Biocatalytic synthesis of ribonucleoside analogues using nucleoside transglycosylase-2

Ribonucleosides are essential building blocks used extensively in antiviral and oligonucleotide therapeutics. A major challenge in the further development of nucleoside analogues for therapeutic applications is access to scalable and environmentally sustainable synthetic strategies. This study uses the type II nucleoside 2'-deoxyribosyltransferase from <i>Lactobacillus leichmannii</i> (<i>Ll</i>NDT-2) to prepare a suite of ribonucleoside analogues using naturally-occurring uridine and cytidine sugar donors. Crystal structure and mutational analyses are used to define the substrate tolerance of the nucleobase exchange and the 2'-substituent of the nucleoside sugar donor. Nucleobase profiling identified acceptance of both purine and pyrimidine nucleobases. Finally, the scalability of the approach is showcased, enabling the preparation of ribonucleosides on millimolar scales. This biocatalytic strategy opens up opportunities to establish chemoenzymatic routes to prepare nucleoside analogues incorporating 2' modifications that are of therapeutic importance.