After optimizing the synthesis under hypoxic and alkaline reaction conditions in small-scale experiments, we aimed to synthesize and purify the four 2-Seleno pyrimidines in semipreparative scale. Therefore, the optimized reaction conditions were up-scaled to a volume of 50 mL with 5 mM 2-Se-nucleobase. The observed product formations in the larger scale of 41-47% after 3 (2b), 4 (1b) or 5 h (1a, 2a) (Table ) were in good accordance with the small-scale experiments. Purification of the target compounds from these reaction mixtures, however, proved rather challenging due to the presence of large quantities of unreacted sugar donor. In fact, our attempts to purify any of the products by preparative HPLC were unsuccessful as the obtained material persistently contained sugar donor starting material. Therefore, we applied an initial silica chromatography step on normal phase to remove most of the sugar donor, followed by a second purification step via preparative HPLC. Using this two-step process, we obtained 4.9-29.5 mg of the target nucleosides, corresponding to isolated yields of 6-40% (Table , Figure ). Despite the incomplete conversions and product losses during purification, our biocatalytic route compares favorably to its chemical counterparts where yields of less than 10% are typically achieved.