (1H, b s, amide NH), 8.2 (2H, d, Ar 3-H and 5-H), 8.25 (2H, d, Ar 2-H and 6-H). UV/vis (CH2Cl2-10% EtOH): λmax 413 nm ( 383 mM-1 cm-1) 507 (25.7), 584 (10.8). PEG-C11-H2TTP. 1H NMR (CDCl3): δ -2.8 (2H, s, pyrrole NH), 1.31-1.33 (18H, m, CCH2C), 2.4-2.6 (2H, m, NCH2C), 2.7 (9H, s, CH3), 3.4-3.8 (425H, m, OCH2CH2O and CCH2COO), 6.3 (1H, b s, amide NH), 7.54 (6H, d, tolyl 3-H and 5-H), 7.95-8.30 (10H, m, (methoxycarbonyl)phenyl 2-H, 3-H, 5-H, and 6-H and tolyl 2-H and 6-H), 8.86 (8H, m, β-pyrrole). UV/vis (CH2Cl210% EtOH): λmax 413 nm ( 382 mM-1 cm-1), 508 (26.5), 584 (8.82). PEG-C0-MnPFPP. MnPFPPCOOH (50 mg, 0.051 mmol) was dissolved in benzene (5.1 mL), and thionyl chloride (0.37 mL, 5.10 mmol) was added. The mixture solution was brought to reflux for 1 h. The solvent was removed under reduced pressure. The residue was redissolved in benzene (2 mL) and once again taken to dryness under reduced pressure to remove traces of thionyl chloride. The acid chloride was dissolved in chloroform (5.1 mL), and PEG-NH2 (208 mg, 0.0408 mmol) in

Poly(ethylene glycol)-Linked Porphyrin Derivatives chloroform (4.08 mL) containing a few drops of triethylamine was added dropwise. The solution was stirred at room temperature for 1 h, and the solvent was removed under pressure. The sample was purified by Sephadex LH-20 gel chromatography (EtOH). The yield was 91.1% (225 mg). UV/vis (CH2Cl2-10% EtOH): λmax 459 nm ( 80.2 mM-1 cm-1), 554 (8.50). PEG-C5-MnPFPP. UV/vis (CH2Cl2-10% EtOH): λmax 459 nm ( 100 mM-1 cm-1), 556 (10.6). PEG-C11-MnPFPP. UV/vis (CH2Cl2-10% EtOH): λmax 459 nm ( 126 mM-1 cm-1), 557 (13.4). PEG-C11-MnTTP. UV/vis (CH2Cl2-10% EtOH): λmax 480 nm, 584, 621.5. Zinc Complexes of PE-Linked Fluorinated Porphyrins: PE-C11-ZnPFPP. ZnPFPPCONH(CH2)11COOH (153 mg, 0.129 mmol) was dissolved in CHCl3 (12.9 mL) containing triethylamine (0.1 mL) and ECC (0.1 mL) at room temperature. The solution was stirred for 30 min and reacted with PE (178 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 10% methanol-chloroform as eluent, giving 187 mg (71.4% yield). 1H NMR (CDCl3): δ 0.82 (6H, b s, CCH3), 1.2 (56H, m, PE CH2), 1.5-1.6 (18H, b s, spacer CH2), 2.2 (4H, b s, PE COCH2), 2.4 (2H, b s, CCH2NHCOPh), 3.5 (b s, PE NCH2CH2OP), 3.95 (6H, b s, PE CH2OPO2OCH2 and PE CH2OCO), 4.4 (1H, b s, PE CHOCO), 5.2 (1H, b s, PE NH), 7.5 (1H, b s, NHCOPh), 7.85 (2H, m, Ar), 8.75 (2H, m, Ar), 8.78 (2H, m, β-pyrrole), 8.9 (6H, m, β-pyrrole). UV/vis (CH2Cl2-10% EtOH): 426 nm, 554.5. MS(FAB): m/z 1918 (MH+) (64Zn). 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 (0.10 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 published.3b Liposomes containing PE-linked porphyrins were prepared by previously reported methods for incorporating metalloporphyrins into egg PC vesicles. For an example, the preparation will be described. 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 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. Preparation of Vesicles with Externally Incorporated Polymer-Linked Porphyrins. Preformed vesicles containing internally trapped ferricyanide were mixed with an aqueous solution of a PEG-linked porphyrin. The porphyrin penetrated into the vesicle membrane, but because of the impermeability of the lipid bilayer to the polymer, the porphyrins were incorporated only from the external surface of the vesicles. The vesicles were deoxygenated as described above.

