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.     

Effect of acyclovir [9-(2-hydroxyethoxymethyl)guanine] on Epstein-Barr virus DNA replication.

The effect of acyclovir [9-(2-hydroxyethoxymethyl)guanine] on Epstein-Barr virus (EBV) DNA replication in the lymphoblastoid cell lines P3HR-1 and Raji is reported. Acyclovir at a concentration of 100 microM completely inhibited EBV DNA synthesis in superinfected Raji cells, but did not inhibit DNA synthesis in mock-infected cells. The number of EBV genome equivalents per cell in the virus-producing cell line P3HR-1 was significantly reduced by acyclovir, whereas the number of latent EBV genomes in Raji cells was not affected by the drug. In situ cytohybridization performed on untreated P3HR-1 cultures revealed the presence of relatively large amounts of EBV DNA in 15 to 20% of the cells. After a 100 microM drug treatment, no P3HR-1 cells contained levels of EBV DNA detectable by in situ cytohybridization. Indirect immunofluorescence studies demonstrated that during treatment with 100 microM acyclovir for 7 days, the percentage of P3HR-1 cells expressing viral capsid antigen was reduced. The EBV DNA remaining in P3HR-1 cells after treatment with 100 microM acyclovir (approximately 14 genomes per cell) had the properties of covalently closed circular DNA with an average molecular weight of 108 X 10(6), as determined by contour length measurements.     

Phosphorylation of acyclovir [9-(2-hydroxyethoxymethyl)guanine] in Epstein-Barr virus-infected lymphoblastoid cell lines.

The extent of phosphorylation of 9-(2-hydroxyethoxymethyl)guanine (acyclovir [ACV]) in fresh peripheral leukocytes, in Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines, and in herpes simplex virus type 1-infected lymphoblastoid (P3HR-1) and monkey kidney (Vero) cells was determined by high-pressure liquid chromatography, Mono-, di-, and triphosphorylated derivatives of [8-14C]ACV were detected at low levels at various times after superinfection of Raji cells with EBV. The extent of phosphorylation appeared to be related to the concentration of ACV in the medium. Small amounts of ACV mono-, di-, and triphosphates were formed in fresh peripheral leukocyte preparations from EBV- seropositive and -seronegative donors. Comparable ACV monophosphate levels were detected in EBV-negative BJAB and the EBV-positive BJAB/GC cell lines; however, no di- or triphosphate derivatives were detected. Comparable ACV-monophosphate levels were detected in both P3HR-1 and HSV-infected P3HR-1 cell lines; however, larger amounts of ACV di- and triphosphorylated derivatives were detected in the HSV-infected P3HR-1 cells. ACV was converted to the triphosphate to a greater extent in HSV-infected Vero cells than in mock-infected Vero cells or in HSV-infected P3HR-1 cells. ACV or its phosphorylated derivatives were converted to guanine nucleotides to a greater extent in lymphoblastoid cells than in fibroblasts (Vero). In conclusion, neither the productive replication of EBV nor the presence of latent viral DNA is required for ACV monophosphate formation in B lymphoblastoid cells. ACV triphosphate, however, was detected only in cells infected productively with EBV.     

On the role of N7 atoms of guanosine in tRNA-Phe (E. coli) in interaction with ribosomes]

The influence of alkylation of phe-tRNAPhe (E. coli) with 2',3'-O-[4-(N-2-chloroethyl-N-methylamino)-benzylidene]-uridine - 5' - methylphosphate on its ability to participate in non-enzymatic complex formation with ribosomes and poly-U was investigated. Phe-tRNAsPhe, containing alkylated guanosines at different positions, including anticodone, are active in binding with ribosomes. It is concluded, that N7 nitrogens of guanosine of the tRHAPhe are not elements, significant for the interaction with ribosomes.     

Alternating d(G-C)3 and d(C-G)3 hexanucleotides containing 7-deaza-2''-deoxyguanosine or 8-aza-7-deaza-2''-deoxyguanosine in place of dG.

