Editing Synthesis of Lipids (section)

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

Step (a): Oleylmethanesulfonate<br>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<br>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<br>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. 

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