Substituted 9-aminoacridine-4-carboxamides tethered to platinum(II)diamine complexes: chemistry, cytotoxicity and DNA sequence selectivity.

Three platinum complexes in which substituted (7-OMe, 9-NH(2); 7-F, 9-NH(2); and 7-H, 9-NH(CH(2))(2)OH) 9-aminoacridine-4-carboxamides were tethered to a platinum(II)diamine moiety were synthesised and characterised at the chemical and biological level. These variants showed a decrease in cytotoxicity, as measured by IC(50) values in HeLa cells, when compared with the parent 7-H, 9-NH(2) compound. The 7-F and 9-NH(CH(2))(2)OH substituents gave rise to a small decrease in cytotoxicity, and the 7-OMe substituent resulted in a larger decrease in cytotoxicity. Their binding to purified pUC19 plasmid DNA was investigated and it was found that the addition of 7-F, 9-NH(CH(2))(2)OH and especially the 7-OMe substituents, resulted in reduced DNA binding. This correlated well with the IC(50) cytotoxicity values. However, the DNA sequence selectivity was unaffected by the addition of these moieties.

The DNA sequence selectivity of maltolato-containing cisplatin analogues in purified plasmid DNA and in intact human cells.

Cisplatin analogues with an attached DNA binding moiety have a higher affinity for DNA, but often suffer from poor aqueous solubility. In this study we examined the DNA sequence specificity of more soluble cisplatin analogues containing the maltolato leaving group in both purified DNA and in intact human cells. In both environments the DNA sequence specificity of these analogues was very similar to cisplatin. However, in purified DNA a higher concentration of the two maltolato-containing analogues was needed to achieve a similar level of DNA damage as cisplatin. This difference in reactivity was not observed in intact cells as the two maltolato-containing complexes were capable of producing a similar level of damage as cisplatin at comparable concentrations. This was consistent with the IC(50) values obtained for both cisplatin and the maltolato compounds which were also similar. This study indicated that maltolato can be utilised as the leaving group to increase the aqueous solubility of cisplatin analogues without reducing their biological activity.

Tuning the gas phase redox properties of copper(II) ternary complexes of terpyridines to control the formation of nucleobase radical cations.

Electrospray ionization (ESI) tandem mass spectrometry (MS/MS) of ternary copper(II) complexes of [Cu(terpyX)(M)]2+ (where terpyX = is a substituted 2,2':6',2''-terpyridine ligand; M = the nucleobases: adenine, guanine, thymine and cytosine) was examined as a means of forming radical cations of nucleobases in the gas phase. The following substituents were examined: 4'-NMe2-2,2':6',6''-terpyridine; 4'-OH-2,2':6',6''-terpyridine; 4'-F-2,2':6',6''-terpyridine; 2,2':6',6''-terpyridine; 4'-Cl-2,2':6',6''-terpyridine; 4'-Br-2,2':6',6''-terpyridine; 4'-CO2H-2,2':6',6''-terpyridine; 4'-NO2-2,2':6',6''-terpyridine and 6,6''-dibromo-2',2:6',2''-terpyridine. Each of the ternary complexes [Cu(terpyX)(M)]2+ was mass selected and subjected to collision induced dissociation (CID) in a quadrupole ion trap. The types of fragmentation reactions observed for these complexes depend on the nature of the substituent on the terpyridine ligand, while the yields of the radical cations of the nucleobases follow the order of their ionization energies (IEs): G (lowest IE) > A > C > T (highest IE). In general, radical cation formation is favoured for electron withdrawing substituents (e.g. NO2) while loss of the neutral nucleobase is favoured for electron donating substituents (e.g. NMe2). Loss of the protonated nucleobase is a major fragmentation pathway for the OH substituted terpyridine system, consistent with its ability to bind to a metal centre as a deprotonated ligand. Crystal structure determinations of (6,6''-dibromo-2',2:6',2''-terpyridine)bis(nitrato)copper(II) and diaqua(4'-oxo-2,2':6',6''-terpyridine)copper(II) nitrate monohydrate were performed and correlated with the ESI results.

Peptide derivatization as a strategy to form fixed-charge peptide radicals.

