TITLE : Binding Interactions between Nickel Schiff Base Complexes and Quadruplex DNA

BODY: Abstract Body: The clinical success of cisplatin, and platinum based drugs,as anti-cancer agents constitutes the most important contribution to the use of metals in medicine.The major concerns associated with these anti-cancer metallodrugs, include problems with resistance, toxicity and other side effects.The main cellular target for platinum drugs is genomic DNA and the major antitumor activity results from inhibition of DNA replication.Hence,considerable efforts are still ongoing to find more effective DNA targeting drugs with fewer side effects. Evaluation of the ‘chemical nucleases’,which can cleave DNA by several pathways,namely nucleobase oxidation, phosphate ester hydrolysis and deoxyribose sugar oxidation could be another approach to design new antineoplastic metallodrugs targeting cellular DNA.The most efficient chemical nucleases contain transition metal ions, like redoxactive Cu, Fe, or the redox-inactive Zn in their active sites.Cu(II) has a long history of medical use, and its prospective antitumor properties have attracted attention recently because it is thought to be less toxic than nonessential metals, such as Pt.The mechanism involved in the cleavage of DNA is probably initiated by the intercalation of the highly planar Cu(II) complex.The use of structurally different ligands is expected to provide a better understanding of the factors that are crucial for the DNA cleavage activity of these type of complexes, allowing a more rational approach for the development of Cu(II) complexes with potential anti-cancer properties. These studies inspired us to synthesize mixed ligand Cu(II) complexes of the fluoroquinolone drug moxifloxacin and ferrocene conjugated amino acids and investigate their DNA binding & nuclease activities. Apoptosis (programmed cell death) is desirable in chemotherapy and therefore induction of apoptosis is one of the considerations in development of anticancer drugs.The realization that apoptosis is a key factor that contributes to antitumor activity of chemotherapeutic drugs, has led us to evaluate the cytotoxic behaviour of the synthesized complexes and their mechanism of action. CONTROL ID: 1706374 TITLE: A Message Regarding Nucleic Acids. A Suitable Primary Binding Site Facilitates Metal Ion Coordination to a Hydroxyl Group! AUTHORS/INSTITUTIONS: H. Sigel, A. Sigel, Department of Chemistry/Inorganic Chemistry, University of Basel, Basel, SWITZERLAND| CURRENT CATEGORY: Metals and Nucleic Acids ABSTRACT BODY: Abstract Body: Knowledge on the binding of biologically relevant divalent metal ions (M(II)) to hydroxyl groups is scarce despite its importance, e.g., for ribozymes [1]. For an overview on the existing literature we joined forces with several colleagues [2] and it turned out that a suitable primary binding site (PBS) in a favorable steric orientation is crucial for a M(II)-hydroxyl group interaction. We studied the position of the intramolecular isomeric equilibrium between an open form, in which M(II) is solely coordinated to PBS, and a closed or chelated form, in which M(II) is also bound to the hydroxyl group. We concentrated on the alkaline earth ions as well as on Mn(II), Co(II), Ni(II), Cu(II), and Zn(II). The following PBSs were considered: Phosph(on)ate, carboxylate, amino, imidazole or pyridyl groups. For 5-membered chelates the extent of chelate formation increases in the given order of the PBSs. The formation degrees of the chelates vary widely, i.e., from a few to nearly 100%. For complexes formed with hydroxyacetate (= glycolate = HO–CH2–COO– = HOAc–), one obtains for Mg(HOAc)+ and Zn(HOAc)+ formation degrees of the chelates of 71±5% and 91±1%, respectively. Interestingly, the formation degree of the closed Ca(HOAc)+ isomer is with 83±3% larger than the one of Mg(HOAc)+. Maybe here is the reason for the atypical strong influence of Ca(II) on group II intron ribozyme catalysis and folding [3]. The relevance of the indicated results for biological systems is obvious, especially because a reduced solvent polarity favors M(II)-hydroxyl group interactions [2]. Naturally, the M(II)-hydroxyl binding gives rise to enhanced complex stabilities, log delta, from which the formation degrees can be calculated [4].BODY: Abstract Body: Knowledge on the binding of biologically relevant divalent metal ions (M(II)) to hydroxyl groups is scarce despite its importance, e.g., for ribozymes [1]. For an overview on the existing literature we joined forces with several colleagues [2] and it turned out that a suitable primary binding site (PBS) in a favorable steric orientation is crucial for a M(II)-hydroxyl group interaction. We studied the position of the intramolecular isomeric equilibrium between an open form, in which M(II) is solely coordinated to PBS, and a closed or chelated form, in which M(II) is also bound to the hydroxyl group. We concentrated on the alkaline earth ions as well as on Mn(II), Co(II), Ni(II), Cu(II), and Zn(II). The following PBSs were considered: Phosph(on)ate, carboxylate, amino, imidazole or pyridyl groups. For 5-membered chelates the extent of chelate formation increases in the given order of the PBSs. The formation degrees of the chelates vary widely, i.e., from a few to nearly 100%. For complexes formed with hydroxyacetate (= glycolate = HO–CH2–COO– = HOAc–), one obtains for Mg(HOAc)+ and Zn(HOAc)+ formation degrees of the chelates of 71±5% and 91±1%, respectively. Interestingly, the formation degree of the closed Ca(HOAc)+ isomer is with 83±3% larger than the one of Mg(HOAc)+. Maybe here is the reason for the atypical strong influence of Ca(II) on group II intron ribozyme catalysis and folding [3]. The relevance of the indicated results for biological systems is obvious, especially because a reduced solvent polarity favors M(II)-hydroxyl group interactions [2]. Naturally, the M(II)-hydroxyl binding gives rise to enhanced complex stabilities, log delta, from which the formation degrees can be calculated [4]. Supported by the Department of Chemistry, University of Basel. [1]R.K.O. Sigel, A.M. Pyle, Chem. Rev. 107 (2007) 97-113. [2]F.M. Al-Sogair, B.P. Operschall, A. Sigel, H. Sigel, J. Schnabl, R.K.O. Sigel, Chem. Rev. 111 (2011) 4964-5003. [3]M. Steiner, D. Rueda, R.K.O. Sigel, Angew. Chem. Int. Ed. 48 (2009) 9739-9742. [4]H. Sigel, L.E. Kapinos, Coord. Chem. Rev. 200-202 (2000) 563-594. (No Image Selected) CONTROL ID: 1707767 TITLE: Binding Interactions between Nickel Schiff Base Complexes and Quadruplex DNA AUTHORS/INSTITUTIONS: K.J. Davis, C. Richardson, J.L. Beck, S.F. Ralph, School of Chemistry, University of Wollongong, Wollongong, New South Wales, AUSTRALIA| CURRENT CATEGORY: Metals and Nucleic Acids ABSTRACT BODY: Abstract Body: Quadruplex DNA (qDNA) is a less common nucleic acid secondary structure present in non-coding regions at the ends of chromosomes known as telomeres. Since many base pairs are lost from the ends of DNA strands during replication, telomeres function to protect chromosomes during this process. However, when DNA becomes so short that it can no longer function as the template for protein synthesis, the cell enters apoptosis, or programmed cell death. In contrast, approximately 85% of tumour cells possess elevated levels of the enzyme telomerase, which is responsible for maintaining the length of telomeres and contributes to tumour cell immortality. The normal substrate for telomerase is the single stranded overhang regions present at the end of telomeres. These regions are rich in guanines, and consequently prone to forming qDNA structures. Drugs that can bind selectively to existing qDNA structures or induce formation of such structures may be able to inhibit telomerase and act as novel anti-cancer agents. One group of compounds that has shown promise in this area are substituted Schiff base complexes of various metals. The work presented here further explores the potential of nickel(II) Schiff base complexes as selective qDNA binders, and inhibitors of telomerase, by varying the number and position of aromatic ring systems in the Schiff base structure, as well as the identity of side chains designed to interact with the qDNA grooves. One complex of interest is (1), which includes the meso-1,2-diphenylethylenediamine unit as part of its structure. This complex binds poorly to duplex DNA, but is able to bind to a tetramolecular qDNA structure, indicating that it is possible to engender selectivity for qDNA structures through the usage of the above non-planar moiety. We are currently using a variety of methods, including electrospray ionisation mass spectrometry (ESI-MS) and CD spectroscopy, to explore the interactions between different types of qDNA and the nickel complexes.BODY: Abstract Body: Quadruplex DNA (qDNA) is a less common nucleic acid secondary structure present in non-coding regions at the ends of chromosomes known as telomeres. Since many base pairs are lost from the ends of DNA strands during replication, telomeres function to protect chromosomes during this process. However, when DNA becomes so short that it can no longer function as the template for protein synthesis, the cell enters apoptosis, or programmed cell death. In contrast, approximately 85% of tumour cells possess elevated levels of the enzyme telomerase, which is responsible for maintaining the length of telomeres and contributes to tumour cell immortality. The normal substrate for telomerase is the single stranded overhang regions present at the end of telomeres. These regions are rich in guanines, and consequently prone to forming qDNA structures. Drugs that can bind selectively to existing qDNA structures or induce formation of such structures may be able to inhibit telomerase and act as novel anti-cancer agents. One group of compounds that has shown promise in this area are substituted Schiff base complexes of various metals. The work presented here further explores the potential of nickel(II) Schiff base complexes as selective qDNA binders, and inhibitors of telomerase, by varying the number and position of aromatic ring systems in the Schiff base structure, as well as the identity of side chains designed to interact with the qDNA grooves. One complex of interest is (1), which includes the meso-1,2-diphenylethylenediamine unit as part of its structure. This complex binds poorly to duplex DNA, but is able to bind to a tetramolecular qDNA structure, indicating that it is possible to engender selectivity for qDNA structures through the usage of the above non-planar moiety. We are currently using a variety of methods, including electrospray ionisation mass spectrometry (ESI-MS) and CD spectroscopy, to explore the interactions between different types of qDNA and the nickel complexes. Figure 1. (a) ESI mass spectrum of a solution containing a 3:1 ratio of (1) and a tetramolecular qDNA molecule. (b) ESI mass spectrum of a solution containing a 3:1 ratio of (1) and a duplex 16mer DNA molecule. CONTROL ID: 1707958 TITLE: Metal-dependent Stabilization of Artificial DNA Junction Structures AUTHORS/INSTITUTIONS: Y. Takezawa, J. Duprey, S. Yoneda, M. Shionoya, Department of Chemistry, Graduate School of Science, The University of Tokyo, Tokyo, JAPAN| CURRENT CATEGORY: Metals and Nucleic Acids ABSTRACT BODY: Abstract Body: DNA molecules have been expected to serve as promising components for nanoscale molecular architectures due to their well-ordered structure and highly programmable nature. We have developed metal-mediated base pairs as additional functional building blocks of DNA structures, in which natural hydrogen bonds between nucleobases were replaced by metal coordination bonds, and have demonstrated significant stabilization of the duplexes. In this study, we synthesized a novel metal-conjugated DNA structure involving a three-way junction, which is one of the key components of 2D and 3D DNA nanoarchitectures. We designed a DNA three-way junction structure with a preorganized metal binding site at its core. Bidentate bipyridine (bpy) ligands were attached to the nucleosides positioned at the center of each strand via Cu-catalyzed Huisgen cycloaddition (Ubpy). The melting profiles of the resulting three-way junction were investigated in the presence of various transition metal ions. The results showed that the addition of equimolar amounts of Ni2+ ion remarkably stabilized the junction structure by 12 °C compared to the unmodified junction. In contrast, three-way junctions with only one or two bpy ligands showed little stabilization upon Ni2+ addition. Thus, the stabilization was attributed to metal-mediated crosslinking among three DNA strands through the formation of a Ni(bpy)3 2+ complex at the junction point as further evidenced by the UV spectral changes. Furthermore, CD spectroscopy indicated the predominant formation of the Λ-isomer that could be a result of the chiral DNA environment at the core of the threeway junction. The “metal-locked” DNA junction motif presented here will have future applications in the field of DNA nanoarchitectures as a building block for stabilization and conformation switching of whole DNA structures by reversible metal complexation. [1] Y. Takezawa, M. Shionoya, Acc. Chem. Res. 2012, 45, 2066. [2] J.