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.     

A high melting cis-[Pt(NH3)2[d(GpG)]]adduct of a decanucleotide duplex

The [cis-Pt(NH3)2(d(GCCGGATCGC)-N7(4), N7(5))]-d(GCGATCCGGC) duplex has been prepared with Tm = 49 degrees C (vs 58 degrees C for the unplatinated form). NMR of the ten observable imino protons supports a kinked structure with intact base pairing of the duplex on the 3'-side of the d(GpG).cis-Pt chelate (relative to the platinated strand) The modification of the B-DNA type CD spectrum, due to the platinum chelate, is comparable to that observed for the platination (at a 0.05 Pt:base ratio) of the Micrococcus Lysodeikticus DNA (72% GC).     

Synthesis and 1H NMR spectroscopic characterization of trans-[Pt(NH3)2[d(ApGpGpCpCpT)-N7-A(1),N7-G(3)]]

The reaction of trans-[Pt(NH3)2Cl2] with the sodium salt of [d(ApGpGpCpCpT)]2 in aqueous solution at 37 degrees C was monitored by reversed-phase high-performance liquid chromatography and UV spectroscopy. Two intermediates, most likely monofunctional adducts, were observed, which subsequently formed one predominant single-stranded product, as well as several polymeric species proposed to be interstrand cross-linked products. The single-stranded adduct was structurally characterized by 1H NMR spectroscopy. From the pH dependence of the chemical shifts, two-dimensional homonuclear chemical shift correlation (COSY) spectroscopy, and one- and two-dimensional nuclear Overhauser effect (NOESY) experiments, the platinum(II) moiety was found to be coordinated to the N7 positions of adenine(1) and guanine(3), with the intervening guanine(2) base destacked from its neighboring residues. This intrastrand 1,3 adduct induces changes in the backbone torsion angles and causes the deoxyribose ring of adenine(1) to switch from a C2'-endo to a predominantly C3'-endo conformation. The other deoxyribose rings retain B DNA type conformations. The structure of trans-[Pt(NH3)2[d(ApGpGpCpCpT)-N7-A(1),N7-G(3)]] differs from those previously reported for cis-DDP 1,2- and 1,3-intrastrand oligonucleotide adducts but is consistent with the structures of trans-DDP 1,3-intrastrand adducts of two previously reported trinucleotides.     

cis-Platinum induced distortions in DNA. Conformational analysis of d(GpCpG) and cis-pt(NH3)2[d(GpCpG)], studied by 500-MHz NMR

Proton NMR studies at 500 MHz in aqueous solution were carried out on the G-G chelated deoxytrinucleosidediphosphate platinum complex cis-Pt(NH3)2[d(GpCpG], on the uncoordinated trinucleotide d(GpCpG) and on the constituent monomers cis-Pt(NH3)2[d(Gp)]2, cis-Pt(NH3)2[d(pG)]2, d(Gp), d(pCp) and d(pG). Complete NMR spectral assignments are given and chemical shifts and coupling constants are analysed to obtain an impression of the detailed structure of d(GpCpG) and the distortion of the structure due to chelation with [cis-Pt(NH3)2]2+. Platination of the guanosine monophosphates affects the sugar conformational equilibrium to favour the N conformation of the deoxyribose ring. This feature is also apparent in ribose mononucleotides and is possibly caused by an increased anomeric effect. In cis-Pt(NH3)2[d(pG)]2 the phase angle of pseudorotation of the S-type sugar ring is 20 degrees higher than in 'free' d(pG) which might be an indication for an ionic interaction between the positive platinum and the negatively charged phosphate. It appears that d(GpCpG) reverts from a predominantly random coil to a normal right-handed B-DNA-like single-helical structure at lower temperatures, whereas the conformational features of cis-Pt(NH3)2[d(GpCpG)] are largely temperature-independent. In the latter compound much conformational freedom along the backbone angles is seen. The cytosine protons and deoxyribose protons exhibit almost no shielding effect as should normally be exerted by the guanine bases in stacking positions. This is interpreted in terms of a 'turning away' of the cytosine residue from both chelating guanines. Conformational features of cis-Pt(NH3)2[d(GpCpG)[ are compared with the 'bulge-out' of the ribose-trinucleotide m6(2)ApUpm6(2)A.     

