FT-IR Spectroscopy and PXRD
FT-IR spectrum of the copper (II) complex revealed all the characteristic bands which were in accordance with the results of the X-ray single crystal analysis. Previous work has suggested that the frequency of separation (Δν) between the two asymmetric & symmetric V(COO) is related to the nature of the carboxylate coordination [25]. In this complex two bands in 1571 cm⁻1 and 1408 cm⁻1 indicates the antisymmetric and symmetric COO- stretching vibrations of carboxylate moiety respectively. ∆ν = 163 cm-1 indicates the bridging mode of benzoate moiety. The Cu–O absorption bands in 516-515 cm⁻1 range for complex 1, confirm the coordination of the carboxylate ligand through oxygen [S1]. The appearance of C=N stretching band at 1609 cm-1 instead of its normally observed characteristic region (1625–1610 cm⁻1) [26-28] indicated the involvement of nitrogen of pyridine in bonding with the copper (II) ion. This was further supported by the appearance of a new medium intensity band in the region 465–463 cm⁻1, attributable to Cu–N vibration. The aromatic C=C and C–H stretching vibrations were observed in 1512–1498 and 3069–3034 cm⁻1regions, respectively. A broad band at 1121 cm-1 assignable to ۷sym (CF3) vibration in complex. The powder X- ray diffraction data [S2] was recorded in the range of 2θ = 10-50o. Sharp peak shows the crystalline nature of the bulk sample. The stimulated X-ray diffraction and PXRD data are completely matched with the single crystal X ray diffraction pattern, shows purity of the bulk sample.
Description of Crystal Structure
The crystallographic characterization reveals that the complex 1 crystallize in the monoclinic space group P1 21/c1. As the center of the paddle-wheel dimer, for the crystal structure of complex 1, is situated on a crystallographic inversion center, only half of the complex molecules are within the asymmetric units shown in Fig. 1. The molecular structure comprised of four μ-benzoate ligands and two ACTP ligands at the two copper (II) ions (shown in Fig. 2), formulated as [Cu2 (C6H5COO)4 (ACTP)2] have the typical structure of the widely known copper carboxylates, with a central Cu2(μ-O,O′−O2CR)4 [R= -C6H5, -CH3 etc.] core . Each copper ion is coordinated in ‘syn-syn mode’ from four different benzoate groups in the equatorial plane with Cu-Oeq distances at around 1.96 Å. This typical arrangement around each cupric center, allowing a rather short intermetallic distance, typically around 2.6612 Å for complex 1 and leaving two axial positions available for terminal ligands. These axial positions of each copper atom belong to nitrogen of substituted pyridine moiety from each ACTP ligand, which act as ancillary ligands. The difference in Cu–Cu distances is attributable to the relatively higher basic strength of the N-donor ligands in complex, resulting in slight decrease in their Cu–O bond ionic character [29]. Thus, the properties of Cu (II) complex, arising as a result of the inter-copper (II) ions separation can be tuned by changing the attached ligand and its donor atoms. As expected, the 2.245(2) Å of Cu-N bond lengths of these ancillary ACTP ligands are significantly longer than those of the four basal benzoate ligands of 1.956(2) Å–1.980(2) Å, due to the weaker bonds and the Jahn–Teller effect of the d9, Cu2+ ions and generate a square pyramidal coordination geometry. Due to centrosymmetric in nature, all trans bond lengths are equal to each other along with their trans bond angles. The maximum bond angles around each Cu atoms in complex O6- Cu1- O3=167.43°, which are shorter than 180° and the little expansion of the O–Cu–N angle from 90°, which is typical in a paddlewheel structure. The distortion of the coordination polyhedral is also evident from the values of the bond angles (Table-2). The small variation in the structure from reported complexes is due to the difference in the electronic and other properties of the group attached to the phenyl ring in the respective complexes. Moreover, the intramolecular hydrogen bonding interaction of complex 1 is linked by the amide groups of ancillary ACTP ligands with the nearest carboxylic group of benzoate ligands creating the 2.046 Å of O3…H102-N3, to stabilize the structure. From the packing diagrams it is also very clear that the complex prefers an isolated dimeric structure with intramolecular hydrogen bonds (S3) rather than a tetrameric or polymeric structure & thus inter dimer interaction is absent in complex.
Electronic spectra
The electronic spectrum was taken in 200-1100 nm range with 1 x 10-5 (M) methanolic solutions of the complex. The intense band is observed around 200-310 nm range, shown in Fig. 4, which is due to intra ligand charge transfer transition. Intense signal display charge transfer transitions in origin at around 280 nm is due to - * transition and peaks in the range 305 nm may be due to the n to * transition which may be assigned as charge-transfer absorption (oxygen-to-copper; LMCT) and could be indicative of a dimeric structure of the complex. Moreover, the complex shows a strong absorption band due to the d-d charge transfer transition at 755 nm. Thermo gravimetric analysis
The thermal stability of the polycrystalline sample was investigated using TGA at a heating rate of 10 oC / min in N2 atmosphere over the temperature range from 30°C to 500°C. The complex shows no weight loss up to 110oC. After that the complex shows two step weight loss in the temp rage 110-206oC and 206 -295oC with a weight loss of 52% and 34% corresponds to two ACTP groups and then the loss of remaining benzoate moieties respectively [S4]. No further weight loss observed upon heating up to 500oC. The black residue is identified as CuO upon qualitative analysis.
