Flow-Synthesis of Nucleosides Catalyzed by an Immobilized Purine Nucleoside Phosphorylase from Aeromonas hydrophila : Integrated Systems of Reaction Control and Product Purification

A purine nucleoside phosphorylase from Aeromonas hydrophyla (AhPNP) was covalently immobilized in a pre-packed stainless steel column containing aminopropylsilica particles via Schiff base chemistry upon glutaraldehyde activation. The resulting AhPNP-IMER (Immobilized Enzyme Reactor, immobilization yield approximate to 50%) was coupled on-line through a 6-way switching valve to an HPLC apparatus containing an analytical or a semi-preparative chromatographic column. The synthesis of five 6-modified purine ribonucleosides was carried out by continuously pumping the reaction mixture through the AhPNP-IMER until the highest conversion was reached, and then directing the reaction mixture to chromatographic separation. The conditions of the AhPNP-catalyzed transglycosylations (2:1 ratio sugar donor:base acceptor; 10mM phosphate buffer; pH7.5; temperature 37 degrees C, flow rate 0.5mLmin(-1)) were optimized by a fractional factorial experimental design. Coupling the bioconversion step with the product purification in such an integrated platform resulted in a fast and efficient synthetic process (yield=52-89%; <10mg) where sample handling was minimized. To date, AhPNP-IMER has retained completely its activity upon 50 reactions in 10 months.