SU508211A3 - Method for preparing antibiotic 3 (N-4-aminobutyl) -aminopropylaminobleomycin (bleomycin a5) - Google Patents

Description

. and concentrate. The resulting residue is dissolved in 2 ml of aqueous methanol and loaded onto a column filled with 10 ml of neutral alumina, then eluted with 8 ° C aqueous methanol. After that, fractions containing bleomycin are collected, and they are concentrated to dryness to obtain 62 mg from the noxell bpeomyain (efficacy 12OOMKr / wr: total efficacy mg). The yield from the culture filtrate is 45%. The obtained Bpoii KT is divided into separate blmicinous cubic components with an aqueous solution of ammonium formate on a CM-C fadex in a column, determining that it will contain 90.8% of bleomyin A. This method of separation stands in green. 50 mg of the indicated product is dissolved. 0.5 ml of water and loaded into a column filled with 5 ml of Adex O-25, then eluted with distilled water. The resulting deep-green fraction containing bleomycids, syrdide, is suspended in 0.02 H aqueous solution of ammonium chloride, added to a mixture of 5 ml of GM-Sephadex, and eluted with M aqueous pacTiBopoM ammonium chloride. Thereafter, the obtained Acne for 4 hours, containing the desired substance, is passed through a column filled with 2 ml of activated carbon to adsorb the desired substance, which is eluted with a mixture of acetone and 0.02 N hydrochloric acid. The eluate is then concentrated to dryness, to give 15 m of bleomycin A (efficacy 2540 µg / total efficacy 38.1 mg). The yield of the product is 78.24%. Yu mg of the obtained substance is dissolved in 1 mg of methanol, and gaseous sulfur hydrogen is passed into the solution for 10 minutes. Then the solution is filtered, the filtrate is concentrated to dryness, to obtain 7 mg of copper-free hydrochloric bleomycin A (efficacy 25OO µg / mg,, 5,,, overall efficacy of 17.5 mg). The copper-free product is hydrolyzed with 6 N aqueous hydrochloric acid, subjected to electrophoresis on paper, and spermidine determined. Example 2. To medium containing,%; 6.4 pure jelly, 0.5 glucose, 3.5 soy bean powder, 0.75 strong corn decoction, 0.3 sodium chloride, 0.1 secondary potassium phosphate, O, O5 Zinc sulfate, 0.01 copper sulfate , 0.2 sodium nitrate. O, O1 silicon and O, O1 Toyo No. 1 (trade name of surface active agent), add-3- (: N-4 aminobutyl-aminoprocylamine in the amount of O, 4 mg / ml, setting the pH to 6.5. 12 L of the treated medium loaded into a glass fermenter with a capacity of 2O l and sterilized. In this environment, 8OO ml of cultured htc-spiaj tom & ct Ci-Wiu (ATCG Nsf 15O03) is inoculated, which is shaken for 48 hours to culture in that medium the same composition as above, and cultured for 163 hours at 27 ° C with stirring at a speed of 350-370 rpm, introducing air in a minute a volume equal to the volume of the medium. l culture liquid is filtered, the filtrate (efficiency 122.2 µg / ml, total efficiency 611.0 mg) is extracted, purified by the method of example 1 and 180 mg of hydrochloric bleomycin (efficiency 120O, O MCH) MG; total efficiency 216 mg). The yield from the culture filtrate is 35.4%. The resulting product is dissolved in 10 ml of distilled water, the solution is loaded onto a 50 ml column with EM-SefadekGOM, suspended in 0, O2 N aqueous solution of ammonium formate for faccia | containing bleomycin A. This. -, . About the fraction is passed through a column filled with 1O micron aRtHBHpciBaHHoro coal, and an adsorbide of Ekg is washed with water, eluted with a 1: 1 mixture (by volume) of acetone with 0.02 N aqueous hydrochloric acid. Then, the resulting active fraction of the eluate is concentrated to dryness, obtaining 44.7 mg of bleomycin A, as a cobalt salt (efficacy of 2543 µg / mg, total efficacy of 113.7 mg). The yield of hydrochloric acid is 52.6%. This substance is hydrolyzed with 6 N hydrochloric acid, treated with 10% sodium hydroxide solution, and gas chromatographed; using a column filled with Fluopac 8O (trade name), covered with Silicone 5F 96 (trade name). This is the peak of spermidine. 30 mg of the obtained bleomycin A < / RTI > hydrochloric acid (effective 2543 µg / mg, total efficacy (strength 76.28 mg) is dissolved in 50 ml of methanol, hydrogen sulfide gas is introduced into the resulting solution for 30 min to obtain a copper sulphide precipitate. This precipitate filtered and washed with methanol. Then the washings were combined and the filtrate was evaporated to dryness to obtain 25 mg of copper-free bleomycin A as a cobalt (efficacy 2LOO μg / mg, efficacy 62.5 mg). Bwisofl from copper containing substance is 83i3%

