Chemo-enzymatic synthesis of α-d-pentofuranose-1-phosphates using thermostable pyrimidine nucleoside phosphorylases

Abstract

α-d-pentofuranose-1-phosphates (Pentose-1Ps) are key intermediates in nucleoside metabolism and important precursors for the enzymatic synthesis of modified nucleosides. To date, Pentose-1Ps are mainly produced by chemical approaches which have numerous disadvantages. Therefore, several enzymatic methods employing mesophilic enzymes have been developed but are not widely applied due to their limited substrate spectrum. Here we report the use of thermostable nucleoside phosphorylases for the chemo-enzymatic synthesis of modified Pentose-1Ps (2-deoxy-2-fluoro-α-d-ribofuranose-1-phosphate, α-d-arabinofuranose-1-phosphate, and 2-deoxy-2-fluoro-α-d-arabinofuranose-1-phosphate), which are interesting building blocks for the synthesis of modified nucleosides. After optimizing the synthesis protocol using the natural substrates uridine and thymidine, grams of modified Pentose-1Ps were purified as their Ba-salts with over 95% purity. Their structures were confirmed by NMR spectroscopy and the temperature and pH stability of natural and modified Pentose-1Ps in aqueous solution was -evaluated. Four of the Pentose-1P-Ba salts were stable with no visible degradation up to 60 °C and pH above 5, while 2-deoxy-α-d-ribofuranose-1-phosphate was less stable. The presented protocol provides an easy, fast, and environmentally-friendly method to produce grams of modified Pentose-1P-Ba salts of high purity.