JPH0965891A - Production of optically active alpha-methylalkanoic acid derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce an optically active α-methylalkanoic acid to be used as a raw material for production of various liquid crystals or as an intermediate for synthesizing optically active medicines and agrochemicals by making microorganisms having ability to asymmetrically hydrolyze ester linkage act on a racemic α-methylalkanoic ester. SOLUTION: The objective optically active derivative to be used as a raw material for production of various liquid crystals or as an intermediate for synthesis of optically active medicines and agrochemicals is effectively produced position-selectively by making a cultured product, microbial bodies or microbial body-treated product of microorganisms [e.g. Escherichia coli MR-2103 (FERM BP-3835)] which have ability to asymmetrically hydrolyze ester linkages act on a racemic α-metylalkanoic ester of formula I (R is a 1-6C alkyl ; n is an integer of 1-3) (e.g. racemic methyl α-methylhexanoate) to produce an optically active α-methylalkanoic acid of formula II (C* is an asymmetric carbon atom) and/or an antipode ester of the optically active α-methylalkanoic acid of formula III, and then by separating the product ester.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an optically active α-methylalkanoic acid represented by the following general formula (II), which is useful as a synthetic intermediate for various liquid crystal raw materials or optically active pharmaceuticals and agricultural chemicals, and / or Of the optically active α- represented by the general formula (III)
The present invention relates to a method for producing an enantiomer ester of methylalkanoic acid.

[0002]

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[0003]

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[0004]

PRIOR ART General formula (II):

[0005]

[Chemical 6]

Or general formula (III):

[0007]

[Chemical 7]

Among the optically active α-methylalkanoic acid derivatives represented by, for example, for optically active α-methylhexanoic acid ester, a method of esterifying α-methylhexanoic acid stereoselectively using lipase has been reported. (EKHeinz, Tetrahedron: Asymmetry, 2 (3), 165-168
(1991)). However, considering industrial production, neither the yield nor the optical purity is satisfactory.

[0009]

The object of the present invention is to provide an optical compound represented by the above general formula (II) having a high optical purity which is expected to be used as a synthetic intermediate for various liquid crystal raw materials or optically active pharmaceutical and agricultural chemicals. Active α-methylalkanoic acid and general formula (III)
Another object of the present invention is to provide a method for efficiently and regioselectively producing an enantiomer ester of an optically active α-methylalkanoic acid represented by

[0010]

That is, the present invention provides the following general formula (I):

[0011]

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(Wherein R represents an alkyl group having 1 to 6 carbon atoms and n represents an integer of 1 to 3), the ester bond is asymmetrical to the racemic α-methylalkanoic acid ester. The following general formula (II) is obtained by reacting a culture of a microorganism having the ability to hydrolyze, a bacterial cell or a treated product of the bacterial cell:

[0013]

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An optically active α-methylalkanoic acid represented by: and / or the following general formula (III):

[0015]

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A method for producing the enantiomer ester of the optically active α-methylalkanoic acid represented by

In the present invention, R in the above formula may be any as long as it serves as a substrate for an enzymatic reaction, and examples thereof include methyl group, ethyl group, propyl group, isopropyl group, butyl group and isobutyl. Examples thereof include groups.

The microorganism used in the present invention asymmetrically hydrolyzes the ester bond of the racemic α-methylalkanoic acid ester represented by the general formula (I) to give an optically active α-methylalkanoic acid and its enantiomer. Anything that has the ability to produce an ester can be used.
As a representative, Pseudomonas
Examples include microorganisms belonging to the genus Escherichia. Specifically, Pseudomonas
as putida) MR-2068 (FERM BP-3846), Esericia coli
(Escherichia coli) MR-2103 (FERM BP-3835). Escherichia coli MR-2103 (FERM
BP-3835) is a Pseudomonas pu
tida) MR-2068 (FERM BP-3846) -derived esterase gene-transformed strain.

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 appropriately mixed with components such as a carbon source, a nitrogen source, vitamins, and minerals that can normally be used by microorganisms 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 ester may be added to the culture solution after culturing the microorganism in advance. 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 cell may be used. It is also possible to immobilize on a suitable carrier, carry out the reaction, and then collect and reuse. Here, as the enzyme, various lipases, proteases and esterases derived from microorganisms can be used.

As the reaction medium, for example, ion-exchanged water,
A buffer is used. The ester concentration in the reaction medium or culture medium is preferably 0.1 to 70% by weight, more preferably 5 to 40%. Methanol, acetone,
It is also possible to add a surfactant or the like 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. In this case, it is preferable to maintain the pH at an optimum level with a suitable neutralizing agent. The reaction temperature is 5
70 degreeC is preferable and 10-60 degreeC is more preferable.

