JPS6217599B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides new antibiotics herbicidin C, E, and F.
and G substance and its production method. Herbicidin C, E, F and G substances of the present invention have the following formula: However, in the formula, in the herbicidin C substance, R ¹ and R ² both represent hydrogen atoms, and in the same E substance, R ¹ is 1-methylethylcarbonyl.

[Formula] R ² represents methyl, and in the substance F, R ¹ is 1-methyl-1-propenylcarbonyl.

[Formula] R ² represents methyl, and in the same substance G, R ¹ is 1-methyl-1-propenylcarbonyl

[Formula] R ² represents a hydrogen atom. Herbicidin C, E, F and G substances of the present invention have the following physical and chemical properties and biological properties (1) Appearance

【table】 (2) Melting point or decomposition point Herbicidin C 165-169℃ (decomposition) 〃E 172～173℃ 〃 F 150-154℃ (decomposition) 〃 G 142-146℃ (decomposition) (3) Elemental analysis (%)

【table】 (4) Ultraviolet absorption spectrum

[Table] (5) Infrared absorption spectra The absorption spectra of herbicidin C, E, F, and G substances measured in potassium bromide tablets are shown in Figures 1, 2, 3, and 4, respectively ( 6) Nuclear magnetic resonance absorption spectrum The spectrum of herbicidin C tetraacetate measured in deuterated chloroform at 60 MHz is shown in Figure 5. Figure 6 shows the spectrum of herbicidin E measured at 100 MHz in a mixed solvent of heavy acetone and heavy methanol. FIG. 7 shows the spectrum of herbicidin F measured at 60 MHz in deuterated acetone. In Figure 7, 2.05ppm
The signal is due to deuterated acetone used as a solvent. FIG. 8 shows the spectrum of herbicidin G methyl ester measured at 60 MHz in a mixed solvent of deuterated chloroform and deuterated methanol. In Figure 8, the signal at 3.18 ppm is due to heavy methanol used as a solvent. (7) Molecular weight Herbicidin E 537 (FD mass spectrum) Herbicidin F 535 (FD mass spectrum) (8) Solubility Herbicidin C: methanol, ethanol,
Soluble in acetone Herbicidin E: Soluble in hot water, hot methanol Herbicidin F: Soluble in water, methanol, ethanol, acetone, ethyl acetate, methylene chloride Herbicidin G: Methanol, ethanol,
Soluble in acetone and ethyl acetate (9) Stability Herbicidin C, E, F, and G are unstable in acids and alkalis (10) Silica gel chromatography Silica gel plate for thin layer chromatography (glass plate, containing fluorescent indicator) , Merck & Co., Ltd.) and developed with chloroform:methanol (7:3).
When observed under ultraviolet light, herbicidin C, E,
The R _f values of F and G substances are 0.30 and 0.30, respectively.
0.69, 0.72 and 0.53. (11) Biological activity Herbicidin C, E, F and G substances all have algicidal and antibacterial activity. Regarding algicidal activity, the minimum inhibitory concentration of Herbicidin C, E, F, and G substances against Anacystis nidulans is 100 to 200μ.
g/ml. In addition, herbicidin C, E, F and G substances have the following biological activities. Herbicidin C: inhibits the germination of Komatsuna at 12.5γ/ml and has antibacterial activity against Candida lipolyteica and Chloechera opiculata. Herbicidin E: inhibits the germination of Komatsuna at 25γ/ml and has antibacterial activity against Trichophyton mentaglophytes, Candida lipolyteica, and Stachybotrys atra. Herbicidin F: Has antibacterial activity shown in Table 1

