DD252616A1 - PROCESS FOR THE PRODUCTION OF CERULENINE ON MICROBIOLOGICAL PATHS - Google Patents

Abstract

The invention relates to a process for the production of cerulenin by microbiological means. The microorganism used for the production is the mutant of a species belonging to the Fungi imperfecti and bears the designation IP 45 and is deposited in the ZIMET depository under the registration number ZIMET 43807. The aim of the invention is the production of the antibiotically and enzyme-inhibiting cerulenin which is of potential use in the pharmaceutical research and industry on the one hand for the production of hybrid antibiotics and on the other hand for the selection of high-performance strains of polyketide-derived antibiotics. The object is achieved in that cerulenin is formed by aerobic submerged fermentation of the strain ZIMET 43807 in media with suitable carbon, nitrogen sources and mineral salts and Zeosorb molecular sieves, isolated from the culture filtrate by suitable methods and subsequently purified.

Description

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Field of application of the invention

The invention relates to a process for the preparation of a microbial active ingredient, which exhibits antibiotic activity against fungi, yeasts and gram-positive bacteria and, moreover, as an inhibitor of fatty acid synthetase and Polyketidsynthetase as a research tool in hybrid biosynthesis for the production of new hybrid antibiotics can be performed in the Chemotherapy of tumor, virus and microorganism-induced diseases in humans and livestock are of potential use. The antibiotic and enzyme inhibitor is an optically active keto-epoxydodecadienamide called cerulenin, which is becoming increasingly important in the microbiological and pharmaceutical industries because of its ready-to-use for the targeted selection of high-performance antibiotic strains. The field of application of the invention is thus in the pharmaceutical research and industry.

Characteristic of the known technical solutions

Cerulenin was first isolated and described in 1960 as an antifungal antibiotic from fungal cultures of the species Cephalosporium caerulens (Hata, T. et al., Jpn. Bacteriol., 15, 1057). Overviews are available on the recovery and properties of cerclenin (Omura, S., 1976. Bacteriol, Rev., 40, 681; Omura, S., 1981. In Lowenstein, JM, Methods in Enzymology, Vol. 72, 520-532, Masuma, R. et al., 1982. J. Antib. 35, 184). It is known that so far only a few types of fungi are capable of biosynthesis of cerulenin.

Helicoceras oryzae, H. celtidis, H. plantaginis, H. nymphaerum are known generators of helicocerin identical with Cerulenin (Engl. Pat. 1,154,133 1969). Cephalosporium caerulens (JP 65/9588 1965), Acrocylindrium oryzae (JP 70/21638 1970) and Sartoriafumigata (JP 72 241561972) are other formers of cerulenin, which together have the disadvantage that during fermentation in addition to the desired Cerulenin several severely separable steroidal Antibiotics, in particular helolic acid, be synthesized, which complicate the isolation of Cerulenin.

It is also known that the addition of zeolites during fermentation promotes cerulenin biosynthesis in cephalosporium cornulens (JP 57206380 1982).

Because of the numerous uses and importance of cerulenin as a specific enzyme inhibitor, several methods for the total synthesis of (±) cerulenin (Boeckmann, RK and Thomas, EW, 1979. J. Am. Chem. Soc., 101, 897; Corey, EJ and Williams, DR, 1977. Tetrahedron Lett., 1977, 3847; Jakubowski, AA et al., 1982. J. Org. Chem., 47, 122), and a process for producing optically active (+) - cerulenin (Sveta, N. et al., 1979. Tetrahedron Lett., 1979, 2039). However, the syntheses lead to a series of chemical reactions with sometimes only moderate yields, so that a synthesis does not currently appear economical.

While the basic structure of the antibiotic has been known for some time (Omura, S. et al., 1969. Chem. Pharm. Bull., 1969, 2361; Arison, B. et al., 1974. J. Antib., 27, 28 ), the absolute configuration of cerulenin as (2R, 3S) -2,3-epoxy-4-keto-7.10.trans, trans-dodecadienamide (Figure 1) has been elucidated only in recent years (Sveta, N. et al., 1979. Tetrahedron Lett., 1979, 2039; Poughny, JR and Sinary, P., 1978. Tetrahedron Lett., 1978, 3031; Vigneron, JP and Blanchard, JM, 1980. Tetrahedron Lett. 1980, 1739).

Object of the invention

The invention aims to produce the antibiotic and enzyme inhibitory cerulenin. Thus, the range of known manufacturing processes for the increasingly important in pharmaceutical research and industry cerulenin is to be extended by an original process that avoids the disadvantages of known production methods.

