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Publication numberUS3770586 A
Publication typeGrant
Publication dateNov 6, 1973
Filing dateMar 24, 1971
Priority dateMar 24, 1971
Publication numberUS 3770586 A, US 3770586A, US-A-3770586, US3770586 A, US3770586A
InventorsL Kominek
Original AssigneeUpjohn Co
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Process for the microbiological 1-dehydrogenation of certain 4,9-(11)-pregnadienes
US 3770586 A
Abstract
An improved process for the production of 11 beta ,21-dihydroxy-1,4,17(20)-pregnatrien-3-one and 6 alpha -methyl-11 beta ,21-dihydroxy-1,4,17(20)-pregnatrien-3-one. The novel process of this invention provides a method whereby high substrate levels of 11 beta ,21-dihydroxy-4,17(20)-pregnadien-3-one and 6 alpha -methyl-11 beta ,21-dihydroxy-4,17(20)-pregnadien-3-one are bioconverted with Septomyxa affinis to obtain corresponding 1-dehydro compound in high yield by employing as the substrate a 21-hydrocarbon dicarboxylic acid ester alkali metal salt of either 11 beta ,21-dihydroxy-4,17(20)-pregnadien-3-one or 6 alpha -methyl-11 beta ,21-dihydroxy-4,17(20)-pregnadien-3-one to obtain the corresponding 1-dihydro compound.
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Description  (OCR text may contain errors)

United States Patent [191 Kominek 1 Nov. 6, 1973 [75] Inventor:

[73] Assignee: The Upjohn Company, Kalamazoo,

Mich.

[22] Filed: Mar. 24, 1971 [21] Appl. No.: 127,797

Related U.S. Application Data [63] Continuation-impart of Ser. No. 819,990, April 28,

Leo A. Kominek, Kalamazoo, Mich.

[52] U.S. Cl 195/51 E [51] Int. Cl. C07c 167/14 [58] Field of Search 195/51 E [56] References Cited UNITED STATES PATENTS 3,020,297 2/1962 Lincoln et al 195/51 E 2,902,4ll 9/1959 Murray et al. 195/51 E Primary Examiner-Alvin E. Tanenholtz Attorney-John Kekich and Ward F. Nixon [57] ABSTRACT An improved process for the production of 113,21- dihydroxy-l ,4, l 7( 20)-pregnatrien-3-one and 6amethyl-l 13,21-dihydroxy-l ,4,l7(20)-pregnatrien- 3-one. The novel process of this invention provides a method whereby high substrate levels of 113,21- dihydroxy-4,l7(20)-pregnadien-3-one and oer-methyl- 1 13,2 1 -dihydroxy-4, 1 7( 20 )-pregnadien-3-one are bioconverted with Septomyxa affinis to obtain corresponding l-dehydro compound in high yield by employing as the substrate a 2l-hydrocarbon dicarboxylic acid ester alkali metal salt of either 1 13,2l-dihydroxy- 4,17(20)-pregnadien-3-one or 6a-methyl-1 13,21- dihydroxy-4,17(20)-pregnadien-3-one to obtain the corresponding l-dihydro compound.

The 1 13,21 -dihydroxy-1 ,4,l 7(20)-pregnatrien-3-one and 6a-methyl-1 13,21-dihydroxy-1,4,17(20)-pregnatrien- 3-one produced by the process of this invention are each known in the art. They are useful intermediates for producing valuable steroid products, for example, prednisolone and 6a-methyl-prednisolone, respectively.

10 Claims, No Drawings PROCESS FOR THE MICROBIOLOGICA l-DEIIYDROGENATION OF CERTAIN 4,9-(1 l )-PREGNADIENES CROSS REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of application Ser. No. 819,990, filed Apr. 28, 1969, now abandoned.

BACKGROUND OF THE INVENTION The 1,2-dehydrogenation of llfl,2l-dihydroxy- 4,17(20) pregnadiene-3-one and 6a-methyl-llB,2ldehydroxy-4,l7(20)-pregnadien-3-one by Septomyxa affinis to obtain the corresponding l-dihydro compounds is well known in the art, for example, see US. Pat. Nos. 2,902,410 and 2,902,411.

In the l,2 -dehydrogenation of 1lB,21-dihydroxy- 4,17(20)-pregnadien-3-one and 6a-methyl-l 13,21- dihydroxy-4,17(20)-pregnadien-3-one by the microorganism Septomyxa affinis, it is essential that the selected substrate be dissolved in the aqueous medium. It was shown by H. J. Koepsell, Biotechnol. Bioeng. IV, 57 (1962) that about 0.18 to 0.2 grams per liter of 11- B,21-dihydroxy-4,17(20 )-pregnadien-3-one is soluble in an aqueous medium and that only dissolved substrate is 1,2-dehydrogenated by the microorganism Septomyxa affinis. The reference also shows that precipitated substrate will not be 1,2-dehydrogenated by the microorganism. Heretofore, the maximum substrate level of 11B,21-dihydroxy-4,l7(20 )-pregnadien-3-one which could be bioconverted using Septomyxa afiinis was about 0.5 grams per liter and the maximum substrate level of 6a-methyl-1lfi,2l-dihydroxy-4,17(20)- pregnadien-3-one was about 2.0 grams per liter. Even though, for example, 0.5 gram per liter is considerably above the solubility level of the steroid, it was discovered by Chen et al., I.C.&E. Process Design and Development, IV, 421-425 (1962), that bioconversion could be effectively carried out at substrate levels up to about 0.5 g. per liter by taking advantage of the supersaturation phenomenon which is achieved by adding the substrate dissolved in a solvent such as acetonepropylene glycol and by slow or intermittent feeding of the substrate during the bioconversion.

