JPH05192190A - Production of optically active carboxylic acid derivative - Google Patents

Abstract

PURPOSE:To obtain the subject compound in high yield and optical purity at a low cost by treating a specific compound with microorganisms of the genus Corynebacterium, etc., or its processed product. CONSTITUTION:A cultured product or treated bacterial cell (C) capable of converting a racemic nitrile (A) of formula I [R1 is (un)substituted aryl, heterocyclic group or cycloalkyl group; R2 is halogen atom, hydroxy, (un)substituted alkyl, alkoxy or NH-Boc group; (n) is 1-5] into the optically active carboxylic acid derivative (B) of formula II is selected from Corynebacterium nitrilophillus strain and microorganisms of the genus Alcaligenes. The component A and the component B are added to an aqueous medium such as water in amounts of 0.3-30-wt.% and 0.05-20wt.%, respectively, and made to react with each other at 5-80 deg.C and pH 4-11. The obtained reaction product is extracted and purified to obtain an optically active carboxylic acid derivative (e.g. 2-hydroxy-3- phenylpropionic acid).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing an optically active carboxylic acid derivative. The optically active substance obtained by the method of the present invention is a raw material for producing optically active pharmaceutical agents such as ACE inhibitors and renin inhibitors, agricultural chemicals such as herbicides and fungicides and their raw materials, raw materials for ferroelectric liquid crystals, and further optical It is a compound useful as a resolving agent.

[0002]

As a method for producing an optically active carboxylic acid derivative, for example, a method for producing optically active 2-hydroxy-4-phenylbutyric acid is obtained by reducing 2-oxo-4-phenylbutyric acid with lactate dehydrogenase. Method (JP-A-2-398
93) and a method of optically resolving using an optically active amine (JP-A-1-308244). In addition, as a method for producing an optically active α-halogenocarboxylic acid, an optically active α-halogenocarboxylic acid is used.
-Method of obtaining ureidocarboxylic acid by diazotization treatment in the presence of halogen ions in an acidic aqueous solution of mineral acid (JP-A 1-2)
99249) and the like are known.

The technique of using the above internal dehydrogenase requires expensive nicotinamide adenine dinucleotide as a coenzyme. In addition, the optical resolution method using an optically active amine is not an industrial technology because it involves many technical problems such as recovery of expensive optical resolution agents in addition to multistage purification operations. It is difficult to obtain a product with high optical purity by the method using a synthesis technique, because the optically active material as a raw material is expensive and a racemate occurs during the reaction.

The present inventors have already invented and applied for a patent for a method for enzymatically producing an optically active carboxylic acid from a racemic nitrile compound (JP-A-2-
84198, JP-A-3-224496). However, the optically active carboxylic acid derivative of the chemical formula 2 of the present invention is not necessarily high in activity and is not satisfactory in optical purity.

[0005]

The present invention provides an optically active carboxylic acid derivative useful as an industrial raw material for medicines,
It is intended to provide a production method for maintaining a high optical purity in a high yield by the action of a microorganism or a preparation thereof from a corresponding racemic nitrile.

[0006]

Means for Solving the Problems In order to solve the above problems, the present inventors have advanced the search for a microorganism capable of producing a specifically optically active carboxylic acid derivative, and as a result,
The present inventors have found a microorganism having the ability to convert a racemic nitrile represented by the chemical formula 1 into an optically active carboxylic acid derivative represented by the chemical formula 2. The microorganism found in the present invention did not metabolize and decompose the produced carboxylic acid or racemize, and thus found that the optical purity can be kept extremely high, and completed the present invention.

That is, the present invention allows a racemic nitrile represented by the chemical formula 1 to act with a microorganism belonging to the genus Corynebacterium or the genus Alcaligenes or a preparation thereof,
A method for producing an optically active carboxylic acid derivative, which comprises obtaining an optically active carboxylic acid derivative represented by Chemical Formula 2.

In the formula 1, R ₁ represents a substituted or unsubstituted aryl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted cycloalkyl group, and R ₂ represents a halogen atom, a hydroxy group, a substituted or It represents an unsubstituted alkyl group, a substituted or unsubstituted alkoxy group, or a -NH-Boc group. Further, n represents an integer of 1 to 5.

