A dinuclear biomimetic Cu complex derived from l-histidine: synthesis and stereoselective oxidations.

A dinuclear copper(ii) complex derived from the chiral N6 ligand (2S,2'S)-N,N'-(ethane-1,2-diyl)bis(2-((1-methyl-1H-imidazol-4-ylmethyl)-amino)-3-(1-trityl-1H-imidazol-4-yl)propanamide) (EHI) was synthesized and studied as a catalyst in stereoselective oxidation reactions. The ligand contains two sets of tridentate binding units, each of them giving rise to a coordination set consisting of a pair of 5- and 6-membered chelate rings, connected by an ethanediamide linker. Stereoselectivity effects were studied in the oxidations of a series of chiral l/d biogenic catechols and the pair of l/d-tyrosine methyl esters, in this case as their phenolate salts. The oxidation of ß-naphthol has also been studied as a model monooxygenase reaction. The catechol oxidation was investigated in a range of substrate concentrations at slightly acidic pH and exhibited a marked dependence on the concentration of the [Cu2EHI]4+ complex. This behavior has been interpreted in terms of an equilibrium between a monomeric and a dimeric form of the catalyst. Binding studies of l- and d-tyrosine were performed as a support for the interpretation of the stereoselectivity effects observed in the reactions. In general, [Cu2EHI]4+ exhibits a binding preference for the l- rather than the d-enantiomer of the substrates, but it appears that in the catecholase reaction the oxidation of the d-isomer occurs at a faster rate than for the l counterpart. The same type of enantio-discriminating behavior is observed in the oxidation of l-/d-tyrosine methyl esters. In this case the reaction produces a complex mixture of products; the main product consisting of a trimeric compound, likely formed by radical coupling reactions, has been isolated and characterized. The oxidation of ß-naphthol yields an additional product of the expected quinone but labeling experiments with 18-O2 show no oxygen incorporation into the product, confirming that the oxidation likely proceeds through a radical mechanism.

Protective effects of novel metal-nonoates on the cellular components of the vascular system.

At the cardiovascular level, nitric oxide (NO) controls smooth muscle functions, maintains vascular integrity, and exerts an antihypertensive effect. Metal-nonoates are a recently discovered class of NO donors, with NO release modulated through the complexation of the N-aminoethylpiperazine N-diazeniumdiolate ligand to metal ions, and thus representing a significant innovation with respect to the drugs traditionally used. In this study, we characterized the vascular protective effects of the most effective compound of this class, Ni(PipNONO)Cl, compared with the commercial N-diazeniumdiolate group derivate, diethylenetriamine/nitric oxide (DETA/NO). Ni(PipNONO)Cl induced a concentration-dependent relaxation of precontracted rat aortic rings. The ED50 was 0.67 µM, compared with 4.3 µM obtained with DETA/NO. When tested on cultured microvascular endothelial cells, Ni(PipNONO)Cl exerted a protective effect on the endothelium, promoting cell proliferation and survival in the picomolar range. The administration of Ni(PipNONO)Cl to vascular smooth muscle cells reduced the cell number, promoting their apoptosis at a high concentration (10 µM). Inhibition of smooth muscle cell migration, a hallmark of atherosclerosis, was accompanied by cytoskeletal rearrangement and loss of lamellipodia. When added to isolated platelets, Ni(PipNONO)Cl significantly reduced ADP-induced aggregation. Since atherosclerosis is accompanied by an inflammatory environment, cultured endothelial cells were exposed to interleukin (IL)-1ß. In the presence of IL-1ß, Ni(PipNONO)Cl inhibited cyclooxygenase-2 and inducible nitric oxide synthase upregulation, and reduced endothelial permeability and the platelet and monocyte adhesion markers CD31 and CD40 at the plasma membrane. Overall, these data indicate that Ni(PipNONO)Cl exerts vascular protective effects relevant for vascular dysfunction and prevention of atherosclerosis and thrombosis.

Exploiting micelle-driven coordination to evaluate the lipophilicity of molecules.

We present a systematic study based on the calculation of complexation constants between a Zn-complex solubilized in Triton X-100 micellar solutions and a series of linear mono- and dicarboxylic acids, under physiological pH conditions, that allowed the evaluation of the lipophilicity of these molecules. This empirical lipophilicity parameter describes conveniently the partition of organic molecules between hydrophobic microdomains and water. The results can be used to predict the lipophilicity of molecules with similar structure and allows the distinction of intrinsic contributions of the carboxylates and of the methylene groups to the lipophilicity of the molecule.

Antibacterial activity of glutathione-coated silver nanoparticles against Gram positive and Gram negative bacteria.

