Chemical-enzyme integrated catalyst for oxygen functionalization reaction of inert C-H bond as well as preparation and application of chemical-enzyme integrated catalyst

Abstract

The invention discloses a chemical-enzyme integrated catalyst for oxygen functionalization reaction of an inert C-H bond. The chemical-enzyme integrated catalyst is obtained by simultaneously fixing a rhodium (III) complex and non-specific peroxygenase AaeUPO by a covalent organic framework (COF) material with a macroporous structure inside, wherein trialdehyde phloroglucinol and 5, 5 '-diamino-2, 2'-dipyridyl are used as ligands, polystyrene spheres are used as hard templates to prepare holes, and a covalent organic framework (COF) material with a macroporous structure is prepared, and is recorded as macro-COF. The chemical-enzyme integrated catalyst provided by the invention not only realizes efficient conversion of in-situ production of hydrogen peroxide to drive AaeUPO to catalyze an inert C-H bond oxygen functionalization reaction, but also can effectively inhibit damage of high-concentration hydrogen peroxide to an AaeUPO active center.

Description

Technical Field

[0001] The invention relates to the technical field of catalysts, in particular to a chemical-enzyme integrated catalyst for oxygen functionalization reaction of inert CH bonds, and the preparation of the catalyst. Background Art

[0002] Inert C(sp 3 )-H bond, due to its high dissociation energy, its functionalization usually requires catalysis. Among them, chemical catalysis has been gradually restricted in its application in recent years due to its disadvantages such as low reaction efficiency, poor selectivity and environmental unfriendliness. Biocatalysis has received more and more attention in the field of organic synthesis due to its advantages of mild reaction conditions and high selectivity.

[0003] Nonspecific peroxygenases (UPOs, EC1.11.2.1) are a class of heme oxygenases from fungi. They can use hydrogen peroxide (H2O2) as an oxidant to directly perform two-electron transfer to achieve regional and stereospecific oxygen functionalization. As an efficient oxygenation reaction biocatalyst, they have excellent catalytic activity and broad substrate specificity in the enantioselective hydroxylation of C-H bonds. Among them, AaeUPO obtained by recombinant fermentation of Pichia pastoris can directly use H2O2 as an electron acceptor to oxygen functionalize C-H bonds. It has the advantages of simple electron transfer process, high catalytic activity and a wide substrate spectrum. However, AaeUPO is easily inactivated when the H2O2 concentration is too high, and has defects such as poor thermal stability and pH stability. These problems have seriously hindered its application in various reactions of catalyzing the oxygen functionalization of inert C-H bonds. Although existing studies have proposed some solutions, such as using in-situ continuous production of H2O2 to ensure continuous supply of H2O2 while effectively reducing local concentration and avoiding the inactivation of AaeUPO, even if H2O2 is continuously synthesized by enzyme catalysis, chemical catalysis, photocatalysis and electrocatalysis and then in-situ driven AaeUPO catalyzes the oxygen functionalization reaction of CH bonds, there are still disadvantages such as complex reaction system, high risk and poor economy. Therefore, the development of a system that can continuously and efficiently catalyze the direct oxygen functionalization reaction of inert CH bonds is of great significance to the development of the field of organic chemical synthesis. Summary of the invention

[0004] In order to address the deficiencies in the prior art, the present invention has developed a chemical-enzyme integrated catalyst, which uses a covalent organic framework COF material with a macroporous structure inside as a carrier, and simultaneously fixes metal sites for catalyzing the reduction of oxygen to H2O2 and a non-specific peroxygenase AaeUPO for catalyzing the oxygen functionalization of CH bonds. This not only achieves the in situ production of hydrogen peroxide to drive the efficient conversion of the inert CH bond oxygen functionalization reaction catalyzed by AaeUPO, but also simplifies the reaction process to achieve a one-pot reaction, effectively inhibiting the destruction of the active center of AaeUPO by high-concentration hydrogen peroxide during the catalytic reaction.

[0005] To achieve the above-mentioned purpose, the present invention provides a chemical-enzyme integrated catalyst for oxygen functionalization reaction of inert CH bonds, which is obtained by simultaneously fixing a rhodium (III) complex and a non-specific peroxygenase on a covalent organic framework COF material having a macroporous structure inside; wherein trialdehyde phloroglucinol and 5,5'-diamino-2,2'-bipyridine are used as ligands, and polystyrene balls are used as templates to prepare pores, thereby preparing a covalent organic framework COF material having a macroporous structure, which is denoted as macro-COF.

