Unprecedented monofunctional metalation of adenine nucleobase in guanine- and thymine-containing dinucleotide sequences by a cytotoxic platinum-acridine hybrid agent

We have investigated the reactions of [PtCl(en)(ACRAMTU-S)](NO(3))(2) (2) (en = ethane-1,2-diamine; ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea, acridinium cation, 1), the prototype of a new class of cytotoxic DNA-targeted agents, with 2'-deoxyguanosine (dGuo) and random-sequence native DNA by in-line liquid chromatography/mass spectrometry (LC/MS) and NMR spectroscopy ((1)H, (195)Pt) to identify the covalent adducts formed by platinum. In the mononucleoside model system, two adducts are observed, [Pt(en)(ACRAMTU)(dGuo)](3+) (P1, major) and [Pt(en)(dGuo)(2)](2+) (P2, minor). The reaction, which proceeds significantly slower (half-life 11-12 h at 37 degrees C, pH 6.5) than analogous reactions with cisplatin and reactions of 2 with double-stranded DNA, results in the unexpected displacement of the sulfur-bound acridine ligand in approximately 15% of the adducts. This reactivity is not observed in double-stranded DNA, rendering 1 a typical nonleaving group in reactions with this potential biological target. In enzymatic digests of calf thymus DNA treated with 2, three adducts were identified: [Pt(en)(ACRAMTU)(dGuo)](3+) (A1, approximately 80%), [Pt(en)(ACRAMTU)[d(GpA)]](2+) (A2, approximately 12%), and [Pt(en)(ACRAMTU)[d(TpA)]](2+) (A3, approximately 8%). A1 and P1 proved to be identical species. In the dinucleotide adducts A2 and A3, complex 2 covalently modifies adenine at GA and TA base steps, which are high-affinity intercalation sites of the acridine derivative 1. A2 and A3, which may be formed in the minor groove of DNA, are the first examples of monofunctional adenine adducts of divalent platinum formed in double-stranded DNA. The analysis of the adduct profile indicates that the sequence specificity of 1 plays an important role in the molecular recognition between DNA and the corresponding conjugate, 2. Possible biological consequences of the unusual adduct profile are discussed.     

Metal-intercalator-mediated self-association and one-dimensional aggregation in the structure of the excised major DNA adduct of a platinum-acridine agent

The crystal structure of the excised major DNA monoadduct, [Pt(en)(ACRAMTU-S)(dGuo-N7)]3+ ("dGuo*"; en = ethane-1,2-diamine; ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea, acridinium cation; dGuo = 2'-deoxyguanosine), of a platinum-acridine cytotoxic agent is reported. The adduct dGuo*, previously identified in enzymatic digests of native DNA treated with this drug, is partially deprotonated and dimerizes through formation of a rare GG- mismatch base pair, which is sandwiched between the planar chromophores of the acridine nonleaving groups linked to platinum. NMR evidence exists that indicates that the dimeric form persists in neutral aqueous solution. The one-dimensional pi-stack produced by the dimers in the solid state is reminiscent of a coordinative-intercalative DNA binding mode.     

Electrospray ionization tandem mass spectrometric studies of competitive pyridine loss from platinum(II) ethylenediamine complexes by the kinetic method

The competition between pyridine ligand loss in square planar Pt(II) complexes has been examined using the doubly and singly charged ions of complexes consisting of platinum(ethylenediamine) coordinated to two different substituted pyridines. Collision induced dissociation (CID) of [Pt(en)Py(1)Py(2)](2+) (where Py(1) = one of ten different substituted pyridines and Py(2) = pyridine) results in loss of the protonated pyridines to yield the singly charged platinum ions [Pt(en)Py(1)-H](+) and [Pt(en)Py(2)-H](+). In contrast, fragmentation of [Pt(en)Py(1)Py(2)-H](+) results in neutral pyridine loss to yield the ions [Pt(en)Py(1)-H](+) and [Pt(en)Py(2)-H](+). In the latter case, the correlation between relative losses of each pyridine compared to their gas-phase proton affinities is poor. A novel chloride ion abstraction reaction occurs for the fragmentation of [Pt(en)Py(1)Py(2)](2+) when Py(1) = o-C(5)H(4)CIN and Py(2) = C(5)H(5)N, to yield the [Pt(en)(Cl)Py(2)](+) and [o-C(5)H(4)N](+) pair of ions. In order to model this process the competition between nitrogen and chlorine binding in [Pt(NH(3))(3)(o-NC(5)H(4)Cl)](2+) has been examined using density functional theory (DFT) calculations at the B3LYP/LANL2DZ level of theory. Both adducts are minima with the N adduct being more stable than the Cl adduct by 22.7 kcal mol(-1). Furthermore, the Cl adduct exhibits a significant stretching of the C-Cl bond (to 1.935 A), consistent with the observed chloride ion abstraction reaction, which is endothermic by 9.0 kcal mol(-1) (relative to the N adduct).     

