- Regio- And Stereo-chemical Effects in the Hydroboration of Δ2-Steroidal Allylic and Homoallylic Alcohols
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A comparison between the hydroboration of δ2-steroidal 1x-allylic and 5x-homoallylic alcohols reveals that whereas both have a stereochemical directing effect, only the allylic alcohol modifies the regiospecificity of the reaction.
- Hanson, James R.,Liman, Mansur D.,Nagaratnam, Sivajini
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- Estradiol-17β inhibition of androgen uptake, metabolism and binding in epididymis of adult male rats in vivo: A comparison with cyproterone acetate
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The effects of estradiol-17β on androgen uptake, metabolism and binding were studied in rat epididymis in vivo in comparison with cyproterone acetate. Steroids (250 ug/100 g body weight) were injected 5 min prior to 3H-testosterone in castrate rats. Estradiol-17 β inhibited 3H-testosterone uptake into epididymal cytosol by 58% as compared to 38% by cyproterone acetate. 3H-Testosterone uptake into epididymal nuclei was inhibited 95% by estradiol-17 β and 83% by cyproterone acetate. Total bound radioactivity in cytosol fractions was reduced to a greater extent by estradiol-17 β than cyproterone acetate when either 3H-testosterone or 3H-dihydrotestosterone was injected. Binding of 3H-dihydrotestosterone to nuclear receptors was completely abolished by estradiol-17 β ; whereas approximately 20% binding remained in the nuclear extract after cyproterone acetate treatment. Metabolism of 3H-testosterone in vivo was also altered by estradiol-17 β, resulting in diminished conversion to 3H-dihydrotestosterone. Cyproterone acetate, on the other hand, did not affect 3H-testosterone metabolism. Estradiol-17 β and cyproterone acetate inhibited in vitro binding of 3H-dihydrotestosterone to the intracellular cytoplasmic receptor, but not the intraluminal androgen binding protein (ABP). These data suggest that estradiol-17 β may have a more potent antiandrogenic effect on the epididymis than cyproterone acetate due to inhibition of 5 α reduction of testosterone as well as binding to the androgen receptor.
- Tindall,French,Nayfeh
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- Comparative studies of androgen metabolism in theca and granulosa cells of human follicles in vitro
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In eight separate experiments, theca and granulosa were isolated from human follicles (5-25 mm in diameter) , and their capacities to metabolize radiolabelled testosterone in 24 hour cultures were assessed. Theca metabolized testosterone primarily to androstenedione, however significant aromatization to estradiol-17β and to estrone was also observed. Granulosa metabolized testosterone primarily to estradiol-17β and estrone, while smaller quantities were converted to androstenedione. In seven of these experiments, the intermediate of aromatization, 19-hydroxytestosterone, was identified. In six of these experiments, theca, when compared to granulosa, produced more androstenedione but less estradiol-17β and estrone. 5α-Reduced androgens were non-detectable or produced in small quantities. In a single experiment, metabolism of androstenedione was compared to metabolism of testosterone by both theca and granulosa. Theca metabolized androstenedione to testosterone in smaller quantities than testosterone to androstenedione. Granulosa metabolized androstenedione to testosterone in higher quantities than testosterone to androstenedione. Both theca and granulosa aromatized androstenedione more readily than testosterone.
- Moon,Duleba
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- CONJUGATION OF ANDROGENS AND ESTROGENS BY HUMAN BREAST TUMORS IN VITRO
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The metabolism of 3H-androstenedione (Δ4-A) and 3H-estriol (E3) was studied in 12 human breast tumors.Part of each tumor was analyzed for estrogen receptor content.Aliquots of tumor homogenates were incubated for 2 hr separately with 3H-Δ4-A and 3H-E3 in the presence of appropriate cofactors.No distinct differences emerged in the profiles of the unconjugated metabolites of 3H-Δ4-A, the major compounds in the approximate order of descendence being androsterone, androstanedione, testosterone, 5α-androstane-3α,17β-diol, epiandrosterone, and dihydrotestosterone.One tumor homogenate from an infiltrating lobular carcinoma converted 3H-Δ4-A to glucosiduronate metabolites (11percent), of which androsterone, 6.4percent; testosterone, 1.6percent; and androstanediol, 0.6percent predominated.The homogenate of this tumor and two other tumors converted 3H-E3 to 3H-E3-3S.Conversions of E3 to E3-3S in the other tumor homogenates were less than 0.6percent.No correlation between receptor content and the capability of the tumor to conjugate Δ4-A or E3 evolved.However, correlations between steroid hormone metabolism and tumor histopathology may exist.
