Synthesis and characterization of trans-[Pt(NH3)2Cl2] adducts of d(CCTCGAGTCTCC).d(GGAGACTCGAGG)

The reaction of trans-diamminedichloroplatinum(II) (trans-DDP), the inactive isomer of the anticancer drug cisplatin, with the single-stranded deoxydodecanucleotide d(CCTCGAGTCTCC) in aqueous solution at 37 degrees C was monitored by reversed-phase HPLC. Consumption of the dodecamer follows pseudo-first-order reaction kinetics with a rate constant of 1.25 (4) x 10(-4) s-1. Two intermediates, shown to be monofunctional adducts in which Pt is coordinated to the guanine N7 positions, were trapped with NH4(HCO3) and identified by enzymatic degradation analysis. These monofunctional adducts and a third, less abundant, one are rapidly removed from the DNA by thiourea under mild conditions. When allowed to react further, the monofunctional intermediates formed a single main product that was characterized by 1H NMR spectroscopy and enzymatic digestion as the bifunctional 1,3-intrastrand cross-link trans-[Pt(NH3)2[d(CCTCGAGTCTCC)-N7-G(5),N7-G(7]]). Binding of the trans-[Pt(NH3)2]2+ moiety to the guanosine N7 positions decreases the pKa at N1 and leads to destacking of the intervening A(6) base. The double-stranded trans-DDP-modified and unmodified DNAs were obtained by annealing the complementary strand to the corresponding single strands and then studied by 31P and 1H NMR and UV spectroscopy. trans-DDP binding does not induce large changes in the O-P-O bond or torsional angles of the phosphodiester linkages in the duplex, nor does it significantly alter the UV melting temperature. trans-DDP binding does, however, cause the imino protons of the platinated duplex to exchange rapidly with solvent by 50 degrees C, a phenomenon that occurs at 65 degrees C for the unmodified duplex. A structural model for the platinated double-stranded oligonucleotide was generated through molecular dynamics calculations. This model reveals that the trans-DDP bifunctional adduct can be accommodated within the double helix with minimal distortion of the O-P-O angles and only local disruption of base pairing and destacking of the platinated bases. The model also predicts hydrogen bond formation involving coordinated ammine ligands that bridge the two strands.     

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.     

Intrastrand cross-links are not formed in the reaction between transplatin and native DNA: relation with the clinical inefficiency of transplatin.

The reaction between trans-diamminedichloroplatinum(II) and single-stranded oligonucleotides containing the sequence d(GXG) (X being an adenine, cytosine or thymine residue) yields trans-[Pt(NH3)2[(GXG)-GN7,GN7]] intrastrand cross-links. These cross-links do not prevent the pairing of the platinated oligonucleotides with their complementary strands but they decrease the thermal stability of the duplexes. The thermal stability is not much affected by the chemical nature of the X residue and its complementary base. By gel electrophoresis, it is shown that the trans- [Pt(NH3)2[d(GTG)-GN7,GN7]] cross-link bends the DNA double helix (26 degrees) and unwinds it (45 degrees). The pairing of the platinated oligonucleotides with their complementary strands promotes the rearrangement of the 1,3-intrastrand cross-links into interstrand cross-links. At a given temperature, the nature of the X residue, its complementary base and of the base pairs adjacent to the adducts do not dramatically affect the rate of the reaction. To know whether trans-[Pt(NH3)2[d(GXG)-GN7,GN7]] cross-links do not rearrange in some sequences, the location of these adducts was searched in double-stranded DNA after reaction with trans-diamminedichloroplatinum(II) by means of the 3'-5' exonuclease activity of T4 DNA polymerase. At low level of platination, trans-[Pt(NH3)2[d(GXG)-GN7,GN7]] cross-links were not detected. Monofunctional adducts and interstrand cross-links were mainly formed. These results are discussed in relation with the clinical inefficiency of trans-diamminedichloroplatinum(II).     

Linkage isomerization reaction of intrastrand cross-links in trans-diamminedichloroplatinum(II)-modified single-stranded oligonucleotides.

