Stability Of Copper Complexes Research Articles

Abstract 4901: The role of protein disulfide isomerase and copper in the paraptotic cell death of clinically investigated anticancer thiosemicarbazones

Abstract α-N-Heterocyclic thiosemicarbazones (TSCs) have long been investigated as anticancer compounds. Triapine is one of the best-known TSCs for anticancer therapy and is currently tested in a clinical phase III trial. To further improve the anticancer activity of TSCs, novel derivatives (such as DpC and COTI-2) have been developed and clinically investigated for their activity against solid tumors. These novel TSCs belong to a subclass (incl. the tetra-methylated Triapine derivative, Me2NNMe2) that are terminally disubstituted and demonstrated enhanced anticancer activity in vitro and in vivo. The improved activity of these TSCs correlated with their ability to form stable copper complexes as well as induce paraptosis, a form of programmed but caspase-independent cell death, that is characterized by the formation of vesicles originating from the endoplasmic reticulum (ER). However, the molecular events behind paraptosis induction are still not fully elucidated. Consequently, the aim of this study was to investigate the molecular signaling behind paraptosis as well as the reasons behind the induction of this quite unknown form of cell death by TSCs. A whole-genome gene expression analysis of cells treated with a terminally disubstituted TSC revealed an upregulation of thiol homeostasis-related genes, copper metabolism, and metallothionein genes as well as ER stress response genes. In addition, we found an upregulation of the copper-sensitive and thiol-containing protein disulfide isomerase (PDI), which was confirmed on protein level. In cell-free activity studies, PDI was indeed inhibited by terminally disubstituted TSCs, although only in form of their copper complexes. In agreement, removal of copper ions by specific chelators greatly reduced their anticancer activity as well as paraptotic potential in cell culture. This inhibition was hypothesized to be induced by a general disruption of the thiol redox homeostasis affecting also other responsive proteins and resulting in a more oxidative environment, especially in the ER. In confirmation, thiol-containing small molecules could reduce the compound’s anticancer activity in viability assays. Furthermore, glutathione as well as PDI were detected predominantly in their oxidized form in cells treated with terminally disubstituted TSCs. Consequently, terminally disubstituted TSCs, which are characterized by higher anticancer activity, induce paraptosis due to their high copper complex stability. The formation of the complex results in the interaction with thiol-containing proteins and subsequently in the disruption of the thiol-redox homeostasis, which leads to paraptotic cell death. Overall this work shed light on the paraptotic cell death and will be of interest for future clinical development of anticancer TSCs, as paraptosis is hypothesized to overcome apoptosis-resistance of cancer cells. Citation Format: Sonja Hager, Walter Berger, Christian R. Kowol, Eva A. Enyedy, Petra Heffeter. The role of protein disulfide isomerase and copper in the paraptotic cell death of clinically investigated anticancer thiosemicarbazones. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4901.

Developing bulky P-alkene ligands: stabilization of copper complexes with 14 valence electrons

The superbulky P-alkene ligand 21 is prepared from dibenzoazepine and stabilizes the highly unsaturated cationic Cu(i)-complex 26.

Optimization of Copper Stain Removal from Marble through the Formation of Cu(II) Complexes in Agar Gels.

Copper complexes with different ligands (ethylenediaminetetraacetic acid, EDTA, ammonium citrate tribasic, TAC, and alanine, ALA) were studied in aqueous solutions and hydrogels with the aim of setting the optimal conditions for copper stain removal from marble by agar gels, with damage minimization. The stoichiometry and stability of copper complexes were monitored by ultraviolet-visible (UV-Vis) spectroscopy and the symmetry of Cu(II) centers in the different gel formulations was studied by electron paramagnetic resonance (EPR) spectroscopy. Cleaning effectiveness in optimized conditions was verified on marble laboratory specimens through color variations and by determining copper on gels by inductively coupled plasma-mass spectrometry (ICP-MS). Two copper complexes with TAC were identified, one having the known stoichiometry 1:1, and the other 1:2, Cu(TAC)2, never observed before. The stability of all the complexes at different pH was observed to increase with pH. At pH 10.0, the gel’s effectiveness in removing copper salts from marble was the highest in the presence of ALA, followed by EDTA, TAC, and pure agar gel. Limited damage to the marble surface was observed when gels with added EDTA and TAC were employed, whereas agar gel with ALA was determined to be the most efficient and safe cleaning material.

