JPH0220290A - Production of trans-4-cyanocyclohexanecarboxylic acid amide - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a novel method for producing trans-4-cyanocyclohexanecarboxylic acid amide. The object of the present invention is to industrially produce trans-4-cyanocyclohexanecarboxylic acid amide represented by the following formula (II) N 0NH2, which is useful as a raw material for producing trans-4-aminomethylcyclohexanecarboxylic acid amide having an antiulcer effect. , and to produce it in high yield.

(Prior art) Regarding 4-cyanocyclohexanes,
14506, 1,4-dicyanocyclohexane represented by the following formula (bi)CN is hydrolyzed with aqueous ammonia to obtain the following formula (Ill)CN OR (wherein, R represents a hydroxyl group or an amino group). There is a description of a method for producing a mixed solution containing 4-cyanocyclohexanecarboxylic acid and 4-cyanocyclohexanecarboxylic acid amide represented by:

The present inventors also obtained a compound of formula (II) by reacting trans-4-cyanocyclohexanecarboxylic acid amide represented by the following formula (IV) with ammonia in a suitable solvent.

(Japanese Patent Application No. 61-180035) (Problems to be Solved by the Invention) All conventional manufacturing methods use synthetic techniques, and therefore require high temperature and high pressure reaction conditions. Furthermore, since many by-products are produced, the process of isolating and producing the target product is extremely complicated. Furthermore, in conventional production methods, it is difficult to produce trans-4-cyanocyclohexanecarboxylic acid amide from trans-form raw materials while maintaining the steric configuration.

(Means for Solving the Problems) As a result of intensive studies to solve the above problems, the present inventors have found that it is possible to maintain the steric configuration at room temperature and normal pressure, and to achieve extremely efficient We have discovered a new manufacturing method that utilizes the biochemical effects of microorganisms. That is, this is a method for producing trans-4-cyanocyclohexanecarboxylic acid amide represented by the following formula (1) from trans-1,4-dicyanocyclohexane represented by the following formula (1) by the action of microorganisms.

Generally, the biochemical action, ie, enzyme, that converts nitrile groups into carboxamide groups is known as nitrile hydratase. [Fermentation and Industry, Volume 41, 382
-388 pages (1983)] However, no reaction is known at all for converting a nitrile compound containing a cyclohexane ring into the corresponding carboxylic acid amide as a substrate. Furthermore, it is also completely unknown to convert only one of the two nitrile groups of the compound of formula (1) into a carboxamide group while maintaining the steric configuration.

Therefore, the present inventors converted the compound of formula (1) into
We searched soil isolates and publicly preserved strains for microorganisms that have the ability to convert into compounds, discovered microorganisms that have this conversion ability, and completed the present invention.

Next, a method of implementing the present invention will be explained.

■Bacterial strains used The microorganisms used in the present invention belong to the genera Corynebacterium, Rhodococcus, Pseudomonas, and Rhodopseudomonas.Specific strains belonging to these genera include Corynebacterium sp. C5 isolated from soil
No. 31), and publicly preserved strains Rhodococcus sp. ATCCI9070, Pseudomonas marginalis I FO3925, and Rhodopseudomonas sphaeroides ATCC11167. The soil distribution strain 05 has been deposited with the above-mentioned number at the Institute of Fine Technology, and its mycological properties are as shown below.

(a) Morphology 1, cell shape and size; bacilli, 0°4-0°6X2
．． 5-6.0μm02, Presence or absence of cell polyplasia; exhibits long culm shape (1 day), develops with snapping, and later (3 days) shows short culm shape (0,4-0 .6 x 0.6-12 μm).

3. Presence or absence of motility; No 4. Presence or absence of spores; No 5. Duram staining; + 6. Anti-acidity; Round,
Smooth, dry, hemispherical, white to pale pink.

