Uzawa Hirotaka

Determination of the lipase stereoselectivities using circular dichroism (CD); lipases produce chiral di-O-acylglycerols from achiral tri-O-acylglycerols

Abstract A general method was described to determine the optical purity of 1,2 (or 2,3)-di-O-acylglycerols via a key compound, 3 (or dibenzoyl-sn-glycerol (3 or 3). The chiral di-O-acylglycerols were first silylated and the acyl groups were removed by the Grignard degradation to 3 (or 1) O-tert-butyldimethylsilyl-sn-glycerol and subsequent benzoylation lead to the key compound 3 or 3 without racemization. The optical purity was determined from the strong exciton Cotton effect of 3 (+) or 3 (−) at 238 nm in the concentration of ca. 1 mM. The method was successfully applied to determine the stereoselectivities of lipases (EC 3.1.1.3) from three origins, bacteria, mammal and fungus such as Pseudomonas (AP, 89% optical purity, sn-1 preference), porcine pancreatin (PPL, 9.3% optical purity, sn-3 preference) and Candida (CC, sn-2 preference) using tripalmitin. The similar studies were extended to tri-O-benzoylglycerol (6) and tri-O-(cyclohexanecarbonyl)glycerol (5). All the enzymes showed high stereoselectivities with tri-O-benzoylglycerol. PPL and AP showed high and low stereoselectivities with tri-O-(cyclohexanecarbonyl) glycerol, while low and high stereoselectivities with tri-O-palmitoylglycerol, respectively. The results show that the stereoselectivities are ruled by the origins of lipases and acyl groups. The structures of the recognition site might be associated with enantioselectivities of the enzymes.

A convenient evaluation of the stereoselectivity of lipase-catalyzed hydrolysis of tri-O-acylglycerols on a chiral-phase liquid chromatography

Abstract A general method was developed using chiral-phase chromatography in order to evaluate the stereoselectivities of lipases-catalyzed hydrolysis of tri-O-acylglycerols independent of acyl groups. 1,2-Di-O-acyl-sn-glycerols or its enantiomer 2,3-di-O-acyl-sn-glycerols in the enzymatic reaction mixtures were derivatized to the key compound, l,2-di-O-benzoyl-3-O-tert-butyldimethylsilyl-sn-glycerol 2 - (+) or its enantiomer 2 - ′ (-) , respectively. The enantiomers were separated on a chiral-phase HPLC, and the method was highly sensitive to determine the stereoselectivities of upases.