Results and Discussion The synthetic sequence leading to the compounds PEGCn-MPFPP (n ) 0, 5, 11; M ) H2, Mn), PEG-C11-MTTP (M ) H2, Mn), PEG-C0-MPFPPBr8 and PEI-Cn-MPFPP (n ) 0, 11; M ) H2, Mn) (Scheme 1) followed these steps: The porphyrins, 5-(4-carboxyphenyl)-10,15,20-tri-p-tolylporphyrin (TTPCOOH) or 5-(4-carboxyphenyl)-10,15,20-tris(pentafluorophenyl)porphyrin (PFPPCOOH, PFPPBr8COOH, TTP(CH2)11COOH, and PFPP(CH2)nCOOH (n ) 5, 11), were prepared as described in our previous papers.3 The preparation of the manganese complex of these porphyrins was accomplished as described previously.3 TTPCONH(CH2)11COOH, PFPPCONH(CH2)nCOOH,

Langmuir, Vol. 12, No. 2, 1996 453

Figure 1. UV-vis spectra of PEG-C5-H2PFPP in CHCl3 (s), 0.01 M Bistris buffer pH 7.0 (- - -), and egg PC vesicle in the buffer at 25 °C (- - -). The ratio of porphyrin/lipids was 1/3000.

PFPPBr8COOH, and their manganese complexes were treated with ethyl chloro formate in chloroform at low temperature and reacted with PEG or PEI to give PEGC11-MTTP (M ) H2, Mn), PEG-Cn-MPFPP (n ) 0, 5, 11; M ) H2, Mn), or PEI-Cn-MPFPP (n ) 0, 11; M ) H2, Mn), followed by chromatographic separation (silica gel, 10% methanol-chloroform). PEI-C0-H2TTP was prepared as described in the previous paper.3 PEG-CnMTTP (n ) 11; M ) H2, Mn) are very soluble in water at various pHs and in typical organic solvents such as ethanol and chloroform. However, PEI-Cn-MnPFPP (n ) 0, 11) is soluble in water but becomes occasionally insoluble in many solvents. The reasons for this insolubility are not clear, but one of the reasons is likely that the primary amines on the PEI moiety were partially cross-linked with the p-fluoro portions of phenyl groups on the porphyrin ring. Characterization and verification of the synthesized compounds were done by means of NMR, UV/visible absorption, and fluorescence emission spectroscopies. The 1 H NMR spectra of PEG-C11-H2TTP, PEG-Cn-H2PFPP (n ) 0, 5, 11), PEI-Cn-H2PFPP (n ) 0, 11), and PEGC0-H2PFPPBr8 unambiguously support the assigned structure as described in the Experimental Section. The integration of these NMR spectra indicated one porphyrin group per polymer. The visible spectra of PEG-CnMPFPP (n ) 0, 5, 11; M ) H2, Mn), PEG-C11-MTTP (M ) H2, Mn), PEI-Cn-MPFPP (n ) 0, 11; M ) H2, Mn), and PEG-C0-MPFPPBr8 (M ) H2, Mn) in chloroform and water showed the presence of a characteristic porphyrin or manganese porphyrin as shown in the Experimental Section. The data for the polymer-linked free base porphyrins and manganese porphyrins agree well with previous spectral data for tetratolylporphyrin (TTP), tetrakis(pentafluorophenyl)porphyrin (PFPP), and their manganese complexes. Figure 1 shows the visible spectra of PEG-C5-PFPP in CHCl3, water, and egg PC vesicle. The broad absorbance at the Soret band can be seen in the water spectrum but cannot be seen in the egg PC vesicle spectrum, and the spectrum in CHCl3 was found to be more nearly like that in egg PC vesicle. Thus, the absorbance spectral data indicate that when the PEGlinked porphyrin complexes are added to the vesicle suspensions, the environment of the porphyrin is more like an apolar environment, consistent with the bilayer interior, than an aqueous environment. Similar spectroscopic behavior was obtained for the rest of the PEGlinked H2PFPP, for PEG-C11-H2TTP, and for their manganese derivatives. Fluorescence emission spectra of PEG-Cn-H2PFPP were also recorded in water, CH2Cl2-10% EtOH, and egg PC vesicle. The solutions containing PEG-linked porphyrin derivatives were excited at wavelengths corresponding to the maxima of the respective Soret bands. The porphyrin produces a normal porphyrin fluorescence, and a higher fluorescence intensity was observed in the egg PC vesicle, more similar to the

