DD146051A5 - MICROBIOLOGICAL PROCESS FOR THE PRODUCTION OF SEKUNDAERIC GLYCOSIDES - Google Patents

Abstract

The invention relates to a novel process for the microbiological production of secondary digitalis glycosides (digitoxin, gitoxin and digoxin) from the corresponding primary glycosides (lanatoside A, B and C) and / or from the 3 '' '- acetyl derivatives of the secondary glycosides (acetyldigitoxin etc.); These starting materials are fermented with the submerged culture of the new microorganism strain Streptomyces griseolus MNG 168 or with an enzyme preparation prepared from this culture.

Description

12 509 58

- I-

Microbiological process for the production of secondary glycosides. ..

let the invention:

The invention relates to a microbiological "process for the preparation of secondary digitalis glycosides of

1 2

general formula I in which R and R each represent a hydrogen atom or a hydroxyl group

As is known, in the glycoside mixture obtained from the leaves of Digitalis lanata, besides the primary glycosides or lanatoside compounds (lanatoside A, B and C) and the secondary glycosides (digitoxin, gitoxin and digoxin), the acetyl derivatives of the latter are also present (acetyldigitoxin, acetylgitoxin and acetyldigoxin) ,

When working up the leaves by any known methods such mixtures of glycosides are always obtained, of which not all components can equally be used as pharmaceuticals. It is therefore appropriate to search for methods by which the individual components of the glycoside mixture in the for therapeutic purposes on best suitable forms can be brought.

The compounds of the lanatoside series differ from the secondary glycosides as follows: Whereas in the secondary glycosides only three digoxotoxic units attach to the aglycone part of the molecule, the compounds of the lanatoside series contain, in addition to these sugars, a glucose bound to the third digitoxose , where the 3 ' ^! 'Hydroxyl group of the third digitoxose is esterified by acetic acid'

.To split off the glucose in ß-position or the 3 ' ^! 'Acetyl group of lanatosides are already several methods known. Thus Z ₀ B. the 3 '''' acetyl group can be cleaved off by very mild alkaline treatment «

In the case of acidic or alkaline hydrolysis of lanatoside compounds, the bond present between the aglycone and the first digitoxase is generally also split; thus, not only the desired termic cleavage is achieved, but also the further cleavage, which is undesirable here to Geninen (Digito.xigenin, Gitoxigenin and Digoxigenin), - so that in this way secondary Herzglykoside from Lanatosiden can not be received The desired secondary Herzglykoside can be made from Lanatosiden rather only by complicated chemical ways. ·

Although it can be achieved by an appropriate Ferrnentierung the leaves of Digitalis lanata that the compounds of the secondary series are enriched in Glykosidgemisch; but in this Pail the lanatoside C valuable as a medicine can not be obtained from the glycoside mixture.

Microbiological processes have been known for a long time to cleave compounds of the lanatoside series. Thus, terminal glucose can be cleaved off with the help of mold fungus cultures or β-glucosidases prepared therefrom. A ₀ Stoll and his coworkers first have the lanatoside-cleaving activity of mold fungi Heid ₁₁ Chim, Acta, A, 397-401 (1951)) "The terminal glucose of lanatosides could be isolated from the culture of various mold fungi (mostly of Aspergillus species). be split off by treatment with acetone dry powder. Thus, it is known (CS-PS 108 046) 5 to use an enzyme preparation obtained from Aspergillus orizae for the production of acetylated secondary glycosides. However, the acetylase activity of the mold fungi cultures is not comparable with their β-glucosidase activity; therefore, this method is generally used only for the preparation of secondary series acetylated compounds.

Object of the invention

The aim of the present invention is the preparation of compounds of the secondary series of lanatosides, with the help of such microorganisms which in addition to the ß-glucosidase activity also have a corresponding acetylase activity _f so that the substrate 'at high speed and in high concentrations in such a way that both the glucose and the acetyl group are split off in one and the same

Furthermore, it is still required of the microorganisms usable for this purpose that the two activities mentioned, i. the ß-glucosidase and the acetylase Akti-Vitat, should be practically the same strength and that during the reaction, no intermediates (acetyl derivatives of the secondary series, or »desacetyl-lanatoside, also known as purpurea-glycosides) to accumulate. It is thus desired that the microorganisms to be used should be capable of producing secondary glycosides from both the acetyl derivatives of the secondary glycosides (with the benefit of acetylase activity) and from the purpurea glycosides (where β-glycoside activity is the key Role plays).

Explanation of the Weseris of the invention;

It has now been found that it is possible to achieve the results corresponding to the above requirements with the aid of a gene belonging to the genus Streptomyces, characterized by an outstandingly high enzyme activity distinctive microorganism strain. This new microorganism strain was identified as Streptomyces griseolus and deposited by the notifier in the Hungarian National Microorganism Strain Collection of the Hungarian National Hygiene Institute under the Ur * MNG 168.

