The new antitumor compound, cis-[Pt(NH3)2(4-methylpyridine)Cl]Cl, does not form N7,N7-d(GpG) chelates with DNA. An unexpected preference for platinum binding at the 5'G in d(GpG)

The reaction of the antitumor active agent cis-[Pt(NH3)2(4-mepy)Cl]Cl (4-mepy stands for 4-methylpyridine) with d(GpG) has been investigated by 1H magnetic resonance spectroscopy. Initially, two mononuclear complexes cis-Pt(NH3)2(4-mepy)[d(GpG)-N7(1)] 1 and cis-Pt(NH3)2(4-mepy)[d(GpG)-N7(2)] 2 are formed in an unexpected ratio 65:35, as determined by 1H NMR and enzymatic digestion techniques. Both products react further with a second equivalent of cis-[Pt(NH3)2(4-mepy)Cl]Cl forming the dinuclear platinum complex [cis-Pt(NH3)2(4-mepy)]2[mu-d(GpG)- N7(1),N7(2)] 3. With [Pt(dien)Cl]Cl and [Pt(NH3)3Cl]Cl similar complexes are formed. No evidence was found for the formation of chelates cis-Pt(NH3)(4-mepy) [d(GpG)-N7(1),N7(2)], which would be formed upon ammonia release from the mononuclear complexes 1 and 2. Even addition of strong nucleophiles, like sodium diethyldithiocarbamate, thiourea, cysteine, or methionine, before or after reaction, do not induce the formation of a chelate. Under all conditions the N-donor ligands remain coordinated to Pt in 1,2 and 3. In addition, the results of bacterial survival and mutagenesis experiments with E. coli strains show that the in vivo formation of bifunctional adducts in DNA, comparable to those induced by cis-Pt(NH3)2Cl2, by treatment of cells with cis-[Pt(NH3)2(4-mepy)Cl]Cl is unlikely. Also, a mechanism of binding and intercalation is not supported by experimental data. All experiments suggest that the mechanism of action of this new class of antitumor agents must be different from that of cis-Pt(NH3)2Cl2.     

The reaction of Pt-antitumor drugs with selected nucleophiles. II. Preparation and characterization of coordination compounds of Pt(II) and L-histidine

Various His-Pt(II) coordination compounds were prepared by reaction of K2PtCl4 or cis-[Pt(NH3)2Cl2](cis-DDP) with His and analyzed by 1H and 13C NMR spectroscopy, electrophoresis, and ion-exchange chromatography. His may be coordinated to Pt by the imidazol iminogroup and/or the alpha-aminogroup; the carboxygroup remains always free. Both bidentate as well as monodentate ligands were identified. Cis-DDP reacts with His to give a mixture of compounds where all these possibilities are present: cis-diamine-(histidine-N,N-)Pt(II) and three different types of cis-diammine-bis(histidine). HCl trans cleavage of compounds with bidentate His ligands leads to a mixture of two compounds having His ligated to Pt by an amino or imin group. The methods applied are suitable for analyzing reactions of His with cis-DDP under model conditions similar to physiological conditions.     

Reaction of cis- and trans-[PtCl2(NH3)2] with reduced glutathione inside human red blood cells, studied by 1H and 15N-[1H] DEPT NMR

Reactions of cis- and trans-[PtCl2(NH3)2] with glutathione (GSH) inside intact red blood cells have been studied by 1H spin-echo nuclear magnetic resonance (NMR). Upon addition of trans-[PtCl2(NH3)2] to a suspension of red cells, there was a gradual decrease in the intensity of the resonances for free GSH, and new peaks were observed that were assignable to coordinated GSH protons in trans-[Pt(SG)Cl(NH3)2], trans-[Pt(SG)2(NH3)2], and possibly the S-bridged complex trans-[[NH3)2PtCl)2SG]+. Formation of trans-[Pt(SG)2(NH3)2] inside the cell was confirmed from the 1H NMR spectrum of hemolyzed cells, which were ultrafiltered to remove large protein molecules; the ABM multiplet of the coordinated GSH cys-beta CH2 protons was resolved using selective-decoupling experiments. Seventy percent of the total intracellular GSH was retained by the ultrafiltration membrane, suggesting that the mixed complex trans-[Pt(SG)(S-hemoglobin)(NH3)2] also is a major metabolite of trans-[PtCl2(NH3)2] inside red cells. The reaction of cis-[PtCl2(NH3)2] with intracellular GSH was slower; only 35% of the GSH had been complexed after a 4-hr incubation compared to 70% for the trans isomer. There was a gradual decrease in the intensity of the GSH 1H spin-echo NMR resonances, but no new peaks were resolved. This was interpreted as formation of high-molecular weight Pt:GSH and mixed GS-Pt-S(hemoglobin) polymers. By using a 15N-[1H] DEPT pulse sequence, we were able to study the reaction of cis-[PtCl2(15NH3)2] with red cells at concentrations as low as 1 mM. 15NH3 ligands were released, and no resonances assignable to Pt-15NH3 species were observed after a 12-hr incubation.     

