JPS63198998A - Optically active carboxylic acid and production of antipode ester thereof - Google Patents

Abstract

PURPOSE:To obtain the titled compound useful as an intermediate raw material for synthesizing a preformed chemical mediator industrially and advantageously, by treating a specific ester with an enzyme capable of asymmetrically hydrolyzing ester bond, a culture solution or a treated material of a bacterium. CONSTITUTION:An ester [e.g. (+ or -)-beta-chloroisobutyric acid methyl ester, etc.] shown by formula I (R1 and R2 are alkyl; X is halogen) is treated with an enzyme capable of asymmetrically hydrolyzing ester bond, a culture solution of a bacterium, a cell or a treated material of the cell to give the aimed optically active carboxylic acid (e.g. beta-chloroisobutyric acid, etc.) shown by formula II or an antipode ester thereof. A bacterium belonging the genus Acinetobacter, Pseudomonas, Escherichia coli, Staphylococcus, Alcaligenes, Micrococcus, Agrobacterium etc., is preferably used as the bacterium.

Description

Detailed Description of the Invention The present invention provides a novel method for producing an optically active carboxylic acid represented by the general formula % (in which R1 represents an alkyl group or a halogen atom) and its enantiomer ester. Regarding.

The carboxylic acid and its enantiomer ester are useful as intermediate raw materials for synthesizing various physiologically active substances having optical activity. Conventionally, the method for producing these optically active forms has been to first synthesize a racemic form by organic synthesis and then resolve it using various optical resolving agents, that is, to physicochemically separate one optically active form and its counterpart. A method of fractionating into a palm form (see JP-A-58-74625) or a method of halogenating an optically active β-hydroxycarboxylic acid (see JP-A-60-78929) is known. However, these methods require a large amount of expensive resolving agent, the resolving agent is easily mixed into the product as an impurity, and the resolving process is extremely complicated.
Alternatively, there are problems in that an excessive amount of halogenating agent is required, and hydrogen halide and sulfur dioxide gas are generated as reaction by-products, so that this method is not satisfactory as an industrial production method.

The present inventors resolved the drawbacks of the above-mentioned production method and proceeded with research on a more efficient production method of optically active carboxylic acid (I) and its ester. As a result, the present inventors developed an enzyme or microorganism capable of hydrolyzing an ester. It has been found that the optically active carboxylic acid of formula (1) and its enantiomer ester can be efficiently produced by using the following method.

The present invention produces an ester represented by the general formula % (in which R1 and R2 are an alkyl group and An optically active carboxylic acid represented by the general formula % (in which R1 and This is a method for producing esters.

Examples of the alkyl groups for the substituents R1 and R2 in the compounds of formulas I and II include methyl, ethyl, and the like. Examples of the halogen atom for the substituent X include a chlorine atom, a bromine atom, and an iodine atom.

The enzyme used in the present invention is an enzyme that has the ability to asymmetrically hydrolyze the ester bond of the compound of formula Even enzymes classified as proteases can be used in the present invention if they have ester hydrolyzing ability. This ester hydrolase is not particularly limited in origin, purity, etc., and can be used in animal and plant homogenates, microbial cells, crushed products thereof,
It may also be in the form of an extract or the like.

Examples of microorganisms that produce this enzyme include:
, Litho7's, Aspergillus, Nocardia, Streptomyces, Trichoderma, Candida, Rhodotorula, Torulopsis, Bacillis, alkaline earth metals, Pseudomonas, Brevibacterium, Entero Examples include microorganisms belonging to the genus Bacter, Chromobacterium, Arthrobacter, Microbacterium, Mycobacterium, Satucharomyces, and Penicillium. Examples of commercially available enzymes derived from these microorganisms include lipase from the genus Mucor (Lipase M-AP, manufactured by Amano Pharmaceutical Co., Ltd.), lipase from the genus Aspergillus (Lipase AP, manufactured by Amano Pharmaceutical Co., Ltd.), and lipase from the genus Candida (Sigma). (manufactured by Osaka Bacteria Research Institute), lipase from the genus Rhizopus (Lipase Saiken, Osaka Bacteria Research Institute), etc. Examples of enzymes derived from animal tissues include esterase derived from pig liver, α-chymotrypsin derived from pancreas, and pancreatin.

