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

• Published in: Analytica chimica acta Vol. 1139; pp. 119 - 128
• Main Authors: Stachelska-Wierzchowska, A., Wierzchowski, J.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-12-2020
• Subjects: Adenine; Blood activity; Escherichia coli - metabolism; Fluorescent/fluorogenic substrates; Humans; Kinetics; Nucleoside and nucleobase analogs; Nucleosides - analogs & derivatives; Purine-Nucleoside Phosphorylase; Nucleoside and nucleobase analogs; R1P; XoX; MTAP; PNP; Fluorescent/fluorogenic substrates; DFPP-DG; Blood activity; HGPRT; EDTA; Purine-nucleoside phosphorylase; ADA
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2020.09.018

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.
Fluorescence changes of the substrate (40 μM) observed in 1000-fold diluted human blood lysate. Time step was 5 min. Red curve represents 100% reaction completed. [Display omitted]
•Nucleobase analogs, ribosylated at positions other than N (9), are substrates for PNP.•They are highly fluorescent and spectra markedly differ from those of the aglycons.•The PNP activity can be observed in 1000-fold diluted hemolysates.•The substrates are highly selective for human vs. bacterial PNP forms.