A novel 1,3,5-triaminocyclohexane-based tripodal ligand forms a unique tetra(pyrazolate)-bridged tricopper(ii) core: solution equilibrium, structure and catecholase activity.

Copper(ii) complexes of a polydentate tripodal ligand L × 3HCl (L = N,N',N''-tris(5-pyrazolylmethyl)-cis,cis-1,3,5-triaminocyclohexane) were characterized in both solution and solid states. Combined evaluation of potentiometric, UV-VIS, and EPR data indicated the formation of two mononuclear (CuHL, CuL) and three trinuclear (Cu3H-xL2, x = 2, 3, 4) complexes. The high stability and spectroscopic properties of the CuL species indicate a coordination of two pyrazole rings in addition to the three secondary amino groups of L in a square pyramidal geometry. In parallel with the formation of trinuclear species, intense charge transfer bands appear at around 400-500 nm, which indicate the formation of pyrazolate-bridged complexes. The crystal structure of [Cu3H-4L2](ClO4)2·5H2O (1) reveals the formation of a unique trinuclear complex that features a tetra(pyrazolate)-bridged linear tricopper(ii) core. The CuCu interatomic distances are around 3.8 Å. The two peripheral copper(ii) ions have a slightly distorted square pyramidal geometry. The four pyrazole rings bound to the peripheral copper(ii) ions are deprotonated and create a flattened tetrahedral environment for the central copper(ii), i.e. the formation of the trinuclear complexes is under the allosteric control of the two peripheral copper(ii) ions. The triply deprotonated trinuclear complex is an efficient catechol oxidase mimic with a surprisingly low pH optimum at pH = 5.6. Since the mononuclear CuL species is not able to promote the oxidation of 3,5-di-tert-butylcatechol, we assume that the central copper(ii) ion of the trinuclear complex with an unsaturated coordination sphere has a fundamental role in the binding and oxidation of the substrate. The experimental and structural details were further elaborated by a series of hybrid density functional theory calculations that support the presence of an antiferromagnetically coupled ground state. However, the magnitude and the pattern of spin coupling are dependent on the composition of the functionals. The optimized theoretical structures highlight the role of the crystal packing effects in inducing asymmetry between the two peripheral copper(ii) sites.

Vanadium(IV/V) complexes of Triapine and related thiosemicarbazones: Synthesis, solution equilibrium and bioactivity.

The stoichiometry and thermodynamic stability of vanadium(IV/V) complexes of Triapine and two related α(N)-heterocyclic thiosemicarbazones (TSCs) with potential antitumor activity have been determined by pH-potentiometry, EPR and (51)V NMR spectroscopy in 30% (w/w) dimethyl sulfoxide/water solvent mixtures. In all cases, mono-ligand complexes in different protonation states were identified. Dimethylation of the terminal amino group resulted in the formation of vanadium(IV/V) complexes with considerably higher stability. Three of the most stable complexes were also synthesized in solid state and comprehensively characterized. The biological evaluation of the synthesized vanadium complexes in comparison to the metal-free ligands in different human cancer cell lines revealed only minimal influence of the metal ion. Thus, in addition the coordination ability of salicylaldehyde thiosemicarbazone (STSC) to vanadium(IV/V) ions was investigated. The exchange of the pyridine nitrogen of the α(N)-heterocyclic TSCs to a phenolate oxygen in STSC significantly increased the stability of the complexes in solution. Finally, this also resulted in increased cytotoxicity activity of a vanadium(V) complex of STSC compared to the metal-free ligand.

Strong effect of copper(II) coordination on antiproliferative activity of thiosemicarbazone-piperazine and thiosemicarbazone-morpholine hybrids.

