JPS6221354B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to tridecane derivatives, their production methods and their uses. As the producing microorganism of the tridecane derivative of the present invention (hereinafter sometimes referred to as SI-2245 substance), a microorganism capable of accumulating a sufficient amount of the tridecane derivative in its culture is used. There is a Streptomyces sp. SI-2245 strain that was isolated from soil in Shimabara City, Nagasaki Prefecture by the present inventors. The mycological properties of this strain are shown below. Morphological properties Spore-forming hyphae are simply branched, and their morphology is usually spiral. More than 10 spores form a chain. Spore size is 0.63~0.75μ×1
~1.3μ, and its surface is smooth. It does not have flagellated spores or sporangia. The epiphyte location of the sporophyte is on the aerial hyphae. Also, sclerotia are not formed. Growth conditions on various media The conditions after culturing at 30°C for one month are as follows.

【table】

[Table] Physiological properties (1) Growth temperature range Growth temperature: 20-40℃, suitable growth temperature: 30-38℃ Produces reddish-brown pigment at 20℃. (2) Liquefaction of gelatin (3) Good hydrolysis and decomposition of starch (4) Coagulation and peptonization of skim milk No coagulation or peptonization (browning) (5) Production of melanin-like pigments peptone and Turns black on both yeast/iron agar and tyrosine agar Assimilation of carbon source (on Pridham-Gotliep agar) Carbon source growth L-arabinose ++ D-xylose + D-glucose ++ D-fructose ++ Seuucrose ++ Inositol ++ L-Rhamnose ++ Raffinose ++ D-Mannitol ++ No additives ± The above-mentioned Streptomyces sp. SI-
The mycological characteristics of strain 2245 are summarized as follows. (1) Grows on gray aerial hyphae (2) Aerial hyphae are simply branched and spiral-shaped (3) Spore surface is smooth (4) More than 10 spores are linked (5) Melanin-like pigment (6) Skimmed milk does not coagulate, but peptonization occurs slightly. (7) Decomposes starch well (8) Does not produce any special pigments other than light yellow or light brown pigments (9) Utilizes various sugars Known strains with the above properties include Streptomyces. Streptomyces resistomycificus
[International Journal of Systematic
Bacteriology, vol. 2, p. 165, 1968]. However, SI-2245 according to the present invention
When the strain grows on sucrose/nitrate, glycerin/asparagine, or yeast/malt agar media, the color of the underside is pale yellow, whereas that of Streptomyces resistomyciphicus is brown to reddish brown. Furthermore, the pigment produced on the above medium is pale yellow in the SI-2245 strain, whereas it is pale brown in Streptomyces resistomyciphicus. Therefore, strain SI-2245 is thought to be a strain of Streptomyces resistomyciphicus, but is considered to be an unidentified species.
Therefore, the present inventor named this strain Streptomyces sp. SI-2245 strain. This strain is Streptomyces sp.
