JPH0673464B2 - Process for producing optically active cis-imidazolidinedicarboxylic acid derivative - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention has the general formula (I) (In the formula, R represents a lower alkyl group. Bzl represents a benzyl group. The 4- and 5-positions are 4S and 5R coordinates.) An optically active cis-imidazolidinedicarboxylic acid The present invention relates to a method for producing a derivative.

That is, the invention has the general formula (II) (In the formula, R represents a lower alkyl group. Bzl represents a benzyl group.) The cis-imidazolidinedicarboxylic acid diesters (hereinafter abbreviated as diesters) include a alkalophilic Bacillus genus. By connecting an esterase derived from an esterase-producing bacterium belonging to the genus Bacillus or an esterase-producing bacterium belonging to the genus Bacillus including alkalophilic Bacillus, the optically active cis-imidazolidinedicarboxylic acid derivative represented by the general formula (I) (hereinafter , Abbreviated as half-esters).

In the general formulas (I) and (II), the lower alkyl group includes methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, n-hexyl groups and the like.
Means C ₁ -C ₆ alkyl group.

The optically active hearth esters (I) obtained by the present invention are extremely important compounds as synthetic intermediates for biotin and other pharmaceuticals.

Conventionally, as a method for producing optically active half esters (I) by microorganisms, Chromobacter (Chromobacter
Only those belonging to the genus ium (Japanese Patent Laid-Open No. 190395/1983) were known.

Therefore, the present inventors have diligently studied a method for producing an optically active half ester (I) from a diester (II) using a microorganism newly isolated from the natural world. As a result, when an esterase-producing bacterium belonging to the genus Bacillus including the alkalophilic Bacillus genus or an esterase derived from an esterase-producing bacterium belonging to the genus Bacillus including the alkalophilic Bacillus is used, the optically active half-esters (I) are The inventors have found that they can be obtained and completed the present invention.

In the method of the present invention, the alkalophilic Bacillus (Baci
The esterase derived from an esterase-producing bacterium belonging to the genus Bacillus including the genus llus) or an esterase-producing bacterium belonging to the genus Bacillus including the alkalophilic Bacillus is hydrolyzed by contacting it with a diester (II). At this time, the reaction proceeds in good yield under mild conditions, and the target compound half ester (I) of the present invention can be obtained.
Moreover, the semi-hydrolysis reaction proceeds stereoselectively or specifically.

That is, it is considered that two types of optical antipodes are produced during semihydrolysis of the diesters (II) which are meso isomers. However, when the method of the present invention is used, one of the two types The so-called asymmetric hydrolysis occurs in which only the palm body is excessively produced, and in some cases, only one type of antipode is produced.

The half-esters (I) selectively obtained by the method of the present invention are those having 4S, 5R coordination and have an optically active d
-Can be converted to biotin. For example, an example thereof is described in JP-A-58-190395.

The microorganism used in the present invention is Bacillus sp. VA-47 strain belonging to the genus Bacillus.
No. 8730) or alkalophilic Bacillus sp. VA-62
Strains (Microtech Lab. No. 8729) and irradiation of the microorganisms with ultraviolet rays, X-rays, γ rays, etc., chemical mutation treatment agents, phage contact, transformation, transduction, gene recombination by conjugation, genes by cell fusion Incorporates artificial mutants or natural mutants obtained by mutation treatment such as recombination and gene transfer using plasmids, and converts diesters (II) belonging to the genus Bacillus to optically active half-esters (I) The strain is generally incorporated.

The above-exemplified strains are all new strains, and the main mycological properties are as follows.

(A) Morphological properties (broth agar medium and broth medium) (B) Growth conditions in various media (1) Meat agar plate culture (30 ° C, 2 days) (2) Beef broth agar slope culture (30 ℃, 2 days) (3) Broth liquid culture (30 ℃, 3 days) (4) Meat broth gelatin stab culture (22 ℃ 1 month) (5) Litmus milk culture (30 ℃, 1 month) (C) Physiological properties As shown in the above bacteriological properties, these two strains are spore-forming bacilli that grow under aerobic conditions, and therefore, the Barjays Manual of Determinative Bacteriology (Bergey ’S Manual of Determin
It is clear that the bacterium belongs to the genus Bacillus according to the search formula of the 8th edition. Further, when comparing the strains of the same genus with the VA-47 strain, similar strains include Bacillus fastidiosus (Bacillus fastidiosus) and Bacillus alkalophilus (Bacillus alkalophilus). VA-47 with a diameter of 1.5 to 2.5 μm and a length of 3 to 6 μm
It differs from the VA-47 strain in that it is larger than the strain and lacks the ability to reduce nitrate. Bacillus alcalophilus is VA-47 because it is alkalophilic, does not grow at pH 7, and has no nitrate reducing ability.
Different from stocks.

