Jiří Kozelka

A Complete Kinetic Study of GG versus AG Platination Suggests That the Doubly Aquated Derivatives of Cisplatin Are the Actual DNA Binding Species

The hairpin-stabilized double-stranded oligonucleotides d(TATGGTATT4ATACCATA) (I) and d(TATAGTATT4ATACTATA) (II) were allowed to react with the three aquated forms of the antitumor drug cisplatin (cis-[PtCl2(NH3)2], 1) which are likely candidates for DNA binding, that is, cis-[PtC1(NH3)2(H2O)]+ (2), cis-[Pt(NH3)2(H2O)2]2+ (3), and its conjugate base cis-[Pt(OH)(NH3)2(H2O)]+ (4). The reaction between I and [Pt(NH3)3(H2O)]2+ (5) was also studied for comparison. All reactions were monitored by HPLC. The platination reactions of I and II were carried out in NaClO4 (0.1M) at 293 K and at a constant pH of 4.5 +/- 0.1 for 2, 3, and 5. The data relative to the platination by 4 were obtained from measurements in unbuffered NaClO4 solutions (0.1M) at a starting pH close to neutrality, where 3 and 4 are present in equilibrium. In this case, a fit function describing the pH-time curve allowed the determination of the actual concentrations of 3, 4, and the dihydroxo complex. The platination rate constants characterizing the bimolecular reactions between either I or II and 2, 3, and 4 were individually determined along with the rate constants for hydrolysis of the chloro-monoadducts and for the chelation reactions of the aqua-monoadducts. The reactivity of compounds 2-5, which have the general formula cis-[Pt(NH3)2(H2O)(Y)]2+/-, decreases in the order 3>4>5>>2, that is, Y= H2O > OH- >NH3 >> Cl-, which is the order of decreasing hydrogen-bond donating ability of Y. Deprotonation of 3 to 4 reduces the reactivity of the platinum complex only by a factor of approximately equals 2, and both complexes discriminate between the different purines of I and II in the same manner. Whereas 3 and 4 react approximately three times faster with the GG sequence of I than with the AG sequence of II, 2 shows a similar reactivity towards both sequences. In view of the well-established preferential binding of cisplatin to GG sequences of DNA in vivo and in vitro, this result suggests that the actual DNA platination species are derived from double hydrolysis of cisplatin.

Kinetic aspects of interactions between DNA and platinum complexes

Abstract Kinetic studies on reactions between oligonucleotides and platinum complexes related to the antitumor drug cisplatin are described. These studies were motivated by the sequence-selectivity observed for the reaction between cisplatin and DNA. Comparison of oligonucleotide platination rate constants measured for the three complexes cis -[PtCl(NH 3 ) 2 (H 2 O)] + , cis -[Pt(NH 3 ) 2 (H 2 O) 2 ] 2+ , and [Pt(NH 3 ) 3 (H 2 O)] 2+ suggests that the hydrogen bond donating capacity of the platinum ligands enhances the platination rate. The binding preferences of cis -[Pt(NH 3 ) 2 (H 2 O) 2 ] 2+ and cis -[PtCl(NH 3 ) 2 (H 2 O)] + indicate that the latter species is unlikely to be the major species interacting with DNA in vivo. This conjecture is corroborated by reactivity and availability considerations. We also address the sequence-dependence observed for the hydrolysis of chloro-monoadducts and for the conversion of aqua-monoadducts to diadducts, and discuss possible mechanisms.

Kinetic study of azole-bridged dinuclear platinum(II) complexes reacting with a hairpin-stabilized double-stranded oligonucleotide

The cytotoxic dinuclear platinum(II) complexes [[cis-Pt(NH(3))(2)](2)(mu-OH)(mu-pz)](NO(3))(2) (pz=pyrazolate) (1) and [[cis-Pt(NH(3))(2)](2)(mu-OH)(mu-1,2,3-ta-N1,N2)](NO(3))(2) (1,2,3-ta=1,2,3-triazolate) (2), were allowed to react with the hairpin-stabilized double-stranded oligonucleotide d(TATGGCATT(4)ATGCCATA), to determine the amounts of intrastrand and interstrand DNA adducts. The reaction kinetics was investigated by reversed-phase HPLC, and the resulting products were analyzed using mass spectroscopy combined with enzymatic digestion, and Maxam-Gilbert sequencing. The reaction of 1 results in the formation of the 1,2-intrastrand d(GG) adduct as the major final product. The two most abundant products of 2 were identified as isomeric 1,2-intrastrand d(GG) adducts differing probably in platinum coordination to the triazole ring. No GG-interstrand crosslinks were detected with either compound. d(GGC)-d(GCC) sequences of DNA do thus not appear to represent significant targets for forming interstrand crosslinks with either 1 or 2.

