JPS6117479B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a method for optical resolution of DL-phenylglycinamide derivatives. Prior Art In general, the asymmetric hydrolysis method for amino acids is as follows:
A DL-amino acid ester decomposition method using esterase, an N-acyl-DL-amino acid decomposition method using acylase, and a DL-amino acid amide decomposition method using amitase are known. Furthermore, as an example of selectively hydrolyzing DL-amino acid amides with an enzyme, aromatic amino acid amides such as phenylalanine amide can be hydrolyzed using papain, bromelain, or fuicin. Problems to be solved by the invention Since phenylglycine is not a natural amino acid,
Since selective hydrolysis by enzymes is not easy, phenylglycine has traditionally been treated with N by acylase.
Optical resolution by -acyl-DL-amino acid decomposition method is only occasionally observed. A splitting method that utilizes the biosynthetic reaction of microbial cells previously discovered by the present inventors,
Special Publication No. 45-8634, Special Publication No. 8635, Special Publication No. 45-8635, Special Publication No. 1973
-6414, JP-A-49-50191 and JP-A-49-
No. 80290 falls under this category. Furthermore, there is little precedent for DL-phenylglycinamide, and asymmetric hydrolysis by enzymes of animal origin has only recently been recognized. JP-A-51-9786 discloses asymmetric hydrolysis of DL-phenylglycinamide using leucine aminopeptidase present in pig kidney. However, the asymmetric hydrolysis of DL-phenylglycinamide derivatives by a microbial amitase of the present invention is a novel optical resolution method described at the end of the literature. Means for Solving the Problem As a result of various searches regarding soil bacteria, the present inventors found that the genus Azotobacter, the genus Thiobacillus,
Alternatively, it has been found that only L-amino acids are asymmetrically hydrolyzed by contacting a culture obtained by culturing a strain belonging to the genus Necromonas or a treated product thereof with DL-phenylglycinamide. Furthermore, when we tried using a DL-phenylglycinamide derivative that has a substituent on the phenyl nucleus, which makes this reaction difficult to act enzymatically, we found that asymmetric hydrolysis of the amide unexpectedly occurred. As a result of intensive research, the present invention was completed. Effect According to the method of the present invention, any phenylglycinamide derivative with or without a substituent on the phenyl nucleus can be used as a substrate. The substituent on the phenyl nucleus may be any group that does not significantly reduce the asymmetric hydrolysis ability, but preferred examples include a halogen atom, a hydroxyl group, a methoxy group, an acetoxy group, or an alkylsulfonylamino group. These may be used alone or in appropriate combinations. That is, the present invention was completed based on the knowledge of a wide range of asymmetric hydrolysis abilities originating from microorganisms, and includes the genus Azotobacter, the genus Thiobacillus,
Or, utilizing the fact that a culture belonging to the genus Necromonas or a treated product thereof has the effect of asymmetrically hydrolyzing a DL-phenylglycineamide derivative to produce a D-phenylglycineamide derivative and an L-phenylglycineamide derivative. It is characterized by optical resolution into a D-phenylglycine amide derivative and an L-phenylglycine derivative. Specific examples of microorganisms belonging to the Azotobacter genus, Thiobacillus genus, and Necromonas genus used in the present invention include Azotobacter No. 5686, Thiobacillus
No. 5886 and Necromonas No. 23709 are mentioned, and the mycological properties of each are as shown below. 1 Morphological characteristics and physiological properties of Azotobacter No.5686

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[Table] Based on the above mycological properties, Bergey's Manual of Bacteria Identification
Determinative Bacteriology), Skerman's The Genera of Bacteria
Bacteria), International Journal of Bacteria Taxonomy (International
Journal of Systematic Bacteriology), the taxonomic genus of this strain was determined as follows. Strain No. 5686 Young cells (15-20 hours) are rod-shaped and have flagella, but older cells (25-40 hours) are egg-shaped and flagella easily fall off, showing pleomorphism. The plants were grown on a nitrogen-free basal medium containing 0.05% dipotassium hydrogen phosphate, 0.03% magnesium sulfate, 0.02% sodium chloride, 0.01% calcium chloride, 0.001% ferrous sulfate, 10 ppm sodium molybdate, and 2% glucose. When cultured in this nitrogen-free medium with the addition of ammonia nitrogen or nitrate nitrogen, the plants grew more rapidly than in a nitrogen-free medium to which no nitrogen was added. Even after consuming these bound nitrogens, if an energy source remains, it can fix nitrogen in the air and increase its strength, which is why it was identified as the genus Azotobacter. It has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microtechnology Research Institute Deposit No. 1661. 2 Morphological characteristics and physiological properties of Thiobacillus No.5886

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[Table] Based on the above mycological properties, Bergey's Manual of Bacteria Identification
Determinative Bacteriology), Skerman's The Genera of Bacteria
Bacteria), International Journal of Bacteria Taxonomy (International
Journal of Systematic Bacteriology), the taxonomic genus of this strain was determined as follows. Strain No. 5886 Can oxidize thiosulfate and assimilate carbon dioxide.
