Chemienzymatic synthesis of uridine nucleotides labeled with [15N] and [13C].
UTP, labeled with 15N and 13C (at all carbon atoms of the ribose moiety), was obtained enzymatically from [15N]uracil and [13C6]glucose. Eleven enzymes and suitable substrates reconstituted a metabolic pathway in which glucose was first transformed to 5-phosphoribosyl-1-pyrophosphate. The latter compound plus uracil yielded UMP in a second step by the reaction catalyzed by uracil phosphoribosyltransferase. UMP was subsequently phosphorylated to the corresponding di- and triphosphate. ATP, required for five phosphorylation reactions, was regenerated from creatine phosphate, whereas NADP+ necessary for the oxidation of glucose 6-phosphate and 6-phosphogluconate was recycled by glutamate dehydrogenase and excess of ammonia and alpha-oxoglutarate. Despite the number and complexity of the enzymatic steps, the synthesis of [15N, 13C]UTP is straightforward with an overall yield exceeding 60%. This method, extended and diversified to the synthesis of all natural ribonucleotides, is a more economical alternative for obtaining nucleic acids for structural analysis by heteronuclear NMR spectroscopy.