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

Abstract

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.

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

“…Glucuronylsynthase enzyme solution (0.58 mL, 1.09 g L –1 , final concentration 0.2 mg mL –1 ) and α‐D‐glucuronyl fluoride 5 (2.3 mg, 10.8 µmol, 5 equiv. ) dissolved in sodium phosphate buffer (220 μL, 50 mM, pH 7.5) were added and the reaction incubated without agitation at 37 °C for 2 d. The reaction was then subjected to solid‐phase extraction to remove non‐volatile salts and other reaction components . A Waters (Rydalmere, Australia) Oasis WAX SPE cartridge (60 mg, 3 cc) was conditioned with methanol (1 mL) and milliQ water (3 mL).…”

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

“…Mono‐sulfation of the 3α/β,17β‐diols to give 3α‐RSS , and mixed 3α‐RSS/3β‐RSS proceeded in high conversion, using established methodology . On the other hand, enzymatic glucuronylation afforded only the 3β‐isomer 3β‐RSG . This was highlighted by the reaction of a mixture of 3α/β‐hydroxy isomers 3α‐RS/3β‐RS , which afforded 2α,17α‐dimethyl‐5α‐androstane‐3β,17β‐diol 3‐glucuronide 3β‐RSG as the sole product after purification by SPE.…”

“…Solid‐phase extraction (SPE) was performed using Waters (Rydalmere, Australia) Oasis WAX 6 cc cartridges (PN 186004647), or Waters Sep‐Pak C18 (3 cc, 500 mg) cartridges (PN WAT020805) as specified. Escherichia coli glucuronylsynthase, and α‐D‐glucuronyl fluoride were prepared according to literature methods . Escherichia coli NADP‐dependent G6PDH was expressed with an N‐terminal hexa‐histidine tag purified according to previously described methods .…”

“…according to literature methods. 41 Escherichia coli NADP-dependent G6PDH was expressed with an N-terminal hexa-histidine tag purified according to previously described methods. 42 The E. coli BL21 (DE3) with pETMCSIII-g6pdh was kindly provided by Professor David Ollis at the Research School of Chemistry, Australian National University.…”

A review of designer anabolic steroids in equine sports

“…No unconjugated furazadrol metabolites were observed. The identity of furazadrol 17‐sulfate, furazadrol 17‐glucuronide, and C17‐epifurazadrol 17‐glucuronide was confirmed by comparison to synthetic reference materials, and the identity of the other metabolites was tentatively assigned through analysis of the LC‐HRAM product‐ion spectra. The sites of hydroxylation for minor metabolites were not identified in this study.…”