- Raju, Uma,Blaustein, Ancel,Levitz, Mortimer
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- A molecularly imprinted receptor for separation of testosterone and epitestosterone, based on a steroidal cross-linker
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A series of molecularly imprinted polymers have been prepared and investigated as stationary phases in high performance liquid chromatography for the separation of testosterone and epitestosterone using non-polar mobile phases. The polymers were imprinted using 5α-dihydrotestosterone as template, and all retain testosterone more strongly than its 17α-OH epimer. The best polymer was prepared using trifluoromethylacrylic acid as functional monomer (interacting with the template via hydrogen bonds), divinylbenzene as 'inert' cross-linker, and chloroform as porogen. It also included a steroid-based cross-linker, which may interact with the template via van der Waals interactions to lend additional 'shape selectivity'. A 250 × 4.6 mm column packed with this polymer gave baseline resolution of testosterone and epitestosterone (15 μg each) in under 20 min. Preparation of the steroid based cross-linker included the selective reduction of 5α- dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) to the 3α,17β-diol using K-selectride.
- Gavrilovi?, Ivana,Mitchell, Karen,Brailsford, Alan D.,Cowan, David A.,Kicman, Andrew T.,Ansell, Richard J.
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- Glucosiduronidation and esterification of androsterone by human breast tumors in vitro
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The metabolism of 3H-androsterone was studied in homogenates (fortified with uridine 5'-diphosphoglucuronic acid and andenosine 3'- phosphate 5'-phosphosulfate) of eighteen breast tumors, one muscle underlying the primary breast carcinoma and metastatic axillary lymph nodes from a patient with suspected primary breast cancer. The major metabolites identified were less polar than androsterone. On saponification these lipoidal derivatives afforded androsterone as the only product (3 to 48%). Unmetabolized androsterone and lesser quantities of epiandrosterone, 5α-andorstane-3α, 17β-diol and 5α-androstane-3,17-dione comprised the free steroid fraction. Androsterone glucosiduronate was isolated (0.17-4.1%) from eight breast tumor homogenates and fromthe node tissue incubation (17%). There was no apparent correlation between glucuronyltransferase activity and histolpathology or estrogen receptor content.
- Raju,Kadner,Levitz,Kaganowicz,Blaustein
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- Formation of 5α-dihydrotestosterone from 5α-androstane-3α,17β-diol in prostate cancer LAPC-4 cells – Identifying inhibitors of non-classical pathways producing the most potent androgen
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5α-Dihydrotestosterone (5α-DHT) possesses a great affinity for the androgen receptor (AR), and its binding to AR promotes the proliferation of prostate cancer (PC) cells in androgen-dependent PC. Primarily synthesized from testosterone (T) in testis, 5α-DHT could also be produced from 5α-androstane-3α,17β-diol (3α-diol), an almost inactive androgen, following non-classical pathways. We reported the chemical synthesis of non-commercially available [4-14C]-3α-diol from [4-14C]-T, and the development of a biological assay to identify inhibitors of the 5α-DHT formation from radiolabeled 3α-diol in LAPC-4 cell PC model. We measured the inhibitory potency of 5α-androstane derivatives against the formation of 5α-DHT, and inhibition curves were obtained for the most potent compounds (IC50 = 1.2–14.1 μM). The most potent inhibitor 25 (IC50 = 1.2 μM) possesses a 4-(4-CF3-3-CH3O-benzyl)piperazinyl methyl side chain at C3β and 17β-OH/17α-C[tbnd]CH functionalities at C17 of a 5α-androstane core.
- Boutin, Sophie,Roy, Jenny,Maltais, René,Poirier, Donald
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- STUDIES ON THE KINETICS OF THE INTERACTION OF 7α-HYDROXYTESTOSTERONE WITH THE STEROID 5α-REDUCTASE
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Microsomal preparations from adult male rat testicular interstitial cells were incubated with tritiated testosterone.Added 7α-hydroxytestosterone, (7α,17β-dihydroxy-4-androsten-3-one), at levels which appear to exist in the adult testis, inhibited production of labelled 5α-reduced steroids in a graded fashion.This interaction is not competitive and occurs only at high substrate levels, such as those found in steroid-producing organs.Relationships to pubertal changes in steroid metabolism are discussed.
- Mittler, James C.
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- STUDY OF THE DIRECT EFFECT OF LHRH AGONIST ON TESTICULAR 17-HYDROXYLASE AND 5α-REDUCTASE ACTIVITIES IN NON-HYPOPHYSECTOMIZED ADULT RATS TREATED WITH AN ANTI-LUTEINIZING HORMONE SERUM
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In order to study both direct and pituitary-mediated mechanisms of action of the LHRH analogue 6, des-Gly-NH210>LHRH ethylamide upon testicular steroidogenesis in adult rat, we compared the effects of the agonist when administered alone or concomitantly with an anti-LH serum to non-hypophysectomized rats.Testicular steroid contents and in vitro progesterone and testosterone metabolism were determined.Anti-LH serum administration was able to prevent 5α-reductase stimulation by the agonistic peptide, but not the inhibition of 17-hydroxylase activity.These data suggest that modulation of 17-hydroxylase involves both direct and pituitary-mediated processes, while 5α-reductase stimulation is mainly if not only due to a pituitary-mediated mechanism.
