Mixed culture bioconversion of 16-dehydropregnenolone acetate to androsta-1,4-diene-3,17-dione: optimization of parameters

Bioconversion of 16-dehydropregnenolone acetate (16-DPA) to androsta-1,4-diene-3,17-dione (ADD), an intermediate for the production of female sex hormones, by mixed culture of Pseudomonas diminuta MTCC 3361 and Comamonas acidovorans MTCC 3362 is reported. Various physicochemical parameters for the bioconversion of 16-DPA to ADD have been optimized in shake flask cultures. Nutrient broth inoculated with actively growing co-culture proved ideal for bacterial growth and bioconversion. A temperature range of 35-40 degrees C was most suitable; higher or lower temperatures adversely affected the bioconversion. Dimethylformamide below 2% concentration was the most suitable carrier solvent. Maximum conversion was recorded at 0.5 mg mL(-1) 16-DPA. A pH of 5.0 yielded a peak conversion of 62 mol % in 120 h incubation period. Addition of 9alpha-hydroxylase inhibitors failed to prevent further breakdown of ADD to nonsteroidal products. 16-DPA conversion in a 5 L fermenter followed a similar trend.     

Microbial conversion of pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione acetates by Nocardioides simplex VKM Ac-2033D

The conversion of pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione 21-acetate (I) and 17,21-diacetate (VI) by Nocardioides simplex VKM Ac-2033D was studied. The major metabolites formed from I were identified as pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II) and pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione (IV). Pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione (III) and pregna-1,4,9(11)-triene-17alpha,20beta,21-triol-3-one (V) were formed in minorities. Biotransformation products formed from VI were pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17,21-diacetate (VII), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione (IV), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17-acetate (VIII), pregna-1,4,9(11)-triene-17alpha,20beta,21-triol-3-one (V). The conversion pathways were proposed including 1(2)-dehydrogenation, deacetylation, 20beta-reduction and non-enzymatic migration of acyl group from position 17 to 21. The conditions providing predominant accumulation of pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II) from I and pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17-acetate (VIII) from VI in a short-term biotransformation were determined.     

Steroid derivatives

Mycobacterium flavum was used to effect the transformation of 16β-methyl-16,17-oxido-7β,11α-dihydroxypregn-4-ene-3,20-dione (I) and the final products were isolated and identified as 16β-methyl-16,17-oxido-7β,11α-dihydroxypregna-1,4-diene-3,20-dione (II) and 16β-methyl-16,17-oxido-11α-hydroxypregna-1,4,6-triene-3,20-dione (IV), and the intermediate product as 16β-methyl-16,17-oxido-11α-hydroxypregna-4,6-diene-3,20-dione (III).     

Biotransformation of progesterone to 14 alpha-hydroxypregna-1,4-diene-3,20-dione, a novel fungal metabolite, by Colletotrichum antirrhini

The fermentation of progesterone by Colletotrichum antirrhini SC 2144 was examined. Instead of 15 alpha-hydroxyprogesterone, the reported product, this fungus converted progesterone to androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione, 14 alpha-hydroxyandrosta-1,4-diene-3,17-dione, 11 alpha-hydroxypregn-4-ene-3,20-dione, 14 alpha-hydroxypregn-4-ene-3,20-dione, and a hitherto undescribed compound, 14 alpha-hydroxypregna-1,4-diene-3,20-dione.     

Preparation of androsta-1,4-diene-3,17-dione from sterols using Mycobacterium neoaurum VKPM As-1656 strain

A product of microbiological cleavage of the sterols side chain, androsta-1,4-diene-3,17-dione, is toxic for bacteria, in particular, actinobacteria of the genera Mycobacterium and Arthrobacter. Sterols were transformed into androsta-1,4-diene-3,17-dione by culturing the M. neoaurum VKPM An-1656 strain in a high yield, provided that a sorbent was used for elimination of contact between the bacterial cells and the product. Unlike the cholesterol side chain, the more branched chains of phytosterols were cleaved in the presence of M. neoaurum at a high rate only under turbulent stirring of the culture medium, which intensified the formation of hydrocarbonate ion from NaNI3 in situ.     

Pregnanes in the root bark of Nerium odorum

Neridienone-A (12β-hydroxy-pregna-4,6,16-triene-3,20-dione), neridienone-B (20β,21-dihydroxy-pregna-4,6-diene-3,12-dione), 12β-hydroxy-pregna-4,6-diene-3,20-dione, 12β-hydroxy-pregn-4-ene-3,20-dione and 12β-hydroxy-16α-methoxy-pregna-4,6-diene-3,20- dione were obtained from the root bark of Nerium odorum.     

