DE1792818C2 - Tenebrimycin antibiotic IV and a process for its preparation - Google Patents

Description

The invention relates to the tenebrimycin antibiotic tenebrimycin IV with that specified in claim 1 Identification and a method for its production (claim 2).

Tenebrimycin IV is made as a factor in a novel antibiotic complex that comprises the seven factors Tenebrimycin I, Γ, II, III, IV, V and Vl includes, of each of which exhibits antiblotic activity. The complex is available as a free base in water and in dimethylsulfoxld soluble, slowly soluble in methanol and insoluble in acetone, higher alcohols, dioxane, ethyl acetate, Diethyl ether, acetonitrile, methyl isobutyl ketone and hydrocarbon solvents. The free base complex is stable at freezing temperatures, at room temperature and at 37 ° C for a period of at least 8 weeks within a pH range of 1 to 11 that are distinguishable Bands in the infrared absorption spectrum over a range from 2 to 15 μm are as follows: 3.02 (broad), 3.15, 5.82, 6.26, 7.43, 8.8 (broad) and 9.7 (very broad) pm, positive reaction in the Anthron and Elson Morgan tests, with the Lowry protein test low positive reaction and negative reaction, in the biuret and Sakaguchl test. This antibiotic complex is described in DE-PS 16 17 451.

Tenebrimycin IV is a basic substance with titratable groups with pK'a values of about 5.3, 6.8, 7.8 and 9.0 as determined by electrometric titration in water. The specific rotation of tenebrimycin IV, determined for a 1% aqueous solution at 25 ° C, is + 114 °.

The infrared absorption spectrum of tenebrimycin IV as a mineral oil gauze is shown in the figure.

The distinguishable bands in the infrared absorption spectrum over the range from 2 to 15 μm are the following: 3.05, 3.17, 3.85, 6.27, 7.46, 8.75, 9.7, 10.5, 11.1 and 12.85 µην

The tetraacetyl derivative of tenebrimycin IV is a crystalline solid with a melting point of 265 to 267 ° C. The specific rotation [a] jo _{ls { +} 109 °, if the concentration of the derivative \% in aqueous solution leads to a column with IRC -50 is packed in the ammonium circuit, then elulert with dilute acid, which combines and concentrates the fractions containing anti-biotic activity, adjusts the pH of the concentrate to 11 and the basic concentrate is poured into acetone and cools, the resulting inactive precipitate by filtration removed and the pH of the filtrate is reduced to 3.5 by adding 20% sulfuric acid to precipitate the antibiotic complex as a sulfate salt, with the sulfate salt or another acid addition salt of the antibiotic complex, if necessary after removing colored impurities, a column packed with IRC-50 in the ammonium circuit, loaded, washed with water, tenebrimycin I, Γ, Π and III with 0.1 η ammonium hydroxide and d on the tenebrimycin antibiotic IV eluted.

Acid addition salts of tenebrimycin IV can be prepared by conventional methods. Either organic or inorganic acids can be used to form salts. A convenient method of making such salts is by adding a solution of the salt-forming acid to an aqueous solution of the antibiotic. In the case of insoluble acid addition salts, the salt separates out of the solution and can be easily separated using conventional methods such as filtration or centrifugation.
Should it happen that the desired addition salt does not readily precipitate, the precipitation can be promoted by either concentrating the solution to a smaller volume or adding a water-miscible solvent, such as acetone or the like. The acid addition salt can easily be converted back to the free base form of the antibiotic by passing an aqueous solution of the salt over an anionic exchange resin in the hydroxyl cycle. Tenebrimycin IV can be distinguished from the various factors that form the mentioned tenebrimycin complex and from other antibiotics with similar properties by paper chromatographic methods, e.g. B. by the following: Chromatography was performed in the conventional descending manner at 23 ° C ± 1 ° on Whatman No. 1 paper, 190x464 mm.

The results are shown in Table I. The constant of migration For each of the antibiotics mentioned, it is better to express with an R ^ constant than through the conventional R / value. The R ,, value expresses that

Ratio of the distance traversed by the antibiotic with respect to the end of the sheet and not the solvent front.

This method of expressing the constant was chosen in view of the fact that the solvent front past the end of the leaf for the period that is used for most solvent systems was, had advanced.

Table I.

