JPH09308497A - Production of optically active gamma-butyrolactone derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain the subject compound useful as a medicine intermediate, a homoserine derivative, etc., by bringing a specific γ-butyrolactone derivative into contact with a cell (treated material) of a fungus belonging to the genus Fusarium, Cylindrocarpon or Gibberella. SOLUTION: A compound of formula I (R is hydroxyl group or amino group) (e.g. α-hydroxy-γ-butyrolactone, etc.) or its salt is brought into contact with a culture product of a bacterium belonging to the genus Fusarium [e.g. Fusarium oxysporum, (IFO 5,942)], Cylindrocarpon [e.g. Cylindrocarpon tonkinense (IFO 30,561)] or Gibberella [e.g. Gibberella fujikuroi (IFO 6,349)], a cell of the fungus or a treated cell and only one of optically active isomers is selectively hydrolyzed to give the objective optically active unreacted y∥γbutyrolactone derivative of formula II or formula III simply, efficiently and industrially advantageously.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a pharmaceutical intermediate and a novel method for producing an optically active γ-butyrolactone derivative useful as a derivative of homoserine which is an amino acid.

[0002]

As a method for producing an optically active γ-butyrolactone derivative, for example, an optical resolution method using quinine or brucine as a resolving agent in the method for producing optically active α-hydroxy-γ-butyrolactone [J. Amer. Che
m. Soc. , 2675 (1921)], and a method of making a racemic mixture with an alkaloid and separating by fractional crystallization [J. Amer. Chem. Soc. , 1576 (19
48), J. Amer. Chem. Soc. , 2497
(1951)], a method using optically active malic acid as a raw material [J. Org. Chem. , 1040 (1988)] and the like are known. Further, as a method for producing an optically active α-amino-γ-butyrolactone, a method of introducing an acyl group into an amino group and biologically hydrolyzing it [Bio
l. Chem. , 212 (1953)], a method using methionine as a raw material [Microchem. J. , 226
(1989)], a method using aspartic acid as a raw material [J. Org. Chem. , 4763 (1990)] and the like are known.

[0003]

However, the optical resolution method requires an expensive resolving agent such as quinine or brucine, and the .gamma.-butyrolactone derivative is not easily recovered after the resolution. have. The fractional crystallization method is operationally complicated and is not industrially suitable. Further, the method using optically active malic acid as a raw material includes a reduction step with diborane in the middle of the method, and is industrially difficult to manufacture. Biological hydrolysis methods such as those in the production of optically active α-amino-γ-butyrolactone require the introduction of an acyl group in the amino group beforehand. When methionine is used as a raw material, methyl mercaptoacetic acid is produced as a by-product, which causes a problem of malodor. The method using aspartic acid as a raw material includes a reduction step with diborane, and is industrially difficult to manufacture.
Thus, at present, a method for easily and industrially producing optically active γ-butyrolactone is not yet known.

[0004]

Means for Solving the Problems The inventors of the present invention have a general formula (I)

Embedded image (R represents a hydroxyl group and an amino group.) As a result of intensive research on asymmetric hydrolysis of a compound represented by the formula (I) or a salt thereof, the use of a specific microorganism enables efficient reaction of the compound represented by the general formula (I). By selectively hydrolyzing only the R form of the compound, the compound of the general formula (II)

[0005]

Embedded image (R is the same as the above.) By separating the unhydrolyzed S-form represented by the formula (III) from the reaction system,

[0006]

Embedded image The hydrolyzate of R-form represented by (R is the same as above) is efficiently lactoned to give a compound of general formula (II) and (IV).

[0007]

[Chemical 12] It was found that an optically active γ-butyrolactone derivative represented by (R is the same as above) can be obtained.

The present invention has been made based on such findings. That is, the present invention provides (1) the general formula (I)
(R represents a hydroxyl group, an amino group.) Or a salt thereof, which is a culture of a microorganism belonging to a genus selected from the group consisting of Fusarium, Sylindrocarpon, and Gibberella, and its cells. Or an optically active γ-represented by the general formula (II) (R is the same as the above) or the general formula (IV), which is characterized in that it is brought into contact with treated cells.
Method for producing butyrolactone derivative or salt thereof, (2) compound represented by general formula (I) (R is the same as above) or salt thereof is selected from the group consisting of genus Fusarium, genus cylindrocarpon and genus Gibberella. The unreacted compound of the general formula (II) (R is the same as above) or a salt thereof is separated from the reaction system by contacting it with a culture of a microorganism belonging to the genus, a bacterial cell thereof, or a treated bacterial cell. Thereby producing a (S) -γ-butyrolactone derivative or a salt thereof, and (3)
A culture of a microorganism belonging to a genus selected from the group consisting of the genus Fusarium, the genus Cylindrocarpon and the genus Gibberella, wherein the compound represented by the general formula (I) (R is the same as above) or a salt thereof is used. A compound of the general formula (III) (R is the same as above) or a salt thereof is obtained by contacting it with a body or a treated microbial cell, and the compound or salt thereof is obtained, and the compound of the general formula (IV) (R is the same as above) or the salt thereof is obtained. Provided is a method for producing a (R) -γ-butyrolactone derivative or a salt thereof by converting the salt into a salt. INDUSTRIAL APPLICABILITY In the present invention, γ-, which has an extremely high optical purity in a short reaction treatment step
It has many advantages such as the ability to obtain a butyrolactone derivative or a salt thereof.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention can be represented by the following reaction formula.

