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A dicopper complex with distant metal centers. Structure, magnetic properties, electrochemistry and catecholase activity
Available online at www.sciencedirect.com JOURNAL OF
Inorganic Biochemistry Journal of Inorganic Biochemistry 102 (2008) 1227–1235 www.elsevier.com/locate/jinorgbio
A dicopper complex with distant metal centers. Structure, magnetic properties, electrochemistry and catecholase activity Laura Gasque a,*, Vı´ctor Manuel Ugalde-Saldı´var a, Ingrid Membrillo a, Juan Olguı´n a, Edgar Mijangos a, Sylvain Berne`s b, Ignacio Gonza´lez c a
Universidad Nacional Auto´noma de Me´xico, Facultad de Quı´mica, Departamento de Quı´mica Inorga´nica, Cd. Universitaria, 04510 Me´xico D.F., Mexico b DEP, Facultad de Ciencias Quı´micas, UANL, Guerrero y Progreso S/N, Col. Trevin˜o. Monterrey, N.L., CP 64570, Mexico c Universidad Auto´noma Metropolitana-Iztapalapa, Departamento de Quı´mica, A´rea de Electroquı´mica, Apartado Postal 55-534, 09340 Me´xico D.F., Mexico Received 31 August 2007; received in revised form 14 November 2007; accepted 14 December 2007 Available online 11 January 2008
Abstract The crystal structure and magnetic properties of a dinuclear copper(II) complex of the ligand [2,8-dimethyl-5,11-di-(dimethylethyleneamine) 1,4,5,6,7,10,11,12-octahydroimidazo [4,5-h] imidazo [4,5-c] [1,6]diazecine] dimeim have been investigated. Also, its catecholase activity has been explored in different solvent mixtures: MeCN/H2O and OH/H2O, each at several pH values. In CH3OH/H2O, where the activity was superior, the optimal pH value for the catalytic activity was found to be lower than in CH3CN/H2O. The study of the complex’s electrochemical behavior (cyclic voltammetry) which was also investigated in these various media, revealed that although an increase in pH in both solvent mixtures results in an increase both in Me oxidizing power (E1/2) and reversibility (ipa/ipc) the change of solvent system seems to be a more influencing factor. The superior catalytic activity found in MeOH/H2OpH=8.0, is associated with a significantly more reversible behavior displayed in this medium. Potentiometric determination of the overall formation constant and three successive pKas for the complex, suggest the formation of stable hydroxo complexes which could be the catalytically active species. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Copper; Dinuclear; Electrochemistry; Catecholase
1. Introduction Due to the presence of one or more copper ions bound to imidazole residues in the active site of many enzymes, a great number of dicopper complexes involving heteroaromatic nitrogen donors have been described together with their structural and magnetic properties, as well as their catalytic activity towards oxidation reactions [1–13]. In the first systematic study on the catalytic activity of model copper complexes towards the oxidation of 3,5-ditert-butylcatechol (DTBC) which contemplated both mononuclear complexes and dinuclear complexes, Nishida et al. [14] found that in some cases mononuclear complexes could be better catalysts that dinuclear ones, and stated *
Corresponding author. Tel./fax: +52 55 56 22 38 11. E-mail address: [email protected] (L. Gasque).
0162-0134/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2007.12.032
that electrochemical data did not correlate in a direct manner to catalytic activity; the main conclusion in their paper was that distorted tetrahedral complexes were far more active than square planar ones. Later, comparing the reactivity of several mononuclear complexes with tripodal ligands, Malachowski [5] concluded that both a convenient redox potential and a good steric match between substrate and catalyst are necessary for catalytic activity to be observed. Since the crystal structure of catechol oxidase was described by Krebs and co-workers in 1998 [15,16], mononuclear complexes have been relegated, while most relevant studies have focused mainly on obtaining dinuclear complexes with Cu–Cu distances in the range of that found in this enzyme, or in its structural analogues, tyrosinase ˚ . This Cu–Cu and hemocyanin, known to be around 2.9 A distances are generally achieved with bridging OH ligands
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which is also a structural feature of these enzymes in their met state. Model compounds with labile hydroxo bridges and different Cu–Cu distance values have been tested as catalysts for the oxidation of 3,5-di-tert-butylcatechol (DTBC) to the corresponding quinone, showing that activity is significantly enhanced once the bridge is formed as pH is ˚ increased, as well as when Cu–Cu distances approach 3 A [1,2,10,17–19]. However, Yang et al. [20] described the catecholase activity of a series of related phenoxo bridged dicopper complexes with Schiff base ligands derived from salicyladehyde and amino acids in which even though the ˚, Cu–Cu distance in all the complexes is very close to 3 A the catalytic activities vary almost 20-fold among the complexes with different amino acid moieties, suggesting the relevance of other structural factors besides Cu–Cu distance. In Sreenivasulu’s work [21], eight different com˚ and 3.06 A ˚ plexes with Cu–Cu distances between 2.99 A exhibited very different catalytic activities. It is important to point out that in complex presented ˚ , with virtually no steric here, Cu–Cu distance is ca. 7.4 A possibility to approach each other enough so that both Cu atoms can bind to a single catecholate unit, as required in the most widely accepted mechanism [8]. However, the observed catalytic activity is similar or even superior to several dinuclear complexes described in the literature with ˚ [8,10,11,20,22–27]. An Cu–Cu distances close to 3 A remarkable feature is that in spite of this rather large separation between the two copper atoms, its frozen solution EPR spectrum suggests they are magnetically coupled. Throughout the specialized literature, the effect of the solvent on the catecholase activity of copper complexes, has been generally overlooked, with no justification given for one choice or another. The most widely used solvents have been acetonitrile, methanol, and water, as well as mixtures of them. The effect of pH variations has indeed been taken in consideration in a limited number of papers, such as those of Belle [18] and Neves [10,22], who have associated the increase in catalytic activity at higher pH values with the existence of hydroxo complex species. Therefore we considered it worthwhile to investigate the differences in catalytic behavior when varying these two factors. The observed increase in catalytic activity at higher pH values can be associated with the existence of complex hydroxo species, [OH–Cu–dimeim–Cu–OH] detected by the determination of the equilibrium constants, while the different performance observed in the two different solvent mixtures employed, can be explained by a corresponding different electrochemical behavior in these media. 2. Experimental 2.1. Synthesis of the complex Caution: Perchlorate salts of metal complexes with organic ligands are potentially explosive.
