Study On Bioenzyme Catalyzed Chiral Intermedediate Of Ticagrelor

Ticagrelor is an antiplatelet drug with good market advancement.Compared with similar drugs(such as Clopidogrel),Ticagrelor can inhibit platelet faster and more strongly.(S)-2-Chloro-1-(3,4-difluorophenyl)ethanol and ethyl(1R,2R)-2-(3,4-difluorophenyl)-1-cyclopropanecarboxylate both are the key chiral intermediates of Ticagrelor,the enzymatic synthesis of these two compounds were studied separately.A novel lipase(TDL)was found in Thermomyces dupontii by gene mining.It was cloned and expressed,and the protein was purified.The results showed that the protein had a molecular weight of 30 kDa,which was consistent with the predicted value.The activity of the protein was verified.The results showed that the reaction was carried out at 30 0 C for 24 h in 1 ml of pH=7 phosphate buffer system,and 3 g/L of Thermomyces dupontii Lipase(TDL)catalyzed 1 g/L of substrate 2-(3,4-difluorobenzene).Ethyl 1-cyclopropanecarboxylate is active and exhibits an expected S selectivity,eep≥99%.The gene sequence of carbonyl reductase(Rhky-ADH)from Rhodococcus kyotonensis is obtained by gene mining.After cloning and expression,the plasmid is transformed into E.coli vector to obtain an engineered strain containing the carbonyl reductase gene sequence.Induction of expression,whole cells of genetically engineered bacteria are obtained,and the whole cell is subjected to catalytic reduction reaction of the substrate 2-chloro-1-(3,4-difluorophenyl)ethanone.The results shows that 100 mg of wet cells acted on 6 mg.The substrate is added with 0.5 g/L coenzyme NADH and reacted in 1 ml phosphate buffer system of pH=7.Rhky-ADH can catalyze the production of the target product of 2-chloro-1-(3,4-difluorophenyl)ethanone.(S)-2-Chloro-1-(3,4-difluorophenyl)-1-ethanol,conversion≥99%,eep≥99%.The Rhky-ADH whole cell catalysis conditions are optimized.The results shows that Rhky-ADH is a coenzyme NADH-dependent protein with a coenzyme concentration of 0.5 g/L;A substrate concentration of 80 g/L,a wet weight of 100 g/L,and 15%(v/v)isopropanol as a cosolvent,pH=8.0,T=25℃,showing the best reactivity,and the conversion rate is>90%.The results of-pH tolerance and temperature tolerance gold necklace shows that the activity is good at pH=7～9.After incubation for 8h at 25 ℃,the residual activity is more than 80%.It is not easy to be inactivated at normal temperature;Metal ions have little effect on enzyme activity,and Mn2+ can increase relative activity by about 8%.Rhky-ADH substrate adaptability and active sites are studied.The results show that Rhky-ADH has very good activity against acetophenone and its derivatives,and has very good stereoselectivity.When the Rhky-ADH active site is studied,it is found that Rhky-ADH has four amino acid residues Tyr,Lys,Ser and Asn,and any one of the residues is mutated to alanine,and the mutant protein loses its activity.Meet the characteristics of the short-chain dehydrogenase family.