Bivalence metal complexes of antithyroid drug carbimazole; synthesis, characterization, computational simulation, and biological studies

New metal complexes of carbimazole (CMZ) were synthesized with a molar ratio of 1:2. The bivalence complexes were characterized by various techniques, such as elemental analyses and UV–visible spectra, magnetic, and thermal analysis. The metal-ligand stability constant obtained from the molar ratio experiment showed high copper complex stability among the prepared metal compounds at room temperature (Kf =6.6 × 105). The infrared spectra (IR) showed that the CMZ ligand coordinated to the metal through the sulfur atom and the oxygen ether group. The spectroscopic results suggested tetrahedral geometry for all complexes except Ni(II) and Zn(II) complexes with a square planar arrangement. The proposed structures were optimized using density functional theory (DFT/B3LYP) and 6–311G*(d,p) basis sets. The geometrical optimization parameters and molecular electrostatic potentials were calculated. Biological investigation started with DNA binding spectroscopy, which revealed a high binding ability of the Cu(II) and Ni(II) complexes to the CT-DNA. A docking study using different protein receptors was implemented to study the proposed drug's molecular behavior. The in vitro cytotoxicity evaluation of the complexes toward breast human cancer (Mcf7) and human colon cancer (CaCo-2) cell linesindicated that the Cu(II)-CMZ complex has high potential anticancer activities against those cell lines.

Abstract 1939: Improved activity and paraptosis-induction of anticancer thiosemicarbazones requires high copper(II) complex stability and slow reduction kinetics

Abstract Triapine, which is currently tested in a clinical phase III trial, is the best-studied thiosemicarbazone (TSC) for anticancer therapy. With regard to the mode of action, anticancer activity of TSCs is frequently linked to their ability to chelate essential metal ions such as copper and iron. As Triapine monotherapy showed promising results mainly against hematological diseases, novel TSC derivatives have been developed and clinically investigated for their activity against solid tumors. These novel TSCs belong to a subclass with enhanced anticancer activity in vitro and in vivo which were recently discovered to induce paraptosis, a form of programmed but caspase-independent cell death. As these TSCs are characterized by a up to 1000-fold higher activity in cell culture compared to Triapine, the aim of this study was to elucidate the mechanism of action as well as the underlying structural and chemical requirements of these effects. For this purpose, a panel of structurally related Triapine derivatives was examined for anticancer activity (as metal-free ligand and copper(II) complex), paraptosis-inducing potential as well as solution stability and redox properties of their copper(II) complexes. Correlation studies between these chemical and biological properties revealed that the increased anticancer activity and paraptosis-inducing potential of the nanomolar active TSCs is related to a higher copper(II) complex solution stability and slower reduction rate. Unexpectedly, the TSCs with lower activity produced higher superoxide levels in a cell-free setting. This paradox could be explained by their lower copper(II) complex stability and increased readiness to be reduced, which resulted in a fast reduction of intracellular complexes and release of the metal-free ligand. Although this process resulted in the generation of superoxide, cell damage seemed to be prevented by rapid upregulation of the superoxide dismutase (in vitro and in vivo). In contrast, the copper complexes of the highly active TSCs are stable enough to reach intracellular targets such as the ER-resident protein disulfide isomerase, whose inhibition is crucial for paraptosis induction by TSCs. In conclusion, copper complex stability is a crucial parameter of TSC activity, influencing the (intracellular) formation or dissociation of copper complexes. This intracellular stability of complexes, affects their mechanism of action as well as cell death induction. Overall, this study points out the importance of the redox parameters in order to understand and predict the TSC anticancer activity as well as their mechanism of action and, thereby, will pave the way for the development of improved anticancer agents. Citation Format: Sonja Hager, Veronika F. Pape, Vivien Pósa, Bianca Montsch, Lukas Uhlik, Gergely Szakàcs, Szilárd Tóth, Nikolett Jabronka, Bernhard K. Keppler, Walter Berger, Christian R. Kowol, Éva A. Enyedy, Petra Heffeter. Improved activity and paraptosis-induction of anticancer thiosemicarbazones requires high copper(II) complex stability and slow reduction kinetics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1939.

