A simple method for the small scale synthesis and solid-phase extraction purification of steroid sulfates

Steroid sulfates are a major class of steroid metabolite that are of growing importance in fields such as anti-doping analysis, the detection of residues in agricultural produce or medicine. Despite this, many steroid sulfate reference materials may have limited or no availability hampering the development of analytical methods. We report simple protocols for the rapid synthesis and purification of steroid sulfates that are suitable for adoption by analytical laboratories. Central to this approach is the use of solid-phase extraction (SPE) for purification, a technique routinely used for sample preparation in analytical laboratories around the world. The sulfate conjugates of sixteen steroid compounds encompassing a wide range of steroid substitution patterns and configurations are prepared, including the previously unreported sulfate conjugates of the designer steroids furazadrol (17β-hydroxyandrostan[2,3-d]isoxazole), isofurazadrol (17β-hydroxyandrostan[3,2-c]isoxazole) and trenazone (17β-hydroxyestra-4,9-dien-3-one). Structural characterization data, together with NMR and mass spectra are reported for all steroid sulfates, often for the first time. The scope of this approach for small scale synthesis is highlighted by the sulfation of 1μg of testosterone (17β-hydroxyandrost-4-en-3-one) as monitored by liquid chromatography-mass spectrometry (LCMS).We thank the Australian Research Council (LP120200444) for financial support

Diastereoselective Osmium-catalyzed Vicinal Oxyamination of Acyclic Allylic Alcohol Derivatives

The osmium-catalyzed oxyamination of chiral acyclic allylic alcohol derivatives bearing mono- and 1,1-di-substituted double bonds with benzyl N-(4-tosyloxy)carbamate proceeds with high regioselectivity and moderate levels of diastereoselectivity favoring the anti product. The observed stereoselectivity shows a clear and systematic trend with anti:syn ratios increasing in line with the size of substituent at both the allylic stereocenter and double bond alpha-carbon. The stereoinduction is in accord with the sense of diastereoselectivity predicted by Kishi’s empirical rule and a previously reported transition state model for the osmium-catalyzed dihydroxylation of allylic alcohol derivatives. In contrast, allylic alcohol derivatives bearing trisubstituted double bonds show low or no reactivity in the oxyamination reaction affording the syn product in low yield in the cases examined.Directorate General of Higher Education Indonesia (DIKTI

A review of designer anabolic steroids in equine sports

In recent years, the potential for anabolic steroid abuse in equine sports has increased due to the growing availability of designer steroids. These compounds are readily accessible online in 'dietary' or 'nutritional' supplements and contain steroidal compounds which have never been tested or approved as veterinary agents. They typically have unusual structures or substitution and as a result may pass undetected through current anti-doping screening protocols, making them a significant concern for the integrity of the industry. Despite considerable focus in human sports, until recently there has been limited investigation into these compounds in equine systems. To effectively respond to the threat of designer steroids, a detailed understanding of their metabolism is needed to identify markers and metabolites arising from their misuse. A summary of the literature detailing the metabolism of these compounds in equine systems is presented with an aim to identify metabolites suitable for incorporation into screening protocols by anti-doping laboratories. The future of equine anti-doping research is likely to be guided by the incorporation of alternate testing matrices into routine screening, the improvement of in vitro technologies that can mimic in vivo equine metabolism, and the improvement of instrumentation or analytical methods that allow for the development of untargeted screening, and metabolomics approaches for use in anti-doping screening protocols. Copyright © 2016 John Wiley & Sons, Ltd.ARC Linkage Project LP120200444 – Strategies for the detection of designer steroids in racehorse

The Escherichia coli glucuronylsynthase promoted synthesis of steroid glucuronides: improved practicality and broader scope

A library of steroid glucuronides was prepared using the glucuronylsynthase derived from Escherichia coliβ-glucuronidase, followed by purification using solid-phase extraction. A representative range of steroid substrates were screened for synthesis on the milligram scale under optimised conditions with conversions dependent on steroid substitution and stereochemistry. Epiandrosterone (3β-hydroxy-5α-androstan-17-one) provided the highest conversion of 90% (84% isolated yield). The previously unreported glucuronide conjugates of methandriol (17α-methylandrost-5-ene-3β,17β-diol), cholest-5-ene-3β,25-diol and the designer steroid trenazone (17β-hydroxyestra-4,9-dien-3-one) were prepared on a multi-milligram scale suitable for characterisation by (1)H and (13)C NMR spectroscopy. The glucuronide conjugate of d5-etiocholanolone (2,2,3,4,4-d5-3α-hydroxy-5β-androstan-17-one), a target developed by the World Anti-Doping Agency as a certified reference material, was also prepared on a milligram scale. The improved E. coli glucuronylsynthase method provides for the rapid synthesis and purification of steroid glucuronides on a scale suitable for a range of analytical applications.Australian Research Council (DP110101235

