JPS6212228B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antibiotic A-11079-B ₂ that exhibits an excellent growth-inhibiting effect on plant pathogenic mycelial fungi, and a method for producing the same. For more details, please see the new antibiotic A-11079-B _2- producing actinomycete Amphiulariella sp.
A 11079 (Ampullariella sp.A 11079) was cultured in a medium, and the new antibiotic A-
11079-New antibiotic that collects B ₂ A-11079-
The present invention relates to a method for producing _B2 and the antibiotic. The novel antibiotic A-11079-B ₂ of the present invention is a weakly basic compound having the following physicochemical properties. Elemental analysis Experimental value C=47.59 H=4.64 N=25.10 Theoretical value C=47.31 H=4.66 N=25.09 Molecular weight (value from mass spectrum) 279 (This antibiotic A-
The tetraacetyl form of 11079-B ₂ is 447) Molecular formula C ₁₁ H ₁₃ N ₅ O ₄ Melting point (value from thermal analyzer) 226℃ (decomposition) 〓〓〓〓〓
Light intensity [α] ²¹ _D = -43.6° (C = 0.67%, H ₂ O) Ultraviolet absorption spectrum Antibiotic A-11079-B ₂ measured in aqueous solution
The ultraviolet absorption spectrum of (concentration 16γ/ml) is the first
As shown in the figure, λmax263mμ, E ¹ % ₁ cm=
It has maximum absorption at 538.9. In addition, in the ultraviolet absorption spectrum at PH3 at the same concentration,
λmax259mμ, maximum absorption _{at E1%1cm} ⁼ 510.8,
Furthermore, in the ultraviolet absorption spectrum at PH10 at the same concentration, it has a maximum absorption of λmax 263 mμ and E ¹ % ₁ cm = 534.4. Infrared absorption spectrum The infrared absorption spectrum of antibiotic A-11079-B ₂ by KBr method is as shown in Figure 2.
3450～3100, 2920, 2740, 2680, 1680, 1630,
1600，1560，1500，1460，1420，1380，1360，
1330, 1300, 1240, 1200, 1170, 1100, 1050,
1020, 960, 920, 900, 880, 830, 780, 720, 680
It has an absorption band at each wavelength of cm ^-1 . Nuclear magnetic resonance spectrum The nuclear magnetic resonance spectrum of antibiotic A-11079-B ₂ at 100 MHz in deuterated dimethyl sulfoxide is shown in Figure 3 (internal standard DSS) Solubility In water, dimethyl sulfoxide, dimethyl formamide, Soluble, poorly soluble in methanol, insoluble in ethyl acetate, chloroform, benzene, hexane, color reaction Potassium permanganate decolorization reaction, periodic acid reaction, Tollens reaction positive, ferric chloride reaction, ninhydrin reaction, anisaldehyde reaction , Morritsch reaction is negative, Acid-base distinction Weak base Color property White (colorless plate-like crystals) Rf value (using silica gel f spot film manufactured by Tokyo Kasei Co., Ltd.) n-butanol:acetic acid:water (6:1:1) Rf=0.33 n-butanol: concentrated ammonia water: water (10:
0.5:2) Rf=0.19 n-propanol: concentrated ammonia water: water:
(10:1:1) Rf=0.30 Acetone: Water (10:1) Rf=0.43 Ethyl acetate: Methanol: Water (10:2:1) Rf=0.27 Chloroform: Methanol: Acetic acid (10:2:
1) Rf=0.20 Based on these physicochemical properties, antibiotic A
-11079- It was confirmed that the planar structure of B ₂ is shown by the following formula. Next, the biological properties of the new antibiotic A-11079- _B2 will be described. Growth inhibitory effect on plant pathogenic fungi A 100γ/ml solution of the new antibiotic A-11079-B ₂ was prepared using a paper disc with a diameter of 7 mm.
31.4 for Helminthosporium oryzae M 0306
showing an inhibition zone of mm diameter, Cladosporium herbarum
For M4278, it shows a 14.0mm diameter inhibition zone,
12.0 for Altenaria kikuchiana M 4588
It showed an inhibition zone of mm diameter. Acute toxicity The acute toxicity of antibiotic A-11079- _B2 in mice was 55 mg/Kg when administered intraperitoneally. Based on the above properties, this substance has an adenine skeleton in its structure and has 3 parts other than the adenine structure.
Angustomycin A is an example of a similar compound in which the presence of hydroxyl groups is clear and the chemical formula is C ₁₁ H ₁₃ N ₅ O ₄ .
Angustomycin A has a positive optical rotation and is recognized as a different substance because its NMR and mpIR patterns are significantly different. Therefore, this substance, antibiotic A-11079-B ₂ , is a new compound. It is recognized that 〓〓〓〓〓
The antibiotic A-11079-B ₂ of the present invention can also be used in the form of salts with normally pharmaceutically acceptable mineral acids or organic acids, such as hydrochloride, acetate, tartrate, citrate, It can be used in the form of salts such as succinate. Furthermore, the actinomycete that produces the novel antibiotic A-11079-B ₂ of the present invention is an actinomycete isolated from the soil of a green onion field in Myoko Kogen-cho, Nakakukujo-gun, Shintara Prefecture, and belongs to the genus Ampullariella. And, Ampillaryella S.P.A-11079
(Reception number 4494 of the National Institute of Microtechnology Deposit Application Form,
Microorganism accession number FERM-
P No. 4494), and its mycological properties are described below. [] Morphological properties On starch/inorganic salt agar medium, 30℃, 10-15
The findings observed after culturing for one day are as follows. The basal hyphae are curved, elongate with branches, and have a diameter of
It is 0.5-0.8μ and forms a few undeveloped aerial mycelia. The sporangium grows on the sporangium stalk produced from the basal hyphae, and the shape of the sporangium is cylindrical or bottle-shaped, and the size is 5-15 x 10-25μ, and there are many sporangia inside. The children are arranged vertically in parallel chains. Sporangium is a zoospore that is motile in water, rod-shaped, and 0.5-1.0 x 1.0-2.0 in size.
μ, and has a polar, tufted flagellum. [] Composition of diaminopimelic acid Meso and hydroxy forms of diaminopimelic acid were detected by whole cell analysis. [] Growth status on various media The findings observed after culturing on various media at 30°C for 20 days are shown in Table 1. No formation of aerial mycelium was observed, except for the formation of a few undeveloped aerial mycelium on starch/mineral salt agar and oat meal agar. Color display is in color.
Color Harmony
Manual) 4th edition, 1958 (Container
Corporation of America).

