JPH02121994A - Physiologically active substance ksb-1939l3 compound and production thereof - Google Patents

Abstract

NEW MATERIAL:A KSB-1939L3 compound expressed by the formula (R represents 2-methylpropanoyl or butanoyl). The KSB-1939L3alpha in which R is 2-methylpropanoyl possesses the following physicochemical properties. Color; colorless. Elementary analysis (%); C, 68.41; H, 8.14; O, 23.45. Molecular formula; C35H50O9. Molecular weight; 614(FDMS). Color reaction on the silica gel thin layer; iodo reaction +, sulfric acid reaction +, ninhydrin reaction -, anisaldehyde reaction -, ferric chloride reaction -, 2,4-dinitrophenylhydrazine reaction +. Solubility; insoluble in water, soluble in methanol, acetone, ethyl acetate, chloroform and hexane; neutral, etc. USE:An insecticide, miticide or vermicide. PREPARATION:A KSB-1939L3 compound-producing bacterium belonging to Genus Streptomyces [example; Streptomyces hygroscopicus KSB-1939 (FERM BP-1901)] is cultured preferably with submerged tank culture method at 26-30 deg.C for 5-10 days.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a novel physiologically active substance KSB-1939L, which exhibits insecticidal, acaricidal, and anthelmintic activity, and a method for producing the compound.

(Prior art) It is undeniable that Qm agents are indispensable for stably securing high yields, but they can also cause light to the human body through their residue in crops and soil. We cannot deny the possibility that it may cause harmful effects such as toxicity and environmental pollution.
The development of even safer agricultural chemicals is desired, and microbial products are now 5! ! It has been developed as a drainage agent.

So far, insecticidal substances originating from microorganisms include filamentous [Agricultural and Biological Chemistry (Agricultural and Biological Chemistry)]
Agriculturalland Biologica
l Chemistry) Volume 26, Page 36 (196
2)], delta-endotoxin, a crystalline protein produced by Bacillus bacterium [Agriculture and Horticulture Vol. 47, No. 1
No. 1, p. 1507 (1972)], pyrone-based piericidins produced by actinomycetes (Agricultural and Biological Chemistry Vol. 30, No. 517)
(196fE)], tetranactin, which belongs to the macrotetrolide group of electron transport inhibitors whose mother nucleus is pyrrolidone [Journal Antibiotyphoid Fever (Journal Antibiotyphoid)]
al Antibiotics) Volume 24A, No. 418
(1971)] 5 Milbemycin, a macrolide similar in life to the compound of the present invention [The Journal of Antibiotics
Antibiotics) Volume 29, Page 76 (19
1976)], abamemethitine antimicrovial
Anti+oi
crobial Agents and Chsmoth
erpy) Volume 15, Page 361 (1979)), L
L-F28249α [Journal of Chemical Society Chemical Communication (Jour
nal of Chemical 5ociet
y Chemical Communicatio
n) No. 402 (1987)] is an intellectual property.

(Problems to be Solved by the Invention) An object of the present invention is to provide a novel natural physiologically active substance.

(Means for Solving the Problems) The present inventors isolated bacterial strains from various soils for the purpose of searching for novel physiologically active substances produced by microorganisms, and as a result of continuing research on the metabolites produced by the strains, they found that We discovered that active substances exhibiting strong insecticidal, acaricidal, and anthelmintic effects were produced in the culture of KSB-1939 strain isolated in

That is, the physiologically active substance of the present invention is represented by the formula (wherein O1+ R represents a 2-methylpropanoyl group or a butanoyl group).

The three compounds of the physiologically active substance KSB-1939r of the present invention include α and β.

KSB-1939L of the present invention, α is the general formula above, and R is 2
-Indicates a methylpropanoyl group and has the following physical and chemical properties.

■ Color of substance: colorless ■ Elemental analysis value: C; 68.41% H; 8.14% 0; 23.45% ■ Molecular formula: C3s l1sn Os1 ■ Molecular weight
: 614 (FDMS) ■Ultraviolet absorption spectrum:
Shown in Figure 1.

■Infrared absorption spectrum: Shown in Figure 2.

■Nuclear magnetic resonance spectrum: Shown in Figure 3.

