The utility of electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) for the characterisation of ligand-oligonucleotide adducts is demonstrated with adducts formed between the oligonucleotide 5'-CACGTG-3' and both a platinating agent, cis-diamminedichloroplatinum(II) (cisplatin), and an alkylating ligand, n-bromohexylphenanthridinium bromide (phenC6Br). We have demonstrated previously that negative ion MS/MS spectra of alkylated oligonucleotides show a highly specific fragmentation pathway that enables the site of binding of the ligand to be readily identified. In comparison, the positive ion ESI-MS/MS spectra reported here also show a single major fragmentation pathway, but the dominant ion is the protonated ligand-base adduct. MS/MS of this ion confirms the site on binding of the ligand to the guanine base. MS/MS spectra of cisplatin adducts show much less specific fragmentation than alkylated adducts, particularly in the negative ion mode. This suggests that the ESI-MS/MS spectra of ligand-DNA adducts are strongly influenced by the extent to which the ligand weakens the glycosidic bond in the residue to which it is bound. For platinating agents, which do not labilise the glycosidic bond, additional experiments involving MS/MS of source-generated product ions were required to enable isomeric adducts to be distinguished.

DNA alkylation is a key cellular event in the mechanism of action of clinical anticancer drugs, chemical mutagens, and carcinogens. 1 The nitrogen and oxygen atoms of purines are preferentially alkylated, with the affinity for a particular base depending on the sequence of neighboring bases. It is most likely that the sequence selectivity of the agent is important in determining the nature of the biological response. 2 Methods based on molecular biology have been used extensively to determine the sequence selectivity of alkylation. 3 Such assays are extremely sensitive and may be quantitative; however, they do not yield direct information concerning the structure of the ligand-DNA adducts. NMR spectroscopy 4 and X-ray crystallography 5 enable detailed structural characterization but are time consuming and require large amounts (micromoles) of material. Mass spectrometry should complement these techniques by providing a rapid and sensitive means of determining the sequence selectivity of alkylation and, at the same time, provide some structural detail about the adducts. 6 Several early studies concerned with the sequencing of ligand-DNA adducts by mass spectrometry have involved tandem mass spectrometry (MS/ MS) following fast atom bombardment (FAB) ionization. 7 This approach is limited, however, by the relatively poor efficiency of FAB ionization for oligonucleotides. 8 In contrast, electrospray ionization (ESI) has allowed mass measurement of relatively large oligonucleotides, including modified oligonucleotides, 9 an intact plasmid DNA, 10 and noncovalent drug-DNA complexes. 11 Tandem mass spectrometry following electrospray ionization (ESI-MS/MS) has been used for the location of modified bases within oligonucleotides and for the characterization of synthetic oligonucleotides which contain "unnatural" bases in their sequence. 12 To date, however, there have been no reports of the use of ESI-MS/MS for the sequencing of covalent, ligand-oligonucleotide adducts. 13 Hedamycin (Figure 1) is a naturally-occurring antitumor antibiotic that binds to double-stranded DNA by both intercalation and alkylation. DNA sequencing shows that alkylation occurs exclusively at guanine bases, but with a distinct preference for guanines located in 5′-CGT-3′ and 5′-CGG-3′ sequences. 14 NMR-derived solution structures of hedamycin covalently bound to double-stranded oligonucleotides show that the anthrapyrantrione chromophore of the ligand threads the double helix, placing the two amino sugars in the minor groove and the alkylating bis-epoxide side chain in the major groove. 15 Our previous studies of hedamycin-DNA binding by ESI-MS have confirmed that hedamycin significantly increases duplex stability and provided strong evidence for the formation of specifically, base-paired duplexes in the gas phase. 16 We demonstrate here that ESI-MS/MS can be used to determine unequivocally the sequence selectivity of base alkylation by a DNA-binding antitumor drug, using hedamycin as a model.ESI-MS/MS spectra of multiply-charged anions of u...

The sequence specificity of DNA damage caused by cis-diamminedichloroplatinum(II) (cisplatin) and four analogues in human (HeLa) cells was studied using Taq DNA polymerase and a linear amplification system. The primer extension is inhibited by the drug-DNA adducts, and hence the sites of these lesions can be analyzed on DNA sequencing gels. The repetitive alphoid DNA was used as the target DNA in human cells. A comparison was made between adduct formation in human cells and in purified DNA. The sequence-specific position and relative intensity of damage was similar in both systems for cisplatin, dichloro(ethylenediammine)platinum(II) (PtenCl2), and N-[3-N-(ethylenediamino)propyl]acridine-4-carboxamidedichloropl atinum(II) (4AcC3PtenCl2). However, no DNA damage could be detected in cells for trans-diamminedichloroplatinum(II) (transPt) or N-[3-N-(ethylenediamino)propyl]acridine-2-carboxamide-dichloroplat inum(II) (2AcC3PtenCl2) despite the ability of these latter analogues to damage purified DNA. Cisplatin, PtenCl2, and 4AcC3PtenCl2, which significantly damaged DNA inside cells, also show antitumor activity in mouse models. However, transPt and 2AcC3PtenCl2, which did not detectably damage DNA inside cells, did not show such antitumor activity. This correlation between intracellular DNA damaging ability and in vivo antitumor activity indicates the potential use of the human cells/Taq DNA polymerase/linear amplification technique as a convenient method for screening new cisplatin analogues for useful chemotherapeutic activity.

Cisplatin analogues were synthesised that consisted of platinum(II) diamine complexes tethered via a polymethylene chain ( n = 3, 5, 8 and 10) to a phenanthridinium cation. Both chloro and iodo leaving groups were examined. DNA adduct formation was quantitatively analysed using a linear amplification system with the plasmid pGEM-3Zf(+). This system utilised Taq DNA polymerase to extend from an oligonucleotide primer to the damage site. This damage site inhibited the extension of the DNA polymerase. The products were electrophoresed on a DNA sequencing gel enabling adduct formation to be determined at base pair resolution. The damage intensity at each site was determined by densitometry. The platinum phenanthridinium complexes were shown to damage DNA at shorter incubation times than cisplatin. To produce similar levels of damage, an 18 h incubation was required for cisplatin compared to 30 min for the n = 3 platinum phenanthridinium complexes; this indicates that the intercalating chromophore causes a large increase in the rate of platination. A reaction mechanism involving direct displacement of the chloride by the N-7 of guanine may account for the rate increase. These results indicate that further development of these compounds could lead to more effective cancer chemotherapeutic agents.

A series of acridine-2- and -4-carboxamide-linked analogues of PtenCl2 has been prepared and evaluated for biological activity against several tumor cell lines in vitro and in vivo. The platinum complexes were generally more cytotoxic than the corresponding ligands against wild-type P388 leukemia cells in vitro, with acridine-4-carboxamide complexes being the more effective. In contrast to cisplatin and PtenCl2, the complexes were equally active in vitro against both wild-type and cisplatin-resistant P388 lines. The 4-carboxamide complexes showed high levels of in vivo activity (ILS greater than 100%) against wild-type P388 using a single-dose protocol, and one compound was also significantly active in vivo in a cisplatin-resistant line, against which cisplatin and PtenCl2 are inactive.