A novel thymidine phosphorylase to synthesize (halogenated) anticancer and antiviral nucleoside drugs in continuous flow

Abstract

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.

Scheme 1. Two-step one-pot enzymatic reaction for the synthesis of halogenated pyrimidine nucleosides using thymidine as sugar donor.

Fig. 1. Monitoring the transglycosylation conversions to produce halogenated nucleoside analogues using free HeTP. The reaction mixture contained 10 mM of the halogenated nucleobase and 4 eq. of thymidine in 20 mM phosphate buffer at pH 7.5. The reactions were triggered with 2 units of free HeTP and incubated at 37 °C and shaking.