O−H⋅⋅⋅PtII: Hydrogen Bond with a Strong Dispersion Component

An MP2 ab initio study of the interaction between a H2O molecule and trans-[Pt(OH)2(NH3)2] revealed a HO−H⋅⋅⋅PtII hydrogen bond (see picture) with a strong dispersion component (ca. 4 kcal mol−1). This dispersion interaction is independent of the charge on the complex and is likely to be ubiquitous in aqueous solutions of PtII complexes.

Effect of platinum N7-binding to deoxyguanosine and deoxyadenosine on the H8 and H2 chemical shifts. A quantitative analysis

Abstract Platinum coordination to the N7 atom of a purine nucleotide generally shifts the H8 and H2 resonances downfield. This downfield shift originates from the “inductive effect,” i.e., the effect of diminishing the electron density on the H8 or H2 atoms and of modifying the ring current in the purine base, and from conformation-dependent effects of the environment. The determination of the latter can yield valuable structural information. In order to separate the inductive effect from the effects of the environment, we have therefore measured the H8 and H2 chemical shifts for a series of complexes [Pt(deoxyribonucleoside-N7) LL′L″] n , where L, L′, and L″ are Cl − , H 2 O, NH 3 , a nonaromatic amine, or a trialkylphosphane. In these mononucleoside compounds, the H8 and H2 downfield shifts mainly reflect the inductive effect. This inductive effect was found to depend on the ligand trans to the nucleoside and on the complex charge, but not appreciably on the ligands cis to the nucleoside. For cis -[Pt(NH 3 ) 2 d(GpG)] + and cis -[Pt(NH 3 ) 2 d(ApG)] + crosslinks encountered in DNA adducts of the antitumor drug cis -diamminedichloroplatinum(II), our analysis predicts an inductive effect of 0.52 ± 0.01, 0.68 ± 0.02, and 0.09 ± 0.03 ppm for the GH8, AH8, and AH2 protons, respectively. For these dinucleotide adducts and their ribonucleotide analogues, the estimates of the inductive effect have enabled us to calculate the δ H8 values for minimum-energy conformations, and to determine the sense of the helicoidal arrangement of the bases.

Hydration and `inverse hydration' of platinum(II) complexes: an analysis using the density functionals PW91 and BLYP

Abstract The interaction between two platinum complexes and water was investigated using the DFT functionals PW91 and BLYP. Previous MP2 and HF calculations have shown that when a H 2 O molecule approaches Pt with its O atom, the interaction is governed by electrostatics and the MP2 and HF interaction energy curves nearly coincide, whereas for the approach with H pointing towards Pt, there is a significant dispersion component portrayed only by MP2 calculations. Here we show that both PW91 and BLYP satisfactorily reproduce the MP2 curves, PW91 slightly exaggerating and BLYP slightly underestimating the dispersion-induced energy minima. This result is discussed in view of the different behavior of the two exchange functionals at large density gradients.

Cytotoxic activity of platinum (II) complexes with tri-n-butylphosphine. Crystal structure of the dinuclear hydrazine-bridged complex, cis,cis-[PtCl(PBu3n)2(μ-N2H4)PtCl(PBu3n)2] (ClO4)2 · 2CHCl3

A series of platinum(II) tri-n-butylphosphine complexes having the formulas cis-[PtCl2L2], NEt4[PtCl3L], [PtCl(en)L]Cl, [Pt(en)L2](ClO4)2, sym-trans-[Pt2Cl4L2], [Pt2Cl2L4](ClO4)2, trans,trans-[PtCl2L(mu-N2H4)PtCl2L] trans,trans-[PtCl2L(mu-en)PtCl2L], and cis,cis-[PtClL2(mu-N2H4)PtClL2](ClO4)2 (L = tri-n-butylphosphine; en = ethylenediamine) have been synthesized and their cytotoxic activity in vitro and in vivo has been studied. The solution behavior of the novel dinuclear diamine-bridged platinum(II) complexes has been investigated by means of UV and 31P NMR spectroscopy. For the ionic hydrazine compound cis,cis-[PtClL2(mu-N2H4)PtClL2](ClO4)2, an x-ray structure determination is reported. Crystal data: space group P2(1)/a, a = 17.803(1), b = 18.888(3), c = 12.506(3) A, beta = 107.97(2) degrees, Z = 2, R = 0.052, RW = 0.058. The platinum coordination is approximately square-planar, with the bond lengths Pt-Cl = 2.358(5), Pt-N = 2.15(1), Pt-P(trans to Cl) = 2.260(5), and Pt-P(trans to N) = 2.262(6) A. All investigated compounds were cytotoxic in vitro against L1210 cells and showed no cross-resistance to cisplatin. On the other hand, no antitumor activity was observed vs L1210 leucemia in DBA2 mice.