Embedded image (* Indicates optically active carbon.)

That is, according to the present invention, the general formula (I)
The compound or salt thereof is contacted with a culture of a microorganism belonging to a genus selected from the group consisting of Fusarium, Sylindrocarpon, and Gibberella, its bacterial cells, or treated bacterial cells, and a compound of the general formula (II) By separating the unhydrolyzed product of S-form represented by or the salt thereof from the reaction system, and the obtained hydrolyzate of the general formula (III) or its salt is then subjected to lactonization. By converting to the general formula (IV) or a salt thereof, an efficient method for producing an optically active γ-butyrolactone derivative or a salt thereof is provided.

The present invention will be described in more detail below. In the present invention, as a microorganism that asymmetrically hydrolyzes the compound of the general formula (I) selectively in the R form, a microorganism having an asymmetric hydrolysis function belonging to the genus Fusarium, the genus Cylindrocarpon and the genus Gibberella is industrially used. Specifically, it is used as a microorganism for producing a compound of general formula (II) or a compound of general formula (IV) via a compound of general formula (III). Among the microorganisms belonging to each of the above genera, particularly excellent R-form-selective asymmetric hydrolysis decomposability is found. Among them, Fusarium oxysporum (IFO)
5942), Fusarium semitectam (IFO302
00), etc., belong to the genus Cylindrocarpon as Syrindrocarpon tonkinens (IFO30561), Syrindrocarpon sclerotigenum (IFO3185).
5) and the like, as the Gibberella genus, Gibberella fujikuroi (IFO6349), Gibberella fujikuroi (IFO6)
603) and the like are preferable.

In the method of the present invention, various conditions for culturing a microorganism vary depending on the strain to be used. Regarding the medium, carbon sources include sugars such as glucose and sucrose, alcohols such as ethanol and glycerol, and oleic acid. , Fatty acids such as stearic acid and esters thereof, oils such as rapeseed oil, soybean oil, etc., and nitrogen sources such as ammonium sulfate, ammonium nitrate, sodium nitrate, peptone, casamino acid, corn steep liquor, bran, yeast extract, etc. ,
As inorganic salts, magnesium sulfate, sodium chloride,
Examples include calcium carbonate, potassium monohydrogen phosphate, potassium dihydrogen phosphate, and the like, and a medium containing malt extract, meat extract, or the like as another nutrient source. In addition, those generally used in this field can be appropriately selected and used, and, for example, vitamins, minerals and the like can be optionally added. The culture is carried out aerobically, and is usually carried out at a culture time of about 1 to 7 days, a medium pH of 3 to 9, a culture temperature of 10 to 50 ° C.

In the method of the present invention, the microorganism used is
Any form such as a culture obtained by culturing a strain in a liquid medium, cells separated from the culture solution, or dried cells obtained by treating the cells or the culture, or immobilized cells Can also be used, and it is also possible to use a microorganism by genetic recombination. It is also possible to increase the enzyme activity of microorganisms by gene recombination and mutation treatment. Genetic recombination is, for example, Molecular
Cloning 2nd ed. J .; Shambr
look et al. , Cold Spring Ha
rbor Lab. It can be performed according to the method described in Press, 1989. Examples of breeding methods by mutagenesis include methods for inducing mutations, for example, methods utilizing physical mutagens such as ultraviolet rays, γ-rays, and X-rays, sodium nitrite, alkylating agents, hydroxylamine, nitroso. Examples include those that use chemical mutagens such as urea and nitrosoguanidine.
The operation can be performed in a batch system, a semi-batch system, or a continuous system. The concentration of γ-butyrolactone used is usually 10 to 500 g / l. The reaction temperature is
Usually, it is 10 to 50 ° C, more preferably 20 to 3 ° C.
0 ° C. In the case of a batch system, the reaction time is usually several hours to 1 day. The pH of the reaction system is usually about 3 to 9, but more preferably 6 to 8. As a result of selective asymmetric hydrolysis of general formula (I) by microorganisms, general formula (II)
The compound of I) is produced, the pH of the reaction solution decreases, and the reaction rate also decreases. To increase the reaction rate,
It is preferable to maintain the pH of the reaction solution at the optimum pH of the lactone hydrolase of each microorganism. At that time, as the inorganic salt for maintaining the pH, there is used a hydroxide or carbonate of an alkali metal or an alkaline earth metal, as well as aqueous ammonia.

