GB2028334A - Microbial or enzymatic production of digitalis tridigitoxosides - Google Patents

Abstract

A process for the preparation of secondary digitalis glycosides of general formula (I), <IMAGE> (wherein R1 and R2, which may be the same or different, each represents a hydrogen atom or a hydroxy group) comprises fermenting a primary glycoside of formula (II), <IMAGE> (wherein R1 and R2are as defined above, R3 represents a hydrogen atom or an acetyl group and R4 represents a hydrogen atom or a D-glucose unit, with the proviso that at least one of R3 and R4 is other than a hydrogen atom) with a culture of a Streptomyces griseolus strain deposited at the Hungarian National Collection of Microorganisms (Budapest, Hungary) on 6th December 1977 under No. MNG 168 or a mutant thereof or with an enzyme preparation produced therefrom and separating the product of formula (I) thus obtained from the fermentation broth.

Description

SPECIFICATION Microbiological process for the preparation of secondary digitalis glycosides This invention relates to a new microbiological process for the preparation of secondary digitalis glycosides of the general formula (I),

wherein R, and R2 represent hydrogen or hydroxy, from primary digitalis glycosides.

As known, the cardiac glycoside mixtures obtained from the leaves of Digitalis lanata contain primary glycosides or lanatoside derivatives (lanatoside A, B and C), secondary glycosides (digitoxin, gitoxin, digoxin), as well as the acetylated derivatives of the latter (acetyldigitoxin, acetylgitoxin and acetyldigoxin). With no respect to the conditions of processing the leaves, mixtures of glycosides are obtained. Since not all components of the mixture can be applied as medicament, methods enabling these compounds to be converted into each other or into the therapeutically most favourable form, respectively, are very important.

The compounds of the lanatoside series differ from those of the secondary series only in that to the aglycone moiety of the secondary compounds three digitoxose sugar units are attached, whereas the lanatoside compounds contain a further glycose unit attached to the third digitoxose, furthermore the hydroxy group in position 3"' of the third digitoxose is acetylated.

Several methods have been disclosed for removing the P-glucose moiety and the 3"' acetate group of lanatosides. The acetate group in position 3"' can be removed selectively by treating the compounds with alkalies under very mild conditions.

The mild acidic and alkaline hydrolysis of lanatoside compounds generally involves the splitting of the bond between the aglycone and the first digitoxose sugar as well, i.e. not only the required terminal glucose is removed; therefore genins (digitoxigenin, gitoxigenin or digoxigenin) are also formed. For this reason it is difficu It to prepare secondary cardiac glycosides from lanatosides by chemical methods.

The compounds of the secondary series can be enriched in the leaves of Digitalis lanata by approp riatefermentation methods; in such instances, however, the pharmaceutically valuable lanatoside C cannot be separated.

Microbiological processes are also known for decomposing the compounds of the lanatoside series. The terminal glucose unit can be split off by utilizing cultures of mould strains or 8-glycosidase enzymes obtained therefrom. The lanatosidesplitting effect of moulds was first studied by A. Stoll et al. (Helv. Chim. Acta 34,397-40111951/). According to their results the terminal glucose unit of lanatosides could be split off using acetone-dry powders of cultures of several mould strains (mainly Aspergillus strains). F. Pitra et al. applied an enzyme isolated from Aspergillus oryzae to produce acetylated secondary products (Czechoslovakian patent specification No. 108,046).Taking into account, however, that the p-glycosidase activity of Hyphomycetes cultures is much higher than their acetylase effect, this method is applied generally for the production of acetylated compounds belonging to the secondary series.

The invention aims at the elaboration of a new method for producing compounds of the secondary series from lanatosides utilizing microorganisms which possess both p-glycosidase and acetylase activities and are able to convert the substrate in high concentrations at a high rate, removing both the glucose and the acetyl group in a single fermentation step. The microorganism should possess approximately the same acetylase and p-glycosidase activities, and intermediates (acetyl derivatives of the secondary compounds or deacetyl lanatosides/purpurea glycosides/) may not accumulate during the conversion process. The microorganism should be ableto convert secondary glycosides from both the appropriate acetyl compounds (utilizing the acetylase activity) and purpurea glycosides (utilizing the ss-glycosidase activity).

