Enzymatic Resolution Of (R,S)-Tetrahydrofuran-2-carboxylic Acid

Tetrahydrofuran-2-carboxylic acid,is also known as 2-tetrahydrofurfuric acid.The optically pure tetrahydrofuran-2-carboxylic acid is an important intermediate for the preparation of various pharmaceuticals and chemical raw materials.At present,the method for industrial resolution of(R,S)-tetrahydrofuran-2-carboxylic acid is mainly chemical method.For example,it is separated by using ephedrine.The method has the disadvantages of high toxicity,complicated steps and large environmental pollution.There are few reports using enzymatic resolution of(R,S)-tetrahydrofuran-2-carboxylic acid.In this paper,we performed a study on the enzymatic resolution of(R,S)-tetrahydrofuran-2-carboxylic acid and(R,S)-tetrahydrofuran-2-carboxylic acid methyl ester.The racemic tetrahydrofuran-2-tarboxylic acid was used as a substrate,and different lipases were used to catalyze the esterification reaction of racemic tetrahydrofuran-2-tarboxylic acid and n-butanol.Novozyme 435 is the selected lipase with the best stereoselectivity.The reaction was preferably run at 30℃ in pyridine with an acid:alcohol molar ratio of 1:2.Under this condition,when the substrate concentration was 0.1 mol/L and the enzyme amount was 20 g/L,the conversion rate was 60%,with an enantiomer excess of 70% towards the product(R)-tetrahydrofuran-2-carboxylic acid butyl ester in a reaction time of 12 h.The racemic tetrahydrofuran-2-carboxylic acid methyl ester was used as a substrate to screen lipases capable of stereoselective hydrolysis of it.Finally,thelipase derived from Aspergillus oryzae was selected to have high stereoselectivity,and the lipase preferentially hydrolyzed(S)-tetrahydrofuran-2-carboxylic acid methyl ester,leaving optically pure(R)-tetrahydrofuran-2-carboxylic acid methyl ester.The optimum conditions for the hydrolysis of racemic tetrahydrofuran-2-carboxylic acid methyl ester by the lipase were found at 30 ℃ in phosphate buffer(pH 7.0,0.2 M).Under this condition,when the substrate concentration was 0.1 mol/L and the enzyme amount was 20 g/L,the conversion rate was 74%,with an enantiomer excess of 100% towards the unreacted of(R)-tetrahydrofuran-2-carboxylic acid methyl ester in a reaction time of 52 h.Immobilization of the enzyme increases the catalytic activity of the enzyme and allows the enzyme to be reused.There are many methods for immobilization of enzymes.The paper used adsorption method to immobilize A.oryzae lipase.The immobilization of A.oryzae lipase was carried out on the macroporous adsorption resin and the ion exchange resin,and the anion exchange resin Q was an ideal immobilization carrier.In phosphate buffer(pH 7.0,0.02 M)and at 30 ℃,when the amount of enzyme was 20 g/L and the amount of Q was 200 g/L,the lipase protein could be immobilized on the resin for about 5 h.The catalytic efficiency of the immobilized lipase was greatly improved compared with the free lipase,and the stereoselectivity was retained.When the substrate concentration was 0.1 mol/L and the immobilized enzyme amount was 100 g/L,the conversion rate was 76.3%,with an enantiomer excess of 99.1% towards the unreacted of(R)-tetrahydrofuran-2-carboxylic acid methyl ester in a reaction time of 5 h.Moreover,the immobilized lipase could still maintain high activity and stereoselectivity for using about 10 batches.