Modified nucleosides, such as 5-fluoro-2'-deoxyuridine (Floxuridine),2',3'-dideoxyinosine (Didanosine), arabinosyladenine (Vidarabine), arabinosyl-2-fluoroadenine (Fludarabine), are important antiviral and antitumor agents. These nucleosides are routinely prepared by multi-step syntheses which negatively affect the outcome of the process in terms of yield, purity and costs and with an high environmental impact. Alternatively, nucleosides can be prepared via a one-pot enzymatic process catalyzed by nucleoside phosphorylases (NPs) in fully aqueous medium. NPs catalyze the phosphorolysis of a nucleoside leading to the formation of a sugar-1-phosphate; in presence of a second nucleobase, the enzyme can catalyze the synthesis of a new nucleoside (transglycosylation). However, due to the narrow substrate specificity of NPs, the number of compounds that can be prepared by this approach is limited. Aim of this study was to set up a collection of immobilized NPs characterized by different specificity towards variously modified nucleosides. The performance of known enzymes like thymidine phosphorylase (TP) from Escherichia coli and uridine phosphorylase (UP) from Bacillus subtilis were investigated; in addition, new NPs, selected by using a bioinformatic approach (phylogenetic and comparative analysis) or by microbiological screening, were considered. The most promising enzymes were immobilized by ionic or covalent binding and post-immobilization cross-linking was considered to achieve the multimeric structure stabilization. TP from E. coli and UP from B. subtilis efficiently synthesized 2'-deoxyribonucleosides; NPs from Clostridium perfringens, Aeromonas hydrophila and Citrobacter koseri were used for the synthesis of 2',3'-dideoxy- and arabino- nucleosides (Didanosine and Vidarabine). All the immobilized biocatalysts were stable at pH 10; in this condition, both substrates and products could be solubilized in high concentration, necessary to develop a preparative synthesis.

Microbial nucleoside phosphorylases as efficient biocatalysts for the synthesis of antiviral and antitumoral nucleosides

SERRA, IMMACOLATA;UBIALI, DANIELA;ALBERTINI, ALESSANDRA;TERRENI, MARCO
2010

Abstract

Modified nucleosides, such as 5-fluoro-2'-deoxyuridine (Floxuridine),2',3'-dideoxyinosine (Didanosine), arabinosyladenine (Vidarabine), arabinosyl-2-fluoroadenine (Fludarabine), are important antiviral and antitumor agents. These nucleosides are routinely prepared by multi-step syntheses which negatively affect the outcome of the process in terms of yield, purity and costs and with an high environmental impact. Alternatively, nucleosides can be prepared via a one-pot enzymatic process catalyzed by nucleoside phosphorylases (NPs) in fully aqueous medium. NPs catalyze the phosphorolysis of a nucleoside leading to the formation of a sugar-1-phosphate; in presence of a second nucleobase, the enzyme can catalyze the synthesis of a new nucleoside (transglycosylation). However, due to the narrow substrate specificity of NPs, the number of compounds that can be prepared by this approach is limited. Aim of this study was to set up a collection of immobilized NPs characterized by different specificity towards variously modified nucleosides. The performance of known enzymes like thymidine phosphorylase (TP) from Escherichia coli and uridine phosphorylase (UP) from Bacillus subtilis were investigated; in addition, new NPs, selected by using a bioinformatic approach (phylogenetic and comparative analysis) or by microbiological screening, were considered. The most promising enzymes were immobilized by ionic or covalent binding and post-immobilization cross-linking was considered to achieve the multimeric structure stabilization. TP from E. coli and UP from B. subtilis efficiently synthesized 2'-deoxyribonucleosides; NPs from Clostridium perfringens, Aeromonas hydrophila and Citrobacter koseri were used for the synthesis of 2',3'-dideoxy- and arabino- nucleosides (Didanosine and Vidarabine). All the immobilized biocatalysts were stable at pH 10; in this condition, both substrates and products could be solubilized in high concentration, necessary to develop a preparative synthesis.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11571/521047
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