Catecholase activity investigations using in situ copper complexes continuing Schiff base derivatives with a theoretical calculation

Introduction

Transition metal ions have an important role in biochemistry and biomimetic systems and may provide the basis of models for active sites of biological targets [1]. The presence of copper (II), iron (II) and zinc (II) is crucial in many biological processes. These metals are present in metallo-enzymes that involve the coordination of a metal ion as an active site and in which the metal plays the role of catalyst [2]. They also participate in the electron transport of O₂, and the degradation of the superoxide ion O₂⁻ [3-5]. The coordination chemistry specially of dinuclear Cu(II) complexes has been developed very well with the goal of  miming the active sites of the type 3 copper proteins/enzymes such as hemocyanin, tyrosinase and catechol oxidase. The oxidation of a wide range of o-diphenols to o-quinones is catalyzed by the catechol oxidases [6]. Thus, to understand more about this biological process and to search for new  ligands  that  are  able  to  contribute  with  other  metals  miming  bio-organism puzzles, we examined a series of Schiff base compounds . These compounds have a wide range of applications, for example, catalyst, nonlinear optical materials, electroluminescent materials and others [7]. The synthesis of novel Schiff bases and the research of their physiological activities have become important due to their biological activities [8–10]. In continuation of our biometric work [11], we focused on the study of catecholase activity by using these ligands with different transition metals especially the copper via the oxidation of catechol to o-quinone.

Experimental

Chemistry of the studied ligands

All reagents were purchased from commercial sources and used without further purification.  The ligands were prepared, and characterized according to literature [12], via an efficient condensation of an amine and dehydroacetic acid L0 in ethanol. The structures are shown in Scheme 1.

Scheme1: Chemical structure of ligands  Click here to View Scheme

 

Method of Catalytic Measurements

The measurements were made spectrophotometrically on UV–Vis spectrophotometer (in the COSTE: Centre de l’Oriental des Sciences et Technologies de l’Eau), following the appearance of o-quinone over time at 25 °C (390 nm absorbance maximum, ε = 1,600 L.mol^-1.cm^-1 in methanol [13]). The complexes were prepared in situ by successively mixing 0.30 mL of a solution  (2×10^-3 M) of CuX₂, nH₂O (X = Cl^–, Br⁻, NO₃⁻, CH₃COO⁻ or SO₄²⁻) with 0.15 mL of a solution (2×10^-3 M) of ligand, then adding 2 mL of a solution of catechol at a concentration of 10^-1 M. After that, the evolution of product absorbance was followed at 390 nm according to time after regulation in zero [11] (Scheme 2).

Scheme2: The oxidation model reaction of the catechol to o-quinone  Click here to View scheme

 

Results and Discussion

Catalytic Activity Studies

After realization of catechol oxidation reactions catalyzed by copper complexes with ligands L0–L7, the evolution of o-quinone absorbance was followed at 390 nm on the UV spectrometer UV–vis, we note that the catalytic activity varies from a complex to the other one, the obtained results  for Cu(CH₃COO)₂  salt  are summarized in below  (Fig. 1)( more figures about the absorbance of o-quinone with different metal salts  in the supplementary material) and the V_max rates of the catechol oxidation were calculated in a time range of 5 min where the slope of the absorbance graph is maximal and collected in Table 1. In this part, we take one equivalent of ligand for two equivalents of metallic salt. In addition, the results obtained with L0 is not recorded here because it is too weak as if we were used the catechol alone.

Figure1: Catechol Oxidation in the presence of different ligands L1-L7 and the metallic salt Cu(CH3COO)2  in methanol  Click here to View figure

 

Table1: Maximum rates of catechol oxidation in methanol  Click here to View table

 

According to the obtained  results, we notice that all copper complexes, formed in situ from ligands L1–L7 and copper salts (Cu(CH₃COO)₂ ,CuBr₂,CuCl₂, Cu(NO₃)₂,CuSO₄) , catalyze the oxidation reaction of catechol to o-quinone, but with different rates which varying from 0.18 µmol. min^-1.L^-1 for the complex formed from ligand L6 and the metallic salt CuSO₄ (weak catalyst) to 62.25 µmol.min^-1.L^-1 for the complex formed from ligand L2 and metallic salt Cu(CH₃COO)₂ (best catalyst). At this step, these results can be explained by  the intervention of two factors: firstly, the nature of the core group on the para position of the  benzene ring, in fact and in general , the  donor  group  can  increase  the  complexing  properties  of  the  ligand by the enrichment of the benzene ring, and promote stability in the complex. However, in presence of an electron attractor group, a disadvantage complex formation occurs by reducing the electron density   on   the   sites   of   chelation   and,   therefore,   reducing   the   rate   of correspondence between the substrate and the complex. Secondly, the nature of counter-ion, so if it is strongly bonded to the metal as in the case of NO₃⁻, Cl⁻ and Br⁻ ions, the binding of the substrate on the metal will be difficult, therefore reducing the catalytic power of the corresponding complex.  Otherwise,  the bonds formed between the metal and its counter-ion are not as strong (case of the CH₃COO⁻ ion),  so  the  substrate  can  be  replaced  easily,  which  positively affects the rate of the reaction. In this part, we use different parameters to study the catalytic activity of the product L2 which gave the best results.

