On the specificity of rat-liver lysophospholipase

1. 1. A study on the specificity of rat-liver lysophospholipase activity (EC 3.1.1.5) revealed that both 1-acyl-sn-glycero-3-phosphorylcholine and 2-acyl-sn-glycero-3-phosphorylcholine are deacylated. From both positional isomers the unsaturated analogs appeared to be degraded at higher rates. 2. 2. Circumstantial evidence is presented indicating that 2-acyl-sn-glycero-3-phosphorylcholine can be attacked directly by this lysophospholipase activity without a prior migration of the fatty acyl constituent. 3. 3. Compounds lacking the free hydroxyl group present in lysophosphatidyl-cholines, e.g. acyl-ethylene glycolphosphorylcholine and 1-acyl-propane diol-3-phosphorylcholine, also fall in the enzyme's range of specificity. 4. 4. Mono-acyl derivatives of sn-glycero-1-phosphorylcholine, sn-glycero-2-phosphorylcholine, as well as sn-glycero-3-phosphorylcholine, were found to be degraded. 5. 5. Inhibition of lysophospholipase activity by various agents exhibited the same effect on the deacylation of both 1-acyl- and 2-acyl-sn-glycero-3-phosphoryl-choline. 6. 6. The degradation of mono-acyl-phosphatidylcholine appeared to be strongly inhibited in the presence of phosphatidylcholine

On the synthesis of plasmalogens

The chemical synthesis is described of (rac)-trans-1-(n-hexadec-1′-enyloxy)-2-oleoylglycerol-3-phosphorylcholine (plasmalogen). This synthesis made use of a specific degradation of (rac)-trans-1-(n-hexadec-1′-enyloxy)-2,3-dioleoyl glycerol with pancreatic lipase (EC 3.1.1.3). This enzyme, which catalyses the hydrolysis of fatty acid ester bonds attached to the primary hydroxyl groups of glycerol, cannot split vinyl ether linkages, thereby yielding (rac)-trans-1-(n-hexadec-1′-enyloxy)-2-oleoyl glycerol. The latter compound was converted into a plasmalogen by a reaction with 2-bromoethyl-phosphoric acid dichloride and trimethylamine. A partial synthesis of cis-1-(n-alk-1′-enyloxy)-2-oleoyl-glycerol-3-phosphorylcholine was developed by application of this method to cis-1-(n-alk-1′-enyloxy)-2,3-dioleoyl glycerol. The preparation of cis-1-(n-alk-1′-enyloxy)-2,3-dioleoyl glycerol was made by acylation of cis-1-(n-alk-1′-enyloxy)glycerol obtained from ox-heart plasmalogen after degradation with phospholipase C (EC 3.1.4.3) and alkaline hydrolysis. The I.R. spectra of both plasmalogens were completely identical with each other and differed from the spectra of lecithins only by the presence of a vinyl ether absorption at 1660 cm−1. The N.M.R. spectrum of the acetylated synthetic (rac)-1-(n-hexadec-1′-enyloxy)glycerol as well as of the synthetic plasmalogen revealed a trans configuration of the vinyl ether linkage. Degradation with phospholipase A (EC 3.1.1.4) of the synthetic (rac)-plasmalogen gives a 50% conversion into free fatty acid and trans-1-(n-hexadec-1′-enyloxy)glycerol-3-phosphorylcholine while the partial synthetic enantiomeric plasmalogen was converted completely by this enzyme. The same degree of conversion was observed when both plasmalogens were degraded with phospholipase C to (rac)-trans-1-(n-hexadec-1′-enyloxy)-2-oleoyl glycerol respectively cis-1-(n-alk-1′-enyloxy)-2-oleoyl glycerol. When these plasmalogenic diglycerides were incubated with pancreatic lipase no further breakdown was observed indicating the absence of positional isomers. Both plasmalogens could be transformed by phospholipase D (EC 3.1.4.4) into 1-(n-alk-1′-enyloxy)-2-oleoyl-glycerol-3-phosphate. Purified pancreatic lipase did not degrade the plasmalogen

