Tarita Biver

Anticancer activity and DNA binding of a bifunctional Ru(II) arene aqua-complex with the 2,4-diamino-6-(2-pyridyl)-1,3,5-triazine ligand.

The synthesis and full characterization of the new aqua-complex [(η(6)-p-cymene)Ru(OH2)(κ(2)-N,N-2-pydaT)](BF4)2, [2](BF4)2, and the nucleobase derivative [(η(6)-p-cymene)Ru(9-MeG)(κ(2)-N,N-2-pydaT)](BF4)2, [4](PF6)2, where 2-pydaT = 2,4-diamino-6-(2-pyridyl)-1,3,5-triazine and 9-MeG = 9-methylguanine, are reported here. The crystal structures of both [4](PF6)2 and the chloro complex [(η(6)-p-cymene)RuCl(κ(2)-N,N-2-pydaT)](PF6), [1](PF6), have been elucidated by X-ray diffraction. The former provided relevant information regarding the interaction of the metallic fragment [(η(6)-p-cymene)Ru(κ(2)-N,N-2-pydaT)](2+) and a simple model of DNA. NMR and kinetic absorbance studies have proven that the aqua-complex [2](BF4)2 binds to the N7 site of guanine in nucleobases, nucleotides, or DNA. A stable bifunctional interaction (covalent and partially intercalated) between the [(η(6)-p-cymene)Ru(κ(2)-N,N-2-pydaT)](2+) fragment and CT-DNA has been corroborated by kinetic, circular dichroism, viscometry, and thermal denaturation experiments. The reaction mechanism entails the very fast formation of the Ru-O-(PO3) linkage prior to the fast intercalation of the 2-pydaT fragment. Then, a Ru-N7-(G) covalent bond is formed at the expense of the Ru-O-(PO3) bond, yielding a bifunctional complex. The dissociation rate of the intercalated fragment is slow, and this confers additional interest to [2](BF4)2 in view of the likely correlation between slow dissociation and biological activity, on the assumption that DNA is the only biotarget. Furthermore, [2](BF4)2 displays notable pH-dependent cytotoxic activity in human ovarian carcinoma cells (A2780, IC50 = 11.0 μM at pH = 7.4; IC50 = 6.58 μM at pH = 6.5). What is more, complex [2](BF4)2 is not cross-resistant with cisplatin, exhibiting a resistance factor, RF(A2780cis), of 0.28, and it shows moderate selectivity toward the cancer cell lines, in particular, A2780cis (IC50 = 3.0 5 ± 0.08 μM), relative to human lung fibroblast cells (MRC-5; IC50 = 24 μM), the model for healthy cells.

Equilibria and kinetics of the intercalation of Pt-proflavine and proflavine into calf thymus DNA

The interaction of the cis-platinum derivative of proflavine [[PtCl(tmen)(2)][HNC(13)H(7)(NHCH(2)CH(2))(2)]](+) (PRPt) with CT-DNA is investigated by spectrophotometry and T-jump relaxation in 0.11M NaCl, pH 7.0, and 25 degrees C. The DNA-proflavine (PR) system is investigated under the same conditions. Static measurements indicate that base-dye interactions prevail and their analysis reveals that the site size for PRPt (n=2.6) is twice that found for PR (n=1.3). One relaxation effect is observed for the DNA/PR system and two effects for the DNA/PRPt system, the faster of them being similar to that of DNA/PR. The kinetics of the process are discussed in terms of the three-step sequence D+S <= => DS(I) <= => DS(II) <= => DS(III), where PR and the aromatic residues of PRPt intercalate into DNA by the same mechanism. The third step represents the penetration of platinum residues between base-pairs and is associated to remarkable enthalpy and entropy changes. Further mechanistic details are discussed.

Solvent effects on the thermodynamics and kinetics of coralyne self-aggregation.

The role of solvent effects on the thermodynamics and kinetics of the coralyne self-aggregation process has been investigated in ethanol-water mixtures of different compositions. The changes in the UV/visible spectra of coralyne and FAB/LSIMS mass spectrometry agreed well with the formation of a dimer species. 1D and 2D 1H experiments have allowed one to look into the features of the self-aggregation process and to determine the equilibrium constant and the deltaH0 and deltaS0 values for the aggregate formation in 0-50% ethanol-water mixtures. The kinetics of self-aggregation has been investigated by the T-jump chemical relaxation method, and the results have been interpreted in terms of dimer formation. The dependence of the relative viscosity of coralyne solutions on the dye concentration was studied in different ethanol-water mixtures. Finally, it was found that coralyne behaves as a solvatochromic indicator which is preferentially solvated according to the sequence ethanol > ethanol-water > water. All of the results concur in elucidating the relevant role of the hydrophobic interaction process of coralyne stack formation.

