ENZYMATIC SYNTHESIS OF NOVEL 1,2,4-TRIAZOLE-3-CARBOXAMIDE NUCLEOSIDES: STUDY OF SUBSTRATE SPECIFICITY OF E. coli NUCLEOSIDE PHOSPHORYLASES
Abstract
Enzymatic synthesis of nucleosides using E. coli uridine (UP) and purine nucleoside (PNP) phosphorylases is an excellent alternative to chemical methods. We have investigated the substrate specificity of the UP/PNP cascade towards a novel acceptor, N-(3-fluorophenyl)-1H-1,2,4-triazole-3-carboxamide (TCA-Ph-3F), using natural and modified carbohydrate donors - uridine (Urd), arabinofuranosyluracil (Ara-U), and 3'-deoxyuridine (3'-dU) (see Scheme). The conditions of the transglycosylation reaction : 5 mM potassium phosphate, pH 7.0, 50 °C, donor:acceptor ratio 9:1. The reaction progress was monitored by RP-HPLC. Ribosylation with Urd was highly efficient: 1.7 U/mL of UP and 1.4 U/mL of PNP yielded 100% conversion in 1 h, whereas an excess (51 U/mL of UP and 42 U/mL of PNP) caused product degradation to 0% by 41 h due to the reverse phosphorolysis. In contrast, under common conditions (5.1 U/mL of UP and 4.2 U/mL of PNP, 24 h), transfer of modified sugars was slow due to strict enzyme specificity, yielding only 6% conversion for Ara-U and 14% for 3'-dU. Optimization (51 U/mL of UP and 42 U/mL of PNP, 72 h) overcame this kinetic barrier, raising conversions to 62% (Ara-U) and 82% (3'-dU). This should allow a successful synthesis of modified triazole nucleosides in high yields.
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