Immobilised-enzyme microreactors for the identification and synthesis of conjugated drug metabolites

The study of naturally circulating drug metabolites has been a focus of interest, since these metabolites may have different therapeutic and toxicological effects compared to the parent drug. The synthesis of metabolites outside of the human body is vital in order to conduct studies into the pharmacological activities of drugs and bioactive compounds. Current synthesis methods require significant purification and separation efforts or do not provide sufficient quantities for use in pharmacology experiments. Thus, there is a need for simple methods yielding high conversions whilst bypassing the requirement for a separation. Here we have developed and optimised flow chemistry methods in glass microfluidic reactors utilising surface-immobilised enzymes for sulfonation (SULT1a1) and glucuronidation (UGT1a1). Conversion occurs in flow, the precursor and co-factor are pumped through the device, react with the immobilised enzymes and the product is then simply collected at the outlet with no separation from a complex biological matrix required. Conversion only occurred when both the correct co-factor and enzyme were present within the microfluidic system. Yields of 0.97 & PLUSMN; 0.26 & mu;g were obtained from the conversion of resorufin into resorufin sulfate over 2 h with the SULT1a1 enzyme and 0.47 & mu;g of resorufin glucuronide over 4 h for UGT1a1. This was demonstrated to be significantly more than static test tube reactions at 0.22 & mu;g (SULT1a1) and 0.19 & mu;g (UGT1a1) over 4 h. With scaling out and parallelising, useable quantities of hundreds of micrograms for use in pharmacology studies can be synthesised simply. On-chip continuous-flow synthesis of metabolites from glucuronidation and sulfonation reactions to enable synthesis of analytical standards and study drug metabolism.

Automated summary

The study focuses on naturally circulating drug metabolites and the development of synthesis methods using glass microfluidic reactors for efficient conversion of resorufin into resorufin sulfate and resorufin glucuronide through sulfonation and glucuronidation reactions. The use of surface-immobilised enzymes in flow chemistry allows for simple collection of the product without the need for complex separation from a biological matrix. The yields obtained from the flow chemistry methods are significantly higher than those from static test tube reactions, indicating the potential for scalable synthesis of metabolites for pharmacology studies.