JP2946054B2 - Process for producing optically active (R)-(-)-3-halo-1,2-propanediol - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial application field) The present invention relates to optically active (R)-(-)-3-halo-1,2
It relates to a process for producing propanediol. (R)-(-)
It is known that -3-halo-1,2-propanediol is useful as a raw material for synthesizing various pharmaceuticals and physiologically active substances, for example, as a raw material for synthesizing L-carnitine (Japanese Patent Application Laid-Open No. 57-1979).
165352).

(Prior art and problems) Regarding the production of optically active (R)-(-)-3-halo-1,2-propanediol, a method of obtaining D-mannitol as a raw material (Japanese Patent Laid-Open No. 57-165352) See) Methyl-
A method of obtaining from 5-chloro-5-deoxy-α-L-arabinofuranoside (see Chemistry and Industry, P. 533, 15, July, 1978) is known.
The steps of these chemical synthesis methods are complicated, and there are many problems in industrial production. Further, as a biological technique, a microorganism is allowed to act on racemic (R, S) -3-halo-1,2-propanediol to produce (S)-(+)-3-halo-
1,2-propanediol is selectively metabolized to form (R)-
(-)-3-Halo-1,2-propanediol method (see JP-A-62-158494), a method wherein a bacterium belonging to the genus Pseudomonas is racemic (R, S) -2,3- (R)-(-)-3 by acting on dichloro-1-propanol
A method for fractionating chloro-1,2-propanediol (see Japanese Patent Application Laid-Open No. 62-69993) is known. However, in these methods, a racemic form can be obtained as a raw material (R)-. The yield of (-)-3-halo-1,2-propanediol relative to the starting material is less than 50%, which cannot be an economically advantageous production method.

The present applicant has proposed (R)-(-)-which does not have these problems.
As a method for producing 3-halo-1,2-propanediol, an optically active (R)-(-)-3- is obtained from 1,3-dihalo-2-propanol which is an inexpensive prochiral compound by the action of a dehalogenase. A patent application was previously filed for a method for producing halo-1,2-propanediol (Japanese Patent Application No. Hei.
In order to make this method industrially more advantageous, a technique for further improving the optical purity of the (R)-(-)-3-halo-1,2-propanediol produced is disclosed. Development was desired.

(Summary of the Invention) The inventors of the present invention have developed an optically active (R)-(-)-3-halo-1 from an inexpensive prochiral compound 1,3-dihalo-2-propanol by the action of a dehalogenase. 2−
As a result of intensive studies on the method of producing propanediol with high optical purity, 1,1,
Optical activity (R) from 3-dihalo-2-propanediol
In producing-(-)-3-halo-1,2-propanediol, the presence of a specific heterocyclic compound, a halogen ion or the compound and a halogen ion in the reaction system allows high optical purity (R )-(−)-3-halo-1,
It has been found that 2-propanediol can be easily obtained, and the present invention has been completed.

That is, the present invention provides a method for dehydrohalogenation in the presence of at least one heterocyclic compound represented by the following general formulas [I], [II] and [III] and / or a halogen ion in the presence of 1,3- Optical activity (R)-characterized by producing optical activity (R)-(-)-3-halo-1,2-propanediol from dihalo-2-propanol.
A process for producing (−)-3-halo-1,2-propanediol,
It is.

(Detailed Description of the Invention) The dehalogenase referred to in the present invention is 1,3-dihalo-2.
-An enzyme capable of finally converting one halogen atom of propanol to a hydroxyl group. Specifically, for example, N-4701 strain of Microbacterium sp.
Enzymes produced by the strains −653 and N-1074 can be exemplified. These microorganisms, respectively,
No. 2644 (Microbacterium sp. N-4701), Microtechnical Laboratory No. 2642 (Corynebacterium sp. N-653), and Microtechnological Laboratory No. 2643 (Corynebacterium sp. N-1074) And deposited at the Institute of Microbial Industry and Technology (MIC), whose microbiological properties are as follows.

