JP3010850B2 - Process for producing (S)-(+)-3-halo-1,2-propanediol and / or (S)-(-)-2,3-dihalo-1-propanol - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention is useful as an intermediate for synthesizing optically active pharmaceuticals, agricultural chemicals and other physiologically active substances.
(+)-3-halo-1,2-propanediol and / or
Or (S)-(-)- 2,3-dihalo-1-propano
The method of manufacturing the steel.

[0002]

2. Description of the Related Art Optical activity (S)-(+)-3-
The production method of halo-1,2-propanediol is (R)-
Method using 1,2-O-isopropylideneglycerol as a raw material (Chem.-Biol.Interaction)
ns, 13 , 193 (1976)), 1,2,5,6-
Method obtained from diacetone-D-mannitol (Che
m. -Biol. Interactions, 41 ,
95 (1982)), a method of synthesizing methyl-6-chloro-6-deoxy-α-D-glucopyranoside (Ch
emistry and Industry 1978
Year, p. 533; West German Patent No. 2,743,858) is known. However, these chemical synthesis methods have complicated steps and are not efficient as industrial production methods. In addition, as a biochemical method, (±) -3-halo-1,2-propanediol is allowed to act on a microorganism to selectively produce (S)-(+)-3-halo-diol.
A method of leaving only 1,2-propanediol (JP-A-62-122596, JP-A-63-36798)
), A method for asymmetric esterification of a racemate (JP-A-3-
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-1
-As a method for producing 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 and the remaining (S)-(−)-
Method for fractionating 2,3-dihalo-1-propanol (JP-A-61-132196, JP-A-62-40298)
JP-A-1-300899) is known. However, in these methods, the usable substrate concentration is about 0.2% or less in a racemic form, which is considered to be a difficult point in terms of efficiency for industrial implementation.

[0004]

SUMMARY OF THE INVENTION We have found that (S)-(+)-3-halo-1,2-propanediol and (S)-(-)-
As a result of studying to overcome the problems of the conventional production technology of 2,3-dihalo-1-propanol, Pseudomonas spp. Newly isolated from soil was converted to (±) -2,3-dihalo- 1- propanol. By acting, the (R) form is converted to (S)-
It has been found that unreacted (S)-(-)-2,3-dihalo-1-propanol remains in the reaction solution after being converted to (+)-3-halo-1,2-propanediol. As a result of repeated studies, the present invention has been completed. The present inventors have reported that Pseudomonas bacteria convert (R)-(+)-2,3-dihalo-1-propanol to (S)-(+)-3-halo-1,2-propanediol. For the first time by the Japanese Patent Laid-Open No. 61-132196.
Pseudomonas genus bacteria disclosed in (1) are (R)-
(R)-(-)-3-Halo-1,2-propanediol is produced from (+)-2,3-dichloro-1-propanol (Japanese Patent Application Laid-Open No. 62-69993), and is used in the present invention. It is different from bacteria. That is, the present invention
From (±) -2,3-dihalo-1-propanol to (S)
Not only is this a method for producing-(+)-3-halo-1,2-propanediol, but also (S)-(-)-
It is an efficient method that can produce 2,3-dihalo-1-propanol. Further, it is an industrially superior method that can carry out the reaction at a substrate concentration higher than 0.2%.

[0005]

DETAILED DESCRIPTION OF THE INVENTION The microorganism producing the dehalogenase used in the present invention is a microorganism belonging to the genus Pseudomonas, for example, MH-12 newly isolated by the present inventors.
Strains and MH-27 strains. These microorganisms were deposited at the Patented Microorganisms Depositary Center of the Institute of Microbial Engineering, National Institute of Advanced Industrial Science and Technology. The deposit numbers are as follows. MH-12: No. 12323, MH-27: No. 12324, No. 12324 The taxonomic properties of both strains are as follows.

1. Morphology of bacteria grown on gravy agar medium Both strains are rod-shaped and have a size of 0.7-0.8.times.1.3-2.0 .mu.m. It has one extra flagella in nature. Does not produce spores, is gram negative and has no acid resistance. MH-12 produces round, conical, full-edge, smooth colonies that are buttery and shiny. M
H-27 forms a round, conical, wavy colony around, and the colony is buttery and has a dull luster. The colonies are cream colored in both strains. Both strains grow at pH 5-10 and grow well at 20-37 ° C. MH-12 is 40
Although it does not grow at 40 ° C, MH-27 does grow at 40 ° C.

