Synthesis of Adenine Nucleosides by Transglycosylation using Two Sequential Nucleoside Phosphorylase-Based Bioreactors with On-Line Reaction Monitoring by using HPLC.

Uridine phosphorylase from Clostridium perfringens ( CpUP, EC 2.4.2.3) was immobilized covalently in an aminopropylsilica monolithic column (25 mm×4.6 mm) upon functionalization with glutaraldehyde. Imino bonds that result from the reaction between the enzyme and the support were reduced chemically to afford a 66 % yield (13 mg) determined spectrophotometrically. The CpUP immobilized enzyme reactor (IMER) was connected to a silica particle-based IMER that contained a purine nucleoside phosphorylase from Aeromonas hydrophila ( AhPNP, EC 2.4.2.1), which was developed previously and used successfully for the fast synthesis of some purine ribonucleosides by a 'one-enzyme' transglycosylation. CpUP-IMER and AhPNP-IMER were connected to a HPLC system by a six-way switching valve. In this set-up, the synthesis of 2′-deoxyadenosine (dAdo, 8), adenosine (Ado, 9), and arabinosyladenine (araA, 10) by a 'two-enzyme' transglycosylation is coupled directly to on-line reaction monitoring. Under the optimized transglycosylation conditions (2:1 ratio sugar donor/base acceptor; 10 m m 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 approximately 90 %, and araA was synthesized in 20 % yield. [ABSTRACT FROM AUTHOR]