JPH0549485A - Production of (s)-(+)-3-halo-1,2-propanediol and/or (s)-(-)-2,3-dihalo-1-propanol - Google Patents

Abstract

PURPOSE:To obtain the title compound useful for medicines, etc., in high yield by treating a (+ or -)2,3-dihalo-1-propanol with a bacterium belonging to the genus Pseudomonas to form an optically active (S)-(+)-diol isomer and fractioning and recovering the residue. CONSTITUTION:A substrate containing both an (R)-(+)-2,3-dihalo-1-propanol and a (S)-(-)-2,3-dihalo-1-propanol is treated with a bacterium such as Pseudomonas MH-12 (FERM P-12,323) belonging to the genus Pseudomonas, capable of acting on an (R)-(+)-2,3-dihalo-1-propanol to form an (S)-(+)-3- halo-1,2-propanediol or its treated material and the formed (S)-(+)-3-halo-1,2- propanediol and/or the (S)-(-)-3-halo-1,2-propanodiol not reacting and left is collected to give the optically active (S)-(-)-2,3-dihalo-1-propanol and/or (S)-(+)-3-halo-1,2-propanediol.

Description

Description: The present invention is useful as an intermediate for the synthesis of optically active drugs, agricultural chemicals and other physiologically active substances (S)-
(+)-3-halo-1,2-propanediol and /
Alternatively, it relates to a method for producing (S)-(−)-3-halo-1,2-propanediol. Optical activity (S)-(+)-3-
The method for producing halo-1,2-propanediol is (R)-
A method using 1,2-O-isopropylidene glycerol as a raw material (Chem.-Biol.Interactio
ns, 13 , 193 (1976)), 1, 2, 5, 6-
Method obtained from diacetone-D-mannitol (Che
m. -Biol. Interactions, 41 ,
95 (1982)), a method for synthesizing methyl-6-chloro-6-deoxy-α-D-glucopyranoside (Ch
emissary and Industry 1978
Pp. 533; West German Patent No. 2743858). However, these chemical synthesis methods have complicated steps and are not efficient as industrial production methods. As a biochemical method, microorganisms are allowed to act on (±) -3-halo-1,2-propanediol to selectively (S)-(+)-3-halo-
A method of leaving only 1,2-propanediol (JP-A-62-122596, JP-A-63-36798).
No.), a method of asymmetrically esterifying a racemate (Japanese Patent Application Laid-Open No. 3-30083)
No. 53886) is known, but the yield is low,
There are drawbacks such as low optical purity, and further improved methods are required. Further, (S)-(-)-2,3-dihalo-
As a method for producing 1-propanol, (R)-(+)-
A strain of the genus Pseudomonas having an ability to assimilate 2,3-dihalo-1-propanol is allowed to act on (±) -2,3-dihalo-1-propanol to leave (S)-(−)-.
A method for separating 2,3-dihalo-1-propanol (Japanese Patent Laid-Open Nos. 61-132196 and 62-40298).
And Japanese Patent Laid-Open No. 1-300899). However, the concentration of the substrate that can be used in these methods is about 0.2% or less in the racemic form, which is considered to be a difficulty in terms of efficiency in industrial practice. SUMMARY OF THE INVENTION We have (S)-(+)-3-halo-1,2-propanediol and (S)-(-)-.
As a result of research to overcome the problems of the conventional production technology of 2,3-dihalo-1-propanol, Pseudomonas sp. Newly isolated from soil was found to be (±) -2,3-dihalo-1,2- It acts on propanol to turn the (R) form into (S)
It was found that unreacted (S)-(-)-2,3-dihalo-1-propanol remained in the reaction solution after being converted into-(+)-3-halo-1,2-propanediol. As a result of repeated studies, the present invention has been completed. The present inventors have found that Pseudomonas bacteria convert (R)-(+)-2,3-dihalo-1-propanol into (S)-(+)-3-halo-1,2-propanediol. It was first discovered by the above-mentioned Japanese Patent Application Laid-Open No. 61-13219.
Pseudomonas bacteria disclosed in No. 6 is (R)-
(R)-(-)-3-halo-1,2-propanediol is produced from (+)-2,3-dichloro-1-propanol (JP-A-62-69993), which is used in the present invention. It is different from fungi. That is, the present invention is
From (±) -2,3-dihalo-1-propanol (S)
Not only is it a method for producing-(+)-3-halo-1,2-propanediol, but at the same time (S)-(-)-
It is an efficient method that can produce 2,3-dihalo-1-propanol. Furthermore, it is an industrially excellent method that can carry out the reaction at a substrate concentration higher than 0.2%. DETAILED DESCRIPTION OF THE INVENTION The dehalogenase-producing microorganism used in the present invention is a microorganism belonging to the genus Pseudomonas. For example, the MH-12 newly isolated by the present inventors.
Strains and MH-27 strains. These microorganisms have been deposited at the Patent Microorganism Depositary Center, Institute for Microbial Engineering, Institute of Industrial Technology. The deposit numbers are as follows: MH-12: Microtechnical Research Institute No. 12323 MH-27: Microtechnical Research Institute No. 12324 The taxonomic properties of both strains are as follows. 1. Morphology of bacteria grown on broth agar medium Both strains are rod-shaped and have a size of 0.7 to 0.8 × 1.3 to 2.0 μm. Sexually with one flagella. It does not sporulate, is Gram-negative and has no acid resistance. MH-12 produces round, conical, full-edge, smooth colonies, which are buttery and shiny. M