Effect of Ligand Concentration on the Catecholase Activity

In this study, the evolution of the  oxidation of catechol to the o-quinone it’s followed  in the presence of variable equivalents of ligand L2 and the metallic salt  Cu(CH₃COO)₂. Three different concentration are used: One equivalent of L2 with one equivalent of             Cu(CH₃COO)₂; Two equivalent of L2 with one equivalent of Cu (CH₃COO)₂ and One equivalent of L2 with two equivalent of Cu (CH₃COO)₂.

Figure2: Catechol Oxidation in the presence of variable equivalents of ligand L2 and the metallic salt Cu(CH3COO)2  Click here to View figure

 

From the figure above, when the increasing of the concentration of the metal salt in first  35 min happens, the absorbance increase as will, after that, the absorbance decrease due to the precipitation of the  a complex in the cuvette. Also the calculation of the oxidation rates, confirmed the absorbance values founded.  From  these observations, we can conclude, that the efficiency of our catalyst increase in presence of an excess of metal relative to the ligand, this result can be explained by the presence of two Cu(II) ions into the enzyme catecholase  structure as site active, which  granted firstly as an idea that our complex must contain two copper ion to in order to make  its catalytic function happens in good way, and secondly as an idea about our model complexes which could be seen as functional or structural  models  for  catechol oxidases  or  related  copper containing  enzymes.

Effect of the Acetate Salts

From the precedent results, what would be observed mainly is that the best results are showed with Cu (CH₃COO)₂. Therefore, for identify if the CH₃COO⁻ ion have an effect on the catalytic activity, we were used in this study different acetate salts with different metal (Cu, Zn, Ni and Mn).

Figure3: Catechol oxidation in the presence of metal complexes formed with L2(1 equivalent of ligand for 2 equivalents of metallic salt)   Click here to View figure

 

The obtained results, showed very clearly that Cu (CH₃COO)₂ give the best results with a oxidation rate equal  to 62.25 µmol.min^-1.L^-1, followed by Ni (CH₃COO)₂ with a value of    5.93 µmol. min^-1.L^-1, when the other salts give a very low rate < 1 µmol. min^-1.L^-1. As we can see here, the acetates has no effect on the oxidation rate in these conditions, but the difference between the rate values it is due to the metal ion which is very logical, because the reduction potential of (Cu ²⁺)(E⁰ =0.34 V) is more higher or favorable than the other metals tested, which make it more reactive than  (Ni²⁺)(E⁰ =-0.25 V),(Zn²⁺)(E⁰ =-0.76 V) and (Mn²⁺)(E⁰ =-1.18 V).

Solvent Effect

The effect of the solvent on the oxidation reaction with ligand L2 and Cu (CH₃COO)₂ salt were studied in different solvents (MeOH, DMF, DMSO and CH₃CN). As a simple comparison, we observe that the absorbance in MeOH  is higher than in other solvents with a maximal value of 2.044 after 45 min of experience, before decreasing after that due to the precipitation of a complex in the cuvette, which is not the case in other solvents specially the DMF because this complex is very soluble in this solvents. So, the methanol is better solvent than the others for this reaction, and that can be explained by its protic and polar nature, because this kind of solvents are very known by their strong solvation of various anions X⁻ (in our case X⁻ = Cl⁻, Br⁻ NO₃⁻, CH₃COO⁻ and SO₄²⁻) through hydrogen  bonds,  which  make them  isolated and therefore are not very reactive in this type of solvents. The copper cations are also solvated by the electronegative pole of the solvent but is weaker than the solvation by a hydrogen bond, and became moderately reactive.

On the other hand, we observe that the positive charge is diffused, whereas the negative charge is concentrated on a single atom in the dipolar and aprotic solvents   ( like DMF, DMSO,CH₃CN,…) which mean that kind of solvents  are very strongly solvating the (Cu²⁺) cations  making them little or no reactive. For this, the formation of the copper complex is very difficult in this case, which negatively and indirectly affects the oxidation rate of the catechol to the o-quinone.

Figure4: Catechol oxidation in the presence of copper complexes formed with L2 in different solvents. (1 equivalent of ligand for 2 equivalents of metallic salt)  Click here to View figure

 

Substitution effect

The effect of substitution was studied for three ligands L7, L8 and L9 respectively substituted in Para, Meta and Ortho by a NH₂ group (Scheme 3), the  Cu (CH₃COO)₂ is used as metal salt and the  methanol as solvent.

Scheme3: Structure of used ligands  Click here to View Scheme

 

Figure5: Catechol oxidation in the presence of copper complexes formed with L7, L8 and L9. (1 equivalent of ligand for 2 equivalents of metallic salt) Click here to View figure

 

Table2: Oxidation rate of catechol oxidation in methanol   Click here to View table

 

Comparing the unsubstituted ligand L2 with L7, L8, and L9, which have the same molecular formula, we note clearly that the existence of a substituted group on the benzene ring play an important role in the decrease of the catalytic activity of our compounds. As we can see from the  Fig. 5 and the  Table 2 the ligands substituted in para and meta position gives  the best result with Cu (CH₃COO)₂ with a rate  of  23.62 and 24.07 µmol.min^-1.L^-1 successively  . For the two ligands L7 and L8, the shape of the curve of absorbance versus time is the same in the first 35 min, but after that, the absorbance in the presence of L7 remains fixed during the rest of the time unlike the ligand L8, which decreases due to the formation of a complex in the cuvette. However, the absorbance in the presence of L9 is weak probably due to the steric effect caused by the amino group, which prevents the (Cu²⁺) cations from complexing better with the ligand, which is not the case in the two other ligands, where the NH₂ group is more far from the coordination site proposed.

Theoretical Study

In this part, we examined a theoretical study to find a correlation between our experimental values and the calculated values obtained by the quantum mechanical calculations [14, 15] of the V_max (Table 3) and the molecular orbitals energy HOMO and LUMO.

Table 3. Oxidation rate of catechol oxidation in presence of the ligand and the copper metallic salt Cu(CH₃COO)₂

Ligands	Vmax (µmol.min-1.L-1)
L1	43.75
L2	62.25
L3	34.12
L4	40.25
L5	38.50
L6	3.62
L7	23.62
L8	24.07
L9	5.86

 

Method of Calculation

The semi-empirical methods are done on Hyperchem program version 8.0.6 for Windows [16] running on a Windows seven with a core i5 vPro. The initial geometry optimization of the studied molecules were performed with the molecular mechanics (MM+) force field [17, 18], where the lowest energy conformations are obtained. Further, Geometry optimization was done by performing the semi-empirical molecular orbital theory at the level parametric method (AM1) [19], within restricted Hartree–Fock (RHF) level [20]. The Polak–Ribier algorithm was used for the optimization [21]. The convergence is set to 0.01 kcal/mol.

Theoretical Calculations

The molecular orbitals energy: LUMO (lowest unoccupied molecular orbital) as an electron acceptor (EA) represents the ability to obtain an electron and HOMO (highest occupied molecular orbital) represents the ability to donate an electron (ED). The HOMO and LUMO energy gap explains the fact that eventual charge transfer interaction is taking place within the molecule. The values of the difference between the HOMO and LUMO orbitals (LUMO-HOMO), known as energy band gap (ΔE), are also given in Table 4. As it is shown in Fig. 6a and Fig. 6b (spatial distribution of the HOMO and LUMO of all molecules is presented in the supplementary material (Fig. 6c and Fig. 6d)), the HOMO orbitals are localized on the benzene ring and the imine function, distribution of molecular orbitals of LUMO is localized on pyrane ring.

 

Table4: Molecular orbitals energies of ligands L0-L9 calculated with AM1 Click here to View table

 

Figure6a: The HOMO orbitals of L1  Click here to View figure

 

Figure6b: The LUMO orbitals of L1  Click here to View figure

 

Figure 6 C Click here to View figure

 

Figure 6D  Click here to View figure

 

Correlation

A study was conducted using IBM SPSS Statistics v 21 to find a correlation between the maximal reaction rate (V_max) and the HOMO energy. The results were given in Table 5 and Fig. 7. Correlation quantifies the extent to which two quantitative variables, here V_max and E_HOMO. When high values of V_max are associated with high values of E_HOMO a positive correlation exists.  However, a negative correlation obtained, when high values of V_max are associated with low values of E_HOMO.

Table5: Correlation between Vmax and EHOMO  Click here to View table

 

A strong correlation is observed between the maximal reaction rate (V_max) and HOMO energy, so the correlation is significant (r = -0.794; P = 0.019), here the correlation is negative.

Figure7: Dependence of Vmax to EHOMO  Click here to View figure

 

Figure 7B  Click here to View figure

 

These results show the good linear correlation between the maximal reaction rate and HOMO energy. This correlation proves the existence of a relationship between the catalytic activity, which is presented by V_max, and HOMO energy, so we can estimate V_max by study of the HOMO energy for this kind of ligands.

Conclusion

In conclusion, we have found that our compounds L1–L7 present a variant capacity to catalyze the oxidation reaction of the catechol to o-quinone at ambient conditions using the oxygen of atmosphere as oxidant. The catalytic activity of these complexes formed in situ is influenced by the nature of counter anion in metallic salt, ligand and the metallic salt concentration, nature of solvent and the nature of substitute group on the benzene ring. In addition, the Quantum mechanical and statistical calculations showed a good correlation between the maximal reaction rate and the  HOMO energy witch proves the existence of a strong relationship between the catalytic activity and the HOMO energy. It seems interesting to continue this work, firstly, on the synthesis of these complexes and test their reactivity as metallo-enzymes in the field of biomimetic catalysis. Secondly, trying to find a general Model of Cu:O₂ intermediates and understanding their action mechanism in the oxidation reaction of the catechol to o-quinone.

Acknowledgement

Our thanks to the Mohamed First University for its financial support.

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