- Carmichael, Rejean,Belanger, Alain
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- Role of human 3α-hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C3) in the extrahepatic metabolism of the steroidal aromatase inactivator Formestane
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The clinical use of the steroidal aromatase inhibitor Formestane (4-hydroxandrostenedione, 4-OHA) in the treatment of advanced ER+ breast cancer has been discontinued, and therefore, interest in this remarkable drug has vanished. As a C-19 sterol, 4-OHA can undergo extensive intracellular metabolism depending on the expression of specific enzymes in the corresponding cells. We used the metabolites 4β-hydroxyandrosterone, 4β-hydroxyepiandrosterone and its 17β-reduced derivative as standards for the proof of catalytic activity present in the cell culture medium and expressed by the isolated enzymes. All of the aldo-keto reductases AKR1C1, AKR1C2, AKR1C3 and AKR1C4 catalysed the reduction of the 3-keto-group and the Δ4,5 double bond of 4-OHA at the same time. Molecular docking experiments using microscale thermophoresis and the examination of the kinetic behaviour of the isolated enzymes with the substrate 4-OHA proved that AKR1C3 had the highest affinity for the substrate, whereas AKR1C1 was the most efficient enzyme. Both enzymes (AKR1C1and AKR1C3) are highly expressed in adipose tissue and lungs, exhibiting 3β-HSD activity. The possibility that 4-OHA generates biologically active derivatives such as the androgen 4-hydroxytestosterone or some 17β-hydroxy derivatives of the 5α-reduced metabolites may reawaken interest in Formestane, provided that a suitable method of administration can be developed, avoiding oral or intramuscular depot-injection administration.
- Wan, Runlan,Kong, Xi,Yang, Youzhe,Tao, Siwen,Chen, Youyou,Teichmann, Alexander Tobias,Wieland, Frank Heinrich
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- A 5 α- androstanedione method for the preparation of
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The invention discloses a preparation method of 5alpha-androstanedion. The method comprises steps of a reduction reaction, a hydrogenation reaction and an oxidation reaction. The chemical synthesis method for preparing 5alpha-androstanedion avoids the disadvantages of long middle route, high danger, low yield and serious environmental pollution of a traditional production method. Compared to the traditional production method, the method provided by the invention has easily controlled reaction conditions, simple operations, high yield and reduced cost, and the yield of refined product is more than 70% in weight. The raw materials used in the method can be obtained by fermentation of phytosterols, which have wide source and low price; therefore, the method has the characteristics of easily available raw materials and low cost.
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Paragraph 0038; 0039
(2017/01/12)
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- Rapid probing of the reactivity of P450 monooxygenases from the CYP116B subfamily using a substrate-based method
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Developing a detailed understanding of the reactivity of self-sufficient Type IV P450 monooxygenases, four types of O-methylated substrates were designed as probes, including monoterpenes, cycloalkanes, aromatic compounds and steroids, and the efficiency of their oxyfunction was determined using a colorimetric assay which was based on the reaction between the enzymatic demethylation product, formaldehyde, and Purpald dye. The activity-based fingerprints of new P450RpMO, P450ArMO and P450CtMO (CYP116B members) indicated that CYP116B P450s preferentially oxidize substrates with aromatic components. Moreover, the hydroxylated products were detected based on the preference results. This rapid and efficient strategy, when coupled with GCMS, enables the exploration of the reactivity of other CYP116B members.
- Li, Ren-Jie,Xu, Jian-He,Yin, Yue-Cai,Wirth, Nicolas,Ren, Jiang-Meng,Zeng, Bu-Bing,Yu, Hui-Lei
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supporting information
p. 8928 - 8934
(2016/10/13)
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- The Stereoselective Reductions of Ketones to the Most Thermodynamically Stable Alcohols Using Lithium and Hydrated Salts of Common Transition Metals
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A simple method is presented for the highly stereoselective reductions of ketones to the most thermodynamically stable alcohols. In this procedure, the ketone is treated with lithium dispersion and either FeCl2·4H2O or CuCl2·2H2O in THF at room temperature. This protocol is applied to a large number and variety of ketones and is both more convenient and efficient than those commonly reported for the diastereoselective reduction of five- and six-membered cyclic ketones.