2. Physiological properties Both strains do not produce soluble pigments, weakly peptone litmus milk and do not liquefy gelatin. Both strains are positive for denitrification, catalase, oxidase and urease, and do not produce indole or hydrogen sulfide. It is negative for methyl red reaction and Voges-Proskauer reaction and utilizes citric acid, nitrate and ammonium salt, does not decompose arginine and does not accumulate polyhydroxybutyric acid. O
The F test is of the oxidized type and the cleavage of the aromatic ring is of the ortho type. MH-12 reduces nitrate but not MH-27. D-glucose, D-mannose, D
-Uses fructose, maltose, sucrose, lactose, trehalose oxidatively and does not use starch. Acid production from L-arabinose, D-xylose, D-galactose, D-sorbitol, D-mannitol, inositol, glycerin is weak.

[0008] The above mycological properties are classified (Berge
y's Manual of Systematic
When examined according to Bacteriology, Vol. 2 (1986), all of these strains were identified as bacteria belonging to the genus Pseudomonas, but there was no corresponding strain.

As a medium composition for culturing the above microorganisms, any medium can be used as long as these microorganisms can grow. For example, glucose, fructose, sugars such as sucrose, acetic acid, organic acids such as citric acid, alcohols such as ethanol and glycerol as carbon sources, and nitrogen sources as peptone, meat extract, yeast extract, protein hydrolysate, organic Acid ammonium salts, amino acids, ammonium sulfate, ammonium chloride and the like can be used. In addition, inorganic salts, trace metal salts, vitamins and the like are used as needed. In order to induce a 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.

The above microorganisms may be cultured by a conventional method, for example, aerobically at pH 4 to 10 and at a temperature of 20 to 40 ° C. for 10 to 96 hours. As a reaction method for 2,3-dihalo-1-propanol, a method in which a substrate is added to a culture solution of a microorganism cultured as described above or a suspension of cells obtained by centrifugation or the like, (For example, a method of adding a substrate to a suspension of cell bodies such as crushed cells, crude enzymes, and purified enzyme, etc.) or cells immobilized by a conventional method or processed cells, etc. There is a method in which a substrate is added to a culture solution and a reaction is performed simultaneously with the culture.

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

The (S)-thus formed in the reaction solution.
(+)-3-Halo-1,2-propanediol and (S)-(-)-2,3-dihalo-1-propanol were obtained by removing cells from the reaction solution by centrifugation or the like. Extraction is carried out with a solvent such as n-butanol, dichloromethane, ethyl acetate, etc., the extract is dehydrated with anhydrous magnesium sulfate or the like, and the concentrated oil is dissolved in water, and then added with diethyl ether or chloroform (S )
-Extract (-)-2,3-dihalo-1-propanol. 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. These can be further purified, if necessary, using a known method such as silica gel chromatography.

[0013]

The present invention will be described more specifically with reference to the following examples. In the examples, the determination of the optical purity was determined by using 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 extract a high-performance liquid. Determined by chromatography.

[0014]

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 0.1% (v / v), CaCO ₃ 1.0%, pH
Pseudomonas sp. 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 with shaking at 220C per minute for 24 hours at 20C. 15 ml of this culture solution was inoculated into a 2 liter Erlenmeyer flask containing 300 ml of the same medium, and cultured under the same conditions as before.
The cells were cultured with shaking for 4 hours. 300 ml of this culture was inoculated into a 5-liter jar fermenter containing 3 liters of a medium having a (±) -2,3-dichloro-1-propanol concentration of 0.15% in the composition of the previous medium. C., 800 rpm, and aeration and aeration were performed for 72 hours at the same volume as the liquid volume per minute. After the cells were separated from the culture broth by centrifugation, a concentration of 0.2% (±) -2,3-
0.1 pH of 7.0 including dichloro-1-propanol.
1 M phosphoric acid-sodium hydroxide buffer, or pH
Into a 2-liter Erlenmeyer flask was added 1.2 liters of a suspension suspended in a 0.1 mol Tris-HCl buffer solution of 9.0 to the same concentration as that in the culture solution obtained by the jar fermenter, and the mixture was allowed to stand at 26 ° C. To react. (S)-(+)-3-Chloro-1,2-propanediol formed in the reaction solution with the elapse of the reaction time and the remaining (S)-
The concentration (v / v) and the optical purity ((S) volume%) of (-)-2,3-dichloro-1-propanol were as shown in Table 1. Table 1 shows the product (S) -3-chloro-
The optical rotation of 1,2-propanediol extracted and concentrated with ethyl acetate and dissolved in methanol is also shown.

[Table 1]

[0015]

Example 2 Pseudomonas sp. MH-
The same operation as in Example 1 was carried out except that 27 was used. Reaction 4
In 8 hours, (S) -3-chloro-1,2-propanediol was formed to a concentration of 0.04% (v / v) and the (S)
The body volume was 96.5%. Residual (S) -2,3-
The concentration of dichloro-1-propanol is 0.10% (v /
In (v), the (S) content was 100%.

