Self-Assembled Lipidporphyrin Bilayer Vesicles. Microstructure and

Nov 8, 1994 - ^obs(faSt) = ^on(°2)[°2f + K4-°2> ..... lb vesicle was gradually blue-shifted with reducing ... Rodriguez, H.; Chin, D.; Bird, G. R. ...
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Langmuir 1995,11, 1877-1884

1877

Self-hisembledLipidporphyrin Bilayer Vesicles. Microstructure and Dioxygen Binding in Aqueous Medium Eishun Tsuchida," Teruyuki Komatsu, Kenji Arai, Kazuhiro Yamada, Hiroyuki Nishide, and Jiirgen-Hinrich Fuhrhopt Department of Polymer Chemistry, Waseda University, Tokyo 169, Japan, and Institut fur Organische Chemie der Freie Universitat Berlin, 14195 Berlin, Germany Received November 8, 1994. I n Final Form: January 25, 1995@ An amphiphilic tetraphenylporphyrinatometal (Zn, Fe) derivative having four dialkylglycerophosphocholine groups on one side of the ring plane (lipidporphyrin)was easily dispersed in an aqueous medium to give a red, stable dispersion. Based on electronmicroscopy,lipidporphyrinsthemselves produced spherical unilamellar vesicles with a diameter of ca. 100 nm. The thickness of the membrane (9.5 f 0.5 nm) corresponded to a bilayer of the lipidporphyrin (4.6 nm); the ratio of the lipidporphyrin molecules in the outer and inner layers was 1.57. The lipidporphyrinatozinc(I1) vesicle displayed some characteristics of a J-aggregate: (i) a red-shifted Soret band (425 438 nm), (ii) no fluorescence quenching, and (iii) a reduced triplet-state lifetime. Simple exciton calculations indicate that the porphyrin squares located in the outer and inner layers formed a J-aggregate with an average angle of 47". A vesicle composed of the lipidporphyrinatoiron(I1) coordinated with an alkylimidazole reversibly formed a stable dioxygen adduct. The 02-binding equilibrium and kinetic parameters were determined. This vesicle has the ability to act as a totally synthetic 0 2 carrier under physiological conditions (pH 7.4, 37 "C).

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Introduction One of the most significant challenges in hemoprotein and porphyrin chemistry is to construct functional multiporphyrin assemblies composed of a large number of self-organized metalloporphyrin amphiphiles in aqueous medium. Hemoproteins or membrane-bound enzymes include highly-ordered metalloporphyrin arrangements as active sites and allow sequential reactions cooperatively through the porphyrin moieties. The profile of multistep 0 2 binding to hemoglobin (Hb), for example, revealed a n allosteric phenomenon.1,2 In the primary steps of photosynthesis, chlorophyll assemblies undergo vectorial and extremely fast electron transfer^.^,^ Such characteristics of oriented porphyrinic arrays should be mimicked by synthetic porphyrins which then may act as molecular devices the functions of which are different from those of natural systems, i.e., as super-hemoproteins, multi-photosensitizers, molecular wires. Consequently, the elucidation of the organization process, microstructure, and electronic properties of the highly-ordered porphyrin assemblies is a topic of current interest. So far, there have been some reports on micelles or phospholipid vesicle embedding porphyrin complexes as hemoprotein m~dels.~-lO We have also found a phosphoInstitut fur Organische Chemie der Freie Universitat Berlin. Abstract published in Advance ACS Abstracts, May 1, 1995. (1)Franklin, B.; Bernard, G. F. In Hemoglobin: Molecular, Genetic and Clinical Aspects; Dyson,J.,Ed.; W. B. Saunders Co.: Philadelphia, 1987;p 37. (2)Imai, K.; Yonetani, T. J . Biol. Chem. 1975,250,7093. (3)Mathis, P.;Rutherford, A. W. In Photosynthesis; Amesz, J. Ed.; Elsevier: New York, 1987;p 63. (4)Deisenhofer, J.;Epp, 0.;Miki, K.; Huber, R.; Michel, H.; Nature +

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1985,318,618. ( 5 ) Chang, C. K.; Traylor, T. G. Proc. Natl. Acad. Sci. U S A . 1975, 72. - , 1166. ~~

(6)Geibel, J.; Cannon, J.; Campbell, D.; Traylor, T. G. J . Am. Chem. SOC.1978,100, 3575. (7)Traylor, T. G.; Chang, C. K.; Geibel, J.; Berzinis, G. A.;Mincey, T.; Cannon, J. J . Am. Chem. SOC.1979,101, 6716. (8)Esch, J.; Roks, M. F. M.; Nolte, R. J . M. J . A m . Chem. SOC.1986, 108,6093. (9)Groves, J. T.; Neumann, R. J . Am. Chem. SOC.1987,109,5045. (10)Groves, J.T.; Neumann, R. J . A m . Chem. SOC.1989,111,2900.

lipid vesicle embedding porphyrinatoiron( 11) derivatives in its bilayer, which can form a stable 0 2 adduct under physiological conditions (in aqueous solution, pH 7.4,37 "C)and function as a totally synthetic 0 2 carrier.11-15 On the other hand, the electrical and spectroscopic properties of porphyrins in a Langmuir-Blodgett monolayer film have often been investigated, and the correlation between the porphyrin arrangement and the unusual electronic feature has been elucidated.16 At present, however, few studies have been reported on a self-organized amphiphilic porphyrin assembly in aqueous solution, and further, no definitive case of its chemical reaction coupled with the aggregate microstructure of the porphyrins has been clarified to date. We have recently found that some porphyrin amphiphiles can be selforganized in water to produce some long-lived types of structures, such as fibers, tubes, v e ~ i c l e s . l ~ -In ~ l particular, amphiphilic tetraphenylporphyrin derivatives having four dialkylglycerophosphocholine groups on one side of the ring plane (lipidporphyrin) formed a spherical unilamellar vesicle, which can bind dioxygen reversibly under physiological conditions.22 We report herein the detailed synthetic procedure for the lipidporphyrin and the microstructure of its assembly in aqueous solution; a (11)Tsuchida, E.; Nishide, H.; Yuasa, M.; Sekine, M. Chem. Lett. 1983,473. (12)Tsuchida, E.; Nishide, H. Top. Curr. Chem. 1986,132, 64. (13)Tsuchida, E.;Nishide, H.;Yuasa,M. J . Chem.Soc., Dalton Trans. 1985,275. (14)Tsuchida, E.; Nishide, H.; Yuasa, M.; Hasegawa, E.; Eshima, K.; Matsushita, Y. Macromolecules 1989,22,2103. (15)Tsuchida, E.; Komatsu, T.; Arai, K.; Nishide, H. J . Chem. SOC., Dalton Trans. 1993,2465. (16)Schick, G. A.; Schreiman, I. C.; Wagner, R. W.; Lindsey, J. S.; Bocian, D.F.J . Am. Chem. SOC.1989,111, 1344. (17)Komatsu, T.;Nakao, K.;Nishide, H.;Tsuchida, E. J . Chem. SOC., Chem. Commun. 1993,728. (18)Komatsu, T.; Arai, K.; Nishide, H.; Tsuchida, E. Chem. Lett. 1993,1949. (19)Tsuchida, E.;Komatsu, T.; Kumamoto, S.; Toyano, N.; Nishide, H.J . Chem. Soc., Chem. Commun. 1993,1731. (20)Fuhrhop, J.-H.; Demoulin, C.; Bottcher, C.; Kbning, J.; Siggel, U.J . A m . Chem. SOC.1992,114,4159. (21)Fuhrhop, J.-H.; Bindig, U.; Siggel, U. J . Am. Chem. Soc. 1993, 115, 11036. (22)Komatsu, T.; Arai, K.; Nishide, H.; Tsuchida, E. J . Chem. Soc., Chem. Commun. 1993,731.