More specifically, examples of the aryl group of R ₁ include a phenyl group and a naphthyl group. As the heterocyclic group, nitrogen as a hetero atom,
A heterocyclic ring containing 1 to 3 of at least one of oxygen and sulfur and composed of 3 to 15 carbons is preferable. Examples of such a heterocycle include thiophene and indole. The cycloalkyl group preferably has 3 to 8 carbon atoms, and particularly has 3 carbon atoms.
Those of from 6 to 6 are preferable.

The hydrogen bonded to carbon and nitrogen of the above-mentioned aryl group, heterocyclic group and cycloalkyl group may be substituted with various substituents. Examples of such a substituent include halogen such as fluorine, chlorine, iodine and bromine, a hydroxy group, a thiol group, a nitro group, an amino group, and an alkoxy group having 1 to 8 carbon atoms. Hydrogen bonded to carbon and nitrogen in these substituents may be further substituted with the above-mentioned substituents.

Examples of the halogen atom for R ₂ include fluorine, chlorine, iodine and bromine. An alkyl group,
The alkoxy group preferably has 1 to 8 carbon atoms, and particularly preferably has 1 to 3 carbon atoms. Hydrogen bonded to carbon of the alkyl group and the alkoxy group may be substituted with the above-mentioned substituent.

Typical examples of the compound of the chemical formula 2 which can be obtained by the production method of the present invention are shown below. 2-hydroxy-3
-Phenylpropionic acid, 2-hydroxy-4-phenylbutyric acid, 2-hydroxy-5-phenylpentanoic acid, 2-bromo-4-phenylbutyric acid, 2-chloro-4-
Phenyl butyric acid, 2-bromo-3-phenylpropionic acid, 2-chloro-3-phenylpropionic acid, 2-methoxy-3-phenylpropionic acid, 4- (3-tolyl)
2-hydroxybutyric acid, 4- (2-trifluoromethylphenyl) -2-hydroxybutyric acid, 4- (4-fluorophenyl) -2-hydroxybutyric acid, 4- (2-chlorophenyl) -2-hydroxybutyric acid, 4 -(4-Bromophenyl) -2-hydroxybutyric acid, 4- (2-hydroxyphenyl) -2-hydroxybutyric acid, 2-hydroxy-4-cyclohexylbutyric acid, 2-hydroxy-4-thiophenebutyric acid, 2- (Boc- Amino) -3
-R or S form of cyclohexylpropionic acid, or (+) or (-) form. The compound represented by the chemical formula 1, which is the starting material compound in the present invention, can be produced by a known method. [For example, Ann. de Chimie
20 , 97 (1933)]

The microorganism used in the present invention is a microorganism selected from the microorganisms belonging to the genus Corynebacterium and the genus Alcaligenes. Specifically, the following microorganisms can be used. Corynebacterium nitrilophilus ATCC 21419, Corynebacterium sp. KO-2-4 (FERM BP-23
53), Corynebacterium sp. HP-226
8 (FERM P-1263), Corynebacterium sp. HP-643 (FERM P-1263)
0), Alcaligenes SP HP-458 (FE
RM P-12632), Alcaligenes SP
HP-611 (FERM P-12633), Alcaligenes SP HP-797 (FERM P-12)
634), Alcaligenes SP HP-817
(FERM P-12635), Alcaligenes SP HP-938 (FERM P-12636).

Corynebacterium sp. KO-2
-4 has been internationally deposited with the Institute of Microbial Science and Technology with the above number, and mycological properties are described in JP-A-3-224496. HP-2268, HP-643, HP-4
58, HP-611, HP-797, HP-817, H
The P-938 strain was newly isolated as a nitrile-assimilating bacterium from soils in Miyazaki and Kumamoto prefectures, and all of them have been deposited at the Institute of Microbial Science and Technology with the above numbers. The mycological properties of these 7 strains are as shown in Tables 1-8.

[0015]

[Table 1]

[0016]

[Table 2]

[0017]

[Table 3]

[0018]

[Table 4]

[0019]

[Table 5]

[0020]

[Table 6]

[0021]

[Table 7]

[0022]

[Table 8]

The above-mentioned mycological properties are based on the Bergy's Manual of Determinative Bacteriology, 8th Edition (1974), and "Manual of Clinical Microbiology, 4th Edition (1985)". Classified.

