Mechanism research and analysis method for catalyzing oxidation of 2, 4-tert-butylphenol by using double-copper biomimetic copper oxide intermediate

Abstract

The invention belongs to the technical field of computational chemistry, and particularly discloses a mechanism research and analysis method for catalyzing oxidation of 2, 4-tert-butylphenol by using a double-copper biomimetic oxygen copper intermediate [Cu (II) 2O2TMP32] < 2 + >, which is based on a density functional theory (DFT), adopts a gas phase model to calculate a QM reaction mechanism, and performs structure optimization on the intermediate and a transition state; a PCM continuous medium solvation model is adopted, single-point energy correction is carried out, heat and enthalpy correction is obtained by calculating frequency analysis, the whole catalytic reaction potential energy surface is calculated by considering the Van der Waals effect, so that a possible reaction channel is obtained, and on the basis of QM calculation, possible intermediates and transition states are searched again under a QM / MM solvent model by adopting a QM / MM method, so that the calculation efficiency is improved. And determining the reaction speed step and the reaction mechanism. A complete theoretical method for simulating the system is established, a basis is provided for treating the system to develop a new solvent model, and meanwhile, an important theoretical basis and a technical reference are provided for design and development of efficient novel copper biomimetic catalytic phenol.

Description

Technical Field

[0001] The present invention belongs to the technical field of computational chemistry, and relates to the study of the oxidation reaction mechanism of phenols catalyzed by tyrosinase, and particularly to a double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] 2+ Analytical methods for mechanistic studies of catalytic oxidation of 2,4-tert-butylphenol. Background Art

[0002] Tyrosinase is a natural enzyme with a binuclear copper active center, which plays an important role in the catalytic process. Due to the complex structure and function of enzymes and the high research cost, scientists have proposed tyrosinase biomimetic compound catalysts, which use simplified biomimetic compound catalysts to simulate the structure and activity of enzymes, and have become one of the research hotspots in the field of enzyme catalysis.

[0003] Tyrosinase has the property of catalyzing phenolic compounds, so it can be used to decontaminate wastewater or contaminated soil containing phenolic compounds. For example, tyrosinase has the catalytic property of catalyzing the conversion of phenol to o-benzoquinone. In the catalytic cycle of tyrosinase catalyzing the conversion of monophenol to o-benzoquinone, copper ions undergo three different forms. This is due to the different ways in which oxygen molecules bind to the dicopper center during the catalytic process. Many studies have confirmed that the different ways in which oxygen molecules bind make the binuclear copper oxygen center of tyrosinase present different forms, namely oxidized state (Eoxy), reduced state (Emet), deoxygenated state (Edeoxy) and inactivated state (Edeact).

[0004] The binuclear copper center of tyrosinase acquires the property of catalyzing the conversion of phenols to o-benzoquinone by activating oxygen. The basic mechanism of tyrosinase catalyzing the conversion of phenol to o-quinone is currently the most recognized mechanism. The substrate is first deprotonated, and after the OO of the binuclear copper oxygen center is cleaved, a copper oxygen radical is generated. The recombination of the two radicals results in the carbon at the ortho position of the substrate coordination to bond with one oxygen of the binuclear copper oxygen center. The coordinated substrate transfers an electron to a copper center and is converted to a semiquinone with a mixed valence state of Cu(I)-Cu(II). A second electron transfer to the copper site transfers the H proton on the C that is bonded to O to the other O of the bridging binuclear copper oxygen center to generate the final product o-benzoquinone and water. The formation and detection of intermediates during the reaction and their influence on catalytic activity are the core issues in studying the catalytic properties of tyrosinase. Due to the complex structure of the enzyme, it is very difficult and costly to directly study the enzyme itself. Therefore, scientists have developed biomimetic models to elucidate the mechanism of enzyme action in biological systems, which can also be used as effective catalysts. The action of the enzyme active center is highly dependent on the structure of the metal coenzyme. This shows that when synthesizing a bionic model system, it is necessary to select appropriate ligands and coordinate them in a suitable configuration.

[0005] However, the specific catalytic mechanism of tyrosinase biomimetic compounds has not been clearly explained. Many studies on the catalytic mechanism of tyrosinase have laid the foundation for the present invention to study the reaction mechanism. In recent years, Schneider et al. synthesized a new complex and explained its synthesis process in an article, and proposed a copper-mediated mechanism for the conversion of monophenol to o-quinone starting from a simple copper (I) salt. Importantly, the catalytic cycle involved only contains monomeric, monocationic Cu (II) complexes, which are particularly stable under weakly coordinated anions, consistent with the increased activity of this species observed when this type of anion is used. This provides clues for studying the mechanism of binuclear copper tyrosinase biomimetic compounds catalyzing substrates. Summary of the invention

[0006] In view of the shortcomings of the prior art, the object of the present invention is to provide a double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] 2+ The present invention discloses a mechanism research and analysis method for catalyzing the oxidation of 2,4-tert-butylphenol. The present invention is based on density functional theory (DFT), uses a gas phase model to calculate the QM reaction mechanism, and on the basis of the QM calculation, uses a QM / MM method to search for possible intermediates and transition states under a QM / MM solvent model, determines the reaction rate-determining step, and determines the reaction mechanism. The present invention establishes a complete theoretical method that can be used to simulate such systems, provides a basis for developing new solvent models for processing such systems, and also provides an important theoretical basis and technical reference for the design and development of efficient new copper biocatalysts for phenol.

[0007] The present invention is achieved through the following technical solutions:

[0008] Bis-copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] 2+ The method for analyzing the mechanism of catalytic oxidation of 2,4-tert-butylphenol comprises the following steps:

[0009] (I) Construction of gas phase model

[0010] Preliminary construction of a double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] 2+ The gas phase model is used as a possible initial state calculation model to calculate the QM reaction mechanism. The QM calculation is completed using the GAUSSIAN09 program. The optimal model is selected based on the calculation results by comprehensively considering the configuration stability and energy level.

[0011] (II) Design of gas phase model reaction pathway

[0012] (1) Determine the order of substrate binding and OO bond cleavage;

[0013] (2) Construction of CO-bonded intermediate 3;

[0014] (3) construct the transition state TS3 of CO bond formation;

[0015] (4) construct intermediate 4 for the breaking of CH bond and the forming of OH bond;

[0016] (5) Construct the transition state TS4 of CH bond breaking and OH bond forming;

[0017] (6) construct products;

[0018] (III) Solvent model correction

[0019] (1) PCM continuous medium model calculation;

[0020] (2) Construct the initial QM / MM model;

[0021] (3) Setting of QM / MM calculation parameters and calculation of reaction mechanism;

[0022] (4) Comparison between PCM model and QM / MM model;

[0023] (iv) Analyze the reaction mechanism and pathway.