BRIEF SUMMARY OF THE INVENTION In the process for the 1,2-dehydrogenation of 113,2- 1-dihydroxy-4,l 7(20 )-pregnadien-3-one and 6amethyl-l 113,2l-dihydroxy-4,17(20)-pregnadien-3-one using Septomyxa affinis, it is essential that the substrate be in solution. It has now been discovered that the selected substrate can be made highly water soluble and thus readily 1,2-dehydrogenated with the microorganism Septomyxa affinis by employing as the starting substrate a 2l-hydrocarbon dicarboxylic acid ester alkali metal salt of l 1 )3, 2l-dihydroxy-4,l7(20 )-pregnadien-3-one or Getmethyl-l 13,21-dihydroxy-4,l7(20)-pregnadien-3-one. By virtue of the greatly increased solubility of the selected substrate, the fermentative 1,2-dehydrogenation can now be effectively carried out at substrate levels greatly above those heretofore possible using prior art methods.

The improved process of the invention is of considerable economic importance because the cost of the fermentation process is inversely related to the level of substrate that can be efficiently converted to the desired product. The principle advantages of the present process over the prior art are: (1) greatly increased product per batch, resulting in substantially reduced labor and equipment requirements; (2) lower cost per pound of product, and (3) precipitation of the substrate is no longer a problem and therefor intermittant substrate feeding is unnecessary and no special techniques or equipment are required for this purpose.

DETAILED DESCRIPTION OF THE INVENTION The improved process of this invention comprises subjecting an alkali metal salt of a 2l-hydrocarbon dicarboxylic acid ester of 1lB,2l-dihydroxy-4,l7(20)- pregnadien-3-one or 6a-methyl-11B,2l-dihydroxy- 4,17(20)-pregnadien-3-one to the activity of Septomyxa affinis. In the improved process of this invention when the 2l-acyl radical attached to the selected substrate is that of a saturated hydrocarbon dicarboxylic acid of three or four carbon atoms, i.e., malonic or succinic, the 2l-acylate group is removed during the bioconversion process giving the corresponding 1- dehydrogenated 21-free alcohol. When the 2l-acyl radical attached to the selected substrate is that of a saturated hydrocarbon dicarboxylic acid of five to 12 carbon atoms or that of an unsaturated hydrocarbon dicarboxylic acid of three to 12 carbon atoms, the acyl group is generally not removed during the bioconversion process and the product obtained is the corresponding l-dehydrogenated 21-acylate. The attached 21-acyl radical can be easily removed if desired by hydrolysis under acidic conditions in accordance with procedures well known in the art, for example, by acidification with a mineral acid such as hydrochloric or sulfuric.

The process of this invention provides an improved means for obtaining 1118,21-dihydroxy-l,4,l7(20)- pregnatrien-S-one and 6a-methyl-1 113,21-dihydroxyl,4,l7(20)-pregnatrien-3-one, each of which are known in the art as useful intermediates in the production of valuable steroid products such as prednisolone and 6a-methylprednisolone, respectively.

Starting materials for the improved process of this invention are alkali metal salts of 115,2l-dihydroxy- 4,17(20)-pregnadien-3-one 21-acylates and 6amethyl-l 113,2ldihydroxy-4,17(20)-pregnadien-3-one 21-acylates, wherein the acyl radical of each is that of a hydrocarbon dicarboxylic acid of from three to 12 carbon atoms inclusive. These starting materials are prepared in accordance with the procedures known in the art, for example, US. Pat. Nos. 3,209,000 and 3,025,311.

Suitable acylating agents are hydrocarbon dicarboxylic acids, containing from three to 12 carbon atoms, inclusive, or the anhydrides or acid halides thereof. For example, a saturated dibasic acid, e.g., malonic, succinic, glutaric, adipic, pimelic, suberic, 0:,B-dimethylsuccinic, a,B-diethylsuccinic, and the like; dibasic un-' saturated acids, e.g., maleic, citraconic; o-, m-, pphthalic acidjand the like; or the acid anhydrides and acid halides thereof.

