Platination Pathways for Reactions of Cisplatin with GG Single-Stranded and Double-Stranded Decanucleotides

摘要

The antieuncer drug cispiatin. cis-[PtCl_2(NH_3)_2]_2 disrupts cellular replication and transcription by the iormauon of Pl-DNA adducis, largely guanme guanine (GGi and adenine guanme (AG) intrastrand cross-links. Therefore it is-important to understand the mechanisms of DNA piatination and the perturbations of DNA structure whieh these adducts cause. The rate-determining step in GG platination is believed to he hydrolysis of cisplatm. followed by monofunctional binding to N7 of either the 3'- or 5'-G, and finally macroeyclie ring closure to give the bifunctional GG adduct. However, direct detection of aquated cisplatm during the course of DNA reactions has proved to be difficult. None was seen by Bancroft et al. by ~(195)Pt NMR spectroscony. even with enriched ~(195)Pt and high concentrations of DNA oligomers (ca. 14 32 mM). These workers were able to detect monofunctional adducts and determine the rate of ring closure by independent studies ot reactions of the aqua-chloro complex with DNA. in analternative approach. Chottard et al. have recently determined the rates of attack of "diaqua cisplatin" (Cl replaced by H_2O) on GG oligonucleotides and rates of ring closure using an HPLC trapping method. We report here that the major species in the pathways of platination of both single- and double-stranded GG oligonucleotides by ~(15)N-cisplatin can all be detected simultaneously by [~1H, ~(15)N] NMR spectroscopv. We have determined directly, for the first time, the lifetime of the aqua-chloro intermediate present at only micromolar concentrations, and investigated differences in the platination pathways for single- and double-stranded DNA. Both ~1H and ~(15)N NMR resonance signals are sensitive to cisplatin-mduced helix melting. The method will begenerally applicable to a wide range of DNA substrates and platinum ammine and amine complexes, and promises to provide new insights into the factors which influence Pt-DNA recoenition.