Journal of Inorganic Biochemistry 146 (2015) 77–88

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Synthesis, characterization, hydrolase and catecholase activity of a dinuclear iron(III) complex: Catalytic promiscuity Tiago P. Camargo a, Fernanda F. Maia a, Cláudia Chaves a, Bernardo de Souza a, Adailton J. Bortoluzzi a, Nathalia Castilho b, Tiago Bortolotto b, Hernán Terenzi b, Eduardo E. Castellano c, Wolfgang Haase d, Zbigniew Tomkowicz e, Rosely A. Peralta a,⁎, Ademir Neves a,⁎⁎ a

Departamento de Química, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazil Centro de Biologia Molecular Estrutural, Departamento de Bioquímica, Universidade Federal de Santa Catarina, Florianópolis 88040-900, Brazil c Instituto de Física, Universidade de São Paulo, 13360-979 São Carlos, SP, Brazil d Institut für Physikalishe Chemie, Technische Universität Darmstadt, Petersenstraße 20, D-64287 Darmstadt, Germany e Institute of Physics, Reymonta 4, Jagiellonian University, PL-30-059 Krakow, Poland b

a r t i c l e

i n f o

Article history: Received 6 November 2014 Received in revised form 24 February 2015 Accepted 24 February 2015 Available online 5 March 2015 Keywords: Dinuclear iron(III) complex Catalytic promiscuity DNA cleavage

a b s t r a c t Herein, we report the synthesis and characterization of the new di-iron(III) complex [(bbpmp)(H2O)(Cl)FeIII (μ-Ophenoxo)FeIII(H2O)Cl)]Cl (1), with the symmetrical ligand 2,6-bis{[(2-hydroxybenzyl)(pyridin-2-yl) methylamino]methyl}-4-methylphenol (H3bbpmp). Complexes 2 with the unsymmetrical ligand H2bpbpmp — {2-[[(2-hydroxybenzyl)(2-pyridylmethyl)]aminomethyl]-6-bis(pyridylmethyl) aminomethyl}-4-methylphenol and 3 with the ligand L1 = 4,11-dimethyl-1,8-bis{2-[N-(di-2-pyridylmethyl)amino]ethyl}cyclam were included for comparison purposes. Complex 1 was characterized through elemental analysis, X-ray crystallography, magnetochemistry, electronic spectroscopy, electrochemistry, mass spectrometry and potentiometric titration. The magnetic data show a very weak antiferromagnetic coupling between the two iron centers of the dinuclear complex 1 (J = −0.29 cm−1). Due to the presence of labile coordination sites in both iron centers the hydrolysis of both the diester model substrate 2,4-BDNPP and DNA was studied in detail. Complex 1 was also able to catalyze the oxidation of the substrate 3,5-di-tert-butylcatechol (3,5-DTBC) to give the corresponding quinone, and thus it can be considered as a catalytically promiscuous system. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Iron containing enzymes have several vital functions in living systems since they participate in many different types of reactions such as electron transfer, hydrolysis of phosphate esters and gene expression. They are extremely versatile and for this reason, there is a huge interest in the commercial application of these systems in biotechnology. However, their structures, mechanisms and functions are not yet fully understood [1]. In general, enzymes are known for their high efficiency and specificity but, in many cases, a single catalytic site can catalyze more than one chemical transformation. This is called catalytic promiscuity and this is a phenomenon reasonably well described for biological systems. For example, it has been shown that an aminopeptidase obtained from Streptomyces griseus containing a dinuclear CuII active site is capable of hydrolyzing phosphate esters and peptides, but it also oxidizes catechols ⁎ Corresponding author. Tel.: +55 48 37213627. ⁎⁎ Corresponding author. Tel.: +55 48 37213605. E-mail addresses: [email protected] (R.A. Peralta), [email protected] (A. Neves).

http://dx.doi.org/10.1016/j.jinorgbio.2015.02.017 0162-0134/© 2015 Elsevier Inc. All rights reserved.

with activity close to that of the native enzyme [2]. It has been proposed that this enzyme and its possible variants could serve as unique dinuclear systems to provide further insight into the structure–mechanism correlations in metal-centered hydrolytic and oxidation chemistry. Catechol oxidase (CO) is a dinuclear copper enzyme that catalyzes the two-electron-transfer reaction during the oxidation of a wide range of catechols to the corresponding o-quinones by O2 in a process known as catecholase activity, while hydrolases belong to a class of metalloenzymes (metal = Zn, Cu, Fe, Mn, Ca) that catalyze the hydrolysis of several type of substrates including phosphate esters, peptides and nuclei acids [3]. A generic scheme of both reactions is shown in Scheme 1. In fact such dinuclear hydrolytic and oxidative metalloenzymes have been used as appropriate starting points in the development of specific classes of synthetic metal complexes, known as synthetic hydrolases and synthetic catechol oxidases. During the last two decades extensive studies on synthetic model systems have been reported, aiming to mimic the structural and/or functional properties of these metalloenzymes, such as metal–metal distances, redox potentials, and geometry around each metal center, with the presence of labile sites essential for binding of the substrate and/or available nucleophiles

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Scheme 1. Schematic representation of catechol oxidation (above) and hydrolase reaction (below).

to initiate the catalytic process [4,5]. In some cases, such information can be very helpful in clarifying the most probable mechanism associated with the native enzyme. Although coordination compounds have been designed in order to mimic these catalytic properties [6,7], with few exceptions, most of the examples of synthetic catecholases described in the literature are related to dinuclear CuII biomimetics, while di-iron systems have been rarely explored. One such interesting example has been recently reported by Neves and co-workers, in which it was possible to demonstrate that a dinuclear FeIIIFeIII complex is able to catalyze both the cleavage of phosphodiester bonds and the oxidation of catechols to the corresponding quinones [8]. In addition, it has also been reported that synthetic mononuclear iron(III) complexes show catalytic activity in the oxidative cleavage of 3,5-di-tert-butylcatechol by O2 to yield specifically the intradiol product (1,2-dioxygenase activity) [7,9]. In order to better understand promiscuous synthetic systems and their structure/activity correlation, we report herein the X-ray structure and solution studies for the new complex [(bbpmp)(H2O)(Cl)FeIII (μ-Ophenoxo)FeIII(H2O)(Cl)]Cl, (1). The complex is able to cleave DNA through a mixed-type hydrolytic/oxidative mechanism, but also shows significant catalytic activity in the oxidation of the model substrate 3,5-di-tert-butylcatechol (3,5-DTBC), thus proving to be a promising complex exhibiting catalytic promiscuity. It is worth noting here that the maximum hydrolase activity catalyzed by 1 occurs at pH 6.5 while catecholase activity reaches its maximum at pH 9.0. 2. Experimental 2.1. Chemicals All reagents, materials, gases and solvents of high purity grade used in the synthesis procedures were purchased from commercial sources and used without prior purification. Spectroscopic solvents were used for solution studies and deuterated solvents were used for the analysis of nuclear magnetic resonance. Bis(2,4-dinitrophenyl)phosphate [10] and the ligand H3bbpmp were synthesized according to the synthetic routes described in the literature [11]. 2.2. Physical measurements Infrared spectra were obtained on a FTIR spectrophotometer-2000, Perkin Elmer, in the region of 4000–400 cm−1. The samples were prepared by dispersion in KBr. Elemental analysis was performed on a Perkin-Elmer CHN 2400 analyzer (USP-São Paulo). A crystal with dimensions of 0.09 × 0.13 × 0.27 mm3 was selected from a crystalline sample of complex 1 for crystallographic analysis. X-ray diffraction data were measured on a Kappa-CCD diffractometer equipped with a molybdenum tube (MoKα λ = 0.71073 Å) and