The ligand dimeim was prepared according to reported procedure [28], the complex [Cu2(dimeim)(H2O)4](ClO4)4, (1) was obtained by gradually adding 1 mmol (0.496 g) of solid dimeim 6H2O to 2 mmol of Cu(ClO4)2 dissolved in 30 mL of water. Blue crystals suitable for X-ray diffraction were collected after a week. Anal. Calcd. for [Cu2(H2O)4 (C20H36N8)] (ClO4)4: % C, 24.37, % H, 4.50, % N, 11.37. Found: % C, 24.55, % H, 4.57 % N, 11.42. 2.2. X-ray crystallography X-ray diffraction data for 1 were measured on a single crystal using a Siemens P4 diffractometer (non-CCD detector [29]). A summary is given in Table 1, and details may be found in the archived CIF deposited with the CCDC, along with complete geometric parameters (deposition number CCDC 639267). The structure was solved and refined using SHLEXS97 and SHLEXL97 software [30]. One of the ClO 4 anions is disordered, a common feature for uncoordinated small anions with a spherical shape. The disorder was modeled considering that this anion rotates about its bond Cl1–O14. Atoms O11, O12 and O13 are distributed over two sites with fixed occupancy factors 0.57 (sites A) and 0.43 (sites B). The actual disorder is however probably more complex, as reflected in prolate displacement ellipsoids for O atoms in this anion. We were unable to refine these sites further, and restraints were thus applied to O atoms: atoms were restrained so that their Uij components approximate to isotropic behavior, and O atoms closer ˚ were restrained to have the same Uij compothan 1.7 A nents (default ISOR and SIMU commands in SHELXL97, respectively). C- and N-bonded H atoms were placed at calculated positions (riding model), while H atoms of water Table 1 Crystal data and structure refinement details for compound 1 1 Chemical formula Mr Cell setting, space group Temperature (K) ˚) Wavelength (A ˚) a, b, c (A a, b, c (°) ˚ 3) V (A Z, Z0 Dx (Mg m3) l (mm1) Crystal size (mm) Tmin, Tmax 2h range (°) Reflections measured Independent reflections Completeness Data/restrains/parameters Goodness-of-fit on F2 Final R1, wR2 [I > 2r(I)] Final R1, wR2 (all data) ˚ 3) Dqmax, Dqmin (e A
C20H44Cl4Cu2N8O20 985.51 Monoclinic, P21/n 295(1) 0.71073 9.4515(11), 16.4506(14), 12.2424(10) 90, 103.274(8), 90 1852.6(3) 2, 1/2 1.767 1.526 0.60 0.12 0.10 0.779, 0.858 4.22–55.00 5372 4261 (Rint ¼ 0:029) 100.0% to 2h = 55.00° 4261/84/288 1.015 0.053, 0.105 0.103, 0.123 0.457, 0.366
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molecules O1 and O2 were found in a difference map and refined with a regularized geometry [restraints: O–H = ˚ ]. 0.85(1) and H H = 1.33(2) A
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were computed from the measured equilibrium constants and plotted with MEDUSA [33]. 3.2. Catecholase activity
2.3. Magnetic measurements Room temperature molar magnetic susceptibility was determined with a Johnson–Metthey Mark I magnetic susceptibility balance. In the 4–300 K range, magnetization (M) of the crystalline solids was measured with a SQUID Quantum Design MPMS-5 at 5000 G. Molar magnetic susceptibilities were corrected for the diamagnetic molar contribution of the solid product. (Sample mass: 41 mg, molar mass: 985.51, diamagnetic contribution: 4.426E4). EPR spectra of MeOH/H2O solutions at 77 K were recorded in the X band (9.85 GHz) using a Bruker ER200-SRC spectrometer with a cylindrical cavity. Spectra for two different samples were recorded, the first prepared from the isolated solid [Cu2(dimeim)(H2O)4](ClO4)4, and the second one from equivalent amounts of Cu(ClO4)2 and dimeim. 3. Solution studies Dimeim 6H2O was prepared according to the reported method [28], and purity verified by elemental analysis. The reagents used for the solution studies were of analytical reagent grade and the solutions prepared with distilled and deionized water. An approximately 0.1 M stock solution of Cu(II) prepared from analytical grade Cu(NO3)2 was standardized by atomic absorption. CH3CN and CH3OH for the kinetic and electrochemical studies were spectrophotometric grade. Buffers for the kinetic and electrochemical studies were MES (2-(N-morpholino)ethanesulfonic acid, pH 5.5–6.5), HEPES (4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid, pH 7.0–8.0) and TRIS (tris(hydroxymethyl-)amino-methane, pH 8.5–9.5). 3.1. Equilibrium studies A Methrom 702 Titrino set including digital pH meter coupled to a PC, fitted with a Methrom Ag/AgCl combined electrode, was used for potentiometric titrations. Each problem solution was contained in a 75 mL jacketed glass cell thermostated at 25.00(±0.05) °C by a circulating constant-temperature water bath. Ionic strength was imposed to 0.1 M with KNO3. Titrations of the ligand in the absence or presence of metal ions in aqueous solution were conducted in a standard way. Cell solutions (50 mL) were purged with a purified nitrogen stream. Experimental runs were carried out by adding increments of standardized ca. 0.1 M carbonate free NaOH to a solution containing dimeim plus other components (KNO3, copper nitrate and an excess of standardized HNO3). Protonation constants and stability constants from the direct titrations were calculated from the potentiometric titration data with the Hyperquad program [31,32]. Species distribution diagrams
The catecholase activity of [Cu2(dimeim)]4+ was monitored by UV/Vis spectroscopy by the increase of the absorption band at 400 nm from the formation of 3,5-ditert-butyl-o-benzoquinone. The catalytic activity of the complex was determined by the method of initial rates, measuring the absorption as a function of time over the first 40 s. The mean value of three experiments was determined; the maximum deviation from the mean value did not exceed 10%. For pH dependence studies, catalyst aqueous solutions were prepared with 0.3 M MES (pH 5.5, 6.0 and 6.5), HEPES (pH 7.0, 7.5, 8.0 and 8.5) and TRIS (pH 9.0) and standardized NaOH or HNO3 and were mixed with equal volumes of DTBC solution in MeOH or MeCN. Since the complex displays different solubility in the two solvent mixtures studied, for pH dependence studies, in MeCN/H2O, cell concentrations were: 0.150 mM for the copper catalyst and 10.02 mM for DTBC. In MeOH/ H2O, cell concentrations were: 0.03 mM for the copper catalyst and 2.0 mM for DTBC. Kinetic parameters were determined in MeCN/ H2OpH=7.5, MeOH/H2OpH=7.5, MeCN/H2OpH=8.5 and MeOH/H2OpH=8.0 For catalyst concentration dependence studies, [DTBC] was kept constant at 10.02 mM in MeCN/H2O and at 2.03 mM in MeOH/H2O, while in the first case catalyst concentration varied from 50 to 301 lM and in the second, from 10 to 60.1 lM. For substrate dependence studies, in MeCN/H2O catalyst concentration was held constant at 150.3 lM while catechol concentration was varied from 3.51 to 15.03 mM; in MeOH/H2O catalyst concentration was held constant at 30 lM while catechol concentration was varied from 0.33 mM to 3.33 mM. The reaction rate was found to be linearly dependent on the complex concentration, while for substrate dependence saturation kinetics were observed. A Michaelis–Menten analysis of the data was applied and Vmax, Km and kcat determined from a Lineweaver–Burk plot. 3.3. Electrochemical studies Cyclic voltammetric studies were performed on the [Cu2(dimeim)]4+ complex with an AUTOLAB PGSTAT 100 potentiostat/galvanostat in MeCN/H2OpH at pH values of 7.5, 8.0 and 8.5, and in MeOH/H2OpH at pH values of 7.5 and 8.0. The experiments were carried out in a conventional three-electrode cell composed of: a reference electrode Ag/AgCl(s)/0.1 M KCl in the corresponding solvent mixture; a platinum wire as auxiliary electrode and a glassy carbon disk (surface area 7.1 mm2) as working electrode. In order to refer all potential measurements to the Ferricenium/Ferrocene (Fc+/Fc) system according to IUPAC convention [34], voltammograms were obtained for approximately 103 M ferrocene (Fc) in background
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electrolyte. Cyclic voltammograms (CVs) were performed at a potential range of 1.4 V to 0.5 V vs Fc+–Fc at a sweep rate of 100 mV/s and were initiated from open circuit potential (Ei=0). The 103 M complex solutions were prepared in situ by mixing the required amounts of the ligand and Cu(NO3)2 in the 50% solvent: H2OpH. The buffer also played the role of supporting electrolyte. The presence of oxygen was avoided by bubbling nitrogen through all the experiments both in the solution and in the cell.
4. Results and discussion 4.1. Crystal structure (1,6)Diazecine-based ligands, like dimeim, are known to favor the formation of dinuclear rather mononuclear complexes with Cu(II), since peripheral N atoms are separated by a large distance, impeding coordination to a single metal center. Such molecules must contain suitable N or O donor sites in order to serve as ligands, for example imidazole heterocycles, amines or carboxylic acids as substituents on the diazecine N atoms, etc. The ligand used in the present work, (Scheme 1) namely 2,8-dimethyl-5, 1-bis(dimethylaminoethyl)-1,4,5,6,7,10,11,12-octahydroimidazo[4,5-h]imidazo[4,5-c] [1,6]diazecine (dimeim) has proven to efficiently coordinate Cu(II), using water and acetato [28] water and sulfato [35], methanol and nitrato, or halides and water [36] as ancillary ligands. A number of counterions were used, which do not seems to have any influence on the geometry of the cation. The cation in complex 1, [Cu2(dimeim)(H2O)4]4+, is placed on an inversion center (Z0 = 1/ 2), and the five-coordinated Cu(II) ion displays a squarepyramidal environment, the axial position being occupied by a water molecule (Fig. 1). The metal center is placed ˚ above the N3/N7/N10/O2 least-squares plane. 0.236 A The 10-membered central (1,6)diazecine ring adopts a chair ˚. conformation, with a total puckering amplitude of 1.44 A The conformation observed for dimeim in 1 is very similar to that observed in other reported Cu2–dimeim complexes, confirming the rigid character of this ligand. For instance, imidazole rings are invariably parallel (by symmetry), and
N
N
NH
N N
HN
N
N
Scheme 1. Structure of the ligand dimeim.
