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 coli P-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 (3 beta-hydroxy-5 alpha-androstan-17-one) provided the highest conversion of 90% (84% isolated yield). The previously unreported glucuronide conjugates of methandriol (17 alpha-methylandrost-5-ene-3 beta-17 beta-diol), cholest-5-ene-3 beta,25-diol and the designer steroid trenazone (17 beta-hydroxyestra-4,9-dien-3-one) were prepared on a multi-milligram scale suitable for characterisation by H-1 and C-13 NMR spectroscopy. The glucuronide conjugate of d(5)-etiocholanolone (2,2,3,4,4-d(5)-3 alpha-hydroxy-5 beta-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.