RU2731712C2 - Microbiological method of producing 11α-acetoxyprogesterone - Google Patents

Abstract

FIELD: chemical or physical processes.SUBSTANCE: invention relates to a method of producing 11 α-acetoxyprogesterone. Method includes transformation of 11 α-hydroxyprogesterone strain of mycelial fungus Aspergillus nidulans, deposited in the Russian National Collection of Industrial Microorganisms under registration number F-1069. For transformation, a spore suspension of the fungus is inoculated into a rocking flask with a growth medium, is grown till the amount of biomass is equal to 1 g/100 ml by weight of dry mycelium while stirring. Then solution 11 is added to the growth medium α-hydroxyprogesterone in dimethylsulphoxide or dimethyl formamide until concentration thereof in the medium is from 2 to 6 % (vol) and to substrate concentration in range of 0.1–1.0 g/l, incubated on a shaker at temperature of 37±0.5 °C for 4±0.5 days in conditions of limited aeration at rate of 90–100 rpm for 216 hours or in conditions of intensive aeration at rate of 240–250 rpm for at least 168 hours. After incubation, the products are extracted by extraction and purified to produce target product 11 α-acetoxyprogesterone.EFFECT: invention ensures selectivity of formation of the end product of not less than 80 %.6 cl, 9 dwg, 4 ex

Description

Technology area

The present invention relates to the field of biotechnology and relates to a new method of using the known strain of the filamentous fungus Aspergillus nidulans VKPM F-1069 for acetylation of steroid hydroxyl-containing compounds, in particular, compounds of the pregnane series (progesterone derivatives) and can be used in industrial biotechnology, as well as in pharmaceutical industry for the production of steroid medicines.

State of the art

The filamentous fungus Aspergillus nidulans is one of the most well-known eukaryotic genetic systems and is widely used as a model system for deciphering the biology of the cell cycle, pathogenicity, drug resistance, human diseases, primary and secondary metabolism of other microorganisms. Despite this, strains of the fungus A. nidulans as steroid-transforming microorganisms have been little studied. There are reports that this species has 11α-monooxygenase activity and is able to transform progesterone with the formation of predominantly 11α-hydroxy-derivative. Thus, M.J. Henry and H.D. Sisler [M.J. Henry, and H.D. Sisler. Effects of sterol biosynthesis-inhibiting (SBI) fungicides on cytochrome P-450 oxygenations in fungi. Pesticide Biochemistry and Physiology, 1984, Vol. 22 (3), 262-275], studying the effect of fungicides that inhibit ergosterol biosynthesis in fungi on cytochrome P450-dependent hydroxylation of progesterone, reported that A. nidulans (Eidam) Winter (strain 003) hydroxylates progesterone at an initial concentration of 100 μg / ml to form a mixture containing 11α-hydroxyprogesterone (50%), 6β-hydroxyprogesterone (5%), 6β, 11α-dihydroxyprogesterone (14%) and unconverted progesterone (31%).

It is also known that the A. nidulans fungus culture (from the collection of the Culture Center of the Laboratory of Microbiological Chemistry, National Research Center, Cairo, local habitat), having steroid-11α-hydroxylating activity, can transform progesterone not only into 11α-hydroxyprogesterone, but also into 21-hydroxyprogesterone (aka 11-deoxycorticosterone) [A.N. El-Refai, and K.M. Ghanem. Some physiological relations of progesterone conversion by Aspergillus nidulans. Egyptian Journal of Microbiology, 1987, Vol. 22 (2), 327-338; A.H. El-Refai, and K.M. Ghanem. Microbial response to steroids. Egyptian Journal of Microbiology, 1989, Vol. 23 (1), 1-11]. The authors introduced progesterone into the growth medium in a 96% ethanol solution with a final solvent concentration of 1%. The transformation of progesterone was carried out with a load of 1 g / L at a temperature of 30 ± 2 ° C on a shaker (200 rpm, amplitude 7 cm) for 48 hours. The authors observed the formation of a dihydroxylated derivative - 6β, 11α-dihydroxyprogesterone. It was concluded that at pH values close to neutral, 6β-hydroxylation is a secondary process and 6β, 11α-dihydroxyprogesterone is formed from the primary formed 11α-hydroxyprogesterone [A.N. El-Refai, and K.M. Ghanem. Some physiological relations of progesterone conversion by Aspergillus nidulans. Egyptian Journal of Microbiology, 1987, Vol. 22 (2), 327-338].

, М. Tadra, and J.

. Microbial transformations of steroids XXIV. Separation of androstane 17-hydroxy-epimers. Folia Microbiol. 1964, Vol. 9(6), 380-382]. При этом авторы отметили, что ацетилирование происходит только с 17β-гидроксипроизводным, тогда как соответствующий 17α-эпимер остается нетронутым. В этих условиях не происходит ацетилирования молекулы стероида с гидроксигруппой в положениях 11α, 11β, 20β и 21. Известен способ ферментативного ацетилирования стероидных 3-олов [US 3597320, 1971], в котором описана способность микромицетов родов Penicillium sp. АТСС 16501, Spicaria sp. АТСС 20152 и Spicaria sp. АТСС 20153 проводить ацетилирование вторичной гидроксильной группы при атоме С3, не затрагивая при этом вторичную 11β-гидроксильную группу. Также описана способность штамма Trichoderma glaucum ацетилировать первичную гидроксильную группу при атоме С21 в молекуле 9α-фтор-11β,21-дигидрокси-16α,17α-изопропилидендиокси-прегн-4-ен-3,20-диона и ряда других производных гидрокортизона и преднизолона [С.Е. Holmlund, L.I. Feldman, N.E. Rigler, B.E. Nielsen, and R.H. Evans Jr. Microbial esterification of steroids. J. Am. Chem. Soc., 1961, Vol. 83(11), 2586-2587]. Однако информация о способности мицелиальных грибов рода Aspergillus ацетилировать стероидные спирты в литературных источниках отсутствует.It is known that microorganisms are able to acetylate steroidal secondary and primary alcohols. Thus, the ability of some types of yeast and yeast-like organisms (Saccharomyces fragilis, S. lactis, Candida pseudotropicalis and Torulopsis sphaerica) to acetylate the secondary 17β-hydroxyl group of testosterone [A.