High Copper Complex Stability and Slow Reduction Kinetics as Key Parameters for Improved Activity, Paraptosis Induction, and Impact on Drug-Resistant Cells of Anticancer Thiosemicarbazones.

Aims: Due to their significant biological activity, thiosemicarbazones (TSCs) are promising candidates for anticancer therapy. In part, the efficacy of TSCs is linked to their ability to chelate essential metal ions such as copper and iron. Triapine, the best-studied anticancer TSC, has been tested clinically with promising results in hematological diseases. During the past few years, a novel subclass of TSCs with improved anticancer activity was found to induce paraptosis, a recently characterized form of cell death. The aim of this study was to identify structural and chemical properties associated with anticancer activity and paraptosis induction of TSCs. Results: When testing a panel of structurally related TSCs, compounds with nanomolar anticancer activity and paraptosis-inducing properties showed higher copper(II) complex solution stability and a slower reduction rate, which resulted in reduced redox activity. In contrast, TSCs with lower anticancer activity induced higher levels of superoxide that rapidly stimulated superoxide dismutase expression in treated cells, effectively protecting the cells from drug-induced redox stress. Innovation: Consequently, we hypothesize that in the case of close Triapine derivatives, intracellular reduction leads to rapid dissociation of intracellularly formed copper complexes. In contrast, TSCs characterized by highly stable, slowly reducible copper(II) complexes are able to reach new intracellular targets such as the endoplasmic reticulum-resident protein disulfide isomerase. Conclusion: The additional modes of actions observed with highly active TSC derivatives are based on intracellular formation of stable copper complexes, offering a new approach to combat (drug-resistant) cancer cells.

Synthesis and Study in Solution of a New Dansyl-Modified Azacryptand

We report the synthesis of a new asymmetric azacryptand (L1), characterized by three p-xylyl spacers, one of which carries a dansyl side arm. The fluorescent sensor has been studied by potentiometric, UV-Vis, and emission studies in MeOH : water 3 : 2 mixture (0.07 M NaNO3), determining, in particular, the protonation constants of the free ligand and metal ion complexation equilibria. Interestingly, the obtained results revealed that the new receptor is fluorescent at neutral pH with a typical emission band of the dansyl group. Metal addition induced a partial quenching of the dansyl emission band; this behavior is more pronounced with Cu(II) that reduces the receptor’s emission by 60%. With all the studied cations, quenching follows the formation of a dimetallic complex. Similar studies on the model compound L2 confirmed that fluorescence quenching is mainly driven by a static mechanism, attributable to the formation of the inclusion dicopper complex [L1Cu2]4+. In order to test the stability of copper complexes under physiological conditions, spectrofluorimetric titrations with Cu(II) were performed in water buffered at pH = 8 (HEPES 0.07 M) and the values of binding constants, K11 and K12, were determined.

Synthesis, characterization and biological studies of homo and hetero-binuclear 13-membered pentaaza bis (macrocyclic) complexes

A new series of homo and hetero binuclear 13-membered pentaaza bis (macrocyclic) complexes, [MM′LX4], [M=Cu(II), M′=Cu(II), Co(II), Ni(II) and Sn(II); L=ligand and X=Cl or NO3] have been synthesized by the template reaction of dichloro/dinitrato diphenyl sulphone 1,3,6,9,12-tetra hydro pentaazacyclo pantane copper (II) complexes with formaldehyde, triethylenetetraamine, and respective metal salts in 1:2:1:1 molar ratio. The complexes have been characterized by elemental analyses, molar conductance measurements, ESI-mass, 1H, 13C and 119Sn NMR, IR, electronic and EPR spectral studies. The results of elemental analyses, ESI-mass and conductivity measurements confirmed the stoichiometry of the complexes while the characteristic absorption bands and resonance peaks in IR and NMR spectra confirmed the formation of macrocyclic frameworks of the complexes. These studies showed octahedral geometry around the metal ion. The thermal stability of copper complexes was also studied by TGA and DTA analyses. Some complexes of this series were also studied for their in vitro anticancer activity against cancer cells lines: Hep3B, MCF7, and HeLa. The recorded IC50 values for the tested complexes show moderate to good cytotoxicity against these cancer cell lines.

