and Heterobinuclear Cu2+ and Zn2+ Complexes ... - ACS Publications

Article Cite This: Inorg. Chem. 2017, 56, 13748-13758

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Homo- and Heterobinuclear Cu2+ and Zn2+ Complexes of Ditopic Aza Scorpiand Ligands as Superoxide Dismutase Mimics Lluís Guijarro,† Mario Inclán,† Javier Pitarch-Jarque,† Antonio Doménech-Carbó,‡ Javier U. Chicote,§ Sandra Trefler,§ Enrique García-España,*,† Antonio García-España,§ and Begoña Verdejo*,† †

Instituto de Ciencia Molecular, c/Catedrático José Beltrán 2, 46980 Paterna, Valencia, Spain Departamento de Química Analítica, Universidad de Valencia, c/Dr. Moliner 50, 46100 Burjassot, Valencia, Spain § Unitat de Recerca, Hospital Joan XXIII, Institut d’Investigació Sanitària Pere Virgili, Universitat Rovira i Virgili, 43007 Tarragona, Spain Downloaded via UNIV OF TOLEDO on June 29, 2018 at 17:57:03 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Two polytopic aza-scorpiand-like ligands, 6-[7(diaminoethyl)-3,7-diazaheptyl]-3,6,9-triaza-1-(2,6-pyridina)cyclodecaphane (L1) and 6-[6′-[3,6,9-triaza-1-(2,6-pyridina)cyclodecaphan-6-yl]-3-azahexyl]-3,6,9-triaza-1-(2,6-pyridina)cyclodecaphane (L2), have been synthesized. The acid−base behavior and Cu2+, Zn2+, and Cu2+/Zn2+ mixed coordination have been analyzed by potentiometry, cyclic voltammetry, and UV−vis spectroscopy. The resolution of the crystal structures of [Cu 2 L2Cl 2 ](ClO 4 ) 2 ·1.67H 2 O (1), [Cu 2 HL2Br 2 ](ClO 4 ) 3 · 1.5H2O (2), and [CuZnL2Cl2](ClO4)2·1.64H2O (3) shows, in agreement with the solution data, the formation of homobinuclear Cu2+/Cu2+ and heterobinuclear Cu2+/Zn2+ complexes. The metal ions are coordinated within the two macrocyclic cavities of the ligand with the involvement of a secondary amino group of the bridge in the case of 1 and 3. Energydispersive X-ray spectroscopy confirms the 1:1 Cu2+/Zn2+ stoichiometry of 3. The superoxide dismutase (SOD) activities of the Cu2+/Cu2+ and Cu2+/Zn2+ complexes of L1 and L2 have been evaluated using nitro blue tetrazolium assays at pH 7.4. The IC50 and kcat values obtained for the [Cu2L1]4+ complex rank among the best values reported in the literature for Cu-SOD mimics. Interestingly, the binuclear Cu2+ complexes of L1 and L2 have low toxicity in cultures of mammalian cell lines and show significant antioxidant activity in a copper-dependent SOD (SOD1)-defective yeast model. The results are rationalized by taking into account the binding modes of the Cu2+ ions in the different complexes.

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INTRODUCTION In recent years, the design of new polytopic ligands has aroused great interest because of their potential applications in fields such as molecular recognition, molecular devices, pharmaceutical chemistry, and enzyme mimicking.1−3 Indeed, metal sites of enzymes are characterized by coordinative unsaturation, which permits the approach and binding of the target substrate to the metal center. In this respect, several families of [1 + 1] polyazacyclophane receptors with different aromatic spacers and polyamine chains containing up to six amino groups have been synthesized in our group during the past few years.4 Although several of these receptors can bind simultaneously more than one metal ion, their low number of donor atoms prevents the coordination spheres of the metal ions from being filled, giving rise to coordinative unsaturation. These arrangements are favorable for the recognition and/or activation of additional guests as exogenous ligands in mixed complexes. Here we report the two new binucleating ligands 6-[7(diaminoethyl)-3,7-diazaheptyl]-3,6,9-triaza-1-(2,6-pyridina)cyclodecaphane (L1) and 6-[6′-[3,6,9-triaza-1-(2,6-pyridina)cyclodecaphan-6-yl]-3-azahexyl]-3,6,9-triaza-1-(2,6-pyridina)cyclodecaphane (L2) (see Chart 1) designed to achieve © 2017 American Chemical Society

Chart 1

different degrees of coordinative unsaturation in the bound metal ions. In L1, a pyridinophane tetraazamacrocyclic ligand containing a pendant aminoethyl chain (L4) has been linked Received: July 10, 2017 Published: October 31, 2017 13748