■Imperial reaction Iodine reaction on a thin layer of Nijiri gel; + Sulfuric acid reaction; + Ninhydrin reaction; - Anisaldehyde reaction; - Ferric chloride reaction; - 2,4-dinitrophenylhydrazine reaction: + ■ Solubility: Insoluble in water , methanol, acetone, acid resistant ethyl,
Soluble in chloroform and hexane.

0 Basic, acidic, neutral classification: Neutral Furthermore, KSB-1939L and β of the present invention have the above general formula, where R represents a butanoyl group, and have the following physical and chemical properties.

■Substance color: colorless Elemental analysis value: C; 68.40% H; 8.14% 0; 23.45% ■Molecular formula: C,,H,. ○.

- Molecular weight: 614 (FDMS) +5) Ultraviolet absorption spectrum; shown in Figure 4.

■Infrared absorption spectrum: Shown in Figure 5.

■Nuclear magnetic resonance spectrum: Shown in Figure 6.

・9-color reaction Iodine reaction on a thin layer of silica gel; + Sulfuric acid reaction; + Ninhydrin reaction; - Anisaldehyde reaction; - Ferric chloride reaction; - 2,4-dinitrophenylhydrazine reaction; + ■Solubility 8 In water Insoluble, methanol, acetone, ethyl acetate,
Soluble in chloroform and hexane.

[Phase] Basic, acidic, neutral classification: Neutral then KSB-1
939L, the structural analysis of the compound will be explained.

From the analysis of the physical and chemical properties of the KSB-1939L3 compound obtained according to the examples, especially the ultraviolet absorption spectrum, proton and carbon nuclear magnetic resonance spectra, it was found that this substance is a 16-shell macrolide, and the quality analysis data (
There is no known macrolide substance corresponding to the molecular weight of 614 determined by El and FD), suggesting its novelty.

Milbemycins, avanuctins, LL-F28249α, etc., which are considered to be structurally related to the compounds of the present invention, are all substances called insecticidal macrolides produced by LA bacteria belonging to Streptomyces, and regarding their structural analysis. , Abstract Papers No. 309 for milbemycin.
-316 pages [18th Symposium Chemical Natural Products Kyoto (SymposiumCh
chemical natural products, K
yoto) (held from 17th to 19th IO, 1974)
] and Tetrahedr
on Letters), pp. 711-714 (1975
Abamectin was published in the Journal of
American Chemical Red Society (Journal)
of American Che+5ical 5oc
iety) Volume 103, pp. 4216-4221 (19
1981), and LL-F28249 is described in the above-mentioned literature.

As a result of examining the various data of KSB-1939L and the compound of the present invention with reference to the structural analysis data shown in these documents, we found that: ■ The basic skeleton was found in The Journal of Antibiotics, Volume 33, Page 1121. (1980) with the lactone ring described above [! ], and ■ there is no sugar side chain found in abamectin at position 13 of the macrolide ring.

■It has a methyl group at the 24th position and a methyl group at the 25th position on the spiro ring. ■The characteristics of the KSB-1939LJ compound are in the side chain connected to the carbon at the 4th position, and L3α is located at the 4th position of the macrolide ring. 2-methylpropanoyloxymethyl group is connected to carbon, L
, β was analyzed to have a butanoyloxymethyl group connected thereto, and was determined to be a new substance represented by the above-mentioned formula (1).

An example of a bacterium producing KSB-1939L, a compound of the present invention, is actinomycete strain KSB-1939, which was isolated from the soil of Shingu City in the Wakayama region by the present inventors. KSB-
The mycological properties of the 1939 strain are as follows.

This strain was identified using the methods of the International Streptomyces Program (ISP) and Mr. Waxman's method, and the description is provided by ISP.
This was done according to the example.

(1) Morphological properties Substrate hyphae grow while branching, and hyphal fragmentation is not observed.Aerial hyphae are actively attached to glucose-asparagine agar medium, oatmeal agar medium, etc., and spore formation is also good. .

Aerial hyphae extend into the air while branching, and uniaxially branch spore chains on short main axes or branches. Spore chains usually exhibit a coiled helix.

When cultured for a long period of time, the tips of the spore chains turn into black sticky lumps, and in further advanced parts, the entire aerial hyphae turn into 1 (- brown sticky lumps).
(glucose-asparagine agar, oatmeal agar), the width of the spore chain is 0.5 to 0.8 microns, and the spore surface is wrinkled and the spore divisions are unclear, so detailed individual spores cannot be observed. Although the shape and number of connections are unknown, it is suggested that the IO cell Y- or more connections are present. In addition, spore f- does not exhibit JtjJ properties, and spore-n, sclerotia, etc. are not observed.

