SU269821A1 - OENTENTIO- -t, * "TYLE SECURITY 'EMBLEOTEKA - Google Patents

Description

The invention relates to methods for producing agricultural antibiotics, mainly antibiotics, used to protect cultivated plants from phytopathogenic fungi.

A method of producing antibiotic venturicidin A (AA368) is known, in which the strains N.C.I.V. are used as a fungus. 9198, N.C.I.V. 9199 and N.C.I.V. 9200 Streptomyces aureofaciens.

A method for the preparation of antibiotics venturicidin B and / or venturicidin X is proposed, in which S. aureofaciens strain NRRL 3399 (National Regional Research Laboratory USDA) is used as a fungus.

It is also stored under the name Tj 342 in the company's laboratories, and is also deposited at the Microbiological Institute at the University of Tübingen (Switzerland).

The strain is isolated from a soil sample, in the vicinity of Kozoussa (Guinea). For species, see HiJtter et al. Arch. f. Mikrobie, 39, 173 (1961) it belongs to the species S. aureofaciens Duggar, 1948.

The main characteristics of the strain NRRL 3399:

2 spores of ellipsoidal shape, size (0.6-0.9) X (0.8-1.4) microns, with a smooth surface;

aerial mycelium initially whitish, later - from brown-gray to golden-gray;

spore chains are branched monopodially in the form of open, free spirals of 4-6 turns;

on a peptone-containing medium, the melanoid pigment does not appear.

The optimal growth temperature for the strain ranges from 33 to, while for other members of the species S. aureofaciens it ranges from 27 to 30 ° C. In addition to antibiotics, ventricidal X p B species S. aureofaciens produces the well-known antibiotic ventzfidin (antibiotic AL 368, see DAS 1 184458), which will be referred to as "venturicidin A." on silica gel. It is also possible to convert venturicidin A to venturicidin B.

The method of producing antibiotics venturicidin X and / or B is as follows. The culture of S. aureofaciens NRRL 3399, which gives venturicidin X and (nl) venturicidin B, is grown under aerobic conditions in water.

a solution containing sources of carbon and nitrogen, as well as inorganic salts, as long as the solution does not show a significant antibiotic effect. Then venturicidin X and / or B are isolated and, if desired, venturicidin A also obtained is transferred to venturicidin B. The resulting antibiotics can be converted to their derivatives.

As sources of carbon and nitrogen, glucose, sucrose, fructose, starch, mannitol, and amino acids can be used, such as glycine, pentides, proteins, and their degradation products, such as peptone or tryptone, meat extracts, water extract from grain (corn, wheat) , distillation residues in the production of alcohol, yeast, seeds (especially plants of rasa, soybean, and cotton), ammonium salts, and nitrates. Of inorganic salts, the nutrient solution may contain chlorides, carbonates, sulfates, nitrates, phosphates of alkali and alkaline earth metals, magnesium, zinc, manganese and iron.

The culture is conducted under aerobic conditions while agitating or moving with air or oxygen in special vessels or fermenters. The most suitable temperature is 27-37 ° С. Cultural fluid shows a significant antibacterial effect on average after 24 hours.

It is mainly cultivated in several stages: first, a liquid nutrient medium is prepared, which is then inoculated. First of all, a spore mycelium, obtained after two weeks of cultivation on solid nutrient medium, is re-inoculated into a liquid nutrient medium and allowed to grow for another 48 hours. Antibiotics from the culture filtrate are isolated by known methods, taking into account their chemical, physical and biological properties.

Botrytis cinerea is a very good test organism for testing the effects of antibiotics in individual isolation stages, as well as in the culture medium. The hyphae of this microorganism morphologically change greatly under the action of antibiotics (a strong branching, starting from a certain point, so that the mite-like forms appear).

This test is carried out, for example, in the form of diffusion on the plates as follows.

In the middle of the malt-agar plates in a circular square with a diameter of 5 mm, B. cinerea is inoculated, and these plates are subjected to incubation at 24 ° C for two to three days. During this time, the mycelium develops up to 20--30 mm. The solution of the controlled substance is applied by means of filter circles with a diameter of 6 mm at a distance of approximately 5 mm from the edge of the target. After that, the nlastinki is subjected to incubation for 24- 36 hours at a temperature of 24 ° C. In active solutions, a morphological change is observed at a distance of up to 24 mm from the filter

circles. Solutions with a concentration of 10 μg lA of antibiotics still give markedly modified hyphae.

