JPH0799983A - Production of p-nitrobenzyl alcohol malonic acid monoester by bacterium - Google Patents

Abstract

PURPOSE:To efficiently obtain p-nitrobenzyl alcohol malonic acid monoester. CONSTITUTION:P-Nitrobenzyl alcohol cyanoacetic ester is hydrolyzed with a bacterium belonging to the genus Pseudomonas or Rhodococcus to synthesize p-nitrobenzyl alcohol malonic acid monoester without forming p-nitrobenzyl alcohol malonic acid diester at all.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing p-nitrobenzyl alcohol malonic acid monoester by a microorganism.

[0002]

2. Description of the Related Art The production of p-nitrobenzyl alcohol malonic acid monoester, which is a raw material for the production of pharmaceuticals, is aimed at by a chemical method in which p-nitrobenzyl alcohol and malonic acid are heated and dehydrated in the presence of an acid catalyst. It is known that a monoester can be produced.

[0003]

However, in such a method, a diester is produced as a by-product at the same time as the monoester. The diester produced as a by-product is required to be completely removed in order to reduce the product purity of the intended monoester. Further, malonic acid is relatively expensive, and if a large amount of diester is by-produced, it is industrially disadvantageous. Therefore, it is necessary to hydrolyze malonic acid into a monoester, resulting in poor efficiency.

[0004]

From these circumstances, as a result of intensive studies on a more efficient method for producing p-nitrobenzyl alcohol malonic acid monoester, the present inventors have found that p- The desired p-nitrobenzyl alcohol malonic acid monoester can be produced in high purity without any by-product of diester by allowing microorganisms to act on nitrobenzyl alcohol cyanoacetate as a raw material to hydrolyze the cyano group. I found a thing.

That is, the present invention provides (1) a method for producing a p-nitrobenzyl alcohol malonic acid monoester, which comprises allowing a microorganism to act using p-nitrobenzyl alcohol cyanoacetic acid ester as a raw material, and (2)
The microorganism described in (1) is Pseudomonas sp.
Omonas), genus Rhodococcus (Rhodococc)
The method according to (1), which is a bacterium of (us).

The manufacturing method of the present invention will be described in detail. The reaction is the bacterial cells of the cultivated microorganism, or a treated bacterial cell product (crushed bacterial cells, crude / purified enzyme, immobilized bacterial cell / enzyme, etc.)
Is contacted with p-nitrobenzyl alcohol cyanoacetate which is a raw material in a solvent. The amounts used and the reaction conditions will be described in detail below. The reaction solvent is preferably water, but physiological saline,
A buffer can be used to keep the pH of the solvent stable. The type of buffer solution may be phosphate, trisaminomethane-HCl, citrate, glycine soda, etc., but is not particularly limited. The concentration of the buffer solution may be 1M to 0.001M, but may be within the range where the effect as the buffer solution is exhibited. When water is used, the reaction is promoted by adding a metal chelating agent such as EDTA. Further, when using the treated product of the above-mentioned bacterial cells, the addition of an antioxidant such as dithiothreitol or mercaptoethanol enhances the reaction efficiency. Others, water and methanol, ethanol, acetone,
A mixed solvent such as acetonitrile can also be used.
The pH should be kept at pH 5-8. Reaction temperature is 0 ℃
It is good to carry out in the range of -80 ° C, preferably 5 ° C-40 ° C. The reaction time requires about 30 minutes to 3 days depending on the concentration of the raw material. The starting material, p-nitrobenzyl alcohol cyanoacetic acid ester, is unstable in neutral to alkaline water, and it is desirable that the reaction be completed in a short time. The concentration of p-nitrobenzyl alcohol cyanoacetic acid ester as a raw material is 0.1% to 10% (% by weight) with respect to the solvent,
It is preferable to set it to 0.3% to 2% to obtain good results. After completion of the reaction, take out p-nitrobenzyl alcohol malonic acid monoester dissolved in water as a salt by acid precipitation or extraction with an organic solvent such as hexane, toluene, benzene, ethyl acetate, diethyl ether or chloroform. You can Cultivation of the microorganism used in the present invention can be carried out according to known culture methods.
As the medium, a normal medium containing a carbon source such as assimilable glucose and glycerin, a nitrogen source such as ammonium sulfate, inorganic phosphorus, and inorganic nutrients essential for growth can be used. Further, it is also possible to use a medium to which a natural medium such as yeast extract or meat extract is added. Isovaleronitrile, benzonitrile, p-nitrobenzonitrile, nitriles such as cyanoacetic acid, amides such as ε-caprolactam, dimethylacetamide, and nicotinamide, and iron, cobalt at a concentration that does not significantly inhibit growth from the early stage to the middle stage of culture, High enzyme activity may be obtained by adding a metal such as manganese or zinc as an enzyme inducer. The medium to be used may have a pH of 5 to 9 and a culture temperature of 5 to 40 ° C., preferably 25.
~ 35 ° C. The culture may be aerobically carried out for about 1 to 7 days and may be continued until the desired enzyme activity is maximized.