As a means of generating fixed-charge peptide radicals in the gas phase we have examined the collision-induced dissociation (CID) chemistry of ternary [Cu(II)(terpy)(TMPP-M)]2+ complexes, where terpy = 2,2':6'2''-terpyridine and TMPP-M represents a peptide (M) modified by conversion of the N-terminal amine to a [tris(2,4,6-trimethoxyphenyl)phosphonium]acetamide (TMPP-) fixed-charge derivative. The following modified peptides were examined: oligoglycines, (Gly)n (n = 1-5), alanylglycine, glycylalanine, dialanine, trialanine and leucine-enkephaline (YGGFL). The [Cu(II)(terpy)(TMPP-M)]2+ complexes are readily formed upon electrospray ionization (ESI) of a mixture of derivatized peptide and [Cu(II)(terpy)(NO3)2] and generally fragment to form transient peptide radical cations, TMPP-M+*, which undergo rapid decarboxylation for the simple aliphatic peptides. This is contrasted with the complexes containing the unmodified peptides, which predominantly undergo fragmentation of the coordinated peptide. These differences demonstrate the importance of proton mobility in directing fragmentation of ternary copper(II) peptide complexes. In the case of leucine-enkephaline, a sufficient yield of the radical cation was obtained to allow further CID. The TMPP-YGGFL+* ion showed a rich fragmentation chemistry, including CO2 loss, side-chain losses of an isopropyl radical, 2-methylpropene and p-quinomethide, and *a1 and *a4 sequence ion formation. In contrast, the even-electron TMPP-YGGFL+ ion fragments to form *a(n) and *b(n) sequence ions as well as the [*b4 + H2O]+ rearrangement ion.

Metal-mediated formation of gas-phase amino acid radical cations.

The results from an investigation of the collision-induced dissociation (CID) of the ternary complexes [Cu(II)(terpy)(AA)](2+) are presented (terpy = 2,2':6',2' '-terpyridine; AA = one of the twenty common amino acids). These complexes show a rich gas-phase chemistry, which depends on the identity of the amino acid. For the histidine-, lysine- and tryptophan-containing complexes, oxidative dissociation of the amino acid is observed, yielding the amino acid radical cation. The results of further mass selection and CID of these amino acid radical cations are presented. The CID of the series [Fe(III)(salen)(AA)](+) (where salen = N,N'-ethylenebis(salicylideneaminato)) is also examined. These complexes undergo loss of the neutral amino acid in all cases, although the radical cation of arginine is also produced and its subsequent fragmentation examined. B3-LYP/6-31G(d) computations were carried out to test aspects of the proposed fragmentation mechanism of the histidine and arginine radical cations.

Syntheses, crystal structure and cytotoxicity of diamine platinum(II) complexes containing maltol.

The cationic complexes (1,2-diaminoethane)(maltolato)platinum(II) ([Pt(en)(ma)]+) and (1R,2R-1,2-diaminocyclohexane)(maltolato)platinum(II) ([Pt(R,R-DACH)(ma)]+) have been prepared and the structure of [Pt(R,R-DACH)(ma)]NO3 has been determined by single crystal X-ray diffraction. The geometry of the metal in [Pt(R,R-DACH)(ma)]NO3 is essentially square planar and the maltolate ligand has a geometry similar to other chelate complexes involving this ligand. The cytotoxicities of the compounds have been assessed in the human cell lines HeLa and K562 and the IC50 values are approximately 32 microM in HeLa cells and 26 microM in K562 cells. In these cell lines the cytotoxicity of cisplatin is higher than the maltolate complexes by a factor of 2 to 3 whereas the cytotoxicity of carboplatin is lower than the maltolate complexes.

Can radical cations of the constituents of nucleic acids be formed in the gas phase using ternary transition metal complexes?