graphite monochromator at room temperature. All reflections were corrected for Lorentz and polarization effects. Gaussian absorption correction was also applied to all measured intensities. The structure was solved by direct methods using the SHELXS97 program and the data were refined by full-matrix least-squares methods on F2, using the SHELXL97 program [12,13]. All non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms bonded to C atoms were placed at their idealized positions using standard geometric criteria. The H atoms of the coordinated water molecules were located from the Fourier difference map and treated using a riding model with Uiso(H) = 1.2Ueq(O). On the other hand, H atoms of the uncoordinated water molecules could not be found. An ORTEP plot of the molecular structure was generated using the PLATON program [12, 13]. Selected crystal data obtained are as follows: C35H43Cl3Fe2N4O8, FW 865.78 g/mol, orthorhombic, Pna2 1, a = 20.6940(4) Å, b = 10.6140(6) Å, c = 17.7040(8) Å, V = 3888.6(3) Å 3 , Z = 4, T = 296(2) K, μ = 1.006 mm− 1, ρcalc = 1.479 Mg/m3, reflections collected 20,568, unique 7240 (Rint = 0.0925), R1 = 0.0744, and wR2 = 0.1977. Crystallographic data for complex 1 (excluding structure factors) have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC-1027986. These data can be obtained free of charge via www.ccdc.cam.ac.uk. Electrospray ionization mass spectrometry (ESI-MS) of 1, dissolved in an ultrapure acetonitrile solution (500 nM), was performed on an amaZon X Ion Trap MS instrument (Bruker Daltonics) with an ion spray source using electrospray ionization in positive-ion mode. The ion source condition was an ion spray voltage of 4500 V. Nitrogen was used as the nebulizing gas (20 psi) and curtain gas (10 psi) and samples were directly infused into the mass spectrometer at a flow rate of 180 μL/h. The scan range was m/z 200–1200. The simulated spectrum was calculated using the Mmass software [14,15]. The ligand 1H NMR spectrum was obtained on a Brucker-FT 400 MHz spectrophotometer and the chemical shifts were recorded in ppm using tetramethylsilane (TMS) as the internal reference (TMS, δ = 0.00 ppm) and deuterated chloroform as the solvent. Electronic spectra of 1, in water solution, were obtained on a Perkin-Elmer Lambda-750 spectrophotometer, at pH values of 2.0, 4.2 and 6.8. The redox behavior of 1 was investigated by cyclic voltammetry on a BAS potentiostat–galvanostat, model Epsilon, and the experiments were performed in aqueous solution at pH values of 2.0, 4.2 and 6.8 under an argon atmosphere. In these experiments, NaCl (0.2 M) was used as the supporting electrolyte and measurements were taken in an electrolytic cell with three electrodes: working electrode — carbon; auxiliary electrode — platinum and reference electrode — Ag/AgCl (Ag/AgCl vs. NHE = +197 mV). The potentiometric studies of 1 were carried out with a pH meter fitted with blue glass and calomel reference electrode calibrated to read –log [H +] directly, designated as pH, in an methanol/water

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(50/50% v/v) solution. All measurements were carried out in a thermostated cell under argon atmosphere at 25.00 ± 0.05 °C. Preceding each titration, the acidity of the metal complex solution (0.02 mmol/ 50 mL) was adjusted to pH near 2.5 with HCl and the ionic strength was maintained at 0.100 M with KCl, under an argon flow to eliminate the presence of atmospheric CO2. Solutions were titrated with the addition of fixed volumes of standard CO2-free KOH solution (0.100 M) in a mixture of methanol/water 50/50% (v/v) prepared with deionized water and analytical grade methanol. The pKw was considered to be the reference value of 14.30 [16]. The titrations were performed in triplicate and the values presented are the average of three experiments. The equilibrium constants were calculated using the BEST7 [17] program and the species distribution diagrams for the species present in solution as a function of pH were obtained using the SPE program [17]. Variable-temperature magnetic susceptibility data of the powdered sample were obtained in the range 2–300 K and in a magnetic field of 1000 Oe with a SQUID magnetometer using a slightly pressed polycrystalline sample of 1. Diamagnetic susceptibility corrections for the ligand susceptibility were made using Pascal's constants [18,19]. 2.3. Kinetic assays The catalytic activity of complex 1 was evaluated through the oxidation of the substrate 3,5-di-tert-butylcatechol (3,5-DTBC) in methanol/ water (32:1) and hydrolysis in acetonitrile/water (50:50) of the substrate bis(2,4-dinitrophenyl)phosphate (2,4-BDNPP), which was synthesized according to the method described by Bunton [10]. Kinetic experiments were performed in triplicate under conditions of excess substrate and monitored on a UV–Vis Varian Cary 50 BIO spectrophotometer coupled to a thermostatic bath. The change in absorbance was measured under reaction conditions at 400 nm (ε = 1645 M− 1·cm− 1), this change being due to the formation of 3,5-di-tert-butylquinone associated with the activity of catecholase, and also at 400 nm (ε = 12,100 M− 1·cm− 1) [20,21], this change being related to the release of the anion 2,4-dinitrophenolate due to the activity of hydrolase. In order to determine the pH value related to the maximum hydrolase or catecholase activity, firstly the role of pH dependence in terms of the kinetic behavior was determined for the catecholase activity over the pH range of 5.50 to 10.00 at 25 °C. In this procedure 100 μL of aqueous buffer solution ([B]final = 3 × 10− 2 M–MES (2(N-morpholino)ethanesulfonic acid) pH 5.50 to 6.50; TRIS pH 7.00 to 9.00 and CHES (2-(cyclohexylamino)ethanesulfonic acid) pH 9.50 to 10.00), 200 μL of a complex solution ([C]final = 2.9 × 10−5 M) and 2900 μL of methanol saturated with oxygen were placed in a 1 cm-path optical glass cell. Lastly, 100 μL of a methanolic solution of substrate ([S]final = 5.6 × 10−3 M) was added and the reaction was monitored for 20 min at 25 °C. The kinetic experiments with catecholase under an excess of substrate were performed as follows: [1] = 4.41 × 10−5 M; [3,5-DTBC] = 0.04 M; buffer TRIS 0.06 M pH = 9.0 and methanol saturated with oxygen were added to quartz or glass cuvettes with 1 cm optical path at 25 °C. The reaction was initiated with the addition of methanolic solution of 3,5-DTBC ([S]final = 1.0 × 10−3–15 × 10−3 M). In all experiments, the final volume of the reaction mixture in the cuvette was 1.7 mL. Corrections for spontaneous oxidation of the substrate 3,5-DTBC were performed under identical conditions without the addition of the catalyst. The initial rates were obtained from the slope of the absorbance versus time during the first 10 min of reaction. To determine if the phenolic ligand was able to oxidize the substrate 3,5-DTBC, reaction mixtures were prepared as above but instead of complex 1 it was added the same amount of the ligand, and it was observed that the ligand does not promote the catechol oxidation. To determine if hydrogen peroxide (or water) was formed as the reduced oxygen species during the 3,5-DTBC oxidation, reaction mixtures were prepared as described above in the kinetic experiments. After 1 h of reaction an equal volume of water was added and the quinone formed was extracted three times with dichloromethane (2 × 3mL).