The acylation of either l13,21-dihydroxy-4,l7(20)- pregnadien-S-One or 6a-methyl1113,21-dihydroxy- 4,17(20)-pregnadien-3-one is carried out using the acylating agent as solvent, or preferably in the presence of a suitable inert solvent, such as tetrahydrofuran, benzene, xylene, dioxane, methylene chloride, ether, dimethyl formamide, and the like, particularly when the acylating agent is a solid, and frequently in the presence of a catalyst, such as p-toluene-sulfonic acid, or an amine, preferably pyridine. Completion of the reaction can take from a few minutes to 24 hours, depending on the temperature and the solvent employed. If the acylating agent is the free acid, the reaction is preferably carried out in the presence of an esterification catalyst, for example, p-toluenesulfonyl chloride, trifiuoroacetic anhydride, p-toluenesulfonic acid, trifluoroacetic acid, sulfuric acid, and the like,

The thus obtained 21-acylates of llB,2l-dihydroxy- 4,17(20)-pregnadien-3-one and 6a-methyl-l 13,21- dihydroxy-4,17(20)-pregnadien-3-one are recovered from the reaction mixture by conventional methods, such as, for example, precipitation by dilution with water or a dilute inorganic acid and collection by filtration or extraction with a water-immiscible solvent such as methylene chloride, ethyl acetate, benzene, ether, and the like, direct crystallization, chro-matography, counter-current extraction or a combination of these, followed by final crystallization if desired.

The 2l-acylates thus obtained are converted to water-soluble alkali metal salts, i.e., sodium, potassium or lithium salts, according to procedures well known in the art for the preparation of water-soluble salts, e.g., the preparation of water-soluble salts of hydrocortisone hemiacylates from hydrocortisone 2l-hemiacylates, disclosed in US. Pat. No. 3,025,311.

In carrying out the improved process of this invention, an alkali metal salt of a 11 B,2l-dihydroxy- 4,l7(20)-pregnadien-3-one 2l-acylate or 6a-methyl- 1 15,21-dihydroxy-4,l7(20)-pregnadien-3-one 2 l acylate is subjected to the activity of Septomyxa affinis. Representative strains of Septomyxa affinis useful in the process of this invention include, for example:

Septomyxa afiinis, ATCC 6737,

Septomyxa afiinis, ATCC 13,414,

Septomyxa affinis, ATCC 13,425,

Septomyxa affinis, CBS

and the like, which are available from known public sources, such as the American Type Culture Collection (ATCC), Washington, DC and the Centraalbureau voor Schimmelcultures (CBS), Baarn, Holland.

The operational conditions and reaction procedures for the bioconversion process of this invention are advantageously those known in the art of bioconversion as illustrated in Weintraub et al., US. Pat. No. 2, 902,410 and Murray et al., U. S. Pat. No. 2,902,411.

In the practice of this invention, the bioconversion can be effected by a growing or resting culture of the microorganism or by spores, washed cells or enzymes of the microorganism.

Culture of the microorganism for the purpose and practice of this invention is in or on a medium favorable to its development. Sources of nitrogen and carbon should be present in the culture medium and an adequate sterile air supply should be maintained during the conversion, for example, by the conventional techniques of exposing a large surface of the medium or by passing air through a submerged culture.

Nitrogen in assimilable form can be provided by sources normally employed in such processes, such as cornsteep liquor, cottonseed meal, soybean meal, yeast extracts, Torula yeast, peptone, soluble or insoluble vegetable or animal protein, lactalbumein, casein, whey, distillers solubles, amino acids, nitrates and ammonium compounds, such as ammonium tartrate, nitrate, sulfate and the like.

Available carbon can also be provided by sources normally used in bioconversions such as carbohydrates, e.g., glucose, fructose, sucrose, lactose, maltose, dextrins, starches, meat extracts, peptones, amino acids, proteins, fatty acid, glycerol, sodium lactate, whey and the like. These materials may be used either in a purified state or as whey concentrate, cornsteep liquor, grain mashes, cottonseed meal, and the like, or as mixtures of the above. Many of the above sources of carbon can also serve as a source of nitrogen.

The medium can also contain naturally present or added mineral constituents, such as calcium, copper, iron, potassium phosphorus, magnesium, and the like, such as potassium phosphate, calcium chloride, ferrous sulfate, magnesium sulfate, etc.

The medium can desirably have a pH before inoculation of between about pH 4 to about 8 though a higher or lower pH can be used. However, it is advantageous to maintain the pH within the range of about 5.5 to about 6 during the growth period. A temperature between about 25" to 32 C. is preferred for growth of the microorganism but higher or lower temperatures within a relatively wide range are suitable.