, M. Tadra, and J.

... Microbial transformations of steroids XXIV. Separation of androstane 17-hydroxy-epimers. Folia Microbiol. 1964, Vol. 9 (6), 380-382]. The authors noted that acetylation occurs only with the 17β-hydroxy derivative, while the corresponding 17α-epimer remains intact. Under these conditions, acetylation of a steroid molecule with a hydroxy group at positions 11α, 11β, 20β and 21 does not occur. There is a known method for enzymatic acetylation of steroid 3-ols [US 3597320, 1971], which describes the ability of micromycetes of the genera Penicillium sp. ATCC 16501, Spicaria sp. ATCC 20152 and Spicaria sp. ATCC 20153 carry out acetylation of the secondary hydroxyl group at the C3 atom, without affecting the secondary 11β-hydroxyl group. Also described is the ability of the Trichoderma glaucum strain to acetylate the primary hydroxyl group at the C21 atom in the molecule 9α-fluoro-11β, 21-dihydroxy-16α, 17α-isopropylidenedioxy-pregn-4-ene-3,20-dione and a number of other derivatives of hydrocortisone and prednisolone [ S.E. Holmlund, LI Feldman, NE Rigler, BE Nielsen, and RH Evans Jr. Microbial esterification of steroids. J. Am. Chem. Soc., 1961, Vol. 83 (11), 2586-2587]. However, there is no information on the ability of filamentous fungi of the genus Aspergillus to acetylate steroidal alcohols in the literature.

It is known from the prior art that acetylation of secondary alcohols of steroidal structure, in particular 11-hydroxylated steroids, is possible only chemically using acetic acid anhydrides (anhydride or acid chloride) as acetylation agent. For example, the acetylation of 11α-hydroxyprogesterone is carried out chemically using acetic anhydride [D.H. Peterson, N.S. Murray, S.H. Eppstein, L.M. Reineke, A. Weintraub, P.D. Meister, and H.M. Leigh. Microbiological Transformations of Steroids. I. Introduction of Oxygen at Carbon-11 of Progesterone. J. Am. Chem. Soc., 1952, Vol. 74 (23), 5933-5936].

Thus, it follows from the prior art that the production of 11α-acetoxyprogesterone by transformation of 11α-hydroxyprogesterone by cultures of filamentous fungi in general, in particular, of the genus Aspergillus, specifically Aspergillus nidulans, has not been previously performed.

11α-Acetoxyprogesterone (11α-hydroxypregn-4-en-3,20-dione acetate, CAS No. 2268-98-6) is a physiologically active derivative of progesterone (pregn-4-en-3,20-dione, CAS No. 57-83 -0) - a native steroid hormone synthesized by the corpus luteum of the ovary, the adrenal cortex (cortex), seminal vesicles and the placenta. Preparations based on progesterone and its derivatives are used as medicines in medicine and veterinary medicine.

11α-Acetoxyprogesterone, like 11α-hydroxyprogesterone, being a progesterone derivative with gestagenic activity, can be used as an agent that reduces the risk of preterm birth, as well as as an active ingredient in combination with natural or synthetic estrogens, both in preparations for oral contraception, so and in preparations for hormone replacement therapy in postmenopausal women.

In addition, 11α-hydroxyprogesterone acetate can be used not only as a progestogenic agent, as mentioned above, but also as an antiandrogenic agent to reduce sebaceous secretions in patients suffering from seborrhea, alopecia, acne vulgaris and other skin diseases associated with hyperandrogenization. Thus, cosmetic compositions for hair and scalp care are known, containing 11α-acetoxyprogesterone in an amount of 1.4-1.8% by weight. [DE 2757024, 1979]. It was noted that this compound, when applied topically, has no hormonal side effects. Cosmetic compositions can be formulated as hair lotions or oils, skin creams, oils or emulsions for application to the skin, shampoos, aerosol sprays, and the like.

11α-Acetoxyprogesterone can be used as a starting product in the synthesis of other drugs for medical use. If necessary, it can be hydrolyzed by any convenient method known in the art to form 11α-hydroxyprogesterone. For example, a chemical method is known for producing 11α-hydroxyprogesterone by solvolysis of 11α-acetoxyprogesterone by the action of bis (tributyltin) oxide [MG Perez, and MS Maier. Mild deprotection of steroid esters by bis (tributyltin) oxide. Tetrahedron Letters, 1995, Vol. 36 (19), 3311-3314] or by the traditional method of chemical solvolysis under the conditions of basic catalysis: in methanol in the presence of KOH [T. Kubota, and F. Hayashi. Studies on A-norsteroids - VI. Directing effects of C ₁₁ substituents on the addition of osmium tetroxide to steroidal Δ ^1,4 -3-ketones. Tetrahedron, 1967. Vol. 23, 995-1006]. The formed 11α-hydroxyprogesterone can be used, for example, in the synthesis of 11-ketoprogesterone (ketogestin, CAS No. 516-15-4), which is further converted to cortisone or 11β-hydroxyprogesterone, the precursor of native hydrocortisone, by methods known in the art. A chemical method for the transition from 11α-hydroxyprogesterone to 11-ketoprogesterone with a yield of more than 90% and then to 11β-hydroxyprogesterone is described in patent [US 2015376225, 2015, examples 1 and 7, respectively]. In addition, 11α-hydroxyprogesterone can be used as a starting compound in the synthesis of promising analogs of the neurosteroid anesthetic alfaxolone [PY Savechenkov, DC Chiara, R. Desai, AT Stern, X. Zhou, AM Ziemba, AL Szabo, Y. Zhang, JB Cohen , SA Forman, KW Miller, KS Bruzik. Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands. European Journal of Medicinal Chemistry, 2017, Vol. 136, 334-347].