Antibiotic venturicidin X is not a glycoside, unlike venturicidins A and B, which are 2-deoxy-O-rhamnose glycosides (venturicidin B) or 3-o-carbaminyl-2-deoxy-O-rhamnose (venturicidin A), respectively. . It contains no sugar and is not identical also with the aglycone of venturicidin A and B (SvNeOO). Venturicidin X has not yet been obtained as a crystalline substance, but as a uniform, amorphous powder by thin-layer chromatography. It is a fat-soluble, neutral, colorless substance, soluble, by the way, in methanol, ethanol, acetone, ethnyl acetate, chloroform and methylene chloride.

Elementary analysis,%: C 66.35; H 9.18; About 24.90.

Mol B. (osmometric method based on vapors, methylene chloride) is 701. These data correspond to the gross formula

CsgHsiOi, (mol. Century 709; C 66.077o; H 9.10%; O 24.83%) and CsgHciOio (mol. Century 692; C 67.60%; H 9.31%; O 23.09 %). b1,5 ° (in chloroform).

The IR spectrum in potassium bromide shows ribbons at 3480, 2965, 2942, 2875, 1702, 1643, 1452, 1382, 1300 (shoulder), 1279, 1232, 1222, 1184, 1169 (shoulder), 1129, 1095, 1052, 984 , 910, 880, 870, 850, 780, 752, 718 cl-i (see Fig. 1). The NMR spectrum in deuterochloroform, 60 ms, is depicted in FIG. 2

The ultraviolet spectrum in pure alcohol indicates the following maximum data l 220 nm (lege 4.47; shoulder); nm (Ieg8 4.50); i 232.5 nm (, 47); nm (iegs 4.25); . 295 nm (, 53, shoulder).

For thin-layer chromatography on silica gel (ethyl acetate flow agent), the Rf value is 0.80, i.e., much higher than that of venturicidins A and B (venturicidin A 0.49; venturicidin B 0.41).

In the hydrogenation of venturicidin X on a palladium angle in methanol, approximately 2 moles of hydrogen are absorbed. Tetrahydroventuricidin X forms colorless crystals with t. Nl. 156-157 ° C. Elementary analysis,%: C 67.39; 67.11; P 9.91; 9.89; a pier at. 765 (homometric method using vapors in ethyl acetate). This data corresponds to the gross formula CgHcgO;., (Calculated C 67.21%; H 9.84%; mol. V. 697). Veron tna gross for. Mule Venturicidin X CsgneiOio.

The rotation of the tetrahydron derivative ajgfg is 48.5 ° (from 0.968% in chloroform).

