Formation and structure of reaction products of cis‐PtCl₂(NH₃)₂ with d(ApG) and/or d(GpA) in di‐, tri‐ and penta‐nucleotides

Abstract

The reaction products of c i~-P t c l , ( N H )~)~ with several deoxyribonucleotides containing d(ApG) and/or d(GpA) have been studied. The various reaction products were separated by high-performance liquid chromatography and characterized by means of absorbance at 254 nm in combination with atomic absorption spectroscopy and 300-MHz 'H-NMR (pH dependence of the non-exchangeable base-protons, TI relaxation time determinations). For the larger fragments the results from these techniques were confirmed by enzymatic degradation studies of the platinated fragments.The (2)] were found, 4% of the reacted material consisted of a 1 mol Pt/2 mol dinucleotide product, and 3% of an unidentified 1 : 1 product. From the main product two rotamers were found to occur: at room temperature, 81% anti,unti and 19% anti,syn product is present. With d(GpA) about equal amounts of N1,N7and N7,N7 products were found; for both products the anti,unti and anti,syn conformations were found, respectively. Upon reaction of C~S -P~C I~( N H~)~ with d(pApG) and d(pGpA) only the N7,N7 products were found;at room temperature and pH > 1.5 these products were present in anti,anti conformation. However, for the d(pApG)-platinum chelate at -20°C a small amount (< 5%) of a second product could be observed in NMR. For the d(pGpA)-platinum chelate a second N7,N7-coordinated product was observed when the pH of the NMR sample was lowered to 1.1 (at this pH the free 5'-phosphate group is protonated). With the larger fragments d(ApGpA), d(pApGpA) and d(TpApGpApT) the intra-molecular competition between the formation of the d(ApG) or the d(GpA) chelates could be studied. Using these nucleotides no NZcoordinated products or rotamers were observed. In the case of d(ApGpA) and d(TpApGpApT) the d(GpA) chelate (67% and 75% respectively) was favoured over the d(ApG) chelate, while with d(pApGpA) about equal amounts of both chelates were formed. Since Rosenberg's discovery [l] of the antitumor activity of cisplatin [cis-PtC12(NH3)Z, also abbreviated as cis-Pt] much research has been aimed at elucidating its working mechanism [2,3]. At this moment there is a wide consensus about cellular DNA being the ultimate target [4]. This prompted many researchers to investigate the reaction products formed between cis-Pt and DNA in vitrn [5 -71, in living cells [S, 91, as well as in material obtained from cisplatin-treated patients [ 101. The major product formed appeared to be cis-Pt coordinated via the N7 atoms of a d(GpG) sequence (over 60% of the total Pt was coordinated in this mode), followed by the d(ApG)N7,N7 chelate (20%). Less frequently occurring lesions are d(GpNpG) chelates [5,6, 111, in which cis-Pt is coordinated to two guanines separated by a third base, a cross-link between two guanines of opposite strands [12] and Correspondence to J. Recdijk, Gorlaeus Laboratoria, Rijks-

Single d(ApG)/cis-diamminedichloroplatinum(II) adduct-induced mutagenesis in Escherichia coli.

“…The spectrum of the nonexchangeable protons exhibits two signals at 8.96 and 9.11 ppm (from trimethylsilyl-3-propionic acid d4-2,2,3,3). These downfield-shifted resonances, together with the fast H-2H exchange of the first one, are comparable to those observed for the adenine (H8) and guanine (H8) protons of previously described cisDDP{d(ApG)} chelates (33,34).…”

“…Limited data are available concerning the structure of the cisDDP{d(ApG)} lesion. At least at the dinucleotide level, the structure of both d(GpG) and d(ApG) adducts is similar (33,34). Chemical probes analysis (50,53) shows that the local distortion of DNA concerns the 5' side of the d(ApG) adduct, whereas the 3' guanine is still paired.…”

Alterations in the d(CpGpT) structure in solution as a result of [PtCl(diethylenetriamine)]+ binding

“…Probably this change in sugar conformation directs the sugar-phosphate backbone to a better position for chelation by hydrogen bonding to phosphates and bases. The syn preference for the guanine residue is only found in small DNA fragments [9,10]. Upon extension to larger fragments, e.g.…”

Head‐to‐Head Right‐Handed Cross‐Links of the Antitumor‐Active Bis(μ‐N,N′‐di‐p‐tolylformamidinato)dirhodium(II,II) Unit with the Dinucleotides d(GpA) and d(ApG)

“…The cisplatin34 and Ru(II)‐dimethyl sulfoxide81 d(ApG) dinucleotide adducts are also right‐handed conformers and their metalated fragments have been isolated with both d(ApG) and d(GpA) dinucleotides , a fact that can be attributed to the conformational freedom of the smaller DNA fragments 54. It is well‐established, however, that cisplatin forms 1,2‐intrastrand adducts with d(ApG) but not with d(GpA) in full‐length double‐stranded DNA 51.…”

“…Moreover, the 1,2-ApG cross-links have been extensively studied, [50][51][52] as they are the next most abundant intrastrand adducts found for cisplatin (after the 1,2-GpG) that are recognized by the HMG proteins. [53] For cisplatin, GpA cross-links have been detected with dA C H T U N G T R E N N U N G (GpA) dinucleotides [54,55] but not with double-stranded DNA. [51,52] In the case of dirhodium compounds bridged by carboxylate groups, single-stranded DNA oligonucleotides containing mixed AG and GA purine sites have been studied by mass spectrometry, [11] but to our knowledge, herein is the first detailed NMR study of the products between the mixed- Table 1) by analysis of 2D NMR spectroscopic data (vide infra); the aromatic resonance at d = 7.81 ppm is attributed to the 3'-A H2 proton, an assignment also confirmed by its longer T 1 relaxation time as compared to H8.…”