In this study, 2-formylpyridine thiosemicarbazones and three different heterocyclic pharmacophores were combined to prepare thiosemicarbazone­piperazine mPip-FTSC (HL1) and mPip-dm-FTSC (HL2), thiosemicarbazone­morpholine Morph-FTSC (HL3) and Morph-dm-FTSC (HL4), thiosemicarbazone­methylpyrrole-2-carboxylate hybrids mPyrr-FTSC (HL5) and mPyrr-dm-FTSC (HL6) as well as their copper(II) complexes [CuCl(mPipH-FTSC-H)]Cl (1 + H)Cl, [CuCl(mPipH-dm-FTSC-H)]Cl (2 + H)Cl, [CuCl(Morph-FTSC-H)] (3), [CuCl(Morph-dm-FTSC-H)] (4), [CuCl(mPyrr-FTSC-H)(H2O)] (5) and [CuCl(mPyrr-dm-FTSC-H)(H2O)] (6). The substances were characterized by elemental analysis, one- and two-dimensional NMR spectroscopy (HL1­HL6), ESI mass spectrometry, IR and UV­vis spectroscopy and single crystal X-ray diffraction (1­5). All compounds were prepared in an effort to generate potential antitumor agents with an improved therapeutic index. In addition, the effect of structural alterations with organic hybrids on aqueous solubility and copper(II) coordination ability was investigated. Complexation of ligands HL2 and HL4 with copper(II) was studied in aqueous solution by pH-potentiometry, UV­vis spectrophotometry and EPR spectroscopy. Proton dissociation processes of HL2 and HL4 were also characterized in detail and microscopic constants for the Z/E isomers were determined. While the hybrids HL5, HL6 and their copper(II) complexes 5 and 6 proved to be insoluble in aqueous solution, precluding antiproliferative activity studies, the thiosemicarbazone­piperazine and thiosemicarbazone­morpholine hybrids HL1­HL4, as well as copper(II) complexes 1­4 were soluble in water enabling cytotoxicity assays. Interestingly, the metal-free hybrids showed very low or even a lack of cytotoxicity (IC50 values > 300 µM) in two human cancer cell lines HeLa (cervical carcinoma) and A549 (alveolar basal adenocarcinoma), whereas their copper(II) complexes were cytotoxic showing IC50 values from 25.5 to 65.1 µM and 42.8 to 208.0 µM, respectively in the same human cancer cell lines after 48 h of incubation. However, the most sensitive for HL4 and complexes 1­4 proved to be the human cancer cell line LS174 (colon carcinoma) as indicated by the calculated IC50 values varying from 13.1 to 17.5 µM.

Copper(II) complexes with highly water-soluble L- and D-proline-thiosemicarbazone conjugates as potential inhibitors of Topoisomerase IIα.

Two proline-thiosemicarbazone bioconjugates with excellent aqueous solubility, namely, 3-methyl-(S)-pyrrolidine-2-carboxylate-2-formylpyridine thiosemicarbazone [L-Pro-FTSC or (S)-H2L] and 3-methyl-(R)-pyrrolidine-2-carboxylate-2-formylpyridine thiosemicarbazone [D-Pro-FTSC or (R)-H2L], have been synthesized and characterized by elemental analysis, one- and two-dimensional (1)H and (13)C NMR spectroscopy, and electrospray ionization mass spectrometry. The complexation behavior of L-Pro-FTSC with copper(II) in an aqueous solution and in a 30% (w/w) dimethyl sulfoxide/water mixture has been studied via pH potentiometry, UV-vis spectrophotometry, electron paramagnetic resonance, (1)H NMR spectroscopy, and spectrofluorimetry. By the reaction of copper(II) acetate with (S)-H2L and (R)-H2L in water, the complexes [Cu(S,R)-L] and [Cu(R,S)-L] have been synthesized and comprehensively characterized. An X-ray diffraction study of [Cu(S,R)-L] showed the formation of a square-pyramidal complex, with the bioconjugate acting as a pentadentate ligand. Both copper(II) complexes displayed antiproliferative activity in CH1 ovarian carcinoma cells and inhibited Topoisomerase IIα activity in a DNA plasmid relaxation assay.

L- and D-proline thiosemicarbazone conjugates: coordination behavior in solution and the effect of copper(II) coordination on their antiproliferative activity.

