Chiral Resolution Of Intermediates Of Ticagrelor And Ezetimibe Using Lipase As Catalyst

Biocatalysis of chiral intermediates using lipase as catalyst offers an excellent technology to prepare the single-isomer chiral drugs and more and more people turn their attention from chemical resolution to biocatalysis.Burkholdria cepacia lipase A (BCLA) and Candida antarctica lipase B (CALB) were widely used in resolution of enantiomers, due to their enantio-, chemo- and regio-selective properties. Lipase have been increasingly applied to the synthesis of a wide variety of organic compounds replacing traditional chemical processes, so we researched the chiral resolution of intermediates of ticagrelor and ezetimibe using lipase as catalyst.In this study, the gene encoding Burkholdria cepacia lipase A (BCLA) was cloned into pET-28(a+), which was expressed as an infusion protein in Escherichia coli. The enzyme activity from these inclusion bodies was recovered by solubilization, purification and refolding with the assistant of a lipase-specific foldase-BCLB. Using intermediate 3 of ticagrelor as the typical substrate to optimize reaction conditions, it turned out that the highly optimal temperature of BCLA was 60℃ and the optimum pH was 8.0. Interestingly, the enzyme activity was significantly increased for 1.1- and 1.27-fold in the presence of Co2+ and Mn (1mM). Burkholderia cepacia lipase A (BCL-A) was used to catalyze the asymmetric hydrolysis reaction under the optimal condition. We found it successly give (1S, 4R)-N-(benzylcarbamoyl)-4-aminocyclopent-2-en-l-ol in>99% enantiomeric excess (ee) with conversion yield up to 45.7%.Besides, the enantiomer of optically pure (S)-5-(4-Fluoro-phenyl)-5-hydroxy-pentanoic acid [(S)-FOA alcohol] which was an important chiral building block in the syntheses of Ezetimibe. In this research, several 5-(4-Fluoro-phenyl)-5-oxo-pentanoic acid (FOA ketone) derivatives have been investigated, in which Candida antarctica lipase B (CALB) was used to catalyze the asymmetric hydrolysis reaction in aqueous media. As a result, only rac-1 successfully gave (S)-5-(4-Fluoro-phenyl)-5-hydroxy-pentanoic acid in>99% enantiomeric excess (ee) with the yield 46.7%.At the same time, we first found that the enantioselective ring-opening alcoholysis reaction of rac-1 can proceed with different alcohols using Candida antarctica lipase B as catalysis, which means CALB is of great value in the application to industrial production.