N-4701 strain Form Polymorphic bacillus Peripheral cells of colony Does not elongate Gram stain + Spores Not observed Motility + Flagella pole-side hair Color of colony Yellow orange Oxidase + Catalase + OFO Growth by anaerobic-Whole cells Presence of meso-diaminopimelic acid in the hydrolyzate of cellulosic acid-Diamino acid of cell wall Lysine Glycolyl test + (glycolyl type) Starch decomposition + Gelatin liquefaction-Nitrate reduction-Utilization of arginine + Hydrogen sulfide production-Urea decomposition-Heat resistance in skim milk medium Sex 60 ° C, 30 minutes-Acid production Inulin + glycerol-glucose + sucrose + trehalose + raffinose + N-653 strain Form of polymorphic bacilli Peripheral cells in colonies No elongation Gram stain + No spores Motility + Oxidase-catalase + OFO Growth under anaerobic-meso in hydrolyzate of hydrochloric acid of whole cells Presence of diaminopimelic acid-Cell wall diamino acid diacetylbutyrate glucolyl test-Degradation of (acetyl type) starch-Liquefaction of gelatin + Degradation of cellulose-Degradation of urea-Acid resistance-Heat resistance in skim milk medium 63 ° C, 30 minutes-72 ° C, 15 minutes-N-1074 strain Form Polymorphic bacillus Peripheral cells of colony No elongation Gram stain + Spores Not observed Motility + Oxidase-Catalase + OFO Growth under anaerobic-Hydrochloride hydrolyzate of all cells Presence of meso-diaminopimelic acid-Diamino acid diaminobutyrate glucolyl test on cell wall-(Acetyl type) Starch degradation + Gelatin liquefaction-Cellulose degradation-Urea degradation-Acid resistance-Heat resistance in skim milk medium 63 ° C for 30 minutes-72 ℃, 15 minutes-The above microbiological properties were determined using the Burgess Manual of Systematic Tertology Vol.2 (1986) [B
ergy's manual of Systematic Bacteriology Vol.2 (19
86)], strain N-4701 was identified as belonging to the genus Microbacterium, and strains N-653 and N-1074 were identified as bacteria belonging to the genus Corynebacterium.

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,
Sugars such as sucrose and maltose; organic acids such as acetic acid and citric acid; alcohols such as ethanol and glycerol; and nitrogen sources such as peptone, meat extract, yeast extract, protein hydrolysates, and general natural nitrogen sources such as amino acids In addition, various inorganic and organic acid ammonium salts can be used, and in addition, inorganic salts, trace metal salts, vitamins, and the like are appropriately used as needed. At this time, in order to induce high enzyme activity, 1,3-dichloro-2-propanol, 3-
It is also effective to add chloro-1,2-propanediol or the like to the medium.

The cultivation of the microorganism may be performed by a conventional method, for example, pH 4-1.
0, aerobically at a temperature of 20-45 ° C for 10-96 hours.

1,3-Dihalo-2-propanol used in the present invention is
1,3-dichloro-2-propanol, 1,3-dibromo-2
-Propanol and the like.

As the heterocyclic compound used in the present invention, for example, pyridine, α-picoline, β-picoline, γ-picoline,
2-hydroxypyridine, 3-hydroxypyridine, 4
-Hydroxypyridine, 2,3-dimethylpyridine, 2,4-
Dimethylpyridine, piperidine, 2-methylpiperidine, 3-methylpiperidine, 4-methylpiperidine, N
-Methylpiperidine, morpholine, N-methylmorpholine and salts thereof. These can be used alone or in combination.

Examples of the halogen ion include a fluorinated ion, a chlorinated ion, a bromide ion, and an iodide ion, and each of them can be used alone or in combination.

In the present invention, (R)-(-)-3 formed by using the above heterocyclic compound or halogen ion alone.
Although it is possible to improve the optical purity of halo-1,2-propanediol, (R)-can be more effectively used by further using the heterocyclic compound and a halogen ion in combination.
The optical purity of (-)-3-halo-1,2-propanediol can be improved.

As a method for obtaining (R)-(-)-3-halo-1,2-propanediol by reacting a dehalogenase with 1,3-dihalo-2-propanol, the enzyme is derived from a microorganism. In some cases, a method of adding a substrate to a culture solution of a microorganism obtained as described above or a suspension of cells obtained by centrifugation or the like, a method of treating a cell (eg, a crushed cell, a cell extract, etc.) ) Or a method in which a substrate is added to a suspension of cells immobilized by a conventional method or processed cells.