Sequence-selective metalation of double-helical oligodeoxyribonucleotides with PtII,MnII, and ZnII ions

Reactions of [PtCl(dien)](+) (dien=diethylenetriamine), Mn(2+) and Zn(2+) ions with three different double-helical oligodeoxyribonucleotides, which contain the central sequence GGXY (XY=AT, TA or CC) have been monitored by NMR spectroscopy. 2 D [(1)H, (15)N] HSQC/HMQC NMR spectroscopy using (15)N-labeled Pt(dien) shows that the rate of formation of 3'-G-N 7 and 5'-G-N 7 platinated adducts is highly sequence dependent. The relative rates of platination of 5'-G versus 3'-G are largest for the sequence -GGCC-, for which only a small fraction of the 3'-G adduct is formed; for -GGTA-, the rate of 5'-G platination is about eight times that of 3'-G, and for -GGAT- the ratio is 1.2. These values are in qualitative agreement with those obtained for G-N 7/Mn(2+) selectivity as determined by paramagnetic line broadening of the adjacent G-H 8, and also G-N 7/Zn(2+) selectivity as determined by G-H 8 chemical shift changes. Fluctuation in the nucleophilicity of G-N 7 may be explained by variation of the pi-stacking interaction between base residues along the double helix. The reaction mixtures containing platinated 3'-G and 5'-G fractions were separated by HPLC. Since the duplexes are self-complementary, the platinated single strands were readily annealed to duplexes with twofold symmetry and analyzed by 2 D [(1)H, (1)H] NOESY NMR spectroscopy. Unexpectedly, the 5'-G-H 8 resonance signals of both 5'-G and 3'-G platinated duplexes showed large downfield shifts in the range delta=0.3-0.6 ppm, while the 3'-G-H 8 resonance signals in both cases exhibited no, or only slight, upfield shifts. Resonance signals for several other protons in the central region undergo large chemical shift variations induced by platination, indicating that monofunctional binding to DNA leads to appreciable conformational changes.     

Selectivity of purine alkylation by a quinone methide. Kinetic or thermodynamic control?

The alkylation reaction of 9-methyladenine and 9-methylguanine (as prototype substrates of deoxy-adenosine and -guanosine), by the parent o-quinone methide (o-QM), has been investigated in the gas phase and in aqueous solution, using density functional theory at the B3LYP/6-311+G(d,p) level. The effect of the medium on the reactivity, and on the stability of the resulting adducts, has been investigated by using the C-PCM solvation model to assess which adduct arises from the kinetically favorable path, or from an equilibrating process. The calculations indicate that the most nucleophilic site of the methyl-substituted nucleobases in the gas phase is the guanine oxygen atom (O(6)) (DeltaG()(gas) = 5.6 kcal mol(-)(1)), followed by the adenine N1 (DeltaG)(gas) = 10.3 kcal mol(-)(1)), while other centers exhibit a substantially lower nucleophilicity. The bulk effect of water as a solvent is the dramatic reduction of the nucleophilicity of both 9-methyladenine N1 (DeltaG)(solv) = 14.5 kcal mol(-)(1)) and 9-methylguanine O(6) (DeltaG)(solv) = 17.0 kcal mol(-)(1)). As a result there is a reversal of the nucleophilicity order of the purine bases. While O(6) and N7 nucleophilic centers of 9-methylguanine compete almost on the same footing, the reactivity gap between N1 and N7 of 9-methyladenine in solution is highly reduced. Regarding product stability, calculations predict that only two of the adducts of o-QM with 9-methyladenine, those at NH(2) and N1 positions, are lower in energy than reactants, both in the gas phase and in water. However, the adduct at N1 can easily dissociate in water. The adducts arising from the covalent modification of 9-methylguanine are largely more stable than reactants in the gas phase, but their stability is markedly reduced in water. In particular, the oxygen alkylation adduct becomes slightly unstable in water (DeltaG(solv) = +1.4 kcal mol(-)(1)), and the N7 alkylation product remains only moderately more stable than free reactants (DeltaG(solv) = -2.8 kcal mol(-)(1)). Our data show that site alkylations at the adenine N1 and the guanine O(6) and N7 in water are the result of kinetically controlled processes and that the selective modification of the exo-amino groups of guanine N2 and adenine N6 are generated by thermodynamic equilibrations. The ability of o-QM to form several metastable adducts with purine nucleobases (at guanine N7 and O(2), and adenine N1) in water suggests that the above adducts may act as o-QM carriers.     

Which one among the Pt-containing anticancer drugs more easily forms monoadducts with G and A DNA bases? A comparative study among oxaliplatin, nedaplatin, and carboplatin

The platination processes of DNA bases with second- and third-generation Pt(II) anticancer drugs have been investigated using density functional theory (DFT) combined with the conductor-like dielectric continuum model (CPCM) approach, in order to describe their binding mechanisms and to obtain detailed data on the reaction energy profiles. Although there is no doubt that a Pt-N7 bond forms during initial attack, the energetic profiles for the formation of the monofunctional adducts are not known. Herein, a direct comparison between the rate of formation of the monofunctional adducts of the second- and third-generation anticancer drugs with guanine (G) and adenine (A) DNA bases has been made in order to spotlight possible common or different behavior. The guanine as target for platination process is confirmed to be preferred over adenine for all the investigated compounds and for both the hydrolyzed forms considered in our investigation. The preference for G purine base is dominated by electronic factors and promoted by a more favorable hydrogen-bonds pattern, confirming the important role played by H-bonds in determining both structural and kinetic control on the purine platination process.     