The ultraviolet spectrum in pure alcohol shows maximum values at 212 nm (, 88) and /.275 nm (Ieg8 2.20). Cho), 2960, 2940, 2875, 1732, 1708, 1697, 1625, 1457, 1411, 1395, 1381, 1370, 1339, 1270, 1242, 1225, 1215, 1190, 1170 (shoulder), 1133, 1089, 1052 ( shoulder), 1029, 1011, 989, 933, 908, 882, 849, 839, 808, 796, 773, 746, 729, 695, 593, clg (see Fig. 3). NMR-cneKxp in COIL of tetrahydroventuricidin X. is shown in FIG. 4. Venturicidin X and its tetrahydropic arsenic may be acylated by the usual method, for example, using acid halides and especially halogen anhydrides, such as aliphatic, araliphatic, aromatic or heterocyclic carboxylic acids (especially lower hydrochloric acids), such as acetic acid. When acetylating tetrahydroventuricidin X, an acetylene derivative is obtained as a colorless oil. The infrared spectrum of this compound in deuterochloroform is shown in FIG. 5. The IR spectrum in chloroform shows tapes at 3460, 1733, 1705 (shoulder), 1457 n 1370 slg i. NMD spectrum in CDCls shows signals at 6 2.02 ppm (s, 3N); 2.08 ppm (s, ca. 9H). (Hereinafter, the reduction of ppm is denoted H / 1 million). In acidic methanolysis, approximately five reaction products are obtained, of which three are chromatographically in pure form as colorless oils. No methyl glycosides are obtained. The NMR spectra of the products show SNS-sigals that can arise from the methanolysis of one of the lactone compounds. The presence of a (macrocyclic) lactone ring is also indicated by the corresponding -COO-CH sigpal 6 5.0 ppm group, which is found in the unhydrogenated and hydrogenated venturi X. The three remaining signals in this area, which only the unnowned product shows, belong to the hydrogen atoms in the field double bond. The bladder at b 2.5 ppm (Fig. 2) should be in the C-methyl group, since this signal is not in the spectrum of the hydrogenated product, either. Due to the ultraviolet spectrum that corresponds to the single conjugated diene, one can be considered as one partial formula I a and I b part of the structure. In addition to this diene system, there is another unconjugated keto group (shoulder at approximately 295 nm in the ultraviolet spectrum of venturicidin X and approximately 275 nm in gidrirovannom product). About NMr patterns of the skeleton resembling classical erythnomycin type macrolides are derived from NMR patterns. Formula C.i3H7iN6i2 is available for venturicidin A. The structure of the compound is not clear. In connection with the study of the new antibiogic venturicidia B, the structures of the partial fragments of venturicidin B have been clarified. The formula C oToOc is derived from the analyzes. Thus, venturicidin B on CONH is less than venturicidin A. On the basis of the infrared spectrum (Figs. 6 and 8), it can be assumed that venturicidin A is a venturicid ester on B, since the maximum at 1605 cm 1 is not present in venturcidin B. This is also confirmed by the fact that venturicidin B produces a tri-o-acetyl derivative, whereas venturcidin A is only a di-o-acetyl derivative. This assumption is confirmed by the production of a single sugar-like cleavage product from venturicidin A and B and an explanation of their interrelationship and construction. Venturcidin. - in methanolysis with an acid catalyst of irregular conditions, among the most severe, crystalline methylglucoside CsHisNOs (I) gives. By hydrolysis with hydroxide of oari, otscheilen caroamoyl group and methylglucoside C-HmO (I) dideoxyhexose was obtained. The following construction is derived from the N.MR spectrum for methylglucoside CgHisKOs. In this case, the distribution of individual signals is supported by the spin rating (see Example 1). The signal at b 4.86 ppm is attributed to the proton in the third position. Its chemical displacement does not correspond to the displacement of hydrogen around the acloxgroup. The signal should be assigned to hydrogen atom H-4 at b 3.21 ppm. At C-4, the same way, there should be a free hydroxyl group, while the carbamoyl residue is at C-3. Signal H-1 (b 4.73 ppm) double doble with low coupling constant Jle ,, 8 CpS and Jle, 2a 1,5 CpS. From this conclusion, Tm, on the one hand, is the equatorial position of H-1, on the other hand, the position of the 2CH2-group, which also follows the release of the spin. The permanent couplings J2, 11.5 cps, Lz, 4 9 cps and J4.5 9 cps prove the axial position of the hydrogen atoms at C-3, C-4 and C-5. The doublet at 6 1.33 for one CHa-CH group shows that the compound is 6-deoxysacchar. The venturicidine cleavage product A is thus a 3-o-carba moli-2-deoxyramine. A-Methyl-2-deoxy-B-rhamnoside