Synthesis of (±)-Panduratin A and Related Natural Products Using the High Pressure Diels-Alder Reaction

Panduratin A and 4-hydroxypanduratin A are synthesised in 6 steps via a high pressure Diels-Alder reaction. This sequence has also allowed the synthesis of isopanduratin A, 4-hyrdoxyisopanduratin A, panduratin H, and panduratin I. In addition the regioselectivity of the Diels-Alder reaction has been investigated.We thank the Australian Research Council for financial support. M.L.C thanks the National Facility of the National Computational Infrastructure for supercomputing time

Pseudomonas aeruginosa arylsulfatase: a purified enzyme for the mild hydrolysis of steroid sulfates

The hydrolysis of sulfate ester conjugates is frequently required prior to analysis for a range of analytical techniques including gas chromatography-mass spectrometry (GC-MS). Sulfate hydrolysis may be achieved with commercial crude arylsulfatase enzyme preparations such as that derived from Helix pomatia but these contain additional enzyme activities such as glucuronidase, oxidase, and reductase that make them unsuitable for many analytical applications. Strong acid can also be used to hydrolyze sulfate esters but this can lead to analyte degradation or increased matrix interference. In this work, the heterologously expressed and purified arylsulfatase from Pseudomonas aeruginosa is shown to promote the mild enzyme-catalyzed hydrolysis of a range of steroid sulfates. The substrate scope of this P. aeruginosa arylsulfatase hydrolysis is compared with commercial crude enzyme preparations such as that derived from H. pomatia. A detailed kinetic comparison is reported for selected examples. Hydrolysis in a urine matrix is demonstrated for dehydroepiandrosterone 3-sulfate and epiandrosterone 3-sulfate. The purified P. aeruginosa arylsulfatase contains only sulfatase activity allowing for the selective hydrolysis of sulfate esters in the presence of glucuronide conjugates as demonstrated in the short three-step chemoenzymatic synthesis of 5α-androstane-3β,17β-diol 17-glucuronide (ADG, 1) from epiandrosterone 3-sulfate. The P. aeruginosa arylsulfatase is readily expressed and purified (0.9 g per L of culture) and thus provides a new and selective method for the hydrolysis of steroid sulfate esters in analytical sample preparation.We thank the Australian Government Anti-Doping Research Pro-gram for financial support

Detection and metabolic investigations of a novel designer steroid: 3-chloro-17α-methyl-5α-androstan-17β-ol

In 2012, seized capsules containing white powder were analyzed to show the presence of unknown steroid-related compounds. Subsequent gas chromatography–mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) investigations identified a mixture of 3α- and 3β- isomers of the novel compound; 3-chloro-17α-methyl-α-androstan-17β-ol. Synthesis of authentic reference materials followed by comparison of NMR, GC-MS and gas chromatography-tandem mass spectrometry (GC-MS/MS) data confirmed the finding of a new ‘designer’ steroid. Furthermore, in vitro androgen bioassays showed potent activity highlighting the potential for doping using this steroid. Due to the potential toxicity of the halogenated steroid, in vitro metabolic investigations of 3α-chloro-17α-methyl-α-androstan-17β-ol using equine and human S9 liver fractions were performed. For equine, GC-MS/MS analysis identified the diagnostic 3α-chloro-17α-methyl-5α-androstane-16α,17β-diol metabolite. For human, the 17α-methyl-α-androstane-3α,17β-diol metabolite was found. Results from these studies were used to verify the ability of GC-MS/MS precursor-ion scanning techniques to support untargeted detection strategies for designer steroids in anti-doping analyses.Synthesis and in vitro metabolic investigations of 3α/β-chloro-17α-methyl-5α-androstan -17β-ol was suppo rted by the Austr a-lian Research Council Linkage Grant (LP120200444) Strat egies for the detection of designer ster oids in ra cehorses