It has been found that the culture of a microorganism belonging to the Streptomyces genus and possessing extremely high enzymatic activities fully meets the above requirements.

According to the present invention there is provided a process for the preparation of secondary digitalis glycosides of general formula (I), as defined above which comprises fermenting a primary glycoside of formula (II), An item of the information required by Rule 17(1)(a)(iii) of the Patents Rules 1978 was not contained in the application as filed, but was supplied later in accordance with Rule 17(2).

(wherein R1 and R2 are as defined above, R3 represents a hydrogen atom or an acetyl group and R4 represents a hydrogen atom or a D-glucose unit, with the proviso that at least one of R3 and R4 is other than a hydrogen atom) with a culture of a Streptomyces griseolus strain deposited at the Hungarian National Collection of Microorganisms (Budapest, Hungary) on 6th December 1977 under No. MNG 168 our a mutant thereof or with an enzyme preparation produced therefrom and separating the product of formula (I) thus obtained from the fermentation broth.

When this primary glycoside of formula (II) is for mented with a culture of the Streptomyces griseolus, the culture is preferably a submerged culture.

According to one preferred method of the invention the conversion is performed with an immobilized enzyme preparation.

The Streptomyces strain utilized according to the invention was isolated from soil. The strain has the following taxonomical characteristics: Morphology: The sporophores have sympodial branchings with straight ends, no helix or loop occurs. When examined with light microscope, the spores are ellipsoidal, 0.7-0.9 x 0.9-1.1 CL in size.

On sucrose-nitrate agar the growth is colourless, turning later grey; the aerial mycelium is thin, white, later olive green. A slight brown pigment diffuses into the agar.

On tyrosine agar the strain is melanine negative.

Both the growth and the aerial mycelium are of loose structure. Light brown soluble pigment diffuses into the agar.

On nutrient agar the growth is compact, strong, initially grey and turns later brown. The aerial mycelium is thin, powdery, light grey. No soluble pigment is formed.

On Bennettagarthe growth is beige-coloured, turning brown upon ageing. The aerial mycelium is thin but compact, porous in structure, bluish-grey.

No soluble pigment is formed.

On glucose-aspartic agar the growth is grey, dark grey, then sporadically turns black. Aerial mycelium is formed only sporadically and is mouse-grey in colour. A small amount of brown soluble pigment diffuses into the agar.

On bouillon agar the growth is thick, grey, and wrinkled. Aerial mycelium and soluble pigment do notform.

On calcium malate agarthe growth is quick, light grey, turning later dark grey. The aerial mycelium is bluish-grey, with sporadical olive green spots. A very small amount of brownish soluble pigment diffuses into the agar.

On potato-glucose agar the growth is of medium rate, but strongly effuse, and brown. The aerial mycelium is velvet-like, bluish-green in the centre, and spreads cobwebby at the edge of the colony. A small amount of light brown soluble pigment diffuses into the agar.

On corn meal agar the growth is light brown, the aerial mycelium is powdery, thin, initially light grey, turning later bluish-grey. No soluble pigment forms.

On glycerol-nitrate agar the growth is grey, greyish brown, finally almost black, and strongly wrinkled. The aerial mycelium is plastery, powdery, and uniformly ash-grey. A small amount of a pronounced brown, soluble pigment diffuses into the agar.

On Sabouraud glucose (glucose-pepton) agar the growing substrate mycelium is light brown, the aerial mycelium is thin, light grey, plastery dry. A light brownish soluble pigment is formed.

On potato slices the growth is strong and fast, coarsely wrinkled, initially grey and later black. The aerial mycelium is ash-grey, the soluble pigment is brown.

On Loeffler's coagulated whey medium a brown growth and a strong proteolytic activity can be observed.

