Equilibrium constants for the phosphorolysis of the sugar donors have been described recently for a broad temperature range, but the corresponding values for 2-Se-nucleosides have not been reported yet. Since these constants are Final concentrations of PyNP-04 of 50.4 µg mL -1 (around 4 U, A) and 24.6 µg mL -1 (around 2 U, B) were applied. Experimental conversion was determined by HPLC as the conversion of 2 to 2b. The equilibrium constant of phosphorolysis was calculated based on the experimental data of the 5-fold sugar donor excess (A). Predictions of the conversion using different sugar donor to nucleobase ratios were carried out as described previously. Similar results were obtained for the other products (Table ). Please see the externally hosted Supporting Information for raw data and calculations. Table . Equilibrium state thermodynamic calculations were used to determine appropriate reaction conditions for the synthesis of 1a-2b Product Product formation [%] at equilibrium for 5-fold sugar donor excess Equilibrium constant of phosphorolysis Product formation [%] at equilibrium for 10-fold sugar donor excess calculated experimental (1 mM nucleobase) experimental (5 mM nucleobase) Experimental conversion was determined by HPLC. Equilibrium constants were calculated with the experimental data of the 5-fold sugar donor excess. Predictions of the conversion using 10-fold sugar donor excess were carried out as described previously. Please see the externally hosted Supporting Information for raw data and calculations. required for yield optimization via equilibrium thermodynamics, we performed small-scale transglycosylation reactions to determine these indirectly. Using a five-fold excess of the sugar donor over the Senucleobase and 0.09 equivalents of phosphate, equilibrium conversions between 30 and 35% were observed (Table , Figure for 2b as a visual example). This yielded equilibrium constants of the phosphorolysis of the Senucleosides 1a-2b in the range of 5 to 10 (Table ). Interestingly, these equilibrium constants surpass those of other natural and modified pyrimidine nucleosides (0.1-0.8) by around an order of magnitude. While the reason for these high equilibrium constants is unclear to date, we hypothesize that the increased electron density near the glycosylation site may contribute to a weaker C1'-N1 bond.