Long Range 1,4 and 1,6-Interstrand Cross-Links Formed by a Trinuclear Platinum Complex. Minor Groove Preassociation Affects Kinetics and Mechanism of Cross-Link Formation as Well as Adduct Structure

Reported here is a comparison of the kinetics of the stepwise formation of 1,4- and 1,6-GG interstrand cross-links by the trinuclear platinum anticancer compound <sup>15</sup>N-[{<i>trans</i>-PtCl(NH<sub>3</sub>)<sub>2</sub>}<sub>2</sub>{μ-<i>trans</i>-Pt(NH<sub>3</sub>)<sub>2</sub>(H<sub>2</sub>N(CH<sub>2</sub>)<sub>6</sub>NH<sub>2</sub>)<sub>2</sub>}]<sup>4+</sup>, (1,0,1/t,t,t (<b>1</b>) or BBR3464). The reactions of <sup>15</sup>N-<b>1</b> with the self-complementary 12-mer duplexes 5‘-{d(ATAT<b>G</b>TACATAT)<sub>2</sub>} (<b>I</b>) and 5‘-{d(TAT<b>G</b>TATACATA)<sub>2</sub>} (<b>II</b>) have been studied at 298 K, pH 5.3 by [<sup>1</sup>H,<sup>15</sup>N] HSQC 2D NMR spectroscopy. The kinetic profiles for the two reactions are similar. For both sequences initial electrostatic interactions with the DNA are observed for <b>1</b> and the monoaqua monochloro species (<b>2</b>) and changes in the chemical shifts of certain DNA <sup>1</sup>H resonances are consistent with binding of the central charged {PtN<sub>4</sub>} linker unit in the minor groove. The pseudo first-order rate constants for the aquation of <b>1</b> to <b>2</b> in the presence of duplex <b>I</b> (3.94 ± 0.03 × 10<sup>-5</sup> s<sup>-1</sup>), or <b>II </b>(4.17 ± 0.03 × 10<sup>-5</sup> s<sup>-1</sup>) are ca. 40% of the value obtained for aquation of <b>1</b> under similar conditions in the absence of DNA. Monofunctional binding to the guanine N7 of the duplex occurs with rate constants of 0.25 ± 0.02 M<sup>-1</sup> s<sup>-1</sup> (<b>I</b>) and 0.34 ± 0.02 M<sup>-1</sup> s<sup>-1</sup> (<b>II</b>), respectively. Closure to form the 1,4- or 1,6-interstrand cross-links (<b>5</b>) was treated as direct from <b>3 </b>with similar rate constants of 4.21 ± 0.06 × 10<sup>-5</sup> s<sup>-1</sup> (<b>I</b>) and 4.32 ± 0.04 × 10<sup>-5</sup> s<sup>-1</sup> (<b>II</b>), respectively. Whereas there is only one predominant conformer of the 1,6 cross-link, evidence from both the <sup>1</sup>H and [<sup>1</sup>H,<sup>15</sup>N] NMR spectra show formation of two distinct conformers of the 1,4 cross-link, which are not interconvertible. Closure to give the major conformer occurs 2.5-fold faster than for the minor conformer. The differences are attributed to the initial preassociation of the central linker of <b>1</b> in the minor groove and subsequently during formation of both the monofunctional and bifunctional adducts. For duplex <b>I</b>, molecular models indicate two distinct pathways for the terminal {PtN<sub>3</sub>Cl} groups to approach and bind the guanine N7 in the major groove with the central linker anchored in the minor groove. To achieve platination of the guanine residues in duplex <b>II</b> the central linker remains in the minor groove but <b>1</b> must diffuse off the DNA for covalent binding to occur. Clear evidence for movement of the linker group is seen at the monofunctional binding step from changes of chemical shifts of certain CH<sub>2</sub> linker protons as well as the Pt−NH<sub>3</sub> and Pt−NH<sub>2</sub> groups. Consideration of the <sup>1</sup>H and <sup>15</sup>N shifts of peaks in the Pt−NH<sub>2</sub> region show that for both the 1,4 and 1,6 interstrand cross-links there is a gradual and irreversible transformation from an initially formed conformer(s) to product conformer(s) in which the amine protons of the two bound {PtN<sub>3</sub>} groups exist in a number of different environments. The behavior is similar to that observed for the 1,4-interstrand cross-link of the dinuclear 1,1/t,t compound. The potential significance of preassociation in determining kinetics of formation and structure of the adducts is discussed. The conformational flexibility of the cross-links is discussed in relation to their biological processing, especially protein recognition and repair, which are critical determinants of the cytotoxicity of these unique DNA-binding agents.