In the present paper, we study the mechanism of antibacterial activity of glutathione (GSH) coated silver nanoparticles (Ag NPs) on model Gram negative and Gram positive bacterial strains. Interference in bacterial cell replication is observed for both cellular strains when exposed to GSH stabilized colloidal silver in solution, and microbicidal activity was studied when GSH coated Ag NPs are (i) dispersed in colloidal suspensions or (ii) grafted on thiol-functionalized glass surfaces. The obtained results confirm that the effect of dispersed GSH capped Ag NPs (GSH Ag NPs) on Escherichia coli is more intense because it can be associated with the penetration of the colloid into the cytoplasm, with the subsequent local interaction of silver with cell components causing damages to the cells. Conversely, for Staphylococcus aureus, since the thick peptidoglycan layer of the cell wall prevents the penetration of the NPs inside the cytoplasm, the antimicrobial effect is limited and seems related to the interaction with the bacterial surfaces. Experiments on GSH Ag NPs grafted on glass allowed us to elucidate more precisely the antibacterial mechanism, showing that the action is reduced because of GSH coating and the limitation of the translational freedom of NPs.

Water soluble extended naphthalene diimides as pH fluorescent sensors and G-quadruplex ligands.

Extended naphthalene diimides (NDIs) fused to 1,4-dihydropyrazine-2,3-dione, containing two solubilizing moieties, have been synthesized. Fluorescence spectra of the new NDIs were remarkably affected by pH, as the second deprotonation of the dihydropyrazinedione moiety (pK(a) 6.9) switched off the emission. Binding to a G-quadruplex folded oligonucleotide and stoichiometry were evaluated by FRET melting assay and CD analysis. G-quadruplex binding was strongly enhanced shifting from pH 7.4 to pH 6.0 as a consequence of the dihydropyrazinedione moiety protonation. Cytotoxicity studies using two human telomerase-positive cell lines (HT29 and A549) revealed that the best G-quadruplex ligand was very active against the colon cell line, with an EC(50) of 300 nM.

Optical method for predicting the composition of self-assembled monolayers of mixed thiols on surfaces coated with silver nanoparticles.

With a simple optical method, based on UV-vis absorption spectra on glass slides, it is possible to predict the composition of self-assembled monolayers of mixed thiols, grafted on monolayers of silver nanoparticles. Glass slides are modified with the layer-by-layer technique, first forming a monolayer of mercaptopropyltrimethoxysilane, then grafting a monolayer of silver nanoparticles on it. These surfaces are further coated by single or mixed thiol monolayers, by dipping the slides in toluene solutions of the chosen thiols. Exchange constants are calculated for the competitive deposition between the colorless 1-dodecanethiol or PEG5000 thiol and BDP-SH, with the latter being a thiol-bearing molecule containing the strongly absorbing BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) moiety, synthesized on purpose. The constants are calculated by determining the fraction of BDP-SH deposited on the surface from a solution with a given molar fraction, directly measuring the absorption spectra of BDP-SH on the slides. Then, the exchange constant for the competitive deposition between 1-dodecanethiol and PEG5000 thiol is calculated by combining their exchange constants with BDP-SH. This allows to predict the fraction of the two colorless thiols coating the silver nanoparticles slides obtained from a toluene solution with a given molar fraction, for example, of PEG5000 thiol. The correctness of the calculated surface fraction is verified by studying the coating competition between 1-dodecanethiol and a PEG5000 thiol remotely modified with a strongly absorbing fluorescein fragment.

Nanoscale phase separation in coated Ag nanoparticles.

In this paper we report the structural investigation of cysteine and glutathione capped Ag nanoparticles (NPs) by means of transmission electron microscopy (TEM), synchrotron X-ray diffraction (XRD) and pair distribution function (PDF) analysis. The combined use of these probes allowed us to observe the presence of two crystal structures in the coated AgNPs, i.e., the cubic and the hexagonal crystal structures of Ag. In particular, it was possible to demonstrate that the coated AgNPs are a nanoscale phase separated system where the two phases coexist within the single grain. In addition, the relative bulk amount of the fcc and hcp phases has been estimated and a possible correlation with the capping agent proposed.

A fluorescent molecular sensor for pH windows in traditional and polymeric biocompatible micelles: comicellization of anionic species to shift and reshape the ON window.