- Kennedy, Nicole,Cohen, Theodore
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p. 8134 - 8141
(2015/09/02)
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- Substrate specificity and inhibitor sensitivity of rabbit 20α-hydroxysteroid dehydrogenase
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In this study, we examined the substrate specificity and inhibitor sensitivity of rabbit 20α-hydroxysteroid dehydrogenase (AKR1C5), which plays a role in the termination of pregnancy by progesterone inactivation. AKR1C5 moderately reduced the 3-keto group of only 5α-dihydrosteroids with 17β- or 20α/β-hydroxy group among 3-ketosteroids. In contrast, the enzyme reversibly and efficiently catalyzed the reduction of various 17- and 20-ketosteroids, including estrogen precursors (dehydroepiandrosterone, estrone and 5α-androstan-3β- ol-17-one) and tocolytic 5β-pregnane-3,20- dione. In addition to the progesterone inactivation, the formation of estrogens and metabolism of the tocolytic steroid by AKR1C5 may be related to its role in rabbit parturition. AKR1C5 also reduced various non-steroidal carbonyl compounds, including isatin, an antagonist of the C-type natriuretic peptide receptor, and 4-oxo-2-nonenal, suggesting its roles in controlling the bioactive isatin and detoxification of cytotoxic aldehydes. AKR1C5 was potently and competitively inhibited by flavonoids such as kaempferol and quercetin, suggesting that its activity is affected by ingested flavonoids.
- Endo, Satoshi,Arai, Yuki,Hara, Akira,Kitade, Yukio,Bunai, Yasuo,El-Kabbani, Ossama,Matsunaga, Toshiyuki
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p. 1514 - 1518
(2013/10/08)
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- Rabbit 3-hydroxyhexobarbital dehydrogenase is a NADPH-preferring reductase with broad substrate specificity for ketosteroids, prostaglandin D2, and other endogenous and xenobiotic carbonyl compounds
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3-Hydroxyhexobarbital dehydrogenase (3HBD) catalyzes NAD(P) +-linked oxidation of 3-hydroxyhexobarbital into 3-oxohexobarbital. The enzyme has been thought to act as a dehydrogenase for xenobiotic alcohols and some hydroxysteroids, but its physiological function remains unknown. We have purified rabbit 3HBD, isolated its cDNA, and examined its specificity for coenzymes and substrates, reaction directionality and tissue distribution. 3HBD is a member (AKR1C29) of the aldo-keto reductase (AKR) superfamily, and exhibited high preference for NADP(H) over NAD(H) at a physiological pH of 7.4. In the NADPH-linked reduction, 3HBD showed broad substrate specificity for a variety of quinones, ketones and aldehydes, including 3-, 17- and 20-ketosteroids and prostaglandin D2, which were converted to 3α-, 17β- and 20α-hydroxysteroids and 9α,11β- prostaglandin F2, respectively. Especially, α-diketones (such as isatin and diacetyl) and lipid peroxidation-derived aldehydes (such as 4-oxo- and 4-hydroxy-2-nonenals) were excellent substrates showing low Km values (0.1-5.9 μM). In 3HBD-overexpressed cells, 3-oxohexobarbital and 5β-androstan-3α-ol-17-one were metabolized into 3-hydroxyhexobarbital and 5β-androstane-3α,17β-diol, respectively, but the reverse reactions did not proceed. The overexpression of the enzyme in the cells decreased the cytotoxicity of 4-oxo-2-nonenal. The mRNA for 3HBD was ubiquitously expressed in rabbit tissues. The results suggest that 3HBD is an NADPH-preferring reductase, and plays roles in the metabolisms of steroids, prostaglandin D2, carbohydrates and xenobiotics, as well as a defense system, protecting against reactive carbonyl compounds.
- Endo, Satoshi,Matsunaga, Toshiyuki,Matsumoto, Atsuko,Arai, Yuki,Ohno, Satoshi,El-Kabbani, Ossama,Tajima, Kazuo,Bunai, Yasuo,Yamano, Shigeru,Hara, Akira,Kitade, Yukio
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p. 1366 - 1375
(2013/11/19)
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- The regio- and stereo-selective reduction of steroidal 4-en-3-ones using Na2S2O4/NaHCO3 and CuCl/NaBH 4
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This paper describes the regio- and stereoselective reduction of a.
- Wang, Chunli,Chen, Xiaoyu,Huang, Yaoqing,Yang, Jesse,Chen, Ying
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p. 1339 - 1346
(2013/11/19)
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- Conversion of human steroid 5β-reductase (AKR1D1) into 3β-hydroxysteroid dehydrogenase by single point mutation E120H: Example of perfect enzyme engineering
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Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C enzymes are essential for bile acid biosynthesis and steroid hormone metabolism. AKR1D1 catalyzes the 5β-reduction of Δ4-3- ketosteroids, whereas AKR1C enzymes are hydroxysteroid dehydrogenases (HSDs). These enzymes share high sequence identity and catalyze 4-pro-(R)-hydride transfer from NADPH to an electrophilic carbon but differ in that one residue in the conserved AKR catalytic tetrad, His120 (AKR1D1 numbering), is substituted by a glutamate in AKR1D1. We find that the AKR1D1 E120H mutant abolishes 5β-reductase activity and introduces HSD activity. However, the E120H mutant unexpectedly favors dihydrosteroids with the 5α-configuration and, unlike most of the AKR1C enzymes, shows a dominant stereochemical preference to act as a 3β-HSD as opposed to a 3α-HSD. The catalytic efficiency achieved for 3β-HSD activity is higher than that observed for any AKR to date. High resolution crystal structures of the E120H mutant in complex with epiandrosterone, 5β-dihydrotestosterone, and Δ4-androstene-3,17-dione elucidated the structural basis for this functional change. The glutamate-histidine substitution prevents a 3-ketosteroid from penetrating the active site so that hydride transfer is directed toward the C3 carbonyl group rather than the Δ4-double bond and confers 3β-HSD activity on the 5β-reductase. Structures indicate that stereospecificity of HSD activity is achieved because the steroid flips over to present its α-face to the A-face of NADPH. This is in contrast to the AKR1C enzymes, which can invert stereochemistry when the steroid swings across the binding pocket. These studies show how a single point mutation in AKR1D1 can introduce HSD activity with unexpected configurational and stereochemical preference.
