DK143712B - PROCEDURE FOR PREPARING ANTIBIOTIC SUBSTANCES 890A1 AND 890A3 - Google Patents

Description

(19) DENMARK V £ S / (sIm

| J | (12) PUBLICATION OF 143712 B

DIRECTORATE OF THE PATENT AND TRADEMARKET SYSTEM

(21) Application No. 4971/76 (51) int.Cf.3 C 12 P 17/18 (22) Filing day 3 · Nov. 1976 C 07 D 487/04 (24) Race day 3 Nov. 1976 (41) Aim. available May 22, 1977 (44) Presented 28. s ep. 1 981 (86) International application no.

(86) International filing day (85) Continuation day - (62) Master application no. -

(30) Priority 21 Nov. 1975i 634, 00, US

(71) Applicant MERCK & CO. INC., Rahway, US.

(72) Inventor Patrick Joseph _Cassidy, US: Robert Thomas Goegelman, US: Edward Olley Stapley, US: Sebastian Hernandez, ES.

(74) Associate Engineer Hofman-Bang & Boutard.

(54) Process for the preparation of the antibiotics 89OAI and 89ΟA3-

The present invention relates to a process for the preparation of novel antibiotics, 8904 and 890A, which are isomeric compounds of the formula set forth in claim 1.

The discovery of the remarkable antibiotic properties of penicillin has greatly stimulated interest in this area, which has resulted in the emergence of other valuable antibiotics such as other penicillins, cephalosporins, strep-> 4-tomycin, bacitracin, tetracyclines, chloramphenicol and erythro - ^ mycines. In general, none of the said antibiotic drugs are; are active against all clinically important pathogenic bacteria.

For example, some of the substances are essentially only active against Gram-positive bacteria. The common use of available antibiotic agents to treat bacterial infections has also resulted in the emergence of resistant bacterial species, which has become a very serious problem.

As a result, there is still interest in finding new antibiotics that are active against a wider range of patho genes as well as against resistant strains of particular microorganisms. The present invention has for its object to provide the aforementioned novel antibiotics which are very effective in inhibiting the growth of various Gram-positive and Gram-negative microorganisms. This is achieved by the method according to the invention, which is peculiar to the characterizing part of claim 1.

A culture of productive strains of the species Streptomyces flavo-griseus is included in the culture collection of Northern Regional Laboratories, Northern Utilization Reasearch and Development Division, Agricultural Research Service, U.S. Department of Agriculture, Peoria, 111., under designations NRRL 8139 and 8140.

Streptomyces flavogriseus NRRL 8139 produces both antibiotics 89OA- ^ and 890A-3J which are isolated in a pure state from fermentation liquids. Streptomyces flavogriseus NRRL 8140 gives the antibiotic 890A- ^ without any detectable amount of 890A ^.

The morphological and cultural characteristics of Streptomyces flavogriseus NRRL 8139 are shown in the following table.

Morphologl - Sporophores are branched, straight or curved chains of spores that form tubercles. The chains contain more than 10 spores. The spores are spherical to oval - 0.9 / 1 x 1.2µ (970x).

Cultural properties Oatmeal stock

Vegetative growth - Back yellow tan, parchment-like growth;

Aerial mycelium - Light gray edged with medium gray Soluble pigment - Nothing.

3 T43712

Czapek Dox Agar (Sucrose Nitrate Agar)

Vegetative growth - Back brown edged with dark brown ·;

Aerial mycelium - Medium gray, velvety;

Soluble pigment - Slight brown staining of medium, Egg albumin agar.

Vegetative growth - Back yellowish 'tan edged with brown;

Aerial mycelium - Medium gray mixed with yellowish gray (2dc) and grayish-yellow (2db);

Soluble pigment - Light yellow tan.

Glycerol-Asparagine Agar

Vegetative growth - Back yellow tan, flat, widespread;

Aerial mycelium - Velvety, light gray with a strong yellowish tone to gray (2dc);

Soluble pigment - Nothing.

Inorganic salt-starch eggs

Vegetative growth - Reverse - brown;

Aerial mycelium - Medium gray, velvety;

Soluble pigment - Light yellow tan.

Yeast extract dextrose + salt agar

Vegetative growth - Reverse - brown edged with very dark brown; Aerial mycelium - Dark gray mixed with light gray, velvety; Soluble pigment - Nothing.

Yeast extract-malt extract agar

Vegetative growth - Reverse - dark brown;

Aerial mycelium - Dark gray, velvety;

Soluble pigment - Nothing.

Skim milk agar

Vegetative Growth - Tan;

Aerial mycelium - sparse, greyish;

Soluble pigment - Slight tan of medium;

Casein Hydrolysis - Good.

Litmus milk

Vegetative growth - Moderate growth, dark tan;

Aerial Mycelium - Nothing;

Color - Purple;

Coagulation and / or peptonization - Complete peptonization; becomes alkaline, pH = 8.2.

4 143712

Skimmed milk

Vegetative growth - Moderate growth, tan;

Aerial Mycelium - Nothing;

Soluble Pigment - Tan;

Coagulation and / or peptonization - Complete peptonization; becomes alkaline, pH = 8.0.

Tyrosine agar

Vegetative growth - Reverse - dark brown;

Aerial mycelium - Dark gray;

Soluble pigment - slight brown tinting of medium;

Decomposition of tyrosine - None.

Pepton Iron Yeast Extract Agar Vegetative Growth - Tan;

Aerial mycelium - Thrifty, greyish;

Soluble Pigment - Nothing;

Melanin - Nothing; ^ S production - None.

nutrient

Vegetative growth - Reverse - light grayish brown with dark grayish brown;

Aerial mycelium - Light gray edged with dark gray;

Soluble pigment - Nothing.

Næringsstivelsesagar

Vegetative growth - Tan edged with gray;

Aerial mycelium - Medium gray edged with dark gray;

Soluble Pigment - Nothing; . Hydrolysis of starch - Good.

Næringsgelatine agar

Vegetative growth - Colorless edged with dark gray;

Aerial mycelium - Greyish white;

Soluble Pigment - Nothing;

Melting Gelatin - Good.

Potato pieces * Vegetative growth - Good growth, strong "wrinkles; Aerial mycelium - Gray to greenish gray;

Soluble pigment - Slight tan of medium.

Loeffler's blood serum

Vegetative growth - Cream colored;

Aerial Mycelium - Nothing;

Soluble Pigment - Nothing;

Melting - None.

5 143712

Gelatinestik

Vegetative growth - Cream colored;

Aerial Mycelium - Nothing;

Soluble Pigment - Nothing;

Melting Gelatin - Good.

All the above data were determined after three weeks of cultivation at 28 ° C, unless otherwise stated. The pH of the media used in these studies was approximately neutral, namely pH = 6.8 - 7.2. The color indications in the specification are consistent with the definitions of the Color Harmony Manual, 4th Edition (1958), Container Corporation of America, Chicago, Illinois.

Streptomyces flavogriseus NRRL 8139 was also tested for its ability to utilize or assimilate various carbohydrates. For this purpose, the microorganism was grown on a synthetic base medium (Pridham and Gottlieb) containing 1% of the carbohydrate at 28 ° C for three weeks. The pH of the media used in the study was approximately neutral (6.8 - 7.2). Table I shows the utilization of these carbohydrate sources by Streptomyces flavogriseus NRRL 8139, with + indicating good growth, - poor growth and - no growth on the carbohydrate concerned. ·

TABLE I

Glucose + Maltose +

Arabinose + mannitol +

Cellulose - Mannose +

Fructose + Raffinose

Inositol - Rhamnose +

Lactose + Sucrose -

Xylose + The amount of growth depending on the temperature and oxygen demand of the microorganism is as follows:

Temperature range (Yeast extract dextrose + salt agar); 28 ° C - Good 37 ° C - Good vegetative growth; no air hypher 50 ° C - no growth 6 143712

Oxygen needs (yeast extract dextrose + salt agar culture);

Aerobic.

The morphological and cultural characteristics of Streptomyces flavogriseus NRRL 8140 are shown in the following table.

Morphology - Sporophores are branched, straight curved chains of spores that form tufts. The chains are more than 10 spores in length. The spores are spherical to oval - 0.9u x 1.2µ (970x).

Growing Properties

Oatmeal Agar

Vegetative growth - Reverse - yellowish-tan edged with dark brown; Aerial mycelium - Light gray edged with medium gray;

Soluble pigment - Nothing.

Czapek Dox agar (sucrose nitrate agar)

Vegetative growth - Reverse - brown edged with dark brown;

Aerial mycelium - Medium gray, velvety;

Soluble pigment - Nothing.

Egg albumin agar

Vegetative growth - Reverse - grayish tan with sections of vigorous yellowish tan

Aerial mycelium - Sections of medium gray, greyish-white and yellowish-gray (2dc);

Soluble pigment - Very light tan.

Glycerolasparagin agar

Vegetative growth - Yellow tan;

Aerial mycelium - sparse, greyish;

Soluble pigment - Nothing.

Inorganic salt-starch eggs

Vegetative growth - Back - greyish cream;

Aerial mycelium - Medium gray, velvety;

Soluble pigment - Nothing.

Yeast extract dextrose + salt agar

Vegetative growth - Reverse - dark brown;

Aerial mycelium - Dark gray mixed with light gray, velvety; Soluble Pigment - No Yeast Extract-Malt Extract Agar

Vegetative growth - Reverse - dark brown; 7 143712

Aerial mycelium - Dark gray, velvety;

Soluble pigment - Nothing.

Pepton Iron Yeast Extract Agar Vegetative Growth - Tan;

Aerial Mycelium - Nothing;

Soluble Pigment - Nothing;

Melanin - Nothing; E ^ S Production - None.

nutrient

Vegetative growth - Light tan;

Aerial Mycelium - Nothing;

Soluble pigment - Nothing.

Næringsstivelsesagar

Vegetative growth - Cream colored;

Aerial Mycelium - Nothing;

Soluble Pigment - Nothing;

Hydrolysis of starch - Good.

Næringsgelatineagar

Vegetative growth - Cream colored;

Aerial Mycelium - Nothing;

Soluble Pigment - Nothing;

Melting Gelatin - Good.

Gelatinestik

Vegetative Growth - Tan;

Aerial Mycelium - Nothing;

Soluble Pigment - Nothing;

Melting Gelatin - Complete.

Skummetmælkagar

Vegetative Growth - Tan;

Aerial Mycelium - Nothing;

Soluble Pigment - Nothing;

Casein Hydrolysis - Good.

Litmus milk

Vegetative growth - Tan growth ring;

Aerial Mycelium - Nothing;

Color - Brownish-purple;

Coagulation and / or peptonization - Complete peptonization, becomes alkaline, pH = 8.0.

8 163712

Skimmed milk

Vegetative growth - Tan, moderate growth;

Aerial Mycelium - Nothing;

Soluble pigment - Light brown;

Coagulation and / or peptonization - Complete peptonization, becomes alkaline, pH = 8.5.

potato Pieces

Vegetative growth - Good, tan;

Aerial mycelium - Very sparse, whitish;

Soluble pigment - Nothing.

Loeffler's blood serum

Vegetative growth - Cream colored;

Aerial Mycelium - Nothing;

Soluble Pigment - Nothing;

Melting - None.

Tyrosine agar

Vegetative Growth - Tan;

Aerial Mycelium - Nothing;

Soluble pigment - Slight tan of medium;

Decomposition of tyrosine - Very weak.

All the above data were determined after three weeks of cultivation at 28 ° C, unless otherwise stated. The pH of the media used in these studies was approximately neutral, namely pH = 6.8 - 7.2. The color indications in the description are in accordance with the definitions in the Color Harmony Manual, 4th edition (1958),

Container Corporation of America, Chicago, Illinois.

Streptomyces flavogriseus NRRL 8140 was also tested for its ability to utilize or assimilate various carbohydrates.

