JPH10229891A - 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 3-20C alkyl group) is treated with the cultured product, a cell or a cell treated material of a microorganism 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]

DISCLOSURE OF THE INVENTION The present inventors have found that when a culture, a microbial cell or a processed cell of a microorganism having nitrilase activity is allowed to act on cyanoacetate, malonic acid monoester is selectively formed. As a result, the inventors have found that a high-purity malonic acid monoester can be produced without side reactions such as hydrolysis of an ester bond, and the present invention has been completed.

The present invention is 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 3 to 20 carbon atoms. General formula (2): HOOCCH ₂ COOR (2) wherein the cyanoacetic ester is treated with a culture, fungus body or treated product of a microorganism having nitrilase activity and hydrolyzed. R is as described above.) The 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 3 to 20 carbon atoms, preferably 3 to 10 carbon atoms.
And more preferably 3 to 6. Specifically, 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.

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, n-propyl cyanoacetate, isopropyl cyanoacetate,
N-butyl cyanoacetate, tert-butyl cyanoacetate, 2-ethylhexyl cyanoacetate, allyl cyanoacetate, benzyl cyanoacetate and the like.

The microorganism used in the present invention is not particularly limited as long as it has nitrilase activity.
Rhodococcus sp., Pseudomonas (Pseud
omonas), Brevibacterium,
Genus Nocardia, Arthrob
acter) genus, Bacillus genus, Escherichia (Esche
richia), Micrococcus, Streptomyces, Aeromonas
as), Mycoplana, Cellulomonas (C
Microorganisms belonging to the genus ellulomonas, the genus Erwinia, the genus Candida, and the like, and include microorganisms having nitrilase activity.

More specifically, Rhodococcus rhodochrous ATCC 33025, Pseudomonas synxanta IAM 123
56, Brevibacterium acetylicum
macetyl) IAM 1790, Nocardia asteroides IFO 3384, Arthrobacter oxydans IFO 12138, Bacillus subtilis ATCC 21697,
Escherichia coli IFO 3301, Micrococcus luteus ATCC 38
3, Streptomyces griseus (Streptomyces gr
iseus) IFO 3355, Aeromonas Punctata
as punctata) IFO 13288, mycoplana dimorpha (M
ycoplanadimorpha) ATCC 4297, Cellulomonas fimi
(Cellulomonas fimi) IAM 12107, Erwinia herbicola IFO 12686, Candida
Gillian Mondi (Candida guilliermondii) IFO 056
6 and the like. These microorganisms are known as American Type Culture Collection (ATCC), Institute of Applied Microbiology (IAM), University of Tokyo, and Fermentation Research Institute (I
FO).

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 having nitrilase activity as described above in a culture medium as it is, or a cell obtained by collecting cells from the culture by centrifugation or the like, Alternatively, a treated bacterial cell 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] Rhodococcus rhodochrous ATCC 33025
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. One ml of the obtained cell culture was inoculated into 100 ml of the following sterilized medium A 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 = 5.6. To this cell suspension was added 1.00 g of n-propyl cyanoacetate as a substrate 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,
All the substrates were converted to mono-n-propyl malonate when analyzed by (detection: UV 220 nm). After the reaction,
Remove the cells by centrifugation, add 2N hydrochloric acid, and adjust the pH.
It was adjusted 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 1.06 g of mono-n-propyl malonate (yield: 89.9%).

[Example 2] 1.01 g in the same manner as in Example 1 except that isopropyl cyanoacetate was used as a substrate.
Was obtained (yield 88.6%).

Example 3 1.05 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 93.3%). In addition, HP
40% acetonitrile, 60% as mobile phase for LC analysis
Water and 0.1% phosphoric acid were used.

Example 4 1.04 g of mono-t-butyl malonate was obtained in the same manner as in Example 3 except that t-butyl cyanoacetate was used as a substrate (yield 92.4%).

Example 5 1.04 g of monoallyl malonate was obtained in the same manner as in Example 3 except that allyl cyanoacetate was used as a substrate (yield: 87.1%).

Example 6 The procedure of Example 3 was repeated except that 2-ethylhexyl cyanoacetate was used as the substrate.
00 g of mono-2-hexylhexyl malonate was obtained (yield 91.7%).
%).

Example 7 A reaction was carried out in the same manner as in Example 3 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 evaporated by evaporation.
g of monobenzyl malonate was obtained (80.2% yield).

Examples 8 to 16 n-Propyl cyanoacetate was added to the cell suspension obtained in the same manner as in Example 1 so as to have a concentration of 5 to 50% by weight. Allowed to react for hours. The reaction yield after completion of the reaction was measured by liquid chromatography. Table 1 shows the results.

[0028]

[Table 1]

Example 20 To 100 ml of the bacterial cell suspension obtained in the same manner as in Example 1, 5 g of n-propyl cyanoacetate was added and reacted at 25 ° C. Thereafter, a total of 30 g of ethyl cyanoacetate was dividedly added while measuring the substrate concentration so that the substrate concentration in the reaction solution did not exceed 5% by weight. After 30 hours, the reaction yield was 100%.

[0030]

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 (3)

[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 3 to 20 carbon atoms). General formula (2): HOOCCH ₂ COOR (2) wherein the cyanoacetic ester is treated with a culture, fungus body or treated product of a microorganism having nitrilase activity and hydrolyzed. R is as described above.). 2. The method for producing a malonic acid monoester according to claim 1, wherein the microorganism having nitrilase activity is a microorganism belonging to the genus Rhodococcus. 3. The method according to claim 1, wherein the ester is continuously added during the hydrolysis reaction while maintaining the concentration of the cyanoacetate in the reaction solution in the range of 0.01 to 10% by weight. Method for producing malonic acid monoester.