- Chen, Mo,Drury, Jason E.,Christianson, David W.,Penning, Trevor M.
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experimental part
p. 16609 - 16622
(2012/07/30)
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- Characterization of rabbit aldose reductase-like protein with 3β-hydroxysteroid dehydrogenase activity
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In this study, we isolated the cDNA for a rabbit aldose reductase-like protein that shared an 86% sequence identity to human aldo-keto reductase (AKR)1 1B10 and has been assigned as AKR1B19 in the AKR superfamily. The purified recombinant AKR1B19 was similar to AKR1B10 and rabbit aldose reductase (AKR1B2) in the substrate specificity for various aldehydes and α-dicarbonyl compounds. In contrast to AKR1B10 and AKR1B2, AKR1B19 efficiently reduced 3-keto-5α/β-dihydro-C19/C21/C24-steroids into the corresponding 3β-hydroxysteroids, showing Km of 1.3-9.1 μM and kcat of 1.1-7.6 min-1. The stereospecific reduction was also observed in the metabolism of 5α- and 5β- dihydrotestosterones in AKR1B19-overexpressing cells. The mRNA for AKR1B19 was ubiquitously expressed in rabbit tissues, and the enzyme was co-purified with 3β-hydroxysteroid dehydrogenase activity from the lung. Thus, AKR1B19 may function as a 3-ketoreductase, as well as a defense system against cytotoxic carbonyl compounds in rabbit tissues. The molecular determinants for the unique 3-ketoreductase activity were investigated by replacement of Phe303 and Met304 in AKR1B19 with Gln and Ser, respectively, in AKR1B10. Single and double mutations (F303Q, M304S and F303Q/M304S) significantly impaired this activity, suggesting the two residues play critical roles in recognition of the steroidal substrate.
- Endo, Satoshi,Matsunaga, Toshiyuki,Kumada, Sho,Fujimoto, Airi,Hara, Akira,Ohno, Satoshi,El-Kabbani, Ossama,Hu, Dawei,Toyooka, Naoki,Mano, Jun'Ichi,Tajima, Kazuo
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p. 23 - 30,8
(2020/08/20)
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- Stereospecific reduction of 5β-reduced steroids by human ketosteroid reductases of the AKR (aldo-keto reductase) superfamily: Role of AKR1C1-AKR1C4 in the metabolism of testosterone and progesterone via the 5β-reductase pathway
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Active sex hormones such as testosterone and progesterone are metabolized to tetrahydrosteroids in the liver to terminate hormone action. One main metabolic pathway, the 5β-pathway, involves 5β-steroid reductase (AKR1D1, where AKR refers to the aldo-keto reductase superfamily), which catalyses the reduction of the 4-ene structure, and ketosteroid reductases (AKR1C1-AKR1C4), which catalyse the subsequent reduction of the 3-oxo group. The activities of the four human AKR1C enzymes on 5β-dihydrotestosterone, 5β-pregnane-3,20-dione and 20α-hydroxy-5β-pregnan-3-one, the intermediate 5β-dihydrosteroids on the 5β-pathway of testosterone and progesterone metabolism, were investigated. Product characterization by liquid chromatography-MS revealed that the reduction of the 3-oxo group of the three steroids predominantly favoured the formation of the corresponding 3α-hydroxy steroids. The stereochemistry was explained by molecular docking. Kinetic properties of the enzymes identified AKR1C4 as the major enzyme responsible for the hepatic formation of 5β-tetrahydrosteroid of testosterone, but indicated differential routes and roles of human AKR1C for the hepatic formation of 5β-tetrahydrosteroids of progesterone. Comparison of the kinetics of the AKR1C1-AKR1C4-catalysed reactions with those of AKR1D1 suggested that the three intermediate 5β-dihydrosteroids derived from testosterone and progesterone are unlikely to accumulate in liver, and that the identities and levels of 5β-reduced metabolites formed in peripheral tissues will be governed by the local expression of AKR1D1 and AKR1C1-AKR1C3. The Authors Journal compilation 2011 Biochemical Society.
