The effect of polar 2'-substituents on the oxidation of nucleic acids at the 1'-C-H bond was investigated. Oligonucleotides with the sequence 5'-ATI CCC2'-X TTI CIT AT-3' (I = inosine, -X = -H, -NH2, - OCH3, -F) were synthesized and subjected to oxidation by Ru(tpy)(bpy)O2+ (tpy = 2,2':6',2 "-terpyridine; bpy = 2,2'-bipyridine). High-resolution electrophoresis revealed a similar cleavage pattern for all of the sites in the oligonucleotides except for the 2'-substituted site, which varied in the extent of cleavage relative to the other sites according to H > NH2 > OCH3 > F. Because the cleavage was monitored following piperidine treatment, this analysis could not be applied to the 2'-hydroxy oligonucleotide. The extent of cleavage was a linear function of oxidant concentration, and the slope of this plot was used as a relative rate after normalizing to the adjacent, unsubstituted 5'-cytosine. When the relative rates were determined using other nucleotides in the sequence for normalization, the results did not change outside the quoted error limits. The log of the relative rates was a linear function of the polar Hammett parameter, F, and gave a slope of rho = -1.4 +/- 0.1. Rate constants on 2'-H and 2'-OH monophosphates from previous studies by stopped-flow spectrophotometry fall on the line defined by the electrophoresis data, supporting the assumption that the C-H bond activation is rate-limiting. The observed Hammett correlation supports an accumulation of positive charge at the 1'-carbon, as would be expected for hydride transfer. These results provide a basis for the oxidation resistance of 2'-substituted oligonucleotides.