On cellulose as the sole carbon source the strain grows well.

Nitrate reduction : positive.

Gelatineliquefaction: positive.

Milk: total coagulation and strong peptonization can be observed.

On blood agar a wrinkled, olive green growth, light grey areal mycelium and a weakp-haemolysis can be observed.

Assimilation of carbon sources: D-glucose: + D-sorbit: 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: + glycerol: ++ On the basis of the above properties the strain belongs to the species Streptomyces griseolus (Waksman), Waksman et Henrici 1948 (Waksman: The Actinomycetes, Vol. 2/The Williams Wilkins Company, Baltimore/, 1961, pp.222-223).

The microorganisms utilized in the process of the invention are cultivated by methods generally applied for the cultivation of Actinomycetes. Cu Itivation is performed most preferably in submerged cultures. The culture medium may contain assimilable carbon and nitrogen sources, inorganic salts and other additives (biotic substances, antifoaming agents, etc.) dissolved or suspended in water.

As carbon source e.g. malt, glucose, maltose, sucrose or other sugars, fats, fatty acids, amino acids, and peptides can be utilized. Of the assimilable nitrogen sources e.g. nitrogen compounds of microbial, vegetable or animal origin, and/or inorganic nitrogen compounds, such as yeast extract, pepton, meat extract, hydrolysed caseine, soybean meal, corn steep liquor, ammonium salts and nitrates are to be mentioned. From economical aspects it is most preferred to apply culture media containing glucose as carbon source and soybean meal and corn steep liquor as nitrogen sources. The components of the culture medium are dissolved or suspended in tap water and the resulting mixture is sterilized for 20 to 45 minutes at 121"C. When reducing sugars are applied in the culture medium, they are sterilized preferably separately.

The strains according to the invention are cultivated at 22 to 370C, preferably at 28"C. The microorganism is not too sensitive to the rate of aeration and stirring. A culture capable of quick conversion can be obtained by aerating the culture, filled into a 100-litre fermenter, at a rate of 100 litres of air/minute and stirring it at 400 r.p.m.; similar results are obtained, however, with other aeration and stirring rates not significantly differing from the above values.

Ths substrates applied in the process of the invention are dissolved in a water-miscible organic solvent which exerts no or only a slight adverse effect on the function of biological systems (such as in ethanol, methanol, dimethyl formamide, dimethyl sulfoxide, acetone ortetrahydrofuran; preferably in methanol), the solution is sterilized by filtration, and then it is added to the grown colture possessing maximum enzymatic activity. The conversion is performed in a vessel equipped with stirring and aerating means at a temperature between 22 to 37"C, preferably at 28-320C.

The conversion process can be separated from the fermentation process when an enzyme preparation is produced first from the culture ready for conversion. The enzyme preparation can be produced by the well known technique of producing "acetone dry powders". The resulting "acetone dry powder" retains about 70% of the original ss-glycosidase and acetylase enzymatic activities. The activity of the enzyme preparation does not decrease upon storage for one month in a refrigerator (4 to 60C).

At the end of the conversion the secondary glycosides are extracted from the fermentation broth with a water-immiscible organic solvent (such as chloroform, dichloroethane, ethyl acetate, methyl isobutyl-ketone, etc.), preferably with chloroform.

The secondary glycosides bound to the mycelia can be dissolved with a water-miscible organic solvent, such as ethanol, methanol, acetone, etc., preferably with methanol. The progress of the conversion can be monitored by thin layer chromatography.

The process of the invention is elucidated in detail by the aid of the following non-limiting Examples.

Example I Sterile physiological saline solution is poured onto a one-week culture of Streptomyces griseolus MNG 168 (slant culture on potato-glucose-agar), and the agarsurface is scraped with a sterile loop. The sus pension obtained from the scraping is applied to inoculate a culture medium (one litre, filled into a 3 litre flask) of the following composition: glucose: 2%, soybean meal: 1%, corn steep liquor: 0.5% (wet weight, calculated for 50% dry substance content), pepton: 0.2%, calcium carbonate: 0.3 /O. The culture is cultivated on a shaker at 28"C for 36 to 48 hours.

