JP3960667B2 - β-carbamoylisobutyric acid and process for producing the same - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to β-carbamoylisobutyric acid useful as a production intermediate for pharmaceuticals, agricultural chemicals and the like, and a method for producing the same.
[0002]
[Prior art]
As β-substituted isobutyric acids, for example, β-cyanoisobutyric acids are known (US Pat. No. 3,644,467, US Pat. No. 3,644,468, JP-A-9-67330). British Patent 808,835, U.S. Pat. No. 2,810,742, JP-A-8-67330, etc.). However, β-carbamoylisobutyric acid and its production method are not known.
[0003]
[Problems to be solved by the invention]
An object of the present invention is to provide β-carbamoylisobutyric acid and a method for producing the same.
[0004]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the present inventors have found a microorganism having the ability to hydrate the cyano group of β-cyanoisobutyric acid to convert it to an amide, that is, a nitrile hydration ability. Completed the invention.
[0005]
That is, the present invention relates to the general formula (1):
H ₂ NOCCH ₂ C ^* H (CH ₃ ) COOR ¹ (1)
(In the formula, R ¹ is hydrogen or an alkyl group having 1 to 6 carbon atoms. The carbon atom marked with * is an asymmetric carbon atom.)
It is (beta) -carbamoyl isobutyric acid represented by these.
[0006]
Further, the present invention provides a compound represented by the general formula (2):
NCCH ₂ C ^* H (CH ₃ ) COOR ¹ (2)
(In the formula, R ¹ is hydrogen or an alkyl group having 1 to 6 carbon atoms. The carbon atom marked with * is an asymmetric carbon atom.)
Hydrated in the presence of nitrile hydratase or a culture of microorganisms having the ability to produce nitrile hydratase, microbial cells or treated cells, It is a manufacturing method of (beta) -carbamoyl isobutyric acid as described in Formula (1).
[0007]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
In the general formulas (1) and (2), the alkyl group having 1 to 6 carbon atoms represented by R ¹ may have a linear or branched structure. Specific examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl, n-pentyl and the like.
[0008]
Further, the β-carbamoylisobutyric acids of the present invention include not only racemic β-carbamoylisobutyric acids but also optically active β-carbamoylisobutyric acids. This optically active β-carbamoylisobutyric acid is an effective production intermediate particularly for optically active pharmaceuticals and optically active agricultural chemicals.
[0009]
For the production of the β-carbamoylisobutyric acid of the present invention, β-cyanoisobutyric acid represented by the above general formula (2) is used as a raw material. When racemic β-cyanoisobutyric acid is used as a raw material, racemic β-carbamoylisobutyric acid is obtained. In addition, when optically active β-cyanoisobutyric acid is used as a raw material, optically active β-carbamoylisobutyric acid is obtained.
[0010]
Racemic β-cyanoisobutyric acid can be produced by a conventionally known general method. For example, racemic methyl β-cyanoisobutyrate can be produced by reacting methyl methacrylate and hydrocyanic acid in the presence of an alkaline catalyst (UK Patent 808,835, US Patent 2,810,742). U.S. Pat. No. 3,644,467, JP-A-8-291158, JP-A-9-67330). Racemic β-cyanoisobutyric acid can be produced, for example, by hydrolyzing racemic β-cyanoisobutyric acid methyl ester with an ester hydrolase.
[0011]
Optically active β-cyanoisobutyric acid is an asymmetric compound in the presence of racemic β-cyanoisobutyric acid ester in the presence of an ester asymmetric hydrolase, or a culture, cell or treated product of a microorganism having the enzyme-producing ability. It can be produced by hydrolysis.
Hereinafter, a method for producing optically active β-cyanoisobutyric acids will be described.
[0012]
The ester asymmetric hydrolase used is not limited to the type of enzyme and its source as long as it has an activity to asymmetrically hydrolyze the ester bond of racemic β-cyanoisobutyric acid ester. Such enzymes include lipases, esterases and proteases. As the enzyme, for example, a microorganism-derived enzyme 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. Specific examples include Pseudomonas putida FERM BP-3846, Escherichia coli FERM BP-3835, and the like. Escherichia coli FERM BP-3835 is a strain transformed with a gene encoding an esterase derived from Pseudomonas putida FERM BP-3846.
