Fluorescence emission properties of 8-azapurines and their nucleosides, and application to the kinetics of the reverse synthetic reaction of purine nucleoside phosphorylase

Abstract

An extensive study has been made of the fluorescence emission properties of the neutral and ionic forms in aqueous medium of the azapurine nucleosides, 8-azaadenosine (8-azaAdo), 8-azainosine (8-azaIno), 8-azaguanosine (8-azaGuo), and their aglycons. The fluorescence of 8-azaGuo at pH 7 originates from its anionic species (p K a = 8.05, φ = 0.55), as is also the case for 8-azaIno (p Ka = 8.0, φ = 0.02), whereas 8-azaAdo is a strong emitter ( φ = 0.06) as the neutral species. By contrast the corresponding free 8-azapurines are only weakly fluorescent in aqueous medium, with the exception of 8-azaguanine (8-azaG). Examination of the emission properties of N-substituted 8-azaguanines demonstrated that the observed blue emission of the neutral form of 8-azaG ( φ = 0.05 to 0.33, dependent on λ exc) originates from a minor tautomer of the compound, the N(8)-H form, present to the extent of 10–15%; while the principal N(9)-H tautomer is virtually nonfluorescent. The 8-azapurines are substrates of purine nucleoside phosphorylase (PNP), leading to their irreversible conversion to the corresponding nucleosides in the synthetic pathway of this enzyme. The fluorescent properties of these compounds, together with spectrophotometric methods, were applied to determine the basic kinetic parameters for synthesis of 8-azapurine nucleosides by PNP from mammalian (calf spleen) and bacterial ( Escherichia coli) sources. The fluorimetric method was also used to determine the kinetic parameters for the second substrate, α- d-ribose 1-phosphate, and for the analytical titration of the latter in solution. The pH optimum of the reverse synthetic PNP reaction with 8-azapurines as substrates is below pH 7, due to their enhanced acidity in comparison with natural purines. The 8-azapurine nucleosides, but not their aglycons, are reasonably good inhibitors of phosphorylysis of Ino and Guo by E. coli PNP. The most effective is 8-azaIno ( K i ∼ 20 μM), also the only one to inhibit phosphorolysis by the calf spleen enzyme ( K i ∼ 40 μM). The nature of this inhibition is apparently uncompetitive.