Enzymatic Preparation Of Chiral 3-cyclohexene-1-carboxylic Acid

Chiral 3-cyclohexene-1-caroxylic acid is an important chemical and pharmaceutical intermediate,which is widely used in chiral medicine,chiral additive,chiral auxiliaries and and other fields.The aim of this thesis was to study the preparation of chiral 3-cyclohexene-1-caroxylic acid by enzymatic catalysis,including the following two routes:1.resolution of racemic 3-cyclohexene-1-caroxylate using microbial esterase;2.enzymatic asymmetric synthesis of chiral 4-cyclohexene-1,2-dicarboxylic acid,1-methyl ester.Firstly,we screened a R-configuration hydrolase-producing strain with best enantioselectivity toward 3-cyclohexene-1-caroxylate from soil samples and our laboratory by using 3-cyclohexene-1-caroxylate as the sole carbon source.By morphological observation and 16S rDNA analysis,the selected strain was identified as Acinetobacter sp,and thus was named as Acinetobacter sp 192.The reaction conditions of enzymatic steroselective hydrolysis resolution of racemic 3-cyclohexene-1-caroxylate by Acinetobacter sp192 were investigated in this paper,and the optimal reaction conditions acquired are as follows:in 0.2M pH 7.5 potassium phosphate buffer(KPB)system,40oC,1 M NaOH as the neutralizer,substrate concentration was4%(v/v)with Acinetobacter sp 192 loading of 10 g/L.After 5 h titrimetric reaction,the e.e._s value was>99.9%,the e.e._p value was 75%,the yield was 40.7%,and the E value was 50.33.Alcoholysis of cis-1,2,3,6-tetrahydrophthalic anhydride as the substrate was made to synthesize 4-cyclohexene-1,2-dicarboxylic acid,dimethyl ester in this paper.Then enzymatic asymmetric hydrolysis of4-cyclohexene-1,2-dicarboxylic acid,dimethyl ester or enzymatic asymmetric alcoholysis of cis-1,2,3,6-tetrahydrophthalic anhydride was made to obtain the chiral 4-cyclohexene-1,2-dicarboxylic acid,1-methyl ester,the best results for the synthesis of chiral 4-cyclohexene-1,2-dicarboxylic acid,1-methyl ester through asymmetric hydrolysis of4-cyclohexene-1,2-dicarboxylic acid,dimethyl ester by Novozyme 435were obtained under the conditions like:40℃,pH 8.5,substrate concentration was 10%(v/v),1%(w/v)Novozyme 435.After 27 h titrimetric reaction,the e.e._P value of(1S,2R)-4-cyclohexene-1,2-dicarboxylic acid,1-methyl ester was 90%.After extraction and purification,decarboxylation of(1S,2R)-4-cyclohexene-1,2-dicarboxylic acid,1-methyl ester was made to prepare(R)-3-cyclohexene-1-caroxylate,and(R)-3-cyclohexene-1-caroxylic acid was obtained by hydrolysis of(R)-3-cyclohexene-1-caroxylate,the e.e._P value of final product was>99.5%,the product was identified by gas chromatography-mass spectrometry(GC-MS).