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Article

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

Supporting Info

John W. Cox,^† Susan J. Berners-Price,*^‡ Murray S. Davies,^‡ Yun Qu,^† and Nicholas Farrell*^†

Contribution from the School of Science, Griffith University, Nathan, Brisbane, Queensland 4111, Australia, and Department of Chemistry, Virginia Commonwealth University, Richmond, Virginia 23284-2006

J. Am. Chem. Soc., 2001, 123 (7), pp 1316–1326

DOI: 10.1021/ja0012772

Publication Date (Web): January 25, 2001

†

Virginia Commonwealth University.

Address correspondence to this author.

‡

Griffith University.

Abstract

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(μ-NH₂(CH₂)_nNH₂)]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 ¹⁵N-1 has been studied at 298 K, pH 5.4, by [¹H,¹⁵N] 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) × 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 H-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) × 10^-⁵ s^-¹) followed by closure of 4 to form 5 (rate constant = (2.7 ± 1.5) × 10^-³ s^-¹). When closure is treated as direct from 3 (G/Cl) the rate constant is (3.39 ± 0.04) × 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 ¹H and ¹⁵N shifts of the interstrand cross-link 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 ESI-FTICR mass spectroscopy to be a cross-linked adduct of ¹⁵N-1 and the duplex. The potential significance of these findings to the antitumor activity of dinuclear platinum complexes is discussed.