CATECHOLASE ACTIVITY AND SUBSTITUENT EFFECT OF NEW HOMOLEPTIC COPPER(II) CHALCONE COMPLEXES

Three new neutral complexes of copper(II) containing chalcone ligands derived from 2'-hydoxyacetophenone and 4-substituted benzaldehydes were synthesized. Complexes were prepared by solution synthesis and characterized by spectroscopy. The catalytic activity of complexes was examined in the reaction of 3,5-di-tert-butylcatehcol (DTBC) oxidation. The kinetics of DTBC catalytic oxidation by copper(II) complexes ( 1 – 3 ) was investigated spectrophotometrically under pseudo-first-order conditions. Catalytic parameters, the maximum reaction rate (v max ), Michaelis-Menten constant (K M ), catalytic efficiency, catalytic reaction rate constant (k cat ), turnover number (TON), and turnover frequencies (TOF) for complexes 1 – 3 in DTBC oxidation were collected. The studied complexes 1 and 2 were found to have moderate catalytic activity, while complex 3 does not show catalytic properties.


INTRODUCTION
The ability of copper proteins to process dioxygen at ambient conditions has inspired numerous research groups to study their structural, spectroscopic, and catalytic properties.Catechol oxidase (CO), also known as o-diphenol oxidase, is a member of the type-3 copper proteins [1].COs are found in plant tissues and some insects and crustaceans.CO catalyzes exclusively the oxidation of catechols (i.e., o-diphenols) to the corresponding oquinones which can rapidly polymerize to form melanin, a dark pigment thought to protect a damaged tissue from pathogens and grants damaged fruits their dark brown coloration [2].A great number of mononuclear and dinuclear copper(II) complexes have been investigated as biomimetic catalysts for catechol oxidation, regarding the binding of catechol substrate in the first step of the catalytic cycle [3][4][5][6][7].While no clear relation between the catalytic activity and the redox potential of the copper species has emerged, dinuclear copper complexes are generally found to be more reactive than mononuclear compounds, and a steric match between the dicopper site and the substrate is assumed to be advantageous [8].The presence of carbonyl oxygen, phenolic oxygen, and/or heteroatom(s) in a heterocyclic ring system makes chalcones excellent chelating ligands for metal coordination.The metal complexes of bidentate chalcone have shown great potential in antiviral, antimalarial, antimicrobial, antioxidant, therapeutic, and catalytic applications [9][10][11][12][13].Recent studies present that copper(II) chalcone complexes show evidence of catalysis in the oxidation reaction of catechol to o-quinone under atmospheric dioxygen [2,14,15].The catalyzing potential of two novel copper complexes of chalcone derivatives in the oxidation reaction of catechol to o-quinone was investigated by Kahrović et al [16].We report the synthesis of three novel neutral copper(II) complexes (1-3) containing chalcone ligands including the study of their catalytic activity towards the 3,5-di-tert-butylcatechol oxidation.

Physical Measurements
Infrared spectra were recorded as KBr pallets in the 4000 -400 cm -1 region on Perkin Elmer BX FTIR.Electronic spectra were collected in DMSO (5×10 -5 mol dm -3 ) in the 260 -900 nm range on a Perkin Elmer Lambda 35.

Catalytic Activity
The catecholase activity of complexes 1 -3 was examined by taking DTBC as a model substrate.Catalysis of the DTBC oxidation reaction by copper(II) complexes was estimated spectrophotometrically, in DMSO solution at room temperature under aerobic conditions.Six experiments were performed for each of the complexes, during which the concentration of the observed complexes (1.25×10 -5 mol dm -3 , 1000 μL) and the hydrogen peroxide co-oxidant (30 % w / w, 25 μL) was kept constant, while the concentration of the DTBC substrate was varied (1×10 -2 mol dm -3 , 500 -25 μL, and DMSO was added up to a volume of 2000 μL).Over 10 min, every 30 seconds, an increase in absorption intensity at 400 nm was monitored, originating as a consequence of the formation of 3,5-di-tertbutylquinone (DTBQ).Michaelis-Menten method of enzymatic kinetics was applied to obtain Lineweaver-Burk plot and values of KM, vmax, and kcat.The conversion of the reaction rate units from A/s to M/s was done using ε = 2818 mol −1 dm 3 cm −1 for 3,5-DTBQ in DMSO with an absorption maximum at 400 nm [18].
3. RESULTS AND DISCUSSION 3.1.Syntheses Complexes 1 -3 contain copper(II) metal centers to which various chalcones (HL 1 , HL 2, or HL 3 ) are bound as ligands.The synthesis was carried out under reflux in methanolic solutions, in which both ligand and starting compound, binuclear copper(II) acetate dihydrate, were soluble, while obtained products had poor solubility and therefore can be isolated by filtration.The molar ratio of binuclear copper(II) acetate and appropriate chalcone was 1:4.This stoichiometry was aimed to ensure the coordination of two bidentate anionic chalcone ligands through the carbonyl oxygen atom and the oxygen atom of the deprotonated hydroxyl group (O, O donor atoms) to the copper metal center (Fig. 1.).Complexes of copper(II) with chalcone ligands were characterized based on infrared and electronic spectroscopy.

