Semi-Rational Design Of Carbonyl Reductases And Synthesis Of Montelukast Sodium/Ezetimibe Intermediate

Montelukast sodium is the leukotriene receptor antagonist drugs for the treatment of asthma. Ezetimibe is a novel cholesterol absorption inhibitor. Currently the demand for these two chiral drugs is rising. Chiral alcohol is an important cornerstone in the synthesis of chiral drugs. In recent years, domestic and international scientists are working on asymmetric reduction of chiral alcohols using biocatalysts. Natural biocatalyst exhibited better activity and selectivity on natural substrates. However, most drugs are non-natural substrates, researchers have focused on the modification of the enzyme to improve the activity, stability and selectivity. In this paper, site-directed mutagenesis of the carbonyl reductase （kred） was performed. In addition, we used a new catalyst in the synthesis of Ezetimibe chiral intermediates.The carbonyl reductase （kred） were inserted into the pET22b and pPIC9K, then expressed protein in Escherichia coli and Pichia pastoris, resp-ectively. The crude enzyme was used as biocatalysts for asymmetric reduction of（E）-methyl-2-（3-（3-（2-（7-chloro-quinolin-2-yl）vinyl）phenyl）-3-oxopropyl）benzoate.Homology modeling of kred using swiss-model was built with the R alcohol dehydrogenase （SMTL id Izk4.1） selected as a template. The coenzyme and substrate binding pockets were analysed. Based on the structural characteristics of the enzyme, site-directed mutagenesis was performed on the enzyme.We firstly synthsized the ezetimibe chiral intermediates using E. coli whole cells as biocatalysts. The product is （4S）-3-[（5S）-5-（4-fluoro phenyl）-5-hydroxy-pentanoyl]-4-phenyl-1,3-oxazolidin-2-one in 99.99% enantiomeric excess （ee） with the yield up to 70%. Characterization results showed that the optimal reaction temperature is 35℃, optimal pH is 7. Ca²⁺, Fe²⁺, K⁺, Mn²⁺ promoted activity and Mg²⁺, Zn²⁺, Cu²⁺, Fe³⁺ inhibited activity.