[0006] The present invention utilizes porous organic polymer materials such as COF as carriers. On the one hand, it benefits from the high stability of the material ligands connected by covalent bonds. On the other hand, it takes advantage of the easy regulation of the COF structure and composition. By introducing ligands with nucleophilic groups such as bipyridine, the metal site complex is immobilized. At the same time, nonspecific peroxygenase is fixed by cross-linking and adsorbed in the macropores, thereby retaining high catalytic activity while improving stability and reusability.

[0007] As a limitation of the above technical solution, the macro-COF material anchors the rhodium (III) complex [Rh(Cp*)Cl2]2 through the coordination effect of the bipyridine unit, and fixes the nonspecific peroxygenase AaeUPO in the macroporous structure through cross-linking.

[0008] As a limitation of the above technical solution, macro-COF and [Rh(Cp*)Cl2]2 are mixed in a mass ratio of 50:(4-24) to prepare Rh-macro-COF; Rh-macro-COF is mixed with AaeUPO enzyme solution with a concentration of 50-100 U / mL in a dosage ratio of (1-10) mg:1 mL to prepare AaeUPO@Rh-macro-COF.

[0009] As a limitation of the above technical solution, the pore size of the macro-COF material is between 100 and 400 nm.

[0010] As a limitation of the above technical solution, the molar ratio of trialdehyde phloroglucinol to 5,5'-diamino-2,2'-bipyridine is 0.3 mmol: (0.1-1) mmol.

[0011] Further improve the preparation conditions of chemical-enzyme integrated catalysts and optimize the activity and stability of the catalysts.

[0012] At the same time, the present invention provides a method for preparing the chemical-enzyme integrated catalyst for the oxygen functionalization reaction of the inert CH bond as described above, comprising the following preparation steps:

[0013] a. Obtain polystyrene spheres used as templates, denoted as colloidal PS spheres;

[0014] b. Evenly mix colloidal PS spheres, p-toluenesulfonic acid monohydrate and 5,5'-diamino-2,2'-bipyridine, then add trialdehyde phloroglucinol, continue stirring to complete the reaction, fully dry the reaction solution, then wash with water to remove p-toluenesulfonic acid monohydrate, and then use tetrahydrofuran to perform Soxhlet extraction to remove colloidal PS spheres to obtain macro-COF; preferably, the dosage ratio of colloidal PS spheres, p-toluenesulfonic acid monohydrate, 5,5'-diamino-2,2'-bipyridine and trialdehyde phloroglucinol is (100-1000) mg: (300-700) mg: (0.1-1) mmol: 0.3 mmol;

[0015] c. Add macro-COF to a methanol suspension of [Rh(Cp*)Cl2]2, disperse by ultrasonication, fully shake the reaction, filter, wash with water, and dry to obtain Rh-macro-COF; preferably, the ratio of macro-COF to [Rh(Cp*)Cl2]2 and methanol is 50 mg: (4-24) mg: (30-70) mL;

[0016] d. Mix Rh-macro-COF and AaeUPO enzyme solution, add ammonium sulfate solution for precipitation treatment, then add glutaraldehyde solution for cross-linking, and finally wash with potassium phosphate buffer and centrifuge to obtain AaeUPO@Rh-macro-COF; preferably, the concentration of AaeUPO enzyme solution is (50-100) U / mL, and the dosage ratio of Rh-macro-COF to AaeUPO enzyme solution is (1-10) mg:1 mL; preferably, the volume concentration of ammonium sulfate solution is (50-100)%.

[0017] As a limitation of the above technical solution, the preparation of the colloidal PS spheres in step a is as follows: polyvinyl pyrrolidone is dispersed in water, and nitrogen is continuously bubbled for 10 to 30 minutes, and then styrene that has been washed with NaOH solution and water to remove the stabilizer is added, stirred for 15 minutes to mix, and refluxed at 60 to 95° C. for 10 to 60 minutes, and then potassium persulfate and water are added to initiate the polymerization of styrene. The polymerization process is continuously stirred at 60 to 95° C. at a speed of less than 500 rpm, and the obtained product is poured into a culture dish and dried at 80° C. for 12 hours to obtain colloidal PS spheres with a solid content of 100 g / L; preferably, the dosage ratio of styrene, polyvinyl pyrrolidone and potassium persulfate is 25 to 30 g: 1 to 2 g: 0 to 1 g.