The invention is therefore a novel process for the microbiological production of secondary digitalis glycosides of the general formula I defined above, wherein

1 2

R and R represent hydrogen atoms or hydroxyl groups, which is characterized in that primary glycosides of the general formula II in which R and R ^{2 are} each one. A hydrogen atom or a hydroxyl group ₉ R ^ is a hydrogen atom or an acety! Group and R is a hydrogen atom or a D-glucose group, but R and R can not both simultaneously be hydrogen, with a

- 5

submerged culture of the strain Streptomyces griseolus ItETG 168 or with an enzyme preparation prepared from such a culture, and the resulting secondary glycosides are isolated from the fermentation fluid 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 with the enzyme preparation bound in this way-carried out

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

Morp ^ oJ ^ o ^ iej ^ The spore carriers show sympodial branches, have just endings; Spirals or slings do not occur. In the light microscope, the spores are egg-shaped, their size is 0.7-0.9 χ 0.9-1.1, u,

Growth on different nutrient media:

§ ^ ££ ^ ^ §X2 zi3i £ Eä £ z ^ £ Si! L £. Growth colorless, later gray; the aerial mycelia are thin, white, later olive green. Weak brown pigment diffuses into the agar.

Tyro sin agar: melanin negative _o The aerial mycelia and the entire growth show loose structure ,, A light brown soluble pigment diffuses into the agar *

Mycalidae, compact, strong growth, gray at first, later brownish. "The aerial mycelia are thin, dusty, light gray, no soluble pigment."

Bennett-Asar: growth of cream, darkens with time to brownish. The aerial mycelia are thin but compact

with dusty structure and blue-gray color o

Glucose-Asparaffin-Aprar:, V / aft around gray, dark gray, later in places black »Air mycelia are only partially formed, with mouse-gray color. Little brown soluble pigment diffuses into the agar.

Bouillon ÄffarI ₁ Growth thick, gray, becomes wrinkled. Neither aerial mycelia nor soluble pigment are formed.

Calcium malate ^ A ^ ar ^ Rapid growth, light gray, later dark gray The aerial mycelia are blue-gray, sometimes olive-green in spots * Very little brownish, soluble pigment diffuses into the agar.

Potato Glucose Agari Growth shows medium

- ~ Tt ~ - ~ - · ---- "-. °

sehwindigkeit but spreads greatly from, with brown color * The aerial mycelia are velvety, blue-gray in the middle, crawling on the edge of the colony spiderweb-like apart _β Little, DIFFUN light brown soluble pigment ~. dig into the agar _e

Maismeh1-Ag arj _ Growth light brown, the aerial mycelia are dusty, thin, at first light gray, later bluish gray. No soluble pigments

& ^ £ ΐ ^ ΐ ^ 2Λ ^ 3 ^ Αβ2 ^ 1 ,, Growth gray, greyish brown, last black, strongly wrinkled ,, The aerial mycelia are gypsum-like, dusty, evenly greyish-gray. V / enig, but pronounced brown soluble pigment diffuses into the agar »

^ ^The growing

Ground mycelium is light brown, the aerial mycelia are thin, light gray, gypsum-like * dry, producing a pale brown soluble pigment «

- 7 - « ^i: .4 P ^ II

Potato slice; Strong and rapid growth, with rough surface; gray at first, later black. "The aerial mycelia are ashen gray, the soluble pigment brown.

Löffler'scher curdled whey-Hähr ^^ brown growth, strong proteolytic activity,

Cellulose as the sole carbon source; good growth. Hitrat re d uk ti ο'η: _ι positive »Gelatin liquefaction: positive.

Milk strong coagulation and strong peptonization are observed. , -.... ·.

zi ^ Säiü. Growth wrinkled, olive green; Aerial mycelia light gray; weak beta ~ hemolysis _o

AssimilationvonKohlenstoffquellen:

D-glucose:. , + D ~ sorbitol: weak

D-galactose: ++ soluble starch: ++

Sucrose: + D-sylose +

Maltose :. <+ L-Rhamnose:. -i-

Lactose: + galactites

Raffinose: "+ D-mannitol; +

L-sorbitol: - inositol: +

Cellobiose: + D-fructose; +

Trehalose: ++ D-mannose: + glycerin

Due to the above characteristics of the new strain to the genus Streptomyces griseolus (Waksman) Waksman et Henrici heard in 1948 .. (VGI Waksman. .- The Actinomyce'tes, Voie 2, The Williams Wilkins Company, Baltimore _1963ij?