(2) Diaminopimelic acid analysis LL-diaminopimelic acid of whole bacterial cell hydrolyzate! 2. Wet cell wall type! It is a type.

(3) Growth status after 3 weeks of culture in various media is shown in Table 1.

Table 1 (4) Physiological properties ■Growth temperature range Optimum temperature range 20-40℃ 25-35℃ (Liquification of gelatin: + ■Hydrolysis of starch: + ■Coagulation of skim milk: + Peptonization: + ■ Production of melanin-like pigments Tyrosine agar medium Peptone yeast iron agar medium Nidributone yeast broth: ■ Reduction of nitrate: + (5) Assimilation of each carbon source (Brideham-Godelive agar medium) 2) L-arabinose , D-kinlose, D-glucose, D-fructose, sucrose, L-rhamnose, raffinose,
Assimilate all carbon sources of r)-mannitol, L-inositol.

The above mycological properties were published in the International Journal
of Systematic Bacteriology (Inter
National Journal of 5ysti*
atic Bacterio14y) Volume 22, No. 30
As a result of identification with reference to the description on page 7-311 (1972), the KSB-1939 strain showed slight differences in the assimilation of sucrose and raffinose, but the basic properties (morphology and differences in various media) growth condition fi) is Streptomyces bigroscopicus (StrepLomyces).
hygroscopicus).

Streptomyces vigroscopicus KSB-19
It was named 39. The KSB-1939 strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, on June 26, 1988, as part of the Microbiological Research Institute No. 1901 (FERM BP-190).
It has been deposited as 1). Note that the KSB-1939 strain is prone to change in properties, as seen in the case of other actinomycetes. Any fusion or genetic f recombinant that depends on the KSB-1939 strain to produce the KSB-1939L3 compound can be used in the present invention.

Further, the production method of the present invention comprises culturing the KSB-1939 strain and extracting and purifying the physiologically active substance from the culture.

That is, during culturing, the KSB-1939 strain is cultured in a medium containing vine nutrients that are commonly used by microorganisms. As Sakae 1fX, known materials conventionally used for culturing actinomycetes can be used. For example, as a carbon source, glucose, glycerin, maltose, lactose, starch syrup, dextrin, sterilized powder, animal and vegetable oils, etc. can be used. Also! Y
As the raw material, beast, soybean flour, wheat germ, cornstarch liquor, peanut flour, cottonseed flour, peptone, meat extract, yeast extract, ammonium sulfate, sodium nitrate, urea, etc. can be used. In addition, sodium, potassium, calcium, magnesium, cobalt, chlorine,
It is also effective to add inorganic salts capable of producing phosphoric acid, sulfuric acid and other ions. It also helps the growth of bacteria.

Organic and inorganic substances that promote the production of KSB-1939L can also be appropriately added.

The most suitable culture method is a culture method under aerobic conditions, especially a deep tank culture method. The appropriate temperature for culturing is 15
-37°C, but in many cases, it is cultured at around 26-30°C.

The production of KSB-1939L and the compound varies depending on the medium and culture conditions, but it takes 5 to 10 minutes for both shaking culture and tank culture.
Its culture reaches its maximum during the day.

KSB-1939L, a compound produced in this way, can be extracted and purified from the culture according to its physicochemical properties, and in particular, it can be extracted and purified efficiently by the following method.

That is, KSB-1939L, since most of the compound is produced in the cultured bacterial cells, the cultured cells are separated by filtration or centrifugation, the bacterial cells are separated, and the bacterial cells are extracted with acetone or alcohol. , After concentrating the extract under reduced pressure, it is dissolved in a transferable solvent such as ethyl acetate or chloroform, or
Immediately, the culture or concentrate is extracted with a transferable solvent as described above. When the inverted solution is concentrated and dried, KSB-193
9L, a powder or oily crude product containing the compound can be obtained.

Crude product is KSB-1939L, multiple KS containing compounds
Because it is obtained as a mixture of insecticidal related substances of the B-1939 series.