0743-7463/95/2411-1877$09.00/0 0 1995 American Chemical Society

Tsuchida et al.

1878 Langmuir, Vol. 11, No. 6, 1995

model for the arrangement of the lipidporphyrin molecules in the structure is proposed. Furthermore, we determined the 02-binding equilibrium and kinetic parameters of the lipidporphyrinatoiron(I1) vesicle and compared its 02binding properties to those of Hb and myoglobin (Mb).

Experimental Section Materials and General Methods. Succinic anhydride, thionyl chloride, 2-bromoethanol, and 12 were purchased as a special grade from Kanto Chemical Co. and used without further purification. 4-(Dimethylamino)pyridine (DMAP), 2,2,2-trichloroethoxycarbonyl chloride, 2-cyanoethyl NjV-diisopropylphosphoramidochloridite, and 2,g-lutidinewere purchased as a special grade from Tokyo Kasei Co. and used without further purification. Zinc powder was purchased as a special grade from Merck Co. and used without further purification. Tetrahydrofuran (THF) and triethylamine were purified immediately before use by distillation from sodium. Acetonitrile (CH3CN) was purified before use by distillation from diphosphorus pentoxide. Dichloromethane (CH2C12)was purified before use by distillation from calcium hydride. DMF was distilled in vacuo under argon and stored over 4A molecular sieves. Anhydrous trimethylamine was purchased from Nitto Kagaku Co. The water used was doubly deionizedusing a Millipore-Qsystem. 1-Dodecylimidazole (DIm)and 1-dodecyl-2-methylimidazole (DMIm)were prepared according to the previously reported pr0cedure.~3 Infrared spectra were recorded with a JASCO FT/IR-5300 spectrometer. The 'H-NMR and COSY ('H-IH, 1H-13C) spectra were measured with samples dissolved in CDC13 or a mixture of CDCldCD30D at room temperature using a JEOL GSX-400 instrument. Chemical shifts are expressed in ppm downfield from Me4Si as an internal standard. Absorption spectra were recorded with a Shimadzu W-2200 spectrophotometer. Elemental analysis was performed on a Yanagimoto MT3 CHN corder. Thin-layer chromatography (TLC) was carried out on 0.2 mm precoated plates of silica gel 60 F-254 (Merck). Purification was performed by silica gel 60 (Merck) flash column chromatography. Fluorescence emission spectra was recorded on a JASCO FP-770. The molecular area of the lipidporphyrin amphiphile was estimated by its surface pressure (n)-area (A) curve using a Nippon Laser & Electronics LB24OS-MWA.

'H-NMR (CDCl3,400 MHz): 6 = 0.9 (3H, t, CH3),1.2-1.5 (32H, m, -CH2-), 2.7 (4H, t, -(C=O)CHz-), 3.5-4.2 (7H, m, glycerol, -OCHz-), 4.8 (2H, s, CHzCC13). IR (KBr): v 1761 (C=O (carbonate)), 1746 (C=O (ester)), 1717 (C=O (carboxyl)) cm-l. Anal. Calcdfor C~8H4908C13:C, 54.24; H, 7.97. Found: C, 54.05; H, 8.12.

5a,lOa,lSa,20a-Tetrakis[o-[2,2-dimethyl-20-[[3-[ [2-(stear[(2,2,2-trichloroethoxy)carbonylloxylpropoxylyloxy)-3-[ carbonyllpropanoylloxy]eicosanamidol phenyllporphine (4a). Thionyl chloride (1.18 mL, 16.4 mmol) was added to a dry benzene solution of 3 (2.57 g, 4.1 mmol) and the mixture