Fig. 1. Structure of cation [Cu2(dimeim)(H2O)4]4+ in 1, with displacement ellipsoids at the 30% probability level for non-H atoms. Labeling scheme is given for the asymmetric unit, while non-labeled atoms are generated by symmetry 2 x, y, 2 z. Selected coordination bond lengths and angles: Cu1–O1 2.251(3), Cu1–O2 2.001(3), Cu1–N3 2.009(3), Cu1–N7 ˚ ; O2–Cu1–N7 168.28(16), N3–Cu1–N10 2.069(3), Cu1–N10 2.034(4) A 162.06(14), N3–Cu1–N7 83.31(12), N7–Cu1–N10 85.74(14)°.
least-squares planes passing through these rings are sepa˚ in 1, which compare well with other comrated by 2.570 A ˚ plexes: 2.680 A in the aqua/acetato-complex [28,35] ˚ in the aqua/sulfato-complex [35] or 2.688 A ˚ in 2.681 A the aqua/chloro-complex [36]. This dimension roughly measures the thickness of dimeim, which results to be more or less constant regardless of ancillary ligands or anions used. As a consequence, metal–metal intra-anion separa˚ for 1, while tion remains also unchanged, 7.5153(11) A the largest observed separation in this series is ˚ , for the molecular complex [Cu2(dimeim)7.5709(18) A (SO4)2(H2O)2] [35]. 4.2. Magnetic susceptibility and electronic paramagnetic resonance Using a Gouy balance, the room temperature magnetic moment of [Cu2(dimeim)(H2O)4](ClO4)4 was found to be 1.26 BM per copper atom, considerably lower than expected for non-coupled Cu(II). The magnetic susceptibility of the complex was then measured in the temperature range 4–300 K. The temperature dependence of vm and vmT of the Cu2–dimeim unit is shown in Fig. 2. Upon cooling, vm increases, reaching maximum at 12 K and then rapidly decreases with temperature, clearly indicating the presence of an antiferromagnetic interaction. From a plot of 1/v vs T in the interval of 100–300 K, a straight line is obtained and a Curie constant of C = 0.847 emu mol1 K and a Weiss constant h = 7.6 K. The isotropic value calculated from the EPR spectrum of the solid sample at 77 K is g = 2.086. To estimate the magnitude of the antiferromagnetic coupling, the magnetic susceptibility data (4–300 K) were fitted to the modified Bleaney–Bowers equation [37] for two interacting copper(II) ions (S = 1/2) with the Hamiltonian in the form H = 2J S1 S2. The susceptibility equation for such a dimeric system can be written as follows:
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Fig. 2. Magnetic susceptibility data vs temperature for compound (1) and least-squares fitting curve adjusted to the Bleaney–Bowers equation.
v¼
Ng2 l2b Ng2 l2b 2 q þ TIP ð1 qÞ ð1 qÞ þ 3 þ e2J =kT kT 2kT
The g value was fixed to the value obtained from EPR. The magnetic data were fitted in the temperature range 4–300 K leading to the following parameters: J = 7.11 cm1, q = 0.003 and TIP = 0.001 emu mol1. A similar compound was described by Mendoza-Diaz et al. [38], for which a value of J = 6.87 cm1 was obtained when the data were fit with this same model. The EPR spectrum of a powdered solid simple of [Cu2(dimeim)(H2O)4](ClO4)4 at 77 K, is consistent with an isotropic Cu(II) center, with g = 2.086 (Supplementary material). However, when the spectrum is measured from a frozen MeOH/H2O solution, (Fig. 3) a well-resolved
hyperfine structure in the gk region is observed, with seven discernible lines with an average splitting of 78 G (gk = 2.294 and g\ = 2.073). Identical spectra were obtained whether the solution was prepared dissolving a sample of the isolated solid [Cu2dimeim(H2O)4](ClO4)4, or from equivalent amounts of Cu(ClO4)2 and dimeim. Similar spectra have been previously assigned to a weak exchange interaction between copper(II) centers in dinuclear com0 ˚ [8,38–40], in plexes with Cu–Cu separations of 7.3–8 A which as in the present case, no DMs = ±2 has been observed at half field. This spectrum was successfully simulated using the WINEPR from Brucker Co. The found parameters are consistent with the coupling of two Cu(II) (S = 3/2) nuclei, the value of A = 78 G (gk = 2.290 and g\ = 2.060), coincides with the obtained experimentally (Supplementary material). 5. Equilibrium studies In order to gain some insight into the stability of the dinuclear complex in solution, as well as on its hydrolyzing properties, a series of potentiometric titrations were carried out, and the corresponding data adjusted with Hyperquad to yield the desired equilibrium constants. For the ligand, four successive protonations were detected with the titration of dimeim, with corresponding pKa values of 10.06, 9.17, 7.04 and 5.88. The refinement of complexation constants indicates that [Cu2(dimeim)(H2O)4]4+ is completely formed and is the only copper containing species in the pH range between 6 and 8; from that point on, the formation of the several hydrolyzed species, becomes appreciable. The corresponding hydrolysis equilibriums are [Cu2 L(H2 O)6 ]4þ ¡ [Cu2 L(H2 O)5 (OH)]3þ + Hþ
Fig. 3. EPR spectrum of [Cu2dimeim(H2O)4](ClO4)4 in MeOH/H2O frozen solution at 77 K.
[Cu2 L(H2 O)5 (OH)]3þ ¡ [Cu2 L(H2 O)4 (OH)2 ]2þ + Hþ
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[Cu2 L(H2 O)4 (OH)2 ]2þ ¡ [Cu2 L(H2 O)3 (OH)3 ]þ + Hþ Table 2 shows the value of the overall formation constant as well as the copper acidity constants corresponding to three successive hydrolyses and a species predominance diagram as a function of pH is shown in Fig. 4. 5.1. Catalytic activity Variation in catecholase activity of the complex as a function of pH was investigated in two solvent mixtures, MeCN/H2O and MeOH/H2O and compared to the sole pH effect on the reaction rate without the presence of a catalyst. The results of these experiments are shown in Fig. 5a and b, respectively. In both solvent mixtures kinetic parameters were determined at pH 7.5, to be able to compare the activity with other catalysts developed by the group [41]. A second pH value was chosen for each solvent system as that which gave the largest increment in activity compared to the activity of the medium without the catalyst. The increase in catalytic activity at higher pH values may be associated with the predominance of the hydrolyzed forms of the catalyst, predicted from the species predominance diagram. (Fig. 4). This optimal pH value was 8.5 for MeCN/H2O and 8.0 for MeOH/H2O, suggesting a complete formation of the catalytically active species under these conditions. Table 2 Overall complexation constants and pKa values for Cu2–dimeim system in aqueous solution at 298 K ½Cu2þ H L
½Cu2 LðH2 OÞ6i ðOHÞi ½Hþ ½Cu2 LðH2 OÞ6iþ1 ðOHÞi1
z bxyz ¼ ½Cu2þxx ½Hy y ½L z
K ðiÞ a ¼
Log b2 0 1 = 20.78 Log b2 1 1 ¼ 12:43 Log b2 2 1 ¼ 3:43 Log b2 3 1 ¼ 6:61
– pK ð1Þ a = 8.35 pK ð2Þ a = 9.00 pK ð3Þ a = 10.04
Since dielectric constant values for methanol and acetonitrile are very similar (33 and 37 respectively, compared to 80 for water [42]), a similar decrease in the pKa value for the complex would be expected in both of the studied solvent mixtures. However, the lower optimal pH value found for MeOH/H2O suggests a qualitatively different participation of the two solvent mixtures during the catalytic process. Complete kinetic studies were carried out on the basis of the Michaelis–Menten model, with the evaluation of the kinetic parameters (Vmax, KM, kcat) at both pH values from the corresponding Lineweaver–Burk plots. As can be seen from data in Table 3, in both solvent mixtures an increase in pH causes a slight improvement in catalyst performance, while a much more noticeable enhancement is observed when solvent is changed from MeCN/H2O to MeOH/ H2O; at pH 7.5 catalytic efficiency is 20 times better in the latter than in the former. The kcat/KM value obtained for this complex in MeOH/H2OpH=8.0 is better than most of the dinuclear copper complexes whose catecholase activity has been reported recently [20,21,25,27,43], suggesting the action of a mechanism that depends on other factors different from the Cu–Cu distance.