HP-458, HP-611, HP-79
7, HP-817 and HP-938 strains are all aerobic,
It is a Gram-negative bacillus. In addition, it has flagella and has motility, catalase positive, oxidase positive, O
Since it was F test, it was identified as the genus Alcaligenes.

HP-643 is an aerobic, Gram-positive, catalase-positive spore-free bacillus, which does not set flagella and is not motile. Furthermore, since it has a polymorphism such that it is rod-shaped in the early stage of development, accompanied by snapping, and later ruptured into a short rod-shaped, it is clear that it belongs to coryneform type. Also,
It was identified as a bacterium belonging to the genus Corynebacterium due to its lack of cellulose degrading ability, no acid resistance, and no absolute aerobic ability.

HP-2268 is a facultatively anaerobic gram-positive, catalase-positive spore-free bacillus that does not grow flagella and is not motile. Moreover, since it has multiple formations, it belongs to the coryneform type. In addition, it was identified as a bacterium belonging to the genus Corynebacterium because it has no cellulose decomposing ability, is not anti-acidic, and has an OF test of F.

The reaction method in the present invention is carried out by contacting a microorganism or a preparation thereof with a racemic nitrile represented by the chemical formula 1. The microorganism or its preparation is specifically a culture obtained by culturing the above-mentioned microorganism, a treated bacterial cell collected from the microorganism (for example, a disrupted bacterial cell or an enzyme separated and extracted from the bacterial cell), and a bacterial cell. Alternatively, the treated cells are immobilized by an appropriate method.

Cultivation of the microorganism used in the present invention can be carried out according to a known method. The medium to be used is available as a known nutrient source for general microorganisms, glucose, glycerin, ethanol, sucrose, dextrin, acetic acid, carbon sources such as ethyl oleate, ammonium sulfate, ammonium chloride, nitrogen sources such as ammonia, Organic nutrient sources such as yeast extract, malt extract, peptone and meat extract and inorganic nutrient sources such as phosphoric acid, magnesium, potassium, iron, cobalt, manganese and lanthanum can be used in appropriate combination. In addition, a cyano compound such as isobutyronitrile or an amide compound such as caprolactam may be added as a substance that increases the reaction activity of the microorganism in the present invention. The pH of the medium may be selected in the range of 4 to 10, and the culture temperature is 15 ° C to 50 ° C, preferably 25 ° C.
To 40 ° C. The number of days of culture may be 1 to 10 days until the activity becomes maximum.

The reaction conditions in the present invention will be described below.
The reaction medium for carrying out the reaction comprises water, an aqueous medium such as a buffer solution or a culture solution, a mixed medium of an aqueous medium and a water-soluble organic solvent such as dimethylsulfoxide and methanol, and further, an aqueous medium and a water-soluble organic solvent. Two-phase media can be used. The nitrile represented by the chemical formula 1 is added to the reaction medium in a powder or liquid state as it is, or after being dissolved in a suitable solvent. Further, a suitable surfactant may be added to the reaction medium in order to obtain higher optical purity. As the surfactant, those that do not inhibit or inactivate the enzyme are desirable, and specifically, Amate 105 (Kao Corporation), Emulgen 320P (Kao Corporation), Ripearl OCJ (Lion Corporation), Tween. 80, Tween 20, lecithin and the like. The amount of these surfactants to be used varies depending on the type of substrate and the type of reaction medium, but is usually 0.01% to 10% by weight, particularly preferably 0.1% to 2% by weight relative to the reaction system. is there.

The addition concentration of the nitrile represented by the chemical formula 1 is 0.01 to 80% by weight, particularly preferably 0.3.
% To 30% and does not have to be completely dissolved in the reaction medium. When bacterial cells are used in the reaction, the bacterial cell concentration may usually be in the range of 0.05% by weight to 20% by weight. The reaction temperature is 5 ° C to 80 ° C, preferably 15 ° C to 50 ° C, and the reaction pH is 4 to 11, preferably 6 to 10. The consumed nitrile represented by the chemical formula 1 may be continuously or intermittently replenished and added so that the concentration in the reaction solution is maintained within the above range. The reaction may be stopped within the range in which the optical purity of the optically active carboxylic acid derivative formed does not decrease.