In carrying out the improved process of this invention, it is advantageous to pre-induce the steroid ldehydrogenase by adding a steroid l-dehydrogenase promotor, such as pro-gesterone, 3-keto-bisnor-4- cholen-22-al, 3-ketobisnor-cholenic acid, androstenedione, 1601,17-epoxy-l6-methyl-progesterone, and the like. The selected l-dehydrogenase promotor can be added simultaneously with the substrate, during the mycelial growth period, after the growth period and before addition of the substrate or after addition of the substrate. It is, however, especially advantageous to add the l-dehydrogenase promotor, 3-ketobisnor-4- cholen-22-al is preferred, after the mycelial growth period is complete and before addition of the substrate. The conditions can be the same as those found suitable for growth of the microorganism. The quantity of promotor can vary over a wide range as illustrated by Murray et al., US. Pat. No. 4,902,411. In the practice of this invention, it is preferred to carry out the preinduction at a temperature of about 28 C. and at a pH within the range of about 5.5 to 6.0 for a period of about 8 hours using the steroid promotor at a concentration of about 0.05 g. per liter of medium.

The selected substrate can be added to the medium in any suitable form, either as a dry powder or as an aqueous solution. However, because of the high soluability of the alkali metal salts of the dibasic acid esters, it is convenient to add the substrate as an aqueous solution. The bioconversion can be carried out at substrate concentrations as high as 20 g. per liter of medium. The preferred range of concentrations of the substrate in the medium is from about 2 to about 15 g. per liter.

The temperature during the fermentation can be the same as that found suitable for growth of the microorganism. It need be maintained only within such range as supports life, active growth or the enzyme activity of the microorganism. A range of 20 to 35 C. is preferred. Maintenance of a low pH is essential for optimum yields, a range of from about 5.5 to about 6 is preferred. Aeration can be effected by surface culture or preferably by use of submerged fermentation conditions with air sparging, in accordance with methods well known in the art. The time required for the bioconversion can vary considerably. The range of about 2 to 300 hours is practical but not limiting; about 72 to 150 hours is generally satisfactory.

Alternatively, l,2-dehydrogenation of the selected substrate can be effected by subjecting it to the activity of enzymes prepared from the microorganism, to the action of spores of the microorganism, and to the action of isolated cells of the microorganism. Isolated enzyme preparations can be prepared in accordance with the general procedure disclosed by Zuidweg et al., Biochim. Biopy. Acta 48, 131-133 (1962). The bioconversion can be effected with spores in accordance with the general process disclosed in U.S. Pat. Nos. 3,031,379 and 3 ,031,382. The separation of washed cells from the fermentation medium is well known in the art, see for example, U. S. Pat. No. 2,831,789. When enzymes, spores or washed cells are used, it is, of course, advantageous to separate them from the growth medium after pre-inductionwith a steroid promotor.

After completion of the fermentation, the resulting l,2-dehydrogenated product is recovered from the fermentation beer by conventional methods. For example, when the product is the free alcohol, i.e., 1113,21- dihydroxy-l ,4,17(20 )-pregnatrien-3-one or 6a-methyll 113,21-dihydroxy-l ,4,17(20)-pregnatrien-3-one, the whole beer can be extracted with a water-immiscible organic solvent such as methylene chloride, chloroform, carbon tetrachloride, ethylene chloride, trichloroethylene, ether, butyl acetate, amyl acetate, benzene, and the like or the beer and mycelia can be separated by conventional methods such as centrifugation or filtration, and then separately extracted with suitable solvents. The mycelia can be extracted with either water-miscible or water-immiscible solvents or in cases where little or no product is contained in the mycelium, it can be merely washed with water and the wash water added to the beer filtrate. The beer, free of mycelia, can then be extracted with water-immiscible solvents such as those listed above. The extracts are combined, dried over a drying agent such as anhydrous sodium sulfate, and the solvent removed by conventional methods such as evaporation or distillation at atmospheric or reduced pressure.

When the product of the bioconversion is a 21- acylate, i.e., a llB,21-dihydroxy-l,4,17(20 )-pregnatrien-3-one 21-acylate alkali metal salt, or 6a-methyl ll- [3,21-dihydroxy-l ,4,l7(20)-pregnatrien-3-one 2 l acylate alkali metal salt, the product is generally readily soluable and in solution in the fermentation beer, in these instances it is convenient to remove the mycelia by conventional methods as disclosed above, and the beer free of mycelia is acidified and allowed to stand at room temperature 12 to 24 hours) until the acyl group is hydrolyzed to give the corresponding 2l-free alcohol. The free alcohol thus obtained, can berecovered from the beer by extraction with a water-immiscible solvent as described herein above.

The l lfl,21-dihydroxy-l ,4 l 7 20 )-pregnatrien-3 -one and 6a-methyl-l 13,21 -dihydroxy-l ,4, l 7(20)-pregnatrien-3-one, obtained by the improved process, can be further purified by conventional methods such as chromotography or by recrystallization from a suitable organic solvent.

The following examples are intended to illustrate the process of this invention. The examples are for the purpose of illustrating the best mode contemplated of carrying out the invention and to supplement the foregoing disclosure with additional descriptions of the manner and process of carrying out the invention so as to further enable workers skilled in the art to do so, but they are not to be construed as limiting.