The synthesis of alternating hexamers (8-13) derived from d(C-G)3 or d(G-C)3 but containing c7z8Gd (2) or c7Gd (3) instead of dG is described employing phosphoramidite-chemistry. Apart from the isobutyryl group the dimethylaminomethylene residue was used for the nucleobase-protection of 3. The methyl- and the cyanoethyl-phosphoramidites of 3 (5a-c) were synthesized. They were employed together with those of c7G or c7z8Gd in automated oligonucleotide synthesis. Tm-values as well as thermodynamic data of the oligomers 9, 10, 12, and 13 indicated that duplexes were destabilized if c7Gd replaced dG, whereas c7z8Gd stabilized the duplex structure. In contrast to d(C-G)3 which underwent salt-dependent B-Z transition, CD-spectra of oligomers containing c7Gd or c7z8Gd in place of dG showed retained B-conformation.     

A calorimetric study of the triple helix formation: interaction of poly(C) - guanosine - protonated poly(C).]

The triple helix formation of poly(C) - guanosine - poly(C+) was investigated by the help of an LKB scanning micro-calorimeter. The existence of the triple helix could also be shown by recording the melting curves. The ultraviolet absorption at different wave lengths namely 275 nm, 260 nm, and 245 nm was plotted as a function of the temperature. Furthermore formation of the triple helix was shown by plotting the ultraviolet absorption at 245 nm during the increasing addition of guanosine solution to a fixed amount of poly(C) in the solution. Finally the formation of the triple helix was demonstrated by plotting the ultraviolet absorption at 245 nm of a certain mixture of the components while the pH value of the solution was continuously lowered. All these methods show that the monomer interacts with the polymer double helix to form a triple helix. The calorimetric measurements show that the reaction enthalpy is concentration dependent. Above a threshold concentration a rapid increase of the reaction enthalpy is observed. This increase occurs in a very narrow concentration interval. Above this interval a final value of the reaction enthalpy is reached. The amount of the reaction enthalpy for the interaction of guanosine with poly(C) - poly(C+) double helix is 5.5 Kcal (mol base triplet)-1.     

An efficient method for the synthesis of [2-(15)N]guanosine and 2'-deoxy[2-(15)N]guanosine derivatives.

Nucleophilic substitution reaction of 6-chloro-2-fluoro-9-beta-D-ribofuranosyl-9H-purine derivative, prepared from guanosine, with potassium [15N]phthalimide at 40 degrees C for 9 h in DMF, followed by hydrolysis, afforded [2-(15)N]guanosine derivative efficiently. The corresponding 2'-deoxy derivative was also synthesized through a similar procedure.     

7-Deaza-2'-deoxyadenosine and 3-deaza-2'-deoxyadenosine replacing dA within d(A6)-tracts: differential bending at 3'- and 5'-junctions of d(A6).d(T6) and B-DNA.

7-Deaza-2'-deoxyadenosine (1, c7Ad) and 3-deaza-2'-deoxyadenosine (2, c3Ad) have been incorporated into d(AAAAAA) tracts replacing dA at various positions within oligonucleotides. For this purpose suitably protected phosphonates have been prepared and oligonucleotides were synthesized on solid-phase. The oligomers were hybridized with their cognate strands. The duplexes were phosphorylated at OH-5' by polynucleotide kinase and self-ligated to multimers employing T4 DNA ligase. Oligomerized DNA-fragments were analyzed by polyacrylamide gel electrophoresis and the bending was determined from anomalies of electrophoretic mobility. Replacement of dA by c3Ad decreased the bending more than replacement by c7Ad. Reduction of bending was much stronger when the modified nucleosides replaced one or several dA residues at the 3'-site of an d(AAAAAA)-tract whereas replacement at the 5'-site showed no significant influence [1, 2].     

A calorimetric approach to the study of the interactions of cytidine-3'-phosphate with bovine seminal ribonuclease

A calorimetric study at 25 degrees C is reported on the interaction between allosteric bovine seminal ribonuclease and cytidine-3'-phosphate. The results are compared with those obtained under identical experimental conditions for the interaction of pancreatic ribonuclease A and the same nucleotide. The analysis of the data provides evidence that the binding sites of seminal ribonuclease for cytidine-3'-phosphate are not equivalent, in agreement with previous equilibrium dialysis studies. A model with two sites with different affinities toward the nucleotide, the site with higher affinity resembling the binding site of pancreatic ribonuclease, is proposed. The values calculated for the thermodynamic parameters provide an insight of the forces involved in the interaction of the two enzymes with the nucleotide.     