DOI: 10.1021/acs.inorgchem.7b01756 Inorg. Chem. 2017, 56, 13748−13758

Article

Inorganic Chemistry Scheme 1. Synthetic Route for L1 and L2

(2,6-pyridina)cyclodecaphan-6-yl]-3-azahexyl]-3,6,9-triaza-1-(2,6pyridina)cyclodecaphane (L2) was carried out following the general procedures described in the literature for the preparation of analogous receptors, which consist of the reaction of the tosylated polyamine (a) with N-tosylaziridine in a 1:2 molar ratio in refluxing acetonitrile (CH3CN) to obtain L1·Ts.5 Treatment with hydrogen bromide (HBr)/acetic acid (AcOH) and phenol (PhOH) leads to removal of the tosyl groups to give the final product L1. On the other hand, synthesis of L2 is achieved by the reaction of the pertosylated polyamine L1·Ts with 2,6-bis(bromomethyl)pyridine in a 1:1 molar ratio using potassium carbonate (K2CO3) as the base in refluxing CH3CN and subsequent detosylation (Scheme 1) . Synthesis of L1·Ts. To a solution of a (3.02 g, 3.92 mmol) in dry CH3CN (50 mL) was added dropwise N-tosylaziridine (1.85 g, 9.42 mmol) in dry CH3CN (100 mL). Then the reaction mixture was refluxed under a dinitrogen atmosphere for 24 h. The solvent was removed under reduced pressure, and the obtained yellow oil was purified by chromatography [98:2 dichloromethane (CH2Cl2)/ methanol (MeOH)] in silica gel (40−63 μm). 1H NMR (300.0 MHz, CDCl3): δ 7.9−7.7 (m, 12H), 7.4−7.2 (m, 14H), 5.9−5.8 (m, 2H), 4.3 (s, 4H), 3.1−3.0 (m, 6H), 2.9−2.8 (m, 6H), 2.5−2.3 (m, 27H), 1.7−1.6 (m, 2H). 13C NMR (75.4 MHz, CDCl3): δ 155.4, 144.0, 144.0, 143.4, 139.1, 137.5, 136.2, 130.4, 130.3, 130.1, 130.1, 128.1, 127.6, 124.3, 56.9, 55.0, 54.0, 52.6, 52.6, 50.3, 47.3, 41.1, 31.3, 21.9. Synthesis of L1. A total of 1.20 g (1.03 mmol) of L1·Ts and 4.0 g (43.7 mmol) of PhOH were dissolved in 50 mL of 33% HBr/AcOH. The mixture was heated at 90 °C with stirring for 24 h. The solid obtained was filtered off and washed with EtOH. Excess AcOH was removed with an anionic exchange resine (Amberlite IRA-402), and the obtained oil was precipitated with HCl in dioxane, obtaining L1 as its hydrochloride salt. Yield: 80%. 1H NMR (300.0 MHz, D2O): δ 7.9 (t, J = 8 Hz, 1H), 7.4 (d, J = 8 Hz, 2H), 4.6 (s, 4H), 3.3−3.2 (m, 16H), 3.1−3.0 (m, 4H), 2.9 (t, J = 5 Hz, 4H), 1.9−1.8 (m, 2H). 13C NMR (75.4 MHz, D2O): δ 152.6, 136.4, 125.9, 60.2, 57.9, 55.9, 55.0, 51.9, 47.1, 46.3, 43.7, 38.8, 27.5. Anal. Calcd for C20H40N8·6HCl·4H2O: C, 35.1; H, 7.9; N, 16.7. Found: C, 35.2; H, 7.9; N, 16.4. MS (ESI): m/z 393 ([M]+). Synthesis of L2·Ts. A total of 1.35 g of L1·Ts (1.16 mmol) and K2CO3 (1.60 g, 11.60 mmol) were suspended in refluxing CH3CN (200 mL). To this mixture was added dropwise over 2 h 2,6bis(bromomethyl)pyridine (0.31 g, 1.16 mmol) in 60 mL of CH3CN. This suspension was refluxed for a further 24 h and then filtered off. The solution was vacuum evaporated to dryness, and the obtained oil was purified by chromatography (90:10 CH2Cl2/MeOH) in silica gel (40−63 μm). 1H NMR (300.0 MHz, CDCl3): δ 7.8−7.6 (m, 12H), 7.4−7.2 (m, 15H), 4.3 (s, 8H), 3.2−2.9 (m, 12H), 2.5 (t, 2H), 2.5−2.2

through a propylenic spacer to the central amine group of a diethylentriamine fragment (L1). In the second one, L4 has been connected to a second pyridinophane tetraazamacrocycle also through a propylenic spacer. The different kinds and number of nitrogen atoms in both ligands, eight in L1 and nine in L2, can lead to significant chemical differences regarding their coordination behavior and possible activation of guest species. As a matter of fact, if one of the metals binds to the macrocyclic core of the asymmetric L1 ligand, the second one should bind to the open-chain site in a coordinatively unsaturated mode. In the case of L2, while each macrocycle can bind a metal ion, the amino group of the spacer will be binding just to one of them, generating again asymmetric coordination sites. Coordinative unsaturation and asymmetry are, as commented on above, recurrent features in many metalloenzymes that play key roles in different biological processes. The SOD1 and SOD3 (SOD = superoxide dismutase) families of enzymes, the first one cytosolic and the second one extracellular, have dinuclear active centers formed by Cu2+ and Zn2+ ions interconnected by an imidazolate bridge, which, although frequent in synthetic complexes, is a distinctive feature of these enzymes. Cu2+ completes its coordination sphere with three monodentate imidazole groups coming from histidine residues, while Zn2+, apart from the bridging imidazolate, is coordinated by two other histidines and by a monodentate carboxylate coming from an aspartate residue. The catalytic role is essentially played by the Cu2+ ion, while the Zn2+ ion can be either removed or replaced by other metal ions without experiencing a very significant loss in activity. Here, besides analyzing the ability of these ligands to form homo- and heterobinuclear Cu2+ and Zn2+ complexes, both in solution and in the solid state, we have explored their performance in vitro as SOD mimics using the McCord− Fridovich method and a SOD-deficient yeast model. We analyze how the presence of either only Cu2+ or Cu2+/Zn2+ ions in the complexes, the different nuclearities, and the types of coordination centers influence the SOD mimicking behavior.

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EXPERIMENTAL SECTION

Synthesis of the ligands 6-[7-(diaminoethyl)-3,7-diazaheptyl]-3,6,9triaza-1-(2,6-pyridina)cyclodecaphane (L1) and 6-[6′-[3,6,9-triaza-113749