-L. H. A. Duprey, Y. Takezawa, M. Shionoya, Angew. Chem., Int. Ed. 2013, 52, 1212.BODY: Abstract Body: DNA molecules have been expected to serve as promising components for nanoscale molecular architectures due to their well-ordered structure and highly programmable nature. We have developed metal-mediated base pairs as additional functional building blocks of DNA structures, in which natural hydrogen bonds between nucleobases were replaced by metal coordination bonds, and have demonstrated significant stabilization of the duplexes. In this study, we synthesized a novel metal-conjugated DNA structure involving a three-way junction, which is one of the key components of 2D and 3D DNA nanoarchitectures. We designed a DNA three-way junction structure with a preorganized metal binding site at its core. Bidentate bipyridine (bpy) ligands were attached to the nucleosides positioned at the center of each strand via Cu-catalyzed Huisgen cycloaddition (Ubpy). The melting profiles of the resulting three-way junction were investigated in the presence of various transition metal ions. The results showed that the addition of equimolar amounts of Ni2+ ion remarkably stabilized the junction structure by 12 °C compared to the unmodified junction. In contrast, three-way junctions with only one or two bpy ligands showed little stabilization upon Ni2+ addition. Thus, the stabilization was attributed to metal-mediated crosslinking among three DNA strands through the formation of a Ni(bpy)3 2+ complex at the junction point as further evidenced by the UV spectral changes. Furthermore, CD spectroscopy indicated the predominant formation of the Λ-isomer that could be a result of the chiral DNA environment at the core of the threeway junction. The “metal-locked” DNA junction motif presented here will have future applications in the field of DNA nanoarchitectures as a building block for stabilization and conformation switching of whole DNA structures by reversible metal complexation. [1] Y. Takezawa, M. Shionoya, Acc. Chem. Res. 2012, 45, 2066. [2] J.-L. H. A. Duprey, Y. Takezawa, M. Shionoya, Angew. Chem., Int. Ed. 2013, 52, 1212. CONTROL ID: 1712972 TITLE: DNA recognition by lanthanide-peptide complexes AUTHORS/INSTITUTIONS: L. Ancel, C. Gateau, C. Lebrun, P. Delangle, CEA SCIB, Grenoble, Isère, FRANCE| CURRENT CATEGORY: Metals and Nucleic Acids ABSTRACT BODY: Abstract Body: Lanthanide ions are powerful luminescent tools thanks to their unique spectroscopic properties (long lived exited state, narrow band emission). Because of the similarity of their ionic radii with Ca 2+[1], studies were done on the design of peptides scaffold able to coordinate Ln3+[2] ions We demonstrated that hexapeptides containing two unnatural amino acids bearing aminopolyacetate side chains (tridentate or pentadentate) and a tryptophan residue as Tb3+sensitizer provide lanthanide-peptide complexes sufficiently stable to avoid dissociation in water at physiological pH. [3,4]BODY: Abstract Body: Lanthanide ions are powerful luminescent tools thanks to their unique spectroscopic properties (long lived exited state, narrow band emission). Because of the similarity of their ionic radii with Ca 2+[1], studies were done on the design of peptides scaffold able to coordinate Ln3+[2] ions We demonstrated that hexapeptides containing two unnatural amino acids bearing aminopolyacetate side chains (tridentate or pentadentate) and a tryptophan residue as Tb3+sensitizer provide lanthanide-peptide complexes sufficiently stable to avoid dissociation in water at physiological pH. [3,4] Compounds that bind DNA, an essential biomolecule that is storing and dispensing genetic data required for life, are extremely useful as biochemical tools for visualization of DNA both in vitro and inside the cell. That’s why, we combined our lanthanide peptide complexes with DNA recognition units for the detection of DNA double helix. We present here hexapeptide sequences containing DNA intercalating units. The intercalators were inserted in the peptide sequences in place of tryptophan and are expected to sensitize the Eu3+ cation luminescence. Taking advantage of Ln3+luminescence properties, we demonstrated that the intercalating unit are able to sensitize Eu3+and that a unique EuP complex is formed in water at physiological pH. Luminescence, DNA melting temperature and absorbance measurements demonstrate that the EuP complexes bind ct-DNA through an intercalative process as efficient as the free intercalator core. Hence, the time resolved luminescence of europium in these novel complexes efficiently senses the interaction with DNA.[5] Acknowledgements: Financial support Rhône-Alpes Region and CEA Grenoble 1 E.Pidccock, G.R.Morre, J. Biol. Inorg. Chem, 2001, 6, 479 2 M.Nitz, K.JFrantz, R.L.Maglathlin, B.Imperiali, ChemBioChem, 2003, 4, 272 3 F.Cisnetti, C.Gateau, C.Lebrun, P. Delangle, Chem. Eur. J., 2009, 15, 7456 4/ A.Niedzwiecka, F.Cisnetti, C.Lebrun, P.Delangle, Inorg Chem, 2012, 51, 5458 5/ L.Ancel, C.Gateau, C.Lebrun, P.Delangle, Inorg. Chem.,2013 , 52, 552 CONTROL ID: 1714042 TITLE: Structural insights into complex between human telomeric quadruplex DNA and N-methylmesoporphyrin IX AUTHORS/INSTITUTIONS: L.A. Yatsunyk, S.T. Miller, J.M. Nicoludis, S.P. Barrett, Chemistry and Biochemistry, Swarthmore college, Swarthmore, Pennsylvania, UNITED STATES| CURRENT CATEGORY: Metals and Nucleic Acids ABSTRACT BODY: Abstract Body: N-methyl mesoporphyrin IX (NMM) is an excellent G-quadruplexes (GQ) ligand with a wide range of applications in biology and chemistry. Here, we present the X-ray crystal structure of a complex between NMM and human telomeric DNA dAGGG(TTAGGG)3, Tel22, determined in two space groups, P21212 and P6, at 1.65 and 2.15 Å resolution, respectively. The former is the highest resolution structure of the human telomeric GQ DNA reported to date. The biological unit contains a Tel22 dimer of 5'-5' stacked parallel-stranded quadruplexes capped on both ends with NMM, supporting the spectroscopically determined 1:1 stoichiometry. NMM is capable of adjusting its macrocycle geometry to closely match that of the terminal G-tetrad required for efficient π-π stacking. The out-ofplane N-methyl group of NMM fits perfectly into the center of the parallel GQ core where it aligns with potassium ions. In contrast, the interaction of the N-methyl group with duplex DNA or antiparallel GQ would lead to steric clashes that prevent NMM from binding to these structures, thus explaining its unique selectivity. Based on