JPH10229890A - Production of malonic acid derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a producing method excellent in productivity of a malonic acid monoester useful as a synthetic intermediate for various chemicals, pharmaceutical agents, agrochemicals, etc. SOLUTION: A cyanoacetic acid ester represented by the formula NCCH2 COOR (R is an alkenyl group, an aryl group, an aralkyl group or a 1-20C alkyl group) is treated with a cultured product, a cell or a cell-treated material of a microorganism belonging to the genus Corynebacterium or Gordona and having nitrilase activity to produce the objective malonic acid monoester represented by the formula HOOCCH2 COOR (R is mentioned above).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing a malonic acid monoester useful as a synthetic intermediate for various chemical products, medicines, agricultural chemicals and the like.

[0002]

2. Description of the Related Art As a method for producing a malonic acid monoester, a method of chemically hydrolyzing a malonic acid diester is generally used. However, in this method, after completion of the reaction,
It is difficult to isolate the malonic acid monoester as a product from unreacted malonic acid diester and by-product malonic acid, and it is not possible to obtain high-purity malonic acid monoester.

[0003] As a method for obtaining high-purity malonic acid monoester, a method using Meldrum's acid as a raw material is known (for example, Matoba Katsuhide et al., Chem. Pharm. Bu.
ll., 31 (8), 2955 (1983), or Rigo B. et al., Tetrahe.
dron Lett., 30 (23), 3073 (1989)). However, this method is not practical because it uses expensive Meldrum's acid, and is not suitable for industrial production.

Further, as a method for obtaining a high-purity malonic acid monoester, a method is known in which an enzyme or a microorganism having the ability to hydrolyze an ester bond is allowed to act on the malonic acid diester (Japanese Patent Application Laid-Open No. 8-173174). . However, malonic diester as a raw material is disadvantageous in cost. Therefore, development of a method for producing high-purity malonic acid monoester having excellent productivity has been desired.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing malonic acid monoester which is useful as a synthetic intermediate for various chemical products, medicines, agricultural chemicals and the like, and has excellent productivity.

[0006]

Means for Solving the Problems The present inventors have added cyanine acetate to Corynebacterium.
When a microorganism, which belongs to the genus or Gordona genus and has nitrilase activity, is allowed to act on a culture, cells or treated cells of malonate, malonic acid monoester is selectively produced, and secondary reactions such as hydrolysis of ester bond are caused. The present inventors have found that a high-purity malonic acid monoester can be produced without any reaction, and completed the present invention.

The present invention provides a compound represented by the general formula (1): NCCH ₂ COOR (1) (wherein R represents an alkenyl group, an aryl group, an aralkyl group, or an alkyl group having 1 to 20 carbon atoms). Wherein the cyanoacetic acid ester is treated with a culture, fungus or treated product of a microorganism belonging to the genus Corynebacterium or Gordona and having nitrilase activity, and hydrolyzed. ): HOOCCH ₂ COOR (2) (wherein, R is as defined above) and a method for producing a malonic acid monoester represented by the formula:

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
In the above general formula (1) or (2), the alkyl group represented by R may have either a straight-chain or branched structure. The alkyl group has 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms.
And more preferably 2 to 6. Specifically, methyl, ethyl, n-propyl, isopropyl, n-butyl,
sec-butyl, tert-butyl, isobutyl, n-pentyl,
Isopentyl, hexyl, octyl, 2-ethylhexyl, decyl, dodecyl, tetradecyl, hexadecyl,
Octadecyl, icosyl and the like are exemplified.

The alkenyl group represented by R may have a linear or branched structure, and preferably has 2 to 6 carbon atoms.
It is. Specific examples include a vinyl group and an allyl group. Examples of the aryl group represented by R include a phenyl group.

Examples of the aralkyl group represented by R include a benzyl group. Among the cyanoacetates represented by the general formula (1), typical compounds include, for example, methyl cyanoacetate, ethyl cyanoacetate, and cyanoacetate.
n-propyl, isopropyl cyanoacetate, benzyl cyanoacetate and the like.