Androgenic and anti-androgenic effects of progesterone derivatives with different halogens as substituents at the C-6 position.

The pharmacological activities of four pregnane derivatives: 17alpha-hydroxy-16beta-methylpregna-4,6-diene-3,20-dio ne (7), 17alpha-acetoxy-16beta-methylpregna-4,6-diene-3,20-dio ne (8), 17alpha-acetoxy-6-bromo-16beta-methylpregna-4,6-diene- 3,20-dione (10), and 17alpha-acetoxy-6-chloro-16beta-methylpregna-4,6-diene -3,20-dione (11), were determined. The derivatives were evaluated on gonadectomized male hamster flank organs and seminal vesicles. The results indicate that topical applications of testosterone (T) on the flank organs increased the diameter of the pigmented spot. Similarly, the same phenomenon occurred on the glands treated with compound 11, whereas compound 10 decreased the size of the spot significantly. In this study, we determined the effects of several new steroids on the conversion of T to DHT in flank organs and seminal vesicles. The results show that compound 10 inhibited T conversion to DHT, but compound 11, at a dose of 200 microg, stimulated T conversion in both flank organs and seminal vesicles. However, when 2 mg of compound 11 was applied, it inhibited the conversion of T to DHT, suggesting that this compound also represses gonadotropin release. The difference between compounds 10 and 11 involves the electronegativity of the halogen at the C-6 position of the progesterone skeleton. These data clearly indicate that by decreasing the electronegativity of the halogen at C-6 (compound 10), 5alpha-reductase is inhibited in both tissues and at different pHs. On the other hand, when the electronegativity of the halogen atom was increased (11), there was a much lower inhibitory effect on the conversion of T to DHT.     

A complete proton and carbon-13 NMR analysis of fluocinonide, a fluorinated corticosteroid

This paper presents a complete analysis of the proton and carbon-13 NMR spectra of 21-acetoxy-6 alpha,9-difluoro-11 beta-hydroxy-16 alpha,17-(1-methylethylidene) bis-(oxy) pregna-1,4-diene-3,20-dione, a potent anti-inflammatory fluorosteroid. The 300 MHz proton spectrum was analyzed using a combination of the two-dimensional homonuclear chemical shift correlation (COSY) technique and one-dimensional NOE difference spectra. Exact coupling constants and chemical shifts were obtained by spectral simulation and iteration. The carbon-13 spectrum was assigned from the proton spectrum via a two-dimensional heteronuclear chemical shift experiment, and long-range fluorine-proton couplings were confirmed by a fully coupled heteronuclear COSY-type experiment.     

Efficient biotransformation of cholesterol to androsta-1,4-diene-3,17-dione by a newly isolated actinomycete <Emphasis Type="Italic">Gordonia neofelifaecis</Emphasis>

A newly isolated actinomycete, Gordonia neofelifaecis (NRRL B-59395) from the faeces of Neofelis nebulosa, was used to selectively degrade the side-chain of cholesterol. The intermediates were purified and characterized. Quantitative analysis of the accumulated metabolites from cholesterol side-chain cleavage was conducted during the biotransformation. The results showed that the profile of accumulated intermediates was different from those of other reported microorganisms. Among the five metabolites, androsta-1,4-diene-3,17-dione (ADD) was the main product of the side-chain degradation, with a high conversion rate (87.2%), indicating its potential for industrial production of ADD. At the end of transformation, the substrate cholesterol was completely consumed. The effect of some factors on the bioconversion was also investigated. To our best knowledge, this is the first report regarding cholesterol side-chain cleavage using bacteria belonging to Gordonia.     

21-Acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione conversion by Nocardioides simplex VKM Ac-2033D

The conversion of 21-acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione (I) by Nocardioides simplex VKM Ac-2033D was studied purposed selective production of its 1(2)-dehydroanalogues—value precursors in the synthesis of modern glucocorticoids starting from 9-hydroxyandrostenes. 21-Acetoxy-pregna-1(2),4(5),9(11),16(17)-tetraene-21-ol-3,20-dione (II), pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione (III) and pregna-1(2),4(5),9(11),16(17)-tetraene-21-ol-3,20-dione (IV) were revealed as metabolites, and the structures were confirmed by mass spectrometry and ¹H nuclear magnetic resonance (NMR) spectroscopy. The metabolic pathways of I by N. simplex included 1(2)-dehydrogenation and deacetylation. The sequence of the reactions was shown to depend on the transformation conditions. The presence of both soluble and membrane associated steroid esterases in N. simplex was demonstrated using cell fractionation. Unlike inducible 1(2)-dehydrogenase, steroid esterase was shown to be constitutive. The conditions providing selective accumulation of II from I by whole N. simplex cells were determined.     

