JPH1180113A - Optically active beta-cyanoisobutyric acids and their production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new compounds useful as an intermediate for producing optically active pharmaceuticals, optically active agrochemicals and the like. SOLUTION: The compounds are represented by the formula NCCH2 C*H (CH3 )COOR<1> (R<1> is H or a 1-6C alkyl; C* is an asymmetric carbon) e.g. optically active methyl β-cyanoisobutyrate. The compounds represented by the formula are obtained by adding a racemic β-cyanoisobutyric acid ester represented by the formula NCCH2 C*H(CH3 )COOR<2> (R<2> is a 1-6C alkyl; C* is an asymmetric carbon which is a substrate at 5-40 wt.% concentration to a reactional medium (e.g. ion-exchanged water or a buffer solution), dissolving or suspending the racemic ester therein and asymmetrically hydrolyzing the racemic ester in the presence of an asymmetric esterase or a cultured product of a microorganism (e.g. Pseudomonas putida or Escherichia coli) having the ability to produce the enzyme, a microbial cell or a treated microbial cell (e.g. a microbial cell treated by acetone, toluene or the like, a lyophilized microbial cell and a milled microbial cell) which is a catalyst preferably at 20-60 deg.C and pH 6-8.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an optically active drug,
The present invention relates to novel optically active β-cyanoisobutyric acids useful as intermediates for producing optically active pesticides and the like, and a method for producing the same.

[0002]

2. Description of the Related Art As a method for producing racemic β-cyanoisobutyrate, US Pat. No. 3,644,467, US Pat. No. 3,644,468, and US Pat. No. 2,810,742 are known. , UK Patent No. 808,835
JP-A-8-291158, JP-A-9-67
The method described in Japanese Patent Publication No. 330 is known. However, these optically active substances and methods for producing them are not known.

[0003]

An object of the present invention is to provide an optically active β-cyanoisobutyric acid which is an effective intermediate for producing optically active pharmaceuticals and agriculturally active agricultural chemicals, and a method for producing the same.

[0004]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that an enzyme capable of optically selectively hydrolyzing racemic β-cyanoisobutyrate and an enzyme capable of hydrolyzing the same. The present inventors have found a microorganism capable of producing an enzyme and completed the present invention. That is, the present invention provides a compound represented by the general formula (1): NCCH ₂ C ^* H (CH ₃ ) COOR ¹ (1) (wherein, R ¹ represents hydrogen or an alkyl group having 1 to 6 carbon atoms. The carbon atom is an asymmetric carbon atom.) Is an optically active β-cyanoisobutyric acid.

Further, the present invention provides a compound of the general formula (2): NCCH ₂ C ^* H (CH ₃ ) COOR ² (2) (wherein R ² represents an alkyl group having 1 to 6 carbon atoms).
The carbon atoms marked with * are asymmetric carbon atoms. Asymmetric hydrolysis of the racemic β-cyanoisobutyrate represented by the formula (1) in the presence of an ester asymmetric hydrolase or a culture, fungus or processed product of a microorganism capable of producing the enzyme. A general formula (3): NCCH ₂ C ^* H (CH ₃ ) COOR ² (3) (wherein R ² is as defined above. The carbon atom marked with * is an asymmetric carbon atom The optically active β- represented by
Formula (4) which is a cyanoisobutyric acid ester and / or its enantiomer: NCCH ₂ C ^* H (CH ₃ ) COOH (4) (In the formula, the carbon atom marked with * is an asymmetric carbon atom.) This is a method for producing optically active β-cyanoisobutyric acid represented by the formula:

[0006]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
In formulas (1) to (3), the alkyl group having 1 to 6 carbon atoms represented by R ¹ or R ² may have any of a straight-chain structure and a branched structure. Specifically, methyl,
Examples include ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl, n-pentyl and the like.

The optically active compound of the general formula (1) of the present invention can be produced by the following method. As a raw material, a racemic β-cyanoisobutyrate represented by the above general formula (2) is used. This racemic β-cyanoisobutyrate 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 (GB 808,835, US Pat. No. 2,811).
0, U.S. Pat. No. 3,644,467;
291158, JP-A-9-67330).

The enzyme used in the present invention may be any enzyme having an activity of asymmetrically hydrolyzing the ester bond of racemic β-cyanoisobutyrate represented by the general formula (2), and the source of the enzyme. Regardless. 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. Pseudomonas (Pseudomonas) is a typical example of a microorganism having an ester asymmetric hydrolysis ability.
And microorganisms belonging to the genus Escherichia. Specifically, Pseudomonas putida (P
seudomonas putida) FERM BP-3846, Escherichia coli
(Escherichia coli) FERM BP-3835 and the like. still,
Escherichia coli FERM BP-3835 is a strain transformed with a gene encoding an esterase from Pseudomonas putida FERM BP-3846.