Paper chromatography of tenebrimycins and known antibiotics with similar properties

Solvent system: 1 B. C. and Ri values E. F. G A. _ _ D. 0.32 0 0 Tenebrimycin complex _ - - 0.65 - - - Tenebrimycin I. 0.20 - - - - - - Tenebrimycin Γ 0.27 0.17 0.32 - - - - Tenebrimycin II 0.40 0.34 0.41 - - - - Tenebrimycin III 0.40 0.20 0.32 - - - - Tenebrimycin IV 0.49 0.22 0.35 - - - - Tenebrimycin V 0.55 0.31 0.38 - - - - Tenebrimycin VI 0.71 0.35 0.41 - 0.42 0 0 Kanamycin 0.35 0.26 0.32 0.65 0.29 0 0.05 Kanamycin B 0.57 0.51 0.39 0.70 0.75 0.62 0.51 0 0.1 Gentamycin 0.83 0.95 - 0.25 0.34 0.35 0 0 Catenulin 0.44 0.12 0.19 0.65 _ 0 0.05 Neomycin 0.60 _

The solvent systems used and the duration of the chromator graph are given below.

A = n-butanol saturated with v / ater, plus 2% p-toluene

sulfonic acid, for 40 hours
B = 80% aqueous ethanol, plus 1.5% sodium chloride, door 40 hours, buffered on paper, with 0.95 m

Sulfate bisulfate
C = propanoi, pyridine, acetic acid and water

(15: 10: 3: 12) for 40 hours
D = water saturated with methyl isobutyl ketone, plus 1%

p-toluenesulfonic acid for 6 hours
E = Propanoi and water (1: 1) for 24 hours

Paper buffered with 0.75 m phosphate, pH 4.0
F = n-butanol saturated with water, door 18 hours
G = n-butanol saturated with water, plus 2% p-toluenesulfonic acid and 2% piperidine, for 18 hours.

These chromatographic data indicate the following: While for each individual solvent system some of the factors that make up the tenebrimycin complex as identical to or identical to each other Other known antibiotics appear, as evidenced by chromatography with a variety of solvent systems clearly their non-identity.

Tenebrimycin IV has an inhibitory effect on the growth of microorganisms that are animal or plant life are pathogenic. The inhibitory concentrations of Tenebrimyclns IV against a number of organisms are shown in the following table. The minimal inhibition concentrate ions were determined by the agar dilution method.

Table II

Test organism minimal inhibition concentration ^a ) (μΕ / ml) Tenebrimycin IV Stephylococcus aureus 3055 6.25 Bacillus subtilis <1.56 Mycobacterium avium <1.56 Streptococcus faecalis <1.56 Lactobacillus casei 25th Leuconostoc citrovorum 25th Escherichia coli No. 1 12.5 Escherichia coli No. 2 12.5 Proteus sp. No. 1 50 Proteus sp. No. 2 12.5 Pseudomonas sp. No. 2 100 Pseudomonas sp. No. 5 6.25 Klebsiella-Aerobacter No. 14th 3.12 Klebsiella-Aerobacter No. 15th 6.25 Salmonella sp. No. 1 25th Vibrio mechnikovii 12.5 Agrobacterium tumefaciens 25th Erwinia amylovora <1.56 Pseudomonas solanacearum 12.5 Xanthomonas phaseoli 12.5

The minimum household ration was reduced to the free base.

The acute toxicity of tenebrimycin IV was on Mice for sure.

The LD; _U value. expressed in mg / kg of free base for tenebrimycin IV given intravenously to mice is about 220.

The antibiotic complex containing tenebrimycin IV is produced by cultivating a certain actinomycete strain under aerobic conditions in a suitable culture medium until this medium contains substantial antiblotic activity. That Tenebrimycin IV can be obtained by applying certain isolation and cleaning measures (see. Claim 2).

Because of the uncertainty of taxonomic studies with regard to the genus Sireptümyces, a! 5 Element of doubt cling to the classification of a newly discovered organism. Nevertheless - as far as it is can be proven - is not the same as the organism used to manufacture the antibiotic a genus of Streptomyces described by Waksman in "The Actinomycetes", Vol. II, The Williams and Wilkens Company (1961) to ensure culture comparisons.