H-27 forms a colony with a circular shape, a conical shape, and a wavy edge, and the colony is buttery and has a dull gloss. The colonies of both strains are cream colored. Both strains grow at pH 5-10 and grow well at 20-37 ° C. 40 for MH-12
It does not grow at ℃, but MH-27 grows at 40 ℃. 2. Physiological properties Neither strain produces soluble pigment, litmus milk is weakly peptone, and gelatin is not liquefied. Both strains are positive for denitrification, catalase, oxidase, and urease, and do not produce indole or hydrogen sulfide. Methyl red reaction, Voges-Proskauer reaction negative, citric acid, nitrate, ammonium salts are used, arginine is not decomposed, and polyhydroxybutyric acid is not accumulated. O
The F test is the oxidative type and the aromatic ring cleavage is the ortho type. MH-12 reduces nitrates, but MH-27 does not. Both bacteria are D-glucose, D-mannose, D
-Fructose, maltose, sucrose, lactose, trehalose are used oxidatively and starch is not used. Acid production from L-arabinose, D-xylose, D-galactose, D-sorbitol, D-mannitol, inositol, and glycerin is weak. The above-mentioned mycological properties are classified into a classification document (Berge).
y's Manual of Systematic
When analyzed according to Bacteriology Vol. 2 (1986), none of these strains was identified as a bacterium belonging to the genus Pseudomonas, but no concordant strain was found. As the medium composition for culturing the above-mentioned microorganisms, any medium can be used so long as these microorganisms can normally grow. For example, sugars such as glucose, fructose and sucrose as carbon sources, 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 hydrolyzate, organic Acid ammonium salts, amino acids, ammonium sulfate, ammonium chloride and the like can be used, and in addition, inorganic salts, trace metal salts, vitamins and the like can be appropriately used. To induce high converting enzyme activity, epihalohydrin, 1,3-dihalo-2-
It is also useful to add propanol, 3-halo-1,2-propanediol and the like to the medium. Cultivation of the above microorganisms may be carried out by a conventional method, for example, aerobically culturing for 10 to 96 hours at a pH of 4 to 10 and a temperature of 20 to 40.degree. As a reaction method for 2,3-dihalo-1-propanol, 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, treated cells (For example, crushed cells, crude enzyme, cell extracts such as purified enzyme) or a method of adding a substrate to a suspension of cells immobilized or cells treated by a conventional method, at the time of culturing a microorganism There is a method in which the substrate is added to the culture solution and the reaction is performed simultaneously with the culture. The substrate concentration in the reaction solution is not particularly limited, but is preferably 0.1 to 10 (W / V)%, and the substrate can be added to the reaction solution all at once or in divided additions. The reaction temperature is 5 to 50 ° C, and the reaction pH is 4-1.
It is preferable to carry out in the range of 0. The reaction time varies depending on the substrate concentration, cell concentration, other reaction conditions, etc., but it is preferable to set the conditions so that it is usually completed in 1 to 120 hours. Thus, (S)-produced in the reaction solution.
(+)-3-Halo-1,2-propanediol and (S)-(-)-2,3-dihalo-1-propanol are removed from the reaction solution by a method such as centrifugation to remove cells, Extraction is performed with a solvent such as n-butanol, dichloromethane, ethyl acetate, the extract is dehydrated with anhydrous magnesium sulfate, and the concentrated oily substance is dissolved in water, followed by diethyl ether or chloroform (S )
-(-)-2,3-Dihalo-1-propanol is extracted. The extract was concentrated to give (S)-(−)-2,3-dihalo-1-propanol and (S)-from the aqueous layer.
(+)-3-Halo-1,2-propanediol can be obtained. If necessary, these can be further purified by a known method such as silica gel chromatography. The present invention will be described in more detail with reference to the following examples. In the examples, the optical purity was determined by measuring the 3-
After extracting and fractionating halo-1,2-propanediol and 2,3-dihalo-1-propanol, each fraction was converted into (R)-(−)-alpha-methoxy-alphafluoromethylphenylacetate to obtain a high-performance liquid. Determined by chromatography. EXAMPLE 1 Glucose 2%, peptone 0.5%, meat extract 0.3%, yeast extract 0.3%, sodium chloride 0.25%, (±) -2,3-dichloro-1 - propanol _{0.1% (v / v),} CaCO 3 1.0%, pH
A Pseudomonas bacterium MH-12 was inoculated into a 300 ml Erlenmeyer flask containing 30 ml of a sterilized medium having a composition of 7.0, and cultured by shaking at 20 ° C. and 220 rpm for 24 hours. 15 ml of this culture solution was inoculated into a 2 liter Erlenmeyer flask containing 300 ml of the same medium, and the culture was performed under the same conditions as before.
