JP3144024B2 - Process for producing (S) -2- (4-chlorophenyl) -3-methylbutyric acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing (S) -2- (4-chlorophenyl) -3-methylbutyric acid represented by the formula (7). (S) obtained by the method of the present invention
2- (4-Chlorophenyl) -3-methylbutyric acid is a compound useful as a raw material for insecticides.

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[0002]

2. Description of the Related Art (S) -2- (4-chlorophenyl)-
As a method for producing 3-methylbutyric acid from racemic 2- (4-chlorophenyl) -3-methylbutyronitrile or 2- (4-chlorophenyl) -3-methylbutyramide by a biochemical action, A method using Brevibacterium is known (Japanese Patent Laid-Open No. 1-2401).
99).

[0003]

At present, (S) -2- (4-chlorophenyl) -3 is prepared using Brevibacterium.
A method for producing -methylbutyric acid is known, but its production rate is low, and the optical purity of (S) -2- (4-chlorophenyl) -3-methylbutyric acid obtained is not sufficient.
Therefore, a higher generation rate and a higher optical purity (S)
There is a need for a method for producing -2- (4-chlorophenyl) -3-methylbutyric acid.

[0004]

Means for Solving the Problems The present inventors have set out 2- (4-chlorophenyl) -3- to solve the above-mentioned problems.
From methylbutyronitrile or 2- (4-chlorophenyl) -3-methylbutyramide, (S) -2- (4
Microorganisms that efficiently produce (-chlorophenyl) -3-methylbutyric acid were widely searched from nature. As a result, they found that the bacterium B21C9 strain isolated from the soil in Misawa City, Aomori Prefecture was a microorganism meeting such a purpose, and made further studies to complete the present invention.

According to the present invention, racemic 2- (4-chlorophenyl) -3-methylbutyronitrile or racemic 2- (4-chlorophenyl) -3-methylbutyronitrile can be obtained in a short time under the condition of normal temperature and normal pressure using a microorganism. (S) -2- (4-Chlorophenyl) -3-methylbutyric acid can be produced using 2- (4-chlorophenyl) -3-methylbutyramide as a raw material.
That is, according to the present invention, racemic 2- (4-chlorophenyl) -3-methylbutyronitrile or 2- (4-chlorophenyl) -3-methylbutyronitrile
Obtaining (S) -2- (4-chlorophenyl) -3-methylbutyric acid having a production rate of 43 to 50% and an optical purity of 100% from (4-chlorophenyl) -3-methylbutyramide within 40 hours (However, the generation rate =
(S) -2- (4-chlorophenyl) -3-methylbutyric acid / racemic 2- (4-chlorophenyl) -3-methylbutyronitrile or 2- (4-chlorophenyl) -3
-Defined as methyl butyramide x 100. Therefore, the present invention is very industrially effective. Less than. The present invention will be described in more detail. That is, the present invention belongs to the genus Pseudomonas, and represents 2- (4-chlorophenyl) -3-methylbutyronitrile represented by Formula 8 or 2- (4-chlorophenyl) -3-methyl represented by Formula 9 A microorganism having an ability to convert butylamide to (S) -2- (4-chlorophenyl) -3-methylbutyric acid represented by Formula 10 is used, wherein (S) -2- (4- Chlorophenyl) -3-methylbutyric acid.

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[0005] The microorganism used in the present invention belongs to the genus Pseudomonas and is 2- (4-chlorophenyl) -3-methylbutyronitrile or 2- (4-chlorophenyl)-.
It is a microorganism having the ability to convert 3-methylbutyramide to (S) -2- (4-chlorophenyl) -3-methylbutyric acid. Particularly preferred is Pseudomonas sp. Strain B21C9 (Microtechnical Research Institute No. 3737) described in the present specification. The mycological properties of this strain are as follows. (A) Form 1) Cell shape and size: Shape; rod size; 0.5 to 0.8 × 0.8 to 2 μm 2) Presence or absence of cell polymorphism: None 3) Presence or absence of motility: Yes, polar flagella 4) Presence or absence of spores: No 5) Gram stain: Negative 6) Acid resistance: No (b) Growing state 1) Broth agar Plate culture: Circumferential, convex, glossy, light brown 2) Broth agar Slope culture: glossy, light brown 3) Liquid broth culture: Growing uniformly turbid 4) Broth gelatin stab culture: not liquefied 5) Litmus milk: does not coagulate and turns blue (c) Physiological properties 1) Nitrate reduction: +2) Denitrification: -3) MR test: -4) VP test: -5) Indole formation: -6) Hydrogen sulfide formation: +7) Starch hydrolysis: -8) Utilization of acid: medium of Coser; + medium of Christiansen; + 9) utilization of inorganic nitrogen source: NaNO ₃ ; + (NH ₄ ) ₂ SO ₄ ; +10) Pigment production: King A medium;-King B medium; + (fluorescent dye) 11) Urease: +12) Oxidase: +13) Catalase: +14) Range of growth: pH; 8 temperature; 3-39 ° C 15) Attitude to oxygen: aerobic 16) OF test: O (D) Other properties 1) Accumulation of poly-β-hydroxybutyric acid: +2) Arginine dihydrolase: +3) GC content: 61.2%