The stability of trans-(Pt(NH3)2[d(CGAG)-N7-G,N7-G]) adducts, resulting from cross-links between two guanine residues at d(CGAG) sites within single-stranded oligonucleotides by trans-diamminedichloro-platinum(II), has been studied under various conditions of temperature, salt and pH. The trans-(Pt(NH3)2[d(C GAG)-N7-G,N7-G]) cross-links rearrange into trans-(Pt(NH3)2[d(CGAG)-N3-C,N7-G]) cross-links. The rate of rearrangement is independent of pH, in the range 5-9, and of the nature and concentration of the salt (NaCl or NaCIO4) in the range 10-400 mM. The reaction rate depends upon temperature, the t1/2 values for the disappearance of the (G,G) intrastrand cross-link ranging from 120 h at 30 degrees C to 70 min at 80 degrees C. The linkage isomerization reaction occurs in oligonucleotides as short as the platinated tetramer d(CGAG). Replacement of the intervening residue A by T has no major effect on the reaction. The C residue adjacent to the adduct on the 5' side plays a key-role in the reaction; its replacement by a G, A or T residue prevents the reaction occuring. No rearrangement was observed with the C residue adjacent to the adduct on the 3' side. It is proposed that the linkage isomerization reaction results from a direct attack of the base residue on the platinum(II) square complex.     

Bending studies of DNA site-specifically modified by cisplatin, trans-diamminedichloroplatinum(II) and cis-[Pt(NH3)2(N3-cytosine)Cl]+

Duplex oligonucleotides containing a single intrastrand [Pt(NH3)2]2+ cross-link or monofunctional adduct and either 15 or 22 bp in length were synthesized and chemically characterized. The platinum-modified and unmodified control DNAs were polymerized in the presence of DNA ligase and the products studied on 8% native polyacrylamide gels. The extent of DNA bending caused by the various platinum-DNA adducts was revealed by their gel mobility shifts relative to unplatinated controls. The bifunctional adducts cis-[Pt(NH3)2[d(GpG)]]+, cis-[Pt(NH3)2[d(ApG)]]+, and cis-[Pt(NH3)2[d(G*pTpG*)]], where the asterisks denote the sites of platinum binding, all bend the double helix, whereas the adduct trans-[Pt(NH3)2[d(G*pTpG*)]] imparts a degree of flexibility to the duplex. When modified by the monofunctional adduct cis-[Pt(NH3)2(N3-cytosine)(dG)]Cl the helix remains rod-like. These results reveal important structural differences in DNAs modified by the antitumor drug cisplatin and its analogs that could be important in the biological processing of the various adducts in vivo.     

Instability of the monofunctional adducts in cis-[Pt(NH3)2(N7-N-methyl-2-diazapyrenium)Cl](2+)-modified DNA: rates of cross-linking reactions in cis-platinum-modified DNA.

Single- and double-stranded oligonucleotides containing a single monofunctional cis-[Pt(NH3)2(dG)(N7-N-methyl-2-diazapyrenium)]3+ adduct have been studied at two NaCl concentrations. In 50 mM and 1 M NaCl, the adducts within the single-stranded oligonucleotides are stable. In contrast, they are unstable within the corresponding double-stranded oligonucleotides. In 50 mM NaCl, the bonds between platinum and guanine or N-methyl-2,7-diazapyrenium residues are cleaved and subsequently, intra- or interstrand cross-links are formed as in the reaction between DNA and cis-DDP. In 1 M NaCl, the main reaction is the replacement of N-methyl-2,7-diazapyrenium residues by chloride which generates double-stranded oligonucleotides containing a single monofunctional cis-[Pt(NH3)2(dG)Cl]+ adduct. The rates of closure of these monofunctional adducts to bifunctional cross-links have been studied in 60 mM NaClO4. Within d(TG.CT/AGCA), d(CG.CT/AGCG) and d(AG.CT/AGCT) (the symbol.indicates the location of the adducts in the central sequences of oligonucleotides), the half-lifes (t1/2) of the cis-[Pt(NH3)2(dG)Cl]+ adducts are respectively 12, 6 and 2.8 hr and the cross-linking reactions occur between guanine residues on the opposite strands. Within d(AG.TC/GACT), d(CG.AT/ATCG) and d(TGTG./CACA) or d(TG.TG/CACA) t1/2 are respectively 1.6, 8 and larger than 20 hr and the intrastrand cross-links are formed at the d(AG), d(GA) and d(GTG) sites, respectively. The conclusion is that the rates of conversion of cis-platinum-DNA monofunctional adducts to minor bifunctional cross-links are dependent on base sequence. The potential use of the instability of cis-[Pt(NH3)2(dG)(N7-N-methyl-2-diazapyrenium)]3+ adducts is discussed in the context of the antisense strategy.     

H8 chemical shifts in oligonucleotide cross-linked at a GpG sequence by cis-Pt(NH3)2(2+): a clue to the adduct structure.