Two enantiomerically pure thiosemicarbazone-proline conjugates with enhanced aqueous solubility, namely, 2-hydroxy-3-methyl-(S)-pyrrolidine-2-carboxylate-5-methylbenzaldehyde thiosemicarbazone [L-Pro-STSC or (S)-H(2)L] and 2-hydroxy-3-methyl-(R)-pyrrolidine-2-carboxylate-5-methylbenzaldehyde thiosemicarbazone [D-Pro-STSC or (R)-H(2)L] have been synthesized and characterized by elemental analysis, spectroscopic methods (UV-vis and (1)H and (13)C NMR), and electrospray ionization mass spectrometry. The metal complexation behavior of L-Pro-STSC, stoichiometry, and thermodynamic stability of iron(II), iron(III), copper(II), and zinc(II) complexes in 30% (w/w) dimethyl sulfoxide/H(2)O solvent mixture have been studied by pH-potentiometric, UV-vis-spectrophotometric, circular dichroism, electron paramagnetic resonance, (1)H NMR spectroscopic, and spectrofluorimetric measurements. By the reaction of CuCl(2)·2H(2)O with (S)-H(2)L and (R)-H(2)L, respectively, the complexes [Cu[(S)-H(2)L]Cl]Cl and [Cu[(R)-H(2)L]Cl]Cl have been prepared and comprehensively characterized. An X-ray diffraction study of [Cu[(R)-H(2)L]Cl]Cl showed the formation of a square-planar copper(II) complex, which builds up stacks with interplanar separation of 3.3 Å. The antiproliferative activity of two chiral ligands and their corresponding copper(II) complexes has been tested in two human cancer cell lines, namely, SW480 (colon carcinoma) and CH1 (ovarian carcinoma). The thiosemicarbazone-proline conjugates L- and D-Pro-STSC show only moderate cytotoxic potency with IC(50) values of 62 and 75 µM, respectively, in CH1 cells and >100 µM in SW480 cells. However, the corresponding copper(II) complexes are 13 and 5 times more potent in CH1 cells, based on a comparison of IC(50) values, and in SW480 cells the increase in the antiproliferative activity is even higher. In both tested cell lines, L-Pro-STSC as well as its copper(II) complex show slightly stronger antiproliferative activity than the compounds with a D-Pro moiety, yielding IC(50) values of 4.6 and 5.5 µM for [Cu(L-Pro-STSC)Cl]Cl in CH1 and SW480 cells, respectively.

Evaluation of the binding of oxovanadium(IV) to human serum albumin.

The understanding of the biotransformations of insulin mimetic vanadium complexes in human blood and its transport to target cells is an essential issue in the development of more effective drugs. We present the study of the interaction of oxovanadium(iv) with human serum albumin (HSA) by electron paramagnetic resonance (EPR), circular dichroism (CD) and visible absorption spectroscopy. Metal competition studies were done using Cu(II) and Zn(II) as metal probes. The results show that V(IV)O occupies two types of binding sites in albumin, which compete not only with each other, but also with hydrolysis of the metal ion. In one of the sites the resulting V(IV)O-HSA complex has a weak visible CD signal and its X-band EPR spectrum may be easily measured. This was assigned to amino acid side chains of the ATCUN site. The other binding site shows stronger signals in the CD in the visible range, but has a hardly measurable EPR signal; it is assigned to the multi metal binding site (MBS) of HSA. Studies with fatted and defatted albumin show the complexity of the system since conformational changes, induced by the binding of fatty acids, decrease the ability of V(IV)O to bind albumin. The possibility and importance of ternary complex formation between V(IV)O, HSA and several drug candidates - maltol (mal), picolinic acid (pic), 2-hydroxypyridine-N-oxide (hpno) and 1,2-dimethyl-3-hydroxy-4(1H)-pyridinone (dhp) was also evaluated. In the presence of maltol the CD and EPR spectra significantly change, indicating the formation of ternary VO-HSA-maltol complexes. Modeling studies with amino acids and peptides were used to propose binding modes. Based on quantitative RT EPR measurements and CD data, it was concluded that in the systems with mal, pic, hpno, and dhp (V(IV)OL(2))(n)(HSA) species form, where the maximum value for n is at least 6 (mal, pic). The degree of formation of the ternary species, corresponding to the reaction V(IV)OL(2) + HSA -->/<-- V(IV)OL(2)(HSA) is hpno > pic ≥ mal > dhp. (V(IV)OL)(n)(HSA) type complexes are detected exclusively with pic. Based on the spectroscopic studies we propose that in the (V(IV)OL(2))(n)(HSA) species the protein bounds to vanadium through the histidine side chains.