[0024] A further improvement of the present invention is:

[0025] When the gas phase model is constructed in step (1):

[0026] (1) The B3LYP-D3 method in symmetry-breaking density functional theory is used to optimize the geometric configurations of possible transition states, intermediates, and products in the reaction process. The gas-phase reaction mechanism is determined by making a potential energy profile based on the structure and energy, and confirmed by vibration frequency analysis. Specifically:

[0027] In the reaction model, the B3LYP / 6-31G(d,p)&LANL2D levels were used to perform geometry optimization and calculate frequency analysis for all intermediates and transition states;

[0028] The input files for the optimization calculation are as follows:

[0029] Intermediate optimization input file: #p opt ub3lyp / 6-31G(d,p)&LANL2D geom=connectivitypseudo=read;

[0030] Transition state optimization input file: #p opt = (ts, modredundant, noeigen) ub3lyp / 6-31G (d, p) & LANL2D geom = connectivity pseudo = read guess = read;

[0031] All intermediates have no imaginary frequencies, and the transition state has exactly one imaginary frequency;

[0032] The input file for the higher-level single-point energy calculation is as follows: #p ub3lyp / ub3lyp / cc-pvtz geom=connectivity pseudo=read;

[0033] The spin-coupling characteristics of the system are described by adding symmetry-breaking corrections to the existing density functional as follows: In a binuclear metal system, if the spin quantum numbers of the two metals are S A and S B , respectively, then the spin-coupling interaction of the system can be described by the Heisenberg Hamitonian,

[0034] H spin = 2J S AS B (1)

[0035] where J is the Heisenberg spin-coupling parameter. For this system, a highest-spin state HS can be defined. In the HS state, S = 1; the HS state is a pure state and can be described by a single determinant, and the spin electrons are arranged in parallel in the two nuclei. Similarly, a symmetry-breaking state BS can also be defined. In the BS state, S = 0; the BS state is not a pure spin state but a linear combination of a series of pure spin states. The total quantum number S of these pure spin states ranges from S = |S A -S B | to S = |S A +S B | in increments, and the energy of each spin state is

[0036] E(S) = E GS + S(S + 1)J / 2 (2)

[0037] where EGS is the ground-state energy; from (1) and (2), we can obtain

[0038] E HS (S = S MAX ) - E GS (S = |S A -S B |) = S MAX *(S MAX + 1)J / 2 (3)

[0039] and

[0040] E HS (S = S MAX ) - E BS = S 2MAX *J / 2 (4)

[0041] From (3) and (4), we can get the Heisenberg spin coupling parameter and ground state energy:

[0042] E GS =[(S MAX +1)*E BS -E HS ] / S MAX (5)

[0043] And from EGS and J value, the energy of any spin state can be calculated from (2).

[0044] Furthermore, the step (1) of determining the order of substrate binding and OO bond cleavage comprises the following:

[0045] According to the experimental data, the model was built in Gaussview software to preliminarily construct the double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] 2+ ; The distance between the two core coppers is pulled to When the binuclear copper center is placed in the TMP3 ligand, the distances between CuA and CuB and O on the ligand are The optimized structure calculations were performed using the B3LYP functional and the 6-31G(d,p)&LANL2D basis set to obtain the bond lengths, bond angles and dihedral angles of the optimized complexes.

[0046] The distance between the binuclear copper CuA-CuB in the optimized structure is The distance between OA and OB is The distances between CuA and OA, OB, and OC are The distances between CuB and OA, OB, and OD are

[0047] In order to obtain more accurate energy, single-point energy calculations with a larger basis set are performed based on the previous structural optimization.

[0048] (1) Constructing intermediate 1 that binds to the substrate after the OO chemical bond is broken: Based on the optimized calculation model, adjust the key bond length and the distance between the OA-OB bonds to The other settings remain unchanged, and the geometry is optimized using the popular B3LYP-D3 method in symmetry-breaking density functional theory. The structure of the optimized intermediate 1 is obtained, and the distance between CuA and CuB is The distances between CuA and CuB and OA and OB are In order to obtain more accurate energy, single-point energy calculations with a larger basis set were performed based on the previous structural optimization;

[0049] (2) Constructing the transition state TS1 after the breaking of the OO chemical bond and the binding to the substrate: Using the flexible scanning method, by analyzing the possible structural changes in the key chemical bond change, further calculating the energy of each step of the structure, obtaining the potential energy curve of the key chemical bond change process, and using the structure corresponding to the highest energy point on the potential energy curve as the initial transition state structure to complete the preliminary modeling;

[0050] By adjusting the key bond length and the distance between the OA-OB bonds, the transition state model was geometrically optimized and the frequency was calculated to obtain a transition state structure with only one imaginary frequency. Further analysis showed that the vibration direction of the imaginary frequency was consistent with the OO bond breaking, proving that the transition state search was correct.

[0051] Change the OA-OB key from Adjust to The adjusted transition state model is subjected to transition state geometry optimization to obtain the optimized structure, and the frequency calculation result has only one imaginary frequency; and the vibration direction of the imaginary frequency is consistent with the OO bond breaking, proving that the transition state search is correct;

[0052] In order to obtain more accurate energy, single-point energy calculations with a larger basis set were performed based on the previous structural optimization;

[0053] (3) Constructing intermediate 2 in which the OO bond breaks after binding to the substrate: Based on the optimized calculation model, the key bond length is adjusted and the distance between the OA-OB bonds is adjusted to The other settings remain unchanged, and the geometry is optimized using the popular B3LYP-D3 method in symmetry-breaking density functional theory. The optimized structure of intermediate 2 is obtained, and the distance between CuA and CuB is The distance between CuA and OA is The CuA-OB distance is The distance between CuB-OA is The distance between CuB and OB is In order to obtain more accurate energy, single-point energy calculations with a larger basis set are performed based on the previous structural optimization.

[0054] (4) Constructing the transition state TS2 after the cleavage of the OO chemical bond and the binding to the substrate: Using the flexible scanning method, by analyzing the possible structural changes in the key chemical bond change, further calculating the energy of each step of the structure, obtaining the potential energy curve of the key chemical bond change process, and using the structure corresponding to the highest energy point on the potential energy curve as the initial transition state structure to complete the preliminary modeling;

[0055] By adjusting the key bond length and the distance between the OA-OB bonds, the transition state model was geometrically optimized and the frequency was calculated to obtain a transition state structure with only one imaginary frequency. Further analysis showed that the vibration direction of the imaginary frequency was consistent with the OO bond breaking, proving that the transition state search was correct.

[0056] Change the OA-OB key from Adjust to The adjusted transition state model is subjected to transition state geometry optimization to obtain the optimized structure, and the frequency calculation result has only one imaginary frequency; and the vibration direction of the imaginary frequency is consistent with the OO bond breaking, proving that the transition state search is correct;

[0057] In order to obtain more accurate energy, single-point energy calculations with a larger basis set were performed based on the previous structural optimization;

[0058] Through energy analysis, the energy of intermediate 2 is lower than that of intermediate 1, that is, the OO chemical bond is broken after the substrate binds.

[0059] Furthermore, the step (2) of constructing the CO-bonded intermediate 3 includes the following contents: a CO-bonding reaction occurs during the process, and based on the optimized intermediate 2, the key bond length is adjusted, and the distance between the CA-OB bonds is adjusted to The other settings remain unchanged, and the geometry is optimized using the popular B3LYP-D3 method in symmetry-breaking density functional theory. The structure of the optimized intermediate 3 is obtained, and the distance between CuA and CuB is The distance between OA and OB is The distances between CuA and OA, OB, and OC are The distances between CuB and OA, OB, and OD are

[0060] In order to obtain more accurate energy, single-point energy calculations with a larger basis set are performed based on the previous structural optimization.

