JPH05219965A - Production of optically active halohydrin - Google Patents

Abstract

PURPOSE:To easily obtain retinoids having high optical purity in high efficiency by reacting a 1,3-dihalo-2-propanol with a microorganism of the genus Aureobacterium. CONSTITUTION:The objective (R)-(-)-3-halo-1,2-propanediol is produced by aerobically culturing a microbial strain belonging to the genus Aureobacterium, e.g. DH-095 strain (FERM P-12360) at 20-45 deg.C and pH4-10 for 10-96hr and reacting the obtained culture liquid or microbial cell suspension with a 1,3- dihalo-2-propanol at 5-50 deg.C and pH4-10.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing an optically active halohydrin. Manufactured according to the present invention
(R)-(-)-3-Halo-1,2-propanediol, (R)-(-)-4-halo-3-hydroxybutyronitrile and (R) -epihalohydrin are various pharmaceuticals and physiological agents. Synthetic raw materials for active substances, for example, L-carnitine, dihydroxybutanoic acid, 4-amino
It is known to be useful as a raw material for the synthesis of 3-hydroxybutanoic acid and the like (see JP-A-57-165352).

[0002]

[Prior Art and Problems] Optically Active (R)-(-)-3-Halo-1,2-
Regarding the production of propanediol, a method of obtaining D-mannitol as a raw material (see JP-A-57-165352),
A method of obtaining from methyl-5-chloro-5-deoxy-α-L-arabinofuranoside (see Chemistry and Industry, p. 533, 15, July, 1978) and the like are known.

Regarding the production of optically active (R)-(-)-4-halo-3-hydroxybutyronitrile, (R)-(-)-2,3-dichloro- which was produced microbiologically. A known method is to obtain (R)-(-)-4-chloro-3-hydroxybutyronitrile by reacting with cyanide after converting 1-propanol to (R) -epichlorohydrin by a chemical synthetic method. (Japanese Patent Laid-Open No. 63-316
(See Japanese Patent No. 758). However, these chemical synthesis methods have complicated steps, and there are many problems in making an industrial production method.

Further, by using a microbiological method, (R)-(-)-3-
As a method for producing halo-1,2-propanediol,
By allowing a microorganism to act on racemic (R, S) -3-halo-1,2-propanediol and selectively metabolizing (S)-(+)-3-halo-1,2-propanediol. A method of leaving (R)-(-)-3-halo-1,2-propanediol (see JP-A-62-158494), a bacterium belonging to the genus Pseudomonas in racemic (R,
S) -2,3-Dichloro-1-propanol to act on (R)-(-)
Although a method for separating -3-chloro-1,2-propanediol is known (see Japanese Patent Laid-Open No. 62-69993), since these are methods for optically resolving a racemic raw material, The molar yield of (R)-(-)-3-halo-1,2-propanediol that can be obtained with respect to the raw material is 50% or less, which is not an economically advantageous production method.

Therefore, the present applicant has previously proposed that the action of an enzyme derived from a microorganism causes (R)-(-)-3- with a theoretical optical yield of 100% from prochiral 1,3-dihalo-2-propanol. Halo-1,2-
Method for producing propanediol (see Japanese Patent Application Laid-Open No. 2-291280) and racemic epihalohydrin (R)-
A method for producing (-)-3-halo-1,2-propanediol (see JP-A-2-283295), and (R)-(-)-4-from 1,3-dihalo-2-propanol Method for producing halo-3-hydroxybutyronitrile (see JP-A-3-53889) and method for producing (R)-(-)-4-halo-3-hydroxybutyronitrile from epihalohydrin Patent application for 3-53890).

However, the optical purity of the optically active halohydrin produced by these inventions cannot be said to be high, and there has been a demand for a search for a microorganism which produces halohydrin of higher optical purity.

[0007]

SUMMARY OF THE INVENTION The present inventors have earnestly studied a method for industrially producing an optically active halohydrin from 1,3-dihalo-2-propanol or epihalohydrin, and as a result, belong to the genus Aureobacterium. For microorganisms, 1,
3-dihalo-2-propanol and epihalohydrin (R)-
Ability to convert to (-)-3-halo-1,2-propanediol, (R)-(-)-4-halo-3 from 1,3-dihalo-2-propanol and epihalohydrin in the presence of cyanide The ability to form hydroxybutyronitrile, and also 1,3-dihalo-2-
The ability to convert propanol to (R) -epihalohydrin was found and the present invention was completed. Until now, it was not known at all that the microorganisms belonging to the genus Aureobacterium have the above-mentioned conversion ability.