The organism is a spiral, carrying, aerobic to microaerophilic Streptomyces with elongated smooth-walled spores. It's unique in that it is inhibited by relatively low intensities of artificial light. Because of this property of his the name Streptomyces tenebrarius sp. n. chosen for this organism.

The organism was isolated from a soil sample by suspending parts of the soil sample in sterile distilled water and spreading the suspension on nutrient agar. The seed nutrient agar plates were ^{incubated at 25 to 35 C} until growth was ensured. At the end of the incubation period, colonies of the antibiotic-producing organisms were transferred to agar slants using a sterile platinum inoculating loop. The agar slant was then incubated to obtain appropriate inoculation levels for the preparation of the antibiotic. The strain of the organism used to make the antibiotic complex has been deposited with the American Type Culture Collection in Washington, D.C. under culture number: ATCC 17920.

The procedures used in the taxonomic experiments on S. tenebrarius ATCC 17920 are the same as those commonly used in the taxonomy of actinomycetes. Cultivation characteristics were observed after incubation for hours. The morphology was determined on Chapek's Pepion agar and Bennett's agar during a 2- to 7-day incubation. Effects on milk and the reduction of nitrate were observed after 7 and 14 days, hydrogen sulfide production after 24 to 48 hours and carbon utilization after 10 days. Unless otherwise stated, the cultures were incubated ^{at 37 ° C.} Carbon recovery studies were carried out according to the procedure described by Pridham and Gottlieb, J. Bact., 56, 107 (1948). ^M.

The results of the taxonomic tests are summarized in the following sections. The payment in brackets refer to the color blocks according to Maerz and Paul, "Dictionary of Color", McGraw Book Company (1950). The colors that are symbolized by the coloring blocks are in the ISCC-NBS Color names have been transferred, as they are in "Circular 553". U.S. Department of Commerce, National Bureau of Standards, 1955, are included.

Microscopic morphology,

Breeding characteristics and physiology

by S. tenebrarius ATCC 17920

Morphology:

On Czapek's peptone agar, branched spore shapes form in irregular clumps on the Aerial mycelium. Isolated spores are rare. The intact aerial mycelium can easily be detached from the substrate. Maturity Spore chains usually form 5 to 6 open spirals. The spores are elongated to cylindrical. Under the When viewed with an electron microscope, the spores appear smooth and measure 0.7 to 1.3 by 2.0 to 2.1 microns. ScIerotien were observed on Bennett's medium.

Colony characteristics on:
Czapek's agar:

Little growth rate, poor aerial mycelium; pale orange-yellow (11-A2); Sporulation good; Back pale orange-yellow (11-A2); soluble pigment light pink (1-sheet). Czapek's peptone:

Growth abundant; Aerial mycelium abundant; light yellowish brown with white spots (11-A4); Profuse sporulation; Back light gray-red (4-Hl); soluble pigment gray-pink (4 sheets).

Calcium malate agar:

Moderate growth, moderate aerial mycelium, pale orange-yellow (11-A2); Moderate sporulation; Back gray-pink (4-Dl); soluble pigment gray-pink (4-sheets).
Tyrosine Agar:

Growth sparse, aerial mycelium sparse; pale orange-yellow (9-B2); Reverse pale red-yellow (10-B3); scanty sporulation; no soluble pigment.
Inorganic Salts Starch Agar:

Moderate growth, moderate aerial mycelium, brownish-pink with white areas (11-A4); Profuse sporulation; back pale yellow (11-B2) soluble pigment pale yellow (11-B2).

Glucose asparagine agar:

Moderate growth, moderate aerial mycelium, pale yellow (9-D2) with white areas (10-A1); moderate sporulation; Reverse pale yellow (11-B2); no soluble pigment.
Tomato paste oatmeal:

Abundant growth, abundant aerial mycelium, light yellowish brown (12-B5); profuse sporulation; Reverse side dark gray-reddish brown (40-J2); soluble pigment dark purple red (47-H1).
Yeast extract:

Abundant growth, abundant aerial mycelium, pale orange-yellow (11-A2) with white areas: profuse sporulation; Back moderately yellow (11-J6); no soluble pigment.
Nutrient agar:

Poor growth, poor white (10-Al) aerial mycelium; poor sporulation; Reverse gray-greenish-yellow (12-12); no soluble pigment.
Bennett's agar.