[0018] As a limitation of the above technical solution, the diameter of the colloidal PS spheres is adjusted by changing the dosage of potassium persulfate and / or the polymerization reaction temperature, thereby adjusting the pore size of the macro-COF material.

[0019] The preparation of the chemical-enzyme integrated catalyst of the present invention is simple and convenient to operate, has high reliability, and is conducive to the extensive manufacture and application of the catalyst.

[0020] In addition, the present invention also provides the use of the chemical-enzyme integrated catalyst for the oxygen functionalization reaction of the inert CH bond as described above, which is to use the chemical-enzyme integrated catalyst to catalyze the direct oxygen functionalization reaction of the inert CH bond and complete it through a one-pot process.

[0021] The chemical-enzyme integrated catalyst of the present invention successfully realizes the one-pot method of in-situ hydrogen peroxide production to drive the oxygen functionalization reaction of the inert CH bond of AaeUPO catalysis, which can play a protective role on the active center of AaeUPO, effectively inhibit the destruction of high-concentration hydrogen peroxide, improve the stability of the enzyme, and make the enzyme less likely to be inactivated. The chemical-enzyme integrated catalyst of the present invention usually carries out the catalytic reaction under normal temperature, normal pressure and near-neutral pH conditions, which reduces energy consumption and equipment requirements, and reduces environmental pollution. At the same time, the mild reaction conditions also help protect the reactants and products and avoid unnecessary losses and degradation.

[0022] As a limitation of the above technical solution, the chemical-enzyme integrated catalyst is used to catalyze the conversion of 4-ethylbenzoic acid (EBA) to synthesize 4-(1-hydroxyethyl)benzoic acid (HEBA), and the yield reaches more than 99% after 9 hours of reaction.

[0023] The chemical-enzyme integrated catalyst of the present invention is used for the hydroxylation reaction of CH bonds to convert EBA into HEBA, so that the HEBA yield after 9 hours of reaction is greater than 99%, or even above 99.5%.

[0024] In summary, the chemical-enzyme integrated catalyst of the present invention uses a covalent organic framework COF material with high stability and adjustable structure and composition as a carrier, utilizes the bipyridine unit in the Bpy ligand of the COF material to anchor the rhodium (III) complex, and uses sodium formate as a hydrogen donor to synthesize hydrogen peroxide in situ by chemical catalysis; uses polystyrene balls (PS balls) as hard templates to prepare macroporous macro-COF materials, and fixes AaeUPO aggregates in the macropores of macro-COF by cross-linking to catalyze the oxygen functionalization of inert CH bonds; the constructed chemical-enzyme integrated catalyst (AaeUPO@Rh-macro-COF) not only has high catalytic activity, realizes the efficient conversion of the inert CH bond oxygen functionalization reaction driven by AaeUPO catalyzed by in situ production of hydrogen peroxide, but also has high stability and reusability, can effectively inhibit the destruction of the active center of AaeUPO by high concentration hydrogen peroxide, and is beneficial to practical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 , Scanning electron microscope image of colloidal PS spheres of Example 1.

[0026] Figure 2 , Scanning electron microscope image of macro-COF material in Example 1.

[0027] Figure 3 , Fourier infrared spectrum of macro-COF material in Example 1.

[0028] Figure 4 , X-ray diffraction pattern of macro-COF material in Example 1.

[0029] Figure 5 , N2 adsorption-desorption isotherms and pore size distribution diagram of macro-COF material in Example 1.

[0030] Figure 6 , Rh 3d fine spectrum of Rh-macro-COF material in Example 1.

[0031] Figure 7 N2 adsorption-desorption isotherms of three materials: TpBpy-COF, macro-COF, and Rh-macro-COF.

[0032] Figure 8 , Pore size distribution diagrams of three materials: TpBpy-COF, macro-COF, and Rh-macro-COF.

[0033] Fig. 9 , CLSM images of AaeUPO@Rh-macro-COF.

[0034] Fig.10 , A is the HPLC of the AaeUPO@Rh-macro-COF catalytic reaction system of Example 8; B is the HPLC of the substrate standard in the reaction of EBA to HEBA; C is the HPLC of the product standard in the reaction of EBA to HEBA.

