The enzymatic and chemical synthesis of nucleoside analogues using N-deoxyribosyltransferase from Lactobacillus leichmannii

Doctoral thesis English OPEN
Hicks, Nicola
  • Subject: QR

N-deoxyribosyltransferase catalyses the transfer of 2-deoxy-I 2.3, -dideoxyand 2,5-dideoxyribose sugars between purine and pyrimidine bases. In this work, a crude extract of N-deoxyribosyltransferase from Lactobacillus leichmannii was used in the chemoenzymatic synthesis of nucleoside analogues with potential antiviral activity, in particular, 9-P-D-2', 3'- dideoxyribofuranosyl 2-aminopurine which had anti-HIV activity. The immobilisation of a crude extract of N-deoxyribosyltransferase on a variety of support matrices is described. The most successful matrices were octyl Sepharose and Poly(acrylamide-co-N-acryloxysuccinimide) (PAN- 550) gel. However, the PAN method gave higher and more sustained levels of activity, was easier to handle and incubate, and provided a general batch reactor method for the large scale synthesis of nucleosides such as 2-thio-2'-deoxyuridine. As the synthesis of Y-subs titute d-2,3'-dide oxynucleo sides was not possible by the enzymatic transfer method, an investigation into the conformations adopted by the nucleosides, which were and were not glycosyl donor nucleosides in the transfer reaction, was undertaken using variable temperature 1H NMR. Little is known about the active sites of the N-deoxyribosyltransferases from lactobacilli, but major factors accounting for this lack of reactivity may be steric hindrance or dipolar effects that inhibit the binding of a substrate to the transferase. Another factor which may be important is the conformation and flexibility of the deoxyribose ring, because a substrate which adopts a "rigid", unfavourable shape may not bind to the active site of the transferase and may be inactive as a substrate. Analysis of the variable temperature 1H NMR data led to information about the conformer populations, the equilibrium constants and, the free energy, enthalpy and entropy of each system. The temperature dependance of the coupling constants yielded quantitative information with regard to the position of the dynamic equilibrium between the two principal conformers of the sugar ring and determined the flexibility of sugar rings in nucleosides. It was found that only "'flexible" sugar rings without extremes of conformation were active as glycosyl donors in our system. The modified nucleoside, (±)-l-[(2'P, 4'p)-2"-(hydroxymethyl)-4'- dioxolanyllthymine, [(±)-Dioxolane-T], in which the Y-carbon atom is replaced by oxygen, was prepared. Its ability to act as a glycosyl donor for Ndeoxyribosyltransf erases from lactobacillus was investigated but unfortunately its extreme conformation prevented it from being an active glycosyl donor.
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