17α- and 17β-boldenone 17-glucuronides: Synthesis and complete characterization by H and C NMR

Boldenone is an androgenic anabolic steroid intensively used for growth promoting purposes in animals destined for meat production and as a performance enhancer in athletics. Therefore its use is officially banned either in animals intended for consumption or in humans. Because most anabolic steroids are completely metabolized and usually no parent steroid is excreted, metabolite identification is crucial to detect the illegal use of anabolic steroids either in humans or in livestock. 17α- and 17β-boldenone 17-glucuronides were synthesized, purified and characterized in order to provide suitable standards for the identification and quantification of these metabolites.     

2006年第17届国际生物学奥林匹克竞赛(7)

理论试卷B 生态学(7题,12分) 为了解犰狳的食性,一个科研小组进行了一项植被调查,并比较犰狳粪便中的残留物. 43 科学家们在向日葵、玉米和天然草场中直线行进,他们每50 m取样1次,一个样方是1 m2,记录植被的种类、密度、覆盖情况和物候情况.以下哪种技术被用到了?     

Syntheses of (14β,17α)-14-Hydroxy- and (14β,17α)-2,14-Dihydroxyestradiols and Their Activities

Structure 1 is proposed for the Inagami-Tamura endogenous digitalis-like factor (EDLF), and (14β,17α)-14-hydroxy- and (14β, 17α)-2,14-dihydroxyestradiols (2 and 3) were synthesized as models for studies on 1. The latter compound was remarkably potent in inducing a contractile response in isolated rat aorta and guinea pig left atrium.     

A review of mechanistic studies on aromatase (CYP19) and 17α-hydroxylase-17,20-lyase (CYP17)

In the conventional P-450 dependent hydroxylation reaction, the Fe(III) resting state of the enzyme, by a single electron transfer, is reduced to Fe(II), which reacts with O(2) to produce a Fe(III)-O-O intermediate. The latter following the transfer of another electron furnishes a ferric-peroxyanion, Fe(III)-O-O(-), which after protonation leads to the fission of the O-O bond resulting in the formation of Fe(V)O, the key player in the hydroxylation process. Certain members of the P-450 family, including CYP17 and CYP19, catalyze, at the same active site, not only the hydroxylation process but also an acyl-carbon bond cleavage reaction which has been interpreted to involve the nucleophilic attack of the ferric-peroxyanion, Fe(III)-O-O(-), on the acyl carbon to furnish a tetrahedral intermediate which fragments, leading to acyl-carbon cleavage. Evidence is presented to show that in the case of CYP17 the attack of Fe(III)-O-O(-) on the target carbon is promoted by cytochrome b(5), which acts as a conformational regulator of CYP17. It is this regulation of CYP17 that provides a safety mechanism which ensures that during corticoid biosynthesis, which involves 17α-hydroxylation by CYP17, androgen formation is avoided. Finally, a brief account is presented of the inhibitors, of the two enzymes, which have been designed on the basis of their mechanism of action. Article from the Special issue on 'Targeted Inhibitors'.     

Synthesis of 7-(tetrahydropyran-2-yl)- and 7-(4-acetoxytetrahydropyran-2-yl)-ε-rhodomycinone and 7-(tetrahydropyran-2-yl)-ε-pyrromycinone

The ¹³C.n.m.r spectra of water-soluble and -insoluble glucans synthesized by enzymes isolated from six strains of Streptococcus mutans are interpreted. The glucans are shown to be composed primarily of α(1→3)- and α-(1→6)-linked glucosyl residues, and the relative abundance of each linkage is estimated from peak areas. Treatment of water-insoluble glucans with dextranase is found to result in water-soluble and -insoluble products, the former enriched in α-(1→6)-linkages and the latter in α-(1→3)-linkages. The structural conclusions arrived at by ¹³C-n.m.r. spectroscopy are consistent with data from methylation analysis and ¹H-n.m.r. spectroscopy.     

ADP-ribosylation factor like 7 (ARL7) interacts with α-tubulin and modulates intracellular vesicular transport

ADP-ribosylation factor (ARF) like 7 (ARL7, also named ARL4C) is a member of ARL family and recent studies showed that it is involved in the AI-dependent cholesterol secretion process. Yet its biological function remains largely unknown. Using a MALDI-TOF/MS analysis, we identified α-tubulin interacted with ARL7. The interaction was confirmed by GST pull-down assay and co-immunoprecipitation in renal carcinoma cell 786-O in which we found the endogenous ARL7 is expressed. This is the second ARL member found interacting with tubulin after ARL8. In addition, ARL7Q72L, a GTP-binding form, promoted the transferrin transport from early endosome to recycling endosome significantly. The above data suggested that ARL7 might modulate the intracellular vesicular transport via interaction with microtubules.     

