| Abstract
| - The enzymatic synthesis of thymidine from 2-deoxy-d-ribose-5-phosphate is achieved, in a one-pottwo-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 overproducingE. coli cells (12000 units/84 mg protein) and is used without further purification. In the secondstep thymine is coupled to the sugar-1-phosphate. The thermodynamically unfavorable equilibriumis shifted to the product by addition of MnCl2 to precipitate inorganic phosphate. In this way theoverall 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 functionalgroup transformations of the enzymatically prepared 2‘-deoxy-uridine. The method has beendemonstrated 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 uracilare tetralabeled at the (1,3-15N2,2,4-13C2)-atomic positions. All compounds are prepared withoutany scrambling or dilution of the labeled material and are thus obtained with a very high isotopeenrichment (96−99%). In combination with the methods that have been developed earlier it isconcluded that each of the 13C- and 15N-positions and combination of positions of the pyrimidinedeoxynucleosides can be efficiently labeled starting from commercially available and highly 13C- or15N-enriched formaldehyde, acetaldehyde, acetic acid, potassium cyanide, methylamine hydrochloride, and ammonia.
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