Characterisation of estrone-nucleic acid adducts formed by reaction of 3,4-estrone-o-quinone with 2'-deoxynucleosides/deoxynucleotides using capillary liquid chromatography/electrospray ionization mass spectrometry

Xenobiotic and endobiotic molecules can react with DNA leading to formation of so-called DNA adducts. This modified DNA can be repaired enzymatically, but, if not, these modifications are believed to be responsible for the initiation of carcinogenic processes. Hence, we studied the interaction of 2'-deoxynucleosides and 2'-deoxynucleotides with 3,4-estronequinone (3,4-E(1)Q), a metabolite of estrone (E(1)) and a supposed carcinogen. These estrone-nucleic acid adducts were analysed by capillary liquid chromatography (CapLC) coupled to electrospray ionization mass spectrometry (ESI-MS). Knowledge of their behaviour from in vitro studies is a prerequisite for detecting adducts in in vivo studies. Our initial attempts to synthesise nucleos(t)ide adducts of 3,4-E(1)Q in an aprotic solvent (dimethylformamide) yielded no adducts. However, under acidic aqueous conditions, adducts were obtained. With dGuo, a dGuo adduct was found in addition to a Gua adduct. Earlier publications on adduct formation in protic solvents failed to report formation of any adduct with dAdo. A N(3)-Ade adduct was reported upon reaction of 3,4-E(1)Q with Ade base and with DNA. With dAdo, we obtained two nucleoside adducts and six Ade adducts due to loss of 2'-deoxyribose. Thus, contrary to general belief that only 2,3-E(1)Q can form stable adducts, we showed formation of substantial amounts of intact DNA adducts with 3,4-E(1)Q in addition to deglycosylated adducts. Adducts were also obtained with dGMP and dAMP, but no phosphate alkylation was found. Adducts of dCyd, dCMP, dThd, and dTMP were not detected. Using chromatographic-MS data a structural relationship between the 2'-deoxynucleoside, 2'-deoxynucleotide and base adducts was found in the various reaction mixtures. The adducts of dGuo and dGMP reaction mixtures were alkylated at the same N(7)-position of the nucleobase, as indicated by the occurrence of a rapid deglycosylation reaction. In dAdo and dAMP reaction mixtures, 14 adducts were detected; their relationships from the LC and MS data reduced the number of structures to six adenine base alkylated adducts with respect to alkylation between N(1), N(3), N(7) and/or N(6) in the adenine and C(1), C(2) and/or C(6) in 3,4-E(1)Q. We could infer, in addition, whether they had an A ring attachment or a C(6) attachment on the estrone moiety.     

Studies on the photoactivation of two cytotoxic trans,trans,trans-diazidodiaminodihydroxo-Pt(IV) complexes

Light-activation of metal ion complexes to cytotoxic species is of interest due to the potential use in anticancer therapy. Two platinum complexes, trans,trans,trans-[Pt(IV)(N(3))(2)(OH)(2)(NH(3))(2)] (3) and trans,trans,trans-[Pt(IV)(N(3))(2)(OH)(2)(py)(NH(3))] (4) were irradiated with either UV (λ = 366 nm) or white fluorescent light and the various photochemical and photobiological phenomena were characterized. HPLC coupled to UV/Vis and MS detection was used to identify photochemical species resulting from irradiation of 4 with UV and white light. These studies showed that various Pt(IV) and Pt(II) products formed during the photolysis. The mass spectra of Pt(IV) complexes showed Pt ions in both the positive as well as the negative mode while Pt(II) complexes resulted in only positively charged Pt(III) ions. Since cellular DNA is considered to be a key target for platinum antitumor drugs, the irreversible platination of calf thymus DNA by the photoactivated Pt(IV) complexes was followed by Atomic Adsorption spectrometry (AAS). The effect of adding chloride or biological reducing agents glutathione (GSH) and ascorbic acid on the rates of DNA platination where also studied. Upon activation by light, both compounds show similar binding behaviour to DNA, but the rates of DNA platination for 3 were faster than for 4. Both chloride and GSH protected DNA from platination by the photoactivated compounds; consistent with the trapping of reactive aqua-Pt species. The presence of ascorbate increased the level of platinum bound to DNA for photoactivated 4 but not for 3. Without photoactivation, little or no DNA platination was observed, either with or without ascorbate or GSH. Cytotoxicity studies with two human cancer cell lines underline the photochemotherapeutic potential of these compounds. Striking is the increase in cytotoxic potency with the replacement of an ammine by a pyridine ligand.     

Effect of thioethers on DNA platination by trans-platinum complexes

Increasing evidence indicates that sulfur-containing molecules can play important roles in the activity of platinum anticancer drugs. Although nuclear DNA is retained to be the ultimate target, these platinum compounds can readily react with a variety of other substrates containing a soft donor atom, such as proteins, peptides, and low molecular weight biomolecules, before reaching DNA. In a recent study it was demonstrated that the DNA platination rate of a trans-geometry antitumor drug was dramatically enhanced by methionine binding, thus suggesting that the thioether could serve as a catalyst for DNA platination. In this work we performed detailed studies on the reactions of a widely investigated and very promising trans-platinum complex having two iminoethers and two chlorido ligands, trans-EE, with methionine (Met) and guanosine 5'-monophosphate (GMP). The results show that in the reaction of trans-EE with methionine the bisadduct is the dominant species in the early stage of the reaction. The reaction is also influenced by chloride concentration: at low NaCl the bis-methionine adduct is formed in preference, whereas the monoadduct is favored at high NaCl concentration. Not only the monomethionine complex, trans-PtCl(E-iminoether)(2)(AcMet), but also the bis-methionine adduct, trans-Pt(E-iminoether)(2)(AcMet)(2), which has already lost both leaving chlorides, can react with GMP to form the ternary platinum complex trans-Pt(E-iminoether)(2)(AcMet)(GMP). The latter reaction discloses the possibility of direct coordination to DNA of a platinum-protein adduct, in which the two carrier ligands remain intact; this is not the case of cis-oriented platinum complexes, like cisplatin, for which formation of a ternary complex is usually accompanied by loss of at least one carrier ligand. Interestingly, isomerization from S to N coordination of one methionine takes place in the bis-methionine complex at neutral pH, while the monoadduct appears to be stable. The shift from S to N coordination of one methionine in the trans-bis-methionine adduct can easily account for the obtainment of the cis isomer in the bis-chelated Pt(Met-S,N)(2) end product.     