[0035] Fig.11 , Scanning electron microscope (SEM) images of TpBpy-COF material.

[0036] Fig.12 , Control Examples 1, 2 and Example 1 catalyze the reaction progress curves of converting EBA into HEBA. DETAILED DESCRIPTION

[0037] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0039] Example

[0040] This example relates to the preparation of a chemical-enzyme integrated catalyst.

[0041] Example 1

[0042] The preparation of the chemical-enzyme integrated catalyst is as follows:

[0043] a. Preparation of colloidal PS spheres: Take 28g of styrene and wash it with 10wt% NaOH solution and deionized water in turn to remove the stabilizer. Disperse 1.2g of polyvinyl pyrrolidone (PVP) in 240mL of water, and continue bubbling with nitrogen for 10 to 30min. Then add the washed styrene and stir for 15min to mix. Then reflux at 75℃ for 10 to 60min. Then add 0.4g of potassium persulfate (K2S2O8) and 40mL of water to initiate the polymerization of styrene. The polymerization reaction is stirred at 75℃ at a speed of <500 rpm for 24h. Pour the product into a culture dish and dry it at 80℃ for 12h to obtain colloidal PS spheres with a solid content of 100g / L. Its morphology is as follows: Figure 1 As shown, the diameter of the colloidal PS spheres is about 200 nm, and the microspheres have a smooth surface, uniform size, and complete morphology.

[0044] b. Preparation of macro-COF: 5 mL of colloidal PS spheres with a solid content of 100 g / L was taken and mixed with 475.5 mg of p-toluenesulfonic acid monohydrate (PTSA, 2.5 mmol) and 83.8 mg of 5,5'-diamino-2,2'-bipyridine (Bpy, 0.45 mmol) by vortex for 10 minutes, and then 63 mg of trialdehyde phloroglucinol (Tp, 0.3 mmol) was added and shaken for 10 minutes until there was an obvious color change from milky white to orange-yellow. The reaction solution was poured into a petri dish to evaporate the water, and then transferred to an oven at 80°C for 1 day to obtain PS spheres @TpBpy; then the PS spheres @TpBpy were thoroughly washed with 100°C water to remove PTSA, and then Soxhlet extraction was performed with tetrahydrofuran (THF) to remove the template PS spheres to obtain macro-COF.

[0045] The morphology of macro-COF was observed by scanning electron microscopy, such as Figure 2 As shown, the interior of the carrier is honeycomb-shaped as a whole, and the pore size of the large pores is measured to be about 200nm. These pores are arranged in order in three-dimensional space to form a macroscopically visible porous structure, which can be called macro-COF. It has excellent molecular transport properties, high porosity and good chemical stability. Compared with traditional microporous or mesoporous COF materials, macro-COF has a larger pore size and a more ordered structure.

[0046] The functional groups of macro-COF were characterized by FT-IR. Figure 3 As shown. macro-COF at 3450cm -1 、1602cm -1 、1574cm -1 and 1262cm -1 The characteristic peaks of -NH, -C=O, -C=C- and -CN- appeared at the position. This is because the enol-keto tautomerism occurred after Tp reacted with Bpy to form imino COF. The FT-IR results showed that macro-COF was successfully synthesized.

[0047] X-ray diffraction analysis of macro-COF, such as Figure 4 As shown, macro-COF shows a strong peak at 2θ = 3.62°, which is consistent with the characteristic diffraction peak position reported in the literature. This result confirms that macro-COF still has a good crystal shape even after removing the PS template.

[0048] The macro-COF was characterized by N2 adsorption-desorption to determine its specific surface area and pore size distribution. Figure 5As shown in Figure 2, macro-COF exhibits a type IV isotherm and has an obvious hysteresis loop at a higher relative pressure, indicating that there is a mesoporous structure (1.1-2.7 nm) in the macro-COF material with a specific surface area of ​​256 m 2 / g.

[0049] c. Preparation of Rh-macro-COF: 50 mg of macro-COF was added to 50 mL of methanol suspension containing 17 mg of [Rh(Cp*)Cl2]2, and the suspension was fully dispersed by ultrasonication for 5 to 10 min, and then placed on a shaker for 24 hours to fix the rhodium (III) complex. After the suspension was fixed, the solid was collected by filtration, washed with a large amount of deionized water, and vacuum dried at 60°C for 12 hours to obtain Rh-macro-COF.

