JPH06261787A - Production of optically active beta-amino acid - Google Patents

Abstract

PURPOSE:To industrially obtain at a low cost an optically active beta-amino acid compound as an important intermediate for medicines. CONSTITUTION:Dihydropyrimidinase derived from microbes capable of stereospecifically cleavingly hydrolyzing dihydrouracil ring is made to act on a 6-substituted dihydrouracil compound in an aqueous medium at pH7-10 to convert this compound into an N-carbamoyl-beta-amino acid compound rich in a stereoisomer which is then collected to obtain the objective optically active N-carbamoyl-beta-amino acid compound. And, this compound is decarbamoylated into a beta-amino acid compound to obtain the other objective optically active beta-amino acid compound.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing optically active β-amino acids and their derivatives.

Β-Amino acids are important intermediates in medicine, for example β-lactam derivatives.

[0003]

2. Description of the Related Art As a method for producing optically active β-amino acids, a method in which a racemic methyl ester is resolved with tartaric acid (Tetrahedron Letters)
Letters) 27 , 2789, (1972)),
Method for optically resolving racemic benzyloxycarbonyl compound with quinine (Burethane of the Chemical Society of Japan (Bull. Chem.
Soc. Japan) 52 , 3326 (197)
9)), the well-known so-called Arnto-Iistelte (Ar
ndt-Eistert) method for carbon-carbon one-carbon addition of the corresponding α-amino acid (Chem. Letters 151 (197).
3)), a method of hydrogenating a phenethylamine Schiff base of an acetylacetic acid derivative (JP-A-60-139655), and a method of adding phenethylamine to a crotonic acid derivative (Journal of Organic Chemistry (J. Org. Chem.) 57 ). , 2396
(1992)), a method of deriving from 3-hydroxybutyric acid (Helvetica Kimika Actor (Helv. Chi
m. Acta) 70 , 1326 (1987)), L-
Method to derive from asparagine (Tetrahedron Letters 31 , 5717 (1990), Journal of
Organic Chemistry 56 , 1355 (199
1)), a method for asymmetrically reducing a 3-aminoacrylic acid derivative with an asymmetric ruthenium complex (tetrahedron asymmetry (Tetrahedron Asymmetr
y) 2 , 464 (1991)) and the like are known.

[0004]

The optical resolution method using tartaric acid or quinine is industrially complicated because the β-amino acids are converted into monotan derivatives and then optically resolved with tartaric acid or quinine and further hydrolyzed. , Has operational difficulties. Next, the method using the Arnto-Eistert synthesis has a drawback that it uses toxic and explosive diazomethane. Further, the method using phenethylamine is not a satisfactory method because phenethylamine is expensive. Furthermore, 3-hydroxybutyric acid and L
-The method of deriving from asparagine requires many manufacturing steps and complicated operations because it consists of several steps of chemical reaction. Further, the asymmetric ruthenium complex is expensive and difficult to handle.

The present inventors have conducted extensive studies to develop an inexpensive and industrial production method of optically active β-amino acids, and as a result, 6-substituted dihydrouracils were used as a raw material and dihydropyrimidinase was used. The present invention has been completed by finding a very effective manufacturing method of optical resolution.

[0006]

The gist of the present invention is expressed by the following equation.

[0007]

[Chemical 5]

(In the formula, X is an alkyl group having 1 to 4 carbon atoms,
formula

[0009]

[Chemical 6]

(Wherein R represents hydrogen, a hydroxyl group or a lower alkoxy group having 1 to 4 carbon atoms), a phenyl group or a substituted phenyl group, a thienyl group, a furyl group, a pyridyl group, a naphthyl group or an indolyl group. That is, the present invention has the general formula (I):

[0011]

[Chemical 7]

(In the formula, X is an alkyl group having 1 to 4 carbon atoms,
formula

[0013]

[Chemical 8]

(Wherein R represents hydrogen, a hydroxyl group or a lower alkoxy group having 1 to 4 carbon atoms), a phenyl group or a substituted phenyl group, a thienyl group, a furyl group, a pyridyl group, a naphthyl group or an indolyl group. The 6-substituted dihydrouracils represented by the formula (1) are derived from a microorganism having the ability to stereolytically cleave and hydrolyze the dihydrouracil ring.
C. 3.5.2.2, also known as hydantoinase) at pH 7
The compound of formula (II):

[0015]

[Chemical 9]

An optically active N-carbamoyl which is characterized in that it is converted into N-carbamoyl-β-amino acids rich in stereoisomers represented by the formula (wherein X is the same as above) and the stereoisomers are collected. -Method for producing β-amino acids and optically active N-carbamoyl- represented by formula (II)
decarbamoylation of β-amino acids to give formula (III)

[0017]

[Chemical 10]

(Wherein X is as defined above) β
-Amino acids, and a method for producing optically active β-amino acids.