2245, and its accession number is FERM P-6197. In the present invention, in addition to the above-mentioned strains, all mutant strains obtained by mutating by artificial mutation means that have the ability to produce the tridecane derivative of the present invention can be used. The tridecane derivative of the present invention, SI-2245 substance, can be obtained by culturing the above-mentioned tridecane derivative-producing bacteria and collecting the tridecane derivative from the culture. Cultivation is carried out using a medium containing nutrients that can be used by microorganisms, such as glucose, sucrose, starch, starch syrup, dextrin, glycerin, etc., can be used as carbon sources. Further, as the nitrogen source, ammonium nitrate, ammonium sulfate, meat extract, corn stave liquor, peptone, dried yeast, corn gluten, soy flour, and other organic or inorganic nitrogen compounds can be used. In addition, inorganic salts such as common salt, phosphates, calcium, zinc, magnesium, iron, etc. can be added as appropriate, and substances that support the growth of microorganisms and are useful for the production of tridecane derivatives can be added as appropriate. Cultivation is carried out under aerobic conditions, usually 25-35
C., preferably 28 DEG to 32 DEG C., for 3 to 6 days, preferably 4 to 5 days, to maximize the amount of tridecane derivative accumulated. Tridecane derivatives mainly accumulate within the culture filtrate. Since the tridecane derivative of the present invention has the physicochemical properties described below, extraction and purification are carried out in consideration of these properties. That is, an organic solvent such as ethyl acetate is added to the filtrate obtained by filtering the solid content from the culture, and the mixture is stirred and extracted. The extract is concentrated by an appropriate means, and the filtrate is separated and purified using a silica gel column. A purified tridecane derivative can be obtained by a method such as adsorption with an exchange resin or activated carbon, followed by elution, followed by separation and purification using a silica gel column as described above. The tridecane derivative thus obtained has the following structural formula () 1R, 3S, 4R, 5R, 6R, 8S, 10S, indicated by
12S, 13S-4-acetoxyl, 12-(3-adenyl), 3,5-dihydroxy, 13-methoxy, 6
-methoxycarbonyl, 2,7,11 ^{-trioxatricyclo[8,3,0,03.8 ]} tridecane, and has the following physical and chemical properties. (a) Elemental analysis values C: 48.29%, H: 5.08%, N: 13.94%, O: 32.69% (b) Molecular weight 495 by mass spectrometry (c) Molecular formula C ₂₀ H ₂₅ N ₅ O ₁₀ (d) Ultraviolet absorption Spectrum As shown in Figure 1, λ ^H2O _nax 260
(158.2), λ ⁰ . ^05N . ^HCl _nax 258 (151.4)
, λ ⁰ . ^05N . ^NaOH _nax
260 (130.1). (e) Infrared absorption spectrum As shown in Figure 2, 3600, 3450,
2950, 1760, 1740, 1640, 1610, 1570, 1220,
There is an absorption maximum between 1200 and 1000. (f) Solubility Soluble in water, methanol, acetone,
Poorly soluble in benzene, chloroform, and n-hexane (1) Color reaction Positive for ninhydrin (yellow) (1) Melting point 176-178℃ (1) Color and shape of substance Colorless orthorhombic crystal (2) Basic, acidic, Distinction between neutral Weak basic (according to electrophoresis) Specific rotation [α] ²¹ _D = +24 (C = 0.1, methanol) The tridecane derivative of the present invention was subjected to a multiple dilution method using a potato glucose agar medium. The minimum inhibitory concentrations for various microorganisms determined by the following are as follows.