[0050] like Figure 6 As shown in the Rh 3d fine spectrum of Rh-macro-COF, two significant characteristic peaks at 309.6 eV and 314.4 eV can be clearly observed, which correspond to the 3d 5 / 2 and 3D 3 / 2 The contribution of this study demonstrates the specific electronic structure characteristics of the rhodium element in Rh-macro-COF.

[0051] In order to understand the specific surface area and pore size distribution of the material, we performed N2 adsorption and desorption characterization and comparison on the TpBpy-COF material synthesized by the solvothermal method (see Comparative Example 1 for the specific synthesis method) and the macro-COF material and Rh-macro-COF material of Example 1. The results are shown in Figure 7 , Figure 8 As shown in the figure, the specific surface areas of TpBpy-COF, macro-COF, and Rh-macro-COF gradually decreased, especially the specific surface area of ​​Rh-macro-COF dropped sharply to 63 m2 / g, and the average pore size and pore volume also decreased, proving that (1) compared with TpBpy-COF, macro-COF formed a macroporous structure; (2) compared with macro-COF, Rh-macro-COF successfully loaded the Rh complex in the pores.

[0052] d. Preparation of AaeUPO@Rh-macro-COF: 6 mg of Rh-macro-COF was mixed with 1 mL of AaeUPO enzyme solution (enzyme concentration was 90 U / mL), and 1 mL of 80% (NH4)2SO4 solution at 0°C (as a precipitant) was added for precipitation treatment for 1 h. Then, 2.5% glutaraldehyde solution (as a cross-linking agent) was added for cross-linking reaction for 35 min. Finally, the mixture was washed with PBS buffer (0.05 M, pH = 5.0) and centrifuged to obtain AaeUPO@Rh-macro-COF.

[0053] To prove the successful immobilization of AaeUPO, confocal laser scanning (CLSM) was performed on AaeUPO@Rh-macro-COF. Using Rh-macro-COF as a carrier, AaeUPO stained with rhodamine B was cross-linked and fixed on the carrier, and the fluorescence of AaeUPO@Rh-macro-COF was detected, as shown in Figure 2. Fig. 9 As shown, the labeled enzyme molecules exhibited red fluorescence and were evenly distributed on the Rh-macro-COF, indicating the successful immobilization of AaeUPO.

[0054] Embodiment 2-3

[0055] The chemical-enzyme integrated catalysts of Examples 2 and 3 are different from those of Example 1 only in the ratio of the amounts of Tp and Bpy ligands used in the catalyst preparation, which is as follows:

[0056] In Example 2, in the step of preparing macro-COF, the amount of Bpy used was 55.86 mg (0.3 mmol), the amount of Tp used was 63 mg (0.3 mmol), and the other operations and conditions were the same as in Example 1.

[0057] In Example 3, in the step of preparing macro-COF, the amount of Bpy used was 111.73 mg (0.6 mmol), the amount of Tp used was 63 mg (0.3 mmol), and the other operations and conditions were the same as in Example 1.

[0058] Embodiments 4 to 6

[0059] The chemical-enzyme integrated catalysts of Examples 4 to 6 differ from Example 1 only in the pore size of the macro-COF material, as follows:

[0060] In Example 4, in the step of preparing colloidal PS spheres, the reflux temperature was changed to 95°C, and the polymerization temperature was changed to 95°C. Other operations and conditions were the same as in Example 1. The pore size of the obtained colloidal PS spheres was measured to be 130 nm.

[0061] In Example 5, in the step of preparing colloidal PS spheres, the reflux temperature is changed to 60°C, the amount of K2S2O8 is changed to 0.5g, and the polymerization temperature is changed to 60°C. Other operations and conditions are the same as in Example 1. The pore size of the obtained colloidal PS spheres is measured to be 320nm.

[0062] In Example 6, in the step of preparing colloidal PS spheres, only the amount of K2S2O8 was changed to 0.1 g, and the other operations and conditions were the same as in Example 1. The pore size of the obtained colloidal PS spheres was measured to be 360 ​​nm.

