Non-typical nucleoside analogs as fluorescent and fluorogenic indicators of purine-nucleoside phosphorylase activity in biological samples

Abstract

Several novel non-typical nucleoside analogs were examined as potential fluorescent indicators of purine-nucleoside phosphorylase (PNP) activity in human blood. The substrates included N⁷-riboside of 8-aza-2,6-diaminopurine, N⁶-riboside of 1,N⁶-etheno-adenine and N²-riboside of N²,3-etheno-2-aminopurine. Reaction rates and apparent Michaelis' constants were determined in 1000-fold blood lysates and compared with those for reference compounds, guanosine and 7-methylguanosine. It was concluded that the most promising for assaying human PNP in biological material was N⁶-riboside of 1,N⁶-etheno-adenine and N²-riboside of N²,3-etheno-2-aminopurine was optimal for the E. coli PNP, both offering at least 10-fold improvement in sensitivity relative to conventional assays. Other potential applications of this approach are discussed.

Keywords: Blood activity; Fluorescent/fluorogenic substrates; Nucleoside and nucleobase analogs; Purine-nucleoside phosphorylase.