[0061] Furthermore, the step (3) of constructing the transition state TS3 of CO bond formation includes the following contents: using a flexible scanning method, by analyzing the possible structural changes in the key chemical bond changes, further calculating the energy of each step structure, obtaining the potential energy curve of the key chemical bond change process, and using the corresponding structure of the highest energy point on the potential energy curve as the initial transition state structure to complete preliminary modeling;

[0062] By adjusting the key bond length and the distance between the CA-OB bonds, the transition state model was geometrically optimized and the frequency was calculated to obtain a transition state structure with only one imaginary frequency. Further analysis showed that the vibration direction of the imaginary frequency was consistent with CO bonding, proving that the transition state search was correct.

[0063] Change the CA-OB key from Adjust to The adjusted transition state model is subjected to transition state geometry optimization to obtain an optimized structure, and the frequency calculation result has only one imaginary frequency; and the vibration direction of the imaginary frequency is consistent with CO bonding, proving that the transition state search is correct;

[0064] In order to obtain more accurate energy, single-point energy calculations with a larger basis set are performed based on the previous structural optimization.

[0065] Furthermore, the step (4) of constructing the intermediate 4 of the CH bond breaking and the OH bond forming includes the following contents: the CH bond breaking and the OH bond forming reactions occur in the process, a hydrogen atom in the ligand phenol migrates to the OA in the binuclear copper oxygen center, the CA-HA bond breaks first, and then the OA-HA bond forms, and on the basis of the optimized intermediate 3, the key bond length is adjusted, and the distance between the OA-HA bonds is adjusted to The other settings remain unchanged, and the geometry is optimized using the popular B3LYP-D3 method in symmetry-breaking density functional theory. The structure of the optimized intermediate 4 is obtained, and the distance between CuA and CuB is The distance between OA and OB is The distances between CuA and OA, OB, and OC are The distances between CuB and OA, OB, and OD are

[0066] In order to obtain more accurate energy, single-point energy calculations with a larger basis set are performed based on the previous structural optimization.

[0067] Furthermore, the step (5) of constructing the transition state TS4 of the breaking of the CH bond and the forming of the OH bond comprises the following contents: adopting a flexible scanning method, by analyzing the possible structural changes in the key chemical bond changes, further calculating the energy of each step structure, obtaining the potential energy curve of the key chemical bond change process, and using the corresponding structure of the highest energy point on the potential energy curve as the initial transition state structure to complete the preliminary modeling;

[0068] By adjusting the key bond length and the distance between the CA-OB bonds, the transition state model was geometrically optimized and the frequency was calculated to obtain a transition state structure with only one imaginary frequency. Further analysis showed that the vibration direction of the imaginary frequency was consistent with CO bonding, proving that the transition state search was correct.

[0069] Reset the OA-HA key from Adjust to The adjusted transition state model is subjected to transition state geometry optimization to obtain an optimized structure, and the frequency calculation result has only one imaginary frequency; and the vibration direction of the imaginary frequency is consistent with the CH bond breaking and OH bond forming, proving that the transition state search is correct;

[0070] In order to obtain more accurate energy, single-point energy calculations with a larger basis set are performed based on the previous structural optimization.

[0071] Furthermore, the step (6) of constructing the product includes the following: adding a proton to participate in the reaction, so that a diamine-CuB(II)-OH2 monomer is separated from the binuclear copper center to obtain a product CuA(I)-semibenzoquinone monomer, and optimizing the geometric configuration using the more popular B3LYP-D3 method in the symmetry breaking density functional theory method to obtain the structure of the optimized product, and the bond length of CuA-OB is The bond length of CuA-OC is The bond length of CuA-N1 is The distance between CuA and N2 is

[0072] In order to obtain more accurate energy, single-point energy calculations with a larger basis set are performed based on the previous structural optimization.

[0073] Furthermore, the specific content of the solvent model correction in step (iii) is:

[0074] (1) PCM continuum model calculation: Based on the optimization of the gas phase ground state structure, the PCM polarized continuum solvation model in the GAUSSIAN09 program package was used, and single-point energy correction was performed using a calculation level equivalent to that of the gas phase. The heat and enthalpy corrections were then obtained through calculation frequency analysis, and the initial material energy was calculated considering the van der Waals effect.

[0075] Consider the effect of polymorphic reactivity on the energy of intermediates and transition states, determine the spin states of intermediates and transition states and the energy of each spin state;

[0076] (2) Construction of the initial QM / MM model:

[0077] In the VMD program, two solvent boxes, non-polar and polar solvents, are constructed, and the optimized cluster model solute molecules are placed in the solvent boxes;

[0078] Molecular force field parameters: The force field parameters of non-polar and polar solvent molecules are generated using the antechamber module of AMBER; the solute molecules use the covalent bond model to describe the coordination between the Cu ion and the ligand, and the parameters of the Cu ion center and the ligand are constructed using the MCPB module in the AMBER program, and the solvent small molecule parameters are calculated using the antechamber module in AMBER;

[0079] Molecular dynamics simulation: The solute molecules are fixed, and the solvent molecules are subjected to 1.5 ns of dynamics simulation to allow the solvent to reach full equilibrium; on this basis, a 10 ns molecular dynamics simulation is performed on the entire system; the dynamics simulation is completed using the Sander module of AMBER, and the NPT ensemble simulation temperature is set to 300K and the pressure is 1 atmosphere;

[0080] Collect the trajectories of molecular dynamics simulations and select a series of structures as the initial models for subsequent QM / MM calculations;

[0081] (3) Setting of QM / MM calculation parameters and calculation reaction mechanism:

[0082] In QM / MM calculations, the core region of the catalytic reaction, including biomimetic compounds and several solvent molecules, is defined as the QM region, which is described by density functional theory and medium basis set B3LYP functional and pseudopotential basis set; the part that is far away from the active center and does not directly participate in the reaction is described by AMBER force field; it is planned to include different numbers of solvent molecules in the QM region to discuss the effect of the solvent in the QM region on the catalytic reaction process; in QM / MM calculations, the effect of the solvent environment on the QM region is described by the electrostatic embedding scheme; QM / MM calculations are mainly completed using the CHEMSHELL program, and the reaction minimum is geometrically optimized using the DL-FIND optimizer in the CHEMSHELL program, and the optimized initial material is confirmed by frequency calculation.

[0083] (4) Comparison between PCM model and QM / MM model:

[0084] On the basis of QM calculation, the possible initial materials were re-searched by adopting QM / MM method under QM / MM solvent model and compared with the results of PCM continuum model, and the effects of different calculation methods and different polar solvents on the reaction were obtained.

