Syntheses and properties of circular dichroism active phospholipids

Chemistry and Physics of Lipids, 49 (1988) 69--79 Elsevier Scientific Publishers Ireland Ltd.

69

Syntheses and properties of circular dichroism active phospholipids* T a k a k o N i s h i y a a, Y u k i h i s a O k u m u r a b a n d T h o m a s M i n g Swi C h a n g a ~Artificial Cells and Organs Research Centre, McCAll University, 3655 Drummond Street, Montreal PQ, H3G I Y6 (Canada) and bDepartment of Synthetic Chemisuy, Kyoto University Sakyo-ku, Kyoto 606 (Japan) (Received April 20th, 1988; revision received July 6th, 1988; accepted July 12th, 1988) A group of circular dichroism (CD) active phospholipids has been synthesised, in which one or both acyl chains has been replaced with a cinnamoyl or azobenzene chromophore-containing acid. Studies on the structure, CD activity and thermodynamic property of liposome membranes composed of CD active phospholipids were carried out. CD active liposomes were found to be stable, normal liposomes of appro~dmately 550 ,~ diameter based on the electron micrograph and dynamic light scattering, and to have thermodynamic property similar to the conventional phospholipid membranes without serious perturbation by aromatic bulk groups based on DSC. Liposomes composed of phosphofipid having two lrans-azobenzene chromophores showed an extremely large CD enhancement even well above To. This CD enhancement was drastically changed by the presence of c/s-azohenzene chromophore and cis-cis isomer content after irradiation was higher than the theoretical value, suggesting the importance of interchromophore interaction in the liposome membranes.

Keywords: circular dichroism active phospholipid; circular dichroism active liposome; circular dichroism enhancement; lipid-lipid interaction; azobenzene-chromophore; ciunamoyl-chromophore.

Introduction

Chemistry of liposomes has attracted increasing attention from scientists [1]. Among a variety of important aspects in the membrane chemistry and physics, dynamic changes in microscopic membrane structure seem to be especially important for understanding of membrane characteristics. The interaction between membranespanning integral proteins and the phospholipids making up the quasi-two-dimensional lipid bilayer membrane [2--5] and the effect of proteins upon the membrane structure changes have been studied by thermal analysis [6--9], fluorescence technique [2,10], spin label technique [11--13] NMR technique [14,15] and CD technique [16,17]. Correspondence to: Dr. T. Nishiya. *This paper is dedicated to the memory of Professor Iwao Tabushi.

Recently, we have chosen and prepared lipids 8--10 (Scheme 1) as the target molecules from which CD active liposomes would be constructed. Lipid 9 has some advantages in the study of the membrane dynamics. Firstly, it is miscible with natural lipids to form very stable bilayer membrane liposomes probably due to the structural similarity [21], i.e. lipid 9 and egg lecithin have a mutual affinity in the liposome membranes without phase separation at 1 °(2 up to 30 mol% of 9. This is a remarkable contrast to azobenzenecontaining ammonium amphiphile (11) which forms the aggregates by phase separation at only 5 mol% in ammonium bilayer membrane [18,19]. The second advantage is the CD activity, which is extremely sensitive to alignment and also to internal motion of chromophores introduced in a close proximity of the chiral carbon of the choline skeleton [21--24]. We now wish to report the syntheses and properties of CD active phospholipids, 8--10, which

0009-3084/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland.

70

Experimental __,

--~

,, o-(-~c~

OCIoH21

OC,o.,,),

General method

OC10H2I

1

2

3

4

6

5

or

egg leCithin

LO~__O'~'NM'3 5+ 6

~R "0-

7

[

O2C1sH31 OH + Oi~)~o ~ NMe3

'o-

R:

80, 9

5

)

fO2C 16H31 [-OR 0 + LO.-.- O ..~,NMe3 10

%

OH -~C,C'~-~OCloH21 for 8

-C?~N'N'~OCIIH17

for 9 lind 10

Scheme 1.

have appropriate chromophores of large absorbance near the asymmetric carbon. Applying the sythetic method shown here, CD active phospholipids having CD band at appropriate wavelength with proper ellipticity can be designed and synthesised, so that the motional membrane changes caused by lipid-protein interactions can he investigated without interfering effects of the perturbation from the proteins, as previously reported [22]. The perturbation of the liposome structure and of the membrane dynamics by the aromatic bulk groups then becomes a question of interest. In this report, the structure, CD activity and thermodynamic property of the liposome systems composed of CD active phospholipids are discussed.