- Jin, Yi,Mesaros, A. Clementina,Blair, Ian A.,Penning, Trevor M.
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supporting information; experimental part
p. 53 - 61
(2012/06/15)
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- Liquid chromatography-mass spectrometry (LC-MS) of steroid hormone metabolites and its applications
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Advances in liquid chromatography-mass spectrometry (LC-MS) can be used to measure steroid hormone metabolites in vitro and in vivo. We find that LC-electrospray ionization (ESI)-MS using a LCQ ion trap mass spectrometer in the negative ion mode can be used to monitor the product profile that results from 5α-dihydrotestosterone (DHT)-17β-glucuronide, DHT-17β-sulfate, and tibolone-17β-sulfate reduction catalyzed by human members of the aldo-keto reductase (AKR) 1C subfamily and assign kinetic constants to these reactions. We also developed a stable isotope dilution LC-electron capture atmospheric pressure chemical ionization (ECAPCI)-MS method for the quantitative analysis of estrone (E1) and its metabolites as pentafluorobenzyl (PFB) derivatives in human plasma in the attomole range. The limit of detection for E1-PFB was 740. attomole on column. Separations can be performed using normal-phase LC because ionization takes place in the gas phase rather than in solution. This permits efficient separation of the regioisomeric 2- and 4-methoxy-E1. The method was validated for the simultaneous analysis of plasma E2 and its metabolites: 2-methoxy-E2, 4-methoxy-E2, 16α-hydroxy-E2, estrone (E1), 2-methoxy-E1, 4-methoxy-EI, and 16α-hydroxy-E1 from 5. pg/mL to 2000. pg/mL. Our LC-MS methods have sufficient sensitivity to detect steroid hormone levels in prostate and breast tumors and should aid their molecular diagnosis and treatment.
- Penning, Trevor M.,Lee, Seon-Hwa,Jin, Yi,Gutierrez, Alejandro,Blair, Ian A.
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experimental part
p. 546 - 555
(2011/12/02)
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- Transformation of some 3α-substituted steroids by Aspergillus tamarii KITA reveals stereochemical restriction of steroid binding orientation in the minor hydroxylation pathway
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Aspergillus tamarii contains an endogenous lactonization pathway which can transform progesterone to testololactone in high yield through a sequential four step enzymatic pathway. In this pathway testosterone is formed which primarily undergoes oxidation of the C-17β-alcohol to a C-17 ketone but, can also enter a minor hydroxylation pathway where 11β-hydroxytestosterone is produced. It was recently demonstrated that this hydroxylase could monohydroxylate 3β-hydroxy substituted saturated steroidal lactones in all four possible binding orientations (normal, reverse, inverted normal, inverted reverse) on rings B and C of the steroid nucleus. It was therefore of interest to determine the fate of a series of 3α-substituted steroidal analogues to determine stereochemical effect on transformation. Hydroxylation on the central rings was found to be restricted to the 11β-position (normal binding), indicating that the 3α-stereochemistry removes freedom of binding orientation within the hydroxylase. The only other hydroxylation observed was at the 1β-position. Interestingly the presence of this functional group did not prevent lactonization of the C-17 ketone. In contrast the presence of the 11β-hydroxyl completely inhibited Baeyer-Villiger oxidation, a result which again demonstrates that single functional groups can exert significant control over metabolic handling of steroids in this organism. This may also explain why lactonization of 11β-hydroxytestosterone does not occur. Lactonization of the C-17 ketone was not significantly affected by the 3α-alcohol with significant yields achieved (53%). Interestingly a time course experiment demonstrated that the presence of the 3α-acetate inhibited the Baeyer-Villiger monooxygenase with its activity being observed 24 h later than non-acetate containing analogues. Apart from oxidative transformations observed a minor reductive pathway was revealed with the C-17 ketone being reduced to a C-17β-alcohol for the first time in this organism.
- Christy Hunter,Khuenl-Brady, Hedda,Barrett, Patrice,Dodd, Howard T.,Dedi, Cinzia
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experimental part
p. 171 - 176
(2011/02/22)
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- Metal coordination-directed hydroxylation of steroids with a novel artificial P-450 catalyst
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A novel catalyst has been synthesized in which a manganese-porphyrin unit is linked to two 2,2′-bipyridyl groups and two pentafluorophenyl groups in trans fashion on its four meso positions. Relative to a previous catalyst in which the manganese-porphyrin had four 2,2′-bipyridyl groups, the new catalyst, in the presence of Cu2+ ions as coordinating linkers, catalyzes the oxidation of a steroid substrate with much better regioselectivity and higher turnover numbers.