[0013]
The microorganism can be cultured in a liquid medium or a solid medium. As the medium, a medium in which components such as a carbon source, a nitrogen source, vitamins, and minerals that can normally be utilized by microorganisms are appropriately blended is used. In order to improve the hydrolytic ability of microorganisms, a small amount of ester can be added to the medium. The culture is performed at a temperature and pH at which the microorganism can grow, but may be performed under the optimal culture conditions for the strain to be used. In order to promote the growth of microorganisms, aeration and agitation may be performed.
[0014]
In addition to the purified ester asymmetric hydrolase, the culture obtained by culturing the above-mentioned microorganism capable of producing an ester asymmetric hydrolase in a medium is used as it is or centrifuged from the culture. A microbial cell obtained by a collection operation such as separation or a treated product thereof can also be used. Cell processed products can be obtained from cells treated with acetone, toluene, etc., freeze-dried cells, crushed cells, cell-free extracts, cell-free extracts by gel filtration, ion exchange chromatography, etc. Crude enzyme and the like. The microbial cell or enzyme can be used by being comprehensively immobilized on a cross-linked acrylamide gel or the like, or physically and chemically immobilized on a solid support such as an ion exchange resin or caustic soil. This facilitates recovery and reuse after the reaction.
[0015]
A commercially available product can be used as the ester asymmetric hydrolase. Specifically, lipase OF (trade name, manufactured by Meika Sangyo Co., Ltd., derived from Candida genus), durazyme (trade name, manufactured by NOVO, genus Bacillus), sabinase (trade name, manufactured by NOVO Co., genus Bacillus) Flavorzyme MG (trade name, manufactured by NOVO, derived from Aspergillus), lipase A-6 (trade name, manufactured by Amano, derived from Aspergillus), lipase M (trade name, manufactured by Amano, manufactured by Mucor) Neulase F (trade name, Amano Pharmaceutical Co., Ltd., Rhizopus genus), Lipase type VII (trade name, SIGMA Co., Candida genus), Acylase I (trade name, SIGMA Co., Aspergillus genus), Tripsin Type II (trade name, manufactured by SIGMA, derived from porcine pancreas), Protease type XVI (trade name, manufactured by SIGMA, derived from Bacillus), Palatase (trade name, manufactured by NOVO), etc. are used. Door can be.
[0016]
The optically selective hydrolysis of the racemic β-cyanoisobutyric acid ester can be carried out as follows. That is, a racemic β-cyanoisobutyric acid ester as a substrate is added to the reaction medium and dissolved or suspended, and an enzyme or microorganism culture as a catalyst is added. However, this catalyst may be added before the substrate is added to the reaction medium. Then, the racemic β-cyanoisobutyric acid ester is hydrolyzed 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 is reacted. However, depending on the case, the reaction may be completed with less than half of the substrate or excessively reacted with more than half of the substrate.
[0017]
As the reaction medium, for example, ion exchange water, a buffer solution or the like is used.
The substrate concentration in the reaction solution is not particularly limited as long as it is in the range of 0.1 to 70% by weight, but it is 5 to 40% by weight in consideration of the solubility and reactivity of the racemic β-cyanoisobutyric acid ester serving as the substrate. A range is preferred.
The reaction temperature is preferably 5 to 70 ° C, more preferably 20 to 60 ° C.
[0018]
The pH of the reaction solution depends on the optimum pH of the asymmetric hydrolysis ability of the enzyme or microorganism to be used, but in general, when carried out within the range of pH 6 to 8, it can suppress a decrease in optical purity due to chemical hydrolysis reaction. It is preferable because it is possible. As the reaction proceeds, the pH of the reaction solution is lowered due to the produced carboxylic acid. Therefore, it is desirable to carry out the reaction while maintaining the optimum pH with a suitable neutralizing agent.
In addition, the substrate concentration, medium, temperature, pH and other reaction conditions as described above are appropriately selected under conditions that advantageously yield the desired optically active compound in consideration of the reaction yield, optical yield, and the like. Is desirable.
