Mechanistic and stereochemical investigation of fatty acid and polyketide biosynthesis using chiral malonates

Article abstract of DOI:10.1016/S0040-4020(01)86493-0

Chiral malonates, (R)-[1-¹³C;2-²H]malonate and (S)-[1-¹³C;2-²H]malonate, were synthesised and characterised as malonyl-CoA derivatives with yeast fatty acid synthase using mass spectrometric analysis of the palmitic acid produced. The chiral malonates were used to investigate the steric course of fatty acid synthase from rat liver and the fatty acid synthase and 6-methylsalicylic acid synthase from Penicillium patulum. Malonic acid, activated in the form malonyl-CoA, is a key building block for the biosynthesis of many naturally occurring compounds including fatty acids, flavanones and polyketides. The methylene carbon atom of the malonyl-CoA, with its two hydrogen atoms, is of particular interest since it is at this position that many of the crucial mechanistic events occur during the assembly of these compounds. Detailed stereochemical information about the fate of the hydrogen atoms at this position during the incorporation of malonyl-CoA into these natural products is thus an essential requirement for the understanding of the mechanism and steric course of the enzymic steps involved. This problem was originally addressed by elegant studies in which the tritiated enantiomers of malonyl-CoA were synthesised and used to elucidate the mechanism of yeast fatty acid synthase. Difficulties encountered in this work with the extreme lability of the methylene protons suggested that the use of malonyl-CoA derivatives had severe limitations. This prompted us to devise an alternative strategy based on the synthesis of the more stable chiral malonates although this approach, as will be clear, introduces cryptic stereochemistry which complicates the analytical methodology. This stems from the fact that malonic acid exhibits pro-pro-chiral stereochemistry, having the structure Ca₂b₂. Thus even if the malonic acid is made chiral, by labeling the paired constituents isotopically with ¹³C and ²H in the same molecule, the labeled groups will remain essentially indistinguishable to an enzyme. Therefore each chiral malonate will yield a unique pair of malonyl-CoA derivatives, even when activated enzymically. Once formed, each of the malonyl-CoA derivatives will be recognised as a pro-chiral molecule by an enzyme and the labeled substituents will be manipulated stereospecifically. Such aspects have been discussed thoughtfully elsewhere. This paper discusses the synthesis of (R)-[1-¹³C;2-²H]malonate and (S)-[1-¹³C;2-²H]malonate and their use to study the mechanism and stereochemistry of fatty acid synthases and the polyketide synthase 6-methylsalicylic acid synthase.