JPH10287620A - Optically active methyl succinate and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an optically active methyl succinic acid diester which is an effective intermediate for producing an optically active pharmaceutical or an optically active agricultural chemical, wherein the two ester residues are different, and a method for producing the same. . SOLUTION: R ¹ OOCCH ₂ C ^* H (CH ₃ ) COOR
² (wherein, R ¹ is an alkyl group having 1 to 6 carbon atoms,
R ² is an alkyl group having 1 to 6 carbon atoms different from R ¹ . The carbon atoms marked with * are asymmetric carbon atoms. ) An optically active methyl succinic acid diester represented by the formula: R ¹ OO
A racemic methyl succinic acid diester represented by CCH ₂ CH (CH ₃ ) COOR ² (wherein R ¹ and R ² are as defined above) is used to prepare a culture, a bacterium, A process for producing an optically active methyl succinate ester, comprising asymmetric hydrolysis in the presence of a processed body or cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an optically active drug,
The present invention relates to a novel optically active methyl succinate useful as an intermediate for producing an optically active pesticide and the like, and a method for producing the same.

[0002]

2. Description of the Related Art As a method for producing an optically active methyl succinic diester, there is known a method for asymmetric hydrolysis of a racemic methyl succinic diester with an enzyme, as described in JP-A-8-285. . However, the optically active methyl succinic acid diester described in this publication has the same ester residue at the 1-position and 4-position,
An optically active methyl succinic acid diester in which two ester residues are different from each other and a method for producing the same are not known.

[0003]

The object of the present invention is to provide an optically active methyl succinic acid diester which is an effective intermediate for producing optically active pharmaceuticals and agriculturally active agricultural chemicals, wherein the two ester residues are different, and It is to provide a manufacturing method thereof.

[0004]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found a microorganism capable of optically hydrolyzing racemic methyl succinic diester. Was completed.

That is, the present invention provides a compound represented by the general formula (1): R ¹ OOCCH ₂ C ^* H (CH ₃ ) COOR ² (1) (wherein R ¹ is an alkyl group having 1 to 6 carbon atoms;
R ² is an alkyl group having 1 to 6 carbon atoms different from R ¹ . The carbon atoms marked with * are asymmetric carbon atoms. ) Is an optically active methyl succinic acid diester.

Further, the present invention provides a compound represented by the following general formula (2): R ¹ OOCCH ₂ CH (CH ₃ ) COOR ²
(2) A racemic methyl succinic acid diester represented by the formula (wherein R ¹ and R ² are as defined above) are obtained by subjecting a culture, a microbial cell, or a treated microbial cell of a microorganism having an ester asymmetric hydrolytic ability Wherein the optically active methyl succinic acid diester represented by the above general formula (1) and its enantiomer represented by the following general formula (3): R ¹ OOCCH ₂ C ^* H (CH ₃ ) COOH (3) (wherein R ¹ is as defined above; the carbon atom marked with * is an asymmetric carbon atom.) A method for producing an optically active methyl succinic acid monoester represented by the formula: It is.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
In the general formulas (1) to (3), the alkyl group having 1 to 6 carbon atoms represented by R ¹ or R ² may have either 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. However, in each of the general formulas (1) and (2),
R ¹ and R ² are different groups.

In the optically active methyl succinic acid diester represented by the general formula (1), R ¹ is a tert-butyl group and R ² is a methyl group.
tert-butyl-1-methyl ester, and the above general formula (3)
In the optically active methyl succinic acid monoester represented by the formula, optically active methyl succinic acid-4-tert-butyl ester in which R ¹ is a tert-butyl group utilizes the difference in reactivity between the 4-position and the 1-position ester groups. As a result, it is possible to derive various optically active compounds, which is a very useful compound.

The optically active compound of the present invention can be produced by the following method. As a raw material, a racemic methyl succinic acid diester represented by the general formula (2) is used.
This racemic methyl succinic acid diester is
The -1-monoester can be produced by catalytic reduction of a diester obtained by reacting an anhydrous alcohol with an appropriate dehydrating agent.

The microorganism used in the present invention has a general formula
Any compound having the ability to asymmetrically hydrolyze the ester bond of the racemic methyl succinic acid diester represented by (2) can be used. Representatives of such microorganisms include Pseudomonas (Pseudom
onasu) or a microorganism belonging to the genus Escherichia. Specifically, Pseudomonas putida (Pseudomonas putida) FERM BP 3846, Escherichia
Escherichia coli FERM BP 3835 and the like.
Escherichia coli FERM BP 3835 is a strain transformed with an esterase gene derived from Pseudomonas putida FERM BP 3846.

The culture of the microorganism used in the present invention 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. To promote the growth of microorganisms,
Aeration stirring may be performed.

