title: The quantitative determination of metabolites of 6-mercaptopurine in biological materials. VII. Chemical synthesis by phosphorylation of 6-thioguanosine 5'-monophosphate, 5'-diphosphate and 5'-triphosphate, and their purification and identification by reversed-phase/ion-pair high-performance liquid chromatography and by various enzymatic assays.
abstract: A fast and reliable two-step method has been established for the chemical synthesis of 6-thioguanosine 5'-monophosphate, 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate starting from the ribonucleoside. In the first step, 6-thioguanosine dissolved in triethyl phosphate, at high yield reacts with phosphorus oxide trichloride to 6-thioguanosine 5'-monophosphate which is purified by anion-exchange chromatography on DEAE-Sephadex using a step gradient of hydrochloric acid. In the second step, 6-thioguanosine 5'-monophosphate dissolved in water, reacts with phosphoric acid in the presence of pyridine/dicyclohexyl carbodiimide and is converted to 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate which are separated from each other and from the 6-thioguanosine 5'-monophosphate by anion-exchange chromatography on DEAE-Sephadex using a gradient of ammonium bicarbonate. Material from each step of the preparation procedure is separated by reversed-phase HPLC chromatography and analyzed for its free ribonucleoside content, 5'-monophosphate, 5'-diphosphate, 5'-triphosphate and small amounts of unidentified phosphorylated compounds. The purity of the final preparations and the identity of each 6-thioguanosine 5'-phosphate are proven by highly specific enzymatic peak-shifting/HPLC analyses using alkaline phosphatase, 5'-nucleotidase, pyruvate kinase, nucleoside diphosphate kinase and combined hexokinase/glucose 6-phosphate dehydrogenase.
authors.affiliation: Institut für Physiologische Chemie, Johannes Gutenberg-Universität, Mainz, F.R.G.
authors.forename: H J
authors.initials: HJ
authors.lastname: Breter
authors.forename: H
authors.initials: H
authors.lastname: Mertes
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004798:Enzymes; D006150:Guanine Nucleotides / Q000032:analysis* / Q000138:chemical synthesis / Q000302:isolation & purification; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000032:analysis / Q000138:chemical synthesis / Q000302:isolation & purification; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000032:analysis / Q000138:chemical synthesis / Q000302:isolation & purification; D007700:Kinetics; D015122:Mercaptopurine / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D013873:Thionucleotides / Q000032:analysis* / Q000138:chemical synthesis / Q000302:isolation & purification
chemical_list: D004798:Enzymes; D006150:Guanine Nucleotides; D013873:Thionucleotides; D006153:Guanosine Diphosphate; C003964:6-thioguanylic acid; C032829:6-thioguanosine 5'-diphosphate; C032827:6-thioguanosine 5'-triphosphate; D006160:Guanosine Triphosphate; D015122:Mercaptopurine