JPS6228959B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to octane derivatives, their production methods and their uses. As the producing microorganism of the octane derivative of the present invention (hereinafter sometimes referred to as SI-4228 substance), a microorganism that can accumulate a sufficient amount of the octane derivative during its culture is used. There is a Streptomyces sp. SI-4228 strain that was isolated from a forest in Miyakonojo City, Miyazaki Prefecture. The mycological properties of this strain are shown below. Morphology (1) Branching method of spore-forming hyphae Simple branching (2) Morphology of spore-forming hyphae Linear (3) Number of spores Chain of 10 or more spores (4) Surface chain of spores Smooth (5) Spore size 0.75 μm×1.25μm (6) Flagellated spores None (7) Sporangium None (8) Sporophyte settlement position On aerial hyphae (9) Sclerotium formation None Growth status on various media (1) Nutrient agar medium Growth: Slight Bad, the back side is pale yellow around the edges (2.5Y, 8.5/6, Munsell Book of
By color. (same below), transparent in the center Aerial mycelium: not attached Soluble pigment: not produced (2) Glycerin/asparagine agar medium Growth: somewhat poor, back side pale reddish brown (10R,
3/4) Aerial mycelium: Slightly reddish gray, growing all over in powder form (5YR, 6/1) Soluble pigment: Not produced (3) Seuculose/nitrate agar medium Growth: Normal growth The underside is shrimp brown at the periphery (7.5R, 2/6), and bright reddish brown at the center (7.5R, 4/6) Aerial mycelium: Sparsely attached, but relatively well and slightly red at the periphery. Flavored gray (2.5YR, 7/2) Soluble pigment: Pale reddish brown (4) Glucose-asparagine agar medium Growth: Slightly poor, underside ocher (10YR,
5/6) Aerial mycelium: Slightly reddish gray (5YR, 7/2), grown in powdery form over one surface (5YR, 7/2) Soluble pigment: Not produced (5) Starch/inorganic salt agar medium Growth: Ordinary It grows, and the underside is ivory-colored at the periphery (5Y, 8.5/6) and white at the center Aerial mycelium: It grows in powdery form on the entire surface and is grayish-white (10R, 8/1) Soluble pigment: Not produced ( 6) Tyrosine agar medium Growth: Normal growth, brownish-brown underside (2.5YR, 2/2) Aerial mycelium: Vigorously epiphytic on one side, gray (10R,
5/1) and velvety Soluble pigment: Light brown (7) Yeast/malt agar medium Growth: Normal growth, ocher underside (10YR, 6/6) Aerial mycelium: Sparsely attached, but Relatively well settled in the peripheral areas and gray (10YR, 6/
1) Soluble pigment: Pale yellow (8) Oatmeal agar medium Growth: Normal growth, ocher color on the periphery of the underside (7.5YR, 5/6), colorless in the center Aerial mycelium: Sparsely attached However, it adheres relatively well in the peripheral areas and is gray (5YR, 7/1) Soluble pigment: pale yellow Physiological properties (1) Growth temperature range Growth temperature: 20-45℃, suitable growth temperature: 35-42℃
(Both bacterial cells and medium turn yellow at around 40℃) (2) Liquefaction of gelatin Liquefaction〓〓〓〓〓
(3) Hydrolysis of starch Strongly degraded (4) Coagulation and peptonization of skim milk No coagulation or peptonization (browning) (5) Production of melanin-like pigments Peptone/yeast/iron agar medium, tyrosine agar All of the media turn black. Assimilable carbon source growth L-arabinose ++ D-xylol ++ D-glucose ++ D-fructose ++ Seuclose ++ Inositol ++ L-rhamnose ++ Raffinose ++ D-mannitol ++ No addition ++ Above Streptomyces sp. SI-4228
The mycological characteristics of the strain are summarized as follows. (1) Epiphytes with gray aerial hyphae (2) Aerial hyphae are simply branched and linear (3) Spore plane is smooth (4) Spores are linked with 10 or more spores (5) Melanin-like Produces pigments (6) Does not coagulate or peptonize skimmed milk (7) Decomposes starch well (8) Does not produce characteristic pigments other than light yellow or light brown pigments (9) Various sugars A known strain with the above properties is Streptomyces phaeopurpureus (International