To this end, the microorganism was grown on a synthetic base medium (Pridham and Gottlieb) containing 1% of the carbohydrate at 28 ° C for three weeks. The pH of the medium used was approximately neutral (6.8 - 7.2). Table II shows the utilization of these carbohydrate sources by Streptomyces flavogriseus NRRL 8140, with + indicating good growth, - poor growth and - no growth on the carbohydrate concerned.

9 143712

TABLE II

Glucose + Maltose +

Arabinose + mannitol +

Cellulose - Mannose + -f

Fructose + Raffinose - J.

Inositol - Rhamnose +

Lactose + Sucrose -

Xylose + The amount of growth depending on the temperature and oxygen demand of the microorganism is as follows:

Temperature range (yeast extract-dextrose + salt agar); 28 ° C - Good 37 ° C - Moderate vegetative growth; no air hypher 50 ° C - No growth

Oxygen needs (yeast extract dextrose + salt agar culture);

Aerobic.

The subject antibiotics, 890A ^ and 890A ^, are prepared by aerobic fermentation under controlled conditions of suitable nutrient media seeded with strains of the organism Streptomyces flavo-griseus. Aqueous media of the same kind used for the preparation of other antibiotics are suitable for the preparation of 890A ^ and 890A ^. Such media contain sources of carbon, nitrogen and inorganic salts that are assimilable by the microorganism.

Generally, the usual carbohydrates such as sugars, e.g. dextrose, glucose, fructose, maltose, sucrose, xylose or mannitol, and starches such as dextrin or grains, e.g. oats, rye, cornstarch, cornmeal and the like, either alone or in combination as sources of assimilable carbon in the nutrient medium. The exact amount of the carbohydrate used in the medium depends in part on the other constituents of the medium, but usually between 1 and 6% by weight of carbohydrate is used in the medium. Said carbon sources may be used individually or several such carbon sources may be combined in the medium. Usually, many proteinaceous materials can be used as a source of nitrogen in the fermentation process. Suitable nitrogen sources include, for example, yeast hydrolysates, primary yeast, soybean meal, cotton flour, casein hydrolysates, cornstarch liquid, broth or tomato paste. The nitrogen sources can be used either alone or in combination, using in amounts between 0.2 and 6 pot. by weight of the aqueous medium. Among the inorganic nutrient salts which can be introduced into the culture medium are the usual salts which can release ions of sodium, potassium, ammonium, calcium, magnesium, phosphate, sulfate, chloride and carbonate. Trace metals such as cobalt, manganese and iron should also be present.

The fermentation can be carried out at temperatures between 20 and 37 ° C, but optimum culture results are obtained between 23 and 28 ° C. The pH of the nutrient should initially be between 6.0 and 8.0.

Although the antibiotics 890A ^ and 890A ^ are formed both in surface and submerged cultures, it is preferred to carry out the fermentation in the submerged state.

Small-scale antibiotic fermentation is conveniently carried out by growing in a suitable nutrient medium with the antibiotic-producing culture, and after transferring to a production medium, fermentation is carried out at a constant temperature of ca. 28 ° C in a shaker for several days.

The fermentation is started in a sterilized flask containing nutrient medium over one or more stages of seed culture development. The nutrient medium for seed culture may consist of a combination of the above carbon and nitrogen sources. The seed flask is shaken at a constant temperature of 28 ° C over a day or until growth is satisfactory and a portion of the growth formed is used for grafting either by a next step of seed culture or the production medium. Intermediate steps of any used seed flasks are developed in the usual manner, ie. a portion of the flask contents from the last inoculation stage is used for inoculation of the production medium. The inoculated flasks are shaken at constant temperature for several days and, after the end of the incubation period, the flask contents are centrifuged or filtered.

For large-scale production, it is preferred to carry out the fermentation in suitable tanks equipped with stirrer and aeration means for the fermentation medium. In this method, the nutrient medium is worked up in the tank and sterilized by heating to temperatures up to 120 ° C. After cooling, the sterilized medium is seeded with a pre-cultured portion of the production culture and fermentation is carried out for a period of, for example, 1-6 days with stirring and / or aeration of the nutrient medium while maintaining the temperature at 24-28 ° C. This method of production of antibiotics 890A ^ and 890A ^ is particularly suitable for the preparation of large quantities of antibiotic.

Physical and chemical properties of antibiotic 89 (^ and 890A ^ Properties of antibiotic 890A- ^

Antibiotic S90A- ^ is an acidic substance which moves towards the po -.....

sitive pole by electrophoresis at neutral pH.

The sodium salt of antibiotic 89 (^ is a white powder when lyophilized by an aqueous solution and it is very easily soluble in water.

The ultraviolet absorption spectrum has an A.max of 299.5 nm and a p.min of 242 nm. E% at 300 nm for a solution of the sodium salt of antibiotic 890A-1 in water at neutral pH is 208 for a pure sample; the ratio of absorption values at 300 nm to 245 nm is 4.25, and the ratio More than 92% of the absorption at 300 nm can be removed by reaction with hydroxylamine and a corresponding decrease is observed by reaction with cysteine.

The circular dichroism spectrum of 890A ^ exhibits a positive maximum at 290.5 nm with a specific ellipticity at 5270 degrees-ml per decimeters-grams, a zero point for ellipticity at 250 nm and a negative minimum at 214 nm, with specific ellipticity At -10,910 degrees-ml per decimeter-gram.

The following table indicates the 100 MHz NMR signals for QSOk 1 sodium salt relative to the internal standard, sodium 2,2-dimethyl-2-silapentane-5-sulfonate, herein referred to as DSS; chemical shifts are given in ppm and coupling constants are in Hz; the apparent multiplicity is indicated.

1.35 (d, J = 6.5) 1.98 (d); 3.63 (d of d, J = 5.2 and J = 9.8); 4.02-4.26 (m); 3.18 (d of d, J = ^ 11 and J = 10); 3.41 (t, J = 6); 2.97 (d of t, J = 3.5 and J = 6).

The antibiotic potency of 890A1, measured on Vibrio percolans ATCC 8461, is defined as 250 units per HAEA ^ 00 unit.

Properties of antibiotic 890A ^

Antibiotic 890A ^ is an acidic substance that moves toward the positive pole by electrophoresis at neutral pH.

The sodium salt is a white powder when lyophilized by an aqueous solution.

The ultraviolet absorption spectrum has a maximum at 300.5 nm and a minimum at 243 nm. E% at 300 nm of a solution of the sodium salt of antibiotic 89OA ^ at neutral pH is 375 for a pure sample; the ratio of absorption at 300 nm to 245 nm is 3.54. More than 90% of the absorption at 300 nm can be removed by reaction with hydroxylamine; and a corresponding decrease is observed in reaction with cysteine.

The circular dichroism spectrum of 890AA has a positive maximum at 294 nm with a specific ellipticity of 9127 degrees-ml per second. decimeter-grams, a zero point for the ellipticity at 249 nm, and a specific ellipticity of -15.053 degrees-ml per decimeter-gram at 220 nm.

The following table lists 100 MHz NMR signals for 890A ^ sodium salt in D20 relative to the internal standard DSS; chemical shifts are given in ppm, and coupling constants in Hz; the apparent multiplicity is indicated.

1.29 (J = 6.5); 1.98 (s); 3.42 (d of d, J = 5 and J = 2.4); <4.01-4.28 (m); 3.14 (d of d, J = 5 and J = 9); 3.39 (t, J = 6.5); 2.92 (d of t, J = ^ 4 and J = 6).

The antibiotic potency of 890Α, measured on Vibrio percolans ATCC 8461, is 31 units per day. HAEA ^ qq unit.

Mass spectral analysis of 890A ^ and 890A ^

Mass spectral data for 890A and 890A are obtained from trimethylsilyl derivatives made from ammonium salts of the antibiotics with bis-trimethylsilyl trifluoroacetamide in dimethylformamide. Conversion of sodium salts of the antibiotics to ammonium salts is carried out with the ammonium salt of an acidic ion exchange resin.

Trimethyl silylation of 890AA and 890A ^ results in three different derivatives: a di- and a trimethylethylsilyl derivative (molar weight 458 and 530 respectively) and a small amount of tetra-trimethylsilyl derivative of a hydrolyzed product (molar weight 620), wherein the β-lactam ring is open.

The values for the major mass spectral fragments are given below: di-trimethylsilyl derivative: 443.1495; 301.1034; 300.0962; 241.0590 and 86.0610.

trimethylethylsilyl derivative: 515,1931; 373,1422 and 158,1000.

tetra-trimethylsilyl derivative (low resolution signal only observed): 620 and 605.

143712 14

Antibiotics 890A ^ and 890A ^ are isomers and have a molecular structure as follows: ch3-ch (oh) --0 I γ-s-ch2-ch2-nh-c-ch3

COOH

Furthermore, antibiotics 89OA- and 89OA ^ have the following antibiotic spectrum profiles.

The test to determine the antibiotic spectrum profile was performed using a drop of 0.015 ml on the surface of 100 x 15 mm petri dish containing 5 ml of grafted nutrient agar and 0.2% yeast extract. Antibiotic 890A3 is tested at a concentration of 33 pg / ml and antibiotic 890A3 is tested at a concentration of 182 pg / ml. The results in terms of the diameters in millimeters for zone inhibition are shown in subsequent Table III.

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Antibiotic 890A-1 exhibits in vivo activity against Gram-negative and Gram-positive organisms and is therefore well-suited for controlling bacterial infections in animals and humans. To determine activity in vivo, antibiotic 890AA is diluted with dilution with 0.15M NaCl, 0.01M sodium phosphate, pH = 7.0, to obtain five quadruplicate concentrations of the drug for testing. White Swiss female mice with a mean weight of 21 g were infected intraperitoneally with the test organism, suspended in the fluid. The number of injected organisms was determined by standard plate counting technique. At the time of infection and again six hours later, some of the mice were treated intraperitoneally with the antibiotic. Five mice were used for each concentration of the drug tested. An additional two mice that were not infected were tested with the antibiotic to determine if the amount of the injected agent was toxic. Five control mice for each of several dilutions of the infecting culture were included in each sample to calculate the number of organisms that were lethal to 50% of the infected, untreated noses (LD ^ q).

This calculation was performed using survival data on the seventh day after infection, at which time the amount of drug capable of protecting 50% of infected mice (ED ^ q) was also calculated.

All such infected animals, which were not treated with antibiotics, died within 48 hours of infection. The effectiveness of antibiotic sgOA ^ with an activity of 5.5 units per pg to Salmonella schottmuellari MB-2837 is shown in the following table: ED ^ q x 2 dose

Enh./mla^ Route Dilution Enh./ml pgb ^ ........ 11 ............ ...... —..... "I. ............... Ill I · ™ · I .. IH I., ........

908 ip 1:32 14 3 ^ 1 unit / ml = the concentration which causes a 25 mm zone of inhibition against Vibrio percolans ATCC 8461.

The pg value is calculated from a purity of 890A ^ of 5500 units / mg, 18 143712

Antibiotics 89 OA ^ and 89 O A ^ are valuable antibiotics that are active against various Gram-positive and Gram-negative bacteria and therefore can be used in human and veterinary medicine. The present compounds may be used alone or in combination with each other as antibacterial drugs for treating infections caused by Gram-positive or Gram-negative bacteria, e.g. against Staphylococcus aureus, Proteus mirabilis, Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae. The antibiotics can further be used as animal feed additives, for food preservation and as disinfectants. For example, they can be used in aqueous compositions at concentrations of 0.1 to 100 parts of antibiotic per day. million parts of solution or preferably in concentrations of 1 to 10 parts of antibiotic per ml. million parts solution to destroy and inhibit the growth of harmful bacteria on medical and dental instruments and as bactericides for industrial use, e.g. in water-based paints and in paper mill water to inhibit the growth of harmful bacteria.

The subject antibiotics may be used in various pharmaceutical compositions as the sole ingredient or in combination either with one or more other antibiotics or one or more pharmaceutically active substances. As an example of the first possibility, mention can be made that an aminocyclitol antibiotic such as gentamycin can be administered simultaneously to obtain the greatest safety against the development of resistant organisms. As an example of the latter possibility, it should be mentioned that diphenoxylate and atropine can be added in dosage form to cure gastroenteritis. The antibiotic can be used in capsule form or as tablets, powders or liquid solutions or as suspensions or elixirs. It can be administered orally, topically, intravenously or intramuscularly. Furthermore, antibiotics 890A ^ and 89OA ^ can be used in combination.

