^1H NMR Studies of Tris(phenanthroline) Metal Complexes Bound to Oligonucleotides: Characterization of Binding Modest
The binding of Ru(phen)_3^(2+), Rh(phen)_3^(3+), and Co(phen)_3^(3+) to the oligonucleotides d(GTGCAC)_2 and 5'-pd(CGCGCG)_i has been examined by ^1H NMR spectroscopy as a function of temperature, concentration, and chirality of the metal complex. The duplex oligonucleotides act as chiral shift reagents for the metal complexes; phenanthroline protons associated with each enantiomer are resolved upon binding to the oligomer. The spectral titrations, consistent with photophysical studies, indicate that the complexes bind to the oligomer through two modes: one assigned as intercalation favoring the A-isomer, and the other assigned as the surface-bound interaction favoring the Λ-isomer. The ligand protons are perturbed in a manner that implies sensitivity of particular protons to binding mode; specifically, the H4,7 protons appear to be altered most for the Λ-enantiomer while the H5,6 protons are perturbed more for the Λ-enantiomer. The NMR chemical shift variations appear particularly sensitive to this surface-bound interaction, which, on the basis of a comparison of binding and photophysical parameters for Ru(phen)_3^(3+), appears more prominant in binding to oligonucleotides than that to polynucleotides. With respect to oligonucleotide proton shifts, the adenine H2 proton, positioned in the minor groove of the helix, shows the largest upfield shifts with metal binding, and more dramatically with Λ-isomers. The major groove thymine methyl protons (TMe) shift downfield to a lesser extent, and more so for Λ-isomers. The different binding modes also differ with respect to their dynamics of association; the longitudinal relaxation rates of Δ- and Λ-4,7 phenanthroline protons of Rh(phen)_3^(3+) are 0.88 and 1.14 s, respectively, in the presence of d(GTGCAC)_2. In contrast to studies with the substitutionally inert metal complexes, addition of racemic Co(phen)_3^(3+) to the oligonucleotide solution yields unequal populations of enantiomers, owing to the rapid racemization of the cobalt complex in the presence of oligomer and reequilibration to that form which favors binding. Duplex melting has also been monitored by ^1H NMR spectroscopy; the complexes increase the duplex melting temperature by ~5 ºC. In the case of Co(phen)_3^(3+), with increasing temperature, as the helix melts, a reequlibration of the enantiomers occurs, indicating that the chiral discrimination arises from enantioselective interactions with the helix rather than with the single-stranded oligonucleotides.
© 1990 American Chemical Society. Received June 30, 1989; Revised Manuscript Received October 19, 1989. This work was supported by NIH Grant GM33309.