By using the method of the present invention, an inexpensive microbial enzyme is used to convert 6-substituted dihydrouracils as a substrate to convert them into optically active N-carbamoyl-
β-amino acids can be obtained. Furthermore, N-carbamoyl-β-amino acids can be converted to the corresponding β-amino acids in high yield by diazotization and decarbamoylation in a mineral acid acidic aqueous medium.

As the 6-substituted dihydrouracils used as a starting material in this reaction, it is appropriate to use a DL-form usually obtained by chemical synthesis. For example, a 6-substituted dihydrouracil is a method of reducing the corresponding 6-substituted uracil (Journal of Organic Chemistry).
49 , 5256 (1984)) or a method of condensing an aldehyde with malonic acid and urea (Izvesty Acibilis Otto de Renja Academy Nauku).
st. Sibir. Otdel. Akad. N
auk S. S. S. R) 7 , 72 (1961)), a method of condensing urea and 2-alkenoic acid at high temperature (Journal of Organic Chemistry 26 , 187).
7 (1961)) and the like, and can be easily and inexpensively synthesized.

The microorganism serving as a source of dihydropyrimidinase has the ability to stereolytically cleave and hydrolyze the dihydrouracil ring of 6-substituted dihydrouracils. Such microorganisms are selected from wild strains existing in nature, strains preserved in public microorganism-holding institutions, or microorganisms artificially mutated therefrom, by examining the presence or absence of the above-mentioned ability. Is.

As a method of detecting this ability, for example, the following method is used. First, 2 ml of microbial culture
Cells were collected by centrifugation and collected in 2 ml of 0.9%
(Wt%, the same below) After washing with saline, centrifuge again to collect bacterial cells. These separated cells (wet weight 40-2
(00 mg) is added to 2 ml of a DL-6-methyl-dihydrouracil aqueous solution having a concentration of 0.5%, and the reaction is carried out for 10 to 40 hours while maintaining the pH at 7 to 10 and the temperature at 40 to 80 ° C. After the reaction, appropriately dilute and add a concentrated hydrochloric acid solution of p-dimethylaminobenzaldehyde to develop color, centrifuge the solution to remove insoluble materials such as bacterial cells, and then generate N-carbamoyl-β-aminobutyric acid by HPLC. To determine the amount of N-carbamoyl-β-aminobutyric acid produced in the reaction solution.

As a result of the detection as described above, for the strains showing a relatively high conversion rate, the experimental scale was increased,
DL-6-Methyl-dihydrouracil is hydrolyzed again, and the cells found to have high optical purity of the produced N-carbamoyl-β-aminobutyric acid are adopted as the microorganisms used in the present invention.

The microorganism used in the present invention is selected from bacteria, actinomycetes, fungi, yeasts and imperfect bacteria by the above detection method. Among them, since the optical yield and the conversion rate of 6-substituted dihydrouracils are high,
Bacteria derived from the genus Bacillus, Pseudomonas or Alcaligenes can be preferably used.

The stereoselectivity of the dihydropyrimidinase referred to here is that it selectively acts on one of the stereoisomers (R isomer, S isomer) at the asymmetric carbon at the 6-position of 6-substituted dihydrouracils. is there. The selectivity varies depending on the microorganism that is the enzyme source and the type of substrate, and includes extremely strict selectivity to the extent that there is a difference in reaction rate depending on stereoisomers. Regarding this point, it is possible to confirm which stereoisomer and what degree of selectivity it has by the above-mentioned ability detection method, and use it for each D-β-amino acid depending on the purpose.

The method of the present invention utilizes the action of dihydropyriminase produced by microorganisms. This enzyme is produced in the cells by culturing the microorganisms in a normal medium containing a natural nutrient source. Can be accumulated. Culturing is usually carried out in a liquid medium, but it can also be carried out by solid surface culturing. The culture usually contains a carbon source and a nitrogen source which can be assimilated, and inorganic salts and nutrients necessary for the growth of each microorganism, and further various pyrimidine nucleobases or their derivatives, or various hydattoins. It is desirable to add 05 to 0.8% to adaptively enhance the desired dihydropyrimidinase. Pyrimidine nucleobases having a high dihydropyrimidinase-inducing effect include uracil, cytosine and thymine, and their derivatives include dihydrouracil and dihydrothymine. Among the hydantoins, hydantoin, DL-5-methylhydantoin and the like are preferable. However, common to many microorganisms, uracil is practically preferred. The culture condition is 20 to 65 depending on the optimum growth condition of the microorganism to be used.
C., pH 4 to 11, culture time of several hours to several days are used. Aeration and agitation may be performed during the culture to promote the growth of microorganisms.

