Interaction of DNA with Simple and Mixed Ligand Copper(II) Complexes of 1,10-Phenanthrolines as Studied by DNA-Fiber EPR Spectroscopy.

The interaction of simple and ternary Cu(II) complexes of 1,10-phenanthrolines with DNA has been studied extensively because of their various interesting and important functions such as DNA cleavage activity, cytotoxicity towards cancer cells, and DNA based asymmetric catalysis. Such functions are closely related to the DNA binding modes of the complexes such as intercalation, groove binding, and electrostatic surface binding. A variety of spectroscopic methods have been used to study the DNA binding mode of the Cu(II) complexes. Of all these methods, DNA-fiber electron paramagnetic resonance (EPR) spectroscopy affords unique information on the DNA binding structures of the complexes. In this review we summarize the results of our DNA-fiber EPR studies on the DNA binding structure of the complexes and discuss them together with the data accumulated by using other measurements.

DNA molecular recognition and cellular selectivity of anticancer metal(II) complexes of ethylenediaminediacetate and phenanthroline: multiple targets.

By inhibiting only two or three of 12 restriction enzymes, the series of [M(phen)(edda)] complexes [M(II) is Cu, Co, Zn; phen is 1,10-phenanthroline; edda is N,N'-ethylenediaminediacetate] exhibit DNA binding specificity. The Cu(II) and Zn(II) complexes could differentiate the palindromic sequences 5'-CATATG-3' and 5'-GTATAC-3', whereas the Co(II) analogue could not. This and other differences in their biological properties may arise from distinct differences in their octahedral structures. The complexes could inhibit topoisomerase I, stabilize or destabilize G-quadruplex, and lower the mitochondrial membrane potential of MCF7 breast cells. The pronounced stabilization of G-quadruplex by the Zn(II) complex may account for the additional ability of only the Zn(II) complex to induce cell cycle arrest in S phase. On the basis of the known action of anticancer compounds against the above-mentioned individual targets, we suggest the mode of action of the present complexes could involve multiple targets. Cytotoxicity studies with MCF10A and cisplatin-resistant MCF7 suggest that these complexes exhibit selectivity towards breast cancer cells over normal ones.

Biological and cytoselective anticancer properties of copper(II)-polypyridyl complexes modulated by auxiliary methylated glycine ligand.

A series of ternary copper(II)-1,10-phenanthroline complexes with glycine and methylated glycine derivatives, [Cu(phen)(aa)(H(2)O)]NO(3)·xH(2)O 1-4 (amino acid (aa): glycine (gly), 1; DL: -alanine (DL: -ala), 2; 2,2-dimethylglycine (C-dmg), 3; sarcosine (sar), 4), were synthesized and characterized by FTIR, elemental analysis, electrospray ionization-mass spectra (ESI-MS), UV-visible spectroscopy and molar conductivity measurement. The determined X-ray crystallographic structures of 2 and 3 show each to consist of distorted square pyramidal [Cu(phen)(aa)(H(2)O)](+) cation, a nitrate counter anion, and with or without lattice water, similar to previously reported structure of [Cu(phen)(gly)(H(2)O)]NO(3)·1½H(2)O. It is found that 1-4 exist as 1:1 electrolytes in aqueous solution, and the cationic copper(II) complexes are at least stable up to 24 h. Positive-ion ESI-MS spectra show existence of only undissociated [Cu(phen)(aa)](+) species. Electron paramagnetic resonance, gel electrophoresis, fluorescence quenching, and restriction enzyme inhibition assay were used to study the binding interaction, binding affinity and selectivity of these complexes for various types of B-form DNA duplexes and G-quadruplex. All complexes can bind selectively to DNA by intercalation and electrostatic forces, and inhibit topoisomerase I. The effect of the methyl substituents of the coordinated amino acid in the above complexes on these biological properties are presented and discussed. The IC(50) values (24 h) of 1-4 for nasopharyngeal cancer cell line HK1 are in the range 2.2-5.2 µM while the corresponding values for normal cell line NP69 are greater than 13.0 µM. All complexes, at 5 µM, induced 41-60 % apoptotic cell death in HK1 cells but no significant cell death in NP69 cells.

