Synthesis Of Aromatic Amide Chiral Pesticides Catalyzed By Stereoselective Lipase

Biocatalysis had the advantages of mild conversion conditions,high stereoselectivity,and ample source of catalyst,and has been increasingly used in the synthesis of chiral pharmaceutical and chiral pesticides.Aromatic amide chiral pesticides is a group of new chiral pesticides with effectively herbicidal and fungicidal activity,and their enzymatic synthesis is the focus of the study in pesticide synthesis.As the most representative biocatalyst,lipase has occupied a pivotal position in this field.In the thesis,in order for the synthesis of chiral aromatic amide pesticides,the lipase as biocatalyst was discovered,manufactured,applied on the enzymatic preparation of key chiral intermediates,and further employed on the chemical synthesis of aromatic amide chiral pesticides.In the first part,the screening systems for producing aromatic amide chiral pesticide intermediates were developed based on the lipase-catalyzed hydrolysis and acylation,respectively.Thereafter,the developed screening systems were employed for screening of the library including 292 microbial strains and 8 commercial lipases.Finally,the lipase Novozym 435 was selected for producing?acylation reaction??S?-1-?4-chlorophenyl?ethylamine and the microbial strain WZZ009 was obtained for producing?hydrolysis reaction??R?-MAP-acid.Secondly,aiming for?S?-1-?4-chlorophenyl?ethylamine production,the optimized enzymatic reaction conditions were obtained through the comparison of different reaction systems and conditions using Novozym 435 as biocalyst:methyl tert-butyl ether as reaction solvent,2-tetrahydrofurfuryl acid methyl ester as the acylating agent,0.8%?w/v?substrate concentration,the volume ratio of?R,S?-1-?4-chlorophenyl?ethylamine:2-methyl-tetrahydrofurfuryl=1:2.2,40°C as reaction temperature,200rpm as shaking speed.Under these optimized reaction conditions,the immobilized lipase can be reused for 10 times.After 20 h reaction,the e.e._s value?S?-1-?4-chlorophenyl?ethylamine was>99%,the conversion reached about 54%,and the enantiomeric ratio E reached 70.The?S?-acetamide sample was further prepared by chemical synthesis using?S?-1-?4-chlorophenyl?ethylamine as substrate.The yield was 83.5%and the purity of the?S?-acetamide sample was 95.3%determined by HPLC.The structure of the?S?-acetamide sample was approved by NMR and mass spectrometry.According to morphological observation,physiological and biochemical tests,16S rDNA sequencing,BIOLOG identification and phylogenetic analysis,the lipase-producing bacterium WZZ009 was identified as Pseudochrobactrum asaccharolyticum,named P.asaccharolyticum WZZ009,and deposited at Wuhan University Culture Collection.The effects of medium composition and culture conditions on lipase production of P.asaccharolyticum WZZ009 were also analyzed.The optimized medium was listed as follows:glucose 6 g/L,yeast extract 16 g/L,NaCl 0.5 g/L,MgSO₄ 0.25 g/L.The optimized culture conditions were listed as follows:initial pH 7.0,temperature 30°C,shaking speed 150 rpm.After 24 h culture under the above conditions,the biomass of P.asaccharolyticum WZZ009 reached 3.8g/L and its enzyme activity reached 118.2 U/g CDW.The effects of substrate concentrations,reaction temperatures and pH values on the?R?-MAP-acid preparation using WZZ009 whole cells were investigated.The optimized catalytic conditions were listed as follows:50 g/L of lyophilized cells,0.256 mol/L substrate concentration,0.2 M phosphate buffer?pH 7.2?,30?,200 rpm,reaction time 48 h.Under the optimized conditions,the e.e._p value and conversion reached up to above 98.0%and nearly 50%,respectively.When the lyophilized cells were reused for three consecutive times,the conversion maintained>40%and the product e.e._p remained above 98%.Therefore,the catalyst production capacity was increased from 0.47 g?product?/g?catalyst?to 1.31g?product?/g?catalyst?.In the last part,the whole chemical-enzymatic process for preparation of mefenoxam was studied with a combination of P.asaccharolyticum WZZ009 fermentation in 500L fermenter and resolution reaction of?R,S?-DMPM in 100 L reactor.Finally,the production of the target compound reached kg-scale?1.2 kg?R?-MAP-acid,42.8%yield?by resolution reaction.Furthermore,the unused?S?-DMPM was successfully racemized,making the full use of substrate possible.?R?-DMPM was synthesized using enzymatically prepared?R?-MAP-acid as the starting building block?⁹0.0%yield?,and was further synthesized to obtain the final product?R?-metalaxyl?⁹9.0%yield?.The effects of mefenoxam on Zebrafish embryo growth was also investigated to determine its environmental compatibility,which results demonstrated that the optically pure mefenoxam showed better environmental compatibility than that of the racemic mefenoxam.