Silica-immobilized enzymes for multi-step synthesis in microfluidic devices

The combinatorial synthesis of 2-aminophenoxazin-3-one (APO) in a microfluidic device is reported. Individual microfluidic chips containing metallic zinc, silica-immobilized hydroxylaminobenzene mutase and silica-immobilized soybean peroxidase are connected in series to create a chemo-enzymatic system for synthesis. Zinc catalyzes the initial reduction of nitrobenzene to hydroxylaminobenzene which undergoes a biocatalytic conversion to 2-aminophenol, followed by enzymatic polymerization to APO. Silica-immobilization of enzymes allows the rapid stabilization and integration of the biocatalyst within a microfluidic device with minimal preparation. The system proved suitable for synthesis of a complex natural product (APO) from a simple substrate (nitrobenzene) under continuous flow conditions.