JP3970898B2 - Process for producing optically active α-methylalkanedicarboxylic acid-ω-monoester and its enantiomer diester - Google Patents

Description

  The present invention relates to a method for producing useful specific optically active α-methylalkanedicarboxylic acid-ω-monoesters and enantiomers thereof which serve as synthetic intermediates for various pharmaceuticals, agricultural chemicals and the like.

  In recent years, the demand for optically active substances as synthetic intermediates of physiologically active substances such as pharmaceuticals and agricultural chemicals is rapidly increasing, and active researches on the synthesis of optically active substances using various techniques have been actively conducted.

  Among the α-methylalkanedicarboxylic acid derivatives represented by the general formula (2), for example, α-methylsuccinic acid monoester can be obtained by partial hydrolysis of methylsuccinic acid diester with an alkali catalyst or the like. However, in this case, the reaction product is a mixture of α-methyl succinic acid, α-methyl succinic acid-1-monoester, α-methyl succinic acid-4-monoester and α-methyl succinic acid diester. It is difficult to obtain selectively and with high purity. Furthermore, when a racemic mixture is used as a substrate, optical resolution cannot be expected in such a reaction mode.

On the other hand, Barnett, Morris, Biochem. J. 40 , 451 (1946) generally uses itaconic anhydride as shown in the following reaction formula (4) as a method for selectively synthesizing α-methyl succinic acid monoester. A method of using as a raw material is disclosed.

  However, the α-methylsuccinic acid monoester obtained by such a method is a racemate, and there is a problem that an optically active substance cannot be obtained in such a reaction mode.

In T. Morimoto et al., Chem. Pharm. Bull. 41 (6), 1149 (1993), itaconic acid or dimethyl itaconate was asymmetrically reduced to obtain optically active α-methyl succinic acid or optically active α-methyl succinic acid dimethyl. However, it is difficult to say that the method is industrially advantageous because an expensive asymmetric catalyst must be used.

  On the other hand, in Eryka Guibe-Jampel et al., J. Chem. Soc., Chem. Commun., 1080, 1987, α-methyl succinic acid diester is hydrolyzed with porcine pancreatic lipase to produce α-methyl succinic acid-1-monoester. How to get it has been reported. However, although the monoester obtained by this method has high optical purity and regioselectivity, it is difficult to say that it is an industrially advantageous method because an expensive animal-derived enzyme is used.

  Japanese Patent Application Laid-Open No. 2-195890 describes a method of obtaining α-methylsuccinic acid-4-monoester by hydrolyzing α-methylsuccinic acid diester using an enzyme derived from a microorganism. In this method, although 4-monoester has high regioselectivity of 95 to 98%, stereoselective hydrolysis is hardly achieved. When racemic diester is used as a raw material, the optical purity of the product is 16 % E. e. There is a problem that it is only a degree.

  The present invention provides a method for efficiently producing optically active α-methylalkanedicarboxylic acid-ω-monoester and its enantiomer diester regioselectively with high optical purity without the above-mentioned problems. The purpose is to do.

That is, the present invention provides a culture, a fungus body, or a fungus treatment of a microorganism having an ability to asymmetrically hydrolyze an ester bond to a racemic α-methylalkanedicarboxylic acid diester represented by the following general formula (1). (R) -form α-methylalkanedicarboxylic acid-ω-monoester represented by the following general formula (2) and (S) -form α-methylalkanedicarboxylic acid represented by the following general formula (3) It exists in the method of manufacturing an acid diester.

  By the method of the present invention, it is possible to efficiently produce an optically active α-methylalkanedicarboxylic acid-ω-monoester having high optical purity and its enantiomer diester. Separation and purification of the produced carboxylic acid diester and carboxylic acid monoester are easy, and this is an industrially advantageous method.

  In the present invention, as a racemate of the α-methylalkanedicarboxylic acid diester represented by the general formula (1) that can be used as a substrate, R is a methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl. Group, pentyl group or hexyl group, and n is 1 or 2.

  The microorganism used in the present invention has the ability to asymmetrically hydrolyze the ester bond of α-methylalkanedicarboxylic acid diester to produce optically active α-methylalkanedicarboxylic acid-ω-monoester and its enantiomer diester If there is no restriction in particular. Typical examples include microorganisms belonging to the genus Pseudomonas and the genus Escherichia. Specific examples include Pseudomonas putida MR-2068 (FERM BP-3846) and Escheri-chia coli MR-2103 (FERM BP-3835). Escherichia coli MR-2103 (FERM BP-3835) is a strain transformed with an esterase gene derived from Pseudomonasput-ida MR-2068 (FERM BP-3846).

  The microorganism used in the present invention can be cultured in a liquid medium or a solid medium. As a culture medium, what mix | blended suitably components, such as a carbon source, a nitrogen source, a vitamin, a mineral which a microorganism can normally utilize, is used. In order to improve the hydrolytic ability of microorganisms, a small amount of ester can be added to the medium. The culture is performed at a temperature and pH at which the microorganism can grow, but it is preferably performed under the optimal culture conditions of the strain to be used. In order to promote the growth of microorganisms, aeration and agitation may be performed.

  When performing the hydrolysis reaction, an ester may be added to the medium at the start or during the cultivation, or the microorganism may be cultured in advance and then the ester may be added to the culture solution. In addition, the cells of the grown microorganisms may be collected by centrifugation or the like and added to a reaction medium containing an ester. The cells treated with acetone, toluene or the like may be used.

