Chemistry and Physics of Lipids, 61 (1992) 209-218

209

Elsevier Scientific Publishers Ireland Ltd.

Chain substituted polymerizable ether lipids: synthesis of sorbyl and diacetylenic ether glycerophosphocholine Y o u n - S i k L e e a n d D a v i d F. O ' B r i e n C.S. Marvel Laboratories, Department of Chemistry, University of Arizona, Tucson, AZ 85721 (USA) (Received July 15th, 1991; revision received January 27th, 1992; accepted February 18th, 1992)

Three novel polymerizable ether lipids, 1,2-O-bis[lO(2",4'-hexadienoyloxy)decyl]-rac, 1,2-O-bis(lO,12-tricosadiynyl)-rac, and (-)-2,3-O-bis(lO,12-tricosadiynyl)-sn-glycet-o-l-phosphocholine, were synthesized from 3-O-benzyl-rac, 3-O-trityl-rac and (-)-I-Otrityl-sn-glycerol as starting materials, respectively. All the reactions employed in these multi-step syntheses are straightforward giving an overall yield of 21% for the sorbyl, 42% for the racemic diacetylenic and 44% for the chiral diacetylenic lipid. All the lipids form bilayer assemblies on hydration and show transitions from gel to liquid-crystalline phases at 11.4°, 27.6 ° and 30.0°C, respectively. Bilayer assemblies of each are photoreactive and are readily polymerized by irradiation with 254 nm light. Tubules of the chiral diacetylenic ether lipid were observed.

Key words: ether lipid; phospholipid; polymerizable ether lipid; sorbate; diacetylene; glycerophosphocholine; tubules

Introduction

Polymerizable lipids have received much attention in the last decade as one method of increasing stability and controlling permeability of liposomes. The polymerization of lipid assemblies was first demonstrated in monolayers, liposomes and extended bilayers. During the 1970s several groups reported that reactive fatty acids could be polymerized in a monolayer. Early in the 1980s methacryloyl [1] and diacetylenic lipids [2-4] were described. A variety of reactive groups, e.g., dienoyl, sorbyl, lipoyl, styryl have been reported in recent years [5-7]. These polymerizable groups have been introduced into different regions of lipids: at the chain terminus, near the middle of the hydrophobic chains, or in the hydrophilic head group. Polymerization in the chains decreases the mobility of the lipid chains, whereas polymerization at the head group has less effect on the hydrophobic interior of the membrane but alters the membrane Correspondence to: David F. O'Brien, Laboratories, Department of Chemistry, Arizona, Tucson, AZ 85721, USA.

C.S. Marvel University of

water interface. Hydrophilic spacer groups between the polymerizable groups and the lipid backbone and chains have been used to retain the fluidity of the polymerized monolayers and bilayers [8]. Ether lipids are widely distributed in human and animal tissues. Many tumor cells contain a higher concentration of ether lipids than normal tissues [9]. Recently, many previously unknown ether lipids have been isolated and characterized. Some naturally occurring and synthetic ether lipids are used in clinical diagnosis and medical research [10]. In contrast to ester iipids, relatively few studies on ether lipids are found in the literature. Recently, the effects of the ether linkage on phospholipid bilayer assemblies have been studied. In particular, comparisons between dipalmitoyl (DPPC) and dihexadecylphosphatidylcholine (DHPC), have been reported [11-14]. The two compounds have similar thermotropic behavior even though DHPC shows a slightly higher transition temperature, Tm (45.5°C) than the corresponding ester analogue, DPPC (41.4°C). The increase in the Tm of the ether lipid was attributed to either an extra methylene unit [15] or lack of H-

0009-3084/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

210 bonding between ether lipid molecules [16,17]. Ruocco et al. reported a significant difference between DHPC, which shows an interdigitated bilayer (detected by DSC and X-ray diffraction) and DPPC which shows no indication of the interdigitated phase [12]. A wide variety of polymerizable diacyl gtycerophosphocholines is known in the literature, but very few polymerizable ether lipids have been described and none of these are dialkyl glycerophosphocholines. Ringsdorf and coworkers reported polymerizable ether lipids with methacryloyls located in the hydrophilic head groups [18,19]. There is no report in the literature about synthesis and characterization of ether phospholipids which contain polymerizable groups in the hydrophobic chains. We have been interested in phospholipids with polymerizable groups, e.g., sorbyl or diacetylene in the hydrophobic chains, since these lipids may be readily photopolymerized to alter bilayer membrane properties [5,6,20,21]. This report describes the synthesis of three polymerizable ether phospholipids: one containing sorbyl groups at the end of the chains and the others with diacetylenic groups in the middle of the chains.

