Peptides Derived from Angiogenin Regulate Cellular Copper Uptake.

The angiogenin protein (ANG) is one of the most potent endogenous angiogenic factors. In this work we characterized by means of potentiometric, spectroscopic and voltammetric techniques, the copper complex species formed with peptide fragments derived from the N-terminal domain of the protein, encompassing the sequence 1-17 and having free amino, Ang1-17, or acetylated N-terminus group, AcAng1-17, so to explore the role of amino group in metal binding and cellular copper uptake. The obtained data show that amino group is the main copper anchoring site for Ang1-17. The affinity constant values, metal coordination geometry and complexes redox-potentials strongly depend, for both peptides, on the number of copper equivalents added. Confocal laser scanning microscope analysis on neuroblastoma cells showed that in the presence of one equivalent of copper ion, the free amino Ang1-17 increases cellular copper uptake while the acetylated AcAng1-17 strongly decreases the intracellular metal level. The activity of peptides was also compared to that of the protein normally present in the plasma (wtANG) as well as to the recombinant form (rANG) most commonly used in literature experiments. The two protein isoforms bind copper ions but with a different coordination environment. Confocal laser scanning microscope data showed that the wtANG induces a strong increase in intracellular copper compared to control while the rANG decreases the copper signal inside cells. These data demonstrate the relevance of copper complexes' geometry to modulate peptides' activity and show that wtANG, normally present in the plasma, can affect cellular copper uptake.

Controlled Dissociation of Iron and Cyclopentadienyl from a Diiron Complex with a Bridging C3 Ligand Triggered by One-Electron Reduction.

The one-electron reduction of a diiron cationic complex revealed unique features: cleavage of the diiron structure occurred despite a multidentate bridging C3 ligand and was accompanied by the clean dissociation of one η5-cyclopentadienyl ring and one iron as isolated units. Thus, the iron(II)-iron(II) µ-vinyliminium complex [Fe2Cp2(CO)(µ-CO){µ-η1:η3-C3(Et)C2HC1N(Me)(Xyl)}][SO3CF3] ([1a]SO3CF3) reacted with cobaltocene in tetrahydrofuran (THF), affording the iron(II) vinylaminoalkylidene [FeCp(CO){C1N(Me)(Xyl)C2HC3(Et)C(═O)}] (2a) in 77% yield relative to the C3 ligand. Analogously, [FeCp(CO){C1N(Me)(Xyl)C2HC3(CH2OH)C(═O)}] (2b) was obtained in 64% yield from the appropriate diiron precursor and CoCp2. The formation of 2a is initiated by the one-electron reduction of [1a]+, followed by a reversible intramolecular rearrangement terminating with the irreversible release of CpH (NMR and gas chromatography-mass spectrometry) and Fe [electron paramagnetic resonance (EPR) and magnetometry]. The key intermediate iron(I) ferraferrocene (3) was detected by EPR and IR spectroelectrochemistry, while the related species 3-H-3 was isolated after the addition of a hydrogen source and then identified by X-ray diffraction. A plausible mechanism for the route from [1a]+ to 3 was ascertained by density functional theory calculations. The dication [1a]2+, displaying both carbonyl ligands in terminal positions, and the anion [3]- were electrochemically generated. The functionalized diiron compounds 4 (52% yield) and 5 (62%) were afforded through the activation of O2 and S8 by a radical intermediate along the reductive pathway of [1a]+. The reaction of [Fe2Cp2(CO)(µ-CO){µ-η1:η3-C(SiMe3)CHCN(Me)(Xyl)}][SO3CF3] ([1c]SO3CF3) with CoCp2 in THF afforded [Fe2Cp2(C≡CSiMe3)(CO)(µ-CO){µ-CNMe(Xyl)}] (6) in 65% yield.

Oxidative Dimerization of Triarylamines Promoted by WCl6, Including the Solid State Isolation and the Crystallographic Characterization of a Triphenylammonium Salt.

