Synthesis of Lipids

Synthesis of DOSPA

DOSPA is a typical cytofectin and contains a cationic
head group attached by a linker to a hydrophobic moiety.
The synthesis of the hydrophobic part, 2, from 3-(dimeth-
ylamino)-1,2-propanediol (1) is depicted in Scheme 1,
and it is identical to the first step of the synthesis of
DOTMA. 15 Compound 3 was made by alkylation of 2
with N-(2-bromoethyl)phthalimide. The phthalimide
(Pht) group is readily removed by refluxing of 3 together
with hydrazine and methanol to give 4 with an 80% yield.
The free amine group of 4 is the site for linking of the
additional portion of the cationic head group.

The additional portion of the cationic head group,
tetra(tert-butoxycarbonyl)spermine-5-carboxylic
acid
(Boc 4 SperCOOH), was synthesized in three steps as
described previously by Remy et al., 1994 (Scheme 2). 16
Instead of using DMF as the solvent in the first step,
methanol was used to perform the addition of acrylonitrile
to L -ornithine tetramethylammonium salt. The substitu-
tion of methanol for DMF increased the yield to 85%.
After the hydrogenation and the protection of the amine
with a Boc group, the cationic head group precursor 8 has
only a free acid group, which can be coupled with the free
amine group of the hydrophobic part 4 under the standard
coupling condition.

As shown in Scheme 3, by using PyBOP, HOBt and DI-
PEA as standard coupling reagents, the hydrophobic part
4 and the protected cationic head group 8 can be linked to-
gether to give 9 with a yield of 70%. Treatment of 9 with
4 M HCl in dioxane led to the removal of the Boc group
of 9 to give the final product, DOSPA (10), 17 with a yield
of 65%.

Synthesis of DOTMA

DOTMA Synthesis and Liposome Preparation. DOTMA
{N-[1-(2,3,-dioleyloxy)propyl]-N,N,N-trimethylammonium
chloride} was prepared as outlined in Fig. 1. A mixture of
3-(dimethylamino)-1,2-propanediol (Aldrich; 1.19 g, 10
mmol), potassium tert-butoxide (3.36 g, 30 mmol), and oleyl
p-toluenesulfonate (12.7 g, 30 mmol) in xylenes (50 ml) was
stirred at room temperature and reduced pressure (30 torr; 1
torr = 133 Pa) for 30 min and then was heated to 50TC with
stirring for an additional 15 min. The reaction vessel was
purged with nitrogen, and the mixture was heated to reflux
(-4140'C) for 3 hr. After cooling, the reaction mixture was
diluted with hexane (100 ml), and the resulting solution was
extracted with water (twice, 50 ml each). The organic layer
was concentrated, and the residue was chromatographed
over silica gel by elution with a mixture of hexanes and
diethyl ether (1:2) to afford the intermediate 2,3-dioleyloxy-
1-(dimethylamino)propane as a colorless oil. Quaternization
was carried out by condensation of methyl chloride (50 ml)
into a Parr pressure apparatus cooled to -78°C and contain-
ing this compound (10 g). The sealed vessel was heated
behind a safety shield at 70°C for 48 hr. After cooling to 0°C,
the reaction vessel was opened, and the excess methyl
chloride was allowed to evaporate under a stream of nitrogen
in a well-ventilated hood. The crude residue was recrystal-
lized from acetonitrile to afford DOTMA as an off-white
solid, mp 35-38°C. Full details of the synthesis of DOTMA
and analogs will be published elsewhere (T.R.G. and P.L.F.).
A solution of dioleoyl phosphatidylethanolamine (PtdEtn;
10 mg) and DOTMA (10 mg) in chloroform (1 ml) was
evaporated to dryness under a stream of nitrogen, and
residual solvent was removed under vacuum overnight.
Liposomes were prepared by resuspending the lipids in
deionized water (2 ml) and sonicating to clarity in a closed
vial. Sterile preparations of liposomes are stable for at least
6 months at 4°C.