The microorganism used in the present invention is a microorganism that has the ability to asymmetrically hydrolyze the ester bond of the compound of formula (1),
For example, Acinetobacter spp.
r), Pseudomonas sp.
), Escherichia spp.
Staphylococcus (5taphylococcus)
), Alcaligenes f4 (Alcaligenes
), Micrococcus
, Agrobacterium spp.
Examples include microorganisms that belong to traps, such as m). These microorganisms are used as a culture solution containing them, isolated bacterial cells, or treated bacterial cells.

Cultivation of these microorganisms is usually carried out by liquid culture, but it can also be carried out by solid culture. The medium contains carbon sources, nitrogen sources, vitamins, etc. that can be normally assimilated by microorganisms.
A mixture containing minerals and other ingredients as appropriate is used.

In order to improve the hydrolytic ability of microorganisms, it is preferable to add a small amount of ester to the medium. Cultivation is carried out at a temperature of 10-50°C and a pH range of 2-11.

Aeration and stirring may be performed to promote the growth of microorganisms.

In carrying out the invention, the ester (
II) is treated with an asymmetric hydrolase.

The raw material ester (n) may be added to the medium at the beginning or during the cultivation of the microorganism, or 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. In this case, for convenience of handling, the bacterial cells are dried, such as freeze-dried bacterial cells, spray-dried bacterial cells, bacterial cells treated with organic solvents such as acetone, toluene, etc., or bacterial cell destruction materials, bacterial cell extracts, etc. It can also be used as a treated bacterial cell product. Ester hydrolase can also be used as an immobilized enzyme.

As the reaction medium, for example, a buffer solution containing ion-exchanged water, an inorganic acid salt such as sodium phosphate, and/or an organic acid salt such as sodium acetate is used. An organic solvent such as methanol, acetone, etc. can also be added to the reaction solution to improve the solubility of the ester.

The concentration of ester in the reaction medium or culture solution is 0°01-5
0% is preferred. The ester may be added batchwise or continuously during the reaction. At that time, it can also be added in a suspended state in water.

The pH of the reaction solution is in the range of 2-11, preferably in the range of 5-8. When the pH of the reaction solution decreases due to the generated carboxylic acid as the reaction progresses, it is preferable to add a neutralizing agent to maintain the optimum pH. The reaction temperature is 5-5
The temperature is 0°C, and the reaction time can be changed as appropriate depending on the amount of enzyme, microbial cells, etc.

Separation and purification of the product from the reaction solution can be carried out by conventional methods such as extraction, recrystallization, column chromatography, and distillation.

Examples 1 to 4 (to) 1.0 g of methyl β-chloroisobutyrate and enzyme 5
001n9 in M/10 phosphate buffer (pH 7.0) 100
The mixture was added to 11 Ll and reacted by shaking at 50°C.

After reacting for 24 hours, the pH of the reaction solution was adjusted to 10, and methyl β-chloroisobutyrate was extracted with an equal volume of chloroform. Next, the pH of the aqueous layer was adjusted to 2.0 with concentrated hydrochloric acid, and then β-chloroisobutyric acid was extracted with an equal volume of chloroform. The optical rotation of the chloroform extract was measured using a digital automatic arm photometer (Model PM-101) manufactured by Union Giken. The results are shown in Table 1. The enzymes used were Lipase Cyken 100 (Osaka Bacteria Research Institute χ Neurose F (manufactured by Ohno Pharmaceutical Co., Ltd.), Pancreatin (manufactured by Ohno Pharmaceutical Co., Ltd.), and Lipase Type ■ (manufactured by Sigma Co., Ltd.).

Table 1) α in CHCl when using a 100 ml cell. shows.

