Synthesis of 2′‐Deoxynucleosides by Transglycosylation with New Immobilized and Stabilized Uridine Phosphorylase and Purine Nucleoside Phosphorylase

Abstract

Multimeric uridine phosphorylase (UP) and purine nucleoside phosphorylase (PNP) of Bacillus subtilis have been expressed from genes cloned in Escherichia coli, purified, characterized, immobilized and stabilized on solid support. A new immobilization strategy has been developed for UP onto Sepabeads coated with polyethyleneamine followed by crosslinking with aldehyde-dextran. PNP has been immobilized onto glyoxyl-agarose. At pH 10 and 45 8C these derivatives catalyzed the transglycosylation of 2'-deoxyuridine to 2'-deoxyguanosine in high yield (92%). Under the same conditions the not immobilized enzymes were promptly inactivated.

Production, Characterization and Synthetic Application of a Purine Nucleoside Phosphorylase from Aeromonas hydrophila

“…[12] In fact, its mm is estimated to be ca. 120 kDa and its specificity is directed to 6-oxopurines; [7] its tetrameric quaternary structure is consistent with the exception accounted by Bzowska et al for Bacilli sp. within the low mm family.…”

“…This enzyme, previously produced and characterized by some of the present authors, [5,7] differs from Ah PNPII and can be classified as a PNPI. [12] In fact, its mm is estimated to be ca.…”

“…within the low mm family. [7,12] Bs PNPI was found to maintain 50% of its activity after 10 h in 50 mM phosphate buffer containing 20% glycerol at room temperature and pH 7.5. The enzymatic assay for evaluating Bs PNPI activity as well as for monitoring the phosphorolysis reactions with the synthesized nucleoside analogues was performed as previously described for Ah PNPII.…”

A Versatile Synthesis of 5′‐Functionalized Nucleosides Through Regioselective Enzymatic Hydrolysis of Their Peracetylated Precursors

“…2.4.2.1) from Bacillus subtilis by a procedure previously developed by our group for the synthesis of some purine 2Ј-deoxynucleosides. [24] In the case of doxifluridine (14), a further advantage of the transglycosylation is the obtainment of the fluorinated nucleoside 14 only in the final step: this minimizes the need for manipulation of toxic materials such as 5-fluorouracil and doxifluridine itself. This aspect should not be underestimated in view of process hazard evaluation.…”

One-pot process of 2′-deoxyguanylic acid catalyzed by a multi-enzyme system

“…Fernández-Lucas et al [8,9] utilised water-organic cosolvents as non-conventional media to improve the solubility of guanine, and dGR was prepared from 2'-deoxyuridine catalysed by immobilised 2'-deoxyribosyltransferase (NDT-II) from Lactobacillus reuteri. The immobilisation of uridine phosphorylase (UPase) and purine nucleoside phosphorylase (PNPase) were efficient biocatalysts in the synthesis of dGR by the enzymatic transglycosylation of 2'-deoxyuridine and guanine in aqueous solution [10,11]. Under identical conditions, guanosine, which is more soluble in water than guanine, was used as source of guanine to obtain dGR, but the maximum yield of dGR was still less than 20%.…”