Reactions of 9-substituted guanines with bromomalondialdehyde in aqueous solution predominantly yield glyoxal-derived adducts

Reactions of 9-ethylguanine, 2'-deoxyguanosine and guanosine with bromomalondialdehyde in aqueous buffers over a wide pH-range were studied. The main products were isolated and characterized by (1)H and (13)C NMR and mass spectroscopy. The final products formed under acidic and basic conditions were different, but they shared the common feature of being derived from glyoxal. Among the 1 : 1 adducts, 1,N(2)-(trans-1,2-dihydroxyethano)guanine adduct (6) predominated at pH < 6 and N(2)-carboxymethylguanine adduct (10a,b) at pH > 7. In addition to these, an N(2)-(4,5-dihydroxy-1,3-dioxolan-2-yl)methylene adduct (11a,b) and an N(2)-carboxymethyl-1,N(2)-(trans-1,2-dihydroxyethano)guanine adduct (12) were obtained at pH 10. The results of kinetic experiments suggest that bromomalondialdehyde is significantly decomposed to formic acid and glycolaldehyde under the conditions required to obtain guanine adducts. Glycolaldehyde is oxidized to glyoxal, which then modifies the guanine base more readily than bromomalondialdehyde. Besides the glyoxal-derived adducts, 1,N(2)-ethenoguanine (5a-c) and N(2),3-ethenoguanine adducts (4a-c) were formed as minor products, and a transient accumulation of two unstable intermediates, tentatively identified as 1,N(2)-(1,2,2,3-tetrahydroxypropano)(8) and 1,N(2)-(2-formyl-1,2,3-trihydroxypropano)(9) adducts, was observed.     

ESI-MS and thermal melting studies of nanoscale platinum(II) metallomacrocycles with DNA

The hydrophilic, long-chain diamine PEGda (O,O'-bis(2-aminoethyl)octadeca(ethylene glycol)), when complexed with cis-protected Pt(II) ions afforded water-soluble complexes of the type [Pt(N,N)(PEGda)](NO(3))(2) (N,N = N,N,N',N'-tetramethyl-1,2-diaminoethane (tmeda), 1,2-diaminoethane (en), and 2,2'-bipyridine (2,2'-bipy)) featuring unusual 62-membered chelate rings. Equimolar mixtures containing either the 16-mer duplex DNA D2 or the single-stranded D2a and [Pt(N,N)(PEGda)](2+) were analyzed by negative-ion ESI-MS. Analysis of D2-Pt(II) mixtures showed the formation of 1 : 1 adducts of [Pt(en)(PEGda)](2+), [Pt(tmeda)(PEGda)](2+) and the previously-described metallomacrocycle [Pt(2)(2,2'-bipy)(2){4,4'-bipy(CH(2))(4)4,4'-bipy}(2)](8+) with D2; the dinuclear species bound to D2 most strongly, consistent with its greater charge and aromatic surface area. D2 formed 1 : 2 complexes with the acyclic species [Pt(2,2'-bipy)(Mebipy)(2)](4+) and [Pt(2,2'-bipy)(NH(3))(2)](2+). Analyses of D2a-Pt(II) mixtures gave results similar to those obtained with D2, although fragmentation was more pronounced, indicating that the nucleobases in D2a play more significant roles in mediating the decomposition of complexes than those in D2, in which they are paired in a complementary manner. Investigations were also conducted into the effects of selected platinum(II) complexes on the thermal denaturation of calf thymus DNA (CT-DNA) in buffered solution. Both [Pt(2)(2,2'-bipy)(2){4,4'-bipy(CH(2))(6)4,4'-bipy}(2)](8+) and [Pt(2,2'-bipy)(Mebipy)(2)](4+) stabilized CT-DNA. In contrast, [Pt(tmeda)(PEGda)](2+) and [Pt(en)(PEGda)](2+) (as well as free PEGda) caused negligible changes in melting temperature (ΔT(m)), suggesting that these species interact weakly with CT-DNA.     

Force field for platinum binding to adenine

The antitumor drug cis-diamminedichloroplatinum(II) (cisplatin) binds preferentially to GpG and ApG sequences of DNA, forming N7,N7 intrastrand chelates. Molecular modeling of the intrastrand adducts have been handicapped, so far, by the lack of force-field data describing the Pt–guanine and Pt–adenine binding. We used ab initio calculations with relativistic pseudopotentials to evaluate three important parameters for the platinum–adenine model complex [Pt(NH₃)₃(Ade)]²⁺: (1) the force constant for the Pt? N7 bond bending out of the adenine plane; (2) the energy profile for the torsion about Pt? N7; (3) a set of fractional atomic charges that reproduce the ab initio potential for a number of space points placed around the adduct. A population analysis and comparative study on the tetrammine complex [Pt(NH₃)₄]²⁺ have shown that for platinum adenine is a better σ-donor than NH₃, but its capacity as a π-acceptor is weak. © 1993 John Wiley & Sons, Inc.     