Journal of Systematic Bacteriology, Volume 3, No.
358 pages, 1968). however,
SI-4228 on oatmeal, glycerin, asparagine, sucrose, nitrate, etc. agar media
The underside of the plant's growth is pale yellow or light brown, whereas Streptomyces pheoppurpureus is reddish-brown. In addition, the pigment produced on the above medium is also
The SI-4228 strain is light yellow to light brown in color, while Streptomyces phaeopurpureus is light brown to reddish brown in color. Therefore, based on these facts, strain SI-4228 is considered to be a strain of Streptomyces phaeopurpureus, but it is considered to be an unidentified species. Therefore, the present inventor named this strain Streptomyces sp. SI-4228 strain. This strain is Streptomyces sp.
It has been deposited with FERMP-6198 as strain 4228, and its accession number is FERMP-6198. In the present invention, in addition to the above-mentioned bacterial strains, any mutant strain obtained by mutating by artificial mutation means that has the ability to produce the octane derivative of the present invention can be used. The octane derivative of the present invention can be obtained by culturing the above-mentioned octane derivative-producing bacteria and collecting the octane derivative from the culture. Cultivation is carried out using a medium containing nutrients that can be used by microorganisms; for example, 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 steep 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, and iron may be added as necessary, and substances useful for supporting the growth of microorganisms and producing octane derivatives may be added as appropriate. Cultures are carried out under aerobic conditions, usually 25-34
C., preferably 28 DEG to 32 DEG C., for 3 to 10 days, preferably 4 to 6 days, to maximize the accumulation of octane derivatives. Octane derivatives are accumulated not only in the culture solution but also in the bacterial cells. Since the octane 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 culture to perform extraction, and the resulting extract is concentrated by an appropriate means and then transferred using benzene. Next, the culture solution to be separated and purified using a silica gel column is subjected to ion exchange resin or activated carbon adsorption, followed by elution, followed by separation and purification using a silica gel column as described above, and further gel filtration. Purified octane derivatives can be obtained by methods such as crystallization.
Ru. The octane derivative obtained in this way has the following structural formula: 2-ethyl-4,8-dihydroxy-
6-[1-[2,4-dihydroxy-3-formyl-5-(1-methoxy-2-methylpropyl)
Benzoyl]-butyl]1,5-dioxaspiro[2,5]octane, and has the following physical and chemical properties. (a) Elemental analysis values C: 62.1%, H: 7.4%, N:
0% (b) Molecular weight 480 (by vapor pressure method, solvent: chloroform) (c) Infrared absorption spectrum
As shown in FIG. (d) Ultraviolet absorption spectrum
As shown in FIG. (e) Nuclear magnetic resonance spectrum
As shown in FIG. (f) Specific rotation [α] ²¹ _D = +54
(C=0.1, methanol) (g) Solubility Soluble in methanol, ethanol, acetone, ethyl acetate, benzene, ether, chloroform, carbon tetrachloride, insoluble in water, n-hexane (h) Neutral (electrophoresis method (Li) Color reaction Positive for ferric chloride, 2,4-dinitrophenylhydrazine, negative for ninhydrin (N) Melting point 114-116℃ (L) Color of substance White needle-like crystals Regarding octane derivatives Potato - The minimum inhibitory concentration for various microorganisms determined by the multiple dilution method using a glucose agar medium is as follows.

