Enzymatic Synthesis of Highly Fluorescent 8-Azapurine Ribosides Using a Purine Nucleoside Phosphorylase Reverse Reaction: Variable Ribosylation SitesReportar como inadecuado




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1

Department of Biophysics, University of Varmia & Masuria, 4 Oczapowskiego St., 10-719 Olsztyn, Poland

2

Division of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland

3

Division of Physical Chemistry, Rudjer Bošković Institute, POB 180, HR-10002 Zagreb, Croatia





*

Author to whom correspondence should be addressed.



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. View Full-Text

Keywords: 8-azapurines; nucleosides; enzymatic synthesis; fluorescence; purine-nucleoside phosphorylase 8-azapurines; nucleosides; enzymatic synthesis; fluorescence; purine-nucleoside phosphorylase





Autor: Alicja Stachelska-Wierzchowska 1,* , Jacek Wierzchowski 1, Beata Wielgus-Kutrowska 2 and Goran Mikleušević 3

Fuente: http://mdpi.com/



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