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Inorganic Chemistry (m, 26H), 1.5 (m, 2H). 13C NMR (75.4 MHz, CDCl3): δ 152.8, 141.4, 136.6, 133.8, 127.8, 125.0, 121.7, 54.7, 51.7, 51.3, 49.0, 48.6, 46.3, 45.3, 41.0, 29.6, 20.9. Synthesis of L2. A total of 0.89 g (0.70 mmol) of L2·Ts and 4.20 g (44.50 mmol) of PhOH were dissolved in 45 mL of 33% HBr/AcOH. The mixture was heated at 90 °C for 24 h under stirring. The solid obtained was filtered off and washed with EtOH, obtaining L2 as its hydrobromide salt. Yield: 70%. 1H NMR (300.0 MHz, D2O): δ 7.8 (t, J = 8 Hz, 2H), 7.3 (d, J = 8 Hz, 4H), 4.6 (s, 8H), 3.3−3.1 (m, 10H), 3.0−2.9 (m, 4H), 2.8−2.6 (m, 10H), 1.9−1.8 (m, 2H). 13C NMR (75.4 MHz, D2O): δ 149.2, 140.1, 122.6, 52.5, 51.3, 50.8, 49.9, 46.6, 45.4, 43.8, 21.7. Anal. Calcd for C27H45N9·6HBr·4H2O: C, 30.8; H, 5.6; N, 11.9. Found: C, 30.9; H, 5.2; N, 11.4. MS (ESI): m/z 496 ([M]+). Synthesis of [Cu2L2Cl2](ClO4)2·1.67H2O (1). Under an argon atmosphere, to a solution of L2 (10 mg, 0.0095 mmol) in water (2 mL) was added 2 equiv of Cu(ClO4)2·6H2O, and the pH was subsequently adjusted to pH 7. Slow evaporation of the solution afforded the formation of blue crystals suitable for X-ray diffraction in 72% yield. Synthesis of [Cu2HL2Br2](ClO4)3·1.5H2O (2). Under an argon atmosphere, to a solution of L2 (10 mg, 0.0095 mmol) in water (2 mL) were added 2 equiv of Cu(ClO4)2·6H2O, and the pH was subsequently adjusted to 4. Slow evaporation of the solution afforded the formation of blue crystals suitable for X-ray diffraction in 75% yield. Synthesis of [CuZnL2Cl2](ClO4)2·1.64 H2O (3). Under an argon atmosphere, to a solution of L2 (10 mg, 0.0095 mmol) in water (2 mL) were added 1 equiv of Cu(ClO4)2·6H2O and 1 equiv of Zn(ClO4)2·6H2O, and the pH was subsequently adjusted to 7. Slow evaporation of the solution afforded the formation of light-blue crystals suitable for X-ray diffraction in 67% yield. Caution! Perchlorate salts of compounds containing organic ligands are potentially explosive and should be handled with care. Electromotive Force (EMF) Measurements. The potentiometric titrations were carried out at 298.1 ± 0.1 K using 0.15 M NaCl as the supporting electrolyte. The experimental procedure (buret, potentiometer, cell, stirrer, microcomputer, etc.) has been fully described elsewhere.6 The acquisition of the EMF data was performed with the computer program PASAT.7 The reference electrode was an Ag/AgCl electrode in a saturated KCl solution. The glass electrode was calibrated as a hydrogen-ion concentration probe by the titration of previously standardized amounts of HCl with CO2-free NaOH solutions and the equivalent point determined by Gran’s method,8 which gives the standard potential, E°′, and the ionic product of water [pKw = 13.73(1)]. The computer program HYPERQUAD was used to calculate the protonation and stability constants.9 The pH range investigated was 2.0−11.0, and the concentrations of the metal ions and ligands ranged from 1 × 10−3 to 5 × 10−3 M with M/L molar ratios varying from 2:1 to 1:2. The different titration curves for each system (at least two) were treated either as a single set or as separated curve without significant variation in the values of the stability constants. Finally, the sets of data were merged together and treated simultaneously to give the final stability constants. For determination of the stability constants of the mixed system, the molar ratio Cu2+/Zn2+/L employed was 1:1:1. In the calculations, the stability constants of the binary systems previously determined were first introduced as fixed parameters, and the data were refined to obtain the constants of the mixed systems. Finally, the data of the titrations corresponding to the binary Cu2+/L2 and Zn2+/L2 and ternary Cu2+/Zn2+/L2 systems were fitted together, leaving free all of the constants of the Cu2+/L2, Zn2+/L2, and Cu2+/Zn2+/L2 systems, obtaining values that do not differ significantly from those obtained from the treatment of the binary systems alone. The distribution diagrams were calculated using the program HySS.10 NMR Measurements. The 1H and 13C NMR spectra were recorded on a Bruker Avance AC-300 spectrometer operating at 300.0 MHz for 1H and at 75.4 MHz for 13C. The chemical shifts are given in parts per million referenced to the solvent signal.

Crystallographic Analysis. Analysis of single crystals of 1−3 was carried out with an Oxford Diffraction Supernova diffractometer using Mo Kα radiation (λ = 0.71073 Å) at 120 K. The structure was solved by direct methods using SHELXT11 and refined by full-matrix least squares on all F2 using SHELXL13 with the OLEX212 suite. Molecular drawings were produced with Mercury.13 The crystal data, data collection parameters, and results of analysis are listed in Table S1. Some soft SHELXL restraints (DELU, ISOR, SIMU, and DFIX) had to be used to correct the geometry of the disordered parts and the thermal parameters of the corresponding atoms of 1−3. In 3, the presence of diffused solvent water molecules in the voids between metal complexes could not be modeled even with restraints. Consequently, to avoid this residual electron density, OLEX2 Solvent Mask was used to calculate the void space and electron count and to get a new HKL file. According to the OLEX2 Solvent Mask results and the different experimental evidence, a total number of 1.64 water molecules (12.2 electrons) were considered per unit cell. CCDC 1507630, 1507631, and 1507632 contain the supplementary crystallographic data for this paper. Electrochemical Measurements. Cyclic voltammetric experiments were performed on 50 mM tris(hydroxymethyl)aminomethane (Tris) and 0.15 M NaCl aqueous solutions at pH 7.4 using a 10−3 M receptor concentration. Electrochemical experiments were performed with BAS CV 50W and Metrohm PGSTAT 101 Autolab instruments in a conventional three-compartment cell with a glassy carbon working electrode. Prior to each voltammetric run, the electrode was polished with an aqueous suspension of alumina on a soft surface, rinsed with water, and dried. An Ag/AgCl (3 M NaCl) reference electrode and platinum wire auxiliary electrode completed the three-electrode configuration. The cyclic voltammograms were recorded at scan rates of 50−500 mV s−1. Energy-Dispersive X-ray Spectroscopy (EDX). EDX analysis of 3 was performed by using the EDX device XL-30 ESEM (Philips). Data acquisition was performed with an acceleration voltage of 20 kV, a focal distance of 10 mm, and an accumulation time of 60 s. Experiments were carried out by selecting windows containing at least one single crystal, and the experimental data were given as an average of at least three measurements. SOD Activity. SOD-like activity was determined by using the nitro blue tetrazolium (NBT) reduction method.14,15 Assay was carried out in a 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer at pH 7.4 and at room temperature. The kinetics of the NBT reduction to monoformazan was recorded by reading the absorbance change at 560 nm with time. Several concentrations of every compound were prepared in a Tris buffer at pH 7.4. A reaction mixture of ca. 50 μM NBT and 175 μM xanthine in a HEPES buffer was prepared. A total of 800 μL of the reaction mixture plus 100 μL of the compound in a Tris buffer was added to a 1 mL cuvette. The reaction starts when 100 μL of a solution of xanthine oxidase was added. The xanthine oxidase concentration was optimized to find the appropriate amount that causes a linear absorbance variance during the reading time (2 min). The nonlinear initial induction period is rejected if it exists. The inhibition of the NBT reduction was computed16 as (S0 − S)/S0, where S0 and S are the slopes (dA/dt) of the blank (uninhibited) and of the compound, respectively. The IC50 values were either read or interpolated from the inhibition versus concentration plots.17 MTT Assay. T24 and 253J transitional-cell human bladder carcinoma cells and VERO green monkey kidney cells [American Type Culture Collection (ATCC), Rockville, MD] were cultured at 37 °C in a 5% CO2 in air atmosphere. VERO cells were grown in Eagle’s minimum essential medium and T24 and 253J in McCoy’s 5A media supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were seeded at 2.5 × 104 cells/mL in 96-well plates and treated with L1, L2, and their metal complexes at different concentrations (10, 50, and 100 μM) for 48 h as previously described.18 SOD-Deficient Yeast Assay. The in vivo SOD-like activity of the metal complexes of L1 and L2 was evaluated using a Saccharomyces 13750


