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 β-d and β-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 β-d and β-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.