[Table] 〓〓〓〓〓

【table】 [] Physiological properties Physiological properties are as shown below. 1 Assimilation of carbon sources

【table】

[Table] 〓〓〓〓〓

[Table] 2 Growth temperature range: 10-45℃ 3 Skim milk: Positive for both peptonization and coagulation 4 Production of melanin-like pigment: Tyrosine agar medium; negative Peptone/yeast/iron agar medium; positive 5 Starch hydrolysis: positive 6 Cellulose hydrolysis: Negative 7 Casein hydrolysis: Positive 8 Gelatin liquefaction: Positive 9 Tyrosine decomposition: Negative 10 Xanthine decomposition: Negative 11 Hypoxanthine decomposition: Negative 12 Hydrogen sulfide production: Negative 13 Nitrate reduction : Negative From the above, the characteristics of this fungus A 11079 strain can be summarized as follows: The sporangium is attached to the sporangium stalk produced from the branched basal hyphae, and the sporangium is cylindrical or pin-shaped. The sporangiospores are rod-shaped, have tufted polar hairs, and contain mesodiaminopimelic acid.・Berger's manual of determination
bacteriology) 8th edition 1974, pages 707-708, it was identified as belonging to the genus Ampyulariella, and based on the above, this bacterium was classified as Ampyulariella sp.・It was named A 11079. The novel antibiotic A-11079-B ₂ of the present invention can be obtained as follows. That is, for example, the Amphiularia sp. A 11079 strain described above is cultured in a medium. The medium used in this case may be an artificial medium or a natural medium, and may also be a solid medium or a liquid medium, but a liquid medium is particularly suitable for mass production. Antibiotic A-11079 as a medium component
- Carbon sources, nitrogen sources, inorganic salts, and others that can be assimilated by B ₂ -producing bacteria are used. For example, carbon sources include glucose, sucrose, glycerin, soluble starch, molasses, etc., and nitrogen sources include peptone, corn steep liquor, soybean flour,
Examples include meat extract, rice bran, casein hydrolyzate, nitrate, ammonium salt, etc. Other inorganic salts include salt, phosphate, calcium, magnesium, iron, manganese, and other trace nutrients may be added as appropriate. good. If necessary, silicone oil, animal or vegetable mineral oil such as soybean oil, etc. may be added as an antifoaming agent. In order to culture the production strain in a medium containing these nutritional substances, a surface culture method or a liquid culture method may be used. When using the liquid culture method, it is advantageous to perform aerated agitation culture, and in this case, the culture temperature is around 25-30℃, which is the optimum temperature for the bacteria used, and the number of days for culture varies depending on the conditions, but usually It takes about 2 to 4 days, and the pH of the culture medium is preferably kept near neutral to slightly acidic. The culture thus obtained was treated with antibiotic A
-11079-B ₂ , and in order to collect the target antibiotic from this culture, a method normally used to collect the metabolites produced by the microorganism from the culture of the microorganism. Separation means may be used as appropriate. For example, since this antibiotic is a water-soluble, weakly basic substance, it is possible to separate and collect it by, for example, inoculating the culture solution with an appropriate adsorbent to adsorb the active ingredient, and then eluting it with an appropriate solvent. be used to advantage. Activated carbon, cation exchange resin, activated alumina, silica gel, etc. are used as the adsorbent, and elution solvents include aqueous solutions of water-soluble organic solvents, such as aqueous methanol, acetone, dioxane, etc., depending on the adsorbent used. Aqueous solutions of acids, alkalis, or salts are used as appropriate. Furthermore, by utilizing the properties of this antibiotic as a weakly basic substance, it can be applied to electrical separation and purification. For example, Amberlight IRC-
For cation exchange resins such as 50 (product name: manufactured by Rohm and Haas Company), Amberlite IR-120 (product name: manufactured by Rohm and Haas Company), or Dowex 50 (product name: manufactured by Dow Chemical Company). After adsorption, the active ingredient can be eluted using an appropriate acid, alkali or salt aqueous solution. In addition, the combination of adsorbent and ion exchange resin