[0085] Furthermore, the specific process of analyzing the reaction mechanism and path in the step (iv) is as follows: analyzing the bond length, bond angle and stereochemical changes around the central metal ion of the transition state and intermediates in each reaction, using the DL-FIND optimizer in the CHEMSHELL program to geometrically optimize the transition state and the minimum point, and confirming the transition state and intermediate optimized on the potential energy surface by frequency calculation. In order to obtain more accurate energy, based on the optimization of the gas phase ground state structure, the PCM polarized continuum solvation model in the GAUSSIAN09 program package is used, and a single point energy correction is performed using a calculation level equivalent to the gas phase, and then heat and enthalpy corrections are obtained by calculating frequency analysis, and the entire catalytic reaction potential energy surface is calculated considering the van der Waals effect, thereby obtaining the reaction path, and finally drawing a potential energy surface diagram; according to the reaction path and the potential energy diagram, the path reaction principle is analyzed.

[0086] The invention is based on density functional theory (DFT), adopts gas phase model to calculate QM reaction mechanism, optimizes the structure of intermediates and transition states, and uses vibration frequency analysis to confirm whether the transition state is correct, and performs single point energy calculation of a larger basis set on the basis of structural optimization to obtain more accurate energy; adopts PCM continuous medium solvation model in solvent model, and uses calculation level equivalent to gas phase to perform single point energy correction, and then obtains heat and enthalpy correction by calculation frequency analysis, and considers van der Waals effect to calculate the whole catalytic reaction potential energy surface, so as to obtain possible reaction channels, and finally, on the basis of QM calculation, adopts QM / MM method to search for possible intermediates and transition states again under QM / MM solvent model, determines reaction rate-determining step, determines reaction mechanism, and compares with the result of PCM continuous medium model, and obtains the influence of different calculation methods and solvents of different polarity on reaction. A set of complete theoretical methods that can be used to simulate such system is established, and a basis is provided for developing new solvent model for treating such system, and an important theoretical basis and technical reference are also provided for the design and development of efficient novel copper biocatalyst phenol.

[0087] The beneficial effects of the present invention are:

[0088] The present invention provides a double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] 2+The catalytic mechanism of 2,4-tert-butylphenol oxidation was studied by using the B3LYP-D3 method, a popular method in symmetry-breaking density functional theory, to optimize the geometric configuration. In order to obtain more accurate energy, based on the optimization of the gas phase ground state structure, the PCM polarized continuum solvation model in the GAUSSIAN09 program package was used, and a single-point energy correction was performed using a calculation level equivalent to the gas phase. The heat and enthalpy corrections were then obtained by calculating the frequency analysis, and the van der Waals effect was considered to calculate the potential energy surface of the entire catalytic reaction. The biomimetic compound oxygenated copper intermediate [Cu(II)2O2TMP32] 2+ The microscopic properties of the structure were analyzed and the microscopic kinetics of its catalytic 2,4-tert-butylphenol oxidation reaction mechanism were studied. A set of research methods for the double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] was designed. 2+ Theoretical calculation method for the catalytic mechanism of 2,4-tert-butylphenol oxidation revealed the reaction path and reaction mechanism of the catalytic oxidation reaction, which is a double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] 2+ It provides a theoretical basis for further application in the field of catalytic oxidation and provides an important theoretical basis and technical reference for the design and development of efficient new copper biocatalysts for phenol. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 is the reaction mechanism diagram;

[0090] Figure 2 is the calculated path potential energy profile at the B3LYP level of theory;

[0091] Wherein, relative free energy G, kcal / mol, closed shell singlet state is represented by C, open shell triplet state is represented by T, open shell singlet state is represented by AF). DETAILED DESCRIPTION

[0092] The present invention is described in detail below in conjunction with specific embodiments.

[0093] Example 1

[0094] (I) Construction of a double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] 2+ Computational modeling of the catalytic reaction of 2,4-tert-butylphenol: construction of a double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] based on the reaction information provided by the experiment 2+ The computational model.

[0095] (1) Preliminary construction of a double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] 2+The gas phase model is used as a possible initial state calculation model. The B3LYP-D3 method, which is a popular symmetry-breaking density functional theory method, is used to optimize the geometric configuration. The optimal model is selected based on the calculation results by comprehensively considering the configuration stability and energy level.

[0096] According to the experimental data, the model was built in Gaussview software to preliminarily construct the double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] 2+ ; The distance between the two core coppers is pulled to When the binuclear copper center is placed in the TMP3 ligand, the distances between CuA and CuB and O on the ligand are The optimized structure calculations were performed using B3LYP functional and pseudopotential basis set to obtain the bond lengths, bond angles and dihedral angles of the optimized complexes. The QM calculations were mainly performed using the GAUSSIAN09 program.

[0097] The distance between the binuclear copper CuA-CuB in the optimized structure is The distance between OA and OB is The distances between CuA and OA, OB, and OC are The distances between CuB and OA, OB, and OD are

[0098] (2) In order to obtain more accurate energy, single-point energy calculations are performed on a larger basis set based on the previous structural optimization.

[0099] (II) Design of gas phase model reaction pathway:

[0100] (a) Determine the order of substrate binding and OO bond cleavage

[0101] Tyrosinase biomimetic compound oxygenated copper intermediate [Cu(II)2O2TMP32] 2+ A key issue in the catalytic oxidation of 2,4-tert-butylphenol to o-benzoquinone is whether the cleavage of the O-O chemical bond occurs before or after binding to the substrate.

[0102] (a1) Construction of intermediate 1 that binds to the substrate after the cleavage of the OO chemical bond:

[0103] (1) Based on the optimized calculation model, the key bond length is adjusted to adjust the distance between the OA-OB bonds to The other settings remain unchanged, and the geometry is optimized using the popular B3LYP-D3 method in symmetry-breaking density functional theory. The structure of the optimized intermediate 1 is obtained, and the distance between CuA and CuB is The distances between CuA and CuB and OA and OB are

[0104] (2) In order to obtain more accurate energy, single-point energy calculations are performed on a larger basis set based on the previous structural optimization.

[0105] (a2) Construction of the transition state TS1 after the breaking of the OO chemical bond and the binding with the substrate:

[0106] (1) By using the flexible scanning method, the possible structural changes in the key chemical bond changes are analyzed, the energy of each step of the structure is further calculated, and the potential energy curve of the key chemical bond change process is obtained. The corresponding structure of the highest energy point on the potential energy curve is used as the initial transition state structure to complete the preliminary modeling.

[0107] By adjusting the key bond length and the distance between the OA-OB bonds, the transition state model was geometrically optimized and the frequency was calculated to obtain a transition state structure with only one imaginary frequency. Further analysis showed that the vibration direction of the imaginary frequency was consistent with the breaking of the OO bond, proving that the transition state search was correct.

[0108] Change the OA-OB key from Adjust to The adjusted transition state model was subjected to transition state geometry optimization to obtain the optimized structure, and the frequency calculation result had only one imaginary frequency; and analysis of the vibration direction of the imaginary frequency was consistent with the OO bond breakage, proving that the transition state search was correct.

[0109] (2) In order to obtain more accurate energy, single-point energy calculations are performed on a larger basis set based on the previous structural optimization.