Experimental Procedures Materials Benzene and tetrahydrofuran were distilled from sodium and chloroform from P205 prior to use. 1,2-Isopropylidene-rac-3-glycerol (solketal) (98%), 1,10-decanediol (99%), benzylchloride (97%), palladium (10%) on activated carbon, zinc (dust, 325 mesh), 2,2,2-trichloroethyl chloroformate (98%), (S)-(+)-dimethyl-l,3-dioxolane-4methanol (98%), 10-chloro-l-decanol (90%), ptoluenesulfonic acid monohydrate (99%), 2,4hexadienoic acid (99+%), oxalyl chloride (99+%), 2-bromoethanol (98%), phosphorus oxychloride (99%), trimethylamine 25 wt.% solution in water, methanesulfonyl chloride (98%), 3,4-dihydro-2Hpyran (97%), lithium aluminium hydride (95+%), sodium (99%) and triphenylmethyl chloride (98%) were purchased from Aldrich Chemical Co. (Milwaukee, WI). 10-Chloro-l-decanol was further purified via flash column chromatography

(hexane/EtOAc, 90:10). 10.12-Tricosadiynoic acid was purchased from Farchan Laboratories (Gainesville, FL), dissolved in CHC13 then filtered to remove polymeric acid. Silica gel (grade 60, 230-400 ASTM mesh) for column chromatography was purchased from American Scientific Products (McGaw Park, IL). Precoated silica gel TLC plates were purchased from Analtech INewark, DE).

Methods Any compound containing a UV-sensitive group was handled under yellow light. Reactions which required anhydrous conditions were run under a dry nitrogen atmosphere. Thin layer chromatography (TLC) was used to monitor each reaction and to check the purity of products. ~HNMR spectra were obtained on a 250-MHz Bruker WM 250 spectrometer in chloroform-d with tetramethylsilane as an internal reference. Infrared spectra were taken on a Perkin-Elmer 983 spectrometer. Visible absorption spectra were recorded on a Hewlett-Packard 8452A diode array spectrophotometer. Melting points were not corrected. Optical rotation was measured with an Autopol III, Auto Polarimeter using a quartz cell of 10 cm in length. FAB mass spectra were obtained using a AMD modified 311A equipped mass spectrometer with a cesium gun. A Microcal Inc., model MC-2 differential scanning calorimeter was used to determine the phase transition temperatures of bilayer assemblies of the ether lipids in t 0 mM Na2HPO4 and 150 mM NaC1 (pH 7.40). The phase transition temperatures were measured at the point of maximum excess heat capacity.

l O-Chloro- l-tetrahydropyranyldecane (1-1) p-Toluenesulfonic acid monohydrate (25 mg) was added to a solution of 1.93 g (10.0 mmol) of 10-chloro-l-decanol and 1.50 g (17.8 mmol) of 3,4-dihydro-2H-pyran in 20 ml of T H F at room temperature. The mixture was stirred for 2 h and the solvent was removed under reduced pressure. The residue was separated by flash column chromatography (hexane/EtOAc, 95:5, Rf= 0.35) to give 2.43 g of the product as a viscous liquid (88%). 1H-NMR (CDCI3) 6 1.28-1.45 (m, t2H, (CH2)6) 1.50-1.65 (m, 6H, 4-CH2 of THP, CH2-

211 CH2-C1 and CH2-CH20), 1.70-1.90 (m, 4H, 3-, 5-CH 2 of THP), 3.35 (t, 1H, CH20), 3.39 (t, 1H, CH20), 3.51 (t, 2H, CH2CI), 3.73 (td, IH, 6-CH 2 of THP), 3.83-3.91 (m, 1H, 6-CH2 of THP), 4.57 (t, 1H, 2-CH of THP).