DNA-fiber EPR spectroscopy as a tool to study DNA-metal complex interactions: DNA binding of hydrated Cu(II) ions and Cu(II) complexes of amino acids and peptides.

DNA-fiber EPR spectroscopy and its application to studies of the DNA binding orientation and dynamic properties of Cu(II) ions and their complexes with amino acids and peptides are reviewed. Cu(II) ions bind in at least two different binding modes; one mode was mobile while the other mode fixed the orientation of the coordination plane. The hydroxyl groups of L-Ser and L-Thr fixed the coordination plane of their respective Cu(II) complexes parallel to the DNA base pair plane, whereas Cu(II) complexes of Lys and Arg induced several binding modes, depending on the tertiary structure of the DNA and the chirality of the amino acids. Unusually broadened signals observed for the His complex were assigned to a mono-L-His complex stacked stereospecifically along the DNA double helix. In comparison, Cu(II). Xaa-Xaa' -His type complexes oriented in the minor groove with different affinities and extents of randomness depending on the Xaa-Xaa' sequence and the chirality of Xaa or Xaa' while the C-terminal Xaa residues in Cu(II).Arg-Gly-His-Xaa (Xaa=L-Leu or L-Glu) decreased the stereospecificity and the stability of the complexes bound to DNA. In contrast to Xaa-Xaa'- His complexes, the coordination planes of Cu(II).Gly-L-His-Gly and Cu(II).Gly-L-His-L-Lys complexes were found to lie parallel to the DNA-fiber axis. Dinuclear Cu(II).carnosine complexes were also shown to bind to DNA stereospecifically.

DNA-fiber EPR investigation of the influence of amino-terminal residue stereochemistry on the DNA binding orientation of Cu(II).Gly-Gly-His-derived metallopeptides.

DNA fiber EPR was used to investigate the DNA binding stabilities and orientations of Cu(II).Gly-Gly-His-derived metallopeptides containing D- vs. L-amino acid substitutions in the first peptide position. This examination included studies of Cu(II).D-Arg-Gly-His and Cu(II).D-Lys-Gly-His for comparison to metallopeptides containing L-Arg/Lys substitutions, and also the diastereoisomeric pairs Cu(II).D/L-Pro-Gly-His and Cu(II).D/L-Pro-Lys-His. Results indicated that L-Arg/Lys to D-Arg/Lys substitutions considerably randomized the orientation of the metallopeptides on DNA, whereas the replacement of L-Pro by D-Pro in Cu(II).L-Pro-Gly-His caused a decrease in randomness. The difference in the extent of randomness observed between the D- vs. L-Pro-Gly-His complexes was diminished through the substitution of Gly for Lys in the middle peptide position, supporting the notion that the epsilon-amino group of Lys triggered further randomization, likely through hydrogen bonding or electrostatic interactions that disrupt binding of the metallopeptide equatorial plane and the DNA. The relationship between the stereochemistry of amino acid residues and the binding and reaction of M(II).Xaa-Xaa'-His metallopeptides with DNA are also discussed.

Effects of N-alkyl and ammonium groups on the hydrolytic cleavage of DNA with a Cu(II)TACH (1,3,5-triaminocyclohexane) complex. Speciation, kinetic, and DNA-binding studies for reaction mechanism.

cis,cis-1,3,5-Triaminocyclohexane (c-TACH), its N-alkyl-derivatives (alkyl = methyl, ethyl), and trans,cis-1,3,5-triaminocyclohexane (t-TACH) were prepared, and speciation and DNA cleaving property of Cu(II) complexes of these ligands were investigated. All of the complexes efficiently promote the hydrolytic cleavage of supercoiled plasmid DNA under physiological conditions without further additives. The DNA cleavage rate (V(obs)) trend at pH values between 8 and 9 is N-Me(3) = N-Et(1) < t-TACH < c-TACH < N-Et(2) < N-Et(3). At pH 7, the trend is c-TACH < N-Et(3) = N-Et(2) < N-Et(1) < N-Me(3) << t-TACH. The cleavage rate constants at 35 degrees C, for the c-TACH complex are 3 x 10(-1) h(-1) at pH 8.1 and 2 x 10(-1) h(-1) at pH 7.0 ([DNA] = 7 microM, [Cu(II)-complex] = 105 microM). The hydrolytically active species at pH > 8 is CuL(H(2)O)(OH)(+) in which L coordinates to Cu(II) as a tridentate ligand for all complexes except for t-TACH. The hydrolytically active species at pH 7 is CuLH(H(2)O)(3)(3+) or CuLH(H(2)O)(4)(3+) in which LH coordinates as bidentate ligand. DNA-binding constants of c-TACH and t-TACH complexes are presented and the effects of N-alkyl and ammonium groups are discussed in light of the proposed reaction mechanism.