[0019]
Such an asymmetric hydrolysis reaction produces optically active β-cyanoisobutyric acid. In addition, the unreacted residual substrate becomes an optically active β-cyanoisobutyric acid ester mainly composed of the enantiomer of the generated optically active β-cyanoisobutyric acid.
[0020]
After completion of the reaction, the optically active β-cyanoisobutyric acid ester can be separated from the reaction solution by extraction with a solvent such as hexane or ethyl acetate. At that time, the microbial cells, enzymes and the like used as the catalyst are removed in advance by an operation such as centrifugation and filtration, and then the solvent extraction is performed, whereby the extraction operation can be performed more easily. On the other hand, the extraction residual liquid is adjusted to pH 1-2 with an acid such as sulfuric acid or hydrochloric acid, and then extracted with a solvent such as hexane or ethyl acetate to obtain the enantiomer optically active β-cyanoisobutyric acid. The extracted product and solvent can be easily separated by a known method such as distillation.
[0021]
The nitrile hydratase used in the production of the β-carbamoylisobutyric acid of the present invention has the ability to hydrate and convert the cyano group of the β-cyanoisobutyric acid represented by the general formula (2) as a raw material into an amide ( As long as it has nitrile hydration ability), the kind of enzyme and its production source are not limited. As the nitrile hydratase, for example, those derived from microorganisms capable of producing nitrile hydratase can also be used.
[0022]
Representative examples of such nitrile hydratase producing microorganisms include Corynebacterium genus, Nocardia genus, Pseudomonas genus, Bacillus genus, bacterium ( Bacteridium genus, Brevibacterium genus, Micrococcus genus, Rhodococcus genus, Aeromonas genus, Citrobacter genus, Agrobacterium genus, Erwinia Examples include microorganisms belonging to the genus, Enterobacter genus, Streptomyces genus, Rhizobium genus and the like.
[0023]
Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium sp. (FERM P-4445) (see Japanese Patent Publication No. 56-17918).
[0024]
Examples of microorganisms belonging to the genus Nocardia include Nocardia sp. (FERM P-4447) (see Japanese Patent Publication No. 56-17918).
[0025]
Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas sp. (FERMP-6220) (see Japanese Examined Patent Publication No. 59-37951).
[0026]
Examples of microorganisms belonging to the genus Bacillus include Bacillus sp. (FERM P-2717) (see Japanese Examined Patent Publication No. 62-21519).
[0027]
Examples of microorganisms belonging to the genus Bacteridium include Bacterium sp. (FERM 2719) (see Japanese Examined Patent Publication No. 62-21519).
[0028]
Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium sp. (FERM P-2721).
[0029]
Examples of microorganisms belonging to the genus Micrococcus include Micrococcus sp. (FERM P-2720) (see Japanese Examined Patent Publication No. 62-21519).
[0030]
Examples of microorganisms belonging to the genus Rhodococcus include Rhodococcus sp. (FERM P-1046) (see JP-A-62-91189).
[0031]
Examples of microorganisms belonging to the genus Aeromonas include Aeromonas sp. (FERM P-12389) (see JP-A-5-30983).
[0032]
Examples of microorganisms belonging to the genus Citrobacter include Citrobacter freundii (FERM P-12390) (see JP-A-5-30984).
[0033]
Examples of microorganisms belonging to the genus Agrobacterium include Agrobacterium tumefaciens (IAM 13129) (see JP-A-5-103681).
[0034]
Examples of microorganisms belonging to the genus Erwinia include Erwinia nigrifluens (MAFF 03-01435) (see JP-A-5-161696).
[0035]
Examples of microorganisms belonging to the genus Enterobacter include Enterobacter sp. (FERM BP-3955) (see JP-A-5-236975).
[0036]
Examples of microorganisms belonging to the genus Streptomyces include Streptomyces sp. (FERM BP-3954) (see JP-A-5-236976).
[0037]
Examples of microorganisms belonging to the genus Rhizobium include Rhizobium loti (IAM 13588) (see JP-A-5-236977).