was stirred for 2 h. The excess thionyl chloride and benzene were removed in uucuo t o yield an oily product. Then a dry THF solution (200 mL) of 5a,10a,15a,2Oa-tetrakis[o-(2,2-dimethyl20-hydroxyeicosanamido)phenyllporphine24 (TPP-(cl@H)4,1.05 g, 0.52 mmol) and D W (0.5 g, 4.1 mmol) was added dropwise to the crude acid chloride at room temperature. The mixture was further reacted for 12 hand then extracted with CHC13. The organic layer was washed with water and aqueous NaHC03. After drying over anhydrous Na2S04, the organic layer was concentrated and the residue was chromatographed on a silica gel flash column using CHClddiethyl ether (20:1, v/v) as the eluent. The major band was collected and dried a t room temperature in vacuo,giving a purple product (4a)(1.51g, 56%). Rf=0.34 (CHCldether, 20:1, v/v). 'H-NMR (CDC13,400MHz): 6 = -2.6 (2H, s, inner HI, -0.2 (24H, s, dimethyl), 0.7-1.3 (260H, m, -CH2-), 1.5-1.6 (16H, m, -OCH2CH2-, -(C=O)OCH2CH2-), -2.6 (16H, t, -(C=O)CH2-), 3.5 (8H, t, -OCH2-), 3.74.3 (28H, glycerol, -C(=O)OCHZ-), 4.8 (8H, s, CHzCC13), 7.1 (4H,s, amide), 7.5-8.7(16H,m, phenyl), 8.8(8H, s,pyrroleP-H). IR (KBr): v 3437 (NH (amide)), 1763 (C=O (carbonate)),1740 ((3-0 (ester)), 1694 (C=O (amide))cm-l. W - v i s (CHC13): Amax = 644, 588, 545, 513, 419 nm. Anal. Calcd for C244H390N8036C112.8H20: C, 63.96; H, 8.9;N, 2.45. Found: C, 63.82; H, 8.72; N, 2.38. Zinc Insertion into 4a. ZnClz (0.25 g, 1.8 mmol) and 2,6lutidine (0.1 mL, 0.86 mmol) were added to a dry THF solution (4 mL) of 4a (0.2 g, 45 pmol) under argon, and the mixture was reacted for 1h a t room temperature. The resultingsolution was evaporated to dryness and the residue was extractedwith CHC13. The organic layer was washed with water several times. After drying over anhydrous Na~S04, the organic layer was evaporated l-o-[(2,2,2-Trichloroethoxy~carbonyll-2-o-stearylgly- to dryness and the residue was chromatographed on a silica gel flash column using CHzClddiethyl ether (20:1, v/v) as the eluent. cero1(2). To a dry CH2C12 solution (300 mL) of2-o-stearylglycerol The major band was collected and then dried at room temperature (10.0 g, 0.29 mmol) and pyridine (11.8 mL, 0.15 mol), 2,2,2in uucuo to give a red-purple zinc(I1)complex (4b)(0.16 g, 79%). trichloroethoxycarbonyl chloride (4.0 mL, 0.29 mmol) dissolved Rf=0.41 (CHzClddiethyl ether, 20:1, v/v). 'H-NMR (CDC13,400 in CHzCl2 (80 mL) was added dropwise for 2.5 h a t room MHz): 6 = -0.4 (24H, s, dimethyl), 0.6-1.3 (260H, m, -CH2-, temperature and reacted for 12 h. After a 5% HC1 aqueous CHZCH~), 1.5-1.6 (16H, m, -OCHzCH2-, -(C=O)OCH2CH2-), solution (20 mL) was added, the mixture was further stirred for 2.4 (16H, t, -(C=O)CHz-), 3.5 (8H, t, -OCHz-),3.6-4.2 (28H, 1h. CHCl3(200 mL) was addedto the suspension, and the organic glycerol, -C(=O)OCHz-), 4.7 (8H, s, CHzCC13),7.0 (4H, s, amide), layer was separated, washed first with dilute aqueous HC1 and 7.5-8.7 ( X H , m, phenyl), 8.8 (8H, s, pyrrole P-HI. IR (KBr): v then twice with water, and dried over anhydrous NazS04. The 3437 (NH (amide)),1763 (C=O (carbonate)),1740 (C-0 (ester)), solution was reduced to a small volume on a rotary evaporator, 1694 (C=O (amide)) cm-l. W - v i s (CHC13): ,A = 591, 555, and the residue was recrystallized using hexane. The precipi513,484,425 nm. Anal. Calcdfor C2&388N8036C112Zn: c,65.11; tated unreacted product was filtered off, and the filtrate was H, 8.69; N, 2.49. Found: C, 64.98; H, 8.95; N, 2.69. dried in uucuo, giving a white solid (2)(13.6 g, 90%). Rf= 0.55 5a,l0a,lSa,2Oa-Tetrakis[o-[2,2-dimethyl-20-[[3-[ [2-(stear(CHCldethyl acetate, 2:1, v/v). 'H-NMR (CDC13, 400 MHz): 6 yloxy)-3-hydroxypropoxy]carbonyllpropanoyl]oxy]= 0.8 (3H, t, CH3), 1.2-1.5 (32H, m, -CH2-), 3.5-4.2 (7H, m, eicosanamidolphenyllporphinatozinc(II)(Sb). Zinc powder glycerol, -OCH2-),4.7 (2H, s, CHzCC13). IR(KBr): v 3445 (OH), (0.9g 13.8mmol)wasaddedtoaTHF/aceticacid(l:lv/v)solution 1767 (C=O (carbonate)) cm-l. Anal. Calcd for C24H45(40 mL) of 4b (1.33 g, 0.3 mmol) in an argon chamber and the 05.1.2H20: C, 53.22; H, 8.82. Found: C, 53.06; H, 8.5. mixture stirred for 1 h at room temperature. After filtration of 3-~~1-~[(2,2,2-Trichloroethoxy)carbonylloxyl-2-~stearthe zinc powder, the filtrate was neutralized with aqueous yloxy)-3-glyceroxylcarbonyllpropanoicAcid(3). Adry THF NaHC03 and extracted with CHC13. The organic layer was solution (30 mL) of 2 (6.0 g, 11.5 mmol) was added dropwise to washed with water and dried over anhydrous Na2S04. The a THF solution (15 mL) of succinic anhydride (2.3 g, 23.1 mmol) solution was reduced to a small volume, and the residue was and DMAP (0.28 g, 2.3 mmol) at 40 "C for 30 min under argon. chromatographed on a silica gel flash column using CHCldCHsThe mixture was reacted at 40 "C for 12 h and then dried on a OH (50:1, v/v) as the eluent. The major band was collected and rotary evaporator. The residue was extracted with CHzClz and dried at room temperature in vucuo, affording a red-purple the organic layer was washed with water and aqueous NaHC03. product (Sb)(0.69 g, 55%). R f = 0.16 (CHCldCH30H, 50:1, v/v). After drying over anhydrous Na2S04, the organic layer was 'H-NMR(CDC13,400MHz): 6 = -0.4 (24H, s, dimethyl), 0.6-1.3 concentrated. Then CHC13 was added to the residue and the (260H, m, -CHz-, CHZCH~), 1.4-1.6 ( E H , m, -OCH2CH2-, precipitated excess succinic anhydride was filtered. The filtrate -(C=O)OCH2CHz-), 2.5 (16H, t, -(C=O)CH2-), 2.9-3.8 (28H, was evaporated and the residue was recrystallized with hexane glycerol, -0CHz-1, 4.0 (8H, t, -C(=O)OCH2-), 7.0 (4H, s, to give 3 (5.24 g, 51%). Rf=0.26 (CHCldethyl acetate, 2:1, v/v). ~

(23)Tsuchida,E.; Nishide, H.; Yuasa, M.;Hasegawa, E.; Matsushita, Y. J.Chem. SOC.,Dalton Trans. 1984, 1147.

(24) Matsushita,Y.; Hasegawa, E.; Eshima, K.; Tsuchida, E. Chem.

Lett. 1983, 1387.