Explanation of the essence of the invention

The invention has for its object to provide a technologically simple method that allows cerulenin using a previously unknown microorganism strain exclusively and without difficult to separate, steroidal companion antibiotics from easily accessible and inexpensive starting materials in good yields. According to the invention the object is achieved in that under mutagenesis from a belonging to the fungi imperfecti population IP 45 is grown under aerobic and sterile culture conditions in a liquid nutrient medium with carbon, nitrogen sources and mineral salts. The microorganism used in this process has been deposited in the ZIMET Depository for Microorganisms, in the Central Institute for Microbiology and Experimental Therapy of the GDR Academy of Sciences, 6900 JENA, Beutenbergstrasse 11, under the registration number ZIMET 43807, and is distinct from known cerulenin-formers by morphological-physiological characteristics and in that during the fermentation no helolic acid and other steroidal antibiotics are synthesized as difficult to separate by-products. Cultures of strain IP 45 grown on suitable agar media are inoculated into liquid, previously sterilized nutrient media and the resulting mycelium is added in a manner known per se at a temperature between 25 and 37 ° C for a period of 2 to 4 days, preferably 2 days cultured acidity at the beginning of the fermentation process between pH 6.2 and pH 6.6 and at the end of the process between pH 5.0 and pH 6.6.

Glucose, glycerin and sucrose can be used as carbon sources in the suture media. Nitrogen sources include dry yeast, meat peptone and sodium nitrate. Good results can be achieved with the addition of mineral salts and Zeosorb molecular sieves. The latter promote the course of fermentation as selective adsorbents and exchangers. In complex media, additions of calcium carbonate, sodium phosphate or potassium phosphate have proved to be advantageous. The fermentation of the cerulenin ZIMET 43807 can be carried out in breast bottles and round-bottomed flasks of various contents, in the glass fermentor and in V2A steel tanks. Isolation of the cerulenin is carried out by extracting the antibiotic from the culture filtrate with organic, water-immiscible or poorly-miscible solvents, reextracting the extracts with a water-immiscible solvent, drying over anhydrous sodium sulfate, drying to dryness is then taken up in an organic nonpolar solvent and purified by column chromatography. From the crystalline crude product, which results after distilling off the solvent of the combined antibiotic fractions of the column chromatography, the cerulenin is obtained by recrystallization as a pure crystalline compound. Properties of cerulenin

Colorless, microcrystalline substance of carbon tetrachloride. M.p .: 90-93 ⁰ C, molecular formula: C ₂ H ₁₇ NO _3, MW: 223, la / g ^5: -12 ° C (chloroform, c = 1.0). Solubility: Readily soluble in chloroform, carbon tetrachloride, benzene and ethyl acetate, moderately soluble in water.

Mass spectrum m / z: M ⁺ gef .: 223.1185; Re: 223.1208; Fragmentation m / z: 206 (M ⁺ -NH ₃ ); 179 (M ⁺ -CONH ₂ , base peak); 142 (M ⁺ -C ₆ H ₉ ). IR spectrum (KBr) Cm " ¹ : 3380.3210 (-NH ₂ , amide); 1720 (C = O); 1663 (C = O, amide); 1610 (C = C); 967 ( ^H C = C _H ) ¹³ C-NMR spectrum: The data from the ¹³ C-NMR analysis are summarized in Table 1. They are characteristic of cerulenin (Funabashi, H. et al., 1983. Tetrahedron Letters, 24, 2673) of the structure (2R, 3S) -2,3-epoxy-4-keto-7,10-trans, transdodecadienamide (formula, Fig. 1).

The advantages of the production method described here for the enzyme inhibitor cerulenin are summarized

- In the provision of a microorganism, in addition to cerulenin no other, difficult to separate from this drug substances in the fermentation medium or enriches in the mycelium;

- the ability to make available, in good yields and using cheap feedstock, a biofematic chemical of significant importance to the pharmaceutical research and industry, for the construction of hybrid antibiotics with a novel structure and mode of action as well as for the selection of industry High performance strains of potential use;

In a stereospecific biosynthesis of the optically active cerulenin, which is obtained in a high yield during the fermentation with the configuration relevant for the biochemical action of the enzyme inhibitor;

- Simple'lsolierung of cerulenin from the culture filtrate of the builder without the need for the separation of helolic acid or other steroidal antibiotics.

embodiments

The invention is explained in more detail by the following embodiments, but not limited. 1. Two 500 ml steep breast bottles each contain 80 ml of the following pre-breeding medium:.