Synthesis, characterization, DNA interaction and potential applications of gold nanoparticles functionalized with Acridine Orange fluorophores.

Two new water-soluble gold nanoparticles (AO-TEG-Au and AO-PEG-Au NPs) are prepared and characterized. They are stabilized by thioalkylated oligoethylene glycols and functionalized with fluorescent Acridine Orange (AO) derivatives. Despite the different core sizes (11.8 and 3.9 nm respectively) and shell composition, they are both well dispersed and are stable in water, even if some self-aggregation is observed in the case of AO-TEG-Au NPs. However, AO-PEG-Au NPs show much lower emission efficiency with respect to AO-TEG-Au NPs. Spectrophotometric and spectrofluorometric experiments indicate that both types of nanoparticle are able to bind to calf thymus DNA, either by external binding or partial intercalation. Preliminary FACS flow cytometry tests seem to indicate that the AO-TEG-Au nanoparticle is able to cross the cell membrane where it is absorbed by Chinese hamster ovary (CHO) cells at the picomolar concentration level.

Solvent Effects on the Kinetics of the Interaction of 1-Pyrenecarboxaldehyde with Calf Thymus DNA

The kinetics of the interaction of a fluorescent probe, 1-pyrenecarboxaldehyde, with calf thymus DNA has been studied in different water/alcohol mixtures (ethanol, 2-propanol, and ter-butanol) at 25 degrees C, by using the stopped flow technique. The kinetic curves are biexponential and reveal the presence of two processes whose rates differ by about 1 order of magnitude on the time scale. The dependence of the reciprocal fast relaxation time on the DNA concentration is linear, whereas the concentration dependence of the reciprocal slow relaxation time tends to a plateau at high DNA concentrations. The simplest mechanism consistent with the kinetic results involves a simple two-step series mechanism reaction scheme. The first step corresponds to the formation of a precursor complex, (DNA/Py)(I), while the second one corresponds to full intercalation of the pyrene dye between the DNA base pairs. The values of the rate constants of both steps decrease as water activity decreases. The results have been discussed in terms of solvation of the species and changes in the viscosity of the solution.

Kinetic and equilibrium studies on the polyazamacrocycle neotetren: metal–complex formation and DNA interaction

The macrocyclic polyamine 2,5,8,11,14-pentaaza[15]-[15](2,9)[1,10]phenanthrolinophane (neotetren) is studied in its ability to coordinate Cu(ii) even at very low pH values and to interact, as a metal complex, with DNA. The kinetics and equilibria for 1 : 1 and 2 : 1 metal-ligand complexes formation are studied by the stopped-flow method and UV spectrophotometry. Differently protonated complexes are formed, with rate constants much lower than that of water exchange at copper(II) and other Cu(II)/amine systems, this behaviour being ascribed to ring effects and intra-molecular hydrogen bonds. Concerning the DNA/copper(II)-neotetren complexes interaction, analysis of data suggests an intercalative mode of binding. The kinetic results for both DNA/CuL and DNA/Cu(2)L systems agree with the sequence D + S <-->D,S <-->DS where the metal complexes (D) react with the DNA sites (S) leading to fast formation of an externally bound form (D,S) which is converted into an intercalated complex (DS). A very slow process is also detected and ascribed to a conformational change in the polynucleotide secondary structure where the metal centre plays a crucial role. Chromatographic experiments demonstrate that both the investigated Cu(II)/L complexes are able to cleave DNA, but only in the presence of hydrogen peroxide.

Preparation of organometallic ruthenium-arene-diaminotriazine complexes as binding agents to DNA.

The reactions of two diaminotriazine ligands 2,4-diamino-6-(2-pyridyl)-1,3,5-triazine (2-pydaT) and 6-phenyl-2,4-diamino-1,3,5-triazine (PhdaT) with ruthenium-arene precursors led to a new family of ruthenium(II) compounds that were spectroscopically characterized. Four of the complexes were cationic, with the general formula [(η(6)-arene)Ru(κ(2)-N,N-2-pydaT)Cl]X (X=BF(4), TsO; arene=p-cymene: 1·BF(4), 1·TsO; arene=benzene: 2·BF(4), 2·TsO). The neutral cyclometalated complex [(η(6)-p-cymene)Ru(κ(2)-C,N-PhdaT*)Cl] (3) was also isolated. The structures of complexes 2·BF(4) and 3·H(2)O were determined by X-ray diffraction. Complex 1·BF(4) underwent a partial reversible-aquation process in water. UV/Vis and NMR spectroscopic measurements showed that the reaction was hindered by the addition of NaCl and was pH-controlled in acidic solution. At pH 7.0 (sodium cacodylate) Ru-Cl complex 1·BF(4) was the only species present in solution, even at low ionic strength. However, in alkaline medium (KOH), complex 1·BF(4) underwent basic hydrolysis to afford a Ru-OH complex (5). Fluorimetric studies revealed that the interaction of complex 1·BF(4) with DNA was not straightforward; instead, its main features were closely linked to ionic strength and to the [DNA]/complex ratio. The bifunctional complex 1·BF(4) was capable of interacting concurrently through both its p-cymene and 2-pydaT groups. Cytotoxicity and genotoxicity studies showed that, contrary to the expected behavior, the complex species was biologically inactive; the formation of a Ru-OH complex could be responsible for such behavior.