The closest in essence to the proposed method for producing 11α-acetoxyprogesterone by acetylation of 11α-hydroxyprogesterone is the chemical method [D.H. Peterson, N.S. Murray, S.H. Eppstein, L.M. Reineke, A. Weintraub, P.D. Meister, and H.M. Leigh. Microbiological Transformations of Steroids. I. Introduction of Oxygen at Carbon-11 of Progesterone. J. Am. Chem. Soc., 1952, Vol. 74 (23), 5933-5936], which consists in obtaining 11α-acetoxyprogesterone by acetylation of 11α-hydroxyprogesterone (20 mg) by the action of acetic anhydride (0.6 ml) in pyridine medium (0.6 ml). The mixture was kept for 16 hours at room temperature. At the end of the reaction, the reaction mixture was diluted with 25 ml of water, kept for 1 h, and cooled to initiate crystallization. The crystals were separated by filtration, washed with water and dried. Received 16.1 mg of 11α-acetoxyprogesterone with so pl. 176-177 ° C. Thus, the molar yield of 11α-acetoxyprogesterone, obtained by the authors by precipitating its crystals from the reaction mixture with a ~ 20-fold amount of water, was 71.4%.

Disclosure of invention

The technical problem solved by the claimed invention is the development of an ecologically cleaner microbiological method for producing 11α-acetoxyprogesterone by acetylation of 11α-hydroxyprogesterone using filamentous fungus while ensuring the yield of the target product is not less than 80%.

The technical result of the claimed invention is the biocatalytic esterification of steroidal alcohol without the use of chemical acetylating reagents and organic bases traditionally used as catalysts and solvents, with almost complete conversion of the original substrate and the selectivity of the formation of the target product of at least 85%. The ecological significance of the method lies in the elimination of the above reagents and organic solvents, the use of which requires the development of additional energy-intensive methods for the regeneration of solvents, purification of industrial effluents, and purification of the target product. It should be noted that the modification of steroid compounds using microorganisms belongs to the field of white biotechnology, since it allows not only to reduce environmental pollution, save raw materials and energy resources, but also to reduce the cost of the final product. This is consistent with current trends in the study of biotechnological processes, including the search for new biocatalysts capable of performing the reactions most important for the steroid industry with a high degree of selectivity (including the search for biotechnological methods that are alternative to chemical ones).

Also, the technical result is the expansion of the range of methods for acetylation of the secondary hydroxyl group at the C11 atom of the progesterone molecule, due to the ability of the A. nidulans VKPM F-1069 strain according to the present invention to acetylate the secondary hydroxyl group at the C11 atom of the progesterone molecule, as an alternative to the chemical acetylation method.

The technical problem posed is solved by the method of producing 11α-acetoxyprogesterone by acetylation of 11α-hydroxyprogesterone by microbiological transformation using the wild type A. nidulans strain VKPM F-1069 under conditions of intense aeration or under conditions of limited aeration.

The used A. nidulans strain was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) under registration number F-1069 (synonym FGSC A4; ATCC 3863, 12996, 26451; CBS 112.46; NRRL 194), previously it was not used for biotransformation of steroid compounds, its acetylating ability discovered for the first time. This strain was first used for biotransformation of 11α-hydroxyprogesterone to 11α-acetoxyprogesterone.

The essence of the claimed invention concerning the production of 11α-acetoxyprogesterone by biotransformation of 11α-hydroxyprogesterone is that, in order to increase the yield and environmental friendliness of the process, the esterification is carried out in an aqueous medium using a wild-type filamentous fungus strain A. nidulans VKPM F-1069 under conditions of intense aeration or under conditions of limited aeration until almost complete conversion of 11α-hydroxyprogesterone.

The advantages of the proposed method for producing 11α-acetoxyprogesterone by biotransformation of 11α-hydroxyprogesterone over the chemical method known from the prior art for acetylation of the secondary 11α-hydroxyl group are as follows:

- biotechnological acetylation of 11α-hydroxyprogesterone is an environmentally safer and more cost effective process;

- the process is carried out without the use of aggressive chemical acetylation reagents, such as acetic anhydride, without the use of organic bases, for example, pyridine, as a solvent and at the same time an acetylation catalyst, as well as other possible organic solvents and catalysts for chemical esterification known from the prior art, for example, mineral and organic acids;

- biocatalytic esterification of 11α-hydroxyprogesterone, as an alternative to the chemical method of acetylation, is an environmentally cleaner and safer method: in the prototype method for the esterification reaction, a 30-fold amount of pyridine and a 30-fold amount of acetic anhydride by volume to the weight of the starting compound are used, and to precipitate crystals of the reaction product, a 20-fold amount of water is applied to the total volume of pyridine and acetic anhydride and the precipitate is washed with water; this requires the development of additional methods for solvent recovery and industrial wastewater treatment;

- biocatalytic acetylation proceeds regionally without the formation of a possible by-product of enolacetylation of the Δ ⁴ -3-keto group of the 11α-hydroxyprogesterone molecule - 3,11α-diacetoxypregna-3,5-dien-20-one, the content of which, when using the chemical method of acetylation, can take place and reach 20-30%, which significantly complicates the purification of the target 11α-acetoxyprogesterone and leads to losses of its output;

- biocatalytic acetylation proceeds with almost complete conversion of the initial substrate and the selectivity of the formation of the target product not less than 85% under conditions of intense aeration and not less than 89% under conditions of limited aeration. The yield of the isolated crystalline 11α-acetoxyprogesterone is at least 80% and 85%, respectively.