As the dihydropyrimidinase used for the hydrolysis of 6-substituted dihydrouracils, the microorganisms cultivated as described above are used in the form of culture, cells or treated cells. Usually, a culture of a microorganism can be used as it is for the reaction, but if the components in the culture interfere, the cells isolated from the culture may be used. The microbial cell reaches the purpose of use even if it is a viable microbial cell, but it can also be used as a lyophilized microbial cell for convenience of storage or handling. It is also possible to use not a microbial cell itself, but a microbial cell processed product such as a microbial cell crushed product or a microbial cell extract. Furthermore, the above-mentioned bacterial cells or treated bacterial cells may be immobilized by a known method. In addition, it goes without saying that the dihydropyrimidinase derived from a microorganism can be used even if it is purified by a commonly used method.

In order to allow the dihydropyrimidinase to act on the 6-substituted dihydrouracils in the form of a culture of microorganisms, cells or treated cells, usually a method of mixing the two in an aqueous medium is used. The amount of dihydropyrimidinase may be appropriately selected depending on the type of substrate and is not particularly limited.

There is no particular limitation on the concentration of the 6-substituted dihydrouracils in the aqueous medium. At a high concentration of about 1 to 30%, the 6-substituted dihydrouracils are not completely dissolved, but the 6-substituted dihydrouracils are successively dissolved as the reaction proceeds, which is not a problem.

When practicing the stereoselective hydrolysis of 6-substituted dihydrouracils in an aqueous medium, p is practically preferred.
The range of H is 7 to 10, and particularly preferably 8 to
It is 9. If the reaction is performed under such conditions using a microorganism having a high dihydropyrimidinase activity, the target product can be obtained in good yield. If the pH is less than 7, the reaction rate is extremely low, and if the pH exceeds 10, unfavorable by-products are produced. pH 7
The reason why 10 is preferable is that the optimum pH of dihydropyrimidinase, which is a microbial enzyme used in the present invention, is 8
-9, and the increased solubility of the substrate with increasing pH, results in an increased conversion rate of 6-substituted dihydrouracils to N-carbamoyl-β-amino acids.

Since the pH of the aqueous medium decreases as the reaction progresses, it is desirable to continuously add a neutralizing agent during the reaction to maintain the pH at the optimum level. Ammonia, caustic soda, caustic potash, sodium carbonate, etc. are suitable as the neutralizing agent. In addition, the reaction can be carried out by adding an organic solvent or a surfactant to the aqueous medium depending on the purpose.

As the reaction temperature, a temperature suitable for the enzyme of the microorganism used is adopted, but it is usually in the range of 20 to 70 ° C. The reaction time depends on the type of substrate, concentration, type of enzyme, concentration,
Since it depends on the reaction conditions such as temperature, it is selected so that the substrate corresponding to the desired stereoisomer is sufficiently converted. It is usually several minutes to several days.

The produced N-carbamoyl-β-amino acids and unreacted 6-substituted dihydrouracils can be easily separated. That is, since the 6-substituted dihydrouracils have low solubility in water, the unreacted 6-substituted dihydrouracils precipitated as a solid may be filtered off by cooling the reaction solution. Further, if necessary, it may be purified using an ion exchange column or a reverse layer column.

In order to decarbamoylate N-carbamoyl-β-amino acids to convert them into β-amino acids while maintaining their configuration, for example, a diazotization treatment is carried out by acting nitrite in an acidic mineral acid aqueous medium. The method is based on the so-called VanSlyke method.
This method is applied to, for example, Japanese Patent Publication No.
No. 8-4707.

This decarbamoylation reaction can be accomplished by reacting N-carbamoyl-β-amino acids with nitrite in an aqueous medium under acidic conditions. According to this, N
The optical activity of -carbamoyl-β-amino acids is retained, and optically active β-amino acids can be obtained.

The mineral acid acidic aqueous medium is preferably a strong mineral acid such as hydrochloric acid or sulfuric acid having a normality of 0.4 or more, and particularly an aqueous medium in which sulfuric acid is present in order to avoid side reactions. As the nitrite, an alkali metal or alkaline earth metal nitrite is preferably used because it is available at a low cost. Nitrite may be allowed to act on N-carbamoyl-β-amino acids in an approximately equivalent amount, whereby the corresponding β-amino acids can be obtained in high yield. Since the decarbamoylation reaction is accompanied by a large amount of heat generation, it is preferable to gradually add nitrite, and the temperature is usually 0 to 40 ° C.
It is performed in the range of. The reaction generates nitrogen gas and carbon dioxide gas, but after the addition of the entire amount of nitrite, the gas generation ends in 15 minutes to several hours. After that, the reaction is carried out for about 30 minutes to 15 hours.