Unless stated otherwise, all reagents and chemicals were obtained commercially and used without further purification. THF was purified by distillation from sodium under a N2 atmosphere. The commercially available chemicals or materials used in this work are as follows: 4-(dimethylamino)pyridine, ethyl chloroformate and 0.1 M tetrabutylammonium hydroxide in MeOH (Aldrich); 1-palmitoyl-L- a-lysophosphatidylcholine (Sigma); silica gel for chromatography, Type 60, 70---230 mesh and silica analytical TLC plate, Type 60, F-254 (Merck); AG-501-X8 resin (BioRad Laboratories). 400 MHz Fourier transform 1H-NMR spectra were recorded with JEOL GX-400 instrument. Infrared spectra were obtained on a Hitachi 26050 spectrophotometer. Electronic spectra were measured with a Union SM-401 high-sensitivity spectrometer and a varian CARY 219 spectrophotometer. CD spectra were measured with a Jobin-Yvon Dichrographe III CD spectrophotometer (10 mm 2 quartz cell was used) and a Jasco J-500 spectropolarimeter (cylindrical cell of pathlength 10 mm was used). Optical rotation was measured by a Perkin Elmer 243 polarimeter. The electron micrograph of the liposomes was obtained with a Hitachi H-500 instrument. Liposome size determination was carried out by dynamic light scattering technique with Hiac/Roico Model Nicomp 270 instrument. For differential scanning calorimetry (DSC), the liposome solution was sealed in Ag sample pan and DSC thermograms were obtained with a heating rate of 2*C/rain with a Perkin Elmer Model DSC-4 instrument.

trans-p-n-Decyloxycinnamic acid (1) Compound 1 was prepared by a reported procedure [25] with slight modification. A mixture of p-hydroxyhenzaldehyde (7.9g), anhydrous potassium carbonate (39 g) and n-decyl bromide (22.9 g) in 60 ml cyciohexanone was heated at 130"C for 2 h with vigorous agitation. After cool-

71 ing, the solution was decanted from the potassium carbonate, which was washed with ether. The washings were added to the cyclohexanone extract, and both solvents were distilled off under reduced pressure. The residual p-n-decyloxybenzaldehyde was then distilled under reduced pressure and obtained as a colourless liquid. Yield 58%, b.p. 163°C/1 mmHg (b.p. 185°C/4 mmHg, yield 65--75% [25]). A mixture of 5.4 g of p-ndecyloxybenzaldehyde, 2.1 g of malonic acid and 1 ml of piperidine in 15 ml of toluene was refluxed for 3 h, the H 2 0 formed being removed by use of a phase-separating head. After distilling off the solvent, the residual solid was dissolved in 20 ml of a 1:1 (v/v) mixture of H 2 0 and pyridine and the mixture was refluxed for 1 h. The mixture was poured on ice-conc. HCI and the collected precipitate was extracted with CHCI3. CHCI3 layer was washed with HeO twice and dried over MgSO4 overnight. Removal of CHC13 afforded 5.2 g (86%) of 1. m.p. 167-170°C. NMR (CDCi3) 8; 0.9 (m, 3H, CH3), 1.32 (br s, 16H, (CHe)), 4.0 (t, 2H, OCH2), 6.33 (d, J = 1 6 H z , 1H, 0----C-----CH), 6.93 (d, J = 8 Hz, 2H, aromatic), 7.54 (d, J = 8 Hz, 2H, aromatic), 7.82 (d, J = 16 Hz, 1H, O~--CH~---C). IR (KBr); Vc_--o 1685, ~'c:=c 1600, ~'~o-=c.2 1170, 1240cm -1.

trans-p- n- Decyloxycinnamic anhydride (2) Compound 2 was prepared according to the literature [26]. To a solution of 1 (0.805 g) dissolved in 15ml of T H F was added 0.5 ml of triethylamine. The resulting solution was cooled to - 2 0 ° C , and a solution of ethyl chioroformate (0.3ml) in 10ml of T H F w a s a d d e d to it in a dropwise fashion. The mixture was then stirred for 2 h, then warmed to room temperature, and stirred for an additional 20-rain period. The contents were then cooled to -200C and a solution of 0.805 g of 1 and 0.5 ml of triethylamine dissolved in 15 ml of T H F was added dropwise. After the product mixture was stirred overnight at room temperature, the solvent was removed under reduced pressure (room temperature). The crude anhydride thus remained as a solid was dissolved in ether (250 ml) and the ether layer was washed