In addition, when compared to strains of the same genus as the VA-62 strain, similar bacteria include Bacillus brevis and Bacillus alkalophilus, but Bacillus brevis has the highest growth temperature.
No starch hydrolyzing power at 40-60 ℃, and salt resistance is NaCl5
It differs from the VA-62 strain in that it is at most%. Bacillus alcalophilus grows only in alkaline medium.
Although it is the same as the VA-62 strain, it differs from the VA-62 strain in that it does not form spores with swelling and has no nitrate reducing ability.

Based on the above, the present inventors recognized the VA-47 strain and the VA-62 strain as new strains belonging to the genus Bacillus and named them Bacillus sp. VA-47 and Bacillus sp. VA-62. These two strains are available under the Accession No.
Deposited as No. 29.

As the medium for culturing the microorganism used in the present invention, a carbon source, a nitrogen source, which is commonly used for culturing microorganisms that is usually performed
Various media containing inorganic substances and the like can be used. Examples of the carbon source of the medium include glucose, fructose, sucrose, starch, glycerin, molasses and other carbohydrates, gluconic acid, pyruvic acid, acetic acid, citric acid,
Organic acids such as, glycine, glutamic acid, alanine,
Examples include amino acids such as asparagine and hydrocarbons such as n-paraffin. Examples of the nitrogen source include meat extract, peptone, yeast extract, dry yeast, taste liquid, soybean powder,
Or its hydrolysates, casein or its hydrolysates, various amino acids, various ammonium salts, nitrates and the like. Examples of inorganic substances are phosphates or salts of magnesium, potassium, calcium, sodium, iron, manganese and the like.

Culturing is usually carried out aerobically, and shaking culture or aeration-agitation culture are preferred. The appropriate number of days for culture is usually from half a day to 7 days. The culture temperature of Bacillus sp. VA-47 strain is usually preferably 25 to 37 ° C, and the pH of the culture solution is 6.5 to 8
A degree is preferable. The culture temperature of Bacillus sp. VA-62 strain is preferably 20 to 30 ° C, and the pH of the culture solution is preferably 8 to 10.

In order to obtain the optically active half-esters (I) by culturing this strain, the method of adding the diesters (II) to the culture solution from the beginning or after the growth, continuing the culture, and conducting the half-hydrolysis is the most preferable method. It's simple. Substrate concentration is 0.1-10%
The degree is appropriate. In order to isolate and purify the optically active half-esters (I) accumulated in the culture, the isolation and purification means usually used for the isolation and purification of organic compounds may be applied.

The optically active half-esters (I) are also obtained by a method of semi-hydrolyzing using microbial cells collected from a culture solution of the strain by centrifugation or filtration, or by sonicating or lysozyme treatment of microbial cells. It is also possible to perform a method in which the esterase is released by freezing, centrifugation is performed under cooling, ammonium sulfate fractionation, precipitation dialysis, and the like, and half-hydrolysis is performed using the esterase. In these cases, the diesters (II) are suspended or dissolved in an aqueous solution or a buffer solution preferably adjusted to pH 6 to 9, and the microbial cells or esterases are preferably added to the diesters (II) in an amount of 0.001 to The reaction proceeds by adding 0.1 weight ratio and stirring at preferably 10 to 40 ° C., and the reaction is usually completed in 1 hour to several days.

As mentioned above, the aqueous solution is sulfuric acid, hydrochloric acid, mineral acid such as phosphoric acid, acetic acid,
Organic acids such as citric acid, inorganic bases such as sodium hydroxide, potassium hydroxide and sodium carbonate, triethylamine,
It means an aqueous solution to which an organic base such as pyridine and a salt such as sodium acetate and sodium chloride are added.

The buffer solution is potassium dihydrogen phosphate / sodium hydroxide, potassium dihydrogen phosphate, sodium monohydrogen phosphate,
It means a general buffer solution such as potassium hydrogen phthalate / hydrochloric acid, glycine / sodium chloride / sodium hydroxide, etc., and there is no particular limitation as long as it does not interfere with the reaction.

