JPH1180103A - Beta-carbamoylisobutyric acids and their production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce β-carbamoylisobutyric acids which are an effective intermediate for producing medicines, agrochemicals or the like and to provide a method for producing the β-carbamoylisobutyric acids. SOLUTION: The β-carbamoylisobutyric acids are represented by general formula (1) H2 NOCCH2 C*H(CH3 )COOR<1> [R<1> is hydrogen or a 1-6C alkyl group; the carbon atom marked with * is asymmetric carbon atom]. The method for producing the β-carbamoylisobutyric acids comprises hydrating β- cyanoisobutyric acids represented by general formula (2) NCCH2 C*H(CH3 ) COOR<1> (R<1> is the same as described above) in the presence of a cultured product, a microbial cell or a treated substance of the microbial cell of a nitrile hydratase or a microorganism capable of producing the nitrile hydratase.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to β-carbamoylisobutyric acids useful as intermediates for producing pharmaceuticals, agricultural chemicals and the like, and a method for producing the same.

[0002]

2. Description of the Related Art As β-substituted isobutyric acids, for example,
-Cyanoisobutyric acids are known (U.S. Pat.
44,467, U.S. Pat. No. 3,644,468, JP-A-9-67330, and British Patent No. 80
8,835, U.S. Pat. No. 2,810,742, JP-A-8-67330, etc.). However, β-carbamoylisobutyric acids and a method for producing the same are not known.

[0003]

The object of the present invention is to provide a β-
An object of the present invention is to provide carbamoyl isobutyric acid and a method for producing the same.

[0004]

The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, have found that the ability of hydrating the cyano group of β-cyanoisobutyric acid to amide, that is, nitrile water Competent microorganisms have been found and the present invention has been completed.

That is, the present invention provides a compound represented by the general formula (1): H ₂ NOCCH ₂ C ^* H (CH ₃ ) COOR ¹ (1) (wherein R ¹ is hydrogen or an alkyl group having 1 to 6 carbon atoms) The carbon atoms marked with * are asymmetric carbon atoms.), Are β-carbamoylisobutyric acids.

Further, the present invention provides a compound of the general formula (2): NCCH ₂ C ^* H (CH ₃ ) COOR ¹ (2) (wherein R ¹ is hydrogen or an alkyl group having 1 to 6 carbon atoms). Β-cyanoisobutyric acids represented by * are carbon atoms treated with nitrile hydratase or a nitrile hydratase or a microorganism having the ability to produce nitrile hydratase. Characterized in that it hydrates in the presence of a substance,
-A method for producing carbamoyl isobutyric acids.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
In the general formulas (1) and (2), the alkyl group having 1 to 6 carbon atoms represented by R ¹ may have any of a straight-chain structure and a branched structure. Specifically, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-
Butyl, isobutyl, n-pentyl and the like are exemplified.

The β-carbamoyl isobutyric acids of the present invention include not only racemic β-carbamoyl isobutyric acids but also optically active β-carbamoyl isobutyric acids.
These optically active β-carbamoylisobutyric acids are particularly effective intermediates for producing optically active pharmaceuticals and optically active agricultural chemicals.

In the production of β-carbamoylisobutyric acids of the present invention, β-cyanoisobutyric acids represented by the above general formula (2) are used as a raw material. When racemic β-cyanoisobutyric acids are used as a raw material, racemic β-carbamoylisobutyric acids are obtained. In addition, optically active β-
When cyanoisobutyric acids are used, optically active β-carbamoylisobutyric acids are obtained.

[0010] Racemic β-cyanoisobutyric acids can be produced by a conventionally known general method. For example, racemic methyl β-cyanoisobutyrate can be produced by reacting methyl methacrylate with hydrocyanic acid in the presence of an alkaline catalyst (GB 808,835).
JP, U.S. Pat. No. 2,810,742, U.S. Pat. No. 3,644,467, JP-A-8-29115
No. 8, JP-A-9-67330). Racemic β-cyanoisobutyric acid can be produced, for example, by hydrolyzing racemic β-cyanoisobutyric acid methyl ester with an esterase.