5.2. Electrochemical studies The significant difference found for the behavior of the catalyst in the two studied solvent mixtures, motivated the performance of a systematic electrochemical study of the dicopper complex in both MeOH/H2O and MeCN/ H2O, at different pH values. Only redox signals associated to the Cu(II)/Cu(I) couple will be discussed, due to their relevance to the catalytic process. Initial studies dealt with [Cu2(dimeim)]4+ in MeCN/ H2OpH=7.5 (Fig. 6). Peak Ic, with Ecp = 0.524 V, corresponds to the Cu(II) ? Cu(I) reduction process, but the
Fig. 4. Species predominance diagram for Cu2–dimeim system as a function of pH at 298 K.
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Fig. 6. Typical cyclic voltammograms in MeCN/H2OpH mixtures at different pH values for Cu2(dimeim)4+ (a) pH 7.5 and (b) pH 8.5.
Fig. 5. The pH effect on the catecholase activity of Cu2–dimeim in (a) MeCN/H2O and (b) MeOH/H2O (concentrations in MeOH/H2O are five times lower).
Table 3 Kinetic parameters (Vmax, Km, kcat) in MeCN:H2OpH at pH 7.5 and 8.5 and in MeOH:H2OpH at pH 7.5 and 8.0, from the corresponding Lineweaver–Burk plots, for the catecholase activity of Cu–dimeim system Medium
Vmax 106 (M s1)
Km 103 (M)
kcat 102 (s1)
kcat/Km (M1 s1)
MeCN:H2OpH=7.5 MeCN:H2OpH=8.5 MeOH:H2OpH=7.5 MeOH:H2OpH=8.0
4.63 9.77 3.05 3.25
11.90 4.19 1.96 1.59
3.09 6.51 10.18 10.83
2.60 15.53 51.99 67.88
Due to different solubilities, concentrations in MeOH/H2O was five times lower than in MeCN/H2O.
low intensity and the position of the corresponding oxidation signal, Ia for which Epa = 0.389 V, is indicative of a practically irreversible system. This irreversibility is confirmed by the value of the quotient, ipa/ipc = 0.13. The breadth of signal Ic, can be indicative of overlapped reduction signals, which suggests the presence of at least two differentiable Cu(II) sites, which are reduced consecutively at very similar potential values. This electrochemical behavior has been associated [8,44] with electronic commu-
nication between two metal centers, which in this case is suggested by the EPR spectrum (Fig. 3). Peak width is measured by DEp/2 = (Epc Epc/2), where Epc is the cathodic peak potential and Epc/2 is the corresponding half-peak potential. For this signal, the obtained value of DEp/2 = 0.135 V can be interpreted as two overlapping one-electron signals. To rule out uncompensated resistance effects, the DEp/2 value was determined for the Fc/Fc+ system and found to be 54 mV (Supplementary material). The presence of two oxidation signals, Ia and IIa is indicative of the partial decoordination of the ligand upon reduction of the metal ion: Ia is associated with the proI cess ½CuI2 dimeim ! ½CuII 2 dimeim and IIa with 2½Cusolv ! II ½Cu2 dimeim. Voltammograms obtained in MeCN/H2O at higher pH values are essentially similar, but indicative of slightly stronger oxidizing power and a more reversible behavior. Relevant electrochemical parameters for all the studied systems are shown in Table 4. The voltammograms obtained MeOH/H2O (see Fig. 7) show that the two main parameters associated with catalytic efficiency, E1/2 and ipa/ipc, are more favorable than in MeCN/H2O. While E1/2 values at the two studied pH values are almost equal, a significant increase in reversibility is observed at the higher (Table 4). This increase in reversibility is a direct consequence of a larger intensity in peak Ia, indicative of a better stability of the reduced form of the complex ½CuI2 dimeim in MeOH/ H2O than in MeCN/H2O. This stability is further improved at higher pH values, as can be inferred from the current intensity increase in Fig. 7b compared to that in 7a.
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Table 4 Electrochemical parameters for Cu2–dimeim and Cu(NO3)2 compounds, in different solution media Medium
E1/2 signal (Ia–Ic) CuII M CuI
Epc signal (Ic) CuII ? CuI
DEp/2 signal (Ic) CuII ? CuI
Epa signal (Ic) CuI ? CuII
ipc/ipa signal (Ia)
MeCN/H2OpH=7.5 MeCN/H2OpH=8.0 MeCN/H2OpH=8.5 MeOH/H2OpH=7.5 MeOH/H2OpH=8.0
0.429 0.426 0.398 0.347 0.346
0.524 0.520 0.506 0.493 0.467
0.135 0.113 0.113 0.144 0.146
0.334 0.332 0.290 0.201 0.225
0.13 0.20 0.25 0.49 0.56
All potential values are in volts and were referred to Fc+–Fc system.
dized species, which explains the increase in reversibility, which correlates with a better activity. Acknowledgements L.G. thanks Prof. Roberto Escudero for variable temperature magnetization measurements, and CONACyT (34847-E) and DGAPA-UNAM (IN106003) for economic support, and Prof. Bob Johnson for his thorough revision of the manuscript. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jinorgbio. 2007.12.032. References
Fig. 7. Typical cyclic voltammograms in MeOH/H2OpH mixtures at different pH values for Cu2(dimeim)4+ (a) pH 7.5 and (b) pH 8.0.
5.3. Concluding remarks A significant catecholase activity was found for this dicopper complex in spite of the steric impossibility of both copper atoms to act simultaneously on one molecule of substrate. Considerable differences were found for the catalytic activity shown in the different solvent media tested. The electrochemical studies performed on all the corresponding media gave some insight into the different catalytic behavior. Although an increase in pH in both solvent mixtures results in an increase both in oxidizing power (E1/2) and reversibility (ipa/ipc) the change of solvent system seems to be a more influencing factor. The very poor reversibility obtained in MeCN/H2O may be caused by the great affinity of Cu(I) for CH3CN, which encourages the decoordination of the ligand upon the reduction of the complex. Since Cu(I) exhibits no comparable affinity for MeOH and/or H2O, in this case, the coordination to three nitrogen donor atoms seems to be a preferred situation leading to similar coordination environments for the reduced and the oxi-
[1] J. Ackermann, F. Meyer, E. Kaifer, H. Pritzkow, Chem. Eur. J. 8 (2002) 247–258. [2] P. Gentschev, N. Moller, B. Krebs, Inorg. Chim. Acta 300 (2000) 442–452. [3] M. Gupta, P. Mathur, R.J. Butcher, Inorg. Chem. 40 (2001) 878–885. [4] C.H. Lee, S.T. Wong, T.S. Lin, C.Y. Mou, J. Phys. Chem. B 109 (2005) 775–784. [5] M.R. Malachowski, B.T. Dorsey, M.J. Parker, M.E. Adams, R.S. Kelly, Polyhedron 17 (1998) 1289–1294. [6] M.R. Malachowski, H.B. Huynh, L.J. Tomlinson, R.S. Kelly, J.W.F. Jun, Dalton Trans. (1995) 31–36. [7] E. Monzani, G. Battaini, A. Perotti, L. Casella, M. Gullotti, L. Santagostini, G. Nardin, L. Randaccio, S. Geremia, P. Zanello, G. Opromolla, Inorg. Chem. 38 (1999) 5359–5369. [8] E. Monzani, L. Quinti, A. Perotti, L. Casella, M. Gullotti, L. Randaccio, S. Geremia, G. Nardin, P. Faleschini, G. Tabbi, Inorg. Chem. 37 (1998) 553–562. [9] A. Neves, L.M. Rossi, A.J. Bortoluzzi, A.S. Mangrich, W. Haase, R. Werner, J. Braz. Chem. Soc. 12 (2001) 747–754. [10] A. Neves, L.M. Rossi, A.J. Bortoluzzi, B. Szpoganicz, C. Wiezbicki, E. Schwingel, Inorg. Chem. 41 (2002) 1788–1794. [11] J. Reim, B. Krebs, Dalton Trans. (1997) 3793–3804. [12] K. Selmeczi, M. Reglier, M. Giorgi, G. Speier, Coord. Chem. Rev. 245 (2003) 191–201. [13] F. Zippel, F. Ahlers, R. Werner, W. Haase, H.F. Nolting, B. Krebs, Inorg. Chem. 35 (1996) 3409–3419. [14] N. Oishi, Y. Nishida, K. Ida, S. Kida, Bull. Chem. Soc. Jpn. 53 (1980) 2847–2850. [15] T. Klabunde, C. Eicken, J.C. Sacchettini, B. Krebs, Nat. Struct. Biol. 5 (1998) 1084–1090. [16] P. Gentschev, M. Lu¨ken, N. Mo¨ller, A. Rompell, B. Krebs, Inorg. Chem. Commun. 4 (2001) 753–756.