EXAMPLE 1 An aqueous 3 liter medium containing 10 g./l. of glucose, 20 g.ll. of cornsteep liquor and a minimal amount of an anti-foaming agent is adjusted to pH 5.0 sterilized at 121 C. for about 30 minutes, and inoculated with 150 ml. of a 24 hour vegetative growth of Septomyxa affinis, ATCC 6737. The medium is then agitated at 300 r.p.m. and aerated with sterile air at the rate of 1. liter per minute. After culturing for 16 hours at 28 C., 0.15 g. of 3-ketobisnor-4-cholen-22-al is added, the pH is adjusted to 5.5 and the incubation is continued for an additional period of about 8 hours. At the end of the 8 hour induction period, the pH is adjusted to 7.0, 15 g. (5 g./l.) of 1 13,2l-dihydroxy-4,17(20)-pregnadien- 3-one 2l-hemisuccinate potassium salt is added and the fermentation is continued for 72 hours with agitation at 300 r.p.m., and with aeration at the rate of 0.5 liter per minute. The mycelia are then removed by filtration and extracted with three 750 ml. portions of acetone. The beer filtrate is extracted two times, with a volume of butyl acetate equal to one-fifth the volume of the filtrate. The butyl acetate and acetone extracts are combined, and the solvent removed by distillation to give 9.225 g. (yield 87% of theory) of llB,21-dihydroxyl ,4,17(20)-pregnatrien-3-one.

EXAMPLE 2 An aqueous medium, containing 15 g./l. of glucose, 2.75 g./l. of ammonium sulfate, 1.0 g./l. of cornsteep liquor, 0.5 g./l. of dibasic potassium phosphate, 1.3 g./l. of calcium chloride, 0.7 g./l. of potassium chloride, 0.05 g./l. of ferrous sulfate and a minimal amount of an anti-foam agent is adjusted to pH 5.7, sterilized at 121 C. for 30 minutes and inoculated with 5% of the final volume of a 24 hour vegetative growth of Septomyxa affinis, ATCC 6737. The medium is agitated at 200 r.p.m. and aerated with a sterile air supply at the rate of 0.06 v.v.m. The culture is allowed to incubate'for about 20 hours at 28 C., while maintaining the pH at 5.7 ($0.3) by the addition of 5% aqueous sodium hydroxide solution. At the end of the growth period, 3-ketobisnor-4-cholen-22-al is added to a final concentration of 0.05 g./1. and the fermenation is continued for an additional 8 hours at a temperature of 28 C. and an aeration rate of 0.01 v.v.m. The pH is maintained at 5.7 (10.3) by the addition of 18% aqueous sulfuric acid as necessary. When the 8 hour induction period is complete, 1 13,21 -dihydroxy-4,l7(20)-pregnadien-3-one -2l-hemisuccinate potassium salt is added to a final concentration at 5 g./l. and the fermentation is continued under the same conditions for an additional period of 72 hours. The pH of the medium is then raised to 7.0 by the addition of 5% aqueous sodium hydroxide solution and the medium is stirred for about 1 hour. Papergram analysis of a sample of the whole beer shows a 90.2% yield of l1B,2l-dihydroxy-l,4,17(20)-pregnatrien-3-one. Extraction and purification of this material by conventional techniques well known in the art yields 1 113,21-dihydroxy-l,4,l7(20)-pregnatriene-3-one, m.p. l84-l91 C., [11],, 108.8.

EXAMPLE 3 The process of Example 2 is repeated on a larger scale to give a 89.0% yield as determined by papergram analysis of a sample of the whole beer. Conventional purification gives 1 13,21-dihydroxy-l ,4,17(20)- pregnadien-S-one, m.p. l88.5l93 C., [01],, 111.3.

EXAMPLE 4 An aqueous medium having the same composition as described in Example 2, above, is adjusted to pH 5.7, sterilized at 121 C. for 30 minutes and inoculated with 5% of the final fermentation volume of a 24 vegetative growth of Septomyxa affinis, ATCC 6737. The medium is agitated at 150 r.p.m. with an aeration rate of 0.2 v.v.m. of sterile air and allowed to grow for about 20 hours at 28 C., while maintaining the pH at 5.7 (-10.3 At the end of the growth period, 3-keto-bisnor-4- cholen -22-al is added to a final concentration of 0.05 g./l. and the fermentation is continued for 8 hours at the same temperature, agitation rate, and pH but with the aeration rate lowered to 0.04 v.v.m. When the 8 hour induction period is complete, 11B,2l-dihydroxy- 4,17(20)-pregnadien-3-one 21-hemmsuccinate potassium salt is added to a final concentration of 10 g./l. and the fermentation is continued under the same conditions for an additional period of 87 hours. Papergram analysis of a sample of the whole beer shows an 84.7% yield of 11B,21-dihydroxy-1,4,17(20)-pregnatrien- 3-one.