Lipidporphyrin Bilayer Vesicle amide), 7.5-8.7 (16H, m, phenyl), 8.8 (BH, s, pyrrole B-H). IR (KBr): v 3432 (NH (amide)), 1738 (C=O (ester)), 1692 (C=O (amide))cm-l. W - v i s (CHC13): Am, = 591,554,514,484,424 nm. Anal. Calcd for C Z ~ ~ H ~ ~ ~ N ~ O ZC,~71.98; Z ~ . ~H,H10.21; ZO: N, 2.89. Found: C, 72.03; H, 10.47; N, 3.01. Dematalation of 5b. A THF solution (80 mL) of 1N HCl(8 mL) was added t o a solution of 5b (0.96 g, 0.18 mmol) and the mixture was stirred for 5 min at room temperature. The solution was neutralized by adding NaHC03 and extracted with CHC13. The organic layer was washed with water and dried over anhydrous Na2S04. The solution was reduced to a small volume, and the residue was chromatographed on a silica gel flash column using CHCldCH30H (50:1, v/v) as the eluent. The major band was collected and then dried a t room temperature in uucuo to give a purple free-base porphyrin (5a)(0.34 g, 83%). Rf = 0.18 (CHCldCH30H, 50~1, v/v). lH-NMR(CDC13,400MHz):6 = -2.6 (2H, s, inner H), -0.3 (24H, s, dimethyl), 0.7-1.3 (260H, m, -CHz-, CHZCH~), 1.5-1.6 (16H, m, -OCH2CHz-, -(C=O)OCHzCH2-), 2.6 (16H, t, -(C=O)CHz-), 3.5-4.2 (36H, m, glycerol, (C=O)OCHz,-OCH2-),7.1(4H, s, amide), 7.5-8.7 (16H, m, phenyl), 8.8 (8H, s, pyrrole P-HI. IR (KBr): v 3434 (NH (amide)), 1736 (C=O (ester)), 1690 (C=O (amide)) cm-l. Wvis (CHC13): I,, = 645,587,545,512,418nm. Anal. Calcd for Cz32H386N80~8'2H20:C, 73.88;H, 10.42;N, 2.97. Found: C, 74.02; H, 10.09; N, 3.12. Iron(II1) Bromide Complex ( 5 ~ ' ) . A dry THF (50 mL) solution of Sa (0.11g, 30pmol)was added dropwiseto FeBrynHzO (0.65 g) under dry argon and the mixture was heated to reflux. The reaction was finished after 6 h under argon. The mixture was extracted with ethyl acetate and the resulting solution was chromatographed on a silica gel column using benzene/CHsOH (4:1,v/v) as the eluent. The elution was treatedwith concentrated HBr (0.1 mL) and dried at room temperature for several hours in uacuo, to give a dark purple crystalline product (5c') (0.11 g, 97%). Rf = 0.26 (CHCldethyl acetate, 2:1, v/v). IR (KBr): v 3445 (OH), 3432 (NH (amide)), 1736 (C=O (ester)), 1692 (C=O (amide))cm-l. W - v i s (CHC13): I,, = 678,651,578,508,417 nm. Anal. Calcd for C~32H384NsO~8FeBr4HzO: C, 70.7; H, 10.02; N, 2.84. Found: C, 70.92; H, 10.14; N, 2.95. 2-Cyanoethyl S-Bromoethyl NJV-Diisopropylphosphoramidite (6). 2-Cyanoethyl Nfl-diisopropylphosphoramidochloridite (0.45mL, 2.0 mmol)was added to a dry CHzClz solution of 2-bromoethanol (0.21 mL, 3.0 mmol) and triethylamine (1.1 mL, 8.0 mmol). The mixture was further stirred for 3 h. The suspension was extracted with CHC13 and the organic layer was washed with water and aqueous NaHC03. After drying over anhydrous NazS04, the organic layer was evaporated to dryness and the residue was chromatographed on a silica gel column using ethyl acetate as the eluent. The major band was collected and dried in uucuo to afford a pale yellow oil (6) (0.63 g, 97%). Rf = 0.85 (ethyl acetate). lH-NMR (CDC13,400 MHz): 6 = 1.01.1(12H, d, CH3), 2.5 (2H, t, CHZCN),3.3-3.8 (8H, O(CHz)zBr, -OCHz-, N[CH(CH3)2]2). IR (NaC1): v 2253 (CN), 1184 (CH2Br), 1024 (PO) cm-l. Introduction of PhosphateTriester to Hydroxy Groups of 5b. Compounds 5b (75 mg, 20pmol) a n d 6 (0.21 g, 0.63 mmol) were dissolved in dry THF/CHsCN( l : l , v/v) solution (6 mL), and tetrazole (56 mg, 0.79 mmol) was added to the solution under argon. The mixture was stirred for 3 h at room temperature and then THF/H20 (7:1, v/v) solution (3.2 mL) containing IZ(0.1 mg, 0.79 mmol) and 2,6-lutidine (0.8 mL, 6.9 mmol) was added. After stirring for 30 min, aqueous Na2S03 was added and solution was brought to dryness on a rotary evaporator. The residue was extracted with CHC13 and the organic layer was dried over anhydrous Na2S04. The solution was evaporated and chromatographed on a silica gel flash column using CHCl&H30H (20:1, v/v) was the eluent. The major band was collected and then dried a t room temperature in uucuo to give a red-purple product (7b)(87 mg, 92%). R f = 0.54 (CHCldCH30H, 20:1, v/v). lH-NMR (CDC13,400MHz): 6 = -0.4 (24H, m, dimethyl), 0.61.3(260H,m, -CHz-, CH&H3), 1.5-1.6 (16H, m, -OCHzCH2-, -(C=O)OCH2CHz-), 2.6 (16H, t,-(C=O)CHz-), 2.7 (BH,t, CHzCN), 3.5 (8H, t, -OCHz-), 3.6-4.3 (52H, m, glycerol, -C(=O)OCHZ-, O(CHz)zBr, OCH~CHZCN), 7.0 (4H, s, amide), 7.4-8.7 (16H, m, phenyl), 8.8 (8H, s, pyrrolep-H). IR (KBr): v 3432 (NH (amide)), 2255 (CN), 1738 (C=O (ester)), 1692 (C=O (amide)) cm-l. UV-vis (CHC13): A, = 594, 555, 514, 484, 425 nm.

Langmuir, Vol. 11, No. 6, 1995 1879

5qlO~l5~20a-Tetrakis[0-[2,2-dimethyl-20-[ [3-[[a-(stearyloxy)-3-[[[2-(trimethylammonio)ethoxylphosphonoloxylpropoxylcarbonyl]propanoylloxyleicosanamidolphenyl] porphinatozinc(I1) (lb). A dry DMF solution (15 mL) containing dissolved 7b (86.5 mg, 18pmol) and trimethylamine (5 mL) was sealed in a pressure bottle and allowed to react for