L. Gasque et al. / Journal of Inorganic Biochemistry 102 (2008) 1227–1235 [17] H. Borzel, P. Comba, H. Pritzkow, Chem. Commun. (2001) 97–98. [18] C. Belle, C. Beguin, I. Gautier-Luneau, S. Hamman, C. Philouze, J.L. Pierre, F. Thomas, S. Torelli, Inorg. Chem. 41 (2002) 479–491. [19] S. Torelli, C. Belle, S. Hamman, J.L. Pierre, E. Saint-Aman, Inorg. Chem. 41 (2002) 3983–3989. [20] C.T. Yang, M. Vetrichelvan, X.D. Yang, B. Moubaraki, K.S. Murray, J.J. Vittal, Dalton Trans. (2004) 113–121. [21] B. Sreenivasulu, F. Zhao, S. Gao, J.J. Vittal, Eur. J. Inorg. Chem. (2006) 2656–2670. [22] C. Fernandes, A. Neves, A.J. Bortoluzzi, A.S. Mangrich, E. Rentschler, B. Szpoganicz, E. Schwingel, Inorg. Chim. Acta 320 (2001) 12– 21. [23] J. Mukherjee, R. Mukherjee, Inorg. Chim. Acta 337 (2002) 429–438. [24] I.A. Koval, C. Belle, K. Selmeczi, C. Philouze, E. Saint-Aman, A.M. Schuitema, P. Gamez, J.L. Pierre, J. Reedijk, J. Biol. Inorg. Chem. 10 (2005) 739–750. [25] J. Anekwe, A. Hammerschmidt, A. Rompel, B. Krebs, Z. Anorg. Allg. Chem. 632 (2006) 1057–1066. [26] C.H. Weng, S.C. Cheng, H.M. Wei, H.H. Wei, C.J. Lee, Inorg. Chim. Acta 359 (2006) 2029–2040. [27] X.B. Wang, J. Ding, J.J. Vittal, Inorg. Chim. Acta 359 (2006) 3481– 3490. [28] L. Gasque, J. Olguin, S. Bernes, Acta Crystallogr. Sect. E 61 (2005) M274–M276. [29] XSCAnS (release 2.21) Users Manual, Siemens Analytical X-ray Instruments Inc., Madison, WI, USA, 1996. [30] G.M. Sheldrick, SHELX97 Users Manual, University of Go¨ttingen, Go¨ttingen, Germany, 1997.
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Inorganic Biochemistry Journal of Inorganic Biochemistry 102 (2008) 1227–1235 www.elsevier.com/locate/jinorgbio
A dicopper complex with distant metal centers. Structure, magnetic properties, electrochemistry and catecholase activity Laura Gasque a,*, Vı´ctor Manuel Ugalde-Saldı´var a, Ingrid Membrillo a, Juan Olguı´n a, Edgar Mijangos a, Sylvain Berne`s b, Ignacio Gonza´lez c a
Universidad Nacional Auto´noma de Me´xico, Facultad de Quı´mica, Departamento de Quı´mica Inorga´nica, Cd. Universitaria, 04510 Me´xico D.F., Mexico b DEP, Facultad de Ciencias Quı´micas, UANL, Guerrero y Progreso S/N, Col. Trevin˜o. Monterrey, N.L., CP 64570, Mexico c Universidad Auto´noma Metropolitana-Iztapalapa, Departamento de Quı´mica, A´rea de Electroquı´mica, Apartado Postal 55-534, 09340 Me´xico D.F., Mexico Received 31 August 2007; received in revised form 14 November 2007; accepted 14 December 2007 Available online 11 January 2008
Abstract The crystal structure and magnetic properties of a dinuclear copper(II) complex of the ligand [2,8-dimethyl-5,11-di-(dimethylethyleneamine) 1,4,5,6,7,10,11,12-octahydroimidazo [4,5-h] imidazo [4,5-c] [1,6]diazecine] dimeim have been investigated. Also, its catecholase activity has been explored in different solvent mixtures: MeCN/H2O and OH/H2O, each at several pH values. In CH3OH/H2O, where the activity was superior, the optimal pH value for the catalytic activity was found to be lower than in CH3CN/H2O. The study of the complex’s electrochemical behavior (cyclic voltammetry) which was also investigated in these various media, revealed that although an increase in pH in both solvent mixtures results in an increase both in Me oxidizing power (E1/2) and reversibility (ipa/ipc) the change of solvent system seems to be a more influencing factor. The superior catalytic activity found in MeOH/H2OpH=8.0, is associated with a significantly more reversible behavior displayed in this medium. Potentiometric determination of the overall formation constant and three successive pKas for the complex, suggest the formation of stable hydroxo complexes which could be the catalytically active species. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Copper; Dinuclear; Electrochemistry; Catecholase
1. Introduction Due to the presence of one or more copper ions bound to imidazole residues in the active site of many enzymes, a great number of dicopper complexes involving heteroaromatic nitrogen donors have been described together with their structural and magnetic properties, as well as their catalytic activity towards oxidation reactions [1–13]. In the first systematic study on the catalytic activity of model copper complexes towards the oxidation of 3,5-ditert-butylcatechol (DTBC) which contemplated both mononuclear complexes and dinuclear complexes, Nishida et al. [14] found that in some cases mononuclear complexes could be better catalysts that dinuclear ones, and stated *
Corresponding author. Tel./fax: +52 55 56 22 38 11. E-mail address: [email protected] (L. Gasque).
0162-0134/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2007.12.032
that electrochemical data did not correlate in a direct manner to catalytic activity; the main conclusion in their paper was that distorted tetrahedral complexes were far more active than square planar ones. Later, comparing the reactivity of several mononuclear complexes with tripodal ligands, Malachowski [5] concluded that both a convenient redox potential and a good steric match between substrate and catalyst are necessary for catalytic activity to be observed. Since the crystal structure of catechol oxidase was described by Krebs and co-workers in 1998 [15,16], mononuclear complexes have been relegated, while most relevant studies have focused mainly on obtaining dinuclear complexes with Cu–Cu distances in the range of that found in this enzyme, or in its structural analogues, tyrosinase ˚ . This Cu–Cu and hemocyanin, known to be around 2.9 A distances are generally achieved with bridging OH ligands
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which is also a structural feature of these enzymes in their met state. Model compounds with labile hydroxo bridges and different Cu–Cu distance values have been tested as catalysts for the oxidation of 3,5-di-tert-butylcatechol (DTBC) to the corresponding quinone, showing that activity is significantly enhanced once the bridge is formed as pH is ˚ increased, as well as when Cu–Cu distances approach 3 A [1,2,10,17–19]. However, Yang et al. [20] described the catecholase activity of a series of related phenoxo bridged dicopper complexes with Schiff base ligands derived from salicyladehyde and amino acids in which even though the ˚, Cu–Cu distance in all the complexes is very close to 3 A the catalytic activities vary almost 20-fold among the complexes with different amino acid moieties, suggesting the relevance of other structural factors besides Cu–Cu distance. In Sreenivasulu’s work [21], eight different com˚ and 3.06 A ˚ plexes with Cu–Cu distances between 2.99 A exhibited very different catalytic activities. It is important to point out that in complex presented ˚ , with virtually no steric here, Cu–Cu distance is ca. 7.4 A possibility to approach each other enough so that both Cu atoms can bind to a single catecholate unit, as required in the most widely accepted mechanism [8]. However, the observed catalytic activity is similar or even superior to several dinuclear complexes described in the literature with ˚ [8,10,11,20,22–27]. An Cu–Cu distances close to 3 A remarkable feature is that in spite of this rather large separation between the two copper atoms, its frozen solution EPR spectrum suggests they are magnetically coupled. Throughout the specialized literature, the effect of the solvent on the catecholase activity of copper complexes, has been generally overlooked, with no justification given for one choice or another. The most widely used solvents have been acetonitrile, methanol, and water, as well as mixtures of them. The effect of pH variations has indeed been taken in consideration in a limited number of papers, such as those of Belle [18] and Neves [10,22], who have associated the increase in catalytic activity at higher pH values with the existence of hydroxo complex species. Therefore we considered it worthwhile to investigate the differences in catalytic behavior when varying these two factors. The observed increase in catalytic activity at higher pH values can be associated with the existence of complex hydroxo species, [OH–Cu–dimeim–Cu–OH] detected by the determination of the equilibrium constants, while the different performance observed in the two different solvent mixtures employed, can be explained by a corresponding different electrochemical behavior in these media. 2. Experimental 2.1. Synthesis of the complex Caution: Perchlorate salts of metal complexes with organic ligands are potentially explosive.