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Iida et al.

Table 1. Fluorescence Emission Wavelength (nm) and Relative Fluorescence Yielda for PEG-Linked Fluorinated Porphyrins CH2Cl2-10% EtOH PEG-linked porphyrin derivatives PEG-C0-H2PFPP PEG-C5-H2PFPP PEG-C11-H2PFPP

H2O (0.01 M Bistris) pH 7.0

λmax wavelengths (nm)

yield (%)

λmax wavelengths (nm)

yield (%)

643.5 648.0 643.5

85.9 93.2 93.7

644.0 650.0 648.0

61.6 57.8 60.3

707.0 707.5 707.0

705.0 704.0 709.0

egg PC vesicle (0.01 M Bistris) pH 7.0 λmax wavelengths (nm) 645.0 648.0 645.0

709.0 708.0 709.0

yield (%) 100 100 100

a The solution of the porphyrin derivatives was adjusted to have equal absorbance of 0.20 at the Soret band λ max. The ratio of porphyrin derivatives/lipids was 1/3000. Values are normalized to the highest yields which were found for the vesicle samples and set at 100.

Figure 2. Separation of porphyrin derivatives and PC vesicle containing porphyrin derivatives by gel chromatography. The egg PC aqueous solution (lipid 24 mM, 0.01 M Bistris buffer, pH 7.0) was sonicated, and then PEI-linked porphyrin dissolved in the buffer was added to be 0.13 mM. The sample was applied to a Sephadex G-150 column (5 × 1.5 cm id) and eluted with the buffer at room temperature. Each fraction was 0.45 mL. (b), PEI-C0-H2PFPP; (9), PEI-C0-H2TTP.

fluorescence intensity in CH2Cl2-10%EtOH. Table 1 summarizes the relative fluorescence intensities for the emission bands for polymer-linked porphyrins in water, CH2Cl2-10% EtOH, and egg PC vesicle. It can be seen that the highest fluorescence yield was observed for the polymer-linked fluorinated porphyrins in vesicles, again closest to the yield 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. Gel Filtration. To further examine the interaction of the polymer-linked fluorinated porphyrin complexes with the lipid bilayer, we attempted to remove the fluorinated porphyrin complexes from the external vesicle surface by gel filtration. The polymer-linked fluorinated porphyrin complexes were first allowed to bind the external surface of the bilayer of preformed vesicles. The proof of insertion into the bilayer was noted by the sharpening and increased intensity of the porphyrin Soret band and an increase in the fluorescence yield. A total of 2-3 mL of the porphyrinembedded vesicles was then applied to a Sephadex G-50 column (45 cm × 1.0 cm id) and eluted with 10 mM Bistris buffer, pH 7.0, and the vesicle fraction was collected. The visible spectra of vesicles containing the porphyrins were measured before and after gel filtration, and the shape of the Soret band was similar. The porphyrin concentrations were estimated for each fraction from the absorbance of the Soret band. Figure 2 illustrates the separation of the porphyrin derivatives and the PC vesicle containing porphyrin derivatives by gel chromatography. These data indicate that the fluorinated porphyrin portion of PEIC0-H2PFPP was almost completely immersed into the egg PC vesicle while about 30% of the porphyrin portion

