With a solid understanding of the thermodynamics and kinetics of the phosphorolysis of 1a by TtPyNP, we proceeded to diversify this scaffold by subjecting the sugar phosphate 3 to subsequent enzymatic catalysis with different nucleobases in situ. Using this transglycosylation approach (Figure and Scheme 1, center), we aimed to access a variety of 4ʹ-methylated nucleosides from 1a in a one-pot manner. After confirming the stability of 3 through equilibrium shift experiments (Figure ), we subjected 1a to phosphorolysis using only minimal phosphate in the presence of different pyrimidine nucleobases 2b-2e belonging to a panel of 5-substituted uracil analogs (Figures and). Analysis of the reaction mixtures by HPLC revealed consumption of 1a and the respective uracil analog with concurrent formation of new products (Figure ), which HRMS analysis identified as the nucleoside products arising from glycosylation of 2b-2e with 3. Equilibrium state thermodynamic calculations based on transglycosylation experiments with different sugar donor concentrations revealed apparent equilibrium constants of phosphorolysis of 0.12-0.73 for these products 1b-1e (Figure and). The trifluoromethylated pyrimidine 2f could also be converted, although the instability of the starting material and product in aqueous solution Geobacillus thermoglucosidasius (PNP). Please see the externally hosted supplementary information for raw data and calculations. precluded us from obtaining equilibrium data (Figure ). A similar elaboration of in situ generated 3 with purine nucleobases proceeded smoothly using the promiscuous purine nucleoside phosphorylase from Geobacillus thermoglucosidasius. Notably, the adenosine analogs 1g-1i were generated in much higher conversions, corresponding to equilibrium constants of phosphorolysis of 0.01-0.02, reflecting the more favorable thermodynamics typically observed for 6-aminopurines. The guanosine and inosine analogs 1j and 1k could also be accessed, although with lower conversions indicative of higher equilibrium constants (Figure ). These experiments not only confirmed that nucleoside transglycosylations with the methylated precursor 1a can deliver a range of modified nucleosides in a one-pot manner, but also that the equilibrium thermodynamics of this system largely resemble those of the well-described ribosyl nucleosides. These findings further indicated that these transglycosylations would offer themselves to rational reaction engineering using established principles of thermodynamic reaction control to predict and maximize conversions in these reactions. Indeed, thermodynamic calculations based on the obtained equilibrium constants suggested that 1b could, for instance, be obtained in 84% conversion from 1a using 4 equivalents of nucleobase, which we confirmed experimentally (Figures and). Similarly, 1i could be obtained in quantitative conversion with 4 equivalents of 2i, in agreement with our predictions. As a proof of synthetic utility, we subjected 1a to transglycosylation with 5 equivalents of 2e and obtained the iodinated 1e in 68% conversion (61% predicted) and ca. 40% isolated yield.