C7Hi4O4 build II: R CH3 III: RH hydroglycosposite The same compound is obtained during the methanolysis of venturicidine B. Hydrolysis II produces free sugar III, which shows optical rotation in water a + 19.9 ° (final value after 90 minutes). Aglycone venturicidin A and B in its pure form has not yet been obtained. Based on the thin-layer chromatography reaction and NMR-cneKTpOB, both antibiotics are assumed to contain the same aglycone. Venturicidin B and venturicidin X have a good antimicrobial, in particular phytopathogenic, effect that is more effective than that of venturicidin A. In the table. Table 1 shows the spectrum of action of venturicidin A, B, and X in various concentrations on various fungi on diffusion plates (diameter of filter circles impregnated with a solution of antibiotics is 6 mm). Numbers in brackets indicate incomplete braking, dashes - the experiment was not conducted. Tab. 1 also indicates that antibiotics with proven maximum concentrations (10 mg / ml)) do not act on Saccharomyces cerevisiae. This has implications for the use of Yeuruticidin X and / or B as fungicides in vineyards. The residues of organic fungicides used in vineyards in the crop are estimated at 10 ppm. However, an amount equal to 3-5 ppm, already inhibits fermentation (Vegeler and. Geelanener, Zeitschr. Fui Obst- und Weinbau, 103, 494-504, 1967). Since the border of ventricidia X and B on yeast is much higher, it is recommended to use them as fungicides in vineyards. When fighting against phytopathogenic gyres, for example, Aspergillus niger, Alternaria tennis, Botrytis cinerea, Cercospora melonis, Didymella Lycopersici, Fusarium nivale, Glemerella cingulata, Helminthosporium avenae usf. and others, antibiotics can be used in the form of liquid agents (solutions, suspensions, or emulsions), or in solid form (granules, powders and sprayables). To obtain directly soluble antibiotic solutions, for example, fractions of mineral oils from the high to medium boiling range are taken: diesel oil or kerosene, coal tar oil and vegetable or animal oils, as well as hydrocarbons such as alkylated naphthalenes, tetrahydronaphtadine, in some cases when using mixtures of xylenes, cyclohexanols, ketones, then chlorinated hydrocarbons, such as trichloroethane and tetrachloroethane, trichlorethylene or tritetrachlorobenzene. Successfully use organic solvents with so on. above 100 ° C. Aqueous forms of antibiotics are prepared from emulsion concentrates, pastes or wettable powders. Neumogenic products, such as condensation products of aliphatic alcohols, amines or carboxylic acids, with a long-chain SI residue with 10–20 carbon atoms with ethylene oxide, a condensation product of octadecyl alcohol and 25–30 mol of ethylene oxide, or a condensation product are used as emulsifiers and dispersion agents. soybean fatty acid and 30 mol of ethylene oxide, or the condensation product of technical oleylamine and 15 mol of ethylene oxide, or the condensation product of dodecyl mercaptan and 12 mol of ethylene oxide. Anionic emulsifiers that can be consumed include sodium dodecyl ester of sulfuric acid, sodium dodecyl ester of benzenesulphonic acid, potassium or triethanolamine of oleic or abietic acid, or mixtures of these acids, or sodium salt of oleic or abietic acid, or mixtures of these acids, or in a mixture of salts of salts of oleic or abietic acid, or in a mixture of these acids, or a sodium salt of oleic or abietic acid, or a mixture of these acids, or a sodium salt of oleic or abietic acid; Quaternary ammonium compounds, such as cetylpyridinium bromide or dioxyethyl benzyl dodecyl ammonium chloride, can be used as cationic dispersing agents. For the manufacture of powders and sprays, talc, kaolin, bentonite, calcium carbonate, calcium phosphate, as well as coal, cork, wood flour and other substances of plant origin can be used as a solid base. The preparations are also made in the form of granulators. By adding substances that improve adhesion, resistance to rinsing, distribution and introduction into another medium, you can expand the scope of application of the drugs. Fatty acids, resin, glue, casein or alginates can be added. Example 1. 300 ml of a nutrient solution containing 20 g of soybean flour (full of fat) and 20 g of mannitol per 1 liter, the pH value of which is brought to 7.5 before potassium hydroxide solution, is poured into an Erlenmeyer flask, sterilized, cooled and inoculated with S. aureofaciens NRRL 3399 bacterial culture. The suspension is then incubated at 27 ° C., continuously shaken on the machine. After about 48 hours