1,2-O-Bis( lO-tetrahydropyranyldecyl)-3-O-benzylrac-glycerol (1-2) A racemic mixture of 3-O-benzylglycerol was prepared from racemic 1,2-isopropylideneglycerol by the method of Howe and Malkin [22]. 3-0Benzyl-rac-glycerol (0.400 g, 2.20 mmol) and KOH powder (2.40 g) in 80 ml of benzene were refluxed with Dean Stark trap for 2 h. 1-Hydropyranyl10-chlorodecane, 1-1 (2.44 g, 8.80 mmol) in 20 ml of benzene was added dropwise. The mixture was refluxed for 14 h, cooled to room temperature, diluted with 100 ml of EtOAc and washed with 5% HCI solution. The organic layer was dried over sodium sulfate, then concentrated under reduced pressure. The residue was purified by flash column chromatography (hexane/EtOAc, 90:10, Rj= 0.27) to obtain 1.08 g of the product as a viscous liquid (74%). IR (neat) 3027 (aromatic C - H ) cm-X; 1H-NMR (CDCI3) ~5 1.28-1.45 (m, 24H, (CH2)6) 1.50-1.90 (m, 20H, 3-, 4- and 5-CH2 of THP, OCH2-CH2), 3.33-3.64 (m, 12H, OCH2, O C H 2 - C H O - C H 2 0 ), 3.75 (td, 2H, 6-CH2 of THP), 3.83-3.92 (m, 2H, 6-CH2 of THP), 4.58 (s, 2H, CH2-C6Hs), 4.59 (t, 2H, 2-CH of THP), 7.29-7.37 (rn, 5H, C,Hs).

1,2-O-Bis( lO-tetrahydropyranyldecyl)-rac-glycerol (I-3) Compound 1-2 (2.10 g, 3.17 mmol) and 0.90 g of 10% Pd/C in 30 mt of EtOAc was shaken at 2 atm of hydrogen for 18 h. The reaction mixture was filtered, concentrated and separated by flash column chromatography (hexane/EtOAc, 80:20, R/= 0.23) to yield 1.30 g of the product as a viscous liquid (90%). IR (neat) 3472 (OH) cm-I; IH-NMR (CDCI3) 6 1.26-1.40 (m, 24H, (CH2)6), 1.50-1.94 (m, 20H, 3-, 4- and 5-CH 2 of THP, OCH2-CH2), 3.35-3.67 (m, 13H, OCH2, OCH2C H O - C H 2 0 ) , 3.70-3.80 (m, 2H, 6-CH 2 of THP), 3.84-3.92 (m, 2H, 6-CH2 of THP), 4.58 (t, 2H, 2-CH of THP).

1,2- O-Bis ( lO-hydroxydecTl)-rac-glycerol-3-fl, [3,[3trichloroethylcarbonate (I--4) A solution of 2,2,2-trichloroethyl chloroformate (0.475 g, 2.24 mmol) in 10 ml of chloroform was added dropwise to an ice-cold solution of compound 1-3 (0.806 g, 1.41 mmol) and 0.201 g of pyridine in 9 ml of chloroform [23]. The starting material disappeared completely within 4 h at room temperature. The reaction mixture was evaporated to dryness, dissolved in diethyl ether and washed with water twice. The ether layer was dried over sodium sulfate, followed by concentration in vacuo. The oily residue was dissolved in 10 ml of THF/MeOH (1:1), followed by addition ofptoluenesulfonic acid monohydrate (0.125 g). The solution was stirred at room temperature for 4 h, then the solvent was evaporated. The residue was dissolved in diethyl ether and washed with water twice. The ether layer was dried, concentrated and purified by flash column chromatography (CHCI3/MeOH from 99:1 to 95:5, Rf= 0.47 with 95:5) to obtain 0.67 g of the product as an oil (82%). IR (neat) 3359 (OH), 1763 (OCO2) cm-I; JH-NMR (CDCI3) 6 1.18-1.43 (m, 24H, (CH2)6) , 1.45-1.62 (m, 8H, OCH2-CH2), 3.39-3.70 (m, I1H, OCH2, O C H z - C H O - C H 2 0 - C O 2 ) , 4.224.38 (m, 2H, O C H z - C H O - C H 2 0 - C O 2 ) , 4.75 (s, 2H, OCH2-CC13).

1,2- O-Bis [l O(2 ', 4 '-hexadieno ylo x y ) decTl]-racglycerol-3-fl,fi,[3-trichloroethylcarbonate (1-5) Sorbyl chloride was prepared from 2,4-hexandienoic acid and oxalyl chloride using the procedure previously reported [21]. A solution of sorbyl chloride (97.1 mg, 0.744 mmol) in 5 ml of chloroform was added dropwise to an ice-cold solution of compound I--4 (0.154 g, 0.266 mmol) and pyridine (0.325 g, 4.11 mmol) in 5 ml of chloroform. The mixture was stirred for 24 h at room temperature. After evaporation of the solvent, the residue was dissolved in diethyl ether and washed with water, 10'7o HCI, 5% NaHCO3 and water. The residue was purified by flash column chromatography (hexane/EtOAc from 95:5 to 80:20, RU= 0.50 with 80:20) to yield 0.135 g of the product as an oil (66%). IR (neat) 3010 ( = C - H ) , 1759 (OCO2) , 1710 (CO2) cm l; IH_NM R (CDCI3) 6 1.15-1.40 (m, 24H, (CH2)6), 1.47-t.70

