Editing Synthesis of Lipids (section)

== Synthesis of DL-1,2-dioleoyl-3-propyltrimethylammonium chloride (DOTAP).<br>  ==

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 the'''silica 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. 

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=== Method 2  ===

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

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

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[[Image:DOTAP Synthesis.png]] 

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