In conclusion, we identified and characterized TtPyNP as a biocatalyst for the diversification of 4ʹmethylated nucleosides. Reversible phosphorolysis of a methylated precursor 1a yields stable the pentose-1phosphate 3 which can be employed as a sugar synthon to access a range of modified nucleosides in one pot. Our investigations revealed that sufficient space near the active site in the open conformation of PyNPs appears crucial for binding and conversion of 1a. Furthermore, the equilibrium thermodynamics of the phosphorolysis of 4ʹ-methylated nucleosides largely resemble those of ribosyl nucleosides, indicating that substitutions distant from the anomeric position have only minor effects on the conversions in these systems. Leveraging principles of thermodynamic reaction control enabled us to access a spectrum of 4ʹ-methylated nucleosides bearing different pyrimidine and purine bases in transglycosylation reactions. Lastly, we expect that other 4ʹ-modified nucleoside analogs can be obtained with such biocatalytic systems in a similar fashion (probably with comparable equilibrium thermodynamics), although bulkier 4ʹ-substitutions will likely require some extent of protein engineering to improve activity.