title: Enzymatic synthesis and phosphorolysis of 4(2)-thioxo- and 6(5)-azapyrimidine nucleosides byE. colinucleoside phosphorylases.
abstract: Thetrans-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 andE. colipurine 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 eitherE. colithymidine (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 and2STd 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 of2SUra and 5-azacytosine with dRib-1P (Ba salt) catalyzed by the accordant UP and PNP in Tris∙HCl buffer gave2SUd 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 byE. colinucleoside phosphorylases is discussed.
authors.affiliation: Institute of Bioorganic Chemistry, National Academy of Sciences, Acad. Kuprevicha 5/2, 220141 Minsk, Belarus.
authors.forename: Vladimir A
authors.initials: VA
authors.lastname: Stepchenko
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 GSP, Moscow B-437, Russia.
authors.forename: Anatoly I
authors.initials: AI
authors.lastname: Miroshnikov
authors.affiliation: Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology, Heisenbergstraße 11, D-48149 Münster, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Seela
authors.affiliation: Institute of Bioorganic Chemistry, National Academy of Sciences, Acad. Kuprevicha 5/2, 220141 Minsk, Belarus.
authors.forename: Igor A
authors.initials: IA
authors.lastname: Mikhailopulo
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