[0006] The above properties are described by Barge's bacterial taxonomy (Be
A search by rgey's Manual of Systematic Bac-teriology) reveals that the genus belongs to the genus Pseudomonas, but nothing corresponding to the species is described. In addition, a known Pseudomonas strain reported to have an activity of converting a nitrile compound (Japanese Patent Publication No.
-37951, and JP-B-62-257386). Therefore, the present inventors identified this strain as a new strain belonging to Pseudomonas and named it Pseudomonase sp. P21B9.
This strain has been deposited with the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology as Microfabrication Article No. 3737 (FERMBP-3737).

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. Examples of the carbon source include glucose, glycerin, and molasses.
Nitrogen sources include peptone, yeast extract, soy flour, corn steep liquor, cottonseed flour, dried yeast, casamino acid,
Examples include ammonium chloride, ammonium nitrate, ammonium sulfate, and urea. Inorganic substances include salts of potassium, sodium, magnesium, iron, manganese and the like, and phosphates such as potassium chloride, sodium chloride,
Magnesium sulfate, ferrous sulfate, manganese sulfate, potassium phosphate, sodium phosphate and the like. Further, as a substance which promotes the reaction activity in the present invention, a cyano compound such as crotononitrile or an amide compound such as crotonamide may be added. Culture is usually performed aerobically, and aeration and agitation culture is appropriate. Culture temperature is 20-3
5 ° C., preferably 25-30 ° C., and the pH is preferably 6-8. The culture time varies depending on various conditions.
About 3 days is preferable.

[0008] In the present invention, the production of (S) -2- (4-chlorophenyl) -3-methylbutyric acid) using the above cultured cells is performed as follows. According to the above-described method, the culture solution in which the cells were cultured for 1 to 3 days, or the cells separated from the culture solution were suspended in water or a buffer solution such as a phosphate buffer or a tris-hydrochloric acid having a pH of 6 to 9, and 2
-(4-Chlorophenyl) -3-methylbutyronitrile or 2- (4-chlorophenyl) -3-methylbutyramide is added to carry out the reaction. The concentration of the cells was 0.
It is suitably from 0.01 to 10% by weight. 2- (4-chlorophenyl) -3-methylbutyronitrile, or 2- (4
-Chlorophenyl) -3-methylbutyramide is added at a concentration of about 0.01 to 10% by weight, preferably 0.1 to 10% by weight.
5% by weight. They can be added as liquid or powder, and do not need to be completely dissolved in the reaction solution. During the reaction, the reaction solution is preferably stirred and dispersed by an arbitrary method. For example, a commonly used stirrer or a reciprocating shaker can be used. The reaction temperature is 15 to 65 ° C, preferably 20 to 40.
C., the reaction pH is 4 to 11, preferably 6 to 9. The reaction is usually carried out for 1 to 40 hours. (S) -2
Most preferably, the reaction is stopped after the production rate of-(4-chlorophenyl) -3-methylbutyric acid reaches 50%. However, even when the reaction is stopped before the production rate reaches 50%, there is no problem in the optical purity of the produced (S) -2- (4-chlorophenyl) -3-methylbutyric acid, and the reaction time is reduced. (S) -2 generated even if extended
Since there is no problem in the optical purity of-(4-chlorophenyl) -3-methylbutyric acid, the reaction time can be arbitrarily set. (S) -2- (4-
Chlorophenyl) -3-methylbutyric acid is recovered by adding hydrochloric acid to the reaction solution to adjust the pH to 1 to 2, then extracting the reaction product, unreacted substrate and intermediate with an appropriate solvent, and then performing silica gel chromatography. And recovering (S) -2- (4-chlorophenyl) -3-methylbutyric acid, which is a reaction product, using the method described above.