In the present invention, a culture obtained by culturing the above microorganism in a medium may be used as it is, or a cell obtained by collecting cells from the culture by centrifugation or a processed product thereof may be used. it can. 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 enzyme or purified enzyme. Enzymes isolated from the microorganisms are lipase, protease,
Esterase and the like. In addition, microbial cells or enzymes,
Entrapping and immobilizing in a cross-linked acrylamide gel, etc.
Physically on solid carriers such as ion exchange resin
It can be used after being chemically immobilized, so that it can be recovered and reused after the reaction.

The optically selective hydrolysis of the racemic methyl succinic diester represented by the general formula (2) can be carried out as follows. Racemic methyl succinic acid diester as a substrate is added to the reaction medium and dissolved or suspended. Before or after the substrate is added to the reaction medium, a culture of a microorganism serving as a catalyst is added. Then, the reaction is carried out while controlling the reaction temperature and, if necessary, the pH of the reaction solution until half of the methyl succinate diester is hydrolyzed. As the reaction medium, for example, ion-exchanged water, a buffer, or the like is used. The substrate concentration of the reaction solution is not particularly limited in the range of 0.1 to 70% by weight, but is preferably 5 to 40% by weight in consideration of the solubility and productivity of the racemic methyl succinate diester serving as the substrate. The reaction temperature is preferably 5 to 70C, more preferably 20 to 60C. The pH of the reaction solution is optimal for the asymmetric hydrolysis ability of the microorganism used
Although it depends on the pH, it is generally preferable to carry out the reaction within a pH range of 6 to 8 because a decrease in optical purity due to a chemical hydrolysis reaction can be suppressed. As the reaction proceeds, the pH of the reaction solution decreases due to the generated carboxylic acid,
In this case, it is desirable to maintain the optimum pH with a suitable neutralizing agent.

After the completion of the reaction, the cells of the microorganism of the present invention are removed by removing the cells of the microorganisms used as the catalyst by centrifugation, filtration, etc., and extracting with a solvent such as hexane or ethyl acetate. An optically active methyl succinic diester of the reaction can be obtained. On the other hand, the extraction residue is adjusted to pH 1 to 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 methyl succinic acid monoester. In order to further improve the optical purity of methyl succinic acid monoester such as methyl succinic acid-4-tert-butyl ester, a method of recrystallization using an aliphatic hydrocarbon solvent having 5 to 10 carbon atoms is effective.

[0015]

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.

Reference Example 1 Racemic methyl 4-s-tert-butyl-1-succinate
50 g of Itaconic acid-1-methyl ester of Teru (Iwata Chemical) was dissolved in 100 ml of toluene, the resulting solution was charged into a glass autoclave, and 20 g of an ion exchange resin (RCP-160H, Mitsubishi Chemical) was added. Next, 29 g of isobutylene was added under cooling and reacted at room temperature for 5 hours. Thereafter, the ion-exchange resin is separated from the reaction mixture by filtration, and the remaining solution is added in an equal amount.
Washed twice with 10% aqueous sodium carbonate solution. The organic layer was dried over anhydrous sodium sulfate and concentrated. Thus, itaconic acid-4-tert-butyl-1-methyl ester 4
8.9 g were obtained.

The whole amount of the obtained 4-tert-butyl-1-methyl itaconate was dissolved in 50 ml of methanol, and then dissolved in 50 ml of methanol.
% Palladium / carbon (Wako Pure Chemical Industries) 0.25 g was added. Hydrogen was added thereto to a concentration of 1 kg / cm ² and reacted for 2 hours. After completion of the reaction, the catalyst was filtered off, and the remaining solution was concentrated under reduced pressure. Thus, 44.2 g of racemic methyl succinic acid-4-tert-butyl-1-methyl ester was obtained.

Example 1 Synthesis of Optically Active Methylsuccinic Acid Derivative Escherichia coli FERM BP 3835
Was inoculated into 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 solution was centrifuged, and the whole amount of the obtained cells was washed with ion-exchanged water and then suspended in 50 ml of 50 mM phosphate buffer (pH 7.0).
5 g of racemic methyl succinic acid-4-tert-butyl-1-methyl ester was added to the cell suspension, and the mixture was 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 NaOH solution. After completion of the reaction, the cells were removed by centrifugation, and unreacted 4-tert-butyl-1-methylmethylsuccinate was extracted with ethyl acetate. The organic layer was dehydrated by adding anhydrous sodium sulfate, and the solvent was distilled off.
g of optically active methyl succinic acid-4-tert-butyl-1-methyl ester is treated with trifluoroacetic acid, and then hydrolyzed with a three-fold molar aqueous solution of sodium hydroxide to obtain optically active methyl succinic acid. Column: Chiralcel OD (manufactured by Daicel), moving bed: hexane / isopropanol / TFA = 90/10 / 0.1, flow rate: 0.5
When the optical purity was measured at ml / min｝, the (S) form was 95.5%
was ee. Add 2N hydrochloric acid to the raffinate aqueous layer and adjust the pH to 2.0.
The reaction product, optically active methyl succinic acid-4
-tert-butyl ester was extracted with ethyl acetate. The organic layer was dehydrated by adding anhydrous sodium sulfate, and the solvent was distilled off to obtain 1.9 g of optically active methyl succinic acid-4-tert-butyl ester. This was measured for optical purity in the same manner as described above, and was found to be the (R) form 99.5% ee.