P. 22-223) ·

- 8th -

For culturing the microorganisms used in the method according to the invention, the methods generally used in the cultivation of ray fungi can be used. Cultivation is most beneficial in submerged culture. The culture medium suitable for cultivation may contain dissolved or suspended sources of carbon and nitrogen which can be assimilated by the fungi, as well as inorganic salts and other customary additives (biosubstances, antifoam agents, etc.). - ·

As a carbon source can malt, glucose, maltose _? Sucrose or other sugars, fats, fatty acids, amino acids and peptides are used as assimilable sources of nitrogen nitrogen compounds microbial, animal or vegetable Herkunt and / or inorganic nitrogen compounds ^ such as yeast extract, Peptcfn, meat extract, 'hydrolysiert.es casein, soybean meal, corn' spring water, ammonium salts and nitrates. "

Taking into account also economic aspects can be used most advantageously glucose as carbon source and soybean meal and / or corn steep liquor · as a nitrogen source in the culture medium "The components of the medium are dissolved in tap water or" suspended and sterilized together at 121 ⁰ C for 20 to 45 minutes , with the exception of the reducing sugars, as these are expediently sterilized separately.

The cultivation of the microorganisms used in the process according to the invention can be carried out at 22 to 37 ° C, preferably at 28 C. These microorganisms are not particularly sensitive to the aeration and the number of revolutions of the agitator; In general, good cultures ready for rapid reaction can be obtained in a 100-per-minute aeration rate of 100 l / min with a stirring speed of 400 rpm

become; however, similar good results could be achieved, even with not much deviating other aeration speeds and agitator revolutions.

The substrates to be reacted 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 harmful to biological systems, advantageously in methanol, the solution is sterilized by filtration and then The reaction is carried out in a ventilated vessel equipped with a stirrer at 22 to 37 ° C., preferably at 28 ° C. to 32 ° C.

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

After completion of the reaction, the present in the fermentation liquid secondary glycosides with a V / ater-immiscible organic solvent (öloroform, dichloroethane _f ethyl acetate ,. Methylisobuty! Ketone uswo), especially with chloroform, 'from the fermentation liquor extracted The detachment of the Mycelium-bound secondary glycosides can be facilitated by the addition of water-miscible organic solvents (methanol, ethanol, acetone, etc.), especially methanol. During production, the progression of the

Implementation be followed by means of thin-layer chromatography.

Out of hand. ice.p : ± elej__

The practice and the details of the invention. Methods are illustrated by the following examples. ·

Example _i 1

A monochromatic culture of the strain Streptomyces griseolus MG 168 grown on an oblique potatoglucose agar is sprayed with sterile saline and the surface of the agar is scratched with a sterile platinum loop. The microorganism suspension obtained in this way is inoculated with an inoculum medium of the following composition: 2 % Glucose, 1 % soybean meal, 0.5% llaisquellwasser (wet weight, calculated on 50 % dry substance), 0.2% peptone, 0.3 % calcium carbonates total volume 1 liter sterilized in 3 1 Erlenmeyer flasks. The culture is shaken on a shaker at 28G for 36 to 48 hours. Three such inoculum cultures are prepared in parallel and the 3 liter inoculum cultures thus obtained are seeded in a 100 liter perforating device equipped with an agitator Injected medium of the same composition used during the fermentation * During the fermentation, the medium is stirred at 400 U / min and aerated with 100 l / min air flow. As an anti-foaming agent edible oil is used "Fermentation is continued C 36 to 48 hours at 28 ⁰ ,, to achieve the required to implement Enz.ymaktivität * '*''

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500 g of lanatoside A are dissolved in 10 l of hot methanol, the solution is filtered off through a Seitz filter and then added under sterile conditions and stirring the perraentation liquid prepared on the above Y / eise The passage of the Unisetzung is using thin-layer chromatography (on Merck "silica gel G · ¹¹ plates, eluent 55: 35 ^ 10 mixture of ethyl acetate, cyclohexane and abs ,, ethanol) tracked The stain of the substrate disappears after 48 to 60 hours and on the plate only the resulting compound of the secondary series, the digitoxin is visible « After complete reaction, the fermentation liquid is filtered * The mycelium is stirred with 80 l of methanol for 2 hours, filtered and stirred again with additional 80 l of methanol for 2 hours The methanolic-aqueous washings are combined, concentrated in vacuo at a maximum of 40 ^° C. to 5 liters and allowed to crystallize at ⁰ ° C. for 24 hours. "The crystals obtained washed with 70% aqueous methanol (crude product I), the mother liquors and washing liquids of the crystallization are extracted three times with 1/2 vol of ₀ chloroform and the combined extracts evaporated to dryness (crude product II) _e

The filtrate of the fermentation liquid is also extracted three times with 1/2 _{t of} VoI ₆ chloroform and the combined extracts are evaporated to dryness (crude product III).

