Transformation of 4-androsten-3,17-dione by growing cultures and cell extracts of Clostridium paraputrificum.

Growing cultures of Clostridium paraputrificum transformed 4-androsten-3,17-dione to 3 alpha-hydroxy-5 beta-androstan-17-one in a sequential manner with 5 beta-androstan-3,17-dione as an intermediate. The addition of 1.5 mM menadione to log-phase cultures which had formed 5 beta-androstan-3,17-dione resulted in a partial reoxidation of this steroid to 4-androsten-3,17-dione. However, this treatment also resulted in transient inhibition of culture growth. Resumption of growth was accompanied by complete reduction of 4-androsten-3,17-dione to 5 beta-androstan-3,17-dione. Cell extracts of C. paraputrificum were capable of carrying out these reductive transformations in the absence of added cofactors. However, Sephadex G-25 treated extracts required NADH or NADPH for these reactions. A flavin nucleotide, either FAD (plus NADH or NADPH) or FMN (plus NADH) was highly stimulatory for 4-androsten-3,17-dione reduction to 5 beta-androstan-3,17-dione. NADH was the preferred reduced pyridine nucleotide for reduction of the C4-C5 double bond, while time-course measurements suggested that NADPH was the preferred donor for reduction of the 3-keto group.     

Microbial transformation of 3-hydroxy-5,6-cyclopropanocholestanes--an alternative route to 6-methylsteroids

Incubation of 3beta-hydroxy-5,6alpha-cyclopropano-5alpha-cholestane (4), 3beta-hydroxy-5,6beta-cyclopropano-5beta-cholestane (5), and 3beta-hydroxy-5,6alpha-cyclopropano-5alpha-cholest-7-e ne (6) with Mycobacterium sp. (NRRL B-3805) gave a mixture of side chain cleaved 17-keto steroids as the major products in 52, 57, and 69% yields, respectively. Among these 17-keto steroids, the cyclopropyl ring eliminated product, androst-4-ene-3,17-dione (9), was isolated in 6, 4, and 8% yields, respectively. A cyclopropyl ring migration product, 6alpha,7alpha-cyclopropanoandrost-4-ene-3,17-dione (16), was isolated from the incubation mixture of 6 in 4% yield, also 10% yield of 16 was obtained when 5, 6alpha-cyclopropano-5alpha-androst-7-ene-3,17-dione (12) was incubated. The cyclopropyl ring opening and subsequent reduction followed by oxidation of the two major biotransformation products, 5, 6beta-cyclopropano-5beta-androsta-3,17-dione (10) and 5, 6alpha-cyclopropano-5alpha-androsta-3,17-dione (7), gave 6beta- and 6alpha-methylandrost-4-ene-3,17-dione in 60, and 45% yields, respectively.     

Studies on anabolic steroids--11. 18-hydroxylated metabolites of mesterolone, methenolone and stenbolone: new steroids isolated from human urine.

New metabolites of mesterolone, methenolone and stenbolone bearing a C18 hydroxyl group were isolated from the steroid glucuronide fraction of urine specimens collected after administration of single 50 mg doses of these steroids to human subjects. Mesterolone gave rise to four metabolites which were identified by gas chromatography/mass spectrometry as 18-hydroxy-1 alpha-methyl-5 alpha-androstan-3,17-dione 1, 3 alpha,18-dihydroxy-1 alpha-methyl-5 alpha-androstan-17-one 2, 3 beta,18-dihydroxy-1-alpha-methyl-5 alpha-androstan-17-one 3 and 3 alpha,6 xi,18-trihydroxy-1 alpha-methyl-5 alpha-androstan-17-one 4. These data suggest that mesterolone itself was not hydroxylated at C18, but rather 1 alpha-methyl-5 alpha-androstan-3,17-dione, an intermediate metabolite which results from oxidation of mesterolone 17-hydroxyl group. In addition to hydroxylation at C18, reduction of the 3-keto group and further hydroxylation at C6 were other reactions that led to the formation of these metabolites. It is of interest to note that in the case of both methenolone and stenbolone, only one 18-hydroxylated urinary metabolite namely 18-hydroxy-1-methyl-5 alpha-androst-1-ene-3,17-dione 5 and 18-hydroxy-1-methyl-5 alpha-androst-1-ene-3,17-dione 6 were both detected in post-administration urine specimens. These data indicate that the presence of a methyl group at the C1 or C2 positions in the steroids studied is a structural feature that seems to favor interaction of hepatic 18-hydroxylases with these steroids. These data provide further evidence that 18-hydroxylation of endogenous steroids can also occur in extra-adrenal sites in man.     