212

(m, 8H, OCH2-CH2), 1.82 (d, 6H, CH3), 3.38-3.73 (m, 7H, OCH2, O C H ; - C H O - C H 2 0 ) , 4.09 (t, 4H, CO2-CH2) , 4.21-4.39 (m, 2H, OCH2-CHO-CH2), 4.74 (s, 2H, OCH2-CCI3), 5.74 (d, 2H, O2C-CH=), 6.07-6.22 (m, 4H, CH=CH-CH3), 7.16-7.28 (m, 2H, O2CCH=CH).

1,2-O-Bis[lO(2', 4' -hexadienoyloxy ) decyl]-racglycerol (1-6) Compound I-5 (0.248 g, 0.323 mmol) was dissolved in a mixture of acetic acid (6 ml) and diethyl ether (4 ml). The mixture was cooled in an ice-bath and 0.60 g of activated zinc was added [24]. The suspension was stirred for 5 h at room temperature. After filtration, the crude product was purified by column chromatography (hexane/EtOAc, 80:20, Rf= 0.20) to yield 0.167 g of the product as a viscous liquid (88%). IR (neat) 3488 (OH), 3016 ( = C - H ) , 1709 (CO2) cm-l; tHN M R (CDCI3) 6 1.20-1.45 (m, 24H, (CH2)6) , 1.50-1.71 (m, 8H, OCH2-CH2), 1.85 (d, 6H, CH3) , 3.42-3.80 (m, 9H, O C H 2 - C H O - C H ; O , OCH2-CH2), 4.12 (t, 4H, CH2-O2C), 5.77 (d, 2H, O2C-CH=), 6.07-6.22 (m, 4H, C H = C H - C H 3 ) , 7.16-7.28 (m, 2H, O 2 C - C H = C H ).

mixture of 2 ml of CHCI 3, 3.0 ml of i-PrOH, and 3.0 ml of CH3CN, followed by addition of 4.0 ml of 25% aqueous trimethylamine solution. The mixture was stirred for 15 h at 56°C. The reaction mixture was transferred into a separatory funnel containing 20 ml of water. The mixture was extracted with chloroform several times using methanol to break emulsions. The chloroform solution was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (CHCI3/MeOH/H20, 65:25:4, RI= 0.26) to yield 0.192 g of the lipid (67%). Mass spectrum, Calculated mol. wt. for C40H72010PN: 757.5. Found: m/z 758.4 (MH+), 224, 184, 166; IH-NMR (CDCI3) 6 1.28-1.39 (m, 24H, (CH2)6), 1.53-1.72 (m, 8H, OCH2-CH2), 1.85 (d, 6H, CH3), 3.44-3.62 (m, 16H, OCH2, O C H 2 - C H O - C H 2 0 - P , +N(CH3)_3), 3.78 (broad m, 2H, CH2-OP), 3.97 (broad m, 2H, OCH2-CH2-N+), 4.12 (t, 4H, CH2-O2C), 4.37 (broad m, 2H, O.CH;-CH2-N+), 5.77 (d, 2H, O2C-CH=), 6.07-6.22 (m, 4H, C H = C H - C H 3 ) , 7.16-7.28 (m, 2H, O2C-CH=CH); Xmax= 258 nm, CH3OH , e = 50 700; DSC, T m = 11.4°C (c = 1.63 mg/ml).

10,12-Tricosadiyn-l-methylsulfonate (I1-I) 1,2-O-B&[IO(2',4'-hexadienoyloxy) decyl]-racglycero-3-phosphocholine (I-7) 2-Bromoethyl phosphoric acid dichloride was prepared from 2-bromoethanol and phosphorus oxychloride using the procedure of Hansen [25]. The phosphorylation of compound I-6 was performed by following the method developed by Eibl and Nicksch [26]. A solution of bromoethyl phosphoric acid dichloride (1.30 g, 0.537 mmol) in 5 ml of diethyl ether was cooled in an ice-bath. After addition of triethylamine (0.110 g, 1.10 mmol), the reaction mixture was kept at room temperature for 10 min and compound I-6 (0.198 g, 0.334 mmol) in 5 ml of diethyl ether was added dropwise while stirring. The mixture was stirred for 2 h then concentrated under reduced pressure. The residue was dissolved in 5 ml of THF, followed by addition of 1 M Na2CO 3 (10 ml) and stirred for 3 h. The reaction mixture was extracted with diethyl ether. The ether layer was dried over sodium sulfate, concentrated and dissolved in a