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The aqueous layer was acidified with H2SO4 to pH = 2 and 1 mL of a 0.3 M aqueous KI solution was added. In the presence of hydrogen peroxide occurs the reaction H2O2 + 2I− + 2H+ → 2H2O + I2, and with an excess of iodide ions, the triiodide ion is formed according to the reaction I2(aq) + I− → I− 3 . The reaction rate is slow but increases with increasing concentrations of acid. The increase in the I− 3 absorption band at 353 nm (ε = 26,000 M−1 cm−1) was monitored by UV/visible (UV/Vis) spectroscopy [22–25]. To determine the role of oxygen, the reaction was also carried out in the absence of oxygen under the same kinetic conditions. The absorbance of only 1 equivalent of quinone per mol of complex was found. The pH dependence of the hydrolase activity catalyzed by 1 was investigated in the pH range of 3.50–9.50 at 25 °C. Reactions were performed as follows: 1500 μL of aqueous buffer solution (0.1 M-MES pH 3.50–6.50; HEPES pH 7.00–8.50; CHES pH 9.00–10.00) with ionic strength I = 0.1 M, LiClO4, 400 μL of an acetonitrile solution of 1 ([1]final = 3.9 × 10−5 M) and 300 μL of acetonitrile were placed in a cuvette. The reaction was initiated by adding 800 μL of an acetonitrile solution of substrate ([S]final = 5 × 10−3 M). Experiments under conditions of excess substrate were performed as follows: 1.5 mL of an aqueous solution of MES buffer pH 6.50 ([B]final = 5 × 10− 2 M), 400 μL of a solution of 1 in acetonitrile ([1]final = 4.9 × 10−5 M) and acetonitrile were added to a quartz cuvette at 25 °C. The reaction was initiated with the addition of volumes ranging from 150 μL to 1100 μL of a solution of the 2,4-BDNPP substrate in acetonitrile ([S]final = 9.4 × 10−4–15 × 10−3 M). In all experiments for the hydrolysis reaction, the final volume of the reaction mixture in the cuvette was 3.0 mL. Corrections for the spontaneous hydrolysis of the substrate 2,4-BDNPP were performed under identical conditions without the addition of the complex. The initial rates were obtained from the slope of the absorbance versus time curve obtained for the first 15 min of reaction. 2.4. DNA cleavage The plasmid pBSK II (2961 bp, Stratagene) was used as a DNA substrate for the DNA cleavage assays. The plasmid was transformed into DH5α Escherichia coli competent cells, amplified as previously described [26] and then purified from E. coli cells according to the manufacturer's instructions (Qiagen Plasmid Maxi Kit protocol). The DNA cleavage promoted by 1 was evaluated by analyzing the conversion of the intact supercoiled form of pBSK II DNA (F I) to the open circular (F II) and linear (F III) forms, which represent the plasmid forms containing single and double-strand breaks, respectively [27,28]. DNA cleavage reactions were conducted using 330 ng of pBSK II DNA (~ 20 μM in bp) in 10 mM MES buffer (pH 6.5). The cleavage reaction was initiated by addition of 1 (at different concentrations depending on the assay) up to 16 h at 50 °C. At the end of the reaction time, the mixtures were quenched by adding 5 μL of a loading buffer solution (50 mM TRIS–HCl pH 7.5, 0.01% bromophenol blue, 50% glycerol, and 250 mM EDTA) and then subjected to electrophoresis in a 1.0% agarose gel containing 0.3 μg mL− 1 of ethidium bromide in 0.5 × TBE buffer (44.5 mM TRIS, 44.5 mM boric acid, and 1 mM EDTA at pH 8.0) at 90 V for 100 min. The resulting gels were visualized and digitized using a DigiDoc-It gel documentation system (UVP, USA). The proportion of plasmid DNA in each band was quantified using KODAK Molecular Imaging Software 5.0 (Carestream Health, USA). The quantification of supercoiled DNA (F I) was enhanced by a factor of 1.47, since the ability of ethidium bromide to intercalate into this DNA topoisomeric form is decreased relative to open circular and linear DNA [29]. To verify the mechanism by which 1 cleaves the DNA, external agents were added to the reaction mixtures before each complex. These agents included sodium chloride and lithium perchlorate (to increase the ionic strength of the reaction medium) and also different inhibitors of reactive oxygen species (ROS) [30–32], such as DMSO (10% v/v), an HO• scavenger, KI (10 mM), which induces the

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disproportionation of peroxide-type species, superoxide dismutase (SOD, 20 units), which scavenges O2•−, and NaN3 (10 mM), an 1O2 scavenger [30–33]. 2.5. Synthesis procedures The ligand H3bbpmp (Chart 1, left) was synthesized according to the synthetic routes described in the literature [11]. The purity of the ligand was confirmed by 1H NMR spectroscopy. The values of δH (400 MHz; CDCl3), in ppm were: 2.2 (s, 3H, CH3); 3.74 (s, 4H, CH2); 3.79 (s, 4H, CH2), 3.83 (s, 4H, CH2); 6.75 (t, 2H, CHar); 6.80 (d, 2H, CHar); 6.87 (s, 2H, CHar); 7.00 (d, 2H, CHar); 7.13 (t, 2H, CHar); 7.19–7.21 (4H, CHar); 7.64 (t, 2H, CHar), 8.60 (d, 2H, CHar); 10.64 (s, 2H, OHphenol). Complex 1 was synthesized by adding a methanolic solution containing two equivalents of FeCl3·5H2O, dropwise, to a methanolic solution of H3bbpmp. The reaction mixture was maintained under mild agitation and heating for ten minutes and it was subsequently filtered off and kept at room temperature for slow evaporation of the solvent. Elemental analysis and the infrared spectrum of 1 confirmed the complex formation. IR (KBr), in cm−1: ν (C–Har and C–Haliph) 3050–2843; ν (C = N and C = C) 1596, 1482; ν (C–Ophenol) 1297; ν (C–N) 1101; δ (C–HAr) 758. Anal. for [Fe2(bbpmp)(Cl)2(OH2)2]Cl.6H2O; Fe2C35H49 Cl3N4O11: Calc. (%) C:45.70; H:5.37; N:6.09, found (%) C:45.98; H:5.22; N:6.33. Crystals of 1 suitable for X-ray analysis were obtained by slow evaporation of a methanolic solution of the complex. 3 . Results and discussion 3.1. Synthesis The ligand H3bbpmp was synthesized according to the method described by Brito et al. and its 1H NMR spectrum verified that its purity was sufficient for the synthesis of 1 [11]. The reaction of one equivalent of H3bbpmp with two equivalents of FeCl3·5H2O in methanol produced crystals suitable for the X-ray analysis of 1 (Fig. 1). Infrared spectra of the ligand and of 1 are in agreement with the coordination of the ligand to the FeIII in its deprotonated form. 3.2. X-ray structure of 1 An ORTEP diagram showing the structure of 1 and the main bond distances and angles is shown in Fig. 1. The structure of 1 consists of discrete dinuclear [(bbpmp)(H2O)(Cl)FeIII(μ-Ophenoxo)FeIII(H2O)(Cl)]+ cations, uncoordinated chloride anions, and three water of crystallization molecules in the asymmetric crystallographic unit. No crystallographic symmetry is imposed on the cation, resulting in distinct ferric sites being observed in the complex. In the dinuclear cation the FeIII centers are single bridged by the phenolate oxygen atom of the bbpmp3 − ligand. The tridentate N2O-donor pendant arms of the dinucleating ligand in a meridional arrangement, one chloro and one water molecule complete the octahedral environment of each FeIII center. Interestingly,