The origin of the anomalous H8 chemical shifts observed in 1H-NMR spectra of oligonucleotides cross-linked at a GpG sequence with cis-[Pt(NH3)2]2+ has been investigated and clarified. The main contributions that distinguish the H8 resonances of the two platinum-ligating guanines from other GH8 signals and from each other are: (a) the inductive effect of platinum binding which we have recently quantified as a downfield shift of 0.48 +/- 0.07 ppm (M. H. Fouchet, D. Lemaire, J. Kozelka and J.-C. Chottard, unpublished results); (b) the ring-current effect of one GpG guanine on the H8 resonance of the other guanine, which is negative (shielding) for the 5'-H8 and positive (deshielding) for the 3'-H8 in single-stranded adducts, but has the opposite sign in double-stranded adducts; (c) a deshielding polarization effect of the phosphate 5' to the GpG unit. The different signs of the ring-current effects in single-stranded and double-stranded oligonucleotides originate from the orientation of the guanines in the cis-[Pt(NH3)2(Gua)2]2+ moiety (Gua, guanine), which is left-handed helicoidal in single strands and right-handed helicoidal in double strands. In the platinated dinucleotides (cis-[Pt(NH3)2(GpG)]+, cis-[Pt(NH3)2(d(GpG))]+ and cis-[Pt(NH3)2(d(GpG))]), the guanines assume either the left-handed or the right-handed arrangement, depending on the sugar moiety (ribose or deoxyribose), protonation state at N1 and, in the solid state, on crystal forces. This work shows that chemical shifts contain valuable structural information which is complementary to that extracted from correlated spectroscopy and nuclear Overhauser spectroscopy data.     

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.     

Model platinum nucleobase and nucleoside complexes and antitumor activity: X-ray crystal structure of [PtIV(trans-1R,2R-diaminocyclohexane)trans-(acetate)2(9-ethylguanine)Cl]NO3.H2O

A series of platinum(II) and (IV) monoadducts of the type [Pt(II)(DACH)LCl]NO3 and [Pt(IV)(DACH)trans-(X)2LCl]NO3 (where DACH=trans-1R,2R-diaminocyclohexane, L=adenine, guanine, hypoxanthine, cytosine, adenosine, guanosine, inosine, cytidine, 9-ethylguanine (9-EtGua), or 1-methylcytosine and X=hydroxo or acetato ligand) have been synthesized and characterized by elemental analysis and by 1H and 195Pt nuclear magnetic resonance (NMR) spectroscopy. The crystal structure of the model nucleobase complex [Pt(IV)(trans-1R,2R-diaminocyclohexane)trans-(acetate)2(9-EtGua)Cl]NO3.H2O was determined using a single crystal X-ray diffraction method. The compound crystallized in the monoclinic space group P2(1), with a=10.446(2) A, b=22.906(5) A, c=10.978(2) A, Z=4, and R=0.0718, based upon the total of 11,724 collected reflections. In this complex, platinum had a slightly distorted octahedron geometry owing to the presence of a geometrically strained five-member ring. The two adjacent corners of the platinum plane were occupied by the two amino nitrogen of DACH, whereas, the other two equatorial positions occupied by chloride ion and 9-ethylguanine. The remaining two axial positions were occupied by the oxygen atoms of acetato ligands. The DACH ring was in a chair configuration. An intricate network of intermolecular hydrogen bonds held the crystal lattice together. Some of these synthesized models of DACH-Pt-DNA adducts have good in vitro cytotoxic activity against the cisplatin-sensitive human cancer ovarian A2780 cell line (IC50=1-8 microM). Interestingly, a substituted nucleobase (9-ethylguanine) adduct was over 6-fold more potent than regular adducts. The cross-resistance factor against the 44-fold cisplatin-resistant 2780CP/clone 16 cells was about 3-9; thus, the cytotoxicity of adducts was indicative of low potency, but the resistance factors were also substantially low. These results suggest that DNA adducts of DACH-Pt are cytotoxic with low cross-resistance.     

Replication inhibition and translesion synthesis on templates containing site-specifically placed cis-diamminedichloroplatinum(II) DNA adducts.

