Chromomycin dimer-DNA oligomer complexes. Sequence selectivity and divalent cation specificity

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Abstract
This paper reports on a solution NMR characterization of the sequence selectivity and metal ion specificity in chromomycin-DNA oligomer complexes in the presence of divalent cations. The sequence selectivity studies have focused on chromomycin complexes with the self-complementary d(A1-A2-G3-G4-C6-C6-T7-T8) duplex containing a pair of adjacent (G3-G4) .cntdot. (C5-C6) steps and the self-complementary d(A1-G2-G3-A4-T5-C6-C7-T8) duplex containing a pair of separated (G2-G3) .cntdot. (C6-C7) steps in aqueous solution. The antitumor agent (chromomycin) and nucleic acid protons have been assigned following analysis of distance connectivities in NOESY spectra and coupling connectivities in DQF-COSY spectra for both complexes in H2O and D2O solution. The observed intermolecular NOEs establish that chromomycin binds as a Mg(II)-coordinated dimer [1 Mg(II) per complex] and contacts the minor-groove edge with retention of 2-fold symmetry centered about the (G3-G4-C5-C6) .cntdot. (G3-G4-C5-C6) segment of the d(A2G2C2T2) duplex. By contrast, complex formation is centered about the (G2-G3-A4-T5) .cntdot. (A4-T5-C6-C7) segment and results in removal of the two fold symmetry of the d(AG2ATC2T) duplex. Thus, the binding of one subunit of the chromomycin dimer at its preferred (G-G) .cntdot. (C-C) site assists in the binding of the second subunit to the less preferred adjacent (A-T) .cntdot. (A-T) site. These observations suggest a hierarchy of chromomycin binding sites, with a strong site detected at the (G-G) step due to the hydrogen-bonding potential of acceptor N3 and donor NH2 groups of guanosine that line the minor groove. The divalent cation specificity has been investigated by studies on the symmetric chromomycin-d(A2G2C2T2) complex in the presence of diamagnetic Mg(II), Zn(II), and Cd(II) cations and paramagnetic Ni(II) and Co(II) cations. A comparative NOESY study of the Mg(II) and Ni(II) symmetric complexes suggests that a single tightly bound divalent cation aligns the two chromomycins in the dimer through coordination to the C1 carbonyl and C9 enolate ions on the hydrophilic edge of each aglycon ring. Secondary divalent cation binding sites involve coordination to the major-groove N7 atoms on adjacent guanosines in G-G steps. This coordination is perturbed on lowering the pH below 6.0, presumably due to protonation of the N7 atoms. The midpoint of the thermal dissociation of the symmetric complex is dependent on the divalent cation with the stability for reversible transitions decreasing in the order Mg(II) > Zn(II) > Cd(II) complexes. We have also measured the real-time proton to deuterium exchange kinetics of the aglycon C8-hydroxyl proton in the symmetric chromomycin-d-(A2G2C2T2) complex after dissolution in D2O solution. The hydrogen exchange lifetimes in the absence of added catalysts decreases in the order Ni(II) >> Mg(II) > Zn(II) > Cd(II) complexes. The same trend is detected for divalent cation exchange between the tightly bound and free cations, with the bound Ni(II) exhibiting the longest and the bound Cd(II) the shortest lifetime in the symmetric complex. These results establish that the divalent cation exchange does not occur through direct penetration of the cation into the complex but rather following dissociation of the divalent cation coordinated chromomycin dimer from its duplex binding site.