Asymmetric Synthesis Of Key Intermediate Of Duloxetine By Biocatalytic System Involving Recombinant Carbonyl Reductase

Optically active alcohols are valuable and promising chiral building blocks widely employed in the production of pharmaceuticals.Biocatalysis has become a green way to synthesize chiral alcohols due to its exquisite regio-and enantioselectivity.In this study,for the synthesis of(S)-N,N-dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine(DHTP),the important intermediate of duloxetine,the target enzyme carbonyl reductase CR2 was selected from a variety of recombinant carbonyl reductases generated from different microorganisms to catalyze asymmetric reduction of 3-(Dimethylamino)-1-(thiophen-2-yl)propan-1-one(DTP).Then the CR2 cell-free system was constructed and the relevant reaction conditions were optimized.To further improve the catalytic efficiency of NADPH-dependent CR2,the fusion enzyme CR2-Linker-GDH was constructed for asymmetric reduction of DTP.Consequently,(S)-DHTP(>99.9% e.e.)with yield of 55.66% was obtained from 20 g·L-1 DTP within 8 h reaction.Then the process of the asymmetric reaction of DTP catalyzed by CR2-Linker-GDH was studied,and the product yield was further improved when regulation of pH and fed-batch strategy were employed during the reaction.Among the available recombinant carbonyl reductases,CR2 and KRD exhibited the catalytic activity for asymmetric synthesis of(S)-DHTP from DTP.Compared to KRD,CR2 showed higher catalytic performance and was selected as the functional enzyme of this study.Then the CR2-involved cell-free catalytic system was constructed and the related enzymatic catalytic conditions were optimized.The optimum initial reaction pH was pH 8.0 and the optimum reaction temperature was 30 °C.Various co-substrates for cofactor regeneration were imposed in the asymmetric reduction reaction and glucose gave the most positive effect.The effects of concentrations of glucose and cofactor on enzymatic reaction were investigated.The results showed that the yield of(S)-DHTP was increased when the concentration of glucose and coenzyme was 120 g·L-1 and 0.1 mmol·L-1,respectively.Consequently,(S)-DHTP(>99.9% e.e.)with yield of 61.70% was obtained from 10 g·L-1 DTP within 8 h reaction,employing 120 g·L-1 glucose and 0.1 mmol·L-1 NADP+ to drive the cofactor regeneration under 30 °C and pH 8.0.To enhance cofactor regeneration and subsequent(S)-DHTP production,the fusion enzyme CR2-Linker-GDH involving NADPH-dependent CR2 and glucose dehydrogenase(GDH)was constructed.Kinetic parameters indicated the comparable affinities of CR2-Linker-GDH towards DTP and glucose.By using the CR2-Linker-GDH cell-free system,the concentration of DTP was improved from 10 g·L-1 to 20 g·L-1 in the reaction.After optimization of the reaction conditions,optimum initial reaction pH and reaction temperature were obtained as pH 8.4 and 40 °C,respectively.Otherwise,40 g·L-1 of glucose and 0.1 mmol·L-1 of NADP + were applied to further improve the production yield.Consequently,the developed CR2-Linker-GDH catalyzing system was achieved involving 20 g·L-1 DTP,40 g·L-1 glucose,and 0.1 mmol·L-1 NADP+,and under the optimized catalytic conditions at 40 °C and pH 8.4,optically pure(S)-DHTP(>99.9%e.e.)with the yield of 55.66% was obtained with increased substrate concentration of 20 g·L-1 DTP after reaction for 8 h,using the fusion enzyme CR2-Linker-GDH,giving a space-time yield of 1.39 g·L-1·h-1.The reaction process of asymmetric reduction catalyzed by CR2 and CR2-Linker-GDH were tracked,respectively.It was found that p H value of the reaction system decreased significantly during the reaction process.In order to regulate the pH of the reaction system,pH automatic control system was used to adjust pH value of the system and the production yield of the asymmetric synthesis was improved.On the other hand,reaction process of asymmetric reduction of DTP at different concentrations was investigated and substrate inhibition was obviously observed for the fusion enzyme system.In order to relieve substrate inhibition,fed-batch strategy was employed in the reaction.Substrate and glucose were both fed at intervals of 2 h for 4 times.Consequently,by conducting reaction pH regulation and fed-batch simultaneously in 100 mL reaction system,the catalytic efficiency of the enzyme was further improved and optically pure(S)-DHTP(>99.9%e.e.)was synthesized with the yield of 71.86% from 20 g·L-1 DTP.