Copper(II)-binding ability of stereoisomeric cis- and trans-2-aminocyclohexanecarboxylic acid-L-phenylalanine dipeptides. A combined CW/pulsed EPR and DFT study.

With the aim of an improved understanding of the metal-complexation properties of alicyclic ß-amino acid stereoisomers, and their peptides, the complex equilibria and modes of coordination with copper(II) of L-phenylalanine (F) derivatives of cis/trans-2-aminocyclohexanecarboxylic acid (c/tACHC), i.e. the dipeptides F-c/tACHC and c/tACHC-F, were investigated by a combination of CW and pulsed EPR methods. For the interpretation of the experimental data, DFT quantum-chemical calculations were carried out. Simulation of a pH-dependent series of room-temperature CW-EPR spectra revealed the presence of EPR-active complexes ([Cu(aqua)](2+), [CuL](+), [CuLH(-1)], [CuLH(-2)](-), and [CuL(2)H(-1)](-)), and an EPR-inactive species ([Cu(2)L(2)H(-3)](-)) in aqueous solutions for all studied cases. [CuLH](2+) was included in the equilibrium model for the c/tACHC-F-copper(II) systems, and [CuL(2)], together with two coordination isomers of [CuL(2)H(-1)](-), were also identified in the F-tACHC-copper(II) system. Comparison of the complexation properties of the diastereomeric ligand pair F-(1S,2R)-ACHC and F-(1R,2S)-ACHC did not reveal significant differences. Considerably lower formation constants were obtained for the trans than for the cis isomers for both the F-c/tACHC and the c/tACHC-F pairs in the case of [CuLH(-1)] involving tridentate coordination by the amino, the deprotonated peptide, and the carboxylate groups. A detailed structural analysis by pulsed EPR methods and DFT calculations indicated that there was no significant destabilization for the complexes of the trans isomers. The lower stability of their complexes was explained by the limitation that only the conformer with donor groups in equatorial-equatorial ring positions can bind to copper(II), whereas both equatorial-axial conformers of the cis isomers are capable of binding. From a consideration of the proton couplings obtained with X-band (1)H HYSCORE, (2)H exchange experiments, and DFT, the thermodynamically most stable cyclohexane ring conformer was assigned for all four [CuLH(-1)] complexes. For the F-cACHC case, the conformer did not match the most stable conformer of the free ligand.

Histidine-rich branched peptides as Cu(II) and Zn(II) chelators with potential therapeutic application in Alzheimer's disease.

Two histidine-rich branched peptides with one lysine as a branching unit have been designed and synthesized by solid-phase peptide synthesis. Their complex formation with Cu(II) and Zn(II) as well as their ability to attenuate the metal-ion induced amyloid aggregation has been characterized. Both peptides can keep Cu(II) and Zn(II) in complexed forms at pH 7.4 and can bind two equivalents of metal ions in solutions with excess metal. The stoichiometry, stability and structure of the complexes formed have been determined by pH potentiometry, UV-Vis spectrophotometry, circular dichroism, EPR and NMR spectroscopy and ESI-MS. Both mono- and bimetallic species have been detected over the whole pH range studied. The basic binding mode is either a tridentate {N(amino), N(amide), N(im)} or a histamine-type of coordination which is complemented by the binding of far imidazole or amino groups leading to macrochelate formation. The peptides were able to prevent Cu(II)-induced Aß(1-40) aggregation but could not effectively compete for Zn(II) in vitro. Our results suggest that branched peptides containing potential metal-binding sites may be suitable metal chelators for reducing the risk of amyloid plaque formation in Alzheimer's disease.

The role of terminal amino group and histidine at the fourth position in the metal ion binding of oligopeptides revisited: Copper(II) and nickel(II) complexes of glycyl-glycyl-glycyl-histamine and its N-Boc protected derivative.

