JP3168202B2 - Method for producing (R)-(-)-4-halo-3-hydroxybutyronitrile - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing an optically active halohydrin. (R)-(-)-4-halo-3-hydroxybutyronitrile produced according to the present invention is a raw material for synthesizing various drugs and physiologically active substances, for example,
It is known that it is useful as a raw material for synthesizing L-carnitine, dihydroxybutanoic acid, 4-amino-3-hydroxybutanoic acid and the like (see JP-A-57-165352).

[0002]

2. Description of the Related Art With respect to production of optically active (R)-(-)-4-halo-3-hydroxybutyronitrile, microbiologically produced (R)-(-)-2,3-dichloro- There is known a method of converting (R) -epichlorohydrin from 1-propanol to (R) -epichlorohydrin by a chemical synthesis method and reacting with cyanide to obtain (R)-(-)-4-chloro-3-hydroxybutyronitrile. (JP-A-63-316758)
Reference). However, these chemical synthesis techniques have complicated processes, and there are many problems in industrial production.

[0003] Accordingly, the applicant of the present invention has previously described 1,3-dihalo-2.
A method for producing (R)-(-)-4-halo-3-hydroxybutyronitrile from propanol (see JP-A-3-53889) and a method for producing (R)-(-)-4-halo- from epihalohydrin Method for producing 3-hydroxybutyronitrile (JP-A-3-5389)
No. 0).

However, the optical purity of the optically active halohydrins produced by these inventions cannot be said to be high, and there has been a need to search for microorganisms that produce halohydrins with higher optical purity.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above problems.

[0006]

DISCLOSURE OF THE INVENTION The present inventors have conducted intensive studies on a method of industrially and more advantageously producing optically active halohydrin from 1,3-dihalo-2-propanol and epihalohydrin. Microorganisms belonging to the genus Aum, from 1,3-dihalo-2-propanol or epihalohydrin in the presence of cyanide ions to (R)-(-)-4-halo-3-
The inventors have found the ability to produce hydroxybutyronitrile and have completed the present invention. Heretofore, it has not been known at all that microorganisms belonging to the genus Oleobacterium have the above conversion ability.

That is, the present invention is a method for producing an optically active halohydrin represented by the following (1) and (2).

(1) Due to the action of a microorganism belonging to the genus Oleobacterium, 1,3-dihalo is present in the presence of cyanide.
(R)-(-)-4-characterized by producing (R)-(-)-4-halo-3-hydroxybutyronitrile from 2--2-propanol.
A method for producing halo-3-hydroxybutyronitrile.

(2) By the action of a microorganism belonging to the genus Oleobacterium, (R)-(-)-4-halo- is derived from (R, S)-or (R) -epihalohydrin in the presence of cyanide. Characterized by producing 3-hydroxybutyronitrile
A method for producing (R)-(-)-4-halo-3-hydroxybutyronitrile.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The microorganism used in the present invention includes, for example, strain DH-095 which is a microorganism belonging to the genus Aureobacterium newly isolated from soil by the present inventors. This microorganism is FERM P-12360 (Au
reobacterium sp. DH095) has been deposited with the Institute of Biotechnology and Industrial Technology, the Ministry of International Trade and Industry, and its microbiological properties are as follows.

Mycological properties of strain DH095 Form Polymorphic Bacillus Gram stain + spore-motility + oxidase-catalase + color tone of colony Yellow rod-coccus cycle-No elongation of cells around colony Oxygen Attitude toward aerobic cell wall diamino acids ornithine glucoril test + (glucolyl type) cell wall sugar composition arabinose-galactose + quinone MK-12

[0012] The above mycological properties are described by the Berges Manual of Systematic Bacteriology (Berge
y's Manual of Systematic Bacteriology) Vol. 2 (198
When searched according to 6), strain DH095 was identified as a bacterium belonging to the genus Aureobacterium.