Lithocholic Acid Side-chain Cleavage to Produce 17-Keto or 22-Aldehyde Steroids by Pseudomonas putida strain ST-491 Grown in the Presence of an Organic Solvent,Diphenyl Ether

We devised a method to screen for microorganisms capable of growing on bile acids in the presence of organic solvents and producing organic solvent-soluble derivatives. Pseudomonas putida biovar A strain ST-491 isolated in this study produced decarboxylated derivatives from the bile acids. Strain ST-491 grown on 0.5% lithocholic acid catabolized approximately 30% of the substrate as a carbon source, and transiently accumulated in the medium androsta-1,4-diene-3,17-dione in an amount of corresponding to 5% of the substrate added. When 20% (v/v) diphenyl ether was added to the medium, 60% of the substrate was converted to 17-keto steroids (androst-4-ene-3,17-dione-like steroid, androsta-1,4-diene-3,17-dione) or a 22-aldehyde steroid (pregna-1,4-dien-3-on-20-al). Amounts of the products were responsible for 45, 10, and 5% of the substrate, respectively. In the presence of the surfactant Triton X-100 instead of diphenyl ether, 40% of the substrate was converted exclusively to androsta-1,4-diene-3,17-dione.     

Microbiological hydroxylation of androst-1,4-dien-3,17-dione by Neurospora crassa

Nine hydroxy-derived androstadiene compounds were isolated from the fermentation broth of Neurospora crassa when incubated in the presence of androst-1,4-dien-3,17-dione (ADD; I) for 7 days. Hydroxylations at 6β, 7β, 11α, 14α- positions and 17-carbonyl reduction of the substrate were the characteristics observed in this biotransformation. Their structures were determined by spectroscopic methods as 17β-hydroxyandrost-1,4-dien-3-one (II), 14α-hydroxyandrost-1,4-dien-3,17-dione (III), 6β-hydroxyandrost-1,4-dien-3,17-dione (IV), 11α-hydroxyandrost-1,4-dien-3,17-dione (V), 6β,17β-dihydroxyandrost-1,4-dien-3-one (VI), 7β-hydroxyandrost-1,4-dien-3,17-dione (VII), 14α,17β-dihydroxyandrost-1,4-dien-3-one (VIII), 6β,14α-dihydroxyandrost-1,4-dien-3,17-dione (IX), and 11α,17β-dihydroxyandrost-1,4-dien-3-one (X). A new steroid substance, 6β,14α-dihydroxyandrost-1,4-dien-3,17-dione (IX), was also characterized during this study. The best fermentation condition was found to be 7-day incubation at 25°C and pH values of 5.0–6.0 in the presence of 0.05 g 100 mL^?1 of the substrate. At a concentration above 0.075 g 100 mL^?1, the biotransformation was completely inhibited.     

Synthesis and analytics of 2,2,3,4,4-d5-19-nor-5alpha-androsterone--an internal standard in doping analysis

A short and efficient synthesis of pentadeuterated 2,2,3,4,4-d5-19-nor-5alpha-androsterone 7 starting from 19-norandrost-4-ene-3,17-dione 1 by a d1-L-Selectride mediated stereo- and regioselective reduction of the 3-keto group is presented. The use of compound 7 as internal standard for the detection of anabolic steroids via mass spectrometric techniques such as gas chromatography-mass spectrometry (GC-MS) is discussed.     