[0009] The cultivation of the microorganisms can be carried out 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.

In the present invention, a culture obtained by culturing a microorganism having the ability to produce an ester asymmetric hydrolase in a medium, as well as a purified enzyme, as it is, or from the culture, Cells obtained by a cell collection operation such as centrifugation or a cell treated product thereof 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.

As the ester asymmetric hydrolase, commercially available products can be used. Specifically, lipase OF (trade name, manufactured by Meito Sangyo Co., derived from Candida), durazyme (trade name, manufactured by NOVO, derived from Bacillus), sabinase (trade name, manufactured by NOVO, derived from Bacillus), Flav
ourzyme 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), Newase F (Trade name, manufactured by Amano Pharmaceutical, derived from Rhizopus sp.), Lipase type VII (trade name, S
Acylase I (trade name, SIGMA, Aspergillus), Trip
Sin type II (trade name, manufactured by SIGMA, derived from pig pancreas), Protease type XVI (trade name, manufactured by SIGMA, derived from Bacillus), Palatase (trade name, manufactured by NOVO) and the like can be used.

The optically selective hydrolysis of the racemic β-cyanoisobutyrate represented by the general formula (2) 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 the amount of the reaction is reacted. In some cases, the reaction may be terminated with less than half the amount of the substrate, or may be caused to exceed the half amount of the substrate.

As the 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 asymmetric hydrolysis.
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. In addition, it is desirable that the substrate concentration, the medium, the temperature, the pH, and the reaction conditions as described above are appropriately selected under conditions in which the desired optically active compound is advantageously obtained in consideration of the reaction yield and the optical yield.

By such asymmetric hydrolysis reaction, optically active β-cyanoisobutyric acid represented by the above formula (4) is produced. The unreacted residual substrate is generated optically active β
An optically active β-cyanoisobutyric acid ester represented by the above general formula (3) having an enantiomer of cyanoisobutyric acid as a main component is obtained.

After completion of the reaction, the unreacted optically active β-cyanoisobutyrate, which is the compound of the present invention, can be separated from the reaction solution by extraction with a solvent such as hexane or ethyl acetate. 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.

Further, the obtained optically active β-cyanoisobutyric acid ester can be converted into β-cyanoisobutyric acid while maintaining the optical activity by hydrolyzing in a usual manner. The optically active β-cyanoisobutyric acid can be converted into β-cyanoisobutyrate by maintaining the optical activity by esterification by a usual method.
Therefore, β-cyanoisobutyric acid having an arbitrary configuration can be obtained.

[0018]

[Example 1]

Synthesis of optically active β-cyanoisobutyric acids Escherichia coli FERM BP 3835
With 50 ml of 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 to collect the cells. The whole amount of the obtained cells was washed with ion-exchanged water. After washing, the cells were suspended in 50 ml of 50 mM phosphate buffer (pH 7.0). Racemic β-cyanoisobutyric acid methyl ester was added to the cell suspension in an amount of 5%.
g was added and reacted at 30 ° C. for 20 hours. During this time, the pH of the reaction solution was adjusted to 7.0 using a 1N aqueous solution of NaOH. 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, 1.2 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: Chiralcel OD (manufactured by Daicel), moving bed: hexane / isopropanol / TFA = 90/10 / 0.1,
When the optical purity was measured at a flow rate of 0.5 ml / min),
(S) The body was 97.5% ee.

2N hydrochloric acid was added to the aqueous phase, which was the extraction residue in the above-mentioned extraction with ethyl acetate, to adjust the pH to 2.0, and the optically active β-cyanoisobutyric acid as the reaction product was extracted with ethyl acetate. The organic phase was dehydrated by adding anhydrous sodium sulfate, and the solvent was distilled off. Thus, 2.5 g
Of optically active β-cyanoisobutyric acid was obtained. When the optical purity of the obtained optically active β-cyanoisobutyric acid was measured in the same manner as described above, the (R) form was 31% ee.

The physical properties of (S) -β-cyanoisobutyric acid methyl ester are shown below. ⁽¹ H-NMR spectrum (FIG. 1)) solvent: CDCl _3, internal _{standard: TMS δ H 1.23~1.26 (3H,} d, -CH 3) δ H 2.70~2.73 (2H, m, —CH ₂ —) δ _H 2.84 to 2.92 (1H, m, —CH—) δ _H 3.67 (3H, s, —CH ₃ )

[0022] ⁽¹³ C-NMR spectrum (FIG. 2)) solvent: CDCl _3, internal _{standard: TMS δ C 16.24 (-CH 3} ) δ C 20.48 (-CH 2 -) δ C 35.57 ( _{-CH-) δ C 118.79 (-CN)} δ C 173.70 (-COOCH 3)

(Optical Purity) (S) Form 97.5% ee (Specific Optical Rotation) [α] _D ²⁵ = −9.97 (neat)