Colonies on thiosulfate plate agar turn yellow or brown due to the influence of precipitated sulfur. Liquid medium ammonium sulfate 0.01%, magnesium sulfate
0.01%, calcium chloride 0.01%, potassium phosphate 0.8
%, ferrous chloride 0.002%, manganese sulfate 0.002%
(PH6.6) as a basic medium and thiobacillus added as an energy source, it was identified as belonging to the genus Thiobacillus. It has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microtechnology Research Institute Deposit No. 1665. 3 Morphological characteristics and biochemical properties of Necromonas No. 23709 a; Morphology (gravy agar slant, 30℃, 24 hours) (1) Bacterial shape: Short rod Size: 0.5-0.6 x 1.5-2.0 (2 ) Presence or absence of bacterial pleomorphism: Most of them are solitary (3) Presence or absence of motility: Motile (4) Presence or absence of spores: None (5) Presence or absence of flagella: 1 long (5μ) polar hair (6 ) Gram staining: Negative (7) Acid-fastness: None (8) Plasma: None b: Growth status in each medium (1) Broth agar medium (30℃, 24 hours): Good growth, smooth surface, pale yellow gloss (2) Meat juice liquid culture (30℃, 24 hours): Good growth, no precipitation, no gas generation (3) Meat juice gelatin puncture culture: Liquefied in layers (4) Potato medium (30℃, 5 days): Good growth, Pale yellow, translucent gloss (5) Meat juice agar slant medium (30℃, 24 hours): Good growth, smooth surface, pale yellow gloss (6) Litmus milk: Alkaline, peptonization c: Physiological properties (1) Growth Range: PH5.0-9.0 for growth, optimum PH7.0-8.0, temperature for growth 10℃-38℃, optimum temperature 28℃ (2) Attitude towards oxygen: aerobic (3) Nitrate reduction: ( +) (4) MR test: (+) (5) VP test: (+) (6) Ammonia production: (+) (7) Indole production: (-) (8) Hydrogen sulfide production: (+) ( 9) Utilization of citric acid: (-) (10) Hydrolysis of starch: Weak (11) Utilization of inorganic nitrogen source: Weak (12) Formation of pigment: (-) (13) Urease: (-) (14) Oxidase (-) (15) Catalase: (+) (16) Methylene blue reduction: (+) (17) Milk coagulation: (-) (18) Salt tolerance: 5% (19) O-F test: Type F ( llngh and Leifson Test) (20) Liquefaction of casein: (-) (21) Denitrification reaction (-) (22) Fermentability of carbohydrates Growth Gas production Acid production L-arabinose + - + D-xylose ± - - D- Glucose = - + D-Mannose = - + D-Fructose = - + D-Galactose = - + Maltose = - + Sucrose = - - Lactose = - - Trehalose = - - Raffinose = - - D-sorbit + - + Inosytes − − − Glycerol + − + Salicin + − − α-Methylglycium 〓 − ＋ Coside Inulin + − − Dextrin 〓 − ＋ Starch 〓 − ＋ Cellulin − − − Ethanol 〓 − ＋ Malonic acid + − − Hippuric acid − − − Tartaric acid − − − Acetic acid + − − Lactic acid − − − D-mannite − − − The taxonomic position of the soil isolated bacteria with the above mycological properties is shown in Virgies Manual.