A new approach is presented to obtain fluorescent sensors for pH windows that work in water and under biomimetic conditions. A single molecule that features all-covalently linked components is used, thus making it capable of working as a fluorescent sensor with an OFF/ON/OFF response to pH value. The components are a tertiary amine, a pyridine, and a fluorophore (pyrene). The forms with both protonated bases or both neutral bases quench the pyrene fluorescence, whereas the form with the neutral pyridine and protonated amine groups is fluorescent. The molecular sensor is also equipped with a long alkyl chain to make it highly hydrophobic in all its protonated and unprotonated forms, that is, either when neutral or charged. Accordingly, it can be confined at any pH value either in traditional (i.e., low-molecular-weight) nonionic surfactant micelles or inside polymeric, biocompatible micellar containers. Relevant for future applications in vivo, thanks to its strong hydrophobicity, no leakage of the molecular sensor is observed from the polymeric biocompatible micelles. Due to the proximity of the pyridine and amine functions in the molecular structure and the poor hydration inside the micelles, the observed pK(a) values are low so that the ON window is positioned at very low pH values. However, the window can be shifted to biologically relevant values by comicellization of anionic species. In particular, in the micelles of the nonionic surfactant TritonX-100, a shift of the ON window to pH 4-6 is obtained by addition of the anionic sodium dodecyl sulphate surfactant, whose negative charge promotes the stability of the protonated forms of the pyridine and amine fragments. In the case of the polymeric micelles, we introduce the use of the amphiphilic polystyrene sulfonate anionic polyelectrolyte, the comicellization of which induces a shift and sharpening of the ON window that is centered at pH 4.

Synthesis, characterization and antibacterial activity against Gram positive and Gram negative bacteria of biomimetically coated silver nanoparticles.

In the present work, we describe a simple procedure to produce biomimetically coated silver nanoparticles (Ag NPs), based on the postfunctionalization and purification of colloidal silver stabilized by citrate. Two biological capping agents have been used (cysteine Cys and glutathione GSH). The composition of the capped colloids has been ascertained by different techniques and antibacterial tests on GSH-capped Ag NPs have been conducted under physiological conditions, obtaining values of Minimum Inhibitory Concentration (MIC) of 180 and 15 µg/mL for Staphylococcus aureus and Escherichia coli, respectively. The antibacterial activity of these GSH capped NPs can be ascribed to the direct action of metallic silver NPs, rather than to the bulk release of Ag(+).

Multicomponent polymeric micelles based on polyaspartamide as tunable fluorescent pH-window biosensors.

PHEA-PEG(5000)-C(16) is a polyaspartamide polymer with appended hydrophilic PEG(5000) functions and hydrophobic n-C(16) units forming biocompatible micelles with a CAC as low as 1.8 × 10(-7) M. The protonation and acidity constants of the polymer's amino and carboxylic groups have been determined by potentiometric titrations at five different concentrations higher than CAC, finding concentration-independent values. Viscosity and polarity of the micellar core have been investigated by means of fluorescent probes, finding local values comparable to those of pure toluene and to the core of sodium dodecyl sulphate micelles, independently on the protonation degree of the polymer. The fluorophore pyrene, the lipophilic N,N'-dimethyl-N"-dodecylamine and 2-dodecylpyridine self-assemble in the hydrophobic core of PHEA-PEG(5000)-C(16) micelles originating a micellar device that behaves as a rare "off-on-off" fluorescence sensor for pH windows, with no interference by the amino and carboxylic functions of the polymer. The "on" state of the sensor includes the physiological 6-8 pH interval, and can be finely shifted in both directions of the pH axis by comicellization of charged cosurfactants. Dialysis experiments demonstrate that the micellar device exibits an efficient retention ability of all molecular components, including cosurfactants, thus candidating for in vivo use.

A micellar multitasking device: sensing pH windows and gauging the lipophilicity of drugs with fluorescent signals.

A multitasking fluorescent device can be obtained by forming micelles of Triton X-100, containing a lipophilic macrocyclic Cu(2+) complex and the coordinating fluorophore Coumarin 343 (C343), which features a COOH moiety. At low pH the two micellised components do not interact, and the fluorescence of Courmarin 343 (C343) is intense. At intermediate pH, C343 is deprotonated and coordinates to the Cu(2+) centre in its apical position, with fluorescence quenching. At higher pH the deprotonated C343 is displaced from Cu(2+) by the formation of an OH(-) complex, and the fluorescence is revived. This allows the system to carry out its first task as it behaves as an "on-off-on" fluorescent sensor for pH windows. The "off" part of the window ranges from pH 6 to 8. In this interval, in which the carboxylate form of C343 is apically coordinated to the Cu(2+) complex inside micelles, the device carries out its second task, that is, it behaves as a gauge for lipophilicity. For pHs between 6 and 8, molecules containing a COOH group are in their COO(-) form and distribute between bulk water and micelles proportionally to their lipophilicity. Upon entering the micelle, their COO(-) moiety competes for coordination with C343, displacing it from the Cu(2+) centre, and this results in fluorescence revival, the intensity of which is also proportional to the lipophilicity of the examined molecule. We have chosen the physiological pH value (7.4) as the working pH, and we have examined the lipophilicity of fatty acids and of the widely used family of non-steroidal anti-inflammatory drugs (NSAIDs). The device successfully measures their lipophilicity, expressing it with an "off-on" type fluorescent signal, as demonstrated by the correlation of the fluorescence increase with the logarithmic water/octanol partition coefficient (log P) and with the difference between the pK(a) observed in micelles and that measured in water for NSAIDs.