Moderate growth; moderately white (10-A1 to 10-B1) aerial mycelium; moderate sporulation; Reverse pale yellow (11-C2); no soluble pigment.
Effect on milk:

dark yellow growth ring on the surface; Coagulation and peptonization were observed.
Nitrate reduction:
Positive.
HjS production:

IO

20th

25th

30th

Negative.

Nutrient gelatine - complete liquefaction after 14 days.

Temperature requirements for Czapek's peptone agar:

no growth

good growth, no aerial mycelium

Growth and aerial mycelium moderate but no sporulation

Growth, aerial mycelium, and sporulation very abundant

Growth, aerial mycelium, and sporulation very abundant

Growth, aerial mycelium, and sporulation very abundant

sparse growth

no growth

Thermal dead center:

Spores from a spore suspension heated to 75 ° C remain viable for 15 min. Became the spore suspension When heated to 100 ° C for 15 minutes, no spores were viable any more.

Oxygen tension:

Either aerobic or microaerophilic growth in stick cultures.

Elsenion effect:

A red soluble pigment will only appear if it is present produced by ferric ions and the intensity of pigmentation is proportional to the concentration of the Iron (III) ions in a given range.

40

Effect of hydrogen ion concentration on Czapek's peptone agar observed:

No growth below pH 5.0; from pH 5.0 to 6.0 growth and aerial mycelium good; Growth and sporulation are plentiful at pH 7.0; from pH 8.0 to 8.6, growth and aerial mycelium are good. The soluble pigment is the most intense at pH 5.0 to 6.0 and is low to absent from pH 6.5 to 8.6.

Light reaction observed on Czapek's agar:

Growth and s ⁿ oru! At! On are plentiful in the dark. When plate cultures are incubated 38 cm from a standard 1.5 watt fluorescent cold light source, growth is sparse and no aerial mycelium is present. Growth and aerial mycelium are moderate when the cultures are incubated 35 cm from a cooled, frosted 60 watt tungsten incandescent lamp.

In the table showing the results of the carbon recovery attempts that were performed with S. tenebrarius ATCC 17920 the characters used have the following meaning:

Table III

Carbon utilization of S. tenebrarius strain ATCC 17920

Carbon source Speak to carbon source

L (+) arabinose
L (+) rhamnose
D (-) ribose
D (+) xylose
D (-) fructose
D (+) mannose
D (+) glucose
Lactose
Maltose

Sucrose

+ = positive
(+) = likely
(-) = questionable

- = none (nothing)

65 D (+) trehalose

L (+) raffinose

Cellulose

Inulin I.

i-inositol

Mannitol

d-sorbitol ι

Salicin I.

control

(no carbon)

S. tenebrarius ATCC 17920 also produces the well-known antifungal antibiotic caerulomycin, which also is produced by Streptomyces caeruleus [Cand. J. Mlcroblol. 5, 317 (1959)]. Investigation of a breed of the The latter organism showed that it did not produce tenebrimycin under the conditions required for its Breeding have been described; even under changed conditions it cannot be induced to do so.

Cultivation media that are relatively simple are expediently used for the production of tenebrimycin IV Contain nutrient sources. For example, media useful for the production of tenebrimycin contain an assimilable source of carbon such as glucose, fructose, mannose, maltose, starch, and the like. A particularly preferred source of carbon is glucose. In addition, the media that can be used include an assimilable one Nitrogen sources such as peptones, hydrolyzed casein, yeast, amino acids and the like. In the present Traps are preferred as nitrogen sources: peptone, hydrolyzed casein, and glutamine.

Mineral salts, e.g. B. those soft calcium, magnesium, sodium, potassium, chloride, sulfate, phosphate and carbonate ions can be incorporated into the media with good success, albeit a An excess of phosphate should be avoided as it appears to reduce the yield of antibiotic. A source of growth factors, such as yeast or yeast extract, can also work to good effect Medium can be added.

As it is necessary for the growth and development of other microorganisms, there should also be important trace elements are added to the culture medium to promote the growth of the invention applied organisms. Such trace elements are mostly present as impurities, as random ones Accompanying substances when adding the other components of the medium.