The microorganism used in the present invention is not particularly limited as long as it belongs to the genus Corynebacterium or Gordona and has nitrilase activity. Specifically, Corynebacterium nitrophilus (Corynebacterium nit
rilophilus) ATCC 21419, Gordona t
errae) MA-1 (FERM BP-4535) and the like.

[0012] Among these microorganisms, Gordona terae MA-1 has been deposited with the National Institute of Advanced Industrial Science and Technology under the above deposit number. Corynebacterium nitriophilus is available from the American Type Culture Collection (ATCC).

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 a nitrile compound 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, but it is preferable to perform the culturing under the optimum culturing conditions of 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 belonging to the genus Corynebacterium or Gordona and having nitrilase activity in a medium may be used as it is, or centrifuged from the culture. Can be used. Examples of the treated cells include cells treated with acetone, toluene, etc., crushed cells, cell-free extracts obtained by crushing cells, crude enzymes or purified enzymes separated from cells. The cells or treated cells can be recovered after the reaction by entrapping and immobilizing them in a cross-linked acrylamide gel or by physically and chemically immobilizing them on a solid carrier such as ion-exchange resin or keso earth. It can be reused.

In the present invention, the malonic acid monoester can be produced by the following method. A cyanoacetic acid ester 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. The hydrolysis reaction 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. Reaction temperature is usually 0 to 70 ° C,
The temperature is preferably 10 to 35 ° C., but may be performed at a temperature at which the nitrilase activity of cells and the like increases. The pH of the reaction solution depends on the optimum pH of the microbial enzyme to be used, but it is generally preferable to carry out the reaction within a pH range of 6 to 9, since side reactions due to a chemical hydrolysis reaction can be suppressed. The concentration of the cells or the treated cells in the reaction solution is usually equivalent to 0.01 to 5% by weight as a dry weight. The substrate concentration of the reaction solution is not particularly limited between 0.01 and 70% by weight, but is preferably in the range of 0.1 to 15% by weight.

Further, during the hydrolysis reaction, malonic acid monoester can be accumulated at a high concentration by continuously adding cyanoacetate. At this time, in order to minimize the inactivation of the enzyme by the substrate,
The substrate is added while maintaining the range of 0.01 to 10% by weight, preferably 0.1 to 5% by weight.

After completion of the reaction, the cells of the microorganisms used as the catalyst are removed by centrifugation, filtration or the like.
Unreacted cyanoacetate can be recovered by extraction with a solvent such as hexane or ethyl acetate. After the extraction residue is adjusted to pH 1 to 2 with an acid such as sulfuric acid or hydrochloric acid, the product is extracted with a solvent such as hexane or ethyl acetate to obtain a malonic acid monoester as a product.

[0018]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to Examples, but the scope of the present invention is not limited to these Examples. Example 1 Gordona terae MA-1 (FERM BP-4535) was inoculated into 3 ml of a sterilized LB medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl) and cultured with shaking at 30 ° C. for 24 hours. The following medium A sterilized from 1 ml of the obtained cell culture solution
The cells were inoculated into 100 ml and cultured at 30 ° C. for 48 hours.

[0019] Medium A (pH 7.2) 0.02% Glycerol 1.0% isovaleronitrile 0.2% yeast extract _{KH 2 PO 4 0.1% 0.2%} NaCl MgSO 4 · 7H 2 O 0.02% FeSO 4 · 7H 2 O 10 ppm CoCl 2 · 4H ₂ O 10 ppm CaCl ₂・ 2H ₂ O 1 ppm MnCl ₂・ 4H ₂ O 7 ppm

After completion of the culture, the culture was centrifuged, and the whole amount of the obtained cells was washed with ion-exchanged water and suspended in 100 ml of 50 mM phosphate buffer (pH 7.0). The turbidity of this cell suspension was OD630 = 5.5. 1.00 g of ethyl cyanoacetate was added as a substrate to the cell suspension and reacted at 30 ° C. for 1 hour. The reaction solution was subjected to high performance liquid chromatography (HPLC, column: TSKgel ODS-120A (manufactured by Tosoh Corporation), 4.6 mm ID × 25 cm, mobile phase: 5% acetonitrile, 95% water, 0.1% phosphoric acid, flow rate: 0.5 ml) / min, detection: UV 220 nm), all substrates were converted to monoethyl malonate. After completion of the reaction, the cells were removed by centrifugation, 2N hydrochloric acid was added to adjust the pH to 2.0, and the reaction product monoethyl malonate was extracted with ethyl acetate. The organic layer was dehydrated by adding anhydrous sodium sulfate, and the solvent was distilled off to obtain 1.05 g of monoethyl malonate (yield: 89.9%).