Fig. 1. Molecular structure, selected distances (Å) and angles (°) of cation complex 1. Fe1– Fe2 3.8640(13); Fe1–O3 1.900(5); Fe1–O1W 2.037(5); Fe1–O1 2.133(4); Fe1–N21 2.153(6); Fe1–N1 2.186(5); Fe1–Cl1 2.314(2); Fe2–O51 1.894(5); Fe2–O2W 2.015(5); Fe2–N41 2.130(5); Fe2–O1 2.151(4); Fe2–N2 2.183(6); Fe2–Cl2 2.326(2); O3–Fe1–O1W 95.1(2); O3–Fe1–O1 91.7(2); O1W–Fe1–O1 84.33(18); O3–Fe1–N21 163.8(2); O1W– Fe1–N21 99.5(2); O1–Fe1–N21 82.9(2); O3–Fe1–N1 86.1(2); O1W–Fe1–N1 173.5(2); O1–Fe1–N1 89.22(19); N21–Fe1–N1 78.6(2); O3–Fe1–Cl1 98.55(17); O1W–Fe1–Cl1 90.02(16); O1–Fe1–Cl1 168.71(15); N21–Fe1–Cl1 88.40(18); N1–Fe1–Cl1 96.16(16); O51–Fe2–O2W 99.3(2); O51–Fe2–N41 163.0(2); O2W–Fe2–N41 95.8(2); O51–Fe2–O1 90.27(19); O2W–Fe2–O1 83.73(19); N41–Fe2–O1 83.83(18); O51–Fe2–N2 86.4(2); O2W–Fe2–N2 172.1(2); N41–Fe2–N2 77.9(2); O1–Fe2–N2 90.75(18); O51–Fe2–Cl2 96.61(16); O2W–Fe2–Cl2 90.57(15); N41–Fe2–Cl2 90.79(15); O1–Fe2–Cl2 171.71(14); N2–Fe2–Cl2 94.28(15); Fe1–O1–Fe2 128.8(2); C31–O3–Fe1 133.8(4).

the two terminal phenolate oxygen donor atoms coordinate trans to the pyridyl ligand in both metal centers, and this result contrasts with that + tri-bridged observed in the closely related [FeIII 2 (bbpmp)(μ-OAc)2] complex, in which the terminal phenolate groups are trans rather than cis to the bridging phenolate. Therefore, the distinct coordination observed in the structures arise from the different arrangements of the tridentate pendant arms (amine-pyridyl-phenolate) of bbpmp3 − around the FeIII centers. In 1 they are coordinated in a meridional fash+ the coordination is facial, thus ion while in [FeIII 2 (bbpmp)(μ-OAc)2] allowing the formation of the tribridged {FeIII(μ-phenoxo)(μ-OAc)2FeIII} unit [34]. 3.3. Magnetic susceptibility The variable-temperature magnetic susceptibility for 1 was investigated over the temperature range of 2–300 K and the results are shown in Fig. 2 (χT vs. T), which indicates a very small degree of coupling between the iron(III) centers in the complex. The data were fitted

Chart 1. Ligand H3bbpmp (left) and ligand H2bpbpmp (right).

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using the expression for molar susceptibility vs. temperature from the spin-exchange Hamiltonian H = − 2J·S1S2 and the parameters J = −0.29 cm−1, g = 2.03, χTIP = 60.10−6 cm3 mol−1 for each iron center are in agreement with a very weak antiferromagnetic intramolecular coupling. This value of J is in agreement with the longer μphenoxo-FeIII distances (av. 2.142 Å) in 1 when compared to the corresponding bond lengths in the dinuclear FeIII di-μ-acetato complex (av. 2.054 Å) which shows a stronger antiferromagnetic coupling (J = −6.0 cm−1) [34,35]. In fact, the experimental antiferromagnetic in+ teractions observed for the complexes 1 and [FeIII 2 (bbpmp)(μ-OAc)2] are −1 for 1 and consistent with the expected J values (J = − 1.5 cm + J = −4.0 cm−1 for [FeIII 2 (bbpmp)(μ-OAc)2] ) calculated from the empirical Gorum and Lippard equation ((J = A(e BP(A) ), where A = 8.763 × 1011, B = − 12.663 and P represents a half of the shortest superexchange pathway between two iron centers) [36]. Therefore, the difference of ~ 0.1 Å in the Fe–O–Fe pathway distance (P) of the phenolate bridge, is most probably the main factor responsible for the distinct J values observed for these complexes since this model only employs the structural P parameter for the J prediction [36,37]. 3.4 . Solution studies: potentiometric titration, UV–Vis, electrochemistry and ESI-MS These experiments were performed to assess the physico-chemical properties of 1 and the presence of water molecules coordinated to metal centers when the complex is in solution, in order to determine the relevant catalytic species for catecholase activity and the hydrolysis of diester bonds (vide infra). The potentiometric titration studies of complex 1 were performed in a mixture of MeOH/H2O (50/50% v/v) and the results showed the neutralization of 3 mols of KOH per mol of complex in the pH range of 2.50 to 10.50. Through the treatment of these data (see Section 2.2) three deprotonation constants were obtained, with values of 2.90 ± 0.10, 6.25 ± 0.20 and 7.56 ± 0.20. Scheme 2 shows a proposal for the species in equilibria in solution and the species distribution curves can be seen in Fig. 3 [38]. The first constant, pKa1, can be attributed to the deprotonation of a coordinated water molecule and the formation of a μ-OH bridge between the FeIII centers. For the [(H2O)FeIII(μ-OH)ZnII(H2O)(bpbpmp)]2+ complex (bpbpmp2− is the deprotonated form of the ligand H2bpbpmp shown in Chart 1 — right), the formation of this bridge has been reported to occur at pH 2.93 [38]. On the other hand, this pKa could not be determined experimentally in the pH range 2.4–10.9 for the corresponding [(H2O)FeIII(μ-OH)FeIII(H2O)(bpbpmp)]3+ complex, most probably due to the higher Lewis acidity of the Fe(III) center containing the softer tridentate N3 group of H2bpbpmp [8]. Indeed, it has been assumed that the {FeIII(μ-OH)FeIII} species is formed when the complex is dissolved in 10

6

3

cm K mol

8

χT,

-1

9

7

5 4 0

50

100

150

200

250

300

Temperature, K Fig. 2. Magnetic susceptibility as a function of the temperature for 1.

