KINETICS AND MECHANISM OF THE OXIDATION OF SUGARS AND NUCLEOTIDES BY OXORUTHENIUM(IV) - MODEL STUDIES FOR PREDICTING CLEAVAGE PATTERNS IN POLYMERIC DNA AND RNA

Kinetic parameters for the oxidation of D-ribose, 2-heoxy-D-ribose, adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), 2'-deoxyadenosine 5'-monophosphate (dAMP), cytidine 5'-monophosphate (CMP), 2'-deoxycytidine 5'-monophosphate (dCMP), and thymidine 5'-monophosphate (TMP) by Ru(tpy)(bpyO2+ were determined in pH 7 phosphate buffer (tpy = 2,2',2''-terpyridine, bpy = 2,2'-bipyridine). Plots of k(obs) vs [substrate] were linear for the oxidation of ribose, 2-deoxyribose, TMP, and dCMP, yielding second-order rate constants of 0.029, 0.082, 0.38, and 0.46 M(-1) s(-1), respectively. From the temperature dependence of the rate constant, activation parameters consistent with the oxidation of other organic molecules by hydride transfer were found. For CMP, AMP, and dAMP, k(obs) vs [substrate] plots were curved due to electrostatic binding of Ru(tpy)(bpyO2+ to the dianionic nucleotides, and the data were treated using double-reciprocal plots, yielding effective second-order rate constants of 0.10, 0.39, and 2.5 M(-1) s(-1), respectively. Product analysis by HPLC shows that a quantitative yield of free cytosine is obtained upon oxidation of dCMP based on nucleotide consumed, In TMP oxidations, an 80% yield of free thymine is observed based on Ru(tpy)(bpyO2+ consumed. The kinetics and product analyses are consistent with sugar oxidation at the 1' position, and the increased reactivity of DNA compared to RNA can be understood on the basis of deactivation of the sugar oxidation product by the polar effect of the 2'-hydroxyl. The oxidation of the guanine base in GMP by Ru(tpy)(bpyO2+ proceeds via an oxo, transfer mechanism where the initial step is formation of a bound Ru(III)OR(2+) intermediate. The ratio of the rate-determining rate constant for oxidation of guanine nucleotides to the average rate constant of sugar oxidation predicts the relative yields of base and sugar oxidation on sequencing gels.