The ligand dimeim was prepared according to reported procedure [28], the complex [Cu2(dimeim)(H2O)4](ClO4)4, (1) was obtained by gradually adding 1 mmol (0.496 g) of solid dimeim 6H2O to 2 mmol of Cu(ClO4)2 dissolved in 30 mL of water. Blue crystals suitable for X-ray diffraction were collected after a week. Anal. Calcd. for [Cu2(H2O)4 (C20H36N8)] (ClO4)4: % C, 24.37, % H, 4.50, % N, 11.37. Found: % C, 24.55, % H, 4.57 % N, 11.42. 2.2. X-ray crystallography X-ray diffraction data for 1 were measured on a single crystal using a Siemens P4 diffractometer (non-CCD detector [29]). A summary is given in Table 1, and details may be found in the archived CIF deposited with the CCDC, along with complete geometric parameters (deposition number CCDC 639267). The structure was solved and refined using SHLEXS97 and SHLEXL97 software [30]. One of the ClO 4 anions is disordered, a common feature for uncoordinated small anions with a spherical shape. The disorder was modeled considering that this anion rotates about its bond Cl1–O14. Atoms O11, O12 and O13 are distributed over two sites with fixed occupancy factors 0.57 (sites A) and 0.43 (sites B). The actual disorder is however probably more complex, as reflected in prolate displacement ellipsoids for O atoms in this anion. We were unable to refine these sites further, and restraints were thus applied to O atoms: atoms were restrained so that their Uij components approximate to isotropic behavior, and O atoms closer ˚ were restrained to have the same Uij compothan 1.7 A nents (default ISOR and SIMU commands in SHELXL97, respectively). C- and N-bonded H atoms were placed at calculated positions (riding model), while H atoms of water Table 1 Crystal data and structure refinement details for compound 1 1 Chemical formula Mr Cell setting, space group Temperature (K) ˚) Wavelength (A ˚) a, b, c (A a, b, c (°) ˚ 3) V (A Z, Z0 Dx (Mg m3) l (mm1) Crystal size (mm) Tmin, Tmax 2h range (°) Reflections measured Independent reflections Completeness Data/restrains/parameters Goodness-of-fit on F2 Final R1, wR2 [I > 2r(I)] Final R1, wR2 (all data) ˚ 3) Dqmax, Dqmin (e A
C20H44Cl4Cu2N8O20 985.51 Monoclinic, P21/n 295(1) 0.71073 9.4515(11), 16.4506(14), 12.2424(10) 90, 103.274(8), 90 1852.6(3) 2, 1/2 1.767 1.526 0.60 0.12 0.10 0.779, 0.858 4.22–55.00 5372 4261 (Rint ¼ 0:029) 100.0% to 2h = 55.00° 4261/84/288 1.015 0.053, 0.105 0.103, 0.123 0.457, 0.366
L. Gasque et al. / Journal of Inorganic Biochemistry 102 (2008) 1227–1235
molecules O1 and O2 were found in a difference map and refined with a regularized geometry [restraints: O–H = ˚ ]. 0.85(1) and H H = 1.33(2) A
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were computed from the measured equilibrium constants and plotted with MEDUSA [33]. 3.2. Catecholase activity
2.3. Magnetic measurements Room temperature molar magnetic susceptibility was determined with a Johnson–Metthey Mark I magnetic susceptibility balance. In the 4–300 K range, magnetization (M) of the crystalline solids was measured with a SQUID Quantum Design MPMS-5 at 5000 G. Molar magnetic susceptibilities were corrected for the diamagnetic molar contribution of the solid product. (Sample mass: 41 mg, molar mass: 985.51, diamagnetic contribution: 4.426E4). EPR spectra of MeOH/H2O solutions at 77 K were recorded in the X band (9.85 GHz) using a Bruker ER200-SRC spectrometer with a cylindrical cavity. Spectra for two different samples were recorded, the first prepared from the isolated solid [Cu2(dimeim)(H2O)4](ClO4)4, and the second one from equivalent amounts of Cu(ClO4)2 and dimeim. 3. Solution studies Dimeim 6H2O was prepared according to the reported method [28], and purity verified by elemental analysis. The reagents used for the solution studies were of analytical reagent grade and the solutions prepared with distilled and deionized water. An approximately 0.1 M stock solution of Cu(II) prepared from analytical grade Cu(NO3)2 was standardized by atomic absorption. CH3CN and CH3OH for the kinetic and electrochemical studies were spectrophotometric grade. Buffers for the kinetic and electrochemical studies were MES (2-(N-morpholino)ethanesulfonic acid, pH 5.5–6.5), HEPES (4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid, pH 7.0–8.0) and TRIS (tris(hydroxymethyl-)amino-methane, pH 8.5–9.5). 3.1. Equilibrium studies A Methrom 702 Titrino set including digital pH meter coupled to a PC, fitted with a Methrom Ag/AgCl combined electrode, was used for potentiometric titrations. Each problem solution was contained in a 75 mL jacketed glass cell thermostated at 25.00(±0.05) °C by a circulating constant-temperature water bath. Ionic strength was imposed to 0.1 M with KNO3. Titrations of the ligand in the absence or presence of metal ions in aqueous solution were conducted in a standard way. Cell solutions (50 mL) were purged with a purified nitrogen stream. Experimental runs were carried out by adding increments of standardized ca. 0.1 M carbonate free NaOH to a solution containing dimeim plus other components (KNO3, copper nitrate and an excess of standardized HNO3). Protonation constants and stability constants from the direct titrations were calculated from the potentiometric titration data with the Hyperquad program [31,32]. Species distribution diagrams
The catecholase activity of [Cu2(dimeim)]4+ was monitored by UV/Vis spectroscopy by the increase of the absorption band at 400 nm from the formation of 3,5-ditert-butyl-o-benzoquinone. The catalytic activity of the complex was determined by the method of initial rates, measuring the absorption as a function of time over the first 40 s. The mean value of three experiments was determined; the maximum deviation from the mean value did not exceed 10%. For pH dependence studies, catalyst aqueous solutions were prepared with 0.3 M MES (pH 5.5, 6.0 and 6.5), HEPES (pH 7.0, 7.5, 8.0 and 8.5) and TRIS (pH 9.0) and standardized NaOH or HNO3 and were mixed with equal volumes of DTBC solution in MeOH or MeCN. Since the complex displays different solubility in the two solvent mixtures studied, for pH dependence studies, in MeCN/H2O, cell concentrations were: 0.150 mM for the copper catalyst and 10.02 mM for DTBC. In MeOH/ H2O, cell concentrations were: 0.03 mM for the copper catalyst and 2.0 mM for DTBC. Kinetic parameters were determined in MeCN/ H2OpH=7.5, MeOH/H2OpH=7.5, MeCN/H2OpH=8.5 and MeOH/H2OpH=8.0 For catalyst concentration dependence studies, [DTBC] was kept constant at 10.02 mM in MeCN/H2O and at 2.03 mM in MeOH/H2O, while in the first case catalyst concentration varied from 50 to 301 lM and in the second, from 10 to 60.1 lM. For substrate dependence studies, in MeCN/H2O catalyst concentration was held constant at 150.3 lM while catechol concentration was varied from 3.51 to 15.03 mM; in MeOH/H2O catalyst concentration was held constant at 30 lM while catechol concentration was varied from 0.33 mM to 3.33 mM. The reaction rate was found to be linearly dependent on the complex concentration, while for substrate dependence saturation kinetics were observed. A Michaelis–Menten analysis of the data was applied and Vmax, Km and kcat determined from a Lineweaver–Burk plot. 3.3. Electrochemical studies Cyclic voltammetric studies were performed on the [Cu2(dimeim)]4+ complex with an AUTOLAB PGSTAT 100 potentiostat/galvanostat in MeCN/H2OpH at pH values of 7.5, 8.0 and 8.5, and in MeOH/H2OpH at pH values of 7.5 and 8.0. The experiments were carried out in a conventional three-electrode cell composed of: a reference electrode Ag/AgCl(s)/0.1 M KCl in the corresponding solvent mixture; a platinum wire as auxiliary electrode and a glassy carbon disk (surface area 7.1 mm2) as working electrode. In order to refer all potential measurements to the Ferricenium/Ferrocene (Fc+/Fc) system according to IUPAC convention [34], voltammograms were obtained for approximately 103 M ferrocene (Fc) in background
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electrolyte. Cyclic voltammograms (CVs) were performed at a potential range of 1.4 V to 0.5 V vs Fc+–Fc at a sweep rate of 100 mV/s and were initiated from open circuit potential (Ei=0). The 103 M complex solutions were prepared in situ by mixing the required amounts of the ligand and Cu(NO3)2 in the 50% solvent: H2OpH. The buffer also played the role of supporting electrolyte. The presence of oxygen was avoided by bubbling nitrogen through all the experiments both in the solution and in the cell.