10,12-Tricosadiynoic acid (9.96 g; 28.8 mmol) in 50 ml of diethyl ether was added to a solution of 1.93 g of LiA1H4 in 60 ml of diethyl ether. The mixture was refluxed for 1 h. The reaction mixture was poured into a separatory funnel containing 100 ml of ice-water and acidified with 5% HCI. The ether layer was dried over sodium sulfate, then filtered through a silica gel packed column to give 9.20 g of 10,12-tricosadiyn-l-ol ~is a white solid (96%). mp 49-50°C (tit. [27] mp 49°C); IR (KBr) 3413 and 3345 (OH), 2183 and 2140 ( C - C ) cm-l; IH-NMR (CDC13) 6 0.86 (t, 3H, CH3), 1.24-1.52 (m, 30H, (CH2) 7 and (CH2)8CH3), 2.22 (t, 4H, C H 2 - C = ) , 3.62 (t, 2H, CH2-O). Triethylamine (3.34 g; 33.0 mmol) was added to a solution of 10,12-tricosadiyn-l-ol (5.50 g; 16.5 mmol) in 150 ml of diethyl ether in an ice-bath. After stirring for 5 min, methanesulfonyl chloride (2.84 g; 24.8 mmol) in 20 ml of diethyl ether was added dropwise at ice-temperature. The mixture was stirred for 2 h at room temperature. The white

213 precipitate which formed was removed by filtration. The ether layer was washed with 5% HCI solution (2 x 50 mt), then dried over sodium sulfate and concentrated. The residue was purified by flash column chromatography (hexane/EtOAc, 80:20, Rr= 0.39) to obtain 6.40 g of compound II-I as a white solid (94%). mp 44.0°C; IR (KBr) 2183 and 2140 ( C - C ) , 1336, 1330 and 1173 (S(=O)2) cm-l; IH-NMR (CDCI3) 6 0.87 (t, 3H, CH3), 1.22-t.56 (m, 28H, (CH2)6 and (CH2)8CH3), 1.73 (m, 2H, CH2-CH20), 2.22 (t, 4H, CH2-C=), 3.00 (s, 3H, S-CH3), 4.20 (t, 2H, CH20).

1,2-O-Bis( lO, 12-tricosadiynyl)-rac-3tritylglycerol (II-2a) A racemic mixture of 3-O-tritylglycerol was prepared from racemic 1,2-isopropylideneglyceroi using the procedure reported by Pfeiffer et al. [23]. 3-O-Trityi-rac-gtycerol (0.706 g; 2.11 mmol) and KOH powder (2.30 g; 41.0 mmol) in 80 ml of dry benzene were refluxed for 5 h with Dean Stark trap to remove water formed during the reaction. 10,12-Tricosadiyn- 1-methylsulfonate (2.60 g; 6.33 mmol) in 30 ml of benzene was added dropwise over period of 1.5 h. The mixture was refluxed for 14 h. The reaction mixture was diluted with 100 ml of diethyl ether and washed with water, 5% HC1 and water. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was separated by flash column chromatography (hexane/EtOAc, 98:2, Rf= 0.30) to obtain 1.70 g of the product as a viscous liquid (83%). IR (neat) 3084, 3057 and 3021 (aromatic C-H), 2256 and 2157 (C--C) cm-l; lH-NMR (CDC13) 6 0.88 (t, 6H, CH3), 1.26-t.57 (m, 60H, (CH2)7 and (CH2)8CH3), 2.24 (t, 8H, CH2-C-=), 3.15 (d, 2H, CH20-CPh3), 3.40 (t, 2H, CH20), 3.50-3.56 (m, 5H, OCH 2CHO, CH2-CH20 ).

( S )- (-)-2,3-O-Bis( lO, 12-tricosadiynyl)-l-tritylsn-glycerol (II-2b) (S)-(-)-l-O-Trityl-sn-glycerol was prepared from (S)-(+)-2,2-dimethyl- 1,3-dioxolane-4-methanol by employing the method used for preparation of the racemic 3-O-tritylglycerol [23]. Yield of compound II-2b after purification by flash column

chromatography, 73%; [~]~5 = -3.6 (c = 0.0105, CHCI3).

