CH644635A5 - MICROBIOLOGICAL METHOD FOR PRODUCING SECONDARY GLYCOSIDES FROM PRIMARY DIGITALISGLYCOSIDES. - Google Patents

Description

The invention relates to a microbiological process for the preparation of secondary digitalis glycosides of the general formula I.

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644 635

(X)

wherein R1 and R2 each represent a hydrogen atom or a hydroxyl group.

As is known, the glycoside mixture obtained from the leaves of Digitalis lanata contains not only the primary glycosides or lanatoside compounds (lanatoside A, B and C) and the secondary glycosides (digitoxin, gitoxin and digoxin ^) but also the acetyl derivatives of the latter (acetyldigitoxin, acetylgitoxin and acetyldigoxin) When the leaves are worked up by any known method, mixtures of glycosides are always obtained from which not all components can be used equally as medicaments, so it is advisable to look for methods by which the individual components of the Glycoside mixture can be brought into the most suitable forms for therapeutic purposes.

The compounds of the lanatoside series differ from the secondary glycosides in that while in the secondary glycosides only three digitoxose units are linked to the aglycone part of the molecule, the compounds in the lanatoside series contain a glucose bonded to a third digitoxose in addition to these sugars. wherein the 3 "'hydroxyl group of the third digitoxose is esterified by acetic acid.

Several methods are known for splitting off the glucose in the β position or the 3 "'acetyl group of the lanatosides. For example, the 3"' acetyl group can be split off by very mild alkaline treatment.

In the acidic or alkaline hydrolysis of lanatoside compounds, the bond present between the aglycone and the first digitoxose is generally also cleaved, so that not only the desired terminal glucose cleavage is achieved, but also the aforementioned cleavage into genins, which is undesirable here (Digitoxigenin, gitoxigenin and digoxigenin), so that secondary cardiac glycosides cannot be obtained from lanatosides in this way; The desired secondary cardiac glycosides can only be produced from lanatoside in laborious chemical ways.

It can be achieved by appropriate fermentation of the leaves of Digitalis lanata that the compounds of the secondary series are enriched in the glycoside mixture; in this case, however, this can be

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valuable lanatoside C cannot be obtained from the glycoside mixture.

Microbiological methods have long been known to cleave compounds of the lanatoside series. The terminal glucose can thus be split off with the aid of mold cultures or from β-glucosidase lakes produced therefrom. A. Stoll and his colleagues first investigated the lanatoside-splitting activity of molds (cf. Helv. Chim. Acta 34, 397-401 [1951]. The terminal glucose of lanatosides could be determined with the help of the culture of various types of mold (mostly from Asper gillus species) by treatment with acetone dry powder F. Pitra and co-workers (Czechoslovakian Patent No. 108 046) used an enzyme preparation derived from Aspergillus orizae to produce acetylated secondary glycosides, but the acetylase activity of the mold cultures is not comparable to their ß-glucosidase activity; therefore this method is generally only used for the production of acetylated compounds of the secondary series.

The aim of the present invention was to prepare compounds of the secondary series from lanatosides, with the aid of those microorganisms which, in addition to the β-glucosidase activity, also have a corresponding acetylase activity, so that they move the substrate at high speed and in can implement high concentration such that both the glucose and the acetyl group are split off in the same fermentation. Furthermore, the microorganisms that can be used for this purpose still require that the two activities mentioned, i.e. the β-glucosidase and acetylase activity should be practically equally strong and that no intermediates (acetyl derivatives of the secondary series or desacetyl-lanato-side, also known as Purpurea glycosides) should accumulate during the reaction. It is therefore desirable that the microorganisms to be used should be able to produce secondary glycosides both from the acetyl derivatives of the secondary glycosides (using the acetylase activity) and from the pur-purea glycosides (using the β-glycosidase activity) plays the crucial role).

It has now been found that it is possible to use the above

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To achieve results corresponding to requirements with the help of a strain of microorganisms belonging to the genus Streptomyces and characterized by outstandingly high enzyme activity. This new strain of microorganisms has been identified as Streptomyces griseolus and has been titularized in the Hungarian National Microorganism Strain Collection by the Hungarian National Institute for

Hygiene deposited under No.MNG 168 on December 6, 1977.

The invention is accordingly a new process for the microbiological production of secondary digitalisglycosides of the general formula I defined above, in which R1 and R2 represent hydrogen atoms or hydroxyl groups, which is characterized in that primary glycosides of the general formula II

(XI)

wherein R1 and R each represent a hydrogen atom or a hydroxyl group R3 represents a hydrogen atom or an acetyl group and 35 R4 represents a hydrogen atom or a D-glucose group, but R3 and R4 cannot both simultaneously signify hydrogen, with a submerged culture of the strain Streptomyces griseolus MNG 168 or in contact with an enzyme preparation prepared from such a culture and isolating the secondary glycosides obtained from the fermentation liquid in a manner known per se.