Bergey's Manual of Determinative
Gram-negative heterotrophs with a single flagellum (long polar hair). It is a bacterium that is salt tolerant and ferments glucose, so it is closely related to the genus Aeromonas, but it differs from the genus Aeromonas in that it does not produce gas from carbohydrates. Furthermore, Genera of Bacteria 2nd
Referring to the edition, IW Smith wrote The Journal of General Microbiology for bacteria related to the genus Aeromonas that do not produce gas from carbohydrates.
General Microbiology) Volume 33, pp. 263-274 (1963
Genus
Necromonas). As a result of a detailed study of soil isolated bacteria No. 23709 used in the present invention with the known bacterial species described in Mr. Smith's original report, No.
Bacterium 23709 was confirmed to belong to the genus Necromonas. Bergey's Manual of Determinative Bacteriology (Bergey's Manual of Determinative Bacteriology)
The 7th edition of the Manual of Determinative Bacteriology does not contain any description of the genus Necromonas. This strain has been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiological Laboratory No. 2830. These microorganisms are treated with irradiation with X-rays, ultraviolet rays, etc., nitrous acid, azaserine, 2-aminopurine, ethylethanesulfonic acid, N-methyl-
By treatment with a mutating agent such as N'-nitro-N-nitrosoguanidine, the hydrolytic activity can be increased and used. To utilize their hydrolytic ability, their culture or processed product is usually used. A culture of microorganisms is obtained by a shaking culture method or an aerobic deep culture method with electric stirring. The composition of the liquid medium used for culture includes glucose, glycerin, starch, peptone, yeast extract, cornsteep liquor, etc., and phosphates, magnesium sulfate, sodium chloride, etc. are added as necessary. The culture temperature is preferably 25 to 30°C, and the culture time is preferably 24 to 48 hours. The culture conditions described above are applied by selecting optimal conditions depending on the characteristics of the bacterial strain used. The cultured cells of microorganisms belonging to the Azotobacter genus, Thiobacillus genus, or Necromonas genus and having the ability to asymmetrically hydrolyze DL-phenylglycinamide obtained by culturing in this manner can be used as they are as a centrifuged bacterial mass. Alternatively, a processed product of the obtained cultured cells can also be used. For example, frozen and thawed bacterial mass,
Cells destroyed by ultrasonic waves, etc., substances exuded to the outside of cells with resolving power due to surfactants, or substances with resolving power immobilized on a carrier may also be used. The reaction of this invention is usually carried out by dissolving or suspending the above-mentioned microbial culture or treated product in water or a buffer solution, and adding the substrate DL-phenylglycinamide derivative thereto. The pH of the reaction solution is preferably PH8
-9, substrate concentration 0.5-1%, reaction temperature 28°C, reaction time 3-24 hours. The reaction conditions described above are applied by selecting optimal conditions depending on the type of substrate and microorganism used. The D-phenylglycine amide derivative and L-phenylglycine derivative produced in the reaction solution are separated and purified according to a conventional method. Specifically, D-phenylglycine amide derivatives are adsorbed using weakly acidic ion exchange resins, such as Amberlite IRC-50 and Diaion CR-10, while L-phenylglycine derivatives are adsorbed with strong acids. Adsorption treatment with a neutral ion exchange resin such as Amberlite IR-120 is advantageous. Next, the target product is eluted from these ion exchange resins with aqueous ammonia, and the eluate is concentrated to obtain the target product as a solid. As the recrystallization solvent, lower alcohols such as methanol, ethanol, and isopropanol are preferably used. EXAMPLES Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereby. Example 1 Azotobacter No. 1 was added to a culture solution (PH6.8) containing 2% glucose and 5% consteep liquor.