Interaction of platinum compounds with short oligodeoxynucleotides containing guanine and cytosine

The products resulting from reaction of cis-Pt(NH3)2Cl2 with d(CpCpGpG), d(GpCpG), d(pCpGpCpG), d(pGpCpGpC) and d(CpGpCpG) and from reaction of [Pt(dien)Cl]Cl with d(CpCpGpG) and d(GpCpG) have been characterized with the aid of proton NMR spectroscopy, circular dichroic spectroscopy and Pt analysis. The binding sites of the Pt compounds were determined by pH-dependent NMR spectroscopy. Binding of the two Pt compounds invariably occurs at the guanine N7 atoms. In all compounds containing [cis-Pt(NH3)2]2+ chelates are formed by coordination of platinum to two guanines of the same oligonucleotide. The resulting intrastrand-cross-linked oligonucleotides contain either d(GpG) . cisPt units, or d(GpCpG) . cisPt units. In the latter case the middle cytosine is not coordinated to platinum. As a result the conformational changes originating from these two chelates are different from each other. In the case of [Pt(dien)Cl]Cl as a starting product, two types of oligonucleotide adducts are formed, i.e. those with one Pt atom/molecule and those with two Pt atoms/molecule. The NMR spectra of the adducts containing only one Pt(dien)2+ show that only one adduct is formed, although two guanine bases are present. This indicates a preference for one of the N7 atoms in the molecule.     

5-Fluorouracil-cisplatin adducts with potential antitumor activity

Using 5-fluorouracil (5-FU) and cis-diamminedichloroplatinum(II) (cisplatin, CDDP) as starting compounds, 5-FU-cisplatin adducts cis-[Pt(NH(3))(2)(HFU)Cl] (1) and cis-[Pt(NH(3))(2)(HFU)(2)] (2) were prepared. The obtained complexes were characterized by IR, ES-MS and 1H NMR spectroscopy. Complex 1 reacted with guanosine-5'-monophosphate (5'-GMP) and gave rise to a stable mixed-ligand complex cis-[Pt(NH(3))(2)(HFU)(GMP)] (3), whereas 2 did not undergo a similar reaction. In vitro cell growth inhibition tests of complexes 1 and 2 exhibited moderate antitumor activities against the melanoma B16-BL6 cell line. This work provides the basis for a potential alternative for the combinational use of 5-FU and CDDP in cancer therapy.     

DNA-platinum interactions in vitro with trans- and cis-Pt(NH3)2Cl2.

In the present study the nature and the hydrolysis of DNA-Pt complexes with the platinum compounds, [Pt(dien)Cl]Cl, trans- and cis-Pt(NH3)2Cl2, using potentiometric chloride determinations, have been investigated. The trans-Pt(NH3)2Cl2 and the [Pt(dien)Cl]Cl react with the GC planes at the N7(G) sites, while the cis-Pt(NH3)2Cl2 compound reacts with the GC planes and forms a chelate by using the N7(G) and O6(G) sites. The complex is a specific 1:1 Pt:DNA adduct. The platinum atom in cis-Pt(NH3)2Cl2 liberates both chlorine atoms on chelation. A mechanism for the in vivo antitumor activity of the cis-Pt(NH3)2Cl2 is proposed and the structure activity relationship is discussed.     