with H20 and dried over MgSO4. Removal of ether followed by extensive drying (0.1 mmHg, 24 h at room temperature) afforded 1.48 g (95%) of 2. m.p. 123-125°C. NMR (CDCI3) 8; 0.9 (m, 6H, CH3), 1.33 (br s, 32H, (CH2)), 4.03 (t, 4H, OCH2), 6.43 (d, J = 16 Hz, 2H, d~---C-------CH),6.97 (d, J = 8 Hz, 4H, aromatic), 7.60 (d, J = 8 Hz, 4H, aromatic), 7.88 (d, J = 16 Hz, 2H, 0---CH------C). IR (KBr), Vc=:o 1740, 1705, Vc:=c 1600, Vc-o 1130, ~',---o--c.2 1175, 1245cm -~.

4'- n- Octanoxyazobenzene- 4-carboxylic acid (4) 4-Hydroxyphenylazo benzoic acid was prepared from 4-aminobenzoic acid and phenol via diazocoupling [27]. The mixture of 4-hydroxyphenylazo benzoic acid (4.5 g), potassium carbonate (15 g) and n-octyl bromide (10.3 g) dissolved in 70 ml ether was refluxed for 6 h. After cooling, 3.0 g of K O H was added to the mixture and the mixture was refluxed for 2 h. Precipitation formed was collected by filtration, washed with ether (50 ml x 2) and the solid was dissolved in diluted aqueous HCi. The solution was extracted with ether (200 ml x 4). Combined ether extract was evaporated to obtain 4.9 g (75%) of crude 4, which was recrystallized from acetone, m.p. 183185°C. NMR (DMSO-dt) 8; 0.87 (m, 3H, CH3), 1.30 (br s, 12H, (CH2)), 4.16 (t, 2H, OCH2), 7.23 (d, J = 9 Hz, 2H, aromatic), 8.03 (d, J = 9 Hz, 4H, aromatic), 8.16 (d, J = 9 Hz, 2H, aromatic). IR (KBr); Vc_-o 1680, vN=r~ 1580, l',--o--c,2 1150, 1250cm -1.

4'-Octanoxyazobenzene-4-carboxylic acid anhydride (5) Compound 5 was prepared by the procedures similar to those described for 2 except that all the procedures were carried out at room temperature. Yield: 92%. m.p. 149m152°C. NMR (CDCI3) 8; 0.95 (m, 6H, CH3), 1.37 (br s, 24H, (CH:)), 4.10 (t, 4H, OCH2), 7.08 (d, J = 9 Hz, 4H, aromatic), 8.05 (d, J = 9 Hz, 8H, aromatic), 8.35 (d, J = 9 Hz, 4H, aromatic). IR (KBr); Vc=o 1780, pc--o 1130, VN=N 1580, ~'~--O---C.2 1200, 1250cm -~.

72

L-a-GlycerophosphorylchoUne (7) Egg lecithin was carefully purified according to the literature [28] and stored at - 7 0 ° C under Ar in the dark. Hydrolysis of the lecithin (3.9 g) was carded out by (i) dissolving it in 35 ml of ether, (ii) removing the suspended particles by centrifugation, (iii) adding 3.9 ml of 0.1 M methanol solution of tetrabutylammonium hydroxide, and shaking the mixture occasionally by hand. Precipitation of 7 thus formed was separated from the liquid phase by decantation. 7 was then washed with ether (20 ml x 2) and dried at 25°C, 0.1 mmHg on P205 for 24 h. Compound 7 thus obtained was either used directly in further synthesis or stored in CHC13 at -20°C.