Head-to-head right-handed cross-links of the antitumor-active bis(mu-N,N'-di-p-tolylformamidinato)dirhodium(II,II) unit with the dinucleotides d(GpA) and d(ApG)

Reactions of cis-[Rh(2)(DTolF)(2)(NCCH(3))(6)](BF(4))(2) with the dinucleotides d(GpA) and d(ApG) proceed to form [Rh(2)(DTolF)(2){d(GpA)}] and [Rh(2)(DTolF)(2){d(ApG)}], respectively, with bridging purine bases spanning the Rh-Rh unit in the equatorial positions. Both dirhodium adducts exhibit head-to-head (HH) arrangement of the bases, as indicated by the presence of H8/H8 NOE cross-peaks in the 2D ROESY NMR spectra. The guanine bases bind to the dirhodium core at positions N7 and O6, a conclusion that is supported by the absence of N7 protonation at low pH values and the notable increase in the acidity of the guanine N1H sites (pK(a) approximately 7.4 in 4:1 CD(3)CN/D(2)O), inferred from the pH-dependence titrations of the guanine H8 proton resonances. In both dirhodium adducts, the adenine bases coordinate to the metal atoms through N6 and N7, which induces stabilization of the rare imino tautomer of the bases with a concomitant substantial decrease in the basicity of the N1H adenine sites (pK(a) approximately 7.0-7.1 in 4:1 CD(3)CN/D(2)O), as compared to the imino form of free adenosine. The presence of the adenine bases in the rare imino form is further corroborated by the observation of DQF-COSY H2/N1H and ROE N1H/N6H cross-peaks in the 2D NMR spectra of [Rh(2)(DTolF)(2){d(GpA)}] and [Rh(2)(DTolF)(2){d(ApG)}] in CD(3)CN at -38 degrees C. The 2D NMR spectroscopic data and the molecular modeling results suggest the presence of right-handed variants, HH1R, in solution for both adducts (HH1R refers to the relative base canting and the direction of propagation of the phosphodiester backbone with respect to the 5' base). Complete characterization of [Rh(2)(DTolF)(2){d(GpA)}] and [Rh(2)(DTolF)(2){d(ApG)}] by 2D NMR spectroscopy and molecular modeling supports anti-orientation of the sugar residues for both adducts about the glycosyl bonds as well as N- and S-type conformations for the 5'- and 3'-deoxyribose residues, respectively.     

Reactivity of ammonia ligands of the antitumor agent cisplatin: a unique dodecanuclear Pt4,Pd4,Ag4 platform for four cytosine model nucleobases

The reaction of a potential mono(nucleobase) model adduct of cisplatin, cis-[Pt(NH(3))(2)(1-MeC-N3)(H(2)O)](2+) (6; 1-MeC: 1-methylcytosine), with the electrophile [Pd(en)(H(2)O)(2)](2+) (en: ethylenediamine) at pH approximately 6 yields a kinetic product X which is likely to be a dinuclear Pt,Pd complex containing 1-MeC(-)-N3,N4 and OH bridges, namely cis-[Pt(NH(3))(2)(1-MeC(-)-N3,N4)(OH)Pd(en)](2+). Upon addition of excess Ag(+) ions, conversion takes place to form a thermodynamic product, which, according to (1)H NMR spectroscopy and X-ray crystallography, is dominated by a mu-NH(2) bridge between the Pt(II) and Pd(II) centers. X-ray crystallography reveals that the compound crystallizes out of solution as a dodecanuclear complex containing four Pt(II), four Pd(II), and four Ag(+) entities: [{Pt(2)(1-MeC(-)-N3,N4)(2)(NH(3))(2)(NH(2))(2)(OH)Pd(2)(en)(2)Ag}(2){Ag(H(2)O)}(2)](NO(3))(10) 6 H(2)O (10) is composed of a roughly planar array of the 12 metal ions, in which the metal ions are interconnected by mu-NH(2) groups (between Pt and Pd centers), mu-OH groups (between pairs of Pt atoms), and metal-metal donor bonds (Pt-->Ag, Pd-->Ag). The four 1-methylcytosinato ligands, which are stacked pairwise, as well as the four NH(3) ligands and parts of the en rings, are approximately perpendicular to the metal plane. Two of the four Ag ions (Ag2, Ag2') of 10 are labile in solution and show the expected behavior of Ag(+) ions in water, that is, they are readily precipitated as AgCl by Cl(-) ions. The resulting pentanuclear complex [Pt(2)Pd(2)Ag(1-MeC(-))(2)(NH(2))(2)(OH)(NH(3))(2)(en)(2)](NO(3))(4)7 H(2)O (11) largely maintains the structural features of one half of 10. The other two Ag(+) ions (Ag1, Ag1') of 10 are remarkably unreactive toward excess NaCl. In fact, the pentanuclear complex [Pt(2)Pd(2)AgCl(1-MeC(-))(2)(NH(2))(2)(OH)(NH(3))(2)(en)(2)](NO(3))(3)4.5 H(2)O (12), obtained from 10 with excess NaCl, displays a Cl(-) anion bound to the Ag center (2.459(3) A) and is thus a rare case of a crystallized "AgCl molecule".     

