Measuring stereoselectivity in lipase-catalyzed acidolysis reactions by ultra-high resolution 13C nuclear magnetic resonance

Elucidating the stereoselectivity of lipases in synthetic reactions of triacylglycerols has hitherto been carried out using traditional analytical techniques to determine the composition of the reaction products. These methods are laborious and are not always appropriate for analysis of certain triacylglycerol types. A direct method, utilizing a stereospecific deuterium-labeled triacylglycerol substrate, has been developed where the stereoisomeric composition of the reaction product is determined by ultra-high resolution C-13 nuclear magnetic resonance (NMR) spectroscopy. Through lipase-catalyzed transesterification of deuterium-labeled trilauroylglycerol with oleic acid, chemical shifts were induced in the 13C NMR spectrum by the deuterium atom and olefinic double bonds, enabling unambiguous stereospecific assignment of triacylglycerol species. By this method of analysis, we found an effect of the degree of reaction conversion on the extent of stereoisomerism in the triacylglycerol product. Stereoselectivity was greatest (for sn-1) with lipase from Rhizomucor miehei. Lipases from Rhizopus niveus, Candida rugosa, Carica papaya, and the cutinase from Fusarium sp. were also found to exhibit stereoselectivity, with preference for either sn-1 or sn-3 acyl exchange.