Abstract
Various forms of purine-nucleoside phosphorylase (PNP) were used as catalysts of enzymatic ribosylation of selected fluorescent 8-azapurines. It was found that the recombinant calf PNP catalyzes ribosylation of 2,6-diamino-8-azapurine in a phosphate-free medium, with ribose-1-phosphate as ribose donor, but the ribosylation site is predominantly N7 and N8, with the proportion of N8/N7 ribosylated products markedly dependent on the reaction conditions. Both products are fluorescent. Application of the E. coli PNP gave a mixture of N8 and N9-substituted ribosides. Fluorescence of the ribosylated 2,6-diamino-8-azapurine has been briefly characterized. The highest quantum yield, ~0.9, was obtained for N9-β-d-riboside (λmax 365 nm), while for N8-β-d-riboside, emitting at ~430 nm, the fluorescence quantum yield was found to be close to 0.4. Ribosylation of 8-azaguanine with calf PNP as a catalyst goes exclusively to N9. By contrast, the E. coli PNP ribosylates 8-azaGua predominantly at N9, with minor, but highly fluorescent products ribosylated at N8/N7.
Highlights
Enzymatic ribosylation of purine analogues has been proposed as an alternative to chemical synthesis of a variety of biologically active nucleosides [1,2,3,4]
We examined a possibility of enzymatic synthesis of ribosides of 2,6-diamino-8-azapurine (Figure 1), a highly fluorescent purine analog [17], using as catalysts various forms of purine-nucleoside phosphorylase (PNP, for a review see [18]), trimeric calf PNP and hexameric E. coli PNP, and α-D-ribose-1-phosphate (R1P) as a ribose donor
Phosphate, or if the reaction was allowed to proceed for 24 h, the apparent trans-ribosylation occurred, resulting in much higher proportion of the N8-riboside in the case of calf PNP catalysis, and of N9-riboside in the reaction catalyzed by the E. coli enzyme
Summary
Enzymatic ribosylation of purine analogues has been proposed as an alternative to chemical synthesis of a variety of biologically active nucleosides [1,2,3,4]. 8-azapurine nucleosides are known as fluorescent species, isosteric with natural purine nucleosides, and may substitute for them in many biochemical processes [8,9]. They are suitable for mechanistic investigations, including probing of the active sites of purine-related enzymes and ribozymes [10,11,12,13,14], as well as for analytical purposes [15,16]. The parent purine nucleoside, 2,6-diaminopurine riboside, is known to possess interesting biochemical properties [21,22,23] and was recently applied to mechanistic studies of the hairpin ribozyme [24], which expressed full activity when the crucial guanine residue was replaced by. The 2,6-diamino-8-azapurine (2-amino-8-azaadenine) ribosides and related compounds exhibit very intense fluorescence in neutral aqueous media, as shown below
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