Protein-Protein Stabilization in VIVO/8-Hydroxyquinoline-Lysozyme adduct: A Spectroscopic, Crystallographic, and Computational Study.

The binding of the potential drug [VIVO(8-HQ)2], where 8-HQ is 8-hydroxyquinolinato, with hen egg white lysozyme (HEWL) was evaluated through spectroscopic (electron paramagnetic resonance, EPR, and UV-visible), spectrometric (electrospray ionization-mass spectrometry, ESI-MS), crystallographic (X-ray diffraction, XRD), and computational (DFT and docking) studies. ESI-MS indicates the interaction of [VIVO(8-HQ)(H2O)]+ and [VIVO(8-HQ)2(H2O)] species with HEWL. Room temperature EPR spectra suggest both covalent and non-covalent binding of the two different V-containing fragments. XRD analyses confirm these findings, showing that [VIVO(8-HQ)(H2O)]+ interacts covalently with the solvent exposed Asp119, while cis-[VIVO(8-HQ)2(H2O)] non-covalently with Arg128 and Lys96 from a symmetry mate. The covalent binding of [VIVO(8-HQ)(H2O)]+ to Asp119 is favored by a π-π contact with Trp62 and a H-bond with Asn103 of a symmetry-related molecule. Additionally, the covalent binding of VVO2+ to Asp48 and non-covalent binding of other V-containing fragments to Arg5, Cys6, and Glu7 is revealed. Molecular docking indicates that, in the absence of the interactions occurring at the protein-protein interface close to Asp119, the binding to Glu35 or Asp52 should be preferred. Such a protein-protein stabilization could be more common than what believed up today, at least in the solid state, and should be considered in the characterization of metal-protein adducts.

Non-Covalent and Covalent Binding of New Mixed-Valence Cage-like Polyoxidovanadate Clusters to Lysozyme.

The high-resolution X-ray structures of the model protein lysozyme in the presence of the potential drug [VIVO(acetylacetonato)2] from crystals grown in 1.1 M NaCl, 0.1 M sodium acetate at pH 4.0 reveal the binding to the protein of different and unexpected mixed-valence cage-like polyoxidovanadates (POVs): [V15O36(OH2)]5-, which non-covalently interacts with the lysozyme surface, [V15O33(OH2)]+ and [V20O51(OH2)]n- (this latter based on an unusual {V18O43} cage) which covalently bind the protein. EPR spectroscopy confirms the partial oxidation of VIV to VV and the formation of mixed-valence species. The results indicate that the interaction with proteins can stabilize the structure of unexpected - both for dimension and architecture - POVs, not observed in aqueous solution.

Experimental and Computational Studies on the Interaction of DNA with Hesperetin Schiff Base CuII Complexes.

The interactions with calf thymus DNA (CT-DNA) of three Schiff bases formed by the condensation of hesperetin with benzohydrazide (HHSB or L1H3), isoniazid (HIN or L2H3), or thiosemicarbazide (HTSC or L3H3) and their CuII complexes (CuHHSB, CuHIN, and CuHTSC with the general formula [CuLnH2(AcO)]) were evaluated in aqueous solution both experimentally and theoretically. UV-Vis studies indicate that the ligands and complexes exhibit hypochromism, which suggests helical ordering in the DNA helix. The intrinsic binding constants (Kb) of the Cu compounds with CT-DNA, in the range (2.3-9.2) × 106, from CuHTSC to CuHHSB, were higher than other copper-based potential drugs, suggesting that π-π stacking interaction due to the presence of the aromatic rings favors the binding. Thiazole orange (TO) assays confirmed that ligands and Cu complexes displace TO from the DNA binding site, quenching the fluorescence emission. DFT calculations allow for an assessment of the equilibrium between [Cu(LnH2)(AcO)] and [Cu(LnH2)(H2O)]+, the tautomer that binds CuII, amido (am) and not imido (im), and the coordination mode of HTSC (O-, N, S), instead of (O-, N, NH2). The docking studies indicate that the intercalative is preferred over the minor groove binding to CT-DNA with the order [Cu(L1H2am)(AcO)] > [Cu(L2H2am)(AcO)] ≈ TO ≈ L1H3 > [Cu(L3H2am)(AcO)], in line with the experimental Kb constants, obtained from the UV-Vis spectroscopy. Moreover, dockings predict that the binding strength of [Cu(L1H2am)(AcO)] is larger than [Cu(L1H2am)(H2O)]+. Overall, the results suggest that when different enantiomers, tautomers, and donor sets are possible for a metal complex, a computational approach should be recommended to predict the type and strength of binding to DNA and, in general, to macromolecules.

Magnetic study and DFT analysis of a doubly carboxylato-bridged Co(II) derivative anchored with a 'scorpionate' precursor as a potential electrocatalyst for heterogeneous H2 evolution.

A new doubly carboxylato-bridged Co(II) dinuclear complex, [Co(bdtbpza)(NCS)]2 (1), was obtained in a satisfactory yield by employing a 'scorpionate'-type precursor, bdtbpza {bis-(3,5-di-tert-butylpyrazol-1-yl)acetate}, and was then structurally characterized. Single-crystal X-ray diffraction analysis revealed that, in 1, each Co(II) is penta-coordinated, leading to a distorted trigonal-bipyramidal geometry within the coordination environment of N3O2. Weak antiferromagnetic coupling within the Co(II) ions in 1 was found based on the isotropic spin Hamiltonian H = -J(S1·S2) for the Si = 3/2 system. For evaluating the spin density distribution and the mechanism for the magnetic exchange coupling, DFT analysis was performed, with the calculated result agreeing the experimental magnetic data. A study into electrochemical H2 evolution, involving cyclic voltammetry (CV), controlled potential electrolysis (CPE), and gas chromatographic (GC) analyses of the graphite electrode modified with the cobalt complex in a neutral aqueous solution revealed the high catalytic activity of the complex with a low overpotential toward H2O reduction. The faradaic efficiency of the catalyst was found to be 83.7% and the di-cobalt catalyst-modified electrode displayed quite an interesting H2-evolution activity compared with that of bare electrodes. These results are encouraging for the future potential application of 1 in water splitting.

Interaction with CT-DNA and in vitro cytotoxicity of two new copper(II)-based potential drugs derived from octanoic hydrazide ligands.

Two copper(II) complexes [Cu(Hpmoh)(NO3)(NCS)] (1) and [Cu(peoh)(N3)]2 (2) were designed and synthesized by reaction of Cu(NO3)2·3H2O with hydrazone Schiff base ligands,abbreviated with Hpmoh and Hpeoh. Hpmoh and Hpeoh were prepared by condensation reaction of octanoic hydrazide with pyridine-2-carboxyaldehyde and 2-acetylpyridine, respectively. Complexes 1 and 2 were characterized using different analytical techniques such as FT-IR, UV-Vis, IR, EPR and single X-ray diffraction (XRD) analyses as well as computational methods (DFT). The XRD of 1 and 2 shows a mononuclear or a dinuclear structure with the copper(II) centre adopting a slightly distorted square pyramidal geometry. In water-containing solution and in DMSO, 1 and 2 undergo a partial transformation with formation of [Cu(Hpmoh)(NO3)(NCS)] (1) and [Cu(Hpmoh)(NO3)(H2O/DMSO)] (1a) in one system and [Cu(peoh)(N3)] (2a) in the other one, as supported by DFT calculations. Docking simulations confirmed that the intercalation is the preferred binding mode with DNA for 1, 1a and 2a, but suggested that the minor groove binding is also possible. A significant fluorescence quenching of the DNA-ethidium bromide conjugate was observed upon the addition of complexes 1 and 2 with a quenching constant around 104 M-1 s-1. Finally, both 1 and 2 were examined for anti-cancer activity using MDA-MB-231 (human breast adenocarcinoma) and A375 (malignant melanoma) cell lines through in vitro MTT assay which suggest comparable cancer cell killing efficacy, with the higher effectiveness of 2 due to the dissociation into two [Cu(peoh)(N3)] units.

Metal vs Ligand Oxidation: Coexistence of Both Metal-Centered and Ligand-Centered Oxidized Species.

A series of two-electron-oxidized cobalt porphyrin dimers have been synthesized upon controlled oxidations using halogens. Rather unexpectedly, X-ray structures of two of these complexes contain two structurally different low-spin molecules in the same asymmetric unit of their unit cells: one is the metal-centered oxidized diamagnetic entity of the type CoIII(por), while the other one is the ligand-centered oxidized paramagnetic entity of the type CoII(por•+). Spectroscopic, magnetic, and DFT investigations confirmed the coexistence of the two very different electronic structures both in the solid and solution phases and also revealed a ferromagnetic spin coupling between Co(II) and porphyrin π-cation radicals and a weak antiferromagnetic coupling between the π-cation radicals of two macrocycles via the bridge in the paramagnetic complex.

Stabilization and Binding of [V4 O12 ]4- and Unprecedented [V20 O54 (NO3 )]n- to Lysozyme upon Loss of Ligands and Oxidation of the Potential Drug VIV O(acetylacetonato)2.

High-resolution crystal structures of lysozyme in the presence of the potential drug VIV O(acetylacetonato)2 under two different experimental conditions have been solved. The crystallographic study reveals the loss of the ligands, the oxidation of VIV to VV and the subsequent formation of adducts of the protein with two different polyoxidovanadates: [V4 O12 ]4- , which interacts with lysozyme non-covalently, and the unprecedented [V20 O54 (NO3 )]n- , which is covalenty bound to the side chain of an aspartate residue of symmetry related molecules.

Stable Dication Diradicals of Triply Fused Metallo Chlorin-Porphyrin Heterodimers: Impact of the Bridge on the Control of Spin Coupling to Reactivity.

We report an unexpected rearrangement, controlled by the nature of the bridge, leading to the formation of novel, remarkably stable triply fused dinickel(II)/dicopper(II) chlorin-porphyrin dication diradical heterodimers in excellent yields. Here, a dipyrromethene bridge gets completely fused between two porphyrin macrocycles with two new C-C and one C-N bonds. The two macrocycles exhibit extensive π-conjugation through the bridge, which results in an antiferromagnetic coupling between the two π-cation radicals. In addition, the macrocyclic distortion also favours a rare intramolecular ferromagnetic interaction between the CuII and π-cation radical spins to form a triplet state. The structural and electronic perturbation in the unconjugated dication diradical possibly enables the bridging pyrrolic nitrogen to undergo a nucleophilic attack at the nearby ß-carbon of the porphyrin π-cation radical with a computed free energy barrier of >20 kcal mol-1 which was supplied in the form of reflux condition to initiate such a rearrangement process. UV-vis, EPR and ESI-MS spectroscopies were used to monitor the rearrangement process in situ in order to identify the key reactive intermediates leading to such an unusual transformation.

A Series of Non-Oxido VIV Complexes of Dibasic ONS Donor Ligands: Solution Stability, Chemical Transformations, Protein Interactions, and Antiproliferative Activity.

A series of mononuclear non-oxido vanadium(IV) complexes, [VIV(L1-4)2] (1-4), featuring tridentate bi-negative ONS chelating S-alkyl/aryl-substituted dithiocarbazate ligands H2L1-4, are reported. All the synthesized non-oxido VIV compounds are characterized by elemental analysis, spectroscopy (IR, UV-vis, and EPR), ESI-MS, as well as electrochemical techniques (cyclic voltammetry). Single-crystal X-ray diffraction studies of 1-3 reveal that the mononuclear non-oxido VIV complexes show distorted octahedral (1 and 2) or trigonal prismatic (3) arrangement around the non-oxido VIV center. EPR and DFT data indicate the coexistence of mer and fac isomers in solution, and ESI-MS results suggest a partial oxidation of [VIV(L1-4)2] to [VV(L1-4)2]+ and [VVO2(L1-4)]-; therefore, all these three complexes are plausible active species. Complexes 1-4 interact with bovine serum albumin (BSA) with a moderate binding affinity, and docking calculations reveal non-covalent interactions with different regions of BSA, particularly with Tyr, Lys, Arg, and Thr residues. In vitro cytotoxic activity of all complexes is assayed against the HT-29 (colon cancer) and HeLa (cervical cancer) cells and compared with the NIH-3T3 (mouse embryonic fibroblast) normal cell line by MTT assay and DAPI staining. The results suggest that complexes 1-4 are cytotoxic in nature and induce cell death in the cancer cell lines by apoptosis and that a mixture of VIV, VV, and VVO2 species could be responsible for the biological activity.

Implications of Protein Interaction in the Speciation of Potential VIVO-Pyridinone Drugs.

Vanadium complexes (VCs) are promising agents for the treatment, among others, of diabetes and cancer. The development of vanadium-based drugs is mainly limited by a scarce knowledge of the active species in the target organs, which is often determined by the interaction of VCs with biological macromolecules like proteins. Here, we have studied the binding of [VIVO(empp)2] (where Hempp is 1-methyl-2-ethyl-3-hydroxy-4(1H)-pyridinone), an antidiabetic and anticancer VC, with the model protein hen egg white lysozyme (HEWL) by electrospray ionization-mass spectrometry (ESI-MS), electron paramagnetic resonance (EPR), and X-ray crystallography. ESI-MS and EPR techniques reveal that, in aqueous solution, both the species [VIVO(empp)2] and [VIVO(empp)(H2O)]+, derived from the first one upon the loss of a empp(-) ligand, interact with HEWL. Crystallographic data, collected under different experimental conditions, show covalent binding of [VIVO(empp)(H2O)]+ to the side chain of Asp48, and noncovalent binding of cis-[VIVO(empp)2(H2O)], [VIVO(empp)(H2O)]+, [VIVO(empp)(H2O)2]+, and of an unusual trinuclear oxidovanadium(V) complex, [VV3O6(empp)3(H2O)], with accessible sites on the protein surface. The possibility of covalent and noncovalent binding with different strength and of interaction with various sites favor the formation of adducts with the multiple binding of vanadium moieties, allowing the transport in blood and cellular fluids of more than one metal-containing species with a possible amplification of the biological effects.

Promising anticancer agents based on 8-hydroxyquinoline hydrazone copper(II) complexes.

We report the synthesis and characterization of a group of benzoylhydrazones (Ln) derived from 2-carbaldehyde-8-hydroxyquinoline and benzylhydrazides containing distinct para substituents (R = H, Cl, F, CH3, OCH3, OH and NH2, for L1-7, respectively; in L8 isonicotinohydrazide was used instead of benzylhydrazide). Cu(II) complexes were prepared by reaction of each benzoylhydrazone with Cu(II) acetate. All compounds were characterized by elemental analysis and mass spectrometry as well as by FTIR, UV-visible absorption, NMR or electron paramagnetic resonance spectroscopies. Complexes isolated in the solid state (1-8) are either formulated as [Cu(HL)acetate] (with L1 and L4) or as [Cu(Ln)]3 (n = 2, 3, 5, 6, 7 and 8). Single crystal X-ray diffraction studies were done for L5 and [Cu(L5)]3, confirming the trinuclear formulation of several complexes. Proton dissociation constants, lipophilicity and solubility were determined for all free ligands by UV-Vis spectrophotometry in 30% (v/v) DMSO/H2O. Formation constants were determined for [Cu(LH)], [Cu(L)] and [Cu(LH-1)] for L = L1, L5 and L6, and also [Cu(LH-2)] for L = L6, and binding modes are proposed, [Cu(L)] predominating at physiological pH. The redox properties of complexes formed with L1, L5 and L6 are investigated by cyclic voltammetry; the formal redox potentials fall in the range of +377 to +395 mV vs. NHE. The binding of the Cu(II)-complexes to bovine serum albumin was evaluated by fluorescence spectroscopy, showing moderate-to-strong interaction and suggesting formation of a ground state complex. The interaction of L1, L3, L5 and L7, and of the corresponding complexes with calf thymus DNA was evaluated by thermal denaturation. The antiproliferative activity of all compounds was evaluated in malignant melanoma (A-375) and lung (A-549) cancer cells. The complexes show higher activity than the corresponding free ligand, and most complexes are more active than cisplatin. Compounds 1, 3, 5, and 8 were selected for additional studies: while these complexes induce reactive oxygen species and double-strand breaks in both cancer cells, their ability to induce cell-death by apoptosis varies. Within the set of compounds tested, 8 emerges as the most promising one, presenting low IC50 values, and high induction of oxidative stress and DNA damage, which eventually lead to high rates of apoptosis.

Spectroscopic Characterization and Biological Activity of Hesperetin Schiff Bases and Their Cu(II) Complexes.

The three Schiff base ligands, derivatives of hesperetin, HHSB (N-[2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chromen-4-ylidene]isonicotinohydrazide), HIN (N-[2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chromen-4-ylidene]benzhydrazide) and HTSC (N-[2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chromen-4-ylidene]thiosemicarbazide) and their copper complexes, CuHHSB, CuHIN, and CuHTSC were designed, synthesized and analyzed in terms of their spectral characterization and the genotoxic activity. Their structures were established using several methods: elemental analysis, FT-IR, UV-Vis, EPR, and ESI-MS. Spectral data showed that in the acetate complexes the tested Schiff bases act as neutral tridentate ligand coordinating to the copper ion through two oxygen (or oxygen and sulphur) donor atoms and a nitrogen donor atom. EPR measurements indicate that in solution the complexes keep their structures with the ligands remaining bound to copper(II) in a tridentate fashion with (O-, N, Oket) or (O-, N, S) donor set. The genotoxic activity of the compounds was tested against model tumour (HeLa and Caco-2) and normal (LLC-PK1) cell lines. In HeLa cells the genotoxicity for all tested compounds was noticed, for HHSB and CuHHSB was the highest, for HTSC and CuHTSC-the lowest. Generally, Cu complexes displayed lower genotoxicity to HeLa cells than ligands. In the case of Caco-2 cell line HHSB and HTSC induced the strongest breaks to DNA. On the other side, CuHHSB and CuHTSC induced the highest DNA damage against LLC-PK1.

Diheme cytochromes: Effect of mixed-axial ligation on the electronic structure and electrochemical properties with cobalt porphyrin dimer.

A series of six-coordinate diCo(III) porphyrin dimers, as synthetic analogues of diheme cytochromes, have been reported here having bis(imidazole), bis(pyridine) and mixed thiophenolate-pyridine/imidazole axial ligands. In the X-ray structures of bis(imidazole) and bis(pyridine) complexes, the axial ligands are in perpendicular orientation while they are parallelly oriented in their monomeric analog. The porphyrin rings are also highly ruffle-distorted in dimer but planar in monomer which reflect the effect of intramolecular interaction between two Co(porphyrin) units in dimers. In the X-ray structure of diCo(III) thiophenolate-pyridine mixed-ligated complex, the axial Co-S and Co-N(py) distances are 2.256(1) and 2.063(2) Å, respectively. The Co-N(py) distance of 2.063(2) Å is much longer than the distances of 1.961(3) and 1.972(3) Å observed in bis(pyridine) complex and the Co-S distance is larger than Co-N(py) in the mixed ligated complex which results in a displacement of Co by 0.15 Štowards the pyridine ligand from the mean porphyrin plane. Indeed, this is the first X-ray structure of a metalloporphyrin with mixed thiophenolate-pyridine axial ligands. The effect of mixed-axial ligation is demonstrated by a blue-shift of the Soret band in the UV-visible spectroscopy and also a positive shift of the Co(III)/Co(II) redox couple as compared to their bis(pyridine) analogue. The redox potentials are shifted to a large negative value just upon replacing the metal from iron to cobalt. The present investigation emphasizes the role of axial ligation, metal ions, and also the effect of heme-heme interaction in controlling the spectral and electrochemical properties.

Multiple and Variable Binding of Pharmacologically Active Bis(maltolato)oxidovanadium(IV) to Lysozyme.

The interaction with proteins of metal-based drugs plays a crucial role in their transport, mechanism, and activity. For an active MLn complex, where L is the organic carrier, various binding modes (covalent and non-covalent, single or multiple) may occur and several metal moieties (M, ML, ML2, etc.) may interact with proteins. In this study, we have evaluated the interaction of [VIVO(malt)2] (bis(maltolato)oxidovanadium(IV) or BMOV, where malt = maltolato, i.e., the common name for 3-hydroxy-2-methyl-4H-pyran-4-onato) with the model protein hen egg white lysozyme (HEWL) by electrospray ionization mass spectrometry, electron paramagnetic resonance, and X-ray crystallography. The multiple binding of different V-containing isomers and enantiomers to different sites of HEWL is observed. The data indicate both non-covalent binding of cis-[VO(malt)2(H2O)] and [VO(malt)(H2O)3]+ and covalent binding of [VO(H2O)3-4]2+ and cis-[VO(malt)2] and other V-containing fragments to the side chains of Glu35, Asp48, Asn65, Asp87, and Asp119 and to the C-terminal carboxylate. Our results suggest that the multiple and variable interactions of potential VIVOL2 drugs with proteins can help to better understand their solution chemistry and contribute to define the molecular basis of the mechanism of action of these intriguing molecules.

Interaction with bioligands and in vitro cytotoxicity of a new dinuclear dioxido vanadium(V) complex.

One centrosymmetric bis(µ-oxido)-bridged vanadium(V) dimer with molecular formula [(VVO2)2(pedf)2] (1) has been synthesized from the reaction of VOSO4·5H2O with a Schiff base ligand (abbreviated with pedf-) obtained from 2-acetylpyridine and 2-furoic hydrazide in methanol. Complex 1 was characterized by elemental analysis, UV-visible (UV-Vis), Fourier-transform infrared spectra (FT-IR), cyclic voltammetry (CV), electron paramagnetic resonance spectroscopy (EPR) and electrospray ionization-mass spectrometry (ESI-MS) techniques along with single crystal X-ray diffraction (SCXRD). The FT-IR spectral data of 1 indicated the involvement of oxygen and azomethine nitrogen in coordination to the central metal ion. The crystallographic studies revealed a dinuclear oxovanadium(V) complex with the Schiff base coordinated via the ONN donor set with formation of two five-membered chelate rings resulting in a distorted octahedral geometry. The interaction of 1 with calf thymus DNA (CT-DNA) was investigated by spectroscopic measurements and results suggested that the complex binds to CT-DNA via moderate intercalative mode with a binding constant (Kb) around 103 M-1. In addition, the in vitro protein binding behavior was studied by fluorescence spectrophotometric method using both bovine serum albumin (BSA) and human serum albumin (HSA) and a static quenching mechanism was observed for the interaction of the complex with both albumins that occurs with a Kb in the range (5-6) × 103 M-1. In vitro cytotoxicity of complex 1 on lung cancer cells (A549) and human skin carcinoma cell line (A431) demonstrated that the complex had a broad-spectrum of anti-proliferative activity with IC50 value of 64.2 µM and 56.2 µM.

Solution chemical properties and anticancer potential of 8-hydroxyquinoline hydrazones and their oxidovanadium(IV) complexes.

We report the synthesis and characterization of a family of benzohydrazones (Ln, n = 1-6) derived from 2-carbaldehyde-8-hydroxyquinoline and benzylhydrazides containing different substituents in the para position. Their oxidovanadium(IV) complexes were prepared and compounds with 1:1 and 1:2 metal-to-ligand stoichiometry were obtained. All compounds were characterized by elemental analyses and mass spectrometry as well as FTIR, UV-visible absorption, NMR (ligand precursors) and EPR (complexes) spectroscopies, and by DFT computational methods. Proton dissociation constants, lipophilicity and solubility in aqueous media were determined for all ligand precursors. Complex formation with V(IV)O was evaluated by spectrophotometry for L4 (Me-substituted) and L6 (OH-substituted) and formation constants for mono [VO(HL)]+, [VO(L)] and bis [VO(HL)2], [VO(HL)(L)]-, [VO(L)2]2- complexes were determined. EPR spectroscopy indicates the formation of [VO(HL)]+ and [VO(HL)2], with this latter being the major species at the physiological pH. Noteworthy, the EPR data suggest a different behaviour for L4 and L6, which confirm the results obtained in the solid state. The antiproliferative activity of all compounds was evaluated in malignant melanoma (A-375) and lung (A-549) cancer cells. All complexes show much higher activity on A-375 (IC50 < 6.3 µM) than in A-549 cells (IC50 > 20 µM). Complex 3 (F-substituted) shows the lowest IC50 on both cell lines and lower than cisplatin (in A-375). Studies identified this compound as the one showing the highest increase in Annexin-V staining, caspase activity and induction of double stranded breaks, corroborating the cytotoxicity results. The mechanism of action of the complexes involves reactive oxygen species (ROS) induced DNA damage, and cell death by apoptosis.

Interaction of pharmacologically active pyrone and pyridinone vanadium(IV,V) complexes with cytochrome c.

The interaction between cytochrome c (Cyt) and potential vanadium drugs, formed by 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate (dhp) and maltolate (ma), was studied by ElectroSpray Ionization-Mass Spectrometry (ESI-MS). Since under physiological conditions redox processes are possible, the binding of the complexes in the oxidation state +IV and +V, [VIVO(dhp)2], [VIVO(ma)2], [VVO2(dhp)2]- and [VVO2(ma)2]-, was examined. In all systems VIV,V-L-Cyt adducts are observed, their formation depending on V oxidation state, ligand L and metal concentration. The larger stability of vanadium(IV) than vanadium(V) complexes favors the interaction of the moieties VIVOL2 and VIVOL+ with VIV, while with VV adducts with VVO2L and VVO2+ fragments are observed. The analysis of the protein structure suggests that Glu4, Glu21, Asp50, Glu62, Glu66 and Glu104 are the most plausible candidates for monodentate coordination, while the couples (Asp2, Glu4), (Glu92, Asp93) and (His33, Glu104) for bidentate binding. The values of E1/2 for [VIVO(dhp)2] and [VIVO(ma)2], measured by cyclic voltammetry (CV), 0.53 V and 0.60 V vs. standard hydrogen electrode, indicate that the oxidation of VIV to VV is possible. The presence of a protein can alter the redox behavior and stabilize one of the states, VIV or VV. Overall, the data reinforce the conclusion that, for V drugs, the biotransformation is fundamental to explain their biological action and the analysis should not be limited to the ligand exchange and hydrolysis but also include the redox processes, and that a mixture of VIV and VV species, VIV,V-L-Protein and VIV,V-Protein, could be responsible of the pharmacological effects.

Highly Oxidized Cobalt Porphyrin Dimer: Control of Spin Coupling via a Bridge.

A cobalt porphyrin dimer is constructed in which two Co(II)porphyrins are connected covalently through a redox-active diethylpyrrole moiety via a flexible but "nonconjugated" methylene bridge. Upon oxidation with even a mild oxidant such as iodine, each cobalt(II) center and porphyrin ring undergo 1e- oxidation, leading to the formation of a 4e--oxidized cobalt(III)porphyrin dication diradical complex. Other oxidants such as Cl2 and Br2 also produce similar results. To stabilize such highly oxidized dication diradicals, the "nonconjugated" methylene spacer undergoes a facile and spontaneous oxidation to form a methine group with a drastic structural change, thereby making the bridge fully π-conjugated and enabling through-bond communication. This results in a strong spin coupling between two π-cation radicals which stabilizes the singlet state. The experimental observations are also strongly supported by extensive density functional theory calculations. The present study highlights the crucial role played by the nature of the bridge in the long-range electronic communication.

New mixed ligand oxidovanadium(IV) complexes: Solution behavior, protein interaction and cytotoxicity.

Herein we report the synthesis of five new mononuclear mixed ligand oxidovanadium(IV) complexes [VIVO(L1-3)(LNN)] (1-5) with tridentate O,N,O-donor aroylhydrazones as main ligand (H2L1-3) and N,N-chelating 2,2'-bipyridine (bipy) and 1,10-phenanthroline (phen) as co-ligand (LNN). The complexes were characterized by elemental and thermogravimetric analysis (TGA), IR, UV-vis, and electron paramagnetic resonance (EPR) spectroscopy, electrospray ionization-mass spectrometry (ESI-MS) and cyclic voltammetry (CV). The structure of 1-5 was confirmed by single crystal X-ray analysis and also optimized by density functional theory (DFT) methods. At physiological pH an equilibrium [VIVO(L1-3)(LNN)] + H2O ⇄ [VIVO(L1-3)(H2O)] + LNN, shifted towards left, is established, with water molecule that could be replaced by the biomolecules of the organism. The studies on the interaction with two proteins, lysozyme (Lyz) chosen as a representative model of a small protein, and human serum albumin (HSA) show that two types of binding are possible: a non-covalent binding through the accessible residues on protein surface with [VIVO(L1-3)(LNN)] keeping its octahedral structure, and a covalent binding upon the replacement of water in [VIVO(L1-3)(H2O)] with His-N donors to form VIVO(L1-3)(HSA). In vitro cytotoxicity of ligands and complexes were screened against human cervical cancer (HeLa) (IC50 = 7.39-15.13 µM), colon cancer (HT-29) (IC50 = 11.04-28.20 µM) and mouse embryonic fibroblast (NIH-3T3) cell lines (IC50 = 62.22-87.75 µM) by MTT assay. Particularly, 5 showed higher cytotoxicity than cisplatin and cyclophosphamide, with an IC50 of 7.39 ± 1.21 µM and 11.04 ± 0.29 µM against HeLa and HT-29.

Binding of VIV O2+ , VIV OL, VIV OL2 and VV O2 L Moieties to Proteins: X-ray/Theoretical Characterization and Biological Implications.

Vanadium compounds have frequently been proposed as therapeutics, but their application has been hampered by the lack of information on the different V-containing species that may form and how these interact with blood and cell proteins, and with enzymes. Herein, we report several resolved crystal structures of lysozyme with bound VIV O2+ and VIV OL2+ , where L=2,2'-bipyridine or 1,10-phenanthroline (phen), and of trypsin with VIV O(picolinato)2 and VV O2 (phen)+ moieties. Computational studies complete the refinement and shed light on the relevant role of hydrophobic interactions, hydrogen bonds, and microsolvation in stabilizating the structure. Noteworthy is that the trypsin-VV O2 (phen) and trypsin-VIV O(OH)(phen) adducts correspond to similar energies, thus suggesting a possible interconversion under physiological/biological conditions. The obtained data support the relevance of hydrolysis of VIV and VV complexes in the several types of binding established with proteins and the formation of different adducts that might contribute to their pharmacological action, and significantly widen our knowledge of vanadium-protein interactions.