Adenine → Vidarabine

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For the determination of transglycosylation catalytic activity the mixture of cell lysates containing both UPase and PNPase were prepared by mixing the lysates so as to have UPase:PNPase enzymatic-activity ratio of about 1:1, determined as in Examples 6 and 7. Transglycosylation reaction was carried out at analytical scale in the following conditions: 250 μl of cell lysates (equivalent to 14 units of each UPase and PNPase enzymatic activities) was added to 10 ml of a solution having the following composition: 4 mM 1-β-D-ribofuranosyluracil (uridine nucleoside), 4 mM adenine base, 30 mM potassium phosphate buffer pH7, thermostatically controlled at 60° C. After 1.5 hours at 60° C., the reaction was stopped by diluting the mixture 1:5 and cooling in ice. The percentage of bioconversion of adenine base to 9-β-D-ribofuranosyladenine (adenosine nucleoside) was determined by analyzing an aliquot of the reaction mixture by high performance liquid chromatography (HPLC) with the use of Kromasil 100-5C18 (Akzo Nobel) column of a size of 250×4.6 mm and eluted with a 4% methanol-water solution. The transglycosylation catalytic activity was expressed as units·ml−1 (μmoles of Ara-A formed in 1.5 hours·ml−1 of mixture of cell lysates) or in units·g−1 of moist resin (μmoles of D-ribofuranosyladenine formed in 1.5 hours·ml−1 of cell lysate) and was calculated relative to a standard D-ribofuranosyladenine solution eluted by HPLC in the same conditions. Under these conditions, about 55 percent of adenosine nucleoside was formed (approximately 9 units per ml of cell lysate).

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