Kinetics and mechanism for platination of thione-containing nucleotides and oligonucleotides: evaluation of the salt dependence

Abstract

Reactions of cis-[PtCl(NH(3))(CyNH(2))(OH(2))](+) (Cy=cyclohexyl) with thione-containing single-stranded oligonucleotides d(T(8)XT(8)) and d(XT(16)) (X=(s6)I or (s4)U) and the mononucleotides 4-thiouridine ((s4)UMP) and 6-mercaptoinosine ((s6)IMP) have been studied in aqueous solution at pH 4.1. The reaction kinetics was followed using HPLC methodology as a function of ionic strength in the interval 5.0 mM<or= I<or=300 mM. A two-fold kinetic preference for reaction with the (s6)I moiety over (s4)U is observed in both monomeric and oligomeric systems. The rate for adduct formation with the oligonucleotides d(T(8)XT(8)) and d(XT(16)) decreases with increasing ionic strength of the medium. The effect is most pronounced for adduct formation with the middle positions, e.g. for d(T(8)(s6)IT(8)): k(2,app)=370 M(-1 )s(-1) and 13 M(-1 )s(-1) at I=5.0 and 300 mM, respectively, and slightly less pronounced for adduct formation at the end positions, e.g. for d((s6)IT(16)): k(2,app)=130 M(-1 )s(-1) and 11 M(-1 )s(-1) at I=5.0 and 300 mM, respectively. Analysis of the salt dependence using the Brønsted-Debye-Hückel relationship shows that the reactions with the monomers are well described as an interaction between a monovalent cation and a monovalent anion. In contrast, a similar analysis of the oligonucleotide reactions indicates influence from polyelectrolyte effects. The results support a mechanism in which pre-association on the DNA surface precedes adduct formation, regardless of the exact location of the final binding site.

The polyamines spermidine and spermine retard the platination rate of single-stranded DNA oligomers and plasmids

“…These values thus well reflect the more pronounced salt dependence observed Table 1 Observed pseudo-first-order rate constants for platination of d(T 6 GT 6 ), d(T 12 GT 12 ), and d(T 24 GT 24 ) as a function of monovalent cations; in the absence of polycations, and in the presence of 10 lM spd 3+ and 10 lM spn 4+a,b,c for reactions with the two shorter oligomers. The magnitude of the here obtained values agree well with previous determinations based on kinetic studies monitoring platinum binding to dðT s6 8 IT 8 Þ and dðT s4 8 UT 8 Þ with nW = 0.80 ± 0.02 and 0.74 ± 0.05, respectively [45]. Further, the present nW -values are significantly higher than those recently obtained for folded DNA hairpin structures of an overall length of 13 bases for which nW was determined to ca.…”

“…The observed pseudo-first-order rate constants, k obs , are listed in Table 1, and shown as a function of monovalent cation concentration in Fig. 2a-c. As previously shown for similar systems, the platination rate decreased with increasing cation concentration [32,43,[45][46][47]61]. The salt dependence differs slightly between the three oligonucleotides studied.…”

“…As previously shown by us, the salt dependence on the adduct formation kinetics follows the features expected for an adduct formation in which electrostatically driven preassociation of the reactive platinum species precedes coordinative DNA adduct formation [32,43,45,61]. More specifically, the reaction is likely initiated by the replacement of an electrostatically bound cation at the oligomer surface by the positively charged Pt(II) complex, Eq.…”

“…Finally, the resulting close proximity between the tentative target and the reactive species allows for a kinetically facilitated formation of the final, coordinated metal adduct [31,32,42,43]. The importance of the electrostatic preassociation of the metal complex on the DNA surface has been illustrated previously by us by a series of examples including targets located on both the phosphodiester backbone and the bases [31,32,[44][45][46]. We have also been able to show that multivalent cations such as Mg 2+ and Mn 2+ with higher DNA binding affinity effectively slow down the binding kinetics [43,47].…”

Similar rates for platination of hairpin loops and single-stranded DNA

“…25 Previous studies have shown that the Cl Ϫ ligand trans to the coordinated cyclohexylaminemoiety is the most labile one, 37 however, the other Cl Ϫ ligand is also labile giving rise to a mixture of aqua-complexes with similar reactivity in the solution. 32…”