Based on the solvent screening we explored the further optimization of the synthesis of a high value pyrimidine nucleoside, 3. Nucleoside 3 (EdU) is used extensively in cellular proliferation assays, as a Raman active reporter, and as a bio-orthogonal reactive group for the synthesis of bioconjugates. When 5 equiv. of 8 were used as the 2'-deoxyribosyl donor the transformation resulted in 53% conversion to 3 using 13 as the nucleobase (Entry 1, Figure ). Increasing the enzyme loading from 2 to 4 µg.mL -1 (from a 1 mg.mL -1 stock solution), increased the conversion to a maximum of 66 % (Entries 2-4). Critical to increasing the conversion of 3 was increasing the equivalents of 13 from 1 to 10 whilst maintaining the catalytic loading of LlNDT-2 at 4 µg.mL -1 (Entries 5-6). A further increase to 20 equiv. of 13 resulted in 90% conversion to 3 (Entry 8). The scope of these optimized conditions was used to scale up the synthesis of pyrimidine nucleoside analogues modified at the 5-position (Figure ). 5-modified nucleosides were then prepared on millimole scale to demonstrate the scalability of this biocatalytic reaction. In this way, 3 and 12 were prepared on 1 mmol. scale in 52% (132 mg) and 55% (137 mg) yields respectively. Preparation of 14 and 15 was also demonstrated affording the desired products in 56% (840 mg) and 31% (53 mg), respectively.