Site-Selective Ribosylation of Fluorescent Nucleobase Analogs Using Purine-Nucleoside Phosphorylase as a Catalyst: Effects of Point Mutations

Molecules. 2015 Dec 28;21(1):E44. doi: 10.3390/molecules21010044.


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.

Keywords: 8-azapurines; enzymatic ribosylation; fluorescent nucleosides; purine nucleoside phosphorylase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Azaguanine / analogs & derivatives*
  • Azaguanine / chemistry
  • Catalysis
  • Catalytic Domain
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / metabolism
  • Humans
  • Molecular Structure
  • Point Mutation*
  • Purine-Nucleoside Phosphorylase / chemistry
  • Purine-Nucleoside Phosphorylase / genetics*
  • Purine-Nucleoside Phosphorylase / metabolism


  • Fluorescent Dyes
  • Purine-Nucleoside Phosphorylase
  • Azaguanine