Enantioselectivity of ribonucleotide reductase: a first study using stereoisomers of pyrimidine 2'-azido-2'-deoxynucleosides.
In this paper, the enantioselectivity of ribonucleotide reductase (RNR, EC 1.17.4.1), a pivotal enzyme involved in DNA biosynthesis, was studied using the beta-d and beta-l stereoisomers of 2'-azido-2'-deoxynucleosides of uracil and cytosine. The corresponding 5'-diphosphate derivatives in the d-configuration have been extensively studied as mechanism-based inhibitors of the enzyme. The original l-enantiomers were synthesized and evaluated in vitro. In cell culture experiments, only the cytosine derivative with a d-configuration was found cytostatic and able to deplete dNTP pools in response to RNR inhibition. In the case of the uracil enantiomeric pair, this result correlates with an inefficient intracellular monophosphorylation as demonstrated in testing their substrate properties against human uridine-cytidine kinase 1. Regarding cytosine analogues, human deoxycytidine kinase was found to be able to phosphorylate both enantiomers with comparable efficiency but only the d-stereoisomer was active in human cell culture. The interaction of the beta-d and beta-l stereoisomers of 2'-azido-2'-deoxyuridine 5'-diphosphate with purified Escherichia coli RNR was also examined. Inactivation of the enzyme was only observed in the presence of the d-stereoisomer, demonstrating that RNR exhibits enantiospecificity with respect to the natural configuration of the sugar moiety, as far as 2'-azido-2'-deoxynucleotides are concerned.
Roy
Béatrice
B
UMR 5625 CNRS-UM II, Université Montpellier II, case courrier 008, place E. Bataillon, 34095 Montpellier Cedex 5, France.
Verri
Annalisa
A
Lossani
Andrea
A
Spadari
Silvio
S
Focher
Federico
F
Aubertin
Anne-Marie
AM
Gosselin
Gilles
G
Mathé
Christophe
C
Périgaud
Christian
C
D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D001386:Azides / Q000737:chemistry / Q000494:pharmacology; D002241:Carbohydrates / Q000737:chemistry; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular; D003562:Cytidine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D003842:Deoxycytidine Kinase / Q000235:genetics / Q000378:metabolism; D003853:Deoxyribonucleosides / Q000737:chemistry / Q000494:pharmacology*; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D010766:Phosphorylation; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors*; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured; D014541:Uridine Kinase / Q000235:genetics / Q000378:metabolism
D000970:Antineoplastic Agents; D001386:Azides; D002241:Carbohydrates; D003853:Deoxyribonucleosides; C114286:2'-azido-2'-deoxyuridine; C015010:2'-azidocytidine; D003562:Cytidine; D012264:Ribonucleotide Reductases; D014541:Uridine Kinase; D003842:Deoxycytidine Kinase; D003857:Deoxyuridine