【table】

[Table] As mentioned above, tridecane derivatives do not show antibacterial properties against most microorganisms in vitro, but
It exhibits antibacterial properties specifically against rice leaf blight fungi in vivo. Therefore, the tridecane derivatives of the present invention are useful as antibiotics. This antibacterial substance is suitable as an agricultural fungicide, and is particularly effective against rice leaf blight in smaller amounts than commercially available fungicides. Agricultural fungicides containing tridecane derivatives as active ingredients can be used alone or in combination with other substances, and can be made into powders, granules, and wettable powders by adding other auxiliary ingredients or diluting with suitable carriers. , an aqueous solution, etc. Note that even if the tridecane derivative is a crude product that has not been sufficiently purified, it can be used for this purpose. Moreover, as the above-mentioned carrier, any carrier known in the art can be used. Next, examples of the present invention will be shown. Example 1 A volume of 500 ml was 5 liters of an aqueous solution containing 2% glucose, 0.1% polypeptone, 0.08% NaCl, 0.12% K ₂ HPO ₄ and 0.4% cornstarch liquor adjusted to pH 7.2.
Dispense 100 ml into 50 Kolben tubes, and add Streptomyces sp. SI-2245 strain (FERMP-) to each sterilized medium.
6197) was inoculated one platinum loop at a time. After inoculation, rotary shaking culture (160r.pm) at 30℃ for 72 hours.
Then, the culture was filtered to remove bacterial cells. filtrate
Add ethyl acetate 5 to 4.5 and stir for 10 minutes,
The ethyl acetate layer was separated and dehydrated by adding anhydrous sodium sulfate. Then, concentrate under reduced pressure at 40℃ to obtain a concentrate.
0.25g was obtained. 20g of silica gel (manufactured by Mallinckrodt) with a diameter of 20
The concentrate was dissolved in a small amount of methanol and benzene and placed on the top of the column for adsorption. Next, impurities were removed by sequentially flowing 500 ml of benzene, 500 ml of benzene:ethyl acetate (9:1), and 1200 ml of benzene:ethyl acetate (1:1). Thereafter, ethyl acetate was poured into the solution and fractionated using a fraction collector. Fractions containing tridecane derivatives were collected and concentrated under reduced pressure at 40°C to obtain a concentrate of 0.038
I got g. This concentrate was dissolved in a small amount of methanol and allowed to stand at room temperature for 7 days to obtain 0.02 g of colorless orthorhombic crystals of a tridecane derivative. This substance had the physicochemical properties described above. Example 2 30 volumes of an aqueous solution containing 3.6% glucose, 0.18% polypeptone, 0.14% NaCl, _0.14 % _K2HPO4 , and 0.72% cornstarch liquor adjusted to pH 7.2
After sterilization, 100 ml of a seed culture (Streptomyces sp. SI-2245 strain, FERM P-6197), which had been cultured in Kolben, was inoculated into a 60 jar fermenter. After inoculation, sterile air was aerated at 30 °C per minute and culture was performed with stirring at 200 rpm. The culture was filtered 72 hours after the start of the culture, and the sterilized filtrate was passed through a column with a diameter of 100 mm packed with eight ion exchange resins, Amberlite XAD-2, to adsorb the active ingredients. Then, remove the adsorbed active ingredients with acetone for 30 minutes.
The eluate was concentrated under reduced pressure at 40°C to obtain concentrate 3.
I got g. 50g of silica gel (manufactured by Mallinckrodt) with a diameter of 20
The concentrate was dissolved in a small amount of methanol and benzene and placed on the top of the column for adsorption. Thereafter, ethyl acetate was passed through and fractionated using a fraction collector. Fractions containing tridecane derivatives were collected and concentrated under reduced pressure at 40°C to obtain a concentrate of 0.8
I got g. This concentrate was dissolved in a small amount of methanol and allowed to stand at room temperature for 7 days to obtain 0.58 g of colorless orthorhombic crystals of a tridecane derivative. Example 3 (Aqueous solvent) 2 parts of tridecane derivative, 15 parts of Solpol 2495G (manufactured by Toho Chemical Co., Ltd.), 50 parts of methanol, and 33 parts of water are mixed and dissolved. Dilute this agent 100 to 400 times with water and spray. Example 4 (Wettable powder) 10 parts of tridecane derivative, 0.9 parts of Detargent 60 (manufactured by Lion Corporation), Solpol 800A (manufactured by Toho Chemical Co., Ltd.)
Co., Ltd.) and 87.3 parts of Zeeklite (Zeeklite Kogyo Co., Ltd.) were mixed and ground. Add this agent to water for 500~
Dilute it 2000 times and spray it. Test Example 1 Preventive effect test of tridecane derivative against rice white leaf blight Paddy rice at 5-6 true leaf stage (variety: Kinnanpu, potted)
A drug containing the test compound at a predetermined concentration was sprayed on the leaves, and 24 hours after spraying, a suspension of the test bacteria was inoculated onto the third leaf with a single needle. Thereafter, the plants were kept in a greenhouse, and after 5 days, the degree of disease was calculated by the following method. The results are shown in Table 3. 0: No disease symptoms 1: Disease symptoms are slightly observed around the inoculation site 2: Disease symptoms are less than 1/4 of the length of the inoculation site and the leaf tip 3: Disease symptoms are the same as the length of the inoculation site and the leaf tip 1/4 or more and less than 1/2 4: Disease symptoms are 1/2 or more and less than 3/4 of the length of the inoculation site and leaf tip 5: Disease symptoms are 3/4 or more of the length of the inoculation site and leaf tip = 1×n ₁ +2×n ₂ +3×n ₃ +4×n ₄ +5×n ₅ /
5 x Total number of research stocks (N) x 100 N = n ₀ + n ₁ + n ₂ + n ₃ + n ₄ + n ₅