1,2-O-Bis( lO,12-tricosadiynyl)-rac-glycerol

(II-3a) Compound II-2a (1.00 g; 1.04 mmol) and ptoluenesulfonic acid monohydrate (60 mg) in 20 ml of methanol were stirred for 12 h at room temperature. The solvent was evaporated and the residue purified by flash column chromatography (hexane/EtOAc, 90:10, Rf=0.16) to yield the alcohol as a white solid (80%). mp 42-44°C; IR (KBr) 3407 (OH), 2255 and 2158 (C=C) cm-1; IH-NMR (CDC13) 6 0.88 (t, 6H, CH3), 1.21-1.60 (m, 60H, (CH2)7 and (CH2)8CH3), 2.24 (t, 8H, CH2-C~- ), 3.44-3.53 (m, 9H, OCH~, OCH2CHO-CH20).

( R )- (+)-2,3- O-Bis (10,12-tricosadiynyl)-snglycerol (II-3h) Yield, 93%; mp (c = 0.0151, CHCI3).

46-47°C;

[c~]25 = + 6.2

1,2-O-Bis(10,12-tricosadiynyl)-rac-glycero3-phosphocholine (II-4a) The procedure used for synthesis of compound 1-7 was employed to synthesize compound lI-4a (0.260 g; 0.361 mmol). The yield was 63% after purification by column chromatography (CHC13/ MeOH/H20, 65:25:4, Rf= 0.32). Mass spectrum, Calculated mol. wt. for C54H9606PN: 885.7, Found: m/z 886.7 (MH +) 224, 184, 166; IH-NMR ~5(CDCI3) 0.88 (t, 6H, CH3), 1.26-1.54 (m, 60H, (C__H~H7) and (CHe)sCH3), 2.24 (t, 8H, CH2-C=-) 3.37-3.85 (m, 20H, OCHz-CHO-CH20, OCH 2, CH2-N +, (CH3)3N+), 4.31-4.33 (m, 2H, OPO3CH2CH2-N+); DSC, Tm= 27.6°C (c = 1.39 mg/ml).

( S)-(-)-2,3-O-Bis( lO, 12-tricosadiynyl)-snglycero- l-phosphocholine (II-4b) Yield, 65%; mass spectrum, Calculated tool. wt. for CsnH9606PN: 885.7, Found: m/z 886.6 (MH +) 224, 184, 166; [~]25 = -3.2 (c = 0.00409, CHCI3); DSC, Tm = 30.0°C (c = 1.39 mg/ml). Results and Discussion

1,2-Isopropylideneglycerol has often been used as a starting material for ether lipid synthesis

214

because of its ease of preparation from mannitol and commercial availability [28,29]. The synthesis of the sorbyl ether lipid, I-7, (Scheme I) used 3-0benzyl-rac-glycerol, which is readily prepared in large quantity by a standard two-step procedure from 1,2-isopropylidene-rac-glycerol [22]. The synthesis of optically active !-7 would be possible via this scheme by starting from commercially available (S) or (R)-isopropylideneglycerot. Alkylation of 3-O-benzyl-rac-glycerol was first attempted with 10-tetrahydropyranyldecyl- 1methylsulfonate (prepared from 1,10-decanediol). However, several side products formed during the

H

~

C

I

i. T H P O , ~ c

I

alkylation reaction made isolation of the product difficult. The use of 10-chloro-l-tetrahydropyranyldecane (1-1) in the alkylation of 3-0benzyl-rac-glycerol permitted easy purification of the desired product (1-2). Furthermore, the chloride is more readily prepared by reaction of 10-chloro-l-decanol with dihydropyran than is the sulfonate. Hydrogenolysis of the benzyl group was accomplished in high yield (90%) with Pd/C giving compound I-3. Trichloroethyl chloroformate was used to form the carbonate before acidic hydrolysis of the terminal tetrahydropyranyl ethers to give the diol, I-4. Esterification was then performed with sorbyl chloride in pyridine giving compound 1-5. Activated zinc was used to remove the trichloro-

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CH3(CH2)9-C ~C-C ~C-(CH2)9-OH ~b

~OTHp

CH3(CH2)9-C ~C-C ~C-(CH2)9-OMs

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(+) or (-) PhaCO"y~ol-t OH

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7

a. 3,4-dihydro-2H-pyran, p-TsOH / THF b. 1, KOH / benzene

--C-C~ C ~ 2a, (+); b, (-)