Molecular-Mechanical studies of the mismatched base analogs of d(CGCGAATTCGCG)2:d(CGTGAATTCGCG)2, d(CGAGAATTCGCG)2, d(CGCGAATTCACG)2, d(CGCGAATTCTCG)2, and d(CGCAGAATTCGCG)·d(CGCGAATTCGCG)

Molecular-mechanical simulations have been carried out on “mismatched base” analogs of the DNA double-helical structure d(CGCGAATTCGCG)₂, in which the base pairs CG at the 3 and 10 positions have been replaced by CA, AG, TC, and TG base pairs, as well as an insertion analog in which an extra adenine has been incorporated into one strand of the above structure between bases 3 and 4. The results of these simulations (calculated relative stabilities, structures, and nmr ring-current shifts) have been compared with calorimetric and nmr data. The calculated relative stabilities of the double-helical parent dodecamer and the various “wobble” base pairs qualitatively correlate with the experimental melting temperatures. The base-pairing structure for the GT wobble pair is in agreement with that previously determined from nmr experiments. For the GA base pair, the structure with both bases anti has a slightly more favorable energy from base pairing and stacking than a structure with non-Watson-Crick H-bonding with adenine syn, in agreement with nmr experiments. The CA wobble base is calculated to favor an adenine 6NH₂ …? cytosine N3 H-bond over cytosine 4NH₂ …? adenine N1, again, in agreement with nmr experiments. There is no definitive experimental data on the TC base pair, but the existence of (somewhat long and weak) H-bonds involving cytosine 4NH₂ …? thymine 4CO and cytosine N3 …? thymine HN3 seems reasonable. We find a structure in which the extra adenine base of the insertion analogs sits “inside” the double helix.     

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.     

Stabilization of the non-canonical adenine-adeninium base pair by N(7) coordination of Zn(II)

A new zinc (II) compound with 9-ethyladenine (9-EtA) of formula [Zn(9-EtA-N7)Cl(3)](9-EtAH) has been synthesized and characterized by X-ray diffraction. Its X-structure consists of an Zn(II) anionic complex and 9-ethyladeninium as counteranion. The Zn(II) complex shows a distorted tetrahedral geometry in which three Cl and an 9-EtA coordinates through N(7) position are the ligands. An indirect chelation via intramolecular H-bond between N(6)H and an Cl ligand is present in the complex. The network of [Zn(9-EtA-N7)Cl(3)](9-EtAH) shows interesting features. Thus, self-association of coordinated adenine-adeninium takes place by H-bonding of N(6)-H...N(1) and N(6)-H...N(7), leading to a polymeric ribbon-like 1D supramolecular arrangement. Ab initio calculations have been applied in order to study the stability of the adenine-adeninium interaction due to the coordination of the Zn(II) to the N(7) position and to compare experimental and theoretical structural data.     

Evidence for long-range interactions in DNA. Analysis of melting curves of block polymers d(C15A15)·d(T15G15), d(C20A15)·d(T15G20), and d(C20A10)·d(T10G20)

The influence of one DNA region on the stability of an adjoining region (telestability) was examined. Melting curves of three block DNA's, d(C₁₅A₁₅)·d(T₁₅G₁₅), d(C₂₀A₁₅)·d(T₁₅G₂₀), and d(C₂₀A₁₀)·d(T₁₀G₂₀) were analyzed in terms of the nearest neighbor Ising model. Comparisons of predicted and experimental curves were made in 0.01 M and 0.1 M sodium ion solutions. The nearest neighbor formalism was also employed to analyze block DNA transition in the presence of actinomycin, a G·C specific molecule. The results show that nearest neighbor base-pair interaction cannot predict the melting curves of the block DNA's. Adjustments in theoretical parameters to account for phosphate repulsion assuming a B conformation throughout the DNA's do not alter this conclusion. Changes in the theoretical parameters, which provide good overall agreement, are consistent with a substantial stabilization of the A·T region nearest the G·C block. The melting temperature T A·T for the average A·T pari in d(C₂₀A₁₀)·d(T₁₀G₂₀), with 10 A·T pairs, appears to be 4°C greater than T_A·T for d(C₁₅A₁₅)·d(T₁₅G₁₅) and d(C₂₀A₁₅)·d(T₁₅G₂₀), both with 15 A·T pairs. Actinomycin bound to the G·C end effectively stabilizes the A·T end by 9°C. These results indicate a long-range contribution to the interactions governing DNA stability. A possible mechanism for these interactions will be discussed.     