[Table] 〓〓〓〓〓

[Table] The octane derivative of the present invention is useful as an antibiotic for agricultural and pharmaceutical purposes, and is particularly suitable as an agricultural fungicide. The agricultural fungicide containing the octane derivative of the present invention as an active ingredient has a sufficient control effect, especially against botrytis, anthracnose, sheath blight, and caterpillar blight at a lower dose than existing fungicides. show. Next, examples of the present invention will be shown. Example 1 100 ml of medium 10 containing 3% glucose, 0.1% polypeptone, 0.08% NaCl, 0.12% K ₂ HPO ₄ and 0.4% corn steep liquor adjusted to pH 6.8 was added to 100 Meyer flasks each having a capacity of 500 ml. It was dispensed and sterilized at 120°C for 15 minutes. Streptomyces sp. SI-4228 strain (FERM P) was added to this medium.
-6198) was inoculated in one platinum loop from a slant medium.
After inoculation, culture with rotary shaking at 32℃ for 96 hours (rotation speed
200r.pm). This culture was collected, 6 ethyl acetate was added, and after stirring for 20 minutes, the ethyl acetate layer was collected. The ethyl acetate layer was dehydrated by adding anhydrous sodium sulfate, and then concentrated to dryness under reduced pressure at 40°C. 1 part of benzene was added to the concentrate, and insoluble matter was removed by filtration. The solution was concentrated under reduced pressure to 5 ml. Benzene was added to 30 g of silica gel (manufactured by Merck & Co.) and packed into a 20 mm glass column, and the concentrate was placed on the top of the column to be adsorbed. Next, a solution of n-hexane:acetone (15:1) was flowed, and the octane derivative elution fraction was collected with a fraction collector. This fraction was concentrated under reduced pressure at 40°C to dryness, and then
ml of chloroform was added to dissolve. Sephadex LH-20 was dispersed in chloroform and then packed into a glass column with a diameter of 15 mm and a length of 1 m. A chloroform solution containing an octane derivative was placed on the top of the column, and purification using molecular sieves was performed using chloroform as a developing solution. Fractions containing octane derivatives were collected with a fraction collector, concentrated to dryness, a small amount of acetone was added, and further n-hexane was added, and the mixture was left indoors for 2 days to obtain 45 mg of needle-shaped crystals. This substance had the physicochemical properties described above. Example 2 Glucose 4%, polypeptone 0.1%, yeast extract 0.5%, NaCl 0.08%, adjusted to PH6.8
K ₂ HPO ₄ 0.18%, Corn Steep Liquor
Inject 30% of medium containing 0.6% into a 60% jar fermentor and sterilize it, then use the seed culture (Streptomyces sp. SI) cultured in a Meyer flask.
-4228, FERM P-6198) 200ml was inoculated.
After inoculation, aerate sterile air at 30 °C per minute at 32 °C.
Stirring culture was performed at 600 rpm for 96 hours. After culturing, the sterilized solution was filled with ion exchange resin Amberlite XAD-2, 8, 100 mm in diameter.
The active ingredients were adsorbed through a 1 m long column. Thereafter, the adsorbed active ingredients were eluted by flowing acetone 20°C. Concentrate the eluate under reduced pressure at 50°C.
72 g of solid material was obtained. Add chloroform to the solid, collect the soluble fractions, concentrate to dryness at 40°C,
g of solid material was obtained. On the other hand, Sephadex LH-20 was dispersed in chloroform and packed into a glass column with a diameter of 40 mm and a length of 1.5 m. A solid substance dissolved in a small amount of chloroform was placed on the top of the column, and fractions containing octane derivatives were collected using a fraction collector using chloroform as a developing solution. This fraction was concentrated and purified using Sephadex LH-20 under the same conditions one more time to collect the octane derivative-containing fraction. This fraction was concentrated and dried, and then dissolved by adding a small amount of acetone. Further, n-hexane was added and the mixture was left indoors for 2 days to obtain 220 mg of needle-shaped crystals. Example 3 (Emulsion) 40 parts of an octane derivative, 45 parts of xylene, and 15 parts of Solpol 3005X (manufactured by Toho Chemical Industry Co., Ltd.) are mixed and dissolved. Dilute this agent 2,000 to 40,000 times with water and spray. Example 4 (Hydrating agent) 10 parts of octane derivative, 0.9 parts of Detargent 60 (manufactured by Lion Corporation), Solpol 800A (Toho Chemical Co., Ltd.)
Mix and grind 1.8 parts (manufactured by Gyosha) and 87.3 parts of Siegrite (manufactured by Zeeklite Kogyo). 500ml of this drug with water
Dilute ~10,000 times and spray. Test Example 1 Test of the preventive effect of octane derivatives on green bean gray mold. Green beans 30 days after sowing (variety: Keystone,
A chemical containing the test compound at a predetermined concentration was sprayed on 5 plants (potted plants), and 24 hours after the spraying, a spore suspension of Botrytis Cinerea (1 x 10 ⁶ cells/ml) was applied to a paper disk with a diameter of 8 mm. It was placed on the surface of two leaves per plant. For comparison, bacteria were inoculated using the same method on a strain to which no chemicals were sprayed. Thereafter, the green beans were kept in a constant temperature and humidity chamber at a temperature of 20° C. and a humidity of 100%, and after 5 days, the long axis of necrosis formed on the leaf surface was measured from the underside of the leaf, and the degree of disease was calculated using the method described below. Table 3 shows the test results. Morbidity = n ₁ + n ₂ +... n ₉ + n ₁₀ /N ₁ + N ₂
+...N ₉ +N ₁₀ ×100 n: Long axis of lesion on test leaf (mm) N: Unsprayed leaf 〃

