JPH02291280A - Preparation of optically active (r)-(-)-3-halo-1,2-propanediol - Google Patents

Abstract

PURPOSE:To readily prepare an optically active (R)-(-)-3-halo-1,2-propanediol from an inexpensive prochiral compound, 1,3-dihalo-2-propanol, by treating the raw material with a dehalogenating enzyme. CONSTITUTION:A dehalogenating enzyme originated from a microorganism separated from soil, such as an enzyme originated from a microorganism, N-653 (FERM 10390), belonging to the genus Corynebacterium is prepared. 1,3- Dihalo-2-propanol is treated with the dehalogenating enzyme to provide the objective (R)-(-)-3-halo-1,2-propandiol. The concentration of the substrate in the reaction solution therein is preferably 0.1 to 10(W/V)%. The reaction temperature and the reaction pH are preferably 5 to 50 deg.C and 4 to 10, respectively. The (R)-(-)-3-halo-1,2-propanediol produced and accumulated in the reaction solution is collected and purified with known methods.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to optically active (,R)-(-)-3-halo-1.
The present invention relates to a method for producing 2-propanesiol. (R)-(-
)-3-Halo-l,2-propanediol is known to be useful as a raw material for the synthesis of various pharmaceuticals and physiologically active substances, for example, as a raw material for the synthesis of L-carnitine (Japanese Patent Application Laid-open No. 165352/1983). (See official bulletin). (Prior art and problems) Regarding the production of optically active (R)-(-)-3-halo-1,2-propanediol, there is a method for obtaining it using D-mannitol as a raw material (Japanese Patent Laid-Open No. 165352/1983). reference)
, from methyl-5-chloro-5-deoxy-α-shi-arabinofuranoside (Chemistry and Industry, P. 533, 15. July,
(see 1978), but these chemical synthesis methods involve complicated steps, and there are many problems in using them as industrial production methods. In addition, as a biological method, (S)-(1)-3-halo-1.
2-propanediol is selectively metabolized, (II)-
A method for leaving (-)-3-halo-1,2-propanediol (see JP-A-62-158494), a racemic (R.S)-
By acting on 2.3-dichloro-1-propanol (R
)-(-)-3-chloro-1,2-propanediol is known (see Japanese Patent Application Laid-open No. 62-69993), but in these methods, the racemate is used as the raw material. (R)-(-)3-halo-1.2-
The yield of propanediol based on the raw material is less than 50%, and this cannot be an economically advantageous production method.

(Summary of the invention) Therefore, the present inventors discovered that optically active (R)-(-)-3
-As a result of intensive investigation into a method for industrially producing halo-1,2-propanediol, the present inventors found that it is an inexpensive prochiral compound produced by the action of a dehalogenating enzyme derived from a microorganism isolated from soil. 1.3 dihalo-2-
The present inventors have discovered that optically active (R)-(-)-3-halo-1,2-propanediol can be easily obtained from propanol, and have completed the present invention.