A theoretical and experimental investigation of the spectroscopic properties of a DNA-intercalator salphen-type Zn(II) complex.

The photophysical and DNA-binding properties of the cationic zinc(II) complex of 5-triethylammonium methyl salicylidene ortho-phenylenediiminato (ZnL(2+)) were investigated by a combination of experimental and theoretical methods. DFT calculations were performed on both the ground and the first excited states of ZnL(2+) and on its possible mono- and dioxidation products, both in vacuo and in selected solvents mimicked by the polarizable continuum model. Comparison of the calculated absorption and fluorescence transitions with the corresponding experimental data led to the conclusion that visible light induces a two-electron photooxidation process located on the phenylenediiminato ligand. Kinetic measurements, performed by monitoring absorbance changes over time in several solvents, are in agreement with a slow unimolecular photooxidation process, which is faster in water and slower in less polar solvents. Moreover, structural details of ZnL-DNA binding were obtained by DFT calculations on the intercalation complexes between ZnL and the d(ApT)2 and d(GpC)2 dinucleoside monophosphate duplexes. Two main complementary binding interactions are proposed: 1) intercalation of the central phenyl ring of the ligand between the stacked DNA base pairs; 2) external electrostatic attraction between the negatively charged phosphate groups and the two cationic triethylammonium groups of the Schiff-base ligand. Such suggestions are supported by fluorescence titrations performed on the ZnL/DNA system at different ionic strengths and temperatures. In particular, the values of the DNA-binding constants obtained at different temperatures provided the enthalpic and entropic contributions to the binding and confirmed that two competitive mechanisms, namely, intercalation and external interaction, are involved. The two mechanisms are coexistent at room temperature under physiological conditions.

Left-handed DNA: intercalation of the cyanine thiazole orange and structural changes. A kinetic and thermodynamic approach

The conditions under which different structures of left-handed DNA (poly(dG-me(5)dC)·poly(dG-me(5)dC)) can exist are investigated by spectrofluorometric, spectrophotometric, circular dichroism and calorimetric measurements and the kinetics of the transformations are analysed. The effects of temperature, salt and ethanol content on the transitions are also studied. The left-handed structure obtained by addition of either Mg(2+) ions or EtOH corresponds to Z-DNA, whereas the structure obtained using the mixture Mg(2+)/EtOH corresponds to the aggregate Z*-DNA. Upon addition of the fluorescent cyanine Thiazole Orange (TO), the transition Z → B immediately starts, whereas Z*-DNA retains its left-handed configuration in the presence of TO provided that the ratio [dye]/[polymer] ≤ 0.1. The equilibria and kinetics of the TO binding to Z*-DNA are investigated under the above conditions using the T-jump technique. The reaction mechanism consists of two series steps, the first one being characterized by the formation of an external electrostatic complex and the second corresponding to the dye penetration between the base pairs. A comparison with the B-DNA/TO system is drawn.

Organization of inner cellular components as reported by a viscosity-sensitive fluorescent Bodipy probe suitable for phasor approach to FLIM.

According to the recent developments in imaging strategies and in tailoring fluorescent molecule as probe for monitoring biological systems, we coupled a Bodipy-based molecular rotor (BoMe) with FLIM phasor approach to evaluate the viscosity in different intracellular domains. BoMe rapidly permeates cells, stains cytoplasmic as well as nuclear domains, and its optical properties make it perfectly suited for widely diffused confocal microscopy imaging setups. The capability of BoMe to report on intracellular viscosity was put to the test by using a cellular model of a morbid genetic pathology (Hutchinson-Gilford progeria syndrome, HGPS). Our results show that the nucleoplasm of HGPS cells display reduced viscosity as compared to normal cells. Since BoMe displays significant affinity towards DNA, as demonstrated by an in vitro essay, we hypothesize that genetic features of HGPS, namely the misassembly of lamin A protein within the nuclear lamina, modulates chromatin compaction. This hypothesis nicely agrees with literature data.