Immobilized enzyme reactors based on nucleoside phosphorylases and 2′-deoxyribosyltransferase for the in-flow synthesis of pharmaceutically relevant nucleoside analogues

[Display omitted] •An in-flow enzymatic system based on immobilized NPs and NDT was developed.•LrNDT and CpUP/AhPNP were immobilized on monolithic silica columns.•The two immobilized enzyme reactors (IMERs) catalyzed continuous reactions in flow.•Pure nucleoside analogues were synthesized in suitable amounts for drug discovery.•Both bioreactors are promising alternatives to nucleoside chemical synthesis. In this work, a mono- and a bi-enzymatic analytical immobilized enzyme reactors (IMERs) were developed as prototypes for biosynthetic purposes and their performances in the in-flow synthesis of nucleoside analogues of pharmaceutical interest were evaluated. Two biocatalytic routes based on nucleoside 2′-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT) and uridine phosphorylase from Clostridium perfrigens (CpUP)/purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) were investigated in the synthesis of 2′-deoxy, 2′,3′-dideoxy and arabinonucleoside derivatives. LrNDT-IMER catalyzed the synthesis of 5-fluoro-2′-deoxyuridine and 5-iodo-2′-deoxyuridine in 65–59% conversion yield, while CpUP/AhPNP-IMER provided the best results for the preparation of arabinosyladenine (60% conversion yield). Both IMERs proved to be promising alternatives to chemical routes for the synthesis of nucleoside analogues. The developed in-flow system represents a powerful tool for the fast production on analytical scale of nucleosides for preliminary biological tests.