Conductometric studies on the stability of copper complexes with different oligosaccharides

Abstract Bioactive copper complexes with pullulan or dextran oligosaccharides are the subject of intensive research mainly because of their possible application in veterinary and human medicine. The thermal stability and stability under oxidative conditions of the Cu(II) complexes with reduced low-molar pullulan or dextran were investigated in this paper, using a conductometric method. The influence of ligand constitutions on the stability of the complexes was examined on the basis of ligand property. Forced degradation studies were performed on bulk sample of complexes by using heat (25, 40 and 60°C) and an oxidation agent (0.1, 0.5, 1.0 and 10.0% v/v hydrogen peroxide). It can be concluded, according to the results obtained (by examining conductivity during the forced degradation studies), that Cu(II) complexes show a large pharmaceutical stability for both tests.

Stability constants of Cu(II) complexes of lanthionine.

Complex stability constants for the stepwise interaction of meso- and L-lanthionine (3,3′-thiodialanine) with varying amounts of Cu2- ions have been determined potentiometrically in aqueous solution at 20° and μ- 0.37 (NaN03). The equilibrium could be best described by postulating six stability constants in addition to copper hydrolysis. The difference obtained between individual copper complex stability constants of meso- and L-forms has been attributed both to the presence of two asymmetric centres in the ligand molecule and to the bridging sulphur atom. Thermodynamic quantities for some equilibria have also been determined from the temperature dependence of respective stability constants at 4, 20, 40, and 70°.

Effect of Cu(II) ligands on electroless copper deposition rate in formaldehyde solutions: An EQCM study

The electroless copper deposition rate for 6 CuII complexes decreases in the ligand sequence: nitrilotriacetic acid (NTA) > N,N,N′,N′-tetrakis-(2-hydroxypropyl)-ethylenediamine (Quadrol) > glycerol > L(+)-tartrate ~ sucrose > \(\hbox{\sc {DL}}(_+^-)\)-tartrate. Both CuII complex stability and specific ligand effects were found to influence the Cu deposition process. The specific ligand effects are most obvious in the case of Quadrol (high kinetic activity at a high CuII complex stability), glycerol and sucrose (additional reaction of Cu2O formation by interaction of CuII with ligand). According to the EQCM data for 11 CuII complexes (including data from the former study) the higher kinetic activity is demonstrated by complexes with ligands containing amino groups; this factor is more important for Cu deposition rate than copper complex stability. A potential dependence of the Cu reduction partial current on the electrode potential has been extracted from the EQCM data in the complete electroless plating bath. An increase in CuII reduction rate was found to occur in electroless plating solution for CuII complexes with NTA and Quadrol compared with that in formaldehyde-free solutions. Possible reasons for the acceleration of the partial CuII reduction reaction and the overall process kinetics are discussed using a hypothetical reaction sequence involving intermediate copper oxy-species and active Cu* formation as well as development of the preferred Cu surface structure.

Theoretical study of dipeptide complexes of copper(II)

The l, l-Phe-Leu-Cu(II), l, l-Leu-Phe-Cu(II), l, l-Phe-Met-Cu(II), and l, l-Met-Phe-Cu(II) systems were studied using molecular modelling. The results obtained, which are in good agreement with results obtained by potentiometric studies show a significant increase in stability of copper complexes, when an aromatic residue is located in C-terminal (compared to the l, l-dipeptides containing the same amino acid residues), this phenomenon is attributed to the interaction between the d-orbital of copper and the π-electrons of the aromatic ring.

Steric and hydrogen-bonding effects on the stability of copper complexes with small molecules.