Non-toxic pharmaceutically acceptable salts are prepared from 89 (8 and 890A3, for example, metal salts of alkali metal or alkaline earth hydroxides, carbonates or bicarbonates, such as derived from sodium, potassium, ammonium and calcium salts and salts of primary, secondary or tertiary amines such as monoalkylamines, dialkylamines, trialkylamines, lower alkanolamines, di-lower alkanolamines, lower alkylene diamines, N, N-diaralkyl-lower-alkylene diamines, aralkylamines, amino-substituted lower alkanols, N, N-di-lower alkylamino-substituted lower alkanols, amino-, polyamino- and guanidine-substituted lower alkanoic acids and nitrogen-containing heterocyclic amines Representative examples include salts derived from sodium hydroxide, ammonium hydroxide, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, calcium carbonate, trimethylamine, triethylamine, piperidine, N-ethyl-piperidine, morpholine, quinine, lysine, protamine, arginine, procaine, ethanolamine, morphine, benzylamine , ethylenediamine, Ν, Ν'-dibenzylethylenediamine, diethanolamine, piperazine, dimethylaminoethanol, 2-amino-2-methyl-1-propanol, theophylline and N-methylglucamine.

The salts in question can be prepared in a manner known per se.

For example, the monosalts, such as the monosodium salt, can be obtained by treating an equivalent of sodium hydroxide with an equivalent of one of the antibiotics in a suitable solvent. Also mixed salts with divalent cations can be prepared by combining one mole of a divalent base with one mole of one of the antibiotics plus one equivalent of another acid. Instead, the salts can be obtained by treating an equivalent of a base with a divalent cation, such as calcium hydroxide, with an equivalent of one of the antibiotics.

The fermentation liquids produced by the process of the invention contain the antibiotics 890A-L and 890A2 at a concentration equal to 2 - 170 units per minute. ml when tested by the diffusion test method using Vibrio percolans (ATCC 8461). Antibiotic preparations with at least 5 units per mg of activity of 890A ^ and 890A ^ can be purified and the antibiotic recovered in several ways. One such procedure consists of adsorbing the antibiotic 890A- and 890A ^ on a strong basic anion exchange resin. Typical examples of such highly basic anion exchange resins are those resins having a styrene-divinylbenzene matrix, e.g. polystyrene resin with Dowex 1x2 nuclear quaternary ammonium (manufactured by Dow Chemical Co., Midland, Michigan), on chloride cycle. Other representative members of this class of basic ion exchange resins are the following: Duolite A-40, A-42, A-101, A-102 and A-114 (manufactured by Chemical Process Co., Redwood City, California). Amberlite IRA-400, IRA 401 and IRA-410. Instead, a weak basic anion exchange resin such as Amberlite IRA-68 can be used. (Amberlite resin is manufactured by Rohm and Haas, Washington Square, Philadelphia 5, Pennsylvania).

The adsorbed antibiotic is readily eluted by the anion exchange resin with an aqueous saline solution. The eluate thus formed may be further purified, if desired, by other purification procedures. Thus, the eluate can be purified by passing through a column packed with acrylic ester polymer of intermediate polarity, such as XAD-7 or 8, or through a column packed with a nonpolar polystyrene polymer, cross-linked hydrophobically with divinylbenzene such as XAD-1, 2 and 4, preferably XAD-2. (XAD-1, 2, 4, 7 and 8 are manufactured by Rohm and Haas, Washington Square, Philadelphia 5, Penssylvania). In this process, partial separation of antibiotics 890A ^ and 89OA ^ occurs. High fractions of 89OA- ^ and high contents of 890A ^ are collected separately. These collected fractions are further purified.

A method to obtain further purified antibiotic. 89OA · and 89OA · consist of applying filtration through polyacrylamide having a pore size which excludes molecules with a molecular weight greater than 1800, such as Bio-Gel-P-2 (manufactured by Bio * Rad, Richmond, California). Other gels, such as Sephadex G-10, can also be used for desalination.

The preferred procedure for obtaining high purity antibiotic 890A- and 890AA from fermentation liquids consists in centrifugation of the filtrate to remove solids, adsorption and elution of an ion exchange resin such as Dowex 1x2 on chloride cycle with NaCl at 3 to 5%, thereby concurrently concentrating and partially purifying the antibiotic; passage over a column of specially prepared XAD-2 retains the antibiotic, thereby purifying and desalinating the Dowex 1 x 2 eluate. Antibiotic 890A ^ is retained more than antibiotic 890A1, so that the two antibiotics are partially separated. Fractions containing 890A and 890A are collected and further purified. Chromatography on Dowex 1x2 minus 400 mesh and elution with NaCl and / or NH4 Cl gives a product that is free of most UV absorbent impurities (NH4 Cl is used to obtain some buffering capacity in the eluent); and a desalination on Bio-Gel-P-2 or Sephadex G-10 removes salt introduced by Dowex 1x2 chromatography and further reduces the amount of other impurities in the antibiotic composition.

When low activity liquid is treated by the above procedure, the final material may contain significant amounts (more than 50%) of foreign impurities. These impurities can be reduced by repeating chromatography on Dowex 1x2 minus 400 mesh, eluting with a solution containing sodium chloride and 50% methanol. It is also advisable to conduct another step of XAD-2 chromatography when isolating antibiotic from liquid with low activity.

This results in further purification, removes residual salt and partially separates antibiotic 890A · ^ from 890A ^. Thus, 890A ^ elutes later than 890A ^ and the two can be separated by their different ratios of bioactivity to HAEA ^ qq (see below) -The fractions exhibiting a ratio of bioactivity on Vibrio percolans ATCC 8461 to HAEA ^ qq of 250, contains antibiotic 890Α ^, and they coat-; tions showing a bioactivity ratio on Vibrio percolans ATCC 8461 to HAEA ^ qq of 31 »contain antibiotic 890A ^.

High-content fractions of antibiotic 890A1 can be treated with penicillinase to remove residual traces of antibiotic 890A1 and further purified by chromatography on Dowex 1 x 2 and Sephadex G-10.

For laboratory scale operation (less than 20 liters of sample volume), all chromatographic steps except chromatography on XAD-2 are performed in a cold room at 2-5 ° C. XAD-2 chromatography is carried out at room temperature, unless otherwise stated. The pH of the antibiotic solutions to be stored is adjusted to 7-8 by careful addition of dilute sodium hydroxide or HCl solutions. Aqueous solutions are stored in a refrigerator, preferably in ice water, and 50% methanol solutions are stored at -20 ° C.

A diagram of the purification procedure for obtaining antibiotics 890A ^ and 890A · ^ is shown in subsequent Figures 1 and 2.

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24 143712

Experimental Procedure for Antibiotics 890A ^ and 890A ^ I. Bioassays

In an agar plate disk diffusion method, either Vibrio percolans ATCC 8461 or Salmonella gallinarum MB-1287 is used as the test organism. A purified sample of antibiotic 890Α is used as standard.

Plates containing Vibrio percolans ATCC 8461 are prepared as follows:

A lyophilized culture of Vibrio percolans ATCC 8461 is suspended in 15 ml of a sterilized medium containing 8 g / liter Difco nutrient liquid and 2 g / liter yeast extract in distilled water "Nutrient Yeast Extract" (hereinafter referred to as NBYE). The culture is grown for 16 hours in a rotary shaker at 28 ° C. This culture is used for grafting the surface of slant cultures containing 1.5% agar in NBYE, and the grafted slant cultures are grown for 16 hours at 28 ° C and then stored in a refrigerator.

The cooled slant cultures made from a single lyophilized culture are used for up to four weeks from their preparation as follows: With a graft needle, graft culture graft material, which is dispersed in 50 ml NBYE, is dispersed in a 250 ml conical flask. The culture is grown for 16 hours in a rotary shaker at 28 ° C and then diluted to a 50% transmission concentration at 660 nm. A 53.2 ml portion of this diluted culture is added to 1 liter of NBYE containing 15 g of agar and maintained at 46 ° C. The grafted agar-containing medium is poured into 100 x 15 mm plastic Petri dishes, 5 ml. bowl, cool and maintain at 2-4 ° C for 1 - 5 days prior to use.

Plates containing Salmonella gallinarum MB-1287 are prepared as follows:

A molten tube containing Salmonella gallinarum MB-1287 cells in skimmed milk, frozen and lyophilized and melted under vacuum, is opened and seeded in 15 ml of brain-cardiac infusion fluid. The cells are allowed to shake without growth at 37 ° C for 16 hours, and the carbon is seeded on slant cultures with 0.8% BBL nutrient liquid + 0.2% Difco yeast extract + 1.5% agar. After growing for 16 hours at 37 ° C, inoculating the culture from the slant culture is transferred to a flask containing 50 ml of 0.8% nutrient liquid + 0.2% yeast extract. The flask is shaken for 16 hours and 20 ml of this culture is seeded in a liter of 0.8% nutrient liquid + 0.2% yeast extract + 1.5% agar which is sterilized and cooled to 48 ° C. The inoculated NBYE agar is immediately poured into 100 x 15 mm plastic Petri dishes, 5 ml per ml. bowl, and the plates are maintained at 0-3 ° C until use.

13 mm diameter filter paper slices are dipped into the solution to be tested and placed on agar. Instead, the slices can be filled by pipetting one tenth of a ml of solution onto a dry slice and then placing it on agar. The diameter of the inhibition zone is measured after appropriate culture (9-18 hours at 37 ° C for Salmonella gallinarum MB-1287 or 12-24 hours at 25T for Vibrio percolans ATCC 8461). If necessary, dilutions of the solutions to be tested are carried out with 0.05M potassium phosphate buffer, pH = 7.4 "potassium phosphate buffer" (hereinafter referred to as KPB), or in deionized water.

Calculations of the activities proceed as follows: A slope is determined by measuring the zone diameters for a solution of the antibiotic 8904- or 890A ^ and for a quadruple dilution (in KPB) of this solution. Two slices of each concentration are tested on a single plate and the mean zone size of each concentration is determined. The slope is equal to half the difference between the average zone sizes. The strength is then calculated by the formula:

Strength (units / ml) = (Strength of Standard) / rn n -i _ \

x dilution, XO

where D is the mean diameter of the zones formed by the unknown, D

5 is the mean diameter of the standard zones and "dilution" is dilution equal to that of the unknown prior to the test. If no standard is used, D is assumed to be 25 mm and (the activity of standard)

ISLAND

is added to 1 unit / ml, measured on Vibrio percolans ATCC 8461. Pure 890A ^ is defined as an activity of 250 units per unit. hydroxyl-amine quenchable absorbent at 300 nm, used as standard.

Accordingly, the activity of antibiotic 890A ^ 31 units per hydroxylamine quenchable absorbent unit at 300 nm.

For testing with Salmonella gallinarum MB-1287 plates, a slope is determined in the same way as with Vibrio percolans ATCC 8461 plates. When no standard is used, only the relative activities are calculated. If no slope is measured, a value of 2.3 mm is assumed. Activity calculations proceed as with Vibrio percolans ATCC 8461 samples. If no 890A ^ standard is used, a control of penicillin G at 250 µg / ml can be used to verify that the sensitivity of the organism is in the normal range.

II. Test procedure for determination of ”890 sample units11

The conventional disc-plate test method is used and the discs have a diameter of 12.7 mm. The routinely cast 10 ml plates are seeded with Vibrio percolans (ATCC 8461) and used with cephaloridine as standard. Four concentrations of cephalori-din are standard - 3.12, 6.25, 12.5 and 25 mcg per day. ml at 12.5 mcg per ml as a reference solution. The zone diameters on a standard 5 ml plate are as follows:

Wife, (mcg / ml) Zone diameter (mm) 3.12 16.8 6.25 22.3 12.5 25.0 25 29.6

One unit is defined as the amount of antibiotic that produces a 25 mm zone of inhibition on a 5 ml plate. For this test, a concentration of 12.5 mcg per therefore, cephaloridine is considered equivalent to 1 unit of 890A. Since the slope of the line for cephaloridine is 4.0, calculations are made for the activity of a sample with a slope of 4.0.