It is necessary to perform fractional purification by a known method commonly used for purifying fat-soluble substances, such as column chromatography using a carrier such as silica gel or alumina. By selecting the chromatography conditions, the KSB of the present invention
-1939I7. When fractionating a compound fraction and collecting a purified product, fractionation by high performance liquid chromatography using a normal phase or reverse phase column of the purified product is effective.

In addition, it is also possible to employ methods such as gel filtration, fractionation using an adsorbent, and crystallization.

Next, the present substance will be specifically explained with reference to examples, but the method for producing this substance is not limited thereto.

Example I A piece of the passage slant of strain KSI3-1939 was
Transfer to a 500 mQ Erlenmeyer flask containing 100 mQ of seed culture medium consisting of 1.0% malt extract, 0.4% yeast extract, 0.4% glucose, and 0.2% defatted soybean,
Seed culture was carried out at 28°C for 3 days in a shaking culture machine.Glucose 3%, yeast extract 0.2%, defatted soybean 0.
5%, dipotassium phosphate 0.2%, magnesium sulfate 0
．． 0.05%, Pronal 502 (manufactured by Toho Chemical Industries, Ltd.) 0.05% was placed in a jar fermenter, and after sterilization and cooling, 5 bottles of the aforementioned seed culture were inoculated. Aeration 120Q/min, culture temperature 30
Culture was carried out for 9 days at a temperature of 200 revolutions per minute at 1°C. After the culture was completed, the cells were collected by suction filtration using Celite as an auxiliary agent. The wet bacterial cells were dispersed in IOR's methanol, stirred for 1 hour for extraction, and a clear methanol solution was collected by filtration.The methanol was then removed by vacuum concentration, and 100 ml. Concentrate to methanol 1
After adding 00ml, extraction was carried out twice using 500mΩ of hexane. Approximately IQ of hexane solution was concentrated under reduced pressure to recover 7 g of oily substance. The oily substance was dissolved in a small amount of a developing solution prepared by adding 5 parts by weight of acetone to 1 part by weight of hexane 85F, and silica gel chromatography was performed using a column with an inner diameter of 5 Q mm and a height of 650 mm. KSB-1939L, α, β mixture 3 was collected by collecting the active fractions of 20 mfl fractions and drying them under reduced pressure.
1.1 g of mixed crude powder containing %.

Example 2 2 somg of the crude product described in Example 1 was dissolved in 5 mQ of acetonitrile, and an O
The mixture was injected into a preparative high-performance liquid chromatography system equipped with a DS column. 80% acetonitrile at 10 mQ/min was flowed as an eluent, detected by ultraviolet heat absorption at 240 mm, and the active fraction of the KSB-1939L3 compound was collected. Five fractions were concentrated to dryness to obtain a purity of 80%.
6 mg of KSB-939LJ mixture was obtained. Furthermore,
KSB-193 is used for preparative liquid chromatography equipped with a silica gel column with an inner diameter of 8 mm and a length of 250 mm.
9L, dissolve 6mg of the mixture in a small amount of hexane and inject to make a mixture of hexane and isopropyl alcohol (98:2).
KSB-1939L3a and KSB-1939L3a developed at 4 mQ/min.

By collecting β fractions and drying under reduced pressure, LJα purified product 2m
2 mg of the β-purified product were collected.

The following margin: To use the compound of the present invention as an insecticide and acaricide, the compound of the present invention may be used by itself, but it is usually mixed with a carrier, a surfactant, a dispersant, or an auxiliary agent, etc., and prepared as a powder by a conventional method. , hydrating agent.

It is used in formulations such as emulsions, microgranules, or granules. Supports include talc, bentonite, clay, kaolin, diatomaceous earth, white carbon, vermulite, slaked lime, silica sand,
Solid carriers such as ammonium sulfate and JAM, and toluene, methyl ethyl ketone, methyl naphthalene, isopropyl alcohol,
Liquid carriers such as xylene and cyclohexanone can be mentioned. Examples of surfactants and dispersants include dinaphthylmethane disulfonate, alcohol sulfate ester salt, alkylaryl sulfonate, lignin sulfonate,
Examples include polyoxyethylene glycol ether, polyoxyethylene alkylaryl ether, polyoxyethylene sorbitan monoalkylate, and the like. Examples of adjuvants include carboxymethylcellulose, polyethylene glycol, gum arabic, and the like. These preparations can be diluted at a suitable dose and sprayed, or applied directly.