That is, the present invention is a method for producing an optically active halohydrin shown in the following (1) to (5).

(1) By the action of a microorganism belonging to the genus Aureobacterium, 1,3-dihalo-2-propanol is converted into (R)-
A method for producing (R)-(-)-3-halo-1,2-propanediol, which comprises producing (-)-3-halo-1,2-propanediol.

(2) From (R, S)-or (R) -epihalohydrin by the action of a microorganism belonging to the genus Aureobacterium
A method for producing (R)-(-)-3-halo-1,2-propanediol, which comprises producing (R)-(-)-3-halo-1,2-propanediol.

(3) Due to the action of a microorganism belonging to the genus Aureobacterium, 1,3-dihalo-2 is produced in the presence of cyanide.
-(R)-(-)-4-halo-3-hydroxybutyronitrile is produced from -propanol. ..

(4) By the action of a microorganism belonging to the genus Aureobacterium, (R, S)-or
Characterized by producing (R)-(-)-4-halo-3-hydroxybutyronitrile from (R) -epihalohydrin
Process for producing (R)-(-)-4-halo-3-hydroxybutyronitrile.

(5) A method for producing (R) -epihalohydrin, which comprises producing (R) -epihalohydrin from 1,3-dihalo-2-propanol by the action of a microorganism belonging to the genus Aureobacterium.

[0014]

DETAILED DESCRIPTION OF THE INVENTION Examples of the microorganism used in the present invention include DH-095 strain which is a microorganism belonging to the genus Aureobacterium newly isolated from soil by the present inventor. This microorganism is Microbiology Research Institute No. 12360
No. (Aureobacterium sp. DH095), which has been deposited at the Institute of Microbial Science and Technology (MIC) of the Agency of Industrial Science and Technology, and its mycological properties are as follows.

[0015] Mycological properties of DH095 strain Polymorphic bacillus Gram stainability + spores-mobility + oxidase-catalase + color of colony yellow rod-coccus cycle-extension of peripheral cells of colony Not observed oxygen Attitude toward aerobic cell wall diamino acids Ornithine Glucolyl test + (Glucolyl type) Cell wall sugar composition Arabinose-Galactose + Quinone MK-12

The above-mentioned mycological properties are described in Berges Manual of Systematic Bacteriology (Berge
y's Manual of Systematic Bacteriology) Vol. 2 (198
By searching according to 6), the DH095 strain was identified as a bacterium belonging to the genus Aureobacterium.

As a medium composition for culturing the above-mentioned microorganisms, any medium can be used so long as these microorganisms can normally grow. For example, glucose as a carbon source, fructose, sucrose, sugars such as maltose, acetic acid, organic acids such as citric acid, alcohols such as ethanol and glycerol, peptone as a nitrogen source, meat extract,
In addition to yeast extracts, protein hydrolysates, natural nitrogen sources such as amino acids, various inorganic and organic acid ammonium salts can be used. In addition to these, inorganic salts, trace metal salts, vitamins, etc. are appropriately used as necessary. 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. Cultivation of the above microorganisms may be carried out by a conventional method, for example, pH 4 to 10 and temperature 20.
Incubate aerobically at ~ 45 ° C for 10-96 hours.

As a method for obtaining an optically active halohydrin by allowing a microorganism to act on 1,3-dihalo-2-propanol or epihalohydrin, a microorganism obtained by culturing as described above or a microorganism obtained by centrifugation or the like is used. A method of adding a substrate to a suspension of cells, a treated product of cells (for example, crushed cells, cell extract of crude or purified enzyme, etc.) or cells immobilized by a conventional method, or cell treatment There are a method of adding a substrate to a suspension of substances, a method of adding a substrate to a culture solution at the time of culturing a microorganism, and performing a reaction at the same time as the culture.