According to an advantageous embodiment of the method according to the invention, the enzyme is fixed to the microorganism cells and the hydrolytic reaction is carried out with the enzyme preparation bound in this way.

The microorganism strain used in the method according to the invention was isolated from a soil sample; the tribe showed the following taxonomic properties:

Morphology: The spore carriers show sympodial branches, have straight ends; There are no spirals or loops. In the light microscope, the spores are egg-shaped, their size is 0.7-0.9 x 0.9-1.1 ji. 55 Growth on various nutrient media: sucrose nitrate agar: growth colorless, later gray; the aerial mycelia are thin, white, later olive green. Weak brown pigment diffuses into the agar.

Tyrosine agar: melanin negative. The aerial mycelia and the 60 total growth show loose structure. A light brown soluble pigment diffuses into the agar.

Nutrient agar: compact, strong growth, initially gray, later brownish. The aerial mycelia are thin, dusty, light gray; no soluble pigment. b5

Bennett agar: growth cream-colored, darkens over time to brownish. The aerial mycelia are thin but compact, with a dusty structure and a blue-gray color.

Glucose-asparagine agar: growth gray, dark gray, later black in places. Aerial mycelia are only formed in places, with mouse gray color. Little brown soluble pigment diffuses into the agar.

Bouillon agar: growth thick, gray, wrinkled. Neither aerial mycelia nor soluble pigment are formed.

Calcium malate agar: rapid growth, light gray, later dark gray. The aerial mycelia are blue-gray, in places with olive-green spots. Very little brownish, soluble pigment diffuses into the agar.

Potato-glucose agar: The growth shows medium speed, but spreads strongly, with brown color. The aerial mycelia are velvety in the middle, blue-gray, crawl apart at the edge of the colony like arachnids. Little light brown soluble pigment diffuses into the agar.

Maize flour agar: growth light brown, the aerial mycelia are dusty, thin, initially light gray, later blue-gray. No soluble pigment.

Glycerine nitrate agar: growth gray, gray-brown, lastly black, very wrinkled. The aerial mycelia are plaster-like, dusty, evenly ash gray. Little, but distinctly brown, soluble pigment diffuses into the agar.

Sabouraud-glucose (glucose-peptone) agar: The growing soil mycelium is light brown, the aerial mycelia are thin, light gray, gypsum-like, dry, a weak brown soluble pigment is formed.

Potato slice: strong and rapid growth, with a roughly wrinkled surface; initially gray, later black. The aerial mycelia are ash gray, the soluble pigment brown.

Löffler's curdled whey culture medium: brown growth, strong proteolytic activity.

Cellulose as the only carbon source: good growth.

Nitrate reduction: positive.

Gelatin liquefaction: positive.

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Milk: strong coagulation and peptonization are observed.

Blood agar: growth wrinkled, olive green; Aerial mycelia light gray; weak beta hemolysis.

Assimilation of carbon sources

D-glucose

+

D-sorbitol weak

D-galactose

+ +

soluble starch

+ +

Sucrose

+

D-xylose

+

Maltose

+

L-rhamnose

+

Lactose

+

Galactite

-

Raffinose

+

D-mannitol

+

L-sorbose

-

Inositol

+

Cellobiose

+

D-fructose

+

Trehalose

+ +

D-mannose

+

Glycerin

+ +

Due to the above characteristic properties, the new strain belongs to the species Streptomyces griseolus (Waksman) Waksman et Henrici 1948 (see Waksman: The Actinomycetes, Vol. 2, The Williams Wilkins Company, Baltimore, 1961, pp. 222-223).

The methods which are generally customary in the cultivation of radiation fungi can be used to cultivate the microorganisms used in the process according to the invention. Cultivation is most advantageously carried out in submerged culture. The nutrient medium suitable for cultivation can contain carbon and nitrogen sources dissolved or suspended in water, which can be assimilated by the fungi, as well as inorganic salts and other usual additives (bio-substances, anti-foaming agents, etc.).

Malt, glucose, maltose, sucrose or other sugars, fats, fatty acids, amino acids and peptides can be used as the carbon source. Suitable assimilable nitrogen sources are nitrogen compounds of microbial, animal or vegetable origin and / or inorganic nitrogen compounds, such as yeast extract, peptone, meat extract, hydrolyzed casein, soy flour, corn steep liquor, ammonium salts and nitrates.

Taking economic aspects into account, glucose can be used most advantageously as a carbon source and soy flour and / or corn steep liquor as a nitrogen source in the nutrient medium. The constituents of the nutrient medium are dissolved or suspended in tap water and sterilized together at 121 ° C. for 20 to 45 minutes, with the exception of the reducing sugars, since these are expediently sterilized separately.

