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

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(NH(3))(2))(2)(mu-NH(2)(CH(2))(n)NH(2))](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 (15)N-1 has been studied at 298 K, pH 5.4, by [(1)H,(15)N] 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 [PtN(3)Cl] 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/H(2)O) 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 (1)H and (15)N 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 NH(2) protons have no contacts except with solvent. The NMR data for 5(f) show several distinct NH(2) environments indicative of interactions between the NH(2) 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 (15)N-1 and the duplex. The potential significance of these findings to the antitumor activity of dinuclear platinum complexes is discussed.

Effects of Geometric Isomerism and Anions on the Kinetics and Mechanism of the Stepwise Formation of Long‐Range DNA Interstrand Cross‐Links by Dinuclear Platinum Antitumor Complexes

“…[37] The methodology followed that reported previously in which the stepwise formation of the cross-link was followed by [ 1 H, 15 N] HSQC NMR spectroscopy and 1 H NMR spectra were also acquired to monitor oligonucleotide base pairing through examination of the imino resonances and to verify platinum-binding to guanine N7 by means of the shift of the H8 resonance from that of the unplatinated duplex. [26][27][28] Our previous studies of interstrand cross-linking by both 1,1/t,t [26,27] and 1,0,1/t,t,t [28] have shown that all the reactions follow the same sequence, with evidence for preassociation through electrostatic interactions and hydrogen bonding, aquation followed by monofunctional Pt-GN7 bond formation, and finally fixation of the interstrand cross-link through bifunctional binding. In this case, the reactions were complicated by competing reactions involving the coordination of phosphate (Scheme 1), which were not observed previously.…”

“…[26] For this reason we chose to follow the platination reaction of [ 15 N]1 with the identical self-complementary 12-mer duplex 5′-{d(ATATGTACATAT) 2 } under conditions (15 mM sodium phosphate buffer, pH 5.9, 298 K) that allow best comparison with the results of that study. [26] The use of a higher pH than that employed in the reaction of 1,1/t,t (pH 5.4) takes into account the higher pK a of the aquated form of 1,1/c,c (6.01) [35] than 1,1/t,t (5.62), [36] so that both reactions were carried out at a pH just below the pK a value. A higher ionic strength was used in this study, with the addition of 80 mM NaClO 4 required to form a stable duplex.…”

“…In this case, the reactions were complicated by competing reactions involving the coordination of phosphate (Scheme 1), which were not observed previously. [26,28] Assignment of the species observed during the reactions was made by reference to our recent [ 1 H, 15 N] HSQC NMR spectroscopy study of the aquation of [ 15 N]1 in 15 mM phosphate buffer [35] in which several different aquated and phosphate-bound species were identified. The chemical shifts of all intermediate and bifunctional product species observed during the reaction are summarized in Table 1, representative [ 1 H, 15 N] HSQC NMR spectra are shown in Figure 1, and plots showing changes in the aromatic and imino regions of the 1 H NMR spectra during the reactions are shown in Figure 2.…”

“…[ 1 H, 15 N] HSQC NMR spectroscopy is a powerful method for examining the kinetics of DNA platination reactions [25] and we have previously used this technique to examine the kinetics and mechanism of the formation of interstrand cross-links by the di-and trinuclear platinum complexes 1,1/t,t and 1,0,1/t,t,t (BBR3464). [26][27][28] In contrast with the mononuclear examples of cis and trans-[PtCl 2 (NH 3 ) 2 ], in the dinuclear case both geometries are antitumor active. The extent of formation of interstrand cross-links as well as local conformational distortions on DNA are affected by geometry.…”

“…For the dinuclear 1,1/t,t complex, we were able to follow the stepwise formation of a longrange DNA 1,4-interstrand cross-link in the duplex 5′-{d(ATATGTACATAT) 2 } (I) by using [ 1 H, 15 N] HSQC NMR spectroscopy and derive kinetic parameters for each step in the pathway. [26] Changes in the 1 H and 15 N shifts of the bifunctional interstrand cross-link showed evidence for the irreversible transformation of an initially formed conformer(s) into a product conformer (s). In this work we compare the kinetics of binding of the 1,1/c,c complex to the same DNA duplex.…”