Preparative enzymic synthesis of nucleoside-5'-phosphates.
1. Wheat shoot phosphotransferase has been employed, with p-nitrophenylphosphate as a phosphate donor, to specifically phosphorylate the 5'-position of a variety of nucleosides and nucleoside analogues. The specificity of the enzyme towards the 5'-position of pentose nucleosides is testified to by the complete resistance to phosphorylation of 5'-O-methylcytidine. 2. With the use of ion-exchange chromatography, the foregoing procedure has been applied to the large-scale preparation of nucleoside-5'-phosphates with overall yields of the order of 80-90%. Quantitative recovery of unreacted nucleoside makes it possible to use this method without risk of losses either on a small or large scale with rare nucleosides. It is also applicable to acid- and alkali-labile nucleosides which cannot readily be phosphorylated by chemical procedures. 3. The wheat shoot phosphotransferase also phosphorylated a galactopyranosyl nucleoside, as well as such derivatives as 1-(beta-hydroxyethyl)cytosine and 5-(beta-hydroxyethyl)uracil, showing that the enzyme does not have an absolute requirement for a 5-membered sugar ring, but rather for the presence of a primary hydroxyl group. 4. The phosphorylated derivatives of galactopyranosyluracil, and of both hydroxyethyl pyrimidines, were resistant to 5'-nucleotidase. E. coli alkaline phosphatase converted all three nucleotides quantitatively to the starting compounds. 5. A synthesis of 1-(beta-hydroxyethyl)cytosine is described.
D000469:Alkaline Phosphatase; D002852:Chromatography, Ion Exchange; D002854:Chromatography, Paper; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008722:Methods; D009708:Nucleotidases; D010770:Phosphotransferases / Q000378:metabolism*; D010944:Plants / Q000201:enzymology; D012265:Ribonucleotides / Q000096:biosynthesis*; D012910:Snake Venoms; D013997:Time Factors; D014908:Triticum / Q000201:enzymology
D012265:Ribonucleotides; D012910:Snake Venoms; D010770:Phosphotransferases; D009708:Nucleotidases; D000469:Alkaline Phosphatase