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aqueous solution at pH = 2.4 and deprotonation of the μ-OH bridge leads to the formation of the {FeIII(μ-O)FeIII}species (pKa = 4.22). The second constant (pKa2) can be assigned to the deprotonation of a terminal water molecule and formation of a hydroxo terminal ligand bound to an FeIII center while the third pKa3 is most probably associated with the deprotonation of a water molecule bonded to the second FeIII center. Alternatively, dissociation of the μ-OH bridge to form a {FeIII(μ-O)FeIII}unit (pKa2 = 6.25) is also reasonable if we consider that the two phenolate groups of the ligand bbpmp3− decrease the Lewis acidity of the FeIII centers in 1 when compared to the dinuclear unsymmetrical FeIII complex with the bpbpmp2− ligand. Nevertheless, such an hypothesis is in disagreement with the hydrolase-like activity of 1 (see Section 3.5). Based on the species distribution obtained from the potentiometric titration studies, electronic spectra of 1 were recorded in aqueous solutions at the pH values at which the maximum concentration of each species occurs. As expected, the spectra (Fig. S1) were found to be very sensitive to a change in the solution pH. Under acidic experimental conditions (pH = 2.0) the spectrum shows a well-defined band at 550 nm (ε = 3720 M−1·cm−1), which is assigned to a ligand-to-metal charge transfer (LMCT) process, from the pπphenolate → dπ* orbitals of the FeIII ions within the complex [(H2O)2FeIIIFeIII(OH2)2]3 + (A — Scheme 2). This spectrum is similar to the solid state diffuse reflectance spectrum of 1 (λmax = 555 ± 5 nm, see inset in Fig. S1), which suggests that the geometry and coordination of the ligand bbpmp3− around the FeIII centers is maintained when the complex is dissolved under these experimental conditions (pH = 2). At pH 4.2, [(OH2)FeIII(μ-OH)FeIII(OH2)]2+ (B — Scheme 2) is the predominant species present in solution and the spectrum shows a λmax at 530 nm with ε = 3590 M−1·cm−1. At pH 6.8 the maximum is again shifted to a higher energy value (λmax = 500 nm; ε = 3590 M− 1·cm− 1) due to the formation of the [(OH)FeIII(μ-OH) FeIII(OH2)]+ (C — Scheme 2) complex. The higher energies observed for species B and C when compared to A are most probably due to a lower order of acidity of the ferric centers in B and C, the t2g orbitals in these species having a higher energy than the t2g orbitals in A [35]. These results indicate a significant change in the coordination sphere of the FeIII centers when the pH is increased. A similar behavior (blue shift) was observed when the spectra were recorded under kinetic conditions (Fig. S6 and Fig. S7) showing that the same species are present during the kinetic assays. The redox behavior of complex 1 was investigated by cyclic voltammetry (CV) in aqueous solution in the potential range of − 0.5 to +0.7 V vs. the NHE (normal hydrogen electrode) using 0.2 M NaCl as the supporting electrolyte and distinct pH conditions. As shown in Fig. 4, at pH 2 two irreversible processes at E1pc = + 0.40 V and E2pc = +0.27 V vs NHE are observed. Taking into consideration the potentiometric titration data (Scheme 2), under these experimental conditions (pH = 2.0) the species A = [(H2O)2FeIIIFeIII(OH2)2]3+, in which the FeIII centers are connected only by the μ-phenoxo bridge, is the most probable species present in solution. Thus, the first wave can be attributed to the redox couple FeIII/FeIII ⇌ FeIII/FeII while the second one is assigned to the FeIII/FeII ⇌ FeII/FeII process for the [(H2O)2FeIIIFeIII (OH2)2]3+ complex. When the pH is raised to 4.2 the CV becomes quasi-reversible and the reduction potentials are cathodically shifted by 0.3–0.5 V (Table 1) in agreement with the formation of the μ-hydroxo complex (species B = [(H2O)FeIII(μ-OH)FeIII(H2O)]2+ in Scheme 2). Finally at pH 6.8 a further cathodic shift can be observed as a consequence of the deprotonation of a terminal FeIII-bound water molecule (pKa2 = 6.25) and the generation of the species C = [(OH)FeIII(μ-OH)FeIII(H2O)]+ (Scheme 2). Comparing the reduction potentials of the species [(H2O)FeIII(μOH)FeIII(H2O)]2+ with those of the di-iron complex with two acetate bridges and the same ligand, these values are anodically shifted by 0.3 and 0.5 V for the first and second reduction processes, respectively [11,21]. The higher Lewis acidity of the iron(III) centers in 1 and consequent anodic shift are due to the absence of acetate groups, which increases the electronic density over the metal centers. A similar anodic

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Scheme 2. Proposed equilibria for complex 1.

shift has been observed for the FeIIINiII complexes with two acetate bridges ([FeIII(μ-OAc)2NiII(bpbpmp)]+, E1/2 = − 0.54 V vs NHE) and with one acetate bridge ([FeIII(μ-OAc)NiII(bpbpmp)]+, E1/2 = − 0.18 V vs NHE) for the FeIII/FeII redox process [21]. It is also worth noting that a μ-OH bridge is present in 1 under the experimental conditions applied in the electrochemical analysis. Neves and co-workers observed that in di-iron(III) complexes a μ-OH or a μ-OAc results in a similar electronic density over the metal center, and in this regard the values obtained in this study are comparable with those reported in the literature [34]. Mass spectrometry studies were performed in CH3CN/water (50:50% v/v), the same solvent conditions employed in the hydrolysis kinetic assays. Five main groups of signals were observed at mass to charge ratios (m/z) of 307.6, 343.2, 614.2, 650.2 and 685.1 (Table 2 and Fig. 5). The prominent peak at m/z = 343.2 can be attributed to 2+ (b species — Table 2) in which the complex [FeIII 2 (μ-OH)(bbpmp)] − H2O and Cl ligands are probably dissociated from both FeIII centers during the ionization process. The peak at m/z = 685.1 is most likely as+ sociated with the [FeIII 2 (μ-O)(bbpmp)] (e) species with the loss of one + H from complex b. The other peaks at mass to charge (m/z) ratios of 307.6, 614.2 and 650.2 can be attributed to the mononuclear FeIII species (a), (c) and (d), respectively, containing the whole ligand in its protonated or deprotonated form and a Cl− anion (see Table 2). The assignments of the peaks are shown in Table 2 and the correct isotopic distribution simulations are shown in Fig. S2. Based on all of these results, it seems reasonable to assume that in aqueous solution the dinuclear [(OH)FeIII(μ-OH)FeIII(OH2)]+ and [(OH)FeIII(μ-OH)FeIII(OH)]0 structural units are present as the catalytically active species in the hydrolysis of diester bonds and catechol oxidation, as demonstrated in the reactivity studies (vide infra). Importantly, based on the physico-chemical studies and the X-ray structure of 1, it can be assumed that in aqueous solution the species [(OH 2)FeIII (μ-OH) FeIII(OH2)]2 + (B — Scheme 2) is formed with a meridional to facial rearrangement of the N2O pendant arms of the ligand bbpmp3− in such

a way that the two coordinated water molecules are cis-oriented to each other. 3.5. Reactivity studies 3.5.1. Hydrolysis of phosphate diester bonds The hydrolytic activity of 1 was evaluated in the hydrolysis of the diester substrate 2,4-BDNPP, under conditions of excess substrate at 25 °C. Kinetic parameters were calculated using the method of initial rates, where the reactions were monitored following an increase in absorbance at 400 nm due to the release of 2,4-dinitrophenolate as a product [21,39]. In order to determine the pH of maximum activity, as well as to estimate the active species, studies on the pH dependence of the hydrolysis of 2,4-BDNPP were carried out. The plot of the initial rates (V0) versus pH shows a bell-shaped profile, with a maximum at pH 6.5, as shown in Fig. 6. The kinetic pKa values obtained from sigmoidal fits (Boltzmann equation) of the curve in Fig. 6 were 5.53 ± 0.15 and 7.33 ± 0.20, which are in relatively good agreement with the pKa1 = 6.25 and pKa2 = 7.56 obtained from the potentiometric titration experiments, indicating that [(OH)FeIII(μ-OH)FeIII(OH2)]+ is the catalytically active species in the hydrolysis of the diester. Interestingly, cyclic voltammetric experiments of 1 in MeOH:H2O (1:1), pH = 6.8, Fig. S3 reveals that only one of the reduction potentials (Fig. S3) is cathodically shifted by 100 mV when the less activated substrate bis-p-nitrophenylphosphate (BNPP) is added to 1, thus suggesting its monodentate binding to one of the FeIII centers. Indeed this hypothesis is strongly supported by the fact that this species contains a labile FeIII–OH2 coordination site which can bind and activate the substrate and an adjacent FeIII–OH group which can perform the nucleophilic attack on the phosphorus atom of the substrate [8]. In addition, the pKa values found for 1 are comparable to those reported