[Table] Test Example 2 Preventive effect test against gray mold of eggplant and gray mold of green pepper A prescribed concentration of the drug was applied to five plants each of eggplant (variety: Senryo, potted) and green pepper (variety: Newace, potted) 40 days after sowing. The bacterial flora of Botrytis Cinerea, which had been grown on an agar medium in advance, was
Two leaves were punched out with a mm cork borer and placed on each plant. Thereafter, a test was conducted in the same manner as Test Example 1. The results are shown in Tables 4 and 5.

【table】

[Table] Test Example 3 Preventive effect test against cucumber anthracnose Cucumber (variety: Ochiai, potted) 7 days after sowing 5
A drug containing a predetermined concentration of the test compound is sprayed on the stock,
24 hours after spraying, a spore suspension of Colletotrichum Iagenarium (1×10 ⁶ spores/ml) was sprayed. For comparison, bacteria were also sprayed using the same method on strains that were not sprayed with chemicals. Thereafter, the specimens were kept in a constant temperature and humidity chamber at a temperature of 23° C. and a humidity of 100% for 5 days, and the degree of morbidity was calculated using the following method. The results are shown in Table 6. Disease severity = 1 x a + 2 x b + 3 x c + 4 x d / 5 x total number of inspected leaves (N) a, b, c, d Number of leaves for each score 0: No disease symptoms 1: Disease symptoms less than 10% 2: Disease Symptoms are 10% or more and less than 50% 3: Disease symptoms are 50% or more and less than 75% 4: Disease symptoms are 75% or more〓〓〓〓〓

[Table] Test Example 4 Preventive effect test against sheath blight A chemical solution of a prescribed concentration was sprayed on 10 cut leaves of broad bean 5 weeks after sowing, and the leaves were placed in a humid room at 28°C for 24 hours. Meanwhile, pre-incubate on potato glucose agar medium
Rice sheath blight fungus (Pelli-
cularia sasakii) was punched out using a cork borer with a diameter of 4 mm, inoculated onto broad bean leaves 24 hours after spraying the above chemical solution, and kept in a humid room at 28° C. for 5 days to observe the condition of lesions. The results are shown in Table 7.