III. Hydroxylamine Reaction Both antibiotics 890A ^ and 890A ^ react with hydroxylamine and provide a substance with significantly diminished absorption at 300 nm.

27 143712

This allows a quantitative test for antibiotics 890A- and 890A3.

The solution to be tested is adjusted to 0.05M in potassium phosphate, pH = 7.4 by adding 1/20 volume of a solution containing 0.8M KgHPO ^ and 0.2M KH₂PO0. Then one hundred volumes of 1M hydroxylamine hydrochloride is added and absorption at 300 nm is measured at intervals of half to two minutes. The reaction is carried out at 22-28 ° C. A first order reaction and a half-life determined from the adsorption decrease over the first ten minutes are assumed. From this half-life, the time after which no further absorption reduction can be observed is calculated and the observations are continued beyond this time. If no further decrease is observed beyond that time, the total absorption decrease (corrected for dilution effect and absorption of hydroxylamine) is considered "Hydroxylamine extinguishable absorption at 300 nm (HAEA ^ qq)". If an absorption reduction is observed beyond that time, the magnitude is calculated. of background absorption diminution, and the observed absorption at that time is corrected for background diminution, assuming that the background diminution is linear with time, the corrected value is then noted as HAEA ^ qq.

The number of HAEA ^ qq units is equal to HAEA ^ qq multiplied by the volume in ml.

The process according to the invention will be illustrated in the following with the aid of some exemplary embodiments.

EXAMPLE 1

Shake flask production of a mixture of antibiotics 890A ^ and 890A ^

A portion of a slant culture of Streptomyces flavogriseus NRRL 8139 is used to inoculate a 200 ml aerated conical flask containing 50 ml of medium A of the following composition: 28 143712

Medium A

Dextrose 10.0 g Yeast autolysate (+ Ardamine type pH) 10.0 g

MgSty 7H 2 O 0.05 g ^ Phosphate buffer 2.0 ml

Distilled H2 O, pH = 6.5 1000 ml + Ardamine: Yeast Products Corporation APhosphate Buffer Solution KH2PO4 91.0 g

Na2HPO4 95.0 g

Distilled H2 O 1000 ml

This seed flask is shaken at 28 ° C on a shaker with 220 rpm. per minute (stroke 5 cm) for 2 days until growth is satisfactory.

Three 250 ml conical flasks, each containing 40 ml Medium B with the following composition:

Medium B

Tomato paste 20.0 g

Oatmeal (ground) 20.0 g

Distilled H90, 1000 ml

Adjust the pH to 7.0 with NaOH

are seeded with 2 ml (5¾ per flask of the growth medium from the inoculum flask. These three production flasks are shaken at 28 ° C in a shaker at a speed of 220 rpm (stroke 5 cm) for up to 4 days. After three days centrifuged fluid a 22 mm inhibition zone against Proteus vulgaris, MB838 and a 15 mm zone against Salmonella gallinarum MB1287 using a 6.35 mm test disc on standard test plates.

EXAMPLE 2

Shake flask production of a mixture of antibiotics 890A ^ and 890A ^

A portion of a slant culture of Streptomyces flavogriseus NRRL 8139 is used to inoculate a 250 ml aerated conical flask containing 50 ml of medium A of the following composition:

Medium A

Dextrose 10.0 g Yeast autolysate (+ Ardamine type pH) 10.0 g

MgSO4-7H20 0.05 g ^ Phosphate buffer 2.0 ml

Distilled H 2 O, pH = 6.5 1000 ml + Ardamine: Yeast Products Corporation

Jr '

Phosphate buffer solution kh2po 91.0 g

Na2HPO4 95.0 g

Distilled H2 O 1000 ml

This seed flask is shaken at 28 ° C in a shaker of 220 rpm. per minute (5 cm stroke) in a day until growth is satisfactory. Keep the flask at 4 ° C for 2 days until used.

Three 2000 ml Erlenmeyer flasks, each containing 200 ml of Medium B with the following composition:

Medium B

Tomato paste 20.0 g

Oatmeal (ground) 20.0 g

Distilled H2 O, 1000 ml

pH: adjusts to 7.0 with NaOH

inoculated with 7 ml per ml. flask (3> 5 $) of the growth from the inoculum flask. The three production flasks are shaken at 28 ° 0 and 220 rpm. per minute in a shaker (5 cm stroke) for four days.

30 143712

Tests performed on centrifuged liquid using 6.35 mm slices on standard test plates and the indicated pH values give the following results:

V. percolans S. gallinarum Age AJCC 8461 MB 1287 pH

3 days 22 mm 16 mm 6.1 4 days 26 mm 18 mm 7.5

After four days, the flasks are harvested.

EXAMPLE · 3

Shake flask production of a mixture of antibiotics 890A ^ and 890A ^

A lyophilized culture tube of Streptomyces flavogriseus NRRL 8139 is opened aseptically and the contents are suspended in a tube containing 0.8 ml of sterile Davis saline solution of the following composition:

Davis salts

Sodium citrate 0.5 g k2hpo4 7.0 g KH2PO4 3.0 g (nh4) 2 SO4 1.0 g

MgSO4-7H2O 0.1 g

Distilled H2 O 1000 ml

This suspension is used for grafting oblique cultures and plates of various media. A natural isolate is selected from one of these plates and streaked on a slant culture of medium A having the following composition: 51 143712

Medium A

Dextrose 10.0 g

Pepton 5.0 g Yeast extract 3.0 g

NaCl 12.705 g EC1 0.72 g

PeSO4 (NH4) 2SO4-6H2O 0.0351 g

MgCl2 * 6H2O 5.52 g

CaCl 2 * 2H 2 O 0.728 g

Distilled H 2 O 1000 ml pH = 7.4 (before sterilization)

Agar 25.0 g

This grafted slant culture is grown for 7 days and after spore formation a spore suspension is prepared with 0.8 ml of sterile Davis saline. Pire slant cultures of the second generation of Medium A are seeded from this suspension. The grafted slant cultures are grown for a week at 28 ° C and then stored at 4 - 6 ° C until 14 days later.

Half of the surface growth of one of these second-generation oblique cultures is used to grow aerated 250 ml conical flasks containing 50 ml of Medium B of the following composition;

Medium B

Fermented autolysate (+ Ardamine) 10.0 g

Glucose 10.0 g A Phosphate buffer 2.0 ml

MgSO 4 * 7H 2 O 50 mg

Distilled H2 O 1000 ml

pH: adjusted to 6.5 with HCl or NaOH

+ Ardamine: Yeast Products Corporation 32 143712 ~ * Phospha tpuf fer solution eh2po4 91.0 g

Na2HPO4 95.0 g

Distilled H2 O 1000 ml

Three seed flasks are grown to obtain sufficient graft material for twelve 2-liter production flasks. The seed flasks are shaken for a day at 28 ° C and 220 rpm. minute in a shaker (5 cm stroke). The flasks are removed from the shaker and stored for 4 days at 4 ° C. The contents of these seed flasks are used to inoculate twelve 2-liter non-aerated tapered production flasks (8 ml seed material per flask) containing 250 ml of production medium C of the following composition:

Medium C

Tomato paste 20.0 g

Whole ground oats 20.0 g

Distilled Hr> 0 1000 ml pH is adjusted to 7.0 with NaOH.

After grafting, the production flasks are grown at 28 ° C and shaken at 220 rpm. minute shake (5 cm stroke) for 4 days.

After this time, the flasks are harvested and the material is tested for activity using standard Salmonella gallinarum MB1287 and Yibrio percolans ATCC 8461 test plates using 12.7 mm test discs dipped in the centrifuged liquid.

Indhøstningsforsøg

Harvesting age (hours) 96 pH 7.4

Salmonella gallium (mm zone) 23

Yibrio percolans ATCC 8461 (mm zone) 29 33 143712 The fermentation liquid is filtered and the pH of the filtrate is adjusted to 7.7 - 7.0 with dilute hydrochloric acid. 2.65 liters of this filtrate is adsorbed on a 135 ml IRA68 resin on chloride cycle (a weak "basic crosslinked acrylic polymer anion exchange resin, manufactured by Rohm and Haas Co., Washington Square, Philadelphia 5, Pennsylvania) at 20 ml / min. The adsorbate is washed with 250 ml of water and eluted with 5 # sodium chloride to collect 6 x 100 ml fractions. All fractions are tested against Vibrio percolans ATCC 8461 and Salmonella gallinarum MB 1287 using slice. These samples show that the eluate fractions 1-3 contain 45 $ of the bioactivity applied to the resin.

Eluate fractions 1-3 are combined to give 310 ml of solution.

To this solution is added 15 g of sodium chloride and the solution is cooled to 2 ° C. The cold solution is adjusted to pH = 3.0 with hydrochloric acid and extracted immediately with 150 ml of n-butanol. The extraction is repeated with a further two 150 ml portions of n-butanol. The n-butanol extracts are extracted back in series with 3 x 75 ml portions of water. During the extraction, the aqueous phases are maintained at pH = 7 by the addition of dilute sodium hydroxide. All fractions are tested against Vibrio percolans ATCC 8461 and Salmonella gallinarum MB 1287, and the results are given below as a percentage of starting bioactivity:

Eraction Yield

Supply $ 100

Consumed water $ 12

Consumed n-butyl alcohol No. 1 0 No. 2 0 No. 3 0

Aqueous Back Extract No. 1 25 $ No. 2 37 $ No. 3 5 $

The first and second extract by back extraction are separately lyophilized. The freeze-dried substances are combined to give 984 mg of product.

EXAMPLE 4

Production of a mixture of antibiotics 890A ^ and 890A ^

A tube of lyophilized culture of Streptomyces flavogriseus NRRL 8139 is opened aseptically and the contents are suspended in a tube containing 0.8 ml of sterile DaYis saline solution of the following composition:

Pavis salts

Sodium citrate 0.5 g k2hpo4 7.0 g kh2po4 3.0 g (nh4) 2 SO4 1.0 g

MgSO4-7H2O 0.1 g

Distilled HgO 1000 ml

This suspension is used for grafting 4 slant cultures of medium A with the following composition:

Medium A

Dextrose 10.0 g

Pepton 5.0 g Yeast extract 3.0 g

NaOl 12,705 g KOI 0.72 g

PeSO4 (NH4) 2SO4-6H2O 0.0351 g

MgCl2-6H2O 5.32 g

CaCl2.2H2O 0.728 g

Distilled HgO 1000 ml pH = 7.4 (before sterilization)

Agar 25.0 g

The grafted slant cultures are grown for a week at 27-28 ° C and stored at 4-6 ° C until used 10 days later. Half of the surface growth of one of these oblique cultures is used for inoculation of aerated 25O ml conical flasks containing 50 ml of medium B having the following composition:

Medium B

Fermented autolysate (+ Ardamine) 10.0 g

Glucose 10.0 g A Phosphate buffer 2.0 ml

MgSO4-7H2O 50 mg

Distilled HgO 1000 ml

pH: adjusted to 6.5 with HCl or NaOH

+ Ardamin: Yeast Products Corporation i

Phosphate Buffer Solution KH2PO4 91.0 g

Na2HPO4 95.0 g

Distilled HgO 1000 ml

Three inoculants are seeded to obtain sufficient graft material for 12 two-liter production inoculars. The seed colonies are shaken for a day at 28 ° 0 in a shaker at 220 rpm. minute (5 cm stroke). The flasks are removed from the shaker and stored for 4 days at 4 ° C. The contents of these seed flasks are used to inoculate 12 two liters of non-aerated tapered production flasks (7 ml seed material per flask) containing 250 ml of production medium C of the following composition:

Medium C

Tomato paste 20.0 g

Primary yeast 10.0 g

Dextrin (Amidex) 20.0 g

Deionized H 2 O 1000 ml pH is adjusted to 7.5 with NaOH.