Submerse aerobic culture conditions are especially preferred for the production of tenebrimycin. For the Shake flasks and surface cultures can be used to produce relatively small quantities. For the However, for large-scale production, submerged aerobic growth in sterile containers is preferable. That Medium in the sterile container can be sponsored with a Suspension, but because of the growth

delay that you get when using a sporulated Suspension noted as inoculum becomes vegetative Form of culture preferred. This is how you report the growth retardation and achieve a more effective one Use of fermentation equipment. Accordingly, it is advisable to start with a vegetative Inoculum of the organism by inoculating a relatively small amount of the culture medium with the spore shape of the organism and when a young active vegetative inoculum is obtained was to transfer this vegetative inoculum sterile into the large container. Appropriate becomes a Fraction of the vegetative inoculum, approximately 496 of the mass of the medium into which it is introduced, is used. That Fermentation medium in which the vegetative inoculum is produced can either be the same or different from the medium used for the large-scale production of tenebrimycin.

The organism grows within a relatively wide temperature range. Still, the organisms seem best to grow at temperatures between 30 and 50 ° C. An optimal production of tenebrimycin takes place at temperatures between 37 and 43 ° C. The organisms that produce tenebrimycin are sensitive to light and grow poorly if it is present.

Thus, fermentations are in which the organisms should be used, if possible without visible light.

Consistent with that of submerged breeding operations In common practice, sterile air is passed through the growth medium. To an effective one Growth of organisms and production of tenebrimycin is the volume of air in tank production of tenebrimycin over 0.1 volume of air per minute per volume of the culture medium, but lies preferably much higher. Effective growth of the organisms and optimal yields of tenebrimycin is obtained when the amount of air used is at least one volume of air per minute per volume Growing medium is.

The concentration of tenebrimycin activity in the fermentation medium can be conveniently monitored during the course of the fermentation by examining samples of the culture medium for inhibitory activity on the growth of organisms known to be inhibited in the presence of tenebrimycin. Two of the organisms so applied in tracking the production of tenebrimycin are Klebsiella pneumoniae and ^Μ Mycobacteriurn butyricum.

The former organism is mainly used in the well-known measurement of turbidity, while the latter is used in the shell-and-plate process.

Generally, maximum production of the antibiotic occurs within 4 to 7 days of inoculation of the culture medium when submerged aerobic culture or shake flask culture is used will, and after a little longer time when surface crops were used.

The mycelium and undissolved residues are removed from the fermentation liquor by conventional means, such as filtration or centrifugation. the Antibiotic activity is in the filtered liquid and can be obtained therefrom by using adsorption processes. The adsorbents are the cation exchange resins »IRC-50« (weakly acidic polymethacrylate carboxylic acid resin).

In general, the procedure for obtaining the tenebrimycin from the fermentation liquid is as follows: All of the liquid is filtered with a filter aid after the pH of the mixture is up about pH 2 was lowered by adding an acid such as sulfuric acid, phosphoric acid, hydrogen chloride and the like. The pH of the filtrate is adjusted to a value of about 5.5 by adding a concentrated Base, and then the mixture is filtered again. The filtrate is passed through an ion exchange column led, which is packed with IRC-50 in the ammonium cycle. The column is then filled with distilled water washed and the antiblotlsche activity with diluted Acid elutes. The fractions containing the antiblotl activity are pooled and approximated Concentrated '/ 20 of the original volume. The pH of the concentrate is increased to about 11 by Add concentrated base, and the basic concentrate is then poured into about 6 volumes of acetone, well mixed and chilled. The microbiologically inactive precipitate that separates is removed by filtration removed. The pH of the filtrate is lowered to about 3.5 by adding 2096 sulfuric acid thorough mixing. The Tenebrlmycln complex precipitates in the form of the sulfate salt, while the Caerulomycln remains as a by-product of fermentation in the supernatant layer which is discarded.