2. Juicy agar slant culture; medium growth, smooth, slightly dry,
White or pale pink.

3. Meat juice liquid culture; vigorous growth, moderate turbidity, and precipitate formation with growth.

4. Meat juice gelatin puncture culture: Grows along the spine, no liquefaction.

5. Lidmus milk; no change (c) Physiological properties ■. Reduction of nitrate; - 2. Denitrification reaction; 3. MR test; 4. VP test; 5. Production of indole; 6. Production of hydrogen nitride; 7. Hydrolysis of starch; - 8. Utilization of citric acid (a) Kosar's medium; (b) Christensen's medium - 19. Utilization of inorganic nitrogen sources (a) Nitrate; + (b) Ammonium salt; + 10 , production of pigment (a) King A medium: (b) King B medium; growth, 11, urease; + 12, oxidase; 13, catalase; ] Temperature: O~37°C 15. Attitude towards oxygen: Aerobic 16.0-F test: ~ 17. Production of acids and gases from W types 7 Assimilation 18.10
% skim milk at 72°C for 115 minutes; no 19, ornithine decarboxylation; +20, lysine decarboxylation;
's Manual of Determinative
e Bacteriology (8th edition) and Manual of Clinical Microbiology (Ma.
nual of C11nical old c'r.

biolog V) 4th edition (1985).

Strain 05 is an aerobic, Durum-positive, catalase-positive bacillus that does not produce endospores, does not attach flagella, and initially grows in a long culm shape with snapping, and later develops into a short culm shape. It is clear from the fact that it belongs to coryneform bacteria because of the rupture. In addition, it does not have cellulose separation ability, Durham staining is not barrier pull, it does not have heat resistance to 10% skim milk at 72 ° C for 115 minutes, it is not absolutely aerobic, and the OF test is -.
It is clear that strain C5 belongs to the genus Corynebacterium.

The seeds of strain 05 grow at 37°C, and although they are aerobic in their attitude toward oxygen, they are also facultatively anaerobic, with O-
F test is -, the ability to assimilate glucose is weak, assimilates D-fructose, sucrose, D-mannite and ethanol, has no nitrate reducing ability, does not produce hydrogen sulfide, and does not assimilate citric acid in Cosar medium. First, since it has urease activity and catalase activity, judging from the classification documents mentioned earlier, there are no corresponding species. Therefore, strain 05 is considered to be a new species of the genus Corynebacterium.

■Reaction method In the present invention, the reaction method for converting the compound of formula (1) into the compound of formula (II) includes a method of culturing the microorganism in the presence of the compound of formula (1), and a method of culturing the microorganism in the presence of the compound of formula (1); A culture, bacterial cells or a bacterial cell treatment product collected therefrom (for example, a crushed bacterial cell product, a bacterial cell treated product with an organic solvent, or an enzyme separated and extracted from the bacterial cells), and a compound of formula (1). There are two methods of contact. Alternatively, the bacterial cells, the treated bacterial cells, or the enzyme extracted from the bacterial cells may be immobilized by a known method such as celite, calcium alginate, carrageenan, etc., and then reacted with the compound of formula (I).

■Culture method The microorganisms used in the present invention can be cultured according to known methods. The culture medium to be used can be one known as a nutrient source for general microorganisms, such as blackstrap molasses, a carbon source such as glucose, glycerin, ethanol, or sucrose, a nitrogen source such as ammonium sulfate or urea, or ammonium chloride, meat extract, or yeast extract. , malt extract, peptone, and other organic nutritional sources, phosphoric acid, magnesium, potassium, iron, cobalt, and other organic nutritional sources, and vitamins can be used in appropriate combinations.

Furthermore, a cyano compound such as acetonitrile or trans-1,4-dicyanocyclohexane may be added as a substance that promotes the conversion activity of the microorganism from the compound of formula (1) to the compound of formula (IT). The pH of the medium may be selected within the range of 5 to 10, and the culture temperature is preferably 18 to 38°C, preferably 25 to 32°C. The culture may be carried out for a period of 1 to 8 days until the activity reaches its maximum.

(2) Reaction conditions (2) As the reaction medium, an aqueous medium such as water or a buffer solution, or a mixture of water and an aqueous solvent such as methanol, chloroform, or methylene chloride can be used. Into the reaction medium, the formula (
The compound of 1) is added as a powder or dissolved in a suitable solvent. The concentration of the compound of formula (1) added is 0.0
It is about 1 to 60% by weight, preferably 1.0 to 20% by weight. When using bacterial cells in the reaction, the concentration of bacterial cells may generally be in the range of 0.1 to 8% by weight. Reaction temperature is 4-50°
C1 desirably 15-32°C1 reaction pH is 4-11
, preferably 6°5 to 9.0. The reaction time is usually
An appropriate time may be selected within the range of 0.5 to 100 hours. The consumed compound may be added continuously or intermittently so that the concentration in the reaction solution is maintained within the above range.