the culture is transferred in a ratio of I: 10 to a nutrient solution of the same composition, poured into a fermentation device. Incubation is carried out again at 27 ° C and intensive blowing of air for 24 hours. To control the activity of the cultures at all stages of work, a test is performed on B. cinerea.

S. aureofaciens NRRL 3399 is grown according to the described method in a nutrient solution containing 20 g of malt extract (solids content of about 50%), 20 g of dry corn stillage (Distillers solubles), 5 g of sodium chloride and 1 g of sodium nitrate per 1 liter.

S. aureofaciens NRRL 3399 is grown according to the described method in a nutrient solution containing 20 g of dry maize stillage, 20 g of lactose, 5 g of chlorinated patri and 1 g of sodium nitrate per liter.

S. aureofaciens NRRL 3399 is grown according to the described method in a nutrient solution containing 10 g of glucose, 5 g of peptone, 3 g of meat extract, 5 g of sodium chloride and 10 g of calcium carbonate per liter. The pH value is set to sterilization with potassium hydroxide to 7.2.

When the culture begins to exhibit a strong antibacterial effect, it is cooled and filtered under pressure with the addition of a filtering aid, such as Hyfle-Supercel. The filtrate is extracted twice at the same pH value (typically 7.5-7.8) with half the amount of ethyl acetate; however, substances acting as antibiotics are transferred to ethyl acetate. Significant amounts of antibiotic are also in the filter residue. They are recovered from there by repeated extraction with methanol. The methanol extracts are evaporated to a small volume in vacuo, diluted with water and then extracted several times with ethyl acetate. After evaporation of the ethyl acetate extracts from the culture filtrate and filter residues, an oily residue in an amount of 80-100 g is obtained from 100 l of the culture solution.

40 g of this residue is purified by distribution over Craigy in 190 steps. The solvent system used contains .4.25 liters of carbon tetrachloride, 0.75 liters of chloroform, 4 liters of methanol and 1 liter of water. Venturicidin A, B, and X accumulate in steps 75-130 (maximum in step 100). These fractions are combined and evaporated to dryness in vacuo. 25 g of a pale yellow powder are obtained. 21 g of this substance are distributed with the same solvent system over 600 steps. Venturicidin is in steps 265-396. By evaporation in vacuum from steps 265-308, 5.1 g of venturicidin B and X are obtained, from steps 309-328, 5.4 g of venturicidin B and XXA are obtained, and from steps 329-396 to 10.1 g of venturicidin A and X. Substances given in a thin-layer chromatogram on silica gel 254 (finished tiles from Merck) with

ethyl acetate as a flow agent, the following Rf values (spots are shown by spraying with concentrated sulfuric acid and heating to 140 ° C): veituritcidin A

0.49; Venturicidin B 0.41; Venturicidin X 0.80. Three mixtures are chromatographed on silica gel columns of the company hmerk. From the residue from the extraction of fraction 329-396 with chromatographing 250 g of silica gel (Merck) and

Elute 1.5 liters of a mixture of chloroform and ethyl acetate (1: 1) to obtain 1.85 g of venturicidin X as a yellowish amorphous powder with the characteristics indicated in the general part of the description.

Following this, the residue of the 329-396 fraction is eluted with 1 L of ethyl acetate. At the same time, 5.27 g of venturicidin A is obtained as crystalline crystals from chloroform ether as colorless crystals with a melting point of 200 g. -M19 ° (concentration 0.5 in chloroform). The spectrum of IR to Kwg (in Fig. 6). The NMR spectrum (60 MNg) in CDC1.3 of FIG. 7

Example III. 3 g of venturicidin X are hydrogenated in 150 ml of methanol with 750 mg of 100% palladium on carbon in an atmosphere of hydrogen. After 2 hours, the hydrogenation is replenished. Absorbed 237 liters; liters of hydrogen (20 ° C, 725 mm Hg. Art.), Which corresponds to about 2 mol. The catalyst is filtered off, the solution is evaporated and the residue is chromatographed on 30 g of selngel. Ethyl acetate is eluted with 2.56 g of tetrahydro-derivative venturicidin X as a colorless solvent, crystallized by wetting with ether. The properties of the compound are indicated in the general part of the description.

Example 3. 100 mg of tetrahydroventuricidin X are reacted with 2 ml of acetic anhydride and 2 ml of pyridine for a G hour at a room temperature. Then grow Or evaporated dry. Chromatography on silica gel (10 g) with a mixture of chloroform and ethyl acetate (4: 1) as an eluent gave 84 lg of colorless oil. The properties of this product, acetylirovap tetrahydroveituricidin X are indicated in the general part of the description.