5686 strain was inoculated and cultured with shaking at 29°C for 24 hours. The obtained cells were centrifuged and cryopreserved as resting cells. 10 g of DL-phenylglycinamide was dissolved in 900 ml of water, the pH was adjusted to 8-9, and 100 ml of suspended frozen cells was added. Stir this wave constantly and keep it at 29℃.
It was held for 3 hours. Next, in order to remove cells from this reaction solution, the reaction solution was centrifuged to obtain a supernatant. This solution was adjusted to pH 10 (3N NH ₄ OH) and passed through a column packed with IRC-50 (H type) 1. Subsequently, the column was washed with 3 portions of deionized water, eluted with N.NH ₄ OH, the ninhydrin-positive fraction was concentrated under reduced pressure, and after cooling, the precipitate was dried to obtain 1.8 g of D-phenylglycinamide, [α] ²⁰ _D −
116° (EtoH, C=0.5) was obtained. The mother liquor was concentrated to dryness and extracted with ethanol. From this ethanol extract, 1.5 g of D-phenylglycinamide, [α] ²⁰ _D -100° (EtoH, C = 0.5) was extracted.
I got it. Dissolve 1 g of primary crystals in 30 ml of 10% hydrobromic acid, heat on a hot water bath for 3 hours and dry, add water and neutralize with concentrated ammonia water to produce D-phenylglycine.
0.6 g, [α] ²⁰ _D −157° (N·HCl, C=1) was obtained. The above-mentioned IRC-50 column permeate and water wash were IR
The solution was passed through a column packed with 300 ml of -120 (H type). After washing the column with deionized water 1, 3N
Elute with NH ₄ OH, concentrate the ninhydrin-positive fraction under reduced pressure, adjust the pH to 4.5, cool it, and dry the precipitate to obtain 3.3 g of L-phenylglycine, [α] ²⁰ _D +153° (N HCl , C=1) was obtained. Example 2 DL-parahydroxyphenylglycinamide
Thiobacillus No. 1 was obtained by dissolving 10 g in water 1.9, adjusting the pH to 8-9, and culturing in the same manner as in Example 1.
100 ml of frozen cell suspension of strain 5886 was added. This liquid was maintained at 29° C. for 5 hours while stirring. Next, in order to remove cells from this reaction solution, the reaction solution was centrifuged to obtain a supernatant. This solution was diluted with 3N・NH ₄ OH.
The pH was adjusted to 10, and the solution was passed through a column filled with CR-10 (H type) 1. The column was washed with 3 portions of deionized water and eluted with N.NH ₄ OH. The ninhydrin-positive fraction was concentrated under reduced pressure. After cooling, the precipitate was dried to obtain 4.2 g of D-parahydroxyphenylglycinamide, [α] ²⁰ _D
−125° (N·HCl, C=1) was obtained. The above-mentioned CR-10 column passing liquid and water washing liquid are
The solution was passed through a column packed with 300 ml of IR-120 (H type). After washing the column with deionized water 1
Elute with 3N NH ₄ OH, concentrate the ninhydrin-positive fraction under reduced pressure, adjust the pH to 4.5, cool it, and dry the precipitate to obtain L-parahydroxyphenylglycine 4.2.
g, [α] ²⁰ _D +156° (N·HCl, C=1) was obtained. Example 3 Necromonas No. 3 was obtained by dissolving 10 g of DL-metamesylaminophenylglycinamide in water 1.9, adjusting the pH to 9, and culturing according to Example 1.
