SU833166A3 - Method of preparing antibiotic a-35512 factor b aglucone - Google Patents

Abstract

The antibiotic mixture A-35512 containing the factors A, B, C, E, F, G and H, antibiotic A-35512 factor A, antibiotic A-35512 factor B, antibiotic A-35512 factor C, antibiotic A-35512 factor E, antibiotic A-35512 factor F, antibiotic A-35512 factor G and antibiotic A-35512 factor H and also antibiotic A-35512 factor B aglycone are prepared. This antibiotic mixture and these factors are obtained by submerse culture under aerobic fermentation conditions of the microorganism Streptomyces candidus NRRL 8156 in a nutrient medium which contains assimilable sources of carbohydrates, nitrogen and inorganic salts until a substantial amount of the antibiotic is formed. The antibiotics obtained can be used as antibacterial agents and as growth-promoting agents for increasing the feed utilisation in ruminants and poultry.

Description

This invention relates to the production of antibiotics by culturing microorganisms and isolating the desired product from the culture fluid. More specifically, the method concerns the preparation of an aglyconic factor mixture of antibiotics A-35512, formed by microorganisms of the species Streptomyces candidus MRRL 8156. Antibiotics A-35512 are known, which have properties similar to those of glycopeptide compounds. These v. Substances are well studied enough. Various fractions with antibiotic activity were isolated. Known, for example, ristomipiny, risto-cetin. Various methods are used to obtain these antibiotics — extraction, chromatography, hydrolysis of fll and 2J. However, the known antibiotic substances and methods for their preparation do not concern the treatment of dental caries and acne. The purpose of the invention is to obtain agly cona A-35512. The goal is achieved by the fact that factor-B hydrolyzes 0.05-1.5 with a normal organic or mineral acid over. 0.5–12 h. Aglikbn factor A-35512 B is obtained by fermentation under aerobic conditions of a culture of Streptomyces cand.idus NRRL 8156. 5 microorganisms are grown on medium with a source of carbohydrates, nitrogen, mineral salts to obtain sufficient antibiotic activity. Mixture A-35512, containing factors A, B, C, F, G .H, is separated from the culture medium. .Further, factor B is recovered and from it, aglycone is isolated by acid hydrolysis. Dihydrochloride A-35512 factor B has at least one hydroxyl group capable of participating in etherization. A-35512 dihydrochloride factor B dissolves in water, partially dissolves in alcohols, such as methanol and ethanol, and is insoluble in other less organic solvents, such as benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane, acetonitrile and dioxane. A-35512 dihydrochloride factor B is stable for 72 hours in aqueous solutions having a pH in the range from 3 to 10.

A-35512 hydrochloride factor B of an aglycone is a white amorphous compound having the following composition,%: carbon 54.29; hydrogen 4.34; nitrogen 7.40; chlorine 5.02; oxygen 28.95.

The infrared absorption spectrum of A-35512 hydrochloride factor B of the aglycone v. Paste Nujola has the most significant absorption peaks at the following frequencies, 3440 (bend), 3340 (bend), 3215 (intense), 2950 (bend), 2910 (intense), 2840 ( intensive), 2640 (bend), 1735. (weak), 1655 (intensive), 1590 (moderate), 1500 (intensive), 1460 (intensive), 1378 (moderate),. 1365 (bend), 1298 (moderate), 1215 (moderate), 1155 (moderate), 1120 (bend), 1105 (weak), 1060 (weak), 1040 (weak), 1008 (moderate), 925 (weak), 875 (weak), 765 (bend) and 718 (weak).

Electrometric titration of A-35512 hydrochloride factor B of the aglycone in a 66% aqueous solution of dimethylformamide indicates the presence of three titratable groups with pKd values of - 7.5; 9.25 and 11.0, and for the possible presence of two additional groups with pH values greater than 11.0.

Hydrochloride A-35512 factor In agdikona has a pier. weight about 1282, which is established by titration.

. Hydrochloride A-35512 factor B agley con has the following characteristic rotations: ci |, - 178 (C 5,); ot 365-716,8 vC 5, CH, OH).

The ultraviolet absorption spectrum of aglycone has an absorption maximum of 282 nm (E; / (102.62)) in acidic and neutral methanol, and an absorption maximum of 302 nm (E:, c 182.09) in alkaline methanol.

And With the spectrum of nuclear magnetic resonance A-35512 factor In aglycone in DMSO-dg at 90 ° C has the following characteristics:

No. PPM. Height%

And with the NMR spectrum indicates

that A-35512 factor Vaglykon continues to retain the constituents of 3-amino 2, 3,6-tridioxy-3-e-methyl-1-xylohexapyranose (one of the sugars contained in A-35512 factor B).