After completion of the reaction, the compound of the general formula (II) in the reaction solution which has not undergone hydrolysis is separated by an operation such as extraction with an organic solvent. Examples of the organic solvent include halogenated hydrocarbons such as ethylene chloride, chloroform and trichloroethane, aromatic hydrocarbons such as benzene and toluene, ethers such as diethyl ether and t-butyl methyl ether, and ethyl acetate. Is used, more preferably ethyl acetate. Moreover, column chromatography etc. are mentioned as a separation method other than extraction. A reversed-phase column is used, and as the eluent, water, an alcohol such as methanol or ethanol, or a mixture thereof is used.
It is also possible to separate the compounds of I). The compound of the general formula (III) remaining in the reaction solution can be converted to the compound of the general formula (IV) by acidifying as it is. As the acid, hydrochloric acid, sulfuric acid or the like is used, and more preferably sulfuric acid. The pH, the reaction temperature, and the reaction time may be conditions that allow the compound of the general formula (III) to undergo ring closure and be converted into the compound of the general formula (IV), but more preferably, the pH is 1 or more strong acidic, The reaction temperature is 80 to 130 ° C., and the reaction time is 1 to 6 hours.

The produced compound of the general formula (IV) is recovered by extraction with an organic solvent. The organic solvent used here is the same as the solvent used for extracting the compound of the general formula (II), halogenated hydrocarbons such as ethylene chloride, chloroform and trichloroethane, and aromatic solvents such as benzene and toluene. In addition to ethers such as hydrocarbons, diethyl ether and t-butyl methyl ether, ethyl acetate and the like are used, and ethyl acetate is more preferable. Further, recovery by column chromatography is also possible. On the other hand, the compound of the general formula (III) remaining in the reaction solution is treated with an alkali metal hydroxide or carbonate, the reaction solution is distilled off under reduced pressure, and the dried solid is recrystallized with a solvent. Thus, it can be isolated as an alkali metal salt of carboxylic acid. Examples of the alkali metal hydroxides or carbonates used include lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate and the like, and preferably sodium hydroxide. Further, as the recrystallization solvent, methanol, ethanol, isopropanol, chloroform and the like are used, and preferably methanol. Thus, the present invention provides a simple and efficient method for producing an optically active γ-butyrolactone derivative useful as a pharmaceutical intermediate or an amino acid derivative.

[0016]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

Examples 1 to 6 Glucose 1%, Polypeptone 0.5
%, Yeast extract 0.5%, corn steep liquor 0.
5 ml of liquid medium (pH 6.5) containing 5% was added to a test tube.
Dispense each by 20 minutes at 121 ℃ in an autoclave,
Heat sterilized. From the slope medium to the medium in each test tube
Each of the various strains listed in the table was inoculated and aerobically shake-cultured at 28 ° C. for 3 days. After culturing, cells were obtained by filtration, 2 ml of 0.2M Tris-HCl buffer solution (pH 7.5) with a concentration of 1% of α-hydroxy-γ-butyrolactone was added to each of these cells, and the cells were kept at 30 ° C. for 2 hours. Shake for hours. After the reaction, bacterial cells were removed by filtration, and HPLC (YM
C-Pack ODS-AQ φ6.0 × L300 mm, eluent 3% methanol (0.1% TFA), flow rate 0.8 ml / min, detection wavelength 230 nm, column temperature 30 ° C. α-hydroxy-γ -The amount of butyrolactone reduced and the amount of 2,4-dihydroxy-butyric acid produced were measured. The optical purity of the produced (R) -2,4-dihydroxy-butyric acid was determined by HPLC (MCI GEL φ4.6 × L100 mm, eluent 2 m).
M CuSO ₄ , flow rate 0.4 ml / min, detection wavelength 254 nm,
It was measured at a column temperature of 30 ° C. The results are shown in Table 1.

[0017]

[Table 1]

[0018]

Examples 7 to 12 In accordance with Examples 1 to 6, cells of each strain listed in Table 2 were obtained. Α-Amino-γ-butyrolactone hydrobromide at a concentration of 1% was added to each of these cells.
2 ml of 2M Tris-HCl buffer solution (pH 7.2) was added, and the mixture was shaken at 30 ° C. for 2 hours. After the reaction, the bacterial cells were removed by filtration and subjected to HPLC (YMC-Pack ODS-AQ φ6.0 × L
300 mm, eluent 3% methanol (0.1% TFA),
The decrease amount of α-amino-γ-butyrolactone and the production amount of homoserine in each reaction solution were measured at a flow rate of 0.8 ml / min, a detection wavelength of 230 nm, and a column temperature of 30 ° C. The optical purity of the (R) -homoserine produced was determined by HPLC (M
CI GEL φ 4.6 x L 150 mm, eluent 2 mM CuSO ₄ , flow rate
0.4 ml / min, detection wavelength 254 nm, column temperature 30
(° C). The results are as shown in Table 2.