Occurrence and Fate of Estrone, 17β-estradiol and 17α-ethynylestradiol in STPs for Domestic Wastewater

Estrone (E1), 17β-estradiol (E2) and 17α-ethynylestradiol (EE2) discharged from sewage treatment plants (STPs) into surface waters, are seen as a threat effecting aquatic life by its estrogenic character. Therefore, much research is conducted on the fate and removal of these compounds. Since these compounds are present in influents and effluents in the ng/l range, methods for detection deserve special attention. Most important processes that play a role in the removal of estrogens are: adsorption, aerobic degradation, anaerobic degradation, anoxic biodegradation and photolytic degradation. Halflifes tend to vary and are remarkably shorter when low initial concentrations are applied. In general anaerobic conditions result in longer halflifes then aerobic conditions. EE2 shows far most persistence of the compounds, thereby also the estrogenic effect in vitro is about 2–3-fold higher compared to E2. The three compounds show a higher affinity to sorb to sludge compared to other tested adsorption materials like sediment. Aerobic degradation is far the most efficient in removing these compounds, but adsorption seems to play a significant role in retaining the estrogens inside full-scale STPs. Removal rates in full scale plants depend on the HRT, SRT and loading rates, but lack of information on the exact dependency so far prevents an optimal design able to fully eliminate estrogens from wastewater.     

Cloning and characterization of zebrafish protocadherin–17

Protocadherins constitute the largest subgroup within the cadherin superfamily of cell surface molecules. In this study, we report the molecular cloning and expression analysis of the non-clustered protocadherin-17 (pcdh17) in the embryonic zebrafish nervous system. The zebrafish Pcdh17 protein is highly conserved, exhibiting 73% sequence homology with the human protein. The zebrafish pcdh17 gene consists of four exons spread over 150 kb, and this organization is highly conserved throughout vertebrates. Pcdh17 message is first detectable by 6 h postfertilization in the developing embryo, and the expression is maintained throughout development. Zebrafish embryos express pcdh17 in all of the major subdivisions of the central nervous system, including the telencephalon, diencephalon, mesencephalon, and rhombencephalon. Analysis of the genomic sequence upstream of pcdh17 in several species reveals a pattern of paired CpG islands. While the CpG islands in zebrafish are further upstream than in other teleosts, alignment of the identified sequences reveals a high degree of conservation, suggesting that the sequences may be important for the regulation of pcdh17 expression.     

ACE2/Ang-(1–7) signaling and vascular remodeling

The renin-angiotensin system (RAS) regulates vascular tone and plays a critical role in vascular remodeling, which is the result of a complex interplay of alterations in vascular tone and structure. Inhibition of the RAS has led to important pharmacological tools to prevent and treat vascular diseases such as hypertension, diabetic vasculopathy and atherosclerosis. Angiotensin converting enzyme 2 (ACE2) was recently identified as a multifunctional monocarboxypeptidase responsible for the conversion of angiotensin (Ang) II to Ang-(1–7). The ACE2/Ang-(1–7) signaling has been shown to prevent cellular proliferation, pathological hypertrophy, oxidative stress and vascular fibrosis. Thus, the ACE2/Ang-(1–7) signaling is deemed to be beneficial to the cardiovascular system as a negative regulator of the RAS. The addition of the ACE2/Ang-(1–7) signaling to the complexities of the RAS may lead to the development of novel therapeutics for the treatment of hypertension and other vascular diseases. The present review considers recent findings regarding the ACE2/Ang-(1–7) signaling and focuses on its regulatory roles in processes related to proliferation, inflammation, vascular fibrosis and remodeling, providing proof of principle for the potential use of ACE2 as a novel therapy for vascular disorders related to vascular remodeling.     

Design and synthesis of functionalized piperazin-1yl-(E)-stilbenes as inhibitors of 17α-hydroxylase-C17,20-lyase (Cyp17)

The synthesis of steroid hormones is critical to human physiology and improper regulation of either the synthesis of these key molecules or activation of the associated receptors can lead to disease states. This has led to intense interest in developing compounds capable of modulating the synthesis of steroid hormones. Compounds capable of inhibiting Cyp19 (Aromatase), a key enzyme in the synthesis of estrogens, have been successfully employed as breast cancer therapies, while inhibitors of Cyp17 (17α-hydroxylase-17,20-lyase), a key enzyme in the synthesis of glucocorticoids, mineralocorticoids and steroidal sex hormones, are a key component of prostate cancer therapy. Inhibition of CYP17 has also been suggested as a possible target for the treatment of Cushing Syndrome and Metabolic Syndrome. We have identified two novel series of stilbene based CYP17 inhibitors and demonstrated that exemplary compounds in these series have pharmacokinetic properties consistent with orally delivered drugs. These findings suggest that compounds in these classes may be useful for the treatment of diseases and conditions associated with improper regulation of glucocorticoids synthesis and glucocorticoids receptor activation.     

