Research On Preparation Of Keychiral Aryloxyphenoxy Propionate Herbicides Intermediates With Microorganism Hydrolases

Lipases, with wide substrate and high stereoselective specificity, are playing an increasingly important role in the preparation of chiral pharmaceuticals and agrochemicals intermediates. Ethyl (S)-2-chloro-propionate and ethyl (R)-2-(4-hydroxyphenoxy)propionate are key intermediates of Aryloxyphenoxy propionate herbicides. Chemical catalyst routes often show low efficiency and purity in harsh reaction condtions. And biocatalyst preparation methods generate less waste and byproducts, which can get products with high optical purity in mild conditions. Researchs on enzymatic preparation of these two types of chiral intermediates showed great practical significance for industrial application.An enantioselective hydrolase production strain screening model was developed, which focused on sole carbon source enrichment screening, followed by polarimeter and GC detections. Forty strains with stereoselective hydrolase production were isolated from rich oil soil samples and hydrolase producting strains preserved by our laboratory, which included27strains with (S)-hydrolase production and13strains with (S)-hydrolase production. Based on morphology, physiological tests and16S rDNA gene sequences analyse, a high enantioselective strain was identified as B. pumilus, and was named as Bacillus pumilus WZ015.Based on single factor and response surface experiments, the fermentational medium composition and culture conditions for B.pumilus WZ015were investigated. The optimized medium compositon was as follows (g/L):glucose8.7, yeast extraction9.0, anhydrous MgSO40.5, KH2PO40.78, olive oil3.8. The statisfatory fermentation conditons for bactrial growth and hydrolase production were as follows:intial pH7.0; culutre temperature30℃; inoculum volume6%(v/v); medium volume50mL in250mL flasks. Under these optimal conditions, when B.pumilus WZ015was cultivated for22h, the enzyme actvity which expressed as hydrolysis of p-nitrophenyl acetate actvity reached960U/L of culture broth. It was enhanced by1.6folds compared with before optimized.Conditions for kinetic resolution of ethyl (R,S)-2-chloropropionate catalyzed by B.pumilus WZ015hydrolase were also studied. It showed the optimized resolution conditions as follows:racemic ethyl2-chloropro-pionate (0.47mmol) was dissolved in3mL phosphate buffer(3mL, pH7.3,0.25mol/L), B.pumilus WZ015hydrolase (0.02g/mL) was then suspended in the buffer. The reaction mixture was shaken (200rpm) in a water shaking bath at35℃for150min. The e.e.s value of97.6%and E value of10.3was obtained under this appropriate conditions. The structure of remaining ethyl (S)-2-chloropropionate was confirmed by chromatograms after enzymatic resolution.Finally, chemo-enzymatic preparation of ethyl (R)-2-(4-hydroxy phenoxy) propionate route was established. Racemic ethyl2-(4-hydroxy phenoxy) propionate was synthesized by chemical method using racemic ethyl2-chloropropionate and hydroquinone as raw materials, with yield 72%,90%purity. Enantioseparation of racemic ethyl2-(4-hydroxypheno-xy) propionate was performed on a chiral column by the normal phase HPLC. With this method, the former screened hydrolase production strains and laboratory strains were screened for asymmetric hydrolysing racemic ethyl2-(4-hydroxyphenoxy)propionate. Three strains showed enantioselectivity for racemic ethyl2-(4-hydroxyphenoxy) propionate were isolated. Aspergillus oryzae WZ007, with the highest enantioselectivity hydrolase production, was chosen as the biocatalyst to asymmetric hydrolysis of ethyl (R<S)-2-(4-hydroxyphenoxy) propionate. The optimized reaction conditions were as follows:racemic ethyl2-(4-hydroxyphenoxy) propionate (0.105mmol) was added to phosphate buffer (3mL, pH7.5,0.20M), then A. oryzae WZ007hydrolase (0.02g) was added. The reaction mixture was shaken (200rpm) in a water shaking bath at40℃. After60mins of enzymatic catalyzed reaction, e.e.s value of98.7%, conversion rate of51.8%and E value of117were obtained. The remaining structure of ethyl (R)-2-(4-hydroxyphenoxy) propionate was confirmed by polarimeter and chromatograms. Thus chemo-enzymatic preparation of ethyl (R)-2-(4-hydroxyphenoxy) propionate had achieved the forgein research level, which was initiated in technical area domestically. Meanwhile it simplifyed the operation steps, so it has great prospects for industrial application.