As a medium composition for culturing the above-mentioned microorganisms, generally, any medium can be used as long as these microorganisms can grow. For example, glucose, fructose, sucrose, sugars such as maltose, acetic acid, organic acids such as citric acid, ethanol, alcohols such as glycerol as a carbon source, peptone as a nitrogen source, meat extract,
In addition to natural nitrogen sources such as yeast extract, protein hydrolyzate and amino acid, various inorganic and organic acid ammonium salts can be used. In addition, if necessary, inorganic salts, trace metal salts, vitamins, and the like are appropriately used. At this time, it is also useful to add epichlorohydrin, 1,3-dichloro-2-propanol, 3-chloro-1,2-propanediol and the like to the medium in order to induce high enzyme activity. The cultivation of the microorganism may be performed by a conventional method.
Incubate aerobically for 10 to 96 hours at ~ 45 ° C.

Microorganisms act on 1,3-dihalo-2-propanol or epihalohydrin to produce (R)-(-)-4-halo-3-
As a method for obtaining hydroxybutyronitrile, a method of adding a substrate to a culture solution of a microorganism obtained by culturing as described above or a suspension of cells obtained by centrifugation or the like, a method of treating a cell (for example, Crushed cells, cell extracts such as crude and purified enzymes) or cells immobilized by a conventional method, or
There are a method in which a substrate is added to a suspension of processed cells, a method in which a substrate is added to a culture solution during culture of a microorganism, and a reaction is performed simultaneously with the culture.

In the presence of a cyanide ion, 1,3-dihalo-2-propanol or epihalohydrin is converted to (R)-(-)-4-halo-3-
When hydroxybutyronitrile is produced, the production of by-products can be suppressed by treating the microbial cells with 1,3-dihalo-2-propanol in advance.

The 1,3-dihalo-2-propanol used in the present invention includes 1,3-dichloro-2-propanol and 1,3-dibromo
And epihalohydrin such as epichlorohydrin and epibromohydrin.

The concentration of 1,3-dihalo-2-propanol or epihalohydrin in the reaction solution is not particularly limited, but is preferably 0.1 to 10 (W / V)%. Can be added in batches or added in portions.

When (R)-(-)-4-halo-3-hydroxybutyronitrile is produced, a cyanide ion is present in the reaction solution. However, with respect to this cyanide ion source, a cyanide compound soluble in the reaction solution is used. If it is anything, for example, potassium cyanide which is an alkali metal salt, sodium cyanide,
In addition, acetone cyanohydrin, which is a cyanohydrin, may be used. The amount of the cyanide used is not particularly limited, either, but it is preferable to add an equimolar amount or an excess amount to the substrate 1,3-dihalo-2-propanol or epihalohydrin.

The reaction temperature is 5 to 50 ° C., and the reaction pH is 4 to 10
It is preferable to carry out within the range. The reaction time depends on the substrate concentration,
Although it depends on the cell concentration or other reaction conditions, it is preferable to set the conditions so that the reaction is usually completed in 1 to 120 hours.

Thus, the (R)-produced and accumulated in the reaction solution.
(-)-4-Halo-3-hydroxybutyronitrile can be collected and purified using a known method. For example,
After removing cells by a method such as centrifugation from the reaction solution, extraction is performed with a solvent such as ethyl acetate, and the solvent is removed under reduced pressure to remove (R)-(-)-4-halo-3-hydroxy. A syrup of butyronitrile can be obtained. Further, the syrup can be further purified by distillation under reduced pressure.

Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples.

