We envisioned that nucleoside phosphorylases could provide a biocatalytic platform for late-stage diversification of 4ʹ-modified nucleosides. These enzymes catalyze the reversible phosphorolysis of nucleosides to the corresponding nucleobases and pentose-1-phosphates via an SN2-like mechanism. The reaction sequence involving phosphorolysis of one nucleoside and in situ reverse phosphorolysis to the target nucleoside is generally known as a transglycosylation, and effectively transfers the sugar moiety from one nucleobase to another. While this reactivity is well-established for ribosyl and 2ʹdesoxyribosyl nucleosides and a few 2ʹ-modified nucleosides (Scheme 1, center), there are no examples in the literature of the enzymatic synthesis of 4ʹ-modified nucleosides, except for Merck's recent report of a 5-step enzymatic cascade for the synthesis of the 4ʹ-alkynylated nucleoside drug Islatravir. Therefore, the feasibility of transglycosylation reactions with 4ʹ-modified nucleosides as well as the thermodynamics of such a cascade process are notably underexplored. Herein, we address this gap by reporting on the phosphorolysis and transglycosylation of the simplest 4ʹ-alkylated pyrimidine nucleoside, 4ʹmethyluridine (1a). Following the identification of a suitable biocatalyst, and a characterization of its reactivity with kinetic experiments and docking studies, we explored the thermodynamics of the phosphorolysis of 1a and leveraged this information in transglycosylation experiments to access a range of 4ʹ-methylated pyrimidine and purine nucleosides.