DD251574A1 - PROCESS FOR PREPARING ACLANITY ACID - Google Patents

Abstract

The invention relates to a process for the preparation of Aklanonsaeure microbiological route. The microorganism used for the production of Aklanonsaeure belongs to the species Streptomyces peucetius var. Caesius and bears the name F 23/135. The aim of the invention is the economic production of aconylic acid as starting substance for mutase syntheses and partial syntheses of anthracycline antibiotics with potentially cancerostatic, antiviral, antibacterial, immunosuppressive and ergotropic properties. The object is achieved in that Aklanonsaeure formed by aerobic submerged fermentation of the strain F 23/135 in media with C, N sources and mineral salts, these isolated from the mycelium and subsequently purified.

Description

Field of application of the invention

The invention relates to a process for the preparation of acyclic acid by microbiological means. Aklanonic acid is a suitable precursor for the semi-synthetic or mutasyrfthetic production of various new antibiotically active substances which are of potential use in the chemotherapy of tumor, virus and bacteria-induced diseases in humans and livestock.

Characteristic of the known technical solutions

It is known that aklanonic acid can be prepared by microbiological means (Eckardt, K. et al., 1985. J. Antibiot. 38, 1034-1039; DD 225714, 7.8.85).

However, the microorganism used in this method (Streptomyces spec. ZIMET 43717) has the disadvantage that it is unable to form aerial mycelia and spores. Therefore, a type assignment is not possible. Furthermore, essential steps of a microbiological production process such as stammering, strain breeding and genotypic optimization of the biosynthetic performance are made more difficult.

Object of the invention

The object of the invention is to use a novel microorganism with biosynthetic capacity for aklanonic acid, which, because of its morphological, biochemical and genetic parameters, is more favorably prone to industrial processes than hitherto possible.

Explanation of the essence of the invention

The invention has for its object to produce aklanonic acid as a starting material for partial syntheses or mutasyntheses of anthracycline antibiotics according to a new microbiological process, which avoids the disadvantages of the known technical solutions.

According to the invention, this object is achieved in that in the method, a strain of a good sporulating microorganism of the species Streptomyces peucetius, preferably the strain Streptomyces peucetius var. Caesius 601 F. I., was used. The strain F 23/135 which was deposited in the ZIMET depository for microorganisms in the Central Institute for Microbiology and Experimental Therapy of the Academy of Sciences of the GDR, 6900 Jena, Beutenbergstrasse 11 under the registration number ZIMET 43810 was found to be suitable. This microorganism, which makes it possible to produce aklanonic acid from readily available raw materials in good yield, was surprisingly able to be isolated after mutagen treatment from a Streptomycetes strain known as the ε-rhodomycinone and daunomycin glycoside builder. The microorganism F 23/135 used in the present invention shows very good sporulation ability. This property is beneficial for stammering, stock breeding and genetic manipulation of the product builder.

The cultivation of the strain and its variants takes place under aerobic and sterile conditions. Spore material dried on soil is inoculated onto suitable agar media and subsequently into liquid, previously sterilized nutrient media and the resulting mycelium is incubated in a manner known per se at a temperature between 25 and 34 ^° C., preferably at 28 ^° C., for a period of 2 to 6 days, preferably 4 days, cultivated at an acidity which at the beginning of the fermentation process between pH; 6.2 and pH 7.0 and at the end of the process between pH 7.5 and pH 8.3. The nutrient medium consists of carbon and nitrogen source as well as organic salts. Starch, glucose, glycerin, dextrin, mannitol, sucrose, soybean meal and soybean oil can be used as the carbon source. In addition to the nitrogenous substrates mentioned above, the nitrogen source also includes dry yeast, meat peptone and casein. Good results can be achieved with the addition of mineral salts. The latter favor the course of the fermentation depending on the nutrient medium used. In the case of complex media containing various flours, additions of calcium carbonate, sodium phosphate or potassium phosphate have proven to be advantageous.

The isolation of the aklanonic acid is carried out by methods known per se by extraction with organic solvents, precipitation with petroleum ether and subsequent chromatographic purification. The thus isolated, yellow-orange needles crystallizing substance was analyzed in comparison with authentic aklanonic acid:

a) thin-layer chromatography on Merck TLC aluminum foils silica gel 60 F ₂₅₄ buffered with 0.5 N oxalic acid; System: chloroform-acetone / 10: 2). Same chromatographic behavior as aclanonic acid. Rf: 0.51.

b) Mass Spectrometry: The mass spectrum of the substance is identical to the mass spectrum obtained upon assay of authentic aklanonic acid. (Eckardt, K. et al., 1985. J. Antibiot. 38, 1034-1039).

c) ¹ H-NMR spectrometry: The ^ NMR spectrum of the substance showed all the signals in a characteristic position, which were also found in the spectrum of aklanonic acid.

d) Rearrangement to Aklanon: The substance was dissolved in dimethyl sulfoxide in a manner known per se (Eckardt, K. et al., 1985. J. Antibiot. 38, 1034-1039) and left at room temperature. The rearrangement product obtained was aklanone, which was identified by chromatography on standard material.

e) Infrared Spectroscopy: The infrared spectra of the substance prepared according to the invention and authentic aklanonic acid are identical.

