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© Research
Publication : Analytical biochemistry

Chemienzymatic synthesis of uridine nucleotides labeled with [15N] and [13C]

Scientific Fields
Diseases
Organisms
Applications
Technique

Published in Analytical biochemistry - 10 Dec 1995

Gilles AM, Cristea I, Palibroda N, Hilden I, Jensen KF, Sarfati RS, Namane A, Ughetto-Monfrin J, Bârzu O

Link to Pubmed [PMID] – 8747475

Anal. Biochem. 1995 Dec;232(2):197-203

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.

http://www.ncbi.nlm.nih.gov/pubmed/8747475