Heavy metal-nucleotide interactions. III. The participation of amino groups in the binding of methylmercury (II) to cytidine and adenosine 5'-phosphate in aqueous solution: studies by Raman difference spectrophotometry.

Raman difference spectrophotometry has been used to study the interaction of CH3Hg(II) with cytidine and Ado-5'-P at high pH. In contrast to the binding reactions which occur at lower pH or in non-aqueous solvents such as dimethyl sulfoxide, a proton is transferred from the amino group; and the complexes are CH3HgCydH-1 and CH3HgAdoH-1-5'-P. The spectra are significantly different from those of the cationic complexes. The integrated intensities of ligand modes which shift upon metalation can be used to measure the concentration of unreacted ligand and consequently the extent of the reaction. Equilibrium constants for the reactions CH3HgOH + L yields CH3HgLH-1 + H2O were estimated to be log KCyd equals 0.63 plus or minus 0.05 and log KAdo-5'-P equals 0.85 plus or minus 0.05, in fair agreement with values determined under very different conditions by ultraviolet spectrophotometry. The vibrational spectrum of the ligand in CH3HgCydH-1 is virtually the same as that of UrdH-1- which is isoelectronic. The spectrum of the ligand in CH3HgAdoH-1-5'-P is more similar to the isoelectronic base InoH-1-than to Ado-5'-P, although the resemblance is not so close as in the CydH-1---UrdH-1-case. The structures of these complexes are discussed on the basis of their vibrational spectra and similarities in the spectra of related compounds. It is concluded that the CH3Hg(II) binds to the amino nitrogen at high pH with both cytidine and Ado-5'-P. In neutral solution with excess CH3Hg(II), metalation occurs on the amino groups, on the ring, and also on the ribose.     

Heavy metal-nucleotide interactions. 15. Reactions of calf thymus DNA with the electrophiles methylmercury(II) nitrate, cis-dichlorodiammineplatinum(II), and trans-dichlorodiammineplatinum(II) studied using Raman difference spectroscopy. Evidence for the formation of c-DNA upon metalation

This study details the reactions of the electrophiles CH3Hg(NO3), cis-[PtCl2(NH3)2] (cis-DDP) and trans-[PtCl2(NH3)2] (trans-DDP) with calf thymus DNA using Raman and Raman difference spectroscopy. The order of CH3Hg(II) binding to calf thymus DNA is G > T > C > A. The electrophilic attack of cis- and trans-DDP on calf thymus DNA produces different orders of binding: cis-DDP-G>C approximately AT, trans-DDP-G approximately C approximately AT. The reaction of CH3Hg(II) with DNA results in a decrease in the percentage of B-form DNA. whereas the reactions of cis- and trans-DDP with DNA decrease the percentage of B-DNA and cause the formation of C-DNA structure.     

A laser Raman spectroscopic study of the interaction of the methylmercury cation with AMP, ADP and ATP

The interaction of the CH3Hg+ cation with adenosine 5'-monophosphate, adenosine 5'-diphosphate, and adenosine 5'-triphosphate has been studied in aqueous solution at neutral pH by laser Raman spectroscopy. Metal binding is shown to occur preferentially at the N-1 ring position of adenine, with some indication of coordination to the N-7 site and substitution of a proton on the exocyclic NH2 group of the nucleic base. Binding of the cation to phosphate groups also occurs extensively, with both the -PO2-3 and -PO-2 groups. The equilibrium constants for the binding to the phosphate groups and for N-1 coordination are approx. 70 and 600 M-1, respectively.     

Heavy metal-nucleoside interactions. Binding of methylmercury(II) to inosine and catalysis of the isotopic exchange of the C-8 hydrogen studied by 1-H nuclear magnetic resonance and raman difference spectrophotometry.