D-homo isomers of 17-acetoxy-6-methylpregna-4,6-diene-3,20-dione

The three most readily accessible D-homo isomers of 17-acetoxy-6-methylpregna-4,6-diene-3,20-dione (Ib) have been prepared and characterized in detail. The 17-keto isomers IIb and IIIb were obtained by base-catalyzed rearrangement of Ia followed by reacetylation, and the 17a-ketone IVb by Lewis-Acid treatment of Ia.     

The biotransformation of hyodeoxycholic acid by Pseudomonas sp. NCIB 10590 under anaerobic conditions

The bacterial degradation of hyodeoxycholic acid under anaerobic conditions was studied. The major acidic product has been identified as 6 alpha-hydroxy-3-oxochol-4-ene-24-oic acid whilst the major neutral product has been identified as 6 alpha-hydroxyandrosta-1,4-diene-3,17-dione. The minor acidic products were 3,6-dioxochola-1,4-diene-24-oic acid, 3-oxochol-5-ene-24-oic acid, 3-oxochol-4-ene-24-oic acid, 3-oxochola-1,4-diene-24-oic acid and 6 alpha-hydroxy-3-oxochola-1,4-diene-24-oic acid and the minor neutral products were androst-4-ene-3,17-dione, androst-4-ene-3,6,17-trione, androsta-1,4-diene-3,6,17-trione, androsta-1,4-diene-3,17-dione, 17 beta-hydroxyandrosta-1,4-diene-3-one and 6 alpha-hydroxyandrost-4-ene-3,17-dione. Evidence is presented which suggests that under aerobic conditions, one pathway of hyodeoxycholic acid metabolism exists whilst under anaerobic conditions an extra biotransformation pathway becomes operative involving the induction of a 6 alpha-dehydroxylase enzyme. A biochemical pathway of hyodeoxycholic acid metabolism by bacteria under anaerobic conditions is discussed incorporating a scheme involving such an enzyme.     

Preparation of androsta-1,4-diene-3,17-dione from sterols using <Emphasis Type="Italic">Mycobacterium neoaurum</Emphasis> VKPM Ac-1656 strain

A product of microbiological cleavage of the sterols side chain, androsta-1,4-diene-3,17-dione, is toxic for bacteria, in particular, actinobacteria of the genera Mycobacterium and Arthrobacter. Sterols were transformed into androsta-1,4-diene-3,17-dione by culturing the M. neoaurum VKPM Ac-1656 strain in a high yield, provided that a sorbent was used for elimination of contact between the bacterial cells and the product. Unlike the cholesterol side chain, the more branched chains of phytosterols were cleaved in the presence of M. neoaurum at a high rate only under turbulent stirring of the culture medium, which intensified the formation of hydrocarbonate ion from NaNI₃ in situ.     

Mycobacterium sp．高效转化植物甾醇为雄甾-1，4-二烯-3，17-二酮的诱变选育

采用紫外线、亚硝基胍复合诱变雄甾-4-烯-3,17-二酮（AD）和雄甾-1,4-二烯-3,17-二酮（ADD）的转化产生菌Mycobacterium sp．,结合平板筛选,获得一株遗传性状稳定单产ADD的突变菌株Mycobacterium sp．-11,其ADD质量浓度达到1246ms／L,比原始菌株（484mg／L）提高了150％,经初步优化后发酵液中ADD最高达到1430mg／L,发酵液中ADD质量占ADD、AD两产物质量总和的比例由70％提高到99．1％。     

Bioconversion of steroids by Cochliobolus lunatus--II. 11 beta-hydroxylation of 17 alpha, 21-dihydroxypregna-1,4-diene-3,20-dione 17-acetate in dependence of the inducer structure.