the biochemical data, binding of NMM to Tel22 does not rely on relatively non-specific electrostatic interactions, which characterize most canonical GQ ligands. NMM could serve as an important prototype for the development of truly selective GQ ligands, and our structural data will help inform the further developments in this area.BODY: Abstract Body: N-methyl mesoporphyrin IX (NMM) is an excellent G-quadruplexes (GQ) ligand with a wide range of applications in biology and chemistry. Here, we present the X-ray crystal structure of a complex between NMM and human telomeric DNA dAGGG(TTAGGG)3, Tel22, determined in two space groups, P21212 and P6, at 1.65 and 2.15 Å resolution, respectively. The former is the highest resolution structure of the human telomeric GQ DNA reported to date. The biological unit contains a Tel22 dimer of 5'-5' stacked parallel-stranded quadruplexes capped on both ends with NMM, supporting the spectroscopically determined 1:1 stoichiometry. NMM is capable of adjusting its macrocycle geometry to closely match that of the terminal G-tetrad required for efficient π-π stacking. The out-ofplane N-methyl group of NMM fits perfectly into the center of the parallel GQ core where it aligns with potassium ions. In contrast, the interaction of the N-methyl group with duplex DNA or antiparallel GQ would lead to steric clashes that prevent NMM from binding to these structures, thus explaining its unique selectivity. Based on the biochemical data, binding of NMM to Tel22 does not rely on relatively non-specific electrostatic interactions, which characterize most canonical GQ ligands. NMM could serve as an important prototype for the development of truly selective GQ ligands, and our structural data will help inform the further developments in this area. Figure 1. Biological unit of Tel22-NMM structure. Quadruplex dimer is formed by head-to-head stacking of 5’ Gtetrads from two monomers. This dimer is capped by two NMM molecules; the binding mode is known as endstacking. This is the first crystallographic observation of porphyrin stacking onto a guanine quadruplex. Note, our and others earlier spectroscopic studies suggested this biding mode. CONTROL ID: 1714549 TITLE: Investigating the Interactions of Metal Complexes with G-Quadruplex forming DNA AUTHORS/INSTITUTIONS: H. Pritchard, M.J. Hannon, School of Chemistry, University of Birmingham, Edgbaston, UNITED KINGDOM| CURRENT CATEGORY: Metals and Nucleic Acids ABSTRACT BODY: Abstract Body: Many metal complexes used as anticancer drugs that target DNA are aimed at duplex DNA, for example Cisplatin. DNA is in its duplex form when in ‘sleep mode’ therefore it may be more useful to target structures seen in DNA when processes like replication, protein synthesis and transcription are occurring.BODY: Abstract Body: Many metal complexes used as anticancer drugs that target DNA are aimed at duplex DNA, for example Cisplatin. DNA is in its duplex form when in ‘sleep mode’ therefore it may be more useful to target structures seen in DNA when processes like replication, protein synthesis and transcription are occurring. Of particular interest are the G-quadruplexes found in the promoter regions of the c-myc proto-oncogene. The activated promoter region of c-myc has been linked to the production of growth stimulating genes in many types of cancer. When G-quadruplexes are able to form in this region they can prevent the action of certain proteins which require single stranded DNA binding in order to turn on the activity of the cell. Therefore the work presented here is based on the design of a complex that has the potential to be an efficient G-Quadruplex binder. The basic concept of the complex design was to use a ligand structure that is planar with the ability to π stack onto the top face of a G-quadruplex. Preference for G-quadruplex binding over duplex binding should arise if the ligand is large enough; hence a biisoquinoline unit was chosen. Incorporating a metal into the structure provides favourable electrostatic interactions with the DNA which in this case was achieved by using palladium and platinum respectively. Circular Dichroism results of the palladium complex in the presence of 100 mM KCl show an induced signal at 380 nm when titrating against telomeric DNA. A possible change in conformation of the quadruplex from antiparallel to parallel is also observed. Similar results can also be seen with cmyc DNA. Fluorescent indicator displacement experiments show very promising DC50 values below 0.5 μM for the Htelo DNA (telomeric sequence) and cmyc DNA which are comparable to those quoted in the literature for good G-quadruplex binders. Structure of biisoquinoline complex (alongside a G-quartet) and the palladium complex CD spectrum with Htelo DNA (ratio 0-8:1) in the presence of 100 mM KCl, 10 mM Tris-HCl CONTROL ID: 1714935 TITLE: Palladium(II) complexes with N,S-donor ligand: Synthesis, cytotoxicity, DNA interaction and topoisomerase II inhibition AUTHORS/INSTITUTIONS: F. Rocha, C. Barra, A. Mauro, A. Godoy Netto, Química Geral e Inorgânica, Universidade Estadual Paulista Júlio de Mesquita Fillho, Araraquara, SP, BRAZIL|I. Carlos, Departamento de Análises Clínicas, Universidade Estadual Paulista Júlio de Mesquita Fillho, Araraquara, SP, BRAZIL|S. Garrido, Bioquímica e tecnologia, Universidade Estadual Paulista Júlio de Mesquita Fillho, Araraquara, SP, BRAZIL|L. Nauton, M. El Ghozzi, A. Gautier, CNRS UMR 6296, Clermont–Université, Université Blaise Pascal, Clermont-Ferrand, FRANCE|L. Morel, GreD UMR 6247 CNRS, INSERM U931, Clermont–Université, Université Blaise Pascal, Clermont-Ferrand, FRANCE| CURRENT CATEGORY: Metals and Nucleic Acids ABSTRACT BODY: Abstract Body: Pd(II) complexes are interesting alternative candidates for metal antitumor drugs due to their structural similarities to Pt(II) complexes. A good strategy to afford new biologically active compounds is incorporate N,S–chelating ligands in square-planar complexes. Motivated by the potential uses of these compounds as anticancer agents, we present the synthesis and cytotoxicity of four cationic complexes of the type [PdX(PhT)(PPh3)]X {X = Cl (1 ); Br (2); I (3); SCN (4); PhT = 4–phenyl–3–thiosemicarbazide} and their ability to interact with DNA and topoisomerase II. The synthesis of 1-4 was achieved starting from bisacetonitriledichloropalladium(II). First, triphenylphosphine (PPh3) and PhT displace the labile ligands acetonitrile and one of the two Cl atoms to obtain 1. In a second step, the Cl atoms are easily replaced by Br, I and SCN ions by the addition of two equivalents of their appropriate