EXAMPLE 5 Five 500 ml. shake flasks each containing 100 ml. of

one of the following media:

Medium 1:

Glucose 10 g./l., Torula yeast 12 g./l., ground extracted soybean meal 6.0 g./l., skim milk 7.5 g./l. pH 6.3

Medium 2:

Glucose 10 g./l., comsteep 20 g./l. pH 5.0

Medium 3:

Glucose 10 g./l., Torula yeast 6 g./l., ground extracted soybean meal 5 g./l., comsteep liquor 7 g./l. pH 6.3-6.5

Medium 4:

Glucose l g./l., sodium lactate 3 g./l.,

KCl 0.07 g./l., FeSO '7H O 0.005 g./l. pH 5.5

Medium Glucose l0 g./l., Torula yeast 15 g./l. pH 6.3 are sterilized at 121 C. for 15 minutes, cooled to 28 C., inoculated with a 24-hour vegetative growth of Septomyxa affinis, ATCC 6737, and incubated for approximately 16 hours on a reciprocal shaker at 28 C. Dehydrogenase activity is induced by the addition of 0.005 g. of 3-ketobisnor-4-cholen-22-al and the fermentation is continued for 8 hours under the same conditions. Substrate, 113,21- dihydroxy-4, l 7(20)-pregnadien-3-one 21-hemisuccinate potassium salt, is then added to each in the amounts of 3 and 5 grams per liter as shown in the table, below. The fermentation is continued at 28 C. and samples are taken from each of the flasks at regular intervals and assayed for 115,21- dihydroxy-l ,4,1 7 20 )-preg natrien-3-one (Product) by spectrophotometric and/or papergram analysis. The medium used, fermentation time, and yields in percent of theory of product, 1118 ,21-dihydroxy-1,4,17(20)-pregnatrien-3-one, are shown in the following table:

Medium No. Substrate Time Product (g-l y (0 1 5 2 5 5 93.6 3 s 5 88.6 4 3 3 90.9 5 3 6 89.9

EXAMPLE 6 The bioconversion and assay procedures of Example 5 are repeated using four 500 ml. shake flasks each containing 100 ml. of Medium 2 (Example 5, above). The bioconversion is carried out for 6 days using substrate levels of l lB,21-dihydroxy-4,17(20)- pregnadien-3-one, 2l-hemisuccinate potassium salt, at 2, 3, 5 and 7 g./l. Yields in percent of theory of product, 1 1 B,21-dihydroxy-1,4,17(20 )-pregnatrien-3-one, are shown in the following table:

Substrate Level Yield of Product 2.0 gJl. 96.6% 3.0 gJl. 94.7% 5.0 g./l. 92.5% 7.0 g./l. 91.1%

EXAMPLE 7 Time Product 1 18,2 1 -dihydroxy-l ,4,1 7(20 pregnatrien-S-one ZI-citraconate potassium salt.

1 1B, 2l-dihydroxy-1,4,l7(20)- pregnatnen-3-one 21-hemiphthalate potassium Substrate Citraconate 6 72.2% Hemiphthalate 5 salt. 50.3% 1 1B,21-dihydroxy-l ,4,17(20)- pregnatrien-3-one, 21-hen1imaleate potassium salt. 1 1 B,2l-dihydroxy-l .4,17(20 pre trien-S-one, gallemiglutarate potassium Hemimaleate 6 57. 1 Hemiglutarate 6 EXAMPLE 8 An aqueous 3 liter medium containing 10 g./l. of glucose, 20 g./l. of comsteep liquor and a minimal amount of an anti-foaming agent is adjusted to pH 5.0 sterilized at 121 C. for about 30 minutes, and inoculated with ml. of a 24 hour vegetative growth of Septomyxa affinis, ATCC 6737. The medium is then agitated at 500 r.p.m. and aerated with sterile air at the rate of 0.5 liter per minute. After culturing for 16 hours at 28 C., 0.15 g. of 3-ketobisnor-4-cholen-22-al is added, the pH is adjusted to 5.5 and the incubation is continued for an additional period of about 8 hours. At the end of the 8 hour induction period, the ph is adjusted to 7.0, 14.7 g. (4.9 g./l.) of 6a-methyl-1lfl,21-dihydroxy-4,l7(20)- pregnadien-3-one ZI-hemisuccinate potassium salt in an aqueous dimethylformamide solution is added and the fermentation is continued for 136 hours with agitation at 500 r.p.m., and with aeration at the rate of 0.5 liter per minute. Papergram and thiosemicarbazide assays of samples of the whole beer show yields of 85.5 percent and 90.4%, respectively, of 6a-methyl-1 13,21-

dihdroxy-l ,4, 1 7(20 )-pregnadien-3-one.

