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

Reported here is a detailed study of the kinetics and mechanism of formatio n of a 1,4 GG interstrand cross-link by [{trans-PtCl(NH3)(2)}(2)(mu -NH2(CH 2)(n)NH2)](2+) (1,1/t,t (n = 6), 1), the prototype of a novel class of plat inum antitumor complexes. The reaction of. the self-complementary 12-mer du plex 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 o f (4.15 +/- 0.04) x 10(-5) s(-1). 2 then undergoes monofunctional binding t o the guanine N7 of the duplex to form 3 (G/Cl) with a rate constant of 0.4 7 +/- 0.06 M-1 s(-1). There is an electrostatic interaction between the unb ound {PtN3Cl} group of 3 and the duplex, which is consistent with II-bondin g 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 li kely 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 f orm 5 (rate constant = (2.7 +/- 1.5) x 10(-3) s(-1)). When closure is treat ed 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 an d N-15 shifts Of the interstrand crosslink 5 indicate that the initially fo rmed 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 proton s have no contacts except with solvent. The NMR data for 5(f) show several distinct NH2 environments indicative of interactions between the NH2 proton s and the DNA. HPLC characterization of the final product showed only one m ajor 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 i s discussed.