Comparative Analysis of [Au(en)2]3+ and [Pt(en)2]2+ non Covalent Binding to Calf Thymus DNA

Reactions of the complexes bisethylendiammine gold(III) and bisethylendiammine platinum(II) with calfthymus DNA were comparatively analysed. Both complexes bind DNA non-covalently most probably on the basis of electrostatic interactions. Binding of either complex at low ratios results into modest modifications of B-type DNA conformations, as detected by CD. Far larger CD alterations are observed at high ratios. The gold(III) chromophore is scarcely perturbed by DNA addition Binding of [Au(en)2]Cl3 to calf thymus DNA is reversed by sodium cyanide. By analogy with the case of [Pt(en)2]Cl2 it is suggested that Auen acts as a minor groove binder.


INTRODUCTION
There is considerable interest in evaluating non-covalent interactions oftransition metal complexes to DNA since the detailed knowledge ofthese interactions may contribute to elucidate the mechanisms of metal-DNA recognition 1,2]. For example extensive work has been carried out on binding of the cations hexammine cobalt(Ill) and tris ethylendiarnine cobalt(Ill) to DNA; both complexes were shown to be very effective in recognizing specific DNA sequences and in modifying DNA conformation through formation of electrostatic and hydrogen bond interactions [3][4][5]. The non-covalent interactions of several ruthenium complexes with DNA and with synthetic oligonucleotides have been investigated in detail by Barton and by other groups [6][7][8]. Recently, Collins et al reported on the reaction of [Pt(en)2]Cl with the DNA dodecamer d(CAATCCGGATTG), and demonstrated, by high resolution 1H NMR spectroscopy, that this complex binds non-covalently the oligonucleotide in the minor groove at AT rich regions [9].
In the last years we have been working on the development, the characterization and the pharmacological evaluationofgold(lll) complexes as potential antitumor agents [10][11][12]; among the investigated compounds, the cationic square planar bisethylendiamine gold(Ill) complex ([Au(en)z]Cl3 hereaRer; scheme I) showed favorable cytotoxic properties toward the A2780 human ovarian cancer cell line [2]. All melting measurements were carried out in the 10 "-M NaC104 and 10 .3 M NaCI buffer. Calf thymus DNA was dissolved in the buffer and DNA concentration determined by absorption measurements at 260 nm. DNA concentration was equal to 3.6xl 0 -5 M [nucleotide]. DNA was treated with [Au(en)2]C13 or [Pt(en)z]Cb at mol/bp ratios r 0.1, and each sample was incubated for hour at room temperature.
Thermal denaturation experiments were performed in quartz cuvettes with a Perkin Elmer Lamba 20 Bio spectrophotometer equipped with a thermostated cell as described in reference. Samples were continuously heated with a rate of temperature increase of 0.5C/min while monitoring the absorbance changes at 260 nm. The investigated interval of temperature ranged from 50C to 90C. Upon reaching 90C, samples were cooled back to 40C in order to follow the renaturation process. Values for melting temperatures (Tm) and for the melting interval (AT) were determined according to the reported procedures [14].
CD spectra were recorded at increasing complexthymus DNA ratio after mixing on a Jasco J600 dichrograph operating at room temperature, interfaced with a PC, and analyzed through the standard Jasco software package; either [Au(en)]Cl3 or [Pt(en)z]Clz were added at the ratios r=0.1, r=0.2, r=0.5, r 1.0, r=2.0, r=4.0, r=10.0. DNA concentration (expressed in basepair) was 8.9x10SM in buffer phosphate 50mM, NaC1 4 mM, pH=7.4. The CD technique is indeed very sensitive to even minor conformational changes of the DNA conformation produced by ligand binding 15].

RESULTS AND DISCUSSION
The reaction ofthe bisethylenediamine gold(III) complex with calf thymus DNA was first analyzed through absorption spectroscopy. It is observed that addition of saturating amounts of calf thymus DNA to a buffered solution containing [Au(en)2]Cl3 does not alter the characteristic charge transfer bands of the [Au(en)z]Cl complex (Figure 1). This means that addition of calf thymus DNA does not produce any major perturbation ofthe gold(III) ehromophore. To establish in more detail whether binding of [Au(en)2]Cl3 brings about any significant conformational change of the DNA double helix CD spectra of calf thymus DNA were carried out at increasing [Au(en)2]C13 to calf thymus DNA ratios. The profile of the CD titration of calf thymus DNA with [Au(en)2]C13 is shown in Figure 2.
Upon addition of small amounts of [Au(en)2]Cl3, minor changes of the B-type CD spectrum of calf thymus DNA are observed that reach completion at r 0.1; for higher gold to DNA ratios more important changes of the CD spectnma are detected that are diagnostic of larger conformational changes. Remarkably addition of excess sodium cyanide causes complete reversal of the CD effects implying that the observed changes are a direct consequence of [Au(en)2]Cl3 binding and that they are reversible upon gold removal. For comparison purposes a CD titration of calf thymus DNA with [Pt(en)2]Cl2 was carried out under the same experimental conditions; the resulting spectral profiles are shown in Figure 3.