ZHANG Yong HU Ji-Wei ZHOU Xi CHEN Shi-Ming MA Zhi-Gng CHEN Xue-Mei (School of Chemicl nd Mterils Engineering,Huei Polytechnic University, Hungshi 435003, Chin)(Hungshi Center Hospitl, Hungshi 435001, Chin)

1 INTRODUCTION

Model enzymes are being developed and extensive studies were conducted in order to gain an understanding of the factors underlying the relationship between coordination geometry and the nature of donor ligands. Dinuclear copper proteins like hemocyanin, tyrosinase and catechol oxidase are known as type-3 copper(II) proteins. The active site contains dicopper core in which both copper ions are surrounded by three nitrogen donor atoms of histidine residues[1-2]. It is for this reason that nitrogen donor ligands such as pyridine and imidazole are a logical choice in modeling the copper proteins since the former has pKa values that are close to those found in histidyl moieties in several enzymes[3].Catechol oxidase catalyzes the oxidation of a broad range of catechols to the corresponding o-quinones.The resulting highly reactive quinones autopolymerize to form brown polyphenolic catechol melanins, a process thought to protect the damaged plant from pathogens or insects[4]. Recently, many binuclear copper(II) complexes have been reported as model compounds of catechol oxidase[5-8]. A great number of researches have indicated that various structural factors may affect their catalytic activity,namely the metal-metal distance, type of exogenous bridging ligand, coordination geometry around the metal ion, and flexibility of the ligand[9-10]. We show great interest in catechol oxidase model complexes containing pyridine-rich ligands. Herein, to continue this work, we report the synthesis, crystal structure and catechol oxidase catalytic activity of a dicopper complex [Cu2LCl2](ClO4)2.

2 EXPERIMENTAL

2.1 Materials and physical measurements

All reagents were purchased from commercial companies and directly used unless stated otherwise.The melting point was determined with an XT4A micromelting point apparatus and was uncorrected.The IR spectra were measured on a Perkin-Elmer Spectrum BX FT-IR instrument in tablets with potassium bromide. Elemental analyses were carried out on a Perkin-Elmer 2400 instrument. UVVis spectra were recorded on an Analytik jena Specord 210 spectrophotometer.1H NMR spectra were recorded on a Varian Mercury 400 spectrometer at 400 MHz. Electrospray ionization mass spectra (ESI-MS) were acquired on an Applied Biosystems API 2000 LC/MS/MS system.

2.2 Synthesis of the ligand L

The two-step synthesis of the ligand L is outlined in Scheme 1.

Scheme 1. Synthesis of the ligand L

1) 2-bromo-N,N-bis(pyridin-2-ylmethyl)ethan-1-amine (a)

A mixture of di-(2-picoyl)amine (1.99 g, 10 mmol)and K2CO3(1.38 g, 10 mmol) in acetonitrile (120 mL) was added dropwise to a solution of 1,2-dibromoethane (1.88 g, 10 mmol). The reaction was refluxed for 6 h with stirring. The solvent was evaporated and the residue was poured into water(100 mL) and stirred for 2 min. The product was extracted with CH2Cl2, dried over MgSO4, filtered,and evaporated. The product was purified by flash column chromatography using dichloromethane/ethyl acetate (1:10) as the eluent. Yield 1.51 g(50%). Anal. Calcd. for C14H16N3Br: C, 54.92; H,5.27; N, 13.72. Found: C, 54.58; H, 5.64; N, 13.52.1H NMR (CDCl3, 400 MHz), (ppm): 2.68 (t, 2H),3.88 (t, 2H), 4.06 (s, 4H), 7.25～7.31 (m, 4H), 7.78(m, 2H), 8.49 (d, 2H). ESI-MS m/z: 306.20(M+).

2) 2,2?-(Piperazine-1,4-diyl)bis(N,N-bis(pyridin-2-ylmethyl)ethan-1-amine) (L)

A mixture of the intermediate (a) (1.22 g, 4 mmol),piperazine (0.17 g, 2 mmol) and K2CO3(1.10 g, 8 mmol) in acetonitrile (150 mL) was refluxed and stirred for 24 h. The solution was evaporated at reduced pressure and the crude residue was purified by column chromatography using dichloromethane/methane (5:1) as the eluent. Yield: 0.55 g (52%).Anal. Calcd. (%) for C32H40N8: C, 71.61; H, 7.51; N,20.88. Found (%): C, 71.33; H, 7.62; N, 21.05.1H NMR (CDCl3, 400 MHz), (ppm): 2.18(t, 8H), 2.48 (t,8H), 3.92 (s, 8H), 7.22～7.36 (m, 8H), 7.68 (m, 4H),8.62 (d, 4H). ESI-MS m/z: 536.73 (M+).