Example 2 1.07 g of monoethyl malonate was obtained in the same manner as in Example 1 except that Corynebacterium nitriophilus ATCC 21419 was used as the microorganism (yield: 91.6%).

Example 3 In the same manner as in Example 1 except that methyl cyanoacetate was used as a substrate, 0.97 g of monomethyl malonate was obtained (yield: 81.4%).

Example 4 1.05 g of mono-n-propyl malonate was obtained in the same manner as in Example 1 except that n-propyl cyanoacetate was used as a substrate (yield: 91.3%).

Example 5 The procedure of Example 1 was repeated except that isopropyl cyanoacetate was used as the substrate.
Was obtained (yield 88.7%).

Example 6 1.06 g of mono-n-butyl malonate was obtained in the same manner as in Example 1 except that n-butyl cyanoacetate was used as a substrate (yield 93.4%). In addition, HP
40% acetonitrile, 60% as mobile phase for LC analysis
Water and 0.1% phosphoric acid were used.

Example 7 1.05 g of mono-t-butyl malonate was obtained in the same manner as in Example 6 except that t-butyl cyanoacetate was used as a substrate (yield 92.5%).

Example 8 The procedure of Example 6 was repeated except that allyl cyanoacetate was used as the substrate to obtain 1.02 g of monoallyl malonate (yield: 87.2%).

Example 9 The procedure of Example 6 was repeated except that 2-ethylhexyl cyanoacetate was used as the substrate.
01 g of mono-2-hexylhexyl malonate was obtained (yield 91.8%).
%).

Example 10 A reaction was carried out in the same manner as in Example 6 except that benzyl cyanoacetate was used as a substrate. After completion of the enzyme reaction, 10% of benzyl cyanoacetate was not reacted. Unreacted benzyl cyanoacetate was extracted and removed with ethyl acetate. After the extraction, 2N hydrochloric acid was added to the aqueous layer to adjust the pH to 2.0, and then the reaction product, monobenzyl malonate, was extracted with ethyl acetate. The organic layer was dehydrated by adding anhydrous sodium sulfate, and the solvent was distilled off.
g of monobenzyl malonate were obtained (80.3% yield).

Examples 11 to 19 Ethyl cyanoacetate was added to the cell suspension obtained in the same manner as in Example 1 by 5 to 50% by weight.
The mixture was added to a concentration and reacted at 25 ° C. for 20 hours. The reaction yield after completion of the reaction was measured by liquid chromatography. Table 1 shows the results.

[0031]

[Table 1]

Example 20 To 100 ml of the cell suspension obtained in the same manner as in Example 1, 5 g of ethyl cyanoacetate was added and reacted at 25 ° C. Thereafter, when the substrate concentration in the reaction solution is 5
Weight while measuring the substrate concentration so that it does not exceed
30 g of ethyl cyanoacetate were added in portions. After 30 hours, the reaction yield was 100%.

[0033]

Industrial Applicability According to the present invention, malonic acid monoesters useful as synthetic intermediates for various chemicals, medicines, agricultural chemicals and the like can be produced with high productivity.

Claims (2)

[Claims] 1. A compound represented by the general formula (1): NCCH ₂ COOR (1) wherein R represents an alkenyl group, an aryl group, an aralkyl group, or an alkyl group having 1 to 20 carbon atoms. Cyanoacetate is used in Coryneb
acterium) or a genus of Gordona, which is characterized by being treated with a culture of a microorganism having nitrilase activity, cells or treated cells, and hydrolyzed.
(2): A method for producing a malonic acid monoester represented by HOOCCH ₂ COOR (2) (wherein R is as defined above). 2. The malonic acid according to claim 1, wherein during the hydrolysis reaction, the cyanoacetic acid ester is continuously added while maintaining the concentration of the cyanoacetate in the reaction solution in the range of 0.01 to 10% by weight. Method for producing monoester.