The microorganism used in the present invention is, for example, a novel Pseudomonas bacterium NKN.
Bacterial 003 (Deposit No. FERM P-13850
No.) and a new genus Rhodococcus
s) Bacteria NKN113 bacteria (Ministry of Engineering Research Deposit No. FERM
P-13865). All of these have been deposited at the Institute of Microbial Technology, Institute of Industrial Technology. The characteristics of these two bacteria are shown in Table 1 below.

[0008]

[Table 1]

Based on the above-mentioned mycological properties, BERGEY'S M
ANUAL of Systematic Bacte
As a result of examining the known strains and their differences according to riology Volume 1 and 2, the above-mentioned 2 strains were as follows.

NKN003 bacterium (Ministry of Industrial Science, Deposit No. FER
(MP No. 13850) is a bacterium which is negative for Gram's staining, shows motility, and has polar hairs as flagella and is an aerobic, non-auxotrophic bacillus that produces a fluorescent dye. The characteristics of this bacterium are Pseudomonas (Ps
eudomonas), the size of the somatic cells of this strain is a little small, and except that the acid is generated from D-galactose, it is almost the same as the P. putida species in the above-mentioned document. From the above points, this bacterium was estimated to be a species closely related to P. putida species.

NKN113 bacteria (Ministry of Industrial Research Deposit No. FER
MP-13865) is Gram-staining positive, non-motile, polymorphic, aerobic, catalase-plus bacillus,
Furthermore, since it has a GC content of 69.1% in the DNA and has diaminopimelic acid and mycolic acid in the cell wall components, it belongs to the genus Rhodococcus. Further, the nitrate reduction plus, no acid is generated from sugar, and the above-mentioned test results are in agreement with the description of Rhodococcus equi (R. equi) species in the above document. From the above points, this bacterium was presumed to be related to Rhodococcus equi (R. equi). The p-nitrobenzyl alcohol malonic acid monoester produced by the present invention is used as a protective group in the production of drugs such as carbapenem antibacterial agents.

[0012]

The present invention will be described with reference to the following examples. All percentages are by weight unless otherwise specified. Further, p-nitrobenzyl alcohol cyanoacetic acid ester is used as a raw material, and p-nitrobenzyl alcohol malonic acid monoester is abbreviated as a target product. It should be noted that no diester was formed at all in all the examples. Example 1 Glycerin 1%, Polypeptone 0.5%, Malto Extract 0.3%, Yeast Extract 0.3
20% well water sterilized by autoclave
Add 0.1% isovaleronitrile to ml and add NKN0
03 bacteria were inoculated and the culture was started at 30 ° C. On the second day of culture, 0.2% isovaleronitrile was added, and on the third day of culture, cells were obtained by centrifugation. This fungus body has a pH of 6.5,
Wash with 0.01 M potassium phosphate buffer, pH 6.0,
Suspended in 3 ml of 0.1 M potassium phosphate buffer, 6 mg
Was added and shaken at room temperature for 2.5 hours. When analyzed by liquid chromatography, the amount of the produced target product was 6.2 mg. The amount of p-nitrobenzyl alcohol produced by the decomposition of p-nitrobenzyl alcohol cyanoacetic acid ester as a raw material was as small as 0.02 mg. Furthermore, no unreacted raw material remained. The purity by liquid chromatography is 99.7%,
The melting point was 107-110 ° C. In the 1H-NMR spectrum, 3.55 ppm is the absorption based on methylene hydrogen of malonic acid, 5.30 ppm is the absorption based on hydrogen of benzylmethylene, and 7.5 to 7.6 is the aromatic hydrogen at the ortho position of the nitro group. Doublet absorption of aromatic hydrogen at the meta position of the nitro group was observed in the doublet of 8.2 to 8.3.