Isolation of the produced optically active β-amino acids is easy. That is, the reaction solution may be concentrated and cooled to be recrystallized. Further, if necessary, it may be purified using an ion exchange column or a reverse layer column.

[0038]

EXAMPLES The present invention will be described in more detail with reference to the following examples.

Example 1 Journal of Fermentation Technology (J. Ferment. Tech.) 56 , 49
2 (1978) according to the method described in Pseudomonas putida I.
FO 12996 (former name Pseudomonas strita) IFO
12996), 50 mg of dihydropyrimidinase (hydantoinase) purified from 100 mg of deionized water
1 was added, 500 mg of DL-6-methyldihydrouracil was added with stirring under nitrogen aeration, and the mixture was reacted at 37 ° C. for 8 hours. During the reaction, the pH of the reaction solution was continuously adjusted to 8.7 by using 1.0N-NaOH solution.

After the reaction, the reaction solution was concentrated to 10 ml, and DE
AE Sephadex A-25 (HCO ₃ type, 30 × 10
0 mm) (made by Pharmacia Biotech). Unreacted 6-methyldihydrouracil 300m by eluting with distilled water 300ml and concentrating the eluate
After obtaining g, the solution was further eluted with 500 ml of a 0.2 M ammonium bicarbonate solution, and the eluate was concentrated to decompose and remove ammonium bicarbonate in the solution to obtain 234 mg of N 2.
-Carbamoyl-β-aminobutyric acid was obtained. 41% yield
Met.

To 234 mg of N-carbamoyl-β-aminobutyric acid, 5 ml of a 1M aqueous sulfuric acid solution was added and cooled to 0 ° C. 107 mg of sodium nitrite was added to this aqueous solution with stirring to carry out a decarbamoylation reaction for 2 hours. The reaction solution was DOWEX 50WX8 (10 × 100 m
m) (made by The Dow Chemical Company),
After elution with 30 ml of distilled water to confirm that the pH of the eluate was neutral, it was further eluted with 50 ml of a 1.5 M ammonium hydroxide solution, and the eluate was concentrated (S)- 130 mg of β-aminobutyric acid crystals were obtained. The yield from N-carbamoyl-β-aminobutyric acid is 8
It was 0%. The obtained (S) -β-aminobutyric acid is [α] _D ²⁰ = + 18.7 ° (H ₂ O, c = 1.0), 5
3% ee (reference value [α] _D ²⁰ = −35.2 ° ((R) body), Journal of Chemical Society (J. Chem. Soc.) 3316 (19).
52)). Analysis by optical isomer separation column chromatography (column: Daicel Crown Pack CR
(+), 4.0 x 150 mm (Daicel Chemical Industry Co., Ltd.)
Manufactured), mobile phase: pH 1.5 phosphoric acid aqueous solution, detection wavelength: 2
30 nm, temperature: 2.5 ° C., mobile phase flow rate: 0.4 ml /
min, retention time of (S) form was 5.8 minutes, retention time of (R) form was 6.8 minutes), and it was 56% ee.

Example 2 A liquid medium having the following composition was prepared, and 100 ml of the medium was dispensed into a shake flask with a volume of 500 ml at 120 ° C.
Steam sterilization was performed for 0 minutes.

Medium composition: Meat extract 0.5% Peptone 1.0% Yeast extract 0.5% NaCl 0.15 Uracil 0.1% pH 7.0 Medium with the same composition in a large test tube with a cotton plug. 10 ml
Bacillus species KNK108 cultured at 33 ° C. for 24 hours
(FERM BP-887) inoculated with 1.0 ml, 33
The cells were cultured with shaking at 24 ° C for 24 hours. 100 ml of this culture solution was centrifuged to collect the cells, which was washed with 100 ml of 0.9% saline solution and then centrifuged again to collect the cells.
suspended in ml. Acrylamide 1.5 was added to this suspension.
g and 80 mg of N, N′-methylenebisacrylamide were added and dissolved, and oxygen was driven out through nitrogen gas, and then 1% of 5% β-dimethylaminopropionitrile was added.
And 1 ml of 0.005% riboflavin were added, and the mixture was left at room temperature under a fluorescent lamp. After 1 hour, the produced bacterial cell-containing gel was cut into 3 mm square pieces and washed with 0.9% physiological saline.
A gel immobilizate was prepared. Two 500 mg of this immobilizate
Put in a 00 ml spinner flask and deionize water 100
ml, and under nitrogen aeration, with stirring, add DL-6-methyldihydrouracil 500 mg, and at 37 ° C. 40
Reacted for hours. During the reaction, the pH of the reaction solution was continuously adjusted to 8.7 by using 1.0N-NaOH solution.