EXAMPLE 9 An aqueous medium, containing 25 g./l. of glucose, 4.58 g./l. of ammonium sulfate, 1.0 g./l. of cornsteep liquor, 0.83 g./l. of dibasic potassium phosphate, 1.3 g./l. of calcium chloride, 0.7 g./l. of sodium chloride, 0.05 g./l. of ferrous sulfate and a minimal amount of an anti-foam agent is adjusted to pH 5.7, sterilized at 121 C. for 30 minutes and inoculated with 5% of the final volume of a 24 hour vegetative growth of Septomyxa affinis, ATCC 6737. The medium is agitated at 150 r.p.m. and aerated with a sterile air supply at the rate of 0.2 v.v.m. The culture is allowed to incubate for about 29 hours at 28 C., while maintaining the pH at 5.7 ($0.3) by the addition of 5% aqueous sodium hydroxide solution. At the end of the growth period, 3-ketobisnor-4-cholen-22-al is added to a final concentration of 0.05.g.ll. and the fermentation is continued for an additional 8 hours at a temperature of 28 C. and an aeration rate of 0.2 v.v.m. The pH is maintained at 5.7 (:03) by the addition of 18% aqueous sulfuric acid as necessary. When the 8 hour induction period is complete, 6amethyl-l l 3,21-dihydroxy-4,l7(20 pregnadien-3-one -2l-hemisuccinate potassium salt is added in an aqueous dimethylformamidesolution to a final concentration at 4.9 g./l. and the fermentation is continued under agitation at 300 r.p.m. and aeration at 0.04 v.v.m. for an additional period of 260 hours. Papergram and thiosemicarbozide assays of samples of the whole beer show yields of 84.5% and 93.3%, respectively, of 6a-methyl-l 13,21 -dihydroxy- 1,4,17(20)-pregnatrien-3-one. The pH of the medium is then raised to 7.0 by the addition of 5% aqueous so dium hydroxide solution and the medium is stirred for about 1 hour. Extraction and purification of this material by conventional techniques well known in the art gives a 80.9% yield of 6a-methyl-l13,21-dihydroxy- 1,4, 1 7( 20 )-pregnatriene-3-one.

EXAMPLE 10 I r.p.m. and aerated with a sterile air supply at the rate of 0.2 v.v.m. The culture is allowed to incubate for about 22 hours at 28 C., while maintaining the pH at 5.7 ($0.3) by the addition of 5% aqueous sodium hydroxide solution. At the end of the growth period, 3-ketobisnor-4-cholen-22-al is added to a final concentration of 0.05 g./l. and the fermentation is continued for an additional 8 hours at a temperature of 28 C. and an aeration rate of 0.2 v.v.m. The pH is maintained at 5.7 (1:0.3) by the addition of 18% aqueous sulfuric acid as necessary. When the 8 hour induction period is complete, 6a-methyl-l 13,21 -dihydroxy-4, 17(20)- pregnadien-S-one -2l-hemisuccinate potassium salt is added in an aqueous dimethyl-formamide solution to a final concentration at 4.9 g./l. and the fermentation is continued under agitation at 300 r.p.m. and an aeration rate of 0.04 v.v.m. for an additional period of 144 hours. Papergram and thiosemicarbazide assays of samples of the whole beer show yields of 88.3% and 95.8%,

respectively of Ga-methyl-l 13,2 1 -dihydroxy- 1 ,4,17(20)-pregnatriene-3-one.

EXAMPLE I 1 An aqueous medium, containing 15 g./l. of glucose, 20 g./l. of cornsteep liquor and a minimal amount of an anti-foam agent is adjusted to pH 5.0, sterilized at 121 C. for 30 minutes and inoculated with 5% of the final volume of a 24 hour vegetative growth of Septomyxa affinis, ATCC 6737. The medium is agitated at 150 r.p.m. and aerated with a sterile air supply at the rate of 0.2 v.v.m. The culture is allowed to incubate for about 20 hours at 28 C. At the end of the growth period, 3-ketobisnor-4-cholen-22-al is added to a final concentration of 0.05 g./1. and the fermentation is continued for an additional 8 hours under the same conditions. When the 8 hour induction period is complete, 6a-methyl-1 13,2l-dihydroxy-4,17(20)-pregnadien-3- one-21-hemisuccinate potassium salt is added in an aqueous dimethylformamide solution to a final concentration at 5.6 g./l. and the fermentation is continued under the same conditions for an additional'period of 133 hours. Papergram and thiosemicarbozide assays of samples of the whole beer show yields of 87.7% and 91.2%, respectively of 6a-methyl-l13,2l-dihydroxy- 1,4,17(20)-pregnatrien-3-one. The pH of the medium is then raised to 7.0 by the addition of 5% aqueous sodium hydroxide solution and the medium is stirred for about 1 hour. Extraction and purification of this material by conventional techniques well known in the art yields 6a-methyl-1 13,21-dihydroxy-1 ,4,] 7(20)- pregnatriene-3-one, m.p. l56l57 C., [01],, 98 (CHCl Amax 244 p. (e=14,850); infrared absorption at 3360, 3230, 3140, 1660, 1625, 1615 and 1600 cm NMR peaks (CDCl 1.06, 1.18, 1.46, 4.14, 4.26, 4.38, 5.28, 6.18, 6.34, 7.40 and 7.24 8.

Anal. Calcd. for C H O z C, 77.16; H, 8.83.

Found: C, 76.77; H. 8.54.