100 ml of frozen cell suspension of strain 23709 was added. The solution was maintained at 29° C. for 24 hours with stirring. Subsequently, the reaction solution was centrifuged to remove cells. This supernatant was adjusted to pH 10 with 3N NH ₄ OH, and then mixed with CR-10 (H
The solution was passed through a column packed with Type) 1. After washing the column with deionized water 3, wash it with N・NH ₄ OH.
The ninhydrin-positive fraction was concentrated under reduced pressure, and after cooling, the precipitate was reconsolidated with water to give 3.8 g of D-metamesylaminophenyl glycinamide, [α] ²⁰ _D -85° (N HCl, C= 1) was obtained. The above-mentioned CR-10 column passing liquid and water washing liquid are as follows:
The solution was passed through a column packed with 300 ml of IR-120 (H type), washed with 1 portion of deionized water, eluted with 3N.NH ₄ OH, and the ninhydrin-positive fraction was dried under reduced pressure. Add a small amount of water to adjust the pH to 5, add ethanol, cool overnight, and dry the precipitate to obtain 2 g of L-metamesylaminophenylglycine, [α] ²⁰ _D +98° (N HCl,
C=1) was obtained. Example 4 Freezing resting cells obtained from culture solution 1 of Azotobacter No. 5686 strain were reacted with 10 g of DL-metamesylaminophenyl glycinamide in the same manner as in Example 3, and the resulting reaction solution was treated in the same manner. Do D
- Metamesylaminophenyl glycinamide 3.5
g, [α] ²⁰ _D −85° (N·HCl) and 2.2 g of L-metamesylaminophenylglycine, [α] ²⁰ _D +99° (N·HCl, C=1). Example 5 Freeze-resting cells obtained from culture solution 1 of Thiobacillus No. 5886 strain were reacted with 10 g of DL-phenylglycinamide in the same manner as in Example 1, and the resulting reaction solution was treated in the same manner as D. -1.5 g of phenylglycinamide, [α] ²⁰ _D -116° (EtOH, C = 0.5) and 3.6 g of L-phenylglycine, [α] ²⁰ _D +154° (N HCl, C = 1) were obtained. Ta. Example 6 Freeze-resting cells obtained from culture solution 1 of Necromonas No. 23709 strain were cultured in the same manner as in Example 2, using DL-
D
- phenylglycinamide 4.0 g, [α] ²⁰ _D -126° (N HCl, C = 1) and L-parahydroxyphenylglycine 3.8 g, [α] ²⁰ _D +155° (N.
HCl, C=1) was obtained. Reference examples 1 to 4 DL-paramethoxyphenylglycinamide,
Using 10 g each of DL-paramethoxy metachlorophenylglycinamide, DL-parahydroxy metamethoxyphenylglycinamide, and DL-parahydroxy methachlorophenylglycinamide, an asymmetric hydrolysis reaction was carried out in the same manner as in Example 1. A D-phenylglycinamide derivative having a substituent on the isolated phenyl nucleus was obtained. Furthermore, D-phenylglycine amide was subjected to acid decomposition to obtain corresponding D-phenylglycine derivatives as shown in the following table. Effect of the Invention According to the present invention, D
-Phenylglycine amide derivatives and L-phenylglycine derivatives can be obtained by optical resolution using microorganisms. The present invention provides an industrially advantageous method for producing raw materials for antibiotics.

Claims (1)

[Claims] 1. General formula (In the formula, R ₁ is a hydrogen atom, a hydroxy group, or a methoxy group, and R ₂ is a hydrogen atom, a chlorine atom, a methoxy group, or a methanesulfonylamino group) Phenylglycine having a substituent on the phenyl nucleus An amide compound is asymmetrically hydrolyzed in the presence of a culture of a microorganism that belongs to the genus Azotobacter, genus Thiobacillus, or genus Necromonas and has asymmetric hydrolysis ability, or a treated product thereof, and the corresponding D(-) phenylglycine is obtained. Amide compound and L
An optical resolution method for a phenylglycine amide compound having a substituent on the phenyl nucleus, which comprises fractionally collecting a (+) phenylglycine compound.