Tenebrimycin IV is obtained by further fractionation of the tenebrimycin complex. For fractionation the tenebrimycin is used as an acid addition salt, preferably in the form of the sulfate. Colored contaminants can be removed by treating the solution at about 596 (weight / volume) Stirring activated charcoal. Effective discoloration is usually obtained by mixing the mixture that contains the charcoal contains, stir for about an hour. Then the mixture is filtered, and with the filtrate, which the Containing tenebrimycin sulfate, a column packed with IRC-50 in the ammonium loop is loaded. The pillar is washed with water and the individual antibiotics that make up the tenebrimycin complex become gradually eluted with 0.1 η ammonium hydroxide. In general, the elution is extremely slow, and a very large number of fractions are usually necessary to fractionate the complex accomplish. So requires z. B. complete fractionation from about 250 g of tenebrimycin complex about 700 fractions, each of which about 900 ml of the eluate matters. The first active fractions obtained contain tenebrimycins I, Γ and II. This follows a number of active fractions in which tenebrimycin II is the only antibiotic. A number of inactive Fractions follow the elution of tenebrimycin II before the fractions containing tenebrimycin IV are eluted will. The first fractions containing tenebrimycin IV show traces of tenebrimycin III.

example 1

Preparation of the tenebrimycin complex
by fermentation of S. tenebrarius ATCC 17920

A sporulled culture of S. tenebrarius ATCC 17920 is generated by turning the organism on a modified Bennett's agar slant medium which has the following composition:

Dextrin
Yeast extract
hydrated casein
Meat extract
CoCl ₂ • 6H ₂ O
washed out agar
deionized water

10 g

1 g

2g

Ig

0.01 g 20 g 1000 ml

The pH of the medium is checked before autoclaving adjusted to pH 7. The agar slant is inoculated with spores of S. tenebrarlus ATCC 17920 and for five days Incubated for a long time at 37 ° C and without visible light. That Growth on the agar slant is covered with water and gently scraped to remove the spores and to obtain an aqueous spore suspension.

The spore suspension thus obtained is used to inoculate 800 ml of a medium, the following Composition has:

Dextrose 0.05% Nutrient meal (contains 35 to 45% dispersible protein) 1.5% Dextrin 700 (Potato dextrin with low Chlorine content) 1% Potassium chloride 0.1% hydrolyzed casein 0.3% KH ₂ PO ₄ 0.05% MgSO ₄ · 7H ₂ O 0.5% CaCl ₂ • 2H ₂ O 0.025% deionized water

The inoculated vegetative medium is 16 at 37 ° C Incubated for hours on a rotary shaker running at 250 r.p.m. works and a hub of 6.3.5 mm.

A 50 ml portion of vegetative cultivation is used to inoculate a 44-1 seed container that contains an aqueous medium of the following composition:

Dextrose

Soybean grains

KH ₂ PO ₄

MgSO ₄

KCl

CaCl ₂ • 2H ₂ O

Antifoam agents

1%

1.5%

0.05%

0.5%

0.1%

0.025%

0.025%

The medium of the seed container is sterilized at 120 ° C for about 30 minutes. Stirring at a speed of 370 rpm is started immediately after inoculation and aeration at a rate of 0.0226 m ³ per minute is maintained throughout the incubation period. After completion of the incubators: bat onsperiode the contents of Einsaatbehälters is used i to inoculate a 1,130-liter fermentor "contains a medium of the following composition!:

Dextrose 4.0% refined soybean oil 3.0% Soy flour 3.0% NH ₄ Cl 0.5% CaCl ₂ 0.3% MgSO ₄ 0.2% NH ₄ NO ₃ 0.1% hydrolyzed casein 0.5% Antifoam agents 0.2% deionized water

Before inoculation, the fermentation medium becomes 30MIn. sterilized at 12O ⁰ C. The fermentation is carried out at 37 ° C. with aeration at a throughput of 0.48 m ³ per minute during the entire time from inoculation to harvest. Stirring is started at 125 rpm and increased up to 180 rpm after 12 hours. The fermentation will

Continued for 5 days. The fermented cultivation liquid is filtered to remove the mycelium and other undissolved solids. The filtered liquid contains tenebrimycin at a concentration of 680 units per ml.

Example 2
Isolation of the tenebrimycin complex

The pH of 880 liters of antibiotic fermentation liquid, obtained as described in Example 1, is lowered to about pH 2 by adding about 10 to 20 liters of 20% aqueous sulfuric acid.

After 45 kg of a commercially available filter aid have been added to the acidified liquid, is mixed thoroughly and filtered. The filter cake is washed with water and the washing solution is dem Added filtrate.

