The 2-Se-modified nucleosides 1a-2b were accessed in a one-pot transglycosylation reaction using the pyrimidine nucleoside phosphorylase PyNP-04 as biocatalyst. The thermodynamic characterization was performed as described previously. Reaction equilibria were determined from reactions with 1 mM 2-Se-nucleobase (1 or 2), 5 mM sugar donor (a or b), 5 mM DTT, 50 mM glycine/NaOH buffer pH 9 and 50.4 µg mL -1 (around 4 U) PyNP-04 in a total volume of 1 mL. The nitrogen-saturated reaction mixtures were incubated at 60 °C for the deoxyribosides 1b and 2b and 80 °C for the ribosides 1a and 2a until reaction completion (indicated by no further product formation; within 30 min under these conditions). Samples were diluted to 1 mM sugar donor concentration in MeOH, centrifuged (4 °C, 21,500 g, 20 min) and analyzed by HPLC.