After the reaction, the same treatment as in Example 1 was carried out to give 22
8 mg of N-carbamoyl-β-aminobutyric acid was obtained. The yield was 40%.

Further, a decarbamoylation reaction was carried out in the same manner as in Example 1, and purified to obtain (R) -β-aminobutyric acid 12
I got 8 mg. The yield from N-carbamoyl-β-aminobutyric acid was 80%. The obtained (R) -β-aminobutyric acid is [α] _D ²⁰ = −30.0 ° (H ₂ O, c = 1.
0) and 85% ee. When analyzed by high performance liquid chromatography using the same optical isomer separation column as in Example 1, it was 87% ee.

Example 3 Immobilized product 20 prepared exactly as in Example 2
Place 0 mg in a 200 ml spinner flask, add 50 ml of ion-exchanged water, and DL under aeration with nitrogen while stirring.
200 mg of -6-methyldihydrouracil was added, and 37
The reaction was carried out at 8 ° C for 8 hours. During the reaction, the pH of the reaction solution was continuously adjusted to 8.7 by using 1.0N-NaOH solution.
After the reaction, the same treatment as in Example 1 was carried out to obtain 20 mg of N-carbamoyl-β-aminobutyric acid. The yield was 9%.

Further, a decarbamoylation reaction was carried out in the same manner as in Example 1, and purified to obtain (R) -β-aminobutyric acid 13
I got mg. The yield from N-carbamoyl-β-aminobutyric acid was 92%. The obtained (R) -β-aminobutyric acid was [α] _D ²⁰ = -31.7 ° (H ₂ O, c = 1.
0) and 90% ee. When analyzed by high performance liquid chromatography using the same optical isomer separation column as in Example 1, it was 91% ee.

Example 4 200 m of an immobilized product prepared in exactly the same manner as in Example 2.
g in a 200 ml spinner flask, 100 ml of deionized water was added, and DL-
200 mg of 6-methyldihydrouracil was added, and the temperature was 50 ° C.
And reacted for 20 hours. During the reaction, the pH of the reaction solution was continuously adjusted to 8.7 by using 1.0N-NaOH solution.
After the reaction, the same treatment as in Example 1 was carried out to obtain 90 mg of N-carbamoyl-β-aminobutyric acid. The yield was 32%.

Further, a decarbamoylation reaction was carried out in the same manner as in Example 1, and purified to obtain (R) -β-aminobutyric acid 52.
I got mg. The yield from N-carbamoyl-β-aminobutyric acid was 82%. The obtained (R) -β-aminobutyric acid is [α] _D ²⁰ = −30.0 ° (H ₂ O, c = 1.
0) and 85% ee. When analyzed by high performance liquid chromatography using the same optical isomer separation column as in Example 1, it was 86% ee.

Example 5 A liquid medium having the following composition was prepared and placed in a large test tube.
It was dispensed by 00 ml and steam sterilized at 120 ° C. for 20 minutes.

Medium composition: Meat extract 2.0% Pro extract 2.0% NaCl 0.3% MnCl ₂ .4H ₂ O 20 ppm Uracil 0.2% pH 7.5 Into a large test tube with a cotton plug. Same composition medium 10ml
Alcaligenes faecalis cultured at 37 ° C for 18 hours
Inoculate 1.0 ml of IFO 13111 and incubate at 37 ° C for 24
Shaking culture was performed for a period of time.

The culture solution was centrifuged to collect the cells, and then 0.9
After washing with 100 ml of 100% saline, the cells were collected by centrifugation again and suspended in 150 ml of 50 mM potassium phosphate buffer. 50 ml of this suspension was placed in a 200 ml four-necked round bottom flask, and DL-6-
Add 300 mg of methyldihydrouracil, and add 2 at 37 ℃.
The reaction was allowed for 0 hours. During the reaction, the pH of the reaction solution was continuously adjusted to 8.7 by using 1.0N-NaOH solution. After the reaction, the pH of the reaction solution was adjusted to 7.0 with IN HCl, and the reaction solution was centrifuged to remove unreacted starting materials and insoluble materials such as cells. Further, the same treatment as in Example 1 was carried out to obtain 160 mg of N-carbamoyl-β-aminobutyric acid. The yield was 47%.