[0110] (a3) Construction of intermediate 2 in which the OO bond is cleaved after binding to the substrate:

[0111] (1) Based on the optimized calculation model, the key bond length is adjusted to adjust the distance between the OA-OB bonds to The other settings remain unchanged, and the geometry is optimized using the popular B3LYP-D3 method in symmetry-breaking density functional theory. The structure of the optimized intermediate 1 is obtained, and the distance between CuA and CuB is The distance between CuA and OA is The CuA-OB distance is The distance between CuB-OA is The distance between CuB and OB is

[0112] (2) In order to obtain more accurate energy, single-point energy calculations are performed on a larger basis set based on the previous structural optimization.

[0113] (a4) Construction of transition state TS2 that binds to the substrate after the cleavage of the OO chemical bond:

[0114] (1) By using the flexible scanning method, the possible structural changes in the key chemical bond changes are analyzed, and the energy of each step of the structure is further calculated to obtain the potential energy curve of the key chemical bond change process. The corresponding structure of the highest energy point on the potential energy curve is used as the initial transition state structure to complete the preliminary modeling.

[0115] By adjusting the key bond length and the distance between the OA-OB bonds, the transition state model was geometrically optimized and the frequency was calculated to obtain a transition state structure with only one imaginary frequency. Further analysis showed that the vibration direction of the imaginary frequency was consistent with the breaking of the OO bond, proving that the transition state search was correct.

[0116] Change the OA-OB key from Adjust to The adjusted transition state model was subjected to transition state geometry optimization to obtain the optimized structure, and the frequency calculation result had only one imaginary frequency; and analysis of the vibration direction of the imaginary frequency was consistent with the OO bond breakage, proving that the transition state search was correct.

[0117] (2) In order to obtain more accurate energy, single-point energy calculations are performed on a larger basis set based on the previous structural optimization.

[0118] Through energy analysis, the energy of intermediate 2 is lower than that of intermediate 1, that is, the OO chemical bond is broken after the substrate binds.

[0119] (b) Construction of CO bond-forming intermediate 3:

[0120] (1) The CO bond formation reaction occurs in the process. Based on the optimized intermediate 2, the key bond length is adjusted to adjust the distance between the CA-OB bonds to The other settings remain unchanged, and the geometry is optimized using the popular B3LYP-D3 method in symmetry-breaking density functional theory. The structure of the optimized intermediate 3 is obtained, and the distance between CuA and CuB is The distance between OA and OB is The distances between CuA and OA, OB, and OC are The distances between CuB and OA, OB, and OD are

[0121] (2) In order to obtain more accurate energy, single-point energy calculations are performed on a larger basis set based on the previous structural optimization.

[0122] (c) Construct the transition state TS3 of CO bond formation:

[0123] (1) By using the flexible scanning method, the possible structural changes in the key chemical bond changes are analyzed, the energy of each step of the structure is further calculated, and the potential energy curve of the key chemical bond change process is obtained. The corresponding structure of the highest energy point on the potential energy curve is used as the initial transition state structure to complete the preliminary modeling.

[0124] By adjusting the key bond length and the distance between the CA-OB bonds, the transition state model was geometrically optimized and the frequency was calculated to obtain a transition state structure with only one imaginary frequency. Further analysis showed that the vibration direction of the imaginary frequency was consistent with CO bonding, proving that the transition state search was correct.

[0125] Change the CA-OB key from Adjust to The adjusted transition state model was subjected to transition state geometry optimization to obtain the optimized structure, and the frequency calculation result had only one imaginary frequency; and analysis of the vibration direction of the imaginary frequency was consistent with CO bonding, proving that the transition state search was correct.

[0126] (2) In order to obtain more accurate energy, single-point energy calculations are performed on a larger basis set based on the previous structural optimization.

[0127] (d) Constructing intermediate 4 of CH bond breaking and OH bond forming:

[0128] (1) During the process, a CH bond breaking reaction and an OH bond forming reaction occur. A hydrogen atom in the ligand phenol migrates to the OA in the binuclear copper oxygen center, first the CA-HA bond breaks, then the OA-HA bond forms. Based on the optimized intermediate 3, the key bond length is adjusted, and the distance between the OA-HA bonds is adjusted to The other settings remain unchanged, and the geometry is optimized using the popular B3LYP-D3 method in symmetry-breaking density functional theory. The structure of the optimized intermediate 4 is obtained, and the distance between CuA and CuB is The distance between OA and OB is The distances between CuA and OA, OB, and OC are The distances between CuB and OA, OB, and OD are

[0129] (2) In order to obtain more accurate energy, single-point energy calculations are performed on a larger basis set based on the previous structural optimization.

[0130] (e) Construct the transition state TS4 of CH bond breaking and OH bond forming:

[0131] (1) By using the flexible scanning method, the possible structural changes in the key chemical bond changes are analyzed, and the energy of each step of the structure is further calculated to obtain the potential energy curve of the key chemical bond change process. The corresponding structure of the highest energy point on the potential energy curve is used as the initial transition state structure to complete the preliminary modeling.

[0132] By adjusting the key bond length and the distance between the CA-OB bonds, the transition state model was geometrically optimized and the frequency was calculated to obtain a transition state structure with only one imaginary frequency. Further analysis showed that the vibration direction of the imaginary frequency was consistent with CO bonding, proving that the transition state search was correct.

[0133] Change the OA-HA key from Adjust to The adjusted transition state model was subjected to transition state geometry optimization to obtain the optimized structure, and the frequency calculation result had only one imaginary frequency; and analysis of the vibration direction of the imaginary frequency was consistent with the CH bond breaking and OH bond forming, proving that the transition state search was correct.

[0134] (2) In order to obtain more accurate energy, single-point energy calculations are performed on a larger basis set based on the previous structural optimization.

[0135] (f) Construction product

[0136] (1) A proton is added to participate in the reaction, so that a diamine-CuB(II)-OH2 monomer is separated from the binuclear copper center to obtain the product CuA(I)-semibenzoquinone monomer. The geometric configuration is optimized using the B3LYP-D3 method, which is a popular method in symmetry-breaking density functional theory. The structure of the optimized product is obtained. The bond length of CuA-OB is The bond length of CuA-OC is The bond length of CuA-N1 is The distance between CuA and N2 is

[0137] (2) In order to obtain more accurate energy, single-point energy calculations are performed on a larger basis set based on the previous structural optimization.

[0138] (III) Solvent model correction

[0139] 1. Solvent model:

[0140] 1.1PCM continuous medium model calculation.

[0141] (1) Based on the optimization of the gas phase ground state structure, the PCM polarizable continuum solvation model in the GAUSSIAN09 program package was used, and a single-point energy correction was performed using a calculation level equivalent to that of the gas phase. The heat and enthalpy corrections were then obtained through computational frequency analysis, and the initial material energy was calculated considering the van der Waals effect.

[0142] (2) Consider the effect of polymorphic reactivity on the energy of intermediates and transition states, and determine the spin states of intermediates and transition states and the energy of each spin state.

[0143] 1.2QM / MM model.

[0144] (1) Construction of the initial QM / MM model

[0145] In the VMD program, two solvent boxes (non-polar and polar solvents) were constructed, and the solute molecules (optimized cluster models) were placed in the solvent boxes.

[0146] Molecular force field parameters: The force field parameters of solvent molecules (non-polar: acetonitrile molecules; polar solvent: water) are generated using the antechamber module of AMBER; the solute molecules use a covalent bond model to describe the coordination effect between Cu ions and ligands, and the MCPB module in the AMBER program is used to construct the parameters of the Cu ion center and the ligand, and the antechamber module in AMBER is used to calculate the solvent small molecule parameters.