Raman difference spectrophotometry reveals that CH3HgII binds quantitatively to N(1) of inosine at pH 8, substituting for the proton. When N(1) is saturated, binding occurs at a second site. Measurements of the 1-H nuclear magnetic resonance spectra of both inosine and of CH3Hg-II are in agreement with the N(1) binding and indicate that the second site for mercuriation is N(7). This second binding reaction is observed to increase the rate of exchange of the C(8) hydrogen with solvent, consistent with results observed for alkylation at N(7). Coordination of the electrophilic CH3Hg-II to N(7) increases the acidity of H(8), facilitating OHminus--catalyzed proton abstraction and reprotonation by themedium. For comparison, the reaction of CH3Hg-II with [8-2-H]inosine has been studied. Displacement of the N(1) hydrogen upon mercuriation of inosine causes a significant electron delocalization into the ring, increasing the basicity of N(7), and accounting for the synergic effect in metal binding observed originally by Simpson. In contrast, 1-methylinosine interacts only slightly with CH3Hg-II at pH 8. Coordination appears to be at N(7), since H(8) again is observed to exchange rapidly with solvent protons. In acidic solution, pH less than 2, binding to inosine is almost quantitative and exclusively to N(7). The behavior of CH3Hg-II is compared with that of Pt(II) and with Ni(II), Co(II), AND Zn(II). A brief comparison is made among ultraviolet absorption spectrophotometry, nuclear magnetic resonance (NMR), and Raman difference spectrophotometry for studying reactions of nucleosides and nucleotides.     

Interaction of metal ions with nucleic acids. Interaction of copper(II) with guanosine and its derivatives.

Interaction of copper(II) with guanosine, 2'-deoxyguanosine, 1-methylguanosine, 7-methylguanosine and GMP was studied withe use of spectroscopic and magneto-chemical methods. The main site of copper(II) binding in guanosine is nitrogen N-7; participation of N-1 is not excluded. The involvement of carbonyl oxygen in copper binding or copper chelation to N-7 and 0-6 is rather unlikely. A crystalline complex of copper(II) with GMP [Cu(C10H12O8N5P) .(H2O)3] was obtained, and it was demonstrated that copper(II) is bound with N-7 and the phosphate group.     

Titanium(IV) targets phosphoesters on nucleotides: implications for the mechanism of action of the anticancer drug titanocene dichloride

Abstract Reactions between the anticancer drug titanocene dichloride (Cp2TiCl2) and various nucleotides and their constituents in aqueous solution or N,N-dimethylformamide (DMF) have been investigated by 1H and 31P NMR spectroscopy and in the solid state by IR spectroscopy. In aqueous solution over the pH* (pH meter reading in D2O) range 2.3-6.5, CMP forms one new species with Ti(IV) bound only to the phosphate group. In acidic media at pH*<4.6, three species containing titanocene bound to the phosphate group of dGMP, AMP, dTMP and UMP are formed rapidly. The bases also appear to influence titanocene binding. Only one of these Ti(IV)-bound species can be detected in the pH* range of 4.6-6.5 in each case. The order of reactivity towards Cp2TiCl2(aq) at pH* ca. 3 is GMP>TMP approximately AMP > CMP. At pH* > 7.0, hydrolysis of Cp2TiCl2 predominated and little reaction with the nucleotides was observed. Binding of deoxyribose 5'-phosphate and 4-nitrophenyl phosphate to Cp2TiCl2(aq) via their phosphate groups was detected by 31P NMR spectroscopy, but no reaction between Cp2TiCl2(aq) and deoxyguanosine, 9-ethylguanine or deoxy-D-ribose was observed in aqueous solution. The nucleoside phosphodiesters 3',5'-cyclic GMP and 2',3'-cyclic CMP did not react with Cp2TiCl2(aq) in aqueous solution; however, in the less polar solvent DMF, 3',5'-cyclic GMP coordination to [Cp2Ti]2+ via its phosphodiester group was readily observed. Binding of titanocene to the phosphodiester group of the dinucleotide GpC was also observed in DMF by 31P NMR. The nucleoside triphosphates ATP and GTP reacted more extensively with Cp2TiCl2(aq) than their monophosphates; complexes with bound phosphate groups were formed in acidic media and to a lesser extent at neutral pH. Cleavage of phosphate bonds in ATP (and GTP) by Cp2TiCl2(aq) to form inorganic phosphate, AMP (or GMP) and ADP (or GDP) was observed in aqueous solutions. In addition, titanocene binding to ATP was not inhibited by Mg(II), but the ternary complex titanocene-ATP-Mg appeared to form. These reactions contrast markedly with those of the drug cisplatin, which binds predominantly to the base nitrogen atoms of nucleotides and only weakly to the phosphate groups. The high affinity of Ti(IV) for phosphate groups may be important for its biological activity.     