Fast Interstrand Cross‐linking of Cisplatin–DNA Monoadducts Compared with Intrastrand Chelation: A Kinetic Study Using Hairpin‐Stabilized Duplex Oligonucleotides

The antitumor drug cisplatin forms two kinds of guanine-guanine cross-links with DNA: intrastrand, occurring mainly at GG sites, and interstrand, formed at GC sites. The former are generally more abundant than the latter, at least in experiments with linear duplex DNA. The formation of interstrand cross-links requires partial disruption of the Watson-Crick base pairing, and one could therefore expect the cross-linking reaction to be rather slow. In contrast with this expectation, kinetic measurements reported here indicate that interstrand cross-linking is as fast as intrastrand, or even faster. We have investigated the reactions between two hairpin-stabilized DNA duplexes, containing either a d(TGCA)(2) sequence (duplex TGCA) or a d(G(1)G(2)CA)-d(TG(3)CC) sequence (duplex GGCA), and the diaqua form of cisplatin, cis-[Pt(NH(3))(2)(H(2)O)(2)](2+), in an unbuffered solution kept at pH 4.5 +/- 0.1 and 20 degrees C. Using HPLC as the analytical method, we have determined the platination (first step) and chelation (second step) rate constants for these reaction systems. Duplex TGCA, in which the two guanines are quasi-equivalent, is found to be platinated very slowly (k=0.5 +/- 0.1M(-1)s(-1)) and to form the final interstrand cross-link very rapidly (k=13 +/- 3 x 10(-3) s(-11)). For GGCA, we find that G(1) is platinated rapidly (k=32 +/- 5M(-1)s(-1)) to form a long-lived monoadduct, which is only slowly chelated (k=0.039 +/- 0.001 x 10(-3) s(-1)) by G(2) (intrastrand), while G(2) is platinated one order of magnitude more slowly than G(1) (k=2.0 +/- 0.5M(-1)s(-1)) and chelated fairly rapidly both by G(1) (intrastrand: k=0.4 +/-0.1 x 10(-3) s(-1)) and G(3) (interstrand: k=0.2 +/- 0.1 x 10(-3) s(-1)); finally, G(3) is platinated at about the same rate as G(2) (k=2.4 +/- 0.5M(-1)s(-1)) and chelated very rapidly by G(2) (interstrand: k=10 +/- 4 x 10(-3) s(-1)). These results suggest that the low occurrence of interstrand cross-links in cisplatinated DNA is due to an extremely slow initial platination of guanines involved in d(GC)(2) sequences, rather than to a slow cross-linking reaction.

Isolation of cis-[PtCl(NH3)2(H2O)](ClO4), the monohydrated form of the anti-tumour drug cisplatin, using cation-exchange high-performance liquid chromatography

A novel procedure allowing a quantitative separation of the anti-tumour drug cisplatin and its hydration products, based on cation-exchange high-performance liquid chromatography, is presented. Thanks to this procedure, the monohydrated form of cisplatin, cis-[PtCl(NH3)2(H2O)]+, which is possibly the principal species reacting in vivo with DNA and thus responsible for the anti-tumour activity, could be isolated and characterized in the pure state for the first time.

Asymmetrical complexes [Pt(N,N-dmen)(dGuo)L](2)+ (N,N-dmen=N,N-dimethylethylenediamine; L=H2O, Cl−, dGuo): H8 nuclei experience an orientation-dependent deshielding effect attributable to the paramagnetic anisotropy of the platinum atom

Abstract N7 coordination of deoxyguanosine (dGuo) to platinum(II) is known to shift the NMR signal of the guanine H8 proton downfield. The amplitude of this downfield shift depends on the platinum ligands and their configuration. We show here that the guanine H8 chemical shift variation upon coordination of dGuo to [Pt(N,N-dmen)(H2O)2]2+ (N,N-dmen=N,N-dimethylethylenediamine) or to [PtCl(N,N-dmen)(H2O)]+ is 0.2–0.3 ppm larger when dGuo binds cis to the NMe2 group than when it coordinates the site cis to the NH2 group. This difference can be explained by sterical restrictions imposed by the bulky NMe2 group to a cis-guanine, forcing it into an orientation approximately perpendicular to the coordination plane. In this position, the H8 proton apparently experiences a downfield shift arising from the paramagnetic anisotropy of the platinum(II) atom.