To achieve the above technical result according to the present invention, it is proposed to use the known wild-type strain of Aspergillus nidulans VKPM F-1069 as a biocatalyst for the biotechnological process of acetylation of 11α-hydroxyprogesterone. Thus, the essence of the claimed invention also lies in the use of the known strain of Aspergillus nidulans VKPM F-1069 for a new purpose, namely for acetylation of 11α-hydroxyprogesterone.

Cultural and morphological features of the A. nidulans strain VKPM F1069.

On agar medium forms round colonies with a diameter of 30-35 mm after 7 days of growth. The surface is flat, convex, fluffy. Medium density texture. The edge of the colonies is dense, even. The color of the colonies in the sporulation zone is greenish-white. The reverse side is pale brown. There is no exudate. It is characterized by intense sporulation. The color of the spores is green.

The main property of the strain according to the present invention is the presence of acetylating activity and the ability to esterify 11α-hydroxyprogesterone with the formation of 11α-acetoxyprogesterone in high yield both under conditions of intense and limited aeration.

FIG. 1 shows a scheme for obtaining 11α-acetoxyprogesterone by transformation of 11α-hydroxyprogesterone with the known A. nidulans strain VKPM F-1069.

The transformation of 11α-hydroxyprogesterone proceeds with the formation of 6β, 11α-dihydroxyprogesterone as a by-product. The processes of acetylation of the hydroxyl group of 11α-hydroxyprogesterone and its 6β-hydroxylation are competitive transformation processes, and the process of biocatalytic esterification has advantages, since it does not require intensive aeration. This conclusion is confirmed experimentally. With a transformation duration of 48 hours under conditions of limited aeration, the presence of only 11α-acetoxyprogesterone and untransformed initial substrate in the culture medium was determined.

Brief Description of Drawings

FIG. 1 shows the scheme of transformation of 11α-hydroxyprogesterone by the A. nidulans VKPM F-1069 strain.

FIG. 2 shows the chromatograms and the result of mass spectrometric analysis of 11α-acetoxyprogesterone.

FIG. 3-9 show ¹ H NMR-, ¹³ C NMR-, DEPT-NMR-, HSQC-NMR- and HMBC-NMR spectra of 11α-acetoxyprogesterone.

Implementation of the invention

The wild-type strain Aspergillus nidulans VKPM F-1069 (synonym FGSC A4; ATCC 3863, 12996, 26451; CBS 112.46; NRRL 194) was obtained from the All-Russian Collection of Industrial Microorganisms (VKPM).

11α-Hydroxyprogesterone (II) (CAS # 80-75-1, C ₂₁ H ₃₀ O ₃ ) and 11α-acetoxyprogesterone (C ₂₃ H ₃₂ O ₄ , Mw 372.5 are commercially available and can be purchased, for example, from Steraloids Inc. . (USA) or other manufacturers.

Inorganic salts were purchased from Fluka (Germany). Yeast extract and agar from Difco Becton Dickinson and company (Sparks, USA).

Other reagents and solvents are commercially available and were purchased from Russian manufacturers.

Distilled water was used to prepare media for cultivation and transformation, aqueous solutions of acids, salts, and alkalis. Drinking tap water was used to wash the extracts in organic solvents, unless otherwise stated.

All procedures, unless otherwise stated, were carried out at room temperature, that is, in the range from 20 to 25 ° C. For processes requiring lower temperatures than room temperature, cooling was provided with cold tap water (in the range from 10 to 20 ° C) or a mixture of crushed ice and cold water (in the range from 5 to 10 ° C).

Biotransformation was performed on a New Brunswick ™ Innova® 44 / 44R shaker in a thermostated room at a temperature of 37 ° C with stirring at 240-250 rpm. to ensure the conditions of intensive aeration or in the stirring mode 60-100 rpm. to ensure the conditions of limited aeration.

Labortechnik AG) при остаточном давлении 0,35±0,05 кгс/см2 (35±5 кПа) и температуре воды в бане в диапазоне от 35-50°С в зависимости от природы упариваемого растворителя.Evaporation of solvents in vacuum was carried out using a rotary vacuum evaporator Rotavapor (

Labortechnik AG) at a residual pressure of 0.35 ± 0.05 kgf / cm2 (35 ± 5 kPa) and a water temperature in the bath in the range from 35-50 ° C, depending on the nature of the solvent being evaporated.

Drying of product crystals to constant weight was carried out at a temperature of 35-45 ° C at atmospheric pressure or using a vacuum drying oven at a residual pressure of 0.35 ± 0.05 kgf / cm2 (35 ± 5 kPa).

To determine the pH of the wash water, we used a universal indicator paper with a range of values from 0 to 12 (Lahema, Czech Republic).

The product was purified by filtration through a layer of silica gel on a Schott funnel No. 1 using silica gel of the brand Silica gel 60 (0.040-0.063 mm) (Merck, Germany).