The optically active β-cyanoisobutyric acid can be prepared by converting racemic β-cyanoisobutyrate into an ester asymmetric hydrolase or a culture, cell or treated cell of a microorganism capable of producing the enzyme. And can be produced by asymmetric hydrolysis. Hereinafter, a method for producing optically active β-cyanoisobutyric acids will be described.

The ester asymmetric hydrolase to be used may be any enzyme having the activity of asymmetrically hydrolyzing the ester bond of racemic β-cyanoisobutyrate,
Regardless of the production source. Such enzymes include lipases, esterases and proteases. As the enzyme, for example, an enzyme derived from a microorganism having the ability to produce an ester asymmetric hydrolase can be used. Representative microorganisms having an ester asymmetric hydrolysis ability include microorganisms belonging to the genus Pseudomonas and the genus Escherichia. Specifically, Pseudomonas putida
FERM BP-3846, Escherichia coli
FERM BP-3835 and the like. Escherichia coli
FERM BP-3835 is Pseudomonas putida FERM BP-3
This strain was transformed with a gene encoding esterase from 846.

The cultivation of the microorganism can be performed in a liquid medium or a solid medium. As the medium, a medium appropriately mixed with components such as a carbon source, a nitrogen source, vitamins, and minerals that can normally be used by microorganisms is used. It is also possible to add a small amount of ester to the medium in order to improve the hydrolytic capacity of the microorganism. The cultivation is performed at a temperature and a pH at which the microorganism can grow, and may be performed under the optimum culturing conditions for the strain to be used. In order to promote the growth of the microorganism, aeration and agitation may be performed.

The culture obtained by culturing the above-mentioned microorganism having the ability to produce an ester asymmetric hydrolase in a medium, as well as the purified ester asymmetric hydrolase, may be used as it is or in the culture. Cells obtained from the product by a cell collection operation such as centrifugation or a processed product of the cells can also be used. The treated cells can be obtained from cells treated with acetone, toluene, etc., freeze-dried cells, crushed cells, cell-free extracts, cell-free extracts, and separation operations such as gel filtration and ion exchange chromatography. Crude enzymes. The microbial cells or enzymes can be immobilized on a crosslinked acrylamide gel or the like, or physically and chemically immobilized on a solid carrier such as an ion-exchange resin or keso earth. This facilitates recovery and reuse after the reaction.

Commercially available products can be used as the ester asymmetric hydrolase. Specifically, lipase OF (trade name, manufactured by Meito Sangyo Co., derived from Candida genus), durazyme (trade name, manufactured by NOVO, derived from Bacillus), sabinase (trade name, manufactured by NOVO, derived from Bacillus) , Fl
avourzyme MG (trade name, from NOVO, from Aspergillus), Lipase A-6 (trade name, from Amano Pharmaceutical, from Aspergillus), Lipase M (trade name, from Amano Pharmaceutical, from Mucor), New Rase F (trade name, manufactured by Amano Pharmaceutical Co., derived from Rhizopus sp.), Lipase type VII (trade name, manufactured by SIGMA, derivable from Candida sp.), Acylas
e I (trade name, SIGMA, from Aspergillus), Tripsin type II (trade name, SIGMA, from pig pancreas), Protein type XVI (trade name, SIGM)
A company, Bacillus genus), Palatase (brand name, NOV)
O company) can be used.

The optically selective hydrolysis of the racemic β-cyanoisobutyrate can be carried out as follows. That is, a racemic β-cyanoisobutyric acid ester as a substrate is added to a reaction medium, dissolved or suspended, and a culture of an enzyme or a microorganism serving as a catalyst is added. However, this catalyst may be added before the substrate is added to the reaction medium. Then, the hydrolysis reaction of the racemic β-cyanoisobutyrate is performed while controlling the reaction temperature and, if necessary, the pH of the reaction solution. This hydrolysis reaction is carried out until about half of the substrate has reacted, but depending on the case, the reaction may be terminated with less than half of the substrate, or may be reacted in excess of half of the substrate.