Examples 5 and 6 25 tnl of methyl β-chloroisobutyrate was added to the flask.
M/20 phosphate buffer (pH 7,0') 250 m and Lipase Cyken 100 or pancreatin s, o
1I was added, and the mixture was reacted at 60° C. and pH 7.0 for 64 hours with stirring. pH was adjusted with 10% NaOH. After the reaction was completed, methyl β-chloroinbutyrate was extracted twice with 3QQd of chloroform. Then, the pH of the aqueous layer was adjusted to 2.0 with concentrated hydrochloric acid.
After that, β-chloroisobutyric acid was extracted twice with 300 m/ml of chloroform.

This chloroform extract was subjected to simple distillation under reduced pressure to obtain optically active β-chloroisobutyric acid and methyl ester of the 70-enantiomer. The yield, boiling point and specific rotation of these distillates are shown in Table 2. It was confirmed by NMR and IR analysis that the reaction product obtained by the distillation was β-chloroisobutyric acid or methyl β-chloroisobutyrate.

Examples 7 and 8 Pseudomonas ψfluorescensae FO12055,
Alcaligenes fuecalis IFO13111-2
The strains were placed in a liquid medium (pH 6,8) consisting of 1.0% meat extract, 1.0% peptone, and 10.5% NaC, respectively.
The cells were inoculated into ooml and cultured with shaking at 28°C for 24 hours. 503 ml of the culture solution of each strain thus obtained was centrifuged to collect the bacteria. Transfer the total volume of this bacterial body to 250 ml of M
/20 phosphate buffer (pH 7.0), then 25 ml of methyl (t)-β-chloroisobutyrate was added to
The reaction was allowed to proceed for 48 hours with stirring at a pH of 7.0°C. Tan is 1
Adjusted with 0% NaOH. After the reaction was completed, methyl β-chloroisobutyrate was extracted twice with 300 ml of chloroform.

Next, the pH of the aqueous layer was adjusted to 2.0 with concentrated hydrochloric acid, and then β-chloroisobutyric acid was extracted twice with 300 ml of chloroform.

This chloroform extract was subjected to simple distillation under reduced pressure to obtain optically active β-chloroisobutyric acid and ethyl ester of the 70-enantiomer. It was confirmed by NMR and IR analysis that the obtained products were β-chloroisobutyric acid and methyl β-chloroisobutyrate. The results are shown in Table 3.

Table 3 Examples 9 to 19 The strains shown in Table 4 were mixed with 1.0% meat extract and 1.0% peptone.
% and NaC1O, 5% liquid medium (pH 68)
The cells were inoculated into 100rILt and cultured with shaking at 28°G for 24 hours. After the culture is completed, the culture solution is centrifuged, the resulting bacterial cells are washed with ion-exchanged water, and the total amount is reduced to M/1.
0 phosphate buffer (pH 7,0) at 100 mA'.

To this bacterial suspension, methyl (t)-β-chloroisobutyrate 1
m7! was added, and the mixture was shaken at 60°C for 24 hours to react.

The reaction solution was adjusted to pH 7.0, and methyl β-chloroisobutyrate was extracted with chloroform. Next, the pH of the aqueous layer was lowered to 2.0 with concentrated hydrochloric acid, and then β-chloroisobutyric acid in the aqueous layer was extracted with chloroform.

The optical rotation of the chloroform extracts of methyl β-chloroinbutyrate and β-chloroisobutyric acid at a wavelength of 589 nm was investigated. The results are shown in Table 4.

Table 4

Claims (1)

[Claims] 1. Asymmetric hydrolysis of the ester bond to form an ester represented by the general formula ▲ Numerical formulas, chemical formulas, tables, etc. There are general formulas ▲ mathematical formulas, chemical formulas, tables, etc. ▼ that are characterized by the action of enzymes, microbial culture fluids, microbial cells, or processed products of microbial cells that have the ability to A method for producing an optically active carboxylic acid and its enantiomer ester represented by 2. The method according to claim 1, characterized in that the microorganism used is one of the genus Acinetobacter, Pseudomonas, Escherichia, Staphylococcus, Alcaligenes, Micrococcus, or Agrobacterium. .