Bis (wans-p-n-decyloxycinnamoyO-L-a-phosphatidylcholine (8) Compound 8 was prepared according to the literature [29] with minor modification. To 0.915 g of potassium salt of 1, 3, was added 0.327 g of 7 in methanol and the solvent was evaporated to dryness under reduced pressure. The powder obtained was dried at room temperature, 0.1 mmHg overnight on P205. Three grams of 2 was added to the powder and the mixture was heated at 90°C for 12h under reduced pressure (0.1 mmHg) with stirring. A thick homogeneous oil was obtained which solidified at room temperature. The solid was extracted with CHCI3. After removal of the solvent, crude 8 was obtained, which was then dissolved in a minimum volume of CHCI3 and applied on a silica gel column. Elution was performed using the following solvents in the order: CHC13;9:1 (v/v) CHCIa/MeOH; 1:1 (v/v) CHCI3/MeOH; 1:9 (v/v) CHCI3/MeOH. Fractions were analyzed by TLC (silica gel, 65:25:4 CHCI3/MeOH/H20), and those fractions containing the product were combined and the solvent was evaporated. Lipid 8 thus obtained (0.47 g, 45%) showed TLC spot at Rf 0.36 but no impurity spot. NMR (CDCI3) 3; 0.87 (m, 6H, CH3), 1.26 (br s, 32H, (CH2)), 3.34 (br s, 9H, N(CH3)3), 3.74 (m, 2H, CHeN), 3.87 (t, 4H, @--OCH2),4.08 (m, 2H, CH2OCO), 4.32 (m, 2H, C H 2 0 P (polar chain)), 4.35 (m, 2H, C H 2 0 P

(glycerol)), 5.39 (m, 1H, CHOCO), 6.21 (d, J = 16 Hz, 1H, ~--C=-CH), 6.27 (d, J = 16 Hz, 1H, ~--C~----CH), 6.77 (d, J = 8 Hz, 2H, aromatic), 6.79 (d, J = 8 Hz, 2H, aromatic), 7.34 (d, J = 8Hz, 2H, aromatic), 7.39 (d, J = 8 H z , 2H, aromatic), 7.54 (d, J = 16 Hz, 1H, @--CH--C), 7.60 (d, J = 16 Hz, 1H, ~k--CH--C). IR (KBr); v~----o 1705, Vc_-c 1600, V~-o--cn2 1170, 1245, ~,+ N M e 3 1090, 1060, 9 7 5 c m -~. Elemental anal: calc. for C46H72010NP'H20 H 8.81, C 65.13, N 1.65, P 3.65; found H 8.95, C 64.97, N 1.55, P 3.42. [or]D5= + 38.3 ° (CHCI3). FAB Mass 830 (M + I-I)+.

Bis (4'-n-octanoxyazobenzene-4-carboxyO-L-aphosphatidylcholine (9) Compound 7 (0.28 g) was treated with 3 g of $ and 0.89 g of 6 by the procedures similar to those described for 8 except that temperature applied was 104°C. The yield of the pure 9 was 450rag (44%). NMR (CDCI3) 8, 0.88 (m, 6H, CH3), 1.28 (br s, 24H, (CH2)), 3.34 (hr s, 9H, ]~(CH3)3),3, 80 (m, 2H, CH2 Iq), 3.94 (t, 4H, ~ - O C H 2 ) , 4.25 (m, 2H, C H 2 0 C O ) , 4.37 (m, 2H, C H 2 0 P (polar chain)), 4.54 (m, 2H, CH20P (glycerol)), 5.65 (m, 1H, CHOCO), 6.86 (d, J = 8 Hz, 2H, aromatic), 6.88 (d, J = 8 Hz, 2H, aromatic), 7.70 (d, J = 8Hz, 2H, aromatic), 7.76 (d, J = S H z , 2H, aromatic), 7.78 (d, J = 8 Hz, 4H, aromatic), 7.97 (d, J = 8 Hz, 2H, aromatic), 8.07 (d, J = 8 Hz, 2H, aromatic). IR (KBr); ~ , ~ 1720, ~'s--~ 1580, l'~-o---ci~2 1150, 1260, v~-a4c3 1100, 1060, 970 cm -~. Elemental anal: calc. for CsoI-I6sO10NsP-H20 H 7.39, C 63.38, N 7.39, P 3.27; found H 7.59, C 63.22, N 7.21, P 3.11. [a]2D5 = +90.0 ° (CHCI3). FAB Mass 930 (M + H) +.