17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus: structural and functional aspects

17beta-Hydroxysteroid dehydrogenase (17beta-HSD) activity has been described in all filamentous fungi tested, but until now only one 17beta-HSD from Cochliobolus lunatus (17beta-HSDcl) was sequenced. We examined the evolutionary relationship among 17beta-HSDcl, fungal reductases, versicolorin reductase (Ver1), trihydroxynaphthalene reductase (THNR), and other homologous proteins. In the phylogenetic tree 17beta-HSDcl formed a separate branch with Ver1, while THNRs reside in another branch, indicating that 17beta-HSDcl could have similar function as Ver1. The structural relationship was investigated by comparing a model structure of 17beta-HSDcl to several known crystal structures of the short chain dehydrogenase/reductase (SDR) family. A similarity was observed to structures of bacterial 7alpha-HSD and plant tropinone reductase (TR). Additionally, substrate specificity revealed that among the substrates tested the 17beta-HSDcl preferentially catalyzed reductions of steroid substrates with a 3-keto group, Delta(4) or 5alpha, such as: 4-estrene-3,17-dione and 5alpha-androstane-3,17-dione.     

17β-hydroxysteroid dehydrogenase from the fungus Cochlioboluslunatus: structural and functional aspects

17β-Hydroxysteroid dehydrogenase (17β-HSD) activity has been described in all filamentous fungi tested, but until now only one 17β-HSD from Cochlioboluslunatus (17β-HSDcl) was sequenced. We examined the evolutionary relationship among 17β-HSDcl, fungal reductases, versicolorin reductase (Ver1), trihydroxynaphthalene reductase (THNR), and other homologous proteins. In the phylogenetic tree 17β-HSDcl formed a separate branch with Ver1, while THNRs reside in another branch, indicating that 17β-HSDcl could have similar function as Ver1. The structural relationship was investigated by comparing a model structure of 17β-HSDcl to several known crystal structures of the short chain dehydrogenase/reductase (SDR) family. A similarity was observed to structures of bacterial 7α-HSD and plant tropinone reductase (TR). Additionally, substrate specificity revealed that among the substrates tested the 17β-HSDcl preferentially catalyzed reductions of steroid substrates with a 3-keto group, Δ⁴ or 5α, such as: 4-estrene-3,17-dione and 5α-androstane-3,17-dione.     

Studies on the Microbiological Transformation of Steroids

An attempt was made to clarify how Pellicularia filamentosa f. sp. microsclerotia IFO 6298 capable of hydroxylating C₂₁-steroids at the C-19 position converts C₁₉-steroids, especially monohydroxyderivatives of androst-4-ene-3, 17-dione. Such substrates as 11β-hydroxyandrost-4-ene-3,17-dione (I), androst-4-ene-3, 11, 17-trione (II), androsta-1,4-diene-3, 17-dione (III), 11β-hydroxyandrosta-1,4-diene-3,17-dione (IV), 14α-hydroxyandrost-4-ene-3, 17-dione (V), 15α-hydroxyandrost-4-ene-3, 17-dione (VI) and 9α-hydroxyandrost-4-ene-3, 17-dione (VII) were converted by the organism. All the main and several minor products were then isolated and identified. As a result it is concluded that this organism converts I and II into 14α-hydroxyandrost-4-ene-3,11,17-trione, III and IV into 14α-hydroxyandrosta-1,4-diene-3,1l,17-trione, V into 11α 14α dihydroxyandrost-4-ene-3, 17-dione (main) and 11β, 14α-dihydroxyandrost-4-ene-3, 17-dione (minor, a tentative structure), VI into 11β, 15α-dihydroxyandrost-4-ene-3,17-dione (main) and 15α-hydroxyandrost-4-ene-3,11,17-trione (minor, a tentative structure) and VII into 9α, 14α-dihydroxyandrost-4-ene-3, 17-dione (main) and 6β, 9α-dihydroxyandrost-4-ene-3,17-dione (minor).