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20 h a t 65 "C. The solvents were removed, and the obtained purple solid was gel chromatographed on a Sephadex LH-60 columnusingbenzene/CHsOH (2:1,v/v) as the eluent. The major band was collected and then dried a t room temperature for several hours in uacuo, to afford a red-purple product (lb)(50 mg, 63%). 'H-NMR (CDC13/CD30D, 1:2 V/V,400 MHz): 6 = -0.3 (24H, S, dimethyl), 0.7-1.3 (260H, m, -CHz-), 1.5-1.6 (16H, m, -OCHzCH2-, -(C=O)OCHzCHz-), 2.6 (16H, t, -(C=O)CHz-), 3.2 (36H,s, choline-CHs),3.6-4.3 (52H,glycerol, -C(=O)OCHz-, -OCHz-, O(CHz)zN), 7.0 (4H, s, amide), 7.5-8.7 (16H, m, phenyl), 8.8 (8H, s, pyrrolep-H). IR (NaCl): v 3428 (NH (amide)), 1732(C=O (ester)),1692(C=O (amide)),1244(P=O), 1067 (POC) cm-l. W-vis (benzene/CHsOH,2:l (v/v)): I,, = 594,557,518, 486, 425, 405 nm. Anal. Calcd for C Z ~ ~ H ~ ~ Z N ~ Z O ~ ~ P ~ Z ~ ' ~ C, 65.75; H, 9.81; N, 3.65. Found: C, 65.73; H, 10.03; N, 3.64. Iron(II1)Bromide Complex (IC'). The synthetic procedure for IC' complex was similar to that used for lb, except for using 5c'. Finally, a dark-purple product, IC' (58.5 mg, 68%), was obtained. IR (KBr): v 3432 (NH (amide)), 1734 (C=O (ester)), 1692 (C=O (amide)), 1235 (P=O), 1067 (PO) cm-l. W - v i s (CH30H): Am, = 675,631,575,500,419 nm. Anal. Calcd for C Z ~ Z H ~ ~ Z N ~ Z O ~ ~C, P 64.76; ~F~B H,~9.66; ~ H N, ZO 3.60. : Found: C, 64.51; H, 9.97; N, 3.79. Preparation of an Aqueous Lipidporphyrin Dispersion. An aqueous dispersion of the lipidporphyrin assembly was prepared as follows. An organic solution containing the lipidporphyrin was evaporated to give a thin film, and then deionized water was added. The mixture was homogenized by an ultrasonic generator (Nihon Seiki UP-600,60 W, 3 min) in a hot water bath (60 "C) under a nitrogen atmosphere. The lipidporphyrinatoiron assembly containing DIm or DMIm was prepared by the same method in a 1mmol L-l phosphate buffer solution (pH 7.4). The molar ratios oflipidporphyrinatoiron [Fe(LP)land the axial base were Fe(LP)/DIm, 1/3mol/mol, and Fe(LP)/DMIm,1/10 mol/mol, respectively. The lipidporphyrinatoiron(II1) assemblies were reduced by the addition of a 5-fold excess of aqueous ascorbic acid under a nitrogen atmosphere. Excess ascorbic acid was removed by gel column separation with Sephadex G-25. Transmission Electron Microscopy (TEM). (a) Negative Stained Specimen. An aqueous solution of the lipidporphyrin assembly was mixed with 2% uranyl acetate, and amixed droplet was placed onto a 200 mesh carbon-coated copper grid. The grid was allowed to air-dry for 1h and was observed in a JEOL JEM100 CX a t an acceleration voltage of 100 kV. (b) Freeze-Etching Specimen. A frozen deep-etching replica was prepared using a JEOL JFD-9010. A small droplet of lipidporphyrin dispersion (0.1 mmol L-l) was mounted on a specimen support. It was rapidly immersed into slushy nitrogen (63 K). The probe was shaved gradually at 173 K i n uucuo and then etched at 163 K for 10 min. Immediately thereafter, the etched surface was shadowed with platinum-carbon a t an elevation angle of 20" on the rotating support. Subsequently it was covered by a layer of pure carbon. The replica was floated off from the specimen support and loaded onto a copper grid. (e) Cryomicroscopy. The vitrified specimen was prepared and observed as described e1sewhe1-e.~~ A droplet of the sample (0.1 mmol L-l) was placed onto the holey carbon-coated grid held by a tweezer in the plunging device. The excess fluid was blotted off and the grid was rapidly plunged through a shutter opening into a cryogenic bath of liquid ethane (89 K). The obtained vitrified sample was observed in a Phillips EM300 at an acceleration voltage of 100 kV using a Gatan model 626 coldstage. The particle size ofthe lipidporphyrin assembly was measured by a submicron particle analyzer (Coulter Electronics N4-SD). Differential Scanning Calorimetry (DSC). DSC measurement was conducted with a SEIKO I. SSC-5200instrument. The finely powdered lipidporphyrin (0.5 mg) was added to (25)Ktining, J.;BBttcher, C . ; Winkler, H.; Zeitler, E.; Talmon, Y.; Fuhrhop, J.-H. J.Am. Chem. SOC.1993,115,693.

Tsuchida et al.

1880 Langmuir, Vol. 11, No. 6, 1995

Scheme lo

3

2

5b

5c'

CN(CH2)26PO(CH&Br Tetrazole

I

12

2'6-'utidine

MTPP-C1a OOC(CH&CO CH3(CH2)170

JOPO(CHZ)ZCN] (!J(CH2)@r

(CH3)$N DMF

~

1b IC'

M:Zn(ll) 7b Fe(lll)Br 7c' a

TPP-(ClsOH)4: 5a,10a, 15a,20a-tetrakis[o-(2,2-dimethyl-20-hydroxyeicosanamido~phenyllporphine.

deionized water and the mixture was vortexed at 70 "C for 10 min. The sample solution was placed in the silver sample pan and sealed. The measurement was usually started a t 5 "C, and the temperature was raised a t a rate of 2 "C/min up to 85 "C. Excited-State Lifetime. Lipidporphyrinatozinc(I1)singlet lifetimes were measured using a Horiba NAES-500 single photon counter equipped with a hydrogen lamp (excited a t 300-400 nm, emission monitored at -400 nm). Deoxygenated samples ([Zn(LP)I= 2pmol L-1) were held in a 1cm cuvet, and experiments were carried out a t 25 "C. Triplet lifetime studies were performed using a Unisoku TSP600 time-resolved spectrophotometer system. Laser flash photolysis was carried out using a Continuum Surelite 1-10 Q-switched Nd:YAG laser. This generated a second-harmonic (532 nm) pulse of 6 ns duration with an energy of 200 mJ per pulse; a repetition rate of 10 Hz was used for excitation of the sample solution. The output signal from a photomultiplier was recorded on a Hewlett-Packard digitizing oscilloscope 54510B. For steady state irradiation, a 150 W Xenon arc-lamp was used as the monitor light source. The triplet decay of the lipidporphyrinatozinc(I1) was monitored by transient absorption a t 445 nm. The Zn(LP) concentrations of 5 pmol L-l were used and most experiments were carried out a t 25 0.2 "C. 0 2 - and CO-BindingEquilibrium. 0 2 - and CO-binding to the lipidporphyrinatoiron(I1) complex was expressed by

*

Fe(LP)B

+L

Fe(LP)B(L)

(1)

where LP is lipidporphyrinato-, B is the axial base, and L is the gaseous ligand 0 2 or CO. The 0 2 - and CO-binding affinity (gaseous pressure at half 0 2 - or CO-binding for the porphyrinatoiron(II), (&(L) = l/[K(L)]) was determined by spectral changes at various partial pressures of 0 2 and CO as in previous l i t e r a t ~ r e . ~For ~ , ~visible ~ absorption spectroscopy, Fe(LP) concentrations of 20 pmol L-l were used. The spectra were recorded within the range of 700-360 nm. 0 2 - and CO-Binding Kinetics. 02-Binding kinetics were measured by a competitive rebinding technique provided by Gibson and Traylor using a laser flash photolysis apparatus (see abo~e).~~,~~

where KO, is the association rate constant and k , , ~is the dissociationrate constant for 0 2 and CO, respectively. Photolysis of Fe(LP)B(CO) in the presence of CO and 0 2 gives the five(26)Collman, J. P.; Brauman,J. I.;Doxsee,K. M.; Halbert, T.; Suslick, K. S. Proc. Natl. Acad. Sci. U S A . 1978,75,564. (27)Collman, J. P.;Brauman, J . I.; Iverson, B. L.; Sessler, J. L.; Morris, R. M.; Gibson, Q. H. J.Am. Chem. SOC.1983,105,3052. (28)Traylor,T.G.; Tsuchiya, S.;Campbell, 0.; Mitchell, M.; Stynes, D.;Koga, N.J . Am. Chem. SOC.1986,107,604.