That is, the present invention provides that
Optical activity (R) from 1.3-dihalo-2-propanol
Optically active (R)-(-)-3 characterized by producing -(-)-3-halo-1,2-brobanediol
- A method for producing halo-1,2-propanediol. According to the present invention, since a brochiral substrate is used, it is theoretically possible to obtain the target product with a yield of 100%, which is economically advantageous. (Specific description of the invention) The dehalogenase used in the present invention is 1,3-dihalo2
-It is an enzyme that can ultimately convert one halogen atom of propanol into a hydroxyl group. Specifically, for example, microorganisms belonging to the genus Corynebacterium newly isolated and discovered by the present inventors, strains N-653 and N-1074, and microorganisms belonging to the genus Microbacterium, N-4.
Examples include enzymes produced by strains such as 701. These microorganisms were submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology (Feikoken), respectively.
No. 391 (Corynebacterium sp. N-1074)
It has been deposited as Microbacterium ip. N-4701, and its scientific properties are as shown below. N-653 Morphology Peripheral cells of colony Durham staining Anti-acidity Spore movement Oxidase power Talase OF Growth under anaerobic conditions No growth of polymorphic bacilli + Not observed + + O Cell wall diamino acid glycolyl test Diaminobutyric acid (Acetyl type) Starch decomposition Gelatin liquefaction + Cellulose decomposition Urea decomposition 63°C for 30 minutes 72°C for 15 minutes N-1074 Morphology Surrounding cells of the colony Durham staining Anti-acidity Spore movement Oxidase power Talase OF Under anaerobic conditions Growth of pleomorphic bacilli with no growth and no growth + + Diamino acid glycolyl test on cell wall Starch decomposition Gelatin liquefy Diaminobutyric acid (acetyl type) + Cellulose decomposition Urea decomposition 63°C for 30 minutes 72°C for 15 minutes N-4701 Morphology Peripheral cells of the colony Durham staining Spore movement Color of the vellus colony Oxidase catalase OF Growth under anaerobic conditions Presence of meso-diaminopimelic acid in the hydrolyzate of whole cells Diamino acid polymorphic bacillus elongation in the cell wall None + Not observed + Very yellow-orange side hair + + Lysine glycolyl test + (glycolyl type) Starch decomposition, gelatin liquefaction, nitrate reduction, use of arginine, hydrogen decasulfide production, urea decomposition, heat resistance in skim milk medium, 60°C for 30 minutes, acid Production of inulin + glycerol glucose + sucrose + trehalose + raffinose + The above mycological properties are described in Berger's Manual of
Systemate Inc. Bacteriology Vol. 2 (1
986) (Bergy's Manual of
Systemamouth CBacteriology Vo
l. 2 (1986)), N-
The strains 653 and N-1074 were identified as belonging to the genus Corynebacterium, and the old strain N-47 was identified as belonging to the genus Microbacterium, respectively. As the medium composition for culturing the above-mentioned microorganisms, any medium can be used as long as these microorganisms can grow. For example, carbon sources include glucose, fructose, shakerose, maltose, and other saccharides, acetic acid, citric acid, and other organic acids, ethanol, glycerol, and other alcohols, and nitrogen sources include veptone, meat extract, yeast extract, and protein hydrolysis. In addition to general natural nitrogen sources such as carbon dioxide and amino acids, various inorganic and organic acid ammonium salts can be used, and inorganic salts, trace metal salts, vitamins, etc. can be used as appropriate. At this time, in order to induce high enzyme activity,
1.3-dichloro-2-propanol, 3-chloro-1
．． It is also useful to add 2-propanediol or the like to the medium. The above-mentioned microorganisms may be cultured by a conventional method, for example, at pH 4 to
10, aerobically at a temperature range of 20-40°C 10-9
Incubate for 6 hours. The 1,3-dihalo-2-propanol used in the present invention includes 1,3-dichloro-2-propanol, 1,3-dibromo-2-propanol, and the like.

As a method for obtaining (R)-(-)-3-halo-1,2-propanediol by allowing a dehalogenating enzyme to act on 1.3-dihalo-2-propanol, the enzyme is derived from a microorganism. In this case, a method of adding a substrate to a microorganism culture solution obtained by culturing as described above or a suspension of microorganisms obtained by centrifugation, etc., or a method of adding a substrate to a microorganism culture solution obtained by culturing as described above,
A method in which a substrate is added to a suspension of bacterial cells (crude enzyme, purified enzyme, etc.) or a suspension of bacterial cells or treated bacterial cells fixed by a conventional method; There are methods to carry out the reaction at the same time as culturing. The substrate concentration in the reaction solution is not particularly limited, but may be as low as 0.
．． The amount is preferably 1 to 10 (W/V)%, and the substrate can be added to the reaction solution all at once or in portions. The reaction temperature is preferably 5 to 50'' and the C1 reaction pu is preferably 4 to 10. The reaction time varies depending on the substrate concentration, bacterial cell concentration, and other reaction conditions, but it is usually completed in 1 to 120 hours. It is preferable to set conditions such that (R)-(-)-3-halo-1.2-
Propanediol can be collected and purified using known methods. For example, after removing bacterial cells from the reaction solution using 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-Yakuchi 1,2-propanediol can be obtained. This syrup can also be further purified by distilling it under reduced pressure.

Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited to these Examples. Examples 1-2 Glucose 1%, peptone 0.5%, meat extract 0.3%
, yeast extract 0. A medium containing 3% was adjusted to pH 1.0, dispensed into 500 liter Erlenmeyer flasks in 100-liter portions, sterilized at 120°C for 15 minutes, and then sterilized with a membrane filter. -Chloro-1,2-propanediol aqueous solution at 0. 8 d was added.

Each of the blank strains shown in Table 1 was inoculated into the above medium, and cultured with shaking at 30°C for 48 hours. Each of these culture solutions was centrifuged to collect the bacterial cells, and then added to a 100...M phosphate buffer solution.
After washing once with 00d, 1.0% 1,3-dichloro-2-propanol solution (IM phosphate buffer, p
l1 7. 5) Suspend the bacterial cells in 100a+1,
The reaction was carried out by shaking for 22-23 hours at 'C.

After the reaction, the bacterial cells were removed from the reaction solution by centrifugation, and the amount of 3-chloro-1,2-propanediol produced in the supernatant was determined by gas chromatography to determine the yield from the substrate. Further, this supernatant was extracted four 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 specific rotation of this Sorosob was measured and the results shown in Table 1 were obtained. In addition, the literature value of (R)-(-)-3-chloro-1,3-chloro-1,2-propanediol is as follows.

(α) o = 6.9 (c = 2, H
zO) Also, 3-chloro-1.
After tosylating 2-propanediol in a conventional manner, the optical isomers were analyzed by high performance liquid chromatography using a Daicel column (Chiralcel QC).
) confirmed the existence of the body.

Table 1 Example 3 The N-4701 strain was inoculated into a medium obtained in the same manner as in Examples 1 and 2, and cultured with shaking at 30°C for 48 hours.
Centrifuge 80 d of this culture solution to collect bacterial cells, and
Tris-HCIII buffer of M (pl1 8.0) 80d
After washing once with water, suspend the bacterial cells in 1.0 (W/V)% 1,3-dichloro-2-propanol solution (IM) Lis-11cI buffer, pH 8.0 at 40°C.
The reaction was carried out by stirring at °C for 6 hours. After the reaction, the bacterial cells were removed from the reaction solution by centrifugation, and the produced 3-chloro-12-propanediol in the supernatant was quantified by gas chromatography. As a result, the yield from the substrate was 100%. Further, this supernatant was extracted three times with 5M ethyl acetate, the extract was dehydrated with anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain syrup.

When the specific rotation of this syrup was measured, it was found to be (cr)
n=5. Q5 (C = 1, II2o
)Met.

In addition, after tosylating 3-chloro-1,2-propanediol in the syrup using a conventional method, a Daicel column (
Optical isomer analysis was performed by high performance liquid chromatography using Chiralcel QC), and the presence of the (1?) isomer was confirmed.

Claims (3)

[Claims] (1) 1,3-dihalo- by the action of dehalogenase
Optically active (R)-(-)-3-halo-1,2- characterized in that optically active (R)-(-)-3-halo-1,2-propanediol is produced from 2-propanol.
Method for producing propanediol. (2) The production method according to claim 1, wherein the dehalogenating enzyme is derived from a microorganism. (3) The microorganism is Corynebacterium (Coryneba).
cterium genus or Microbacterium (Mic
3. The production method according to claim 2, which is of the genus Robacterium.