Some useful lipids

Oleic acid

http://www.sigmaaldrich.com/catalog/product/sial/o1008

O1008-1G, $19.20; O1008-5G $60.00; O1008-25G, $191.50

Linoleic acid

http://www.sigmaaldrich.com/catalog/product/sigma/l1376

L1376-1G, $37.40; L1376-5G, $56.20; L1376-10G, $129.00;
L1376-25G, $219.00

Oleylamine, O7805-5G, $32.60;; O7805-100G, $36.10; O7805-500G, $111.50

Cholesterol

Cholesteryl chloroformate

http://www.sigmaaldrich.com/catalog/product/aldrich/c77007, C77007-25G, $61.60; C77007-100G, $143.00

Synthesis of Cholesteryl Iodide and Cholesteryl Chloride

Cholesteryl Iodide.-To 20.0 g (0.13 mole) of sodium iodide in 200 ml of acetone at 35 oC (constant-temperature bath) were added 5.0 g (0.0110 mole) of cholesteryl chloroformate and then sufficient diethyl ether ( ~ 2 ml) 0 to make the solution homogeneous. Precipitation of sodium chloride was observed and, after
70 hr, the solution was evaporated to dryness under reduced pressure. The residue was dissolved using 100 ml of diethyl ether and 100 mi of water. The ether layer was washed with 50 ml of dilute sodium hydrogen sulfite solution and then with 25 ml of water, followed by evaporation to dryness. The residue was recrystallized from acetone to give 2.9 g of a white crystalline solid. A second recrystallization from acetone gave 2.7 g of cholesteryl iodide (49% yield).

Cholesteryl Chloride.-To 50 ml of acetone were added 5.0 g (0.0110 mole) of cholesteryl chloroformate and 10.0 g (0.17 mole) of sodium chloride and the mixture was refluxed for 250
hr. After cooling, the mixture was filtered and the precipitate was washed with 25 ml of diethyl ether. The ether was added to the mother liquor, which was then evaporated to dryness
under reduced pressure, and the residue was recrystallized twice from acetone to give 3.0 g of white crystalline cholesteryl chloride (67% yield)

Synthesis of Dilinoleyl Ketone

http://www.google.com/patents/US20110256175

Synthesis of Linoleyl Bromide (II)

A mixture of linoleyl methane sulfonate (6.2 g, 18 mmol) and magnesium bromide etherate (17 g, 55 mmol) in anhydrous ether (300 mL) was stirred under argon overnight (21 hours). The resulting suspension was poured into 300 mL of chilled water. Upon shaking, the organic phase was separated. The aqueous phase was extracted with ether (2×150 mL). The combined ether phase was washed with water (2×150 mL), brine (150 mL), and dried over anhydrous Na2SO4. The solvent was evaporated to afford 6.5 g of colourless oil. The crude product was purified by column chromatography on silica gel (230-400 mesh, 300 mL) and eluted with hexanes. This gave 6.2 g (approximately 100%) of linoleyl bromide (II). 1H NMR (400 MHz, CDCl3) δ: 5.27-5.45 (4H, m, 2×CH═CH), 3.42 (2H, t, CH2Br), 2.79 (2H, t, C═C—CH2—C═C), 2.06 (4H, q, 2× allylic CH2), 1.87 (2H, quintet, CH2), 1.2-1.5 (16H, m), 0.90 (3H, t, CH3) ppm.
Synthesis of Dilinoleyl Methanol (III)

To a suspension of Mg turnings (0.45 g, 18.7 mmol) with one crystal of iodine in 200 mL of anhydrous ether under nitrogen was added a solution of linoleyl bromide (II) in 50 mL of anhydrous ether at room temperature. The resulting mixture was refluxed under nitrogen overnight. The mixture was cooled to room temperature. To the cloudy mixture under nitrogen was added dropwise at room temperature a solution of ethyl formate (0.65 g, 18.7 mmol) in 30 mL of anhydrous ether. Upon addition, the mixture was stirred at room temperature overnight (20 hours). The ether layer was washed with 10% H2SO4 aqueous solution (100 mL), water (2×100 mL), brine (150 mL), and then dried over anhydrous Na2SO4. Evaporation of the solvent gave 5.0 g of pale oil. Column chromatography on silica gel (230-400 mesh, 300 mL) with 0-7% ether gradient in hexanes as eluent afforded two products, dilinoleyl methanol (2.0 g, III) and dilinoleylmethyl formate (1.4 g, IV). 1H NMR (400 MHz, CDCl3) for dilinoleylmethyl formate (IV) δ: 8.10 (1H, s, CHO), 5.27-5.45 (8H, m, 4×CH═CH), 4.99 (1H, quintet, OCH), 2.78 (4H, t, 2×C═C—CH2—C═C), 2.06 (8H, q, 4×allylic CH2), 1.5-1.6 (4H, m, 2×CH2), 1.2-1.5 (32H, m), 0.90 (6H, t, 2×CH3) ppm.