The contents of three flasks are combined and used to inoculate a broth filled into a pilot-plant fermenter of 100 litres net capacity, equipped with a stirrer.

Edible oil is added to the broth as antifoaming agent, and fermentation is performed at 28"C at an aeration rate of 100 litres/min. and a stirring rate of 400 r.p.m.

After 36 to 48 hours of fermentation the culture pos sesses the required enzymatic activity.

500 g of lanatosideA are dissolved in 10 litres of hot methanol, the solution is filtered through a Seitz-filter, and the filtrate is introduced into the stir red culture under sterile conditions. The progress of the conversion is monitored by thin layer chromatography (Kieselgel G plates; applying a 55:35:10 mixture of ethyl acetate, cyclohexane and abs. ethanol as solvent). After 48 to 60 hours offer- mentation the substrate disappears and only the spot of the resulting secondary compound, i.e.

digitoxin, can be observed on the chromatographic plate. At the end of the conversion the fermentation broth is filtered. The mycelia are admixed with 80 litres of methanol, the suspension is stirred for 2 hours, thereafter it is filtered, and this operation is repeated once again with 80 litres of fresh methanol.

The The aqueous methanol solutions are combined, evaporated to a final volume of 5 litres in vacuo below40"C, and the resulting concentrate is main tained at 0 C for 24 hours. The separated crystals are washed with 70% aqueous methanol. The resulting crystalline substance is termed in the following as "crude product 1".

The mother liquor and the washing liquid used in the crystallization step are combined, extracted thrice with 1/2 volumes of chloroform each, and the extracts are evaporated to dryness. The resulting substance is termed in the following as "crude pro duct II".

The filtrate is extracted thrice with 1/2 volumes of chloroform each, and the extracts are evaporated to dryness. The resulting substance is termed in the following as "crude product Ill".

Crude products II and Ill are combined, dissolved in 5 litres of 50% aqueous methanol, and the solution is shaken with 2 litres of petroleum ether. The pet roleum ether solution is discarded and the aqueous methanol phase is evaporated to dryness. The result ing substance is termed in the following as "semi finished product ll-lll".

Crude product I is admixed with semi-finished pro duct lI-Ill, the mixture is pulverized, dissolved in 5 litres of a 1:1 mixture of benzene and methanol, decolourized with charcoal under stirring, and finally filtered through an aluminium oxide layer. 2.5 litres of tap water are added to the filtrate under stirring, and the resulting mixture is allowed to crystallize at 0 C for 48 hours. The benzene phase is discarded, the crystals are filtered off, washed with benzene and then with 50 /O aqueous methanol, and dried at 50"C until constant weight. 310 g of digitoxin are obtained with a purity grade of at least 97%. When combining and processing the mother liquor and the washing liquids further 25 g of digitoxin can be obtained, thus the total yield is 85%.

Example 2 The microorganism culture ready for conversion, prepared according to Example 1, is filtered, the mycelia are washed with water, and then put into -20 C acetone just covering the mycelia. The suspension is stirred at -20"C for one hour, thereafter it is filtered cold, and the mycelia are dried at room temperature until constant weight. In this way 70 g of an enzyme preparation are obtained.

70 g of this enzyme preparation, stored for a period of maximum one month, are suspended in 80 litres of an 0.01 molar phosphate buffer (pH = 7) under stirring, and the hot, concentrated methanol solution of 350 g of lanatoside A is added to the stirred sus pension. The mixture of the enzyme preparation and the substrate is stirred at 28"C, and the progress of the conversion is monitored by thin layer chromatography. After 48-60 hours the substrate disappears and only the spot characteristic of digitoxin can be observed in the chromatogram. The mixture is processed as described in Example 1 to obtain 236 g (85% of digitoxin.