[Table] Test Example 5 Preventive effect test against rice blast disease A chemical solution of a predetermined concentration was sprayed on 4 pots (10 plants per pot) of rice (variety Norin No. 29) at the 4-leaf stage and air-dried. After air drying, a spore suspension of Pyricularia oryzae (3 x 10 ⁵ spores/ml) was sprayed and kept in a greenhouse at 25°C for 4 days, after which the number of lesions that appeared on the apical leaf surface was counted. The control value was calculated by comparing the measurement with the area without chemical application. The results are shown in Table 8. Control value = [1 - number of lesions in chemical treated area/number of lesions in area without chemical application] x 100

【table】 [Brief explanation of the drawing]

Figure 1 shows an infrared absorption spectrum of an octane derivative by the KBr method, Figure 2 shows an ultraviolet absorption spectrum of an octane derivative in methanol, and Figure 3 shows a nuclear magnetic resonance spectrum of an octane derivative in deuterated chloroform. 〓〓〓〓〓

Claims (1)

[Claims] 1 formula An octane derivative having the structural formula shown and the following properties. (a) Elemental analysis values C: 62.1%, H: 7.4%, N:
0% (b) Molecular weight 480 (by vapor pressure method, solvent: chloroform) (c) Infrared absorption spectrum
As shown in FIG. (d) Ultraviolet absorption spectrum
As shown in FIG. (e) Nuclear magnetic resonance spectrum
As shown in FIG. (f) Specific rotation [α] ²¹ _D = +54
(C=0.1, methanol) (g) Solubility Soluble in methanol, ethanol, acetone, ethyl acetate, benzene, ether, chloroform, carbon tetrachloride, insoluble in water, n-hexane (h) Neutral (electrophoresis method (Li) Color reaction Positive for ferric chloride, 2,4-dinitrophenylhydrazine, negative for ninhydrin (N) Melting point 114-116℃ (L) Color of substance White needle-like crystals 2 Streptomyces sp. Belongs to the formula〓〓〓〓〓
An octane derivative having the structural formula shown by and having the following properties, (a) Elemental analysis values C: 62.1%, H: 7.4%, N:
0% (b) Molecular weight 480 (by vapor pressure method, solvent: chloroform) (c) Infrared absorption spectrum
As shown in FIG. (d) Ultraviolet absorption spectrum
As shown in FIG. (e) Nuclear magnetic resonance spectrum
As shown in FIG. (f) Specific rotation [α] ²¹ _D = +54
(C=0.1, methanol) (g) Solubility Soluble in methanol, ethanol, acetone, ethyl acetate, benzene, ether, chloroform, carbon tetrachloride, insoluble in water, n-hexane (h) Neutral (electrophoresis method (Li) Color reaction Positive for ferric chloride, 2,4-dinitrophenylhydrazine, negative for ninhydrin (N) Melting point 114-116℃ (L) Color of substance Ability to produce white needle-shaped crystals 1. A method for producing an octane derivative, which comprises culturing a microorganism having the following and collecting the above-mentioned octane derivative from the culture. 3. The method according to claim 2, wherein the microorganism belonging to the genus Streptomyces and having the ability to produce octane derivatives is Streptomyces sp. SI-4228 (FERM P-6198). 4 formula An octane derivative having the structural formula shown by and having the following properties, (a) Elemental analysis values C: 62.1%, H: 7.4%, N:
0% (b) Molecular weight 480 (by vapor pressure method, solvent: chloroform) (c) Infrared absorption spectrum
As shown in FIG. (d) Ultraviolet absorption spectrum
As shown in FIG. (e) Nuclear magnetic resonance spectrum
As shown in FIG. (f) Specific rotation [α] ²¹ _D = +54
(C=0.1, methanol) (g) Solubility Soluble in methanol, ethanol, acetone, ethyl acetate, benzene, ether, chloroform, carbon tetrachloride, insoluble in water, n-hexane (h) Neutral (electrophoresis method (Li) Color reaction Positive for ferric chloride, 2,4-dinitrophenylhydrazine, negative for ninhydrin (N) Melting point 114-116℃ (L) Color of substance White needle crystals as active ingredient Contains agricultural fungicides. 〓〓〓〓〓
5 Claim No. 4, which is a botrytis blight control agent
Agricultural fungicides as described in section. 6. The agricultural fungicide according to claim 4, which is an anthrax control agent. 7. The agricultural fungicide according to claim 4, which is a sheath blight control agent. 8. The agricultural fungicide according to claim 4, which is a blast control agent.