Dilinoleylmethyl formate (IV, 1.4 g) and KOH (0.2 g) were stirred in 85% EtOH at room temperature under nitrogen overnight. Upon completion of the reaction, half of the solvent was evaporated. The resulting mixture was poured into 150 mL of 5% HCL solution. The aqueous phase was extracted with ether (3×100 ml). The combined ether extract was washed with water (2×100 mL), brine (100 mL), and dried over anhydrous Na2SO4. Evaporation of the solvent gave 1.0 g of dilinoleyl methanol (III) as colourless oil. Overall, 3.0 g (60%) of dilinoleyl methanol (III) were afforded. 1H NMR (400 MHz, CDCl3) for dilinoleyl methanol (III) δ: ppm.
Synthesis of Dilinoleyl Ketone (V)

To a mixture of dilinoleyl methanol (2.0 g, 3.8 mmol) and anhydrous sodium carbonate (0.2 g) in 100 mL of CH2Cl2 was added pydimium chlorochromate (PCC, 2.0 g, 9.5 mmol). The resulting suspension was stirred at room temperature for 60 min. Ether (300 mL) was then added into the mixture, and the resulting brown suspension was filtered through a pad of silica gel (300 mL). The silica gel pad was further washed with ether (3×200 mL). The ether filtrate and washes were combined. Evaporation of the solvent gave 3.0 g of an oily residual as a crude product. The crude product was purified by column chromatography on silica gel (230-400 mesh, 250 mL) eluted with 0-3% ether in hexanes. This gave 1.8 g (90%) of dilinoleyl ketone (V). 1H NMR (400 MHz, CDCl3) δ: 5.25-5.45 (8H, m, 4×CH═CH), 2.78 (4H, t, 2×C═C—CH2—C═C), 2.39 (4H, t, 2×COCH2), 2.05 (8H, q, 4×allylic CH2), 1.45-1.7 (4H, m), 1.2-1.45 (32H, m), 0.90 (6H, t, 2×CH3) ppm.

Synthesis of 1,2-0-dioleyl-3-dimethylamino propyl-β-hydroxyethylammonium acetate (DORI diether)

Step (a): Oleylmethanesulfonate
In a 500 ml three-necked flask equipped with a dropping funnel, 5.0 g (18.7 mmoles) of oleyl alcohol (Nu Check Prep, Elysian, Minn. 56028) was dissolved in 6.67 ml of dry pyridine and 100 ml of freshly distilled chloroform. The solution was chilled in an ice bath and 3.22 g (28.14 mmoles) of methanesulfonyl chloride (Nu Check Prep) dissolved in 50 ml of dry chloroform was added dropwise during one hour. The reaction mixture was allowed to stir for another 4 hours at room temperature. At the end of the reaction period, 30 ml of ice cold water and 50 ml of diethylether was added. The organic layer was washed twice with 50 ml of 0.5N cold HCL, followed by 50 ml of cold 0.5N sodium bicarbonate. Finally, the organic phase was dried over anhydrous sodium sulfate and evaporated under vacuum on a rotary evaporator. The product was dissolved in 45 ml of absolute ethanol and crystallized at -20° C. Pure long needles of oleylmethanesulfonate were obtained in 90% yield.