20 mg of this copper-free hydrochloric bleomycin per A (efficacy 25OO 1L {g / mg, total efficacy 5O mg) is dissolved in 2 ml of distilled water, loaded onto a column filled with 5 ml of anion-exchange resin Amer 4OO (trade name; sulfate type of ion exchange resin, produces Rohm and Haas KoJ and then eluted with distilled water, the eluate is concentrated to dryness, get 1S.S kg CBOfoflHoro from copper bleomkina A sulfate (efficacy 2500 μg / mg, total Efficiency 38.75 mg), №1 FROM copper-free hydrochloric acid salt

Example 3. The medium contains,% J 4,0 soybean powder. 1.0 Yeast extract, O, 3 sodium chloride, J.O soybean oil, O, O07 O.OOO1 Pe5 O -7 H „0. O.OOOv 4 and

0.0002 ZvtS 7 HgOs PH set to 7.0. Each 120 ml of medium is loaded: into a Sakagushi flask with a capacity of 500 ml and sterilized. On Wednesday inoculant strain MA 267-A.j, allocated ia

the soil of Musashino-sho, Saitama-ken, which fern is identified as a species of I SHef tomuce "ve & ticUtud n which

produces especially bleomycin Hell.

Then 3- (W-4-amnobutyl) -amino-pro-1) 1 amine phosphate, which was nooBeprujT sterilized with a Millipor filter, is added so that its amount is 0.5 mg / ml. The strain is cultivated at 27 ° C for 7 days with stirring at a speed of 130 rpm. Collect 5.2 l of culture liquid, filter ,. the filtrate (efficacy 196 µg / ml, total efficacy 25E mg, 4.5 l) is extracted

and CLEANAGE according to the procedure of Example 1, obtaining 137 MP crude powder of bleomish ng (efficacy 1846, µg / mg, overall efficacy 253 mg). Quiche output is about 28f8%. Cheese product is chromatographed using a digging stick filled with 25 ml of Sephiumus O-5O, and eluted by dissipation in water. Psch glennye blue active fraccoia is collected and concentrated; they are dry, full of 53.5 mg of blue powder with 2413 µg / mg cytocytosis (total activity of 14O mg), are obtained only from bleomkadae A. The raw powder is 55.4%. Gr)

 1cm

At 203, the product is equal to

. 13.6. the sermindin side chain in it was determined by gas chromatography of the product hydrolysate 6 N hydrochloric acid,

Claims (1)

Invention Formula The method of obtaining antibiotic 3- (N 4-aminobutyl) -aminstropylamino-6leomycin (bleomyad Hell) by growing a producer of the genus Stfe6 / i) tomyfle6 on a medium containing nitrogen, carbon and mineral salts, characterized in that, in order to increase the yield of the target product, the cultivation is carried out in the presence of 3- (A (-4-aminobutyl) aminoprosphenamine or N, W-bis (3-aminopropyl) -1,4-diaminobutane,