After grafting, the production flasks are grown at 24 ° C with shaking in a shaker at 220 rpm. per minute (5 cm stroke) for four days and five hours. After this time, the flasks are harvested and the material is tested for activity using Salmonella gallinarum. MB1287 and Vibrio percolans ATCC 8461 specimens with 12.7 mm specimens dipped in the centrifuged liquid. The results are shown in the following table:

Samples after harvest

Harvest age (hours) 101 pH 7.1

Salmonella gallium (mm zone) 31

Vibrio pereolans (mm zone) 43

The fermentation liquid is centrifuged. To the clear centrifugate of 2.2 liters at pH = 6.8, 22 mg (ethylenedinitrilo) tetraacetic acid is added and the pH is adjusted to 7.2. This centrifugate is adsorbed on 150 ml Dowex 1x2 resin in C1 cycle at 15 ml / min, collecting the effluent as one fraction. The adsorbate is washed with 150 ml of deionized water containing 10 µg / ml (ethylenedinitrilo) tetraacetic acid and eluted with 5 $ NaCl containing 10 µg / ml (ethylenedinitrilo) tetraacetic acid, collecting 5 x 75 ml fractions. All fractions are tested against Vibrio pereolans, ATCC 8461, and the results are shown in the following table, expressed as a percentage of the original bioactivity:

Extraction Yield Curd mix $ 100

Drain 0 5 $ NaCl eluates fractions 2 to 4 75 $

Wash water 1 $

Sixty ml of fraction 3 of the 5 $ NaCl eluate is adjusted to pH = 5.2 with dilute hydrochloric acid and percolated through a 100 ml column of XAD-2 which has been washed with five column volumes of 60 $ aqueous acetone (v / v) , five column volumes of deionized water and five columns volume of 5 $ (w / v) NaCl solution containing 25 µM neutral EDTA in deionized water, and washed with 5 column volumes of deionized water. The resin is developed with deionized water containing 10 µg / ml (ethyl dinitrilo) tetraacetic acid, collecting 2 x 25 37 14 3712 ml and 5 x 10 ml fractions, controlling the drain until negative to acidic silver nitrate. A negative silver nitrate reaction is obtained after obtaining the fifth 10 ml fraction. Development with deionized water is continued until 4 x 100 ml fractions are collected. The resin is developed with 60 $ acetone water containing 4 µg / ml (ethylenedinitrilo) tetraacetic acid until 2 x 50 ml fractions are collected. All the fractions are tested against Vibrio percolans ATCC 8461 and the results are as follows:

Sample Added Volume, 50 ml Yield, $ 100

Praction 1 25 0 2 25 0 3 10 0 4 10 0 5 10 1 6 10 4,5 7 10 7 8 100 60 9 100 6 10 100 1 11 100 0 12 50 1 13 50 0

Extraction eight is evaporated to 10 ml and lyophilized to give 34.2 mg of solids.

A 33.2 mg portion of the freeze-dried substances is taken up in 1.5 ml of 1 $ n-butanol water and applied to a column of Bio-Gel P-2 (200-400 mesh) 1.4 x 81.5 cm is pre-set to equilibrate with 1 $ n-butanol in water. The gel is developed with the same solvent at 1 ml / min, collecting 2 ml fractions. Each fraction 29 -46 is tested against Vibrio percolans ATCC 8461. Bioactivity occurs in fractions 36 to 44, with a maximum observed in fraction 39. The fractions 38 to 41 are combined and lyophilized to give 4.0 mg of antibiotic.

EXAMPLE 5

Shake flask production of a mixture of antibiotics 890A ^ and 890A ^

A lyophilized culture tube of Streptomyces flaTOgriseus NRRL 8139 is opened aseptically and the contents are suspended in a tube containing 0.8 ml of sterile Davis saline solution of the following composition:

Davis salts

Sodium citrate 0.5 g

KgHPO ^ 7.0 g KH2PO4 3.0 g (nh4) 2 SO4 1.0 g

MgSO4.7H2O 0.1 g

Distilled HgO 1000 ml

This suspension is used to inoculate four medium A slant cultures of the following composition:

Medium A

Glycerol 20.0 g

Primary yeast 5.0 g

Egg flour 15.0 g

Distilled H2 O 1000 ml

Agar 20.0 g

pH: adjusted to 7.2 with NaOH

The seeded slanting cultures are grown for a week at 27-28 ° C and then stored at 4-6 ° C until use.

One third of the surface growth from two oblique cultures is used to inoculate 6 aerated 250 ml conical flasks containing 50 ml of medium B having the following composition:

Medium B

39 143712

Geera Autolysate (+ Ardamine) 10.0 g

Glucose 10.0 g A Phosphate buffer 2.0 ml

MgSO ^ -7H₂O 50 mg

Distilled H2 O 1000 ml

pH: adjusted to 6.5 with HCl or NaOH

+ Ardamine: Yeast Products Corporation ^ Phosphate Buffer Solution KH2PO4 91.0 g

Na2HPO4 95.0 g

Distilled HgO 1000 ml

The flask is shaken for a day at 27-28 ° C in a shaker at 220 rpm. minute (5 cm stroke). The flask and contents are stored stationary for a day at 4 ° C.

Twenty-two liters of conical flasks, each containing 300 ml of medium C, are seeded at 8 ml per flask of growth from the inoculum flask.

Medium C has the following composition:

Medium C

Tomato paste 20.0 g

Primary yeast 10.0 g

Dextrin (Amidex) 20.0 g

Co212 * 6H2O 5.0 mg

Distilled H 2 O 1000 ml pH: adjusted to 7.2 - 7.4 with NaOH,

After grafting, the production flask is grown at 24 ° C with shaking in a shaker at 212 rpm. per minute (5 cm stroke) for four days and five hours. The flasks are harvested and tested for activity with Salmonella gallinarum MB1287 and Vibrio percolans ATCC 8461 with standard test plates using 12.7mm 40 143712 test discs dipped in centrifuged liquid. Dilute if necessary with 0.02M phosphate buffer, pH = 7.0. The results are given in the following table:

Harvest age hours 101 pH 7.1

Salmonella gallium (mm zone) 31

Yibrio percolans 1/10 fort.

(mm zone) 24.5 890 sample units 46

Four liters of the total fermentation liquid containing 23 units / ml is cooled to 3 ° 0 and centrifuged in 200 ml portions at 9000 rpm. minute for 15 minutes each. To the combined centrifuged liquids (3.6 liters) is added 1 ml of 0.1M neutral EDTA and 10 ml of Tris-HCl buffer, pH = 7.5. The solution of 0.1 OM neutral EDTA is prepared by dissolving 0.1 mole of disodium ethylenediamine tetraacetic acid in 1 liter of deionized water, adjusting the pH to 7 by adding sodium hydroxide.

The centrifuged liquid is adsorbed on a column of Dowex-1 x 2 (Cl-), 50-100 mesh, 5.1 cm x 20 cm dimensions of the layer which has been pre-washed with 1 liter of deionized water. The flow rate during application is 50 - 80 ml per minute. After completion of the application, wash the column with 500 ml of deionized water containing 25 μΜ neutral EDTA and then elute with 1300 ml of 5 $

NaCl in deionized water containing 25 µM neutral EDTA. Nine fractions are collected, counting from the first application of sodium chloride solution. Fractions 1 - 4 contain 100 - 115 ml per ml. and fractions 5-9 contain 170 to 185 ml per ml. fraction. The fractions are tested for bioactivity on plates of Salmonella gallinarum MB1287. Bioactivity occurs in fractions 2 to 9 with a maximum in fractions 4 and 5.

The ultraviolet absorption of appropriate dilutions of each fraction is measured in a Gilford model 2000 spectrophotometer and the ratio of bioactivity to absorption at 220 nm (-A ^ q) is calculated for each fraction. Such fractions with bioactivity / A22Q ratios greater than half the maximum value are collected for further processing. In this way, fractions 41 to 377 are collected containing 30 # of the applied bioactivity.

The collected sodium chloride eluate is evaporated to 100 ml under reduced pressure while maintaining the temperature below 35 ° C during evaporation.

The pH of the concentrate is adjusted to 5.2 by adding 3 ml of 1 molar hydrochloric acid and the concentrate is applied to a column of XAD-2 (3.5 cm in diameter and 57 cm long), which is washed with 3 liters of 60 # aqueous acetone. (v / v), 3 liters of deionized water, 3 liters of 5 # (w / v) sodium chloride solution containing 25 µM neutral EDTA in deionized water, and 1 liter of saturated sodium chloride solution. After washing, the column is transferred to a cold chamber maintained at 3 ° C before the concentrate is applied.

The applied concentrate is left to drain and the active fractions are eluted with 1.3 L of 25 µM neutral EDTA deionized water. Fractions of every 10 ml are collected, counting from the first application of the concentrate to the column.

t

Every fifth fraction is tested for bioactivity on plates of Salmonella gallinarum MB1287, and values for un ^ are using appropriate dilutions. Such bioactivity / λ ratio fractions greater than half the maximum value are collected. Thus, fractions 48 to 95 are collected containing 50 # of the applied activity, representing 17 # of the activity of the filtered liquid.

The collected XAD-2 fractions are evaporated to 50 ml and applied to a column of Dowex-1 x 4 (CI ""), minus 400 mesh, with dimensions of the resin layer of 1.3 x 13 cm. The column is rinsed with 15 ml of deionized water and eluted with 400 ml of 0.08M sodium chloride plus 0.005M ammonium chloride plus 0.0001M ammonia in deionized water. Reactions of each 10 ml are collected at a flow rate of 0.6 ml per ml. minute. Activity corresponding to 100 # of the applied activity is eluted in fractions 23 to 30, with maximum activity being obtained in fraction 26. Fractions 26 and 27 have the highest absorption ratio at 300 nm to absorption at 220 nm (A ^ Q / Ag ) and these two fractions are collected for desalination. The total activity in 26 and 27 is 7390 units or 59 # of the activity applied.

42 143712

To the collected fractions 26 and 27 is added 5 µL 0.01M neutral EDTA.

The cooled fractions are evaporated by rotary evaporation under reduced pressure to 1.95 ml and the concentrate is applied to a column of Bio-Gel P-2, 200 to 400 mesh with dimensions of the layer of 2.2 x 80 cm. The sample is washed with rinsing portions of 1 ml and 2 ml of deionized water and then eluted with deionized water at a flow rate of 0.7 ml per ml. minute. Collect 2.1 - 2.2 ml solutions. The major peak of TJV absorption is found in tubes 89 to 100 containing 74 $ of the applied units, measured at 300 nm (A ^ oo "en <3s) The pen sample contains 18.6 Aq qq units representing $ 5.8 of the bioactivity contained in the starting liquid Fractions 89 to 91 and 99 to 100 are further purified as indicated in Example 6.

EXAMPLE · 6

Shake flask production of antibiotic 890A ^

A lyophilized culture tube of Streptomyces flavogriseus NRRL 8139 is opened aseptically and the contents are suspended in a tube containing 0.8 ml of sterile Davis saline solution of the following composition:

Pavis salts

Sodium citrate 0.5 g k2hpo4 7.0 g EH2PO4 3.0 g (nh4) 2 SO4 1.0 g

MgSO4.7H2O 0.1 g

Distilled H2 O 1000 ml

This suspension is used to inoculate four medium A slant cultures of the following composition:

Medium A

43 143712

Glycerol 20.0 g

Primary yeast 5.0 g

Fishmeal 15.0 g

Distilled H2 O 1000 ml

Agar 20.0 g

pH: adjusted to 7.2 with NaOH

The seeded slant cultures are incubated for a week at 27-28 ° C and then stored at 4-6 ° C until use.

