DD152581A1 - PROCESS FOR PREPARING 20-CARBOXYPREGNA-1,4-DIEN-3-ON - Google Patents

Abstract

The purpose of the microbiological process for the production of 20-carboxypregna-1,4-dien-3-one is to produce this compound in an economically simple manner, taking into account the requirements for reusability of the biomass and simplifying the sterility conditions. By degrading the C & ind17! Side chain of sterols and their derivatives with immobilized microorganisms, 20-carboxypregna-1,4-dien-3-one is obtained as the final product. Cholesterol, ergosterol, or sitosterol are exemplified by a culture of Nocardia erythropolis, is immobilized by adsorption on Festeger Traeger, by crosslinking of polyelectrolytes by polyvalent cations or by combined methods, fermented by a Bath process as a prerequisite for a reactor. In this case, the inhibitor required for the selective degradation of the side chain is used particularly advantageously as part of the matrix. The end product is suitable for the synthesis of pharmaceutically valuable steroids of the C & ind21! Series such as corticosteroids and has even pharmacologically interesting properties.

Description

Dr ^ Peter Atrat

Dr · Clare Hörhold

Dr. Michael Buchar

Dr · Kira A © Koschtscheenko

Title of the invention

Process for the preparation of 20-carboxypregna-1,4-diene »3- * on

Field of application of the invention

The invention relates to a process for the preparation of 20 ** Carboxypregna-1,4-diene «* 3 ** on from sterols by raikrobielle Um-» conversion, 20-Carboxypregna-i, 4 ™ diene-3-On can by known methods The substance itself causes an inhibition of estrogen biosynthesis. "Such inhibitors may be used, for example, in gymnastic treatment of breast cancer and for the control of pertility.

Characteristic of the known technical solution

It is known that sterols such as, for example, cholesterol, sitosterol, stigmasterol, campesterol and ergosterol, as well as derivatives of this class of substances, are tolerated by microorganisms of the genera Arthrobacter, Brevibacterium, Noeardia _f Myoobotovirus, Gorynebacteriuin, and the like under suitable conditions in 20-carboxypregna. 1,4-dien-3 * -On »The problem here is to choose the process conditions so that only the side chain of the sterols is degraded by the microorganisms, but the further ring skeleton degradation is prevented. In the known methods, this problem is essentially solved by three principles. First, by the use of mutants which are unable to dissolve silicates; secondly, by the addition of suitable inhibitors of enzymes, such as B *. Thirdly, heavy metal compounds and cliclimates and, thirdly, by using correspondingly substituted sterols which are not substrates of the Ln-S ^ iTiO, which are known from US Pat. Initiate ring skeletal removal * Allan procedure

It is common that the sterols used as starting material are added to a growing or resting discontinuous culture of a microorganism whose cells are present in non-stabilized form in the fermentation medium.

The fermentation route under these conditions has the following disadvantages: the need for sterile fermentation in the nutrient-rich fermentation medium, fluctuations in the content and activity of the inductive enzyme systems as a function of changes in the fermentation medium, by decreasing the inhibitor or inducer concentration Nutrient consumption, increase of the ion concentration in the required pH regulation u. As a result of the defects indicated, the reproducibility of the results is greatly impaired by the known methods. In addition, the ability of the microorganisms to degrade the sterol side chain is generally greatly reduced after the first fermentation, so that the biomass grown can be used practically only for one or at most for a second fermentation.

Object of the invention

The object of the present invention is the improved production of 20-carboxypregna-1,4-dien-3-one from sterols according to an economically favorable process.

Explanation of the essence of the invention

The invention has for its object to provide a technologically simple microbial process for the preparation of 20-Carboxypregna-1,4-dien-3-one from readily available sterols, which essential shortcomings of the known methods, in particular the discontinuous mode of operation, the heavy exhaustion of Biomass after a. Fermentation and risk factors such as sterility limits or avoids sprouting problems during cultivation and fluctuations in the performance of the culture used for fermentation.

According to the invention, this object is achieved in that one

Side chain degradation capable of microorganisms such. B. from the genera Arthrobacter, Brevibaterium, Corynebacterium and preferably from the genus Nocardia according to methods known per se immobilized by the cells to solid support such . Cellulose or cellulose derivatives or by cross-linking of polyelectrolytes, such as eg alginates with more than one

valent cations, such as, for example, include Al or Co, and therewith fermented with repeated use of the immobilized biomass, sterols or, respectively, derivatives of sterols which have the following general formula:

* 4

wherein R is the hydrocarbon radical containing 8 to 10 carbon atoms of a sterol which may have an additional double bond in the 22-position, the bond . ». о can be a single "double bond and E _{2 is} hydrogen or an organic or inorganic acid radical

The method is advantageously carried out as follows: The immobilized cells of a bacterial strain, such as B. Nocardia erythropolis, in the depository at the Central Institute for Microbiology and Experimental Therapy of the Academy of Sciences of the GDR, 69 Jena, Beutenbergstr »11, under the number IbCET 7185 is taken up in 500 ml shake flask in 50 ml of 0.01 M phosphate buffer pH 8 (biomass 3.5 g wet weight) with 25 mg of the sterol or 'of a sterol derivative, dissolved in dimethylformamide, added

2+

In time, a suitable inhibitor, preferably Cc-ions added in a concentration which is chosen so that not the side chain degradation, but the degradation of the ring skeleton is prevented This is done by adding a cobalt sols in a known manner, but particularly advantageous ™

2+

in that Co z »B. is used directly as cobalt alginate or» polyacrylate as part of the matrix. «The fermentation is carried out at 28 ⁰ O for 24 h according to the principle of

ma performed with shaking · The workup

of the fermentation batch is done in a simple manner as follows: The immobilized cells are separated by filtration or decantation from the fermentation medium _t which was acidified to obtain the 20-Carboxypregna-1, 4-dien-3-one in a known V / else with dilute mineral acid and a suitable solvent such as ethyl acetate or chloroform is extracted. From the concentrated extract a pure product is obtainable by known methods.