4. Results and discussion 4.1. Crystal structure (1,6)Diazecine-based ligands, like dimeim, are known to favor the formation of dinuclear rather mononuclear complexes with Cu(II), since peripheral N atoms are separated by a large distance, impeding coordination to a single metal center. Such molecules must contain suitable N or O donor sites in order to serve as ligands, for example imidazole heterocycles, amines or carboxylic acids as substituents on the diazecine N atoms, etc. The ligand used in the present work, (Scheme 1) namely 2,8-dimethyl-5, 1-bis(dimethylaminoethyl)-1,4,5,6,7,10,11,12-octahydroimidazo[4,5-h]imidazo[4,5-c] [1,6]diazecine (dimeim) has proven to efficiently coordinate Cu(II), using water and acetato [28] water and sulfato [35], methanol and nitrato, or halides and water [36] as ancillary ligands. A number of counterions were used, which do not seems to have any influence on the geometry of the cation. The cation in complex 1, [Cu2(dimeim)(H2O)4]4+, is placed on an inversion center (Z0 = 1/ 2), and the five-coordinated Cu(II) ion displays a squarepyramidal environment, the axial position being occupied by a water molecule (Fig. 1). The metal center is placed ˚ above the N3/N7/N10/O2 least-squares plane. 0.236 A The 10-membered central (1,6)diazecine ring adopts a chair ˚. conformation, with a total puckering amplitude of 1.44 A The conformation observed for dimeim in 1 is very similar to that observed in other reported Cu2–dimeim complexes, confirming the rigid character of this ligand. For instance, imidazole rings are invariably parallel (by symmetry), and
N
N
NH
N N
HN
N
N
Scheme 1. Structure of the ligand dimeim.
Fig. 1. Structure of cation [Cu2(dimeim)(H2O)4]4+ in 1, with displacement ellipsoids at the 30% probability level for non-H atoms. Labeling scheme is given for the asymmetric unit, while non-labeled atoms are generated by symmetry 2 x, y, 2 z. Selected coordination bond lengths and angles: Cu1–O1 2.251(3), Cu1–O2 2.001(3), Cu1–N3 2.009(3), Cu1–N7 ˚ ; O2–Cu1–N7 168.28(16), N3–Cu1–N10 2.069(3), Cu1–N10 2.034(4) A 162.06(14), N3–Cu1–N7 83.31(12), N7–Cu1–N10 85.74(14)°.
least-squares planes passing through these rings are sepa˚ in 1, which compare well with other comrated by 2.570 A ˚ plexes: 2.680 A in the aqua/acetato-complex [28,35] ˚ in the aqua/sulfato-complex [35] or 2.688 A ˚ in 2.681 A the aqua/chloro-complex [36]. This dimension roughly measures the thickness of dimeim, which results to be more or less constant regardless of ancillary ligands or anions used. As a consequence, metal–metal intra-anion separa˚ for 1, while tion remains also unchanged, 7.5153(11) A the largest observed separation in this series is ˚ , for the molecular complex [Cu2(dimeim)7.5709(18) A (SO4)2(H2O)2] [35]. 4.2. Magnetic susceptibility and electronic paramagnetic resonance Using a Gouy balance, the room temperature magnetic moment of [Cu2(dimeim)(H2O)4](ClO4)4 was found to be 1.26 BM per copper atom, considerably lower than expected for non-coupled Cu(II). The magnetic susceptibility of the complex was then measured in the temperature range 4–300 K. The temperature dependence of vm and vmT of the Cu2–dimeim unit is shown in Fig. 2. Upon cooling, vm increases, reaching maximum at 12 K and then rapidly decreases with temperature, clearly indicating the presence of an antiferromagnetic interaction. From a plot of 1/v vs T in the interval of 100–300 K, a straight line is obtained and a Curie constant of C = 0.847 emu mol1 K and a Weiss constant h = 7.6 K. The isotropic value calculated from the EPR spectrum of the solid sample at 77 K is g = 2.086. To estimate the magnitude of the antiferromagnetic coupling, the magnetic susceptibility data (4–300 K) were fitted to the modified Bleaney–Bowers equation [37] for two interacting copper(II) ions (S = 1/2) with the Hamiltonian in the form H = 2J S1 S2. The susceptibility equation for such a dimeric system can be written as follows:
L. Gasque et al. / Journal of Inorganic Biochemistry 102 (2008) 1227–1235
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Fig. 2. Magnetic susceptibility data vs temperature for compound (1) and least-squares fitting curve adjusted to the Bleaney–Bowers equation.
v¼
Ng2 l2b Ng2 l2b 2 q þ TIP ð1 qÞ ð1 qÞ þ 3 þ e2J =kT kT 2kT
The g value was fixed to the value obtained from EPR. The magnetic data were fitted in the temperature range 4–300 K leading to the following parameters: J = 7.11 cm1, q = 0.003 and TIP = 0.001 emu mol1. A similar compound was described by Mendoza-Diaz et al. [38], for which a value of J = 6.87 cm1 was obtained when the data were fit with this same model. The EPR spectrum of a powdered solid simple of [Cu2(dimeim)(H2O)4](ClO4)4 at 77 K, is consistent with an isotropic Cu(II) center, with g = 2.086 (Supplementary material). However, when the spectrum is measured from a frozen MeOH/H2O solution, (Fig. 3) a well-resolved
hyperfine structure in the gk region is observed, with seven discernible lines with an average splitting of 78 G (gk = 2.294 and g\ = 2.073). Identical spectra were obtained whether the solution was prepared dissolving a sample of the isolated solid [Cu2dimeim(H2O)4](ClO4)4, or from equivalent amounts of Cu(ClO4)2 and dimeim. Similar spectra have been previously assigned to a weak exchange interaction between copper(II) centers in dinuclear com0 ˚ [8,38–40], in plexes with Cu–Cu separations of 7.3–8 A which as in the present case, no DMs = ±2 has been observed at half field. This spectrum was successfully simulated using the WINEPR from Brucker Co. The found parameters are consistent with the coupling of two Cu(II) (S = 3/2) nuclei, the value of A = 78 G (gk = 2.290 and g\ = 2.060), coincides with the obtained experimentally (Supplementary material). 5. Equilibrium studies In order to gain some insight into the stability of the dinuclear complex in solution, as well as on its hydrolyzing properties, a series of potentiometric titrations were carried out, and the corresponding data adjusted with Hyperquad to yield the desired equilibrium constants. For the ligand, four successive protonations were detected with the titration of dimeim, with corresponding pKa values of 10.06, 9.17, 7.04 and 5.88. The refinement of complexation constants indicates that [Cu2(dimeim)(H2O)4]4+ is completely formed and is the only copper containing species in the pH range between 6 and 8; from that point on, the formation of the several hydrolyzed species, becomes appreciable. The corresponding hydrolysis equilibriums are [Cu2 L(H2 O)6 ]4þ ¡ [Cu2 L(H2 O)5 (OH)]3þ + Hþ
Fig. 3. EPR spectrum of [Cu2dimeim(H2O)4](ClO4)4 in MeOH/H2O frozen solution at 77 K.
