JP2002223789A - Method for producing optically active 3-hydroxybutyric ester compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing an optically active 3- hydroxybutyric ester compound with the 4-position optionally substituted with a halogen atom. SOLUTION: This method for producing an optically active 3-hydroxybutyric ester compound of the general formula (2) comprises subjecting the cells or treated product thereof of at least one kind of microorganism selected from the group consisting of Penicillium citrinum, Cryptococcus humicolus and Bacillus alvei to act with a 3-oxobutyric ester compound of the general formula (1).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing an optically active 3-hydroxybutyric acid ester compound which may be substituted at the 4-position with a halogen atom.

[0002]

BACKGROUND OF THE INVENTION Optically active 3-hydroxybutyric acid ester compounds which may be substituted at the 4-position with a halogen atom are useful as intermediates for medicinal and agricultural chemicals. It is a compound for which development of a production method is desired. An object of the present invention is to provide a method for producing an optically active 3-hydroxybutyrate compound which may be substituted at the 4-position with a halogen atom.

[0003]

The present inventors have conducted intensive studies to find a method for producing an optically active 3-hydroxybutyric acid ester compound which may be substituted at the 4-position with a halogen atom. As a result, the present inventors have found that Penicillium citrinum is used. citr
inum), Cryptococcus humicola
humicolus) and Bacillus alv
ei) one or more microorganisms selected from the group consisting of a microorganism or a treated product of the microorganisms is a 3-oxobutyrate compound represented by the following general formula (1) and an optically active compound represented by the following general formula (2): It has been found that the compound has the ability to reduce to a -hydroxybutyrate compound, and the present invention has been completed.

That is, the present invention provides a compound represented by the general formula (1):

Embedded image (Wherein, R ¹ represents a C1-C8 alkyl group, and R ² represents a methyl group which may be substituted with a halogen atom.) A 3-oxobutyric acid ester compound represented by the following formula: Penicillium citrinum A cell of one or more microorganisms selected from the group consisting of Cryptococcus humicolus and Bacillus alvei, or a processed product of the cells, characterized by the general formula (2).

Embedded image (Wherein * represents an asymmetric carbon atom, and R ¹ and R ² represent the same meaning as described above) (hereinafter referred to as the production method of the present invention and the production method of the present invention). Note) is provided.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION In the general formulas (1) and (2), the C1-C8 alkyl group represented by R ¹ includes:
Examples include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a t-butyl group, a pentyl group, a hexyl group, a heptyl group, and an octyl group.

Examples of the methyl group optionally substituted by a halogen atom represented by R ² include a methyl group and a methyl group substituted by a halogen atom. Specific examples of the methyl group substituted by a halogen atom include: Examples thereof include a monofluoromethyl group, a monochloromethyl group, a monobromomethyl group, a difluoromethyl group, a dichloromethyl group, a dibromomethyl group, a trifluoromethyl group, and a trichloromethyl group.

In the production method of the present invention, the reaction for converting the 3-oxobutyrate compound represented by the general formula (1) into the optically active 3-hydroxybutyrate compound represented by the general formula (2) is carried out by Penicillium citrinum.
citrinum), Cryptococcus humicolas (Cryptoco)
ccus humicolus) and Bacillus albay (Bacillus)
alvei), using at least one microbial cell selected from the group consisting of: Examples of specific microorganisms or processed cells thereof used in the reaction include, for example, Penicillium citrinum
IFO4631, Cryptococcu humicolas
s humicolus) IFO1527 or Bacillus Albay (Baci
llus alvei) IFO3343t cells or treated cells.

Here, the cultivation of the microorganism used in the production method of the present invention will be described. The microorganism used in the production method of the present invention can be cultured using a medium for culturing various microorganisms appropriately containing a carbon source, a nitrogen source, organic salts, inorganic salts and the like.

[0009] Examples of the carbon source contained in the medium include glucose, sucrose, glycerol, starch, organic acids and molasses. Examples of the nitrogen source include yeast extract, meat extract, peptone, casamino acid, malt extract, and the like. Soy flour, corn steep liquo
r), cottonseed flour, dried yeast, ammonium sulfate and sodium nitrate. Examples of organic and inorganic salts include sodium chloride, potassium chloride, sodium carbonate, monopotassium phosphate, dipotassium phosphate, calcium carbonate, and acetic acid. Ammonium, magnesium sulfate, copper sulfate, zinc sulfate, ferrous sulfate and cobalt chloride.

[0010] Examples of the culture method include solid culture and liquid culture (test tube culture, flask culture, jar fermenter culture, etc.). Culture temperature and pH of culture solution
Is not particularly limited as long as it is within a range in which the microorganisms can grow. For example, the culture temperature may be in the range of 15 to 45 ° C., and the pH of the culture solution may be in the range of 4 to 8. The culture time can be appropriately selected depending on the culture conditions,
Usually, it is 1 to 7 days.