Interaction of cis-[Pt(NH3)2(H2O)2](NO3)2 with ribose deoxyribose diguanosine phosphates

The three diguanosine phosphates GpG (4 X 10(-4) M), d(GpG) (10(-5) M), and d(pGpG) (10(-5) M) have been reacted with cis-[Pt(NH3)2(H2O)2](NO3)2 (1 Pt/dinucleotide) in water at pH 5.5 and 37 degrees C. In each case a single product is formed. The three complexes have been characterized by proton nuclear magnetic resonance (1H NMR) and circular dichroism (CD) analyses. They are N(7)-N(7) chelates of the metal with an anti-anti configuration of the bases. They present a conformational change upon deprotonation of guanine N(1)H whose pKa is ca. 8.7 (D2O). Their CD spectra, compared to those of the free dinucleotides, exhibit an increase of ellipticity in the 275-nm region, which can be qualitatively related to the characteristic increase reported for platinated DNA and poly(dG) . poly(dC). These results are in favor of the hypothesis of intrastrand cross-linking of adjacent guanines, by the cis-PtII(NH3)2 moiety, after a local denaturation of DNA.     

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.     

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.     

Synthesis and characterization of a d(ApG) platinated nonanucleotide duplex.

The nonamer 5'd(CTCAGCCTC) 3' 1 has been reacted with cis-diamminediaquaplatinum(II) in water at pH 4.2. The major reaction product was shown by enzymatic digestion and 1H NMR to be the d(ApG)cis-Pt(NH3)2 chelate [cis-Pt(NH3)2[d(CTCAGCCTC)-N7(4),N7(5)]] 1-Pt. When mixed with its complementary strand 2, 1-Pt forms a B DNA type duplex 3-Pt with a Tm of 35 degrees C (versus 58 degrees C for the unplatinated duplex). The NMR study of the exchangeable protons of 3-Pt revealed that the helix distortion is localized on the CA*G*-CTG moiety (the asterisks indicating the platinum chelation sites) with a strong perturbation of the A*(4)T(15) base pair related to a large tilt of A*(4).     

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.     

DNA fragment conformations. A 1H-NMR conformational analysis of the d(G-G)-chelated platinum-oligonucleotide d(A-T-G-G)cisPt

The conformation of d(A-T-G-G) and d(A-T-G-G)cisPt has been investigated by 1H-NMR at 500 MHz and 90 MHz under various experimental conditions of temperature and concentration. Analysis of the coupling constants between the deoxyribose protons shows that all the sugar rings of d(A-T-G-G) adopt the S(C2'-endo) conformation most of the time. By contrast, in the platinated tetramer, d(A-T-G-G)cisPt, the N(C3'-endo) conformation is highly predominant for the internal dG residue while the S(C2'-endo) conformation is largely favoured for the other residues as in the case of the unplatinated compound. The relaxation time and nuclear Overhauser effect measurements indicate that the orientation of the two guanines of d(A-T-G-G)cisPt is anti in agreement with the previous results obtained for the dimers: r(G-G)cisPt, d(G-G)cisPt. On lowering the temperature from 80 degrees C to 20 degrees C, several proton resonances of d(A-T-G-G)cisPt exhibit large chemical shift and linewidth variations. The most spectacular temperature effect was observed for the internal dG(H1') and dT(H4') protons. All the delta = f(t) curves display a sigmoid form with the same mid-point temperature of 44 +/- 2 degrees C. This mid-point temperature together with the observed chemical shift and linewidth variations were found to be independent of the d(A-T-G-G)cisPt concentration. These results suggest that d(A-T-G-G)cisPt can adopt two different conformations depending on the temperature. The enthalpy for the transition between the high and low temperature conformations is about 84 kJ/mol.     