- Fang, Zhenglai,Breslow, Ronald
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p. 251 - 254
(2007/10/03)
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- Substrate specificity of a mouse aldo-keto reductase (AKR1C12)
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AKR1C12, a mouse member of the aldo-keto reductase (AKR) superfamily, is highly expressed in the stomach and is identical to a protein encoded in an interleukin-3-regulated gene in mouse myeloid cells, but its function remains unknown. In this study, the recombinant AKR1C12 was purified to homogeneity and the specificity for coenzymes and substrates was examined at a physiological pH of 7.4. The enzyme reduced various α-dicarbonyl compounds, several ketosteroids, aldehydes and some ketones using NADH as the preferred coenzyme. In the reverse reaction, the enzyme showed coenzyme preference for NAD +, and oxidized 3α-, 17β- and 20α-hydroxysteroids, and non-steroidal aliphatic and alicyclic alcohols, of which many hydroxysteroids and geranylgeraniol were good substrates, exhibiting low K m and high kcat/Km values. The results, together with the intracellular high ratio of NAD+/NADH, suggest that AKR1C12 functions as a dehydrogenase for the endogenous hydroxysteroids and geranylgeraniol in mouse stomach and myeloid cells.
- Endo, Satoshi,Matsumoto, Kengo,Matsunaga, Toshiyuki,Ishikura, Shuhei,Tajima, Kazuo,El-Kabbani, Ossama,Hara, Akira
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p. 2488 - 2492
(2007/10/03)
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- The microbiological hydroxylation of 3α,17β- and 3β,17α-dihydroxy-5α-androstanes by Cephalosporium aphidicola
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Both 3α,17β- and 3β,17α-dihydroxy-5α-androstanes are hydroxylated at C-6β by the fungus, Cephalosporium aphidicola but the 3α,17β-diol is also hydroxylated at C-7α whereas the 3β,17α-diol is hydroxylated at C-11β.
- Hanson, James R.,Hunter, A. Christy
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p. 216 - 217
(2007/10/03)
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- Acceleration of the reduction of aldehydes and ketones using Mn(dpm)3 catalyst and phenylsilane in the presence of dioxygen
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Saturated ketones and aldehydes are reduced to alcohols by phenylsilane and Mn(dpm)3(cat) in the presence of dioxygen.
- Magnus, Philip,Fielding, Mark R
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p. 6633 - 6636
(2007/10/03)
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- Phosphine effects in the copper(I) hydride-catalyzed hydrogenation of ketones and regioselective 1,2-reduction of α,β-unsaturated ketones and aldehydes. Hydrogenation of decalin and steroidal ketones and enones
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The stereoselectivity and regioselectivity of the catalytic hydrogenation of ketones and α,β-unsaturated ketones and aldehydes using soluble copper(I) hydride catalysts have been investigated as a function of the ancillary phosphine ligand. While a relatively narrow range of aryldialkylphosphine ligands produce active hydrogenation catalysts, some ligands provide higher selectivity for 1,2-reduction of acyclic unsaturated carbonyl substrates than observed using the previously reported dimethylphenylphosphine-stabilized catalyst. The synthetic utility of this class of hydridic hydrogenation catalysts is illustrated by the hydrogenation of decalin and steroidal ketones and enones, the latter giving allylic alcohols with high selectivity. (C) 2000 Elsevier Science Ltd.
- Chen, Jian-Xin,Daeuble, John F.,Stryker, Jeffrey M.
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p. 2789 - 2798
(2007/10/03)
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- Steroid transformations with Exophiala jeanselmei var. lecanii-corni and Ceratocystis paradoxa
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The fungi Exophiala jeanselmei var. lecanii-corni [IMI (International Mycological Institute) 312989, UAMH (University of Alberta Microfungus Collection and Herbarium) 8783] and Ceratocystis paradoxa (IMI 374529, UAMH 8784) have been examined for their potential in steroid biotransformation. The study has determined that E. jeanselmei var. lecanii-corni effected overall anti-Markovnikov hydration on dehydroisoandrosterone, and side-chain degradation on a variety of pregnanes. Both ascomycetes were found to carry out redox reactions of alcohols and ketones as well as 1,4 reduction of α,β-unsaturated carbonyl systems.
- Porter, Roy B.R.,Gallimore, Winklet A.,Reese, Paul B.
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p. 770 - 779
(2007/10/03)
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- The Stereochemistry of an Elimination Reaction accompanying the Hydroboration of a Steroidal Allylic Alcohol
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Deuterium labelling studies have shown that the facile elimination of the 5β-hydroxy group observed in the course of hydroboration of a 5β-hydroxyandrost-3-ene may involve a trans diaxial borane-borinate elimination coupled with a syn transfer of hydrogen from the borinate.
- Hanson, James R.,Liman, Mansur D.,Uyanik, Cavit
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p. 126 - 127
(2007/10/03)
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- Regioselectivity in the Hydroboration of Steroidal δ3-Allylic Alcohols
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The presence of an allylic 5α-hydroxy group in an androst-3-ene increases the proportion of addition of a borane to the adjacent C-4 compared to the unsubstituted steroid and directs the addition to the face of the alkene anti to the hydroxy group with stereochemical effects that may oppose those of the C-10β-methyl group.