As a reaction medium, for example, ion-exchanged water,
A buffer solution or the like is used. The substrate concentration in the reaction solution is 0.1 to
There is no particular limitation as long as it is in the range of 70% by weight, but it is preferably in the range of 5 to 40% by weight in consideration of the solubility and reactivity of the racemic β-cyanoisobutyrate as a substrate. The reaction temperature is preferably 5 to 70C, more preferably 20 to 60C.

The pH of the reaction solution depends on the optimum pH of the enzyme or microorganism to be used for the asymmetric hydrolysis of the enzyme.
It is preferable to carry out in the range of since the decrease in optical purity due to the chemical hydrolysis reaction can be suppressed. As the reaction proceeds, the pH of the reaction solution is increased by the generated carboxylic acid.
Therefore, it is desirable to carry out the reaction while maintaining the optimum pH with a suitable neutralizing agent. As for the above-mentioned substrate concentration, medium, temperature, pH and other reaction conditions, conditions under which the desired optically active compound can be advantageously obtained are appropriately selected in consideration of the reaction yield, optical yield and the like. Is desirable.

By such an asymmetric hydrolysis reaction, optically active β-cyanoisobutyric acid is produced. The unreacted residual substrate becomes an optically active β-cyanoisobutyric acid ester containing the enantiomer of the generated optically active β-cyanoisobutyric acid as a main component.

After completion of the reaction, the reaction solution is extracted with a solvent such as hexane or ethyl acetate to obtain an optically active β-
The cyanoisobutyrate can be separated. At this time, the microbial cells, enzymes, and the like used as the catalyst are removed in advance by an operation such as centrifugation or filtration, and then the solvent is extracted, whereby the extraction operation can be performed more easily. On the other hand, after the extraction residue is adjusted to pH 1 to 2 with an acid such as sulfuric acid or hydrochloric acid, the mixture is extracted with a solvent such as hexane or ethyl acetate to obtain the enantiomer, optically active β-cyanoisobutyric acid. The extracted product and the solvent can be easily separated by a known method such as distillation.

The nitrile hydratase used in the production of β-carbamoylisobutyric acids according to the present invention is obtained by hydrating the cyano group of β-cyanoisobutyric acid represented by the general formula (2) as a raw material and converting it to an amide. The type of enzyme and the production source thereof are not limited as long as the enzyme has the ability to perform (nitrile hydration ability). As the nitrile hydratase, for example, those derived from microorganisms having nitrile hydratase-producing ability can also be used.

Representative microorganisms having such nitrile hydratase producing ability include Corynebacterium spp. And Nocardi (Nocardi).
a) Genus, Pseudomonas genus, Bacillus (B
acillus, Bacteridium, Brevibacterium, Micrococcus, Rhodococcus
Genus, Aeromonas, Citrobacter (C
genus itrobacter, Agrobacteriu
m) genus, Erwinia genus, Enterobacter genus, Streptomyces
s) Microorganisms belonging to the genus, Rhizobium and the like.

Corynebacterium
Examples of microorganisms belonging to the genus include Corynebacterium sp. (FERM P-4445) (JP-B-56-17).
No. 918).

Microorganisms belonging to the genus Nocardia include, for example, Nocardia sp.
-4447) (see Japanese Patent Publication No. 56-17918).

Examples of microorganisms belonging to the genus Pseudomonas include, for example, Pseudomonas sp.
MP-6220) (see Japanese Patent Publication No. 59-37951).

Examples of microorganisms belonging to the genus Bacillus include Bacillus sp. (FERM P-271).
7) (see JP-B-62-21519).

Examples of the microorganism belonging to the genus Bacteridium include, for example, Bacteridium sp.
(FERM 2719) (see Japanese Patent Publication No. Sho 62-21519).

Microorganisms belonging to the genus Brevibacterium include, for example, Brevibacterium
sp. (FERM P-2721) and the like.

Microorganisms belonging to the genus Micrococcus include, for example, Micrococcus sp.
(FERM P-2720) (Japanese Patent Publication No. 62-21519)
Reference).

Examples of microorganisms belonging to the genus Rhodococcus include Rhodococcus sp. (FERM).
P-1046) (see JP-A-62-91189).