A series of the copper(II) complexes with tripodal tetradentate tris(pyridyl 2-methyl)amine-based ligands possessing the hydrogen-bonding 6-aminopyridine units (tapa, three amino groups; bapa, two amino groups; mapa, one amino group) have been synthesized, and their copper(II) complexes with a small molecule such as dioxygen and azide have been studied spectroscopically and structurally. The reaction of their Cu(II) complexes with NaN(3) have given the mononuclear copper complexes with azide in an end-on mode, [Cu(tapa)(N(3))]ClO(4) (1a), [Cu(bapa)(N(3))]ClO(4) (2a), [Cu(mapa)(N(3))]ClO(4) (3a), and [Cu(tpa)(N(3))]ClO(4) (4a) (tpa, no amino group). The crystal structures have revealed that the coordination geometries around the metal centers are almost a trigonal-bipyramidal rather than a square-planar except for 1a with an intermediate between them. The UV-vis and ESR spectral data indicate that the increase of NH(2) groups of ligands causes the structural change from trigonal-bipyramidal to square-pyramidal geometry, which is regulated by a combination of steric repulsion and hydrogen bond. The steric repulsion of amino groups with the azide nitrogen gives rise to elongation of the Cu-N(py) bonds, which leads to the positive shift of the redox potentials of the complexes. The hydrogen bonds between the coordinated azide and amino nitrogens (2.84-3.05 A) contribute clearly to the fixation of azide. The Cu(I) complexes with bapa and mapa ligands have been obtained as a precipitate, although that with tapa was not isolated. The reactions of the Cu(I) complexes with dioxygen in MeOH at -75 degrees C have given the trans-micro-1,2 peroxo dinuclear Cu(II) complexes formulated as [((tapa)Cu)(2)(O(2))](2+) (1c), [((bapa)Cu)(2)(O(2))](2+) (2c), and [((mapa)Cu)(2)(O(2))](2+) (3c), whose characterizations were confirmed by UV-vis, ESR, and resonance Raman spectroscopies. UV-vis spectra of 1c, 2c, and 3c exhibited intense bands assignable to pi(O(2)(2)(-))-to-d(Cu) charge transfer (CT) transitions at lambda(max)/nm (epsilon/M(-1)cm(-1)) = 449 (4620), 474 (6860), and 500 (9680), respectively. The series of the peroxo adducts generated was ESR silent. The resonance Raman spectra exhibited the enhanced features assignable to two stretching vibrations nu((16)O-(16)O/(18)O-(18)O)/cm(-1) and nu(Cu-(16)O/Cu-(18)O)/cm(-1) at 853/807 (1c), 858/812 (2c), 847/800 (3c), and at 547/522 (2c), 544/518 (3c), respectively. The thermal stability of the peroxo-copper species has increased with increase in the number of the hydrogen-bonding interactions between the peroxide and amino groups.

Copper complexation by thiol compounds in estuarine waters

The stability of copper complexes with thiol substances in estuarine waters was determined for the first time using a new procedure based on cathodic stripping voltammetry (CSV). The free thiol concentration was monitored during titrations with copper in the presence of a competing ligand salicylaldoxime (SA); concentrations of copper-complexing ligands and conditional stability constants were determined simultaneously but independently. The decrease in the free thiol concentration with increasing copper concentration was used as an independent measure of the thiol-complex stability. The conditional stability constant of the thiol complexes (log K CuThiol′) was between 12.3 and 14.1, and decreased with increasing salinity. The copper complexing titrations were found to fit to two complexing ligands: L 1 with concentrations between 10 and 33 nM, and L 2 between 14 and 300 nM. The complex stability of most of the thiols was similar to that of CuL 2. Titrations at different detection windows showed a shift in the thiol complex stability suggesting that a second thiol species was present. It is therefore possible that L 1 is also a thiol species. The estimated thiol concentrations can account for up to half of the total ligand concentration at low to intermediate salinities and for all of the ligands at high salinities.