potassium salt to afford 2–4. The formation of the N,S-chelated products was proved by spectroscopic data. IR spectra show an important variation of 30 cm-1 for the νC=S after complexation. Variation of ~ 4 ppm downfield of the chemical shift (1H NMR) was observed for the two N2 protons after complexation. Ultimately, the structure was proved by X–ray diffraction of a crystal of complex 3 (figure 1). The cytotoxicity of the complexes and cisplatin were determined against two murine cell lines, LM3 (mammary adenocarcinoma) and LP07 (lung adenocarcinoma). Compounds 1–4 exhibited good activity that overcame cisplatin’s in the case of LM3. The binding of the complexes with a purine base (guanosine) was investigated by 1H NMR and mass spectrometry, showing that the coordination of guanosine occurred through N7. However, gel electrophoresis assay demonstrated that 1–4 unwind the DNA plasmid only at high concentrations, suggesting that the cytotoxicity mechanisms of 1-4 may not necessarily involve interaction with DNA. Therefore, the ability of complexes to act as topoisomerase II inhibitors was tested and the results showed that the compounds 2-4 inhibited this enzyme at concentrations between 5 – 25 μM.BODY: Abstract Body: Pd(II) complexes are interesting alternative candidates for metal antitumor drugs due to their structural similarities to Pt(II) complexes. A good strategy to afford new biologically active compounds is incorporate N,S–chelating ligands in square-planar complexes. Motivated by the potential uses of these compounds as anticancer agents, we present the synthesis and cytotoxicity of four cationic complexes of the type [PdX(PhT)(PPh3)]X {X = Cl (1 ); Br (2); I (3); SCN (4); PhT = 4–phenyl–3–thiosemicarbazide} and their ability to interact with DNA and topoisomerase II. The synthesis of 1-4 was achieved starting from bisacetonitriledichloropalladium(II). First, triphenylphosphine (PPh3) and PhT displace the labile ligands acetonitrile and one of the two Cl atoms to obtain 1. In a second step, the Cl atoms are easily replaced by Br, I and SCN ions by the addition of two equivalents of their appropriate potassium salt to afford 2–4. The formation of the N,S-chelated products was proved by spectroscopic data. IR spectra show an important variation of 30 cm-1 for the νC=S after complexation. Variation of ~ 4 ppm downfield of the chemical shift (1H NMR) was observed for the two N2 protons after complexation. Ultimately, the structure was proved by X–ray diffraction of a crystal of complex 3 (figure 1). The cytotoxicity of the complexes and cisplatin were determined against two murine cell lines, LM3 (mammary adenocarcinoma) and LP07 (lung adenocarcinoma). Compounds 1–4 exhibited good activity that overcame cisplatin’s in the case of LM3. The binding of the complexes with a purine base (guanosine) was investigated by 1H NMR and mass spectrometry, showing that the coordination of guanosine occurred through N7. However, gel electrophoresis assay demonstrated that 1–4 unwind the DNA plasmid only at high concentrations, suggesting that the cytotoxicity mechanisms of 1-4 may not necessarily involve interaction with DNA. Therefore, the ability of complexes to act as topoisomerase II inhibitors was tested and the results showed that the compounds 2-4 inhibited this enzyme at concentrations between 5 – 25 μM. Figure 1. ORTEP view of complex 3. CONTROL ID: 1714936 TITLE: DNA binding and citotoxicity of Pd(II) complexes bearing 1,10-phenanthroline and thioureas ligands AUTHORS/INSTITUTIONS: C.V. Barra, F.V. Rocha, A.E. Mauro, R.C. Frem, A.V. Godoy Netto, Departamento de Química Geral e Inorgânica, IQ Unesp Araraquara, Araraquara, BRAZIL|L. Morel, GreD UMR 6247 CNRS, INSERM U931, Clermont–Université, Université Blaise Pascal, Clermont-Ferrand, FRANCE|A. Gautier, CNRS, UMR 6296, Clermont–Université, Université Blaise Pascal, Clermont-Ferrand, FRANCE| CURRENT CATEGORY: Metals and Nucleic Acids ABSTRACT BODY: Abstract Body: Metallointercalators represent a promising alternative in cancer chemotherapy. Modifications at the intercalator backbone as well the ancillary ligand may result in changes in the binding modes, drug DNA association constants and cytotoxicity. We present herein DNA binding and cytotoxic studies on a series of complexes of general formulae [Pd(phen)L2] 2+ incorporating the intercalator 1,10-phenanthroline and thiourea ligands (L = thiourea 1, Nmethylthiourea 2, N,N’-dimethylthiourea 3). The binding constants (Kb) for the interaction of the complexes with SS-DNA were determined by UV spectroscopy. The values obtained for Kb were 4.8×10 4 M−1 – 7.0×104 M−1, which falls in the range of what found for metallointercalators. Competitive experiments with ethidium bromide (EB) were investigated by fluorescence spectroscopy and show that all the complexes were able to displace EB from the DNA–EB complex, which is also indicative of an intercalative mode of binding. DNA-unwinding results showed that the complexes can induce the unwinding of the plasmid DNA. In order to verify the ability of the complexes to interact covalently with DNA, guanosine was used as a model system and the reaction was monitored by 1H NMR. As expected, the reaction has not occurred, excluding the possibility of covalent interaction. Compounds were tested against KB (oral carcinoma), MCF7 (human breast carcinoma) and MCF7-R (resistant human breast carcinoma) cell lines. Cytotoxic effects were expressed as cellular viability (Figure). Although the complexes were inactive towards MCF7 cells at 10 μM, they showed good cytotoxic activity towards MCF7-R. This finding suggest that the cytotoxicity mechanisms of the Pd(II) compounds differ from that observed for cisplatin. Complexes 2 and 3 were more active than 1 in both KB and MCF7-R cells. From DNA binding experiments, there appears to be no significant difference between any of the metal complexes, suggesting that DNA binding affinity is not a key determinant of the observed difference in their cytotoxicity.BODY: Abstract Body: Metallointercalators represent a promising alternative in cancer chemotherapy. Modifications at the intercalator backbone as well the ancillary ligand may result in changes in the binding modes, drug DNA association constants and cytotoxicity. We present herein DNA binding and cytotoxic studies on a series of complexes of general formulae [Pd(phen)L2] 2+ incorporating the intercalator 1,10-phenanthroline and thiourea ligands (L = thiourea 1, Nmethylthiourea 2, N,N’-dimethylthiourea 3). The binding constants (Kb) for the interaction of the complexes with SS-DNA