Phospholipids chiral at phosphorus. Synthesis of chiral phosphatidylcholine and stereochemistry of phospholipase D

Abstract

Chirally labeled 1,2-dipalmitoyl-sn-glycero-3-phosphocholines (DPPC) with known configuration were synthesized by N-methylation of chirally labeled 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). Transphosphatidylation of (RP)- and (SP)-[18O]DPPC catalyzed by phospholipase D from cabbage gave (Rp)- and (Sp)-[18O]DPPE, respectively, as indicated by 31P NMR analysis of [18O]DPPE. Therefore, phospholipase D catalyzes transphosphatidylation with overall retention of configration at phosphorus. The steric course of hydrolysis of DPPC catalyzed by the same enzyme was elucidated by the following procedures. Hydrolysis of (RP)-[17O, 18O]DPPC by phospholipase D gave 1,2-dipalmitoyl-sn-glycero-3-[16O, 17O, 18O]phosphate ([16O, 17O, 18O]DPPA) with unknown configuration. The latter compound was then converted to 1-[16O, 17O, 18O]phospho-(R)-propane-1,2-diol by a procedure involving no P-O bond cleavage. The configuration of the phosphoproane-1,2-diol was determined as Rp by 31P NMR analysis following ring closure and methylation. Hydrolysis of DPPC catalyzed by phospholipase D also proceeds with retention of configuration at phosphorus. The results therefore support a 2-step mechanism involving a phsophatidyl-enzyme intermediate in the reactions catalyzed by phospholipase D from cabbage.