title: Site-Selective Ribosylation of Fluorescent Nucleobase Analogs Using Purine-Nucleoside Phosphorylase as a Catalyst: Effects of Point Mutations.
abstract: Enzymatic ribosylation of fluorescent 8-azapurine derivatives, like 8-azaguanine and 2,6-diamino-8-azapurine, with purine-nucleoside phosphorylase (PNP) as a catalyst, leads to N9, N8, and N7-ribosides. The final proportion of the products may be modulated by point mutations in the enzyme active site. As an example, ribosylation of the latter substrate by wild-type calf PNP gives N7- and N8-ribosides, while the N243D mutant directs the ribosyl substitution at N9- and N7-positions. The same mutant allows synthesis of the fluorescent N7-β-d-ribosyl-8-azaguanine. The mutated form of the E. coli PNP, D204N, can be utilized to obtain non-typical ribosides of 8-azaadenine and 2,6-diamino-8-azapurine as well. The N7- and N8-ribosides of the 8-azapurines can be analytically useful, as illustrated by N7-β-d-ribosyl-2,6-diamino-8-azapurine, which is a good fluorogenic substrate for mammalian forms of PNP, including human blood PNP, while the N8-riboside is selective to the E. coli enzyme.
authors.affiliation: Department of Physics and Biophysics, University of Varmia &amp; Masuria in Olsztyn, 4 Oczapowskiego St., 10-719 Olsztyn, Poland. alicja.stachelska@uwm.edu.pl.
authors.forename: Alicja
authors.initials: A
authors.lastname: Stachelska-Wierzchowska
authors.affiliation: Department of Physics and Biophysics, University of Varmia &amp; Masuria in Olsztyn, 4 Oczapowskiego St., 10-719 Olsztyn, Poland. jacek.wie@uwm.edu.pl.
authors.forename: Jacek
authors.initials: J
authors.lastname: Wierzchowski
authors.affiliation: Division of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland. abzowska@biogeo.uw.edu.pl.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Bzowska
authors.affiliation: Division of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland. beata@biogeo.uw.edu.pl.
authors.forename: Beata
authors.initials: B
authors.lastname: Wielgus-Kutrowska
mesh_terms: D001375:Azaguanine / Q000031:analogs & derivatives* / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D015394:Molecular Structure; D017354:Point Mutation; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D011683:Purine-Nucleoside Phosphorylase; D001375:Azaguanine
keywords: 8-azapurines; enzymatic ribosylation; fluorescent nucleosides; purine nucleoside phosphorylase