Copper(II) complexes of 1,10-phenanthroline-derived ligands: studies on DNA binding properties and nuclease activity.

A series of copper(II) complexes of the type [Cu(L)]2+, where L = N,N'-dialkyl-1,10-phenanthroline-2,9-dimethanamine and R = methyl (L1), n-propyl (L2), isopropyl (L3), sec-butyl (L4), or tert-butyl (L5) group, have been synthesized. The interaction of the complexes with DNA has been studied by DNA fiber electron paramagnetic resonance (EPR) spectroscopy, emission, viscosity and electrochemical measurements and agarose gel electrophoresis. In the X-ray crystal structure of [Cu(HL2)Cl2]NO3, copper(II) is coordinated to two ring nitrogens and one of the two secondary amine nitrogens of the side chains and two chloride ions as well and the coordination geometry is best described as trigonal bipyramidal distorted square based pyramidal (TBDSBP). Electronic and EPR spectral studies reveal that all the complexes in aqueous solution around pH 7 possess CuN3O2 rather than CuN4O chromophore with one of the alkylamino side chain not involved in coordination. The structures of the complexes in aqueous solution around pH 7 change from distorted tetragonal to trigonal bipyramidal as the size of the alkyl group is increased. The observed changes in the physicochemical features of the complexes on binding to DNA suggest that the complexes, except [Cu(L5)]2+, bind to DNA with partial intercalation of the derivatised phen ring in between the DNA base pairs. Electrochemical studies reveal that the complexes prefer to bind to DNA in Cu(II) rather than Cu(I) oxidation state. Interestingly, [Cu(L5)]2+ shows the highest DNA cleavage activity among all the present copper(II) complexes suggesting that the bulky N-tert-butyl group plays an important role in modifying the coordination environment around the copper(II) center, the Cu(II)/Cu(I) redox potential and hence the formation of activated oxidant responsible for the cleavage. These results were compared with those for bis(1,10-phenanthroline)copper(II), [Cu(phen)2]2+.

Effect of a conjugated acridine moiety on the binding and reactivity of Cu(II)[9-acridinylmethyl-1,4,7-triazacyclononane] with DNA.

The DNA binding orientation and dynamic behavior of Cu(II) complexes of 1,4,7-triazacyclononane ([9]aneN(3)), 1, and an acridine conjugate, 2, were investigated by DNA fiber EPR (EPR=electron paramagnetic resonance) spectroscopy. Crystal and molecular structure of 2 were determined by X-ray diffraction. It has been shown that 1 binds to DNA in two different modes at room temperature; one species is rapidly rotating and the other is immobilized randomly on the DNA. The introduction of acridine to [9]aneN(3) fixed the [Cu([9]aneN(3))](2+) moiety of 2 in two different environments on the DNA: the g(mid R:mid R:) axis of one species (g( parallel)=2.26) is aligned perpendicularly to the DNA fiber axis whereas that of the other (g( parallel)=2.24) aligns<90 degrees with the DNA fiber axis. The different DNA binding structures of 1 and 2 are reflected also in their different efficiencies of DNA cleavage; 2 was found to be more effective both in oxidative and hydrolytic cleavage reactions.

Interaction of Cu(II)-Arg-Gly-His-Xaa metallopeptides with DNA: effect of C-terminal residues, Leu and Glu.

