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.