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Inorganic Chemistry cerevisae strain defective in the cytoplasmatic copper-dependent SOD enzyme [ATCC: ATCC 96687 (SOD1Δ), MATa ura3-52 trp1-289 his3-Δ1 leu2-3 leu2-112 sod1::URA3). Wild-type BY4741 (MATa, leu2 Δ 0his3 Δ 1, met15 Δ 0, ur3 Δ 0] was used as the control. A solution of each strain with an optical density of 0.7 mU (620 nm) was diluted 1:200 in 5 mL of a SDC medium (0.68% YNB, 0.16% drop-out mixture without leucine, 0.15% leucine, 0.03% metionine, tryptophan, uracil, and hystidine, 0.07% adenosin, and 2% glucose). Yeasts were grown aerobically in 50 mL falcon tubes at 30 °C with shaking at 220 rpm in the presence of 10 μM metal complexes or ligands. The growth at the indicated times was measured turbidimetrically at 620 nm. The antioxidant capacity was determined as the growth ratio between the S. cerevisiae strain (SOD1Δ) with the ligands or their metal complexes and that without them.

Table 2. Logarithms of the Equilibrium Constants for the Interaction of Cu2+ with L1 and L2 Determined at 298.1 K in 0.15 M NaCl

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RESULTS AND DISCUSSION Acid−Base Behavior. Table 1 collects the stepwise protonation constants of L1 and L2, determined in 0.15 M

reactionb

L1

L2

L3

1 2 3 4 5 6

H + L ⇆ HL H + HL ⇆ H2L H + H2L ⇆ H3L H + H3L ⇆ H4L H + H4L ⇆ H5L log βd

9.84(3)c 9.72(1) 9.00(2) 8.55(2) 7.36(2) 44.47(2)

10.19(3) 9.58(3) 8.74(3) 8.24(3) 7.28(3) 44.03(3)

10.13 9.40 8.27 7.02

reactiona

1 2 3 4 5 6 7 8 9 10

CuH2L + H ⇄ CuH3L CuHL + H ⇄ CuH2L CuL + H ⇄ CuHL Cu + L ⇄ CuL CuL + H2O ⇄ CuL(OH) + H 2Cu + L ⇄ Cu2L Cu2L + H ⇄ Cu2HL CuL + Cu ⇄ Cu2L Cu2L + H2O ⇄ Cu2L(OH) + H Cu2L(OH) + H2O ⇄ Cu2L(OH)2 + H

L1 4.22(3) 8.29(5) 9.94(3) 18.96(5) −11.3(1) 32.31(3) 13.35(7) −8.75(5) −10.53(9)

L2 b

5.82(8) 8.21(5) 9.50(3) 18.23(7) −10.5(1) 33.84(5) 4.76(5) 15.62(9) −9.86(5) −10.6(1)

a

Charges omitted for clarity. bValues in parentheses are standard deviations in the last significant figure.

comparison of the stability constants obtained for L1 and L2 with those of the related ligand L3 previously studied26 suggests at least four as the number of nitrogen atoms tightly bound to the first Cu2+ (see Table 2). This is reflected in the first two protonation constants of [CuL]2+, which display values that are, in some cases, even higher than those of the stepwise protonation constants of the free receptor at the stages at which both reactions display the same charges (for instance, [CuL1]2+ + H+ ⇄ [CuHL1]3+, with log K = 9.94(3), and [H2L1]2+ + H+ ⇄ [H3L1]3+, with log K = 8.99(2); entries 3−5 in Table 1 and entries 2−4 in Table 2). An analogous analysis reveals that the third protonation process would occur on a coordinated nitrogen atom. In this sense, the UV−vis spectra of solutions containing Cu2+ and L1 in a 1:1 molar ratio can provide additional information about the role of the pyridine spacer as an anchorage point for the metal ion (see Figure 1). The variation as a function of the pH of the band at 270 nm (attributable to the pyridine ring) shows that coordination of the metal ion by the pyridine nitrogen atom occurs as soon as the 1:1 complexes start to be formed. Indeed, an increase in the intensity of this band is observed as the [CuH3L1]5+ species appears, reaching a plateau when its maximum percentage in solution is achieved. In accordance with the potentiometric studies, this fact suggests that coordination of the metal ion occurs through the macrocyclic cavity, with the amino groups of the pendant arm remaining available for protonation. Then, a further increase in absorbance is observed with the formation of the remaining less protonated 1:1 Cu2+/L1 complexes. These data can be related, in conjunction with analysis of the variations with the pH of the wavelength of the maximum of the d−d band, to a change in the number of nitrogen atoms implicated in coordination of the metal ion, from four in ([CuH3L]5+ to five in [CuH2L]4+; that is, at this stage, the secondary nitrogen atom of the pendant arm should start to bind the metal ion. For a 2:1 Cu2+/L molar ratio, [Cu2L]4+, [Cu2L(OH)]3+, and [Cu2L(OH)2]2+ species have been observed for L1 and L2, while for L1, a [Cu2HL]5+ species was additionally identified. The distribution diagrams show that the binuclear species are predominant in solution throughout the entire pH range investigated (Figure 2). The introduction of a second Cu2+ ion is accompanied, in both ligands, by a reduction in the stepwise formation constants, which is much more pronounced in the case of L1 [Cu + L1 ⇄ CuL1, with log K = 18.96(5), and CuL1 + Cu ⇄ Cu2L1, with log K = 13.35(7); entries 4 and 8,

Table 1. Logarithms of the Stepwise Protonation Constants of L1 and L2 Determined at 298.1 K in 0.15 M NaCla entry

entry

34.84

a

Values obtained for L3 are included for comparison.19 bCharges omitted for clarity. cValues in parentheses are standard deviations in the last significant figure. dlog βc = ∑log K.