Solid-phase synthesis of the self-complementary hexamer d(c7GpCpc7GpCpc7GpC) via the O-3''-phosphoramidite of 7-deaza-2''-deoxyguanosine.

The synthesis of the O-3'-phosphoramidite of a suitably protected 7-deaza-2'-deoxyguanosine (c7G) which is an isostere of 2'-deoxyguanosine is described. The phosphoramidite of the modified nucleoside was used in the synthesis of the self-complementary hexamer d(c7GpCpc7GpCpc7GpC) on functionalized silica gel in a mini-reactor. As expected from the parent hexamer d(GpCpGpCpGpC) the isosteric d(c7GpCpc7GpCpc7GpC) exhibits a rigid secondary structure (22% hypochromicity at 280 nm) and forms a duplex in 1 M aqueous sodium chloride solution. Due to the altered pi-electron system of the pyrrolo[2,3-d]pyrimidine nucleobase, which affects base stacking and hydrogen bonding, the Tm of the modified duplex is decreased by 10 degrees C compared to that of the parent purine hexamer. Moreover, it is expected that the incorporation of c7G influences the pitch of the helix.     

Consequences of methylation on the amino group of adenine. A proton two-dimensional NMR study of d(GGATATCC) and d(GGm6ATATCC)

A two-dimensional 500-MHz 1H-NMR study of two oligonucleotides, d(GGATATCC) and d(GGm6ATATCC), is presented in which we have investigated the effects of adenine methylation. The two-dimensional nuclear Overhauser spectra (NOESY) show that both oligonucleotides adopt a normal right-handed B-type helix and one-dimensional nuclear Overhauser enhancement (NOE) studies demonstrate that any difference in conformation must be small. However methylation drastically slows down the helix in equilibrium coil exchange which becomes slow on a proton NMR time scale. While d(GGATATCC) fits a two-site exchange model, d(GGm6ATATCC) does not and we invoke the presence of a third species which may be an intermediate in helix formation. NMR and ultraviolet spectroscopy show that methylation destabilizes the helix, measured by the melting temperature and enthalpy of dissociation.     

Differential interactions of antitumor antibiotics chromomycin A(3) and mithramycin with d(TATGCATA)(2) in presence of Mg(2+)

The antitumor antibiotics chromomycin A(3) (CHR) and mithramycin (MTR) are known to inhibit macromolecular biosynthesis by reversibly binding to double stranded DNA with a GC base specificity via the minor groove in the presence of a divalent cation such as Mg(2+). Earlier reports from our laboratory showed that the antibiotics form two types of complexes with Mg(2+): complex I with 1:1 stoichiometry and complex II with 2:1 stoichiometry in terms of the antibiotic and Mg(2+). The binding potential of an octanucleotide, d(TATGCATA)(2), which contains one potential site of association with the above complexes of the two antibiotics, was examined using spectroscopic techniques such as absorption, fluorescence, and circular dichroism. We also evaluated thermodynamic parameters for the interaction. In spite of the presence of two structural moieties of the antibiotic in complex II, a major characteristic feature was the association of a single ligand molecule per molecule of octameric duplex in all cases. This indicated that the modes of association for the two types of complexes with the oligomeric DNA were different. The association was dependent on the nature of the antibiotics. Spectroscopic characterization along with analysis of binding and thermodynamic parameters showed that differences in the mode of recognition by complexes I and II of the antibiotics with polymeric DNA existed at the oligomeric level. Analysis of the thermodynamic parameters led us to propose a partial accommodation of the ligand in the groove without the displacement of bound water molecules and supported earlier results on the DNA structural transition from B --> A type geometry as an obligatory requirement for the accommodation of the bulkier complex II of the two drugs. The role of the carbohydrate moieties of the antibiotics in the DNA recognition process was indicated when we compared the DNA binding properties with the same type of Mg(2+) complex for the two antibiotics.     