were determined by UV spectroscopy. The values obtained for Kb were 4.8×10 4 M−1 – 7.0×104 M−1, which falls in the range of what found for metallointercalators. Competitive experiments with ethidium bromide (EB) were investigated by fluorescence spectroscopy and show that all the complexes were able to displace EB from the DNA–EB complex, which is also indicative of an intercalative mode of binding. DNA-unwinding results showed that the complexes can induce the unwinding of the plasmid DNA. In order to verify the ability of the complexes to interact covalently with DNA, guanosine was used as a model system and the reaction was monitored by 1H NMR. As expected, the reaction has not occurred, excluding the possibility of covalent interaction. Compounds were tested against KB (oral carcinoma), MCF7 (human breast carcinoma) and MCF7-R (resistant human breast carcinoma) cell lines. Cytotoxic effects were expressed as cellular viability (Figure). Although the complexes were inactive towards MCF7 cells at 10 μM, they showed good cytotoxic activity towards MCF7-R. This finding suggest that the cytotoxicity mechanisms of the Pd(II) compounds differ from that observed for cisplatin. Complexes 2 and 3 were more active than 1 in both KB and MCF7-R cells. From DNA binding experiments, there appears to be no significant difference between any of the metal complexes, suggesting that DNA binding affinity is not a key determinant of the observed difference in their cytotoxicity. Effect of the complexes on tumor cells viability after 48 h of incubation CONTROL ID: 1715517 TITLE: Introducing Selectivity to Differential Protein Sensors AUTHORS/INSTITUTIONS: L. Motiei, D. Margulies, Weizmann Institute of Science, Rehovot, ISRAEL| CURRENT CATEGORY: Metals and Nucleic Acids ABSTRACT BODY: Abstract Body: Current approaches to disease diagnosis and proteomics are largely based on the ability of monoclonal antibodies to bind protein biomarkers with high affinity and selectivity. In recent years, however, an alternative approach has been developed in which combinations of proteins are identified using cross-reactive sensor arrays that are inspired by the olfactory neural system. Although such devices have been shown to detect proteins quickly and effectively, all of them suffer from a major limitation, namely, their inability to identify proteins within mixtures. We have designed, synthesized, and prepared pattern-based detection arrays that can discriminate between Glutathione S-Transferases (GSTs) in biological mixtures. GSTs were chosen as the protein targets because different expression profiles within this family have been associated with the progression of various diseases. Our fluorescent arrays are based on modified DNA-duplexes that can bind these enzymes with dual interaction modes: specific and non-specific. On one hand, they bind the enzymes' active site with high affinity and selectivity. On the other hand, they possess a nonspecific binding region that enables them to differentiate between closely related isozymes. Our results indicate that these arrays can discriminate between different combinations and concentrations of GSTs even in the presence common serum proteins such as immunoglobulin G and transferrin.BODY: Abstract Body: Current approaches to disease diagnosis and proteomics are largely based on the ability of monoclonal antibodies to bind protein biomarkers with high affinity and selectivity. In recent years, however, an alternative approach has been developed in which combinations of proteins are identified using cross-reactive sensor arrays that are inspired by the olfactory neural system. Although such devices have been shown to detect proteins quickly and effectively, all of them suffer from a major limitation, namely, their inability to identify proteins within mixtures. We have designed, synthesized, and prepared pattern-based detection arrays that can discriminate between Glutathione S-Transferases (GSTs) in biological mixtures. GSTs were chosen as the protein targets because different expression profiles within this family have been associated with the progression of various diseases. Our fluorescent arrays are based on modified DNA-duplexes that can bind these enzymes with dual interaction modes: specific and non-specific. On one hand, they bind the enzymes' active site with high affinity and selectivity. On the other hand, they possess a nonspecific binding region that enables them to differentiate between closely related isozymes. Our results indicate that these arrays can discriminate between different combinations and concentrations of GSTs even in the presence common serum proteins such as immunoglobulin G and transferrin. CONTROL ID: 1715576 TITLE: Dinuclear Ruthenium-Silicon Complexes as DNA and G4 DNA Binding Agents AUTHORS/INSTITUTIONS: J. Henker, S. Glöckner, E. Meggers, FB Chemie, Philipps-Universität Marburg, Marburg, GERMANY| CURRENT CATEGORY: Metals and Nucleic Acids ABSTRACT BODY: Abstract Body: Octahedral metal complexes permit the construction of demanding globular and rigid structures due to their sophisticated stereochemistry and chelation-induced conformational restrictions. Utilizing these features led to the design of kinetically inert metal-based enzyme inhibitors and nucleic acid binders with interesting properties like DNA-light-switch behavior.[1,2] However, general concerns about the toxicity of such complexes prevent their wide use in biomedical research and therapy.[3] Thus finding less harmful substitutions for the metal center is an upcoming challenge.BODY: Abstract Body: Octahedral metal complexes permit the construction of demanding globular and rigid structures due to their sophisticated stereochemistry and chelation-induced conformational restrictions. Utilizing these features led to the design of kinetically inert metal-based enzyme inhibitors and nucleic acid binders with interesting properties like DNA-light-switch behavior.[1,2] However, general concerns about the toxicity of such complexes prevent their wide use in biomedical research and therapy.[3] Thus finding less harmful substitutions for the metal center is an upcoming challenge. An excellent replacement is silicon, because of lower toxicity and its availability in larger amounts. Even though silicon is the higher homologue of carbon, silicon does not show the same structural limitations as a coordination number beyond four is possible. As a consequence, silicon is able to build up pentaand hexacoordinated complexes that are interesting and structurally stable substitutions for a missing hypervalent carbon. In our group, the use of silicon as a coordination center is well investigated and we are able to synthesize octahedral silicon complexes with three bidentate ligands.