NaCl at 298.1 K along with those previously reported for the parent compound L3.19 Distribution diagrams of L1 and L2 are collected in Figure S1. Both receptors present in the pH range of study (2.5−11.0) the same number of stepwise protonation constants as the secondary and the primary or secondary amino groups are in L1 and L2, respectively. The values of the constants follow the expected tendency observed in previous works on this topic20−23 and can be largely interpreted in terms of minimization of electrostatic repulsions.24 Analysis of 13C and 1H NMR experiments provides information about the average protonation sequence, followed by these polyaminic ligands.22,24,25 However, for L1 and L2, upon deprotonation of the different amino groups, all of the proton signals experience significant upfield shifts, making it difficult to easily identify which were the preferential protonation sites at each protonation step. Cu2+ Complexation Studies. The formation of Cu2+ complexes with L1 and L2 was studied in solution by potentiometric measurements and UV−vis spectroscopy. The pH-metric titrations carried out at 298.1 K in 0.15 M NaCl provided the stability constants and model species that are shown in Table 2. For a 1:1 Cu2+/L molar ratio, [CuHxL](2+x)+ mononuclear species with protonation degrees varying from −1 to +3 have been detected for both ligands. In the case of L1, the distribution diagram depicted in Figure 1 shows that, for a 1:1 Cu2+/L molar ratio, mainly mononuclear species are present in solution throughout the pH range covered in the studies (2.5− 11.0). However, for L2, the formation of binuclear species is observed even at 1:1 Cu2+/L molar ratio (see Figure S2). A 13751


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Inorganic Chemistry

Figure 1. UV−vis titration curves for a 1:1 Cu2+/L1 molar ratio determined in 0.15 M NaCl at 298.1 ± 0.1 K with [L1] = 5.0 × 10−4 M overlapped to mole fraction distribution curves for the various protonated forms (solid lines). Changes in the absorbance at 270 nm (left) and changes in the maximum transition d−d band (right).

Figure 2. Distribution diagram for the system (a) 2:1 Cu2+/L1 and (b) 2:1 Cu2+/L2. [L] = 1.0 × 10−3 M and [Cu2+] = 2.0 × 10−3 M.

Table 2]. This fact can be explained by taking into account the available coordination environments. While the first metal ion is coordinated in L1, a priori, by the macrocyclic unit and the secondary amino group at the lateral chain, this receptor provides a poorer environment to the second Cu2+ consisting of two primary amino groups at the end of the extended lateral chain and the tertiary nitrogen atom. However, the second macrocyclic unit present in L2 favors a tighter binding of the second metal than that in L1, yielding a smaller reduction of the second stepwise formation constant. A comparison of the experimental λmax (701 nm) of the spectrum of [Cu2L2]4+ with that derived from the equation proposed by Prenesti et al. to predict the number of equatorial donor atoms (λmax = 625 nm) indicates the presence of at least one axial amine group in each of the coordination sites.27 In the case of L1, the lower number of nitrogen atoms involved in the coordination of the second metal ion is also reflected by the higher tendency to hydrolyze its [Cu2L1]4+ species [pKa values of 8.74(5) and 9.86(5) for L1 and L2, respectively]. This low value might suggest the presence of a hydroxido bridge between the metal centers in the case of L1. Slow evaporation of a solution containing L2 and Cu(ClO4)2·6H2O in a 2:1 Cu2+/L molar ratio at pH 7 and 4 afforded crystals suitable for X-ray diffraction of 1 and 2, respectively (see Figure 3). These crystal structures reveal the active role of the bridge secondary amino group in the coordination of the metal ion. In 1, the first metal ion, Cu1, is coordinated by one of the macrocycles in an octahedral distorted geometry in which the elongated axial positions are occupied by the benzylic nitrogen atoms [Cu1−N2, 2.29(2) Å]. The equatorial plane is defined by the nitrogen atom of the

Figure 3. Representation of the [Cu 2 L2Cl 2 ] 2+ (top) and [Cu2HL2Br2]3+ (bottom) cations. Hydrogen atoms are omitted for clarity.

pyridine ring [Cu1−N1, 1.934(14) Å], the tertiary amino group of the macrocyclic core [Cu1−N3, 2.183(15) Å], the nitrogen atom of the pendant arm [Cu1−N4, 2.050(15) Å], and a chloride anion [Cu1−Cl1, 2.376(4) Å]. Cu2 is coordinated by the second macrocycle, presenting an axially elongated squarepyramidal geometry (τ = 0.01;28 see Figure 3A and Table 3), where the equatorial positions are occupied by the pyridine nitrogen atom [Cu2−N7, 1.960(13) Å], the benzylic nitrogen atoms [Cu2−N6, 2.022(13) Å], and a chloride anion [Cu2− Cl2, 2.256(4) Å]. The elongated axial position is occupied by a tertiary nitrogen atom of the macrocyclic cavity [Cu2−N5, 2.283(12) Å]. 13752


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Inorganic Chemistry Table 3. Selected Bond Distances (Å) of Complexes 1−3a [Cu2(L2)Cl2](ClO4)2·1.67H2O (1) Cu1−N1 Cu1−N2 Cu1−N3 Cu1−N4 Cu1−Cl1 Cu2−Cl2 Cu2−N5 Cu2−N6i Cu2−N6 Cu2−N7 a

[Cu2HL2Br2](ClO4)3·1.5H2O (2)

1.934(14) 2.29(2) 2.183(15) 2.050(15) 2.376(4) 2.256(4) 2.283(12) 2.022(13) 2.022(13) 1.960(13)

Cu1−Br1 Cu1−N1 Cu1−N2 Cu1−N4 Cu1−N3 Cu2−Br2 Cu2−N8 Cu2−N6 Cu2−N9 Cu2−N7

[CuZn(L2)Cl2](ClO4)2·1.64H2O (3)

2.3734(10) 1.943(5) 2.054(5) 2.065(5) 2.287(6) 2.4086(11) 1.930(6) 2.201(5) 2.043(6) 2.047(6)

Zn1−Cl3 Zn1−N1 Zn1−N2 Zn1−N3 Zn1−N4ii Cu1−Cl4 Cu1−N5 Cu1−N6ii Cu1−N7

2.380(4) 2.025(19) 2.11(2) 2.229(18) 2.17(2) 2.295(4) 2.227(12) 2.030(15) 1.956(16)

(i) x, y, −z; (ii) x, y, −z + 1.