A study of interactions of platinum (II) compounds with DNA by means of CD spectra of solutions and liquid crystalline microphases of DNA

The optical properties of the DNA complexes with divalent platinum compounds of the cis-diamine type differing both in the nature of anionic and neutral ligands and in the spatial arrangement about the platinum atom were studied. The platinum compounds cis-[Pt(NH3)2Cl2], [Pt(en)Cl2], [Pt(tetrameen)Cl2], cis-[Pt(NH3)2NO2Cl], and cis-[PtNH3(Bz)Cl2] at small values of r (r is the molar ratio of a platinum compound to DNA nucleotides in the reaction mixture) were found to induce an increase in the amplitude of the positive band in the circular dichroic (CD) spectrum of linear DNA. All the compounds listed except cis-[Pt(NH3)2NO2Cl] caused a sharp decrease of the amplitude of the negative band in the CD spectrum of a liquid crystalline microphase of DNA formed in solution in the presence of poly(ethylene glycol). All these platinum compounds (except [Pt(tetrameen)Cl2]) exhibit biological (antimitotic, antitumour, etc.) activity. The platinum compounds trans-[Pt(NH3)Cl2], trans-[Pt(NH3)2NO2Cl], cis-[PtNH3PyCl2], cis-[Pt(NH3)2(NO2)2], and [Pt(NH3)3Cl]Cl exhibiting a low (if any) biological activity, either induced a decrease of the amplitude of the positive band in the CD spectrum of linear DNA, or did not affect the CD spectrum at all. The effect of these platinum compounds on the CD spectrum of the liquid crystalline microphase of DNA was either weak or absent. It is assumed that the specific biological action of platinum compounds of the cis-diamine type is determined by the polydentate binding to DNA: in addition to the cis-bidentate covalent binding of platinum to DNA nitrogen bases, a hydrogen bond formation between the DNA and cis-amino ligands occurs by means of protons at nitrogen atoms.     

Similar binding of the carcinostatic drugs cis-[Pt(NH3)2Cl2] and [Ru(NH3)5Cl] Cl2 to tRNAphe and a comparison with the binding of the inactive trans-[Pt(NH3)2Cl2] complex - reluctance in binding to Watson-Crick base pairs within double helix.

A comparative study of the binding of square planar cis- and trans-[Pt(NH3)2Cl2] complexes and the octahedral [Ru(NH3)5(H2O)]3+ complex to tRNAphe from yeast was carried out by X-ray crystallography. Both of the carcinostatic compounds, cis-[Pt(NH3)2Cl2] and [Ru(NH3)5(H2O)]3+ show similarities in their mode of binding to tRNA. These complexes bind specifically to the N(7) positions of guanines G15 and G18 in the dihydrouridine loop. [Ru(NH3)5(H2O)]3+ has an additional binding site at N(7) of residue G1 after extensive soaking times (58 days). A noncovalent binding site for ruthenium is also observed in the deep groove of the acceptor stem helix with shorter (25 days) soaking time. The major binding site for the inactive trans-[Pt(NH3)Cl2] complex is at the N(1) position of residue A73, with minor trans-Pt binding sites at the N(7) positions of residues Gm34, G18 and G43. The similarities in the binding modes of cis-[Pt(NH3)2Cl2] and [Ru(NH3)5(H2O)]3+ are expected to be related to their carcinostatic properties.     

Guanine-06 methylation reduces the reactivity of d(GpG) towards platinum complexes.

6-methylated guanine dinucleotides were used to study the influence of hydrogen bonding on the specific binding of the antitumor drug cDDP, cis-PtCl2(NH3)2, to DNA. In this interaction, the guanine-06 site appears to be important in explaining the preference for a pGpG-N7(1),N7(2) chelate, which results from H-bridge formation with the ammine ligand of cDDP. Guanine-06 methylated dinucleotides and the nonmodified dinucleotides were reacted with [Pt(dien)Cl]+, cis-PtCl2(NH3)2, and cis-[Pt(NH3)2(H2O)2]2+ and the reaction products were characterized by 1H NMR using pH titrations. Methylation at guanine-06 clearly reduces the preference for the guanine. In competition experiments monitored by NMR and experiments using UV spectrophotometry a decreasing reactivity towards [Pt(dien)(H2O)]2+ and cis-[Pt(NH3)2(H2O)2]2+ was found, in the order of d(GpG) greater than d(GomepG) greater than d(GpGome) greater than d(GomepGome). The difference in reactivity between 5' guanine methylation and 3' guanine methylation is ascribed to differences in the H-bond formation with the backbone phosphate. The resulting reduced stacking of the bases in both modified dinucleotides, compared to the bases in d(GpG), results in a preference for the 3' guanine over 5'.     

