Development of a biochromatographic integrated system based on a purine nucleoside phosphorylase from A. hydrophila for the flow synthesis of nucleoside analogues

Nucleoside phosphorylases (NPs, EC 2.4.2) are enzymes of the salvage pathway of nucleic acids which catalyze the biosynthesis of natural nucleosides in one step. Specifically, NPs catalyze the reversible cleavage of the glycosidic bond of (deoxy)ribonucleosides in the presence of inorganic orthophosphate (Pi) to generate the nucleobase and α-D-(deoxy)ribose-1-phosphate (R-1-P) (phosphorolysis). If a second nucleobase is added to the reaction medium the formation of a new nucleoside can result (transglycosylation). The use of NPs from different biological sources as catalysts in nucleoside analogue synthesis can be therefore an advantageous alternative to “conventional” chemical synthesis to achieve more straightforward and efficient synthetic routes. A purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) has been recently cloned, over-expressed and used for synthetic applications, also as an immobilized biocatalyst. Moreover, an integrated biochromatographic system based on immobilized AhPNP on the inner surface of a fused silica capillary coupled on line with a chromatographic column has been set up for the assessment, by phosphorolysis, of substrate specificity towards a set of 6-substituted purine ribonucleosides. As a step forward, the developed biochromatographic system has been implemented to synthesize nucleoside analogues through a “on-flow”-based approach. To this aim, AhPNP was covalently immobilized in a 50 Lx4.6 ID mm pre-packed stainless steel column containing aminopropylsilica particles (5 µm, 100 Å). The AhPNP-IMER (Immobilized Enzyme Reactor) was then coupled in-line through a switching valve to a HPLC apparatus containing a semi-preparative chromatographic column with UV-Vis detection. This integrated system allowed to perform the transglycosylation reaction and the product separation in a single chromatographic run, thus resulting in an efficient process where sample handling is minimized. Five modified purine ribonucleosides have been synthesized and purified by this approach at a mg scale. To date, AhPNP-IMER has been shown to retain completely its activity upon 35 reactions.