[3] These complexes are hydrolytically completely stable and can easily be modified by standard organic reactions like nitration or halogenation. Furthermore, we expect that multinuclear silicon complexes provide interesting chemical and physicochemical properties that can be used in different biological applications, for example DNA binding or protein surface recognition. As a starting point for the synthesis of such complexes, we first synthesized different dinuclear complexes composed of a ruthenium and a silicon center bridged by a dppzOH-ligand. These complexes show a strong affinity to DNA, with a binding constant to duplex DNA and G4 DNA of about 106–107 M-1 and thus within the range of the known, strong DNA binding agents [Ru(dppz)(phen)2] 2+ and [Ru(bpy)2(μ-dppz)Ru(bpy)2] 4+.[2,3] [1] Blanck et al., Organometallics 2011, 30, 4598-4606. [2] Lutterman et al., J. Am. Chem. Soc. 2008, 130, 1163-1170. [3] Xiang et al., Chem. Commun. 2012, 48, 7131-7133. CONTROL ID: 1716075 TITLE: Study of Mn(II)-DNA interaction using pulsed high magnetic-field Electron Paramagnetic Resonance. AUTHORS/INSTITUTIONS: E.M. Bruch, L.C. Tabares, S. Un, DSV, CEA-Saclay, Gif-sur-Yvette, Île-de-France, FRANCE| CURRENT CATEGORY: Metals and Nucleic Acids ABSTRACT BODY: Abstract Body: The interaction of DNA with Mn(II) was studied using pulsed high magnetic-field Electron Paramagnetic Resonance techniques (ENDOR and PELDOR-NMR). Several nucleic acids (genomic DNA and dimeric or monomeric oligonucleotides) and free nucleotides were used as models for the interaction. ENDOR was used to study the amount and the nature of 31P from the backbone bound to the manganese and PELDOR-NMR was used to detect the binding to the nitrogen of the bases. The details of the interaction of this metal with nucleic acids, studied mainly by crystallographic techniques, have suggested that the metal interacts with the N7 of a guanosine or via a coordinated water molecule with the phosphate backbone. Also Mn-nucleic acids complexes show a broad 31P ENDOR spectrum with a high splitting. This signal has only been observed in Mn-nucleic acids complexes and has been attributed to a metal bound to a phosphate and the N7 of a guanosine simultaneously. Our results indicate that the metal can interacts directly with both the phosphate backbone via the oxygen and the bases via the nitrogen depending on the relative Mn(II)/nucleic acids ratio. Also we found that the 31P ENDOR spectrum characteristic from nucleic acids doesn’t require the metal binding the base and the backbone simultaneously. On the other hand the interaction with the backbone appears to be direct and independent of the dimerization state of the oligonucleotide. Finally, for the oligonucleotides used in this work, the nature of the interaction appears to be independent of the nucleotide composition. (No Image Selected)BODY: Abstract Body: The interaction of DNA with Mn(II) was studied using pulsed high magnetic-field Electron Paramagnetic Resonance techniques (ENDOR and PELDOR-NMR). Several nucleic acids (genomic DNA and dimeric or monomeric oligonucleotides) and free nucleotides were used as models for the interaction. ENDOR was used to study the amount and the nature of 31P from the backbone bound to the manganese and PELDOR-NMR was used to detect the binding to the nitrogen of the bases. The details of the interaction of this metal with nucleic acids, studied mainly by crystallographic techniques, have suggested that the metal interacts with the N7 of a guanosine or via a coordinated water molecule with the phosphate backbone. Also Mn-nucleic acids complexes show a broad 31P ENDOR spectrum with a high splitting. This signal has only been observed in Mn-nucleic acids complexes and has been attributed to a metal bound to a phosphate and the N7 of a guanosine simultaneously. Our results indicate that the metal can interacts directly with both the phosphate backbone via the oxygen and the bases via the nitrogen depending on the relative Mn(II)/nucleic acids ratio. Also we found that the 31P ENDOR spectrum characteristic from nucleic acids doesn’t require the metal binding the base and the backbone simultaneously. On the other hand the interaction with the backbone appears to be direct and independent of the dimerization state of the oligonucleotide. Finally, for the oligonucleotides used in this work, the nature of the interaction appears to be independent of the nucleotide composition. (No Image Selected) CONTROL ID: 1716084 TITLE: Use of Osmium (III) Complexes to determine influence of base mismatches on DNA-Protein Crosslinking AUTHORS/INSTITUTIONS: K.R. Miller, Z.A. Perez, E.D. Stemp, Physical Sciences and Mathematics, Mount St. Mary's College, Los Angeles, California, UNITED STATES|K.N. Schaefer, Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, UNITED STATES| CURRENT CATEGORY: Metals and Nucleic Acids ABSTRACT BODY: Abstract Body: 8-oxoguanine is a common oxidation product in DNA and can lead to missense mutations. The metallointercalator Ru(phen)2dppz2+ is a useful luminescent probe for DNA that has also found use as a guanineselective oxidizing agent via the flash-quench technique. Here, we introduce its osmium analogue as a way to selectively oxidize 8-oxoguanine in double-stranded DNA as visualized by DNA-protein crosslinking and MaxamGilbert sequencing. With a 3+/2+ couple of 1.15 V, Os(phen)2dppz3+ , this metallointercalator should be able to oxidize 8-oxo-G (~0.7 V) without oxidizing guanine (~1.3V). MALDI mass spectrometry data confirms that oxidizing guanine using Ru(NH3)63+ and Ru(phen)2dppz2+ produces 8-oxoguanine. In plasmid DNA where 8-oxo-G has been incorporated as above, further flash-quench treatment with Os(phen)2dppz2+ and Co(NH3)5Cl2+ leads to crosslinking with histone protein in gel shift experiments. Furthermore, in gel shift experiments with a duplex of the oligonucleotide 5’-ATATGATAT8GATATGATAT -3’ (8 = 8-oxo-G), flash-quench treatment with Ru(phen)2dppz2+ in the presence of histone produces a band of intermediate mobility (presumably 1:1 crosslink) and well-shifted material. In contrast, analogous treatment with Os(phen)2dppz2+ produces only the band of intermediate mobility, consistent with the presence of only a single site that is oxidizable by the osmium complex. A Maxam-Gilbert sequencing gel shows damage only at the 8-oxo-G site upon flash quench treatment with the osmium complex, as expected. Lastly, control