【Example】

Example 1 A medium containing 1% glucose, 0.5% peptone, 0.3% meat extract, and 0.3% yeast extract was adjusted to pH 7.0 and adjusted to 50%.
Dispense 100 ml each into a ml Erlenmeyer flask, sterilize at 120 ° C for 15 minutes, and add 1 ml of a 20 (W / V)% 3-chloro-1,2-propanediol solution that has been sterilized with a membrane filter in advance. did. Inoculate DH095 strain into the above medium, 30 ℃
For 48 hours. 50 ml of this culture was centrifuged to collect the cells, and 50 mM Tris-sulfate buffer (pH
8.0) After washing twice with 50 ml, the cells were suspended in 10 ml of the same buffer to prepare a cell suspension. 2M with 1M potassium cyanide
Tris-sulfate buffer (pH 8.0) 25ml, cell suspension 10m
l, a reaction solution of 3.22 g of 1,3-dichloro-2-propanol (to make the total volume 50 ml with demineralized water, the final concentration of 1,3-dichloro-2-propanol is 500 mM) at 20 ° C. 1
Allowed to react for hours.

After the reaction, the cells were removed from the reaction solution by centrifugation, and the resulting 4-chloro-3-hydroxybutyronitrile in the supernatant was quantified by gas chromatography.
100 mM of 4-chloro-3-hydroxybutyronitrile was produced. Further, the supernatant was extracted three times with 50 ml of ethyl acetate, the extract was dehydrated with anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a syrup.

The 4-chloro-3-hydroxybutyronitrile in this syrup was converted to its ester derivative using (R)-(-)-α-methoxy-α-trifluoromethylphenylacetyl chloride, and was subjected to high performance liquid chromatography. Optical isomer analysis was performed. The resulting 4-chloro-3
-Hydroxybutyronitrile has an optical purity of 72.6% ee
(R)-(-)-4-chloro-3-hydroxybutyronitrile.

Example 2 Cells prepared in the same manner as in Example 1 were treated with 1 M tris-sulfuric acid (pH 8.0) containing 500 mM 1,3-dichloro-2-propanol.
And treated at 20 ° C. for 2 hours, washed three times with 50 ml of 50 mM Tris-sulfuric acid (pH 8.0) and suspended in 50 ml of the same buffer to prepare a treated cell suspension. Using the treated cell suspension prepared as described above as an enzyme source, 25 ml of 2 M Tris-sulfate buffer (pH 8.0) containing 1 M potassium cyanide, 10 ml of the cell suspension, 1.25 g of epichlorohydrin (desalinated water) With total capacity
(The final concentration of epichlorohydrin was adjusted to be 270 mM so as to be 50 ml.) At 20 ° C. for 1 hour.

After the reaction, the cells were removed from the reaction solution by centrifugation, and the resulting 4-chloro-3-hydroxybutyronitrile in the supernatant was quantified by gas chromatography.
124 mM of 4-chloro-3-hydroxybutyronitrile had been produced. Further, the supernatant was extracted three times with 50 ml of ethyl acetate, the extract was dehydrated with anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a syrup. The 4-chloro-3-hydroxybutyronitrile in this syrup was replaced with (R)-
Using (-)-α-methoxy-α-trifluoromethylphenylacetyl chloride as an ester derivative thereof, the optical isomer was analyzed by high performance liquid chromatography. As a result, the generated 4-chloro-3-hydroxybutyronitrile was (R)-(-)-4-chloro-3- with an optical purity of 33.6% ee.
It was hydroxybutyronitrile.

[0027]

According to the present invention, halohydrins of high optical purity can be efficiently produced from each substrate in one step.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁷ identification code FI C12R 1:01) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12P 13/00 C12P 41/00 CA (STN ) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. The method according to claim 1, wherein the action of a microorganism belonging to the genus Oleobacterium causes 1,3-dihalo-2-amine in the presence of cyanide.
(R)-(-)-4-halo, wherein (R)-(-)-4-halo-3-hydroxybutyronitrile is produced from propanol
A method for producing 3-hydroxybutyronitrile. 2. The action of a microorganism belonging to the genus Oleobacterium in the presence of (R, S)-or
(R)-(-)-4-halo-3-hydroxybutyronitrile is produced from (R) -epihalohydrin
A method for producing (R)-(-)-4-halo-3-hydroxybutyronitrile.