Novel binding interactions of the DNA fragment d(pGpG) cross-linked by the antitumor active compound tetrakis(mu-carboxylato)dirhodium(II,II)

Insight into the N7/O6 equatorial binding interactions of the antitumor active complex Rh(2)(OAc)(4)(H(2)O)(2) (OAc(-) = CH(3)CO(2)(-)) with the nucleotide 5'-GMP and the DNA fragment d(pGpG) has been obtained by one- (1D) and two-dimensional (2D) NMR spectroscopy. The lack of N7 protonation at low pH values and the significant increase in the acidity of N1-H (pK(a) approximately 5.6 as compared to 8.5 for N7 only bound platinum adducts), indicated by the pH dependence study of the H8 (1)H NMR resonance for the HT (head-to-tail) isomer of Rh(2)(OAc)(2)(5'-GMP)(2), are consistent with bidentate N7/O6 binding of the guanine. The H8 (1)H NMR resonance of the HH (head-to-head) Rh(2)(OAc)(2)(5'-GMP)(2) isomer, as well as the 5'-G and 3'-G H8 resonances of the Rh(2)(OAc)(2) [d(pGpG)] adduct exhibit pH-independent titration curves, attributable to the added effect of the 5'-phosphate group deprotonation at a pH value similar to that of the N1 site. The enhancement in the acidity of N1-H, with respect to N7 only bound metal adducts, afforded by the O6 binding of the bases to the rhodium centers, has been corroborated by monitoring the pH dependence of the purine C6 and C2 (13)C NMR resonances for Rh(2)(OAc)(2)(5'-GMP)(2) and Rh(2)(OAc)(2) [d(pGpG)]. The latter studies resulted in pK(a) values in good agreement with those derived from the pH-dependent (1)H NMR titrations of the H8 resonances. Comparison of the (13)C NMR resonances of C6 and C2 for the dirhodium adducts Rh(2)(OAc)(2)(5'-GMP)(2) and Rh(2)(OAc)(2) [d(pGpG)] with the corresponding resonances of the unbound ligands at pH 8.0, showed substantial downfield shifts of Deltadelta approximately 11.0 and 6.0 ppm, respectively. The HH arrangement of the bases in the Rh(2)(OAc)(2) [d(pGpG)] adduct is evidenced by intense H8/H8 ROE cross-peaks in the 2D ROESY NMR spectrum. The presence of the terminal 5'-phosphate group in d(pGpG) results in stabilization of one left-handed Rh(2)(OAc)(2) [d(pGpG)] HH1 L conformer, due to the steric effect of the 5'-group, favoring left canting in cisplatin-DNA adducts. Complete characterization of the Rh(2)(OAc)(2[d(pGpG)] adduct revealed notable structural features that resemble those of cis-[Pt(NH(3))(2) [d(pGpG)]]; the latter involve repuckering of the 5'-G sugar ring to C3'-endo (N-type) conformation, retention of C2'-endo (S-type) 3'-G sugar ring conformation, and anti orientation with respect to the glycosyl bonds. The superposition of the low energy Rh(2)(OAc)(2) [d(pGpG)] conformers, generated by simulated annealing calculations, with the crystal structure of cis-[Pt(NH(3))(2) [d(pGpG)]], reveals remarkable similarities between the adducts; not only are the bases almost completely destacked upon coordination to the metal in both cases, but they are favorably poised to accommodate the bidentate N7/O6 binding to the dirhodium unit. Unexpectedly, the two metal-metal bonded rhodium centers are capable of engaging in cis binding to GG intrastrand sites by establishing N7/O6 bridges that span the Rh-Rh bond.     

Organometallic ruthenium(II) diamine anticancer complexes: arene-nucleobase stacking and stereospecific hydrogen-bonding in guanine adducts

