Flow synthesis of nucleoside analogues: development of a biochromatographic integrated system based on a purine nucleoside phosphorylase from Aeromonas hydrophila

Purine nucleoside phosphorylases (PNPs, EC 2.4.2.1) act in the biochemical process of purine salvage/recycling. PNPs catalyze the reversible cleavage of the glycosidic bond of purine (deoxy)ribonucleosides by phosphorolysis producing the purine (B1) and α-D-(deoxy)ribose-1-phosphate. This latter intermediate can react with a new purine (B2) generating a new nucleoside (transglycosylation) (Scheme). PNPs can be thus used as biocatalysts in nucleoside synthesis as an alternative to “conventional” chemical routes. PNP from Aeromonas hydrophila (AhPNP) has been investigated and used by our group in several bioconversions. Recently, AhPNP has been immobilized on the inner surface of a silica capillary: the resulting bioreactor has been coupled on line with a chromatographic column and the system has been used as a tool to assess AhPNP substrate specificity (by phosphorolysis) towards a set of 6-substituted purine ribonucleosides. As a step forward, this biochromatographic system has been implemented and used to synthesize nucleoside analogues (by transglycosylation) through a “flow-based” approach. AhPNP was covalently immobilized (25 mg, 50% yield) in a 50 L x 4.6 ID mm pre-packed stainless steel column containing aminopropylsilica particles (5 µm, 100 Å). The resulting AhPNP-IMER (Immobilized Enzyme Reactor) was coupled on line through a 6-port switching valve to a HPLC apparatus containing a semi-preparative chromatographic column. The conditions of the AhPNP-catalyzed bioconversions were optimized by a DoE approach and then applied to the synthesis of some 6-substituted purine ribonucleosides at a mg scale. Coupling of transglycosylation reaction and product separation resulted in a fast and efficient process (yield=52-89%) where sample handling was minimized. To date, AhPNP-IMER has retained completely its activity upon 50 reactions and 7 months.