[0016]

Example 3 Example 3 was carried out in the same manner as in Example 1 except that (±) -2,3-dibromo-1-propanol was used as a substrate. After 88 hours of reaction, (S) -2,3-dibromo-1
-Propanol remained at a concentration of 0.10% (v / v), and its (S) content was 100%. The concentration of the produced (S)-(+)-3-bromo-1,2-propanediol was 0.41% (v / v), and its (S) content was 97.3%.

[0017]

Example 4 A reaction was performed in the same manner as in Example 1 to obtain a reaction solution for a reaction time of 64 hours. 2,3-dichloro-
0.136% (v / v) of 1-propanol and 0.064% (v / v) of 3-chloro-1,2-propanediol
v). 100 ml of the reaction solution was extracted five times with 100 ml of ethyl acetate, and the extracts were combined, dried over magnesium sulfate, concentrated, and dissolved in 5 ml of ethyl acetate. At this stage, the recovery was 88% for 2,3-diclo-1-propanol, 3-chloro-1,2.
-Propanediol was 23.3%. The oil obtained by concentrating this ethyl acetate solution was dissolved in 10 ml of water. 3 ml of this aqueous solution with 5 ml of diethyl ether
When extracted twice, 2,3-diclo-1-
Propanol was 100% recovered. 3-chloro-1,
93.1% of 2-propanediol was recovered in the aqueous layer. The (S) form ratio of 2,3-dichloro-1-propanol obtained by removing the solvent from the ether extract was 82%. The (S) form ratio of 3-chloro-1,2-propanediol in the aqueous layer was 93.5%.

[0018]

Example 5 2,3-Dichloro-1-propanol and 3
An approximately 1: 1 mixed aqueous solution of -chloro-1,2-propanediol was made. The concentration of 2,3-diclo-1-propanol in the mixed aqueous solution was 0.570% (v / v
v) The concentration of 3-chloro-1,2-propanediol was 0.517% (v / v). 1m of this mixed aqueous solution
1 ml of an extraction solvent was added to 1 liter, homogenized and separated, and the concentration of each component in the extract was analyzed by gas chromatography. The results are as shown in Table 2. That is, 2,3-dichloro-1-propanol and 3-chloro-1,2-propanediol can be fractionated by performing chloroform extraction or diethyl ether extraction from a mixed aqueous solution containing both components.

[Table 2]

[0019]

Example 6 Cells were cultured 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 changed to 26 ° C. and 400 rpm. 3 liters of reaction solution added to 3 liters of 1M Tris-HCl buffer (pH 9.0) containing 2% (v / v) (±) -2,3-dichloro-1-propanol5 The reaction was carried out at a rate of 600 rpm per minute at 35 ° C. with aeration while stirring the liter fermenter at 1.5 liters per minute. After 216 hours of reaction, (S)-(−)-2,3-dichloro-1-propanol remained in the reaction solution at 0.826% (residual rate: 41.3%),
Its (S) content was 100%.

[0020]

According to the present invention, (S) useful as a synthetic intermediate for pharmaceuticals, agricultural chemicals and other optically active physiologically active substances.
-(+)-3-Halo-1,2-propanediol and / or (S)-(-)-2,3-dihalo-1-propanol can be efficiently produced.

Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12R 1:38)

Claims (2)

(57) [Claims] (1) (R)-(+)-2,3-dihalo-1-
(S)-(+)-3-halo- acting on propanol
Shoe having an ability to produce 1,2-propanediol
A bacterium belonging to the genus Domonas or a processed product thereof is (R)-
(S)-(+)-(+)-3-halo-1,2-propanediol is produced by acting on (+)-2,3-dihalo- 1- propanol. )
Method for producing -3-halo-1,2-propanediol. 2. (R)-(+)-2,3-dihalo-1-
(S)-(+)-3-halo- acting on propanol
Shoe having an ability to produce 1,2-prop Njio Lumpur
A bacterium belonging to the genus Domonas or a processed product thereof is (R)-
(+)-2,3-dihalo -1- propanol and (S)-
Contains both (-)-2,3-dihalo-1-propanol
It is allowed to act on the non-substrate, resulting (S) - (+) - remaining without 3-halo-1,2-propanediol and / or reaction (S) - (-) - 2,3- dihalo - (S)-(+)-characterized by fractionating 1-propanol
A method for producing 3-halo-1,2-propanediol and / or (S)-(-)-2,3-dihalo-1-propanol.