Application Of Lipase BCLA And BCLC In Chiral Resolution Of Intermediates Of Vorapaxar And Other Chiral Drugs

In the synthesis process of chiral durg,lipase as a effective bioiogical catalyst was widely used in resolution of enantiomers,due to the properties of efficient,convenience,high purity and yield.Correlational research about mechanism of Burkholdria cepacia lipase is nearly clear,and it has a widely substrate range.More and more people turn their attention to the lipase application of synthesis of chrial compouds,instead of traditional chemical process.In this study,cultivate the genetically engineered E.coli bacteria of Burkholdria cepacia lipase A(BCLA)and chaperone lipase B(BCLB)which were saved in our laboratory,inducible expression respectively.But BCLA was expressed as an infusion protein,so after the process of BCLA purification,denaturation,renaturation under the auxiliary of BCLB,which can help BCLA correctly folding,we get the soluble protein with enzyme activity.Apply it to the chiral resolution of Vorapaxar intermediate(4-acetyloxy-N,N-diphenyl-2-pentynamide),and optimize the reaction conditions.It turned out that the optimal reaction condition was 60 ℃,pH 7,reaction time 4 h,enzyme content 0.014 mg.The enzyme activity reduced in the presence of metal irons,especially Li+ was only 53%compare to the control.In the research of two-phase reaction,we found that no solvents enhanced the enzyme activity.Under the optimal condition,we get the product of(4R)-4-hydroxy-N,N-diphenyl-2-pentynamide,and the enantiomeric excess(ee)of substrate in 94.5%with the recovery percent of 42.86%.We discovered a lipase gene by sequence-structure guided genome mining from genome of Burkholderia cepacia ATCC 25416,named BCLC.Cloned it into different vector to construct genetic engineered strain,and research the activities of varieties substrates of ester.The results truned out it has split activity to vorapaxar intermediate 4-acetyloxy-N,N-diphenyl-2-pentynamide,and the two Ezetimibe intermediates 6-(4-Fluoro-phenyl)-tetrahydro-pyran-2-one,methyl-5-(4-fluorophenyl)-5-oxopentanoate with the enantiomeric excess(ee)78.49%,44.28%,60.86%respectively.