Glucose 2.0%

Peptone 0.5%

Meat extract 0,5%

Dry yeast 0.3%

Sodium chloride 0.5%

Calcium carbonate 0.3%

in tap water

Sterilization: 35 min at 110 ^° C. The acidity after sterilization is at pH 6.3. Each steep breast bottle is inoculated with mycelium fragments of Cerulenin-Former IP 45, which was grown on the following proximal bottom:

Malt extract 3.0%

Agar-agar 1.2%

in tap water

Sterilization: 35min. at 115 ° C. The acidity after sterilization is pH 6.3. The inoculated pre-breeding cultures are 48h. incubated at 28 ° C on a shaker with a frequency of 180 U / min. 8 ml of a pre-breeding culture incubated in this way are used to inoculate 500 ml steep-breasted bottles, each containing 80 ml of the following production medium:

Glycerine 3.0%

Glucose 1.0%

Peptone 0.5%

Sodium chloride 0.2%

Zeosorb molecular sieve

4 A powdery 1.0% '' '

in tap water

Sterilization: 35min. at 115 ° C. The acidity after sterilization is pH 6.3. During the fermentation, the temperature is at 28 ⁰ C and the shaking frequency is 180U / min. With a culture of cerulenin builder IP 45 from a solid medium, inoculate 80 ml of the mentioned liquid pre-breeding medium, which is contained in a 500 ml Steilbrustflasche. It is incubated for 48 hours at 28 ⁰ C on a shaking table with a shaking frequency of 180 U / min. 12 ml of the culture broth thus obtained are used to inoculate 400 ml of the same pre-breeding medium, which is contained in a glass flask. It breeds another 48hrs. at 28 ° C at a shaking frequency of 180 U / min, before the thus obtained 400 ml of culture broth for inoculation of 201 of the described production medium serve. The latter is contained in a 321 Giasfermenter. During the fermentation, which is carried out with a stirring speed of 400 rpm, and with an air supply of 15 l / min, foaming can be controlled by adding small amounts of Antafron. Cultured broth, which is obtained from a fermentation batch in the 321 gypsum fermenter, are centrifuged and the supernatant culture filtrate is exhaustively extracted with chloroform. The combined chloroform extracts are dried over sodium sulfate, the solvent is distilled off in a vacuum rotary evaporator, the oily residue is taken up in a little chloroform and washed on silica gel (0.063-0.2 mm) with chloroform, chloroform / ethyl acetate = 95/5 and chloroform / ethyl acetate = 90 /. 10 chromatographed. Fractions containing thin layer chromatography cerulenin are concentrated to dryness in a vacuum rotary evaporator. This gives 2.7 g of crystalline Cerulenin crude product, which can be recrystallized for ultrafine purification from benzene / petroleum ether and then from carbon tetrachloride.

2. The procedure is as in Example 1 with the difference that the production medium has the following composition:

Glucose 4.0%

Peptone 1.0%

in tap water Sterilization: 35 min at 115 ^° C. The acidity after sterilization is pH 6.3.

3. The procedure is as in Example 1 with the difference that the culture medium has the following composition:

potato extract 2.0% glucose 1.0% peptone 0.1% Agar Agar 1.2% in tap water 35min. at 120 ⁰ C, except nature. Table 1

¹³ C-NMR data of cerulenin (CDCI ₃ ) compared to references by Funabashi et al. / 1 /. The chemical shifts are related to tetramethylsilane as internal standard.

C atom Chemical shift 1) Own measurement 167.3 C-1 167.27 55.4 C-2 55.33 58.4 C-3 58.36 202.0 C-4 , 201.97. 40.9 C-5 40.84 26.0 C-6 25.95 125.8 C-7 125.79 127.8 C-8 127.78 129.2 C-10 129.20 130.7 C-II 130.66 35.5 C-9 35; 41 17.9 C-12 17.82

/ 1 / H. Funabashi, S. Iwasaki and S. Okuda Tetrahedron Letters 24 (1983) 2673-2676

Claims (4)

1. A process for the preparation of Cerulenin microbiological, characterized by the use of the preferred formation of the target product capable of microorganism IP 45 (belonging to the fungi imperfecti species) by fermentation under aerobic and sterile conditions in liquid nutrient media containing carbon, nitrogen Sources, mineral salts and Zeosorb molecular sieve at temperatures of 25 to 37 ° C for a period of 2 to 4 days and isolation and purification of the formed cerulins from the culture filtrate at an acidity of pH 5.0 to pH 6; 6 in. in a manner known per se by extraction with an organic solvent, subsequent concentration, chromatographic purification and recrystallization. 2. Method according to item 1, characterized by carrying out the fermentation process at 28 ° C over a period of 2 days. 3. The method according to item 1 and 2, characterized by the addition of powdered Zeosorb molecular sieve 4A to the fermentation broth. 4. Method according to item 1, characterized by the use of trichloromethane for the extraction of cerulenin from the culture filtrate.