Organometallic ruthenium(II) arene anticancer complexes of the type [(eta(6)-arene)Ru(II)(en)Cl][PF(6)] (en = ethylenediamine) specifically target guanine bases of DNA oligomers and form monofunctional adducts (Morris, R., et al. J. Med. Chem. 2001). We have determined the structures of monofunctional adducts of the "piano-stool" complexes [(eta(6)-Bip)Ru(II)(en)Cl][PF(6)] (1, Bip = biphenyl), [(eta(6)-THA)Ru(II)(en)Cl][PF(6)] (2, THA = 5,8,9,10-tetrahydroanthracene), and [(eta(6)-DHA)Ru(II)(en)Cl][PF(6)] (3, DHA = 9,10-dihydroanthracene) with guanine derivatives, in the solid state by X-ray crystallography, and in solution using 2D [(1)H,(1)H] NOESY and [(1)H,(15)N] HSQC NMR methods. Strong pi-pi arene-nucleobase stacking is present in the crystal structures of [(eta(6)-C(14)H(14))Ru(en)(9EtG-N7)][PF(6)](2).(MeOH) (6) and [(eta(6)-C(14)H(12))Ru(en)(9EtG-N7)][PF(6)](2).2(MeOH) (7) (9EtG = 9-ethylguanine). The anthracene outer ring (C) stacks over the purine base at distances of 3.45 A for 6 and 3.31 A for 7, with dihedral angles of 3.3 degrees and 3.1 degrees, respectively. In the crystal structure of [(eta(6)-biphenyl)Ru(en)(9EtG-N7)][PF(6)](2).(MeOH) (4), there is intermolecular stacking between the pendant phenyl ring and the purine six-membered ring at a distance of 4.0 A (dihedral angle 4.5 degrees). This stacking stabilizes a cyclic tetramer structure in the unit cell. The guanosine (Guo) adduct [(eta(6)-biphenyl)Ru(en)(Guo-N7)][PF(6)](2).3.75(H(2)O) (5) exhibits intramolecular stacking of the pendant phenyl ring with the purine five-membered ring (3.8 A, 23.8 degrees) and intermolecular stacking of the purine six-membered ring with an adjacent pendant phenyl ring (4.2 A, 23.0 degrees). These occur alternately giving a columnar-type structure. A syn orientation of arene and purine is present in the crystal structures 5, 6, and 7, while the orientation is anti for 4. However, in solution, a syn orientation predominates for all the biphenyl adducts 4, 5, and the guanosine 5'-monophosphate (5'-GMP) adduct 8 [(eta(6)-biphenyl)Ru(II)(en)(5'-GMP-N7)], as revealed by NMR NOE studies. The predominance of the syn orientation both in the solid state and in solution can be attributed to hydrophobic interactions between the arene and purine rings. There are significant reorientations and conformational changes of the arene ligands in [(eta(6)-arene)Ru(II)(en)(G-N7)] complexes in the solid state, with respect to those of the parent chloro-complexes [(eta(6)-arene)Ru(II)(en)Cl](+). The arene ligands have flexibility through rotation around the arene-Ru pi-bonds, propeller twisting for Bip, and hinge-bending for THA and DHA. Thus propeller twisting of Bip decreases by ca. 10 degrees so as to maximize intra- or intermolecular stacking with the purine ring, and stacking of THA and DHA with the purine is optimized when their tricyclic ring systems are bent by ca. 30 degrees, which involves increased bending of THA and a flattening of DHA. This flexibility makes simultaneous arene-base stacking and N7-covalent binding compatible. Strong stereospecific intramolecular H-bonding between an en NH proton oriented away from the arene (en NH(d)) and the C6 carbonyl of G (G O6) is present in the crystal structures of 4, 5, 6, and 7 (average N...O distance 2.8 A, N-H...O angle 163 degrees ). NMR studies of the 5'-GMP adduct 8 provided evidence that en NH(d) protons are involved in strong H-bonding with the 5'-phosphate and O6 of 5'-GMP. The strong H-bonding from G O6 to en NH(d) protons partly accounts for the high preference for binding of [(eta(6)-arene)Ru(II)en](2+) to G versus A (adenine). These studies suggest that simultaneous covalent coordination, intercalation, and stereospecific H-bonding can be incorporated into Ru(II) arene complexes to optimize their DNA recognition behavior, and as potential drug design features.     

Combined theoretical and computational study of interstrand DNA guanine-guanine cross-linking by trans-[Pt(pyridine)2] derived from the photoactivated prodrug trans,trans,trans-[Pt(N3)2(OH)2(pyridine)2

Molecular modeling and extensive experimental studies are used to study DNA distortions induced by binding platinum(II)-containing fragments derived from cisplatin and a new class of photoactive platinum anticancer drugs. The major photoproduct of the novel platinum(IV) prodrug trans,trans,trans-[Pt(N(3))(2)(OH)(2)(py)(2)] (1) contains the trans-{Pt(py)(2)}(2+) moiety. Using a tailored DNA sequence, experimental studies establish the possibility of interstrand binding of trans-{Pt(py)(2)}(2+) (P) to guanine N7 positions on each DNA strand. Ligand field molecular mechanics (LFMM) parameters for Pt-guanine interactions are then derived and validated against a range of experimental structures from the Cambridge Structural Database, published quantum mechanics (QM)/molecular mechanics (MM) structures of model Pt-DNA systems and additional density-functional theory (DFT) studies. Ligand field molecular dynamics (LFMD) simulation protocols are developed and validated using experimentally characterized bifunctional DNA adducts involving both an intra- and an interstrand cross-link of cisplatin. We then turn to the interaction of P with the DNA duplex dodecamer, d(5'-C(1)C(2)T(3)C(4)T(5)C(6)G(7)T(8)C(9)T(10)C(11)C(12)-3')·d(5'-G(13)G(14)A(15)G(16)A(17)C(18)G(19)A(20)G(21)A(22)G(23)G(24)-3') which is known to form a monofunctional adduct with cis-{Pt(NH(3))(2)(py)}. P coordinated to G(7) and G(19) is simulated giving a predicted bend toward the minor groove. This is widened at one end of the platinated site and deepened at the opposite end, while the P-DNA complex exhibits a global bend of ～67° and an unwinding of ～20°. Such cross-links offer possibilities for specific protein-DNA interactions and suggest possible mechanisms to explain the high potency of this photoactivated complex.     