Example 4. 4.31 g of residue from evaporation of fraction 265-308 of Example 1 are chromatographed on 350 g of silica gel. A mixture of chloroform and ethyl acetate (1: 1) was eluted with 1.76 g of venturicidin X. Elution with ethyl acetate, 1.23 g of chromatographically homogeneous veituritcidin B were obtained. Co-precipitation from a mixture of ethyl acetate and petroleum ether was obtained

substance in the form of a colorless powder with m. pl.

145-149 ° C; + 100 ° (concentration 0.847

in chloroform). IR spectrum in methyl potassium

in fig. 8, NMR spectrum in CDCls of FIG. 9.

Calculated,%: C 67.16; H 9.40; a pier in 751;

C, 2P-eO11.

Found,%: C 66.86; H 9.29; a pier at 733. Molecular weight is determined osmometrically by pressure in methylene chloride. PRI me R 5. 200 mg venturicidin In ace13

as described in Example 1 for venturicidin A. Crude tri-o-acetylventurucidin B is purified by chromatography on silica gel and eluted with ethyl acetate. An amorphous colorless powder is obtained with t. Nl. . The NMR spectrum in the CDCU shows, among other things, signals at, 99 ppm (s, 3H); 2.01 ppm (S, 3N) and 2.02 ppm (s, 3N). Calculated,%: C 65.73; H 8.73;

C48H7 "Oi4.

Found,%: C 65.92; H 8.54.

Example 6 100 mg of venturicidin X are dissolved in 1 ml of ethyl cellosolve containing 10% triton and made up with distilled water to 1 l. The solution is sprayed with grape vines, for example, to fight an infection with B. cinerea.

The plots of the experimental vineyard after spraying with the second generation gray rot suspension are sprayed four times with the above solution at intervals of 14 days. When compared with the control

14

The areas not exposed to the solution established a good effect of the solution against B. cinerea.

Example 7. The test solution of the biologically active substance was prepared by dissolving 10% venturicid B in ethyl cellosolve, containing 10% Triton X 100, and diluting with distilled water to the required concentration.

. Young leaves of approximately the same size of vine seedlings of the variety “Riesling Silvaner” are sprayed with the test solution and laid out to dry. Another batch of leaves is placed for 48 hours in a solution of active

substances. The leaves treated in this way are infected with an aqueous suspension of conidia from B. cinerea and incubated at room temperature in a humid room. After four days, the surface is removed by eye.

leaf, changed color due to infection by the pathogen. The results are shown in Table. 2, and the 50% copper oxychloride preparation was taken as a reference.

table 2

Example 8. A 0.1% aqueous solution of venturicidin X is prepared, which is prevented from spraying rice plants grown in a greenhouse once. Two days after spraying, the plants are infected with a conidia suspension of Poricularia oryral and examined seven days later to determine the extent of the fungal infection. Compared with non-sprayed control plants, plants are infected with fungi only by 15%.

Subject invention

1. A method of obtaining antibiotics of the venturicidin group based on the culture of the fungus producer Streptomyces aureofaciens Duggar, including cultivation of the fungus on a liquid nutrient medium containing carbon and nitrogen sources and mineral salts, mainly under aerobic conditions by the in-depth method, with possible conversion of a-ntibiotics to their derivatives, characterized in that, in order to obtain vepturicidin B and / or

vepturicidin X, as a fungus-producer take S. phytome S. aureofaciens NRRL 3399 (National Regional Research Laboratory USDA).

2. Method no. P. 1, characterized in that antibiotics are extracted with an organic solvent or a mixture of solvents, preferably with ethyl acetate and / or methanol.

3. Method for ip. 1 and 2, characterized in that antibiotics purify and / or divide the distribution according to the counter-flow principle, which is advantageous by the distribution

according to Graigy in a solvent system containing carbon tetrachloride, chloroform, methanol, water.

4. Method according to paragraphs. 1 and 2, characterized in that the antibiotics are separated by m chromatography, preferably on silica gel.

5. The method according to paragraphs. 1-4, characterized in that the antibiotics are subjected to hydrogenation.

6. Method according to paragraphs. 1-5, characterized in that antibiotics or their products are hydrated 7. The method according to paragraphs. 1-b, characterized in that venturicidin X is converted to its tetrahydro derivative.

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