In the presence of cyanide ions, 1,3-dihalo
When (R)-(-)-4-halo-3-hydroxybutyronitrile is produced from 2-propanol or epihalohydrin, the microbial cells should be treated with 1,3-dihalo-2-propanol in advance. Can suppress the formation of by-products.

The 1,3-dihalo-2-propanol used in the present invention is 1,3-dichloro-2-propanol or 1,3-dibromo.
Examples include -2-propanol, and epihalohydrin includes 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)%, and these substrates are collectively contained in the reaction solution. It can be added at once or dividedly added.

When (R)-(-)-4-halo-3-hydroxybutyronitrile is produced, cyan ions are present in the reaction solution, but this cyan ion source is soluble in the reaction solution. Any cyanide compound may be used, and examples thereof include potassium cyanide and sodium cyanide which are alkali metal salts, and acetone cyanohydrin which is cyanohydrin. The amount of the cyanide compound used is not particularly limited, but the substrate 1,3-dihalo-2 is used.
-It is preferable to add an equimolar amount or an excess amount with respect to 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 in the range of. 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, (R)-which was produced and accumulated in the reaction solution.
(-)-3-Halo-1,2-propanediol, (R)-(-)-4-halo-3
-Hydroxybutyronitrile or (R) -epihalohydrin can be collected and purified using known methods. For example, cells are removed from the reaction solution by a method such as centrifugation, extraction is performed with a solvent such as ethyl acetate, and the solvent is removed under reduced pressure (R)-(-)-3-halo-1. A syrup of 2,2-propanediol, (R)-(-)-4-halo-3-hydroxybutyronitrile or (R) -epihalohydrin can be obtained. Further, this syrup can be further purified by distillation under reduced pressure.

[0025]

[Effect] According to the present invention, halohydrin having high optical purity can be efficiently produced from each substrate in one step.

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

【Example】

Example 1 A medium consisting of 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 100 ml Erlenmeyer flask, sterilize at 120 ° C for 15 minutes, and then add 1 ml of 20 (W / V)% 3-chloro-1,2-propanediol solution that had been sterilized with a membrane filter. did. Inoculate the above medium with the DH095 strain and grow at 30 ° C.
At 48 hours, shaking culture was performed. 50 ml of this culture solution was centrifuged to collect the cells, and 50 mM Tris-sulfate buffer solution (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. Using the bacterial cell suspension obtained as described above as an enzyme source, 25m of a 2M Tris-hydrochloric acid buffer solution (pH 8.0) containing 155mM 1,3-dichloro-2-propanol
l, 10 ml of cell suspension and 15 ml of deionized water (total volume 50 ml) were reacted at 20 ° C. for 8 hours. After the reaction, the bacterial cells were removed from the reaction solution by centrifugation, and the produced 3-chloro-
As a result of quantifying 1,2-propanediol by gas chromatography, 68.5 mM 3-chloro-1,2-propanediol 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. After tosylating 3-chloro-1,2-propanediol in this syrup by a conventional method, chiralcel OC [Daicel Corporation]
Was analyzed by high performance liquid chromatography and found to be 3-chloro-1,2-propanediol having an optical purity of 78.0% ee.

Example 2 Using the bacterial cell suspension prepared in the same manner as in Example 1 as an enzyme source, 25 ml of 2M tris-hydrochloric acid (pH 8.0) containing 216 mM epichlorohydrin, 10 ml of the bacterial cell suspension, and A reaction solution of 15 ml of brine (50 ml in total volume) was reacted at 20 ° C. for 6 hours. After the reaction, the bacterial cells were removed from the reaction solution by centrifugation, and the produced 3-chloro-1,2-propanediol in the supernatant was quantified by gas chromatography.As a result, 97.3 mM 3-chloro-1,2 −
Propanediol was produced. Furthermore, 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 3-chloro-1,2-propanediol in this syrup was tosylated by a conventional method, and the optical isomers were analyzed by high performance liquid chromatography using Chiralcel OC (manufactured by Daicel Corp.). , Optical purity 48.2
It was found to be (R)-(-)-3-chloro-1,2-propanediol with% ee.