Binding of mercury(II) to poly(dA-dT) studied by proton nuclear magnetic resonance

The binding of Hg(II) to poly(dA-dT) has been examined with proton NMR spectroscopy. Addition of HgCl2 between r (Hg2+/nucleotide) = 0 and 0.25 results in loss of the exchangeable imino N3H resonance of thymine, indicating preferential binding at this site. The nonexchangeable base resonances AH8, AH2, and TH6 shift their intensity downfield in a cooperative manner, indicating complexation which is slow on the NMR time scale and changes in the polymer conformation upon binding. At r = 0.25, the polymer is cross-linked, and an increase in temperature does not result in denaturation of the polymer, as evidenced by the thymine proton resonance chemical shifts. The chemical shifts of the AH2 and T(CH3)5 base resonances allow some general conclusions to be made about the stereochemistry of this complex.     

Introduction of interrupted secondary structure in supercoiled DNA as a function of superhelix density: consideration of hairpin structures in superhelical DNA.

PM2 DNA was prepared with different superhelical densities (sigma) in order to examine the relationship betweenn supercoiling and the occurrence of a region(s) of unpaired bases in this DNA. A previous study showed that CH3HgOH reacts with native superhelical PM2 DNA more rapidly than the nicked form II. This evaluation of binding, monitored through the change of sedimentation velocity, was repeated on PM2 DNA I with different superhelical densities. Early binding is detected by an increase in sedimentation velocity and occurs with molecules with sigma' values betwee -0.025 and -0.037. The conversion of form I to form II with the single-strand-specific endonuclease from Neurospora crassa also occurs above a sigma value of -0.025. This data strongly supports the view that supercoiling produces interrupted secondary structure. The question whether the interrupted regions remain single stranded in character or form small intrastrand hairpin regions is considered by examining which model best fits the CH3HgOH- induced sedimentation velocity changes and the standard sedimentation velocity versus the superhelical density curve for the in vitro made DNAs. The hairpin model offers the most satisfactory explanations for all the results of this and previous studies.     

DNA recognition by the helix-turn-helix motif: investigation by laser Raman spectroscopy of the phage lambda repressor and its interaction with operator sites OL1 and OR3.

The lambda repressor provides a model system for biophysical studies of DNA recognition by the helix-turn-helix motif. We describe laser Raman studies of the lambda operator sites OL1 and OR3 and their interaction with the DNA-binding domain of lambda repressor (residues 1-102). Raman spectra of the two DNA sites exhibit significant differences attributable to interstrand purine-purine steps that differ in the two oligonucleotides. Remarkably, the conformation of each operator is significantly and specifically altered by repressor binding. Protein recognition, which involves hydrogen-bond formation and hydrophobic contacts in the major groove, induces subtle changes in DNA Raman bands of interacting groups. These include (i) site-specific perturbations to backbone phosphodiester geometry at AT-rich domains, (ii) hydrophobic interaction at thymine 5CH3 groups, (iii) hydrogen bonding to guanine 7N and 6C = O acceptors, and (iv) alterations in sugar pucker within the C2'-endo (B-DNA) family. These perturbations differ between aqueous OL1 and OR3 complexes of repressor, indicating that protein binding in solution determines the precise DNA conformation. The overall structure of the lambda domain is not greatly perturbed by binding to either OL1 or OR3, in accord with X-ray studies of other complexes. However, Raman markers indicate a change in hydrogen bonding of the OH group of tyrosine-22, which is a hydrogen-bond acceptor in the absence of DNA but a combined donor and acceptor in the OL1 complex; yet, Y22 hydrogen bonding is not altered in forming the OR3 complex. The present results demonstrate qualitatively different and distinguishable modes of interaction of the lambda repressor DNA-binding domain with operators OL1 and OR3 in solution. This application of laser Raman spectroscopy to a well-characterized system provides a prototype for future Raman studies of other DNA-binding motifs under physiological conditions.     