and 3 in Table 4 and entries 3 and 4 in Table 1) suggest that such processes involve uncoordinated nitrogen atoms. An analogous analysis for the protonation of [ZnH2L]4+ reveals that the last protonation process occurs in a coordinated nitrogen atom. For a 2:1 Zn2+/L molar ratio, [Zn2L]4+, [Zn2L(OH)]3+, and [Zn2L(OH)2]2+ species have been observed for L1 and L2 (see Figure S6). As occurs for Cu2+, the value of the constant associated with the entry of the second Zn2+ ([ZnL]2+ + Zn2+ ⇆ [Zn2L]4+) is higher for L2 than for L1, with the difference in this case for the two ligands being even larger. Such a large value of the second stepwise constant makes the [Zn2L2]4+ species prevail in solution from pH 4 to 9. Cu2+/Zn2+ Mixed Complexes. Taking into account the presence of these two metal ions in the CuZn-SOD active site, heterobinuclear complexes of Cu2+/Zn2+/L have been studied. However, only in the case of L2, it was possible to determine the formation of mixed complexes by means of potentiometric studies (see Table 5). For this receptor, as can be seen in the

However, for 2, both metal ions are coordinated with similar axially elongated square-pyramidal geometries (τ = 0.01 and 0.14 for Cu1 and Cu2, respectively) by the nitrogen atoms of the macrocyclic cavities and bromide anions, with the tertiary amino groups occupying the axial positions (see Figure 3B). Table S1 collects selected bond distances and angles. The secondary amino group in the bridge is protonated and the ligand adopts an extended conformation in order to minimize the electrostatic repulsions. The torsion angles along the chain range from −179.9° to −166.3°. Zn2+ Complexation Studies. The stability constants with Zn2+ have also been determined as a first step to analyze the capability of L1 and L2 to form heterobinuclear Cu2+/Zn2+ complexes. Table 4 presents the equilibrium constants determined for the formation of Zn2+ complexes of L1 and L2. For a 1:1 Zn2+/ Table 4. Logarithms of the Stability Constants for the Formation of Mononuclear and Binuclear Complexes of Zn2+/L1 and Zn2+/L2 Determined at 298.1 ± 0.1 K in 0.15 M NaCl a

entry

reaction

1 2 3 4 5 6 7 8 9 10 11

ZnH2L + H ⇄ ZnH3L ZnHL + H ⇄ ZnH2L ZnL + H ⇄ ZnHL Zn + L ⇄ ZnL ZnL + H2O ⇄ ZnL(OH) + H 2Zn + L ⇄ Zn2L 2Zn + L + H2O ⇄ Zn2L(OH) + H 2Zn + L + 2H2O ⇄ Zn2L(OH)2 + H ZnL + Zn ⇄ Zn2L Zn2L + H2O ⇄ Zn2L(OH) + H Zn2L(OH) + H2O ⇄ Zn2L(OH)2 + H

L1

L2

5.36(3) 8.43(6) 9.80(3) 15.99(6)b −10.72(8) 23.88(4) 15.11(5) 3.99(8) 7.89(6) −8.77(5) −11.12(5)

8.35(5) 9.69(5) 17.23(7) −10.29(9) 28.93(3) 20.18(8) 10.19(7) 11.70(8) −8.76(7) −10.0(1)

Table 5. Logarithms of the Stability Constants for the Formation of Heterobinuclear Complexes of Cu2+/Zn2+/L2 Determined at 298.1 ± 0.1 K in 0.15 M NaCl entry

reactiona

L2

1 2 3 4

Cu + Zn + L + H ⇆ CuZnHL Cu + Zn + L ⇆ CuZnL Cu + Zn + L + H2O ⇆ CuZnL(OH) + H Zn + CuL ⇆ CuZnL

37.34(2)b 32.09(4) 22.46(8) 13.86(9)

a


a


L molar ratio, [ZnHxL](2+x) mononuclear species with protonation degrees varying from +2 to −1 have been detected in all cases, while for L1, a triprotonated [ZnH3L]5+ species was additionally identified. As expected, the values of the constants obtained are lower than those obtained for the Cu2+ complexes. Furthermore, as occurred for Cu2+, the presence of protonated species and the high values of the protonation constants of [ZnHL]3+ and [ZnH2L]4+ ([ZnL]2+ + H+ ⇄ [ZnHL]3+, with log K = 9.80(3), [H2L]2+ + H+ ⇄ [H3L]3+, with log K = 8.99(2), [ZnHL]3+ + H+ ⇄ [ZnH2L]4+, with log K = 8.44(5), and [H3L]3+ + H+ ⇄ [H4L]4+, with log K = 8.54(1); entries 2

Figure 4. Distribution diagram for the ternary system Cu2+/Zn2+/L2. [Cu2+] = [Zn2+] = [L2] = 10−3 M. 13753


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Inorganic Chemistry distribution diagram depicted in Figure 4, the Cu2+/Zn2+ mixed species [CuZnHL]5+ and [CuZnL]4+ prevail in solution from pH 4 to 10, with [CuZnL]4+ being the predominant one at physiological pH. Furthermore, [CuZnL(OH)]3+ species coexists in solution with the homobinuclear hydroxylated species. The existence of mixed complexes is supported by isolation of the crystals of a heterobinuclear complex of L2 suitable for X-ray diffraction. Crystals of [CuZn(L2)Cl2](ClO4)2·1.64H2O were obtained by the slow evaporation of an aqueous solution containing L2, Cu(ClO4)2·6H2O, and Zn(ClO4)2·6H2O in a 1:1:1 Cu2+/Zn2+/L molar ratio at pH 7 (Figure 5). Although

Table 6. Elemental Analysis (atom %) of the Three Experiments Performed by Transmission Electron Microscopy (TEM)−EDX Including Average (aver) and Standard Deviation (std) sample