coordinated Fe(LP)B complex which subsequently adds dioxygen in a fast step due to the larger k,,(Oz) and k,ft(Oz)values for than those of CO. Therefore, the analysis of the fast reaction allows direct determination of k,,(Oz) from a plot of eq 3:

The rate constant for the following return (slow process) of the Fe(LPIB(02) to Fe(LP)B(CO)is then given by

and the slope of the plots of k,,(CO)/k,~(slow)vs 0 2 a t fixed [CO] gives the 02-binding constant [K(Oz)laccurately. The required k,,(CO) is simply obtained from photolysis in the absence of 0 2 . Furthermore, the k,a(02) and k,ft(CO)were calculated from kon/ K, respectively.

Results and Discussion Synthesis of Lipidporphyrin. The synthetic proced u r e for the lipidporphyrinatometal complex (Zn, Fe) is summarized in Scheme 1. The acid chloride of 3 w a s allowed t o couple with T P P - ( C I ~ O Ha)n~d~afforded ~ 4a in almost quantitative yields. The (2,2,2-trichloroethoxy)carbonyl (Troc) protecting group of 4b was then selectively removed by zinc in acetic acidPTHF at room temperature (55%). If removal of the protective groups w a s carried out with the free-base porphyrin, decomposition occurred. In the final step, introduction of t h e four phosphocholine moieties as hydrophilic h e a d groups t o 5b could not be achieved by t h e general procedure using 2-chloro-1,3,2dioxaphospholane 2-oxide; only 1-3 phosphocholine groups were introduced at the most. In contrast, a mild-acidcatalyzed phosphorylation strategy based on phosphoramidite coupling w a s successful.29 This procedure permitted easy purification of the nonionic intermediates using silica gel column chromatography. Simultaneous removal of t h e cyanomethyl group a n d displacement of the bromide were accomplished by heating in a DMF solution of trimethylamine, producing the lipidporphyrin having four zwitterionic dialkylphosphocholine groups on t h e ring plane. No atropisomerization of the a4-structure of t h e lipidporphyrin took place, as confirmed by lH-NMR spectra. The chemical shift of the 2,2-dimethyl groups (-0.4 ppm) remained uniform. Iron porphyrin (IC')w a s prepared from 5c' in the same manner as for l b from 5b. (29) Hebert, N.; Just, G. J.Chem. SOC., Chem. Commun. 1990,1497.

Langmuir, Vol. 11, No. 6,1995 1881

Lipidporphyrin Bilayer Vesicle

All porphyrins were characterized by physicochemical measurements (see Experimental Section). R

Lipidporphyrin (1)

M: 2H Zn( II)

a b

Fe(lll)Br

c'

Fe(ll)

c

Morphology and Molecular Packing of the LipidporphyrinAssembly. Lipidporphyrinatozinc(11)(lb) was dispersed in deionized water by sonication ([lb] = 10- 100@molL-*) to give a transparent red solution. The homogeneous dispersion did not changefor several months and there was no precipitation. The aggregate morphology was clearly elucidated by transmission electron microscopy. Figure 1 shows three types of electron micrographs of the l b assembly. The lipidporphyrin formed a spherical unilamellar vesicle with a uniform diamter ofca. 100nm (Figure la,b). The particle diameters also agreed with the average sizes (94f 19 nm) estimated from a light scattering experiment. The thermodynamic stability of the vesicle was checked by electron microscopy. The spherical morphology of the lb vesicles, which had been heated to 85 "C and then cooled to room temperature, remained essentially unchanged. Cryomicroscopy ensured that the specimenis maintained in its original state without any artifacts.30 The thickness of the membrane was estimated to be 9.5 f 0.5 nm in cryomicrographs (Figure IC),which corresponds to twice the length of the side chains of lb (4.6nm). The lipidporphyrin packing geometry of the bilayer vesicle can be estimated from the effective surface area of the lipidporphyrin molecule. The numbers of the lipidporphyrin molecules in the outer and inner layers (no, ni) of the lb vesicle can be calculated from eq 5:

4n12 no = a

ni =

4 d l - d)2 a

(5)

where I is the particle diameter of the vesicle, d is the thickness of the bilayer membrane, and a is the surface area per lipidporphyrin molecule. From the n-A curve of the lb monolayer, the moiecular area per lipidporphyrin was determined to be 287 A2. Accordingly, the obtained molecular area of l b is in good agreement with 4 times the head group area of egg yolk phosphocholine; ca. 70 A2.31 Thq tetraphenylporphyrin square at the bottom of lb (280 A2), which is the root of the four phospholipid (30)Dubochet, J.; Adrian, M.; Chang, J.J.; Lepault, J.; McDowall, A. W.In Cryotechniquesin BiologicalElectronMicroscopy;Steinbrecht, R. A., Zierold, IC, Eds.; Springer-Verlag: Berlin, 1987; p 114. (31)Huang, C.; Mason, J. T.; Proc. Nutl. Acud. Sei. U S A . 1978,75, 308.

Figure 1. Transmission electron micrographs of the lipidporphyrinatozinc(I1) (lb)vesicle in aqueous solution.

groups, has almost the same width as the head groups.

As a result, a cylindrical form of the lipidporphyrin conformer can be inferred. Because the thickness of the bilayer membrane is 9.5 nm (from the cryomicrograph), noand ni are estimated to be 9670 and 6160,respectively. Therefore, the ratio of the numbers of the lipidporphyrinatozinc(I1) molecules in the outer and inner layers (X = nJni) is calculated to be 1.57.

Tsuchida et al.

1882 Langmuir, Vol. 11, No. 6, 1995

'

I

I, ''

Wavelength I nm

Figure 2. Visible absorption spectra of the lipidporphyrinatozinc(I1) vesicle, [lbl = 5 pmol L-l. Vesicle dispersion (-1, benzene/methanol (l:l, v/v) solution (- - -1 at 25 "C.