of PEI-C0-H2TTP was removed.3 These data imply that in the vesicle systems the fluorinated porphyrin moiety is more easily incorporated into the hydrophobic interior of the liposomal membrane in comparison to the TTP moiety. Furthermore, the porphyrin portions of PEGC0-H2TTP and PEG-C0-H2PFPP were almost completely immersed into to the egg PC vesicle while the porphyrin portion of PEG-C0-H2PFPPBr8 was almost removed (data not shown). These results also imply that in the vesicle systems the PEG moiety can be easily incorporated with the lipid bilayer in comparison to the PEI moiety, but the porphyrin moiety of PEG-C0-H2PFPPBr8 is not easy to insert into the hydrophobic interior of the liposomal membrane because of the bromo portions of the porphyrin ring. Release of Calcein. Calcein, a charged water-soluble fluorescence dye, was used for the determination of permeability properties. Calcein (75 mM) loaded in PC vesicles showed quite weak fluorescence, providing a sensitive test of liposome integrity. None of the PEGCn-H2PFPP caused significant calcein efflux from the liposomes under these conditions in spite of the length of the spacer methylene group (data not shown). The fluorescence intensity of free calcein was not affected by the addition of these polymers even at the highest concentration [100 porphyrin moieties/3000 lipids (500 nmol/10 mg egg PC), where it is assumed that 1 liposome consists of 3000 lipids]. These results indicate that the porphyrin moiety is very stable in the lipid bilayer and also that the PEG moiety does not cause any leakages of calcein from the interior phase of the liposome. The Stability of Poly(ethylene glycol)-Linked Manganese Porphyrins against Oxidants. The decomposition of the porphyrin moiety on PEG-linked manganese porphyrins in aqueous solution containing H2O2 (2.8 × 10-1 M) and imidazole (1.4 × 10-3 M) at room temperature was measured spectroscopically to see the effects of the halogen portions of the porphyrin ring on the stability against the oxidant. The data indicated that no or little decomposition of the porphyrin was observed for PEG-C0-MnPFPPBr8 even after 2 h while 80% and 95% decompositions of the porphyrin were observed for PEG-C0-MnPFPP and PEG-C0-MnTTP, respectively, at the same conditions. This result implies that the bromo portions of the pyrrole group as well as the fluoro portions of the phenyl group for the manganese porphyrin play an important role in the stability against the oxidant in aqueous solution. Furthermore, no precipitation was observed for PEG-linked manganese porphyrins in the aqueous solution containing the oxidant after 24 h while precipitation was occasionally observed in the case of PEIlinked manganese fluorinated porphyrins at the same conditions. These results indicated that the fluorinated porphyrins or the PEG moiety are chemically stable against oxidant in comparison to nonfluorinated porphyrins such as TTP or to the PEI moiety. Energy Transfer in Lipid Bilayers. Energy transfer between porphyrins was measured to gain more informa-

Poly(ethylene glycol)-Linked Porphyrin Derivatives

Langmuir, Vol. 12, No. 2, 1996 455 Table 2. The Quantum Efficiency (Φet) of the Energy Transfer and the Energy Transfer Rate (kenergy) PEG-C0-H2PFPPc PEG-C5-H2PFPPc PEG-C11-H2PFPPc ZnPFPP-C2-H2PFPPd

Φeta

kenergyb (s-1)

0.563 0.750 0.770 0.768

0.81 × 109 1.88 × 109 2.09 × 109 2.07 × 109

a Φ ) 1 - I/I . I is the fluorescence intensity of the zinc porphyrin et 0 moiety at 615 nm and I0 is that of the zinc porphyrin at 615 nm. b k energy ) (I0/I - 1)/τ0 where the fluorescence lifetime τ0 of ZnPFPPCOOMe in CH2Cl2-10% EtOH is taken to be 1.6 ns for ZnPFPPCOOMe (see text). c Free base porphyrin/PE-C11-ZnPFPP ) 30/1 in egg PC vesicle. d 5-[4-(10,15,20-tris(pentafluorophenyl)5-porphyrinyl)phenyl]carboxyethyl)amino)carbonyl)phenyl]-10,15,20tris(pentafluorophenyl)zincporphyrin in benzene containing 1% pyridine. Reference 20.