The triphenylammonium salt [NHPh3][WCl6], 1, and the product of the C-C dimerization of triphenylamine, Ph2N(C6H4)2NPh2, 2, were afforded from the reaction between WCl6 and NPh3 in CH2Cl2. Compound 2 was isolated in 43% yield upon hydrolysis of the reaction mixture. The X-ray structure of 1 provides the first crystallographic characterization of the triphenylammonium ion. Combined EPR and DFT studies gave insight into the reaction mechanism, and allowed the identification of WCl5···[Cl(C6H4)NPh2] as a presumable key intermediate. The reactions of WCl6 with 4-bromotriphenylamine, 4,4'-dimethyltriphenylamine, 9-phenylcarbazole, followed by hydrolytic treatment, led to the dimerization products 3-6, in admixture with variable amounts of the parent amines. N,N,N',N'-tetrakis(4-bromophenyl)-[1,1'-biphenyl]-4,4'-diamine, 3, was isolated in 60% yield from the reaction of WCl6 with 4,4'-dibromotriphenylamine.

Coordination Compounds of Niobium(IV) Oxide Dihalides Including the Synthesis and the Crystallographic Characterization of NHC Complexes.

The 1:1 molar reactions of NbOX3 with SnBu3H, in toluene at 0 °C in the presence of oxygen/nitrogen donors, resulted in the formation of NbOX2L2 (X = Cl, L2 = dme, 2a; X = Br, L2 = dme, 2b; X = Cl, L = thf, 2c; X = Cl, L = NCMe, 2d; dme = 1,2-dimethoxyethane, thf = tetrahydrofuran), in good yields. The 1:2 reactions of freshly prepared 2d and 2b with the bulky NHC ligands 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene, Imes, and 1,3-bis(2,6-dimethylphenyl)imidazol-2-ylidene, Ixyl, respectively, afforded the complexes NbOCl2(Imes)2, 3, and NbOBr2(Ixyl)2, 4, in 50-60% yields. The reactions of 2b with NaOR, in tetrahydrofuran, gave NbOCl(OR) (R = Ph, 5; R = Me, 6) in about 60% yields. All the products were characterized by analytical and spectroscopic techniques; moreover DFT calculations were carried out in order to shed light on synthetic and structural features. Compounds 3 and 4, whose molecular structures have been ascertained by X-ray diffraction, represent very rare examples of crystallographically characterized niobium-NHC systems.

Copper(II) complexes with peptides based on the second cell binding site of fibronectin: metal coordination and ligand exchange kinetics.

Copper(ii) complexes with short peptides based on the second cell binding site of fibronectin, PHSFN and PHSEN, have been characterized by potentiometric, UV-vis, CD, EPR and NMR spectroscopic methods. The histidine imidazole nitrogen is the anchoring site for the metal ion binding. Thermodynamic and spectroscopic evidence is given that the side chain oxygen donor atom of glutamyl residue in Ac-PHSEN-NH2 is also involved in the binding up to physiological pH. To determine ligand exchange kinetic parameters after the imidazole nitrogen anchoring, proton relaxation enhancement NMR data have been collected for the two hydrogen atoms of the imidazole ring in the temperature range 293-315 K at pH 5.2 and globally treated within different kinetic models for ligand exchange. The best fitting model involves two steps. In the first one, which is slow, a water molecule disengages a carbonyl or a carboxylate group coordinated to the metal ion in the complex formed by PHSFN or PHSEN, respectively. This stage is one order of magnitude slower for PHSEN, due to entropic effects. In the second step, which is fast, the complex just formed exchanges with the ligand. In this step, no appreciable differences are found for the two cases examined.

Effects of azole treatments on the physical properties of Candida albicans plasma membrane: a spin probe EPR study.

EPR spectroscopy was applied to investigate the effects of the treatment of Candida albicans cells with fluconazole (FLC) and two newly synthesized azoles (CPA18 and CPA109), in a concentration not altering yeast morphology, on the lipid organization and dynamics of the plasma membrane. Measurements were performed in the temperature range between 0°C and 40°C using 5-doxyl- (5-DSA) and 16-doxyl- (16-DSA) stearic acids as spin probes. 5-DSA spectra were typical of lipids in a highly ordered environment, whereas 16-DSA spectra consisted of two comparable components, one corresponding to a fluid bulk lipid domain in the membrane and the other to highly ordered and motionally restricted lipids interacting with integral membrane proteins. A line shape analysis allowed the relative proportion and the orientational order and dynamic parameters of the spin probes in the different environments to be determined. Smaller order parameters, corresponding to a looser lipid packing, were found for the treated samples with respect to the control one in the region close to the membrane surface probed by 5-DSA. On the other hand, data on 16-DSA indicated that azole treatments hamper the formation of ordered lipid domains hosting integral proteins and/or lead to a decrease in integral protein content in the membrane. The observed effects are mainly ascribable to the inhibition of ergosterol biosynthesis by the antifungal agents, although a direct interaction of the CPA compounds with the membrane bilayer in the region close to the lipid polar head groups cannot be excluded.