Phytic acid: divalent cation interactions: III. A calorimetric and titrimetric study of the pH dependence of copper(II) binding

The interaction of the cupric ion with phytic acid as a function of pH has been studied by potentiometric and thermal titration and by the determination of ligand binding. As has been found for the reaction of zinc and calcium cations with phytate, the presence of the Cu(II) ion results in a displacement of the titration curves to more acid values. Evaluation of the parameters that describe such changes in ionization behavior by curve-fit analysis showed that as the Cu(II):phytate mol ratio was increased from one to eight, the pK' values of the ionizable group sets of phytic acid (ranging from 1.59 to 9.79) were consolidated into just two sets with curve-fit (CP) values ca. 1.5 and 3.7. Marked pH hysteresis effects are seen in such systems because of the pronounced acid strength of the Cu(II):aqua ion and the Cu(II) ligand aqua ion complex. The combined heat of binding and precipitation (plus solvation changes, etc.) of Cu(II) to phytate is endothermic (21.8–22.2 kcal mol⁻¹). This is similar in magnitude to that reported for the binding of either Zn(II) or Ca(II) to phytate. In the titration of Cu(NO₃)₂ with KOH, presumably to form Cu(OH)₂, ΔH° was exothermic (−12.5 kcal mol⁻¹). From measurements of free Cu(II) cation concentration in the presence of phytate the binding reaction was found to be stoichiometric with 6 mols Cu(II) bound at pH 6. Binding occurs within the pH range 2–6. An apparent necessary requirement for binding is the availability of the oxo dianion structure formed from the second dissociation step of a phosphoryl group. Curve-fit analysis of the binding data as a function of pH showed that a group or group set with CP value ca. 4 governs the binding reaction(s) at all mol ratios of Cu(II) to phytate examined. It is suggested that the binding of cupric ions to phytate may occur to the equatorial rather than the axial configuration as suggested for Ca(II) binding. A space-filling molecular model to illustrate this has been constructed. Soluble Cu(II):phytate complexes are formed within the pH range from 2 to ca. 3.4. This is supported by the results of difference absorption spectrometry.     

A calorimetric study of the Ni(II)-5' AMP system. A base-stacking stabilization.

The thermodynamic parameters of the interaction between Ni(II) (M) and 5'-AMP in aqueous solution were determined calorimetrically (I = 0.1 tetramethylammonium bromide, 25 degrees C) at physiological pH. The experimental conditions were carefully selected to avoid the polynuclear complex formation. From the Ni(5'AMP)-complex-formation constant (logK1 = 2.55), the Ni(5'AMP)2(2-)-complex-formation constant and the Ni(5'AMP) (ML) and Ni(5'AMP)2(2-) (ML2) complex-formation enthalpies were determined (logK2 = 2.34, delta H1 = -10.0 kJ/mol and delta H2 = 21.6 kJ/mol). These results confirm the formation of the ML2 complex in solution and agree very well with the hypothesis of the 'stacking' between the purine rings, promoted for metal ions.     

Reactivity of phenyldi(2-thienyl)phosphine towards group 7 metal carbonyls: Carbon-phosphorus bond activation

Addition of phenyldi(2-thienyl)phosphine (PPhTh₂) to [Re₂(CO)_10−n(NCMe)_n] (n = 1, 2) affords the substitution products [Re₂(CO)_10−n(PhPTh₂)_n] (1, 2) together with small amounts of fac-[ClRe(CO)₃(PPhTh₂)₂] (3) (n = 2). Reaction of [Re₂(CO)₁₀] with PPhTh₂ in refluxing xylene affords a mixture which includes 2, [Re₂(CO)₇(PPhTh₂)(μ-PPhTh)(μ-H)] (4), [Re₂(CO)₇(PPhTh₂)(μ-PPhTh)(μ-η¹,κ¹(S)-C₄H₃S)] (5) and mer-[HRe(CO)₃(PPhTh₂)₂] (6). Phosphido-bridged 4 and 5 are formed by the carbon-phosphorus bond cleavage of the coordinated PPhTh₂ ligand, the cleaved thienyl group being retained in the latter. Reaction of [Mn₂(CO)₁₀] with PPhTh₂ in refluxing toluene affords [Mn₂(CO)₉(PPhTh₂)] (7) and the carbon-phosphorus bond cleavage products [Mn₂(CO)₆(μ-PPhTh)(μ-η¹,η⁵-C₄H₃S)] (8) and [Mn₂(CO)₅(PPhTh₂)(μ-PPhTh)(μ-η¹,η⁵-C₄H₃S)] (9). Both 8 and 9 contain a bridging thienyl ligand which is bonded to one manganese atom in a η⁵-fashion.