404839-28-7Relevant academic research and scientific papers
One-pot two-step enzymatic coupling of pyrimidine bases to 2-deoxy-D-ribose-5-phosphate. A new strategy in the synthesis of stable isotope labeled deoxynucleosides
Ouwerkerk,Steenweg,De Ruijter,Brouwer,Van Boom,Lugtenburg,Raap
, p. 1480 - 1489 (2007/10/03)
The enzymatic synthesis of thymidine from 2-deoxy-D-ribose-5-phosphate is achieved, in a one-pot two-step reaction using phosphoribomutase (PRM) and commercially available thymidine phosphorylase (TP). In the first step the sugar-5-phosphate is enzymatically rearranged to α-2-deoxy-D-ribose-1-phosphate. Highly active PRM is easily obtained from genetically modified overproducing E. coli cells (12000 units/84 mg protein) and is used without further purification. In the second step thymine is coupled to the sugar-1-phosphate. The thermodynamically unfavorable equilibrium is shifted to the product by addition of MnCl2 to precipitate inorganic phosphate. In this way the overall yield of the β-anomeric pure nucleoside increases from 14 to 60%. In contrast to uracil, cytosine is not accepted by TP as a substrate. Therefore, 2′-deoxy-cytidine is obtained by functional group transformations of the enzymatically prepared 2′-deoxy-uridine. The method has been demonstrated by the synthesis of [2′,5′-13C2]- and [1′,2′,5′-13C3]thymidine as well as [1′,2′,5′-13C3]2′-deoxyuridine and [3′,4′-13C2]2′-deoxycytidine. In addition the nucleoside bases thymine and uracil are tetralabeled at the (1,3-15N2,2,4-13C2)-atomic positions. All compounds are prepared without any scrambling or dilution of the labeled material and are thus obtained with a very high isotope enrichment (96-99%). In combination with the methods that have been developed earlier it is concluded that each of the 13C- and 15N-positions and combination of positions of the pyrimidine deoxynucleosides can be efficiently labeled starting from commercially available and highly 13C- or 15N-enriched formaldehyde, acetaldehyde, acetic acid, potassium cyanide, methylamine hydrochloride, and ammonia.