Step (b): 1,2-O-dioleyl-3-dimethylaminopropylglycerol
Racemic 3-(dimethylamino)-1,2 propanediol (Aldrich Chemical, Milwaukee, Wis.), 1.5 g, 8.3 mmoles, and potassium hydroxide, 3.7 g in 100 ml of freshly distilled dry benzene was refluxed for 2 hours in a 300 ml, three-necked, round bottom flask fitted with a Soxhlet apparatus containing molecular sieves. Oleyl methanesulfonate, 5.78 g, dissolved in 100 ml of dry benzene, was dropped slowly into the reaction mixture and refluxing was continued for another 4 hours. At the end of the reaction period, cold water and diethylether were added. The organic phase was washed with acid and bicarbonate successively as described above. The crude yellow product gave three spots on thin layer chromatography on silica gel G plates, developed with chloroform/acetone/methanol/acetic acid/water, (50/15/5/5/2) by volume. The required compound was purified by silicic acid column chromatography as follows: Approximately 3.0 g of the above material was loaded on the silica CC 7, BioRad (40.0 g) column and sequentially eluted with chloroform (200 ml), chloroform/methanol 5%, (200 ml), 10% (250 ml) and finally with methanol (500 ml). The pure compound was eluted with 10% methanol fractions and gave an Rf value of 0.45 when chromatographed on the silica gel G plates developed in the above system.

Step (c): 1,2-0-dioleyl-3-dimethylamino propyl-β-hydroxyethylammonium acetate
Racemic 1,2-0-dioleyl-3-dimethylaminopropylglycerol, 2.1 g, (3.4 mmol) and 4 ml of 2-bromoethanol (Aldrich Chemical, Milwaukee, Wis.) in 18 ml of dimethylformamide was added in a 100 ml round bottom flask and stirred for 36h at 45° C. At the end of the reaction period, the mixture was concentrated under reduced pressure, and the product was purified by passing through the silica gel column. The compound was dissolved in a small amount of chloroform and loaded on to 30 gms of silica gel 60, 70-270 mesh, packed in a 1×18 column. The pure compound was eluted with 8% methanol in chloroform and gave an Rf value of 0.21 on silica gel G plates developed in the above system. Finally, the bromide salt was converted to acetate by passing the product through a Whatman DE-52 cellulose (acetate form) column. The product was obtained in 50/50 chloroform/methanol eluate. The compound was crystallized in acetonitrile at -20° C.

Synthesis of DL 1,2-0-dipalmityl-3-dimethylamino propyl-β-hydroxyethylammonium acetate (DPRI diether)

This compound was synthesized by substituting palmityl alcohol for oleyl alcohol in the above procedure.

Synthesis of DL-1,2-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium acetate (DORI diester)

To 10 g. of oleoyl chloride in 21 ml of dry dimethylformamide were added 1.6 g of Rac-3-(dimethylamino)-1,2 propanediol and 5 ml of tributyl. The mixture was heated to 60°-65° C. for 48 hours. After the mixture was cooled to room temperature, 50 ml of freshly distilled diisopropylether was added and the mixture was heated to boiling. The reaction mixture was again cooled to room temperature and filtered. Filtrate was evaporated under vacuum and the product was crystallized with acetonitrile. The pure dioleoyl-3-dimethylaminopropylglycerol was further subjected to quaternization by treatment with 2-bromoethanol in dimethylformamide as described in Example 1 above.

Synthesis of DL-1,2-dipalmitoyl-3-dimethylaminopropyl-β-hydroxyethylammonium (DPRI diester)

This compound was synthesized by using palmitoyl chloride instead of oleoyl chloride in the above procedure.

Synthesis of DL-1,2-dioleoyl-3-propyltrimethylammonium chloride (DOTAP).

To 2.0 g of 3-(dimethylamino)-1,2-propanediol, dissolved in 15 ml of freshly distilled chloroform and 10 ml of anhydrous pyridine, chilled to 4° C., was added dropwise 12.6 g of oleoylchloride, dissolved in 50 ml of chloroform, over a period of one hour. The reaction was allowed to stir overnight and then stopped by the addition of 50 ml of cold water and ether. The organic phase was washed twice with 0.5N HCL and 0.5N sodium bicarbonate, and after drying over anhydrous sodium sulfate, evaporated under vacuum. The product was purified by silicic acid column chromatography as described in Example 1. The pure compound was next quaternized with methyl chloride as follows: 500 mg of the pure compound was added into the protein hydrolysis tube and the methyl chloride (Aldrich Chemical, Milwaukee, Wis.) was condensed into the tube by repeated cooling of the tube in liquid nitrogen until filled with 5 ml of methyl chloride. The tube was once again thawed, frozen and evacuated with oil pump to remove any residual air. Finally, the tube was sealed and placed in the heated metal block maintained at 70° C. for 72 hours. After the reaction period, the tube was cooled to 0° C. and then opened to evaporate unreacted methyl chloride. The yellow wax was crystallized from acetonitrile at -20° C. Further purification of the compound was done on thesilica gel 60 column. The pure compound was eluted with 200 ml of 10% methanol in chloroform and gave an Rf value of 0.23 on a silica gel G plates, when developed in the above solvent system.