It was shake-cultured for 4 hours. 300 ml of this culture was inoculated into a 5 liter jar fermenter containing 3 liters of a medium in which the composition of the previous medium had a (±) -2,3-dichloro-1-propanol concentration of 0.15%, and 30 C., 800 rpm, and aeration and aeration were carried out for 72 hours at the same amount as the liquid flow rate per minute. After the cells were separated from the thus obtained culture solution by centrifugation, the concentration was 0.2% (±) -2,3-
PH of 7.0 including dichloro-1-propanol.
1 molar phosphate-sodium hydroxide buffer, or pH
Suspended in 9.0 mol of 0.1M Tris-HCl buffer solution to the same concentration as the culture solution obtained by the jar fermenter, 1.2 liter was put in a 2 liter Erlenmeyer flask and kept at 26 ° C. It was placed and reacted. (S)-(+)-3-chloro-1,2-propanediol and residual (S)-formed in the reaction solution as the reaction time passed.
The concentration (v / v) of (-)-2,3-dichloro-1-propanol and the optical purity (% of (S) body) were as shown in Table 1. Table 1 shows the produced (S) -3-chloro-
The optical rotation of a solution of 1,2-propanediol extracted and concentrated with ethyl acetate and dissolved in methanol is also shown. [Table 1] Example 2 Pseudomonas sp. MH- as a microorganism
It carried out like Example 1 except having used 27. Reaction 4
After 8 hours, (S) -3-chloro-1,2-propanediol was produced at a concentration of 0.04% (v / v), and
The body rate was 96.5%. Remaining (S) -2,3-
The concentration of diclo-1-propanol is 0.10% (v /
In (v), the (S) body ratio was 100%. Example 3 The procedure of Example 1 was repeated, except that (±) -2,3-dibromo-1-propanol was used as the substrate. (S) -2,3-dibromo-1 in 88 hours of reaction
-Propanol remained at a concentration of 0.10% (v / v) and its (S) body ratio was 100%. The concentration of the produced (S)-(+)-3-bromo-1,2-propanediol was 0.41% (v / v), and the (S) body ratio was 97.3%. Example 4 The same procedure as in Example 1 was carried out to obtain a reaction solution having a reaction time of 64 hours. In this reaction solution, 2,3-dichloro-
0.136% (v / v) of 1-propanol and 0.064% (v / v) of 3-chloro-1,2-propanediol.
v) was present. 100 ml of this reaction solution was extracted 5 times with 100 ml of ethyl acetate, the extracts were combined, dehydrated with magnesium sulfate, concentrated, and dissolved in 5 ml of ethyl acetate. At this stage, the recovery rate was 88% for 2,3-dichloro-1-propanol and 3-chloro-1,2-
-Propanediol was 23.3%. The oily substance obtained by concentrating the ethyl acetate solution was dissolved in 10 ml of water. 3 ml of 5 ml of diethyl ether was added to this aqueous solution.
After extraction twice, 2,3-dichloro-1-in ether
100% recovery of propanol. 3-chloro-1,
2-Propanediol was recovered in the aqueous layer by 93.1%. The (S) form ratio of 2,3-dichloro-1-propanol obtained by removing the solvent from the ether extract was 82%. The (S) content of 3-chloro-1,2-propanediol in the aqueous layer was 93.5%. EXAMPLE 5 2,3-Dichloro-1-propanol and 3
An approximately 1: 1 mixed aqueous solution of -chloro-1,2-propanediol was prepared. The concentration of 2,3-dichloro-1-propanol in the mixed aqueous solution was 0.570% (v /
v), the concentration of 3-chloro-1,2-propanediol was 0.517% (v / v). 1m of this mixed aqueous solution
After adding 1 ml of the extraction solvent to 1 and homogenizing and separating, the concentration of each component in the extract was analyzed by gas chromatography. The results are shown in Table 2. That is, 2,3-dichloro-1-propanol and 3-chloro-1,2-propanediol can be separated by performing chloroform extraction or diethyl ether extraction from a mixed aqueous solution containing both components. [Table 2] Example 6 From 3 liters of the culture solution obtained in the same manner as in Example 1 except that the culture conditions in the 5 liter jar fermenter were 26 ° C. and 400 rpm. The cells were added to 3 liters of 1 mol of Tris-hydrochloric acid buffer solution (pH 9.0) containing 2% (v / v) of (±) -2,3-dichloro-1-propanol, and 3 liters of the reaction solution was added. The reaction was carried out by aerating and agitating at a speed of 600 rpm at 35 ° C. while aerating 1.5 liters per minute into the placed 5 liter jar fermenter. After 216 hours of reaction, (S)-(−)-2,3-dichloro-1-propanol remained in the reaction solution at 0.826% (residual ratio 41.3%),
The (S) body rate was 100%. INDUSTRIAL APPLICABILITY According to the present invention, it is useful as a synthetic intermediate for pharmaceuticals, agricultural chemicals and other optically active physiologically active substances (S).
-(+)-3-Halo-1,2-propanediol and / or (S)-(-)-2,3-dihalo-1-propanol can be efficiently produced.

Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display area C12R 1:38)

Claims (1)

Claims: (R)-(+)-2,3-dihalo-1-
Acting on propanol, (S)-(+)-3-halo-
A microorganism having the ability to produce 1,2-propanediol or a treated product thereof is allowed to act on (R)-(+)-2,3-dihalo-1,2-propanol to give (S)-
(S)-(+)-3-halo- characterized by producing (+)-3-halo-1,2-propanediol
A method for producing 1,2-propanediol. 2. (R)-(+)-2,3-dihalo-1-
Acting on propanol, (S)-(+)-3-halo-
A microorganism having the ability to produce 1,2-propanol or a treated product thereof is (R)-(+)-2,3-dihalopropanol and (S)-(-)-2,3-dihalo-1-propanol. And (S)-(+)-3-halo-1,2-propanediol produced and / or remaining unreacted (S)-(-)-.
(S)-(+)-3-halo-1,2-propanediol and / or (S)-(-)-2,3 characterized by fractionating 2,3-dihalo-1-propanol -Method for producing dihalo-1-propanol. 3. The method according to claim 1, wherein the microorganism used is a bacterium belonging to the genus Pseudomonas. 4. The method according to claim 2, wherein the microorganism used is a bacterium belonging to the genus Pseudomonas. 5. 2,3-Dihalo-1-propanol and 3
An aqueous solution containing both -halo-1,2-propanediol is extracted with diethyl ether or chloroform to extract 2,3-dihalo-1-propanol into a solvent to give 3-halo-1,2- A method for fractionating and purifying 2,3-dihalo-1-propanol and 3-halo-1,2-propanediol, which comprises allowing propanediol to remain as an aqueous solution.