One-third of the growth from three oblique cultures is used to inoculate 9 250 ml soft-plate Erlenmeyer flasks containing 50 ml of medium B of the following composition:

Medium B

Fermented autolysate (+ Ardamine) 10.0 g

Glucose 10.0 g A Phosphate buffer 2.0 ml

MgSO 4 * 7H 2 O 50 mg

Distilled Hr, 0 1000 ml

pH: adjusted to 6.5 with HCl or NaOH

+ Ardamin: Yeast Products Corporation El.

Phosphate Buffer Solution kh2po4 91.0 g

Na2HPO4 95.0 g

Distilled HgO 1000 ml

The seed flask is shaken for 1 day at 27-28 ° C in a shaker at 220 rpm. minute (5 cm stroke). The flask contents are stored stationarily for 1 day at 4 ° C.

33 two liter conical flasks, each containing 250 ml Medium C are seeded at 8 ml per ml. flask of the growth from the inoculum.

Medium C

44 143712

Tomato paste 20.0 g

Primary yeast 10.0 g

Dextrin (Amidex) 20.0 g

5.0 mg

Distilled R 50 O 1000 ml

pH: adjusted to 7.2 - 7.4 with NaOH

After grafting, the production crust is grown at 24 ° C with shaking on a shaker at 220 rpm. per minute (5 cm stroke) for 4 days. The flasks are harvested and tested for activity using standard test plates with Salmonella gallinarum MB1287 and Vibrio percolans ATCC 8461 using 12.7 mm test discs dipped in the centrifuged liquid. If necessary, dilute the samples with 0.02M phosphate buffer, pH = 7.0. The results are shown in the following table:

Harvesting aids, hours 96 pH 7.2

Salmonella gallium (mm zone) 29.5

Vibrio percolans 1/10 fort.

(mm zone) 31 890 sample units 40

Seven liters of the total fermentation liquid is cooled to 3 ° C and centrifuged in 200 ml portions at 9000 rpm. minute for 15 minutes each.

To the combined centrifuged liquids, 7 ml of 0.1M neutral EDTA is added and the entire sample is adsorbed on a Dowex-1 x 2 (Cl ”), 50-100 mesh column, layer dimensions 5.1 x 25 cm, at a flow rate of 40 ml per minute. The column is washed with 500 ml of deionized water containing 5 ml of 1M tris-HCl buffer, pH = 7.0, and 25 µM neutral EDTA. The antibiotic is eluted with 1 liter of deionized water containing 50 g of sodium chloride and the column is then washed with 300 ml of deionized water. 100 ml fractions are collected, taking into account the first occurrence of salt in the drain from the column. The bioactivity is shown in fractions 1 to 10 with a maximum in fraction 2. The fractions with the highest ratio for hioactivity / Aggo are combined. Thus, fractions 2 to 5 containing 17 $ of the applied activity are combined.

The combined fractions are evaporated to 110 ml by means of a rotary evaporator under reduced pressure and the pH is adjusted to 5.8 by the addition of 4.2 ml of 1M hydrochloric acid. The set concentrate is applied to a column of XAD-2, layer dimensions 3.8 x 50 cm, which has been previously "washed with 3 liters of 60 $ aqueous acetone (v / v), followed by 3 liters of deionized water, 3 liters 5 $ (w / v) sodium chloride and 1 liter $ 25 (w / v) sodium chloride in deionized water.

The applied concentrate is dripped, the antibiotic eluted with deionized water at a flow rate of 15 ml / min.

Twenty-two fractions of 100 ml are collected, counting from the first application of the sample. The bioactivity appears in fractions 6 to 22 with a maximum in fractions 9 and 10. Fractions with bioactivity / Aggg ratio greater than 0.3 times the maximum value are collected. Thus, fractions 9-20 are combined to continue treatment. To these fractions are added fractions 89, 90, 91, 99 and 100 from the Bio-G-el P-2 column of Example 5. The collected portion is evaporated to 70 ml by rotary evaporation under reduced pressure.

The concentrate is adsorbed on a Dowex-1 x 4 (Cl “) minus 400 mesh column with layer dimensions 2.2 x 27 cm. The column is washed with 50 ml of deionized water and the antibiotic eluted with 2 liters of 0.070M sodium chloride + 0.005M ammonium chloride + 0.0001M ammonia in deionized water at a flow rate of 2 ml per ml. minute.

9.5 ml fractions are collected.

The major peak of the antibiotic is eluted in fractions 142 to 163. Fractions 146 to 157 contained substances with the highest ratios of A ^ Qg / Ag ^ Q, and these were combined for further treatment.

The combined fractions 146-157 are evaporated to 3 ml by rotary evaporation under reduced pressure and the concentrate is adjusted to a pH of 6.5 by adding 5 µl of 1 M ammonia. The concentrate is applied to a column (2.2 x 70 cm) of Bio-Gel P-2 (200-400 mesh) which has been washed with 20 ml of 5M sodium chloride and 500 ml of ionized water. After the concentrate is drained to level, two rinse fluids are each applied to 1 ml of deionized water and left to drain. The column is then eluted with deionized water at a flow rate of 0.6 ml per minute. 2.9 ml fractions are collected.

The main peak of the antibiotic is eluted in fractions 63 to 70. The fractions 64 and 65 have the highest ratio A ^ qq / A ^ q and are collected for further processing. These two fractions contain $ 44 of the UY absorption at 300 nm applied to the Bio-Gel P-2 column.

The combined fractions 64 and 65 are subjected to rotary evaporation under reduced pressure to 2 ml and then frozen and lyophilized in a 14 ml screw-cap ampoule for 8 hours to obtain 2.25 mg of substantially pure antibiotic 890A , 5 A500 units).

EXAMPLE · 7

Shake flask production of antibiotic 890A ^

A lyophilized culture tube of Streptomyces flavogriseus NRRL 8139 is opened aseptically and the contents are suspended in a tube containing 0.8 ml of sterile Davis saline solution of the following composition:

Davis salts

Sodium citrate 0.5 g K2HPO4 7.0 g KH2PO4 3.0 g (nh4) 2 SO4 1.0 g

MgSO4-7H2O 0.1 g

Distilled H2 O 1000 ml 47 143712

This suspension is used to inoculate four medium A slant cultures of the following composition:

Medium A

Glycerol 20.0 g

Primary yeast 5.0 g

Whisk flour 15.0 g

Distilled B ^ O 1000 ml

Agar 20.0 g

pH: adjusted to 7.2 with NaOH

The grafted oblique cultures are grown for a week at 27-28 ° C and then stored at 4-6 ° C before use (no longer than 21 days).

One-third of the growth from four oblique cultures is used to inoculate 12 aerated 250 ml conical flasks containing 50 medium B of the following composition:

Medium B

Fermented autolysate (+ Ardamine) 10.0 g

Glucose 10.0 g of phosphate buffer 2.0 ml

MgSO4-7H2O 50 mg

Distilled H2 O 1000 ml

pH: adjusted to 6.5 with HCl or NaOH

+ Ardamine: Yeast Products Corporation Phosphate Buffer Solution KH2 PO4, 91.0 g

Na2HPO4 95.0 g

Distilled Hr> 0 1000 ml 48 H8712

The seed flasks are shaken for a day at 27-28 ° C in a shaker with 220 rpm. minute (5 cm stroke). Keep the flask and contents up for a day at 4 ° C.

44 2-liter conical flasks, each containing 200 ml of medium C, are seeded at 8 ml per ml. flask of the growth from the inoculum.

Medium C has the following composition:

Medium 0

Tomato paste 20.0 g

Primary yeast 10.0 g

Dextrin (Amidex) 20.0 g

CoCl2 * 6H20 5.0 mg

Distilled HgO 1000 ml pH: adjusted to 7.2 - 7.4 with NaOH.

After grafting, the production flasks are grown at 24 ° 0 with shaking in a shaker at 220 rpm. per minute (5 cm stroke) for 4 days and 5 hours. The flasks are harvested and tested for activity using Salmonella gallinarum MB1287 and Vibrio percolans ATGO 8461 on test plates with 12.7 mm test discs dipped in centrifuged liquid. If necessary, dilute the samples with 0.02M phosphate buffer, pH = 7.0. The results are given in the following table:

Harvest age, hours 101 pH 7.2

Salmonella gallium (mm zone) 55

Vibrio percolans 1/10 fort.

(mm zone) 54,890 sample units 121 A total of 7.0 liters of the fermentation obtained by this fermentation is cooled to 3 ° C and centrifuged in 200 ml portions at 9,000 rpm for 15 minutes separately. To the combined centrifuged liquid is added 1.7 ml of 0.1M neutral EDTA and the portion is maintained at 3 ° C.

49 143712

The above fermentation is repeated under the same conditions, except that 44 2-liter conical flasks are grown with 7 ml per flask of the liquid from the flask. The pH and the test results are given below:

Harvest age, hours 101 pH 7.3

Salmonella gallium (mm zone) 38

Vibrio percolans 1/10 fort.

(mm zone) 39 890 sample beds 92.8 A total of 7.4 liters of the fermentation thus formed is cooled to 3 ° C and centrifuged in 200 ml portions at 9000 rpm. every 15 minutes. To the combined liquid above is added 1.8 ml of 0.1M neutral EDTA.

The above liquid from centrifuged fermentation liquid from the two aforementioned ferments in this example is combined to a total volume of 13 liters and an activity of 80 units per liter. ml by sample on Vibrio percolans ATCC 8461.

The combined supernatant is passed through a column of Dowex-1 x 2 (01 "), 50-100 mesh, with layer dimensions 4.7 cm x 50 cm and a flow rate of 60 ml per minute. The column is washed with 1 liter deionized water and the antibiotic is eluted with 5 L of 5 # (w / v) sodium chloride solution containing 0.1M Tris-HCl buffer, pH = 7.0, and 25 µM neutral EDTA. Reactions of 220 ml are collected at a flow rate of 60 ml per minute and the fractions are tested on Salmonella gallinarum MB1287 plates.

Antibiotic activity is detected in fractions 3 to 26 with a maximum in fractions 5 and 6. Fractions 5 to 9 have the highest ratio of bioactivity / A22o> ° & these fractions are combined for continued treatment. The pH of the collected fractions is 7.8 and contains 24 # of the original bioactivity, measured on Salmonella gallinarum MB1287 plates.

50 143712

The combined fractions 5 to 9 are concentrated to 150 ml of Ted rotary evaporation under reduced pressure and applied to a column (4 »9 cm x 47 cm layer dimensions) of XAD-2 which has been washed with 5 liters of 60 $ (v / v aqueous acetone followed by 5 liters of deionized water and 5 liters of 50 g / liter of sodium chloride in deionized water.

The sample is applied in 20 ml portions, leaving the column to drain each time. After completion of the application, three 20 ml portions of deionized water are introduced into the layer each time, by draining. The sample is eluted with deionized water at a flow rate of 20 ml. minute. All the operations with the XAD column are performed at room temperature (24 ° 0) and the eluted fractions are rapidly cooled in an ice bath immediately after collection. Collect 95- to 230-ml fractions.

The antibiotic activity is shown in fractions 2 to 21, measured by sample on plates with Salmonella gallinarum MB1287, with a maximum in fractions 5 to 7 (comprising 510 to 895 ml of eluted volume, calculated from the first supply of deionized water). Fractions 6 to 21 are collected which have a bioactivity / AggQ ratio within $ 50 of the observed maximum value.

The total bioactivity recovered is 280,000 units, which is $ 112 of the apparently applied activity.

The collected fractions 6 to 21 are evaporated to 68 ml by rotary evaporation under reduced pressure, and then diluted to 112 ml by the addition of deionized water. The concentrate is applied to a column (2.2 cm x 21 cm layer dimensions) of Dowex-1 x 4 (Cl ”) minus 400 mesh. The column is washed with 20 ml of deionized water and the antibiotic eluted with 2 liters of 0.07M sodium chloride + 0.005M ammonium chloride + 0.0001M ammonia in deionized water, at a flow rate of 1.6 ml per ml. minute. Fractions of every 8 ml are collected.

The major peak for absorption at 300 nm is found in fractions 155 to 182 with a maximum at fraction 162. Fractions 157 to 179 with greatest ratios of absorption at 300 nm to absorption at 245 nm are combined for further processing. The combined fractions have a total of 780 absorption units at 300 nm.