The cells of the microorganism obtained by the culturing can be directly used in the reaction of the production method of the present invention. Examples of the method of using the microorganism cells as they are include a method of using the culture solution as it is, a method of collecting the cells by centrifugation of the culture solution, and using a wet cell obtained by washing the cells with a buffer or water. It is.

In the reaction of the production method of the present invention, a treated product of a microorganism obtained by culturing can also be used. The treated cells of microorganisms that can be used in the reaction include cells obtained by culturing cells treated with an organic solvent (acetone, ethanol, etc.), lyophilization, alkali treatment, and cells. Physically or enzymatically crushed ones can be mentioned. Further, the treated bacterial cells also include those treated and immobilized by a known method.

In the reaction of the production method of the present invention, the cells of the microorganism obtained as described above or the treated cells are used.

The reaction is usually carried out in the presence of water. In this case, the water may be in the form of a buffer. And alkali metal salts of acetic acid such as sodium acetate and potassium acetate, and the like. In this case, the pH of the aqueous layer at the time of the reaction can be appropriately changed within the range in which the reaction proceeds, but is usually within the range of pH 3 to 10. The reaction can be further performed in a two-layer system of water and a hydrophobic organic solvent using a hydrophobic organic solvent. Examples of the hydrophobic organic solvent used in this case include esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, ethyl propionate, and butyl propionate, n-butyl alcohol, and n-butyl alcohol.
Amyl alcohol, alcohols such as n-octyl alcohol, benzene, toluene, aromatic hydrocarbons such as xylene, diethyl ether, diisopropyl ether,
Examples thereof include ethers such as methyl-t-butyl ether, halogenated hydrocarbons such as chloroform and 1,2-dichloroethane, and mixtures thereof.

The concentration of the 3-oxobutyric acid ester represented by the general formula (1), which is a starting compound in the reaction, is usually not more than 50% (w / v), and In order to keep the concentration of the 3-oxobutyric acid ester compound represented by the formula (1) substantially constant,
The oxobutyrate compound may be added continuously or sequentially to the reaction system. The reaction temperature is usually in the range of 0 to 60 ° C. If necessary, sugars such as glucose, sucrose and fructose, alcohols such as ethanol, a surfactant and the like can be added to the reaction. The reaction time is usually in the range of 0.5 hours to 10 days, and the reaction is terminated after the addition of the 3-oxobutyric acid ester compound represented by the general formula (1) is completed. The amount of the compound can be confirmed by measuring by liquid chromatography, gas chromatography, or the like. After completion of the reaction, the optically active 3-hydroxybutyric acid ester compound represented by the general formula (2) can be isolated by subjecting the reaction solution to a usual post-treatment such as extraction with an organic solvent and concentration. Can be further purified by column chromatography, distillation, etc.

[0016]

EXAMPLES Hereinafter, the production method of the present invention will be described in detail with reference to examples, but the production method of the present invention is not limited to these examples.

Example 1 A 500 ml Sakaguchi flask was charged with 100 ml of sterilized medium (200 g of potato extract and 20 g of dextrose in 1 L of water), and the cells of Penicillium citrinum IFO4631 were inoculated therein. Bacteria. This was cultured with shaking at 30 ° C under aerobic conditions. Thereafter, the culture was centrifuged to collect the cells, and the collected cells were suspended in 5 ml of physiological saline. 300 ml of cooled acetone was added to the cell suspension, followed by filtration. The obtained cells were vacuum dried to obtain acetone-dried cells of Penicillium citrinum IFO4631. The Penicillium citrinum (Pe
(nicillium citrinum) In 15 mg of acetone-dried cells of IFO4631, (1.5 ml of butyl acetate in which 15 mg of methyl 4-bromo-3-oxobutyrate is dissolved) and (pH 6.5 in which 75 mg of glucose, 9 mg of NADP ⁺ , and 15 U of glucose dehydrogenase are dissolved). 100 mM
(1.5 ml of phosphate buffer) at 30 ° C.
Shake for hours. Thereafter, the reaction solution was centrifuged to separate an organic layer. The content of this organic layer was analyzed by gas chromatography, and it was confirmed that 5.5 mg of methyl 4-bromo-3-hydroxybutyrate was produced. (Content analysis conditions) Column: HR-20M (0.53 mm × 30 m, 1 μm)
m) (Shinwa Kako Co., Ltd.) Column temperature: 120 ° C (5 minutes) → 3 ° C / min → 150 ° C
(5 min) → 10 ° C./min→200° C. (5 min) Carrier gas: helium (flow rate: 20 ml / min) Detector: FID

Next, methyl 4-bromo-3-hydroxybutyrate isolated by concentrating the organic layer under reduced pressure was dissolved in dichloromethane, and trifluoroacetic anhydride was added.
It was left at room temperature for 1 hour. This solution was analyzed under the following optical isomer analysis conditions, and the obtained methyl 4-bromo-3-hydroxybutyrate was 98% e (S) -isomer. e. Was determined. (Optically active substance analysis conditions) Column: Chirasil-DEX CB (0.32 m
m × 25 m, 0.25 μm) (manufactured by CHROMPACK) Column temperature: 80 ° C. (20 minutes) → 7 ° C./min→150° C. Carrier gas: helium (flow rate: 1.25 ml / min) Detector: FID The absolute configuration of the product is (S) -4-bromo-3
-Determined by comparison with a sample of methyl hydroxybutyrate.