【table】

[Table] In addition, Alternaria alternata,
Polysectus cinnavarius, Stachybotrys atla, Trametes sanguinea,
It has antibacterial activity against Candida lipolyteica and Schizosatucharomyces pombe. Herbicidin G: The minimum inhibitory concentration against Trichophyton mentagrophytes is 50 μg/ml, and it also has antibacterial activity against Candida lipolyteica and Schizosatucharomyces pombe. Based on the above-mentioned physicochemical or biological properties of herbicidin C, E, F, and G substances, there are no existing antibiotics that fall under this category, and herbicidin C, E, F, and G substances are new. Recognized as an antibiotic. Further, due to the above-mentioned biological activities, herbicidin C, E, F and G substances are expected to be used as antibacterial agents and algaecides. Herbicidin C, E, F, and G substances of the present invention can be obtained by culturing microorganisms belonging to the genus Streptomyces that can produce these substances, and producing herbicidin C, E, F, and G substances in the culture. Manufactured by collecting. A specific example of a microorganism that can produce herbicidin C, E, F, and G substances is Streptomyces saganonensis No. 4075, and the mycological properties of this strain have already been published in the Journal of
J Antibiotics 29
Vol. 863 (1976), and this bacterial strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, and its microbial accession number is Microbiological Research Institute No. 1821. In addition, Streptomyces saganonensis, which has been artificially mutated from this strain,
M403, M403R, and m17-403 strains have even better productivity of herbicidin C, E, F, and G, and these bacteria have also been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, and their deposit numbers No. 4974, No. 4975, and No. 4975, respectively.
This is No. 4976. As seen in other Streptomyces strains, the bacterial strain used in the present invention is susceptible to changes in properties, such as exposure to ultraviolet rays, X-rays, high frequency,
It can be mutated by artificial mutagenesis methods using radiation, chemicals, etc., and strains of this bacterium that have been mutated by artificial methods are, of course, microorganisms belonging to the genus Streptomyces, including herbicidin C, E, F, and Any substance capable of producing G substance can be used in the method of the present invention. As a method for culturing production bacteria for producing herbicidin C, E, F, and G substances, the above-mentioned bacterial strains are cultured in a medium containing nutrients that can be used by ordinary microorganisms. As the nutrient source, any known nutrient source conventionally used for culturing Streptomyces bacteria can be used. For example, glucose, sucrose, starch, glycerin, starch syrup, molasses, soybean oil, etc. can be used as the carbon source. Further, as the nitrogen source, soybean flour, wheat germ, meat extract, peptone, yeast cells, corn steep liquor, ammonium sulfate, sodium nitrate, etc. can be used. In addition, inorganic salts such as calcium carbonate, common salt, potassium chloride, and phosphates may be added as necessary.
Organic and inorganic substances can be added as appropriate, such as those that aid the growth of the fungus and promote the production of herbicidin C, E, F and G substances. As for the culture method, the liquid culture method, especially the deep culture method, is most suitable, as is the case with general antibiotic production methods. Cultivation is carried out under aerobic conditions, and the appropriate temperature for cultivation is 22-30°C, but in many cases 24°C
Culture at around ℃. The production of herbicidin C, E, F and G was 5% in both shaking culture and tank culture.
Maximum value is reached in ~10 days. The following method is used to assay herbicidin C, E, F, and G substances. Silica gel plate for thin layer chromatography (glass plate, containing fluorescent indicator, manufactured by Merck & Co.),
Add 5 microliters of a sample prepared by appropriately diluting the culture solution, and add chloroform:methanol (7:3).