1-Palmitoyi-2-(4"n-octanoxyazobenzene-4carboxyO-~.-a-phosphatidylcholine (10) Compound 10 was prepared according to the literature [26] with minor modification. Twohundred and fifty milligrams of 1-palmitoyl-L-alysophosphatidylcholine was dissolved in 15 ml of CHCI3 (freshly distilled over P205) at 45 °C. T o

73 the solution, 870mg of $ and 154mg of 4(dimethylamino)pyridine were added, and the mixture was degassed with Ar and stirred at room temperature in the dark for 36 h. After the solvent was evaporated under reduced pressure, the residue was dissolved in 5ml of 4:5:1 CHCI3/MeOH/H20 and the solution was passed through an AG-501-X8 column and the eluent was evaporated under reduced pressure at room temperature. The crude 10 was further purified by silica gel column by the procedures similar to those described for 8. The yield of the pure 10 was 360 mg (85.9%). NMR (CDC13) 8; 0.88 (In, 6H, CH3), 1.24 (br s, 38H, (CH2)), 2.20 (t, 2H, COCH2), 3.28 (br s, 9H, N(CH3)3), 3.70 (m, 2H, CH2N), 3.99 (t, 2H, O--OCH2), 4.12 (m, 2H, C H 2 0 C O ) , 4.30 (m, 2H, C H 2 0 P (polar chain)), 4.44 (m, 2H, C H 2 0 P (glycerol)), 5.47 (m, 1H, CHOCO), 6.93 (d, J = 9.6 Hz, 2H, aromatic), 7.83 (d, J = 9.6 Hz, 2H, aromatic), 7.87 (d, J = 9.6 Hz, 2H, aromatic), 8.10 (d, J = 9.6Hz, 2H, aromatic). IR (KBr); ~'c----o 1720, lPN~_~_s 1 5 8 0 , VO..-O..--CH2 1250, V~e3 970 c m -1 . Elemental anal: calc. for C 4 5 H 7 4 0 9 N 3 P ' 2 H 2 0 H 8.53, C 62.30, N 4.85, P 3.57; found H 8.61, C 62.31, N 4.96, P 3.49. FAB Mass 832 (M + H) ÷.

Irradiation of Uposomes The irradiation of liposome solution was cartied OUt by 355 nm light using 500-W Xenon lamp equipped with a grating monochrometer. After the irradiation of 50% diazofiposome solution by 355 nm light at 50°C or 5°C, the amount of three isomers of 9, trans-tran,~ (both azobenzene chromophores are trans), a'ans-c/s (one azobenzene chromophore is Warts and another is c/s) and cis-cis (both azobenzene chromophores are cis) were determined by HPLC analysis. Thus, the irradiated liposome solution (2.5 ml) was concentrated to dryness in vacuo. The dried lipid was dissolved in 50/~1 of dimethyl formamide (DMF)/MeOI-I/H20 (25:25:4, by vol.) and aliquots (15/~1) were analyzed by HPLC (Waters Z module Model R-Z and Waters Model 116K HPLC pump) using 10 cm reverse phase column (Waters /~-Boundapak Cls, Radial PAK) and DMF/MeOH/H20 (25:25:4, by vol.) as an eluent. All operations were performed in the dark. Three isomers of 9 were monitored by absorbance at 317 nm by Shimazu SPD-6AV UV detector. By the independent measurements, the retention time of cis-cis, cis-trans and trans-trans isomers were determined to be 2.8, 4.0 and 6.2 rain, respectively.

Preparation of CD active iiposomes Results and ¢Fmeusdon Liposomes were prepared from a mixture of lipid in distilled water by ultrasonic irradiation followed by centrifugation and Sepharose 4B gel filtration [21,30]. The 50% diazoliposomes were prepared from a mixture of 9 and egg lecithin with a molar ratio of 1. Dicinnamoylliposomes and monoazoliposomes were prepared from 100% of phospholipid 8 or 10, respectively. For each liposomes, phospholipid concentration of 6.0 × 10 -3 M in distilled water was used. Concentration of pbospholipid 8, 9 or 10 in liposome solution was estimated from their characteristic absorption in C H C I 3 . Thus, the liposome solution was concentrated to dryness in vacuo (1 mmHg for 2 h) and the dried lipid was dissolved in C H C I 3 to measure the absorbance. The preparation and measurements of liposome solution were carried out under dark conditions.