The 11 beta-hydroxylase of the filamentous fungus Cochliobolus lunatus m 118 was induced with the substrate 17 alpha, 21-dihydroxypregna-1,4-diene-3,20-dione 17-acetate (11 beta-deoxyprednisolone 17-acetate) itself, substrate analogues, different pregnane compounds, sterols, intermediates of microbial sterol side-chain degradation or bile acids, together with 24 different steroids in a standardized test system. The resulting 11 beta-hydroxylation rate, leading to prednisolone 17-acetate and prednisolone, respectively, was determined and compared with the hydroxylation rate of non-induced cultures. The transformation yield strongly depended on the inducer structure. The microbial sterol side-chain degradation intermediates (20S)-20-hydroxymethylpregn-4-en-3-one and the corresponding pregna-1,4-diene compound caused the highest induction effects (induction factors 5.1 and 4.9, respectively). The metabolism of (20S)-20-hydroxymethylpregna-1,4-dien-3-one during the cultivation was elucidated. The induction effect decreased with the rising oxidation of the inducer. The significant increase of the 11 beta-hydroxylation rate of 1-dehydro-pregnane substrates by specific induction allows alternative pathways to glucocorticoid partial syntheses.     

A practical large-scale synthesis of 17alpha-acetoxy-11beta-(4-N, N-dimethylaminophenyl)-19-norpregna-4,9-diene-3,20-dione (CDB-2914)

A new practical synthesis of 17alpha-acetoxy-11beta-(4-N, N-dimethylaminophenyl)-19-norpregna-4,9-diene-3,20-dione (CDB-2914) is described. The synthesis gives easily isolable solids at all steps and is amenable to large-scale process.     

Metabolism of megestrol acetate by rat adrenal glands in vitro

Megestrol acetate is metabolized by homogenates of rat adrenal glands to three more polar metabolites Y(2), Y(3) and Y(4). Their structures were investigated by microchemical reactions, paper chromatography, and ultraviolet and infrared spectroscopy. Metabolite Y(3) was identified as 17alpha-acetoxy-11beta-hydroxy-6-methyl-pregna-4,6-diene-3,20-dione, its oxidation product being identical with authentic 17alpha-acetoxy-6-methylpregna-4,6-diene-3,11,20-trione. It is suggested that metabolite Y(2) is 17,18-dihydroxy-6-methylpregna-4,6-diene-3,20-dione and that it also exists in the form of an alpha-ketol formed between the 18-hydroxyl and 20-oxo groups. Metabolite Y(4) is probably a tautomeric form of metabolite Y(2) as they were partially interconverted on chromatography and also have similar properties.     

Studies on the Microbiological Transformation of Steroids

The microbiological reduction of the 20-carbonyl group of steroids has been investigated. Candida pulcherrima IFO 0964 and Sporotrichum gougeroti IFO 5982 converted the following substrates into the corresponding 20β-hydroxy derivatives (yields of the products are indicated in parentheses): Reichstein’s Compound S (60~70%) and 17α,21-dihydroxypregna-l,4-diene- 3,20-dione (40~80%). Rhodotorula glutinis IFO 0395 converted the following substrates into the corresponding 20α-hydroxy derivatives: Reichstein’s Compound S (65%), 17 α,21-dihydroxy- pregna-l,4-diene-3,20-dione (80%), llβ,l7α-dihydroxypregn-4-ene-3,20-dione (45%) and 17α, 19,21 -trihydroxypregn-4-ene-3,20-dione (10%).     

Recognition of D-homoannulation by proton and carbon NMR spectra

One-and two dimensional proton and carbon NMR spectra of the D-homoannulated rearrangement product of triamcinolone (9 alpha-fluoro-11 beta,16 alpha,17 alpha, 21-tetrahydroxy-pregna-1,4-diene-3,20-dione) establish its structure as that of 9 alpha-fluoro-11 beta,16 alpha,17 alpha-trihydroxy-17 beta-hydroxy-methyl-D-homoandrosta-1,4-diene-3,17 alpha-dione. These methods accord ready recognition of D-homoannulation of C21-17-hydroxy-20-ketosteroids.     

The degradation of beta-sitosterol by Pseudomonas sp. NCIB 10590 under aerobic conditions

The bacterial degradation of beta-sitosterol by Pseudomonas sp NCIB 10590 has been studied. Major biotransformation products included 24-ethylcholest-4-en-3-one, androsta-1,4-diene-3,17-dione, 3-oxochol-4-en-3-one-24-oic acid and 3-oxopregn-4-en-3-one-20-carboxylic acid. Minor products identified were 26-hydroxy-24-ethylcholest-4-en-3-one, androst-4-ene-3,17-dione, 3-oxo-24-ethylcholest-4-en-26-oic acid, 3-oxochola-1,4-dien-3-one-24-oic acid, 3-oxopregna-1,4-dien-3-one-20 carboxylic acid and 9 alpha-hydroxyandrosta-1,4-diene-3,17-dione. Studies with selected inhibitors have enabled the elucidation of a comprehensive pathway of beta-sitosterol degradation by bacteria.