100 A

D

B

% species

80

C

60

40

20

0 2

4

6

8

10

pH Fig. 3. Graph of species distribution as a function of pH for complex 1. A = [(H2O)2FeIIIFeIII(OH2)2]3+; B = [(OH2)FeIII(μ-OH)FeIII(OH2)]2+; C = [(OH)FeIII(μ-OH)FeIII (OH2)]+; D = [(OH)FeIII(μ-OH)FeIII(OH)].

Fig. 4. Cyclic voltammogram of 1. Conditions: Solvent — water (pH 2.0, 4.2 and 6.8), electrolyte — 0.2 M NaCl, working electrode — carbon, auxiliary electrode — platinum, and reference electrode — Ag/AgCl.

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Table 1 Reduction potentials for 1 at different pH values. pH

E1pc (V) Fe1

E2pc (V) Fe2

2.0 4.2 6.8

0.402 0.114 0.067

0.271 −0.207 −0.243

for a dinuclear FeIII complex (5.0 and 7.03) containing similar coordination environments around the metal centers [35]. The dependence of the reaction rate on the substrate concentration was investigated at pH 6.5, the optimum pH of activity, as described above. In these experiments it was observed that, initially, the hydrolysis rate increases linearly with the increase in [2,4-BDNPP] (first-order dependence), but with an increasing concentration of the substrate a deviation from linearity, tending toward a saturation curve (zero-order), occurs. This dependence of the initial rates on the substrate concentration suggests that the hydrolysis reaction occurs with the formation of a substrate-complex intermediate. Thus, the Michaelis–Menten model could be applied and the kinetic parameters were obtained from nonlinear fits of the Michaelis–Menten equation. The dependence of the rate of hydrolysis reaction catalyzed by 1 on the substrate concentration is shown in Fig. 7 and the kinetic parameters obtained, together with some data taken from the literature, are given in Table 3 [8,39–41]. In this table it can be observed that 1 is very effective in converting the substrate to products, with an acceleration of 6722 times compared with the uncatalyzed reaction (knc (spontaneous hydrolysis–uncatalyzed reaction turnover number) = 1.8 × 10−7 s−1) [10], under the same pH and temperature conditions. A comparison of the kinetic parameters for the di-iron(III) complex [(OH)FeIII(μ-OH)FeIII(OH2)(bpbpmp)]2+ (2) containing the unsymmetrical ligand (bpbpmp2−) with those for 1 reveals that while the association constant (Kass)of the substrate 2,4-BDNPP for this complex is significantly higher the kcat (turnover number) is around 150 times lower than that obtained for 1. The lower pKa for 2 (4.2) when compared to that for 1 (6.25) involving the formation of the catalytically active species [(OH)FeIII(μ-OH)FeIII(OH2)] is most probably responsible for this difference in reactivity, given that the FeIII-bound hydroxide in 1 is a much stronger nucleophile than the corresponding hydroxide in 2. The lower affinity of 1 for the anionic substrate 2,4-BDNPP can be explained in terms of the formal charge of complexes 1 and 2. In 1, the active species has a charge of 1+ while in 2 the charge is 2+, hence the higher affinity of 2,4-BDNPP for 2. On the other hand the catalytic activity kcat = 1.21 × 10− 3 s−1 for 1 compares favorable to the value of 1.33 × 10− 3 s− 1 for the di-iron(III) complex 3 reported by Schenk et al. However, in this case, a direct comparison in relation to the kinetic parameters KM (Michaelis–Menten constant) and kcat/KM is not appropriate, since, for complex 3, important second coordination sphere effects of the cyclam ligand anchored to the dinuclear catalytic site influence the affinity of the substrate 2,4-BDNPP for the complex [41]. We also tested the ability of 1 to hydrolyze the monoester 2,4-DNPP, one of the products of the hydrolysis of the diester 2,4-BDNPP, and after 6 h only the background reaction was observed, thus indicating that 1 shows only diesterase activity. Also, 6 turnovers in 24 h were observed for this complex at pH = 6.5, which indicates that the intermediate

Fig. 5. MS-ESI spectrum for m/z ratio of 150 to 900 for 1 in CH3CN/H2O (50/50% v/v).

containing the monoester is dissociated to regenerate the catalyst. Finally, the deuterium kinetic isotope effect kH/kD = 0.97 (kH/kD = ratio between the turnover number measured in regular solvent and the turnover number measured in deuterated solvent) obtained under the same conditions as those with H2O supports the hypothesis that no proton transfer is involved in the rate-limiting step of the reaction. Therefore, based on the crystal structure, physico-chemical properties and phosphatase-like activity studies, we propose the following mechanism (Fig. 8) for the hydrolysis of the phosphate diester 2,4BDNPP catalyzed by 1 (active species = [(OH)FeIII(μ-OH)FeIII(OH2)]+). The proposed catalytically active species [(HO)FeIII(μ-OH)FeIII(H2O)] possesses a water molecule in one of the FeIII centers, which is promptly replaced by a substrate molecule, and one hydroxo bound to the other FeIII center, available for the nucleophilic attack on the phosphorus atom. The saturation profile observed in Fig. 7 is in accordance with the formation of a complex–substrate intermediate and hence the rate-limiting step must involve the intramolecular attack of the FeIII– OH group on the phosphorus atom with concomitant release of the product of the reaction (2,4-dinitrophenolate). 3.5.2. Oxidation of 3,5-di-tert-butylcatechol The oxidation of the substrate 3,5-DTBC in the presence of complex 1 was studied using the method of initial rates. The experiments were carried out by monitoring the release of the product 3,5-DTBQ, which

Table 2 ESI-MS data for complex 1 in CH3CN/H2O (50/50% v/v). m/z a = 307.6 (2+) b = 343.2 (2+) c = 614.2 (1+) d = 650.2 (1+) e = 685.1 (1+)

Formula III

Fe C35H35N4O3 FeIII 2 C35H34N4O4 FeIII2C35H34N4O4 FeIIIC35H3N4O3Cl FeIII 2 C35H33N4O4

Species FeIII(Hbbpmp) + H+ FeIII 2 (μ-OH)(bbpmp) FeIII(Hbbpmp) FeIII(Hbbpmp)(Cl) + H+ FeIII 2 (μ-O)(bbpmp)

Fig. 6. pH dependence of reaction rate of hydrolysis of 2,4-BDNPP catalyzed by 1. Conditions: [1] = 3.9 × 10−5 M, [2,4-BDNPP] = 5.0 × 10−3 M, [buffer] = 0.05 M, CH3CN/H2O solution (50/50% v/v) at 25 °C.