Example 2 A 500 ml Sakaguchi flask was charged with 100 ml of sterilized medium (200 g of potato extract and 20 g of dextrose in 1 L of water).
Humicolas (Cryptococcus humicolus) IFO152
7 cells were inoculated. This was cultured with shaking at 30 ° C under aerobic conditions. After that, the culture was centrifuged to collect the cells,
The collected cells were suspended in 5 ml of physiological saline. 300 ml of cooled acetone was added to the cell suspension, followed by filtration. The obtained cells were dried in vacuo, and Cryptococcus humicolus IFO1527 was used.
Of acetone-dried cells were obtained. Cryptococcus humicolus (1.5 ml of butyl acetate in which 15 mg of methyl 4-bromo-3-oxobutyrate is dissolved in 15 mg of acetone-dried cells of IFO1527)
And (1 of pH 6.5 in which 75 mg of glucose, 9 mg of NADP ⁺ , and 15 U of glucose dehydrogenase are dissolved)
(1.5 ml of a 00 mM phosphate buffer).
Shake at 18 ° C. for 18 hours. Then, the reaction solution is centrifuged,
The organic layer was separated. The content of this organic layer was analyzed by gas chromatography to confirm the production of 2.0 mg of methyl 4-bromo-3-hydroxybutyrate.

Next, methyl 4-bromo-3-hydroxybutyrate isolated by concentrating the organic layer under reduced pressure was dissolved in dichloromethane, and trifluoroacetic anhydride was added.
It was left at room temperature for 1 hour. This solution was analyzed under the same conditions as for the analysis of the optically active substance of Example 1, and the obtained 4-bromo-
Methyl 3-hydroxybutyrate is (S) form 88% e. e. Was determined.

Example 3 A sterilized medium (20 g of glucose, 5 g of polypeptone, 1 g of yeast extract, 3 g of yeast extract) was placed in a 500 ml Sakaguchi flask.
g, meat extract 3 g, potassium dihydrogen phosphate 1 g, magnesium sulfate heptahydrate 0.5 g and ammonium sulfate 2 g
Bacillus alvei I previously cultured in a medium of the same composition
0.3 ml of a culture solution of FO3343t was added. This is 3
Shaking culture was performed at 0 ° C. for 2 days. Thereafter, the culture was centrifuged to collect the cells, and the collected cells were washed with physiological saline and then suspended in 5 ml of physiological saline. 300 ml of cooled acetone was added to the cell suspension, followed by filtration. The obtained cells were dried in a vacuum, and the cells were dried under a Bacillus albae (Bacillus a.
lvei) IFO3343t acetone-dried cells were obtained.
The Bacillus alvei IFO3
(1.5 ml of butyl acetate in which 15 mg of methyl 4-bromo-3-oxobutyrate was dissolved) and (75 mg of glucose NAD ⁺ 9 m
g, pH in which 15 U of glucose dehydrogenase was dissolved
6.5 of 100 mM phosphate buffer (1.5 ml) was added and shaken at 30 ° C. for 18 hours. Thereafter, the reaction solution was centrifuged to separate an organic layer. The content of the organic layer was analyzed by gas chromatography, and 4-bromo-3-
Generation of 4.5 mg of methyl hydroxybutyrate was confirmed.

Next, methyl 4-bromo-3-hydroxybutyrate isolated by concentrating the organic layer under reduced pressure was dissolved in dichloromethane, and trifluoroacetic anhydride was added.
It was left at room temperature for 1 hour. This solution was analyzed under the same conditions as for the analysis of the optically active substance of Example 1, and the obtained 4-bromo-
Methyl 3-hydroxybutyrate is (R) form 96% e. e. Was determined.