[Cu2 L(H2 O)5 (OH)]3þ ¡ [Cu2 L(H2 O)4 (OH)2 ]2þ + Hþ
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[Cu2 L(H2 O)4 (OH)2 ]2þ ¡ [Cu2 L(H2 O)3 (OH)3 ]þ + Hþ Table 2 shows the value of the overall formation constant as well as the copper acidity constants corresponding to three successive hydrolyses and a species predominance diagram as a function of pH is shown in Fig. 4. 5.1. Catalytic activity Variation in catecholase activity of the complex as a function of pH was investigated in two solvent mixtures, MeCN/H2O and MeOH/H2O and compared to the sole pH effect on the reaction rate without the presence of a catalyst. The results of these experiments are shown in Fig. 5a and b, respectively. In both solvent mixtures kinetic parameters were determined at pH 7.5, to be able to compare the activity with other catalysts developed by the group [41]. A second pH value was chosen for each solvent system as that which gave the largest increment in activity compared to the activity of the medium without the catalyst. The increase in catalytic activity at higher pH values may be associated with the predominance of the hydrolyzed forms of the catalyst, predicted from the species predominance diagram. (Fig. 4). This optimal pH value was 8.5 for MeCN/H2O and 8.0 for MeOH/H2O, suggesting a complete formation of the catalytically active species under these conditions. Table 2 Overall complexation constants and pKa values for Cu2–dimeim system in aqueous solution at 298 K ½Cu2þ H L
½Cu2 LðH2 OÞ6i ðOHÞi ½Hþ ½Cu2 LðH2 OÞ6iþ1 ðOHÞi1
z bxyz ¼ ½Cu2þxx ½Hy y ½L z
K ðiÞ a ¼
Log b2 0 1 = 20.78 Log b2 1 1 ¼ 12:43 Log b2 2 1 ¼ 3:43 Log b2 3 1 ¼ 6:61
– pK ð1Þ a = 8.35 pK ð2Þ a = 9.00 pK ð3Þ a = 10.04
Since dielectric constant values for methanol and acetonitrile are very similar (33 and 37 respectively, compared to 80 for water [42]), a similar decrease in the pKa value for the complex would be expected in both of the studied solvent mixtures. However, the lower optimal pH value found for MeOH/H2O suggests a qualitatively different participation of the two solvent mixtures during the catalytic process. Complete kinetic studies were carried out on the basis of the Michaelis–Menten model, with the evaluation of the kinetic parameters (Vmax, KM, kcat) at both pH values from the corresponding Lineweaver–Burk plots. As can be seen from data in Table 3, in both solvent mixtures an increase in pH causes a slight improvement in catalyst performance, while a much more noticeable enhancement is observed when solvent is changed from MeCN/H2O to MeOH/ H2O; at pH 7.5 catalytic efficiency is 20 times better in the latter than in the former. The kcat/KM value obtained for this complex in MeOH/H2OpH=8.0 is better than most of the dinuclear copper complexes whose catecholase activity has been reported recently [20,21,25,27,43], suggesting the action of a mechanism that depends on other factors different from the Cu–Cu distance.
5.2. Electrochemical studies The significant difference found for the behavior of the catalyst in the two studied solvent mixtures, motivated the performance of a systematic electrochemical study of the dicopper complex in both MeOH/H2O and MeCN/ H2O, at different pH values. Only redox signals associated to the Cu(II)/Cu(I) couple will be discussed, due to their relevance to the catalytic process. Initial studies dealt with [Cu2(dimeim)]4+ in MeCN/ H2OpH=7.5 (Fig. 6). Peak Ic, with Ecp = 0.524 V, corresponds to the Cu(II) ? Cu(I) reduction process, but the
Fig. 4. Species predominance diagram for Cu2–dimeim system as a function of pH at 298 K.
L. Gasque et al. / Journal of Inorganic Biochemistry 102 (2008) 1227–1235
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Fig. 6. Typical cyclic voltammograms in MeCN/H2OpH mixtures at different pH values for Cu2(dimeim)4+ (a) pH 7.5 and (b) pH 8.5.
Fig. 5. The pH effect on the catecholase activity of Cu2–dimeim in (a) MeCN/H2O and (b) MeOH/H2O (concentrations in MeOH/H2O are five times lower).
Table 3 Kinetic parameters (Vmax, Km, kcat) in MeCN:H2OpH at pH 7.5 and 8.5 and in MeOH:H2OpH at pH 7.5 and 8.0, from the corresponding Lineweaver–Burk plots, for the catecholase activity of Cu–dimeim system Medium
Vmax 106 (M s1)
Km 103 (M)
kcat 102 (s1)
kcat/Km (M1 s1)
MeCN:H2OpH=7.5 MeCN:H2OpH=8.5 MeOH:H2OpH=7.5 MeOH:H2OpH=8.0
4.63 9.77 3.05 3.25
11.90 4.19 1.96 1.59
3.09 6.51 10.18 10.83
2.60 15.53 51.99 67.88
Due to different solubilities, concentrations in MeOH/H2O was five times lower than in MeCN/H2O.
low intensity and the position of the corresponding oxidation signal, Ia for which Epa = 0.389 V, is indicative of a practically irreversible system. This irreversibility is confirmed by the value of the quotient, ipa/ipc = 0.13. The breadth of signal Ic, can be indicative of overlapped reduction signals, which suggests the presence of at least two differentiable Cu(II) sites, which are reduced consecutively at very similar potential values. This electrochemical behavior has been associated [8,44] with electronic commu-
nication between two metal centers, which in this case is suggested by the EPR spectrum (Fig. 3). Peak width is measured by DEp/2 = (Epc Epc/2), where Epc is the cathodic peak potential and Epc/2 is the corresponding half-peak potential. For this signal, the obtained value of DEp/2 = 0.135 V can be interpreted as two overlapping one-electron signals. To rule out uncompensated resistance effects, the DEp/2 value was determined for the Fc/Fc+ system and found to be 54 mV (Supplementary material). The presence of two oxidation signals, Ia and IIa is indicative of the partial decoordination of the ligand upon reduction of the metal ion: Ia is associated with the proI cess ½CuI2 dimeim ! ½CuII 2 dimeim and IIa with 2½Cusolv ! II ½Cu2 dimeim. Voltammograms obtained in MeCN/H2O at higher pH values are essentially similar, but indicative of slightly stronger oxidizing power and a more reversible behavior. Relevant electrochemical parameters for all the studied systems are shown in Table 4. The voltammograms obtained MeOH/H2O (see Fig. 7) show that the two main parameters associated with catalytic efficiency, E1/2 and ipa/ipc, are more favorable than in MeCN/H2O. While E1/2 values at the two studied pH values are almost equal, a significant increase in reversibility is observed at the higher (Table 4). This increase in reversibility is a direct consequence of a larger intensity in peak Ia, indicative of a better stability of the reduced form of the complex ½CuI2 dimeim in MeOH/ H2O than in MeCN/H2O. This stability is further improved at higher pH values, as can be inferred from the current intensity increase in Fig. 7b compared to that in 7a.
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Table 4 Electrochemical parameters for Cu2–dimeim and Cu(NO3)2 compounds, in different solution media Medium
E1/2 signal (Ia–Ic) CuII M CuI
Epc signal (Ic) CuII ? CuI
DEp/2 signal (Ic) CuII ? CuI
Epa signal (Ic) CuI ? CuII
ipc/ipa signal (Ia)
MeCN/H2OpH=7.5 MeCN/H2OpH=8.0 MeCN/H2OpH=8.5 MeOH/H2OpH=7.5 MeOH/H2OpH=8.0
0.429 0.426 0.398 0.347 0.346
0.524 0.520 0.506 0.493 0.467
0.135 0.113 0.113 0.144 0.146
0.334 0.332 0.290 0.201 0.225
0.13 0.20 0.25 0.49 0.56
All potential values are in volts and were referred to Fc+–Fc system.
dized species, which explains the increase in reversibility, which correlates with a better activity. Acknowledgements L.G. thanks Prof. Roberto Escudero for variable temperature magnetization measurements, and CONACyT (34847-E) and DGAPA-UNAM (IN106003) for economic support, and Prof. Bob Johnson for his thorough revision of the manuscript. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jinorgbio. 2007.12.032. References
Fig. 7. Typical cyclic voltammograms in MeOH/H2OpH mixtures at different pH values for Cu2(dimeim)4+ (a) pH 7.5 and (b) pH 8.0.
5.3. Concluding remarks A significant catecholase activity was found for this dicopper complex in spite of the steric impossibility of both copper atoms to act simultaneously on one molecule of substrate. Considerable differences were found for the catalytic activity shown in the different solvent media tested. The electrochemical studies performed on all the corresponding media gave some insight into the different catalytic behavior. Although an increase in pH in both solvent mixtures results in an increase both in oxidizing power (E1/2) and reversibility (ipa/ipc) the change of solvent system seems to be a more influencing factor. The very poor reversibility obtained in MeCN/H2O may be caused by the great affinity of Cu(I) for CH3CN, which encourages the decoordination of the ligand upon the reduction of the complex. Since Cu(I) exhibits no comparable affinity for MeOH and/or H2O, in this case, the coordination to three nitrogen donor atoms seems to be a preferred situation leading to similar coordination environments for the reduced and the oxi-
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