Optical properties of DNA complexes with antitumor compounds of bivalent platinum

The optical properties of the DNA complexes with the compounds of bivalent platinum were studied. The compounds differed by the nature of the anionic and neutral ligands and their spatial arrangement about the platinum atom. It was shown that the same as cis-[Pt (NH3)2Cl2] the platinum compounds with the biological activity, i.e. [Pt (en) Cl2], cis-[PtNH3 (Bz) Cl2] and cis-[Pt (NH3)2NO2Cl] induced at low values of r (a ratio of the number of the platinum moles added to the number of the DNA nucleotide moles in the solution) an increase in the amplitude of the positive band in the spectrum of the circular dichroism (CD) of the linear DNA and a marked decrease in the amplitude of the negative band in the spectrum of the CD of the liquid crystalline microphase of DNA formed in the presence of polyethyleneglycol. By the character of the action on the CD spectrum of the linear and condensed DNA [Pt (tetrameen)Cl2] which had no selective antimitotic effect might be referred to the above platinum compounds. Trans-[Pt (NH3)2NO2Cl], [PtNH3PyCl2], cis-[Pt (NH3)2(NO2)2] and [Pt (NH3)3Cl]Cl having no biological activity either induced only a decrease in the amplitude of the positive band in the CD spectrum of the linear DNA or had no effect on the CD spectrum. The effect of these compounds on the CD spectrum of the liquid crystalline microphase of DNA was slightly pronounced or not observed.     

A circular dichroism study of DNA.platinum complexes. Differentiation between monofunctional, cis-bidentate and trans-bidentate platinum fixation on a series of DNAs

The circular dichroism (CD) spectra of a series of DNA . platinum complexes are presented. The following platinum compounds, [Pt(dien)Cl]Cl, cis-Pt(NH3)2Cl2, cis-Pt(en)Cl2, trans-Pt-(NH3)2Cl2, K[Pt(NH3)Cl3] and K2[PtCl4] were complexed with the DNA extracted from bacteria Micrococcus lysodeikticus (72% dG + dC), Escherichia coli (50% dG + dC), Clostridium perfringens (32% dG + dC) and salmon sperm (41% dG + dC). Strong differences were found between the different DNA . Pt complexes. Three types of spectra clearly demonstrate the different platinum binding modes on DNA. In the first type, the platinum compound, i.e. [Pt(dien)Cl]Cl, is fixed to DNA with only one bond (monofunctional complex formation) and no significant change of the CD positive band of DNA is found. The main feature of the second type is a continuous intensity decrease of the positive band as observed for trans-Pt(NH3)2Cl2 (trans-bidentate complex formation). The third type concerns the cis-bidentate platinum fixation obtained with cis-Pt(NH3)2Cl2, cis-Pt(en)Cl2, K[Pt(NH3)Cl3] and K2[PtCl4]. The CD spectra are in this case characterized by an increase in the positive Cotton effect which is dG + dC-dependent up to an rb value around 0.10 (where rb = number of platinum atoms bound per nucleotide), followed by a decrease until DNA saturation with platinum is reached. A linear decrease in the amplitude of the negative band is detected in all the complexes except in the case of the monofunctional DNA . Pt complexes. For the cis-bidentate and trans-bidentate platinum fixation, a continuous bathochromic shift occurs.     

The influence of methionine-containing peptides on the reaction of carboplatin with 5'-guanosine monophosphate: a comparison with cisplatin