Examples of microorganisms belonging to the genus Aeromonas include Aeromonas sp. (FERM).
P-12389) (see JP-A-5-30983)
And the like.

Examples of microorganisms belonging to the genus Citrobacter include Citrobacter freundii (FERM P-123).
90) (see JP-A-5-30984).

The microorganism belonging to the genus Agrobacterium includes, for example, Agrobacterium tumefaciens.
(IAM 13129) (see JP-A-5-103681).

Examples of microorganisms belonging to the genus Erwinia include Erwinia niglifluens (E.
rwinia nigrifluens) (MAFF 03-01435)
(See Japanese Patent Application Laid-Open No. 5-161496).

Microorganisms belonging to the genus Enterobacter include, for example, Enterobacter sp.
(FERM BP-3955) (JP-A-5-23697)
No. 5).

As a microorganism belonging to the genus Streptomyces, for example, Streptomyces sp.
(FERM BP-3954) (JP-A-5-23697)
No. 6).

As a microorganism belonging to the genus Rhizobium, for example, Rhizobium lti
oti) (IAM 13588) (JP-A-5-236977)
Reference).

The cultivation of the microorganism having the nitrile hydration ability can be performed in a liquid medium or a solid medium. As a medium, a carbon source, a nitrogen source, which microorganisms can usually utilize,
What mix | blends components, such as a vitamin and a mineral suitably, is used. In order to improve the nitrile hydration ability of the microorganism, a small amount of nitrile, amide, organic acid, or urea can be appropriately added to the medium. The cultivation is performed at a temperature and a pH at which the microorganism can grow, and may be performed under the optimum culturing conditions for the strain to be used. In order to promote the growth of the microorganism, aeration and agitation may be performed.

In the present invention, not only the purified enzyme but also the culture obtained by culturing the above-mentioned microorganism having nitrile hydratase producing ability in a medium may be used as it is, or centrifuged from the culture. Or a processed product thereof obtained by the cell collection operation described above.
The treated cells can be obtained from cells treated with acetone, toluene, etc., freeze-dried cells, crushed cells, cell-free extracts, cell-free extracts, and separation operations such as gel filtration and ion exchange chromatography. Crude enzymes. The microbial cells or enzymes can be immobilized on a crosslinked acrylamide gel or the like, or physically and chemically immobilized on a solid carrier such as an ion-exchange resin or keso earth. This also facilitates recovery and reuse after the reaction.

The nitrile hydration of β-cyanoisobutyric acids represented by the general formula (2) can be carried out as follows. That is, β-cyanoisobutyric acid as a substrate is added to a reaction medium and dissolved or suspended, and nitrile hydratase as a catalyst or a culture of a microorganism is added. However,
The catalyst may be added before the substrate is added to the reaction medium.
Then, the hydration reaction of β-cyanoisobutyric acids is performed while controlling the reaction temperature and, if necessary, the pH of the reaction solution. As the reaction medium, for example, ion-exchanged water, a buffer, or the like is used.

The substrate concentration in the reaction solution is 0.1 to 70% by weight.
There is no particular limitation as long as it is between the above ranges, but it is preferably 5 to 40% by weight in consideration of the solubility, productivity and the like of β-cyanoisobutyric acid as a substrate. The reaction temperature is preferably 5 to
70 ° C, more preferably 20 to 60 ° C. The pH of the reaction solution depends on the optimum pH of the hydration ability of the enzyme or microorganism to be used, but it is generally preferable to carry out the reaction within a pH range of 6 to 9.

After completion of the reaction, β generated by an appropriate method
Extracting carbamoylisobutyric acids from the reaction solution; Specifically, when the obtained compound is β-carbamoylisobutyrate, the compound is extracted with a solvent such as hexane or ethyl acetate, and when the obtained compound is β-carbamoylisobutyrate, sulfuric acid, hydrochloric acid, or the like is used. After adjusting the pH to 1 to 2 with an acid, the mixture is extracted with a solvent such as ethyl acetate. At this time, the extraction operation can be performed more easily by removing the microbial cells, enzymes, etc., which have been used as catalysts in advance, by operations such as centrifugation and filtration and then performing solvent extraction. The extracted product and solvent are
It can be easily separated by a known method such as distillation.