Cu

Zn

1 2 3 aver std

2.68 2.26 2.17 2.37 0.27

2.81 2.32 2.25 2.46 0.30

anion [M2-Cl3, 2.380 (4) Å] and the tertiary amino group of the macrocyclic core [M2−N3, 2.229(18) Å]. The equatorial plane is defined by the nitrogen atom of the pyridine ring [M2−N1, 2.025(19) Å], the benzylic nitrogen atoms [M2−N2, 2.11(2) Å; M2−N2B(#1), 2.11(2) Å], and the nitrogen atom of the pendant arm [M2−N4, 2.17(2) Å]. The fact that, also in solution, Zn2+ is 6-coordinated and Cu2+ is 5-coordinated is supported by the very close λmax values of the spectra of solutions in which either [CuZnL]4+ or [CuZnHL]5+ are the predominant species. This suggests that upon deprotonation the central amine group would bind to the Zn2+ ion and not to the Cu2+ ion. Electrochemistry. Figure 7 compares the cyclic voltammetric response of 10−3 M aqueous solutions of Cu2L2 (left) and CuZnL2 (right) at pH 7.4. In the initial cathodic scan voltammograms, both complexes display a reduction peak at −0.55 V (C1) followed by a cathodic wave at −0.80 V (C2), which are followed, in the subsequent anodic scan, by an oxidation peak at −0.45 V (A1) and a high oxidation peak at −0.08 V (ACu). This last peak can be unambiguously attributed to the stripping oxidation of copper metal previously deposited on the electrode surface, thus denoting that the overall electrochemical cycle involves the Cu2+-to-Cu0 interconversion. Interestingly, the peak current of C1 for 10−3 M Cu2L2 is just 2-fold the peak current of the same signal for CuZnL2, thus denoting that in the Cu2L2 complex both metal centers are reduced independently.29 To rationalize the observed voltammetry, it has to be considered that peak A1 increases in height relative to peak C1 with increasing potential scan rate, whereas peak C2 exhibits the opposite effect. This feature suggests that there is an intermediate chemical reaction coupled between two successive electron-transfer processes (ECE mechanism).30,31 Thus, the C1/A1 couple would correspond to the one-electron (per Cu) reduction of Cu2+ to Cu+

Figure 5. Representation of the [CuZnL2Cl2]2+ cation. Hydrogen atoms are omitted for clarity.

Cu2+ and/or Zn2+ ions are indistinguishable by conventional Xray diffraction, EDX allowed us to evaluate the ratio of the different metals in the sample by analyzing the characteristic bands of Cu2+ and Zn2+ in the obtained crystals (Figure 6). Measurement of the three different samples showed an average atomic composition of 2.37% (Cu) and 2.46% (Zn) (Table 6), supporting a 1:1 Cu2+/Zn2+ stoichiometry. These results are in agreement with the potentiometric studies and corroborate the formation of heterobinuclear complexes for L2. Moreover, analysis of the different coordination modes and distances obtained for 3 allows one to assign each metal ion. In this regard, the first metal ion (Cu2+) presents an axially elongated square-pyramidal geometry (see Figure 5 and Table 3), where the equatorial positions are occupied by the pyridine nitrogen atom [M1−N7, 1.956(16) Å], the benzylic nitrogen atoms [M1−N6, 2.030(15) Å; M1−N6(#1), 2.030(15) Å], and a chloride anion [M1−Cl4, 2.295(4) Å]. The elongated axial position is occupied by the tertiary nitrogen atom [M1−N5, 2.227(12) Å]. However, the second metal ion (Zn2+) is coordinated by the other macrocycle in an octahedral fashion, where the elongated axial positions are occupied by a chloride

Figure 6. TEM−EDX image of a [CuZn(L2)Cl2](ClO4)2·1.64H2O crystal (left) and EDX spectra of the three different samples analyzed (right). 13754


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Inorganic Chemistry

Figure 7. Cyclic voltammograms at a glassy carbon electrode of 10−3 M solutions of Cu2L2 (left) and CuZnL2 (right) in 5 × 10−2 M Tris and 0.15 M NaCl aqueous solutions at pH 7.4. Potential scan rates (mV s−1): 500 (blue), 250 (lilac), 100 (green), and 50 (red).

[Cu II 2L2] + 2e− → [Cu I 2L2]

(1)

is followed by a reorganization or dissociation reaction yielding a different Cu+ species, which is subsequently reduced to Cu metal via the process C2: [Cu I 2L2] → k → [Cu I 2L2]*

(2)

[Cu I 2L2]* + 2e− → 2Cu + L2

(3) 4+

A similar scheme applies for the [CuZnL2] complex, for which the initial one-electron reduction of the copper center [Cu IIZn IIL2] + e− → [Cu IZn IIL2]

(4)

is followed by a reorganization or dissociation reaction yielding a different Cu+ species, which is subsequently reduced to Cu metal via the process C2: [Cu IZn IIL2] → k → [Cu IZn IIL2]*

(5)

[Cu IZn IIL2]* + e− → Cu + Zn 2 + + L2

(6)

Figure 8. Cyclic voltammograms at a glassy carbon electrode of 10−3 M solutions of Cu2L1 in 5 × 10−2 M Tris and 0.15 M NaCl aqueous solutions at pH 7.4. Potential scan rates (mV s−1): 500 (blue), 250 (lilac), 100 (green), and 50 (red).

2+

As far as the reduction of the Zn center would occur at clearly more negative potentials, there is no possibility of detecting zinc-localized electrochemistry for the complex under these experimental conditions. The voltammetry of Cu2L1 can be seen in Figure 8. Here, the cathodic response consists of two consecutive signals at −0.40 (C3) and −0.60 V (C4), followed by a weak anodic shoulder at −0.50 V (A4), apparently coupled to the precedent cathodic signal C4, and a stripping peak at −0.10 V (ACu), followed by an oxidation wave at +0.10 V (A5). This electrochemistry can be described in terms of the independent reduction of the two copper centers following different electrochemical pathways. Then two Cu2+-to-Cu+ reduction processes involving the C3/A5 and C4/A4 couples can be distinguished. Although the separation between the cathodic and anodic peak potentials was larger than that expected for a reversible behavior, formal electrode potentials can be approached as the half-sum of the above peak potentials. The obtained values, respectively of −0.15 and −0.50 V vs SCE, suggested that one of the copper centers of Cu2L1 displayed a one-electron reduction clearly more easily than those in Cu2L2 and CuZnL2, a feature consistent with the differences in the SOD activity of such complexes (vide infra).