The interactions of Cu(II)-Arg-Gly-His-Xaa metallopeptides with DNA (where Xaa is L-Leu or L-Glu) were investigated by DNA-fiber EPR spectroscopy, ESI-MS spectrometry, and agarose gel electrophoresis. The average angle between the g// axis of Cu(II)-Arg-Gly-His-Leu and the DNA-fiber axis increased from 45 degrees at room temperature to 90 degrees at -150 degrees C. The Cu(II)-Arg-Gly-His-Glu complex partly dissociated on DNA to several species. The g//value (2.341) of the main species was smaller than that (2.377) observed for free Cu(II) ion bound to DNA. This indicated that the Cu(II) ion was transferred by the peptide to a DNA site where the free Cu(II) ion can hardly reach. ESI-MS spectra of a mixture of the Cu(II) peptide complex and the oligodeoxynucleotide, [d(CGCGTATACGCG)], suggested that the maximum binding stoichiometries of Cu(II) peptide complexes and double stranded oligodeoxynucleotides were 3:1 for Cu(II)-Arg-Gly-His-Leu and 2:1 for Cu(II)-Arg-Gly-His-Glu, respectively. Cu(II)-Arg-Gly-His-Glu completely converted the supercoiled DNA to the nicked-circular form, whereas the cleavage activity was considerably reduced when excess ligand was added. In the presence of excess peptide, nicked DNA formation ratios were 64% for Cu(II)-Arg-Gly-His-Leu and 15% for Cu(II)-Arg-Gly-His-Glu, respectively. The negative charge on Cu(II)-Arg-Gly-His-Glu reduced the affinity of the complex for DNA and enhanced the specificity of the binding.

EPR study of the dynamic behavior of cis,cis-1,3,5-triaminocyclohexane Cu(II) complex on B-form DNA-fibers.

The orientation and the dynamic behavior of [Cu(TACH)](2+)(TACH = cis,cis-1,3,5-triaminocyclohexane) on B-form DNA-fiber have been investigated by electron paramagnetic resonance (EPR) spectroscopy. The complex showed novel EPR spectra indicating that a rapidly moving species (X) is in equilibrium with a stereospecifically oriented species (Y) on the DNA-fiber in the range 20 and -20 degrees C. The thermodynamic parameters Delta H and Delta S of the equilibrium X right arrow over left arrow Y are respectively -51.3 kJmol(-1) and -1.9 x 10(2) JK(-1)mol(-1) for the DNA-fibers prepared at pH 7 and -47.1 kJmol(-1) and -1.7 x 10(2) JK(-1)mol(-1) for the DNA-fibers prepared at pH 9. These results suggest that the equilibrium involved a concerted structural change of the coordination sphere and the hydrogen bonding network around the complex on the B-form DNA-fibers. The orientation of the species Y was completely randomized below -20 degrees C, indicating that the freezing of the water molecules in the DNA-fibers breaks down the hydrogen bonding network which regulated the orientation of the complex in the DNA-fibers. The correlation between the observed dynamic behavior and the hydrolytic ability of the complex was also discussed.

DNA-fiber EPR study of the orientation of Cu(II) complexes of 1,10-phenanthroline and its derivatives bound to DNA: mono(phenanthroline)-copper(II) and its ternary complexes with amino acids.

The orientation of mono(1,10-phenanthroline)copper(II), [Cu(phen)]2+, and the ternary complexes with amino acids, [Cu(phen)X(aa)]n+, where X(aa) stands for an alpha-amino acid, has been investigated by electron paramagnetic resonance (EPR) spectra of the complexes on DNA fibers. It has been revealed that these complexes bind to DNA with several different binding modes. The observation of a species whose g axis is almost parallel to the DNA double helical axis has suggested that the phenanthroline moiety intercalates to DNA. An absence of the intercalated species for the corresponding 2,2'-bipyridine complex has shown that the three-fused aromatic rings in phenanthroline are critical for the intercalative binding of the complexes. The intercalative binding was promoted by 5,6-dimethyl groups on the phenanthroline ring, whereas it was disturbed by 2,9-dimethyl groups, indicating that the planarity of the coordination sphere is important for the intercalative binding. In all cases, the amount of the non-intercalated species was larger than that of the intercalated one. The amino acids in the ternary complexes of glycine, leucine, serine, threonine, cysteine, methionine, and asparagine were partly substituted with some coordinating groups in DNA, whereas the ternary complexes of lysine, arginine, and glutamine remained intact on DNA.