In addition, the structural requirement of substrate for the 19-hydroxylation catalyzed by the organism and the influence of a hydroxyl group on steroid nucleus upon the 11β- and 14α-hydroxylations and the 11β-OH-dehydrogenation was discussed.     

Microbial transformation of androst-4-ene-3,17-dione by Beauveria bassiana

The microbial transformation of androst-4-ene-3,17-dione (I) by the fungus Beauveria bassiana CCTCC AF206001 has been investigated using pH 6.0 and 7.0 media. Two hydroxylated metabolites were obtained with the pH 6.0 medium. The major product was 11alpha-hydroxyandrost-4-ene-3,17-dione (II) whereas the minor product was 6beta,11alpha-dihydroxyandrost-4-ene-3,17-dione (III). On the other hand, four hydroxylated and/or reduced metabolites were obtained with the pH 7.0 medium. The major product was 11alpha,17beta-dihydroxyandrost-ene-3-one (V) and the minor products were 17beta-hydroxyandrost-ene-3-one (IV), 6beta,11alpha,17beta-trihydroxyandrost-ene-3-one (VI) and 3alpha,11alpha,17beta-trihydroxy-5alpha-androstane (VII). The products were purified by chromatographic methods, and were identified on the basis of spectroscopic methods. This fungus strain is clearly an efficient biocatalyst for 11alpha-hydroxylation and reduction of the 17-carbonyl group.     

1-Ene-steroid reductase of Mycobacterium sp. NRRL B-3805

The microbial enzymatic reduction of 1,4-androstadiene-3,17-dione (ADD) to 4-androstene-3,17-dione (AD), testosterone and 1-dehydrotestosterone (DHT) is described. Two reducing activities observed in washed cell suspensions and cell free extracts of Mycobacterium sp. NRRL B-3805 were found to account for these bioconversions. One was a 1-ene-steroid reductase and the other a 17-keto steroid reductase. The first reducing activity was found to appear in the soluble cell fraction whereas the latter could be precipitated by centrifugation. Maximum 1-ene-steroid reductase specific activity was achieved during the exponential growth phase of the organism and significantly increased upon induction with ADD. The 1-ene-steroid reductase was partially purified (30-fold) by ammonium sulfate fractionation, gel-filtration and ion-exchange chromatography, and was eluted from a Sephacryl S-300 column with an Mr = 115,000. The 1-ene-steroid reductase activity was NADPH-dependent and had specificity towards steroid compounds containing C-1,2 double bond with an apparent Km for ADD of 2.2 X 10(-5) M. The reverse reaction catalyzing C-1,2 dehydrogenation could not be detected in our preparations. The results suggest that in Mycobacterium sp NRRL B-3805 and B-3683 the steroid C-1,2 dehydrogenation and 1-ene reduction are two separable activities.     

Microbial 16β-Hydroxylation of Steroids with Aspergillus niger

Microbial 16β-hydroxylation of some steroids with Wojnowicia graminis, Corticium centrifugum and Bacillus megaterium has been reported, but not 16β-hydroxylation of normal 17-oxo steroids with Aspergillus niger. This time, we tried microbial transformation of dehydroepiandrosterone with this fungus, and obtained 4-androstene-3,17-dione, 17β-hydroxy-4-androstene-3,16-dione, 16β,17β-dihydroxy-4-androsten-3-one and a new compound, 16β-hydroxy-4-androstene-3,17-dione. This new compound was also obtained by the fermentation of 4-androstene-3,17-dione and testosterone.     

