Four pharmaceutically relevant nucleoside analogues (5-fluoro-2'-deoxyuridine, 5-chloro-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, and 5-iodo-2'-deoxyuridine) have been synthesized by using a novel thymidine phosphorylase from the halotolerant H. elongata (HeTP). Following enzyme immobilization on microbeads, the biocatalyst was implemented as a packed-bed reactor for the continuous production of halogenated nucleosides, achieving up to 90% conversion at the 10 mM scale with 30 min residence time. Taking the synthesis of floxuridine (5-fluoro-2'-deoxyuridine) as a study case, we obtained the highest space-time yield (5.5 g L-1 h-1) reported to date. In addition, bioinformatic tools such as MD analysis and CapiPy have contributed to shine light on the catalytic performance of HeTP as well as its immobilization, respectively.
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