title: Enzymatic synthesis of highly fluorescent 8-azapurine ribosides using a purine nucleoside phosphorylase reverse reaction: variable ribosylation sites.
abstract: Various forms of purine-nucleoside phosphorylase (PNP) were used as catalysts of enzymatic ribosylation of selected fluorescent 8-azapurines. It was found that the recombinant calf PNP catalyzes ribosylation of 2,6-diamino-8-azapurine in a phosphate-free medium, with ribose-1-phosphate as ribose donor, but the ribosylation site is predominantly N7 and N8, with the proportion of N8/N7 ribosylated products markedly dependent on the reaction conditions. Both products are fluorescent. Application of the E. coli PNP gave a mixture of N8 and N9-substituted ribosides. Fluorescence of the ribosylated 2,6-diamino-8-azapurine has been briefly characterized. The highest quantum yield, ~0.9, was obtained for N9-β-d-riboside (λmax 365 nm), while for N8-β-d-riboside, emitting at ~430 nm, the fluorescence quantum yield was found to be close to 0.4. Ribosylation of 8-azaguanine with calf PNP as a catalyst goes exclusively to N9. By contrast, the E. coli PNP ribosylates 8-azaGua predominantly at N9, with minor, but highly fluorescent products ribosylated at N8/N7.
authors.affiliation: Department of Biophysics, University of Varmia & Masuria, 4 Oczapowskiego St., 10-719 Olsztyn, Poland. alicja.stachelska@uwm.edu.pl.
authors.forename: Alicja
authors.initials: A
authors.lastname: Stachelska-Wierzchowska
authors.forename: Jacek
authors.initials: J
authors.lastname: Wierzchowski
authors.forename: Beata
authors.initials: B
authors.lastname: Wielgus-Kutrowska
authors.forename: Goran
authors.initials: G
authors.lastname: Mikleušević
mesh_terms: D000818:Animals; D001375:Azaguanine / Q000031:analogs & derivatives* / Q000138:chemical synthesis*; D055162:Biocatalysis; D002417:Cattle; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D006031:Glycosylation; D007700:Kinetics; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012267:Ribosemonophosphates / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D012267:Ribosemonophosphates; D011683:Purine-Nucleoside Phosphorylase; D001375:Azaguanine