A theoretical model for the binding of cis-Pt(NH3)2(+2) to DNA

The binding of cis-Pt(NH3)2B1B2 to the bases B1 and B2, i.e., guanine (G), cytosine (C), adenine (A), and thymine (T), of DNA is studied theoretically. The components of the binding are analyzed and a model structure is proposed for the intrastrand binding to the dB1pdB2 sequence of a kinked double helical DNA. Quantum mechanical calculations of the ligand binding energy indicates that cis-Pt(NH3)2(+2) (cis-PDA) binds to N7(G), N3(C), O2(C), O6(G), N3(A), N7(A), O4(T) and O2(T) in order of decreasing binding energy. Conformational analysis provides structures of kinked DNA in which adjacent bases chelate to cis-PDA. Only bending toward the major groove allows the construction of acceptable square planar complexes. Examples are presented for kinks of -70 degrees and -40 degrees at the receptor site to orient the base pairs for ligand binding to B1 and B2 to form a nearly square planar complex. The energies for complex formation of cis-PDA to the various intra-strand base sites in double stranded DNA are estimated. At least 32 kcal/mole separates the energetically favorable dGpdG.cis-PDA chelate from the dCpdG.cis-PDA chelate. All other possible chelate structures are much higher in energy which correlates with their lack of observation in competition with the preferred dGpdG chelate. The second most favorable ligand energy occurs with N3(C). A novel binding site involving dC(N3)pdG(N7) is examined. Denaturation can result in an anti----syn rotation of C about its glycosidic bond to place N3(C) in the major groove for intrastrand binding in duplex DNA. This novel intrastrand dCpdG complex and the most favored dGpdG structure are illustrated with stereographic projections.     

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.     

Formation and structure of reaction products of cis-PtCl2(NH3)2 with d(ApG) and/or d(GpA) in di-, tri- and penta-nucleotides. Preference for GpA chelation over ApG chelation

The reaction products of cis-PtCl2(NH)3)2 with several deoxyribonucleotides containing d(ApG) and/or d(GpA) have been studied. The various reaction products were separated by high-performance liquid chromatography and characterized by means of absorbance at 254 nm in combination with atomic absorption spectroscopy and 300-MHz 1H-NMR (pH dependence of the non-exchangeable base-protons, T1 relaxation time determinations). For the larger fragments the results from these techniques were confirmed by enzymatic degradation studies of the platinated fragments. The smallest of the investigated nucleotides, d(ApG) and d(GpA), both formed a variety of different platinum chelates. In the reaction with d(ApG) 15% cis-Pt(NH3)2-[d(ApG)N1(1),N7(2)] and 78% cis-Pt(NH3)2[d(ApG)N7(1),N7(2)] were found, 4% of the reacted material consisted of a 1 mol Pt/2 mol dinucleotide product, and 3% of an unidentified 1:1 product. From the main product two rotamers were found to occur: at room temperature, 81% anti,anti and 19% anti,syn product is present. With d(GpA) about equal amounts of N1,N7 and N7,N7 products were found; for both products the anti,anti and anti,syn conformations were found, respectively. Upon reaction of cis-PtCl2(NH3)2 with d(pApG) and d(pGpA) only the N7,N7 products were found; at room temperature and pH greater than 1.5 these products were present in anti,anti conformation. However, for the d(pApG)-platinum chelate at -20 degrees C a small amount (less than 5%) of a second product could be observed in NMR. For the d(pGpA)-platinum chelate a second N7,N7-coordinated product was observed when the pH of the NMR sample was lowered to 1.1 (at this pH the free 5'-phosphate group is protonated). With the larger fragments d(ApGpA), d(pApGpA) and d(TpApGpApT) the intra-molecular competition between the formation of the d(ApG) or the d(GpA) chelates could be studied. Using these nucleotides no N1-coordinated products or rotamers were observed. In the case of d(ApGpA) and d(TpApGpApT) the d(GpA) chelate (67% and 75% respectively) was favoured over the d(ApG) chelate, while with d(pApGpA) about equal amounts of both chelates were formed.     

