Optimization of beta-D-glucuronide synthesis using UDP-glucuronyl transferase

The optimization of alkyl and aryl beta-D-glucuronide synthesis using UDP-g lucuronyl transferase and UDP-glucuronic acid acid was undertaken to develo p a synthetic method suitable for preparation of multimilligram quantities of glucuronides as analytical standards. The two most important factors in yield optimization appeared to be having the right amounts of enzyme and co -factor present in the reaction. The enzyme concentration showed a clear op timum. Too much enzyme could seriously reduce yields (by as much as 70% for 4-methyl phenol, for example). The optimal UDPGA concentration appeared to be approximately twice that of the substrate, whatever the latter may have been. Higher substrate concentrations (up to 8 mM) appeared to be benefici al, provided that excessive quantities of co-solvent were not required to s olubilize the substrate. The choice of so-solvent was also important, aceto nitrile and DMSO were considerably better than ethanol. A simple, effective means of isolating and purifying both the glucuronide adn any unreacted st arting material using C-18-derivatized flash silica gel has been demonstrat ed. Since this synthetic approach generally resulted in little loss of unre acted starting material, it should be well suited to substrates that are sc arce and expensive, especially isotopically labeled compounds. (C) 1999 Els evier Science Inc. All rights reserved.