Figure 3. Fluorescence resonance energy transfer from PEC11-ZnPFPP to externally added PEG-C0-H2PFPP in egg PC vesicle at room temperature. The vesicles contained PE-C11ZnPFPP in the ratio 3000 lipids/1 porphyrin. PEG-C0-H2PFPP dissolved in the buffer was added to the solution to be in the ratios PEG-C0-H2PFPP/PE-C11-ZnPFPP ) 0 (s), 2 (- - -), 4 (‚‚‚), before measurement. The excitation wavelength was 426 nm which is the Soret band of PE-C11-ZnPFPP in egg PC vesicle. The fluorescence spectrum of PEG-C0-H2PFPP alone in egg PC vesicle is also illustrated when excited at 414 nm (‚-‚).

increase in I0/I, indicating a large energy transfer between the zinc porphyrin and these PEG-linked free base fluorinated porphyrins. In contrast, PEG-C0-H2PFPP shows a lower energy transfer. Furthermore, PEG-C0H2PFPPBr8 shows no or little energy transfer. The reason is likely that the bromo groups on the porphyrin ring have an inhibiting effect on the insertion of the porphyrin into the lipid bilayers and also that the porphyrin becomes too electron deficient due to the electron-withdrawing effect of the bromo groups on the porphyrin ring. These results indicate that the energy transfer from the zinc porphyrin to the PEG-linked porphyrins depends on the length of the spacer methylene group of the porphyrins serving as an electron acceptor of the energy transfer and the porphyrin structure. Similar results were obtained with the interaction of PEI-Cn-H2PFPP (n ) 0, 11) with the PC vesicle as described previously.1d The most probable mechanism for the observed energy transfer is the Forster dipole-dipole interaction. A value for the rate constant for energy transfer (kenergy) for PEGlinked porphyrins may be calculated using the following equation,25,26

kenergy ) (I0/I - 1)/τ0

Figure 4. Plots of I0/I of PE-C11-ZnPFPP vs the concentration of PEG-linked free base porphyrin in egg PC vesicle, 0.01 M Bistris, pH 7.0. The x-axis is represented by [free base porphyrins]/[PE-C11-ZnPFPP]. (0), PEG-C0-H2PFPP; (4), PEG-C5-H2PFPP; (O), PEG-C11-H2PFPP, (9), PEG-C0-H2PFPPBr8.

tion on the environment of the porphyrin molecules for PEG-Cn-H2PFPP (n ) 0, 5, 11) and PEG-C0-H2PFPPBr8 in the lipid bilayer. Energy transfer from a previously incorporated zinc porphyrin, PE-C11-ZnPFPP, in the vesicle to externally added PEG-linked porphyrins in the same vesicle was measured. PE-C11-ZnPFPP was prepared as described in the Experimental Section.3 With the addition of free base porphyrins, the fluorescence emission of zinc porphyrin on PE-C11-ZnPFPP at 600 nm was decreased and, in contrast, the emission of the free base porphyrins at 660 nm increased as is seen in Figure 3, indicating that energy transfer from the zinc porphyrin to the free base porphyrins in lipid bilayers had taken place. The efficiency of the energy transfer is expressed by the intensity change of the fluorescence emission of the zinc porphyrin at 600 nm, represented by I0/I. Figure 4 illustrates the change of I0/I with addition of the free base porphyrins PEG-Cn-H2PFPP. Both PEG-C11-H2PFPP and PEG-C5-H2PFPP show a large

where τ0 is the fluorescence lifetime of ZnPFPPCOOMe in the absence of a free base porphyrin as an intermolecular quencher, I is the fluorescence intensity of the free base porphyrin at 615 nm, and I0 is the intensity of PE-C0ZnPFPP at 613 nm. The τ0 value is taken 1.6 ns as described in the Experimental Section. Values of the quantum efficiency of the energy transfer (Φet) and the energy transfer rate (kenergy) were obtained using the above values and equation as shown in Table 2. From the data presented in the Table 2, light absorbed by the Zn porphyrin is efficiently transferred to the free base porphyrin, PEG-C11-H2PFPP or PEG-C5-H2PFPP, and is emitted from that center with a quantum yield that must be near that of the free base monomer as was observed with covalenty linked porphyrin dimers such as ZnPFPP-C2-H2PFPP as described previously.1g However, smaller Φet and kenergy values were observed for PEGC0-H2PFPP in comparison to those for PEG-Cn-H2PFPP (n ) 5, 11). These results of energy transfer also support the conclusion that all the fluorinated porphyrin portions of PEG-Cn-H2PFPP are immersed in the hydrocarbon region of the bilayer, in the following order: PEG-C11H2PFPP g PEG-C5-H2PFPP > PEG-C0-H2PFPP, indicating that PEG-linked fluorinated porphyrins with a long aliphatic linker were very immersed in the bilayer. (25) Tamiaki, H.; Nomura, K.; Maruyama, K. Bull. Chem. Soc. Jpn. 1994, 67, 1863-1871. (26) Anton, J.; Loach, P. A.; Govindjee. Photochem. Photobiol. 1978, 28, 235-242.