- Alam, Muzaffar,Hanson, James R.,Liman, Mansur,Nagaratnam, Sivajini
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- The Diastereoselectivity of Zirconium Alkoxide Catalysed Meerwein-Ponndorf-Varley Reductions
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The new variation of the Meerwein-Ponndorf-Verley reduction using 1-ethanol (6) or 1-tetralol (12) (3 equiv.) as the reducting alcohols and Zr(OtBu)4 as the catalyst (0.2 equiv.) is kinetically controlled and highly stereoselective.Preferential axial attack is achieved with the sterically less bulky alcohol 6 in the case of 4-tert-butyl- and 3-methylcyclohexanones (1a -> 96percent axial and 3a -> 93percent axial attack).The combination tetralol (12)/Zr(OtBu)4 behaves as a very bulky reducting agent, and the thermodynamically less stable alcohols 2c (80percent) and 4c (92 - 96percent) are formed preferentially in the reduction of 2a and 4b.The fused bicyclic systems 1-methyl-2-tetralone (14a) and flavanone (15a) and the steroids 16a-18a are reduced with high stereoselectivity to the corresponding cis alcohols 14b and 15b and the β-alcohols 16b-18b.The stereochemical outcome is in agreement with the Felkin-Ahn model. - Key Words: Reduction / Meerwein-Ponndorf-Verley reduction / Catalysis / Zirconium tetra-tert-butoxide / Stereochemistry / Diastereoselectivity / Felkin-Ahn model
- Krohn, Karsten,Knauer, Birgit
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p. 1347 - 1352
(2007/10/02)
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- Facial selectivity in the hydroboration of androst-4-enes
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In the absence of an allylic hydroxy group, the stereochemistry of hydroboration of an androst-4-ene is determined by the presence and stereochemistry of the C-10 methyl group.Allylic hydroxy groups at C-3 direct the hydroboration/oxidation to the anti-face.In the case of the 3α-alcohol, this effect is in opposition to the normal hydration from the α-face of the steroid and leads to the 4β-alcohol.The stereochemistry of 4β,17β-diacetoxy-19-nor-5β-androstane was established by X-ray crystallography.
- Hanson, James R.,Hitchcock, Peter B.,Liman, Mansur D.,Naragatnam, Sivajini
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p. 2183 - 2188
(2007/10/02)
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- Microbiological Transformations. XVI. Transformation of 5α- and 5β-Dihydro, and 1- and 6-Dehydro Derivatives of Testosterone and Androstenedione by Means of Rhodotorula mucilaginosa Strain
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The following transformation of 5α- and 5β-dihydro derivatives of testosterone and androstenedione by means of Rhodotorula mucilaginosa has been observed: reduction of the carbonyl group on C-3 to both possible alcohols in the 5α-series and only to one alcohol (3α) in the 5β-series.In the transformation of 1- and 6-dehydro derivatives of testosterone and androstenedione, reduction in ring A was completely inhibited.The carbonyl or hydroxy groups at C-17 in both series of substrates underwent interconversion.
- Draczynska, Bozena,Tlomak, Elzbieta,Dmochowska-Gladysz, Jadwiga,Siewinski, Antoni
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- Lanthanoids in Organic Synthesis. 6. The Reduction of α-Enones by Sodium Borohydride in the Presence of Lanthanoid Chlorides: Synthetic and Mechanistic Aspects
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Lanthanoid chlorides (LnCl3) are efficient catalysts for the regioselective 1,2-reduction of α-enones by NaBH4 in methanol solution.Optimal conditions of this reaction have been determined.A mechanistic interpretation depicting the role of the Ln3+ ions is given.The major effect of Ln3+ is the catalysis of BH4- decomposition by the hydroxylic solvent to afford alkoxyborohydrides, which may be responsible for the observed regioselectivity.The stereoselectivity of the process is also modified by the presence of the Ln3+ ions, in that axial attack of cyclohexanone systems is enhanced.
- Gemal, Andre L.,Luche, Jean-Louis
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p. 5454 - 5459
(2007/10/02)
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- Kinetic study of HCG induced decrease of microsomal 7α-hydroxylase activity in rat testes
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Microsomes from rat testes were incubated with varying concentrations of 14C labelled testosterone and androstenedione. The production of 7α-hydroxytestosterone and 7α-hydroxyandrostenedione was followed; K(m) and V(m) values were calculated from Lineweaver-Burk curves. A sustained treatment of rats with HCG resulted in a considerable decrease of the maximal 7α-hydroxylation rate (V(m)) whereas the K(m) value was not changed. V(m) of microsomes from normal rats, when incubated with microsomes from HCG-treated animals, was also decreased substantially. It is concluded that HCG-induced depression of 7α-hydroxylation capacity of testicular microsomes is at least in part due to non-competitive inhibition of the enzyme.
- Eechaute,Lacroix,Leusen
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p. 647 - 660
(2007/10/02)
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