Physics of Copper in Silicon

This article reviews the progress made in the studies of copper in silicon over the last several years and puts forward a comprehensive model of the behavior of copper in silicon. Technical aspects of this model are discussed in detail. It is shown that many important aspects of the behavior of copper in silicon are not shared with the other 3d transition metals. The positive charge state of interstitial copper makes its defect reactions Fermi-level-dependent, and results in a noticeable difference in the out-diffusion and precipitation behavior of copper in n-Si and p-Si. The extremely high diffusivity of copper in silicon, which is a consequence of the small ionic radius of copper and its relatively weak interaction with the silicon lattice, makes it highly mobile at room temperature and impacts the stability of copper complexes. Large lattice strains and electrostatic effects in p-Si make the formation of copper-silicide precipitates in the bulk energetically unfavorable, unless the chemical driving force for precipitation is high enough to overcome the nucleation and precipitation barrier. Literature data on the effect of copper on minority carrier lifetime and device yield are analyzed using our improved understanding of the physics of copper in silicon. Finally, the impact of the physics of copper in silicon on the development and characterization of copper diffusion barriers is discussed. © 2001 The Electrochemical Society. All rights reserved.

Thermal equilibrium and stability of copper complexes in silicon crystal

Formation of the photoluminescence (PL) Cu center (1.014 eV) for silicon crystals diffused with Cu at 700 °C was observed. For the samples with a Cu concentration lower than 1×1014 atom/cm3, almost the same amount of the Cu center was formed for the same concentration of Cu for all the cooling conditions employed without forming Cu precipitation, and the Cu center was very stable for a long storage time at room temperature. More frequent Cu precipitation occurred for the samples with higher Cu concentrations. Quenching of the equilibrium states of the Cu complexes at high temperatures did not occur by rapid cooling. Thermal equilibrium and stability of Cu complexes for the samples containing no Cu precipitates differed from those of the samples containing precipitates.

Intracrystalline and Electronic Structures of Copper(II) Complexes Stabilized in Two-Dimensional Aluminosilicate

X-ray absorption spectroscopic studies at the Cu K-edge have been performed for [Cu(en)2]2+, [Cu(cyclam)]2+, and their intercalated forms of two-dimensional layer silicate to examine how the structural and electronic modifications influence the stabilization of copper complexes in the charged interlayer space. According to the EXAFS analysis, copper complex ions in the layer silicate are stabilized at the center of the siloxane ring, and negatively charged layers are likely to act as axial counteranions. It is spectroscopically confirmed that the covalent bonding character between copper and ligand is enhanced by the intercalation into the silicate layers, depending upon the kind of ligands. There is a good linear relationship between the shoulder peak position of Cu K-edge XANES spectra and d−d transition band position of UV−visible spectra, which means that the former reflects the ligand-to-metal charge transfer effect.

π-d Effect in complexation between copper(II) ions and L,L-dipeptides containing aromatic side-chains

The L,L-Phe-Leu-Cu(II), L,L-Leu-Phe-Cu(n) and Gly-L-Phe-Cu(II) systems were studied using potentiometric, calorimetric and spectroscopic measurements. The results obtained from potentiometric and calorimetric studies show a significant increase in stability of copper complexes, when an aromatic residue is located in C-terminal, compared to the L,L-dipeptides containg the same amino acid residues, but in which the phenyl group is N-terminal. This stability increase is suggested as the result of interaction between π-electrons of the aromatic ring and the d-orbitals of the metal ion. An IR spectroscopic study was also carried out for the L,L-Leu-Phe-Cu(II) and Gly-L-Phe-Cu(II) systems.

An improved method for the separation of peptides and α-amino acids on copper-Sephadex

An improved method for the separation of peptides from large amounts of α-amino acids on copper-Sephadex is described. The separation is essentially dependent upon the copper content of Sephadex, the pH of the system, and the concentration of the sample, and it is due to the different stabilities of copper complexes of Sephadex, peptides, and α-amino acids. Sephadex behaves as a solid ligand. Compounds that form weaker complexes with copper than Sephadex apparently move with the solvent front. α-Amino acids that form copper complexes having a stability comparable to that of copper-Sephadex complexes are retained on the column. Peptides form strong complexes, stripping copper from the column. They are only slightly retained. The method is most practical when copper is removed from the eluates with chelating ion-exchanger Dowex A-1. Examples are given for the chromatography of single compounds, model mixtures, and extracts from cheese and yeast.