The pH- and time-dependent reaction of the anticancer drug carboplatin, [Pt(cbdca-kappa(2)O,O')(NH(3))(2)] (cbdca=cyclobutane-1,1-dicarboxylate), with the tripeptides H-glyglymet-OH (glycylglycyl-L-methionine) and Ac-glyglymet-OH at 313 K was investigated by high-performance liquid chromatography, NMR and mass spectrometry. The relative stability of the initial ring-opened kappaS complex [Pt(cbdca-kappaO)(Ac-glyglymet-OH-kappaS)(NH(3))(2)] leads to increased formation of the kinetically favoured kappaS:kappaS' bis-adduct [Pt(Ac-glyglymet-OH-kappaS)(2)(NH(3))(2)](2+) in comparison with cisplatin. As a result a second 1:2 reaction pathway kappaS-->kappaS:kappaS'-->kappa(2)N(M), S:kappaS'-->kappa(3)N(G2),N(M), S:kappaS', where N(M) and N(G2) represent, respectively, metallated methionine and glycine nitrogen atoms, competes with the 1:1 route kappaS-->kappa(2)N(M), S-->kappa(3)N(G2),N(M), S also observed for cisplatin. Cleavage of N-acetylglycine at the backbone C(O)-N bond to the second gly residue (G2) is observed after 100 h for the respective tridentate complexes [Pt(Ac-glyglyH(-1)metH(-1)-OH-kappa(3)N(G2),N(M), S) (Ac-glyglymet-OH-kappaS)] and [Pt(Ac-glyglyH(-1)metH(-1)-OH-kappa(3)N(G2),N(M), S)(NH(3))] at pH <5.2. The longevity of the initial kappaS complex leads to about an eight-fold increase in the rate of formation of the kappaN7:kappaN7' bis-adduct [Pt(5'-GMP-kappaN7)(2)(NH(3))(2)](2-) for the reaction of carboplatin with 5'-GMP(2-) at pH 7 in the presence of Ac-glyglymet-OH. A mixed-ligand kappaS:kappaN7 species [Pt(5'-GMP-kappaN7)(Ac-glyglymet-OH-kappaS)(NH(3))(2)] provides the major precursor for this 1:2 nucleotide complex and kappaN7 coordination of 5'-GMP(2-) is also observed in the kappa(2)N(M),S:kappaN7 complex [Pt(5'-GMP-kappaN7)(Ac-glyglymetH(-1)-OH-kappa(2)N(M),S)(NH(3))(2)](-) formed by substitution of the ammine ligand trans to the methionine sulphur. As the intermediate kappaS:kappaN7 species is formed rapidly within the first 10 h of reaction, these results suggest that the transfer reaction pathway kappaS-->kappaS:kappaN7-->kappaN7:kappaN7' involving kappaS platinated peptides could play an important role in accelerating the rate of DNA binding for carboplatin.     

Mechanism of the reversal reaction of platinated DNA with thiourea studied by platinated 5'-GMP

The reversal reactions of cis-[Pt(NH3)2(5'-GMP)2] 2-(1) and trans-[Pt(NH3)2(5'-GMP)2] 2-(2) with thiourea were examined by reversed phase HPLC and monothioureido intermediate cis-[Pt(NH3)2(5'-GMP) (tu)] (4) was detected. This result suggested that Pt-[5'-GMP-N(7)] bond was more labile than Pt-NH3 bond and the release of ammonia from cis-Pt(II)-DNA base complexes is a result of trans-labilizing effect of sulfur containing molecule displaced with DNA base.     

Identification and characterization of a novel linkage isomerization in the reaction of trans-diamminedichloroplatinum(II) with 5'-d(TCTACGCGTTCT)

The oligonucleotide 5'-d(TCTACGCGTTCT) reacts with trans-diamminedichloroplatinum(II) to yield primarily trans-[Pt(NH3)2[d(TCTACGCGTTCT)-N7-G(6),N7-G(8)]], containing the desired trans-[Pt(NH3)2[d(GCG)]] 1,3-cross-link. A key element of the platination reaction is the use of low pH to suppress coordination at A(4). The product was fully characterized by pH-dependent NMR titrations, enzymatic degradation analysis, and 195Pt NMR spectroscopy. Interestingly, the 1,3-cross-linked adduct is unstable at neutral pH, rearranging unexpectedly to form the linkage isomer trans-[Pt(NH3)2[d-(TCTACGCGTTCT)-N3-C(5),N7-G(8)]]. This rearrangement product is more stable than the initially formed isomer and could be characterized by pH-dependent NMR titrations, enzymatic degradation analysis, liquid secondary ion mass spectrometric analysis of an enzymatically digested fragment, 195Pt NMR spectroscopy, and modified Maxam-Gilbert footprinting experiments. By contrast, the 1,3-intrastrand cross-linked isomer rearranges during the course of both pH titration and enzymatic degradation experiments to form the 1,4-adduct. The equilibrium constant for this rearrangement is approximately 3, favoring the 1,4-adduct. Kinetic studies of the linkage isomerization reaction reveal t1/2 values for the first-order disappearance of the 1,3-intrastrand cross-linked isomer ranging from 129 (at 30 degrees C) to 3.6 h (at 62 degrees C), with activation parameters delta H not equal to = 91 +/- 2 kJ/mol and delta S not equal to = -58 +/- 8 J/(mol.K). Mechanistic implications of these kinetic results as well as the general relevance of this linkage isomerization reaction to platinum-DNA chemistry are briefly discussed.     