The redox chemistry of [Co6C(CO)15](2-): a synthetic route to new co-carbide carbonyl clusters.

The oxidation and reduction reactions of [Co6C(CO)15](2-) have been studied in detail, leading to the isolation of several new Co-carbide carbonyl clusters. Thus, [Co6C(CO)15](2-) reacts in tetrahydrofuran (THF) with oxidants such as HBF4·Et2O and [Cp2Fe][PF6], resulting first in the formation of the previously reported [Co6C(CO)14](-); then, in CH2Cl2, the new dicarbide [Co11C2(CO)23](2-) is formed. The latter may be further oxidized, yielding the isostructural monoanion [Co11C2(CO)23](-), whereas its reduction with (cyclopentadienyl)2Co affords the unstable trianion [Co11C2(CO)23](3-), which decomposes during workup. Oxidation of [Co6C(CO)15](2-) in CH3CN with [C7H7][BF4] affords the same major products, and besides, the new monoacetylide [Co10(C2)(CO)21](2-) was obtained as side-product. Conversely, the reduction of [Co6C(CO)15](2-) in THF with increasing amounts of Na/naphthalene results in the following species: [Co6C(CO)13](2-), [Co11(C2)(CO)22](3-), [Co7C(CO)15](3-), [Co8C(CO)17](4-), [Co6C(CO)12](3-), and [Co(CO)4](-). The new [Co11C2(CO)23](-), [Co11C2(CO)23](2-), [Co10(C2)(CO)21](2-), [Co8C(CO)17](4-), [Co6C(CO)12](3-), and [Co7C(CO)15](3-) clusters were structurally characterized. Moreover, the paramagnetic species [Co11C2(CO)23](2-) and [Co6C(CO)12](3-) were investigated by means of electron paramagnetic resonance spectroscopy. Finally, electrochemical studies were performed on [Co11C2(CO)23](n-) (n = 1-3).

Long-lived radical cation salts obtained by interaction of monocyclic arenes with niobium and tantalum pentahalides at room temperature: EPR and DFT studies.

The 1:3 reactions of the alkoxy arenes 1,4-(MeO)2 C6 H4 and 1,4-F2 -2,5-(MeO)2 C6 H2 with TaF5 in chloroform at 40-50 °C resulted in formation in about 35 % yield of the long-lived radical cation salts [1,4-(MeO)2 C6 H4 ][Ta2 F11 ] (2 a) and [1,4-F2 -2,5-(MeO)2 C6 H2 ][Ta2 F11 ] (2 b), respectively. The non-alkoxy-substituted [arene][M2 X11 ] [M=Ta, X=F: arene=C6 H5 Me (2 c), 1,4-C6 H4 Me2 (2 d), C6 H5 F (2 e), C6 H5 NO2 (2 f); M=Nb, X=F: arene=C6 H5 Me (4 a), 1,4-C6 H4 Me2 (4 b), C6 H5 F (4 c), C6 H5 NO2 (4 d); M=Ta, X=Cl: arene=1,4-C6 H4 Me2 (5)] were obtained from the 3:1 reactions of MX5 with the appropriate arene in chloroform at temperatures in the range 40-90 °C. Compounds 2-5 were detected by EPR spectroscopy (in CHCl3 ) at room temperature, and their gas-phase structures were optimized by DFT calculations. Formation of the M(IV) species [MX4 (NCMe)2 ] [M=Ta, X=F (3 a); M=Nb, X=F (3 b); M=Ta, X=Cl (3 c)] was ascertained by EPR spectroscopy on solutions obtained by treatment of the reaction mixtures with acetonitrile. Non-selective reactions occurred upon combination of 1,4-F2 -2,5-(MeO)2 C6 H2 with AgNbF6 (in CH2 Cl2 ) and 1,4-(MeO)2 C6 H4 with SbF5 .

Coordination environment of highly concentrated solutions of Cu(II) in ionic liquids through a multidisciplinary approach.

The coordination environment around Cu(II) in highly concentrated solutions of copper(II) salts (CuCl(2) and Cu(Tf(2)N)(2)) in two pure ionic liquids bearing the same anion, namely, 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([bmim][Tf(2)N]) and 1-butyl-3-methylimidazolium chloride ([bmim]Cl), is investigated by X-ray photoelectron spectroscopy (XPS), UV/Vis spectroscopy, EPR spectroscopy and DFT calculations. Moreover, the electrochemical behavior of these mixtures is studied. Whereas reversible reduction of Cu(II) to copper metal can be observed in the 1:1 [bmim][Tf(2)N]:Cu(Tf(2)N)(2) solution, 2:1 and 1:1 [bmim]Cl:CuCl(2) mixtures showed one-electron reduction of Cu(II) to Cu(I) with formation of a permanent deposit of CuCl. XPS, UV/Vis and EPR spectra as well as DFT calculations suggest the formation in [bmim]Cl of dynamic coordination complexes arising from the interaction between CuCl(2) and [bmim](+)Cl(-). The two long-lived situations are probably trigonal and deformed tetrahedral copper(II) chloride coordination complexes ([CuCl(3)](-) and [CuCl(4)](2-), respectively).

Impact of Leaf Removal on Phenolics and Antioxidant Activity of Trebbiano Berries (Vitis vinifera L.).

Leaf removal is a canopy management practice widely applied in viticulture to enhance the phenol composition and concentration of grapes, which then results in improved wine quality. Many studies were carried out on red berried varieties, but information on white ones is scanty. The aim of the study was to assess the effect of basal leaf defoliation in post fruit set on the phenol composition, ascorbate level and antioxidant activity of Trebbiano grapes. Electron paramagnetic resonance was also employed to monitor the decay kinetics of 1,1-diphenyl-2-picrylhydrazyl which allowed the identification of antioxidants with different action rates. The results show that defoliation caused an increase in the phenolic acid (hydroxycinnamic and hydroxybenzoic acids) and flavonol concentrations of berries without changes in the composition. Both ascorbate and antioxidant activity were also enhanced in the berries from defoliated vines. Besides increasing the number of fast-rate antioxidants, leaf removal resulted in the formation of intermediate-rate ones. In the Trebbiano variety, leaf removal in the post fruit set may represent an effective strategy to enhance the phenolic composition and the antioxidant defense system of berries.

Contribution of major lipophilic antioxidants to the antioxidant activity of basil extracts: an EPR study.

BACKGROUND: The present research analyses the contribution of some lipid antioxidants to the antioxidant activity of lipophilic extracts from basil by an electron paramagnetic resonance (EPR) study using the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). DPPH assay is considered an easy and accurate method with regard to measuring the antioxidant activity of plant extracts and EPR has already been employed to determine antioxidant activities of lipophilic plant extracts. RESULTS: Lipid extracts were obtained from basil grown hydroponically for 20 or 35 days from sowing and in soil for 35 days from sowing. Fast and slow rate constants were distinguishable in the decay kinetics of DPPH mixture added with lipid extract. Antioxidants with kinetics characterized by a fast decay rate were tocopherols and chlorophylls, whereas those characterized by a slow decay rate were carotenoids, among which were ß-carotene, lutein, ß-apo-8'-carotenal and zeaxanthin. The 20-day hydroponically grown sample, which showed higher contents of tocopherol, chlorophyll and carotenoid molecules, was the sample endowed with the higher content of fast lipophilic antioxidants (FLA) and slow lipophilic antioxidants (SLA). CONCLUSION: The three samples showed different compositions of FLA and SLA, giving rise to different decay kinetics. Despite the differences, in all samples tocopherol contributed about 0.3% to the bulk of FLA, whereas the figure for chlorophyll was about 40%, evidencing the relevant but little-studied role of chlorophyll as an antioxidant.

Phytic acid prevents oxidative stress in seeds: evidence from a maize (Zea mays L.) low phytic acid mutant.