  Further, instead of the microbial cells, a culture such as a culture solution, a crushed microbial cell extract, a microbial cell extract, a crude enzyme, a purified enzyme or other processed microbial cells may be used, and the enzyme or microorganism may be used as a suitable carrier. It is also possible to immobilize and recover and reuse after reacting.

  Here, various lipases derived from microorganisms, proteases, esterases and the like can be used as the enzymes.

  In addition, as a reaction medium, ion-exchange water and a buffer solution are used, for example. The ester concentration in the reaction medium or the culture medium is preferably 0.1 to 70% by weight, more preferably 5 to 40% by weight. It is also possible to add methanol, acetone, surfactant, etc. to the reaction system. The pH of the reaction solution is in the range of 2-11, preferably 5-8. The pH of the reaction solution is lowered by the carboxylic acid produced as the reaction proceeds. In this case, it is desirable to maintain the optimum pH with a suitable neutralizing agent. The reaction temperature is preferably 5 to 70 ° C, more preferably 20 to 60 ° C.

  Separation and purification of the product from the reaction finished solution is performed by extraction with an organic solvent such as ethyl acetate, chloroform, ether, etc., and by applying a conventional method such as distillation or column chromatography, an optically active α-methylalkanedicarboxylic acid diester Can be purified and obtained. By lowering the pH of the aqueous layer after extraction to 2 or less, the enantiomer optically active α-methylalkanedicarboxylic acid-ω-monoester is converted into a free acid and then extracted with an organic solvent such as ethyl acetate. For example, optically active α-methylalkanedicarboxylic acid-ω-monoester can be recovered.

  The optically active α-methylalkanedicarboxylic acid-ω-monoester and the enantiomer diester thus obtained can be derived into a diester or a dicarboxylic acid by a known method.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, it is not limited to these.

Example 1 Production of optically active α-methyl succinic acid monoester and its enantiomer diester.
Inoculate Escherichia coli MR-2103 (FERM BP-3835) in 500 ml of LB medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl) containing 50 μg / ml ampicillin, The culture was shaken at 37 ° C. for 20 hours. After completion of the culture, the culture solution was centrifuged, and the whole amount of the obtained cells was washed with ion-exchanged water and then suspended in 500 ml of 50 mM phosphate buffer (pH 7.0). To this bacterial cell suspension, 50 g of racemic α-methyl succinate was added and reacted at 30 ° C. for 20 hours. During this time, the pH of the reaction solution was adjusted to 7.0 using a 10% NaOH aqueous solution. After completion of the reaction, the cells were removed by centrifugation, and unreacted α-methyl succinate was extracted with ethyl acetate. The organic layer was dehydrated by adding anhydrous sodium sulfate, the solvent was removed by evaporation, and the residue was further purified by distillation to obtain 19.8 g of optically active dimethyl α-methylsuccinate. This optically active α-methyl succinate was measured for its optical purity using an optical resolution column (Chiral Cell OD, manufactured by Daicel Chemical Industries, Ltd.). e. Met. Next, the pH of the aqueous phase was lowered to 2.0 with dilute sulfuric acid, and then the acid content in the aqueous phase was extracted with ethyl acetate. Anhydrous sodium sulfate was added to the organic layer for dehydration, the solvent was removed by evaporation, and the residue was further purified by distillation to obtain 17.2 g of optically active α-methylsuccinic acid-4-monoester. When the optical purity was measured using an optical resolution column (Chiral Cell OD, manufactured by Daicel Chemical Industries, Ltd.), 96% e. e. Met. Further, from ¹ H-NMR, the resulting monoester was only 4-ester, and no 1-ester was present.

Example 2 Production of optically active α-methylglutaric acid monoester and its enantiomer diester.
To the cell suspension obtained in Example 1, 50 g of racemic dimethyl α-methylglutarate was added and reacted at 30 ° C. for 20 hours. During this time, the pH of the reaction solution was adjusted to 7.0 using a 10% NaOH aqueous solution. After completion of the reaction, the cells were removed by centrifugation, and unreacted dimethyl α-methylglutarate was extracted with ethyl acetate. The organic layer was dehydrated by adding anhydrous sodium sulfate, the solvent was removed by evaporation, and the residue was further purified by distillation to obtain 18.2 g of optically active dimethyl α-methylglutarate. This optically active α-methylglutarate dimethyl was measured for its optical purity from ¹ H-NMR spectrum using tris [3- (heptafluoropropylhydroxymethylene)-(+)-camphorato] -europium (III). At 100% e.e. e. Met. Next, the pH of the aqueous phase was lowered to 2.0 with dilute sulfuric acid, and then the acid content in the aqueous phase was extracted with ethyl acetate. Anhydrous sodium sulfate was added to the organic layer for dehydration, the solvent was removed by evaporation, and the residue was further purified by distillation to obtain 18.0 g of optically active α-methylglutaric acid-4-monoester. ^{From 1} H-NMR, the resulting monoester was only a 4-ester, and no 1-ester was present. This optically active α-methylglutaric acid-4-monoester was converted into a diester by a conventional method, and optical purity was determined by the same method as described above. As a result, 96% e.e. e. Met.

Claims (2)

The α-methylalkanedicarboxylic acid diester represented by the following general formula (1) is allowed to act on a culture, cell or treated cell of a microorganism belonging to the genus Pseudomonas, and the following general formula (2) (R) -form α-methylalkanedicarboxylic acid-ω-monoester and (S) -form α-methylalkanedicarboxylic acid diester represented by the following general formula (3) are collected. A process for producing methylalkanedicarboxylic acid-ω-monoester and (S) -α-methylalkanedicarboxylic acid diester.

The microorganism belonging to the genus Pseudomonas is Pseudom.
onas putida).