Electrospray ionization (ESI) tandem mass spectrometry (MS/MS) of ternary transition metal complexes of [M(L(3))(N)](2+) (where M = copper(II) or platinum(II); L(3) = diethylenetriamine (dien) or 2,2':6',2''-terpyridine (tpy); N = the nucleobases: adenine, guanine, thymine and cytosine; the nucleosides: 2'deoxyadenosine, 2'deoxyguanosine, 2'deoxythymine, 2'deoxycytidine; the nucleotides: 2'deoxyadenosine 5'-monophosphate, 2'deoxyguanosine 5'-monophosphate, 2'deoxythymine 5'-monophosphate, 2'deoxycytidine 5'-monophosphate) was examined as a means of forming radical cations of the constituents of nucleic acids in the gas phase. In general, sufficient quantities of the ternary complexes [M(L(3))(N)](2+) could be formed for MS/MS studies by subjecting methanolic solutions of mixtures of a metal salt [M(L(3))X(2)] (where M = Cu(II) or Pt(II); L(3) = dien or tpy; X = Cl or NO(3)) and N to ESI. The only exceptions were thymine and its derivatives, which failed to form sufficient abundances of [M(L(3))(N)](2+) ions when: (a) M = Pt(II) and L(3) = dien or tpy; (b) M = Cu(II) and L(3) = dien. In some instances higher oligomeric complexes were formed; e.g., [Pt(tpy)(dG)(n)](2+) (n = 1-13). Each of the ternary complexes [M(L(3))(N)](2+) was mass-selected and then subjected to collision-induced dissociation (CID) in a quadrupole ion trap. The types of fragmentation reactions observed for these complexes depend on the nature of all three components (metal, auxiliary ligand and nucleic acid constituent) and can be classified into: (i) a redox reaction which results in the formation of the radical cation of the nucleic acid constituent, N(+.); (ii) loss of the nucleic acid constituent in its protonated form; and (iii) fragmentation of the nucleic acid constituent. Only the copper complexes yielded radical cations of the nucleic acid constituent, with [Cu(tpy)(N)](2+) being the preferred complex due to suppression, in this case, of the loss of the nucleobase in its protonated form. The yields of the radical cations of the nucleobases from the copper complexes follow the order of their ionization potentials (IPs): G (lowest IP) > A > C > T (highest IP). Sufficient yields of the radical cations of each of the nucleobases allowed their CID reactions (in MS(3) experiments) to be compared to their even-electron counterparts.

Formation of cationic peptide radicals by gas-phase redox reactions with trivalent chromium, manganese, iron, and cobalt complexes.

The collision-induced dissociation (CID) of a series of gas-phase complexes [M(III)(salen)(P)](+) [where M = Cr, Mn, Fe, and Co; P = hexapeptides YGGFLR, WGGFLR, and GGGFLR; and salen = N,N'-ethylenebis(salicylideneaminato)] has been examined with respect to the ability of the complexes to form the corresponding cationic peptide radical ions, P(+)(*), by homolytic cleavage of the metal peptide bond. This is the first example of the use of gas-phase ternary metal peptide complexes to produce the corresponding cationic peptide radical for a metal other than copper(II). The fragmentation reactions competing with radical formation are highly dependent on the metal ion used. In addition, examination of modified complexes in which the periphery of the salen was substituted allowed evaluation of electronic effects on the CID process, presumably without significant change in the geometry surrounding the metal. This substitution demonstrates that the ligand can be used to tune the dissociation chemistry to favor radical formation and suppress unwanted further fragmentation of the peptide radical that is typically observed immediately following its dissociation from the complex.

Designing copper(II) ternary complexes to generate radical cations of peptides in the gas phase: role of the auxiliary ligand.

A series of ternary copper(II) complexes of the type [Cu(II)(L)(M)](2+), where M represents the hexapeptides GGGFLR, YGGFLR and WGGFLR and L a set of 12 nitrogen donor ligands have been evaluated for their ability to form cationic peptide radicals, M(+)*, in the gas phase. Although the fragmentation chemistry of these ions is complex, two main conclusions emerge: (i) Complexes containing a tri- or tetra-dentate ligand were found to be more effective at producing the peptide radical because in these instances competitive loss of the ligand from the complex is inhibited; (ii) The ligands ought not possess any acidic protons in order to prevent competitive loss of the protonated peptide, [M + H](+). There is significant interaction of the N-terminal aromatic residues in YGGFLR and WGGLFR with the copper(ii) ion in several of the complexes as revealed by the formation of [Cu(I)(L)(p-quinomethide)](+) and [Cu(I)(L)(3-methyleneindoline)](+) fragment ions. Following its dissociation from the ternary complex, CID of the YGGFLR(+)* radical cation shows a dependence on the ligand in the complex from which it was formed. This 'memory effect' most likely reflects differences in the coordinated peptide structure induced by the ligand in the precursor complex which are maintained following dissociation.