[0038]
The culture of the microorganism having the nitrile hydration ability can be carried out in a liquid medium or a solid medium. As a culture medium, what mix | blended suitably components, such as a carbon source, a nitrogen source, a vitamin, a mineral which a microorganism can normally utilize, is used. In order to improve the nitrile hydration ability of microorganisms, a small amount of nitrile, amide, organic acid or urea can be added to the medium as appropriate. The culture is performed at a temperature and pH at which the microorganism can grow, but may be performed under the optimal culture conditions for the strain to be used. In order to promote the growth of microorganisms, aeration and agitation may be performed.
[0039]
Further, in the present invention, not only the purified enzyme but also the culture obtained by culturing the above-mentioned microorganism capable of producing nitrile hydratase in a culture medium as it is or collected from the culture by centrifugation, etc. Bacteria obtained by the operation or a processed product thereof can also be used. Cell processed products can be obtained from cells treated with acetone, toluene, etc., freeze-dried cells, crushed cells, cell-free extracts, cell-free extracts by gel filtration, ion exchange chromatography, etc. Crude enzyme and the like. In addition, microbial cells or enzymes can be used by being comprehensively immobilized on a cross-linked acrylamide gel or the like, or physically and chemically immobilized on a solid support such as an ion exchange resin or caustic soil. This also facilitates recovery and reuse after the reaction.
[0040]
Nitrile hydration of β-cyanoisobutyric acid represented by the general formula (2) can be performed as follows. That is, β-cyanoisobutyric acid as a substrate is added to the reaction medium to be dissolved or suspended, and nitrile hydratase or a microorganism culture as a catalyst is added. However, the catalyst may be added before the substrate is added to the reaction medium. Then, the hydration reaction of β-cyanoisobutyric acid is performed while controlling the reaction temperature and, if necessary, the pH of the reaction solution.
As the reaction medium, for example, ion exchange water, a buffer solution or the like is used.
[0041]
The substrate concentration in the reaction solution is not particularly limited as long as it is between 0.1 and 70% by weight, but considering the solubility and productivity of β-cyanoisobutyric acid as a substrate, it is carried out at 5 to 40% by weight. Is preferred.
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 hydrating ability of the enzyme or microorganism used, but it is generally preferable to carry out the reaction within the range of pH 6-9.
[0042]
After completion of the reaction, β-carbamoylisobutyric acid produced by an appropriate method is extracted from the reaction solution. Specifically, when the obtained compound is β-carbamoyl isobutyric acid ester, extraction is performed with a solvent such as hexane or ethyl acetate, and when the obtained compound is β-carbamoyl isobutyric acid, sulfuric acid, hydrochloric acid or the like is used. After adjusting the pH to 1-2 with an acid, extraction is performed with a solvent such as ethyl acetate. At this time, the extraction operation can be performed more easily by performing solvent extraction after removing microbial cells, enzymes and the like previously used as a catalyst by operations such as centrifugation and filtration. The extracted product and solvent can be easily separated by a known method such as distillation.
[0043]
【Example】
EXAMPLES The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited to the scope of these examples.
[Example 1]
Synthesis of racemic β-carbamoylisobutyric acid methyl ester Rhodococcus rhodochrous strain IFO15564 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-captolactam 0.5%
pH 7.2
[0044]
After completion of the culture, the culture solution was centrifuged, and the entire 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). To 50 ml of this bacterial cell suspension, 2.5 g of racemic β-cyanoisobutyric acid methyl ester was added and reacted at 30 ° C. for 20 hours.
After completion of the reaction, the cells were removed by centrifugation and extracted twice with 20 ml of ethyl acetate. The extract was dried over magnesium sulfate, and then ethyl acetate was distilled off. In this way, 2.5 g of racemic β-carbamoylisobutyric acid methyl ester crystal was obtained.
[0045]
The purity of the obtained racemic β-carbamoylisobutyric acid methyl ester was measured by high performance liquid chromatography (column: ODS-120-A (manufactured by Tosoh Corporation), moving bed: 10% methanol, flow rate: 0.5 ml / min). The result was 98%.
The physical property values of the obtained compound are shown below.