Absorption and Excitation Spectral Properties of the Lipidporphyrin Vesicle. The visible absorption spectrum of the l b vesicle showed a Soret band (,Imm= 438 nm) with a small shoulder a t 408 nm, which was shifted toward the red region relative to that observed in benzene/methanol ( L l , v/v) (Amm = 425 nm). The Q bands remained essentially unaltered (Figure 2). In general, local inhomogeneities in the vesicle would lead to a broadening of all absorption m a ~ i m a The . ~ ~observed ~ ~ ~ spectrum suggests that the Soret band splitting is due to exciton interactions. The red-shifted Soret band can then be attributed to a lateral arrangement of the transition moments of the porphyrin molecule^.^^,^^-^^ This type of "J-aggregate" was further confirmed by its photophysical properties. The fluorescence emission intensity for the lb vesicle was slightly red-shifted relative to that of the corresponding monomer in homogeneous organic solution, although it was not quenched (Figure 3). Concomitantly, the singlet lifetime for the lb vesicle ( t =~ 4.36 ns) was almost the same as that of the lb monomer in benzene/ methanol solution ZF (=2.1 ns). In contrast, the triple lifetime for the lb vesicle was extraordinarily short compared with that of the lb monomer in organic solution. The transition absorption difference spectra for the triplet decay of the lipidporphyrinatozinc(I1)vesicle induced by laser flash photolysis under argon are shown in Figure 4. For a benzene/ methanol nonaggregated solution, the decay of the difference spectra with a maxima a t 455 nm and minimum at 425 nm, which corresponded to the triplet state spectra, was cleanly monophasic (ZT= 4 3 0 , ~ s )On . the other hand, in aqueous solution of the lb vesicle, the triplet state was too short to be measured by our laser flash technique. The decrease in the triplet lifetime implicates additional nonradiative decay channels from the excited states due to formation of a n oriented multiporphyrin arrangement. (32)Gouterman, M. In The Porphyrins; Dolphin, D., Ed. Academic Press: New York, 1978;Vol. 3,p 1. (33)Kim, D.; Holten, D.; Gouterman, M. J.Am. Chem. SOC.1984, 106, 2793. (34)Esch, J.H.; Peters, A.-M.P.;Nolte, R. J. M. J.Chem. SOC.,Chem. Commun. 1990,638. (35)Barber, D.C.;Freitag-Beestron, R. A.; Whitten, D. G . J.Phys. Chem. 1991,95,4074. (36)Nagata, T.; Osuka, A.; Maruyama, K. J.A m . Chem. SOC.1990, 112, 3054. (37)Anderson, H.L.Inorg. Chem. 1994,33,972.

I

I

I

600

700

Wavelength I nm

Figure 3. Fluorescence emission spectra of the lipidporphyrinatozinc(I1)vesicle under argon, [lb] = 5 pmol L-l: Vesicle dispersion (-), benzendmethanol,(l:l,v/v) solution (- - -)at 25 "C. Excited at 425 nm. 0.2

,

II

I

0.1

4

0 Q

0

I

400

450

500

550

600

Wavelength / nm

Figure 4. Transition absorption differential spectra for the lipidporphyrinatozinc(I1)solution under argon, [lb]= 5 pmol L-l, excited at 532 nm. The numbers 1-5 label curves recorded for nonaggregated benzene/methanol (1:1 v/v) solution at 0,

100,200,400,and 800ps after photolysis. The number l'labels the curve recorded for aqueous vesicle at 0 ps after photolysis. The insert shows the absorption change vs time at 455 nm; (a) l b in benzene/methanol (1:lv/v) solution, (b) an aqueous l b vesicle. Similar observations have been reported by Schick and Bocian et al. They showed the split Soret band absorption of a supported monolayer assembly containing 5,10,15,2O-tetrakis[4-(1-octyloxy)phenyl]porphinatozinc(II) with 442 and 400 nm components relative to that ofthe solution. Whitten et al. also reported that 5a,lOa,l5a,20a-tetrakis(0-hexanamidopheny1)porphyrinJ-aggregates in dilute aqueous surfactant solution (i.e.,below the critical micelle concentration)exhibited red-shifted Soret band absorption (Amm 436 nm) and strongly reduced triplet lifetimes.35 DSC measurement of the spherical bilayer vesicle of the lipidporphyrinatozinc(I1)was performed in order to elucidate the crystal-to-liquid-crystal phase transition behavior. The DSC curve of the lb dispersion showed a broadening peak at 56 "C. Interestingly, the Soret band maxima of the lb vesicle showed a temperature dependence in the range of 20-85 "C. The Lmm (438 nm) of the

-

Lipidporphyrin Bilayer Vesicle

Langmuir, Vol. 11, No. 6, 1995 1883 AE3' for the S, and S, transitions) for dimeric interaction through the interlayer was estimated from the apparent A E 2 and AE4 divided by 2l[(nJni) 11,respectively. From the energy shifts h E 3 (-643 cm-l) and A&' (1514 cm-l), r2 is determined to be 5.9 A, and 8 is presumed to be ca. 47". However, in the case ofthe l b vesicle even below the phase transition temperature, the geometric packing of the porphyrin square in the middle of the bilayer is fluid, so that the calculated distance and angle according to this exciton coupling theory are average values. 0 2 - and CO-Binding Properties of Lipidporphyrin Vesicle. The lipidporphyrin vesicle was obtained in a similar manner, in the presence of a small excess molar amount of alkylimidazole (DIm or DMIm) a s a n axial base for the dioxygen adduct. The visible absorption spectrum of the iron(I1) deoxy complex of the lipidporphyrinatoiron( 11) vesicle (Amm = 434,536, and 558 nm) changed to that of its 0 2 adduct on exposure to dioxygen (Ama = 432 and 540 nm). The spectrum changed reversibly in response to 0 2 pressure. The 0 2 adduct changed to the corresponding CO adduct upon bubbling CO gas through the solution (Amm = 431 and 544 nm). The 02-binding affinity [P~2(02); the 0 2 partial pressure a t half 0 2 binding for the porphyrinatoiron(1I)l of the lc-DMIm vesicle was estimated to be 43 Torr at 37 "C, which is slightly lower than that of a red cell suspension. The half-life of the dioxygenated lcDIm vesicle was 50 h under physiological conditions (37 "C, pH 7.4). Most important, one spherical ICvesicle (MW 8.16 x lo7 Da) can bind 1.58 x lo4 mol of 0 2 molecules, although one particle of Hb (MW 6.45 x lo4 Da) binds only four moles of 02. The lipidporphyrinatoiron(I1)vesicle incorporated with the sterically hindered base, DMIm, affords only a fivecoordinated high-spin species owing to the repulsion between the 2-methyl group and the porphyrinring.26The deoxy state of the lc-DMIm vesicle also binds dioxygen reversibly in aqueous medium [A, = 445, 532, 561 (deoxy); 432, 549 (oxy); 430, 532 (carbonyl) nml. The P y ~ ( 0 2was ) 34 Torr at 25 "C, which approximates the Hb model. In order to elucidate the 0 2 - and CO-binding properties of the lipidporphyrinatoiron(I1) vesicle, the kinetics of the binding were explored by laser flash photolysis. Kinetic parameters for the 0 2 and CO binding to the lipidporphyrinatoiron(I1) vesicle are summarized in Table 1.The Pvz(O2) of the lc-DMIm vesicle is almost the same as that of T-state Hb.43r44However, the association and dissociation rate constants for 0 2 of the lipidporphyrinatoiron(I1) vesicle are 102-foldlarger than those of Hb, which are similar to those of the 5a,10a,15a,20a-tetrakis(o-pivalamidophenyl)porphyrinatoiron(II)-(1,2-dimethylimidazole) [Fe(TpivPP)1,2-dMIm]complex in homogeneous toluene solution.27 This has been interpreted to indicate that the 02-binding reaction is not retarded by the diffusion of dioxygen in and through the alkyl moieties of the lipidporphyrin bilayer vesicle. Even in the case of the ICmolecules which are self-assembled to form a highlyordered structure, porphyrinatoiron(I1) moieties can bind