[0147] Molecular dynamics simulation: The solute molecules are fixed, and the solvent molecules are simulated for 1.5 ns to make the solvent fully balanced; on this basis, the entire system is simulated for 10 ns. The dynamics simulation is completed using the Sander module of AMBER, and the NPT ensemble simulation temperature is set to 300K and the pressure is 1 atmosphere.

[0148] Collect the trajectories of molecular dynamics simulations and select a series of structures as the initial models for subsequent QM / MM calculations.

[0149] (2) Setting of QM / MM calculation parameters and calculation of reaction mechanism

[0150] In QM / MM calculations, the core region of the catalytic reaction, including biomimetic compounds and several solvent molecules, is defined as the QM region. The QM region is described using density functional methods and medium basis sets (B3LYP functionals and pseudopotential basis sets); the part that is far away from the active center and does not directly participate in the reaction is described using the AMBER force field. It is planned to include different numbers of solvent molecules (1 to 10) in the QM region to discuss the effect of the solvent in the QM region on the catalytic reaction process. In QM / MM calculations, the effect of the solvent environment on the QM region is described using an electrostatic embedding scheme. Since there is generally no covalent bond between the solvent and the biomimetic compound, there is no need to consider the problem of chemical bond truncation in the QM / MM method. In the initial configuration optimization calculation, the solvent is divided into two parts, which are 1 / 200 nm away from any atom of the solute. (This is an empirical value and can be changed according to specific circumstances) The solvent molecules within will be optimized together with the solute, and Solvent molecules other than the above are frozen and only provide electrostatic fields during the optimization process without directly participating in the optimization. QM / MM calculations are mainly completed using the CHEMSHELL program. The reaction minimum is geometrically optimized using the DL-FIND optimizer in the CHEMSHELL program. The optimized initial product is confirmed by frequency calculation.

[0151] (3) Comparison between PCM model and QM / MM model

[0152] On the basis of QM calculation, the possible initial materials were re-searched by adopting QM / MM method under QM / MM solvent model and compared with the results of PCM continuum model, and the effects of different calculation methods and different polar solvents on the reaction were obtained.

[0153] (IV) Analysis of reaction mechanism and pathway

[0154] The bond lengths, bond angles and stereochemical changes around the central metal ions of the transition states and intermediates in each reaction were analyzed. The DL-FIND optimizer in the CHEMSHELL program was used to geometrically optimize the transition states and minimum points. The optimized transition states and intermediates on the potential energy surface were confirmed by frequency calculation. In order to obtain more accurate energy, the PCM polarized continuum solvation model in the GAUSSIAN09 program package was used on the basis of gas phase ground state structure optimization, and single-point energy correction was performed using a calculation level equivalent to the gas phase. The heat and enthalpy corrections were then obtained by calculating the frequency analysis, and the entire catalytic reaction potential energy surface was calculated considering the van der Waals effect, thereby obtaining the reaction path. Finally, the potential energy surface diagram was drawn, as shown in the figure. Figure 2 shown.

[0155] Specifically, the analysis of the tyrosinase biomimetic compound oxygenated copper intermediate [Cu(II)2O2TMP32] 2+ A key issue in the catalytic oxidation of 2,4-tert-butylphenol to o-benzoquinone is whether the cleavage of the OO chemical bond occurs before or after binding with the substrate. Through calculation, it is found that the cleavage of the OO chemical bond occurs after binding with the substrate, which is low in energy. 2+ The optimized compound is used as the initial compound of the reaction pathway.

[0156] According to the reaction path and the potential energy diagram, analyze the path reaction principle, such as Figure 1 :

[0157] The first step of the reaction is the breaking of the OO chemical bond of the initial binuclear copper oxygen center to generate intermediate 2. In this process, μ-η2:η2-peroxo copper oxygen clusters are transformed into bis-μ-oxo copper oxygen clusters, and the valence state of the Cu ion changes from +2 to +3. The free energy barrier that needs to be broken through when passing through the transition state is 17.84 kcal / mol. This step is the rate-determining step of the reaction. The second step is that an oxygen atom (OB) in the binuclear copper oxygen center electrophilically attracts the carbon on the substrate benzene ring through a transition state TS3 to generate an intermediate 3 bonded with a CO chemical bond. This indicates that the electron is transferred from the substrate to the copper ion, and the valence state of CuA is reduced from +3 in the intermediate to +2. The free energy barrier that needs to be broken through when passing through the transition state is 11.61 kcal / mol. The third step is to transfer the proton HA from CA in the six-membered ring to the bridging oxygen atom OA. The free energy barrier that needs to be broken through when passing through the transition state is 3.25 kcal / mol, which indicates that the proton transfer is very rapid. The last step is to add another proton to participate in the reaction. Adding a proton to the hydroxyl group in intermediate 4 causes a diamine -CuB(II)-OH2 monomer to separate from the binuclear copper center to obtain the product CuA(I)-semibenzoquinone monomer.

[0158] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. Double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] 2+ The method for studying the mechanism of catalytic oxidation of 2,4-tert-butylphenol is characterized in that: The following steps are involved: (I) Construction of gas phase model Preliminary construction of a double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] 2+ The gas phase model is used as a possible initial state calculation model to calculate the QM reaction mechanism. The QM calculation is completed using the GAUSSIAN09 program. The optimal model is selected based on the calculation results by comprehensively considering the configuration stability and energy level. (II) Design of gas phase model reaction pathway (1) Determine the order of substrate binding and OO bond cleavage; (2) Construction of CO-bonded intermediate 3; (3) construct the transition state TS3 of CO bond formation; (4) construct intermediate 4 for the breaking of CH bond and the forming of OH bond; (5) Construct the transition state TS4 of CH bond breaking and OH bond forming; (6) construct products; (III) Solvent model correction (1) PCM continuous medium model calculation; (2) Construct the initial QM / MM model; (3) Setting of QM / MM calculation parameters and calculation of reaction mechanism; (4) Comparison between PCM model and QM / MM model; (iv) Analyze the reaction mechanism and pathway.