In order to assess the proposed electrochemical pathways, chronoamperograms were recorded on solutions of the complexes upon application of a potential slightly more negative than the cathodic peak recorded in cyclic voltammetric experiments. Figure S15 compares the Cottrell plots of the current (i) versus inverse of the square root of time (t−1/2) for 2.0 mM solutions of Cu2L1 and Cu2L2 at pH 7.4. At short times (large t−1/2 values), experimental data tend to straight lines of similar slope, consistent with the one-electron assignment under diffusion control to the initial step of the voltammetric process. At large times (low t−1/2 values), however, experimental data tend to a different straight line whose slope is approximately 2-fold that at short times. Consistent with voltammetric data, this behavior is just that expected for an ECE mechanism consisting of two successive one-electron per Cu, diffusion-controlled transfers coupled to an intermediate chemical reaction, as theoretically described by Feldberg and Jeftic.32 Antioxidant Activity in Vitro. NBT assay allows calculation of the IC50 values for Cu2+ and Cu2+/Zn2+/L systems for receptors L1 and L2 at physiological pH (Table 7). Analysis of the experimental data shows that homobinuclear 13755


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Inorganic Chemistry Table 7. IC50, kcat, and Apparent Formal Potential of the Cu2+/Cu+ Couple (E°′) from Voltammetric Data for L1 and L2 Obtained by the McCord−Fridovich Test and Examples Reported from the Literature system CuL1 Cu2L1 Cu2L2 CuZnL2 free Cu2+ Cu2Zn2-SOD

E°′ (V vs SCE) −0.480 −0.150 −0.510 −0.500 −0.085 +0.076

± ± ± ± ±

0.005 0.005 0.005 0.005 0.005

IC50 (μM) 12.80 0.23 2.93 18.24 1.1 0.010

± ± ± ± ± ±

2.93 0.07 0.62 0.98 0.1 0.002

kcat (M−1 s−1) 2.72 1.52 1.19 1.91

± ± ± ±

0.53 0.42 0.22 0.22

× × × ×

105 107 106 105

43 × 107

[Cu2L1]4+ exhibits a remarkable catalytic activity toward O2− dismutation in comparison with [Cu2L2]4+. The high activity observed for [Cu 2 L1] 4+ can be associated with the coordinatively unsaturated metal centers and the flexibility of the modified pendant arm, which would permit molecular rearrangements along the catalytic pathway.33 Biological Activity. SOD enzymes deactivate in an initial step the deleterious reactive oxygen species (ROS) generated by the normal cellular metabolism of aerobic organisms.30 Although purified and recombinant SOD enzymes have demonstrated pharmacological efficacy in some animal models of ROS-related diseases,34−36 their therapeutic use has been limited by their high production cost, molecular size, and antigenic activity.37 Synthetic SOD mimetic compounds of low molecular weight could compensate for the limitations of purified enzymes because of such properties as the lack of antigenicity, higher stability in solution, longer half-life, and lower production cost.31,32 Before assessing the antioxidant capacity of L1 and L2 and their metal complexes, we checked their toxicity. While the L1 and L2 ligands were toxic, their metal complexes showed very low toxicity at 10 μM in a panel of commercial cell lines (Figure S8). To check the SOD mimetic activity of L1 and L2 metal complexes, we used a SOD1-deficient yeast, which has reduced rates of aerobic growth in a synthetic glucose medium unless an antioxidant is able to enter the cells and compensates for the SOD deficiency.38−42 All L1 and L2 metal complexes increased the growth rate of the SOD-deficient cells. The binuclear (Cu2/L2 and Cu2/L1) complexes were more active than the mononuclear ones (Figure 9). However, to assess the antioxidant activity in living cells, many parameters have to be considered, like the degree of compound accumulation inside the cells, etc. This should explain why the most active compound Cu2/L2 in cells is not the compound showing the highest catalytic activity in the NBT assay in vitro.

Figure 9. Protective effect of L1 and L2 metal complexes in yeast. The aerobic growth of SOD1-deficient yeast strain with 10 μM L1 and L2 and its metal complexes was monitored at 620 nm after 16, 18, and 20 h of incubation. The protective effect of the compounds was assessed with increasing growth ratio of SOD1-deficient yeast strain in the presence of metal complexes.

IC50 = 12.80 μM. This can be ascribed to the fact that the second metal binds to the diethyletriamine side of the molecule in a coordinatively unsaturated fashion, which leads to a decrease in the stability of the Cu2+ complexes. Consequently, the redox potential would be less negative, permitting a better cycling between oxidation states I and II of copper needed for effective superoxide dismutation and removal. Although the existence of binuclear species even for low Cu2+/L molar ratios prevents a similar analysis in the case of L2, the fact that the replacement of one of the Cu2+ ions by one Zn2+ ion yields a remarkable drop in the SOD activity suggests again that the SOD activity is mostly provided by the second, less tightly coordinated, metal ion. In spite of the fact that in the NBT assays the binuclear Cu2+ complexes of L2 show less SOD activity (IC50 = 2.93 μM) than those of L1, antioxidant assays performed with a S. cerevisae strain defective in the cytoplasmatic copper-dependent SOD enzyme show a reverse trend. However, as commented previously, to assess the antioxidant activity in a living cell, many other parameters have to be considered. In view of these results, synthetic modifications of the ligands are currently being done to try to match the maximum efficiencies in NBT assays and cells as well as to explore their activity in higher organisms.

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ASSOCIATED CONTENT

S Supporting Information *

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b01756. Complete characterization of L1 and L2, distribution diagrams, crystallographic data, selected distances and angles, and cyclic voltammograms of L1 and their copper complexes (PDF)

CONCLUSIONS Two new ditopic ligands have been prepared in reasonable yield. While both ligands are able to form binuclear Cu2+ and Zn2+ metal complexes, L2, consisting of two tetraazapyridinophane ligands linked by an (ethylamino)propyl bridge also form mixed Cu2+/Zn2+ complexes, as proven by potentiometric measurements, single-crystal X-ray diffraction, and EDX spectroscopy. The Cu2+ complexes show significant SOD mimetic capacity. In the case of L1, the ligand having a pyridinophane macrocycle linked to the central nitrogen atom of a diethylenetriamine unit by an (ethylamino)propyl bridge, the SOD activity is much more relevant for the binuclear complexes, IC50 = 0.23 μM, than for the mononuclear ones,

Accession Codes

CCDC 1507630−1507632 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033. 13756


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Inorganic Chemistry

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: (+34)963544879. *E-mail: [email protected]. Phone: (+34)963544401. ORCID

Antonio Doménech-Carbó: 0000-0002-5284-2811 Begoña Verdejo: 0000-0003-3022-1835 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support by the Spanish Ministerio de Economiá y Competitividad (Projects Consolider Ingenio CSD-201000065, CTQ2013-48917-C3-1-P, CTQ2016-78499-C6-1-R, FIS PI16/00504, and Unidad de Excelencia MDM 20150038) and Generalitat Valenciana (Project PROMETEOII2015-002) is gratefully acknowledged.

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