Copper(II) and nickel(II) binding properties of two pseudo tetrapeptides, N-Boc-Gly-Gly-Gly-Histamine (BGGGHa) and Gly-Gly-Gly-Histamine (GGGHa) have been investigated by pH-potentiometric titrations, UV-visible-, EPR-, NMR- and ESI-HRMS (electrospray ionization high resolution MS) spectroscopies, in order to compare the role of N-terminal amino group and imidazole moiety at the fourth position in the complex formation processes. Substantially higher stabilities were determined for the ML complexes of GGGHa, compared to those of BGGGHa, supporting the coordination of the terminal amino group and the histamine imidazole of the non-protected ligand. A dimeric Cu(2)H(-2)L(2) species, formed through the deprotonation of peptide groups of the ligands, was found in the GGGHa-copper(II) system. Deprotonation and coordination of further amide nitrogens led to CuH(-2)L and, above pH~10, CuH(-3)L. Experimental data supports a {NH(2), 2 × N(amide),N(im)} macrochelate structure in CuH(-2)L whereas a {NH(2), 3 × N(amide)} coordination environment in CuH(-3)L. The first two amide deprotonation processes were found to be strongly cooperative with nickel(II) and spectroscopic studies proved the transformation of the octahedral parent complexes to square planar, yellow, diamagnetic species, NiH(-2)L and above pH~9, NiH(-3)L. In the basic pH-range deprotonation and coordination of the amide groups also took place in the BGGGHa containing systems, leading to complexes with a {3 × N(amide),N(im)} donor set, and in parallel the re-dissolving of precipitate. Above pH~11, a further proton release from the pyrrolic NH group of the imidazole ring of BGGGHa occurred providing an additional proof for the different binding modes of the two ligands.

Two pyridine derivatives as potential Cu(II) and Zn(II) chelators in therapy for Alzheimer's disease.

Two pyridine derivatives, DMAP and ENDIP, have been investigated as possible metal chelators in the therapy of Alzheimer's disease. Their complex formation with Cu(ii) and Zn(ii) were characterised in detail. In the case of ENDIP a high stability tetradentate ML complex is formed at physiological pH both with Cu(ii) and Zn(ii). DMAP was found to be a weaker metal binder. At physiological pH, it forms a bidentate ML complex with Zn(ii) and MLH(-1) and ML(2) complexes with Cu(ii), depending on the metal ion to ligand ratio. Fluorescence spectroscopy and dynamic light scattering measurements proved that ENDIP effectively competes with aggregated amyloid-beta peptides (Abeta) for both Cu(ii) and Zn(ii) and thus is able to prevent the metal ion-induced amyloid aggregation and to resolubilise amyloid precipitates.

Probing the Cu(2+) and Zn(2+) binding affinity of histidine-rich glycoprotein.

The zinc(II) and copper(II) binding ability of two oligopeptide fragments, Ac-HHPHG-NH(2) and Ac-HHPHGHHPHG-NH(2), derived from the repeat-region of the His-Pro-rich domain of histidine-rich glycoprotein (HRG) and the structure of the formed complexes have been investigated by potentiometry, NMR-, UV-visible-, CD-, SRCD- and EPR spectroscopy. Exclusive coordination of the side-chain imidazoles of the peptides has been observed with both metal ions in the acidic and neutral pH range. While the three His units of the pentapeptide resulted in a modest stability of the ML complexes, the decapeptide with its increased number of His residues offered a high-affinity metal binding site for both metal ions with the participation of at least four nitrogen donors. Due to the high number of potential donor groups, the formation of binding isomers of the protonated and parent complexes is very likely. Both peptides show a synchrotron radiation (SR) CD-pattern resembling to that of the polyproline II structure, similarly to that of the His-Pro-rich domain of the HRG protein. The longer sequence was shown to bind a second metal ion in the slightly acidic pH-range. The determined stability data suggest a remarkable extra stabilization emerging in the decapeptide for the coordination of the second metal ions, as compared to the ML complexes of the pentapeptide. Whether the observed cooperativity has similarities to the cooperative metal binding feature of HRG or the two phenomena have different sources is a question yet to be clarified.