Elution progress was monitored by thin layer chromatography (TLC) using Silica gel 60 F ₂₅₄ plates (Merck, Germany) and chromatographic solvent systems: dichloromethane-acetone 9: 1 (v / v) and dichloromethane-acetone 4: 1 (v / v). The plates were viewed in UV light at a wavelength of 254 nm, then they were sprayed with 1% vanillin solution in 10% aqueous solution of HClO ₄ , developed at a temperature of 100-120 ° C.

The structure and purity of the product was confirmed by at least one of the following methods: TLC (TLC plates Silica gel 60 F ₂₅₄ (Merck, Germany)), mass spectrometry, elemental analysis, nuclear magnetic resonance (NMR), high performance liquid chromatography ( HPLC).

Labortechnik AG).The melting point of the product was determined on an apparatus for determining the melting point M-565 (

Labortechnik AG).

¹ H- and ¹³ C-NMR spectra were determined on a Bruker Avance-400 spectrometer (Bruker BioSpin GmbH) with an operating frequency of 400 MHz and 100.6 MHz, respectively, using deuterated chloroform (99.8% D, Sigma-Aldrich) or deuterated dimethyl sulfoxide (99.9% D, Sigma-Aldrich) as solvent relative to tetramethylsilane (TMS NMR grade ≥99.9%, Sigma-Aldrich) as internal standard, in parts per million (ppm).

High resolution mass spectra (HRMS) were recorded on a Bruker Daltonics micrOTOF-Q II instrument using electrospray ionization (ESI). The measurements were obtained in the positive ion mode with the following parameters: the boundary capillary voltage is 4500 V; mass range from m / z 50 to 3000; external calibration (Electricpray Calibrant Solution, Fluka); atomizer pressure 0.4 bar; flow rate 3 μl / min; nitrogen was used as dry gas (6 l / min); the interface temperature was set at 180 ° C. The sample was injected into the mass spectrometer chamber from an Agilent 1260 HPLC system equipped with an Agilent Poroshell 120 EC-C18 column (3.0 × 50 mm; 2.7 μm) and shielding using an autosampler. The sample was injected in a solution of 50% acetonitrile (LC-MS qualification) in water (MilliQ ultrapure water, Merck Millipore KGaA, Germany), and the column was eluted with a mixture of acetonitrile (A) and water (B) in a concentration gradient with a flow rate of 400 μL / min. in the following gradient parameters: 0-15% A for 6 minutes, 15% -85% A for 1.5 minutes, 85% -0% A for 0.1 minutes, 0% A for 2.4 minutes ... The retention time was as follows: 11α-hydroxyprogesterone 5.2 min; 11α-acetoxyprogesterone - 6.0 min; 6β, 11α dihydroxyprogesterone - 4.2 min.

The formation of 11α-acetoxyprogesterone as a result of the transformation of 11α-hydroxyprogesterone by the culture of A. nidulans VKPM F-1069 was confirmed by the data of ¹ H NMR, ¹³ C NMR, 2DYMP, high-resolution chromatography-mass spectrometry. FIG. Nos. 3-9 show the NMR spectra and FIG. No. 2 - chromatogram and mass spectrum of the obtained 11α-acetoxyprogesterone.

To confirm the formation of 11α-acetoxyprogesterone during the transformation of 11α-hydroxyprogesterone, 11α-acetoxyprogesterone was obtained by chemical synthesis from 11α-hydroxyprogesterone using the acetylation method known from the prior art. In addition, the resulting 11α-acetoxyprogesterone was hydrolyzed by a method known in the art to form the starting 11α-hydroxyprogesterone.

The yields of 11α-acetoxyprogesterone obtained by biotransformation of 11α-hydroxyprogesterone by the strain of the present invention and given in the examples of the claimed invention confirm the effectiveness of the proposed method and its suitability for use in industrial processes. However, the examples are given only to illustrate the biocatalytic ability of the inventive strain to convert 11α-hydroxyprogesterone to 11α-acetoxyprogesterone. It should be noted that the specified acetylating ability of the strain was revealed by us for the first time. The yields can be improved by optimizing the conditions for biotransformation in order to minimize the formation of 6β, 11α-dihydroxyprogesterone and shift the direction of modification of 11α-hydroxyprogesterone towards acetylation. It is known that the composition of products depends on the composition of the medium, the pH of the medium, the age of the culture, on the induction of the original substrate or product [A.N. El-Refai, and K.M. Ghanem Some physiological relations of progesterone conversion by Aspergillus nidulans. Egyptian Journal of Microbiology, 1987, Vol. 22 (2), 327-338]. Therefore, to reduce the activity of the 6β-hydroxylase enzyme and reduce the amount of the formed by-product 6β, 11α-dihydroxyprogesterone, it is necessary to optimize the parameters of the transformation process, for example, the pH of the medium, the age of the culture, the method of adding the substrate, the composition of the medium for transformation, the temperature regime and others prior art. For example, Table 1 shows the effect of the aeration regime on the conversion of 11α-hydroxyprogesterone by the claimed strain and on the selectivity of the formation of 11α-acetoxyprogesterone and 6β, 11α-dihydroxyprogesterone, all other conditions being the same.

et al [А.

, М. Tadra, J.

