Enzymatic synthesis and phosphorolysis of 4(2)-thioxo- and 6(5)-azapyrimidine nucleosides by E. coli nucleoside phosphorylases

Abstract

The trans-2-deoxyribosylation of 4-thiouracil (^4SUra) and 2-thiouracil (^2SUra), as well as 6-azauracil, 6-azathymine and 6-aza-2-thiothymine was studied using dG and E. coli purine nucleoside phosphorylase (PNP) for the in situ generation of 2-deoxy-α-D-ribofuranose-1-phosphate (dRib-1P) followed by its coupling with the bases catalyzed by either E. coli thymidine (TP) or uridine (UP) phosphorylases. ^4SUra revealed satisfactory substrate activity for UP and, unexpectedly, complete inertness for TP; no formation of 2'-deoxy-2-thiouridine (^2SUd) was observed under analogous reaction conditions in the presence of UP and TP. On the contrary, ^2SU, ^2SUd, ^4STd and ^2STd are good substrates for both UP and TP; moreover, ^2SU, ^4STd and 2'-deoxy-5-azacytidine (Decitabine) are substrates for PNP and the phosphorolysis of the latter is reversible. Condensation of ^2SUra and 5-azacytosine with dRib-1P (Ba salt) catalyzed by the accordant UP and PNP in Tris∙HCl buffer gave ^2SUd and 2'-deoxy-5-azacytidine in 27% and 15% yields, respectively. 6-Azauracil and 6-azathymine showed good substrate properties for both TP and UP, whereas only TP recognizes 2-thio-6-azathymine as a substrate. 5-Phenyl and 5-tert-butyl derivatives of 6-azauracil and its 2-thioxo derivative were tested as substrates for UP and TP, and only 5-phenyl- and 5-tert-butyl-6-azauracils displayed very low substrate activity. The role of structural peculiarities and electronic properties in the substrate recognition by E. coli nucleoside phosphorylases is discussed.

Keywords: 4(2)-thioxo- and 6(5)-aza-uacil and -thymine; PM3 and ab initio calculations; enzymatic glycosylation; recombinant E. coli uridine, thymidine and purine nucleoside phosphorylases; substrate properties.

Scheme 1

Enzymatic synthesis of 2-deoxy-β-D-ribofuranosides 1b–5b of the heterocyclic bases 1a–5a. Regents and conditions: dG/base ratio: 1.5:1.0 (mol), 10 mM K,Na-phosphate buffer; 40 °C, 48–72 h; HPLC analysis of the reaction mixtures see Experimental section and Supporting Information File 1.

Scheme 2

Phosphorolysis of nucleosides 1b–5b and related pyrimidine nucleosides (2’-deoxyuridine, thymidine, 2- and 4-thiothymidines) catalyzed by E. coli UP (Figure 1) and TP (Figure 2).

Figure 1

Phosphorolysis of a number of 2’-deoxy-β-D-ribofuranosides of uracil and thymine, and their 6-aza derivatives in comparison with the corresponding 4- and 2-thio derivatives catalyzed by E. coli UP. Reaction conditions: reaction mixture 1.0 mL; 25 mМ K,Na-phosphate buffer (рН 7.0), 20 °C; 2 mМ testing nucleoside. Enzyme: 0.016 units of E. coli UP (substrates drawn with solids lines) or 1.9 units of E. coli UP (substrates drawn with dotted lines); reaction progress was monitored by HPLC (see Supporting Information File 1); yields refer to the percentage of the resulting heterocyclic base.

Figure 2

Phosphorolysis of 2′-deoxyuridine and thymidine, their 4- and 2-thio derivatives and 6-aza-2-thiothymidine (5b) catalyzed by E. coli TP (for reaction conditions, see caption of Figure 1. Enzyme: 6.6 × 10⁻⁴ units of TP was used for all substrates, except for 6-aza-2-thiothymidine, for which 26.5 units of the enzyme was used. Phosphorolysis of 6-aza-2′-deoxyuridine and 6-azathymidine did not exceed a few percent even at high enzyme concentrations and is not presented on the plot.

Figure 3

Supposed monoanionic forms of 4-thiouracil and 2-thiouracil in aqueous medium [–49].

Figure 4

Phosphorolysis of 6-aza-2-thiothymidine (5b), 4-thiothymidine (11a) and 4-thio-2′-deoxyuridine (1b) by E. coli TP for extended time period (for details, see caption of Figure 2).

Figure 5

Structures of 2-thiopyrimidine(9–12) and 5-azacytidine (13 and 14) nucleosides.

Figure 6

Energy minimized structures of N³-(β-D-ribofuranosyl)adenine (left) and 5-aza-2′-deoxycytidine (right) and in the mid both structures are overlapped by the glycosyl bonds (calculations by ab initio, 3-21G level; Polak–Ribiere (conjugate gradient); basis set of parameters; HyperChem 8.1).

Figure 7

Structures of 6-azapyrimidines 15–18 tested for E. coli UP and TP.

Figure 8

Geometry optimized structures (PM3 method) of 5-tert-butyl-6-azauracil (15) and 5-phenyl-6-azauracil (16) (upper structures) vs those of 5-ethyluracil and (E)-5-(2-bromovinyl)uracil (lower structures).

Figure 9

The UV spectra of 4-thio-2′-deoxyuridine (1b).

Figure 10

The UV spectra of 6-aza-2-thiothymidine (5b).