■Separation and purification The produced compound of formula (II) can be easily obtained as highly pure crystals by a known method such as solvent extraction or crystallization after removing insoluble matter such as bacterial cells from the reaction completed liquid. be able to.

(Effects of the Invention) By utilizing the present invention, trans-4-cyanocyclohexanecarboxylic acid amide can be produced at a high concentration under reaction conditions of room temperature and normal pressure. Furthermore, since it can be manufactured while maintaining its steric configuration, it is extremely useful economically.

(Example) Next, the present invention will be explained with reference to Examples, but the present invention is not limited to these Examples in any way.

Example 1 Meat extract 1.0g, peptone 1.0g. Og, glucose 1.
0 g, trans-1,4-dicyanocyclohexane 0.
5g, dipotassium phosphate 0.2g, thorium chloride 0.
1g, magnesium sulfate 0.02g, biotin hydrochloride 1
100 m of sterilized medium containing μg and pH 7.5
Rhodococcus sp. ATCC 19070, which had been previously cultured in the same medium, was inoculated into g. and cultured with shaking at 30°C for 3 days. After the bacterial cells are removed from the culture solution by centrifugation, caustic soda is added to the supernatant to adjust the pH to 12.5, and then 100 mf of chloroform is added to perform extraction. The chloroborum layer was then concentrated to give trans-4-
Cyanocyclohexanecarboxylic acid amide crystal 0.12
I got g.

The main island showed a single peak in high performance liquid chromatography analysis.

The melting point, TR, and NMR1 elemental analysis are shown below, which support the structure of the target product.

Melting point 189-191°C IR (KBr) 3355 cm-' (7=NI-1) 3200 cm-' (7-NH) 2220 cnr' Cr=CH) 1665 mark-' (r=co) NMR (MeOH-d', DMSO-d6)δ; 1
．． 2~3.0 (IOHlm) Elemental analysis theoretical value Analysis value C63,16% 63.00% T-17,89% 7.95% N18.42% 18.50% 010.53% 10.55% High performance liquid chromatography analysis was performed as follows. Analyzer: Waters 6000A pump, Tosoh R1-8 differential refractometer, column: Tsubabac C-
18 (manufactured by Waters), solvent; water/acetonitrile = 9515 (volume ratio) -1-PI C84 (manufactured by Waters) 1.5% by volume.

Example 2 Meat extract 0.1g, peptone 0.1g, glucose 0.
1g, potassium phosphate 0.01g, thorium chloride 0
．． Contains 0.01g, magnesium sulfate 0.005g, ferrous sulfate 0.001g, ammonium sulfate 0.01g, p
Corynebacterium sp. 05 strain, which had been previously cultured in the same medium, was inoculated into 10 mρ of a sterilized medium containing H of 7.0, and cultured at 30°C for 40 hours. After culturing, the bacterial cells were collected by centrifugation, suspended in an Erlenmeyer flask containing 10% water (1+ffi), and trans-1,4-
20 g of dicyanocyclohexane was added and reacted at 30°C with shaking.

After 8 hours, the reaction was terminated and high performance liquid chromatography analysis revealed that 16.4 g of trans-4-cyanocyclohexanecarboxylic acid amide had been produced.

(Conversion rate 72.4%) Hereinafter, the same operation as in Example 1 was performed to obtain 14.0 g of crystals of trans 4-cyanocyclohexanecarboxylic acid amide. (Yield 61.9%) The physical property data of the wood product supported the structure of the target product as in Example 1.

Example 3 Pseudomonas marginalis was grown in the same medium as in Example 1.
After inoculating IF03925 and culturing at 30°C for 3 days, the same post-treatment as in Example 1 was performed to obtain 0°08 g of trans-4-cyanocyclohexanecarboxylic acid amide.

Example 4 Rhodopseudomonas sphaeroides ATCC 11167 was inoculated into the same medium as in Example 1, and after culturing at 32°C for 3 days, the same post-treatment was performed and 0.15 g of trans-4-cyanocyclohexanecarboxylic acid amide was obtained. I got it.

Claims (1)

[Claims] The following formula (I) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Trans-1,4-dicyanocyclohexane represented by (I) is expressed as the following formula (II) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (II) The production of trans-4-cyanocyclohexanecarboxylic acid amide from trans-1,4-dicyanocyclohexane by the action of a microorganism having the ability to convert it into trans-4-cyanocyclohexanecarboxylic acid amide shown by ▼(II) A characterized method for producing trans-4-cyanocyclohexanecarboxylic acid amide.