Next, the present invention will be specifically explained with reference to formulation examples. under! +
i! % in the formulation examples indicates weight percentage.

Formulation Example 1 Powder KSB-1939L, 0.5% α, 5% diatomaceous earth, and 94.5% clay were uniformly mixed and pulverized to obtain a powder.

Formulation example 2 Wettable powder KSFI-1939L□α 2%, diatom ±53%, clay 40%, sodium dinaphthylmethane disulfonate 2%
and 3% sodium ligninsulfonate were uniformly mixed and pulverized to prepare a wettable powder.

Formulation Example 3 Emulsion KSB-1939L, α5%, xylene 30%, polyoxyethylene alkylaryl ether 11%, calcium alkylbenzenesulfonate 4% and methylnaphthalene 50% were uniformly dissolved to prepare an emulsion.

Formulation Example 4 Granules 0.5% of KSB-1939L3α, 2% of sodium salt of lauryl alcohol sulfate, 5% of sodium lignin sulfonate, 2% of carboxymethyl cellulose and 5% of clay 90° are uniformly mixed and ground. 20 parts by weight of water were added to 100 parts by weight of this mixture, kneaded, processed into granules of 14 to 32 mesh using an extrusion type granulator, and then dried to form granules.

(Effects of the Invention) The compounds of the present invention are useful as agricultural insecticides and acaricides, insecticidal medical products, deworming agents for livestock beds, insect repellents and fungicides, and sanitary insecticides and acaricides.

Next, the effects of the compounds of the present invention will be specifically explained using test examples.

Test Example 1 Adult insecticidal test for two-spotted spider mites Pour water into a polyethylene cup with a diameter of 55 mm, cover with a lid with a hole of 10 mm in diameter in the center, and drop absorbent cotton from the hole in the lid to suck up the water. Place a filter paper on top of H, place two green bean leaf disks on top of it, and inoculate each leaf disk with 10 female adult two-spotted spider mites using a brush that constantly supplies water to the disks. After a period of time, remove the abnormal insects, dilute the wettable powder prepared according to Formulation Example 2 to the specified concentration, and make 2mg of the drug solution.
/am” equivalent amount.

The test was conducted in two series, and the substance appeared 2 days after treatment with the drug! 1fi
The survival or death of the mites was determined using a microscope, and the corrected mortality rate was calculated using the following formula. The results are shown in Table 2.

Test Example 2 Brown Planthopper Insecticidal Test A wettable powder prepared in accordance with Preparation Example 2 was diluted with water to a predetermined concentration, and rice grass leaves were immersed in the solution, air-dried, and then placed in a test tube. Ten brown planthopper larvae were released into the container, which was then plugged with absorbent cotton and kept at a constant temperature of 25° C. for 5 days. The exam is
The experiment was carried out in duplicate, and the mortality rate after rearing was calculated. 3rd result
Shown in the table.

Test Example 3 Insecticidal Test for Insecticides of Pygmyus japonica According to Preparation Example 2, a wettable powder expressed as 5+ was diluted with water to a predetermined concentration, and sprouted paddy was immersed in this and air-dried.
It was placed in a sized polyethylene cup. Ten 3rd instar larvae of N. japonica were released into it and reared at a constant temperature of 25° C. for 50 days. The test was conducted in duplicate, and the mortality rate after rearing was calculated. The results are shown in Table 4.

4. Explanation of drawings Figure 1 shows the ultraviolet absorption spectrum of KSB-1939L, α, Figure 2 shows the infrared absorption spectrum of KSB-1939L, α, and Figure 3 shows the proton nuclear magnetism of KSB-1939L, α. Figure 4 shows the resonance spectrum of KSB-1939.
Figure 5 shows the ultraviolet absorption spectra of L and β for KSB-1.
Figure 6 shows the infrared absorption spectra of 939L and β.
The proton nuclear magnetic resonance spectrum of B-1939L and β is shown.

Claims (2)

[Claims] (1) KSB-1939L_3 compound represented by the formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, R represents a 2-methylpropanoyl group or a butanoyl group.) (2) KSB-1939L belonging to the genus Streptomyces
_3 A method for producing a compound, which comprises culturing a producing bacterium and collecting the compound from the culture.