Studies on the microbial transformation of androst-1,4-dien-3,17-dione with Acremonium strictum

The strain of Acremonium strictum PTCC 5282 was applied to investigate the biotransformation of androst-1,4-dien-3,17-dione (I; ADD). Microbial products obtained were purified by preparative TLC and the pure metabolites were characterized on the basis of their spectroscopic features (¹³C NMR, ¹H NMR, FTIR, MS) and physical constants (melting points and optical rotations). The 15α-Hydroxyandrost-1,4-dien-3,17-dione (II), 17β-hydroxyandrost-1,4-dien-3-one (III), androst-4-en-3,17-dione (IV; AD), 15α-hydroxyandrost-4-en-3,17-dione (V), 15α,17β-dihydroxyandrost-1,4-dien-3-one (VI) and testosterone (VII) were produced during this fermentation. Formation of the 15α,17β-dihydroxy derivative of ADD is reported for the first time during steroid biotransformation. The bioconversion reactions observed were 1,2-hydrogenation, 15α-hydroxylation and 17-ketone reduction. From the time course profile of this biotransformation, ketone reduction and 1,2-hydrogenation were observed from the first day of fermentation while 15α-hydroxylation occurred from the third day. Optimum concentration of the substrate, which gave the maximum bioconversion efficiency, was 0.5 mg ml⁻¹ in one batch. The highest yield of the microbial products recorded in this work was achieved within the pH range 6.5–7.3 and at the temperature of 27 °C.     

Nostoc muscorum: a regioselective biocatalyst for 17-carbonyl reduction of androst-4-en-3,17-dione and androst-1,4-dien-3,17-dione

Nostoc muscorum PTCC 1636 was examined for its ability to convert androst-4-en-3,17-dione (AD) and androst-1,4-dien-3,17-dione (ADD) to their 17-hydroxy related derivatives in BG-11 medium. Bioconversion procedures were carried out at 25 °C without shaking. The metabolites obtained were purified using chromatographic methods and characterized as testosterone and 1-dehydrotestosterone on the basis of their spectroscopic features. In both cases, the bioreaction characteristics observed were 17-carbonyl reduction.     

Three-dimensional model of cytochrome P450 human aromatase

A three-dimensional (3-D) structure of human aromatase (CYP19) was modeled on the basis of the crystal structure of rabbit CYP2C5, the first solved X-ray structure of an eukaryotic cytochrome P450 and was evaluated by docking S-fadrozole and the steroidal competitive inhibitor (19R)-10-thiiranylestr-4-ene-3,17-dione, into the enzyme active site. According to a previous pharmacophoric hypothesis described in the literature, the cyano group of S-fadrozole partially mimics the steroid backbone C(17) carbonyl group of (19R)-10-thiiranylestr-4-ene-3,17-dione, and was oriented in a favorable position for H-bonding with the newly identified positively charged residues Lys119 and Arg435. In addition, this model is consistent with the recent combined mutagenesis/modeling studies already published concerning the roles of Asp309 and His480 in the aromatization of the steroid A ring.     

Catalysis of the isomerization of Δ5-3-ketosteroids to Δ4-3-ketosteroids by primary amines: Evidence for an imine intermediate

The isomerization of 5-androstene-3,17-dione and 17β-hydroxy-5-androstene-3-one to 4-androstene-3,17-dione and 17β-hydroxy-4-androstene-3-one, respectively, is catalyzed by primary amines. In the case of the isomerization catalyzed by glycylglycine the reaction proceeds through an intermediate which absorbs maximally at 275 nm. Based on spectral similarities to appropriate model compounds and structural analysis of the intermediate after its reduction by sodium borohydride, the intermediate has been tentatively identified as the Δ⁴-3-imine.     

Biotransformation of 3b-Hydroxyandrost-5-en-17-one by Cell Suspension Cultures of Catharanthus roseus

Catharanthus roseus (L.) G. Don cell suspension cultures were used to transform 3b-hydroxyandrost-5-en-17-one, the products were isolated by chromatographic methods. Their structures were established by means of NMR and MS spectral analyses. Nine metabolites were respectively elucidated as: androst-4-ene-3,17-dione (Ⅰ), 6a-hydroxyandrost-4-ene-3,17-dione (Ⅱ), 6a,17b-dihydroxyandrost-4-en-3-one (Ⅲ), 6b-hydroxyandrost-4-ene-3,17-dione (Ⅳ), 17b-hydroxyandrost-4-en-3-one (Ⅴ), 15a,17b-dihydroxyandrost-4-en-3-one (Ⅵ), 15b,17b-dihydroxyandrost-4-en-3-one (Ⅶ), 14a-hydroxyandrost-4-ene-3,17-dione (Ⅷ), 17b-hydroxyandrost-4-ene-3,16-dione (Ⅸ). It is the first time to obtain the above compounds by biotransformation with Catharanthus roseus cell cultures.     