If necessary, an alcohol solvent such as methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, an ether solvent such as ether, tetrahydrofuran, dioxane, benzene,
Aromatic hydrocarbon solvents such as toluene, acetone, methyl ethyl ketone, ketone solvents such as diethyl ketone, amine solvents such as triethylamine and pyridine, polar solvents such as dimethylformamide, dimethyl sulfoxide, etc.,
Surfactants such as sorbitan monopalmitate and sorbitan monolaurate can also be added.

Hereinafter, the present invention will be described in more detail with reference to Examples, but it goes without saying that the present invention is not limited thereto.

Example 1) Soluble starch 1 g, yeast extract 0.5 g, peptone 0.5 g, K _{2
HPO 4, 0.1g, MgSO 4 ·} 7H 2 O0.02g, CuSO 4 · 5H 2 O0.0005g,
MnCl ₄ / 4H ₂ O 0.0005g, ZnSO ₄ / 7H ₂ O 0.0001g, FeSO ₄ / 7H ₂
A solution consisting of 0.0002 g of O and 100 ml of distilled water was adjusted to pH 7.6 with diluted sodium hydroxide.

This liquid medium was sterilized at 120 ° C for 20 minutes, then inoculated with Bacillus sp. VA-47 strain, and at 28 ° C.
It was shake-cultured for 48 hours. To this was added 400 mg of cis-1,3-dibenzyl-2-oxoimidazolidine-4,5-carboxylic acid dimethyl ester, and the mixture was further shaken at 28 ° C. for 72 hours for reaction.

100 ml of ethyl acetate was added to this reaction solution, which was acidified with diluted hydrochloric acid, and then filtered through Celite. The filtrate was separated, the organic solvent layer was washed with water and dried over anhydrous magnesium sulfate,
It was concentrated under reduced pressure. To the residue was added 20% of 5% heavy sodium hydroxide water, and after dissolution, the mixture was washed with 20 ml of ether and separated.

The aqueous layer was adjusted to pH 2 with diluted sulfuric acid, and the precipitated white crystals were filtered, washed with water and dried. 300 mg of (4S, 5R) -1,3-dibenzyl-5-methoxycarbonyl-2-oxoimidazolidine-4-carboxylic acid was obtained.

The melting point was 149 to 151 ° C ▲ [α] ²⁰ ₃₆₅ ▼ -28.0 ° (C = 1, DMF).

Example 2) Soluble starch 1 g, yeast extract 0.5 g, peptone 0.5 g, K _{2
HPO 4, 0.1g, MgSO 4 ·} 7H 2 O0.02g, CuSO 4 · 5H 2 O0.0005g,
MnCl ₄ / 4H ₂ O 0.0005g, ZnSO ₄ / 7H ₂ O 0.0001g, FeSO ₄ / 7H ₂
A solution consisting of 0.0002 g of O and 100 ml of distilled water was adjusted to pH 9.0 with diluted sodium hydroxide.

This liquid medium was sterilized at 120 ° C for 20 minutes and then inoculated with Bacillus sp. VA-62 strain at 28 ° C.
It was shake-cultured for 48 hours. To this was added 400 mg of cis-1,3-dibenzyl-2-oxoimidazolidine-4,5-carboxylic acid dimethyl ester, and the mixture was further shaken at 28 ° C. for 72 hours for reaction.

100 ml of ethyl acetate was added to this reaction solution, which was acidified with diluted hydrochloric acid, and then filtered through Celite. The filtrate was separated, the organic solvent layer was washed with water and dried over anhydrous magnesium sulfate,
It was concentrated under reduced pressure. To the residue was added 20% of 5% heavy sodium hydroxide water, and after dissolution, the mixture was washed with 20 ml of ether and separated.

The aqueous layer was adjusted to pH 2 with diluted sulfuric acid, and the precipitated white crystals were filtered, washed with water and dried. 320 mg of (4S, 5R) -1,3-dibenzyl-5-methoxycarbonyl-2-oxoimidazolidine-4-carboxylic acid was obtained.

The melting point was 149 to 151 ° C ▲ [α] ²⁰ ₃₆₅ ▼ -28.0 ° (C = 1, DMF).