[0019]

[Table 2]

[0020]

Example 13 Fusarium oxysporum (IFO 594
500 ml containing 300 ml of liquid medium (pH 6.5) consisting of 2) glucose 1%, polypeptone 0.5%, yeast extract 0.5% and corn steep liquor 0.5%.
Using two shake flasks, shaking culture was performed aerobically at 28 ° C. for 4 days. After culturing, the cells obtained by filtration were added with 100 m of a 10% aqueous solution of α-hydroxy-γ-butyrolactone.
1 was added, and the reaction liquid p
While maintaining H at 7, the reaction was carried out at 30 ° C. for 3 hours. After completion of the reaction, bacterial cells were removed by filtration, the obtained reaction completed solution was salted out with ammonium sulfate, and then extracted with ethyl acetate. The obtained ethyl acetate layer was dehydrated with anhydrous magnesium sulfate. Magnesium sulfate was removed by filtration, the obtained ethyl acetate layer was distilled off under reduced pressure, and then distilled under reduced pressure (bp 98 to 99 ° C /
0.17 Torr) and (S) -α-hydroxy-γ-butyrolactone 2.9 g (yield: 29%, optical purity: 98.2)
% Ee [α] _D ²⁵ = −69.2 ° (C 1.15, CHCl ₃ )) was obtained.

[0021]

Example 14 Fusarium oxysporum (IFO 5942) cells obtained by culturing in the same manner as in Example 13 were added to 100 m of a 10% aqueous solution of α-hydroxy-γ-butyrolactone.
1 was added, and the reaction liquid p
While maintaining H at 7, the reaction was carried out at 30 ° C. for 1 hour. After the reaction was completed, the bacterial cells were removed by filtration, the obtained reaction completed solution was salted out with ammonium sulfate, and the unreacted α-hydroxy-γ-butyrolactone was extracted and removed with ethyl acetate, and the resulting aqueous layer was obtained. The pH was adjusted to 1 by adding sulfuric acid, and the mixture was boiled for 5 hours. After cooling, it was neutralized with aqueous ammonia and extracted with ethyl acetate. The obtained ethyl acetate layer was dehydrated with anhydrous magnesium sulfate. Magnesium sulfate was removed by filtration, the obtained ethyl acetate layer was distilled off under reduced pressure, and then distilled under reduced pressure to obtain (R) -α.
-Hydroxy-γ-butyrolactone 2.2 g (yield: 2
2%, optical purity: 93.5% ee [α] _D ²⁵ = + 65.9 °
(C 1.15, CHCl ₃ )) was obtained.

[0022]

Industrial Applicability According to the present invention, an expensive resolving agent is not required, it is suitable for industrial production with a simple operation, has little environmental problems, and is an efficient method, and is useful as a pharmaceutical intermediate. An optically active γ-butyrolactone derivative useful as a derivative of homoserine which is an amino acid can be produced. A method for easily and industrially producing an optically active γ-butyrolactone derivative is provided.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location (C12P 41/00 C12R 1: 645)

Claims (3)

[Claims] 1. A compound of the general formula (I) (R represents a hydroxyl group, an amino group.) Or a salt thereof, which is a culture of a microorganism belonging to a genus selected from the group consisting of Fusarium, Sylindrocarpon, and Gibberella, and its cells. , Or a treated bacterial cell, which is characterized by being represented by the general formula (II): (R is the same as above.) Or the general formula (IV) (R is the same as above.) A method for producing an optically active γ-butyrolactone derivative or a salt thereof. 2. A compound of the general formula (I) (R represents a hydroxyl group, an amino group.) Or a salt thereof, which is a culture of a microorganism belonging to a genus selected from the group consisting of Fusarium, Sylindrocarpon, and Gibberella, and its cells. , Or a non-reacted compound of the general formula (II): A method for producing a (S) -γ-butyrolactone derivative or a salt thereof by separating the compound (R is the same as above) or a salt thereof from the reaction system. 3. A compound represented by the general formula (I): (R represents a hydroxyl group, an amino group.) Or a salt thereof, which is a culture of a microorganism belonging to a genus selected from the group consisting of Fusarium, Sylindrocarpon, and Gibberella, and its cells. , Or by contacting the treated cells with the general formula (III): (R is the same as above) or a salt thereof, which is represented by the general formula (IV): A method for producing a (R) -γ-butyrolactone derivative or a salt thereof by converting the compound (R is the same as above) or a salt thereof.