[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 Glycerol 1%, polypeptone 0.5%, yeast extract 0.3%, malt extract 0.3%, pH 7.2
Inoculated with 1% of Pseudomonas sp. B21C9 strain previously cultured on the same medium in 400 ml of the sterilized medium,
Shaking culture was performed at 26 ° C. for 2 days. After the culture, the cells were collected by centrifugation, and added to a 0.1 M phosphate buffer (pH
7.0) After suspending by adding 40 ml, racemic 2
-(4-Chlorophenyl) -3-methylbutyronitrile (60 mg, 300 μmol) was added, and the mixture was heated at 26 ° C. and 500 r.
The reaction was carried out with stirring at pm. After 20 hours, the reaction was terminated, and the cells were removed by centrifugation.
8 ml of acetonitrile was added and the composition and content of the reaction mixture was determined by gas chromatography. Then, after evaporating the reaction mixture to dryness, the residue is mixed with hexane and ethanol (4: 1, 1 ml in volume ratio).
Eluted. The optical purity of the eluted reaction product was measured by high performance liquid chromatography under the following conditions. Column; SUMIPAX OA-4500 (5μ, 4mm IDx25cm) Solvent: n-hexane / 1,2-dichloroethane / ethanol / acetic acid (350/150
/0.5/0.1) Flow rate; 1.0 ml / min detection; UV (254 nm) Table 1 shows the amount of reaction products contained in the reaction mixture and the optical purity.

【table 1 】

Example 2 40 mL of the cell suspension prepared by the method described in Example 1
Was added with racemic 2- (4-chlorophenyl) -3-methylbutyramide (63 mg, 300 μmol).
The reaction was performed at 6 ° C. and 500 rpm. After the reaction was completed for 40 hours, the composition of the reaction mixture, the amount of the reaction product generated, and the optical purity were measured by the method described in Example 1. The reaction mixture contained 2- (4-chlorophenyl) -3-methylbutyric acid as a product in an amount of 150 μmol, and the ratio of the S-form to the R-form was 100: 0.

Example 3 Glycerol 1%, polypeptone 0.5%, yeast extract 0.3%, malt extract 0.3%, crotonamide 0.3%
400 ml of sterilizing medium containing 25% and pH 7.2
Then, 1% of Pseudomonas sp. B21C9 strain previously cultured in the same medium was inoculated and cultured at 26 ° C. for 2 days.
Hereinafter, by the method described in Example 1, preparation of a cell suspension,
The reaction was performed, and the composition of the reaction mixture, the amount of reaction product produced, and the optical purity were measured. Table 2 shows the results.

[Table 2]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12R 1:38) (72) Inventor Ken Mitsuda 4-2-1 Takashi Takarazuka-shi, Hyogo Sumitomo Chemical Co., Ltd. (56) References JP-A-2-84198 (JP, A) JP-A-6-506343 (JP, A) International Publication No. 92/1062 (WO, A1) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 7/40 C12N 1/20 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. A represented by 2- (4-chlorophenyl) with formalized 1-3-methylbutyronitrile or shown are 2- (4-chlorophenyl) with formalized 2 3 Pseudomonas sp methyl butyl amide, (Pseudomonas sp.)
By reacting the B21C9 strain (Microtechnical Research Laboratories No. 3737), (S) -2- (4-chlorophenyl) -represented by Formula 3
(S) characterized in that 3-methylbutyric acid is produced.
A method for producing 2- (4-chlorophenyl) -3-methylbutyric acid. Embedded image Embedded image Embedded image 2. A method according to claim 1, wherein 2- (4-chlorophenyl) -3-methylbutyronitrile of the formula (4) or 2- (4-chlorophenyl) -3-methylbutyramide of the formula (5) is converted to the compound of the formula (6). shoe with in represented by (S) -2- (4- chlorophenyl) -3-activity to convert to the methyl butyrate
Domonas sp. (Pseudomonas sp.) B21C9 strain (micro
Koken Article No. 3737) . Embedded image Embedded image Embedded image