Synthesis of adenine nucleosides by transglycosylation using two sequential nucleoside phosphorylase-based bioreactors coupled on-line to an HPLC system for reaction monitoring

Uridine phosphorylase from Clostridium perfringens (CpUP, EC 2.4.2.3) was covalently immobilized in an aminopropylsilica monolithic column (25 mm x 4.6 mm) upon functionalization with glutaraldehyde. Imino bonds resulting from the reaction between the enzyme and the support were chemically reduced affording a 66% yield (13 mg), as spectrophotometrically determined. The CpUP-IMER (Immobilized Enzyme Reactor) was connected to a silica particle-based IMER containing a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP, EC 2.4.2.1) which was previously developed and successfully used for the fast synthesis of some purine ribonucleosides by a “one-enzyme” transglycosylation. CpUP-IMER and AhPNP-IMER were coupled to an HPLC system, by a 6-way switching valve; in such a set-up, the synthesis of 2’-deoxyadenosine (dAdo, 8), adenosine (Ado, 9) and arabinosyladenine (araA, 10) by a “two-enzyme” transglycosylation is directly coupled to the on-line reaction monitoring. Under the optimized transglycosylation conditions (2:1 ratio sugar donor:base acceptor; 10 mM phosphate buffer; pH 7.25; temperature 37 °C, flow rate 0.1 mL min-1), defined by a 2(5-2) III experimental design, the conversion of dAdo and Ado was about 90%, whereas araA was synthesized in 20% yield.