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,EC2.4.2.3) was immobilizedcovalentlyinanaminopropylsilica monolithic column (25 mm V 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 a66% yield (13 mg) determined spectrophotometrically.The CpUP immobilized enzyme reactor (IMER) was connected to asilica particle-based IMER that contained apurine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP, EC 2.4.2.1), which was developed previously and used successfully for the fast synthesis of some purineribonucleosidesbya “one-enzyme” transglycosylation. CpUP-IMER and AhPNP-IMER were connected to aHPLC system by asix-way switching valve. In this set-up, the synthesis of 2’-deoxyadenosine (dAdo, 8), adenosine (Ado, 9), and arabinosyla denine (araA, 10)bya “two-enzyme” transglycosylation is coupled directly to on-line reactionmonitoring. Under the optimized transglycosylation conditions (2:1 ratio sugar donor/base acceptor;10mm phosphate buffer;pH7.25;temperature 37 8C, flow rate 0.1 mL min@1), defined by a2(5-2)III experimental design, the conversion of dAdo andAdo was approximately 90 %, and araA was synthesized in 20 %yield.