Kinetic analysis of the stepwise formation of a long-range DNA interstrand cross-link by a dinuclear platinum antitumor complex: Evidence for aquated intermediates and formation of both kinetically and thermodynamically controlled conformers

Reported here is a detailed study of the kinetics and mechanism of formation of a 1,4 GG interstrand cross-link by [{trans-PtCl(NH3)(2)}(2)(mu -NH2(CH2)(n)NH2)](2+) (1,1/t,t (n = 6), 1), the prototype of a novel class of platinum antitumor complexes. The reaction of. the self-complementary 12-mer duplex 5'-{d(ATATGTACATAT)(2)} with N-15-1 has been studied at 298 K, pH 5.4, by [H-1,N-15] HSQC 2D NMR spectroscopy. Initial electrostatic interactions with the duplex are observed for 1 and the monoaqua monochloro species (2). Aquation of 1 to yield 2 occurs with a pseudo-first-order rate constant of (4.15 +/- 0.04) x 10(-5) s(-1). 2 then undergoes monofunctional binding to the guanine N7 of the duplex to form 3 (G/Cl) with a rate constant of 0.47 +/- 0.06 M-1 s(-1). There is an electrostatic interaction between the unbound {PtN3Cl} group of 3 and the duplex, which is consistent with II-bonding interactions observed in the molecular model of the monofunctional (G/Cl) adduct. Closure of 3 to form the 1,4 GG interstrand cross-link (5) most likely proceeds via the aquated (G/H2O) intermediate (4) (pseudo-first-order rate constant = (3.62 +/- 0.04) x 10-5 s(-1)) followed by closure of 4 to form 5 (rate constant = (2.7 +/- 1.5) x 10(-3) s(-1)). When closure is treated as direct from 3 (G/Cl) the rate constant is (3.39 +/- 0.04) x 10-5 s(-1). Closure is ca. 10-55-fold faster than that found for 1,2 GG intrastrand cross-link formation by the diaqua form of cisplatin. Changes in the H-1 and N-15 shifts Of the interstrand crosslink 5 indicate that the initially formed conformer (5(i)) converts irreversibly into other product conformer(s) 5(f). The NMR data for 5(i) are consistent with a molecular model of the 1,4 GG interstrand cross-link on B-form DNA, which shows that the NH2 protons have no contacts except with solvent. The NMR data for 5(f) show several distinct NH2 environments indicative of interactions between the NH2 protons and the DNA. HPLC characterization of the final product showed only one major product peak that was confirmed by ESIFTICR mass spectroscopy to be a cross-linked adduct of N-15-1 and the duplex. The potential significance of these findings to the antitumor activity of dinuclear platinum complexes is discussed.