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Fig. 7. Dependence of the reaction rate of hydrolysis of 2,4-BDNPP on the substrate concentration for complex 1. Conditions: [1]final = 4.9 × 10−5 M, [2,4-BDNPP]final = 1 × 10−3–15 × 10−3 M, [MES]final = 0.05 M (pH = 6.5), I = 0.05 M in CH3CN/H2O (50/ 50% v/v) solution at 25 °C.

shows a strong band at 400 nm. This substrate is commonly used due to its low redox potential, facilitating the oxidation to quinone, and its bulky substituents, which slow down additional oxidation reactions. Under an argon atmosphere only one equivalent of quinone was formed, suggesting the role of oxygen in the re-oxidation of the FeII centers in the catalytic cycle (Fig. S4). The oxidation reaction of 3,5-DTBC was highly dependent on the pH of the solution, as can be observed from the sigmoidal-shaped curve in Fig. 9 (pH range 5.5 to 10). The data were fitted using a Boltzmann model, and a sigmoidal fit of the curve revealed a kinetic pKa of 8.55, which is 1.0 pH unit higher than the value (7.56) obtained from the potentiometric titration experiments. This pKa may be related to the deprotonation of 3,5-DTBC and its bridging coordination to the metal centers [42]. In order to confirm the bidentate bridging coordination mode of 3,5-DTBC, electrochemical studies of 1 with and without the substrate were carried out in MeOH:H2O (2:1), pH = 6.8, as shown in Fig. S5. In the absence of substrate the square wave voltammogram (SWV) shows two reduction potentials at E1pc = 0.18 V and E2pc = −0.07 V vs. NHE. Addition of one equivalent of 3,5-DTBC to the solution of 1 immediately results in an anodic shift of both waves to E1pc = 0.31 V and E2pc = 0.10 V vs. NHE, a result which is consistent with a bridging bidentate binding of the catechol group to the dinuclear FeIII 2 center. Further addition of 3,5-DTBC to the reaction mixture does not change the reduction potentials of the catalyst/substrate adduct. According to the potentiometric titration (Scheme 2), [(OH)FeIII(μ-OH) FeIII(OH)] is the major species at pH values above 7.56. Since the oxidation of the 3,5-DTBC occurs with the simultaneous transfer of two electrons, it is plausible to suppose that this is the active species in the catalytic process. The graph of the initial reaction rates (V0) vs 3,5-DTBC concentrations, at pH 9.0, shows a saturation profile (Fig. 10) suggesting that the oxidation reaction occurs with the formation of an intermediate

substrate/complex. The kinetic parameters obtained from the nonlinear regression of the Michaelis–Menten equation are shown in Table 4. According to the data shown in Table 4, it can be observed that 1 has a lower affinity for the substrate when compared to the dinuclear FeIII complex containing the unsymmetrical ligand bpbpmp2−. This lower affinity of 1 for the deprotonated form of the substrate 3,5-DTBC can be explained in terms of the formal charge of the complexes 1 and III + [FeIII 2 (bpbpmp)] : in 1, the active species is neutral while in [Fe2 (bpbpmp)]+ the charge is 1 +, which explains the higher affinity of + the substrate for [FeIII 2 (bpbpmp)] . On the other hand, the kinetic parameters kcat and kcat/KM for the dinuclear FeIII 2 complexes 1 and 2 are comparable to those reported for dinuclear CuII2 complexes with the same substrate and a similar mechanism, thus suggesting that dinuclear FeIII complexes can also be used as efficient synthetic catechol oxidases [8,43]. The oxidation reaction of 3,5-DTBC catalyzed by 1 was also followed for ~ 24 h and no formation of 3,5-DBSQ radical species could be observed since in the 500–700 nm range the spectrum remains unchanged (Fig. S8) [44]. The accumulation of H2O2 during turnover was confirmed by means of the iodometric I− 3 assay (Fig. S9 and Fig. S10) [22–25], which indicates that re-oxidation of the FeIIFeII species to the active FeIIIFeIII species occurs with the concomitant formation of hydrogen peroxide. Very important to observe here, is the fact that kinetic experiments carried out in the presence of equimolar amounts of H2O2 do not affect the catalytic activity of 1. In fact, such information confirms that molecular dioxygen is responsible for the rapid re-oxidation of the FeIIFeII species. In summary, from the combined physico-chemical data and the catecholase-like experiments we can propose a feasible mechanism for the oxidation of 3,5-DTBC promoted by complex 1 as shown in Fig. 11. At pH 9.0, in the first step of the reaction, we propose that the species [(OH)FeIII(μ-OH)FeIII(OH)] interacts with 3,5-DTBC promoting its deprotonation with subsequent coordination to the FeIII centers followed by the formation of the adduct complex/substrate. In the next step, the intramolecular electron transfer reaction, which is the determinant step of the reaction, occurs with the release of 3,5-DTBQ and the reduction of both FeIII centers. Finally, in the presence of molecular oxygen, the FeII2 complex is re-oxidized and H2O2 is formed, thus completing the catalytic cycle. It is worth noting that this mechanism is distinct from the type III enzyme catechol oxidase in which molecular oxygen is reduced to water [45] but is similar to that proposed for many dinuclear biomimetic copper(II) complexes [5,46,47]. 3.6 . DNA cleavage Initial assays were conducted at the pH at which the highest activity of 1 in the 2,4-BDNPP hydrolysis occurred (i.e., pH 6.5), as described in the kinetics section (section 3.3.1). Herein, the plasmid DNA was treated at different complex concentrations (up to 0.1 mM) and left to react for 16 h at 50 °C. Complex 1 was able to induce single-strand DNA cleavage in a concentration-dependent fashion (Fig. 12). As the complex concentration increases from 10 to 100 μM, the amount of cleaved DNA (F II) also increased, reaching ~80% at the highest concentration assayed. The phosphate groups present in the nucleotide structure of DNA are negatively charged at pH 6.5 and thus it is reasonable to assume that the

Table 3 Kinetic parameters for the hydrolysis of the model substrate 2,4-BDNPP. Complex 1 [FeIII(μ-OH)FeIII(bpbpmp)]2+(2)a 1 b FeIII 2 H2L (3) a

kcat ×103 s−1 1.21 ± 0.12 0.084 1.33

[48]; b[41]; L1 = 4,11-dimethyl-1,8-bis{2-[N-(di-2-pyridylmethyl)amino]ethyl}cyclam.

