Development of an automated synthesis system for preparation of glucuronides using a solid-phase extraction column loaded with microsomes.

An automated synthesis system using a solid-phase extraction (SPE) system and column packed with octadecylsilica (ODS), which was coated with phospholipid and loaded with dog liver microsomes, was developed for synthesis of glucuronides. Preparation of the microsome-immobilized SPE column, glucuronidation of drugs to synthesize the glucuronides and elution of the products were performed by an automated synthesis system. The phospholipid-coated SPE column and then the microsome-immobilized SPE column were readily prepared by allowing a solution containing L-alpha-dipalmitoylphosphatidylcholine to flow through the SPE column, and then by recycling a buffer solution containing dog liver microsomes through the resulting phospholipid-coated SPE column. The microsome-immobilized SPE column exhibiting the uridine diphosphate (UDP)-glucuronosyltransferase activity catalyzed the glucuronidation of mefenamic acid and estradiol to the corresponding glucuronides in the presence of UDP-glucuronic acid, and three glucuronides of mefenamic acid and estradiol were synthesized using the automated synthesis system, by simply recycling a buffer solution containing UDP-glucuronic acid through the microsome-immobilized SPE column loaded with the substrate. We used beta-cyclodextrin as a solubilizing agent for the synthesis of the glucuronides of estradiol that is practically insoluble in aqueous solutions. The productivity of these glucuronides using the microsome-immobilized SPE column was higher than that using the free microsomes (batch method). Furthermore, we developed a fully automated synthesis-isolation system by coupling the automated synthesis system to an automated preparative HPLC system. The automated synthesis system as well as the fully automated synthesis-isolation system should be very useful for synthesizing glucuronides for drug development.