-=C-C= C~

C -C_C = C ~ 3a, (-+); b, (+) e, f , g

-o,. ,o O,P . O ~ , ~ O ~ / ~ I ~ / - - C +N2 / I\

0 ~ . ~ ~ _

--C-C- C ~ C ~-C_C_= C

~

4a, (+_); b, (-)

c. H 2, Pd/C / EtOAc d. CICO2CH2CC13, Pyr / CHCI 3

a. LiAIH4 / Et20 b. CH3SO2C1, Et3N / Et20

e. p-TsOH / MeOH f. C1COCH=CH-CH=CH-CH3, Pyr / CHCI 3

c. 1, KOH / benzene d. p-TsOH / MeOH

g. Zn / HOAc-Et20 h. CI2PO2(CH2)2Br, Et3N / Et20 i. 1M Na2CO 3

e. CI2PO2(CH2)2Br, Et3N/Et20 f. 1M Na2CO 3

j. 25 % aq. (CH3)3N / i-PrOH-CH3CN-CHCI3 Scheme 1.

g. 25 % aq. (CH3)3N / i-PrOH-CH3CN-CHCla Scheme It.

215

ethylene carbonate protecting group. Phosphorylation and amination of intermediate 1-6 were achieved using the procedure of Eibl [26]. The total synthesis of sorbyl ether lipid, 1-7, consists of six well-known reactions from 3-O-bezyl-racglycerol with an overall yield of 21%. The most effective syntheses of the diacetylenic ether lipids (II-4) are outlined in Scheme II. Initially, we examined the use of optically active 3-0benzyl-sn-glycerol followed by deprotection of the benzyl group with iodotrimethylsilane. However, this chemistry caused some reduction of the conjugated triple bond with vinyl protons appearing between 6 and 7 ppm in the IH-NMR spectrum. Furthermore, the phase transition from gel to liquid crystalline phases of the hydrated bilayer assemblies of the lipid occurred over a wide range of temperatures with a fhll width at half height of 2.6°C for the transition peak (T1/2), suggesting that the lipid product was not homogeneous. Therefore, racemic and chiral tritylglycerol were chosen as alternative primary intermediates. They were prepared from racemic and (S)-(-)-l,2-isopropylideneglycerol, respectively, by the procedure of Pfeiffer et al. [23]. 10,12-Tricosadiyn1-methylsulfonate (II-1) was prepared by LiA1H4 reduction of 10,12-tricosadiynoic acid, followed by reaction with methanesulfonyl chloride. The ether linkages of compound 1I-2 were formed from racemic or chiral tritylglycerol and the diacetylenic sulfonate in the presence of potassium hydroxide in refluxing benzene in a yield of 83% for the racemic and 73% tbr the chiral compound. The trityl group was then removed using ptoluenesulfonic acid monohydrate in methanol. The phosphorylation and amination of intermediates 11-3 were again achieved using the standard procedure [26] giving lipids !1-4. This synthesis contains just three steps from tritylglycerol and each reaction was readily accomplished with an overall yield of 42% for the racemic and 44% for the chiral lipid. IH-NMR and IR spectra of the racemic and chiral intermediates and lipids were not distinguishable from each other. Optical rotation of lipid ll-4b, D-isomer (Sconfiguration), was measured to be -3.2 ° in chloroform at 25°C. However, there is no reference in the literature for direct comparison as far

as we know. The optical rotation of the corresponding L-isomer ester diacetylenic analogue was reported to be +5.7 ° in chlorotbrm at 20°C [30]. In order to compare the optical rotation between ester and ether lipids, L-DPPC, received as greater than 99% pure from Avanti Polar-Lipids, lnc (Alabaster, AL) was measured. Optical rotation of L-DPPC at 25°C was found to be +6.8 ° in chloroform-methanol (50:50, v/v) in this laboratory and the optical rotation of D-DPPC at 20°C was reported to be -6.1 ° [30]. The [o~]~5 of 1,DHPC was reported to be +3.2 ° in chloroform-methanol (50:50, v/v) [28]. This result indicates that the optical rotation of an ether lipid is approximately one half of the value of the corresponding ester analogue, which may be due to the difference in the steric arrangements of the first three carbon atoms of the hydrocarbon chains. In the ester lipids, the conformation around the ester bond is restricted by the C=O bond. Note that the starting material for the chiral lipid synthesis, (S)(-)-l-O-trityl-sn-glycerol, showed [o~]~)5 of -17.9 ° in pyridine, which compares to the reported literature values of -15.7 and -16.8 ° under the same conditions [23]. Based on these comparisons of ester and ether lipids a value of - 3 ° for ll-4b indicates a high degree of optical purity of this product. FAB mass spectrometry has proven to be very useful in identifying sn-glycerophosphocholines [31,32] and was used here to confirm the lipid structures obtained from these syntheses. The sorbyl and diacetylenic ether lipids each generate some common fragments (m/z = 224, 184, 166), characteristic for glycerophosphocholine [31]. Hydrated lipid assemblies of each of the three lipids are photosensitive. The sorbyl ether lipid shows the characteristic sorbyl ultraviolet absorption. The absorbance maximum (258 nm) and extinction coefficient (50 700) for methanol solution are comparable to the values of the corresponding ester lipid [21]. Hydrated bilayer assemblies of I-7 at 25°C also show an absorbance maximum at 258 nm (Fig. 1) which decreased when hydrated bilayer assemblies were irradiated with 254 nm light from a low pressure mercury lamp. This loss of monomer absorption was accompanied by the progressive loss of mobility of the lipid on TLC. These

