In this study, we demonstrate enzymatic transglycosylation catalyzed by LlNDT-2 using a 2ʹmodified nucleoside sugar donor either in the arabino and ribo configuration (Figure ). We define the mechanistic basis for substrate acceptance and highlight how the nature of the modification may interact with the active site of the enzyme. Finally, we demonstrate the potential of LlNDT-2 to prepare high value nucleoside analogues on a 1 mmol scale.