The separated immobilized cells can be repeatedly used without noticeable loss of activity in the batch Batsch process, which is the most important requirement for continuous operation in the reactor is met. It is surprising to the person skilled in the art that a complex enzymatic process which is extremely oxygen-intensive, as it is the degradation of the side chain of sterols, can be carried out with microorganisms which are in the immobilized state, especially because only simple transformations have hitherto been carried out in the steroid region how reduction, dehydration or hydroxylation with immobilized microorganisms has become known «

The immobilization of the microorganisms now makes it possible to avoid the disadvantages of the known processes. The microorganisms can be immobilized at the time of a favorable physiological state and thus stabilized. Stabilization of a particular physicochemical state is likewise favored. This provides conditions for carrying out the microbial side kinetin degradation in a continuously operating reactor, which makes economic use of the biomass more economically, sterility problems during the fermentation are immaterial and the reproducibility of the process with respect to yields of 20-carboxypregna -1,4-dien-3 «is guaranteed over a long period of time.

embodiments

1.2g Foeardia erythropolis, IMET 7185 (wet biomass), are dissolved in 200 ml of phosphate buffer. 0.01 M, pH 8 at room temperature for 1 h with 4 g of DEAE-cellulose stirred After repeated decantation and suction baw »centrifugation, the entire batch is taken up in 50 ml of phosphate buffer 0.01 M, pH 8 and with 25 mg of cholesterol, which dissolved in 1 ml of dimethylformamide is added soY / ie with 3 ml of 0.1 M Co (HO ₃ ) ₂ at pH 8. The fermentation is 24 h at 28 ⁰ C with shaking carried out in a 50fr ml flask · After conversion, the fermentation solution is sterilized by filtration or · centrifuging nevertheless immobilized cells separated and acidified with HCl to pH 2 to 3 · The 20-Carboxypregna- 1,4-diene-3-one is extracted in a known manner with chloroform and worked up.

Yield: 60 to 70 # d »Cn. The adsorbed cells are used repeatedly without noticeable loss of activity.

2 * 2 g wet biomass Noeardiiä erythropolis, IME3? 7185, are suspended in 5 ml of phosphate buffer (0.01 M, pH 8) and mixed with 5 ml of a 2% NH 3 solution of alginate in phosphate buffer. With vigorous stirring, the suspension is poured into a pH of 20 adjusted to pH 5-7 The resulting particles are separated by repeated washing and decanting or aspiration of free cells and, as in Example 1 ml. 0.1 M Al ₂ (S (O) and 20 ml 0.1 M 'Co (NOo) ₂ added dropwise

24 ·

described, but without further addition of Co, used. Yield of 20-Carboxypregna ~ 1,4-diene-3 ~ On: 10 to 20 <f> d. lh ·

The biomass adsorbed on DEAS cellulose according to Example 1 is suspended in 5 ml of phosphate buffer, stirred with 10 ml of 2% NH 4 Cl-alginate solution and at pH 5 "× 7 in a mixture of 20 ml of 0.1 M Al ₂ ( SO ₄ ), and 20 ml of 0.1 M Co (N0 ₃ ) _? Are added dropwise with vigorous stirring. "" The resulting particles are ground in the same way, described in Example 2, to give an

brought.

Yield of 20 % carboxypregna-1-diene-O-on: 40 to 50 % of theory,

4 "2 g of moist biomass Nocardia erythropolis, IMET 7185, are stirred with 1 ml of phosphate buffer 0.01 M, pH 8, and 10 ml of 2-fold Жa-polyacrylate are suspended. The resulting mixture is poured into a solution of 20 ml of 0 with vigorous stirring. 1 M Al ₂ (SO ₄ ) and 20 ml of 0.1 M Co (NO ₃ ) are added dropwise. The particles are treated and used as described in Example 2. Yield of 20-carboxypregna-1,4-diene 3-on: 5 to 10 $ d »Th.

Claims (5)

He is looking forward to it The procedure for the preparation of 20 ~ Carboxypregna «1,4-аіеп-З-оп. Sterins through microbial transformation? characterized by the fact that microorganisms capable of side chain degradation of sterols are immobilized by methods known per se and thus sterols or derivatives of sterols are fermented in the presence of an inhibitor, vde Co *, in the case of repeated use of the immobilized biomass. 2 * Method according to item 1, characterized in that one uses microorganisms from the genera Arthrobacter, Bacillus, Bre ·· vibaeterium, Corynebaeteriuin, Mierobacteriuio, Hocardiaj Protaminba-cter and Mycobacterium * 3 »A method according to the points 1 and 2, characterized in _t, DSSS microorganisms by adsorption on solid '= carrier by Vernetzimg of polyelectrolytes or other methods immobilized * 4 * Method according to points 1 to 3, characterized in that the inhibitor "eg Co" is a direct constituent of the carrier, for example, as co-alginate, co-polyacrylate, etc. " 5 * Method according to items 1 to 4 $, characterized in that sterols or "their derivatives of the general formula are used" J **? wherein R. represents the 8 to 10 carbon atoms-containing hydrocarbon radical of stering and "may have an additional double bond position, the binding £ __» in 22 __ · - & may be a single or double bond and Rg represents hydrogen or "an inorganic or" organic Saarereot stands «