The microorganisms used in the process according to the invention can be grown at 22-37 ° C., preferably at 28 ° C. These microorganisms are not particularly sensitive to the ventilation and the speed of the agitator; in general, good cultures which are suitable for rapid reaction can be obtained in a 100 l fermenter with an aeration speed of 1001 / min and a stirring speed of 400 rpm; Similar good results can also be achieved with other ventilation speeds and agitator revolutions that are not too different.

The substrates to be converted in the process according to the invention are dissolved in water-miscible organic solvents (methanol, ethanol, dimethylformamide, acetone, tetrahydrofuran), which are not or only moderately damaging to the function of biological systems, advantageously in methanol, and the solution is sterilized by filtration and then added to the fully developed culture showing maximum enzyme activity. The reaction is carried out in a ventilated vessel equipped with a stirrer at 22 to 37 ° C., preferably at 28 to 32 ° C.

The reaction can also be carried out separately from the fermentation process by producing an enzyme preparation from the culture which is already suitable for the reaction and using this for the reaction. The enzyme preparation can be produced according to the generally known method of producing “dry powders obtained by treatment with acetone”. About 70% of the total ß-glucosidase and acetylase activity of the culture is contained in this acetone dry powder. The activity of such dry enzyme preparations is fully retained when stored at 4 to 6 ° C for one month.

After the reaction has ended, the secondary glycosides present in the fermentation liquid are expediently extracted from the fermentation liquid using a water-immiscible organic solvent (chloroform, dichloroethane, ethyl acetate, methyl isobutyl ketone, etc.), especially chloroform. The detachment of the secondary glycosides bound to the mycelium can be facilitated by the addition of water-miscible organic solvents (methanol, ethanol, acetone, etc.), especially methanol. The progress of the reaction can be followed during the production by means of thin layer chromatography.

The practical examples and the further details of the method according to the invention are illustrated by the following examples.

example 1

A one-week culture of the Streptomyces griseolus MNG 168 strain grown on oblique potato glucose agar is showered with sterile physiological saline and the surface of the agar is scraped open with a sterile platinum loop. An inoculum culture medium of the following composition is inoculated with the microorganism suspension obtained in this way: 2% glucose, 1% soybean meal, 0.5% corn steep liquor (wet weight, calculated on 50% dry matter), 0.2% peptone, 0.3 % Calcium carbonate; Total volume 1 liter sterilized in 3-1 Erlenmeyer flasks. The culture is shaken on a shaker at 28 ° C for 36-48 hours. Three such inoculum cultures are prepared in parallel and the 3-liter inoculum cultures obtained in this way are used to inoculate the nutrient medium of the same composition prepared in a fermenter equipped with a stirrer of 100 liters useful volume. During the fermentation, the medium is stirred at 400 rpm and aerated with 1001 / min air flow. Cooking oil is used as an anti-foaming agent. The fermentation is continued at 28 ° C for 36 to 48 hours to achieve the enzyme activity required for the reaction.

500 g of lanatoside A are dissolved in 10 liters of hot methanol, the solution is filtered off through a Seitz filter and then added to the fermentation liquid prepared in the above manner under sterile conditions and with stirring. The progress of the reaction is monitored using thin layer chromatography (on Merck “Kieselgel-G” plates; mobile phase: 55:35:10 mixture of ethyl acetate, cyclohexane and absolute ethanol). The stain of the substrate disappears after 48 to 60 hours and only the resulting connection of the secondary row, the digitoxin, is visible on the plate. After the reaction is complete, the fermentation liquid is filtered. The mycelium is stirred with 801 methanol for 2 hours, filtered and stirred with a further 80 l of methanol again for 2 hours. The methanolic-aqueous washing liquids are combined, concentrated to 5 liters in vacuo at a maximum of 40 ° C. and left to crystallize at 0 ° C. for 24 hours. The crystals obtained are washed with 70% aqueous methanol (crude product

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I). The mother liquors and washing liquids from the crystallization are extracted three times with each volume of chloroform and the combined extracts are evaporated to dryness (crude product II).

The filtrate of the fermentation liquid is also extracted three times with 1/2 volume of chloroform and the combined extracts are evaporated to dryness (crude product III).

The combined raw products II and III are taken up with 5 liters of 50% aqueous methanol and extracted with 2 liters of petroleum ether. The aqueous-methanolic phase is evaporated to dryness (semi-finished product II-III); the petroleum ether phase is discarded.