[0043]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples, but the scope of the present invention is not limited to the examples. [Example 1] Synthesis of racemic β-carbamoylisobutyric acid methyl ester
Formed Rhodococcus Rodokurosu (Rhodococcus rhodochrous)
The IFO15564 strain was cultured in the following medium (50 ml) at 30 ° C. for 2 days. Medium glucose 1.5% monopotassium phosphate 0.05% dipotassium phosphate 0.05% magnesium sulfate 0.05% yeast extract 0.1% ε-caprolactam 0.5% pH 7.2

After completion of the culture, the culture solution was centrifuged, and the whole amount of the obtained cells was washed with ion-exchanged water and then suspended in 100 ml of 50 mM phosphate buffer (pH 7.5). This cell suspension 5
0 ml of racemic β-cyanoisobutyric acid methyl ester
2.5 g was added and reacted at 30 ° C. for 20 hours. After the reaction,
After removing the cells by centrifugation, the mixture was added with 20 ml of ethyl acetate.
Extracted times. After drying the extract over magnesium sulfate,
Ethyl acetate was distilled off. In this way, 2.5 g of crystals of racemic β-carbamoylisobutyric acid methyl ester were obtained.

The obtained racemic β-carbamoylisobutyric acid methyl ester was subjected to high performance liquid chromatography (column: ODS-120-A (manufactured by Tosoh Corporation), moving bed: 10%
The purity was measured with methanol (flow rate: 0.5 ml / min) to be 98%. The physical properties of the obtained compound are shown below.

[0046] ⁽¹ H-NMR spectrum (FIG. 1)) solvent: CDCl _3, internal _{standard: TMS δ H .09 ~1.12 (3H} , d, -CH 3) δ H .19 ~2.27 (1H, m, - CH _2- ) δ _H .43 to 2.52 (1H, m, -CH _2- ) δ _H .73 to 2.86 (1H, m, -CH-) δ _H .83, 7.36 (2H, s, -NH ₂ )

[0047] ⁽¹³ C-NMR spectrum (FIG. 2)) solvent: CDCl _3, internal _{standard: TMS δ c 6.72 (-CH 3} ) δ c 5.47 (-CH 2 -) δ c 8.46 (-CH-) δ c _{1.45 (-CH 3) δ c 72.90} (-COOCH 3) δ c 75.86 (-COONH 2)

Example 2 Synthesis of racemic β-carbamoylisobutyric acid 2.5 g of racemic β-cyanoisobutyric acid was dissolved in 10 ml of pure water and adjusted to pH = 7.5 with 5N sodium hydroxide. To this solution, 50 ml of the bacterial cell suspension prepared in the same manner as in Example 1 was added, and reacted at 30 ° C. for 40 hours. After completion of the reaction, the cells were removed by centrifugation, the pH was adjusted to 1.0 with sulfuric acid, and the mixture was extracted twice with 20 ml of ethyl acetate. After the extract was dried over magnesium sulfate, ethyl acetate was distilled off. In this way,
2.4 g of racemic β-carbamoylisobutyric acid were obtained.

The obtained racemic β-carbamoylisobutyric acid was subjected to high performance liquid chromatography (column: OD
Purity was measured with S-120-A (manufactured by Tosoh Corporation), moving bed: 10% methanol, flow rate: 0.5 ml / min, and it was 62%. The physical properties of the obtained compound are shown below.

