JPS6120272B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antibiotic ATS-1287 substance and a method for producing the same. The ATS-1287 substance of the present invention has the following physical and chemical properties. (1) Appearance: Colorless to pale yellow viscous oily substance. Its sodium salt is a white to yellowish-white amorphous powder. (2) Melting point, freezing point: It does not show a clear melting point or freezing point, and even sodium salts turn brown while moist at temperatures above 130°C. (3) Solubility: Easily soluble in benzene, chloroform, ether, ethyl acetate, acetone, and alcohols. Slightly soluble in cyclohexane. Insoluble in n-hexane, carbon tetrachloride, petroleum ether, and water. (4) Distinction between basic, neutral, and acidic: acidic substances (5) Specific rotation: [α] ²⁰ _D = -24.5° (C = 0.70,
(6) Ultraviolet absorption spectrum: Shown in Figure 1 (measured in ethanol). (7) Infrared absorption spectrum: Shown in Figure 2. (Measured as a film with a small amount of chloroform) (8) Nuclear magnetic resonance absorption spectrum: Shown in Figure 3.
(In deuterated chloroform, TMS standard) (9) Elemental analysis: C 70.0%±0.5% H 8.7%±0.5% N 0.0%±0.5% (10) Composition formula: C _32-34 H _46-50 O _6-8 (11) Molecular weight: 526-586 (calculated value) (12) Color reaction: Bright red with antimony trichloride. Absorbs brome.
Decolorizes potassium permanganate. Ehrlich reaction,
Anisaldehyde sulfuric acid color development, iodine color development positive.
2,4-dinitrophenylhydrazine false positive.
Negative coloration based on ninhydrin, tollence, and ferric chloride reactions. (13) R _f value in thin layer chromatography:

[Table] Sunol
(14) Stability: Stable for 30 minutes at 100℃ from PH2 to PH9. Unstable at PH10 or higher. The physiological properties of ATS-1287 substance are as follows. (1) Antibacterial activity: The antibacterial activity of the ATS-1287 substance is shown in Tables 1 and 2. ATS-1287 substance is partly fungal,
For example, Schizosatucharomyces bombe IAM 4020
Mucor racemosus Christianus strain is characterized by strong antibacterial activity.
In addition, for Schizosatucharomyces bombe strain IAM 4020, elongating its yeast-like cells is antibacterial, and for Mucor racemosus christianus strain, it is antibacterial to bend the growing hyphae in a wavy manner. It is mentioned as a characteristic associated with activity. Also against other fungi, against human skin pathogens such as Trichophyton mentagrophytes and Microsporum gypsium, and also against plant anthracnose pathogens such as Glomerella singulata and Colletotrichum tolimafolii. It does not have growth inhibiting activity, but has fungistatic action (2) Toxicity ATS-1287 substance is acutely toxic using 4-week-old male ICR/JCL mice, and the 50% lethal dose when administered intravenously is 18 It was ~36mg/Kg.

[Table] Based on the usual agar plate dilution method.

【table】

[Table] Due to the physicochemical or physiological properties of the ATS-1287 substance as described above, it is not applicable to existing antibiotics, and the ATS-1287 substance is recognized as a new antibiotic. The ATS-1287 substance of the present invention is produced by culturing a microorganism belonging to the genus Streptomyces that can produce the ATS-1287 substance, producing the ATS-1287 substance in the culture, and collecting the resulting product. A specific example of a microorganism capable of producing the ATS-1287 substance is strain No. 1287, which belongs to the genus Streptomyces and was isolated from soil by the present inventor.The mycological properties thereof are as follows. It is. Observation results were determined after 2 weeks of culture unless otherwise specified. (1) Morphological characteristics: Strain No. 1287 grows in the form of hyphae on many agar plates, and its basal hyphae do not divide. When aerial hyphae are formed, the hyphae branch irregularly and do not branch in whorls; when spores are formed, spore sacs The presence of hyphae is not observed, and 50 or more strands are formed at the tips of aerial hyphae. The spore chains can be straight, curved in the shape of a hook, or loose and incompletely spiral, which are the morphological characteristics of Reteina cuyulia pertei in the classification of the genus Streptomyces. shows. These forms can be observed particularly well on sucrose nitrate agar, glucose-asparagine agar, oatmeal agar, and the like. The shape of the spore is approximately 1
μ, the short axis is approximately 0.6μ, and the surface is smooth. (2) Properties on various media: As shown in Table 3, the color of aerial mycelium belongs to the gray system. (3) Physiological properties: (a) Optimal growth temperature: 26.5℃ to 37℃ (b) Production of melanin pigment: tryptone yeast extract liquid medium peptone yeast extract iron agar medium tyrosine agar medium It does not produce melanin-like pigments and is considered a non-chromogenic strain. (c) Nitrate reducing property: Negative. (d) Starch hydrolysis ability: Negative. (E)
Protein hydrolysis ability: Coagulation ability of skim milk (37℃) Negative 〃 (26.5℃) 〃 Peptonization ability of skim milk (37℃) Negative 〃 (26.5℃) 〃 Liquefaction ability of gelatin (25℃)〃 Ru. (4) Assimilation of carbon sources by the Pridham-Gotzlief method: Used: D-glucose, D-fructose, L-rhamnose, D-galactose Generally not used: Raffinose Not used: D-xylose, L-arabinose, D -Mannitol, i-inositol,
It is determined to be salicin, sucrose, and cellulose.