Raman spectra of single crystals of r(GCG)d(CGC) and d(CCCCGGGG) as models for A DNA, their structure transitions in aqueous solution, and comparison with double-helical poly(dG).poly(dC)

The self-complementary oligonucleotides [r(CGC)d(CGC)]2 and [d(CCCCGGGG)]2 in single-crystal and solution forms have been investigated by Raman spectroscopy. Comparison of the Raman spectra with results of single-crystal X-ray diffraction and with data from polynucleotides permits the identification of a number of Raman frequencies diagnostic of the A-helix structure for GC sequences. The guanine ring frequency characteristic of C3'-endo pucker and anti base orientation is assigned at 668 +/- 2 cm-1 for both dG and rG residues of the DNA/RNA hybrid [r(GCG)d(CGC)]2. The A-helix backbone of crystalline [r(GCG)d(CGC)]2 is altered slightly in the aqueous structure, consistent with the conversion of at least two residues to the C2'-endo/anti conformation. For crystalline [d(CCCCGGGG)]2, the Raman and X-ray data indicate nucleosides of alternating 2'-endo-3'-endo pucker sandwiched between terminal and penultimate pairs of C3'-endo pucker. The A-A-B-A-B-A-A-A backbone of the crystalline octamer is converted completely to a B-DNA fragment in aqueous solution with Raman markers characteristic of C2'-endo/anti-G (682 +/- 2) and the B backbone (826 +/- 2 cm-1). In the case of poly(dG).poly(dC), considerable structural variability is detected. A 4% solution of the duplex is largely A DNA, but a 2% solution is predominantly B DNA. On the other hand, an oriented fiber drawn at 75% relative humidity reveals Raman markers characteristic of both A DNA and a modified B DNA, not unlike the [d-(CCCCGGGG)]2 crystal. A comparison of Raman and CD spectra of the aqueous [d(CCCCGGGG)]2 and poly(dG).poly(dC) structures suggests the need for caution in the interpretation of CD data from G clusters in DNA.     

Platinum complexes of nucleotides.

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

Characterisation of metal binding sites for 8-azaadenine. Formation and X-ray structural analysis of methylmercury(II) complexes

The compounds [(CH₃Hg)AAdH]NO₃ (1) and [(CH₃Hg)AAd]·4H₂O (2) have been isolated from aqueous 1:1 solutions of CH₃HgOH and 8-azaadenine (AAdH) at respective pH values of 2 and 5. Their structures have been established by X-ray structural analysis. N9 is the metal binding site in both complexes. Alteration of the metal to ligand ratio to 2:1 at a pH of 5 allows the preparation of [(CH₃Hg)₂AAd]NO₃·H₂O (3) in which the base is coordinated at both N3 and N9. The compound [(CH₃Hg)₃AAdH₋₁]NO₃ (4), in which N1, N6 and N9 function as binding sites for the CH₃Hg⁺ cation, is formed in a 3:1 solution at a pH of 6.5. X-ray structural analyses have been performed on 3 and 4. N8 takes part in weak intermolecular secondary bonds to symmetry related Hg9 atoms in all four complexes. The relevance of the structures to an understanding of the basicities of the nitrogen atoms in 8-azaadenine and their alteration upon metal coordination of N9 and N6 is discussed.     

A possible molecular link between the toxicological effects of arsenic,selenium and methylmercury: methylmercury(II) seleno bis(<Emphasis Type="Italic">S</Emphasis>-glutathionyl) arsenic(III)

Using a combination of As and Se K-edge and Hg L_III-edge X-ray absorption spectroscopy, ⁷⁷Se nuclear magnetic resonance spectroscopy, electrospray ionization mass spectrometry and molecular modeling, we have structurally characterized the novel species methylmercury(II) seleno bis(S-glutathionyl) arsenic(III). This species is formed in aqueous solution from CH₃HgOH and the seleno bis(S-glutathionyl) arsinium ion and constitutes an important first step towards characterizing the observed toxicologically relevant interaction between arsenite, selenite and methylmercury which has been previously reported in mammals.     

Dithiocarbamates in heavy metal poisoning: complexes of N,N-di(2-hidroxyethyl)dithiocarbamate with Zn(II), Cd(II), Hg(II), CH3Hg(II), and C6H5Hg(II)

The complexes M(DHDC)2, CH3Hg(DHDC), and C6H5Hg(DHDC) (M = Zn, Cd, Hg; DHDC = N,N-di(2-hydroxyethyl)dithiocarbamate) were prepared and investigated in solution and in the solid state by using 1H and 13C NMR, ir, and Raman spectroscopy. The dithiocarbamate group is anisobidentate and the complexes are associated in solution and the solid state via hydrogen bonding. The possible relation of these structural properties to the behavior of DHDC in the treatment of cadmium poisoning is discussed.     