The raw product I is combined with the semi-finished product II-III, powdered, in 5 liters of a 1; 1 mixture of methanol and benzene dissolved, the solution stirred with activated carbon and filtered through an aluminum oxide layer. The filtrate is mixed with stirring with 2.5 liters of tap water and left at 0 ° C for 48 hours to crystallize. The benzene phase is discarded, the crystals are filtered off, washed with benzene and then with a 1: 1 mixture of methanol and water and finally dried at 50 ° C. to constant weight. In this way, 310 g of digitoxin (purity at least 97%) are obtained. The mother liquors and washing liquids of the crystallization are combined and also worked up in the above manner; a further 25 g of digitoxin are obtained. Overall yield: 85%.

Example 2

The culture of the microorganism strain which is already suitable for the reaction and is obtained in the manner described in Example 1 is filtered off, the mycelium is washed with water and then placed in an amount of acetone cooled to −20 ° C. such that the liquid covers the mycelium mass. The mycelium is stirred for one hour in the acetone kept at −20 ° C., then filtered cold and dried at room temperature to constant weight. In this way, 70 g of dry enzyme preparation are obtained.

70 g of enzyme preparation (which is not older than 1 month) are suspended in 80 liters of 0.01 M phosphate buffer solution (pH = 7) with stirring; then the hot concentrated methanolic solution of 350 g of Lanatosid A is added with further stirring. The mixture is stirred further at 28 ° C. and the progress of the reaction is monitored by thin layer chromatography. After 48 to 60 hours, the stain of the starting material no longer appears; only the digitoxin stain is visible. The reaction mixture is worked up in the manner described in Example 1; 236 g of digitoxin (85% of theory) are obtained.

Example 3

5 g of Lanatoside B are added to 1 liter of enzyme-active fermentation medium obtained according to Example 1. After the reaction has ended, the reaction mixture is worked up in the manner described; 3.37 g of gitoxin (85% of theory) are obtained.

Similar results are obtained if you look at that

Adds lanatoside B to 1 liter of enzyme preparation obtained according to Example 2 and suspended in phosphate buffer solution.

Example 4

5 g of lanatoside C are treated according to Example 3; 3.34 g of digoxin (84% of theory) are obtained.

Example 5

5 g of acetyl digitoxin are treated according to Example 3; 5 g of acetyl digitoxin are treated according to Example 3; 3.95 g (85% of theory) of digitoxin are obtained.

Example 6

5 g of acetylgitoxin are reacted in the manner described in Example 3; 3.92 g (84% of theory) of gitoxin are obtained.

Example 7

5 g of acetyl digoxin are reacted in the manner described in Example 3; 3.90 g of digoxin (84% of theory) are obtained.

Example 8

5 g of deacetyl lanatoside A are reacted in the manner described in Example 3; 3.54 g of digitoxin (84% of theory) are obtained.

Example 9

5 g of deacetyl lanatoside B are reacted in the manner described in Example 3; 3.55 g of gitoxin (84% of theory) are obtained.

Example 10

5 g of deacetyl lanatoside C are reacted in the manner described in Example 3; 3.51 g of digoxin (83% of theory) are obtained.

Example 11

The details of the procedure are as described in Examples 1 or 2, with the difference that, instead of pure lanatoside A, any mixture of lanatoside A, lanatoside B, lanatoside C, deacetyl-lanatoside A, deacetyl-lanatoside B, deacetyl -lanatoside C, acetyldigitoxin, acetylgitoxin and / or acetyldigoxin can be used as starting materials. In each case, corresponding mixtures of digitoxin as the compound of series A, gitoxin as the compound of series B and digoxin as the compound of series C are obtained as end products, the proportions of these three secondary glycosides each corresponding to the proportions in which the compounds of the series A (Lanatoside A, desacetyl-lanatoside A, acetyldigitoxin), the compounds of the series B (Lanatosid B, des-acetyl-lanatoside B, acetyl-gitoxin) and the compounds of the series C (Lanatosid C, deacetyl-lanatoside C, acetyldigoxin) in the glycoside mixture used as the starting material were present. The yield of secondary glycosides is generally 84%.

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Claims (2)

644 635 PATENT CLAIMS 1. Process for the microbiological production of secondary digitalis glycosides of the general formula I (X) wherein R1 and R2 each represent a hydrogen atom or a hydroxyl 30 digitalis glycoside and / or acetyl derivative of secondary group, characterized in that primary digitalis glycosides of the general formula II (IX) wherein R1 and R2 have the above meanings, R3 represents a hydrogen atom or an acetyl group and R4 represents a hydrogen atom or a D-glucose group, but R3 and R4 cannot both mean simultaneously hydrogen, with a submerged culture of the strain Streptomyces griseolus MNG 168 or with one enzyme preparation produced from such a culture is fermented and the secondary glycosides obtained are isolated from the fermentation liquid. 2. The method according to claim 1, characterized in that the enzyme is fixed on the microorganism cells 60 and carries out the hydrolytic reaction with the enzyme preparation fixed in this way.