216

1

2 /

\

/

c

eo 1 E

/'

.3

" ......5 .

0 200

~

3~o Wc]velength (nm)

4-00

Fig. 1. Absorption spectra of liposomesof sorbyl ether lipid (I-7, 5.84 x 10-5 M) in water. The spectra were recorded after exposure to 254 nm light for the following times: curve 1, 0 min; 2, 2 min; 3, 3 min; 4, 10 min and 5, 20 rain.

data indicate that the lipid molecules in bilayer assemblies were photochemically polymerized. The similarity in ~ m a x for 1-7 in methanol and in hydrated bilayer assemblies indicates the two sorbyl chromophores do not interact or aggregate at temperatures above the lipid phase transition temperature [33]. The Tm for extended bilayer assemblies of 1-7 was 11.4°C (Fig. 2). When the racemic diacetylenic ether lipid (II-4a) sample was prepared by cooling the lipid suspension at 0°C and irradiated with the UV light, it turned purple slowly. However, when the lipid sample was frozen in dry ice-isopropanol, then melted by equilibration in an ice-bath, the sample turned bluish-purple immediately upon UV light exposure in the manner typical for polydiacetylenic formation [341. Polymerization behavior of chiral diacetylenic ether lipid ( I I 4 b ) in

extended bilayers was observed to be similar to that of the racemic ether lipid analogue. The phase transition of the extended bilayers of the chiral diacetylenic ether lipid (T m = 30.0°C with TI/2 = 1.2°C) occurs at a few degrees higher than that of the racemic analogue (T m = 27.6°C with I"1/2 = 1.3°C). A similar observation was reported for the diacetylenic ester analogs [30]. The sharp transitions imply a high degree of cooperativity of lipid molecules during the transition. Interestingly, the transition peaks of the diacetylenic ether lipids have a small shoulder which is not resolved, which is similar to reports for the diacetylenic ester analogs [30]. It was reported that a chiral diacetylenic ester analogue isomer formed microstructures such as tubules on slow cooling of the lipid bilayer assemblies below the transition temperature [35].

217 O CO

,-i

ID (kl

__

O OJ

__

tO

:Z:

u

O

1

j

2

_/

Q. (D

O

I

J

__

o

t

1

I

I

iO

2o

30

40

Temp

50

(deg)

Fig. 2. Main phase transitions of extended bilayers of the three lipids: curve 1, offset by 10 kcal, sorbyl (I-7, 1.63 mg/ml); 2, offset by 5 kcal, racemic diacetylenic (ll--4a, 1.39 mg/ml); 3, chiral diacetylenic (lI-4b, 1.39 mg/ml) ether lipid in sodium phosphate buffer (pH 7.4).

The diacetylenic ester and some other diacetylenic ester lipids formed helices and tubules upon precipitation of their alcoholic solutions by the addition of water at temperatures below their Tm [30, 36]. We also observed formation of tubules and helices of the chiral diacetylenic ether lipid (ll-4b) by the precitation method performed at 6-8°C. Further details on these observations will be described in a subsequent paper. In summary, we report here the synthesis of racemic sorbyl and diacetylenic ether lipids from racemic 1,2-isopropylideneglycerol and chiral diacetylenic ether lipid from (S)-l,2-isopropylideneglycerol. These three lipids are the first examples of ether lipids containing polymerizable groups in the hydrophobic chains. The reactions

used in these syntheses are straightforward and convenient; therefore, these ether lipids may be readily obtained. The methods may also be used to synthesize optically active sorbyl ether lipids by starting the synthesis with commercially available (R)- or (S)-l,2-isopropylideneglycerol. Further characterization of these lipids is still in progress and will be reported in a future paper.

Acknowledgement Acknowledgement is made to the donors of the Petroleum Research Fund, administrated by the American Chemical Society, for partial support of this research.

218

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