The sequence of the decomposition reactions, as deduced from TGA studies, is summarized below:
[Cu2 (C6H5COO)4(ACTP)2] → Cu2(C6H5COO)4 + 2 ACTP
[Cu2(C6H5COO)4] → 4 C6H5COO + 2CuO
Electrochemical and EPR studies
Cyclic voltammetry was employed to investigate the redox behavior of the synthesized copper (II) complex. Study concerning the redox behavior of the compound was carried out in methanol solution containing 0.01 M TBAP in the potential range -2.5 to + 2.5 V at a glassy carbon electrode (GCE) versus an Ag/AgCl reference electrode at 25 °C. Cyclic voltammogram shows one oxidation and one reduction peak in the potential range -0.4 to + 1.1 V, shown in Fig. 5. The cathodic peaks appeared at Epc= -0.4V and anodic peaks appeared at Epa=1.1 V, which is attributed to the reduction of the copper (II) ion into copper(I) ion in reversible manner. The E1/2 value for the complex is 0.35V.
An EPR spectrum of copper (II) benzoate based paddle wheel complex was recorded at low temperature and it shows five signals which are typical for the antiferromagnetically coupled copper (II) centers (S5).
Catecholase activity and kinetics
The catecholase activity of complex was determined by 3, 5-di-tertbutylcatechol (3,5-DTBC) as a substrate at room temperature under aerobic condition .The catecholase activity was checked by mixing a methanolic solution of complex (1 x 10-4) with 3, 5-ditert-butylcatechol (100 equiv.) under aerobic conditions at room temperature. There was a gradual increase at 400 nm with addition of 3, 5 DTBC in methanolic solution of the complex (due to the formation of the oxidized product 3,5-DTBQ (Fig. 6). The course of the reaction was checked by UV-vis spectroscopy, by observing the increase in absorbance of the quinone band shows maximum around 400 nm [30]. The time dependent UV-vis spectral scan was performed in pure methanolic solution. The observed rate versus substrate concentration data were then analyzed on the basis of the Michaelis–Menten approach of enzymatic kinetics. The Michaelis−Menten constant (KM) and maximum initial rate (Vmax) were determined by linearization using Lineweaver−Burk plots [31]. The turnover number (kcat) was obtained by dividing the Vmax value by the concentration of the complex [Table 3 and Fig. 6].
The catecholase-like activity of Cu (II) complex was determined by the catalytic oxidation of 3,5-DTBC. 3,5-di-tert-butylquinone (3,5-DTBQ), is highly stable and a characteristic absorption band maxima appeared at around 400 nm (ε = 1900 M−1 cm−1) in a pure methanol solvent system. By choosing initial rate method the rate constant for a particular complex substrate concentration ratio can be obtained by change in absorbance versus time plot. The Cu (II) complex showed high turnover number (Table 4) for the catalytic oxidation of 3,5-DTBC to 3,5-DTBQ under mild conditions by molecular oxygen and the formation of 3,5-DTBQ was identified from ESI-MS+(m/z) study. The observed rate vs. [substrate] plot in methanol solution, as well as Lineweaver-Burk plot, is given in Fig. 6. In methanolic solution of the complex, with addition of 3,5- di-tert-butyl catechol in presence of air, there was a gradual increase in absorbance at 400 nm as shown in Fig. 7.
For complex−substrate intermediate and a mechanistic inference of catecholase activity during the oxidation reaction, we have recorded ESI-MS spectra of the complex after 15 min mixing of 3,5-DTBC in methanol solvent (S6) to check if there was any complex –substrate aggregate formed or not . The peaks at m/z = 243.1 can be assigned to the quinone sodium aggregates [3,5-DTBQ-Na] +. Complex 1 shows a peak 464.35 which can be assigned to corresponding species, [Cu (PhCOO-)(MeOH)2(3,5-DTBC)] and 463.35 due to [Cu (PhCOO-)(MeOH)2(3,5-DTBC)] +, formation of catalyst-substrate intermediates that take part in substrate activation during the oxidation of 3, 5 DTBC to 3,5 DTBQ. Peak at 324.16 may be due to formation of [Cu (PhCOO-)2(Solvent)H2].
Detection of Hydrogen Peroxide in the Catalytic Reactions
Modification of iodometric method is carried out to detect H2O2 quantitatively during the catalytic reaction [32-33] and mixtures of reaction were prepared as in the kinetic experiments. Using dichloromethane after 1 h of the reaction, same volume of water was added to extract the formed quinone. Further oxidation was stopped by acidified with H2SO4 to pH≈2 to the aqueous layer, and 10% solution of KI (1ml) and 3% solution of ammonium molybdate (three drops of) were added. In the presence of hydrogen peroxide I- is oxidised to I2, H2O2 + 2I- + 2H+→ 2H2O + I2, and with an excess of iodide ions, the tri-iodide ion is formed according to the reaction I2(aq) + I-→I3 -. Normally the reaction rate is slow but rate is increases with increasing acid concentrations, and the reaction almost condenses immediately when an ammonium molybdate solution was added to the reaction. The formation of I3 - is detected by UV-vis spectroscopy due to development of the characteristic I3 - band. (λ = 353 nm) [S7].