Separation and purification of the product from the reaction-terminated liquid is carried out by extraction with an organic solvent such as ethyl acetate, chloroform, diethyl ether and the like, and then applying a usual purification method such as distillation or column chromatography.
An optically active α-methylalkanoic acid ester can be purified and obtained. By lowering the pH of the aqueous layer after extraction to 2 or less, the antipodal optically active α-methylalkanoic acid is converted into a free acid, and then extracted with an organic solvent such as ethyl acetate to obtain the optically active α-methylalkanoic acid. The alkanoic acid can be recovered.

The optically active α-methylalkanoic acid ester obtained as described above is hydrolyzed according to a conventional method under conditions such that racemization does not occur, so that the optically active α-methylalkanoic acid ester retains its configuration. Further, the optically active α-methylalkanoic acid can be converted into an optically active α-methylalkanoic acid ester while maintaining the configuration by esterification under conditions that do not cause racemization according to a conventional method. can do.

[0025]

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

[Example 1] Production of optically active α-methylhexanoic acid and its enantiomer ester 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 50 ml and cultured with shaking at 37 ° C. for 20 hours. After 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 50 ml of 50 mM phosphate buffer (pH 7.0). To this cell suspension, 5 g of racemic methyl-α-methylhexanoate was added, and the mixture was reacted at 30 ° C for 20 hours. During this period, p of the reaction solution
H was adjusted to 7.0 using a 1N NaOH aqueous solution. After completion of the reaction, cells were removed by centrifugation and unreacted methyl α-methylhexanoate 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 1.9 g of optically active methyl α-methylhexanoate. When the specific optical rotation of this optically active methyl α-methylhexanoate was measured,
[Α] ²⁵ _D was +16.3.

Next, the pH of the aqueous phase was lowered to 2.0 with diluted sulfuric acid, and 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 1.3 g of optically active α-methylhexanoic acid. When the specific optical rotation of this sample was measured, it was [α] ²⁵ _D = -16.3.

[Example 2] Production of optically active α-methylpentanoic acid and its enantiomer ester To the bacterial cell suspension obtained in Example 1, 5 g of racemic methyl-α-methylpentanoate was added to give 30 The reaction was carried out at 0 ° C for 20 hours. During this period, the pH of the reaction solution was adjusted to 7.0 using a 1N NaOH aqueous solution. After completion of the reaction, cells were removed by centrifugation and unreacted methyl α-methylpentanoate was extracted with hexane. Anhydrous sodium sulfate was added to the organic layer for dehydration, the solvent was removed by evaporation, and the residue was purified by distillation to obtain 1.9.
Thus, g of optically active methyl α-methylpentanoate was obtained.

When the specific optical rotation of this optically active methyl α-methylpentanoate was measured, [α] ²⁵ _D = + 19.5.
Met.

Next, the pH of the aqueous phase was lowered to 2.0 with diluted sulfuric acid, and 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 1.3 g of optically active α-methylpentanoic acid. When the specific optical rotation of this sample was measured, it was [α] ²⁵ _D = -16.8.

[0031]

INDUSTRIAL APPLICABILITY According to the present invention, the optically active α-represented by the above-mentioned general formula (II) having high optical purity, which is expected to be used as a synthetic intermediate for various liquid crystal raw materials or optically active pharmaceuticals and agricultural chemicals.
The enantiomer ester of methylalkanoic acid and the optically active α-methylalkanoic acid represented by the general formula (III) can be efficiently produced regioselectively with high optical purity.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication (C12P 41/00 C12R 1:19) (C12P 7/24 C12R 1:19) (C12P 7/24 C12R 1:38) (C12P 7/40 C12R 1:38) (C12P 7/40 C12R 1:19)

Claims (4)

[Claims] 1. The following general formula (I): In the racemic α-methylalkanoic acid ester represented by
The following general formula (II): [Chemical Formula 2] is obtained by reacting a culture, a microbial cell or a treated product of a microorganism having the ability to asymmetrically hydrolyze an ester bond. An optically active α-methylalkanoic acid represented by and / or the following general formula (III): The method for producing the enantiomer ester of the optically active α-methylalkanoic acid represented by: 2. The optically active α-methylalkanoic acid according to claim 1, wherein the microorganism capable of asymmetrically hydrolyzing an ester bond is a microorganism capable of producing lipase, protease or esterase, and / Or a method for producing an antipodal ester thereof. 3. The optically active α-methylalkanoic acid according to claim 1, wherein the microorganism having the ability to asymmetrically hydrolyze an ester bond is a microorganism belonging to the genus Pseudomonas or the genus Escherichia. ,
And / or a method for producing an antipodal ester thereof. 4. 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. Of the optically active α-methylalkanoic acid, and / or an enantiomer ester thereof.