A series of site-specifically plantinated, covalently closed circular M13 genomes (7250 bp) was constructed in order to evaluate the consequences of DNA template damage induced by the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP). Here are reported the synthesis and characterization of genomes containing the intrastrand cross-linked adducts cis-[Pt(NH3)2[d(ApG)-N7(1),-N7(2)]], cis-[Pt-(NH3)2[d(GpCpG)-N7(1),-N7(3)]], and trans-[Pt(NH3)2[d(CpGpCpG)-N3(1),-N7(4)]]. These constructs, as well as the previously reported M13 genome containing a site-specifically placed cis-[Pt(NH3)2[d-(GpG)-N7(1),-N7(2)]] adduct, were used to study replication in vitro. DNA synthesis was initiated from a position approximately 177 nucleotides 3' to the individual adducts, and was terminated either by the adducts or by the end of the template, located approximately 25 nucleotides on the 5' side of the adducts. Analysis of the products of these reactions by gel electrophoresis revealed that, on average, bypass of the cis-DDP adducts occurred approximately 10% of the time and that the cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]] intrastrand cross-link is the most inhibitory lesion. The cis-[Pt(NH3)2[(GpCpG)-N7(1),-N7(3)]] adduct allowed a higher frequency of such translesion synthesis (ca. 25%) for two of the polymerases studied, modified bacteriophage T7 polymerase and Escherichia coli DNA polymerase I (Klenow fragment). These enzymes have either low (Klenow) or no (T7) associated 3' to 5' exonuclease activity. Bacteriophage T4 DNA polymerase, which has a very active 3' to 5' exonuclease, was the most strongly inhibited by all three types of cis-DDP adducts, permitting only 2% translesion synthesis. This enzyme is therefore recommended for replication mapping studies to detect the location of cis-DDP-DNA adducts in a heterologous population. The major replicative enzyme of E. coli, the DNA polymerase III holoenzyme, allowed less than 10% adduct bypass. Postreplication restriction enzyme cleavage studies established that the templates upon which translesion synthesis was observed contained platinum adducts, ruling out the possibility that the observed products were due to a small amount of contamination with unplatinated DNA. The effects on in vitro replication of a recently characterized adduct of trans-DDP [Comess, K. M., Costello, C. E., & Lippard, S. J. (1990) Biochemistry 29, 2102-2110] were also evaluated. This adduct provided a poor block both to DNA polymerases and to restriction enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

A 1H and 31P NMR study of cis-Pt(NH3)2[d(CpGpG)-N7(2),N7(3)]. The influence of a 5'-terminal cytosine, on the structure of the cis-Pt(NH3)2[d(GpG)-N7,N7] intrastrand cross-link

Proton NMR studies at 300 MHz and 500 MHz have been carried out on the trinucleoside bisphosphate d(CpGpG) and on cis-Pt(NH3)2[d(CpGpG)-N7(2),N7(3)] [abbreviated as d(CpGpGp) . cisPt]. For the Pt adduct, 13C and 31P NMR was also used for characterizing the oligonucleotide. d(CpGpG) appears to revert to a B-DNA-type single helix at lower temperatures. The relatively small concentration dependence of the proton chemical shifts, in comparison with shifts due to intramolecular stacking effects, indicates that the compound is essentially single-stranded. In d(CpGpGp) . cisPt, the first nucleoside, C(1), stacks well on top of the second, G(2), despite the N conformation of the G(2) sugar ring. The platinated GpG part in this trimer adopts largely the same structure as in cis-Pt(NH3)2[d(GpGpG)-N7(1),N7(2)] [den Hartog, J. H. J., et al. (1982) Nucleic Acids Res. 10, 4715-4730]. Main differences however, are changes in H8 chemical shifts and a 0.6-ppm downfield shift of the third nucleotide phosphorus, P(3), in d(CpGpGp) . cisPt with respect to P(2) in d(GpG) . cisPt. The latter shift change is likely to be induced by a structural alteration, caused by stacking of C(1) on top of G(2). Also, the large chemical shift differences between the two H8 protons in d(NpGpG) . cisPt fragments is discussed; the deviation from a mirror symmetry of the two guanine bases seems to be the main origin of this effect. The chemical shift changes, observed in the proton and phosphorus NMR chemical shift temperature and chemical shift pH profiles have been explained in terms of stack-destack equilibria changes.     

