A combination of access to preassociation sites and local accumulation tendency in the direct vicinity of G-N7 controls the rate of platination of single-stranded DNA

Abstract

Adduct formation between cationic reagents and targets on DNA are facilitated by the ability of DNA to attract cations to its surface. The electrostatic interactions likely provide the basis for the documented preference exhibited by cisplatin and related compounds for nuclear DNA over other cellular constituents. As an extension of a previous communication, we here present an investigation illustrating how the rate of adduct formation with the naturally occuring base guanine (G-N7) can be modulated by i) bulk solvent conditions, ii) local nature and size of the surrounding DNA and, iii) increasing DNA concentration. A series of single-stranded DNA oligomers of the type d(T_nGT_m); n = 0, 2, 4, 6, 8, 10, 12, 14, 16 and m = 16 − n or n = m = 4, 6, 8, 12, 16, 24 were allowed to react with the active metabolite of a potential orally active platinum(IV) drug, cis-[PtCl(NH₃)(c-C₆H₁₁NH₂)(OH₂)]⁺ in the presence of three different bulk cations; Na⁺, Mg²⁺, and Mn²⁺. For all positions along the oligomers, a change from monovalent bulk cations to divalent ones results in a decrease in reactivity, with Mn²⁺ as the more potent inhibitor as exemplified by the rate constants determined for interaction with d(T₈GT₈): 10³ × k_obs/s⁻¹ = 6.5 ± 0.1 (Na⁺), 1.8 ± 0.1 (Mg²⁺), 1.0 ± 0.1 (Mn²⁺) at pH 4.2 and 25 °C. Further, the adduct formation rate was found to vary with the exact location of the binding site in the presence of both Na⁺ and Mg²⁺, giving rise to reactivity maxima at the middle position. Increasing the size of the DNA-fragments was found to increase the reactivity only up to a total length of ca. 20 bases. The influence from addition of further bases to the reacting DNA was found to be salt dependent. At [Na⁺] = 0.5 mM a retardation in reactivity was observed whereas [Na⁺] ≥ 4.5 mM give rise to length independent kinetics. Finally, for the first time we have here been able to evaluate the influence from an increasing concentration of non-reactive DNA bases on the adduct formation process. The latter data were successfully fitted to an inhibition model suggesting that non-productive association of the platinum complex with sites distant from G-N7 competes with productive ones in the vicinity of the G-N7 target. Taken together, the kinetics support a reaction mechanism in which access to suitable association sites in the direct vicinity of the target site controls the rate of platination.