Method 2

1. N-[1 -(2,3 -diacyloxy ) propyl ] -N,N,N-trimethyl-ammonium methyl sulfate ( DAcylTAP ) ( general description ) Oleic-, stearic-, palmitic-, myristic-, or lauric
acid (15.8 mmol), was dissolved in 25 ml of THF. N,N’-carbonyldiimidazole (CDI), 2.82 g (17.4 mmol), was added and the mixture was stirred for 30 min at rt. 3-Dimethyl-amino-1,2-propanediol, 750 mg (6.30 mmol) and n-BuLi (15% in hexane) 200 ml (0.315 mmol) were added and the reaction was refluxed for two days. The solvent was removed using a rotatory evaporator. 100 ml of acetone and dimethyl sulfate, 2.0 ml (21.1 mmol) were added and the solution was stirred for further two days at 4°C. The precipitated product was isolated by filtration and recrystallized from acetone. Yield: 30 – 40%.

In analogy with the procedures described by Leventis and Silvius for the synthesis of DOTAP (Leventis and Silvius, 1990) with iodide as counter ion and of its ether analog DOTMA (Felgner et al., 1987), the synthesis is based on the commercially available 3-dimethyl-1,2-propanediol (1). In contrast to the method published by Leventis, who acylated 1 using oleic acid chlorides in pyridine, our reaction sequence applies fatty acid imidazolides as acylating agents. They are prepared in situ from the respective fatty acids and N,N’-carbonyldiimidazole (CDI) (Staab and Mannschreck, 1962). As both the imidazolide formation and the following acylation of 1 (in refluxing THF with a catalytic amount of n-BuLi) occur without formation of insoluble by-products
like pyridine hydrochlorides, neither extraction nor filtration is necessary. The products, the 1,2-diacyl-3-dimethylamino-propanes (2a–e) need not to be isolated, only the solvent has to be replaced by acetone. For subsequent methylation, dimethyl sulfate is added. Due to the solvent characteristics of acetone, the methylated (quaternized) products (3a–e) precipition step from acetone is sufficient. The overall yields range from 30 to 40%, which is only slightly lower than described for the much more complicated original DOTAP-synthesis (49% for DOTAP).

Synthesis of DL 1,2-dipalmitoyl-3-propyltrimethylammonium chloride (DPTMA diester).
This compound was synthesized by using palmitoyl chloride instead of oleoyl chloride in the above procedure.

Synthesis of Mixed Acyl/Ether Derivatives

The alkyl, acyl and mixture of acyl/alkyl derivatives with the same or different aliphatic carbon chain lengths of the above compounds can be synthesized by using known procedures of blocking the primary and or secondary alcohol of the starting materials.

For example, the 1-acyl, 2-alkyl analogue of the above compound was synthesized by benzylation of the primary hydroxyl group of 3-(dimethylamino)-1,2-propanediol (1.0) mol) with 0.9 mol of benzyl chloride to obtain a lyso compound which, on condensation with palmitic or oleic methansulfonate gave 1-O-alkyl, 2-O-benzyl-3-dimethylaminopropyl glycerol. Debenzylation followed by acylation of the resulting compound with palmitic acid chloride and quaternization under similar conditions as described above gave the required compound. The synthesis of alkyl/acyl analogues was achieved by two routes:

(a) 3-(dimethylamino)-1,2-propanediol (1.0 mol) was reacted with 0.7 mol of palmitic methanesulfonate to obtain 1-O-palmityl-2-lyso-3-dimethylaminopropyl glycerol. Acylation of the above lyso compound with oleic anhydride gave the alkyl/acyl derivative.

