Asymmetrically Biocatalytic Synthesis Of Chiral Intermediate Of Sitagliptin

Enantiopure alcohols are important chiral building blocks for pharmaceuticals.The effective method for synthesis of single enantiopure alcohols is becoming increasingly attractive,and the asymmetric bioreduction of prochiral ketones to chiral alcohols is one of the most promising processes.?S?-3-hydroxy-1-?3-?trifluoromethyl?-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7?8H?-yl?-4-?2,4,5-trifluorophenyl?butan-1-one??S?-HTPP?is a key chiral intermediate of Sitagliptin,a leading drug for the treatment of patients with type 2 diabetes.This research mainly aims to develop an effective green process for the production of?S?-HTPP.And in the present study,we have found a novel potent biocatalyst suitable for the biocatalytic process,and the research aboout process optimization was also successfully conducted.The newly isolated soil strain of YHB3-10 was found to enantioselectively reduce prochiral ketone?2Z?-4-oxo-4-[3-?trifluoromethyl?-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7?8H?-yl]-1-?2,4,5-trifluorophenyl?butan-2-one?OTPP?to its corresponding chiral alcohol?S?-HTPP with the optical purity above 99%?ie,ee>99%?.Based on its morphological characteristics,along with partial 16S rDNA sequence analysis,the strain was identified as Pseudomonas aeruginosa.This is the first report about P.aeruginosa that contained S-stereospecific carbonyl reductase capable of reducing OTPP to?S?-HTPP.The culture conditions of Pseudomonas aeruginosa YHB3-10 for the production of crucial carbonyl reductase were optimized by using a one-at-a-time method and a response surface methodology of Box-Behnken Design?BBD?.The optimal medium composition and culture conditions were as follows:maltose 30.45 g/L,peptone 17.31 g/L,NaCl 0.98 g/L,initial fermentation pH of 7.5;8%?v/v?inoculum,90 mL of culture volume in 250 mL Erlenmeyer flask,30°C,200 rpm,cultivation for 28 h.Under the above optimized conditions,the enzyme activity and biomass reached 0.35 U/g_cellell and 2.65 g/L,respectively;the product ee kept above 99%.Subsequently,the biocatlysis in aqueous reaction system was studied.Some key reaction parameters involved in the bioreduction catalyzed by whole cell of Pseudomonas aeruginosa YHB3-10 were also optimized.The results indicated that under the optimal conditions,that is,buffer pH7.5?0.1 M?,112 g_DCW/L of cell concentration,6%?w/v?of glycerol as cosubstrate,40°C,4 g/L?10 mM?of OTPP was bio-reduced to?S?-HTPP with a yield of 91.49%within 24 h.Finally,the bioreduction process was performed in aqueous/organic solution and aqueous/ionic liquid media.The results indicated that the yield varied much,whereas the ee value kept above 99%.Among nine kinds of organic media which include glycerol,two surfactants and six usual organic solution,5%?v/v?of dibutyl phthalate addition gave the best performance with a yield of 75.61%from 9 g/L of OTPP within 24 h.As for sixteen kinds of aqueous/ionic liquid media,the addition of 0.5%?w/v?choline chloride/glycine?ChCl/Gly?could improve the yield to70.11%.As the control,the bioreduction conducted in aqueous buffer solution just obtained a yield of 57.41%.