2.3 Synthesis of the complex

The ligand L (0.54 g, 1 mmol) was dissolved in ethanol (40 mL), then CuCl2·2H2O (0.34 g, 2 mmol)and NaClO4(0.12 g, 1 mmol) were added to the stirred solution at 333 K for 6 h. The solution was filtrated, obtaining blue crystals suitable for X-ray diffraction studies after a week. m.p: 206～208 ℃.Anal. Calcd. (%) for C32H35N17O9Cu: C, 44.38; H,4.04; N, 27.50. Found (%): C, 44.04; H, 4.46; N,27.24. IR (KBr, cm-1): 2940(s) -CH2-; 1635(s),1545(w), 1405(w), 956(w), 835(m), 742(w)pyridine.

2.4 Structure determination of the complex

A blue crystal of the title compound having approximate dimensions of 0.30mm × 0.30mm ×0.20mm was mounted on a glass fiber in a random orientation at 298(2) K. The crystallogra phic data were collected with MoKα radiation (λ = 0.71073 ?)using π and σ scan mode. A total of 7896 reflections were collected in the range of 1.83＜θ＜25.68° at room temperature. The structure was solved by direct methods and semi-empirical absorption corrections were applied. The non-hydrogen atoms were located by direct phase determination and full-matrix least-squares refinement on F2, while the hydrogen atoms for non-water protons were treated using the riding mode. The final cycle of full-matrix least-squares refinement was based on 3675 independent reflections (I ＞ 2σ(I)). All calculations were carried out on a PC using SHELXTL program[11-12]. The final R = 0.0669 and wR = 0.1486(w = 1/[(Fo2) + (0.0600P)2+ 0.5659P],where P = (Fo2+ 2Fc2)/3). S = 1.071,(Δρ)max= 1.303, (Δρ)min= –0.674 e/?3and (Δ/σ)max= 0.000.

2.5 Catechol oxidase mimic activity

The catechol oxidase mimic activity of the complex has been determined using 3,5-di-tert-butylcatechol (3,5-DTBC) as the substrate[13-14]. Kinetic experiments for the oxidation of 3,5-di-tert-butylcatechol were monitored spectrophotometrically on an Analytik jena Specord 210 spectrophotometer.Increase of the characteristic absorption band of the product 3,5-di-tert-butyl-obenzoquinone (3,5-DTBQ)at 400 nm was measured as a function of time.Freshly prepared stock solutions of the complex(1.0×10-3mol·L-1) and 3,5-DTBC (1.0×10-2mol·L-1)were used under aerobic conditions at room temperature. To determine the dependence of the rate on the substrate concentration and various kinetic parameters, a 10?4mol·L-1solution of the complex was treated with 5, 10, 20, 40, 60, 80 and 100 equiv. of substrate. The absorbance versus wavelength(wavelength scan) of the solutions was recorded at a regular time interval of 5 min. A kinetic treatment on the basis of the Michaelis-Menten approach was applied, and the results were evaluated from the Lineweaver-Burk double-reciprocal plots. The kinetic determination in the absence of the title complex was taken as a control.

3 RESULTS AND DISCUSSION

Crystals of the complex were obtained by slow evaporation of an ethanol solution at room temperature. In the crystal structure (Fig. 1), each copper(II) ion is five-coordinated by one chloride ion Cl(1), two pyridine nitrogen atoms (N(2), N(3)) and two amine nitrogen atoms (N(1), N(4)) from the ligand L, forming a distorted tetragonal pyramidal coordination geometry. N(2), N(3), N(4) and Cl(1)make up the square plane of the tetragonal pyramid,and the copper(II) ion deviates from the plane(0.251 ?), while N(1) occupies the apical position.The square plane around each copper(II) ion is parallel, and the Cu··Cu distance is 6.486 ?. The Cu–N bond distances range from 1.995(5) to 2.388(4) ?, and the amino N atoms (N(1), N(4)) are slightly farther away from the copper(II) ion than the pyridine N atoms (N(2), N(3)) (Table 1). The angles around the copper center vary from 81.85(19)° to 168.94(12)°. Each di-(2-picoyl)amine(DPA) moiety of the ligand adopts tridentate modes to bond one copper(II) ion, and the dihedral angles between two pyridine rings of DPA are both 13.17°. As shown in Table 2 and Fig. 2, the uncoordinated anion ClO4ˉ, known as a balancing negative charge, participates in forming hydrogen bonds. The molecules are stabilized by intermolecular C–H··O and C–H··Cl hydrogen bonds, leading to the forma-tion of a three-dimensional network.

