JPH08285A - Production of optically active alpha-methylalkanedicarboxylic acid omega-monoester and its antipode diester - Google Patents

Abstract

PURPOSE:To produce the subject compound useful e.g. as an intermediate for pharmaceuticals, etc., in high efficiency, optical purity and yield by treating a specific racemic alpha-methylalkanedicarboxylic acid diester with a cultured product, etc., of a specific mlcroorganism. CONSTITUTION:This (R)-isomer of an alpha-methylalkanedicarboxylic acid omega- monoester of formula II and (S)-isomer of an alpha-methylalkanedicarboxylic acid diester of formula III are produced by treating a racemic alpha- methylalkanedicarboxylic acid diester of formula I (R is a 1-6C alkyl; (n) is 1 or 2) with a cultured product, cells or treated cells of a microorganism belonging to the genus Pseudomonas or Escherichia and capable of asymmetrically hydrolyzing an ester bond [e.g. Pseudomonas putida MR-2068 (FERM BP-3846)].

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing a specific useful optically active α-methylalkanedicarboxylic acid-ω-monoester and its enantiomer diester, which are useful as synthetic intermediates for various medicines, agricultural chemicals and the like. Regarding

[0002]

2. Description of the Related Art In recent years, the demand for optically active substances as synthetic intermediates for physiologically active substances such as pharmaceuticals and agricultural chemicals has been rapidly increasing, and researches for synthesizing optically active substances using various methods have been actively conducted. ing.

Among the α-methylalkanedicarboxylic acid derivatives represented by the general formula (2), for example, α-methylsuccinic acid monoester can be obtained by partially hydrolyzing the methylsuccinic acid diester with an alkali catalyst or the like. However, in this case, the reaction product is α-methylsuccinic acid, α-methylsuccinic acid-1-monoester,
Since it is a mixture of α-methylsuccinic acid-4-monoester and α-methylsuccinic acid diester, it is difficult to obtain the desired product selectively and in high purity. further,
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) discloses, as a method for selectively synthesizing an α-methylsuccinic acid monoester, a method using itaconic anhydride as a raw material, which is generally represented by the following reaction formula (4).

[0005]

[Chemical 4]

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 by such a reaction mode.

T. Morimoto et al., Chem. Pharm. Bull.
41 (6), 1149 (1993) also reported a method of asymmetrically reducing itaconic acid or dimethyl itaconic acid to obtain optically active α-methylsuccinic acid or optically active α-methylsuccinate, but it was expensive. Since a simultaneous catalyst must be used, it cannot be said to be an industrially advantageous method.

On the other hand, Eryka Guibe-Jampel et al., J. Ch
Em. Soc., Chem. Commun., 1080, 1987 reported a method for obtaining α-methylsuccinic acid-1-monoester by hydrolyzing α-methylsuccinic acid diester with porcine pancreatic lipase. 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.

Further, JP-A-2-195890 describes a method of hydrolyzing α-methylsuccinic acid diester using an enzyme derived from a microorganism to obtain α-methylsuccinic acid-4-monoester. In this method, 4-monoester has a high regioselectivity of 95 to 98%, but stereoselective hydrolysis is hardly achieved, and when a 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 nothing more than a degree.

[0010]

DISCLOSURE OF THE INVENTION The present invention does not have the above-mentioned problems and is capable of regioselecting an optically active α-methylalkanedicarboxylic acid-ω-monoester and its enantiomer diester with high optical purity. The purpose of the present invention is to provide a method of efficiently manufacturing the same.

[0011]

Means for Solving the Problems That is, the present invention provides a racemic form of α-methylalkanedicarboxylic acid diester represented by the following general formula (1), which comprises a microorganism having the ability to asymmetrically hydrolyze an ester bond. The (R) form α-represented by the following general formula (2) is caused by the action of a culture, cells or a treated product of cells.
It is a method for producing a methylalkanedicarboxylic acid-ω-monoester and an (S) -form α-methylalkanedicarboxylic acid diester represented by the following general formula (3).

Embedded image

[Chemical 6]

[Chemical 7]

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

The microorganism used in the present invention asymmetrically hydrolyzes the ester bond of α-methylalkanedicarboxylic acid diester to give an optically active α-methylalkanedicarboxylic acid-ω.
-There is no particular limitation as long as it has an ability to produce a monoester and its enantiomer diester. Representative examples include microorganisms belonging to the genus Pseudomonas and the genus Escherichia. Specifically, Pseudomonas putida MR
-2068 (FERM BP-3846), Escheri-chia
coli) MR-2103 (FERM BP-3835). Escherich-ia coli MR-2103 (FERM BP-3835)
Is Pseudomonas put-ida MR-2
This is a strain transformed with the esterase gene derived from 068 (FERM BP-3846).

The culture of the microorganism used in the present invention can be carried out in a liquid medium or a solid medium. As the medium, a medium in which components such as a carbon source, a nitrogen source, vitamins and minerals which can be normally assimilated by microorganisms are appropriately mixed is used. It is also possible to add a small amount of ester to the medium in order to improve the ability of the microorganism to hydrolyze. The culture is carried out at a temperature and pH at which the microorganism can grow, but it is preferably carried out under the optimum culture conditions of the strain to be used. Aeration and agitation may be performed to promote the growth of microorganisms.