Characterization of a DNA damage-recognition protein from mammalian cells that binds specifically to intrastrand d(GpG) and d(ApG) DNA adducts of the anticancer drug cisplatin

A factor has been identified in extracts from human HeLa and hamster V79 cells that retards the electrophoretic mobility of several DNA restriction fragments modified with the antitumor drug cis-diamminedichloroplatinum(II) (cisplatin). Binding of the factor to cisplatin-modified DNA was sensitive to pretreatment with proteinase K, establishing that the factor is a protein. Gel mobility shifts were observed with probes containing as few as seven Pt atoms per kilobase of duplex DNA. By competition experiments the dissociation constant, Kd, of the protein from cisplatin-modified DNA was estimated to be (1-20) X 10(-10) M. Protein binding is selective for DNA modified with cisplatin, [Pt(en)Cl2] (en, ethylenediamine), and [Pt(dach)Cl2] (dach, 1,2-diaminocyclohexane) but not with chemotherapeutically inactive trans-diamminedichloroplatinum(II) or monofunctionally coordinating [Pt(dien)Cl]Cl (dien, diethylenetriamine) complexes. The protein also does not bind to DNA containing UV-induced photoproducts. The protein binds specifically to 1,2-intrastrand d(GpG) and d(ApG) cross-links formed by cisplatin, as determined by gel mobility shifts with synthetic 110-bp duplex oligonucleotides; these modified oligomers contained five equally spaced adducts of either cis-[Pt(NH3)2d(GpG) or cis-[Pt(NH3)2d(ApG)]. Oligonucleotides containing the specific adducts cis-[Pt(NH3)2d(GpTpG)], trans-[Pt(NH3)2d(GpTpG)], or cis-[Pt(NH3)2(N3-cytosine)d(G)] were not recognized by the protein. The apparent molecular weight of the protein is 91,000, as determined by sucrose gradient centrifugation of a preparation partially purified by ammonium sulfate fractionation. Binding of the protein to platinum-modified DNA does not require cofactors but is sensitive to treatment with 5 mM MnCl2, CdCl2, CoCl2, or ZnCl2 and with 1 mM HgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reactions of cisplatin hydrolytes with methionine, cysteine, and plasma ultrafiltrate studied by a combination of HPLC and NMR techniques

The reactions of cis-[PtCl(NH3)2(H2O)]+ with L-methionine have been studied by 1D 195Pt and 15N NMR, and by 2D[1H, 15N] NMR. When the platinum complex is in excess, the initial product, cis-[PtCl(NH3)2(Hmet-S)]+ undergoes slow ring closure to [Pt(NH3)2(Hmet-N,S)]2+. Slow ammine loss then occurs to give the isomer of [PtCl(NH3)(Hmet-N,S)]+ with chloride trans to sulfur. When methionine is in excess, a reaction sequence is proposed in which trans-[PtCl(NH3)(Hmet-S)2]+ isomerises to the cis-isomer, with subsequent ring closure reactions leading to cis-[Pt(Hmet-N,S)2]2+. Near pH 7, methionine is unreactive toward cis-[PtCl(OH)(NH3)2]. By contrast, L-cysteine reacts readily with cis-[PtCl(OH)(NH3)2] at pH 7, but there were many reaction products, including bridged species. Cis-[PtCl(OH)(NH3)2] reacts with reduced thiols in ultrafiltered plasma but these are oxidized if the plasma is not fresh or appropriately stored. With very low concentrations of the platinum complexes (35.5 microM), HPLC experiments (UV detection at 305 nm) indicate that the thiolate (probably cysteine) reactions become simpler as bridging becomes less important.     

Conformational analysis of the adduct cis-[Pt(NH3)2 d(GpG)]+ in aqueous solution. A high field (500-300 MHz) nuclear magnetic resonance investigation.