Synthesis, characterization and binding with DNA of four planaramineplatinum(II) complexes of the forms: trans-PtL2Cl2 and [PtL3Cl]Cl, where L = 3-hydroxypyridine, 4-hydroxypyridine and imidazo(1,2-alpha)pyridine

Four new trans-planaramineplatinum(II) complexes, three of the form: trans-PtCl2L2, code named CH1, CH2 and CH4 where L = 3-hydroxypyridine, 4-hydroxypyridine and imidazo[1,2-alpha]pyridine, respectively, and one of the form: PtClL3, code named CH3 where L = 3-hydroxypyridine, have been prepared and characterized by elemental analyses and IR, Raman, mass and 1H NMR spectral studies. The interactions of the compounds with salmon sperm and pBR322 plasmid DNAs have been investigated and their activity against human ovarian cancer cell lines: A2780, A2780cisR and A2780ZD0473R have also been determined. The compounds are believed to form mainly monofunctional N7(G) and bifunctional intrastrand N7(G)N7(G) adducts with DNA, causing a local distortion of DNA as a result of which gel mobility of the DNA changes. The compound containing three planaramine ligands per molecule (CH3) is found to be less reactive than the compounds containing two planaramine ligands per molecule (CH1, CH2 and CH4), which in turn are less reactive than compounds containing one of the same planaramine ligands per molecule. The decrease in reactivity is reflected in lower molar conductivity values (indicating lower degree of dissociation), less pronounced changes caused to DNA conformation (indicating decreased level of platinum-DNA binding) and lower activity. The decreased reactivity of the compounds is due to a greater steric crowding produced by the bulky planaramine ligands. Changes in DNA conformation are also found to be a function of the actual nature of the planaramine ligand. The results illustrate structure-activity relationship.     

Structural forms and transitions for the complex of mercury(II) with poly(dG-dC)

Infrared spectroscopy was used to study hydrated double-helical poly(dG-dC) complexed with varying amounts of mercury(II). For one Hg(II) per ten nucleotide residues (r = 0.1), the B structure was stabilized and the B* structure was absent at high hydration. The Z structure did not form as hydration was reduced. For r = 0.2, the B and Z structures coexisted at high hydration and the transition to total Z structure was broad as hydration was reduced. Hg(II) was bound exclusively to the guanine residues probably at N3 or N7 for r less than or equal to 0.25. The cytosine residue did not protonate (at N3) as Hg(II) was bound to guanine. The addition of NaCl together with Hg(II) reduced the binding of Hg(II), stabilized the B structure at the highest hydration and caused a sharp transition between the B and Z structures as hydration was lowered. Hydration with D2O stabilized the Z structure for poly(dG-dC) complexed with HgCl2.     

1H-NMR study of the removal of methylmercury from intact erythrocytes by sulfhydryl compounds

The effectiveness of penicillamine, N-acetylpenicillamine, meso-2,3-dimercaptosuccinic acid, 2,3-dimercaptopropanesulfonic acid, and dithioerythritol for removing methylmercury (CH3Hg(II) from intact human erythrocytes has been studied by 1H-nuclear magnetic resonance spectroscopy. The removal of CH3Hg(II) was monitored by measuring the chemical shift of the resonance for the proton on the alpha-carbon of the cysteinyl residue of intracellular glutathione in 1H-NMR spectra of intact, CH3Hg(II)-containing erythrocytes in suspensions to which the sulfhydryl ligands were added. Because exchange of intracellular glutathione between its free and CH3Hg(II) complexed forms is fast, the chemical shift of the cysteinyl resonance provides a direct, noninvasive measure of the fraction of intracellular glutathione that is complexed. The sulfhydryl ligands were found to remove CH3Hg(II) from intact erythrocytes in the order 2,3-dimercaptosuccinic acid greater than 2,3-dimercaptopropane sulfonic acid greater than dithioerythritol greater than penicillamine approximately N-acetylpenicillamine, which also is the order of the conditional formation constants of the CH3Hg(II) complexes at pH 7.4. All five ligands removed CH3Hg(II) from intact erythrocytes much more rapidly than can be accounted for by a mechanism in which the ligand crosses the membrane, combines with the CH3Hg(II), and then transports it out of the cell. An alternative mechanism is proposed in which the ligand reacts with CH3Hg(II) which is complexed by sulfhydryl groups of the membrane, which in turn react with the intracellular CH3Hg(II) to bring more CH3Hg(II) into the membrane, where it can react with the added sulfhydryl ligand.     