Fig. 1. An ORTEP view of the structure of [Cu2LCl2]2+ (The ellipsoids are at the probability level of 30%,and the noncoordinating ClO4- are omitted for clarity. Symmetry code: A: –x, –y, –z)

Fig. 2. Packing of the complex in a unit cell

Table 1. Selected Bond Lengths (?) and Bond Angles (°)

Table 2. Hydrogen Bond Lengths (?) and Bond Angles (°)

3,5-DTBC can be easily oxidized to the corresponding quinine (3,5-DTBQ) due to its low quinone-catechol reduction potential, so it is often chosen as the substrate to study the catechol oxidase activity of the model compounds[13-14]. The rate constant for the complex was determined by initial rate method. The kinetic study of the oxidation of 3,5-DTBC to 3,5-DTBQ by the complex was investigated using UV-Vis spectroscopy, and the absorbance of the solution was measured in different pH values of methanol solution. Fig. 3 shows a linear relationship of absorbance[ln(Af-Ai)/(Af–At)] versus reaction time (t) in the presence of the complex. Here, Af, Aiand Atare the final, initial and the time t absorbance of the reaction solution, respectively. The results indicated that the oxidation reaction of 3,5-DTBC was of the first-order, and it increased with increasing the pH values. To determine the dependence of the rates on the substrate concentration and various kinetic parameters, solutions of the complex were studied by increasing the concentration of 3,5-DTBC (from 5 to 100 equiv.) under aerobic conditions. A first-order dependence was observed at low concentration of the substrate, whereas saturation kinetics was found at higher concentration of the substrate for the complex (Fig. 4). So, a treatment on the basis of the Michaelis-Menten approach,originally developed for enzyme kinetics, seemed to be appropriate. Several kinetic parameters including Michaelis-Menten constant (KM), the maximum initial rate (Vmax) and the catalytic constant (kcat)were obtained under different conditions, and these data were evaluated from the Lineweaver-Burk plot model, as shown in Table 3.

Fig. 3. Effect of different pH on the catechol oxidase mimic activity of the complex at room temperature

Fig. 4. Initial rates vs. substrate concentration for the 3,5-di-tert-butylcatechol oxidation reaction catalyzed by the complex (Inset shows the Lineweaver-Burk plot)

Table 3. Kinetic Data of the Title Complex

It is found that kcatof the complex toward 3,5-DTBC increased from 8.68 to 30.92 min-1at pH 6.0～9.0. Thus, the catalytic activity of the complex increased with increasing the pH values, and it can also be proved by the results of Fig. 3. It is reported that the model compounds of catechol oxidase showed the best catalytic activity when the Cu··Cu distance is ～3 ?, but the Cu··Cu distance (6.486 ?)of the complex is too long to bind coorperatively with the substrate molecules[15-16]. A probable explanation for its catechol oxidase catalytic reactivity was mainly due to its coordination environment.Each five-coordinated copper(II) ion had an unsaturated vacancy, and it could combine with the substrates[15-17].

In summary, we have prepared a dinuclear copper(II) complex, which was characterized by singlecrystal X-ray diffraction. It is shown that the kinetics of 3,5-DTBC catalyzed by the complex obeyed the Michaelis-Mentent equation, and its catechol oxidase catalytic activity increased with increasing the pH values.

(1) Than, R.; Feldman, A. A.; Krebs, B. Structural and functional studies on model compounds of purple acid phosphatases and catechol oxidases.Coord. Chem. Rev. 1999, 182, 211–241.

(2) Gerdermann, C.; Eicken, C.; Krebs, B. The crystal structure of catechol oxidase: new insight into the function of type-3 copper proteins. Acc.Chem. Res. 2002, 35, 183–191.