Example 4 A sterilized medium (1 L) was placed in a 30-liter jar fermenter.
In water, glucose 20 g, polypeptone 5 g, yeast extract 3 g, meat extract 3 g, potassium dihydrogen phosphate 1 g, magnesium sulfate heptahydrate 0.5 g, ammonium sulfate 2
g) and Bacillus alvei IF previously cultured in a medium of the same composition.
180 ml of a culture solution of O3343t was added. This is 30
Shaking culture was performed at 2 ° C. for 2 days. Thereafter, the culture was centrifuged to collect the cells, the cells were washed with physiological saline, and then suspended in physiological saline. Cooled acetone was added to the cell suspension, followed by filtration. The obtained cells were dried in vacuo, and Bacillusalvei IFO3343t was used.
Of acetone-dried cells were obtained. To 750 mg of acetone-dried cells of Bacillus alvei IFO3343t, 150 mg of ethyl 4,4,4-trifluoro-3-oxobutyrate and (750 mg of glucose, N
AD ⁺ 9 mg, glucose dehydrogenase 2.25 m
g (70 U / mg) dissolved in 100 mM at pH 6.5
Phosphate buffer (30 ml) was added, and the mixture was stirred at 30 ° C. for 27 hours. Thereafter, ethyl acetate was added to the reaction solution, followed by centrifugation. The content of this organic layer was analyzed by gas chromatography to confirm the formation of 114 mg of ethyl 4,4,4-trifluoro-3-hydroxybutyrate. (Content analysis conditions) Column: DB-1 (0.53 mm × 30 m, 1.5 μm)
m) Column temperature: 40 ° C. (5 minutes) → 4 ° C./min→60° C. (0
Min) → 30 ° C./min→290° C. (2 min) Carrier gas: Helium (flow rate: 20 ml / min) Detector: FID

Next, ethyl 4,4,4-trifluoro-3-hydroxybutyrate isolated by concentrating the organic layer under reduced pressure was dissolved in dichloromethane, trifluoroacetic anhydride was added, and the mixture was allowed to stand at room temperature for 1 hour. . This solution was analyzed under the following optical isomer analysis conditions, and the obtained 4,4,4-
Ethyl trifluoro-3-hydroxybutyrate is (R) form 9
9% e. e. Was determined. (Optically active substance analysis conditions) Column: GAMMMA-DEX 120 (0.25 mm
× 30 m, 0.25 μm) (supplico) Column temperature: 80 ° C. (5 minutes) → 5 ° C./min→130° C. → 2
0 ° C./min→200° C. (5 min) Carrier gas: Helium (flow rate: 1.25 ml / min) Detector: FID

[0025]

According to the present invention, an optically active 3-hydroxybutanoic acid ester can be produced.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court ゛ (Reference) (C12P 7/62 (C12P 7/62 C12R 1:01) C12R 1:01) (C12P 7/62 (C12P 7/62 C12R 1:07) C12R 1:07) (C12N 1/14 (C12N 1/14 B C12R 1:80) C12R 1:80) (C12N 1/20 (C12N 1/20 A C12R 1:01) (C12R 1:01) (C12N 1/20 (C12N 1/20 A C12R 1:07) C12R 1:07) (72) Inventor Masanori Shimizu 4-2-1 Takashi Takarazuka-shi, Hyogo Prefecture Sumitomo Chemical Co., Ltd. F term in the company (reference) 4B064 AD64 CA02 CA05 CB18 CD07 CD09 CD15 CD30 CE08 DA16 4B065 AA01X AA15X AA67X BD10 BD30 BD34 BD36 BD44 CA12 CA44 CA47

Claims (3)

[Claims] 1. A compound of the general formula (1) (Wherein, R ¹ represents a C1-C8 alkyl group, and R ² represents a methyl group which may be substituted with a halogen atom.) A 3-oxobutyric acid ester compound represented by the following formula: Penicillium citrinum A cell of one or more microorganisms selected from the group consisting of Cryptococcus humicolus and Bacillus alvei, or a treated product thereof, characterized by the general formula (2): Formula 2 (In the formula, * represents an asymmetric carbon atom, and R ¹ and R ² represent the same meaning as described above.) 2. A compound of the general formula (1) (Wherein, R ¹ represents a C1-C8 alkyl group, and R ² represents a methyl group which may be substituted with a halogen atom.) A 3-oxobutyric acid ester compound represented by the following formula: IFO4631, Cryptococcus humicolus
IFO1527 or Bacillus alvei I
A compound of the general formula (2), wherein a cell or a treated product of any of the microorganisms of FO3343t is allowed to act. (In the formula, * represents an asymmetric carbon atom, and R ¹ and R ² represent the same meaning as described above.) A method for producing an optically active 3-hydroxybutyrate compound represented by the formula: 3. A compound of the general formula (1) (Wherein, R ¹ represents a C1-C8 alkyl group, and R ² represents a methyl group which may be substituted with a halogen atom.) A 3-oxobutyric acid ester compound represented by the following formula: A cell of one or more microorganisms selected from the group consisting of Cryptococcus humicolus and Bacillus alvei, or a processed product of the cells, characterized by the general formula (1). A method for optically reducing the 3-position of a 3-oxobutyric acid ester compound.