On the synthesis of plasmalogens

The chemical synthesis is described of (rac)-trans-1-(n-hexadec-1′-enyloxy)-2-oleoylglycerol-3-phosphorylcholine (plasmalogen). This synthesis made use of a specific degradation of (rac)-trans-1-(n-hexadec-1′-enyloxy)-2,3-dioleoyl glycerol with pancreatic lipase (EC 3.1.1.3). This enzyme, which catalyses the hydrolysis of fatty acid ester bonds attached to the primary hydroxyl groups of glycerol, cannot split vinyl ether linkages, thereby yielding (rac)-trans-1-(n-hexadec-1′-enyloxy)-2-oleoyl glycerol. The latter compound was converted into a plasmalogen by a reaction with 2-bromoethyl-phosphoric acid dichloride and trimethylamine. A partial synthesis of cis-1-(n-alk-1′-enyloxy)-2-oleoyl-glycerol-3-phosphorylcholine was developed by application of this method to cis-1-(n-alk-1′-enyloxy)-2,3-dioleoyl glycerol. The preparation of cis-1-(n-alk-1′-enyloxy)-2,3-dioleoyl glycerol was made by acylation of cis-1-(n-alk-1′-enyloxy)glycerol obtained from ox-heart plasmalogen after degradation with phospholipase C (EC 3.1.4.3) and alkaline hydrolysis. The I.R. spectra of both plasmalogens were completely identical with each other and differed from the spectra of lecithins only by the presence of a vinyl ether absorption at 1660 cm−1. The N.M.R. spectrum of the acetylated synthetic (rac)-1-(n-hexadec-1′-enyloxy)glycerol as well as of the synthetic plasmalogen revealed a trans configuration of the vinyl ether linkage. Degradation with phospholipase A (EC 3.1.1.4) of the synthetic (rac)-plasmalogen gives a 50% conversion into free fatty acid and trans-1-(n-hexadec-1′-enyloxy)glycerol-3-phosphorylcholine while the partial synthetic enantiomeric plasmalogen was converted completely by this enzyme. The same degree of conversion was observed when both plasmalogens were degraded with phospholipase C to (rac)-trans-1-(n-hexadec-1′-enyloxy)-2-oleoyl glycerol respectively cis-1-(n-alk-1′-enyloxy)-2-oleoyl glycerol. When these plasmalogenic diglycerides were incubated with pancreatic lipase no further breakdown was observed indicating the absence of positional isomers. Both plasmalogens could be transformed by phospholipase D (EC 3.1.4.4) into 1-(n-alk-1′-enyloxy)-2-oleoyl-glycerol-3-phosphate. Purified pancreatic lipase did not degrade the plasmalogen

Synthesis of lysophosphoglycerides

The chemical syntheses of 2-stearoyl-glycerol-1-phosphorylcholine, 2-[9,10–3H]-stearoyl- glycerol-3-phosphorylcholine, (rac)-1- and 2-stearoyl-glycerol-3-phosphorylcholine, (rac)-1- and 2-stearoyl-glycerol-3-phosphoryl-(N,N-dimethyl)ethanolamine and (rac)-1- and 2-stearoyl-glycerol-3-phosphate are described. These lysophosphoglycerides were prepared using (rac)-isomeric-0-benzyl-stearoyl-glycerol-3-(benzyl)phosphates as starting products. Hydrogenolysis of this latter compounds yielded the lysophosphatidic acids while reaction of the silver salts of the starting products with 2-bromo-ethyl N,N-dimethylamine picrate or 2-bromo-ethyl trimethylammonium picrate finally led to the isomeric N,N-dimethyl lysophosphatidyl ethanolamines and lysolecithins respectively. The (rac)-isomeric-0- benzyl-stearoyl-glycerol-3-phosphoryl-(N,N-dimethyl)ethanolamines could easily be converted with methyl iodide into the corresponding lecithins. Stereospecific degradation of (rac)-1-0-benzyl-2-stearoyl-glycerol-3-phosphorylcholine with phospholipase A (EC 3.1.1.4) gave 1-0-benzyl-glycerol-3-phosphorylcholine leaving 3-0-benzyl-2-stearoyl- glycerol-1-phosphorylcholine unhydrolysed. The latter product yielded upon hydrogen-olysis 2-stearoyl-glycerol-1-phosphorylcholine. The first product viz. 1-0-benzyl-glycerol- 3-phosphorylcholine was acylated with [9,10–3H]-stearoyl chloride and after removal of the protecting benzyl group 2-[9,10–3H]-stearoyl-lysolecithin was obtained. Infrared spectra of the synthesized lysophosphoglycerides are reported. Some general properties of the lyso compounds e.g. their behaviour towards phospholipase A and their stability are discussed