A 500, 400 and 300 MHz proton NMR study of the reaction product of cis-Pt(NH3)2Cl2 or cis-[Pt(NH3)2 (H2O)2] (NO3)2 with the deoxydinucleotide d(GpG): cis-[Pt(NH3)2 d(GpG)] was carried out. Complete assignment of the proton resonances by decoupling experiments and computer simulation of the high field part of the spectrum yield proton-proton and proton-phosphorus coupling constants of high precision. Analysis of these coupling constants reveal a 100% N (C3'-endo) conformation for the deoxyribose ring at the 5'-terminal part of the chelated d(GpG) moiety. In contrast, the 3'-terminal -pG part of the molecule displays the normal behaviour for deoxyriboses: the sugar ring prefers to adopt an S (C2'-endo) conformation (about 70%). Extrapolating from this model compound, it is suggested that Pt chelation by a -dGpdG- sequence of DNA would require a S to N conformational change of one deoxyribose moiety as the main conformational alteration and lead to a kink in one strand of the double-helical structure of DNA.     

Platinum complexes of nucleotides.

The reaction of [Pt(dien)Cl1Cl (dien = NH2CH2CH2NHCH2CH2NH2) with nucleotides has been studied by nuclear magnetic resonance. It has been found that the CMP (cytidine 5'-monophosp-ate) and GMP (guanosine 5'-monophosphate/coordinate to the platinum atom through N3 and N7, respectively. The reaction of the platinum salt with the nucleotide is complete when one to one ratio of platinum to nucleotide is used and no evidence of phosphate group binding to platinum has been found. No additional binding sites have been detected except the N7 site on the guanylic group of GMP even in the presence of a large excess of [Pt(dien) Cl1Cl. The AMP (adenosine 5'monophosphate] coordinates to the platinum at the N1 and/or N7 sites. The reaction of AMP and platinum is complete is complete at a ratio of four platinum to one AMP.     

Preparation, 195Pt NMR spectra and biological activity of platinum (IV) complexes with dipeptides

Three dipeptide complexes of the form K[Pt(IV) (dipep) Cl(OH)2] and four dipeptide complexes of the form K[Pt(IV)-(Hdipep)Cl2(OH)2] were newly prepared. The 195 Pt NMR peak of the K[Pt(IV) (dipep)Cl(OH)2] complexes appeared at about 1200 ppm and these chemical shifts were about 3150 ppm downfield compared with those of the K[Pt(II) (dipep) Cl] complexes. The chemical shifts of the K[Pt(IV) (Hdipep) Cl2 (OH)2] complexes were at about 900 ppm, i.e., about 3050 ppm downfield compared with those of the K[Pt(II) (Hdipep)Cl] complexes. The H[Pt(IV) (Hdigly) Cl2(OH)2] and K[Pt(IV) (Hdigly) Cl2(OH)2] complexes inhibited the growth of C. albicans at a more diluted concentration than cisplatin at 1 microgram/ml, but the platinum complexes only weakly inhibited the growth of these cells compared with the cisplatin-inhibited growth of Meth-A and Hep-2 cells at 10 micrograms/ml. These results suggested that the platinum complexes selectively inhibited the growth of fungal cells.     

Mono- and polynuclear complexes of Pt(II) with polypyridyl ligands. Synthesis, spectroscopic and structural characterization and cytotoxic activity

An array of poly- and mononuclear complexes of Pt(II) with polypyridyl ligands is reported. The framework complexes [(PtCl(2))(2)(bpp)(2)(micro-PtCl(2))](H(2)O)(2) [bpp=2,3-bis(2-pyridyl)pyrazine], [PtCl(2)(micro-tptz)PtClNCPh]Cl [tptz=2,4,6-tris(2-pyridyl)-1,3,5-triazine], and mononuclear PtCl(2)(NH(2)dpt) [NH(2)dpt=4-amino-3,5-bis(2-pyridyl)-1,2,4-triazole] have been prepared and structurally characterized. Both neutral and ionic complexes are present, with bifunctional and monofunctional Pt(II) moieties, whose size and shape enable them to behave as novel scaffolds for DNA binding. Pt(II) complexes were tested for their biological activity. Cell viability assay and flow cytometric analysis demonstrated that these complexes, particularly [PtCl(2)(micro-tptz)PtClNCPh]Cl, were effective death inducers in human colon rectal carcinoma HT29 cells and their cytotoxic activity was higher than that exerted by cisplatin. Morphological analysis of treated HT29 cells, performed by fluorescence microscopy after Hoechst 33258 staining, showed the appearance of the typical features of apoptosis. Moreover, our results suggested that mitochondria are involved in apoptosis induced by Pt(II) complexes in HT29 cells as demonstrated by dissipation of mitochondrial transmembrane potential.