Structure of CD active liposomes The structure of 50% diazoliposomes, monoazoliposomes and dicinnamoylliposomes were observed by electron microscopy. They clearly demonstrated the liposome structure. A typical example of an electron micrograph of 50% diazoliposomes is shown in Fig. 1. Based on the dynamic light scattering, the average diameter of these liposomes were found to be approximately 550 ,~. The liposome structure remained intact after the CD measurements based on the electron micrograph and dynamic light scattering, indicating the integrity of CD active liposomes. It is also reported that liposomes composed of azobenzenecontaining phospholipids are stable [31], although the morphological change of phospholipids and

74

Fig. 1. Electron micrograph of 50% diazoliposomesstained by 2% uranylacetate (pH 7.0). Bar represents 1000 .~. the increase of m e m b r a n e permeability by photoisomerization were observed. T h e liposomes composed of pure egg lecithin, which have the same average diameter as C D active liposomes, were prepared by the procedures similar to those described for preparation of C D active liposomes, and the phospholipid concentration of the egglecithin liposome solution was adjusted to the same as that of C D active liposome solutions (1.24 × 10 -4 M). This egg lecithin-liposome solution did not exhibit any C D band from 250 to 500 nm under the experimental conditions applied to C D active liposomes. This result shows that light scattering contribution to C D spectra exhibited by C D active liposomes is negligibly small under our experimental conditions.

CD activity of phospholipids One of the most significant characteristics of the present artificial phospholipids is the intense C D ellipticity due to the planar c h o m o p h o r e located very close to the chiral carbon of the choline skeleton, as shown in Table I. Interestingly, C D ellipticity of 9 is much larger than that of 10, indicating that even in CHCI3 solution, C D

TABLE I Electronic and CD spectra of phospholipids in 25 *C Electronic spectra (nm) 8 316 (e = 4.4 x 104) 9 364 (e = 5.0 x 104) 10 364 (e = 1.4 x 104)

CHCI 3

at

CD spectra (nm) 290 (0 = -0.67 x 104 deg cm2/dmol) 335 (0 = +1.00 x 104) 382 (0 = +1.49 x 104) 367 (0 = +0.20 x 104)

ellipticity of 9 is enhanced by the interaction between inter- or intramolecular azobenzene chromophores, as discussed below. Dicinnamoylliposomes, composed of 100% 8, showed the characteristic C D spectrum at 25°C having a negative band at 280 n m ( O = - 2 . 1 8 x 104 d e g - cm2/dmol) and a positive band at 322 nm (O= +2.01 x 104). Ellipticity is strongly dependent on the temperature as shown in Fig. 2. A t low temperature ( 5 - - 1 0 " C ) the ellipticity is almost constant. With elevating temperature (at 10---20°C), ellipticity gradually decreases and it drastically decreases at 25. 40°C. A b o v e 40°C

75 (a)

(a) 1C

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1

K

" -, . " ~ ' - ' ~ . ~ .

0

........

-10

x

a6o

1.E

250

a60

3~0

a,~o

46o

4so

rllTI

(b)

,(=,,,

4~nm

a~ i

~//

I.C

~.

"~ \l ~ .\~

........ ~'~,

(b)

|

3.0

u ~

2.S

o ~x

2.¢1

1.5

o

+b

~

~o

,/o

sb oc

Fig. 2. (a) Temperature-dependent CD spectra of dicin-

namoylliposomes: - - . , I°C; . . . . . . , 15°(2; ---, 25°C; . . . . . ,28°C; oooooo,30°(2; ---, 40°C; - - , 8 in CHCls. (b) Temperature-dependent ellipticity change of dicinnamoylliposomes.

ellipticity again becomes constant. From the observed ellipticity change, phase transition temperature is estimated to be 28 °C. As previously reported [21], 50% diazoliposomes showed temperature-dependent visible and CD spectra, and Tc was estimated to be 25 °C (Fig. 3). The most important finding is that, even well above To, lipid molecules 8 and 9 in the liposome membranes show the enhanced C D spectra. C D ellipticity of lipid 8 and 40°C in the liposome membranes at 322 and 280 nm is a few times larger than that in CHCI3 at 335 and 290 nm, respectively (Fig. 2). C D ellipticity of 50% diazoliposomes at 3 6 8 n m (50°C) is five

Fig. 3. (a) Temperature-dependent CD spectra of 50% diazoliposomes:- - , 1°C; ...... ,20°C;---, 250C; . . . . . , 30°C; ---, 40°C; - - , 9 in CHI3. (b) Temperaturedependent electronic spectra of 50% diazofiposomes: loc; ...... ,20°C; ---, 25°C; . . . . . ,300C; ---, 40°C.

times larger than that of 9 in CHCI3 at 382 nm (Fig. 3). By contrast, the liposomes composed of 100% monoazophospholipid, 10, (monoazoliposomes) did not show the CD enhancement above To. The temperature-dependent C D spectra of monoazoliposomes are shown in Fig. 4. Above 35°C, monoazoliposomes showed no change in C D spectrum and the CD spectrum of monoazoliposomes above 35°C was approximately the same as that of CHCI3 solution of 10. C D enhancement has been observed when chiral molecules having chromophores near the chiral center form a certain kind of aggregate [20,32--41]. It is widely accepted that the CD enhancement arises from a spatially ordered