The metabolism of anabolic-androgenic steroids in the greyhound

BACKGROUND Effective control of the use of anabolic-androgenic steroids (AASs) in animal sports is essential in order to ensure both animal welfare and integrity. In order to better police their use in Australian and New Zealand greyhound racing, thorough metabolic studies have been carried out on a range of registered human and veterinary AASs available in the region. RESULTS Canine metabolic data are presented for the AASs boldenone, danazol, ethylestrenol, mesterolone, methandriol, nandrolone and norethandrolone. The principal Phase I metabolic processes observed were the reduction of A-ring unsaturations and/or 3-ketones with either 3α,5β- or 3β,5α-stereochemistry, the oxidation of secondary 17β-hydroxyl groups and 16α-hydroxylation. The Phase II β-glucuronylation of sterol metabolites was extensive. CONCLUSION The presented data have enabled the effective analysis of AASs and their metabolites in competition greyhound urine samples.Australian Research Council LP077483

Replacing PAPS: In vitro phase II sulfation of steroids with the liver S9 fraction employing ATP and sodium sulfate

In vitro technologies provide the capacity to study drug metabolism where in vivo studies are precluded due to ethical or financial constraints. The metabolites generated by in vitro studies can assist anti-doping laboratories to develop protocols for the detection of novel substances that would otherwise evade routine screening efforts. In addition, professional bodies such as the Association of Official Racing Chemists (AORC) currently permit the use of in-vitro-derived reference materials for confirmation purposes providing additional impetus for the development of cost effective in vitro metabolism platforms. In this work, alternative conditions for in vitro phase II sulfation using human, equine or canine liver S9 fraction were developed, with adenosine triphosphate (ATP) and sodium sulfate in place of the expensive and unstable co-factor 3′-phosphoadenosine-5′-phosphosulfate (PAPS), and employed for the generation of six representative steroidal sulfates. Using these conditions, the equine in vitro phase II metabolism of the synthetic or so-called designer steroid furazadrol ([1′,2′]isoxazolo[4′,5′:2,3]-5α-androstan-17β-ol) was investigated, with ATP and Na2SO4 providing comparable metabolism to reactions using PAPS. The major in vitro metabolites of furazadrol matched those observed in a previously reported equine in vivo study. Finally, the equine in vitro phase II metabolism of the synthetic steroid superdrol (methasterone, 17β-hydroxy-2α,17α-dimethyl-5α-androstan-3-one) was performed as a prediction of the in vivo metabolic profile.Australian Research Council LP12020044

Alternate steroid sulfation pathways targeted by LC-MS/MS 1 analysis of disulfates. Application to prenatal diagnosis of steroid synthesis disorders

The steroid disulfates (aka bis-sulfates) are a significant but minor fraction of the urinary steroid metabolome that have not been widely studied because major components are not hydrolyzed by the commercial sulfatases commonly used in steroid metabolomics. In early studies, conjugate fractionation followed by hydrolysis using acidified solvent (solvolysis) was used for the indirect detection of this fraction by GC–MS. This paper describes the application of a specific LC–MS/MS method for the direct identification of disulfates in urine, and their use as markers for the prenatal diagnosis of disorders causing reduced estriol production: STSD (steroid sulfatase deficiency), SLOS (Smith-Lemli-Opitz syndrome) and PORD (P450 oxidoreductase deficiency). Disulfates were detected by monitoring a constant ion loss (CIL) from the molecular di-anion. While focused on disulfates, our methodology included an analysis of intact steroid glucuronides and monosulfates because steroidogenic disorder diagnosis usually requires an examination of the complete steroid profile. In the disorders studied, a few individual steroids (as disulfates) were found particularly informative: pregn-5-ene-3β,20S-diol, pregn-5-ene-3β,21-diol (STSD, neonatal PORD) and 5α-pregnane-3β,20S-diol (pregnancy PORD). Authentic steroid disulfates were synthesized for use in this study as aid to characterization. Tentative identification of 5ξ-pregn-7-ene-3ξ,20S-diol and 5ξ-pregn-7-ene-3ξ,17,20S-triol disulfates was also obtained in samples from SLOS affected pregnancies. Seven ratios between the detected metabolites were applied to distinguish the three selected disorders from control samples. Our results show the potential of the direct detection of steroid conjugates in the diagnosis of pathologies related with steroid biosynthesis.Spanish Health National System is acknowledged for OP contract (CPII16/00027). Strategic Plan for Research and Innovation in Health (PERIS) of the Catalan government is acknowledge for OK contract (SLT002/16/00007)