The DNA binding site specificity and antiproliferative property of ternary Pt(II) and Zn(II) complexes of phenanthroline and N,N'-ethylenediaminediacetic acid.

The binding site specificity of the ternary complexes, [M(II)(phen)(edda)] (M(II) = Pt(2+) and Zn(2+); phen = 1,10-phenanthroline; edda = N,N'-ethylenediaminediacetic acid), for the self-complementary oligonucleotides (ODNs), ds(C(1)G(2)C(3)G(4)A(5)A(6)T(7)T(8)C(9)G(10)C(11)G(12))(2) (ODN1) and ds(C(1)G(2)C(3)G(4)T(5)A(6)T(7)A(8)C(9)G(10)C(11)G(12))(2) (ODN2), was studied by NMR measurements. The results indicated that [Pt(ii)(phen)(edda)] was partially intercalated between C(3)/G(10) and G(4)/C(9) base pairs of ODN1 and ODN2 in the major grooves, whereas [Zn(II)(phen)(edda)] was bound specifically to the TATA region of ODN2 in the minor groove and to the terminal G(2)/C(11) base pair of ODN1 in the major groove. The preference for the TATA sequence over the AATT sequence in the binding of [Zn(phen)(edda)] was attributed to the wider minor groove width of the TATA sequence. The bindings of the complexes to ct-DNA were also studied by UV, CD, and fluorescence spectroscopy. Additionally, the antiproliferative property of [Pt(II)(phen)(edda)] towards MCF7 breast cancer cells and normal MCF10-A cells was compared with that of [Zn(II)(phen)(edda)].

Synthesis, characterization, DNA-binding study and anticancer properties of ternary metal(II) complexes of edda and an intercalating ligand.

A series of ternary metal(ii) complexes {M(phen)(edda); 1a (Cu), 1b (Co), 1c (Zn), 1d (Ni); H(2)edda = N,N(')-ethylenediaminediacetic acid} of N,N'-ethylene-bridged diglycine and 1,10-phenanthroline were synthesized and characterized by elemental analysis, FTIR, UV-visible spectroscopy and magnetic susceptibility measurement. The interaction of these complexes with DNA was investigated using CD and EPR spectroscopy. MTT assay results of 1a-1c , screened on MCF-7 cancer cell lines, show that synergy between the metal and ligands results in significant enhancement of their antiproliferative properties. Preliminary results from apoptosis and cell cycle analyses with flow cytometry are reported. seems to be able to induce cell cycle arrest at G(0)/G(1). The crystal structure of 1a is also included.

Fabrication of DNA nanowires by orthogonal self-assembly and DNA intercalation on a Au patterned Si/SiO2 surface.

A novel Ru complex bearing both an acridine group and anchoring phosphonate groups was immobilized on a surface in order to capture double-stranded DNAs (dsDNAs) from solution. At low surface coverage, the atomic force microscopy (AFM) image revealed the "molecular dot" morphology with the height of the Ru complex ( approximately 2.5 nm) on a mica surface, indicating that four phosphonate anchor groups keep the Ru complex in an upright orientation on the surface. Using a dynamic molecular combing method, the DNA capture efficiency of the Ru complex on a mica surface was examined in terms of the effects of the number of molecular dots and surface hydrophobicity. The immobilized surface could capture DNAs; however, the optimal number of molecular dots on the surface as well as the optimal pull-up speed exist to obtain the extended dsDNAs on the surface. Applying this optimal condition to a Au-patterned Si/SiO 2 (Au/SiO 2) surface, the Au electrode was selectively covered with the Ru complex by orthogonal self-assembly of 4-mercaptbutylphosphonic acid (MBPA), followed by the formation of a Zr (4+)-phosphonate layer and the Ru complex. At the same time, the remaining SiO 2 surface was covered with octylphosphonic acid (OPA) by self-assembly. The selective immobilization of the Ru complex only on the Au electrode was identified by time-of-flight secondary-ion mass spectrometry (TOF-SIMS) imaging on the chemically modified Au/SiO 2 surface. The construction of DNA nanowires on the Au/SiO 2 patterned surface was accomplished by the molecular combing method of the selective immobilized Ru complex on Au electrodes. These interconnected nanowires between Au electrodes were used as a scaffold for the modification of Pd nanoparticles on the DNA. Furthermore, Cu metallization was achieved by electroless plating of Cu metal on a priming of Pd nanoparticles on the Pd-covered DNA nanowires. The resulting Cu nanowires showed a metallic behavior with relatively high resistance.