迷失的世界(17)

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Steroid modification by filamentous fungus Drechslera sp.: Focus on 7-hydroxylase and 17β-hydroxysteroid dehydrogenase activities

Fungal strain Drechslera sp. Ph F-34 was shown to modify 3-oxo- and 3-hydroxy steroids of androstane series to form the corresponding allylic 7-alcohols and 17β-reduced derivatives thus evidencing the presence of 7α-, 7β-hydroxylase and 17β-hydroxysteroid dehydrogenase (17β-HSD) activities. The growing mycelium predominantly hydroxylated androsta-1,4-diene-3,17-dione (ADD) at the 7β-position, while much lower 7α-hydroxylation was observed. Along with 7β-hydroxy-ADD and its corresponding 7α-isomer, their respective 17β-alcohols were produced.In this study, transformation of ADD, androst-4-en-17β-ol-3-one (testosterone, TS) and 3β-hydroxyandrost-5-en-17-one (dehydroepiandrosterone, DHEA) by resting mycelium of Drechslera sp. have been estimated in different conditions with regard to the inducibility and functionality of the 17β-HSD and 7-hydroxylase enzyme systems. Steroids of androstane, pregnane and cholane series were evaluated as inducers. The inhibitory analysis was provided using cycloheximide (CHX). Steroids were assayed using TLC and HPLC methods, and the structures were confirmed by mass-spectrometry, ¹H and ¹³C NMR spectroscopy data.17β-HSD of the mycelium constitutively reduced 17-carbonyl group of ADD and DHEA to form the corresponding 17β-alcohols, namely, androsta-1,4-diene-17β-ol-3-one (1-dehydro-TS), and androst-5-ene-3β,17β-diol. Production of the 7α- and 7β-hydroxylated derivatives depended on the induction conditions. The inducer effect relied on the steroid structure and decreased in the order: DHEA > pregnenolone > lithocholic acid. β-Sitosterol did not induce hydroxylase activity in Drechslera sp. CHX fully inhibited the synthesis of 7-hydroxylase in Drechslera mycelium thus providing selective 17-keto reduction.Results contribute to the diversity of steroid modifying enzymes in fungi and can be used at the development of novel biocatalysts for production of valuable steroid 7(α/β)- and 17β-alcohols.     

Synthesis and stereochemistry of 7β- and 7α-amino-, acetamido-, hemisuccinamido- and terephthalamido derivatives of testosterone

The reduction of 3-ethylenedioxy-7-oximino-5-androsten-17β-yl acetate and of its 17β-tetrahydropyranyl ether analog with sodium in ethanol, followed by thin-layer chromatography, allowed the isolation of the corresponding 17β-hydroxy- and 17β-tetrahydropyranyioxy-5-en-7β- and 7α-amines which were also characte-rized as 7-acetamides. The acylation of the two epimeric 17β-hydroxy-5-en-7-amines with succinic anhydride followed by selective saponification of the 17β-hemisuccinate group and diazomethane esterification, gave the corresponding 17β-hydroxy-5-en-7β- and 7α-hemisuccinamido methyl esters characterized also as 17β-acetates. On the other hand, the acylation of the two 17β-tetrahydropyranyl-oxy-5-en-7-amines with the acid chloride of terephthalic acid monomethyi ester led to the more rigid 7β- and 7α-terephthalamido methyl ester side-chains. The acidolysis of the 3-ethyleneketal protecting group of the preceding 5-en-7-N-acyl derivatives regenerated the 4-en-3-oxo function while the 17β-tetrahydropyranyl ether group was cleaved simultaneously into the 17β-alcohol. The four desired 7β- and 7α-hemisuccinamido- and terephthalamido carboxylic side-chain derivatives of 17β-hydroxy-4-androsten-3-one (testosterone) were finally obtained by saponification of the corresponding methyl esters.     

17α-ethinylestradiol cometabolism by bacteria degrading estrone, 17β-estradiol and estriol

17alpha-ethinylestradiol (EE2), the active compound of the contraceptive pill, is a recalcitrant estrogen, which is encountered at ng/l levels in wastewater treatment plant (WWTP) effluents and rivers and can cause feminization of aquatic organisms. The aim of this study was to isolate micro-organisms that could remove such low EE2 concentrations. In this study, six bacterial strains were isolated from compost that cometabolize EE2 when metabolizing estrone (E1), 17beta-estradiol (E2) and estriol (E3). The strains belong to the alpha, beta and gamma-Proteobacteria. All six strains metabolize E2 over E1, at mug/l to ng/l concentrations. In 4 days, initial concentrations of 0.5 mug E2/l and 0.6 mug EE2/l were degraded to 1.8 +/- 0.4 ng E2/l and 85 +/- 16 ng EE2/l, respectively. No other metabolites besides E1, E2, E3 or EE2 were detected, suggesting that total degradation and cleavage of the aromatic ring occurred. This is the first study describing that bacteria able to metabolize E2, can subsequently cometabolize EE2 at low mug/l levels.