A Raman spectroscopic study of the interaction of divalent metal ions with adenine moiety of adenosine 5'-triphosphate.

Raman spectra of ATP at various pH values are affected by addition of equimolar solution of divalent metal ions such as Ca2+, Mg2+, Co2+, Cu2+, and Hg2+. The changes in frequency and intensity have been used to construct models describing the nature of metal-adenine and metal-triphosphate interactions under different conditions. The metal ions are found to co-ordinate the triphosphate group in the entire pH range studies (pH to 12). Calcium (II) and magnesium (II) interact strongly with the phosphate moiety at neutral pH, although a weak interaction with the ring occur at low pH values. Around neutrality, several Raman spectral changes are observed to implicate the interaction of cobalt (II) ion with the five-membered ring of the adenine. The changes in Raman frequency are too small to suggest a direct Co(II)-N7 binding. At least six different Cu(II)-ATP species are identified between pH 3 and 12. At pH approximately 7.0 Raman data are explained better by Cu(II) interacting with N7 simultaneously with the amino group of the adenine ring. However, a Cu(II) binding to N3 at pH 10 to 11 is indicated by the enhancement of the 760 and 1360 cm-1 vibrations. At neutral pH, mercury (II) ion shows a direct coordination at N1 while at low pH with N1 blocked by protonation, mercury (II) does not interact with the adenine moiety.     

Raman spectroscopy of mercury (II) binding to two filamentous viruses: Ff (fd, M13, f1) and Pf1

Ff and Pf1 are filamentous bacteriophages. Each contains, in a central core region surrounded by protein, a circular single-stranded DNA molecule, and it is known that the DNA bases are sites of Hg(II) binding. In the present study, Raman spectra were obtained for the two viruses in the presence of increasing amounts of Hg(II), with ratios (m) of Hg(II) added per nucleotide residue in the range 0 less than m less than 2.0. Hg(II) binding to the viruses induces Raman intensity changes in previously assigned Raman lines of viral DNA, demonstrating metal binding to the DNA bases, but also in many lines assigned to protein. The overall structures of the viruses do not change with Hg(II) binding, and the Raman spectra indicate little, if any, change in protein secondary structure. Changes in certain protein Raman lines induced by Hg(II) binding to the DNA for low values of m are attributed to altered interactions between solvent and protein side chains, aliphatic groups being the most affected. The nature of such changes for both viruses suggests DNA-protein linkage. In Pf1, lines assigned to ring vibrations of all four bases are perturbed upon initial addition of Hg(II) to m = 0.25. In Ff, however, lines assigned to base ring vibrations are not perturbed until m greater than or equal to 0.5. The results provide additional evidence for fundamentally different DNA structures in Ff and Pf1.     

Raman spectroscopic detection of the T-Hg II-T base pair and the ionic characteristics of mercury

Developing applications for metal-mediated base pairs (metallo-base-pair) has recently become a high-priority area in nucleic acid research, and physicochemical analyses are important for designing and fine-tuning molecular devices using metallo-base-pairs. In this study, we characterized the Hg(II)-mediated T-T (T-Hg(II)-T) base pair by Raman spectroscopy, which revealed the unique physical and chemical properties of Hg(II). A characteristic Raman marker band at 1586 cm(-1) was observed and assigned to the C4=O4 stretching mode. We confirmed the assignment by the isotopic shift ((18)O-labeling at O4) and density functional theory (DFT) calculations. The unusually low wavenumber of the C4=O4 stretching suggested that the bond order of the C4=O4 bond reduced from its canonical value. This reduction of the bond order can be explained if the enolate-like structure (N3=C4-O4(-)) is involved as a resonance contributor in the thymine ring of the T-Hg(II)-T pair. This resonance includes the N-Hg(II)-bonded state (Hg(II)-N3-C4=O4) and the N-Hg(II)-dissociated state (Hg(II+) N3=C4-O4(-)), and the latter contributor reduced the bond order of N-Hg(II). Consequently, the Hg(II) nucleus in the T-Hg(II)-T pair exhibited a cationic character. Natural bond orbital (NBO) analysis supports the interpretations of the Raman experiments.