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Figure 5. The rate (V0) of transmembrane electron transfer catalyzed by PEG-Cn-MnPFPP (n ) 0, 5, 11) as a function of porphyrin concentration in egg PC vesicle, 0.4 M imidazole buffer, pH 7.0 at 25 °C. (b), PEG-C11-MnPFPP; ([), PEGC5-MnPFPP; (9), PEG-C0-MnPFPP; (2), PEG-C11-H2PFPP; (O), PEG-C11-MnTTP.

Transmembrane Electron Transfer across Egg Yolk PC Vesicles. 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. 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. PEG-Cn-MnPFPP (n ) 0, 5, 11) and PEG-C11-MnTTP catalyzed electron transport was 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. Figure 5 illustrates the rate (V0) of transmembrane electron transport catalyzed by PEG-Cn-MPFPP (n ) 0, 5, 11; M ) Mn, H2) in egg PC vesicles. As is apparent from Figure 5, PEG-C0-MnPFPP and PEG-C5-MnPFPP showed little or no catalytic activity alone, consistent with the rate of control. However, PEG-C11-MnPFPP catalyzed transmembrane electron transport with increasing concentration of the porphyrin in the liposome, in which free base porphyrin, PEG-C11-H2PFPP, also showed little or no catalytic activity. Furthermore, Figure 6 illustrates the effect of externally added PEG-C11MnPFPP on the rate (V0) of transmembrane electron transport catalyzed by PEG-Cn-MnPFPP (n ) 0, 5) and PEG-C11-MnTTP in egg PC vesicles, respectively. As is apparent from Figure 6, an enhanced electron transfer as PEG-C11-MnPFPP was externally added was not observed in the case of the liposomes containing PEG-C11MnTTP; this indicates that electron transfer in the lipid bilayer from PEG-C11-MnPFPP to PEG-C11-MnTTP did not occur. It is also was observed that no enhanced rate of control which corresponded to the rate when PEGC11-MnPFPP is externally added to the liposome not containing the porphyrins, consistent with the rate of control. However, all PEG-Cn-MnPFPP (n ) 0, 5) catalyzed transmembrane electron transport with increasing concentration of externally added PEG-C11MnPFPP in the liposome. Since the rate-limiting step for electron transport by PEG-Cn-MnTTP and PEG-Cn-MnPFPP when n ) 11

Iida et al.

Figure 6. Rate of transmembrane electron transfer vs externally added concentration of PEG-C11-MnPFPP. (O), PEG-C11-MnTTP (4.55 nmol/10 mg egg PC) present on both sides of the vesicle with PEG-C11-MnPFPP on the external side; ([), PEG-C11-MnPFPP present only on the external surface of the vesicle; (2), PEG-C5-MnPFPP (7 nmol/10 mg egg PC) present on both sides of the vesicle with PEG-C11MnPFPP on the external side; (9), PEG-C0-MnPFPP (8 nmol/ 10 mg egg PC) present on both sides of the vesicle with PEGC11-MnPFPP on the external side.