Claims (2)

The amino acid acidisyl hydrochloride A-35512 factor B of the aglycone, which has been further hydrolyzed in acid, indicates that A-3551 factor B aglycone contains glycine and at least three compound amino acid residues. Hydrochloride A-35512 Factor B agli cona has at least one hydroxyl group capable of participating in the esterification process. Hydrochloride A-35512 Factor B aglycone is soluble in water and methanol, but not soluble in less polar organic solvents such as benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane, acetonitrile, and dioxane. Hydrochloride A735512 factor B agly cona in the system butanol-pyridine-acetic acid-water (15: 10: 3: 12) has a Rj equal to 0.80, obtained using thin-layer chromatography on cellulose with an aluminum carrier. Hydrochloride A-35512 factor B agley con has R equal to 0.26, which is obtained by CRI. using thin-layer chromatography on silica gel using methanol-chloroform-concentrated ammonium hydroxide (3: 2: 1) as a solvent. A-3-5512 factor A aglycone (free base) is a white amorphous compound having an elemental composition ( averaged),%: carbon 52.65; hydrogen 4.57; nitrogen 6.91; chlorine 2.94; oxygen 27.04; soda ash 4.70. Infrared Absorption Spectrum A-35512 factor B aglycna. (free base) in a tablet of KB g has significant absorption peaks at the following frequencies, cm: 3360 (intense), 3260 (bend), 2940 (bend), 1735 (bend), 1660 (intense), 1598 (moderate), 1510 (intense) 1440 (moderate), 1295 (weak), 1215 (moderate), 1165 (moderate), 1122 (weak), 1070 (weak), 1018 (intense), 940 (weak) and 920 (weak). Electrometric titration of A-35512 factor B agly.con (free base) in a 66% aqueous solution of dimethylformamide indicates the presence of five titrable groups with pK values approximately equal to 6.2; 8.2; 10.1; 11.4; 12.4, and the possible presence of one or two additional groups with HA values exceeding 12.5. A-35512 factor B aglycone (free base) has the following characteristic rotation: oL -64.5 (C3, DMSO). The ultraviolet absorption spectrum of A-35512 factor B of the aglycone (free base) shows an absorption maximum in neutral and acidic methanol of 282 nm (43.65) and in alkaline methanol, a maximum absorption of 301 nm (67.46). The values of A-35512 factor B of the aglycone (free base) coincide with the values of the hydrochloride A-35512 factor B of the aglycone. P p and Me p. A lyophilized tablet of Streptomycescandfdus NRR78156 is dissolved in 1-2 ml of sterile water. This solution is then used to graft a culture on oblique N 2 agar containing Bacto yeast malt extract. LSP (Difco Laboratories, Detroit .. Michigan). Grafted agar is agitated for seven days at a temperature. Matured to the skin, it is covered with water (2 MP) and scraped with a sterile pipette in order to loosen the spores. A portion (0.1 ml) of this aqueous suspension containing spores is used for grafting another oblique agar LSP No. 2. This grafted agar is grown for seven days at. The culture matured on the agar is covered with water (5 mp) and scraped with a sterile pipette in order to loosen spores. A portion (2.5 ml) of the resulting spore suspension is used as a graft material for grafting 50 ml of vegetative medium for growing a culture having the following composition: Frypt lease soy broth (biological laboratories in Valtimore, Kokisville, Maryland) 30 g, water ( deionized) 1l. The grafted culture medium is incubated at 30 ° C for 48 hours in a 250 mm Erlenmeyer flask located on a shaker having a rotation speed of 250 rpm. The grown inoculum (0.5 ml) is used for the suspension 50, ml of the medium of the following composition, g / l: Tapioca dextrin 25.0 Glucose 10.0 NH4N032.5 KCE1.5 MgS04 -1/1 FeC., 3 ZnCt.2.0, 3, 1.0 L-Glutamic acid, 0.1 DL-Citrulline, OD CaCO-g, .5.0 Deionized water1 l Grafted medium is incubated at 32 ° C for 8-10 days in a 250 ml Erlenmeyer flask chickpeas vibrator. The fermentation is carried out on a culture medium for cultivation, having the following composition, g / l: tapioca dextrin 75.0; molasses 40.0; soluble peptone meat 15.0; about 0.5; CaCO 2.0; H, 2.0 1 l. Branch A-35512 antibiotic mixture. The culture fluid from the fermentation product is filtered using a filter accelerator (Hyf Super-cell diatomaceous earth, John-Manville Sell), with a pH of 6.8-7.2. The transparent filtrate thus obtained is passed through a column containing 10 ml of a polymeric adsorbent {Ambertete XAD- (Rom and Haas) per 100 ml of filtrate at a rate of 150 ml / min. The fractions obtained in this way are tested for biological activity against Sarclna lutea using the known method of sampling on disks. Biologically inactive effluent discarded. Then the columns are washed with water (1/8 of the volume of the culture fluid) at a rate of 150 ml / min. Inactive wash water is discarded. Then the column was eluted with a 50% aqueous solution of methanol (600 l) at a rate of 200 ml / min. The eleate, containing the A-35512 antibiotic mixture, is concentrated under vacuum to a volume of 15 liters, which contains about 200 g of the A-35512 antibiotic mixture per liter. Separation of various factors in A-35512 antibiotic mixture. A-35512 antibiotic mixture (about 3000 g, dissolved in 15 liters of methanol), obtained as described above, is subjected to chromatography on a polyamide