Recovery of the target product in the present invention is carried out as follows. After removing insoluble matters such as bacterial cells from the reaction-terminated solution, the pH is adjusted to alkali, preferably 8.5 to 1
2, diethyl ether, chloroform, hexane,
Unreacted nitrile represented by the chemical formula 1 is extracted and removed with a solvent such as ethyl acetate, and then the pH is made acidic, preferably 1.0 to 2.0, and the nitrile represented by the above formula is extracted with the above solvent to obtain a target product. Collect things. Furthermore, the target product is purified by column chromatography using silica gel or an ion exchange resin, or by a crystallization method.

The reaction mechanism in the present invention is that nitrilase or nitrile hydratase, which is an enzyme for converting nitrile to carboxylic acid, and amidase selectively act on only one isomer of racemic nitrile, that is, It is considered that this is based on the fact that the reaction rate by the enzyme varies greatly depending on the optical isomer. Therefore, when an optically active carboxylic acid is produced according to the present invention, an optically active nitrile or amide remains or is formed as an unreacted substance or a reaction intermediate. Then, these can be collected by a known method. The recovered nitrile or amide is
It can be derived into an optically active compound which is useful as a drug.
Further, the recovered nitrile can be easily racemized by a reaction using an alkali such as ammonia, and can be used as a racemic nitrile as a raw material of the present invention. Further, when R ₂ is a hydroxy group in the chemical formula 1, a corresponding aldehyde and hydrocyanic acid (a salt such as sodium hydrocyanic acid or potassium hydrocyanic acid may be used) can be used as a raw material instead of hydroxynitrile.

[0033]

EXAMPLES Next, the present invention will be described in more detail by way of examples. However, these examples do not limit the scope of the present invention. Example 1 Production of R-2-hydroxy-4-phenylbutyric acid 1% glucose, 0.5% yeast extract, 0.5 peptone
%, 1 potassium phosphate 0.12%, 2 potassium phosphate 0.08%, magnesium sulfate 0.02%, ferrous sulfate 0.003%, sodium chloride 0.1%, isobutyronitrile 0.1 %, PH 7.2 sterilized medium 2
Corynebacterium pre-cultured in the same medium in 00 ml
2% of HP HP-2268 strain was inoculated and 4
It was cultured for 8 hours. After culturing, collect the cells by centrifugation and add 70 ml of 0.05M phosphate buffer (pH 7.5).
700 mg of racemic 2-hydroxy-4-phenylbutyronitrile was added and the mixture was allowed to stand at 32 ° C. for 35 minutes.
Reacted for hours. After completion of the reaction, the cells were removed by centrifugation, the pH of the supernatant was adjusted to 9.5, and 60 ml of chloroform was added to the unreacted 2-hydroxy-4.
-Phenylbutyronitrile was removed by extraction. PH of water layer
After adjusting to 1.5, 60 ml of chloroform was added to extract the desired product. This was concentrated under reduced pressure and then purified by a silica gel column to give R-2-hydroxy-
255 mg of white crystals of 4-phenylbutyric acid were obtained.

[0034]

[Equation 1] Melting point: 114-116 ° C

The substance was single in high performance liquid chromatography, and the IR and NMR spectra also supported the structure. Further, the optical purity determined by high performance liquid chromatography under the following conditions was 95% ee.

Column: CHIRALCEL OD-R
(Daicel Chemical Industries, Ltd.) Solvent: 0.05M 1 potassium phosphate (pH 3.
0): Acetonitrile = 75: 25 Flow rate; 0.3 ml / min Detection: 215 nm

Example 2 Production of R-2-hydroxy-4-phenylbutyric acid In 200 ml of the same sterilizing medium as in Example 1, 2% of Alcaligenes sp. HP-458 strain previously cultivated in the same medium was used.
The cells were inoculated and cultured at 32 ° C. for 30 hours. After the culture was completed, the cells were collected by centrifugation, suspended in an Erlenmeyer flask containing 50 ml of 0.05M phosphate buffer, and then 2.5 ml.
500 mg of racemic 2-hydroxy-4-phenylbutyronitrile dissolved in dimethylsulfoxide was added and reacted at 32 ° C. for 10 hours. When analyzed by high performance liquid chromatography after completion of the reaction, it was found to be 19
0 mg of R-2-hydroxy-4-phenylbutyric acid had formed. The reaction supernatant was adjusted to pH 9.5 and 120 ml
Was extracted with chloroform to recover 240 mg of unreacted 2-hydroxy-4-phenylbutyronitrile. After adjusting the pH of the aqueous layer to 1.5, 60 ml of chloroform was added to extract the desired product. This was concentrated under reduced pressure and then purified by a silica gel column to give R-2
179 mg of hydroxy-4-phenylbutyric acid were obtained. When the optical purity was analyzed by the high performance liquid chromatography shown in Example 1, it was 93.5% ee.