Further, a decarbamoylation reaction was carried out in the same manner as in Example 1, and the product was purified to give (S) -β-aminobutyric acid 11
I got 0 mg. The yield from N-carbamoyl-β-aminobutyric acid was 97%. The obtained (S) -β-aminobutyric acid was [α] _D ²⁰ = + 22.8 ° (H ₂ O, c = 1.
0) and 65% ee. When analyzed by high performance liquid chromatography using the same optical isomer separation column as in Example 1, it was 67% ee.

Example 6 A liquid medium having the following composition was prepared and placed in a large test tube.
Each 000 ml was dispensed and steam sterilization was performed at 120 ° C. for 20 minutes.

Medium composition: Meat extract 3.0% Glucose 1.0% MnCl ₂ .4H ₂ O 20 ppm Uracil 0.2% pH 7.5 Using a large test tube with a cotton plug, 10 ml of the same composition medium
1.0 ml of Pseudomonas putida IFO 12996 cultured at 30 ° C. for 18 hours at 30 ° C.
Shaking culture was performed for 4 hours.

This culture solution was centrifuged to collect the cells, and then 0.9
After washing with 100 ml of 100% saline, the cells were collected by centrifugation again and suspended in 150 ml of 50 mM potassium phosphate buffer. 50 ml of this suspension was placed in a 200 ml four-necked round bottom flask, and DL-6-
Add 300 mg of methyldihydrouracil, and add 2 at 37 ℃.
The reaction was allowed for 0 hours. During the reaction, the pH of the reaction solution was continuously adjusted to 8.7 by using 1.0N-NaOH solution. After the reaction, the pH of the reaction solution was adjusted to 7.0 with IN HCl, and the reaction solution was centrifuged to remove unreacted starting materials and insoluble materials such as cells. Further, the same treatment as in Example 1 was carried out to obtain 150 mg of N-carbamoyl-β-aminobutyric acid. The yield was 45%.

Further, a decarbamoylation reaction was carried out in the same manner as in Example 1, and purified to obtain (S) -β-aminobutyric acid 10
I got 5 mg. The yield from N-carbamoyl-β-aminobutyric acid was 99%. The obtained (S) -β-aminobutyric acid was [α] _D ²⁰ = + 18.0 ° (H ₂ O, c = 1.
0) and 51% ee. When analyzed by high performance liquid chromatography using the same optical isomer separation column as in Example 1, it was 54% ee.

Example 7 Immobilized product 30 prepared in exactly the same manner as in Example 2
Add 0 mg to a 200 ml spinner flask, add 100 ml of ion-exchanged water, and stir under nitrogen aeration while stirring.
300 mg of L-6-phenyldihydrouracil was added,
The reaction was carried out at 37 ° C for 150 hours. During the reaction, 1.0N-N
The pH of the reaction was continuously adjusted to 8.7 with aOH solution. After the reaction, treat in the same manner as in Example 1 to obtain 40 mg.
To give N-carbamoyl-3-amino-3-phenylpropionic acid. The yield was 12%.

Further, a decarbamoylation reaction was carried out in the same manner as in Example 1 and purification was carried out to obtain 30 mg of (S) -3-amino-3-phenylpropionic acid. N-carbamoyl-
The yield from 3-amino-3-phenylpropionic acid is 9
It was 5%. The obtained (S) -3-amino-3-phenylpropionic acid is [α] _D ²⁵ = -3.36 ° (H
₂ O, c = 0.2), 48% ee (reference value [α] _D ²⁵ = + 7.0 ° ((R) form), Journal of Chemical Society 4047 (196).
1)). Analysis by high-performance liquid chromatography using an optical isomer separation column (column: Daicel Chiral Cell WH, 4.6 x 250 mm (Daicel Chemical Industry Co., Ltd.)
Manufactured), mobile phase: 0.25 mM copper sulfate aqueous solution, detection wavelength: 230 nm, temperature: 30 ° C., mobile phase flow rate: 1.5 m
It was 51% ee when the retention time of the (S) form was 21 minutes and the retention time of the (R) form was 32 minutes.

Example 8 40 ml of a suspension obtained by culturing Pseudomonas putida IFO 12996 in exactly the same manner as in Example 6 was placed in a flask and DL-6-phenyldihydrouracil was stirred under aeration of nitrogen with stirring. 200 mg
Was added and reacted at 37 ° C. for 150 hours. During the reaction, 1.
The pH of the reaction was continuously adjusted to 8.7 with 0N-NaOH solution. After the reaction, treat in the same manner as in Example 1,
Obtained 48 mg of N-carbamoyl-3-amino-3-phenylpropionic acid. The yield was 22%.