Structural and dynamic aspects of binding of a prototype lexitropsin to the decadeoxyribonucleotide d(CGCAATTGCG)2 deduced from high-resolution 1H NMR Studies

Structural and dynamic properties of the self-complementary decadeoxyribonucleotide d(CGCAATTGCG)2 and the interaction between a prototype lexitropsin, or information-reading oligopeptide, and the decadeoxyribonucleotide are deduced by using high-resolution 1H NMR techniques. The nonexchangeable and imino proton resonances of d(CGCAATTGCG)2 have been completely assigned by two-dimensional NMR studies. The decadeoxyribonucleotide exists as a right-handed B-DNA. In the 1H NMR spectrum of the 1:1 complex, the selective chemical shifts and removal of degeneracy of AH2(4), AH2(5), T-CH3(6), and T-CH3(7) due to the anisotropy effects of the heterocyclic moieties of the ligand, and with lesser effects at the flanking base sites C(3) and G(8), locate the drug centrally in the decadeoxyribonucleotide. This conclusion is supported by plots of individual chemical shift changes across the decadeoxyribonucleotide. Similarly, imino protons IV and V experience larger shifts and II and III smaller shifts in accord with this conclusion while drug complexation permits the detection of imino proton I. Strong nuclear Overhauser effects (NOEs) between pyrrole H5 and AH2(5), and weaker NOEs to AH1'(5), TH3'(6), and AH2'(5), firmly locate the ligand in the minor groove. Intraligand NOEs between the adjacent heterocyclic moieties indicate that the lexitropsin is subject to propeller twisting about the N6-C9 bond in both the bound and free forms. Nuclear Overhauser effect spectroscopy (NOESY) and correlated spectroscopy (COSY) experiments also indicate that the removal of degeneracy of the C16 methylene protons upon complexation may arise from restricted rotation about the C15-N9, C15-C16, and C16-C17 bonds. Specific hydrogen bonds between amide NH groups on the concave face of the ligand (N4H, N6H, N9H) and adenine N3 or thymine O2 on the floor of the minor groove are in accord with displacement of the hydration shell by the drug. NOE measurements on the decadeoxyribonucleotide in the 1:1 complex confirm it exists as a right-handed helix and belongs to the B family. Exchange NMR effects permit an estimate of a rate of approximately equal to 44 s-1 for the two-site exchange of the lexitropsin between two equivalent sites on the decamer with delta G++ approximately equal to 70 +/- 5 kJ mol-1 at 294 K. Alternative mechanisms for this exchange process are considered.     

Preparation and characterization of oligonucleotides containing S-[2-(N7-guanyl)ethyl]glutathione.

S-[2-(N7-Guanyl)ethyl]glutathione is the major adduct derived from modification of DNA with 1,2-dibromoethane in biological systems and is postulated to be a mutagenic lesion [Humphreys, W. G., Kim, D.-H., Cmarik, J. L., Shimada, T., & Guengerich, F. P. (1990) Biochemistry 29, 10342-10350]. Oligonucleotides containing this modified base were prepared by treatment of oligonucleotides with S-(2-chloroethyl)glutathione and purified by chromatography. The self-complementary oligonucleotide d(ATGCAT), when thus modified at the single guanine, appeared to associate with itself as judged by UV measurements, but CD and NMR measurements indicated a lack of hybridization, with a decrease in the melting temperature of greater than 10 degrees C. The same lack of self-association was noted when d(ATGCAT) was modified to contain an N-acetyl-S-[2-(N7-guanyl)ethyl]cysteine methyl ester moiety. The oligomer d-(C1A2T3G4C5C6T7) was modified to contain a single S-[2-(N7-guanyl)ethyl]glutathione moiety at the central position, and UV, CD, and 1H NMR studies indicated that this oligomer hybridized to its normal complement d(A8G9G10C11A12T13G14), although the binding was considerably weakened by adduction (imino proton NMR spectroscopy in the presence of H2O indicated that the hydrogen bond signals seen in the oligomer were all broadened upon modification). All proton resonances were identified using two-dimensional 1H NMR spectroscopy. Adduct formation affected the chemical shifts of the base and 1', 2', and 2" protons of T3 and C5, the 2" proton of C6, and the 8 and 1' protons of C11, while little effect was observed on other protons. No cross-peaks were detected between the glutathione and oligomer moieties in two-dimensional nuclear Overhauser enhanced NMR studies. These results suggest that a rather local structural perturbation occurs in the DNA oligomer upon modification and that the glutathione moiety appears to be relatively unperturbed by its placement in the duplex. When the cytosine in the normal d(AGGCATG) complement to d-(CATGCCT) was changed to each of the other three potential bases at the central position, no hybridization with the oligomer d(CATGCCT) containing S-[2-(N7-guanyl)ethyl]glutathione was detected. We conclude that these N7-guanyl derivatives destabilize hybridization and that bases other than cytosine do not appear to show preferential thermodynamic bonding to these adducts, at least in the sequences examined to date.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Circular dichroism spectra of the individual rotamers of [Pt(N,N-dmen)(GpG)]+ (N,N-dmen=N,N-dimethylethylenediamine) indicate that the base-base oscillator coupling is not the main source of ellipticity in cis-PtG2L2 head-to-head complexes

