A multisubstrate purine nucleoside phosphorylase from Aeromonas hydrophila catalyzes the high-yield synthesis of modified purine ribosides

Nucleoside phosphorylases (NPs; EC 2.4.2.n) are intracellular enzymes widely distributed in eukaryotes and prokaryotes. NPs act in the salvage of nucleobases catalyzing, in presence of inorganic orthophosphate, the reversible conversion of (deoxy)ribonucleosides to their corresponding free base and a-D-(deoxy)ribose-1-phosphate (phosphorolysis). The reaction reversibility of NPs can be exploited to synthesize new nucleosides by adding a second nucleobase, either natural or not, to the reaction medium (transglycosylation). Thus, NPs provide a competitive synthetic route over the commonly used complex and multistage chemical methods, particularly in terms of stereoselectivity, yield and environmental impact. We have recently reported on the production, characterization and synthetic applications of a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP, E.C. 2.4.2.1). This PNP catalyzed the phosphorolysis of both natural 6-oxo- and 6-aminopurine (deoxy)ribonucleosides, and also accepted several modified purine ribosides as substrates. The synthetic potential of this multisubstrate PNP has been confirmed by the preparation, in very high yield and purity, of 2-amino-6-chloropurine-9-riboside, 6-methoxypurine-9-riboside and 2-amino-6-(methylthio)purine-9-riboside. The substrate specificity investigation has been now enlarged to a new library of modified purines, revealing that AhPNP is able to catalyze the transglycosylation of most the assayed nucleobases. In the view of large scale synthetic applications, AhPNP has been immobilized on solid support affording a reusable and very stable biocatalyst. The immobilized AhPNP has been successfully used in the synthesis of 2-amino-6-chloropurine-9-riboside providing the target compound in 90% yield.