Solution structural study of a DNA duplex containing the guanine-N7 adduct formed by a cytotoxic platinum-acridine hybrid agent

[PtCl(en)(ACRAMTU-S)](NO(3))(2) (PT-ACRAMTU; en = ethane-1,2-diamine, ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea) is a dual metalating/intercalating DNA binding drug conjugate that shows cytotoxicity at micromolar to nanomolar concentrations in a wide range of solid tumor cell lines. In approximately 80% of its adducts, PT-ACRAMTU binds to guanine-N7 in the major groove, selectively at 5'-CG sites [Budiman, M. E. et al. (2004) Biochemistry 43, 8560-8567]. Here, we report the synthesis, physical characterization, and NMR solution structure of a site-specifically modified octamer containing this adduct, 5'-CCTCGTCC-3'/3'-GGAGCAGG-5', where the asterisk indicates the [Pt(en)ACRAMTU)](3+) fragment. The structure was determined by a combination of high-resolution 2-D NMR spectroscopy and restrained molecular dynamics/molecular mechanics (rMD/MM) calculations using 179 NOE distance restraints and refined to an r(6) weighted residual (R(x)) of 9.2 x 10(-)(2) using the complete relaxation matrix approach. An average structure was calculated from the final ensemble of 19 rMD geometries showing pairwise root-mean-square deviations of <1.05 A. The dual binding increases the thermal stability of the octamer compared to the unmodified duplex (DeltaT(m) = 13.2 degrees ). The modified sequence shows structural features reminiscent of both B- and A-type DNA. Watson-Crick hydrogen bonding is intact at and beyond the adduct site. Platinum is bound to the N7 position of G5 in the major groove, and ACRAMTU intercalates into the central 5'-C4G5/C12G13 base-pair step on the 5'-face of the platinated nucleobase. The chromophore's long axis is aligned with the long axes of the adjacent base pairs, maximizing intermolecular pi-pi stacking interactions. PT-ACRAMTU lengthens (rise, 6.62 A) and unwinds (twist, 15.4 degrees ) the duplex at the central base-pair step but does not cause helical bending. No C3'-endo deoxyribose pucker and no significant roll are observed at the site of intercalation/platination, which clearly distinguishes the PT-ACRAMTU-induced damage from the 1,2-intrastrand cross-link formed by cisplatin. Overall, the DNA perturbations produced by PT-ACRAMTU do not appear to mimic those caused by the major cisplatin lesion. Instead, intriguing structural similarities are observed for PT-ACRAMTU's monoadduct and the N7 adducts of dual major-groove alkylating/intercalating antitumor agents, such as the pluramycins.     

Intercalation of the (1R,2S,3R,4S)-N6-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct in the N-ras codon 61 sequence: DNA sequence effects

The structure of the bay region (1R,2S,3R,4S)-N6-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct at X(7) of 5'-d(CGGACAXGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, was determined by NMR. This was the bay region benz[a]anthracene RSRS (61,3) adduct. The BA moiety intercalated above the 5'-face of the modified base pair. NOE connectivities between imino protons were disrupted at T16 and T17. Large chemical shifts at the lesion site were consistent with ring current shielding arising from the BA moiety. A large chemical shift dispersion was observed for the BA aromatic protons. An increased rise of 8.17 A was observed between base pairs A6 x T17 and X7 x T(16). The PAH moiety stacked with the purine ring of A6, the 5'-neighbor nucleotide. This resulted in buckling of the 5'-neighbor A6 x T17 base pair, evidenced by exchange broadening for the T17 imino resonance. It also interrupted sequential NOE connectivities between nucleotides C5 and A6. The A6 deoxyribose ring showed an increased percentage of the C3'-endo conformation. This differed from the bay region BA RSRS (61,2) adduct, in which the lesion was located at position X6 [Li, Z., Mao, H., Kim, H.-Y., Tamura, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1999) Biochemistry 38, 2969-2981], but was similar to the benzo[a]pyrene BP SRSR (61,3) adduct [Zegar I. S., Chary, P., Jabil, R. J., Tamura, P. J., Johansen, T. N., Lloyd, R. S., Harris, C. M., Harris, T. M., and Stone, M. P. (1998) Biochemistry 37, 16516-16528]. The altered sugar pseudorotation at A6 appears to be common to both bay region BA RSRS (61,3) and BP SRSR (61,3) adducts. It could not be discerned if the C3'-endo conformation at A6 in the BA RSRS (61,3) adduct altered base pairing geometry at X7 x T16, as compared to the C2'-endo conformation. The structural studies suggest that the mutational spectrum of this adduct may be more complex than that of the BA RSRS (61,2) adduct.     