. Microbial transformations of steroids XXIV. Separation of androstane 17-hydroxy-epimers. Folia Microbiol. 1964, Vol. 9(6), 380-382] для ацетилирования тестостерона дрожжами и описана в статье А.Н. El-Refai и K.M. Ghanem [А.Н. El-Refai, and K.M. Ghanem. Some physiological relations of progesterone conversion by Aspergillus nidulans. Egyptian Journal of Microbiology, 1987, Vol. 22(2), 327-338] (среда II, pH 6,5), как наиболее благоприятная для конверсии прогестерона в 11α-гидрокси-производное культурой A. nidulans. Исходный субстрат вносили в среду для трансформации в виде раствора в биполярном апротонном растворителе, в качестве которого могут быть использованы диметилсульфоксид или диметилформамид. При этом концентрация растворителя в ферментационной среде может составлять 2-6% по объему. Трансформацию проводили при температуре 37°С, так как ранее нами было установлено, что повышение температуры трансформации прогестерона мицелиальным грибом Aspergillus niger с 28°С до 37°С на среде СМ при прочих равных условиях проведения процесса приводит к существенному подавлению 6β-гидроксилазной активности штамма [О.С. Савинова, П.Н. Сольев, Д.В. Васина, Т.В. Тяжелова, Т.В. Федорова, Т.С. Савинова. Биотрансформация прогестерона аскомицетом Aspergillus niger N402. Биохимия (Москва). 2018. Том 83(1), с. 71-77].For transformation, we used the SM medium of the following composition, g / l: glucose - 40; MgSO ₄ × 7H2O - 1; KN ₂ PO ₄ - 0.74; peptone - 1; yeast extract - 1; L-asparagine 0.7 The CM medium was previously used by A.

et al [A.

, M. Tadra, J.

... Microbial transformations of steroids XXIV. Separation of androstane 17-hydroxy-epimers. Folia Microbiol. 1964, Vol. 9 (6), 380-382] for testosterone acetylation by yeast and described in the article by A.N. El-Refai and KM Ghanem [A.N. El-Refai, and KM Ghanem. Some physiological relations of progesterone conversion by Aspergillus nidulans. Egyptian Journal of Microbiology, 1987, Vol. 22 (2), 327-338] (medium II, pH 6.5), as the most favorable for the conversion of progesterone to 11α-hydroxy-derivative by A. nidulans culture. The initial substrate was introduced into the transformation medium as a solution in a bipolar aprotic solvent, which can be dimethyl sulfoxide or dimethylformamide. In this case, the concentration of the solvent in the fermentation medium can be 2-6% by volume. The transformation was carried out at a temperature of 37 ° C, since we previously found that an increase in the temperature of transformation of progesterone by the filamentous fungus Aspergillus niger from 28 ° C to 37 ° C on CM medium, all other things being equal, leads to a significant suppression of the 6β-hydroxylase activity of the strain [O.S. Savinova, P.N. Soliev, D.V. Vasina, T.V. Tyazhelova, T.V. Fedorova, T.S. Savinova. Biotransformation of progesterone by ascomycete Aspergillus niger N402. Biochemistry (Moscow). 2018.Vol. 83 (1), p. 71-77].

The claimed invention is illustrated by the following examples.

Example 1. General method of biotransformation of 11α-hydroxyprogesterone

Biotransformation

To obtain inoculum (spore suspension), the strains were grown at 37 ° C for 7-10 days on an MPA agar medium (malt extract - 3 g / L, peptone - 1 g / L, agar - 20 g / L). The spore suspension was inoculated into 750 ml shaking flasks containing 100 ml of growth medium (at the rate of ~ 1 × 10 ⁷ spores per 100 ml of medium), and grown on a shaker (240-250 rpm) at 37 ° C for 4 ± 0.5 days before reaching the amount of biomass ~ 1 g / 100 ml (by weight of dry mycelium). Then, 11α-hydroxyprogesterone was added to the growth medium in the form of a solution in dimethyl sulfoxide to a final concentration of 0.5 g / L, while the solvent concentration was 2% (v / v). The transformation was carried out at a temperature of 37 ° C under conditions of intense or limited aeration for 48 h or 216 h, in parallel cultivating the strain without 11α-hydroxyprogesterone as a control. Each experiment was performed in triplicate.

Extraction of transformation products

To extract the transformation products of 11α-hydroxyprogesterone, either the extraction method with the preliminary use of a disintegrator-homogenizer to destroy the cells of the microorganism was used, or the two-stage extraction method - extraction from the previously separated culture fluid and extraction from the mycelium by maceration - followed by combining the extracts.

Option 1.

At the end of the incubation, the fermentation suspension was placed in a Potter-Elwegeim-type disintegrator-homogenizer in portions of 30-40 ml. Homogenization was carried out at room temperature for 4-5 minutes until the cells of the microorganism were completely destroyed. The combined homogenate was extracted with dichloromethane three times in portions of equal volume. The combined extracts of the homogenate were washed with water, dried with Na ₂ SO ₄ , clarified with activated charcoal, and evaporated until the run stopped. The content of products in the residue after evaporation was assessed using quantitative TLC. Any disintegrator-homogenizer known from the prior art, suitable for the destruction of cells of filamentous fungi (ultrasonic, mechanical, etc.) can be used to destroy cells of a microorganism.

Option 2.

At the end of the incubation, the mycelium was filtered off, washed with dichloromethane on the filter. The reaction products from the culture liquid were extracted three times with portions of dichloromethane of equal volume. Steroids adsorbed on the mycelium were recovered by remaceration (three times) using methanol and keeping without stirring for 4 hours. Methanol was evaporated, the residue containing water was extracted with dichloromethane three times. The combined extracts of the culture broth and mycelium were washed with water, dried with Na ₂ SO ₄ , clarified with activated charcoal, and evaporated until the run stopped. The content of products in the residue after evaporation was assessed using quantitative TLC.