76

|

A

B

0

C

--

300

350

4~)0

4,50 nm

Fig. 4. Temperature-dependent CD spectra of monoazoliposomes: , I°C; . . . . . . , 10°C; - - - , 20°C; . . . . . , 30°C; - - - , 35°C; ~ , 10 in CHCI3.

structure of chromophores in the aggregate, since the CD enhancement is greatly reduced or absent under such conditions that disaggregation might occur [32--38]. From these results, it might be concluded that even after the release of phase separation, the interactions between intramolecular cinnamoyl or trans-azobenzene chromophores in liposome membranes still remain and lipids 8 and 9 keep an ordered structure in the liposome membranes even well above T¢. As shown in Fig. 3, temperature-dependent electronic spectra of 50% diazoliposomes exhibited the absorption at 348nm above 40°C. Observed blue shift of 16 nm from 364 nm of CHC13 solution may be attributed to the chromophore stacking which is known for azo dyes in general. At low temperature, the absorption at 304 nm characteristic of the aggregate of highly parallel chromophore orientation [ 19] and also the absorption at 348 run were observed. The optical spectra of liposomes composed of pure monoazophospholipids exhibited a peak at 342 nm above 35°C and at 332 nm at l°C. Based on the electronic and CD spectra, the chromophore interactions in 50% diazoliposomes and monoazoliposomes can be schematically illustrated as shown in Fig. 5. In 50% diazoliposomes, a large proportion of lipid 9 forms the aggregates by phase separation and a small proportion is present as monomer at low temperature

D

Fig. 5. Schematic illustration of the lipid orientation in bilayer. (A) Lipid 9 below Tc; (B) Lipid 9 above Tc; (C) Lipid 10 below To; (D) Lipid 10 above To.

(A in Fig. 5). With elevating temperature, the aggregates convert to monomer in which intramolecular chromophore interactions are still kept (B in Fig. 5). In monoazoliposomes, the two chromophores are placed face-to-face at low temperature (C in Fig. 5). Above To, some chromophores are isolated by the neighboring alkyl chain (D in Fig. 5). It is interesting that the CD ellipticity of state B in Fig. 5 is approximately four times larger than that of state C in Fig. 5, in spite of that the statistical number of face-to-face chromophore interaction among a certain number of phospholipid molecules is the same. This suggests that the intramolecular face-to-face chromophore orientation is more improved than intermolecular one. Effect of trans-to-cis photoisomerization of azobenzene on CD enhancement The extraordinary CD enhancement above Tc in 50% diazoliposomes was drastically diminished in the presence of a small amount of c/s-azobenzene chromophore formed upon irradiation by 355 nm light. CD spectra of 50% diazoliposomes at 50°C in the presence of c/s chromophore are shown in Fig. 6. Only 2.6% of c/s-chromophore reduced the CD ellipticity at 368 nm up to 88% of the one before the irradiation and it was further reduced to 47% in the presence of 11.2% of c/s-chromophore. On the other hand, UV-VIS spectra showed the normal concentrationdependent change, thus the photoisomerization of 6% of trans-azobenzene caused 7% decrease of

77 TABLE II 5

Isomer contents in 50% diazoliposomesafter irradiation (%)

|

50% Diazoliposomes at 50"C

.'7"300.. .-....

0.

•. . . . . . . . . .

N. ~ -.'~.~.

,.q

3~0

460

nm

.11

trans-lrans~ 89.7 ttans-ciss 9.8 cis-cis" 0.5

Effect of c/s-chromophore on CD enhancement: before irradiation, - - - - 2.6% . . . . . 5.4% ...... 11.2%. Percentage numbers refer to c/s content as a percent of the total chromophore numbers.

at 5"C

80.2 17.2 2.6

9 4 . 8 9 2 . 2 93.6 4.4 6.3 6.4 0.8 1.6 0.1

89.8 10.2 0.2

calc. b

cis-cis

",.22

9 in CHCi3 at 25°C

0.27

0.92

0 . 0 5 0 . 1 1 0 . 1 1 0.29

aObserved values. bTheoretical values based on the observed contents of transtrans and trans-c/s.