Further, a decarbamoylation reaction was carried out in the same manner as in Example 1 and purification was carried out to obtain 35 mg of (S) -3-amino-3-phenylpropionic acid. The yield based on N-carbamoyl-3-amino-3-phenylpropionic acid was 91%. The obtained (S) -3-amino-3-phenylpropionic acid is [α] _D ²⁰ = −3.0 ° (H
₂ O, c = 0.2) and 43% ee. When analyzed by high performance liquid chromatography using the same optical isomer separation column as in Example 7, it was 44% ee.

Reference Example Synthesis of 6- (p-methoxyphenyl) dihydrouracil Dissolve 6.4 g of p-anisaldehyde, 7.4 g of malonic acid and 7.1 g of urea in 10 ml of glacial acetic acid and stir 1 while stirring.
The reaction was carried out at 20 ° C for 7 hours. To the reaction solution, 200 ml of distilled water was added, and the mixture was vigorously stirred, and the produced white solid was collected by filtration,
It was washed with water and dried. Furthermore, by recrystallizing from 300 ml of a mixed solvent of ethanol: water = 9: 1, 6-
2.9 g of (p-methoxyphenyl) dihydrouracil was obtained.

The yield was 31% and the mp was 228 to 229 ° C.

¹ HNMR (DMSO-d ₆ δ) 1
0.1 (brs, 1H), 7.9 (brs, 1H),
7.2 (d, 2H), 6.9 (d, 2H), 4.6
(M, 1H), 3.8 (s, 3H), 2.8 (dd, 1
H), 2.6 (dd, 1H) Example 9 Immobilized product 30 prepared exactly as in Example 2.
Add 0 mg to a 200 ml spinner flask, add 100 ml of ion-exchanged water, and stir under nitrogen aeration while stirring.
L-6- (p-methoxyphenyl) dihydrouracil 3
00 mg was added, and it was made to react at 37 degreeC for 150 hours. During the reaction, the pH of the reaction solution was continuously adjusted to 8.7 by using 1.0N-NaOH solution. After the reaction, the same treatment as in Example 1 was performed to obtain 40 mg of N-carbamoyl-3-amino-
3- (p-Methoxyphenyl) propionic acid was obtained. The yield was 12%.

Further, a decarbamoylation reaction was carried out in the same manner as in Example 1, and purified to obtain (S) -3-amino-3-.
30 mg of (p-methoxyphenyl) propionic acid was obtained. The yield from N-carbamoyl-3-amino-3- (p-methoxyphenyl) propionic acid was 95%. The obtained N-CHO form of (S) -3-amino-3- (p-methoxyphenyl) propionic acid is [α] _D ²⁰ =
+ 64.8 ° (MeOH, C = 0.2) and 48% ee (reference value [α] _D ²⁰ = −135.0 ° ((R)
Body) Journal of Organic Chemistry
56 , 1355 (1991). Analysis by high performance liquid chromatography using an optical isomer separation column (column: Daicel chiral cell WH, 4.6 × 250 mm,
Mobile phase: 0.25 mM copper sulfate aqueous solution, detection wavelength: 23
0 nm, temperature: 30 ° C., mobile phase flow rate: 1.5 ml / mi
n, (S) body retention time 11 minutes, (R) body retention time:
After 17 minutes, it was 50% ee.

Example 10 Immobilized product 20 prepared in exactly the same manner as in Example 2
Add 0 mg to a 200 ml spinner flask, add 100 ml of ion-exchanged water, and stir under nitrogen aeration while stirring.
L-6- (2-thienyl) dihydrouracil 200 mg
Was added and reacted at 45 ° C. for 48 hours. During the reaction, 1.0
The pH of the reaction was continuously adjusted to 8.7 with N-NaOH solution. After the reaction, the same treatment as in Example 1 was performed to give 1
00 mg of N-carbamoyl-3-amino-3- (2-
Thienyl) propionic acid was obtained. The yield was 24%.

Further, a decarbamoylation reaction was carried out in the same manner as in Example 1 and purified to obtain (S) -3-amino-3-.
30 mg of (2-thienyl) propionic acid was obtained. The yield from N-carbamoyl-3-amino-3- (2-thienyl) propionic acid was 100%. Got (S)
-3-Amino-3- (2-thienyl) propionic acid has [α] _D ²⁰ = + 13.2 ° (H ₂ O, c = 1.0), 8
6% ee (reference value [α] _D ²⁰ = + 15.3 ° ((S) body), Buretane of Chemical Society of Japan 52 , 3326 (197).
9)). Analysis by high-performance chromatography using an optical isomer separation column (column: Daicel Chiralcel W
H, 4.6 × 250 mm, mobile phase: 0.25 mM copper sulfate aqueous solution, detection wavelength: 230 nm, temperature: 20 ° C., mobile phase flow rate: 1.5 ml / min, retention time of (S) body: 1
2 minutes, (R) body retention time: 26 minutes), 87
% Ee.

