Phospholipid synthesis: effects of solvents and catalysts on acylation

The peptide dinitrophenylprolylthreoninamide (DNP-Pro-Thr-NH2) was used as a model system to develop better acylation conditions for the synthesis of phospholipids using catalyst-activated anhydrides. The acylation rate was found to be inversely related to the polarity of the solvent, chloroform alone resulting in much better rates of reaction than did pyridine, dimethylformamide (DMF) or mixtures of these solvents. Anhydride activated by 4-pyrrolidinopyridine (PPY) was twice as reactive as that activated by 4-dimethylaminopyridine (DMAP). It was shown that the phosphate group of phosphatidylcholine (PC) interferes with the acylation by a process which could be reserved by means of the addition of a 200-fold excess of PPY. This reversal is not due to base catalysis by the PPY; the results suggest that a mixed anhydride may be formed with the phosphate and that this can be reversed by high catalyst concentrations to produce the reactive acylating agent. The acylation rates for lysophosphatidylcholine (lyso PC) using our optimum conditions were found to be approximately 50 times faster than the best rates reported in the literature, the reaction being complete within 5 min even using only a slight excess of anhydride. acyl group migration was assessed during these reactions and no increase in migration of the acyl groups could be detected due to these reaction conditions. The procedures described provide significant improvements over previous methods described for large scale, as well as highly radioactive microscale phospholipid synthesis.