The sample was developed with a mixed solvent of 100%, and the ultraviolet absorption was measured using a dual-wavelength chromatography scanner (sample wavelength 258 nm, reference wavelength 380 nm) together with a spot of each standard product. When collecting herbicidin C, E, F and G substances from the culture, activated carbon, alumina,
Adsorbent such as silica gel, Diaion HP-20
Synthetic adsorbents such as (manufactured by Mitsubishi Kasei Corporation), ion exchange resins, ion exchange gel filters, etc. are used, but the collection method shown below is the most efficient. Cells and other solid matter are separated from the culture solution using a supernatant such as diatomaceous earth, and then the active ingredients in the solution are adsorbed onto Diaion HP-20. After washing the resin part with water, it is eluted using hydrous alcohol. The active fraction in the eluate is concentrated under reduced pressure to distill off the alcohol. The active ingredient is extracted from the obtained concentrate using butanol, methylene chloride, ethyl acetate, etc., and the respective organic solvents are distilled off to obtain a crude powder of the active ingredient. Among the herbicidins, Substance F is obtained in crystal form by recrystallizing the above-mentioned coarse powder with methanol, Substances C and E are obtained by recrystallization with methanol after silica gel chromatography, and Substance G is obtained using Cephadex LH.
-20°C and then recrystallized with ethyl acetate to obtain a crystalline form. Example 1 Streptomyces saganonensis M403 strain was mixed with 2% soluble starch (Lastargen FK),
Soy flour 2%, glucose 1%, KH ₂ PO ₄ 1%,
CaCO ₃ 0.3%, Dasform CB442 0.02% PH6.5
3 days and 1 day in preculture medium adjusted to
Cultured at 27℃, 2% soluble starch, 8% soybean flour
%, glucose 1%, KH ₂ PO ₄ 1%, CaCO ₃ 0.3
%, NH ₄ Cl 0.5%, MgSO ₄ 7H ₂ O 0.02%,
ZnSO ₄・7H ₂ O0.1%, Disform CB442 0.02
% of the main culture medium was transferred with a volume equivalent to 3% of the seed culture broth. The main culture tank has an internal pressure of 1Kg/cm ²
Maintain the temperature at a constant temperature and aerate at an equal rate per minute to remove dissolved oxygen.
Stirring was performed to adjust the concentration to 2 ppm. The culture temperature is
The temperature was kept at 30°C for 2 days, and the temperature was lowered to 24°C from the 3rd day, and the culture was continued for 13 days while adding glucose to maintain the glucose concentration in the culture solution at 1%. Add 10kg of supernatant (Celite) to the culture solution obtained here and filter it under pressure to obtain a solution of 85%
, adsorbed with SV4 to a column containing Diaion HP-20 8.5, and soaked this column with 80% water.
Wash with SV4 followed by 10% methanol 80
Wash with SV4. Then 50% methanol 80
Elute herbicidin A and B with SV2 at SV2, followed by elution of herbicidin E material with 90% methanol 20 at SV2. Here, the total number of fractions containing substance E is 10. This was heated to 50℃ under reduced pressure.
Concentrate to 0.5. This concentrated solution is extracted five times with 0.5 methylene chloride to obtain extract 2.5. After dehydrating this with 250 g of Na ₂ SO ₄ , methylene chloride was distilled off to obtain 18.5 g of an oil. This is silica gel 250 filled with chloroform.
ml column, and with chloroform 1
Wash with SV1, followed by SV1 with chloroform 1 containing 1% methanol. It is then eluted with chloroform 2 containing 2% methanol at SV1. The effective fraction 2 was distilled off at 50°C under reduced pressure, the residue was dissolved in 10 ml of ethyl acetate, 50 ml of n-hexane was added, and the mixture was refrigerated.
600 mg of substance was obtained in crystalline form. Example 2 Streptomyces saganonensis M403R
The strain was cultured in the same manner as in Example 1. However, the main culture medium contains 2% soluble starch and soybean flour.
4.5%, glucose 1%, _KH2PO4 1 _% , CaCO3 _0.3
%, NH ₄ Cl 0.5%, MgSO ₄ 7H ₂ O 0.02%,
ZnSO ₄・7H ₂ O0.1%, Disform CB442 0.02