(b) The primary alcohol group of batyl alcohol (Serdary Research Laboratory) is protected with ts-Cl and NaI and the secondary hydroxyl group is then acylated with oleic anhydride to obtain alkyl/acyl iodohydrin derivative. Further treatment of the iodohydrin derivative with dimethyl amine gave the required product. These compounds are then quaternized by using the procedures described above.

EXAMPLE 8: 3,5-(N,N-di-lysyl)-diaminobenzoyl-3-(DL-1,2-dipalmitoyl-dimethylaminopropyl-β-hydroxyethylamine) (DLYS-DABA-DPRI diester) Step 1: Di-t-Butyloxycarbonyl-3,5-di-aminobenzoic acid (Bis-Boc-DABA)
A quantity of 3,5-Di-aminobenzoic acid (1.52 g; 10 mmol), triethylamine (2.8 ml, 10 mmol) and di-t-butyl-di-carbonate (4.5 g; 22 mmol) (Aldrich Chemical Co, Milwaukee, Wis.), were dissolved in DMF (10 ml) and stirred for 24 hours at room temperature. The solvent was evaporated under vacuum and the product was chromatographed on silica gel using chloroform as eluent to obtain the title compound.

Step 2: Bis-Boc-DABA-DPRI diester
Bis-Boc DABA (3.52 g, 10 mmol) and DPRI (10 mmol) were coupled following the procedure described in procedures 7.3 and 7.4 above.

Step 3: 3,5-(NN-Di-lysyl)-DABA-DPRI diester
Compound #6 (2 mmol) was treated with TFA (10 ml) for 30 min at room temperature to remove the BOC protecting groups. After evaporating the solvent, the product was reacted with Bis-Boc-lysine (5 mmol) using DCC as condensing agent. The product isolated after evaporating the solvent was deprotected using TFA and purified as described in 7.4 above.

EXAMPLE 9: 3,5-(N,N-di-lysyl)-diaminobenzoyl-glycyl-3-(DL-1,2-dipalmitoyl-dimethylaminopropyl-β-hydroxyethylamine) (DLYS-DABA-GLY-DPRI diester) Step 1: Di-t-Butyloxycarbonyl-3,5-di-aminobenzoic acid (Bis-Boc-DABA)
As in Example 7 above.

Step 2: Boc-glycyl-DPRI diester:
Boc-glycine (1.75 g, 10 mmol) and DPRI (10 mmol) were coupled following the procedure described in Steps 3 and 4 of Example 8.

Step 3: Bis-Boc-DABA-glycyl-DPRI diester:
Bis-Boc-DABA (3.52 g, 10 mmol) of the compound from Step 2 above was treated with TFA (10 ml) doe 30 min. at room temperature to remove the Boc protecting group. TFA was evaporated and the product was coupled with Bis-Boc-DABA from Step 1 above as described in Example 8.

Step 4: 3,5-(NN-Di-lysyl)-DABA-glycyl-DPRI diester
The compound from Step 3 above (2 mmol) was treated with TFA (10 ml) for 30 min. at room temperature to remove the BOC protecting groups. After evaporating the solvent, the product was reacted with Bis-Boc-lysine (5 mmol) using DCC as condensing agent. The product isolated after evaporating the solvent was deprotected using TFA and purified as described in Step 4 of Example 8.

Various DORI derivatives corresponding to the DPRI derivatives described in Examples 8 and 9 can be synthesized by substituting DORI in the coupling procedures.

EXAMPLE 10: Synthesis of L-spermine-5-carboxyl-3-(DL-1,2-dipalmitoyl-dimethylaminopropyl-β-hydroxyethylamine) (SPC-DPRI diester)
L-5-carboxytetrabutyloxycarbonylspermine (tetra-BOC-Sper-COOH) (664 mg; 1 mmol) prepared according to published procedures (J.-P. Behr et. al. Proc. Natl. Acad. Sci., USA, 86, pp 6982-6986, 1989) was coupled to DPRI (1 mmol) as described in 8.3. The product was de-protected and purified by chromatography to prepare SPC-DPRI diester.

