Editing Synthesis of Lipids (section)

== Synthesis of Mixed Acyl/Ether Derivatives  ==

<br>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)<br>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<br>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<br>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)<br>As in Example 7 above. 

Step 2: Boc-glycyl-DPRI diester:<br>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:<br>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<br>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)<br>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<br>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<br>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.<br> 

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