7α-Alkyltestosterone derivatives: Synthesis and activity as androgens and as aromatase inhibitors

The 7α-ethyl,propyl,butyl,3'-t-butoxypropyl, allyl,3'-hydroxypropyl 17-acetate, and 3'-chloropropyl 17-acetate derivatives of testosterone and the 7α-3'-t-butoxypropyl,3'-hydroxypropyl,3'-acetoxypropyl, 3'-bromoacetoxypropyl,3'-chloropropyl, and 2'-oxo-3'-bromopropyl derivatives of 4-androstene-3,17-dione were synthesized. The testosterone derivatives were found to lose androgenic and anabolic activity rapidly as the size of the group at the 7 position increased. Many of the compounds were tested as inhibitors of aromatase. The 17-keto compounds were more active than the corresponding alcohols and the enzyme was found to tolerate at least the bulk of a hydroxypropyl group at the C-7α position.     

微生物发酵降解植物甾醇侧链生产17-酮甾体研究进展

微生物发酵降解植物甾醇侧链,生产雄甾-4-烯-3,17-二酮(AD),雄甾-1,4-二烯-3,17-二酮(ADD),和9α-羟基-AD甾体药物中间体的工业生物技术对改变制造甾体激素药物半合成原料薯蓣皂素短缺的现状,实现甾体激素药物半合成原料多元化,合理利用我国甾体植物资源具有重要意义。重点评述了近期微生物法断植物甾醇侧链制AD、ADD和9α-羟基-AD的研究现状,内容包括:1)微生物菌种选育;2)菌种相关的细胞生理,酶学性质和生物催化过程;3)相关酶的细胞定位及生物反应器;4)发酵工艺选择和甾醇原料的合理利用。     

The altered specificity of cortisone reductase with certain retroandrostan-3-one substrates.

The retro steroids 17beta-hydroxy-5beta,9beta,10alpha-androstan-3-one and 5beta,9beta,10alpha-androstane-3,17-dione were good substrates for cortisone reductase in the presence of NADH, and the products corresponded to the respective 3beta-hydroxy compounds, in which the 3beta-hydroxyl group is axial and the absolute configuration is 3S. The analogous natural steroids 17beta-hydroxy-5beta,9alpha,10beta-androstan-3-one and 5beta,9alpha,10beta-androstane-3,17-dione were very poor substrates, and gave the corresponding 3alpha(equatorial,3R)-hydroxy compounds, and, in the latter case, also an appreciable amount of 3beta(axial, 3S)-hydroxy-5beta,9alpha,10beta-androstan-17-one. The natural steroids 17beta-hydroxy-5alpha,9alpha,10beta-androstan-3-one and 5alpha,9alpha,10beta-androstane-3,17-dione were better substrates than the retro steroid 17beta-hydroxy-5alpha,9beta,10alpha-androstan-3-one, but were not such good substrates as the retro steroids 17beta-hydroxy-5beta,9beta,10alpha-androstan-3-one and 5beta,9beta,10alpha-androstane-3,17-dione. Unlike these retro steroid 5beta,9beta,10alpha-androstan-3-ones, the natural steroids 17beta-hydroxy-5alpha,9alpha,10beta-androstan-3-one and 5alpha,9alpha,10beta-androstane-3,17-dione gave the corresponding 3alpha(axial,3R)-hydroxy compounds. The retro steroid 17beta-hydroxy-5alpha,9beta,10alpha-androstan-3-one was not a good substrate, and the product of reaction corresponded to the 3alpha(axial,3R)-hydroxy compound. The nature of substrate recognition by this enzyme is discussed in the light of these structure-activity relationships.