Enantioselectivity and stereoselectivity in the reactions of the enantiomers of the platinum complex [PtCl2(ahaz)] (ahaz = 3(R)- or 3(S)-aminohexahydroazepine) with DNA

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Abstract

The platinum–DNA adduct profile formed by the R- and S-enantiomers of [PtCl2(ahaz)] (ahaz = 3(R)-aminohexahydroazepine or 3(S)-aminohexahydroazepine) on reaction with salmon sperm DNA were characterised using HPLC and GFAAS (graphite furnace atomic absorption spectrometry) analyses. At a platinum to nucleotide ratio (Rt) equalling 0.05, the R-enantiomer forms a substantially larger amount (approximately 60%) of monofunctional adducts than the S-enantiomer (less than 35%). Fewer intrastrand GpG adducts are formed by the R-enantiomer (approximately 21%) than the S-enantiomer (approximately 37%). For both enantiomers, two isomeric GpG adducts, corresponding to the different orientations of the primary amine of ahaz ligand with respect to the O6 atom of the 5′ guanine, were observed in the ratios of 1:1.3 and 1:4.3 for the R- and S-enantiomers, respectively. The reasons for this enantioselectivity and stereoselectivity are discussed.

Introduction

Platinum anticancer drugs such as cisplatin, carboplatin and oxaliplatin represent one of the mainstays of current chemotherapy for the treatment of solid tumours [1], [2]. They are believed to effect their action by binding to DNA and forming a series of bifunctional adducts the most prevalent of which are at GpG and ApG sequences [3], [4], [5], [6], [7]. Failure to repair these adducts for any reason including protection of the adduct by proteins such as HMG (high mobility group) proteins leads to apoptosis and hence effective treatment of the tumour. Variation of the am(m)ine ligands of these complexes is now known to influence the type of cancers effectively treated by each compound with oxaliplatin proving active against colon cancer [1], [8]. This shows that bulky ligands do not necessarily reduce activity and their complexes can be used to provide new insights into the factors that influence the interactions between DNA and platinum complexes [9]. The platinum(II) complex, [PtCl2(ahaz)] (ahaz = 3(R)-aminohexahydroazepine or 3(S)-aminohexahydroazepine), is both chiral and unsymmetric, and therefore has the potential to demonstrate both enantioselectivity and stereoselectivity in its interactions with a chiral target such as DNA. Such effects, which are purely structural in origin, can provide detailed understanding of the role of steric interactions in controlling platinum binding to DNA [9], [10], [11], [12], [13], [14]. We have previously reported that the enantiomers of [PtCl2(ahaz)] have different cytotoxicities in a number of cell lines [15] and it is at least possible that these differences arise from differences in the interactions with DNA. We have also reported differences in the HMG recognition of the adducts formed by the two enantiomers [16].

The DNA adduct profiles for the R- and S-enantiomers of [PtCl2(ahaz)] have been characterised in order to investigate the origins of the difference in their in vitro cytotoxic activities found in some of the cell lines studied. In these experiments, salmon sperm DNA was treated with the platinum complexes, the products enzymatically digested, the digestion products separated by HPLC, and the platinum–DNA adducts identified by their characteristic retention times as well as analysis of the base sequence of the mono- or oligonucleotides cross-linked by the platinum complexes.

Section snippets

Materials

K2[PtCl4] was purchased from Aldrich. The resolved R- and S-ahaz were provided by Dr. R.R. Fenton. P1 nuclease EC 3.1.30.1, alkaline phosphatase, deoxyribonuclease I, EC 3.1.21.1, Type II, salmon sperm DNA, dG, dGpG and dApG were purchased from Sigma.

Instrumentation

The 1H and 13C NMR spectra of the platinum complexes were acquired on a Bruker AMX400 spectrometer. The splitting of proton resonances in the reported 1H NMR spectra are defined as s = singlet, d = doublet and m = multiplet. Infrared data were obtained

HPLC standards for the [PtCl2(R-ahaz)] reactions

The retention times for the standards of the major adducts are listed in Table 1. The reaction between dG and [PtCl2(R-ahaz)] yielded two peaks at 22.1 min and 25.5 min. The corresponding deoxyribonucleoside to platinum ratios for these bands are 2.0 and 0.9, respectively. As the molar ratio of dG to [PtCl2(R-ahaz)] increased, the intensity of the peak at 22.1 min increased (Fig. S3). Therefore, the peak at 22.1 min is assigned to [Pt(R-ahaz)(dG)2] and the peak at 25.5 min is assigned to [Pt(R

Discussion

The enantiomers of [PtCl2(ahaz)] display a remarkable degree of stereoselectivity and enantioselectivity in their interactions with DNA. [PtCl2(R-ahaz)] forms the two isomers of the GpG adduct in approximately equal amounts, but together they account for only about 20% of the DNA platination. This contrasts with the sterically much less demanding cisplatin where the GpG adducts account for up to 65% of the platination [3], [4], [5], [6], [7]. One of the isomers of the S-enantiomer forms at a

Abbreviations

    ahaz

    3(R)-aminohexahydroazepine or 3(S)-aminohexahydroazepine

    DMF

    dimethylformamide

    DRIFTS

    diffuse reflectance infrared Fourier transform spectroscopy

    GFAAS

    graphite furnace atomic absorption spectrometry

    HMG

    high mobility group

Acknowledgments

We acknowledge the Australian Research Council and the University of Sydney Cancer Research Fund for financial support.

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