Design of Salen-type Ni(II) complexes for recognition of DNA base sequence.

The DNA binding properties of cationic salen-type Ni(II) complexes (Fig. 1) have been studied by CD and NMR measurements. The binding constants estimated for poly(dA-dT)2 and poly(dA)poly(dT) revealed that (1) binds more preferentially to poly(dA)poly(dT) than to poly(dA-dT)2, and vice versa for (2). The intermolecular NOE and chemical shift change of the oligonucleotides, d(CGCGAATTCGCG)2 (ODN1) and d(CGCGTATACGCG)2 (ODN2) indicated that the two complexes bound to the minor grooves at AT-rich site of the ODNs from the "en" bridging side. The difference in the sequence specificity between (1) and (2) was attributed to the difference in the width of the minor groove; (2) recognized the wider minor groove of ODN2 'TATA' region. In contrast to (1) and (2), the asymmetric complex (3) bound to the major groove at terminal GC-rich site of ODN1. In this study, we could reveal that hydrophobic interaction is an important factor for base sequence recognition and an appropriate modification of the salen could switch the binding site from minor to major groove of DNA.

Interaction of several Ni(III) complexes of peptides with DNA; analysis by DNA-fiber EPR.

EPR spectra of Ni(III) complexes of GGH, GHG, and GHK were obtained by in-situ oxidation of the Ni(II) complexes in DNA-pellet and on DNA-fibers. A species with an apical coordination of nitrogenous base of DNA was detected for Ni(III) GGH. Both GHG and GHK complexes showed the EPR signals of Ni(III) species when the ligand to metal ion ratio was 2:1. The complexes bind as Ni(III)(N6), NI(III)(N5), and Ni(III)(N4) species on DNA.

The effect of acridine moiety for binding and reaction of Cu(II)[1 ,4,7-triazacyclononane] with DNA.

The orientation and the dynamic behavior of Cu(II) complex of 1,4,7-triazacyclononane ([9]aneN3) and its acridine conjugate on B-form DNA fiber have been investigated by EPR spectroscopy. It has been shown that a Cu([9]aneN3)2+ binds to DNA with two different binding modes at room temperature; one species is rapidly moving and the other is immobilized randomly on the DNA. An introduction of acridine to 1,4,7-triazacyclononane fixed the orientation of all the Cu(II) complex with the g// axis perpendicular to DNA fiber axis, indicating that the tetragonal coordination plane is almost parallel to the DNA double helical axis.

Deglyco-peplomycin metal complexes on DNA fibers: a role of the sugar moiety for the stability and the orientation of the complexes.

Binding structures of metal complexes of deglyco-peplomycin (dPEP) on DNA were investigated by comparing dPEP complexes with those of bleomycin (BLM) using DNA fiber EPR spectroscopy. A low spin species of Fe(III)dPEP observed in the DNA pellet changed irreversibly to several high spin species after the fabrication of the DNA fibers. The g values of the high spin species were different from those of Fe(III)BLM. The high spin species could not be nitrosylated reductively to ON-Fe(II)dPEP, suggesting that some nitrogen atoms coordinated to the Fe(III) were displaced on the DNA fibers. On the other hand, O(2)-Co(II)dPEP remained intact on the fibers similarly to O(2)-Co(II)BLM but with an increased randomness in the orientation on the DNA. In contrast to Cu(II)BLM, a considerable amount of Cu(II)dPEP bound almost randomly on B-form DNA fibers. These results indicated that the sugar moiety in peplomycin or bleomycin is playing an important role in enhancing the stability of the metal-binding domain and in the stereospecificity of the binding on DNA.