N-terminal fragment of the anti-angiogenic human endostatin binds copper(II) with very high affinity.

A histidine-rich peptide HSHRDFQPVLHL-NH(2) (L), identical with the N-terminal fragment of the anti-angiogenic human endostatin has been synthesized. Endostatin is a recently identified broad spectrum angiogenesis inhibitor, which inhibits 65 different tumor types. The N-terminal 25-mer peptide fragment of human endostatin has the same antitumor effect as the entire protein. The zinc(II) binding is crucial for the antitumor effect in both cases. Our peptide may provide all critical interactions for zinc(II) binding present in the N-terminal 25-mer peptide fragment. In addition, the N-terminus of human endostatin has a supposedly high affinity binding site for copper(II), similar to human serum albumin. Since copper(II) is intimately involved in angiogenesis, this may have biological relevance. In order to determine the metal binding properties of the N-terminal fragment of endostatin, we performed equilibrium, UV-visible (UV-vis), CD, EPR and NMR studies on the zinc(II) and copper(II) complexes of L. In the presence of zinc(II) the formation of a stable [NH(2),3N(im),COO(-)] coordinated complex was detected in the neutral pH-range. This coordination mode is probably identical to that present in the zinc(II) complex of the above mentioned N-terminal 25-mer peptide fragment of human endostatin. Moreover, L has extremely high copper(II) binding affinity, close to those of copper-containing metalloenzymes, and forms albumin-like [NH(2),N(-),N(-),N(im)] coordinated copper(II) complex in the neutral pH-range, which may suggest that copper(II) binding is involved in the biological activity of endostatin.

Interaction of imidazole containing hydroxamic acids with Fe(III): hydroxamate versus imidazole coordination of the ligands.

Solution equilibrium studies on Fe(III) complexes formed with imidazole-4-carbohydroxamic acid (Im-4-Cha), N-Me-imidazole-4-carbohydroxamic acid (N-Me-Im-4-Cha), imidazole-4-acetohydroxamic acid (Im-4-Aha), and histidinehydroxamic acid (Hisha) have been performed by using pH-potentiometry, UV-visible spectrophotometry, EPR, ESI-MS, and H1-NMR methods. All of the obtained results demonstrate that the imidazole moiety is able to play an important role very often in the interaction with Fe(III), even if this metal ion prefers the hydroxamate chelates very much. If the imidazole moiety is in alpha-position to the hydroxamic one (Im-4-Cha and N-Me-Im-4-Cha) its coordination to the metal ion is indicated unambiguously by our results. Interestingly, parallel formation of (Nimidazole, Ohydroxamate), and (Ohydroxamate, Ohydroxamate) type chelates seems probable with N-Me-Im-4-Cha. The imidazole is in beta-position to the hydroxamic moiety in Im-4-Aha and an intermolecular noncovalent (mainly H-bonding) interaction seems to organize the intermediate-protonated molecules in this system. Following the formation of mono- and bishydroxamato mononuclear complexes, only EPR silent species exists in the Fe(III)-Hisha system above pH 4, what suggests the rather significant "assembler activity" of the imidazole (perhaps together with the ammonium moiety).

Thermodynamic analysis of the chemical exchange of beta-phosphorylated cyclic nitroxides by using two-dimensional (temperature versus magnetic field) simulation of ESR spectra: the impact of labile solvent-solute interactions on molecular dynamics.

A complete thermodynamic characterization of the chair-to-chair interconversion in beta-diphosphorylated piperidine-N-oxyl radicals was achieved by means of the analysis of temperature-dependent ESR spectra. A new two-dimensional simulation method was developed with the coordinates temperature and magnetic field, in which the entire set of spectra was simulated simultaneously by adjusting the coefficients in the power expansion, giving the temperature-dependent ESR parameters and the thermodynamic and kinetic parameters determining the site populations and exchange rates, respectively. The new method promotes elimination of the ambiguities inherent in the spectroscopic determination of thermodynamic parameters. Labile solvent-solute interactions can strongly influence the chemical exchange, producing a complex network of symmetric and asymmetric interconversions. The solvent dependence of magnetic relaxation was also analyzed.