Development of a biochromatographic integrated system based on a purine nucleoside phosphorylase from Aeromonas 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 [(d)R-1-P] (phosphorolysis).[1] If a second nucleobase is added to the reaction medium the formation of a new nucleoside can result (transglycosylation) (Scheme 1). The use of NPs from different biological sources as catalysts in nucleoside analogue synthesis can be therefore an advantageous alternative to “conventional” chemical routes. A purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) has been recently cloned, over-expressed and used for synthetic applications, also as immobilized biocatalyst.[2,3] Moreover, a bioreactor based on AhPNP immobilized on the inner surface of a silica capillary has been coupled on line with a chromatographic column for the assessment, by phosphorolysis, of substrate specificity towards a set of 6-substituted purine ribonucleosides.[4] As a step forward, this biochromatographic system has been implemented to synthesize, by transglycosylation, nucleoside analogues through a “flow-based” approach. To this aim, AhPNP (25 mg; IU/mg: 39) was covalently immobilized in a 50 L4.6 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 an analytical or a semi-preparative chromatographic column with UV-Vis detection. The bioconversion was firstly characterized by a fractional factorial experimental design using, as a model reaction, the transglycosylation between inosine and adenine. Then, five modified purine ribonucleosides were synthesized and purified at a mg scale. The transglycosylation reaction and the product separation were performed in a single chromatographic run, thus resulting in an efficient process where sample handling is minimized. To date, AhPNP-IMER has been shown to retain completely its activity upon 35 reactions. References [1] Pugmire, M. J.; Ealick, S. E. Structural analyses reveal two distinct families of nucleoside phosphorylases. Biochem. J. 2002, 361, 1–25. [2] Ubiali, D.; Serra, C. D. et al. Production, characterization and synthetic application of a purine nucleoside phosphorylase from Aeromonas hydrophila. Adv. Synth. Cat. 2012, 354, 96–104. [3] Serra, I.; Ubiali, D. et al. Developing a collection of immobilized nucleoside phosphorylases for the preparation of nucleoside analogues: enzymatic synthesis of arabinosyladenine and 2′,3′-dideoxyinosine. ChemPlusChem 2013, 78, 157–165. [4] Calleri, E.; Ubiali, D. et al. Immobilized purine nucleoside phosphorylase from Aeromonas hydrophila as an on-line enzyme reactor for biocatalytic applications. J. Chromatogr. B 2014, DOI: 10.1016/j.jchromb.2013.12.031.