Synthesis of 2-Se pyrimidine nucleosides in semipreparative scale 2-Se nucleosides were synthesized in a reaction volume of 50 mL consisting of 5 mM 2-Se base (1 or 2), 50 mM sugar donor (a or b), 5 mM DTT, 50 mM glycine/NaOH buffer pH 9 and 24.6 µg mL -1 (a: 98.5 U, b: 97.4 U) PyNP-04. The reaction mixture was saturated with nitrogen. Deoxyribosyl derivatives were prepared at 60 °C and ribosyl derivatives at 80 °C. The reactions were stopped after 3 to 5 h by a pH shift to 13 through addition of 10 M NaOH (monitored with a pH-electrode). The pH was shifted back to 9 using 25% HCl and proteins were removed by filtration at room temperature using a vacuum pump and 0.45 µm cellulose nitrate filters (Sartorius, Goettingen, Germany). The filtrate was saturated with nitrogen and stored at 4 °C until purification.