The Application Of Screened Lipase In Resolution Of Threo-P-Methyl-Sulfonylphenyl Serine Ethyl Ester

D-threo-p-methylsulfonylphenyl serine ethyl ester is one of the key intermediates for the synthesis of florfenicol. The present methods to prepare D-threo-p-methylsulfonylphenyl serine ethyl ester are mainly chemical approaches and there are rarely reports concerning enzymatic ways to preparing this desired single enantiomer in the world.Platform of lipases including lipase producing microorganisms screened from the soils and the existing lipases in the lab had been established, which was then taken as the enzyme sources for the asymmetric hydrolysis of D-threo-p-methylsulfonylphenyl serine ethyl ester.19strains of lipase producing microorganisms had been screened from65soil sources employing the improved primary screening medium, among which,9of them were with relative high activities.Series of byproducts had been detected when threo-p-Methylsulfo-nylphenyl serine ethyl ester was spontaneously hydrolyzed in water, or was hydrolyzed by lipases in organic solvent system. Methods of reserve-phase HPLC, normal-phase HPLC,1H NMR and LC-MS with different elution conditions were employed to furthest detect and identify these byproducts, as well as to analyze the effects of factors, such as pH, temperature and initial concentration of reaction material on side reactions. Mechanisms of generating main byproducts had also been investigated and discussed. The results showed that the byproduct, p-methylsulphonyl benzaldehyde, served as the key intermediate during the whole side reaction process. It had also been found that threo-p-methylsulfonylphenyl serine ethyl ester with a proper hydrolytic velocity would serve as a driving force to generate p-methylsulphonyl benzaldehyde, thus to accelerate the side reactions. A feasible approach to suppress side reactions both in water and enzymatic catalysis system of tert butyl alcohol by adding ions was offered.Tert butyl alcohol would be the most suitable organic solvent and Novozyme435was the most ideal lipase for the enzymatic enantioselective hydrolysis of p-methylsulfonylphenyl serine ethyl ester to be conducted. The effects of different factors to enzymatic enantioselective hydrolysis had also been investigated, thus, the most ideal conditions for resolution were identified. Results showed that adding Zn2+, ion with side reactions suppressing effect, to the system would exert a negative effect to the E value.