Extraction of 11α-acetoxyprogesterone

The evaporation residue was dissolved in 2-5 ml of dichloromethane and filtered through a layer of silica gel placed on a Schott filter funnel No. 1 using a 10-fold amount of silica gel to the weight of the starting substrate (the ratio of the diameter of the filter funnel to the height of the silica gel layer 1: 1.5-2). Dichloromethane was used as an eluent; TLC was used to control the elution. At the end of the elution of 11α-acetoxyprogesterone, the silica gel was washed with methanol to wash out 6β, 11α-dihydroxyprogesterone. The eluates were evaporated to dryness. The crystallizing residue was triturated with diethyl ether, the suspension was cooled to a temperature of 5-10 ° C, kept for 2 h, the precipitate was filtered off, dried to constant weight in a vacuum drying oven. The chromatographic purity of 11α-acetoxyprogesterone was confirmed by TLC, HPLC, mass spectrometry and NMR spectroscopy.

Example 2. Biocatalytic acetylation of 11α-hydroxyprogesterone under intense aeration on a shaker (240-250 rpm)

Option 1. Transformation within 48 hours

For the extraction of transformation products, variant 1 of Example 1 was used. From 50 mg of 11α-hydroxyprogesterone loaded on the transformation, after extraction, 51 mg of the dry residue after evaporation was obtained, containing, according to the analysis, 30 mg of 11α-hydroxyprogesterone (60% of the loaded), 17 mg ( 30.16 mol%) 11α-acetoxyprogesterone and 2.5 mg (4.77 mol%) 6β, 11α-dihydroxyprogesterone. According to TLC analysis, there are no other transformation products.

Option 2. Transformation within 216 hours

For the extraction of transformation products, variant 2 of Example 1 was used. From 500 mg of 11α-hydroxyprogesterone loaded on the transformation, 575 mg of the dry residue after evaporation was obtained, containing, according to the analysis, 480 mg (85.17 mol%) of 11α-acetoxyprogesterone and 75 mg (14 , 31 mol%) 6β, 11α-dihydroxyprogesterone. According to TLC analysis, 11α-hydroxyprogesterone is practically absent. The yield of crystalline 11α-acetoxyprogesterone was 451 mg (80.02%, based on the loaded 11α-hydroxyprogesterone).

Example 3. Biocatalytic acetylation of 11α-hydroxyprogesterone under conditions of limited aeration on a shaker (90-100 rpm)

Option 1. Transformation within 48 hours

For the extraction of transformation products, variant 1 of Example 1 was used. From 50 mg of 11α-hydroxyprogesterone loaded on the transformation, after extraction, 50 mg of the dry residue after evaporation was obtained, containing, according to the analysis, 40 mg of 11α-hydroxyprogesterone (80% of the loaded) and 10 mg ( 17.74 mol%) 11α-acetoxyprogesterone. According to TLC analysis, 6β, 11α-dihydroxyprogesterone and other transformation products are absent.

Option 2. Transformation within 216 hours

For the extraction of transformation products, variant 2 of Example 1 was used. From 500 mg of 11α-hydroxyprogesterone loaded onto the transformation, 580 mg of the dry residue after evaporation was obtained, containing, according to the analysis, 506 mg (89.78 mol%) of 11α-acetoxyprogesterone and 49 mg (9 , 35 mol%) 6β, 11α-dihydroxyprogesterone. TLC analysis showed no 11α-hydroxyprogesterone. The yield of crystalline 11α-acetoxyprogesterone was 480 mg (85.17%, based on the loaded 11α-hydroxyprogesterone).

NMR spectra of the obtained 11α-acetoxyprogesterone samples are identical to the spectra of the standard sample and the spectra of the sample obtained by the chemical method of acetylation of 11α-hydroxyprogesterone.

Example 4. Obtaining 11α-acetoxyprogesterone by chemical method

To a solution of 34 mg of 11α-hydroxyprogesterone in 0.2 ml of acetic anhydride was added 1 drop of 60% perchloric acid. The reaction mass was kept at room temperature for 1 h. After the completion of the reaction, the reaction mass was added dropwise to 5 ml of water containing 1 ml of 25% ammonia solution (pH ~ 7.5). The mixture was stirred for 30 minutes, then the reaction mixture was extracted with dichloromethane three times, the extract was washed with water until neutral, and evaporated to dryness. 39 mg of a residue was obtained, from which 27 mg of 11α-acetoxyprogesterone was recovered by preparative chromatography in a yield of 70.45%. T. pl. 173-174 ° C (lit. mp 176-177 ° C [DH Peterson, HC Murray, SH Eppstein, LM Reineke, A. Weintraub, PD Meister, and HM Leigh. Microbiological Transformations of Steroids. I Introduction of Oxygen at Carbon-11 of Progesterone J. Am Chem Soc., 1952, Vol. 74 (23), 5933-5936].

Characterization of biotransformation products

11α-Hydroxyprogesterone obtained by hydrolysis of the acetoxy group of 11α-acetoxyprogesterone obtained according to example 2 under the conditions of the general method of biotransformation of 11α-hydroxyprogesterone by the A. nidulans strain VKPM F-1069.

T. pl. 164-166 ° C (lit. mp 164-165 ° C [K. Yildirim, and A. Kuru. Biotransformation of some steroids by Aspergillus candidus. Journal of Chemical Research, 2015. Vol. 39 (9), 546 -549]). M.w. 330.46.

High Resolution Mass Spectrum C ₂₁ H ₃₀ O ₃ (m / z): calculated for [M + H] ⁺ 331.2268, found 331.2264; calculated for [M + Na] ⁺ 353.2087, found 353.2088. The ¹ H-NMR spectrum is identical to that of the starting substrate.