The physical properties of (R) -β-cyanoisobutyric acid are shown below. ( ¹ H-NMR spectrum (FIG. 3)) Solvent: CDCl ₃ , Internal standard: TMS δ _H 1.21 to 1.24 (3H, d, —CH ₃ ) δ _H 2.63 to 2.66 (2H, m, —CH ₂ —) δ _H 2.71 to 2.79 (1H, m, —CH—) δ _H 10.47 (1H, s, —OH)

( ¹³ C-NMR spectrum (FIG. 4)) Solvent: CDCl ₃ , Internal standard: TMS δ _C 16.29 (—CH ₃ ) δ _C 20.40 (—CH ₂ −) δ _C 35.50 ( _{-CH-) δ C 119.03 (-CN)} δ C 174.83 (-COOH)

(Optical Purity) (R) Form 31% e.e.

Example 2 Synthesis of Optically Active (R) -β-Cyanoisobutyric Acid Derivative 1 g of lipase OF (trade name, manufactured by Meito Sangyo Co., Ltd., Candida sp.) In 50 ml of 50 mM phosphate buffer (pH 7.0) Origin)
Was dissolved. 2.5 g of racemic β-cyanoisobutyric acid methyl ester was added to this solution, and reacted at 30 ° C. for 20 hours. During this time, the pH of the reaction solution was adjusted to 7.0 using a 1N aqueous solution of NaOH. After the reaction, 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, 0.4 g of optically active β-cyanoisobutyric acid methyl ester was obtained.

Further, the obtained optically active β-cyanoisobutyric acid methyl ester can be converted into β-cyanoisobutyric acid while maintaining the optical activity by hydrolyzing in a usual manner. The optically active β-cyanoisobutyric acid can be converted into β-cyanoisobutyrate by maintaining the optical activity by esterification by a usual method. Therefore, an arbitrary configuration can be 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,
When the optical purity was measured at a flow rate of 0.5 ml / min),
(R) was 92.3% ee.

[Examples 3 to 13] In 1 ml of 50 mM phosphate buffer (pH 7.0) in which 2% of racemic β-cyanoisobutyric acid methyl ester was dissolved, 0.02 to 0.
After adding 05 g, the mixture was reacted at 30 ° C. for 20 hours. After 1 ml of ethyl acetate was added to the reaction solution and stirred sufficiently, the organic phase was subjected to high performance liquid chromatography (column: Chiralpak AS
(Manufactured by Daicel), moving bed: hexane / isopropanol / TFA = 90/10 / 0.1, flow rate: 0.5 ml / mi
The optical purity of the optically active β-cyanoisobutyric acid methyl ester produced in the step n) was measured. Table 1 shows the results.

[0031]

[Table 1]

[0032]

According to the present invention, there is provided a novel optically active β-protein which is an effective intermediate for producing optically active pharmaceuticals and agriculturally active agricultural chemicals.
Carboxyaminoisobutyric acids are obtained.

[Brief description of the drawings]

FIG. 1 is a diagram showing a ¹ H-NMR spectrum of (S) -β-cyanoisobutyric acid methyl ester obtained in Example 1.

FIG. 2 is a view showing a ¹³ C-NMR spectrum of (S) -β-cyanoisobutyric acid methyl ester obtained in Example 1.

FIG. 3 is a diagram showing a ¹ H-NMR spectrum of (R) -β-cyanoisobutyric acid obtained in Example 1.

FIG. 4 is a view showing a ¹³ C-NMR spectrum of (R) -β-cyanoisobutyric acid obtained in Example 1.

Claims (2)

[Claims] 1. General formula (1): NCCH ₂ C ^* H (CH ₃ ) COOR ¹ (1) (wherein, R ¹ represents hydrogen or an alkyl group having 1 to 6 carbon atoms. Is an asymmetric carbon atom.) Optically active β-cyanoisobutyric acids. 2. General formula (2): NCCH ₂ C ^* H (CH ₃ ) COOR ² (2) (wherein, R ² represents an alkyl group having 1 to 6 carbon atoms).
The carbon atoms marked with * are asymmetric carbon atoms. Asymmetric hydrolysis of the racemic β-cyanoisobutyrate represented by the formula (1) in the presence of an ester asymmetric hydrolase or a culture, fungus or processed product of a microorganism capable of producing the enzyme. A general formula (3): NCCH ₂ C ^* H (CH ₃ ) COOR ² (3) (wherein R ² is as defined above. The carbon atom marked with * is an asymmetric carbon atom The optically active β- represented by
Formula (4) which is a cyanoisobutyric acid ester and / or its enantiomer: NCCH ₂ C ^* H (CH ₃ ) COOH (4) (In the formula, the carbon atom marked with * is an asymmetric carbon atom.) A method for producing an optically active β-cyanoisobutyric acid represented by the formula: