Elucidation of Binding Site and Chiral Specificity of Oxidovanadium Drugs with Lysozyme through Theoretical Calculations.

This study presents an implementation of the protein-ligand docking program GOLD and a generalizable method to predict the binding site and orientation of potential vanadium drugs. Particularly, theoretical methods were applied to the study of the interaction of two VIVO complexes with antidiabetic activity, [VIVO(pic)2(H2O)] and [VIVO(ma)2(H2O)], where pic is picolinate and ma is maltolate, with lysozyme (Lyz) for which electron paramagnetic resonance spectroscopy suggests the binding of the moieties VO(pic)2 and VO(ma)2 through a carboxylate group of an amino acid residue (Asp or Glu). The work is divided in three parts: (1) the generation of a new series of parameters in GOLD program for vanadium compounds and the validation of the method on five X-ray structures of VIVO and VV species bound to proteins; (2) the prediction of the binding site and enantiomeric preference of [VO(pic)2(H2O)] to lysozyme, for which the X-ray diffraction analysis displays the interaction of a unique isomer (i.e., OC-6-23-Δ) through Asp52 residue, and the subsequent refinement of the results with quantum mechanics/molecular mechanics methods; (3) the application of the same approach to the interaction of [VO(ma)2(H2O)] with lysozyme. The results show that convenient implementation of protein-ligand docking programs allows for satisfactorily reproducing X-ray structures of metal complexes that interact with only one coordination site with proteins and predicting with blind procedures relevant low-energy binding modes. The results also demonstrate that the combination of docking methods with spectroscopic data could represent a new tool to predict (metal complex)-protein interactions and have a general applicability in this field, including for paramagnetic species.

Nonoxido V(IV) Complexes: Prediction of the EPR Spectrum and Electronic Structure of Simple Coordination Compounds and Amavadin.

Density functional theory (DFT) calculations of the (51)V hyperfine coupling (HFC) tensor A have been completed for 20 "bare" V(IV) complexes with different donor sets, electric charges, and coordination geometries. Calculations were performed with ORCA and Gaussian software, using functionals BP86, TPSS0, B1LYP, PBE0, B3LYP, B3P, B3PW, O3LYP, BHandHLYP, BHandH, and B2PLYP. Among the basis sets, 6-311g(d,p), 6-311++g(d,p), VTZ, cc-pVTZ, def2-TZVPP, and the "core properties" CP(PPP) were tested. The experimental Aiso and Ai (where i = x or z, depending on the geometry and electronic structure of V(IV) complex) were compared with the values calculated by DFT methods. The results indicated that, based on the mean absolute percentage deviation (MAPD), the best functional to predict Aiso or Ai is the double hybrid B2PLYP. With this functional and the basis set VTZ, it is possible to predict the Aiso and Az of the EPR spectrum of amavadin with deviations of -1.1% and -2.0% from the experimental values. The results allowed us to divide the spectra of nonoxido V(IV) compounds in three types-called "type 1", "type 2", and "type 3", characterized by different composition of the singly occupied molecular orbital (SOMO) and relationship between the values of Ax, Ay, and Az. For "type 1" spectra, Az ≫ Ax ≈ Ay and Az is in the range of (135-155) × 10(-4) cm(-1); for "type 2" spectra, Ax ≈ Ay ≫ Az and Ax ≈ Ay are in the range of (90-120) × 10(-4) cm(-1); and for the intermediate spectra of "type 3", Az > Ay > Ax or Ax > Ay > Az, with Az or Ax values in the range of (120-135) × 10(-4) cm(-1). The electronic structure of the V(IV) species was also discussed, and the results showed that the values of Ax or Az are correlated with the percent contribution of V-dxy orbital in the SOMO. Similarly to V(IV)O species, for amavadin the SOMO is based mainly on the V-dxy orbital, and this accounts for the large experimental value of Az (153 × 10(-4) cm(-1)).

Speciation of the Potential Antitumor Agent Vanadocene Dichloride in the Blood Plasma and Model Systems.

The speciation of the potential antitumor agent vanadocene dichloride ([Cp2VCl2], abbreviated with VDC) in the blood plasma was studied by instrumental (EPR, ESI-MS, MS-MS, and electronic absorption spectroscopy) and computational (DFT) methods. The behavior of VDC at pH 7.4 in aqueous solution, the interaction with the most important bioligands of the plasma (oxalate, carbonate, phosphate, lactate, citrate, histidine, and glycine among those with low molecular mass and transferrin and albumin between the proteins) was evaluated. The results suggest that [Cp2VCl2] transforms at physiological pH to [Cp2V(OH)2] and that only oxalate, carbonate, phosphate, and lactate are able to displace the two OH(-) ions to yield [Cp2V(ox)], [Cp2V(CO3)], [Cp2V(lactH(-1))], and [Cp2V(HPO4)]. The formation of the adducts with oxalate, carbonate, lactate, and hydrogen phosphate was confirmed also by ESI-MS and MS-MS spectra. The stability order is [Cp2V(ox)] ≫ [Cp2V(CO3)] > [Cp2V(lactH(-1))] > [Cp2V(HPO4)]. No interaction between VDC and plasma proteins was detected under our experimental conditions. Several model systems containing the bioligands (bL) in the same relative ratio as in the blood samples were also examined. Finally, the speciation of VDC in the plasma was studied. The results obtained show that the model systems behave as the blood plasma and indicate that when V concentration is low (10 µM) VDC is transported in the bloodstream as [Cp2V(ox)]; when V concentration is high (100 µM) oxalate binds only 9.2 µM of [Cp2V](2+), whereas the remaining part distributes between [Cp2V(CO3)] (main species) and [Cp2V(lactH(-1))] (minor species); and when V concentration is in the range 10-100 µM [Cp2V](2+) distributes between [Cp2V(ox)] and [Cp2V(CO3)].

Interaction of antidiabetic vanadium compounds with hemoglobin and red blood cells and their distribution between plasma and erythrocytes.

The interaction of V(IV)O(2+) ion with hemoglobin (Hb) was studied with the combined application of spectroscopic (EPR), spectrophotometric (UV-vis), and computational (DFT methods) techniques. Binding of Hb to V(IV)O(2+) in vitro was proved, and three unspecific sites (named α, ß, and γ) were characterized, with the probable coordination of His-N, Asp-O(-), and Glu-O(-) donors. The value of log ß for (VO)Hb is 10.4, significantly lower than for human serum apo-transferrin (hTf). In the systems with V(IV)O potential antidiabetic compounds, mixed species cis-VOL2(Hb) (L = maltolate (ma), 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate (dhp)) are observed with equatorial binding of an accessible His residue, whereas no ternary complexes are observed with acetylacetonate (acac). The experiments of uptake of [VO(ma)2], [VO(dhp)2], and [VO(acac)2] by red blood cells indicate that the neutral compounds penetrate the erythrocyte membrane through passive diffusion, and percent amounts higher than 50% are found in the intracellular medium. The biotransformation of [VO(ma)2], [VO(dhp)2], and [VO(acac)2] inside the red blood cells was proved. [VO(dhp)2] transforms quantitatively in cis-VO(dhp)2(Hb), [VO(ma)2] in cis-VO(ma)2(Hb), and cis-VO(ma)2(Cys-S(-)), with the equatorial coordination of a thiolate-S(-) of GSH or of a membrane protein, and [VO(acac)2] in the binary species (VO)xHb and two V(IV)O complexes with formulation VO(L(1),L(2)) and VO(L(3),L(4)), where L(1), L(2), L(3), and L(4) are red blood cell bioligands. The results indicate that, in the studies on the transport of a potential pharmacologically active V species, the interaction with red blood cells and Hb cannot be neglected, that a distribution between the erythrocytes and plasma is achieved, and that these processes can significantly influence the effectiveness of a V drug.

Interaction of insulin-enhancing vanadium compounds with human serum holo-transferrin.

The interaction of VO(2+) ion and four insulin-enhancing compounds, [VO(ma)2], [VO(dhp)2], [VO(acac)2], and cis-[VO(pic)2(H2O)], where Hma, Hdhp, Hacac, and Hpic are maltol, 1,2-dimethyl-3-hydroxy-4(1H)-pyridinone, acetylacetone, and picolinic acid, with holo-transferrin (holo-hTf) was studied through the combined application of electron paramagnetic resonance (EPR) and density functional theory (DFT) methods. Since in holo-hTf all of the specific binding sites of transferrin are saturated by Fe(3+) ions, VO(2+) can interact with surface sites (here named sites C), probably via the coordination of His-N, Asp-COO(-), and Glu-COO(-) donors. In the ternary systems with the insulin-enhancing compounds, mixed species are observed with Hma, Hdhp, and Hpic with the formation of VOL2(holo-hTf), explained through the interaction of cis-[VOL2(H2O)] (L = ma, dhp) or cis-[VOL2(OH)](-) (L = pic) with an accessible His residue that replaces the monodentate H2O or OH(-) ligand. The residues of His-289, His-349, His-473, and His-606 seem the most probable candidates for the complexation of the cis-VOL2 moiety. The lack of a ternary complex with Hacac was attributed to the square-pyramidal structure of [VO(acac)2], which does not possess equatorial sites that can be replaced by the surface His-N. Since holo-transferrin is recognized by the transferrin receptor, the formation of ternary complexes between VO(2+) ion, a ligand L(-), and holo-hTf may be a way to transport vanadium compounds inside the cells.

V(IV)O versus V(IV) complex formation by tridentate (O, N(arom), O) ligands: prediction of geometry, EPR 51V hyperfine coupling constants, and UV-Vis spectra.

Systems formed using the V(IV)O(2+) ion with tridentate ligands containing a (O, N(arom), O) donor set were described. Examined ligands were 3,5-bis(2-hydroxyphenyl)-1-phenyl-1H-1,2,4-triazole (H2hyph(Ph)), 4-[3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl]benzoic acid (H3hyph(C)), 4-[3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl]benzenesulfonic acid (H3hyph(S)), and 2,6-bis(2-hydroxyphenyl)pyridine (H2bhpp), with H3hyph(C) being an orally active iron chelator that is commercially available under the name Exjade (Novartis) for treatment of chronic iron overload arising from blood transfusions. The systems were studied using EPR, UV-Vis, and IR spectroscopies, pH potentiometry, and DFT methods. The ligands bind vanadium with the two terminal deprotonated phenol groups and the central aromatic nitrogen to give six-membered chelate rings. In aqueous solution the main species were the mono- and bis-chelated V(IV)O complexes, whereas in the solid state neutral non-oxido V(IV) compounds were formed. [V(hyph(Ph))2] and [V(bhpp)2] are hexacoordinated, with a geometry close to the octahedral and a meridional arrangement of the ligands. DFT calculations allow distinguishing V(IV)O and V(IV) species and predicting their structure, the (51)V hyperfine coupling constant tensor A, and the electronic absorption spectra. Finally, EPR spectra of several non-oxido V(IV) species were compared using relevant geometrical parameters to demonstrate that in the case of tridentate ligands the (51)V hyperfine coupling constant is related to the geometric isomerism (meridional or facial) rather than the twist angle Φ, which measures the distortion of the hexacoordinated structure toward a trigonal prism.

Formation of new non-oxido vanadium(IV) species in aqueous solution and in the solid state by tridentate (O, N, O) ligands and rationalization of their EPR behavior.

The systems formed by the V(IV)O(2+) ion with tridentate ligands provided with the (O, N(imine), O) donor set were described. The ligands studied were 2,2'-dihydroxyazobenzene (Hdhab), α-(2-hydroxy-5-methylphenylimino)-o-cresol (Hhmpic), calmagite (H2calm), anthracene chrome red A (H3anth), calcon (H2calc), and calconcarboxylic acid (H3calc(C)). They can bind vanadium with the two deprotonated phenol groups and the imine nitrogen to give (5,6)-membered chelate rings. The systems were studied with EPR, UV-vis and IR spectroscopy, pH-potentiometry, and DFT methods. The ligands form unusual non-oxido V(IV) compounds both in aqueous solution and in the solid state. [V(anthH(-1))2](4-) and [V(calmH(-1))2](2-) (formed in water at the physiological pH) and [V(dhabH(-1))2] and [V(hmpicH(-1))2] (formed in the solid state in MeOH) are hexa-coordinated with geometry intermediate between the octahedron and the trigonal prism and an unsymmetric facial arrangement of the two ligand molecules. DFT calculations were used to predict the structure and (51)V hyperfine coupling tensor A of the non-oxido species. The EPR behavior of 13 non-oxido V(IV) species was put into relationship with the relevant geometrical parameters and was rationalized in terms of the spin density on the d(xy) orbital. Depending on the geometric isomer formed (meridional or facial), d(z)(2) mixes with the d(xy) orbital, and this effect causes the lowering of the highest (51)V A value.

Application of DFT methods to the study of the coordination environment of the VO2+ ion in V proteins.

Density functional theory (DFT) methods were used to simulate the environment of vanadium in several V proteins, such as vanadyl-substituted carboxypeptidase (sites A and B), vanadyl-substituted chloroplast F(1)-ATPase (CF(1); site 3), the reduced inactive form of vanadium bromoperoxidase (VBrPO; low- and high-pH sites), and vanadyl-substituted imidazole glycerol phosphate dehydratase (IGPD; sites α, ß, and γ). Structural, electron paramagnetic resonance, and electron spin echo envelope modulation parameters were calculated and compared with the experimental values. All the simulations were performed in water within the framework of the polarizable continuum model. The angular dependence of [Formula: see text] and [Formula: see text] on the dihedral angle θ between the V=O and N-C bonds and on the angle φ between the V=O and V-N bonds, where N is the coordinated aromatic nitrogen atom, was also found. From the results it emerges that it is possible to model the active site of a vanadium protein through DFT methods and determine its structure through the comparison between the calculated and experimental spectroscopic parameters. The calculations confirm that the donor sets of sites B and A of vanadyl-substituted carboxypeptidase are [[Formula: see text], H(2)O, H(2)O, H(2)O] and [N(His)(||), N(His)(⊥), [Formula: see text], H(2)O], and that the donor set of site 3 of CF(1)-ATPase is [[Formula: see text], OH(Thr), H(2)O, H(2)O, [Formula: see text]]. For VBrPO, the coordination modes [N(His)(||), N(His)(∠), OH(Ser), H(2)O, H(2)O(ax)] for the low-pH site and [N(His)(||), N(His)(∠), OH(Ser), OH(-), H(2)O(ax)] or [N(His)(||), N(His)(∠), [Formula: see text], H(2)O] for the high-pH site, with an imidazole ring of histidine strongly displaced from the equatorial plane, can be proposed. Finally, for sites α, ß, and γ of IGPD, the subsequent deprotonation of one, two, and three imidazole rings of histidine and the participation of a carboxylate group of a glutamate residue ([N(His)(||), [Formula: see text], H(2)O, H(2)O], [N(His)(||), N(His)(||), [Formula: see text], H(2)O], and [N(His)(||), N(His)(||), [Formula: see text], OH(-), [Formula: see text]], respectively) seems to be the most plausible hypothesis.

Is the spin-orbit coupling important in the prediction of the 51V hyperfine coupling constants of V(IV) O2+ species? ORCA versus Gaussian performance and biological applications.

Density functional theory calculations of the (51)V hyperfine coupling (HFC) tensor A, have been completed for eighteen V(IV)O(2+) complexes with different donor set, electric charge and coordination geometry. A tensor was calculated with ORCA software with several functionals and basis sets taking into account the spin-orbit coupling contribution. The results were compared with those obtained with Gaussian 03 software using the half-and-half functional BHandHLYP and 6-311g(d,p) basis set. The order of accuracy of the functionals in the prediction of A(iso), A(z) and dipolar term A(z,anis) is BHandHLYP > PBE0 >> B3PW > TPSSh >> B3LYP >> BP86 > VWN5 (for A(iso)), BHandHLYP > PBE0 >> B3PW > TPSSh > B3LYP >> BP86 > VWN5 (for A(z)), B3LYP > PBE0 ∼ B3PW ∼ BHandHLYP >> TPSSh > BP86 ∼ VWN5 (for A(z,anis)). The good agreement in the prediction of A(z) with BHandHLYP is due to a compensation between the overestimation of A(iso) and underestimation of A(z,anis) (A(z) = A(iso) + A(z,anis)), whereas among the hybrid functionals PBE0 performs better than the other ones. BHandHLYP functional and Gaussian software are recommended when the V(IV)O(2+) species contains only V-O and/or V-N bonds, whereas PBE0 functional and ORCA software for V(IV)O(2+) complexes with one or more V-S bonds. Finally, the application of these methods to the coordination environment of V(IV)O(2+) ion in V-proteins, like vanadyl-substituted insulin, carbonic anhydrase, collagen and S-adenosylmethionine synthetase, was discussed.

Complex formation in aqueous solution and in the solid state of the potent insulin-enhancing V(IV)O2+ compounds formed by picolinate and quinolinate derivatives.

The complexation of V(IV)O(2+) ion with 10 picolinate and quinolinate derivatives, provided with the donor set (N, COO(-)), was studied in aqueous solution and in the solid state through the combined application of potentiometric (pH-titrations), spectroscopic (EPR, UV/vis and IR spectroscopy), and computational (density functional theory (DFT) calculations) methods. Such derivatives, that form potent insulin-enhancing V(IV)O(2+) compounds, are picolinic (picH), 6-methylpicolinic (6-mepicH), 3-methylpicolinic (3-mepicH), 5-butylpicolinic or fusaric (fusarH), 6-methyl-2,3-pyridindicarboxylic (6-me-2,3-pdcH(2)), 2-pyridylacetic (2-pyacH), 2-quinolinecarboxylic or quinaldic (quinH), 4-hydroxyquinoline-2-carboxylic or kynurenic (kynurH), 1-isoquinolinecarboxylic (1-iqcH) and 3-isoquinolinecarboxylic (3-iqcH) acid. On the basis of the potentiometric, spectroscopic, and DFT results, they were divided into the classes A, B, and C. The ligands belonging to class A (3-mepicH, 1-iqcH, 2-pyacH) form square pyramidal complexes in aqueous solution and in the solid state, and those belonging to class B (picH, fusarH, 3-iqcH) form cis-octahedral species, in which the two ligands adopt an (equatorial-equatorial) and an (equatorial-axial) arrangement and one water molecule occupies an equatorial site in cis position with respect to the V═O bond. Class C ligands (6-mepicH, 6-me-2,3-pdcH(2), quinH, kynurH) yield bis chelated species, that in water are in equilibrium between the square pyramidal and trans-octahedral form, where both the ligand molecules adopt an (equatorial-equatorial) arrangement and one water is in trans position with respect to the V═O group. The trans-octahedral compounds are characterized by an anomalous electron paramagnetic resonance (EPR) response, with A(z) value being reduced by about 10% with respect to the prediction of the "additivity rule". DFT methods allow to calculate the structure, (51)V hyperfine coupling constant (A(z)), the stretching frequency of V═O bond (ν(V═O)), the relative stability in aqueous solution, and the electronic structure and molecular orbital composition of bis chelated complexes. The results were used to explain the biotransformation of these potent insulin-enhancing compounds in blood serum.

Interaction of VO2+ ion and some insulin-enhancing compounds with immunoglobulin G.

Complexation of VO(2+) ion with the most abundant class of human immunoglobulins, immunoglobulin G (IgG), was studied using EPR spectroscopy. Differently from the data in the literature which report no interaction of IgG with vanadium, in the binary system VO(2+)/IgG at least three sites with comparable strength were revealed. These sites, named 1, 2, and 3, seem to be not specific, and the most probable candidates for metal ion coordination are histidine-N, aspartate-O or glutamate-O, and serinate-O or threoninate-O. The mean value for the association constant of (VO)(x)IgG, with x = 3-4, is log ß = 10.3 ± 1.0. Examination of the ternary systems formed by VO(2+) with IgG and human serum transferrin (hTf) and human serum albumin (HSA) allows one to find that the order of complexing strength is hTf ≫ HSA ≈ IgG. The behavior of the ternary systems with IgG and one insulin-enhancing agent, like [VO(6-mepic)(2)], cis-[VO(pic)(2)(H(2)O)], [VO(acac)(2)], and [VO(dhp)(2)], where 6-mepic, pic, acac, and dhp indicate the deprotonated forms of 6-methylpicolinic and picolinic acids, acetylacetone, and 1,2-dimethyl-3-hydroxy-4(1H)-pyridinone, is very similar to the corresponding systems with albumin. In particular, at the physiological pH value, VO(6-mepic)(IgG)(OH), cis-VO(pic)(2)(IgG), and cis-VO(dhp)(2)(IgG) are formed. In such species, IgG coordinates nonspecifically VO(2+) through an imidazole-N belonging to a histidine residue exposed on the protein surface. For cis-VO(dhp)(2)(IgG), log ß is 25.6 ± 0.6, comparable with that of the analogous species cis-VO(dhp)(2)(HSA) and cis-VO(dhp)(2)(hTf). Finally, with these new values of log ß, the predicted percent distribution of an insulin-enhancing VO(2+) agent between the high molecular mass (hTf, HSA, and IgG) and low molecular mass (lactate) components of the blood serum at physiological conditions is calculated.

VO2+ complexation by bioligands showing keto-enol tautomerism: a potentiometric, spectroscopic, and computational study.

The interaction of VO(2+) ion with ligands of biological interest that are present in important metabolic pathways--2-oxopropanoic acid (pyruvic acid, pyrH), 3-hydroxy-2-oxopropanoic acid (3-hydroxypyruvic acid, hydpyrH), oxobutanedioic acid (oxalacetic acid, oxalH(2)), (S)-hydroxybutanedioic acid (l-malic acid, malH(2)), and 2,3-dihydroxy-(E)-butanedioic acid (dihydroxyfumaric acid, dhfH(2))--was described. Their complexing capability was compared with that of similar ligands: 3-hydroxy-2-butanone (hydbut) and 3,4-dihydroxy-3-cyclobutene-1,2-dione (squaric acid, squarH(2)). All of these ligands (except l-malic acid) exhibit keto-enol tautomerism, and the presence of a metal ion can influence such an equilibrium. The different systems were studied with electron paramagnetic resonance (EPR) and UV-vis spectroscopies and with pH potentiometry. Density functional theory (DFT) methods provide valuable information on the relative energy of the enol and keto forms of the ligands both in the gas phase and in aqueous solution, on the geometry of the complexes, and on EPR and electronic absorption parameters. The results show that most of the ligands behave like α-hydroxycarboxylates, forming mono- and bis-chelated species with (COO(-), O(-)) coordination, demonstrating that the metal ion is able to stabilize the enolate form of some ligands. With dihydroxyfumaric acid, the formation of a non-oxidovanadium(IV) complex, because of rearrangement of dihydroxyfumaric to dihydroxymaleic acid (dhmH(2)), can be observed. With 3-hydroxy-2-butanone and 3,4-dihydroxy-3-cyclobutene-1,2-dione, complexation of VO(2+) does not take place and the reason for this behavior is explained by chemical considerations and computational calculations.

A quantitative study of the biotransformation of insulin-enhancing VO(2+) compounds.

Potentiometric (pH titrations) and spectroscopic (electron paramagnetic resonance) methods have been used to determine the thermodynamic stability constants of the various VO(2+) complexes formed after the interaction of four insulin-enhancing vanadium compounds, [VO(6-mepic)(2)], cis-[VO(pic)(2)(H(2)O)], [VO(acac)(2)], and [VO(dhp)(2)], where 6-mepic, pic, acac, and dhp indicate the deprotonated forms of 6-methylpicolinic acid, picolinic acid, acetylacetone, and 1,2-dimethyl-3-hydroxy-4(1H)-pyridinone, with high molecular mass [human serum apotransferrin (hTf) and human serum albumin (HSA)] and low molecular mass (lactate) components of blood serum. In particular, log beta values for the formation of (VO)hTf (13.0 +/- 0.5), (VO)(2)hTf (25.5 +/- 0.5), (VO)HSA (9.1 +/- 1.0), (VO) (2) (d) HSA (20.9 +/- 1.0), cis-VO(dhp)(2)(hTf) (25.5 +/- 0.6), cis-VO(dhp)(2)(HSA) (25.9 +/- 0.6), (VO)hTf(lact) (14.5 +/- 0.8), (VO)(2)hTf(lact)(2) (28.5 +/- 0.8), (VO)hTf(pic) (15.6 +/- 0.8), and (VO)(2)hTf(pic)(2) (30.4 +/- 0.8) were determined. The values of the stability constants were used to compare the calculated composition of ternary and quinary systems with that recently proposed by some of us through electron paramagnetic resonance and density functional theory methods (Sanna et al. in Inorg. Chem. 49:174-187, 2010) and to predict the distribution of VO(2+) ion in blood serum when one of the four insulin-enhancing vanadium compounds studied, [VO(carrier)(2)], is administered.

The equilibrium between the octahedral and square pyramidal form and the influence of an axial ligand on the molecular properties of V(IV)O complexes: a spectroscopic and DFT study.

The previously unreported equilibrium in aqueous solution between the V(IV)O square pyramidal and trans octahedral form with an axial water molecule for a number of bidentate ligands with (N,O) and (O,O) donor sets (6-methylpicolinic (6-mepicH) and 6-methyl-2,3-pyridinedicarboxylic (6-me-2,3-pdcH(2)) acids, dipyridin-2-ylmethanol (Hdpmo) and 1,2-dimethyl-3-hydroxy-4(1H)-pyridinone (Hdhp)) has been demonstrated by the combined application of EPR spectroscopy and DFT methods. The EPR spectra suggest that, with increasing ionic strength, the equilibrium is shifted towards the formation of the pentacoordinated species and values of K approximately 4.0 and 7.0 for the systems containing 6-methyl-2,3-pyridinedicarboxylic acid and dipyridin-2-ylmethanol were measured. DFT calculations performed with Gaussian 03 and ORCA software predict the (51)V anisotropic hyperfine coupling constant along the z axis (A(z)), which can be used to demonstrate the presence of an axially bound ligand trans to the V=O bond. The results suggest that an axial donor (charged or not) can lower |A(z)|, in contrast to what was previously believed on the basis of the "additivity rule", and this explains the anomalous behaviour of the V(IV)O complexes formed by N-{2-[(2-pyridylmethylene)amino]phenyl}pyridine-2-carboxamide (Hcapca) and several amidrazone derivatives. The decrease in |A(z)| for the axial binding of a solvent molecule is mainly a result of the reduction of |A(iso)| and this was also observed when the solid [VO(6-methylpicolinato)(2)] was dissolved in DMSO or DMF. The variations in the structural (V=O, V-O and V-N distances, O-V-O and N-V-N angles, and the trigonality index tau) and spectroscopic (|A(z)|, |A(iso)| and nu(V=O)) properties as a function of the axial V-OH(2) distance (R) are also presented. Finally, the electronic structures of the penta- and hexacoordinated complexes are discussed.

New developments in the comprehension of the biotransformation and transport of insulin-enhancing vanadium compounds in the blood serum.

The possible biotransformations in the blood serum of four representative insulin-enhancing vanadium compounds, [VO(6-mepic)(2)], cis-[VO(pic)(2)(H(2)O)], [VO(acac)(2)], and [VO(dhp)(2)], where 6-mepic, pic, acac, and dhp indicate the deprotonated forms of 6-methylpicolinic and picolinic acids, acetylacetone, and 1,2-dimethyl-3-hydroxy-4(1H)-pyridinone, were examined. In particular, the behavior of the quinary systems formed by the insulin-enhancing species, human serum apo-transferrin (hTf), human serum albumin (HSA), and lactate (lact) or citrate (citr) at physiological pH and conditions was studied. The results indicate that, besides the case in which the ligand is very weak like 6-mepic, the carrier can interact in some form with VO(2+) ion until its intake into the cell. In fact with stronger ligands like pic, acac, and dhp, VO(2+) is transported not only by transferrin but also as [VO(carrier)(2)] and as mixed species VO(2+)-hTF-carrier. There are two ways in which the undissociated form of a bis-chelated complex can interact with transferrin, one "specific" when the carrier possesses a carboxylate group and behaves like a synergistic anion, and another "non-specific" when an imidazole nitrogen of a histidine residue from hTf replaces an equatorially coordinated water molecule giving rise to a ternary species with cis-octahedral geometry and cis-VO(carrier)(2)(hTf) stoichiometry. It is found that also albumin can participate in the transport of an insulin-enhancing compound forming a mixed species cis-VO(carrier)(2)(HSA), when the carrier stabilizes in aqueous solution the cis-octahedral form, or the dinuclear compound (VO)(2)(d)HSA, when the carrier forms unstable complexes. These insights were confirmed through density functional theory (DFT) calculations.

Assessing the dependence of (51)V A(z) value on the aromatic ring orientation of V(IV)O(2+) pyridine complexes.

Characterization of V(IV) biomolecules relies strongly on electron paramagnetic resonance (EPR) spectroscopy, particularly the application of the additivity relationship of A(z) values. It has been shown experimentally that the A(z) values of V(IV)O(2+) imidazole species have a critical angular dependence. Density-functional theory (DFT) calculations elucidate the dependence of (51)V A(z) value on the orientation of the aromatic ring in V(IV)O(2+) pyridine complexes, following closely the functional dependence observed for V(IV)O(2+) imidazole species, [A(z)(pyr) = 42.23 + 1.80 x sin(2theta - 90)], with A(z) measured in 10(4) cm(-1). A DFT re-examination of V(IV)O(2+) imidazole complexes gives an equation very similar [A(z)(imid) = 42.35 + 2.34 x sin(2theta - 90)] to that experimentally found. These results generalize the application of the additivity relationship for V(IV)O(2+) complexes containing aromatic nitrogen ligands such as pyridine or imidazole. The increase of the absolute value of A(z), |A(z)|, when the dihedral angle theta between the V=O and N(pyr)-C or N(imid)-C bonds varies from a parallel to a perpendicular orientation is due to an increase of the d vanadium orbital contribution and to a decrease of the pi aromatic system participation in the singly occupied molecular orbital.

On the transport of vanadium in blood serum.

The complexation of the VO(2+) ion in several systems that can model the physiological conditions of its transport in blood serum was studied using electron paramagnetic resonance (EPR) spectroscopy. Particularly, the ternary systems formed by (i) VO(2+) and two high-molecular-mass components of blood serum, human serum apo-transferrin (hTf) and human serum albumin (HSA); (ii) VO(2+), hTf, and bL; and (iii) VO(2+), HSA, and bL, where bL is one of the six most important low-molecular-mass bioligands of the blood serum (bL = lactate, citrate, oxalate, phosphate, glycine, or histidine), were examined. The results indicate that, in aqueous solution, transferrin is a stronger binder than albumin, and at the physiological ratio, most of the VO(2+) ion is present as (VO)(2)hTf, and a small amount as (VO)(2)(d)HSA, the dinuclear species formed by albumin where the two metal ions are interacting and the spin state S is 1. Among the bL ligands, only lactate and citrate are able to bind VO(2+) in the presence of transferrin or albumin, the others not interacting at all. Finally, the quaternary systems formed by (i) VO(2+), hTf, HSA, and lactate and (ii) VO(2+), hTf, HSA, and citrate were studied. In these cases, the results suggest that the predominant species is (VO)(2)hTf, followed by the mixed complexes VO(2+)-hTf-lactate or VO(2+)-hTf-citrate, whereas (VO)(2)(d)HSA and [(VO)(2)(citrH(-1))(2)](4-) are minor components at physiological pH. The conclusions of this study give new insights on how the VO(2+) ion distributes among the blood serum components and is transported in the plasma toward the target sites in the organism.

Interaction of copper(II) with the prion peptide fragment HuPrP(76-114) encompassing four histidyl residues within and outside the octarepeat domain.