2. The double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] according to claim 1 2+ The method for studying the mechanism of catalytic oxidation of 2,4-tert-butylphenol is characterized by: When the gas phase model is constructed in step (1): (1) The B3LYP-D3 method in symmetry-breaking density functional theory is used to optimize the geometric configurations of possible transition states, intermediates, and products in the reaction process. The gas-phase reaction mechanism is determined by making a potential energy profile based on the structure and energy, and confirmed by vibration frequency analysis. Specifically: In the reaction model, the B3LYP / gen level was used to perform geometry optimization and calculate frequency analysis for all intermediates and transition states; The input files for the optimization calculation are as follows: Intermediate optimization input file: #p opt ub3lyp / 6-31G(d,p)&LANL2D geom=connectivitypseudo=read; Transition state optimization input file: #p opt=(ts,modredundant,noeigen)ub3lyp / 6-31G(d,p)&LANL2D geom=connectivitypseudo=read guess=read; All intermediates have no imaginary frequency, and transition states have only one imaginary frequency; The input file for higher level single point energy calculation is as follows: #p ub3lyp / cc-pvtz geom=connectivitypseudo=read; The spin coupling characteristics of the system are described by adding symmetry breaking correction on the basis of the existing density functional theory. The method is as follows: In a binuclear metal system, if the spin quantum numbers of the two metals are S A and S B , then the spin coupling of the system can be described by the Heisenberg Hamitonian, <h2 style=";text-align:left;direction:ltr">H<h2 style=";text-align:left;direction:ltr"> spin <h2 style=";text-align:left;direction:ltr"> =2J<h2 style=";text-align:left;direction:ltr"> S <h2 style=";text-align:left;direction:ltr"> AS<h2 style=";text-align:left;direction:ltr"> B <h2 style=";text-align:left;direction:ltr"> (1) where J is the Heisenberg spin coupling parameter. For this system, a highest spin state HS can be defined. In the HS state, S = 1; the HS state is a pure state and can be described by a single determinant, and the spin electrons are arranged parallelly in two nuclei. Similarly, a symmetry broken state BS can be defined. In the BS state, S = 0; the BS state is not a pure spin state but a linear combination of a series of pure spin states, and the total quantum number S of these pure spin states ranges from S = |S A -S B | to S = |S A +S B | increasing step by step, and the energy of each spin state is E(S)=E GS +S(S+1)J / 2 (2) where EGS is the ground state energy; according to (1) and (2), we can get E HS (S=S MAX )-E GS (S=|S A -S B |)=S MAX *(S MAX +1)J / 2 (3) as well as E HS (S=S MAX )-E BS =S 2 MAX *J / 2 (4) From (3) and (4), we can get the Heisenberg spin coupling parameter and ground state energy: E GS =[(S MAX +1)*E BS -E HS ] / S MAX (5) And from EGS and J value, the energy of any spin state can be calculated from (2).

3. The double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] according to claim 1 2+ The method for studying the mechanism of catalytic oxidation of 2,4-tert-butylphenol is characterized by: The step (1) of determining the order of substrate binding and OO bond cleavage comprises the following: (1) Constructing intermediate 1 that binds to the substrate after the OO chemical bond is broken: Based on the optimized calculation model, adjust the key bond length and the distance between the OA-OB bonds to The other settings remain unchanged, and the geometry is optimized using the popular B3LYP-D3 method in symmetry-breaking density functional theory. The structure of the optimized intermediate 1 is obtained, and the distance between CuA and CuB is The distances between CuA and CuB and OA and OB are In order to obtain more accurate energy, single-point energy calculations with a larger basis set were performed based on the previous structural optimization; (2) Constructing the transition state TS1 after the breaking of the OO chemical bond and the binding to the substrate: Using the flexible scanning method, by analyzing the possible structural changes in the key chemical bond change, further calculating the energy of each step of the structure, obtaining the potential energy curve of the key chemical bond change process, and using the structure corresponding to the highest energy point on the potential energy curve as the initial transition state structure to complete the preliminary modeling; By adjusting the key bond length and the distance between the OA-OB bonds, the transition state model was geometrically optimized and the frequency was calculated to obtain a transition state structure with only one imaginary frequency. Further analysis showed that the vibration direction of the imaginary frequency was consistent with the OO bond breaking, proving that the transition state search was correct. Change the OA-OB key from Adjust to The adjusted transition state model is subjected to transition state geometry optimization to obtain an optimized structure, and the frequency calculation result has only one imaginary frequency; and the vibration direction of the imaginary frequency is consistent with the OO bond breakage, proving that the transition state search is correct; In order to obtain more accurate energy, single-point energy calculations with a larger basis set were performed based on the previous structural optimization; (3) Constructing intermediate 2 in which the OO bond breaks after binding to the substrate: Based on the optimized calculation model, the key bond length is adjusted and the distance between the OA-OB bonds is adjusted to The other settings remain unchanged, and the geometry is optimized using the popular B3LYP-D3 method in symmetry-breaking density functional theory. The optimized structure of intermediate 2 is obtained, and the distance between CuA and CuB is The distance between CuA and OA is The CuA-OB distance is The distance between CuB-OA is The distance between CuB and OB is In order to obtain more accurate energy, single-point energy calculations with a larger basis set are performed based on the previous structural optimization. (4) Constructing the transition state TS2 after the cleavage of the OO chemical bond and the binding to the substrate: Using the flexible scanning method, by analyzing the possible structural changes in the key chemical bond change, further calculating the energy of each step of the structure, obtaining the potential energy curve of the key chemical bond change process, and using the structure corresponding to the highest energy point on the potential energy curve as the initial transition state structure to complete the preliminary modeling; By adjusting the key bond length and the distance between the OA-OB bonds, the transition state model was geometrically optimized and the frequency was calculated to obtain a transition state structure with only one imaginary frequency. Further analysis showed that the vibration direction of the imaginary frequency was consistent with the OO bond breaking, proving that the transition state search was correct. Change the OA-OB key from Adjust to The adjusted transition state model is subjected to transition state geometry optimization to obtain an optimized structure, and the frequency calculation result has only one imaginary frequency; and the vibration direction of the imaginary frequency is consistent with the OO bond breakage, proving that the transition state search is correct; In order to obtain more accurate energy, single-point energy calculations with a larger basis set were performed based on the previous structural optimization; Through energy analysis, the energy of intermediate 2 is lower than that of intermediate 1, that is, the OO chemical bond is broken after the substrate binds.

4. The double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] according to claim 1 2+ The method for studying the mechanism of catalytic oxidation of 2,4-tert-butylphenol is characterized by: The step (2) of constructing the CO-bonded intermediate 3 comprises the following contents: a CO-bonded reaction occurs during the process, and based on the optimized intermediate 2, the key bond length is adjusted, and the distance between the CA-OB bonds is adjusted to The other settings remain unchanged, and the geometry is optimized using the popular B3LYP-D3 method in symmetry-breaking density functional theory. The structure of the optimized intermediate 3 is obtained, and the distance between CuA and CuB is The distance between OA and OB is The distances between CuA and OA, OB, and OC are The distances between CuB and OA, OB, and OD are In order to obtain more accurate energy, single-point energy calculations with a larger basis set are performed based on the previous structural optimization.

5. The double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] according to claim 1 2+ The method for studying the mechanism of catalytic oxidation of 2,4-tert-butylphenol is characterized by: The step (3) of constructing the transition state TS3 of CO bond formation comprises the following contents: By using the flexible scanning method, the energy of each step structure is further calculated through the analysis of possible structural changes in the key chemical bond changes, and the potential energy curve of the key chemical bond change process is obtained. The corresponding structure of the highest energy point on the potential energy curve is used as the initial transition state structure to complete the preliminary modeling. By adjusting the key bond length and the distance between the CA-OB bonds, the transition state model was geometrically optimized and the frequency was calculated to obtain a transition state structure with only one imaginary frequency. Further analysis showed that the vibration direction of the imaginary frequency was consistent with CO bonding, proving that the transition state search was correct. Change the CA-OB key from Adjust to The adjusted transition state model is subjected to transition state geometry optimization to obtain an optimized structure, and the frequency calculation result has only one imaginary frequency; and the vibration direction of the imaginary frequency is consistent with CO bonding, proving that the transition state search is correct; In order to obtain more accurate energy, single-point energy calculations with a larger basis set are performed based on the previous structural optimization.