The asymmetrical platinum complex [PtCl2(N,N-dmen)] (N,N-dmen = N,N-dimethylethylenediamine) reacts with the dinucleotide GpG to form two isomeric chelates of the formula [Pt(N,N-dmen)(GpG)]+ [9]. One of the isomers forms two stable rotamers separable by HPLC, whereas the other apparently prefers one single rotameric form. The favored conformations of these three forms were elucidated by means of molecular mechanics and dynamics techniques. In parallel, we have prepared the adduct, isolated the three rotamers, and recorded their solution circular dichroism (CD) spectra. For the first time we were thus able to correlate the CD features of individual rotamers of a cis-Pt(GpG) chelate with their structures. We show here that the two forms labeled in Inagaki's paper 1'e and 2e have the same right-handed helicoidal arrangement of the guanine bases but display different CD spectra in which the prominent bands have inverted signs. Thus, base-base interactions cannot be the principal cause of the CD of these compounds.     

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.     

NMR based structural studies decipher stacking of the alkaloid coralyne to terminal guanines at two different sites in parallel G-quadruplex DNA, [d(TTGGGGT)]4 and [d(TTAGGGT)]4

BackgroundTelomere elongation by telomerase gets inhibited by G-quadruplex DNA found in its guanine rich region. Stabilization of G-quadruplex DNA upon ligand binding has evolved as a promising strategy to target cancer cells in which telomerase is over expressed.MethodsInteraction of anti-leukemic alkaloid, coralyne, to tetrameric parallel [d(TTGGGGT)]₄ (Ttel7), [d(TTAGGGT)]₄ (Htel7) and monomeric anti-parallel [dGGGG(TTGGGG)₃] (Ttel22) G-quadruplex DNA has been studied using Circular Dichroism (CD) spectroscopy. Titrations of coralyne with Ttel7 and Htel7 were monitored by ¹H and ³¹P NMR spectroscopy. Solution structure of coralyne-Ttel7 complex was obtained by restrained Molecular Dynamics (rMD) simulations using distance restraints from 2D NOESY spectra. Thermal stabilization of DNA was determined by absorption, CD and ¹H NMR.Results and conclusionsBinding of coralyne to Ttel7/Htel7 induces negative CD band at 315/300 nm. A significant upfield shift in all GNH, downfield shift in T2/T7 base protons and upfield shift (1.8 ppm) in coralyne protons indicates stacking interactions. ³¹P chemical shifts and NOE contacts of G3, G6, T2, T7 protons with methoxy protons reveal proximity of coralyne to T2pG3 and G6pT7 sites. Solution structure reveals stacking of coralyne at G6pT7 and T2pG3 steps with two methoxy groups of coralyne located in the grooves along with formation of a hydrogen bond. Binding stabilizes Ttel7/Htel7 by ~ 25–35 °C in 2:1 coralyne-Ttel7/Htel7 complex.General significanceThe present study is the first report on solution structure of coralyne-Ttel7 complex showing stacking of coralyne with terminal guanine tetrads leading to significant thermal stabilization, which may be responsible for telomerase inhibition.