column (Voelm, 100 l) Then the column is eluted with deionized water at a rate of about 80-120 ml / min Fractions are examined using cellulosic thin-layer chromatography or paper chromatography, and the mixture, p-butanol-pyridine-acetic acid-water (15: 10: 3: 12), is used as a solvent, in addition, it is bioavtografied for Sarcina lutea. The first 100 l of the eluate is discarded. Then the flow rate is changed to 160-200 ml / min. In this case, a fraction of 12 l is taken. In this way, twenty 12-liter volumes are selected. At this point, the eluting solvent is changed, or rather. The percentages of its ingredients are as follows: the contents of the container (360 liters of methanol) are siphoned into a container containing 120 liters of water: In a container for water, the mixed solution is mixed and transferred to a column. After that, twenty-four volumes (24 liters each) are collected at a flow rate of 200-300 ml / min. Based on the bioautographic results, groups of volumes are combined and evaporated to dryness under vacuum, as a result of which A-35512 factor B dihydrochloride is obtained. The following enriched factor mixture: Volumes Volume, L - Factor (S) Weight, g Purification of A-35512 factor B. Partially purified dihydrochloride A-35512 factor B (400 g), dissolved in 1.2 l of 50% aqueous methanol solution and chromatography on an alkyne containing column prepared as follows: acid-forming alumina (100 kg, M. Voelm) is stirred in a 50% aqueous solution of methanol. After the mixture is allowed to stand, the solution formed from above is drained and removed. The alumina is again mixed with a 50% aqueous solution of methanol and then placed in a column having a diameter of 13.5 cm. Now the column containing alumina is washed with an aqueous solution of methanol (50%) until; not according to transparent effluent. The column is eluted with a 50% aqueous solution of methanol at a rate of about 8-10 ml / min, while collecting fractions with a volume of about 240-300 ml. Fractions are examined by thin layer chromatography. Based on these data, the fractions are combined, and the yield of purified A-35512 factor B dihydrochloride, depending on the fraction number, is distributed as follows: fractions. 17-21 - 9.6 g; fractions 2229 - 72.0 g; fractions 30-37 - 117.0 g. Each of them crystallizes from a concentrated 50% aqueous solution of methanol at. The A-35512 factor B dihydrochloride purified in this way contains about chlorine. A-35512 factor B dihydrochloride solution in 66% aqueous solution of dimethyl forms and has a pH of 6.5. Getting A-35512 aglycone. A-35512 dihydrochloride of factor B (5.0 g) is dissolved in iv water (200 Ml). This solution is acidified with a 4N hydrochloric acid solution (14 ml). The resulting solution is refluxed for 2 hours. The solution is then cooled and evaporated in vacuo to a volume of 3/4 of the original volume. Then hydrochloric acid (6H) is added dropwise to the resulting solution until a precipitate forms. The resulting precipitate is separated and dried, yielding 3.56 g of crude A-35512 aglucone hydrochloride. Crude A-35512 aglucone is purified by chromatography on alumina, washed with an acid, using a water-methanol (1: 9) solution as a solvent. Elution of the column is followed by analysis by TLC. The eluted fractions containing A-35512 aglycone are mixed and evaporated under vacuum, yielding 398 mg of partially purified product Comparison of the results of thin layer chromatography using a ninhydrin stream for analysis shows that the impurities are not bioactive. A portion of this partially purified A-35512 aglycone (100 mg) is further purified by chromatography over polyamide (4, g, Masheri, Nagel and K °, MI-5C-6), (Brinkman Instrument, Oh, 07 mm) and elute with water. This column is also subjected to analysis using cellulose thin-layer chromatography, as described above. The eluted fractions containing A-35512 factor B aglycone are mixed and lyophilized, resulting in 64 mg of purified A-35512 hydrochloride aglycone factor. The overall yield is 5.08% of the starting A-35512 factor B. Preparation of A-35512 factor B aglyc in the form of a free base. A-35512 hydrochloride factor B aglycone (90 mg) is dissolved in 30 ml of methanol-water (1: 1). This solution is neutralized with an ion exchange resin (3.5 ml of B i O-Rod "AG-3 (X (). The resulting solution is stirred for 15 minutes at room temperature. Then the resin is removed by filtration The filtrate is concentrated under vacuum, while maintaining the temperature: less than 60 ° C to a volume of half the original, then lyophilized, resulting in 68 mg of A-35512 factor B of the aglycone in the form of a free base. activity n Elimination of causative agents of dental caries and therapeutic effect from claims Formula of the invention The method of obtaining the aglycone of the antibiotic A-35512. factor B, from l and h and isch.i and so that, in order to obtain the aglycone A-35512, factor B is hydrolyzed 0.051, 5 with normal organic or mineral acid for 0.5 to 12 h. Information sources, . taken into account in the examination 1. Brazhnikov MG and others. Ristomycin and its use in the clinic. M., Medicine, 1965, pp.21-23. 2. Blinov I. O. and Khokhlov A. S. Paper chromatography of antibiotics, Nauka, 1970, pp. 252-253 (prototype).