Although the substrate concentration in the reaction solution is not particularly limited,
The concentration is preferably 0.1 to 10 (W / V)%, and the substrate can be added to the reaction solution at once or in portions.

The concentration of the heterocyclic compound in the reaction solution is 0.1 to 10 (W
/ V)%, the concentration of halogen ions is preferably 0.1 to 2M,
These additions may be made at the beginning or during the reaction.

The reaction is preferably performed at a temperature of 5 to 50 ° C and a reaction pH of 4 to 10. The reaction time varies depending on the substrate concentration, the cell concentration or other reaction conditions, but it is preferable to set the conditions so that the reaction is usually completed in 1 to 120 hours.

Thus, (R)-(−)-produced and accumulated in the reaction solution.
3-Halo-1,2-propanediol 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)-(−)-3-halo. A syrup of 1,2-propanediol 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.
The present invention is not limited to only these examples.

Example 1 1% glycerol, 0.5% peptone, 0.3% meat extract,
Adjust the medium consisting of 0.3% yeast extract to pH 7.0, 500m
100 ml each was dispensed into an Erlenmeyer flask, sterilized at 120 ° C. for 15 minutes, and added with 0.8 ml of a 25 (W / V)% 3-chloro-1,2-propanediol aqueous solution which was sterilized by a membrane filter.

The above-described medium was inoculated with the microhalogenase-bearing Microbacterium N-4701 strain (manufactured by Jiko Kyojo No. 2644), and cultured at 30 ° C. for 48 hours with shaking. The cells were collected by centrifugation from the culture solution 80 ml, tris -H ₂ SO ₄ buffer 50 mM (pH
8.0) After washing twice with 80 ml, the cells were suspended in 5 ml of the same buffer (pH 8.0) to prepare a cell suspension.

The cell suspension obtained as described above was reacted as a dehalogenase source with stirring at 20 ° C. in the following reaction solution composition.

(Reaction solution composition) 2 (W / V)% 1,3-dichloro-2-propanol solution
25 ml (2M Tris -H ₂ SO ₄ buffer, pH 8.0) Determination of 3-chloro-1,2-propanediol 2.5M potassium halide solution 20ml cell suspension 5ml reaction solution by gas chromatography Done, 3-chloro-
After extracting 1,2-propanediol with ethyl acetate, it is tosylated by a conventional method, and is then subjected to a Daicel column (Chiral Cell).
OC) was used to analyze the optical isomers by high performance liquid chromatography. Table 1 shows the results.

Example 2 Using the cell suspension obtained in the same manner as in Example 1 as an enzyme source, the reaction was carried out by stirring at 20 ° C. with the following reaction solution composition.

(Reaction solution composition) 2 (W / V)% 1,3-dichloro-2-propanol solution
25 ml (2 M Tris-H ₂ SO ₄ buffer, pH 8.0) Heterocyclic compound 0.5 g Cell suspension 5 ml Deionized water 20 ml The produced 3-chloro-1,2-propanediol was analyzed in Example 1. Was performed in the same manner as described above. Table 2 shows the results.

Example 3 A Corynebacterium N-1074 strain having a dehalogenase (Microtechnical Laboratory No. 2643) was cultured in the same manner as in Example 1, and the following reaction was carried out using a cell suspension obtained as an enzyme source. 20 in solution composition
The reaction was carried out with stirring at ℃.

(Reaction solution composition) 2 (W / V)% 1,3-dichloro-2-propanol solution
25 ml (2 M Tris-H ₂ SO ₄ buffer, pH 8.0) 2.5 M potassium bromide solution 20 ml heterocyclic compound 0.5 g cell suspension 5 ml The analysis of the produced 3-chloro-1,2-propanediol was as follows: Performed in the same manner as in Example 1. Table 3 shows the results.

Claims (1)

(57) [Claims] 1. The following general formulas [I], [II] and [III]
In the presence of at least one heterocyclic compound represented by
bacterium or Corynebact
erium), the optically active (R)-(−)-3-halo-1,2-propane is converted from 1,3-dihalo-2-propanol by the action of the cells of the microorganism belonging to the genus Erium), the treated cells thereof, or the immobilized products thereof. A process for producing optically active (R)-(-)-3-halo-1,2-propanediol, which comprises producing a diol.