Example 3 Using the bacterial cell suspension prepared in the same manner as in Example 1 as an enzyme source, 25 ml of 2M Tris-sulfate buffer (pH 8.0) containing 1M potassium cyanide, 10 ml of the bacterial cell suspension, 3-dichloro-2-
3.22 g of propanol (total volume of demineralized water is 50 ml, final concentration of 1,3-dichloro-2-propanol is 500 m
The reaction solution of M) was reacted at 20 ° C. for 1 hour. After the reaction, the cells were removed from the reaction solution by centrifugation, and the produced 4-chloro-3-hydroxybutyronitrile in the supernatant was quantified by gas chromatography, and as a result, 100 mM 4-chloro-
3-Hydroxybutyronitrile had formed. 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 the optical isomer was obtained by high performance liquid chromatography. Was analyzed. As a result, the produced 4-chloro-3-hydroxybutyronitrile was (R)-(-)-4-chloro-3-hydroxybutyronitrile having an optical purity of 72.6% ee.

Example 4 Cells prepared in the same manner as in Example 1 were treated with 1M tris-sulfuric acid (pH 8.0) containing 500 mM 1,3-dichloro-2-propanol.
The cells were suspended in the cells, treated at 20 ° C. for 2 hours, washed with 50 ml of 50 mM Tris-sulfuric acid (pH 8.0) three times, and suspended in 50 ml of the same buffer to prepare a treated cell suspension. Using the treated bacterial 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 bacterial cell suspension, 1.25 g of epichlorohydrin (demineralized water) With total capacity
The final concentration of epichlorohydrin was 270 mM, which was adjusted to 50 ml, and reacted at 20 ° C. for 1 hour. After the reaction, the bacterial cells were removed from the reaction solution by centrifugation, and the produced 4-chloro-3-hydroxybutyronitrile in the supernatant was quantified by gas chromatography.
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. In this syrup
4-chloro-3-hydroxybutyronitrile as (R)-(-)-α-
The optical isomer was analyzed by high performance liquid chromatography as the ester derivative thereof using methoxy-α-trifluoromethylphenylacetyl chloride. As a result, the produced 4-chloro-3-hydroxybutyronitrile was (R)-(-)-4-chloro-3-hydroxybutyronitrile having an optical purity of 33.6% ee.

Example 5 The treated bacterial cell suspension prepared in the same manner as in Example 4 was used as an enzyme source to prepare a 2M solution containing 200 mM 1,3-dichloro-2-propanol.
Tris-sulfate buffer (pH 8.0) 25 ml, cell suspension 10 ml
20 with the reaction solution (to make the total volume 50 ml with demineralized water)
The reaction was carried out at 30 ° C for 30 minutes. After the reaction, bacterial cells were removed from the reaction solution by centrifugation, and the produced epichlorohydrin in the supernatant was quantified by gas chromatography. As a result, 26.5 mM epichlorohydrin 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 epichlorohydrin in this syrup
Using p-toluenesulfonic acid as the tosyl derivative, the optical isomers were analyzed by high performance liquid chromatography. As a result, the produced epichlorohydrin was epichlorohydrin with an optical purity of 52.3% ee.

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Claims (5)

[Claims] 1. From the action of a microorganism belonging to the genus Aureobacterium, 1,3-dihalo-2-propanol is converted into (R)-(-)-.
A method for producing (R)-(-)-3-halo-1,2-propanediol, which comprises producing 3-halo-1,2-propanediol. 2. From (R, S)-or (R) -epihalohydrin by the action of a microorganism belonging to the genus Aureobacterium
A method for producing (R)-(-)-3-halo-1,2-propanediol, which comprises producing (R)-(-)-3-halo-1,2-propanediol. 3. Due to the action of a microorganism belonging to the genus Aureobacterium, 1,3-dihalo-2-
(R)-(-)-4-halo characterized by producing (R)-(-)-4-halo-3-hydroxybutyronitrile from propanol
A process for producing 3-hydroxybutyronitrile. 4. By the action of a microorganism belonging to the genus Aureobacterium, (R, S)-or
Characterized by producing (R)-(-)-4-halo-3-hydroxybutyronitrile from (R) -epihalohydrin
Process for producing (R)-(-)-4-halo-3-hydroxybutyronitrile. 5. A method for producing (R) -epihalohydrin, which comprises producing (R) -epihalohydrin from 1,3-dihalo-2-propanol by the action of a microorganism belonging to the genus Aureobacterium.