A maize mutant defective in the synthesis of phytic acid during seed maturation was used as a tool to study the consequences of the lack of this important reserve substance on seed survival. Data on germinability, free iron level, free radical relative abundance, protein carbonylation level, damage to DNA, degree of lipid peroxidation, alpha- and gamma-tocopherol amount and antioxidant capacity were recorded on seeds of maize B73 and of an isogenic low phytic acid mutant (lpa1-241), either unaged or incubated for 7 d in accelerated ageing conditions (46 degrees C and 100% relative humidity). The lpa1-241 mutant, compared to wild type (wt), showed a lower germination capacity, which decreased further after accelerated ageing. Whole lpa1-241 mutant kernels contained about 50% more free or weakly bound iron than wt ones and showed a higher content of free radicals, mainly concentrated in embryos; in addition, upon accelerated ageing, lpa1-241 seed proteins were more carbonylated and DNA was more damaged, whereas lipids did not appear to be more peroxidated, but the gamma-tocopherol content was decreased by about 50%. These findings can be interpreted in terms of previously reported but never proven antioxidant activity of phytic acid through iron complexation. Therefore, a novel role in plant seed physiology can be assigned to phytic acid, that is, protection against oxidative stress during the seed's life span. As in maize kernels the greater part of phytic acid (and thus of metal ions) is concentrated in the embryo, its antioxidant action may be of particular relevance in this crop.

Stable coordination complexes of α-diimines with Nb(v) and Ta(v) halides.

Uncommon examples of coordination compounds of high valent transition metals with α-diimines were obtained and characterized by IR and NMR spectroscopy, X-ray diffraction and/or DFT analysis. The 1 : 1 molar reactions of NbF5 with a selection of α-diimines afforded the ionic compounds [NbF4(DADR)2][NbF6] (DADR = DADDip, 1a; DADXyl, 1b; DADMes, 1c) in 80-90% yields. The addition of DADDip to NbOCl3 gave NbOCl3(DADDip), 7 (62% yield). [MBr4(DADDip)][MBr6] (M = Nb, 7a; M = Ta, 7b) were afforded in moderate yields from MBr5 and DADDip. The reactions of NbCl5 with DADDip and DADXyl proceeded with non-selective activation of the organic substrate affording complex mixtures of products, including the quinoxalinium salt [{2,6-C6H3(CHMe2)2}N(CH)2NCC(CHMe2)(CH)3C][NbCl6], 2, the Nb(iv) coordination complex NbCl4(DADDip), 3, the N-(monoprotonated) α-diimine salts [DADDip(H)][MCl6], 4a-b, and the iminomethyl-imidazolium [(2,6-C6H3Me2)NCHCHN(2,6-C6H3Me2)CCHN(2,6-C6H3Me2)][NbCl6], 5.

One pot conversion of benzophenone imine into the relevant 2-aza-allenium.

Interaction of common imines with WCl6 in organic solvents revealed very unusual features in the context of imine chemistry. Ph2C[double bond, length as m-dash]NH was converted in one pot into the 2-aza-allenium species [Ph2C[double bond, length as m-dash]N[double bond, length as m-dash]CPh2]+via N2 release, with [Ph2C[double bond, length as m-dash]NH2]+ being a co-product. PhCH[double bond, length as m-dash]NtBu underwent C-H bond activation resulting in the formation of [C[triple bond, length as m-dash]N] containing derivatives, together with [PhCH[double bond, length as m-dash]NHtBu]+.

Alterations of wheat root plasma membrane lipid composition induced by copper stress result in changed physicochemical properties of plasma membrane lipid vesicles.

A response when wheat is grown in excess copper is an altered lipid composition of the root plasma membrane (PM). With detailed characterisation of the root PM lipid composition of the copper-treated plants as a basis, in the present study, model systems were used to gain a wider understanding about membrane behaviour, and the impact of a changed lipid composition.PMs from root cells of plants grown in excess copper (50 microM Cu(2+)) and control (0.3 microM Cu(2+)) were isolated using the two-phase partitioning method. Membrane vesicles were prepared of total lipids extracts from the isolated PMs, and also reference vesicles of phosphatidylcholine (PC). In a series of tests, the vesicle permeability for glucose and for protons was analysed. The vesicles show that copper stress reduced the permeability for glucose of the lipid bilayer barrier. When vesicles from stressed plants were modified by addition of lipids to resemble vesicles from control plants, the permeability for glucose was very similar to that of vesicles from control plants. The permeability for protons did not change upon stress. Electron paramagnetic resonance (EPR) of the lipid vesicles spin probed with n-doxylstearic acid (nDSA) was used to explore the lipid rotational freedom at different depth of the bilayer. The EPR measurements supported the permeability data, indicating that the copper stress resulted in more tightly packed bilayers of the PMs with reduced acyl chain motion.