experiments indicate that with a duplex of the oligonucleotide 5’-ATATGATATGGATATGATAT -3’ flash quench treatment with Ru(phen)2dppz2+ in the presence of histone produces well-shifted material, whereas treatment with Os(phen)2dppz2+ does not produce crosslinked material, as expected since the osmium (III) complex formed should not be capable of guanine oxidation. Taken together, these results show that Os(phen)2dppz2+ is a promising selective oxidant of 8-oxoguanine in double stranded DNA. (No Image Selected)BODY: Abstract Body: 8-oxoguanine is a common oxidation product in DNA and can lead to missense mutations. The metallointercalator Ru(phen)2dppz2+ is a useful luminescent probe for DNA that has also found use as a guanineselective oxidizing agent via the flash-quench technique. Here, we introduce its osmium analogue as a way to selectively oxidize 8-oxoguanine in double-stranded DNA as visualized by DNA-protein crosslinking and MaxamGilbert sequencing. With a 3+/2+ couple of 1.15 V, Os(phen)2dppz3+ , this metallointercalator should be able to oxidize 8-oxo-G (~0.7 V) without oxidizing guanine (~1.3V). MALDI mass spectrometry data confirms that oxidizing guanine using Ru(NH3)63+ and Ru(phen)2dppz2+ produces 8-oxoguanine. In plasmid DNA where 8-oxo-G has been incorporated as above, further flash-quench treatment with Os(phen)2dppz2+ and Co(NH3)5Cl2+ leads to crosslinking with histone protein in gel shift experiments. Furthermore, in gel shift experiments with a duplex of the oligonucleotide 5’-ATATGATAT8GATATGATAT -3’ (8 = 8-oxo-G), flash-quench treatment with Ru(phen)2dppz2+ in the presence of histone produces a band of intermediate mobility (presumably 1:1 crosslink) and well-shifted material. In contrast, analogous treatment with Os(phen)2dppz2+ produces only the band of intermediate mobility, consistent with the presence of only a single site that is oxidizable by the osmium complex. A Maxam-Gilbert sequencing gel shows damage only at the 8-oxo-G site upon flash quench treatment with the osmium complex, as expected. Lastly, control experiments indicate that with a duplex of the oligonucleotide 5’-ATATGATATGGATATGATAT -3’ flash quench treatment with Ru(phen)2dppz2+ in the presence of histone produces well-shifted material, whereas treatment with Os(phen)2dppz2+ does not produce crosslinked material, as expected since the osmium (III) complex formed should not be capable of guanine oxidation. Taken together, these results show that Os(phen)2dppz2+ is a promising selective oxidant of 8-oxoguanine in double stranded DNA. (No Image Selected) CONTROL ID: 1716238 TITLE: Setting up photoinduced charge transfer through metal-modified nucleic acids: Towards molecular wires AUTHORS/INSTITUTIONS: B.S. Dave, S. Johannsen, R. Sigel, Institute of Inorganic Chemistry, University of Zürich, Zürich, SWITZERLAND| CURRENT CATEGORY: Metals and Nucleic Acids ABSTRACT BODY: Abstract Body: Nucleic acids are propitious candidates for the use as templates to design molecular wires and magnets on nano-scale. This is mainly attributed to their superb self-assembly properties and robust structural features. Although the conductivity of natural nucleic acids is insufficient for their direct application in nano-scale electronic architectures, the site-specific functionalization of nucleic acids can overcome this shortcoming.[1] The formation of metal-mediated base pairs is the most prominent method to insert metal ions in a specific manner along the helix of nucleic acid.[2–4] Our goal is to study the electronic properties of such metal-modified nucleic acids. Using metallointercalators as an electron donor-acceptor couple we are setting-up a photoinduced charge transfer experiment with metal-modified RNA duplexes. Our RNA duplexes contain continuous stretches of 2, 3 and 6 uracil-uracil mismatches that form UHg(II)-U base pairs upon addition of Hg(II) ions.[4] As a first step towards setting-up our experiment, we characterize the binding of the donor metallointercalator [Ru(bpy)2dppz]2+ using absorption and emission experiments. Our data suggests an intercalative mode of binding between the metallointercalator and our Hg(II)-modified RNA duplexes. Intercalative binding is a feasible method through which an electron can be injected into the base-stack of nucleic acids without covalently attaching metal complexes to it. These studies are an important step to further development of the charge transfer experiment of metal-modified nucleic acids.BODY: Abstract Body: Nucleic acids are propitious candidates for the use as templates to design molecular wires and magnets on nano-scale. This is mainly attributed to their superb self-assembly properties and robust structural features. Although the conductivity of natural nucleic acids is insufficient for their direct application in nano-scale electronic architectures, the site-specific functionalization of nucleic acids can overcome this shortcoming.[1] The formation of metal-mediated base pairs is the most prominent method to insert metal ions in a specific manner along the helix of nucleic acid.[2–4] Our goal is to study the electronic properties of such metal-modified nucleic acids. Using metallointercalators as an electron donor-acceptor couple we are setting-up a photoinduced charge transfer experiment with metal-modified RNA duplexes. Our RNA duplexes contain continuous stretches of 2, 3 and 6 uracil-uracil mismatches that form UHg(II)-U base pairs upon addition of Hg(II) ions.[4] As a first step towards setting-up our experiment, we characterize the binding of the donor metallointercalator [Ru(bpy)2dppz]2+ using absorption and emission experiments. Our data suggests an intercalative mode of binding between the metallointercalator and our Hg(II)-modified RNA duplexes. Intercalative binding is a feasible method through which an electron can be injected into the base-stack of nucleic acids without covalently attaching metal complexes to it. These studies are an important step to further development of the charge transfer experiment of metal-modified nucleic acids. Financial support by the Swiss National Science Foundation (to SJ and RKOS), the University of Zurich, within the COST Action CM1105 is gratefully acknowledged. 1.S. Liu; G. H. Clever; Y. Takezawa; M. Kaneko; K. Tanaka; X. Guo; M. Shionoya, Angew. Chem. Int. Ed. 2011, 50, 8886. 2.K. Tanaka; M. Shionoya, J. Org. Chem. 1999, 64, 5002. 3.S. Johannsen; N. Megger; D. Böhme; R. K. O. Sigel; J. Müller, Nat. Chem.2010, 2, 229. 4.S. Johannsen; S. Paulus; N. Düpre; J. Müller; R. K. O. Sigel, J. Inorg. Biochem. 2008, 102, 1141.