[0046]
( ¹ H-NMR spectrum (FIG. 1))
Solvent: CDCl ₃ Internal standard: TMS
δ _H .09 to 1.12 (3H, d, —CH ₃ )
δ _H .19 to 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 )
[0048]
[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 caustic soda. To this solution, 50 ml of a 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 extracted twice with 20 ml of ethyl acetate. The extract was dried over magnesium sulfate, and ethyl acetate was distilled off. In this way, 2.4 g of racemic β-carbamoylisobutyric acid was obtained.
[0049]
The purity of the racemic β-carbamoylisobutyric acid obtained was measured by high performance liquid chromatography (column: ODS-120-A (manufactured by Tosoh Corporation), moving bed: 10% methanol, flow rate: 0.5 ml / min). 62%.
The physical property values of the obtained compound are shown below.
[0050]
( 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)
[0052]
Example 3
Synthesis of optically active (S)-[ beta] -carbamoylisobutyric acid derivative Escherichia coli FERM BP 3835 was added to LB medium (1% polypeptone, 0.5% yeast extract, 0.5%) containing ampicillin 50 [mu] g / ml. NaCl) 50 ml × 2 were inoculated and cultured at 37 ° C. for 24 hours with shaking. After completion of the culture, the culture solution was centrifuged, and the whole amount of the obtained bacterial cells was washed with ion-exchanged water and then suspended in 100 ml of 100 mM phosphate buffer (pH 7.0). 4 g of racemic β-cyanoisobutyric acid methyl ester was added to this bacterial cell suspension and reacted at 30 ° C. for 20 hours. During this time, the pH of the reaction solution was adjusted to 7.0 using 1N NaOH aqueous solution. 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. In this way, 1.0 g of optically active β-cyanoisobutyric acid methyl ester was obtained.
[0053]
About the obtained optically active β-cyanoisobutyric acid methyl ester, 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) As a result of measuring the optical purity, the (S) body was 97.0% ee.
[0054]
1 g of the above (S) -β-cyanoisobutyric acid methyl ester was added to 20 ml of the cell suspension prepared in the same manner as in Example 1, and reacted at 30 ° C. for 20 hours. After completion of the reaction, the mixture was extracted twice with 20 ml of ethyl acetate, and the extract was dried over magnesium sulfate, and then ethyl acetate was distilled off. In this way, 0.98 g of (S) -β-carbamoylisobutyric acid methyl ester was obtained.
[0055]
The obtained (S) -β-carbamoylisobutyric acid methyl ester was purified by high performance liquid chromatography (column: ODS-120-A (manufactured by Tosoh Corporation), moving bed: 10% methanol, flow rate: 0.5 ml / min). Was measured to be 93%. Further, 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) Body was 97% ee.
(Specific rotation)
[α] _D ^24.5 = −8.72 ° (neat)
[0056]
【The invention's effect】
According to the present invention, β-carbamoylisobutyric acids useful as production intermediates for various pharmaceuticals and agricultural chemicals can be obtained.
[Brief description of the drawings]
1 is a diagram showing a ¹ H-NMR spectrum of β-carbamoylisobutyric acid methyl ester obtained in Example 1. FIG.
2 shows a ¹³ C-NMR spectrum of β-carbamoylisobutyric acid methyl ester obtained in Example 1. FIG.

Claims (3)

General formula (2):
NCCH ₂ C ^* H (CH ₃ ) COOR ¹ (2)
(In the formula, R ¹ is hydrogen or an alkyl group having 1 to 6 carbon atoms. The carbon atom marked with * is an asymmetric carbon atom.)
Hydrated β-cyanoisobutyric acid represented by nitrile hydratase or a culture of microorganisms having nitrile hydratase-producing ability, microbial cells or processed microbial products,
General formula (1):
H ₂ NOCCH ₂ C ^* H (CH ₃ ) COOR ¹ (1)
(In the formula, R ¹ is hydrogen or an alkyl group having 1 to 6 carbon atoms. The carbon atom marked with * is an asymmetric carbon atom.)
The manufacturing method of (beta) -carbamoyl isobutyric acid represented by these.   The method according to claim 1, wherein the microorganism capable of producing nitrile hydratase is Rhodococcus rhodochrous. The method of claim 1, wherein R ¹ is hydrogen or methyl.

Images