Alteration in the choice of DNA repair pathway with increasing sequence selective DNA alkylation in the minor groove

BACKGROUND: Many conventional DNA alkylating anticancer drugs form adducts in the major groove of DNA. These are known to be chiefly repaired by both nucleotide (NER) and base (BER) excision repair in eukaryotic cells. Much less is known about the repair pathways acting on sequence specific minor groove purine adducts, which result from a promising new class of anti-tumour agents. RESULTS: Benzoic acid mustards (BAMs) tethering 1-3 pyrrole units (compounds 1, 2 and 3) show increasing DNA sequence selectivity for alkylation from BAM and 1, alkylating primarily at guanine-N7 in the major groove, to 3 which is selective for alkylation in the minor groove at purine-N3 in the sequence 5'-TTTTGPu (Pu=guanine or adenine). This increasing sequence selectivity is reflected in increased toxicity in human cells. In the yeast Saccharomyces cerevisiae, the repair of untargeted DNA adducts produced by BAM, 1 and 2 depends upon both the NER and BER pathways. In contrast, the repair of the sequence specific minor groove adducts of 3 does not involve known BER or NER activities. In addition, neither recombination nor mismatch repair are involved. Two disruptants from the RAD6 mutagenesis defective epistasis group (rad6 and rad18), however, showed increased sensitivity to 3. In particular, the rad18 mutant was over three orders of magnitude more sensitive to 3 compared to its isogenic parent, and 3 was highly mutagenic in the absence of RAD18. Elimination of the sequence specific DNA adducts formed by 3 was observed in the wild type strain, but these lesions persisted in the rad18 mutant. CONCLUSIONS: We have demonstrated that the repair of DNA adducts produced by the highly sequence specific minor groove alkylating agent 3 involves an error free adduct elimination pathway dependent on the Rad18 protein. This represents the first systematic analysis of the cellular pathways which modulate sensitivity to this new class of DNA sequence specific drugs, and indicates that the enhanced cytotoxicity of certain sequence specific minor groove adducts in DNA is the result of evasion of the common excision repair pathways.     

Spectral and thermal study on the adduct formation between square planar nickel(II) chelates and some bidentate ligands

Adducts of Ni(II)-square planar complexes [Ni(beta-dik)(Me(4)en)](+), with a series of bidentate ligands (L), where beta-dik=acetylacetonate (acac) and benzoylacetonate (bzac), Me(4)en=N,N,N',N'-tetramethylethylenediamine and L=Me(4)en, 2,2'-bipyridine (bipy), ethylenediamine (en) and oxalate (C(2)O(4)(2-)) have been synthesized and characterized by spectral, thermal and magnetic measurements. Formation constants of the adducts formed from a series of ternary mixed Ni(II) complexes with the general formula [Ni(beta-dik)(diam)](+) with 1,10-phenanthroline (phen), 2,2'-bipyridine (bipy) and pyridine were spectrophotometrically determined. Thermodynamic parameters of the adduct formation between nickel(II) square-planar chelates and pyridine (py), 2,2'-bipyridine (bipy) and acetylacetone (acac) were also spectrophotometrically determined in 1,2-dichloroethane. The thermal stability of the isolated adducts was studied using thermogravimetry and the decomposition schemes of the adducts were given.     

Complexes of platinum(II) containing neutral and deprotonated 9-methyladenine. Synthesis, x-ray structures, and NMR studies on the cyclic trimer cis-[L2Pt[9-MeAd([bond]H)]]3(NO3)3 and the Dinuclear cis-[L2Pt(ONO2)[9-MeAd([bond]H)]PtL2](NO3)2 (L = PMePh2)

The dinuclear hydroxo complex cis-[L(2)Pt(mu-OH)](2)(NO(3))(2) (L = PMePh(2), 1), in CH(2)Cl(2), CH(3)CN, or DMF solution, deprotonates the NH(2) group of 9-methyladenine (9-MeAd) to give the complex cis-[L(2)Pt[9-MeAd(-H)]](3)(NO(3))(3), 2, which was isolated in good yield. The X-ray structure shows that the nucleobase binds symmetrically the metal centers through the N(1),N(6) atoms forming a cyclic trimer with Pt...Pt distances in the range 5.202(1)-5.382(1) A. Dissolution of 2 in DMSO or DMF determines the partial (or total) dissociation of the cyclic structure to form several fragments. A multinuclear NMR analysis of the resulting mixture supports the presence of the mononuclear species cis-[L(2)Pt[9-MeAd(-H)]](+), 3, in which the deprotonated nucleobase chelates the metal center with the N(6),N(7) atoms. Addition of a stoichiometric amount of the nitrato complex cis-[L(2)Pt(ONO(2))(2)] (L = PMePh(2), 4) to a DMSO or DMF solution of 2 affords quantitatively the diplatinated compound cis-[L(2)Pt(ONO(2))[9-MeAd(-H)]PtL(2)](NO(3))(2), 5. The single-crystal X-ray analysis shows that the adenine behaves as a tridentate ligand bridging two cis-L(2)Pt units at the N(1) and N(6),N(7) sites, respectively [Pt(1)-N(1) = 2.109(5) A, Pt(2)-N(6) = 2.095(7) A, Pt(2)-N(7) = 2.126(7) A]. The N(1)-bonded metal center completes the coordination sphere through an oxygen atom of a nitrate group, and its coordination plane is arranged orthogonally with respect the second one. The Pt-O distance [2.109(5) A] is similar to those found in the nitrato complex 4 [2.110 A, average]. The related complex cis-[[L(2)Pt(ONO(2))](2)(9-MeAd)](NO(3))(2), 6, containing the neutral adenine platinated at the N(1),N(7) atoms, was isolated and its stability in solution investigated by NMR spectroscopy. In DMSO, 6 undergoes decomposition forming a mixture of the species 4, 5, and the adenine mono- and bis-adducts cis-[L(2)Pt(9-MeAd)(DMSO)](2+), 7, and cis-[L(2)Pt(9-MeAd)(2)](2+), 8, respectively. This last complex, quantitatively formed upon addition of 9-MeAd (Pt/adenine = 1:2) to the mixture, was also isolated and characterized.