Complex formation processes between the 39-mer residue peptide fragment of human prion protein, HuPrP(76-114), and copper(II) ions have been studied by potentiometric, UV-vis, circular dichroism (CD), electron paramagnetic resonance, and electrospray ionization mass spectrometry methods. This peptide consists of 39 amino acid residues and contains two histidines (His77 and His85) belonging to the octarepeat domain and two histidines (His96 and His111) outside this domain. It was found that HuPrP(76-114) is able to bind 4 equiv of metal ions and all histidyl residues are independent, except nonequivalent metal binding sites in the oligonuclear species. Imidazole nitrogen donor atoms are the primary and exclusive metal binding sites below pH 5.5 in the form of various macrochelates. The macrochelation slightly suppresses, but cannot prevent, the deprotonation and metal ion coordination of amide functions, resulting in the formation of (N(im),N(-)), (N(im),N(-),N(-)), and (N(im),N(-),N(-),N(-))-coordinated copper(II) complexes in the pH range from 5.5 to 9. CD spectroscopy results gave clear evidence for the differences in the metal binding affinity of the histidyl sites according to the following order: His111 > His96 >> His77 approximately His85. Among the oligonuclear complexes, the formation of di- and tetranuclear species seems to be favored over the trinuclear ones, at pH values beyond the physiological ones. This phenomenon was not observed in the complex formation reactions of HuPrP(84-114), a peptide fragment containing only one histidyl residue from the octarepeat. As a consequence, the data support the existence of cooperativity in the metal binding ability of this peptide probably due to the presence of two octarepeat sequences of the dimeric octarepeat domain of HuPrP(76-114) at basic pH values.

Synthesis, characterisation and insulin-mimetic activity of oxovanadium(IV) complexes with amidrazone derivatives.

The complexation of VO(2+) ion by ten acetamidrazone and 2-phenylacetamidrazone derivatives (L) was studied. Sixteen novel VO(2+) complexes were synthesised and characterised through the combined application of analytical and spectroscopic (EPR (electron paramagnetic resonance), FT-IR and diffuse reflectance electronic absorption) techniques. Eight are 1:2 species of composition [VOL(2)]SO(4) x xH(2)O and eight are 1:1 species with formula [VOL(SO(4))](n) x xH(2)O. The experimental data suggest a bidentate coordination mode for L with the donor set formed by the imine nitrogen and the carbonyl oxygen. EPR spectra indicate a square-pyramidal geometry for the 1:1 complexes and a penta-coordinated geometry intermediate between the square-pyramid and the trigonal-bipyramid for the 1:2 species. The hyperfine coupling constant along z axis, A(z), of the 1:2 complexes exhibits a marked reduction with respect to the predicted value (approximately 148x10(-4)cm(-1) vs. approximately 170x10(-4)cm(-1)). IR spectroscopic evidence supports the presence of sulphate as a counter-ion in the 1:2, and as a bridging bidentate ligand in the 1:1 complexes. Insulin-mimetic tests on modified fibroblasts, based on a modified MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide) assay, performed on three of the bis-chelated and eight of the mono-chelated derivatives, indicate that they are biologically active. The similar hydro/lipophilicity and the lack of ligand substituents recognizable by cell membrane receptors prevent substantial differentiation in the insulin-mimetic action.

Speciation in human blood of Metvan, a vanadium based potential anti-tumor drug.

The first report on the anti-cancer activity of the compound Metvan, [VIVO(Me2phen)2(SO4)], where Me2phen is 4,7-dimethyl-1,10-phenanthroline, dates back to 2001. Although it was immediately identified as one of the most promising multitargeted anti-cancer V compounds, no development on the medical experimentation was carried out. One of the possible reasons is the lack of information on its speciation in aqueous solution and its thermodynamic stability, factors which influence the transport in the blood and the final form which reaches the target organs. To fill this gap, in this work the speciation of Metvan in aqueous solution and human blood was studied by instrumental (EPR, electronic absorption spectroscopy, ESI-MS and ESI-MS/MS), analytical (pH-potentiometry) and computational (DFT) methods. The results suggested that Metvan transforms at physiological pH into the hydrolytic species cis-[VO(Me2phen)2(OH)]+ and that both citrate and proteins (transferrin and albumin in the blood serum, and hemoglobin in the erythrocytes) form mixed complexes, denoted [VO(Me2phen)(citrH-1)]2- and VO-Me2phen-Protein with the probable binding of His-N donors. The measurements with erythrocytes suggest that Metvan is able to cross their membrane forming mixed species VO-Me2phen-Hb. The redox stability in cell culture medium was also examined, showing that ca. 60% is oxidized to VV after 5 h. Overall, the speciation of Metvan in the blood mainly depends on the V concentration: when it is larger than 50 µM, [VO(Me2phen)(citrH-1)]2- and VO-Me2phen-Protein are the major species, while for concentrations lower than 10 µM, (VO)(hTf) is formed and Me2phen is lost. Therefore, it is plausible that the pharmacological activity of Metvan could be due to the synergic action of free Me2phen, and VIVO and VVO/VVO2 species.