( ¹ H-NMR spectrum) Solvent: CDCl ₃ , Internal standard: TMS δ _H .039 to 1.063 (3H, w, —CH ₃ ) δ _H .201 to 2.552 (2H, m, —CH ₂ —) δ _H .64 to 3.58 (1H, m, -CH-) δ _H .781 to 7.352 (2H, w, -NH ₂ ) δ _H 2.084 (1H, s, -OH)

[0051] ⁽¹³ C-NMR spectrum) solvent: CDCl _3, internal _{standard: TMS δ c 7.887 (-CH 3} ) δ c 5.678 (-CH 2 -) δ c 7.481 (-CH-) δ c 73.289 (-COONH _{2) δ c} 77.200 (-COOH)

[Example 3] Synthesis of optically active (S) -β-carbamoylisobutyric acid derivative Escherichia coli FERM BP 3835
Was inoculated into 50 ml × 2 LB medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl) containing 50 μg / ml of ampicillin, and cultured with shaking at 37 ° C. for 24 hours. After completion of the culture, the culture was centrifuged, and the whole amount of the obtained cells was washed with ion-exchanged water, and then suspended in 100 ml of 100 mM phosphate buffer (pH 7.0). To this cell suspension was added 4 g of racemic β-cyanoisobutyric acid methyl ester, and the mixture was reacted at 30 ° C. for 20 hours. During this time, the pH of the reaction solution was adjusted using a 1N aqueous solution of NaOH.
Adjusted to 7.0. After completion of the reaction, the cells were removed by centrifugation, and unreacted methyl β-cyanoisobutyrate was extracted with ethyl acetate. The organic phase was dehydrated by adding anhydrous sodium sulfate, and the solvent was distilled off. Thus, 1.0 g of optically active β-cyanoisobutyric acid methyl ester was obtained.

The obtained optically active β-cyanoisobutyric acid methyl ester was subjected to high performance liquid chromatography (column: Chiralpak AS (manufactured by Daicel), moving bed: hexane / isopropanol / TFA = 90/10 / 0.1, flow rate: 0.
The optical purity was measured at 5 ml / min).
% Ee.

Cell suspension prepared in the same manner as in Example 1.
To 20 ml, 1 g of the above-mentioned (S) -β-cyanoisobutyric acid methyl ester was added and reacted at 30 ° C. for 20 hours. After the completion of the reaction, the mixture was extracted twice with 20 ml of ethyl acetate. The extract was dried over magnesium sulfate, and the ethyl acetate was distilled off. Thus, 0.98 g of (S) -β-carbamoylisobutyric acid methyl ester was obtained.

The obtained (S) -β-carbamoylisobutyric acid methyl ester was subjected to high performance liquid chromatography (column: ODS-120-A (manufactured by Tosoh Corporation), moving bed: 10%
The purity was measured with methanol (flow rate: 0.5 ml / min) to be 93%. When the optical purity was measured by high performance liquid chromatography (column: Chiralpak AS (manufactured by Daicel), moving bed: hexane / isopropanol / TFA = 90/10 / 0.1, flow rate: 0.5 ml / min), (S)
The body was 97% ee. (Specific rotation) [α] _D ^24.5 = -8.72 ° (neat)

[0056]

According to the present invention, .beta.-carbamoylisobutyric acids useful as intermediates for producing various pharmaceuticals and agricultural chemicals can be obtained.

[Brief description of the drawings]

FIG. 1 is a diagram showing a ¹ H-NMR spectrum of β-carbamoylisobutyric acid methyl ester obtained in Example 1.

FIG. 2 is a diagram showing a ¹³ C-NMR spectrum of β-carbamoylisobutyric acid methyl ester obtained in Example 1.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI (C12P 41/00 C12R 1:19) C07M 7:00

Claims (3)

[Claims] 1. General formula (1): H ₂ NOCCH ₂ C ^* H (CH ₃ ) COOR ¹ (1) wherein R ¹ is hydrogen or an alkyl group having 1 to 6 carbon atoms. Β-carbamoylisobutyric acids represented by the attached carbon atom is an asymmetric carbon atom). 2. The β- according to claim 1, which is optically active.
Carbamoyl isobutyric acids. 3. General formula (2): NCCH ₂ C ^* H (CH ₃ ) COOR ¹ (2) wherein R ¹ is hydrogen or an alkyl group having 1 to 6 carbon atoms. Β-cyanoisobutyric acid represented by the formula (1), in the presence of nitrile hydratase, or a culture, cells or treated cells of nitrile hydratase-producing microorganisms: 3. The β- according to claim 1 or 2, characterized by hydration.
A method for producing carbamoyl isobutyric acids.