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.
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 alpha-2-deoxy-D-ribose-1-phosphate. Highly active PRM is easily obtained from genetically modified overproducing E. coli cells (12,000 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 MnCl(2) to precipitate inorganic phosphate. In this way the overall yield of the beta-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'-(13)C(2)]- and [1',2',5'-(13)C(3)]thymidine as well as [1',2',5'-(13)C(3)]2'-deoxyuridine and [3',4'-(13)C(2)]2'-deoxycytidine. In addition the nucleoside bases thymine and uracil are tetralabeled at the (1,3-(15)N(2),2,4-(13)C(2))-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 (13)C- and (15)N-positions and combination of positions of the pyrimidine deoxynucleosides can be efficiently labeled starting from commercially available and highly (13)C- or (15)N-enriched formaldehyde, acetaldehyde, acetic acid, potassium cyanide, methylamine hydrochloride, and ammonia.
Ouwerkerk
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Leiden University, Leiden Institute of Chemistry, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
Steenweg
M
M
de Ruijter
M
M
Brouwer
J
J
van Boom
J H
JH
Lugtenburg
J
J
Raap
J
J
D002384:Catalysis; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry; D003853:Deoxyribonucleosides / Q000138:chemical synthesis* / Q000737:chemistry; D003857:Deoxyuridine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry; D004099:Dihydroxyacetone Phosphate / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D030781:Organisms, Genetically Modified; D010770:Phosphotransferases / Q000378:metabolism; D011743:Pyrimidines / Q000737:chemistry*; D012267:Ribosemonophosphates / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013936:Thymidine / Q000138:chemical synthesis* / Q000737:chemistry; D013939:Thymidine Phosphorylase / Q000378:metabolism; D014305:Triose-Phosphate Isomerase / Q000378:metabolism; D014498:Uracil / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry
D003853:Deoxyribonucleosides; D011743:Pyrimidines; D012267:Ribosemonophosphates; D003841:Deoxycytidine; D014498:Uracil; D004099:Dihydroxyacetone Phosphate; C058245:2-deoxyribose 5-phosphate; D013939:Thymidine Phosphorylase; D010770:Phosphotransferases; C036179:phosphoribomutase; D014305:Triose-Phosphate Isomerase; D013936:Thymidine; D003857:Deoxyuridine