Excavation Of Novel Ketoreductases And Its Application Research In Synthesis Of (S)-1-(2,6-Dichioro-3-Fluorophenyl) Ethanol

Chiral alcohols are important intermediates for the synthesis of chiral drugs, flavors, agricultural chemicals and other important substances. Ketoreductases ?KRED? could obtain chiral alcohols by asymmetric hydrogenation reduction of carbonyl compounds. In recent years, the biocatalysis method for preparing chiral alcohols has been more and more widely used. However, many biological catalysts have shortcomings of low catalytic activity, product concentration, and selectivity. Therefore, except to modify existing enzymes, discovering new KRED is still a very important approach to solve the problem. ?S?-1-?2,6-dichloro-3-fluorophenyl? ethanol is a key chiral alcohol intermediate of crizotinib, and has important commercial value. To date, there are two methods which are chemical synthesis and biocatalysis to producing the chiral alchol in market. Between them, the problems existing in the chemical synthesis are that chemical catalyst is usually expensive, the reaction conditions are harsh, and the catalytic efficiency and ee value are not high. The problems existing in the biocatalysis are that the operation steps are tedious, the purification process is complex, and the final yield is not high. So, it has a very important practical value to establish the enzymatic preparation technology of ?S?-1-?2,6-dichloro-3-fluorophenyl? ethanol. The main content of this thesis is about the excavation of novel KRED from soil metagenome and the enzymatic preparation technology of ?S?-1-?2,6-dichloro-3-fluorophenyl? ethanol.According to the results of sequencing and database annotations, we discovered three KRED ?KRED424, KRED432, and KRED433? from soil metagenome. Then, they were cloned, heterogeneously expressed and purified. We investigated their enzymatic properties. The experimental results showed that all the three KRED had the optimal activity at 55? and pH 7.0 and were more stable in alkaline environment. The KRED showed no apparent dependence of metal ions. KRED424 had a broader substrate spectrum compared with KRED432 and KRED433. For further verifying the application potential of the three KRED in asymmetric synthesis of chiral alcohols, we selected three important prochiral carbonyl compounds to evaluate the results of kinetic parameters and asymmetric reduction reaction of the KRED. For ethyl 4-chloroacetoacetate ?COBE?, KRED424 and KRED433 showed useful activity (the kcat/Km values of KRED424 and KRED433 were 94.38 M^-1S^-1 and 232.63 M^-1S^-1, respectively), and KRED433 could obtain an ?R?-alcohol with 94% ee. For ethyl 2-oxo-4-phenylbutyrate ?OPBE?, KRED424 showed a higher enantiomeric excess ?S-type,> 99% ee? than KRED432 and KRED433. For N-Boc-3-pyrrolidone, all the KRED could get an ?S?-type alcohol with an excellent enantioselectivity ?> 99% ee? suggesting their potential for preparation of chiral N-Boc-3-pyrrolidinol with S-type. All the results demonstrate that it is an effective approach to discover efficient biocatalyst from metagenome.We studied the enzymatic preparation technology of ?S?-1-?2,6-dichloro-3-fluorophenyl? ethanol. By using a concentration of 20 mM of 2,6-dichloro-3-fluoroacetophenone, we found a KRED which could convert the substrate completely and obtain an ?S?-type alcohol with an ee value of 99.9% from our enzyme library ?135 enzymes?. Then, the substrate concentration was enlarged from 100 mM,500 mM,1 M,2 M to 3 M. When the substrate concentration was 3 M, the substrate conversion rate could still reached 99.5% in 24 h. At the same time, we developed a simple product purification process. Ethyl acetate was used to extract the alcohol product. Then, a rotary evaporator was used to concentrate the chiral alchol product and the crude liquid product was obtained. Finally, by adding n-hexane with 0.5 mL/g to crystallization, a white powder product was obtained. Product parameters including HPLC purity, ee value, melting point range, and water content met the requirements of industry. The technology opened up a new approach of enzymatic synthesis of ?S?-1-?2,6-dichloro-3-fluorophenyl? ethanol and had a very high application value.