Chemical and biological studies of the major DNA adduct of cis-diamminedichloroplatinum(II), cis-[Pt(NH3)2(d(GpG]], built into a specific site in a viral genome

A duplex Escherichia coli bacteriophage M13 genome was constructed containing a single cis-[Pt(NH3)2(d(GpG]] intrastrand cross-link, the major DNA adduct of the anticancer drug cis-diamminedichloroplatinum(II). The duplex dodecamer d(AGAAGGCCTAGA).d(TCTAGGCCTTCT) was ligated into the HincII site of M13mp18 to produce an insertion mutant containing a unique StuI restriction enzyme cleavage site. A genome with a 12-base gap in the minus strand was created by hybridizing HincII-linearized M13mp18 duplex DNA with the single-stranded circular DNA of the 12-base insertion mutant. The dodecamer d(TCTAGGCCTTCT) was synthesized by the solid-phase phosphotriester method and platinated by reaction with cis-[Pt(NH3)2(H2O)2]2+ (yield 39%). Characterization by pH-dependent 1H NMR spectroscopy established that platinum binds to the N7 positions of the adjacent guanosines. The platinated oligonucleotide was phosphorylated in the presence of [gamma-32P]ATP with bacteriophage T4 polynucleotide kinase and incorporated into the 12-base gap of the heteroduplex, thus situating the adduct specifically within the StuI site in the minus strand of the genome. Approximately 80% of the gapped duplexes incorporated a dodecanucleotide in the ligation reaction. Of these, approximately half did so with the dodecanucleotide covalently joined to the genome at both 5' and 3' termini. The site of incorporation of the dodecamer was mapped to the expected 36-base region delimited by the recognition sites of XbaI and HindIII. The cis-[Pt(NH3)2(d(GpG]] cross-link completely inhibited StuI cleavage, which was fully restored following incubation of the platinated genome with cyanide to remove platinum as [Pt(CN)4]2-. Gradient denaturing gel electrophoresis of a 289-base-pair fragment encompassing the site of adduction revealed that the presence of the cis-[Pt(NH3)2(d(GpG]] cross-link induces localized weakening of the DNA double helix. In addition, double- and single-stranded genomes, in which the cis-[Pt(NH3)2(d(GpG]] cross-link resides specifically in the plus strand, were constructed. Comparative studies revealed no difference in survival between platinated and unmodified double-stranded genomes. In contrast, survival of the single-stranded platinated genome was only 10-12% that of the corresponding unmodified single-stranded genome, indicating that the solitary cis-[Pt(NH3)2(d(GpG]] cross-link is lethal to the single-stranded bacteriophage.     

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.     

Interaction of La (III) and Tb (III) ions with purine nucleotides: evidence for metal chelation (N-7-M-PO3) and the effect of macrochelate formation on the nucleotide sugar conformation.

The interaction of the La (III) and Tb (III) ions with adenosine-5'-monophosphate (5'-AMP), guanosine-5'-monophosphate (5'-GMP), and 2'-deoxyguanosine-5'-monophosphate (5'-dGMP) anions with metal/nucleotide ratios of 1 and 2 has been studied in aqueous solution in acidic and neutral pHs. The solid complexes were isolated and characterized by Fourier transform ir and 1H-nmr spectroscopy. The lanthanide (III)-nucleotide complexes are polymeric in nature both in the solid and aqueous solutions. In the metal-nucleotide complexes isolated from acidic solution, the nucleotide binding is via the phosphate group (inner sphere) and an indirect metal-N-7 interaction (outer-sphere) with the adenine N-1 site protonated. In the complexes obtained from neutral solution, metal chelation through the N-7 and the PO3(2-) group is prevailing. In aqueous solution, an equilibrium between the inner and outer sphere metal-nucleotide interaction has been observed. The ribose moiety shows C2'-endo/anti pucker in the free AMP anion and in the lanthanide (III)-AMP complexes, whereas the GMP anion with C2'-endo/anti sugar conformation exhibits a mixture of the C2'-endo/anti and C3'-endo/anti sugar puckers in the lanthanide (III)-GMP salts. The deoxyribose has O4'-endo/anti sugar pucker in the free dGMP anion and a C3'-endo/anti, in the lanthanide (III)-dGMP complexes.     

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

The trinucleotide d(CpGpT) reacts with [PtCl(dien)]Cl (dien = diethylenetriamine) to yield as a single adduct Pt(dien)[d(CpGpT)-N7(2)]. The structure of this adduct in solution has been analysed with the aid of NMR spectroscopy and compared with that of the unmodified trinucleotide. A change in the population of the S conformer of the guanosine deoxyribose ring and a syn preference of the guanine residue are the most important changes occurring upon platination. As a result the dC-dG stack disappears, whereas the dG-dT stack is hardly affected. The CD spectra of both platinated and free d(CpGpT) confirm the different nature of the two molecules.     

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.     

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)