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

The crude product I is combined with the semi-finished product H-III and pulverized in 5 liters of a 1, 1 mixture

dissolved by methanol and benzene »The solution is stirred with activated charcoal and filtered through an aluminum oxide layer» The filtrate is added with stirring with 2.5 liters of tap water and allowed to stand at O ⁰ G 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 at 50 ⁰ C to constant weight dried on ... this way, 310 g digitoxin (purity at least 97 % ) received the mother liquors and, washing liquids of

crystallization is combined and the above procedure worked up to give a further 25 g digitoxin * total yield; 85 %. ,

Example 2.

Sine obtained according to the manner described in Example 1, for the implementation already suitable culture of the microorganism strain is filtered off, the mycelium washed with water, then placed in such an amount of cooled to -20 C acetone, that the liquid mycelium over-defect "The MyzeX stirred in the acetone kept at -20 C for one hour, then filtered cold and dried at room temperature to constant weight * In this way, 70 g of dry enzyme preparation 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 5 then with continued stirring, the hot concentrated methanolic. Solution of 350 g lanatoside A added "The mixture is further stirred at 28 ⁰ O and follows the progress of the reaction by thin layer chromatography" laughing 45 to 60 hours, the spot of the Ausgangsatoffes no longer shows, it is only the spot of digitoxin visible The reaction mixture is worked up in the manner described in Example 1; it

13

--13- Z 1 4- 8 Pi

236 g digitoxin (85% of theory) are obtained. Example ^ ₁

5 g of Lanatoside B are added to 1 liter of enzyme-active fermentation medium obtained according to Example 1. After completion of the reaction, the reaction mixture is worked up in the manner described; 3.77 g of gitoxin (85 % of theory ) are obtained.

Similar results are obtained when adding the lanatoside B to 1 liter of Example 2 obtained, suspended in phosphate buffer solution enzyme preparation.

Example. 4,

5 g lantoside G are treated according to Example 3; will be 3.34 g of digoxin (84% _o Th.) obtained "

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

5 g of acetylgitoxin are reacted in the manner described in Example 3; there are obtained 3.92 g (84% d ₀ Th.) gitoxin.

5 g of acetyldigoxin are reacted according to the described in Example 3 enclosed manner; 3 _s 90 g of digoxin (84 % d ₀₎ are obtained.

"14"

Example · ..... 8 '·'

5 g of desacetyl-lanatoside A are reacted to the method described in Example 3; 3,54.g digitoxin (84 % d, Th.) are obtained,

Example 9

5 g of desacetyl-lanatoside B are reacted according to the manner described in Example 3; are obtained (84% do Th ₀₎ 3.55 g gitoxin.

Beis piel 10

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

Example .11

It is worked in detail according to the manner described in Examples 1 or 2, with the difference that instead of pure Lanatosid A any mixtures of Lanatosid'A, Lanatosid B, Lanatosid 0, desacetyl-lanatoside A, desacetyl-lanatosid B, Desaltyl-lanatoside Cj "acetyldigitoxin, acetylgitoxin and / or acetyldigoxin are used as starting materials." Final products "corresponding mixtures of digitoxin as series A compound, gitoxin as series B compound and digoxin as series C compound are obtained in each Pail, wherein the proportions of these three secondary glycosides respectively corresponding to those proportions in which the compounds of the series A (Aj lanatoside desacetyl lanatoside A, acetyldigitoxin) _s ~ Ver the _- compounds of the number B (B lanatoside, desacetyl lanatoside B,

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Acetylgitoxin) and the compounds of series C (Lanatosid C, Desacetylvlanatosid 0, acetyldigoxin) were present in the starting material glycoside mixture. The yield of secondary glycosides is generally 84 % <,

- .16 -

al

'Cl %% I

OH

-Ah

ν μ * p κ- stn

Claims (1)

-IS- Invention claim Process for the microbiological production of secondary digitalis glycosides of general formula 1, 12 in which R and R each represent a hydrogen atom or a hydroxyl group, characterized in that primary diglysis glycosides and / or acetyl derivatives of secondary digitalis glycosides of the general formula II 12 3 in which R and R have the above meanings, R is a hydrogen atom or an acetyl group and R is a water Mean that R and R can not both be hydrogen at the same time, are fermented with a submerged culture of the strain Streptomyces griseolus MNG-168 or with an enzyme preparation prepared from such a culture and the secondary glycosides obtained from the fermentation liquid in isolated in a known manner. Process according to item 1, characterized in that the enzyme is fixed on the microorganism cells and the hydrolytic reaction is carried out with the enzyme preparation fixed in this way. ^ ... pages formally