When carrying out the hydrolysis reaction, the ester may be added to the medium at the start or during the culture, or the microorganism may be previously cultured and then the ester may be added to the culture solution. Alternatively, the cells of the grown microorganism may be collected by centrifugation or the like and added to the reaction medium containing the ester. As the bacterial cells, bacterial cells treated with acetone, toluene or the like may be used.

Further, instead of the cells, a culture such as a culture solution, a cell disruption product, a cell extract, a crude enzyme, a purified enzyme or the like treated product may be used. It is also possible to immobilize it on a suitable carrier, carry out the reaction, and then collect and reuse it.

As the enzyme, various lipases, proteases and esterases derived from microorganisms can be used.

As the reaction medium, for example, ion-exchanged water or a buffer solution is used. The ester concentration in the reaction medium or the culture solution is preferably 0.1 to 70% by weight, more preferably 5 to 40% by weight. It is also possible to add methanol, acetone, a surfactant, etc. to the reaction system. The pH of the reaction solution is in the range of 2 to 11, preferably 5 to 8. As the reaction progresses, the pH of the reaction solution decreases due to the carboxylic acid formed, but in this case, it is desirable to maintain the optimum pH with an appropriate neutralizing agent. The reaction temperature is preferably 5 to 70 ° C, more preferably 20 to 60 ° C.

The product is separated and purified from the reaction-finished liquid by extraction with an organic solvent such as ethyl acetate, chloroform, ether, etc., and the conventional method such as distillation or column chromatography is applied to obtain an optically active α-methylalkane. The dicarboxylic acid diester can be purified and obtained. By lowering the pH of the aqueous layer after extraction to 2 or less, the antipodal 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, the optically active α-methylalkanedicarboxylic acid-ω-monoester can be recovered.

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

[0021]

The present invention will be described in more detail with reference to the following examples, but the invention is not limited thereto.

Example 1 Production of optically active α-methylsuccinic acid monoester and its enantiomer diester. Escherichia coli MR-2103 (FERM BP-
3835) in LB medium containing 50 μg / ml of ampicillin (1% polypeptone, 0.5% yeast extract, 0.5% N).
(aCl) was inoculated into 500 ml and cultured with shaking at 37 ° C. for 20 hours. After the completion of the culture, the culture solution was centrifuged, the whole amount of the obtained bacterial 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 dimethyl α-methylsuccinate
Was added and reacted at 30 ° C. for 20 hours. During this period, the pH of the reaction solution was adjusted to 7.0 using a 10% NaOH aqueous solution. After completion of the reaction, cells were removed by centrifugation, and unreacted dimethyl α-methylsuccinate 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 19.8 g of optically active dimethyl α-methylsuccinate. This optically active α-
Dimethyl methylsuccinate was measured for optical purity using an optical resolution column (Chiralcel OD, manufactured by Daicel Chemical Industries, Ltd.), and was 99% 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 give 17.2 g of optically active α-methylsuccinic acid-
4-monoester was obtained. When the optical purity was measured using an optical resolution column (Chiralcel OD, manufactured by Daicel Chemical Industries, Ltd.), it was 96% e. e.
Met. Further, from ¹ H-NMR, the obtained monoester was only 4-ester, and 1-ester was not mixed.

Example 2 Production of optically active α-methylglutarate monoester and its enantiomer diester. To the bacterial cell suspension obtained in Example 1, 50 g of racemic dimethyl dimethyl α-methylglutarate was added and reacted at 30 ° C. for 20 hours. During this period, the pH of the reaction solution was adjusted to 7.0 using a 10% NaOH aqueous solution. After completion of the reaction, cells were removed by centrifugation and unreacted dimethyl α-methylglutarate 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 18.2 g of dimethyl optically active α-methylglutarate. This optically active dimethyl α-methylglutarate is tris [3- (heptafluoropropylhydroxymethylene)-(+)
-camphorato] -europium (III) was used to measure the optical purity from the ¹ H-NMR spectrum.
0% 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. ¹ H
-From NMR, the obtained monoester was only 4-ester, and 1-ester was not mixed. After the optically active α-methylglutaric acid-4-monoester was converted into a diester by a conventional method, the optical purity was determined by the same method as described above. e. Met.

[0024]

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

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12R 1:19)

Claims (3)

[Claims] 1. A culture of a microorganism, a microbial cell or a treated product of a microbial cell, which has the ability to asymmetrically hydrolyze an ester bond to a racemic α-methylalkanedicarboxylic acid diester represented by the following general formula (1): And the following general formula (2)
(R) -form α-methylalkanedicarboxylic acid represented by
A method for producing an ω-monoester and an (S) -form α-methylalkanedicarboxylic acid diester represented by the following general formula (3). Embedded image Embedded image Embedded image 2. The method according to claim 1, wherein the microorganism having the ability to asymmetrically hydrolyze an ester bond is a microorganism belonging to the genus Pseudomonas and the genus Escherichia. 3. The microorganism having the ability to asymmetrically hydrolyze ester bonds is a genetically engineered microorganism transformed with a gene encoding an enzyme that asymmetrically hydrolyzes ester bonds. The method described.