Fig. 6.

the absorbance at 348 nm. These observations suggest that the C D e n h a n c e m e n t in 50% diazoliposomes is due to the trans-azobenzene c h r o m o p h o r e interactions and that one distorted cis-azobenzene c h r o m o p h o r e [42], formed upon irradiation, reduces the trans-azobenzene e h r o m o p h o r e interactions and collapses its surrounding ordered structure which is still kept above To. T h e amount of three isomers, trans-trans, trans-cis and cis-cis, were determined by H P L C analysis after the irradiation of 50% diazoliposome solution by 355 nm light at 50"C and at 5 °C. As shown in Table II, observed cis-cis isomer contents in 50% diazoliposomes after irradiation at 50"C were a few times larger than the theoretical values. In the case of irradiation at 5"C, where a large proportion of lipid 9 forms the aggregates by phase separation and ordered lipid structure is more rigid than at 50°C, observed cis-cis isomer contents in 50% diazoliposomes were approximately 15 times larger than the theoretical values. On the other hand, they were consistent for CHCI3 solution of 9. These results indicate that the photoisomerization of transazobenzene to c/s-azobenzene (trans-* cis isomerization) in trans-cis isomer is much easier

than that in trans-trans isomer, suggesting that the trans--~cis isomerization is controled by the c h r o m o p h o r e interactions and that one cisc h r o m o p h o r e changes the conformation of surrounding trans-chromophores to make their isomerization easy and this effect is preferential on trans-chromophore in the same molecule. Thermodynamic property of C D active liposome membranes

In order to investigate the perturbation of the membrane dynamics by the aromatic bulk groups, Tc and the transition enthalpy (AH) of C D active liposome membranes were obtained by DSC. As shown in Table III, Tc (peak-top temperature) of 50% diazoliposome membranes (27.5°C), monoazoliposome membrane (22.5°C) and dicinnamoylliposome membrane (26.0°C) are consistent with the results of CD. A H for three membranes are very reasonable comparing with A H for D M P C membrane (5.4 kcal/mol)[43-45], TABLE III Tc and AH for CD active liposome membranes. Liposome system

Tc (°C)

AH (kcal/mol)

50% Diazoliposome Monoazofiposome Dicinnamoylliposome

27.5 22.5 26.0

8.6 7.7 8.3

78

DPPC membrane (8.7kcal/mol) [43-45] and DSPC membrane (10.6 kcal/mol) [43-45]. These AH data strongly suggest that the phosphatidylcholines with aromatic chromophore tails, 8, 9 and I0, have the thermodynamic property similar to the conventional phosphatidylcholines without serious perturbation by aromatic rings. It is also reported that the presence of the azobenzenecontaining phospholipid does not grossly perturb the bilayer structure [46]. It is interesting that AH for diazophospholipid, 9, in the mixed membrane with egg lecithin with a molar ratio of 1 (8.6 kcal/mol) is greater than that for monoazophospholipid, 10, in the single-component membrane (7.7 kcal/mol). This establishes beyond doubt that the chromophore orientation is more improved in 50% diazoliposome membrane than in monoazoliposome membrane. The results presented demonstrate that the azobenzene-containing liposomes may be useful tools for the investigation of lipid-lipid or lipidprotein interactions. Further, the effects of photoisomerization such as thechanges in molecular packing and polarity of azobenzene-containing phospholipids in the liposome membranes, might be exploited to control the activity of membranebound enzymes or channel-forming peptides. Acknowledgement Support for this research by Natural Science and Engineering Research Council of Canada (Grant No. 234-95) is gratefully acknowledged. References 1 J.H. Fendler (1982) Membrane Mimetic Chemistry, John Wiley and Sons. 2 D. Chapman (1982) Biol. Membr. 4, 179--229. 3 D. Marsh and A. Watts (1982) in: P.C. Jost and O.H. Griflith (Eds.), Lipid-protein Interactions, Vol 2, Wiley, New York, pp. 53--126. 4 W. Ho~man and C.J. Restall (1984) in: D. Chapman (Ed.), Biomembrane Structure and Function, Vol 4, MacMillan Press, London, pp. 257--318. 5 P.F. Devaux and M. Seigneuret (1985) Biochim. Biophys. Acta 822, 63--126. 6 J.C. Gomez-Fernandez, F.M. Goni, D. Bach, C.J. Restall and D. Chapman (1980) Biochim. Biophys. Acta 598, 502--516.

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