5i 143712

Oise collected fractions are evaporated by rotary evaporation under reduced pressure to 7 ml and the pH is adjusted to 7.5 by the addition of 20 µl of 1M sodium hydroxide. The solution is further evaporated to 5 mm and applied to a column (2.2 x 75 cm) of Bio-Gel P-2, 200-400 mesh. The sample is washed in the colon layer with two rinses each of 1 ml of deionized water, then eluted with deionized water at a flow rate of 0.6 ml per ml. minute.

Ten 3.3 ml fractions followed by 60 fractions of 2.65 ml and ten fractions of 2.0 ml were collected.

The main peak for absorption at 500 nm appears in fractions 60 to 68. The pH of these fractions is adjusted to 7.5 to 8.0 by the addition of 1.5 to 2.5 µl of 0.1 M sodium hydroxide.

Formulations 62, 63, 64 and 65 are separately frozen and lyophilized in 14 ml vials and stored at -20 ° C under vacuum. These four fractions contain a total of 606 absorption units at 300 nm.

To isolate antibiotic 890A ^ released from QQOk · ^, the relatively higher resistance of antibiotic 890A ^ to degradation with penicillinase is utilized as follows:

Bio-Gel fraction 63 is combined with fractions 61, 66 and 67 from the Bio-Gel column. These four combined fractions contain a total of 235 absorption units at 300 nm. To this is added 0.2 ml of 1M tris-HCl buffer, pH = 7.5, and 0.2 ml of penicillinase [Difco "Bacto-Penase", containing 500,000 units per ml. ml (1000 LTJ / ml), where the term LU refers to levy units; 1,000 LU will inactivate 500,000 units of penicillin G]. After 113 minutes at 23 ° C, an additional 0.2 ml of penicillinase is added. After an additional 7 hours at room temperature, an additional 0.2 ml of penicilinase is added and after an additional 2 hours at room temperature, the reaction mixture is cooled on an ice bath. The quenched reaction mixture is diluted to 15 ml by the addition of 0.5 ml of deionized water. The absorption of the diluted reactant is 6.3 at 300 nm, of which 2.64 is extinguishable by reaction with cysteine.

The reaction mixture is adsorbed on a column of Dowex-1 x 4 (Cl-) minus 400 mesh, layer dimensions 2.15 x 40 cm. Antibiotic 890A7) 52 143712 is eluted with 0.07M sodium chloride + 0.005M ammonium chloride + 0.0001M ammonia in deionized water at a flow rate of 3 ml per ml. minute. Fractions are collected every 13.8 ml.

The main peak with absorption at 300 nm is detected in fractions 185 to 203. Fractions 187 to 200 have the highest ratio of Δ300 ^ 245 combined for further processing. The combined fractions have a total of 31 Å ^ QQ units.

The combined fractions are concentrated to 4 ml by rotary evaporation under reduced pressure and the concentrate is applied to a column (2.2 x 70 cm) of Bio-Gel P-2, 200-400 mesh. The antibiotic 890AA is eluted with deionized water at a flow rate of 0.6 ml per ml. minute. Thirty fractions 3.3 ml followed by 50 fractions 2.65 ml are collected.

The principal peak for absorption at 300 nm is detected in fractions 64 to 74 with a maximum in fraction 70. Fractions 66 to 70 are combined and contain 19 A ^ QQ units or 61 µl of the applied units. The combined samples are evaporated to 1.5 ml by rotary evaporation under reduced pressure and the concentrate is frozen and lyophilized to give 5.4 mg of solid containing antibiotic 890A + plus salts.

EXAMPLE · 8

Preparation of antibiotics 890A ^ and 890A ^

A lyophilized culture tube of Streptomyces flavogriseus NRRL 8139 is opened aseptically and the contents are suspended in a tube containing 0.8 ml of sterile Davis saline solution of the following composition:

Davis salts

Sodium citrate 0.5 g K2HPO4 7.0 g KH2PO4 3.0 g (nh4) 2 SO4 1.0 g

MgSO 4 * 7H 2 O 0.1 g

Distilled ftp 1000 ml 53 143712

This suspension is used to inoculate four medium A slant cultures of the following composition:

Medium A

Glycerol 20.0 g

Primary yeast 5.0 g

Whisk flour 15.0 g

Distilled HgO 1000 ml

Agar 20.0 g pH: adjusted to 7.2 with NaOH.

The seeded slanting cultures are grown for a week at 27-28 ° C and then stored at 4-6 ° C until used (no more than 21 days).

Ten ml of medium B with the composition:

Medium B

Fermented autolysate (+ Ardamine) 10.0 g

Glucose 10.0 g A Phosphate buffer 2.0 ml

MgSO4 »7H2O 50 mg

Distilled H 2 O 1000 ml

pH: adjusted to 6.5 with HCl or NaOH

+ Ardamine: Yeast Products Corporation * Phosphate Buffer Solution KH2PO4 91.0 g

Na2HPO4 95.0 g

Distilled HgO 1000 ml is aseptically transferred to one of these slanting cultures, spores and aerial mycelium scraped into suspension and 3.5 ml of this suspension is used for inoculation of 2 liters of conical aerated flasks containing 500 ml medium B. This seed flask is shaken at 28 ° C. a shaker with 160 rpm. per minute (5 cm stroke) for 36 hours, 54 U3712 thus providing satisfactory growth.

The growth from this seed flask is used for inoculation of a 189 liter stainless steel seed tank containing 160 liters of medium B. This tank is operated at 28 ° C, stirring at a rate of 150 rpm. per minute and an air flow of 85 liters per minute. per minute for 24 hours. If necessary, add anti-foaming agent, Polyglyeol 2000 (Dow Chemical Corp.), which must not exceed $ 0.1. The pH values are as follows:

Age, hours 0 12 pH 6.3 6.55 43 liters of the growth from this seed tank is used to seed a 756 liter stainless steel fermentation container containing 467 liters of medium C of the following composition:

Medium C

Tomato paste 20.0 g

Primary yeast 10.0 g

Dextrin (Amidex) 20.0 g

CoCl2 * 6H20 5.0 mg

Distilled H 2 O 1000 ml pH: adjusted to 7.2 - 7.4 with NaOH.

This tank is operated at 25 ° C, stirring at a rate of 146 rpm. per minute and an air flow of 254 liters per minute. per minute for 92 hours. If necessary, additional anti-foaming agent, Polyglyeol 2000, is added but not more than $ 0.1. Antibacterial tests are performed on Salmonella gallinarum MB1287, Vibrio percolans ATCC 8461, and the data are as follows:

Age, hours 0 12 24 56 48 60 72 84 92 pH 6.6 6.7 6.6 6.3 6.0 6.4 6.15 6.5 6.5 MB-1287 mm - - - S - 19 26 28 32 ATCC 8461 mm (1-10) - - - S - 21 24 26 30 890A unit / ml NA NA 6.8 13.5 24.9 24.3 55 143712

The 890A units / ml are "determined as specified in the section headed" II. Test procedure for determining '890 sample units.

475 liters of fermentation liquid is cooled to 5 ° C and centrifuged through a Titan P-9 centrifuge. 23 kg of Celite is added to 370 liters of centrifuged liquid and the suspension is filtered through a Shriver 45 cm filter press. The 345 liters of filtrate are adsorbed on a column containing 26.5 liters of Dowex-1 x 2 (Cl 2), 50-100 mesh, and the column is washed with 38 liters of deionized water. The mixture of antibiotics 890A1 and 890A $ Sodium Chloride + 0.01M Tris-HCl Buffer, pH = 7.0 + 25 µM Neutral EDTA in Deionized Water. Collections of every 19 liters are collected. The mixture of antibiotics 890A ^ and 890A ^ appears in fractions 3 to 6 with maximum activity in fractions 4 and 5. Fractions 4, 5 and 6 containing 8% of the activity of the filtered liquid are combined and evaporated under reduced pressure to 7.6 liters.

The 7.6 liter concentrate is applied to a column containing 38 liters of XAD-2 which has been pre-washed with 190 liters of 60 # aqueous acetone followed by 190 liters of deionized water and 190 liters of 5 # sodium chloride solution. The concentrate is eluted with 142 liters of deionized water.

Three 9.5 liter fractions followed by six 19 liter fractions are collected. Activity occurs in fractions 1 to 6 with maximum activity in fraction 3. Fractions 4 and 5 containing 64 # of the activity applied to the column or 370,000 units are collected.

Fractions 4 and 5 are evaporated to 120 ml under reduced pressure. The pH is adjusted to 6.5 and the concentrate is applied to a column (7 x 50 cm) of XAD-2 which is washed with 8 liters of 60 # aqueous acetone followed by 4 liters of deionized water and 8 liters of 5 # sodium chloride in deionized water. . The sample is drained to the layer level and the column rinsed with three 20 ml portions of deionized water, dripping to the layer level each time. The antibiotic is then eluted with 7 liters of deionized water at a flow rate of 40 ml. minute. Eight fractions of 200 ml followed by 14 fractions of 400 ml are collected. The antibiotic activity appears in fractions 4 to 19 containing 77 # of the bioactivity indicated on the column (measured by Salmonella gallinarum MB1287), with a maximum of activity in fractions 6 to 8.

56 143712

The relationship between the side activity of the fractions on Vibrio percolans A1CC 8461 and HAEA ^ qq for the fractions is determined. Fractions exhibiting a ratio of about 250 indicate the presence of antibiotic 890A.J, and those fractions having a ratio of about 31 indicate the presence of antibiotic 890A Accordingly, fractions 7, 8 and 9 containing mainly antibiotic 890A ^ are combined and further treated as set forth below under heading a) Purification of antibiotic 890Å1 · Fractions 10, 11, 12 and 13 containing mainly antibiotic 890A ^, are united and further treated as set out below under the heading b) Purification of Antibiotic 89OA3.

a) Purification of antibiotic 890A ^

The combined fractions 7, 8 and 9 of the second ZAD-2 column containing 480 hydroxylamine quenchable absorbent units at 300 nm are evaporated to 100 ml under reduced pressure. The sample is applied to a column of Dowex-1 x 4 (Cl ”) -400 mesh resin, layer dimensions 2.15 x 40 cm, and eluted with 4 liters of 0.075M ammonium chloride + 0.001M ammonia in deionized water. Fractions of each 12 ml are collected at a flow rate of 2 ml per minute.

The antibiotic activity, as determined by sample of Salmonella gallium MB1287, is found in fractions 119 to 146, with a maximum in fractions 134 to 140. The ultraviolet absorption at 300 nm, 245 nm and 220 nm is measured for every third fraction in the range. for bioactivity, and hydroxylamine quenchable absorption at 300 nm was measured for several fractions on each side of the peak. Fractions 129 to 141, which have the highest ratio of hydroxylamine extinguishable 300 nm absorption to UV absorption at 220 nm, are combined.

The combined fractions 129 to 141 are concentrated to 4 ml and 0.1 ml of 1M ammonia is added. The concentrate is applied to a column of Bio-Gel P-2, 200-400 mesh, with layer dimensions 2.2 x 70 cm, and eluted with deionized water at a flow rate of 1 ml per ml. minute. 3.1 ml fractions are each collected.

57 143712

The major peak from UY absorption at 300 nm appears in fractions 40 to 47. Fractions 42, 43 and 44 have the highest ratio of hydroxylamine extinguishable 300 nm absorption to UY absorption at 220 nm and these are combined. These combined fractions contain a total of 207 absorption units at 300 nm, of which 133.4 are hydroxylamine quenchable, corresponding to a yield of $ 44 of the applied hydroxylamine quenchable absorption.

The combined Bio-Gel P-2 fractions were diluted with the same volume of methanol and applied to a column of Dowex-1 x 2 (Cl ”) minus 400 mesh, layer dimensions 2.15 cm x 40 cm, which was previously equilibrated with $ 50 (v / v) methanol. The antibiotic is eluted with 2 liters of 0.065M ammonium chloride + 0.001M ammonia in $ 50 methanol at a flow rate of 2 ml per ml. minute. Collect 12.5 ml fractions.

