Catalytic Synthesis Of Chiral Epoxides By Agromyces Mediolanus Epoxide Hydrolase

Chiral phenyl oxirane(SO)and chiral benzyl glycidyl ether(BGE)are important precursors for the synthesis of many drugs.For example,(S)-SO can be used as a raw material for the synthesis of phenylcyclopropylamine structure for the synthesis of anti-tumor,anti-depression and treatment of acute coronary syndrome.(R)-BGE is an important precursor of synthetic antitumor drugs SPIKET-P and lychee lactone,and has important value in anti-leukemia.(S)-BGE is an important structural part of many immunosuppressants and can be used to synthesize the natural product diacetate saffron.The drug has important research value in the treatment of headache,morning sickness and anti-cancer and even in the synthesis of algal pheromone precursors.According to the literature,the gene sequence of Agromyces mediolanus epoxide hydrolase(EH-Am)was synthesized and introduced into E.coli plasmid for expression.The optimal expression conditions were obtained by single factor optimization: the inducer concentration was 0.2 mM,the induction temperature was 28 °C,and the induction time was 8 h.The enzymatic properties of EH-Am were studied and found to have an optimum pH of 8.0 and an optimum reaction temperature of 35 °C.The temperature stability results showed that the half-life at 30 °C was 51.14 h,while the half-life at 50 °C was 4.74 h.First,chiral SO was prepared by resolution of racemic SO by EH-Am.The results showed that when the substrate concentration was 1.06 mM,the ee value of(R)-SO was >99% after 10 h of reaction,and the yield was 25%.The mutual inhibition between the two configuration substrates and the product inhibition were studied.The results show that(S)-SO can significantly inhibit the hydrolysis of(R)-SO,(S)-SO hydrolysis product(S)-1-phenyl-1,2-ethanediol has strong inhibition on(S)-SO hydrolysis.The substrate concentration study showed that the ee value of(R)-SO was lower when the SO concentration was greater than 2.11 mM.By the addition of the substrate,the yield of(R)-SO was increased from 24.8% to 33.2%(substrate concentration 2.11 mM);a certain amount of Tween 20 surfactant was added to the reaction system,The yield of(R)-SO was increased to 34.2%.Chiral BGE was then prepared by resolution of racemic BGE using EH-Am.The results showed that when the substrate concentration was 6.72 mM,the ee value of(S)-BGE was >99% after 6 h of reaction,and the yield was 34%.The mutual inhibition between the two configuration substrates and the product inhibition were investigated.The results show that(R)-BGE can significantly inhibit the hydrolysis of(S)-BGE,(R)-BGE hydrolysis product(R)-3-benzyl-1,2-propanediol has strong inhibition on(R)-BGE hydrolysis.The substrate concentration had a significant effect on the ee value and yield of(S)-BGE.When the substrate concentration was 0.672 mM,the ee value of(S)-BGE was >99%,and the yield was 40.1%.As the substrate concentration increases,the yield of(S)-BGE gradually decreases.When the concentration of racemic BGE was 10.752 mM,the yield of(S)-BGE was only 21.2%;if the substrate concentration was continued to increase,the ee value of(S)-BGE was less than 99%.By the addition of the substrate,when the concentration is 10.752 mM,the yield of(S)-BGE is increased from 21.2% to 26.9%;a certain amount of surfactant Triton X-100 is added to the reaction system,The yield of(S)-BGE was increased to 35.9%.