Example 2 To 20 ml of the same medium as in Example 1, 0.1% isovaleronitrile was added, and NKN113 bacteria were cultured at 30 ° C.
On the 3rd day of culture, 0.2% isovaleronitrile was added, and on the 4th day of culture, cells were obtained by centrifugation. When the reaction was carried out in the same manner as in Example 1 and analyzed, the target product produced was 5.3.
It was mg. The amount of p-nitrobenzyl alcohol produced by the decomposition of p-nitrobenzyl alcohol cyanoacetic acid ester as a raw material was 1.1 mg, which was very small. Furthermore, no unreacted raw material remained in the solvent.

Example 3 To 20 ml of the same medium as in Example 1, 0.15% isovaleronitrile was added, NKN003 was cultured at 30 ° C., and 0.15% isovaleronitrile was added on the first day of the culture. Then, on the second day of culture, the bacterial cells were obtained by centrifugation. The cells were suspended in 3 ml of 0.1 M potassium phosphate buffer having a pH of 6.5, 29.6 mg of the starting material was added, and the mixture was shaken at 27 ° C. for 4.5 hours. When analyzed in the same manner as in Example 1, the amount of the target product produced was 27.2 mg. The amount of p-nitrobenzyl alcohol was as small as 1.4 mg, and other products and raw materials were contained only in trace amounts. This was treated with activated carbon and centrifuged, and concentrated hydrochloric acid was added to the supernatant to adjust the pH to 1.
When set to 6, white sediment was generated. This is filtered and dried to 2
6.0 mg of p-nitrobenzyl alcohol malonic acid monoester was obtained.

Example 4 20% of the same sterilizing medium as in Example 1 was supplemented with 0.1% isovaleronitrile to cultivate NKN003 at 30 ° C., and on the first day of culture, 0.1% isovaleronitrile was added. Was added. On the second day of culture, cells were obtained from 10 ml of this culture medium by centrifugation. The cells were suspended in 20 ml of a 0.2 M phosphate buffer of pH 7.5 containing 10 mM of EDTA, 419 mg of the starting material was added, and the mixture was shaken at room temperature for 1 hour and then left standing at room temperature. When analyzed in the same manner as in Example 1 after 3 days, the amount of the target product produced was 237 mg. In addition, p-nitrobenzyl alcohol produced by decomposition of the raw material was 53 mg, malonic acid (p-nitrobenzyl alcohol) ester-amide as an intermediate product was 75 mg, and unreacted raw material was 55 mg. Insoluble matter is separated by centrifugation, and hydrochloric acid is added to the filtrate to adjust the pH to about 2
And a precipitate was formed. The precipitate was collected by filtration, washed with water and dried to obtain 225 mg of P-nitrobenzyl alcohol-malonic acid monoester. The purity by liquid chromatography was 96.3%. The crude product was suspended in water, ammonia water was gradually added to dissolve it near neutrality, treated with activated carbon, and then acidified with hydrochloric acid to a pH of about 2.0 to purify the product. It was possible to obtain a high-purity product.

Example 5 Glucose 2%, KH ₂ PO ₄ 0.1%, K ₂ HPO ₄
100 μM each in well water containing 0.4%, (NH ₄ ) ₂ SO ₄ 0.26%, and yeast extract 0.03%.
0.1% isovaleronitrile was added to 20 ml of sterilized medium containing ZnCl ₂ , FeSO ₄ , and MnSO ₄ , and NKN003 was cultivated at 30 ° C. and 0.
1% isovaleronitrile was added. On the second day of culture, cells were obtained from 10 ml of this culture medium by centrifugation. The suspension was suspended in 20 ml of the same buffer as in Example 4, 793 mg of the starting material was added, and the mixture was stirred at room temperature for 3 days to give 493 mg.
The desired product was produced. Further, p-nitrobenzyl alcohol was 109.8 mg, malonic acid (p-nitrobenzyl alcohol) ester-amide was 186.4 mg, and unreacted raw material was 9.3 mg. The target compounds obtained in Examples 2 to 5 also showed the same physicochemical data as the target compounds obtained in Example 1.

[0017]

EFFECTS OF THE INVENTION By allowing p-nitrobenzyl alcohol cyanoacetic acid to act on a bacterium belonging to the genus Zhuodomonas or Rhodococcus, p-nitrobenzyl alcohol malonic acid monoester can be efficiently synthesized.

Claims (2)

[Claims] 1. P-nitrobenzyl alcohol cyanoacetate is used for Pseudomonas.
s) or a bacterium belonging to the genus Rhodococcus (Phodococcus) is hydrolyzed by the action thereof to produce a p-nitrobenzyl alcohol malonic acid monoester. 2. The genus Pseudomonas
The method according to claim 1, wherein the bacterium of as) is NKN003 and the bacterium of Rhodococcus is NKN113.