KM mM 7.20 ± 1.08 0.19 1.94

Kass (M−1) 138.7 ± 20.8 5263 515

kcat/KM (M−1s−1) 0.17 0.44 0.68

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85

Fig. 8. Proposed mechanism for the hydrolysis of 2,4-BDNPP catalyzed by 1.

electrostatic interactions between the complex and DNA could contribute to the binding and consequently the cleavage event. To verify this, DNA cleavage reactions were performed with increasing concentrations of NaCl (25 to 250 mM) to alter the ionic strength of the reaction medium (Fig. 13). Even in the presence of the lowest concentration of NaCl assayed (25 mM), the complex activity was fully inhibited. Sodium ions can be electrostatically attracted to the anionic portion of DNA, blocking the interaction between 1 and DNA and preventing cleavage. However, chloride ions, from NaCl, could also bind to the metal centers, disrupting the catalytic mechanism of nucleophile formation. For this reason, the same assays were performed with lithium perchlorate,

since perchlorate ions cannot affect the metal center as proposed for the chlorides (Fig. 14). In this second scenario, the DNA cleavage activity of 1 was also inhibited under strongly ionic conditions, but to a lesser extent than in the case of sodium chloride, which suggests that 1 may bind to DNA via electrostatic interactions, and the chloride ions may have a considerable impact on the metal center. To elucidate whether the mechanism of DNA cleavage performed by 1 could be radical-dependent, different inhibitors of ROS were added to the reaction medium (Fig. 15). The addition of DMSO (10%), KI (10 mM), SOD (20 units) or NaN3 (10 mM) partially reduced the activity of 1, suggesting the involvement of ROS species in the oxidative mechanism of the strand scission event. However, a mixed-type mechanism,

Fig. 9. pH dependence of the rate of oxidation of 3,5-DTBC catalyzed by 1. Conditions: [1] = 2.9 × 10−5 M, [3,5-DTBC] = 5.0 × 10−3 M, [buffer] = 1.0 M, MeOH/H2O solution (32:1 v/v) at 25 °C, buffer = MES, TRIS, and CHES.

Fig. 10. Dependence of the initial rate of oxidation of 3,5-DTBC on the substrate concentration catalyzed by 1. Conditions: [1]final = 4.4 × 10−5 M, [3,5-DTBC]final = 2.0 × 10−3–15.10−3 M, [CHES]final = 6.0 × 10−2M (pH = 9.0); in MeOH/H2O solution (32:1 v/v) at 25 °C.

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Table 4 Kinetic parameters for the oxidation of 3,5-DTBC by 1 and [Fe2 III(bpbpmp)]+. Complex

kcat (×103 s−1)

KM (mM)

Kass (M−1)

kcat/KM (M−1·s−1)

1 [Fe2 III(bpbpmp)]+a

9.54 11.6

7.30 0.71

137.0 1408.5

1.30 16.34

a

Ref. [48].

involving a hydrolytic-like pathway, cannot be ruled out since, even in the presence of ROS inhibitors, there is partial DNA cleavage activity.

4. Conclusions In summary, we synthesized and fully characterized a new dinuclear iron(III) complex 1 with the symmetrical N4O3-donor ligand H3bbpmp. The X-ray structure of 1 shows that the N2O-donor pendant arm of the ligand coordinated to each FeIII center in a meridional fashion, with the μ-Ophenolate unit, one chloro and one water molecule completing the octahedral environment. The dinuclear complex showed a very weak antiferromagnetic coupling (J = −0.29 cm−1), in good agreement with the Gorum and Lippard equation. The complex was also characterized in solution through electronic spectroscopy, electrochemistry, potentiometric titration and ESI-MS. The results obtained allowed us to propose the [(OH)FeIII(μ-OH)FeIII(H2O)]+ and [(OH)FeIII(μ-OH)FeIII(OH)] species as being, respectively, the catalysts for hydrolase-like and catecholaselike activities (catalytic promiscuity). Finally, it was also shown that 1 efficiently promotes the cleavage of plasmid DNA. Detailed studies using ROS inhibitors and increased ionic strength in these experiments suggested that a mixed (oxidative/ hydrolytic) mechanism may operate in the DNA strand scission, in agreement with the catecholase-like and hydrolase-like activities of 1.

Fig. 12. Cleavage of supercoiled DNA by 1 with 16 h of reaction at 50 °C in MES buffer (10 mM, pH 6.5) and different concentrations of complex (10 to 100 μM) (n = 2).

Abbreviations 2,4-BDNPP bis(2,4-dinitro)phenylphosphate 2,4-DNPP 2,4-dinitrophenylphosphate 2,4-DNP 2,4-dinitrophenolate 3,5-DTBC 3,5-di-tert-butylcatechol 3,5-DTBQ 3,5-di-tert-butylquinone 3,5-DBSQ 3,5-di-tert-butylsemiquinone anion radical CHES 2-(cyclohexylamino)ethanesulfonic acid CV cyclic voltammetry EDTA ethylenediamine tetracetic acid ESI-MS electrospray ionization mass spectrometry

Fig. 11. Proposed mechanism of oxidation of 3,5-DTBC promoted by complex 1.

T.P. Camargo et al. / Journal of Inorganic Biochemistry 146 (2015) 77–88

Fig. 13. Effect of ionic strength (NaCl) on cleavage of supercoiled DNA by 1 (A) at 25 μM with 16 h of reaction at 50 °C in MES buffer (10 mM, pH 6.5). Before the addition of 1, different concentrations of NaCl (25 to 250 mM) were added to reaction medium. The sample “−” denotes the reaction in the absence of complex and NaCl.

H2bpbpmp {2-[[(2-hydroxybenzyl)(2-pyridylmethyl)]aminomethyl]6-bis(pyridylmethyl)aminomethyl}-4-methylphenol H3bbpmp 2,6-bis{[(2-hydroxybenzyl)(pyridin-2yl)methylamino]methyl}-4-methylphenol HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 4,11-dimethyl-1,8-bis{2-[N-(di-2L1 pyridylmethyl)amino]ethyl}cyclam LMCT ligand to metal charge transfer MES 2-(N-morpholino)ethanesulfonic acid NHE normal hydrogen electrode PAP purple acid phosphatase ROS reactive oxygen species SOD superoxide dismutase TMS tetramethylsilane

87

Fig. 15. Effect of ROS scavengers on cleavage of supercoiled DNA by 1 (A) at 25 μM with 16 h of reaction at 50 °C in MES buffer (10 mM, pH 6.5). The ROS scavengers used were: DMSO (10%), KI (10 mM), SOD (20 units) and NaN3 (10 mM). The controls (reaction without complex) for each inhibitor show DNA cleavage similar to or even lower than the control presented in the figure (data not shown).

Acknowledgments Financial support for this study was received from the Brazilian governmental agencies CNPq (472956/2013-2), CAPES (STINT Proc 009/2013) and INCT-Catálise. Appendix A. Supplementary data Crystallographic data (without structure factors) for the structure reported in this paper (complex 1) have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC-1027986. Copies of the data can be obtained free of charge from the CCDC (12 Union Road, Cambridge CB2 1EZ, UK; tel: (+ 44) 1223-336-408; 30 fax: (+ 44) 1223-336-003; e-mail: deposit@ccdc. cam.ac.uk; website www.ccdc.cam.ac.uk). Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jinorgbio. 2015.02.017. References

Fig. 14. Effect of ionic strength (LiClO4) on cleavage of supercoiled DNA by 1 (A) at 25 μM with 16 h of reaction at 50 °C in MES buffer (10 mM, pH 6.5). Before the addition of the complex, different concentrations of LiClO4 (25 to 250 mM) were added to the reaction medium. The sample “−” denotes the reaction in the absence of complex and LiClO4.

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Synthesis, characterization, hydrolase and catecholase activity of a dinuclear iron(III) complex: Catalytic promiscuity.

Herein, we report the synthesis and characterization of the new di-iron(III) complex [(bbpmp)(H2O)(Cl)Fe(III)(μ-Ophenoxo)Fe(III)(H2O)Cl)]Cl (1), with ...
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