JP3161000B2 - Process for producing optically active nitrile and optically active amide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing an optically active nitrile compound and an optically active amide compound. The optically active nitrile compound and the optically active amide compound obtained by the method of the present invention are useful as raw materials for agricultural chemicals and pharmaceuticals, or as intermediates.

[0002]

2. Description of the Related Art As a method for producing an optically active nitrile compound, a microorganism having a nitrile hydrolytic ability is allowed to act on a racemic nitrile compound to produce an optically active nitrile compound from the obtained hydrolysis reaction product. A method is known (JP-A-3-39095). On the other hand, as a method for producing an optically active amide compound, a method for producing an optically active carboxylic acid amide by reacting an optically active carboxylic acid with an amino compound (JP-A-61-50)
965) or racemic carnitinamide with D
-Method for producing L-carnitinamide by reacting -carnitinamide hydrolase
-222797) is known.

[0003]

There is known a method for producing an optically active nitrile compound or an optically active amide compound, respectively. However, the racemic nitrile compound is optically selectively hydrated by biochemical action to obtain an optically active amide compound and its enantiomer nitrile, thereby obtaining an optically active nitrile compound and an optically active amide compound. There is no known method for producing the compound.

[0004]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have found that a microorganism which acts on a racemic nitrile compound and optically selectively hydrates it to produce an optically active amide compound. Was widely searched from the natural world. As a result, a microorganism belonging to the Pseudomonas family having such activity was found, and the present inventors completed the present invention after further study.

[0005]

According to the present invention, an optically active nitrile compound and an optically active amide can be obtained from a racemic nitrile as a raw material using a microorganism in a short time under the condition of normal temperature and normal pressure. Compounds can be prepared. The use of the present invention is industrially very advantageous. Less than,
The present invention will be described in more detail. That is, the present invention
A microorganism belonging to the genus Pseudomonas, optically selective hydration of a racemic nitrile compound represented by the following formula 2, and using a microorganism having an ability to convert into an optically active amide compound, The present invention relates to the production of optically active nitrile compounds and optically active amide compounds.

Embedded image (In the formula, R ₁ represents a substituted or unsubstituted aromatic residue, and R ₂ represents a substituted or unsubstituted alkyl group having 1 to 3 carbon atoms.) As the substituted or unsubstituted aromatic residue, And aryl groups such as phenyl and naphthyl. Examples of the substituent include a halogen atom such as a chlorine atom and a bromine atom, an alkyl group such as a methyl group and an ethyl group, an alkoxy group such as a methoxy group and an ethoxy group, an alkoxycarbonyl group such as a methoxycarbonyl group, an acetyl group and a benzoyl group. And a haloalkyl group such as a trifluoromethyl group.

[0006] The microorganism used in the present invention is a microorganism belonging to the genus Pseudomonas and having an ability to convert a racemic nitrile compound into an optically active amide compound. Particularly preferred is Pseudomonas sp. B21C9 strain (Microtechnical Research Institute No. 3737) described in the present specification. This strain has been sent to the Institute of Microbial Industry and Technology by the National Institute of Advanced Industrial Science and Technology
BP-3737). The bacteriological properties of this strain are described in Japanese Patent Application No. 4-33259.

For cultivation of the strain used in the present invention, various media containing a carbon source, a nitrogen source, inorganic substances and the like commonly used for culturing Pseudomonas microorganisms can be used. Glucose, glycerin, molasses and the like can be used as the carbon source. Examples of the nitrogen source include peptone, yeast extract, soybean flour, corn steep liquor, cottonseed flour, dried yeast, casamino acid, ammonium chloride, ammonium nitrate, ammonium sulfate, and urea. As inorganic substances, potassium, sodium, magnesium, iron,
Salts such as manganese, and phosphates such as potassium chloride, sodium chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, potassium phosphate, and sodium phosphate can be used. Further, a cyano compound such as crotononitrile may be added as a substance that promotes the reaction activity in the present invention. Culture is usually performed aerobically, and aeration and agitation culture is appropriate. The culture temperature is 2
0-35 ° C., preferably 25-30 ° C., pH 6-
8 is preferred. The culture time varies depending on various conditions, but is preferably about 1 to 3 days.

In the present invention, the production of an optically active nitrile compound and an optically active amide compound using the above-mentioned cultured cells is performed as follows. According to the method described above, the culture solution cultured for 1 to 3 days, or the cells separated from the culture solution, are suspended in water or an arbitrary buffer, and a racemic nitrile compound is added thereto to carry out a reaction. The appropriate concentration of the cells is 0.01 to 5% by weight. The addition concentration of the racemic nitrile compound is 0.01 to 10% by weight.
Degree, preferably 0.1 to 5% by weight. The racemic nitrile compound can be added as a liquid, and need not be dissolved. During the reaction, the reaction solution is preferably stirred and dispersed by an arbitrary method. For example, a commonly used shaker or a reciprocating shaker can be used. The reaction temperature is 5 to 50 ° C, preferably
10 to 35 ° C., the reaction pH is 4 to 11, preferably 6 to
9 The reaction is usually sufficient for a period of 10 minutes to 24 hours. By arbitrarily setting the reaction time, the ratio of the optical isomers of the nitrile compound and the amide compound can be changed. The optically active nitrile compound and the optically active amide compound are recovered from the reaction solution after the completion of the reaction by adding hydrochloric acid to the reaction solution to adjust the pH to 1 to 2 and then reacting the reaction product and unreacted substrate with an appropriate solvent. And after extracting by-products,
The reaction can be carried out by a method of isolating and recovering an optically active amide compound as a reaction product or an optically active nitrile compound as an unreacted substrate using silica gel column chromatography or the like. Optically active nitrile compounds and optically active amide compounds produced by the above methods are ibuprofen, ketoprofen,
It is useful as a raw material for agricultural chemicals and pharmaceuticals such as naproxen or α- (4-chlorophenyl) -isovaleric acid, or as an intermediate.

