Optimization of β- d-glucuronide synthesis using UDP-glucuronyl transferase

The optimization of alkyl and aryl β- d-glucuronide synthesis using UDP-glucuronyl transferase and UDP-glucuronic acid was undertaken to develop 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 optimum. 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 m m) appeared to be beneficial, provided that excessive quantities of co-solvent were not required to solubilize the substrate. The choice of co-solvent was also important, acetonitrile and DMSO were considerably better than ethanol. A simple, effective means of isolating and purifying both the glucuronide and any unreacted starting material using C-18-derivatized flash silica gel has been demonstrated. Since this synthetic approach generally resulted in little loss of unreacted starting material, it should be well suited to substrates that are scarce and expensive, especially isotopically labeled compounds .