Antioxidants, free radicals, storage proteins, puroindolines, and proteolytic activities in bread wheat (Triticum aestivum) seeds during accelerated aging.

Seeds of bread wheat were incubated at 40 degrees C and 100% relative humidity for 0, 3, 4, 6, and 10 days. The effects of accelerated aging on seed germinability and some biochemical properties of flour (carotenoid, free radical, and protein contents and proteolytic activity) and gluten (free radical content and flexibility) were investigated. Seed germinability decreased during aging, resulting in seed death after 10 days. A progressive decrease of carotenoid content, in particular, lutein, was observed, prolonging the incubation, whereas the free radical content increased in both flour and gluten. A degradation of soluble and storage proteins was found, associated with a marked increase of proteolytic activity and a loss of viscoelastic properties of gluten. On the contrary, puroindolines were quite resistant to the treatment. The results are discussed in comparison with those previously obtained during accelerated aging of durum wheat seeds.

Effects of gamma-irradiation on the free radical and antioxidant contents in nine aromatic herbs and spices.

Nine spice and aromatic herb samples (i.e., basil, bird pepper, black pepper, cinnamon, nutmeg, oregano, parsley, rosemary, and sage) were gamma-irradiated at a dose of 10 kGy according to commercial practices. The effects of the disinfection treatment on the content of organic radicals and some nutrients (namely, vitamin C and carotenoids) in the samples were investigated by chromatographic and spectroscopic techniques. Irradiation resulted in a general increase of quinone radical content in all of the investigated samples, as revealed by electron paramagnetic resonance spectroscopy. The fate of these radicals after storage for 3 months was also investigated. The cellulose radical was clearly observed in a few samples. Significant losses of total ascorbate were found for black pepper, cinnamon, nutmeg, oregano, and sage, whereas a significant decrease of carotenoids content was observed for cinnamon, oregano, parsley, rosemary, bird pepper, and sage.

Antioxidants, free radicals, storage proteins, and proteolytic activities in wheat (Triticum durum) seeds during accelerated aging.

Accelerated aging was performed by incubation of wheat seeds at 40 degrees C and 100% relative humidity for 3, 4, 6, 10, and 14 days. The effects of the treatment on seed germinability and on several biochemical characteristics of flour (carotenoids, free radical and protein contents, and proteolytic activity) and gluten (free radical content and flexibility) were evaluated. A decrease of germinability was found during aging, the germination being completely inhibited after 14 days. The lutein content decreased gradually, without going to zero, while that of free radicals increased. A reduction of soluble proteins and a degradation of glutenins and gliadins were observed, associated with a substantial increase of protease activity and a decrease in gluten flexibility. The results were discussed in reference to those previously obtained by natural aging of wheat seeds of the same species and cultivar.

Direct fluorometric determination of fluorescent substances in powders: the case of riboflavin in cereal flours.

Front-face emission spectra of powders can be recorded with a commercial spectrofluorometer. By combining the emissions of a scatterer powder and of a wheat flour sample, the scattering contribution to the front-face emission spectra of flour is removed, and the fluorescence of the flour is isolated. The fluorescence depends on the concentration of the fluorophores. By choosing convenient measurement parameters and by measuring the emission spectra of flour samples suitably enriched with riboflavin, the fluorescence of riboflavin could be isolated from that of other substances present in flours and the concentration of vitamin B(2) in native substrates could be determined. This method is particularly apt for the measurement of vitamin B(2) in low riboflavin-containing powders such as wheat flours, which are usually analyzed through complex chemical and microbiological methods. The method is essentially phenomenological, in view of the interpretation difficulties connected to the origin of the fluorescence resulting from the absorption of multiply scattered photons.