(3) Dedert, P. L.; Thomson, J. S.; Ibers, J. A.; Marks, T. J. Metal ion binding sites composed of multiple nitrogeneous heterocycles. Synthesis and spectral and structural study of bis(2,2?,2??-tripyridylamine)copper(II)bis(trifluoromethanesulphonate) and its bis(acetonitrile) adduct. Inorg.Chem. 1982, 21, 969–977.

(4) Klabunde, T.; Eicken, C.; Sacchettini, J. C.; Krebs, B. Crystal structure of a plant catechol oxidase – a dicopper center for activation of dioxygen.Nat. Struct. Biol. 1998, 5, 1084–1090.

(5) Apurba, B.; Lakshmi, K. D.; Michael, G. B. D.; Carmen D.; Ashutosh, G. Insertion of a hydroxido bridge into a diphenoxido dinuclear copper(II)complex: drastic change of the magnetic property from strong antiferromagnetic to ferromagnetic and enhancement in the catecholase activity.Inorg. Chem. 2012, 51, 10111–10121.

(6) Peter, C.; Bodo, M.; Amsaveni, M.; Johannes, S. Structure, bonding, and catecholase mechanism of copper bispidine complexes. Inorg. Chem.2012, 51, 9214–9225.

(7) Marion, R.; Zaarour, M.; Qachachi, N. A.; Saleh, N. M.; Justaud, F.; Floner, D.; Lavastre, O.; Geneste, F. Characterization and catechole oxidase activity of a family of copper complexes coordinated by tripodal pyrazole-based ligands. J. Inorg. Biochem. 2011, 105, 1391–1397.

(8) Sukanta, M.; Jhumpa, M.; Francesc, L.; Rabindranath, M. Modeling tyrosinase and catecholase activity using new m-xylyl-based ligands with bidentate alkylamine terminal coordination. Inorg. Chem. 2012, 51, 13148–13161.

(9) Koval, I. A.; Gamez, P.; Belle, C.; Selmeczi, K.; Reedijk, J. Synthetic models of the active site of catechol oxidase: mechanistic studies. Chem.Soc. Rev. 2006, 35, 814–840.

(10) Apurba, B.; Lakshmi, K. D.; Michael, G. B. D.; Guillem, A.; Patrick, G.; Ashutosh, G. Synthesis, crystal structures, magnetic properties and catecholase activity of double phenoxido-bridged penta-coordinated dinuclear nickel(II) complexes derived from reduced Schiff-base ligands:mechanistic inference of catecholase activity. Inorg. Chem. 2012, 51, 7993–8001.

(11) Sheldrick, G. M. SHELXS-97, A Program for the Solution of Crystal Structures. University of G?ttingen, Germany 1997.

(12) Sheldrick, G. M. SHELXL-97, A Program for the Refinement of Crystal Structures, University of G?ttingen, Germany 1997.

(13) Samit, M.; Suraj, M.; Pascale, L.; Sasankasekhar, M. Dinuclear mixed-valence CoIIICoIIcomplexes derived from a macrocyclic ligand: unique example of a CoIIICoIIcomplex showing catecholase activity. J. Chem. Soc., Dalton Trans. 2013, 42, 4561–4569.

(14) Averi, G.; Kazi, S. B.; Sudhanshu, D.; Tanmay, C.; Ria, S.; Ennio, Z.; Debasis, D. A series of mononuclear nickel(II) complexes of Schiff-base ligands having N,N,O- and N,N,N-donor sites: syntheses, crystal structures, solid state thermal property and catecholase-like activity. Polyhedron 2013, 52, 669–678.

(15) Kazi, S. B.; Madhuparna, M.; Averi, G.; Santanu, B.; Ennio, Z.; Debasis, D. Dinuclear copper(II) complexes: solvent dependent catecholase activity. Polyhedron 2012, 45, 245–254.

(16) Zhang, Y.; Meng, X. G.; Liao, Z. R.; Li, D. F.; Liu, C. L. Synthesis, structures and polyphenol oxidase activities of dicopper and dicobalt complexes.J. Coord. Chem. 2009, 62, 876–885.

(17) Sohini, S.; Samit, M.; Sujit, S.; Luca, C.; Eva, R.; Sasankasekhar, M. Triple bridged μ-phenoxo-bis(μ-carboxylate) and double bridged μ-phenoxo-μ1,1-azide/μ-methoxide dicopper(II) complexes: syntheses, structures, magnetochemistry, spectroscopy and catecholase activity.Polyhedron 2013, 50, 270–282.