For example, the culture solution may be charged to a cation exchange resin Amberlite IR-120 (trade name) to adsorb the active ingredient, and then eluted with an alkaline aqueous solution, such as 4N ammonia water. The active fraction is obtained, and after adjusting the pH to neutral or weakly basic, it is charged to an activated carbon packed column and adsorbed, and then eluted with an elution solvent such as 70% methanol to extract its active ingredients. Collect the fraction containing the powder, concentrate it under reduced pressure, and freeze-dry it to obtain a crude powder. For example, dissolve it in a water-saturated n-butanol solvent, and use a developing solvent of n-butanol: concentrated ammonia water: water (10:0.2:1). The product is purified by silica gel adsorption chromatography. The results of separation and purification by each of the above methods are purified by a recrystallization method. The fact that the obtained antibiotic A-11079-B ₂ is a single substance shows a single melting point by recrystallization, and can be analyzed by paper chromatography, thin layer chromatography, paper electrophoresis, etc. in various solvent systems. indicates a single spot.
In addition, confirmation and quantification of active fractions are
It is carried out in a bioassay using Helminthosporium oryzae M0306 as the test bacterium. This antibiotic A-11079-B ₂ is a useful compound with antifungal properties, and since it is a novel nucleic acid-related compound, it is also useful as a synthetic intermediate for various nucleic acid-based physiologically active substances. It is a chemical compound. Next, the present invention will be explained with reference to examples, but the present invention is not limited thereto. Example 1 2% glucose, 2% starch, 1% yeast extract, 1% casein hydrolyzate, calcium carbonate
Dispense 100 ml of a medium (PH6.5) containing 0.2% into a 500 ml Erlenmeyer flask, steam sterilize it at 120°C for 15 minutes, and add 100 ml of culture medium (PH6.5) containing 0.2% to two bottles of this medium. One platinum loopful was inoculated and cultured with shaking at 30°C for 4 days. This was then mixed with a steam-sterilized medium of the same composition as above.
The culture was transplanted into a 30-volume jar fermentor containing 4% glucose, and cultured at 30°C for 48 hours with aeration and stirring at 300 rpm and sterile air at a rate of 20 per minute. 1%, meat extract 0.4%, peptone 0.4%, yeast extract 0.1
%, salt 0.25%, calcium carbonate 0.1% liquid medium 200 (PH6.5), 180 rpm, 130 min.
Culture with aeration was carried out at 30°C for 40 hours with an aeration rate of . Approximately 200 of the resulting culture was then filtered, and the liquid and washings were combined to yield approximately 140 (culture titer 300 mcg/ml). Furthermore, this solution is added to the cation exchange resin Amberlite IR-120 (H ⁺ type).
(Product name) Charge the column with 20 to adsorb the active ingredients, wash with about 100 of water, and then elute with 4N ammonia water.
Discard the solution, collect the next 100, adjust the pH to 8 with concentrated hydrochloric acid, charge this solution to a column of activated carbon 4, wash with water, and add 70% methanol solution 70
The eluate obtained was concentrated under reduced pressure to approximately 1.5, and this was further freeze-dried to obtain 83.6 g of raw powder (purity 29.5%). Next, 41.8 g of this crude material was dissolved in a water-saturated n-butanol solvent and placed in about 2 silica gel columns (diameter 8.3 x 40 cm) filled with n-butanol in advance.
and elute with a solvent of n-butanol: concentrated ammonia water: water (10:0.2:1).
Active fractions No. 53 to 108 were obtained by fractionating 150 ml of each fraction, which were combined, concentrated, and allowed to stand at a low temperature. The precipitate was removed and the white color of antibiotic A-11079-B ₂ was obtained. 18.2g of crystals (96% purity,
Yield: 41.6%). Next, this was dissolved in hot water and left at room temperature to precipitate colorless plate-like crystals of antibiotic A-11079- _B2 .
A pure product of _B2 was obtained.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of antibiotic A-11079-B ₂ , and Figure 2 shows the ultraviolet absorption spectrum of antibiotic A-11079-B 2.
Figure ₃ shows the nuclear magnetic resonance spectrum of antibiotic A-11079- _B2 . 〓〓〓〓〓

Claims (1)

[Claims] 1. The following formula A novel antibiotic A-11079-B ₂ or a pharmaceutically acceptable salt thereof. 2. A culture medium containing assimilated carbon and nitrogen sources and inorganic substances is inoculated with bacteria that produce antibiotic A-11079- _B2 belonging to the genus Amphiulariella, and after culturing, antibiotic A-11079- is extracted from the culture. Novel antibiotic A-11079 characterized by collecting _B2
-Production method of _B2 . 3 The bacterium is Amphiulariella sp.A.
A method for producing the novel antibiotic A-11079- _B2 according to claim 2, which is A-11079.