Spectroscopic Characterization Infrared spectra
The infrared spectra of complexes (1 -3) have bands at similar positions, which indicates their structural similarities.Infrared spectra of copper(II) complexes (1 -3) and free chalcone ligands (HL 1 -HL 3 ) are shown in Fig. 2. Chalcones are bound to the copper(II) metal center as bidentate anionic ligands via the deprotonated hydroxyl oxygen atom and the carbonyl oxygen atom.
After ligand coordination, strong intensity bands assigned to the stretching vibrations of the carbonyl groups (C=O) and C=C stretching vibrations, shifted to lower wavenumbers: 1631 and 1609 cm -1 for 1; 1633 and 1607 cm -1 for 2; 1628 and 1606 cm -1 for 3, compared to the free ligands.The coordination of chalcone ligands is further indicated by the shift of the C-O stretching vibrations to higher wavenumbers in the spectra of complexes (1360 cm -1 for 1 and 1354 cm -1 for 2 and 3) compared to the spectra of free ligands (1339 cm -1 for HL 1 , 1341 cm -1 for HL 2 and 1344 cm -1 for HL 3 ) [19].In the low-frequency region in spectra of all complexes, a new band, attributed to stretching vibrations of the newly formed Cu-O bond, appeared.Cu-O stretching vibration appeared at 574 cm -1 for 1; 577 cm -1 for 2; 577 cm -1 for 3. Table 1 presents the most important bands that confirm the coordination of the ligand to the metal center.

Electronic spectra
Electronic spectra (Fig. 3) of complexes and corresponding ligands were recorded in DMSO solutions.After coordination of the chalcone ligands HL 1 and HL 2 , the bands ascribed to the carbonyl group (314 nm for HL 1 and 338 nm for HL 2 ), appeared on similar positions in the spectra of corresponding complexes 1 and 2, which indicated coordination via a carbonyl oxygen atom.The band in the HL 3 spectrum at 370 nm, after coordination on the copper(II), shifted to a lower wavelength, 361 nm (complex 3), also indicating the participation of the carbonyl group in coordination with the metal center.In the spectra of all three complexes, after coordination of the chalcone ligands, new bands appeared with maxima in the 430-450 nm region.Based on their positions and extinction coefficients they could be identified as charge transfer bands.The mentioned electronic transitions are shown in Table 2.

Spectroscopic Evidence of the Catalysis
Catalytic activities of complexes 1 -3 were investigated by spectroscopic measurements.DTBC was used as a substrate in all experiments due to its low oxidation potential and absence of any concomitant or successive reaction upon its oxidation to DTBQ.In all three cases, the reaction mixture containing DTBC, hydrogen peroxide, and the observed complex showed an absorbance increase of the quinone band near 400 nm (λmax = 400 nm; ε = 2818 mol -1 dm 3 cm -1 ).In the absence of copper(II) complexes, no significant change in the spectrum of the DTBC was observed, which confirms the catalytic nature of this oxidation process.Complexes 1 -3 are stable in DMSO solutions and their activity can be ascribed to the originally formulated complex species.

Kinetic Measurements
Kinetic measurements of the catalytic activity of complexes 1 -3 were carried out in a DMSO solution using DTBC as a substrate.The chemical reaction kinetics of the oxidation of DTBC to DTBQ is pseudo-first-order and was investigated using the method of initial rates.The concentrations of the complexes and cooxidant were kept constant, while the concentration of the substrate was varied.A possible reaction pathway for DTBC oxidation in the presence of complexes 1 -3 is presented in Fig. 4. Michaelis-Menten method of enzymatic kinetics was applied to obtain Lineweaver-Burk plot (Fig. 5.) and values of catalytic parameters, the maximum reaction rate (vmax), Michaelis-Menten constant (KM), catalytic efficiency, catalytic reaction rate constant (kcat), turn over number (TON), and turn over frequencies (TOF) for synthesized complexes.Based on the conducted research, it was concluded that complexes 1 and 2 catalyze the oxidation reaction of DTBC to DTBQ and obey Michaelis-Menten kinetics in the range of observed concentrations, while complex 3 did not act as a catalyst for the mentioned reaction as presented in Fig. 6.This can be explained as a consequence of the significant substituent effect.Complex 3 containing chalcone ligand with electron-donating methoxy substituent in para position HL 3 did not show catalytic properties.As shown in Table 3, complex 2, including a methyl group as a substituent on the chalcone ligand, showed higher catalytic efficiency and greater affinity for binding to the DTBC substrate compared to complex 1 containing chloro substituent.Reaction rate constants kcat are most often used to compare the catalytic properties of observed catalysts.Complexes 1 and 2 could be compared  with other examples of copper complexes possessing catalytic properties (Table 4).The TON value represents the number of moles of substrate that a mole of catalyst can convert before it becomes inactivated and it is given as the ratio of the DTBQ and complex concentration at the defined time (10 min).Although the kcat value for 2 is higher than for 1, a higher TON value suggests that 1 is the more promising catalyst due to the higher conversion rate of DTBC to DTBQ for 3% as compared to complex 2.

CONCLUSION
Three new copper(II) complexes were prepared by solution synthesis from copper(II) acetate and appropriate chalcone ligands (HL 1 , HL 2, and HL 3 ).Infrared spectroscopy suggested coordination of the chalcones as bidentate anionic ligands to the metal center via the deprotonated hydroxyl oxygen atom and the carbonyl oxygen atom.Electron spectroscopy showed the formation of new bands in the spectra of products that were assigned to charge transfer bands of the new copper(II) chalcone complex species.Catalytic measurements revealed moderate catalytic properties of complexes 1 and 2, while complex 3 did not catalyze the DTBC oxidation reaction.TON and TOF values suggested that 1 is the more promising catalyst due to the higher conversion rate of DTBC to DTBQ as compared to complex 2.

Table 1 .
The most important vibrational bands in FTIR spectra of chalcone ligands and complexes of copper(II) 1 -3

Table 2 .
Data on electronic spectra of chalcone ligands and corresponding copper(II) complexes 1 -3.

Table 4 .
Comparison of complexes 1 and 2 with some reported catecholase-like biomimetic complexes.