Example 3 5 g/litre of lanatoside B are added to the fermentation broth prepared as described in Example 1 orto the enzyme preparation suspended in phosphate buffer, prepared as described in Example 2. The resulting gitoxin is separated from the medium in a manner known per se. 3.37 g (85%) of gitoxin are obtained.

Example 4 One proceeds as described in Example 3 with the difference that 5 g of lanatoside C are applied as starting substance. 3.34 g (84%) of digoxin are obtained.

Example 5 One proceeds as described in Example 3 with the difference that 5 g of acetyldigitoxin are applied as starting substance. 3.95 g (85%) of digitoxin are obtained.

Example 6 One proceeds as described in Example 3 with the difference that 5 g of acetylgitoxin are applied as starting substance. 3.92 g (84%) of gitoxin are obtained.

Example 7 One proceeds as described in Example 3 with the difference that 5 g of acetyldigoxin are applied as starting substance. 3.90 g (84%) of digoxin are obtained.

Example 8 One proceeds as described in Example 3 with the difference that 5 g of deacetyl-lanatoside A are applied as starting substance. 3.54 g (84%) of digitoxin are obtained.

Example 9 One proceeds as described in Example 3 with the difference that 5 g of deacetyl-lanatoside B are applied as starting substance. 3.55 g (84%) of gitoxin are obtained.

Example 10 One proceeds as described in Example 3 with the difference that 5 g of deacetyl lanatoside C are applied as starting substance. 3.51 g (83%) of digoxin are obtained.

Example Ii One proceeds as described in Example 1 or 2, with the difference that a mixture of lanatoside A, lanatoside B, lanatoside C, acetyldigoxin, acetylgitoxin, acetyldigoxin, deacetyl lanatoside A, deacetyl lanatoside B and deacetyl lanatoside C is applied as substrate, instead of pure lanatoside A.

The end product is a mixture of digitoxin of the "A" series, gitoxin of the "B" series and digoxin of the "C" series, containing these compounds in a ratio corresponding to that of the compounds of "A" series (lanatoside A, deacetyl lanatoside A, acetyldigitoxin), "B" series (lanatoside B, deacetyl lanatoside B, acetylgitoxin) and "C" series (lanatoside C, deacetyl lanatoside C, acetyidigoxin) present in the starting substrate. The conversion proceeds with a yield of 84%.

Claims (8)

1. Aprocessforthe preparation of secondary digitalis glycosides of general formula (I),

(wherein R1 and R2, which may be the same or different, each represents a hydrogen atom of a hydroxy group) which comprises fermenting a primary glycoside of formula (II),

(wherein R, and R2 are as defined above, R3 represents a hydrogen atom or an acetyl group and R4 represents a hydrogen atom or a D-glucose unit, with the proviso that at least one of Ra and R4 is other than a hydrogen atom) with a culture of a Streptomyces griseolus strain deposited at the Hungarian National Collection of Microorganisms (Budapest, Hungary) on 6th December 1977 under No.MNG 168 our a mutant thereof or with an enzyme preparation produced therefrom and separating the product of formula (I) thus obtained from the fermentation broth.

2. A process as claimed in claim 1 wherein the primary glycoside of formula (II) is fermented with a submerged culture of the Streptomyces griseolus.

3. A process as claimed in claim 1 wherein the primary glycoside of formula (II) is fermented with an immobilized enzyme preparation of the Streptomyces griseolus.

4. A process as claimed in any one of claims 1 to 3 wherein the fermentation is effected at a temperature of from 28 to 32"C.

5. Aprocessforthe preparation of secondary digitalis glycosides of general formula (I) as claimed in claim 1 substantially as herein described.

6. A process for the preparation of secondary digitalis glycosides of general formula I as claimed in claim 1 substantially as herein described in any one of Examples 1 to 11.

7. Secondary digitalis glycosides of general formula (I) as defined in claim 1 whenever prepared by a process as claimed in any one of claims 1 to 6.

8. Each and every novel method, process, composition and product herein disclosed.