Synthesis Of A Key Chiral Intermediate Of Sitagliptin Phosphate Catalyzed By Microbial Carbonyl Reductase

It has become attractive to synthesize chiral drugs through biotransformations. Synthesis of chiral alcohols catalyzed from prochiral ketones by microbial carbonyl reductase has become an important synthesis approach of chiral drugs and their intermediates. In this thesis, it was researched that the asymmetric reduction of 4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-1-(2,4,5-trifluorophenly)butan-2-one(2) to optically active(S)-3-hydroxy-1-(3-(trifluoro-methyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-4-(2,4,5-trifluorophenyl)bu-tan-1-one((S)-1), a chiral intermediate for the production of sitagliptin, was catalyzed by carbonyl reductase of a novel microorganism. And highly efficient and enantioselective synthesis of(S)-1 was achieved.To find carbonyl reductase-producing microbial strains capable of enantioselectively reducing prochiral ketone 2 to chiral alcohol(S)-1, the classified screening approach using acetophenone as the sole carbon source was adopted. Of 89 soil isolates from 125 soil samples tested for their reductive capabilities on acetophenone, only 24 were able to reduce 2 to 1. Among them, the strain 40-1 was selected for further study, due to its excellent enantioselectivity and good conversion. This strain was designated as Pseudomonas pseudoalcaligenes XW-40 based on classic phenotypic informations and 16 S rDNA analysis.The medium composition was optimized by single factors and response surface methodology. The optimized medium and fermentation conditions were as follows: fructose 13 g/L,beef extract 11.2 g/L,NaCl 4.6 g/L,peptone 28 g/L, MgSO4?7H2O 1.2 g/L,KH2PO4 1.0 g/L, initial pH 7.0, shaker speed 180 r/min and fermentation period 36 h, respectively. Under optimized conditions, the enzyme activity reached 326 U/g(dry weight cell). Compared with the initial medium, it enhanced 23%.The effects of bioreduction conditions were also studied. The optimized reduction conditions were as follows: 80 g/L dry weight cells, 10 g/L substrate concentration, 5%(w/v)glucose as co-substrate, 10%(v/v) DMSO as cosolvent, pH 7.0, 30 °C, 180 r/min, 24 h. The product yield reached >99% and the ee value remained >99%. Furthermore, reusability of whole cells for the bioreduction was established to further improve the production of(S)-1, and the yield and ee of product retained 45% and >99%, respectively, after five cycles. Finally, a total yield of 37.4 g/L(S)-1 was obtained using the same batch cells.In order to enhance the substrate concentration and relieve the substrate and product inhibitions, in situ resin adsorption technology was used. The substrate was absorbed into macroporous resin, XAD-7, and it was then added into reaction system. With this method, the initial concentration of substrate increases from 10 g/L to 25 g/L. The yield and ee of product reached 90.5% and>99%, respectively, after 45 h.To improve the stability of the biocatalyst, alginate was used to immobilize the resting cells of Pseudomonas pseudoalcaligenes XW-40. The alginate immobilization preparation conditions were set as follows: 1.5%(w/v) alginate, 16%(w/v) cell concentration, and 3%(w/v) calcium chloride, 6 h of immobilization time. With 10 g/l substrate, the product yield reached 99.8% by immobilized cell after 35 h and the ee value remained >99%.