+

unit: A

hE,\_

I

42:Ak AEi

425

AEZ

.-

430 (441)

unit: nm

Figure5. Model of the exciton calculationsand energysplitting of the Soret band as calculated by exciton theory.

l b vesicle was gradually blue-shifted with reducing absorption from 60 "C and reached 434 nm at 80 "C. This characteristic behavior of the Am= was reversibly observed dependent on the temperature changes. In contrast, the A, of the monomeric l b micelle solution dispersed with Triton X-100 (4 wt %) (=427 nm) was always invariable with the temperature. This indicates that a broad phase transition existed near 55-65 "C in the lipidporphyrin vesicle accompanied by negligible movements of the porphyrin square. Exciton Coupling Model. Arrangement of the porphyrin moiety in the l b vesicle was estimated by exciton coupling c a l c ~ l a t i o n s . ~First, ~ ~ ~ the ~ - ~interaction ~ of a large number of edge-to-edgeneighbors in eachlayer, outer or inner, was considered and then a simple dimer pair calculation involving only the nearest porphyrin units through the interlayers was performed. The molecular exciton shift in each monolayer is given in the general equation for the exciton levels of a square planner lattice with a n in-plane transition dipole and the interlayered (dimeric) one is given in the simple equation

A E = h- 1 M2 ~ (- 31cos2 0) r

(6)

where AE is the spectral shift from monomer absorption, h is Planck's constant, r is the distance between the centers of the interacting transition dipoles, 6 is the tilt angle between the line of the centers, and M is the monomer transition dipole moment calculated from the l b spectrum for the Soret band (8.0 D).42 In the each monolayer, the distance of the transition +pole moments for the in-plane arrangements (r1)is 19 A, estimated on the basis of the close-packing structural model of the four head groups on the lipidporphyrin. The calculated energy splittinglevels for S, and S, of the Soret band are shown in Figure 5. The first splitting is due to in-plane alignment in the individual layer and each of the resulting levels is further shifted because of the interlayer stacking to the nearest porphyrin. In the second step, because no and ni are different, the energy shift ( A E 3 and (38)Kasha, M.Rad. Res. 1963,20,55. (39)Kasha, M.;Rawls, H. R.; El-Bayoumi, M. A. Pure. Appl. Chem. 1965,11, 371. (40)Emerson, E. S.;Conlin, M. A.; Rosenoff, A. E.; Norland, K. S.; Rodriguez, H.; Chin, D.; Bird, G. R. J. Phys. Chem. 1967, 71,2396. (41)Hochstrasser, R. M.;Kasha, M. Photochem. Photobiol. 1964,3, 317.

(42)Transition dipole moment was calculated by integrating plots of excitoncoefficient divided by wave number, E(v)/v, versus wavenumber v and applying the equation M2 = 9.19 x 1 0 - 3 [ ~ ( ~ ) / vdv, l where M is the transition dinole moment in units of Debve. (43)Sharma, k. S.;Schmidt, M. R.; Ran&, H. M. J. Bid. Chem. 1976,251,4267. (44)Steinmier, R.C.; Parkhurst, L. J. Biochemistry 1975,14,1564. (45)Brunori, M.;Schuster, T. M. J. Biol. Chem. 1969,244, 4046. (46)Antonini, F.;Brunori, M. In Hemoglobin and Myoblogin and their Reactions with Ligands; North Holland Publishing Co.: Amsterdam, 1970;p 220.

1884 Langmuir, Vol. 11, No. 6, 1995

Tsuchida et al.

Table 1. Os- and CO-Binding Parameters of Lipidporphyrinatoiron(I1)Vesicles in Phosphate Buffer at 25 "C PH Pvz(OZ)PTO~~ kon(O2)lM' s-' kodOz)ls-' P~/z(CO)PTOIT kOn(C0)lM' s-1 k0dCO)ls-l Fe(LP)DMIm vesicle Hb (T-state)' a Mbb Fe(TpivPP)l,a-dMIm a

7.4 7.0-7.4 7.0-7.4 (toluene)

32 40 0.37-1 38

2.8 x lo8 2.9 x lo6 1-2 107 1.1x 108

1.5 x lo4 1.8 x lo2 10-30 4.6 104

2.8 x

0.3 1.4-2.5 x 9 10-3

3.9 x 106 2.2 105 3-5 x lo5 1.4 x lo6

0.14 0.09 1.5-40 x 0.14

At 20 "C. At 20 "C. From refs 45 and 46.

and release dioxygen more rapidly and than Hb. These results might suggest that the four dialkylphosphocholine groupsoon each ring plane form a n 02-binding pathway (ca.42 Aj from the outside aqueous phase to the 02-binding site of the porphyrinatoiron(II), resulting in acceleration of 0 2 diffusion in the lipidporphyrin bilayer. Thus this lipidporphyrinatoiron(I1j vesicle has the ability to act as a totally synthetic 0 2 carrier under physiological conditions.

lipidporphyrinatoiron(I1) vesicle with a diameter of 92 nm can bind much more 0 2 than natural Hb. Based on these results, the lipidporphyrinatoiron(I1)vesicle including highly-ordered reactive sites with high density is regarded as a model for superhemoglobin. Furthermore, the lipidporphyrin vesicle would also be useful as a new molecular structure in biochemical reactions such as an electron pool for photoenergy conversion and regioselective oxidation utilizing the ordered bilayer.

Conclusions The microstructure of the highly-ordered lipidporphyrin vesicle has been elucidated. Because the thickness of the membrane corresponds to twice the length of the lipidporphyrin, the porphyrin moieties in the outer and inner layers are presumed to be in close contact. The calculated distance value between the twq porphyrin rings a t the middle of the membrane is 4.3 A. The lipidporphyrinatozinc(I1)vesicle has the characteristics of a J-aggregated porphyrin. The lipidporphyrinatoiron(I1) coordinated alkylimidazole vesicle formed a stable dioxygen adduct under physiological conditions. One particle of the

Acknowledgment. This work was partially supported by a Grant-in-Aid for Scientific Research (No. 05403028, 05236103 and 053666) from the Ministry of Education, Science and Culture, Japan, and by the DFG (SFB312). The authors are grateful to Dr. Christoph Bottcher, Fritz Haber Institut der Max Planck Gesellschaft for the cryomicroscopyand Dr. Ulrich Siggel,MaxVolmer Institut fur Physikalische Chemie der Technische Universitat, for his helpful discussions on the excited states of porphyrin aggregates. LA940887A