【table】 [Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of the tridecane derivative in methanol, and Figure 2 shows the infrared absorption spectrum of the tridecane derivative measured by the KBr method.

Claims (1)

[Claims] 1 formula A tridecane derivative represented by and having the following properties. (a) Elemental analysis values C: 48.29%, H: 5.08% N: 13.94%, O: 32.69% (b) Molecular weight 495 by mass spectrometry (c) Molecular formula C ₂₀ H ₂₅ N ₅ O ₁₀ (d) Ultraviolet absorption spectrum As shown in FIG. (e) Infrared absorption spectrum As shown in Figure 2. (f) Solubility Soluble in water, methanol, acetone, slightly soluble in benzene, chloroform, n-hexane (g) Color reaction Positive for ninhydrin (yellow) (h) Melting point 176-178℃ (li) Color of substance and shape Colorless orthorhombic crystal (nu) Distinction between basic, acidic, and neutral Weak basicity (by electrophoresis) (ru) Specific rotation [α] ²¹ _D = +24 (C = 0.1, methanol) 2 Streptomyces belongs to the genus, formula A tridecane derivative having the following properties (a) Elemental analysis values C: 48.29%, H: 5.08% N: 13.94%, O: 32.69% (b) Molecular weight 495 by mass spectrometry (c) Molecular formula C ₂₀ H ₂₅ N ₅ O ₁₀ (d) Ultraviolet absorption spectrum As shown in Figure 1. (e) Infrared absorption spectrum As shown in Figure 2. (f) Solubility Soluble in water, methanol, acetone,
Slightly soluble in benzene, chloroform, and n-hexane (1) Color reaction Positive for ninhydrin (yellow) (1) Melting point 176-178℃ (1) Color and shape of substance Colorless orthorhombic crystal (2) Basic, acidic, Distinction between neutrality Weakly basic (by electrophoresis) (le) Specific rotation, [α] ²¹ _D = +24 (C = 0.1, methanol) A microorganism that has the ability to produce is cultivated, and the above tridecane is extracted from the culture. A method for producing a tridecane derivative, which comprises collecting the derivative. 3. The method according to claim 2, wherein the microorganism belonging to the genus Streptomyces and having the ability to produce tridecane derivatives is Streptomyces sp. SI-2245 (FERMP-6197). 4 formula A tridecane derivative having the following properties (a) Elemental analysis values C: 48.29%, H: 5.08% N: 13.94%, O: 32.69% (b) Molecular weight 495 by mass spectrometry (c) Molecular formula C ₂₀ H ₂₅ N ₅ O ₁₀ (d) Ultraviolet absorption spectrum As shown in Figure 1. (e) Infrared absorption spectrum As shown in Figure 2. (f) Solubility Soluble in water, methanol, acetone, slightly soluble in benzene, chloroform, n-hexane (g) Color reaction Positive for ninhydrin (yellow) (h) Melting point 176-178℃ (li) Color of substance and shape Colorless orthorhombic crystal (nu) Distinction between basic, acidic, and neutral Weak basicity (by electrophoresis) (ru) Specific rotation [α] ²¹ _D = +24 (C = 0.1, methanol) as active ingredient Agricultural fungicide containing as. 5. The agricultural fungicide according to claim 4, which is a rice leaf blight control agent.