The pH of the filtrate is adjusted to pH 5.5 by adding 50% aqueous sodium hydroxide. About 4 kg of a commercially available filter aid will be used added and the mixture stirred thoroughly and filtered. The filtrate is passed through an IRC-50 column sent the dimensions of 10.2 cm to 1.83 m. The column is circulated up with the resin in the ammonium packed to a bed height of 112 to 114 mm. After all of the filtrate has passed through the column, will washed this with about 200 liters of distilled water. The antiblotic activity is then during a period of 12 to 15 hours with about 150 to 175 liters of 0.1 η sulfuric acid eluted and the eluate was removed in 10 liter fractions. Antibiotic activity can be seen in the eluate when its pH value drops to around pH 4 or a little below.

As the eluate progresses, the pH of the eluate continues to drop until it reaches a value of about pH 1.5 achieved.

The fractions which contain antibiotic activity are pooled and _{concentrated under reduced pressure to about V 20 of} the original volume.

The pH of the concentrated eluate is adjusted to pH 11

adjusted by adding 50% aqueous sodium hydroxide, and the basic concentrate then becomes too

6 volumes of acetone added. The mixture is cooled and turned off to allow the precipitate to settle separates without antibiotic activity. The mixture is filtered and the filter cake, the inactive precipitate contains, is washed with water and discarded.

The pH of the filtrate is adjusted to pH 3.5 with thorough mixing of the addition of 20% aqueous Sulfuric acid adjusted. The tenebrimycin complex is excreted as sulfate during treatment and is practically completely deposited if the pH of the solutions is 4 or slightly below Value reached. The mixture is filtered and the filtrate, the caerulomycin as a by-product of the fermentation contains is discarded. The filter cake, which contains the tenebrimycin sulfate, is in a possible small amount of water dissolved and cooled. All solid components that do not dissolve easily are discarded.

17 92 813

fen, like ζ. B. all solids; which during the Precipitate or crystallize during cooling. The clear solution of tenebrimycin sulfate is over a Dowex-1 χ 1 resin board from 10.2 a to 2.13 m in basic Cycle guided. The antibiotic solution is added to the column and 40 to 50 liters are then distilled Water. The drain is taken in 10 liter fractions, the active fractions are pooled and concentrated under vacuum to a thick syrup, which is dried and the dry Tenebrimycin complex results.

Example 3
Separation of the tenebrimycin factors

Tenebrimycin sulfate is obtained by adjusting the pH of a 20% aqueous solution containing 258 g of the tenebrimycin complex in free base form 4.5. by adding concentrated sulfuric acid. The solution of the tenebrimycin sulfate thus produced is decolorized by stirring for about one hour with 5% (w / v) activated charcoal. The solution will be filtered and the Filtrai through a column of 9 × 105 cm sent, which has been packed with about 7 liters of IRC-50 resin in the ammonium circuit. After the filtrate, that contains the antibiotic salt has passed through the column, this is with about 17 liters of deionized water washed out. The elution of the antibiotic activity retained on the column begins with 0.1 η Ammonium hydroxide solution at a flow rate of about 20 ml per min. The eluate is divided into fractions of about 900 ml are collected and each fraction is assessed for its microbiological activity checked. The fractions containing the same antibiotic factor are collected and lyophilized, to obtain the individual dried tenebrimycins. Tenebrimycin IV appears as follows:

Fractions of the antibiotic present

1-34 inactive

35-53 Tenebrimycin I, Γ and II

54-77 tenebrimycin II

78-153 inactive

154-172 tenebrymcin IV with a trace of
Tenebrimycin III

173-249 Tene ¹ rimycin IV

Of course, tenebrimycin IV helianthat can also be produced.

1 sheet of drawings

Claims (2)

Patent claims: 1. Tenebrimycin antibiotic IV, characterized by the following parameters: basic substance, Titratable groups, determined by electrometric Titration in water, with pK ^ values of about 5.3; 6.8; 7.8 and 9.0 IR absorption spectrum as in the figure, the Is part of this claim. 2. Process for the preparation of tenebrimycin IV according to claim 1, characterized in that one Streptomyces tenebrarius ATCC 17920 in a medium containing assimilable sources of carbon, Contains nitrogen and inorganic salts, submerged under aerobic conditions, then adjusted to pH 2 adjusted fermentation liquid filtered with a filter aid, the filtrate adjusted to pH 5.5 after filtration through an ion exchanger