In the absence of obvious pyrimidine nucleoside phosphorylase (PyNP) candidates for the phosphorolysis of 1a, we began our investigation by screening a small panel of PyNPs with known broad substrate spectra. To our surprise, only the PyNP from Thermus thermophilus (TtPyNP) showed measurable conversion of 1a under screening conditions (Figure ). Other broad-spectrum PyNPs, such as those from Geobacillus thermoglucosidasius (GtPyNP) or Bacillus subtilis, displayed no activity with 1a (Figure ). To substantiate the observed conversion of 1a by TtPyNP, we performed a series of control experiments. Reactions either without Scheme 1. Synthesis and biocatalytic diversification of nucleosides with modified sugars. NB = Nucleobase, NP = nucleoside phosphorylase. phosphate, without enzyme or with denatured enzyme gave no conversion. Similarly, no conversion was observed under reaction conditions outside of the working space of TtPyNP (pH 3 or pH 12, Figure ). NMR analysis of a reaction mixture with TtPyNP and 1a corroborated the proposed reactivity and creation of the pentose-1-phosphate 3, as evident from the rise of an additional 1 H NMR signal at 5.57 ppm showing a strong H,P-HMQC signal (Figure ). Consistent with the native reactivity of PyNPs, inversion at the anomeric position was evident by this signal lacking NOE contacts to the 4ʹ-methyl group of 3, while the corresponding anomeric proton in 1a showed clear correlation to the methyl substituent.