【table】

[Table] Based on the above properties, strain No. 1287 was determined to belong to the genus Streptomyces, and was designated as Streptomyces sp. strain No. 1287. Streptomyces sp.
Strain No. 1287 has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, and its microorganism deposit number is Microbiological Research Institute No. 4842. Streptomyces sp. No. 1287 strain is one example of the strain producing the ATS-1287 substance in the present invention, and not only strains obtained by artificially mutating this strain, but also microorganisms belonging to the genus Streptomyces. , ATS-1287 substance production ability can all be used in the method of the present invention. As a method for culturing production bacteria for the ATS-1287 substance, a conventional microbial culture method can be used as appropriate. As long as the medium contains essential nutrients for the growth of the producing bacteria, ATS-
It is possible to produce 1287 substances, and specific examples of culture materials for this purpose include soybean oil-free, glucose, glycerol, etc. as a carbon source, and soy flour, pharmaceutical media, peptone as a nitrogen source. , meat extract, yeast, ammonium salts, etc. can be used, and if necessary, salts such as sodium salts, potassium salts, calcium salts, magnesium salts, phosphates, etc. can also be added. Aerated agitation culture is suitable as a culture format, the culture temperature is preferably 25℃ to 30℃, and the culture time is usually 3 to 5℃.
The ATS-1287 substance is mainly produced within the bacterial cells in the culture. The culture conditions described above can be adapted by selecting optimal conditions depending on the characteristics of the production strain used. To collect ATS-1287 material from culture,
Based on the physicochemical properties of the ATS-1287 substance described above, the means normally used for isolation and purification of the substance may be appropriately selected and adapted. An example of a means for separating and purifying the ATS-1287 substance from a culture is as follows. That is, the ATS-1287 substance mainly exists within the bacterial cells, but the bacterial cells are separated by an appropriate method and can be effectively extracted from the wet bacterial cells using, for example, acetone. After distilling off the acetone, it is effectively extracted with an organic solvent that is immiscible with water, such as ethyl acetate or chloroform. By distilling this off, ATS-
Crude samples of 1287 substances are obtained, but it is further
Purification can be carried out by utilizing the difference in solubility between the ATS-1287 substance and impurities and the difference in distribution between two immiscible liquid phases. Chromatography is a more effective method for purifying ATS-1287 substances. Many methods are possible for chromatography to purify the ATS-1287 substance, but column chromatography using silica gel is one of the suitable methods. An example of an effective method is to add ethyl acetate, methanol, etc. to these and develop and elute. Other methods include column chromatography using silanized silica gel as a carrier and based on reverse phase partitioning using a system such as chloroform-methanol-saline; and ion-exchange chromatography using DEAE cellulose as a carrier in an organic solvent. , or security
Chromatography based on molecular sieves using LH-20 as a carrier can be appropriately selected or used in combination. By the method described above, the ATS-1287 substance can be collected in a pure manner. Examples are shown below, but the present invention is not limited thereto. Example 1 From a seed slant of Streptomyces sp. strain No. 1287, 0.3% glycose, 0.4% Bactotryptone (manufactured by Difco), 0.1% yeast extract (manufactured by Difco), and salt were added to a 500 ml Sakaguchi flask as a preculture. The seeds were inoculated into 75 ml of a medium containing 0.4% water and cultured with shaking at 26.5°C for 48 hours.The main culture was carried out in a 600 tank with 3% soybean oil and 5 ml of soy flour (Ajinomoto S-Sun Meal).
%, dry yeast 0.3%, HKCl 0.4%, _{K2HPO4 0.02}
%, CaCO ₃ 0.3%, PH 7.2% medium 300 was inoculated at 0.75%, and cultured with aeration at 26.5°C for 4 days. Approximately 10 μg of ATS per ml of culture, that is, a total amount of approximately 3 g. -1287 substances were produced, 90% of which were present in the bacterial compartment. The above culture was stopped on the 4th day, the culture was passed with a supernatant, the residue containing the bacterial cells was extracted twice with 100 acetone, the extracts were combined, the acetone was distilled off, and the The residue is further extracted with the same amount of ethyl acetate. After dehydrating the ethyl acetate layer with anhydrous sodium sulfate, ethyl acetate is distilled off. 2.8 g of ATS-1287 was contained in 300 g of residue, and the yield from the culture medium was about 90%. This sample was dissolved in ethyl acetate in step 2, and after removing insoluble matter, it was passed through a 500 ml silica gel column, further washed with ethyl acetate in step 1, the passed liquid and the washing liquid were combined, and the ethyl acetate was distilled off. Add hexane from Step 2 to the remaining oily substance, leave it at a low temperature, and collect the insoluble portion. Contains about 2g of ATS-1287 substance in 60g. A portion of the crude extract prepared in this way was taken and subjected to column chromatography. That is, 10g of the standard sample was taken and adjusted with chloroform.
ATS on a 500ml silica gel (Merck) column
-1287 substance was adsorbed, and chromatography was performed by adding ethyl acetate stepwise to chloroform as a developing solvent. The eluent contains approximately 40
% pure ATS-1287 material was obtained in approximately 85% yield. After silica gel adsorption chromatography, the sample was prepared using silanized silica gel (manufactured by Merck & Co., Ltd.) as a carrier, and chloroform/methanol/0.1M saline solution (33:
Reversed phase partition chromatography was performed using a 400 ml column equilibrated with 66:34) and developed with the same solvent. As a result, approximately 200 ml of almost pure ATS-1287 substance was obtained by thin layer chromatography, but further preparative silica gel thin layer chromatography was performed using a chloroform/methanol solvent system.
Finally, gel filtration was performed using Cephadex LH-20 (manufactured by Pharmacia) with ethanol to obtain 122 mg of pure ATS-1287 substance.
We investigated the physicochemical and physiological properties of substances.