Gas-phase ligand loss and ligand substitution reactions of platinum(II) complexes of tridentate nitrogen donor ligands.

The source of protons associated with the ligand loss channel of HX((n - 1)+) from [Pt(II)(dien)X](n+) (X = Cl, Br and I for n = 1 and X = NC(5)H(5) for n = 2) in the gas phase was investigated by deuterium-labelling studies. The results of these studies indicate that these protons originate from both the amino groups and the carbon backbone of the dien ligand. In some instances (e.g. X = Br and I), the protons lost from the carbon backbone can be even more abundant than the protons lost from the amino groups. The gas-phase substitution reactions of coordinatively saturated [Pt(II)(L(3))L(a)](2+) complexes (L(3) = tpy or dien) were also examined using ion-molecule reactions. The outcome of the ion-molecule reactions depends on both the ancillary ligand (L(3)) as well as the leaving group (L(a)). [Pt(II)(tpy)L(a)](2+) complexes undergo substitution reactions, with a faster rate when L(a) is a good leaving group, while the [Pt(II)(dien)L(a)](2+) complex undergoes a proton transfer reaction.

The interaction of DNA-targeted 9-aminoacridine-4-carboxamide platinum complexes with DNA in intact human cells.

As part of an ongoing drug development programme, this paper describes the sequence specificity and time course of DNA adduct formation for a series of novel DNA-targeted analogues of cis-diaminedichloroplatinum(II) (cisplatin) (9-aminoacridine-4-carboxamide Pt complexes) in intact HeLa cells. The sequence specificity of DNA damage caused by cisplatin and analogues in human (HeLa) cells was studied using Taq DNA polymerase and a linear amplification/polymerase stop assay. Primer extension is inhibited by a Pt-DNA adduct, and hence the sites of these lesions can be analysed on DNA sequencing gels. The repetitive alphoid DNA sequence was used as the target DNA in human cells. The 9-aminoacridine-4-carboxamide Pt complexes exhibited a markedly different sequence specificity relative to cisplatin and other analogues. The sequence specificity of the 9-aminoacridine-4-carboxamide Pt complexes is shifted away from a preference for runs of guanines. The 9-aminoacridine-4-carboxamide Pt complexes have an enhanced preference for GA dinucleotides. This is the first occasion that an altered DNA sequence specificity has been demonstrated for a cisplatin analogue in human cells. A time course of DNA damage revealed that the DNA-targeted Pt complexes, consisting of four 9-aminoacridine-4-carboxamide Pt complexes and one acridine-4-carboxamide Pt complex, damaged DNA more rapidly compared to cisplatin and non-targeted analogues. A comparison of the time taken to reach half the maximum relative intensity indicated that the DNA-targeted Pt complexes reacted approximately 4-fold faster than cisplatin and the non-targeted analogues.

Side-chain radical losses from radical cations allows distinction of leucine and isoleucine residues in the isomeric peptides Gly-XXX-Arg.

Sequencing of peptides via low-energy collision-induced dissociation of protonated peptides typically yields b(n) and y(n) sequence ions. The isomeric residues leucine and isoleucine rarely can be distinguished in these experiments since they give b(n) and y(n) sequence ions of the same m/z. Siu's pioneering work on electrospray ionization of copper complexes of peptides (Chu IK, Rodriquez CF, Lau TC, Hopkinson AC, Siu KWM. J. Phys. Chem. B 2000; 104: 3393) provides a way of forming radical cations of peptides in the gas phase. This method was used to generate M(+ small middle dot) ions of the two isomeric peptides Gly-Leu-Arg and Gly-Ile-Arg in order to compare their fragmentation reactions. Both radical cations fragment to give even electron y(2) and y(1) sequence ions as well as side-chain radical losses of CH(3) and CH(3)CH(2) for isoleucine and (CH(3))(2)CH for leucine. In contrast the [M + H](+) and [M + 2H](2+) ions do not allow distinction between the isomeric leucine and isoleucine peptides.