Tri-Cyclic Nucleobase Analogs and Their Ribosides as Substrates of Purine-Nucleoside Phosphorylases. II Guanine and Isoguanine Derivatives

Abstract

Etheno-derivatives of guanine, O6-methylguanine, and isoguanine were prepared and purified using standard methods. The title compounds were examined as potential substrates of purine-nucleoside phosphorylases from various sources in the reverse (synthetic) pathway. It was found that 1,N2-etheno-guanine and 1,N6-etheno-isoguanine are excellent substrates for purine-nucleoside phosphorylase (PNP) from E. coli, while O6-methyl-N2,3-etheno-guanine exhibited moderate activity vs. this enzyme. The latter two compounds displayed intense fluorescence in neutral aqueous medium, and so did the corresponding ribosylation products. By contrast, PNP from calf spleens exhibited only modest activity towards 1,N6-etheno-isoguanine; the remaining compounds were not ribosylated by this enzyme. The enzymatic ribosylation of 1,N6-etheno-isoguanine using two forms of calf PNP (wild type and N243D) and E. coli PNP (wild type and D204N) gave three different products, which were identified on the basis of NMR analysis and comparison with the product of the isoguanosine reaction with chloroacetic aldehyde, which gave an essentially single compound, identified unequivocally as N9-riboside. With the wild-type E. coli enzyme as a catalyst, N9-β-d- and N7-β-d-ribosides are obtained in proportion ~1:3, while calf PNP produced another riboside, tentatively identified as N6-β-d-riboside. The potential application of various forms of PNP for synthesis of the tri-cyclic nucleoside analogs is discussed.

Keywords

• chemical mutagenesis
• fluorescence
• nucleobase/nucleoside analogs
• purine nucleoside phosphorylase
• tautomerism