This work presents a biocatalytic synthesis of 2-Sepyrimidine nucleosides via transglycosylation from uridine or thymidine. Following a thermodynamic characterization of the phosphorolysis of the target nucleosides on a small scale, four selenium-containing pyrimidine nucleosides were obtained in scale-up experiments where alkaline and hypoxic conditions, as well as high temperatures, enabled sufficient solubility and stability of the starting materials. Although the present route delivers improved yields compared to previous efforts, product purification currently represents a major bottleneck and needs to be addressed in future studies.