6. The double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] according to claim 1 2+ The method for studying the mechanism of catalytic oxidation of 2,4-tert-butylphenol is characterized by: The step (4) of constructing the intermediate 4 of the breaking of the CH bond and the forming of the OH bond comprises the following contents: the process undergoes a CH bond breaking reaction and an OH bond forming reaction, a hydrogen atom in the ligand phenol migrates to the OA in the binuclear copper oxygen center, the CA-HA bond breaks first, and then the OA-HA bond forms, and based on the optimized intermediate 3, the key bond length is adjusted, and the distance between the OA-HA bonds is adjusted to The other settings remain unchanged, and the geometry is optimized using the popular B3LYP-D3 method in symmetry-breaking density functional theory. The structure of the optimized intermediate 4 is obtained, and the distance between CuA and CuB is The distance between OA and OB is The distances between CuA and OA, OB, and OC are The distances between CuB and OA, OB, and OD are In order to obtain more accurate energy, single-point energy calculations with a larger basis set are performed based on the previous structural optimization.

7. The double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] according to claim 1 2+ The method for studying the mechanism of catalytic oxidation of 2,4-tert-butylphenol is characterized by: The step (5) constructing the transition state TS4 of the breaking of the CH bond and the forming of the OH bond comprises the following contents: adopting a flexible scanning method, through analyzing the possible structural changes in the key chemical bond change, further calculating the energy of each step structure, obtaining the potential energy curve of the key chemical bond change process, and using the corresponding structure of the highest energy point on the potential energy curve as the initial transition state structure to complete the preliminary modeling; By adjusting the key bond length and the distance between the CA-OB bonds, the transition state model was geometrically optimized and the frequency was calculated to obtain a transition state structure with only one imaginary frequency. Further analysis showed that the vibration direction of the imaginary frequency was consistent with CO bonding, proving that the transition state search was correct. Change the OA-HA key from Adjust to The adjusted transition state model is subjected to transition state geometry optimization to obtain an optimized structure, and the frequency calculation result has only one imaginary frequency; and the vibration direction of the imaginary frequency is consistent with the CH bond breaking and OH bond forming, proving that the transition state search is correct; In order to obtain more accurate energy, single-point energy calculations with a larger basis set are performed based on the previous structural optimization.

8. The double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] according to claim 1 2+ The method for studying the mechanism of catalytic oxidation of 2,4-tert-butylphenol is characterized by: The step (6) of constructing the product includes the following contents: Adding a proton to participate in the reaction, a diamine-CuB(II)-OH2 monomer is separated from the binuclear copper center to obtain the product CuA(I)-semibenzoquinone monomer. The geometric configuration is optimized using the more popular B3LYP-D3 method in symmetry-breaking density functional theory methods to obtain the structure of the optimized product. The bond length of CuA-OB is The bond length of CuA-OC is The bond length of CuA-N1 is The distance between CuA and N2 is In order to obtain more accurate energy, single-point energy calculations with a larger basis set are performed based on the previous structural optimization.

9. The double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] according to claim 1 2+ The method for studying the mechanism of catalytic oxidation of 2,4-tert-butylphenol is characterized by: The specific content of the solvent model correction in step (iii) is: (1) PCM continuum model calculation: Based on the optimization of the gas phase ground state structure, the PCM polarized continuum solvation model in the GAUSSIAN09 program package was used, and single-point energy correction was performed using a calculation level equivalent to that of the gas phase. The heat and enthalpy corrections were then obtained through calculation frequency analysis, and the initial material energy was calculated considering the van der Waals effect. Consider the effect of polymorphic reactivity on the energy of intermediates and transition states, determine the spin states of intermediates and transition states and the energy of each spin state; (2) Construction of the initial QM / MM model: In the VMD program, two solvent boxes, non-polar and polar solvents, are constructed, and the optimized cluster model solute molecules are placed in the solvent boxes; Molecular force field parameters: The force field parameters of non-polar and polar solvent molecules are generated using the antechamber module of AMBER; the solute molecules use the covalent bond model to describe the coordination between the Cu ion and the ligand, and the parameters of the Cu ion center and the ligand are constructed using the MCPB module in the AMBER program, and the solvent small molecule parameters are calculated using the antechamber module in AMBER; Molecular dynamics simulation: The solute molecules are fixed, and the solvent molecules are subjected to 1.5 ns of dynamics simulation to allow the solvent to reach full equilibrium; on this basis, a 10 ns molecular dynamics simulation is performed on the entire system; the dynamics simulation is completed using the Sander module of AMBER, and the NPT ensemble simulation temperature is set to 300K and the pressure is 1 atmosphere; Collect the trajectories of molecular dynamics simulations and select a series of structures as the initial models for subsequent QM / MM calculations; (3) Setting of QM / MM calculation parameters and calculation reaction mechanism: In QM / MM calculations, the core region of the catalytic reaction, including biomimetic compounds and several solvent molecules, is defined as the QM region, which is described by density functional theory and medium basis set B3LYP functional and pseudopotential basis set; the part far from the active center and not directly involved in the reaction is described by AMBER force field; it is planned to include different numbers of solvent molecules in the QM region to discuss the effect of the solvent in the QM region on the catalytic reaction process; in QM / MM calculations, the effect of the solvent environment on the QM region is described by electrostatic embedding scheme; QM / MM calculations are mainly completed using CHEMSHELL program, and the reaction minimum is geometrically optimized using DL-FIND optimizer in CHEMSHELL program, and the optimized initial material is confirmed by frequency calculation; (4) Comparison between PCM model and QM / MM model: On the basis of QM calculation, the possible initial materials were re-searched by adopting QM / MM method under QM / MM solvent model and compared with the results of PCM continuum model, and the effects of different calculation methods and different polar solvents on the reaction were obtained.

10. The double copper biomimetic oxygenated copper intermediate [Cu(II)2O2TMP32] according to claim 1 2+ The method for studying the mechanism of catalytic oxidation of 2,4-tert-butylphenol is characterized by: The specific process of analyzing the reaction mechanism and path in the step (iv) is as follows: analyzing the bond length, bond angle and stereochemical changes around the central metal ion of the transition state and the intermediate in each reaction, using the DL-FIND optimizer in the CHEMSHELL program to geometrically optimize the transition state and the minimum point, and confirming the transition state and the intermediate optimized on the potential energy surface by frequency calculation. In order to obtain more accurate energy, based on the optimization of the gas phase ground state structure, the PCM polarized continuous medium solvation model in the GAUSSIAN09 program package is used, and a single point energy correction is performed using a calculation level equivalent to the gas phase, and then heat and enthalpy corrections are obtained by calculating frequency analysis, and the entire catalytic reaction potential energy surface is calculated considering the van der Waals effect, thereby obtaining a reaction path, and finally drawing a potential energy surface diagram; according to the reaction path and the drawn potential energy diagram, the path reaction principle is analyzed.

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