EXAMPLE 11: PREPARATION OF LIPOSOME-FORMING DOTAP
The cationic liposome-forming material 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP) may be also prepared as reported by L. Stamatatos, et al. Biochemistry 27:3917-3925 (1988) or H. Eibl et al., Biophysical Chemistry 10:261-271 (1979).

Briefly, Stamatatos et al. report that 1 mmol of 3-bromo-1,2-propanediol (Aldrich, Milwaukee, Wis.) was acylated for 48 hours at 20° C. with 3 mmol of oleoyl chloride (freshly prepared from oleic acid and oxalyl chloride) in dry, alcohol-free diethyl ether (20 ml) containing 5 mmol of dry pyridine. The precipitate of pyridinium hydrochloride was filtered off, and the filtrate was concentrated under nitrogen and redissolved in 10 ml of hexane. The hexane solution was washed 3 times with an equal volume of 1:1 methanol/0.1N aqueous HCOONa, pH 3.0, 3 times with 1:1 methanol/0.1N aqueous NaOH, and 1 time with 1% aqueous NaCl. The crude 3-bromo-1,2-bis-(oleoyloxy)propane was then stirred for 72 hours in a sealed tube with a solution of 15% trimethylamine in dry dimethyl sulfoxide (30 ml) at 25° C. The products of this reaction were dissolved in chloroform (200 ml), which was repeatedly washed with 1:1 methanol/100 mM aqueous HCOONa, pH 3.0, and then evaporated in vacuo to yield a light yellow oil. The material was purified on a column of silicic acid (Bio-Sil A, Bio-Rad Laboratories), eluting with a 0-15% gradient of methanol in chloroform to give the desired product in pure form at 9-10% methanol.

This purified product was a colorless, viscous oil that migrates with an Rf of 0.4 on thin layer chromatography plates (Silica Gel G) that were developed with 50:15:5:5:2 CHCl3 /acetone/CH3 OH/CH3 COOH/H.

EXAMPLE 12: LIPID VESICLE PREPARATION
Dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylethanolamine (DOPE) were purchased from Avanti Polar Lipids, (Pelham, Ala.). DOTMA was synthesized according to U.S. Pat. No. 4,897,355 to Epstein, D. et al. or Felgner, P. L. et al., PNAS 84: 7413-7417 (1987) and DOTAP was synthesized according to Example 10. DOPG/DOPC vesicles were prepared by drying 50 mg of DOPG and 50 mg of DOPC under a stream of nitrogen gas into a sonication vial. The sample was placed on a vacuum pump overnight and was hydrated the following day with deionized water to a concentration of 10 mg/ml total lipid. The sample was sonicated for 2 hours in the capped vial, using the Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting and the bath was circulated at 15° C. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles (MLV) or by extrusion through nuclepore membranes to produce unilamellar vesicles of a discrete size. Other methods are also available and are known to those familiar with the art. DOTMA or DOTAP vesicles were prepared in an exactly analogous manner.

N¹-oleylspermine tritrifluoroacetate salt.

Spermine (6.90 g, 34.09 mmol) and trifluoroacetic acid (TFA, 7.88 ml, 102 mmol) were dissolved in methanol (455 ml) at 0 ^oC. Oleic acid (3.37 g, 11.93 mmol), activated at 0 ^oC
with isobutyl chloroformate (1.49 ml, 11.36 mmol) and triethylamine (3.15 ml, 22.7 mmol), in dichloromethane (91 ml) was added for 1 h and 10 min. After another hour, the ice bath was removed and stirring was continued at room temperature for 19 h. The solvent was removed under reduced pressure and the mixture was partitioned between dichloromethane (400 ml) and 1 M NaOH (aq., 400 ml). The aqueous layer was separated and reextracted with dichloromethane (2 x 400 ml). The combined organic layers were washed
with NaOH (aq., 0.5 M , 2 x 500 ml), dried with Na2SO4 and evaporated in vacuo, yielding 4.77 g of crude amine. Crystallization in CHCl 3 –ethanol (1:1) as the tri-TFA salt (by addition of a slight excess of TFA) yielded 5.44 g (59 %) of product.