The main peak for absorption at 300 nm is found in fractions 59 to 72. Fractions 62 to 68 have the highest ratio - ^ - 30 (/ ^ 245 'and these are combined. The combined fractions contain 93.7 absorption units at 300 nm, of which, 76, 3 is hydroxylamine extinguishable This represents a yield of $ 57 of the applied hydroxylamine extinguishable absorption in the maximum fractions.

The combined fractions are evaporated to 3.4 nil by rotary evaporation under reduced pressure and 100μ11Μ of ammonia is added. The adjusted concentrate is applied to a column (2.15 cm x 70 cm) of Se-phadex G-10 which is pre-washed with 20 ml of saturated sodium chloride solution and 20 ml of 12 $ ammonium chloride and then with 500 ml of deionized water. The sample is eluted with deionized water at a flow rate of 2.15 ml. minute. Four fractions of 10.3 ml followed by 56 fractions of 3.23 ml are collected. The first peak of ultraviolet absorption at 300 nm containing the desalinated antibiotic 890A1 appears in fractions 25 to 47. Fractions 28 to 41 are combined and the pH measured to 3.5. Adjust the pH to 7.3 by adding 8 μΐ of 1M ammonia solution. The total fractions contain 28 absorption units at 300 nm, 18 of which are hydroxylamine quenchable.

100 ml of these combined fractions are taken for reference and the residue is evaporated to 2.0 ml. The pH of the concentrate is adjusted to 7.4 by the addition of 0.7 μΐ of 1M ammonia solution.

143712 58

Sixty microliters of the concentrate is taken out and the residue is frozen and lyophilized in a 14 ml glass vial to give 4 »32 mg of solid-containing antibiotic 890Å ^ plus residual salts.

b) Purification of antibiotic 8901 ^

The combined fractions 10 to 13 obtained from the second XAD-2 column are evaporated to 40 ml and applied to a column (2.15 cm x 40 cm layer dimensions) of Dowex-1 x 4 (Cl ”) minus 400 mesh. The antibiotic is eluted with 3 liters of 0.075M ammonium chloride and 0.001M ammonia in deionized water at a flow rate of 3 ml per ml. minute. 9 ml fractions are collected. The antibiotic activity, tested with Tibrio percolans ATCC 8461, appears in fractions 165 to 234, with maximal activity in fractions 195 "to 201.

Fractions 186 to 213 are combined, containing a total of 472 Aq qq units, of which 312 are quenchable by reaction with hydroxylamine.

These combined fractions are evaporated to 4.2 ml by rotary evaporation under reduced pressure. The concentrate is applied to a column of Bio-Gel P-2 (200-400 mesh) with layer dimensions of 2.15 x 60 cm. The sample is recommended for layer-level drainage and two rinses on each 1 ml of deionized water are applied and dripped. The column is eluted with deionized water at a flow rate of 1 ml.pr. collecting fractions of 2.6 ml each.

Antibiotic 890A ^ is eluted in fractions 38 to 48 with a maximum at fraction 41, determined by hydroxylamine quenchable absorption at 300 nm. Fractions 40 to 43 are collected containing 260 k300 "of widow (s) of which 173 is hydroxylamine quenchable.

To remove residual contamination of antibiotic 890A ^ with antibiotic 890A ^, the combined desalted fractions 40 to 43 are treated with 50 µl of penicillinase [Difco "Bacto-Penase" containing • 500,000 units per ml. ml (1000 ITJ / ml, where the term 1U refers to Devy units; 1,000 ITJ will inactivate 500,000 units of peni cillin G)] and 0.1 ml 1M tris-HCl buffer, pH = 7.5. The reaction 59 is allowed to rise at 23 ° C for 6 hours, then 15 ml of deionized water and 15 ml of methanol are added. This mixture is applied to a column (2.15 x 44 cm layer dimensions) of Dowex-1 x 2 (Cl-) minus 400 mesh packed in 50 $ methanol and washed with two liters of 50 $ (v / v) methanol Antibiotic 890A ^ eluted with two liters of 0.05M sodium chloride + 0.005M ammonium chloride + 0.0001M ammonia in $ 50 methanol 9.2 ml fractions are collected The major peak of UY desorption, measured at 300 nm, is obtained in fractions 74 to 88. Fractions 78 to 85 are collected The total absorbance units at 300 nm in these fractions after removal of the methanol at evaporation under reduced pressure is 97.6, of which 87.5 is extinguishable by reaction with hydroxylamine.

The combined fractions 78 to 85 are evaporated to 3.92 ml by rotary evaporation under reduced pressure and the concentrate is applied to a column of Sephadex G-10 (2.15 x 70 cm layer dimensions) washed with 4 ml of 4M ammonia. followed by equilibrium with 0.15mM ammonia in deionized water. After two washes with 1 ml of 0.02 mM ammonia each, antibiotic 890A ^ eluted with 0.02 mM ammonia at a flow rate of 0.8 ml per ml. minute. 49 fractions of 2.45 ml followed by 20 fractions of 3.33 ml are collected. The main peak for absorption at 300 nm is found in fractions 35 to 53. Fractions 38 to 46 with greatest A ^ QQ / ^^ values are combined for further processing. The combined fractions have a total of 65 desorption units at 300 nm. The collected fractions are subjected to rotary evaporation under reduced pressure to 2.84 ml and divided into two equal portions which are rapidly frozen and lyophilized separately in 14 ml glass vials to obtain antibiotic 890A

One sample contains 32.0 A ^ QQ units in 0.88 mg and the other contains 31.9 A ^ OQ units in 0.82 mg. The first sample containing 32.0 Aq QQ units was subjected to NMR analysis and showed the following peaks: 1.29 (d, J = 6.5); 1.98 (s); 3.42 (d of d, J = 5 and J = 2.4); / ✓ 4.01 - 4.28 (m); 3.14 (d of d, J = 5 and J = 9); 3.39 (t, J = 6.5); 2.92 (d of t, J = ^ 4 and J = 6).

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The above table indicates 100 mHz NMR signals for the 890A 1 sodium salt in D 2 O relative to the internal standard DSS; chemical shifts are given in ppm and coupling constants in Hz; apparent multiplicity is indicated, EXAMPLE · 9

Shake flask production of antibiotic 890A.J

A tube of lyophilized culture of Streptomyces flavogriseus NRRL 8140 is opened aseptically and the contents are suspended in a tube containing 1.5 ml of sterile medium A of the following composition:

Medium A

Yeast extract 10.0 g

Glucose 10.0 g

MgSO4.7H20 0.05 g A Phosphate buffer 2 ml

Distilled HgO 1000 ml

Ar ÆPhos ~ phatpuf fer solution KH2PO0 91.0 g

Na2HPO4 95.0 g

Distilled H2 O 1000 ml

This suspension is used to inoculate a 250 ml triple aerated conical flask containing 54 ml of graft medium B having the following composition:

Medium B

Autolysed Yeast (Ardamine +) 10.0 g

Glucose 10.0 g

MgSO4-7H20 0.05 g A Phosphate Buffer 2 ml

Distilled H2 O 1000 ml 61 143712

The graft flask is plugged with cotton wool and shaken for 1 30 hours at 28 ° C - 1 ° C in a gyrotonic shaker at 220 rpm. minute (5 cm stroke).

50 250 ml of non-aerated tapered production choruses, each containing 40 ml of production medium 0 are seeded with 1 ml per ml. flask of the liquid from the inoculum flask. The production bottles are stuffed with cotton wool.

Medium C

Tomato paste 20.0 g

Primary yeast 10.0 g

Dextrin (Amidex) 20.0 g ΟοΟ ^^ δ ^ Ο 5.0 mg

Distilled HgO 1000 ml pH: adjusted to 7.2 - 7.4 with NaOH.

After grafting, the production flasks are grown at 28 ° C - 1 ° C while shaking in a gyrotonic shaker at 220 rpm. per minute (5 cm stroke) for 3 days. The flasks are tested for activity against standard Vibrio percolans ATCC 8461 test plates with 12.7 ml sample discs dipped in centrifuged fermentation liquid. The samples are diluted with 0.05M phosphate buffer, pH = 7.4. The results are given in the following table:

Harvested age, hours 72 pH 6.4

Vibrio percolans (1/100 dilution) sample 23 mm 890 sample, unit / ml 103

The 890A units / ml are determined as specified in the section entitled "II, Test Procedure for Determination of '890 Sample Units1",

The total fermentation liquid is centrifuged in 200 ml portions in polycarbonate bottles at 9000 rpm. per minute for 15 minutes to obtain 1600 ml of combined centrifuged liquid with an activity of 104 units / ml. To this add 0.5 ml of neutral EDTA.

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The centrifuged liquid is adsorbed on a column of Dowex-1 x 2 (01 "), 50-100 mesh, with layer dimensions 3.8 x 22 cm, using a flow rate of 6 to 20 ml / min. with 100 ml of deionized water and eluted with 1 liter of deionized water containing 50 g of sodium chloride, 0.02M tris-HCl buffer, pH = 7.0, and 25 μΜ neutral EDTA, at a flow rate of 6 ml / min. 10 ml fractions are collected.

Antibiotic. 890A ^ occurs in fractions 13 to 81 with maximum in fractions 25 to 33, calculated from the first addition of salt eluate. fractions 24 to 41 having the highest ratio of bioactivity / A22Q> are combined for further treatment. The combined fractions have a total of 29,000 units or $ 17 of the applied bioactivity.

The Dowex eluate is evaporated to 10 ml, the pH is adjusted to 6.5 with dilute hydrochloric acid and the concentrate is applied to a column of XAD-2, layer dimensions 3.3 x 36 cm, previously washed with 2 liters each of $ 60 aqueous acetone, deionized water and $ 5 (w / v) sodium chloride in deionized water. The sample is eluted with deionized water at a flow rate of 6 ml / min. Fractions of 40 to 260 ml are collected.

Antibiotic activity occurs in fractions 6 to 14, representing from 220 to 2560 ml of eluted volume. The peaks appear in fractions 9 and 10 corresponding to 370 to 590 ml of eluted volume. These factions have 36,600 units, equal to $ 126 of the seemingly inflated activity.

The combined fractions 9 to 12 are evaporated to 100 ml and the concentrate is applied to a column of Dowex-1 x 4 (01 ”), minus 400 mesh, layer dimensions 2.2 x 41 cm, at a flow rate of 2 ml / min. The column is rinsed with 50 ml of deionized water and eluted with 3 liters of 0.07M sodium chloride + 0.005M ammonium chloride + 0.0001M ammonia in deionized water at a flow rate of 2 ml / min. 10.8 ml fractions are collected starting from the first application of eluate.

63 U3712

The major peak of antibiotic 890A1 appears in fractions 181 to 217 with a maximum in fraction 198. Fractions 186 to 210 containing a total of 114 absorption units at 300 nm are collected.

The collected fractions are evaporated to 4.0 ml and the pH is adjusted to 7.3 by the addition of 16 filter 1M sodium hydroxide. The concentrate is applied to a column of Bio-Gel P-2, 200-400 mesh, layer dimension 2.15 x 70 cm, and washed with 3 x 1 ml portions of deionized water and eluted with deionized water at 0.96 ml / min.

Fractions of 3.85 ml are collected. The major peak of antibiotic 890A1 is found in fractions 24 to 44 with a maximum in fractions 33 and 34. Fractions 27 to 38 with the highest A ^ goo / Ag ^ ratio are combined and lyophilized. These combined fractions have a total of 72 A ^ QQ units.

To carry out the lyophilization, the combined fractions were evaporated to 3.0 ml and the pH of the concentrate was adjusted to 7.5 by the addition of 10 filter 0.1 M sodium hydroxide. The sample is divided into two portions of 1.50 ml and the portions are quickly frozen and lyophilized in 14 ml with screw-cap glass ampoules. Each sample contains 1.73 mg of 890A ^ corresponding to 35.8 µg units.