is the oxidation of ITSAH2,3b we cannot kinetically distinguish the subsequent movement of electrons across the lipid bilayer. The mechanism of electron transfer is presumed to occur via the following pathway.3b Electron transfer occurs from external reduced indigotetrasulfonic acid to an oxidized manganese porphyrin which momentarily assumed a folded position relative to its attached PEG 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 the extending configuration relative to the PEG group. Electron transfer from this manganous porphyrin to a manganic porphyrin tethered to a PEG 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 Figures 5 and 6, the rate-limiting step for the PEG-Cn-MnPFPP/PEG-Cn-MnPFPP system was predominantly the oxidation of ITSAH2 at the outside surface of the membrane when n ) 11. As the number of carbons connecting the MnPFPP to PEG was decreased, the ratelimiting step is assumed to shift to the electron transfer reaction between MnPFPP molecules and thus reflect the increasing distance between MnPFPP molecules anchored to their PEG constituents at opposite aqueous interfaces. The minimum distance separating two PEG-linked porphyrins inserted from opposite sides of the membrane can be calculated by subtracting the distance these PEGlinked porphyrins penetrate into the bilayer from the width of the lipid membrane (52 Å) as shown in Figure 7.3b Figure 8 illustrates that the rate of transmembrane electron transfer decreases with increasing separation. According to the data of Figure 8, it is considered that a significant electron transfer occurs at separation distance of less than 5 Å, indicating that the electron transfer occurs when the edge-to-edge separation of the two porphyrins becomes too small in the lipid bilayers.3b

Poly(ethylene glycol)-Linked Porphyrin Derivatives

Figure 7. Schematic diagram of minimum distances separating PEG-Cn-MnPFPP derivatives inserted from opposite sides of a lipid bilayer. Each polymer porphyrin is represented in its extended conformation. The distance from the PEG/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.

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Figure 9. C0/k0 of transmembrane electron transfer catalyzed by PEG-C11-MnPFPP and PEG-C11-MnTTP (16 nmol/10 mg egg PC) as a function of ITSA (S0) concentration in egg PC vesicle in the presence of 0.4 M imidazole at 25 °C, pH 7.0. (9), PEG-C11-MnPFPP; (b), PEG-C11-MnTTP.

enzymatic catalyses as described previously.23,27,28 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 the 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 + k2)/k1. A linear transform of eq 2 is

C0/k0 ) (KM/k2) + (1/k2)S0 Figure 8. Relative electron transfer as a function of the distance separating two PEG-linked porphyrins inserted from opposite sides of the lipid bilayer. The distances are calculated by using the data from Figures 6 and 7.

Effect of the Porphyrin Structure on the Electron Transfer. As is apparent from Figure 5, PEG-C11MnTTP and PEG-C11-MnPFPP catalyzed transmembrane electron transport with increasing concentration of the porphyrins in egg PC liposome, where an enhanced electron transfer was observed for PEG-C11-MnPFPP in comparison to PEG-C11-MnTTP. To further examine the effect of the porphyrin structure on the electron transfer rate, kinetic measurements were made with these manganese porphyrins. The rate-limiting step for this electron transfer reaction is likely to be the oxidation of ITSAH2 by manganese porphyrins at the outside surface of the membrane under the conditions described above. This reaction at the outside surface of the membrane is not simply a bimolecular reaction, but the reaction may be governed by incorporation of ITSAH2 into the catalytic site on the outside surface of the membrane containing manganese porphyrins as observed in enzyme-like catalytic reactions on the surfaces of micelle and vesicle.23 Thus, the kinetics of oxidation of ITSAH2 by manganese porphyrins were analyzed in a format similar to that in

(3)

Fitting experimental data at S0 . C0 to eq 3, one can evaluate k2 and KM. The initial rate constants V0/S0 showed saturation behavior for both PEG-C11-MnPFPP and PEG-C11-MnTTP under the conditions of S0 . C0; Figure 9 illustrates the fit of the data for S0 . C0 to the linear eq 3 as is to be expected. The kinetic constants given in Table 3 were determined. As is shown in eq 1, we can assume that KM is responsible for the complex forming between the substrate and manganese porphyrin on the surface of the membrane and k2 is responsible for the subsequent electron transfer process from substrate to the porphyrin derivative and the release of the substrate from the lipid bilayer surface. Thus, if there are some differences between k2 and KM, we can see the effect of the porphyrin structure on the electron transfer reaction on the surface of the membrane. Comparing catalytic effectiveness between PEG-C11-MnTTP and PEG-C11MnPFPP, we note that the difference of the second-order rate parameter k2/KM between PEG-C11-MnTTP and PEG-C11-MnPFPP is affected by the contribution of KM rather than by that of k2 (see Table 3). Similar kinetic results were observed for the difference of the electron transfer between manganese 5,10,15,20-tetrakis(pen(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.