The above infrared absorption spectra are recorded on a Perkin-Elmer model 421 infrared spectrophotometer. Mineral oil (Nujol) mulls of the solids are used.

The above NMR spectra are recorded on a Varian A-60 spectrophotometer with tetramethylsilane as an internal standard (downfield) and using the solvent indicated.

EXAMPLE 12 Seven 500 ml. shake flasks each containing ml. of an aqueous medium comprising 10 g./l. of glucose ing table, and the fermentation is continued at 28 C. for the time indicated in the said table. At the end of the bioconversion period, samples of each are assayed using thiosemicarbazide to determine the yield of product, 6a -methyl-11B,2l -dihydroxy-l,4,17(20)-pregnatrien-3-one, in each. The yields in percent of theory of product are shown for each flask in the following table:

Substrate Bioconversion Time Product (g.ll.) (hours) yield) 2.45 66 95.8 3.7 66 95.0 4.9 66 93.2 6.1 66 91.7 7.0 66 89.1 10.5 96 88.2 14.0 96 84.1

EXAMPLE 13 The bioconversion and assay procedures of Example 12 are repeated using seven 500 ml. shake flasks each containing 100 ml. of a medium of the same compostion but substituting an equivalent amount of the corresponding sodium salt of 6a-methyl-1 lB,21-dihydroxy- 4,17(20)-pregnadien-3-one, 2l-hemi-succinate as the substrate in each to give substantially equivalent yields of 6a-methyl-1 13,2 1 -dihydroxy-l ,4, 1 7(20 )-pregnatrien-3-one.

I claim:

1. An improved process for the production of 1 13,2- l-dihydroxy-l,4,l7(20)-pregnatrien-3-one and the alkali metal salts of the 21-acylates thereof, wherein the acyl radical is that of a hydrocarbon dicarboxylic acid of three to 12 carbon atoms, inclusive, which comprises subjecting an alkali metal salt of an 1lB,2l-dihydroxy- 4,17(20)-pregnadien-3-one 2l-acylate, wherein the acyl radical has the meaning given above, to the activity of Septomyxa affinis under aerobic fermentation conditions in an aqueous nutrient medium at a substrate level within the range of from 2 to 10 grams per liter of said medium.

2. The process of claim 1, wherein the 1,2- dehydrogenated product is recovered from the fermentation medium.

3-one and the alkali metal salts of the 2 l-acylates thereof, wherein the acyl radical is that of a hydrocarbon dicarboxylic acid of 3 to 12 carbon atoms, inclusive, which comprises subjecting an alkali metal salt of a 60:- methyl-l 118,2 l-dihydroxy-4, l 7( 20)-pregnadien-3-one 2l-acylate, wherein the acyl radical has the meaning given above, to the activity of Septomyxa affinis under aerobic fermentation conditions in an aqueous nutrient medium at a substrate level within the range of from 3 to 15 grams per liter of said medium.

4. The process of claim 3, wherein the 1,2- dehydrogenated product is recovered from the fermentation medium.

5. The process for the production of 113,21- dihydroxy-l,4,]7(20)-pregnatrien-3-one, which comprises subjecting an alkali metal salt of 113,21- dihydroxy-4,17(20)-pregnadien-3-one 21- hemisuccinate at a substrate level within the range of from 2 to 10 grams per liter to the activity of Septomyxa affinis in an aqueous nitrient medium under submerged aerobic fermentation conditions.

6. The process of claim 5, wherein the alkali metal salt is the sodium salt.

7. The process of claim 5, wherein the alkali metal salt is the potassium salt.

8. The process for the production of 6a-methyl- 1 113,2 1 -dihydroxy-l ,4, l 7( 20 )-pregnatrien-3-one, which comprises subjecting an alkali metal salt of 6amethyl-l 15,21dihydroxy-4,l7(20)-pregnadien-3-one 2l-hemisuccinate at a substrate level within the range of from 3 to 15 grams per liter to the activity of Septomyxa affinis in an aqueous nutrient medium under submerged aerobic fermentation conditions.

9. The process of claim 8, wherein the alkali metal salt is the sodium salt.

10. The process of claim 8, wherein the alkali metal salt is the potassium salt.

Patent Citations
Cited PatentFiling datePublication dateApplicantTitle
US2902411 *Jun 28, 1957Sep 1, 1959Upjohn CoUse of second steroid to accelerate 1-dehydrogenation of substrate steroid
US3020297 *Nov 12, 1959Feb 6, 1962Upjohn Co2alpha, 6alpha-dimethylhydrocortisone and the 21-acylates thereof
Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US4088537 *Jun 21, 1976May 9, 1978The Upjohn CompanyΔ1 Dehydrogenation of corticoids without side chain degradation by Septomyxa
US5225335 *Sep 12, 1989Jul 6, 1993The Upjohn CompanyEnzymatic dehydrogenation
Classifications
U.S. Classification435/52, 435/911, 435/61
International ClassificationC12P33/00
Cooperative ClassificationY10S435/911, C12P33/00
European ClassificationC12P33/00