Example 11 Immobilized product 30 prepared in exactly the same manner as in Example 2
Add 0 mg to a 200 ml spinner flask, add 100 ml of ion-exchanged water, and stir under nitrogen aeration while stirring.
Add 300 mg of L-6-ethyldihydrouracil and add 4
The reaction was carried out at 5 ° C for 48 hours. During the reaction, 1.0 N-NaO
The pH of the reaction solution was continuously adjusted to 8.7 using H solution. After the reaction, the same treatment as in Example 1 was carried out to obtain 117 mg of N-carbamoyl-3-aminovaleric acid. Yield is 3
It was 5%.

Further, a decarbamoylation reaction was carried out in the same manner as in Example 1, and purified to obtain (R) -3-aminovaleric acid 3
I got 0 mg. The yield from N-carbamoyl-3-aminovaleric acid was 90%. The obtained (R) -3-aminovaleric acid is [α] _D ²⁰ = -35.0 ° (H ₂ O, c =
1.0) and 91% ee (reference value [α] _D ²⁰ = +
38.5 ° ((S) form), Gazzetta Kimika Italiana (Gazz. Chim. Ital.) 96 , 1
380 (1966)). Analysis by optical isomer separation column chromatography (column: Daicel Crown Pack CR (+), 4.0 × 150 mm, mobile phase: pH
1.5 phosphoric acid aqueous solution, detection wavelength: 230 nm, temperature:
2.5 ° C., mobile phase flow rate: 0.4 ml / min, (R) body retention time: 3.7 minutes, (S) body retention time: 4.9
Min), it was 93% ee.

[0070]

According to the present invention, the general formula (I)

[0071]

[Chemical 11]

(In the formula, X is an alkyl group having 1 to 4 carbon atoms,
formula

[0073]

[Chemical 12]

(Wherein R represents hydrogen, a hydroxyl group or a lower alkoxy group having 1 to 4 carbon atoms), a phenyl group or a substituted phenyl group, a thienyl group, a furyl group, a pyridyl group, a naphthyl group or an indolyl group. The 6-substituted dihydrouracils represented by the formula (1) are hydrolyzed by the action of a dihydropyrimidinase derived from a microorganism having the ability to stereolytically cleave and hydrolyze the dihydrouracil ring. N-
It is converted to carbamoyl-β-amino acids, and the optically active N-carbamoyl-β-amino acids are decarbamoylated to give a compound of formula (III)

[0075]

[Chemical 13]

A stereoselective method for producing an optically active β-amino acid represented by the formula (wherein X is the same as above) can be provided. Therefore, the present invention provides a very effective method for producing β-amino acids, which are important intermediates for pharmaceuticals.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Internal reference number FI Technical indication (C12P 41/00 C12R 1:05) (72) Inventor Junzo Hasegawa 2 Takaoka, Okubo-cho, Akashi-shi, Hyogo Chome 13-4

Claims (6)

[Claims] 1. A compound represented by the general formula (I): (In the formula, X is an alkyl group having 1 to 4 carbon atoms; (In the formula, R is hydrogen, a hydroxyl group or a carbon number of 1 to
4 represents a lower alkoxy group of 4) or 6-substituted dihydrouracils represented by a substituted phenyl group, a thienyl group, a furyl group, a pyridyl group, a naphthyl group or an indolyl group), and a dihydrouracil ring A dihydropyrimidinase derived from a microorganism having the ability to selectively cleave and hydrolyze is allowed to act in an aqueous medium having a pH of 7 to 10 to give a compound of the formula (II): (Wherein X is the same as described above) is converted into N-carbamoyl-β-amino acids rich in stereoisomers, and the stereoisomers are collected to obtain an optically active N-carbamoyl-β-amino acid. Amino acid production method. 2. The production method according to claim 1, wherein a culture, a microbial cell or a treated product of the microbial cell is used as the dihydropyrimidinase derived from the microbial cell. 3. The method according to claim 1, wherein a bacterium of the genus Bacillus, Pseudomonas or Alcaligenes is used as the microorganism. 4. The production method according to claim 1, 2 or 3, wherein the reaction is carried out while keeping the pH at 8-9. 5. The optically active N-carbamoyl-β-amino acid obtained in claim 1 is decarbamoylated while maintaining the configuration, and is represented by the formula (III): A process for producing an optically active β-amino acid, which is a β-amino acid represented by the formula (wherein X is the same as above). 6. The production method according to claim 5, wherein the diazotization treatment is carried out by using nitrite in a mineral acid acidic aqueous medium, and decarbamoylation is carried out.