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Table 3. Kinetic Constantsa for Transmembrane Electron Transfer and Redox Potentialsb of Porphyrin Derivatives

porphyrin PEG-C11-MnPFPP PEG-C11-MnTTP MnPFPPc MnTTPc

redox imidazole k2 105KM 10-4k2/Km potential -1 -1 -1 derivatives (s ) (M) (M s ) (V) vs SCE imidazole imidazole imidazole imidazole

1.2 1.2 0.45 0.50

5.2 6.2 1.9 3.3

2.3 1.9 2.4 1.5

-0.09 -0.18 -0.08 -0.25

on the membrane surface, in which the fluoro portions of the porphyrin ring provide the steric or electrodeficient effect on the porphyrin ring. These results imply that by selection of the proper aqueous redox component and by appropriate choices of porphyrin, a specific reaction at the liposomal membrane surface can be studied. Conclusions

a

Experimental conditions of PEG-linked porphyrin derivatives were as in Figure 9. The concentrations of MnPFPP and MnTTP were 10.5 and 6.1 nmol/10 mg PC in the presence of 0.4 M imidazole at pH 7.0 at 25 °C, respectively. b Redox potentials of PEG-linked porphyrins were measured in DMSO-0.1 M TBAP without imidazole derivatives. c MnPFPP: manganese 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin. MnTTP: manganese 5,10,15,20-tetra-p-tolylporphyrin.

tafluorophenyl)porphyrin (MnPFPP) and manganese 5,10,15,20-tetra-p-tolylporphyrin (MnTTP) (see Table 3), indicating that the KM step is responsible for the difference of the 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 contributed at the KM step. Marcus theory predicts that the electron transfer rate strongly depended on the difference of the redox potential, ∆E°, between electron transfer species.29 Thus, one of the reasons for the increased rate observed may be the difference of ∆E°, in which the manganese fluorinated porphyrins show a high oxidation potential in comparison to nonfluorinated porphyrins as shown in Table 3. However, the kinetic data indicate that the fluorinated porphyrins accelerate the electron transfer reaction on the outside surface of the membrane by contribution of KM rather than k2, implying that a steric interaction of the porphyrin with reduced indigotetrasulfonic acid also plays an important role in the electron transfer at this condition.21 These results indicated that fluoro portions of the phenyl groups on the porphyrin ring may contribute to the specific interaction with substrate (29) Marcus, R. A. Angew. Chem., Int. Ed. Engl. 1993, 32, 11111121.

PEG-linked porphyrin derivatives with spacer methylene groups (Cn) were synthesized. The fluorinated porphyrin moiety on the compounds can be easily embedded into the lipid bilayer of egg PC or DPPC liposomes, and the fluorinated porphyrin moiety is chemically stable against oxidants such as H2O2 even in the absence of the liposome. An energy transfer from the zinc porphyrin moiety of PE-C11-ZnPFPP to the externally added free base porphyrin PEG-Cn-H2PFPP (n ) 0, 5, 11) in lipid bilayers was observed, depending on Cn. Furthermore, the manganese complexes of the PEG-linked porphyrin derivatives catalyzed transmembrane electron transfer in the presence of imidazole, depending not only on the length of Cn of the compounds but also on the structure of the porphyrins. It is considered that a significant electron transfer occurs at separation distance of less than 5 Å when imidazole is present, indicating that the electron transfer occurs only when the edge-to-edge separation of the two porphyrins becomes too small in the lipid bilayers. The kinetic results indicate that the complex forming between the substrate, reduced indigotetrasulfonic acid, and the manganese porphyrin on the surface of the lipid bilayers plays an important role on the electron transfer, in which fluoro portions of the phenyl groups on the porphyrin ring provide the electrodeficient or steric effect on the porphyrin ring. These results indicate that by selection of the proper aqueous redox component and by appropriate choices of the porphyrin, a specific reaction at the liposomal membrane surface can be studied. Thus, these PEG-linked porphyrin complexes are being utilized to systematically examine energy transfer and electron transport in liposomal membrane systems. LA940899W