11α-Acetoxyprogesterone obtained under the conditions of biotransformation of 11α-hydroxyprogesterone by A. nidulans strain VKPM F-1069.

T. pl. 172-174 ° C (lit. mp 176-177 ° C [DH Peterson, HC Murray, SH Eppstein, LM Reineke, A. Weintraub, PD Meister, and HM Leigh. Microbiological Transformations of Steroids. I Introduction of Oxygen at Carbon-11 of Progesterone J. Am Chem Soc. 1952 Vol 74 (23) 5933-5936 Mw 372.5;

High Resolution Mass Spectrum C ₂₃ H ₃₂ O ₄ (m / z): calculated for [M + H] ⁺ 373.2373, found 373.2361; calculated for [M + Na] ⁺ 395.2193, found 395.2183.

¹ H-NMR (300 MHz, DMSO-d ₆ ): 5.67 (s, 1H CH-4), 5.14 (dt, 1H, ³ J _{11, 9} = 10.6 Hz, ³ J _{11, 10} 5.0 Hz, CH-11 ), 2.62 (dd (pseudo-t), 1H, ³ J ₁₇ , _16α 8.9 Hz, ³ J _{17, 16β} 9.1 Hz, CH-17), 2.47-2.35 (m, 2H, CH-2β & CH- 6β), 2.29-2.12 (m, 3H, CH-6α & CH-12β & CH-2α), 2.10-1.96 (m, 1H, CH-16β), 2.05 (s, 3H, CH ₃ -21), 2.01 (s, 3H, CH ₃ -23), 1.91-1.78 (m, 3H, CH ₂ -1 & CH-7α), 1.72-1.60 (m, 3H, CH-16α & CH-15α & CH-8), 1.50 (t, 1H, ³ J _{12α, 12β} 11.6 Hz, CH-12α), 1.44 (t, 1H, ³ J _{9, 8} 10.6 Hz, CH-9), 1.39-1.29 (ddd, 1H, ³ J _{14, 8} 10.7 Hz, ³ J _{14, 15β} 6.4 Hz, ³ J _{14, 15α} 5.6 Hz, СН-14) 1.29 (br.s, 4Н, CH ₃ -19 & СН-15β), 1.17-1.00 (2 × ddd, 2H, ³ J _{7α, 7β} 12.6 Hz, ³ J _{7, 6α} ~ ³ J _{7, 6β} 12.2 Hz, ³ J _{7α, 8} 3.1 Hz, CH ₂ -7), 0.64 (s, 3H, CH ₃ -18).

¹³ C-NMR (300 MHz, DMSO-d ₆ ): 208.61 (s, CO-20), 199.40 (s, CO-3), 170.14 (s, C-5), 170.10 (s, CO-22) 124.48 (s, CH-4), 70.58 (s, CHOH-11), 62.30 (s, CH-17), 55.34 (s, CH-9), 54.39 (s, CH-14), 45.02 (s, CH ₂ -12), 43.54 (s, C-13), 39.75 (s, C-10), 36.79 (s, CH ₂ -1), 34.72 (s, CH-8) 34.19 (s, CH ₂ -2), 33.03 (s, CH ₂ -6), 31.67 (s, CH ₂ -7), 31.37 (s, CH ₃ -21), 24.16 (s, CH ₂ -15), 22.96 (s, CH ₂ -16), 22.02 (s, CH ₃ -23), 18.20 (s, CH ₃ -19), 14.24 (s, CH ₃ -18).

The spectra are identical to those of the standard sample, as well as the spectra of the sample obtained by the chemical method of acetylation of 11α-hydroxyprogesterone.

Claims (6)

1. A method for producing 11α-acetoxyprogesterone, including the transformation of 11α-hydroxyprogesterone by a strain of the filamentous fungus Aspergillus nidulans deposited in the All-Russian Collection of Industrial Microorganisms under registration number F-1069, while for transformation, the spore suspension of the fungus is sown in shaking medium flasks with growth the amount of biomass ~ 1 g / 100 ml by weight of dry mycelium with stirring, then a solution of 11α-hydroxyprogesterone in dimethyl sulfoxide or dimethylformamide is added to the growth medium until their concentration in the medium reaches 2 to 6% (vol.) and until the substrate concentration is in the range of 0 , 1-1.0 g / l, incubated on a shaker at a temperature of 37 ± 0.5 ° C for 4 ± 0.5 days under conditions of limited aeration at a speed of 90-100 rpm for 216 hours or under conditions of intense aeration with speed of 240-250 rpm for at least 168 hours, at the end of incubation, the products are extracted by extraction and purified to obtain the target product 11α-ace toxiprogesterone. 2. The method according to claim 1, characterized in that a spore suspension of the fungus is inoculated into shaking flasks with a growth medium at the rate of ~ 1 × 10 ⁷ spores per 100 ml of medium. 3. A method according to claim 1, characterized in that stirring is carried out at a speed of 240-250 rpm. 4. The method according to claim 1, characterized in that the extraction of transformation products from the fermentation suspension is carried out after the preliminary destruction of the cells of the microorganism using a disintegrator-homogenizer. 5. The method according to claim 1, characterized in that the extraction of transformation products from the fermentation suspension is carried out by a two-stage extraction method - extraction from a previously separated culture liquid and extraction from the mycelium by maceration - followed by combining the extracts. 6. The method according to claim 1, characterized in that the purification of 11α-acetoxyprogesterone from the transformation by-product - 6β, 11α-dihydroxyprogesterone - is carried out by filtration through a layer of silica gel in a dichloromethane solution.

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