[Brief explanation of the drawing]

Figures 1, 2, and 3 are ATS-
Ultraviolet absorption spectrum (in ethanol), infrared absorption spectrum (measured as a film in a small amount of chloroform), and nuclear magnetic resonance absorption spectrum (in deuterium chloroform, TMS standard) of 1287 substances
shows.

Claims (1)

[Claims] 1. New antibiotic ATS having the following physical and chemical substances
-1287 Substance (1) Appearance: Colorless to pale yellow viscous oily substance. Its sodium salt is a white to yellowish-white amorphous powder. (2) Melting point, freezing point: It does not show a clear melting point or freezing point, and even sodium salts turn brown while moist at temperatures above 130°C. (3) Solubility: Easily soluble in benzene, chloroform, ether, ethyl acetate, acetone, and alcohols. Slightly soluble in cyclohexane. Insoluble in n-hexane, carbon tetrachloride, petroleum ether, and water. (4) Distinction between basic, neutral, and acidic: acidic substances (5) Specific rotation: [α] ²⁰ _D = -24.5° (C = 0.70,
(in methanol) (6) Ultraviolet absorption spectrum: Shown in Figure 1. (7) Infrared absorption spectrum: Shown in Figure 2. (8) Nuclear magnetic resonance absorption spectrum: Shown in Figure 3. (9) Elemental analysis: C 70.7%±0.5% H 8.7%±0.5% N 0.0%±0.5% (10) Composition formula: C _32-34 H _46-50 O _6-8 (11) Molecular weight: 526-586 (Calculated value) (12) Color reaction: Bright red with antimony trichloride. Absorbs brome.
Decolorizes potassium permanganate. Ehrlich reaction,
Anisaldehyde sulfuric acid color development, iodine color development positive.
2,4-dinitrophenylhydrazine false positive.
Negative coloration based on ninhydrin, tollence, and ferric chloride reactions. 2. Cultivate ATS-1287 substance-producing bacteria belonging to the genus Streptomyces, and use the culture to determine ATS-1287.
A method for producing an ATS-1287 substance, characterized by collecting the substance.