Smoothly shifting fluorescent windows: a tunable "off-on-off" micellar sensor for pH.

The position of the window in an "off-on-off" fluorescent pH sensor may be shifted at will along a pH axis by changing the overall charge of the micellar container. This is obtained by using non-ionic Triton X-100 as the surfactant, and by increasing the molar fraction of the anionic sodium dodecyl sulfate (chi(SDS)) as co-surfactant, with pyrene as the fluorophore and a lipophilic tertiary amine and a lipophilic pyridine as pH-switchable quenchers. As the negative micellar charge increases by increasing chi(SDS), the observed pKa of the protonated bases move to higher pH values, followed by the dumb-bell shaped "off-on-off" fluorescence intensity vs. pH profile.

The Cu(II) complex of a C-lipophilized 13aneN4 macrocycle with an additional protonable amino group as micellar anion receptor.

Three 13aneN4 macrocyclic ligands have been prepared bearing a -CH(2)NHR side arm (R = H, n-C(5)H(11), n-C(10)H(21)) on a carbon atom. When Cu(2+) is complexed in the macrocyclic ring, the amino group of the side arm undergoes an acid-base protonation equilibrium but it is not able to coordinate apically the metal cation even when it is deprotonated. The Cu(2+) complex with the ligand bearing the longest appended aliphatic chain is fully confined inside Triton X-100 micelles, and its ability to bind and sequestrate a series of anions inside micelles has been studied at two different pH values, i.e. both with protonated and neutral side-arm amino group. The favourable role played by the protonated amino group in the side arm has been demonstrated.

Micelles as containers for self-assembled nanodevices: a fluorescent sensor for lipophilicity.

Potentiometric titrations, fluorescence versus pH titrations, dynamic light scattering and fluorescence polarization anisotropy studies demonstrate that inside the nanodimensioned Triton X-100 micelles, 1-pyrenecarboxylic acid, PCOO(-), forms an apical complex with the Zn(2+) cation encircled by a lipophilic cyclen ligand and hugely increasing its fluorescence. The ability of the Zn(2+)-cyclen-PCOO(-) complex plus its micellar container to act as a fluorescent sensor to evaluate the lipophilicity of molecular species is demonstrated on the fatty acid series CH(3)(CH(2))(x)COOH (x=0-16). At pH 7.4 a decrease in fluorescence is observed on the addition of fatty acids that is directly related to their chain length, that is, to their tendency to enter the micellar containers, where they dislocate PCOO(-) from the Zn(2+) centre. The independent determination of fatty acid pK(a) values in the presence of Triton X-100 micelles confirms that our fluorescent micellar device is capable of sensing their lipophilicity.

Residual and exploitable fluorescence in micellar self-assembled ON-OFF sensors for copper(II).

A set of new ligands, L2H(2)-L5H(2), containing the 1,4,8,11-tetraaza-5,7-dione framework has been prepared, The ligands feature lipophilic substituents either on the carbon atom in the 6 position or on the amino groups, or on both. The solution behaviour of the ligands when included in TritonX-100 micelles has been investigated by means of potentiometric titrations and protonation and complexation constants for the Cu(2+) cation have been determined in micellar medium. Micellar assemblies containing the ligands and pyrene have been prepared, and coupled pH-metric and fluorimetric titrations allowed the determination of the response of the systems as ON-OFF fluorescent sensors for Cu(2+). A correlation between the effective lipophilicity of the ligand and the residual fluorescence (i.e. the fluorescence of the OFF state) was observed, and with the more lipophilic ligand, L3H(2), we obtained a residual fluorescence as low as 8%, with a significant improvement with respect to other published systems. On the other hand, introduction of functionalities on the amino groups of 1,4,8,11-tetraaza-5,7-dione brings the drawback of a small but significant decrease of the exploitable fluorescence, i.e. the fluorescence of the system in the absence of added Cu(2+), at the pH value suitable for full metal complexation.