%, PH6.5, and the number of culture days is 14 days. Add 64 kg of Celite to the culture solution 800 obtained here, and apply pressure to obtain a 600 solution. This is adsorbed on a column packed with 60% Diaion HP-20 using SV4, washed with 600% water using SV4, and then washed with 10% methanol 600%. Continuing
When eluted with 50% methanol, herbicidin C substance is eluted in the first 40 minutes, followed by herbicidin A and B. Next, the fraction 40 containing herbicidin C is concentrated at 55° C. under reduced pressure to obtain a concentrated liquid 6. This is extracted twice with n-butanol (No. 6), and water is added to the extract (No. 14) and concentrated at 55° C. under reduced pressure to obtain 100 g of crude powder. Dissolve 4g of it in 5ml of methanol, adsorb it on a 200ml silica gel column packed with chloroform, and add 500ml of chloroform.
(SV1), followed by elution with 4000 ml of chloroform containing 10% methanol (SV1) to remove herbicidin A and B substances. Subsequently, elution was performed with 2000 ml of chloroform containing 20% methanol to obtain effective category 2. The solvent is distilled off at 50° C. under reduced pressure, and the residue is reconsolidated with 20 ml of methanol to obtain 200 mg of herbicidin C substance as a crystalline powder. Example 3 Streptomyces saganonensis m17−
The 403 strain was cultured in the same manner as in Example 1 for 5 days.
However, the main culture medium contains 2% soluble starch, 2% defatted soybean flour, 1% glucose, 1% KH ₂ PO ₄ ,
CaCO ₃ 0.3%, fresh yeast 2%, NH ₄ Cl 0.5%,
_MgSO4・_7H2O0.02 %, _ZnSO4・_7H2O0.1 %, PH
It is 6.5. 2.4 kg of celite is added to the broth 30 obtained here, and pressure filtration is performed to obtain a liquid 20.
This was adsorbed onto a column packed with 2x Diaion HP-20 (SV5), washed with 10% water (SV5), then washed with 40% methanol 10% (SV5), and then washed with 45% methanol. Elute (SV3) to obtain G substance containing category 15. Next, elute with 70% methanol (SV3) to obtain F substance containing category 20. The F substance-containing fraction is concentrated to 3 at 45°C under reduced pressure. This was extracted three times with methylene chloride in step 3 to obtain the extract in step 9. After dehydration with 1 kg of Glauber's salt, methylene chloride is distilled off, dissolved in 500 ml of methanol, decolorized with 5 g of charcoal powder, and concentrated to obtain 12 g of herbicidin F substance as crystals. The G substance-containing fraction was concentrated to 3 at 45°C under reduced pressure, extracted 3 times with ethyl acetate from 3, and the extract
get it. This was dehydrated with Glauber's salt and the solvent was distilled off to obtain 5.8 g of coarse powder. Dissolve this in 5 ml of methanol, apply it to 700 ml of Sephadex LH-20 filled with methanol, pour methanol through it at SV 0.1, and collect the G substance-containing fraction, which is 300 ml. The solvent was distilled off and the residue was reconstituted with ethyl acetate to obtain 190 mg of herbicidin G substance as crystals.

[Brief explanation of the drawing]

Figures 1, 2, 3 and 4 show infrared absorption spectra of herbicidin C, E, F and G substances, respectively. FIG. 5 shows nuclear magnetic resonance absorption spectra of herbicidin C tetraacetate, FIGS. 6 and 7 show herbicidin E and F, respectively, and FIG. 8 shows herbicidin G methyl ester.

Claims (1)

[Claims] 1. An antibiotic herbicidin C having the following formula:
E, F and G substances. However, in the formula, in the herbicidin C substance, R ¹ and R ² both represent a hydrogen atom, in the herbicidin substance E, R ¹ represents 1-methylethylcarbonyl and R ² represents methyl, and in the same F substance, R ¹ represents 1- Methyl-1-propenylcarbonyl, ^R2 represents methyl, and in the substance G, ^R1 represents 1-methyl-1-propenylcarbonyl, and ^R2 represents a hydrogen atom. 2 Herbicidin C, E, F, and G substance-producing bacteria belonging to the genus Streptomyces were cultured aerobically, and herbicidin C, E,
A method for producing herbicidin C, E, F and G substances, which comprises collecting F and G substances.