Screening And Application Of Stereoselective Carbonyl Reductases For Synthesis Of Chiral Pharmaceutical Intermediates

Chrial alcohols contained an active hydroxy group are important building blocks in the synthesis of chiral pharmaceuticals.The improvement of the synthetic technology of chiral alcohols decided the development of chiral pharmaceuticals.Thus,developing advanced strategies for converting prochiral ketones to chiral alcohols is urgent and significantly.In this study,we screened stereoselective carbonyl reductases through gene mining,tested their substrate scope and catalytic activity,and employed them into the asymmetric synthesis of chiral pharmaceuticals intermediates.Firstly,we screened two carbonyl reductases-producing strains from soil samples and our laboratory data,Burkholderia gladioli ZJB12126 and Rhodosporidium toruloides ZJB2014212 respectively.A tool-box of carbonyl reductases contained 48 carbonyl reductases was constructed through genome hunting and data mining.These recombinant enzymes were investigated for their substrate scope,and finally three carbonyl reductases,BgADH2,BgADH3,and RtSCR9 respectively,exhibited high activity and stereoselectivity as well as wide substrate specificity were selected as the target enzymes for further study.In the second part,BgADH2,BgADH3,and RtSCR9 were purified by one-step affinity chromatography and biochemically characterized.The molecular weights of BgADH2,BgADH3,and RtSCR9 were 114.2 kDa(tetramer),63.4 kDa(dimer),and 123.7 kDa(tetramer)respectively.The optimum pH of BgADH2,BgADH3,and RtSCR9 were 6.5,6.5,and 7.0.The optimum temperature of BgADH2,BgADH3,and RtSCR9 were 35 ?,40 ?,and 35 ?.Half-lives at 35 ? were 17 h,85 h,and 113 h.The deactivation energy were 1.27 kJ/mol,10.6 kJ/mol,and 14.6 kJ/mol respectively.Few metal ions enhanced the activity of these three enzymes,while EDTA has an insignificant effect on the activity.Therefore,BgADH2,BgADH3,and RtSCR9 belong to the non-metal dependent enzymes.The K_m of BgADH2 and RtSCR9 toward NADPH exhibited20-200 fold lower than that against NADH,suggesting the NADPH dependency of BgADH2 and RtSCR9.In contrast,The K_m of BgADH3toward NADPH slight lower than that against NADH,indicating that BgADH3 favor the NADPH.In the third part,asymmetric synthesis of(2S,3R)-methyl-2-benzamideo-methyl-3-hydroxyl-butyrate(MBHB)using recombinant BgADH2 coupled with glucose dehydrogenase/glucose cofactor regeneration.The stereoselective determination of BMOB and MBHB by chiral HPLC was first established on Chiralpak AY-H column.The elution order of isomers was then identified.Using water-toluene biphasic system,60 mM BMOB was completely converted in 8 h at 35 ? and pH 6.5,resulting in 55.1 mM of(2S,3R)-MBHB(yield 91.1%)with99.0%ee and 98.5%de,higher than so far reported.The reaction mechanism and molecular basis of BgADH2 toward BMOB was further predicted.The C=O oxygen of(2S)-BMOB forms hydrogen bonds with both Try151 and Ser138 and it is protonated from Try151-OH,followed by the attack of a hydride from C4-NADPH toward the C=O carbon of(2S)-BMOB.In the fourth part,the BgADH3 was co-expressed in tandem with glucose dehydrogenase,resulting in E.coli/BgADH3-GDH,and subjected it to the asymmetric synthesis of ethyl(R)-4-chloro-3-hydroxybutyrate(CHBE).Different parameters of protein expression were evaluated,IPTG0.5 mM,28 ? and 12 h was considered as the optimal induction condition.The BgADH3 production exhibited 55.8 U/g wet cells,and the GDH production was 66.5 U/g wet cells.The OD₆₀₀ of E.coli/BgADH3-GDH reached 36.6 through fermentation,and the enzymatic activity of BgADH3and GDH were 2347.1 U/L and 2874.2 U/L respectively.Organic solvents were further investigated for the aymmetric reduction,octanol was finally selected in the biphasic system.In 1 L aqueous/octanol biphasic system,as much as 2400 mM of COBE was fully reduced within 20 h by using substrate fed-batch strategy to afford(R)-CHBE with a yield of 91.8% in 99.9%ee.Finally,chemoenzymatic enantioselective synthesis of duloxetine using RtSCR9 was developed.We initially synthesized five thiophene-ring-containing ketones,including 3-(dimethylamino)-1-(2-thienyl)-1-propanone hydrochloride 2a 3-(methylamino)-1-(2-thienyl)-1-propanone hydrochloride 2b,3-chloro-1-(2-thienyl)-1-propanone 2c,3-oxo-3-(2-thienyl)propionic acid ethyl ester 2d,and 3-oxo-3-(2-thienyl)propanenitrile 2e as well as the corresponding racemic alcohols 3a-3e.The RtSCR9 displayed high catalytic activity and excellent enantioselectivity(>99%)toward these ketones.Then,co-expressed strain E.coli/RtSCR9-GDH was constructed haboring RtSCR9 and GDH genes and employed into the asymmetric reduction of 2a to(S)-3a.The optimal conditions involved glucose concentration 6 mmol/(mmol 2a),NADP⁺0.75?mol/(mmol 2a),30 ?,pH6.5,cell dosage 10 g DCW/L,and substrate loading 1000 mM.At a 200-g scale,1000 mM 2a was fully converted,providing(S)-3a in a yield of 92%with 99.9%ee and an space-time yield per gram of biomass(DCW)of 22.9mmol/(L·h·gDCW).From 2-acethylthiophene,an effective chemoenzymatic route is achieved in four steps,shorter than the original pathway(six steps),and the overall yield was 60.2%with 98.5%ee.