[0009]

Next, an embodiment of the present invention will be described. However, this embodiment is merely an example and does not limit the present invention. Example 1 Contains 1% glycerol, 0.5% polypeptone, 0.3% yeast extract, 0.3% malt extract, pH 7.2.
1% of Pseudomonas sp. B21C9 strain previously cultured in the same medium was inoculated into 125 mL of the sterilized medium,
Shaking culture was performed at 26 ° C. for 2 days. The bacterial cells obtained by centrifuging the culture solution were placed in a 50 m 0.1 M phosphate buffer (pH 8.0).
L, and suspended in 2-phenylpropionitrile (1.
269 g, 9.5 mmol) at 150 ° C. at 26 ° C.
The reaction was carried out with shaking at pm. After 15 minutes, 5 mL of 5% hydrochloric acid was added, and then methyl isobutyl ketone was added to extract unreacted substrate and reaction product. When the composition of the extracted unreacted substrate and the reaction product was quantified by gas chromatography, 6.0 mmol of 2-phenylpropionitrile was 3.4 mmol of 2-phenylpropionamide, and no 2-phenylpropionic acid was detected. Was. Next, the reaction mixture was concentrated, spotted on a PLC silica gel plate (20 × 20 cm, 2 mm thick), and developed in a developing solvent (chloroform / ethyl acetate (１ ／)). After development, 2-phenylpropionitrile or 2-phenylpropionitrile, or 2-phenylpropionitrile or 2-phenylpropionitrile
-Phenylpropionamide was eluted with diethyl ether. Then, the diethyl ether was concentrated to obtain 2-phenylpropionitrile or 2-phenylpropionamide. The optical isomer ratio of the obtained 2-phenylpropionitrile or 2-phenylpropionamide is calculated as follows, by chemically converting 2-phenylpropionitrile or 2-phenylpropionamide to 2-phenylpropionic acid. After hydrolysis, it can be measured. 131 mg (1 mmol) of 2-phenylpropionitrile was hydrolyzed in 5 mL of 70% sulfuric acid while cooling under reflux in a water bath. After about 1 hour, the formed 2-phenylpropionic acid was extracted with chloroform, and the ratio of the optical isomers was measured by high performance liquid chromatography under the following conditions. The ratio of the S-form to the R-form was 33.9: 66. It was one. Column: SUMICHIRAL OA-2000 (5 μm, 4 mm IDx25 cm) Solvent: n-hexane / 1,2-dichloroethane / ethanol / acetic acid (490/9/1/1) Flow rate: 1.0 mL / min detection; UV (254 nm) 2- 136 mg (0.9 mmol) of phenylpropionamide was hydrolyzed in 5 mL of an aqueous solution of 84 mg (1.5 mmol) of potassium hydroxide while cooling under reflux in a water bath. After about 2 hours, the formed 2-phenylpropionic acid was extracted with chloroform, and the ratio of the optical isomers was measured by high performance liquid chromatography under the above conditions. The ratio of the S-form to the R-form was 64.7: 35. Was 3.

Example 2 Pseudomonas sp. B21C9 strain previously cultured in 1.2 L of the sterilizing medium described in Example 1 was cultured in the same medium at 1%.
The cells were inoculated and cultured with shaking at 26 ° C. for 2 days. The cells obtained by centrifuging the culture solution were subjected to 0.2M phosphate buffer (pH 8.
0) After suspending in 200 mL, 2-phenylpropionitrile (5.346 g, 40 mmol) was added, and 26
The reaction was carried out at 150 ° C. with shaking at 150 rpm. Twenty minutes later, 30 mL of 5% hydrochloric acid was added, and then diethyl ether was added to extract unreacted substrate and reaction product. When the composition of the extracted unreacted substrate and reaction product was quantified by gas chromatography, 8.3 mmol of 2-phenylpropionitrile was 31.1 mmol of 2-phenylpropionamide, and no 2-phenylpropionic acid was detected. Was. Next, the reaction mixture is concentrated and subjected to silica gel column chromatography (adsorbent; Wakogel).
C200, column; 4 cm ID x 40 cm) and eluted with chloroform / ethyl acetate (1/3). The eluted fraction containing 2-phenylpropionitrile and not containing 2-phenylpropionamide was concentrated to obtain 2-phenylpropionitrile. The 2-phenylpropionitrile was chemically hydrolyzed to 2-phenylpropionic acid using the method described in Example 1, and the optical isomer ratio was measured. The ratio is 12.
It was 5 to 87.5.

Continuation of the front page (56) References JP-A-2-2577893 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 41/00 BIOSIS (DIALOG)

Claims (2)

(57) [Claims] 1. A racemic nitrile compound represented by Formula 1 is acted on by a microorganism belonging to the genus Pseudomonas to optically selectively hydrate to obtain an optically active amide compound and an enantiomeric nitrile compound. A process for producing an optically active nitrile compound and an optically active amide compound, characterized by the following: (In the formula, R ₁ represents a substituted or unsubstituted aromatic residue, and R ₂ represents a substituted or unsubstituted alkyl group having 1 to 3 carbon atoms.) 2. The method of claim 1 wherein the microorganism is Pseudomonas sp. (Ps
2. The method for producing an optically active nitrile compound and an optically active amide compound according to claim 1, wherein the strain is Eudomonas sp.