Example 3 Production of R-2-bromo-4-phenylbutyric acid In 200 ml of the same sterilizing medium as in Example 1, 1% of Alcaligenes spp HP-611 strain previously cultivated in the same medium was used.
The cells were inoculated and cultured at 25 ° C for 40 hours. After the completion of the culture, the cells were collected by centrifugation, suspended in an Erlenmeyer flask containing 90 ml of 0.01 M phosphate buffer (pH 8.0), and then dissolved in 10 ml of dimethyl sulfoxide to prepare racemic 2 -Bromo-4-phenylbutyronitrile 1 g
Was added and reacted at 32 ° C. for 10 hours. After the reaction was completed, the bacterial cells were removed by centrifugation and the pH of the supernatant was adjusted to 8.5.
Adjust to 80 ml of chloroform and add the unreacted 2-
Bromo-4-phenylbutyronitrile was removed by extraction.
Next, after adjusting the pH of the aqueous layer to 1.5, 120 ml of chloroform was added to extract the desired product. This was concentrated under reduced pressure and then purified by a silica gel column to obtain 350 mg of oily R-2-bromo-4-phenylbutyric acid.

[0039]

[Equation 2]

The substance was single in high performance liquid chromatography, and the IR and NMR spectra indicated the structure. Further, the optical purity determined by high performance liquid chromatography under the following conditions was 98.6% ee.

Column: CHIRALCEL OD-R
(Daicel Chemical Industry Co., Ltd.) Solvent: 0.05M 1 potassium phosphate (pH 2.
5): Acetonitrile = 7: 3 Flow rate; 1.0 ml / min Detection: 215 nm

Example 4 Production of R-2-bromo-4-phenylbutyric acid In the same manner as in Example 1, 100 ml of sterilized medium was inoculated with 2% of a microorganism previously cultivated in the same medium and cultured at 30 ° C. for 48 hours. .. After culturing, collect the cells by centrifugation and
After suspending in an Erlenmeyer flask containing 30 ml of M phosphate buffer, 90 mg of racemic 2-bromo-4-phenylbutyronitrile was added and reacted at 32 ° C. for 2 hours. The production amount and optical purity of the produced R-2-bromo-4-phenylbutyric acid were analyzed by high performance liquid chromatography. The results are shown in Table 9.

[0043]

[Table 9]

[0044]

INDUSTRIAL APPLICABILITY By utilizing the present invention, various optically active carboxylic acids are used as raw materials of optically inactive substances.
Since it can be produced using a microorganism under normal temperature and normal pressure reaction conditions, it is very economically advantageous. Furthermore, according to the present invention, an optically active carboxylic acid having an extremely high optical purity of 90% ee or higher can be obtained in good yield. Although the present invention has been described in detail, and particularly in its embodiment with reference to Examples, various changes and modifications may be made within the scope of the present invention without departing from the spirit and scope of the present invention. , Obvious to those in the art.

Claims (1)

[Claims] 1. An optically active carboxylic acid represented by the following chemical formula 2 is obtained by reacting a racemic nitrile represented by the following chemical formula 1 with a microorganism belonging to the genus Corynebacterium or Alcaligenes or a preparation thereof. A method for producing an optically active carboxylic acid derivative, which comprises: [Chemical 1] (In the formula, R ₁ represents a substituted or unsubstituted aryl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted cycloalkyl group, and R ₂ represents a halogen atom, a hydroxy group, a substituted or unsubstituted alkyl group. Represents a group, a substituted or unsubstituted alkoxy group, or a —NH—Boc group, and n represents an integer of 1 to 5.) (In the formula, R ₁ , R ₂ and n are the same as above.)