The physical properties of the obtained compound are shown below.・ (S) -methylsuccinic acid-4-tert-butyl-1-methyl ester ( ¹ H-NMR spectrum) CDCl ₃ , internal standard TMS
(FIG. 1) δ _H 1.19 to 1.24 (3H, d, -CH ₃ ) δ _H 1.44 (9H, s, -tBu) δ _H 2.29 to 2.38 (1H, m, -CH _2- ) δ _H 2.60 to 2.69 ( _{1H, m, -CH 2 -)} δ H 2.83~2.91 (1H, m, -CH-) δ H 3.69 (3H, s, -COOCH 3) (13 C-NMR spectra) CDCl _3, internal standard TMS
(FIG. 2) δ _C 16.90 (-CH ₃ ) δ _C 28.05 (-tBu) δ _C 35.76 (-CH _2- ) δ _C 39.06 (-CH-) δ _C 51.77 (-COOCH ₃ ) δ _C 80.66 (-tBu) ) Δ _C 171.00 (-COOtBu) δ _C 175.84 (-COOCH ₃ )

(Optical purity) Measured by liquid chromatography optical resolution column, see Example. (S) form 95.5% ee

(Α) _D ²⁵ = −2.11 (neat) · (R) -methylsuccinic acid-4-tert-butyl ester ( ¹ H-NMR spectrum) CDCl ₃ , internal standard TMS
(FIG. 3) δ _H 1.23 to 1.25 (3H, d, -CH ₃ ) δ _H 1.44 (9H, s, -tBu) δ _H 2.32 to 2.40 (1H, m, -CH _2- ) δ _H 2.60 to 2.69 ( _{1H, m, -CH 2 -)} δ H 2.86~2.93 (1H, m, -CH-) δ H 9.80 (1H, s, -COOH) (13 C-NMR spectra) CDCl _3, internal standard TMS
(FIG. 4) δ _C 16.69 (-CH ₃ ) δ _C 28.02 (-tBu) δ _C 35.91 (-CH _2- ) δ _C 38.75 (-CH-) δ _C 81.01 (-tBu) δ _C 170.98 (-COOtBu) δ _C 181.53 (-COOH)

(Optical purity) Measured by liquid chromatography optical resolution column, see Example. (R) form 99.5% ee

(Specific optical rotation) [α] _D ²⁵ = + 4.74 (c = 2.30, EtOH)

[0024]

According to the present invention, an optically active methyl succinic acid diester having two different ester residues, which is useful as an intermediate for producing various optically active pharmaceuticals and optically active agricultural chemicals, can be obtained.

[Brief description of the drawings]

FIG. 1 shows (S) -methylsuccinic acid-4 obtained in Example 1.
FIG. 3 is a ¹ H-NMR spectrum of -tert-butyl-1-methyl ester.

FIG. 2 shows (S) -methylsuccinic acid-4 obtained in Example 1.
FIG. 3 is a ¹³ C-NMR spectrum of -tert-butyl-1-methyl ester.

FIG. 3 shows (R) -methylsuccinic acid-4 obtained in Example 1.
FIG. 3 is a ¹ H-NMR spectrum of -tert-butyl ester.

FIG. 4 shows (R) -methylsuccinic acid-4 obtained in Example 1.
FIG. 3 is a ¹³ C-NMR spectrum of -tert-butyl ester.

Claims (2)

[Claims] 1. A general formula (1): R ¹ OOCCH ₂ C ^* H (CH ₃ ) COOR ² (1) wherein R ¹ is an alkyl group having 1 to 6 carbon atoms,
R ² is an alkyl group having 1 to 6 carbon atoms different from R ¹ . The carbon atoms marked with * are asymmetric carbon atoms. ) An optically active methyl succinic acid diester represented by the formula: 2. A general formula (2): R ¹ OOCCH ₂ CH (CH ₃ ) COOR ² (2) (wherein, R ¹ is an alkyl group having 1 to 6 carbon atoms,
R ² is an alkyl group having 1 to 6 carbon atoms different from R ¹ . A) a racemic methyl succinic acid diester represented by the following formula:
Characterized by asymmetrically hydrolyzing in the presence of a bacterial cell or processed product, the general formula _{^{(1): R 1 OOCCH 2}} C * H (CH 3) COOR 2 (1) ( In the formula, R ¹ and R ² are as defined above * is the carbon atom attached is an asymmetric carbon atom) optically active methyl succinate diester represented by, and pairs thereof formula is enantiomer (3):.. R ¹ OOCCH ₂ C ^* H (CH ₃ ) COOH (3) (wherein R ¹ is as defined above. The carbon atom marked with * is an asymmetric carbon atom.) Method for producing monoester.