The equilibrium constants of phosphorolysis of the 2-Sepyrimidine nucleosides were calculated via equilibrium state thermodynamics and numerical solutions. Nucleoside phosphorolysis is a tightly thermodynamically controlled reaction and transglycosylations behave as coupled equilibrium reactions as they comprise a forward and a reverse phosphorolysis. Thus, knowledge of the equilibrium constant of the sugar donor, the concentrations of the starting materials as well as the degree of conversion to the product nucleoside allows calculation of the equilibrium constants of phosphorolysis of the product nucleoside via numerical solutions of the system of coupled equilibrium constraints (see ). These numerical solutions can either be obtained via the Python code described in our previous work or, more conveniently, the Excel sheet presented in the externally hosted Supplementary Information of this publication. Herein, we used the previously published equilibrium constants of phosphorolysis of the sugar donors a and b for calculation. Based on the obtained equilibrium constants, expected conversions for the 2-Senucleosides using a 10-fold sugar donor excess were calculated (please see the externally hosted Supplementary Information for details). To verify the theoretical calculations and to monitor the reaction progression, reactions with 5 mM 2-Se-base, 50 mM sugar donor, 5 mM DTT in 50 mM glycine/NaOH buffer pH 9 saturated with nitrogen and 24.64 µg mL -1 (around 2 U) PyNP-04 in a total volume of 1 mL were performed at 60 °C for the deoxyribosides and 80 °C for the ribosides. Samples were diluted 50-fold in MeOH, centrifuged (4 °C, 21,500 g, 20 min) and analyzed by HPLC.