Thus, with the methods used, no differences could be found between the substance prepared according to the invention and authentic aklanonic acid.

embodiments

By the following embodiments, the invention will be explained, but not limited.

1. Two oOOml bottles each containing 80 ml of the following pre-breeding medium:

Glucose 1.5%

Soy flour. 1.5%

Sodium chloride 0.5%

Calcium carbonate 0.1%

Potassium hydrogen phosphate 0.3%

in tap water

Sterilization: 35 min at 110 ° C. After sterilization, the acidity is at pH 6.3.

Each steep-flask is inoculated with a spore suspension prepared with sterile isotonic saline from a 10 to 14-day culture of strain F 23/135 grown in a test tube on the following culture medium:

Oatmeal 2%

Agar-agar 2%

in tap water

Sterilization: 40 min at 12O ⁰ C. After sterilization, the acidity is adjusted to pH 7.0.

The inoculated Vorzucht-cultures are incubated on a shaker at a frequency of 180 rev / min for 48 hours at 28 ⁰ C. 8 ml of pre-culture incubated in this way are used to inoculate 500 ml steep-breasted bottles, each containing 80 ml of the following production medium:

Glucose 2.0%

Soybean meal 2.0%

Sodium chloride 0.5%

Calcium carbonate 0.3%

in tap water

Sterilization: 35min at 11O ⁰ C. The acidity after sterilization is pH 6.3.

The temperature during the fermentation is 28 ⁰ C and the shaking frequency at 180 U / min. After 72 to 96 hours of fermentation, the highest yield of aclanoic acid is achieved. From the culture solution thus obtained, the mycelium is separated without prior change of the pH and extracted with organic solvents, preferably acetone. After concentration, the extract is mixed with water and acidified to yellow until the color changes. Now, the substance is re-extracted by the addition of organic solvents immiscible with water and precipitated from the extract concentrates by the addition of petroleum ether. By filtering off and washing with petroleum ether, aklanonic acid was obtained as a yellow-brown powder, which can be further purified by column chromatography.

2. With a culture of the strain F 23/135 from a semi-solid medium, as described in Example 1, 400 ml of the liquid pre-emergence medium mentioned in Example 1, which is contained in a 2I glass flask. It is incubated for 48 hours at 28 ⁰ C on a shaker with a frequency of 180 U / min. The thus obtained 400 ml of culture broth serve to inoculate 2Ol of the production medium described in Example 1, which is contained in a 321 glass fermentor. During the fermentation, which can be carried out with a stirring speed of 400 rpm and an air supply of 15 l / min, foaming is controlled by the addition of small amounts of sunflower oil or silicone-containing antifoaming agent. The work-up is carried out as indicated in Example 1.

3. With the media described in Example 1, the pre-breeding is first carried out for 24 hours in a 500 ml Steilbrustflasche with 80 ml and then for another 24 hours in the same medium in a 2I glass flask with 400 ml content, before a 4001 V 2 A steel tank can be inoculated containing the production medium described in Example 1. The stirring speed is 280rpm and the air supply is 400l / min. The pure aklanonic acid is recovered from the fermentation broth by the process steps indicated in Examples 1 and 2.

Claims (3)

1. A process for the preparation of acyclic acid by microbiological means, characterized in that a strain of a well-spurulating strain of the species Streptomyces peucetius under aerobic conditions in liquid nutrient media containing a carbon, a nitrogen source and mineral salts, at a temperature of 25 to 34 C. for a period of from 2 to 6 days, and the aklanic acid formed is extracted from the mycelium by means of customary solvents, subsequently concentrated by conventional methods, precipitated and purified by chromatography. 2. The method according to item 1, characterized in that the microorganism used was obtained from a strain of the species Streptomyces peucetius var. Caesius 601 F. I. 3. The method according to item 1 and 2, characterized in that the strain F23 / 135 is used, which was obtained by mutagen treatment from a known as Bildnervon ε-Rhodomycinon and Daunomycinonglykosense strain of the species Streptomyces peucetius.