Example 3) The test tube containing 10 ml of the liquid medium used in Example 1) was added to 120
5 ml of preculture obtained by sterilizing at ℃ for 20 minutes, inoculating Bacillus sp. VA-47 strain and shaking culturing at 28 ℃ for 2 days
Each of them was inoculated into 4 2L Sakaguchi flasks containing 250 ml of the following medium A, and cultured by shaking at 30 ° C for 40 hours.

The culture was centrifuged at 8000 rpm for 20 minutes under cooling to collect the cells. The cells were washed three times with 100 ml of 0.85% saline (by centrifugation) to obtain 15.7 g of washed wet cells.

Washed wet cells 15.7 g and cis-1,3-dibenzyl-2-oxoimidazolidine-4,5-carboxylic acid dimethyl ester 5.0
Suspend g in pH 8.0 0.01 M phosphate buffer 500 ml and adjust to pH around 8 with 1N aqueous sodium hydroxide solution.
Stir at 170 ° C. for 170 hours.

The reaction solution was centrifuged to separate the cells and the supernatant (the cells were collected). The supernatant was adjusted to pH 2 with diluted sulfuric acid, and the precipitated white crystals were filtered, washed with water and dried.

4.5 g of (4S, 5R) -1,3-dibenzyl-5-methoxycarbonyl-2-oxoimidazolidine-4-carboxylic acid
Was obtained.

The melting point was 145 to 147 ° C. [α] ²⁰ ₃₆₅ ▼ −25.0 ° (C = 1, DMF).

Example 4) A test tube containing 10 ml of the liquid medium used in Example 1) was added to 120
After sterilizing at ℃ for 20 minutes, inoculate with Bacillus sp. VA-62 strain, and shake culture at 28 ℃ for 2 days.
2 L Sakaguchi Flask 5 containing 250 ml of the following medium B
The book was inoculated and shake-cultured at 30 ° C. for 40 hours.

The culture was centrifuged at 800 rpm for 20 minutes under cooling to collect the cells. The cells were washed three times with 100 ml of 0.85% saline (by centrifugation) to obtain 10.5 g of washed wet cells.

Washed wet cells 10.5 g and cis-1,3-dibenzyl-2-oxoimidazolidine-4,5-carboxylic acid dimethyl ester 5.0
Suspend g in pH 8.0 0.01 M phosphate buffer 500 ml and adjust to pH around 8 with 1N aqueous sodium hydroxide solution.
Stir at 100C for 100 hours.

The reaction solution was centrifuged to separate into cells and a supernatant. The supernatant liquid was adjusted to pH 2 with diluted sulfuric acid and the precipitated white crystals were filtered,
It was washed with water and dried.

4.6 g of (4S, 5R) -1,3-dibenzyl-5-methoxycarbonyl-2-oxoimidazolidine-4-carboxylic acid
Was obtained.

The melting point was 146 to 148 ° C ▲ [α] ²⁰ ₃₆₅ ▼ -26.0 ° (C = 1, DMF).

Example 5) The cells recovered in Example 3) and cis-1,3-dibenzyl-
Suspend 5.0 g of 2-oxoimidazolidine-4,5-carboxylic acid dimethyl ester in 500 ml of pH 8.0 0.01 M-phosphate buffer, and adjust the pH to around 8 with 1N aqueous sodium hydroxide solution for 190 hours at 30 ° C. It was stirred.

The reaction solution was centrifuged to separate into cells and a supernatant. The supernatant liquid was adjusted to pH 2 with diluted sulfuric acid and the precipitated white crystals were filtered,
It was washed with water and dried.

4.4 g of (4S, 5R) -1,3-dibenzyl-5-methoxycarbonyl-2-oxoimidazolidine-4-carboxylic acid
Was obtained.

The melting point was 149 to 151 ° C ▲ [α] ²⁰ ₃₆₅ ▼ -28.0 ° (C = 1, DMF).

Claims (1)

[Claims] 1. A general formula (In the formula, R represents a lower alkyl group, Bzl represents a benzyl group.) The cis-imidazolidinedicarboxylic acid diesters represented by the formula (3) are esterase-producing bacteria belonging to the genus Bacillus including Bacillus A general formula characterized by contacting an esterase derived from an esterase-producing bacterium belonging to the genus Bacillus including alkaline genus Bacillus (In the formula, R represents a lower alkyl group. Bzl represents a benzyl group. The 4- and 5-positions are 4S and 5R coordinates.) An optically active cis-imidazolidinedicarboxylic acid Method for producing derivative