[0063] Embodiments 7 to 9

[0064] The chemical-enzyme integrated catalysts of Examples 7 to 9 differ from those of Example 1 only in the amount of the rhodium (III) complex fixed in the catalyst, which is as follows:

[0065] In Example 7, in the step of preparing Rh-macro-COF, the amount of [Rh(Cp*)Cl2]2 was changed to 4 mg, and the other operations and conditions were the same as in Example 1.

[0066] In Example 8, in the step of preparing Rh-macro-COF, the amount of [Rh(Cp*)Cl2]2 was changed to 10 mg, and the other operations and conditions were the same as in Example 1.

[0067] In Example 9, in the step of preparing Rh-macro-COF, the amount of [Rh(Cp*)Cl2]2 was changed to 24 mg, and the other operations and conditions were the same as in Example 1.

[0068] Embodiments 10 to 11

[0069] The chemical-enzyme integrated catalysts of Examples 10 to 11 are different from those of Example 1 only in the COF-enzyme dosage ratio in the catalyst, which is as follows:

[0070] In Example 10, in the step of preparing AaeUPO@Rh-macro-COF, the amount of Rh-macro-COF was changed to 3 mg, and the other operations and conditions were the same as those in Example 1.

[0071] In Example 11, in the step of preparing AaeUPO@Rh-macro-COF, the amount of Rh-macro-COF was changed to 9 mg, and the other operations and conditions were the same as in Example 1.

[0072] Comparative Example 1

[0073] This comparative example is a mixed system of free enzyme AaeUPO enzyme solution and Rh-COF series chemical catalyst, wherein the Rh-COF series chemical catalyst is Rh-TpBpy-COF.

[0074] The chemical catalyst Rh-TpBpy-COF was prepared by a solvothermal method. The specific preparation method is as follows:

[0075] 21.0 mg Tp (0.1 mmol), 27.9 mg Bpy (0.15 mmol), 0.5 mL mesitylene, 0.5 mL dioxane and 0.1 mL HAc (6 M) were added to a Pyrex tube (10 mL), and the mixture was sonicated for 2 min, then degassed by three freeze pump-thaw cycles and sealed under vacuum. The sealed test tube was kept in an oven at 120 ° C for 3 days and then cooled to room temperature. The precipitate was collected by filtration, washed with tetrahydrofuran and acetone, and finally extracted with THF Soxhlet for 24 h to remove unreacted ligands, and then dried in vacuo at 40 ° C for 24 h to obtain TpBpy-COF powder, as shown Fig.11 As shown, the overall structure of the TpBpy-COF material is fibrous, with only micropores (<5nm) possessed by COF itself, and no macroporous structure, so it cannot be used to immobilize enzymes.

[0076] 50 mg of TpBpy-COF was added to 50 mL of methanol suspension containing 17 mg of [Rh(Cp*)Cl2]2, ultrasonically dispersed for 5 to 10 minutes, and then placed on a shaker for 24 hours for immobilization. The obtained solid was filtered, washed with a large amount of deionized water, and vacuum dried at 60°C for 12 hours to obtain Rh-TpBpy-COF.

[0077] Comparative Example 2

[0078] This comparative example is a mixed system of free enzyme AaeUPO enzyme solution and Rh-COF series chemical catalyst, wherein the Rh-COF series chemical catalyst is Rh-NT-COF.

[0079] Chemical catalyst Rh-NT-COF, no template is used to make holes in the preparation of COF material, the specific preparation operation is as follows:

[0080] 5 mL of water, 475.5 mg of PTSA (2.5 mmol) and 83.8 mg of Bpy (0.45 mmol) were vortexed for 10 minutes, and then 63 mg of Tp (0.3 mmol) was added and the mixture was shaken for another 10 minutes. The mixture was poured into a petri dish to evaporate the water, and then transferred to an oven at 80°C for 1 day. After that, it was thoroughly washed with 100°C water to remove PTSA to obtain NT-COF material. Since no template was used, it did not have a macroporous structure and could not be used to immobilize enzymes.

[0081] 50 mg of NT-COF was added to 50 mL of methanol suspension containing 17 mg of [Rh(Cp*)Cl2]2, and after ultrasonic dispersion for 5 to 10 min, it was placed on a shaker for 24 hours for immobilization. The obtained solid was filtered, washed with a large amount of deionized water, and vacuum dried at 60°C for 12 hours to obtain Rh-NT-COF.

[0082] Comparative Example 3

[0083] This comparative example is a chemical-enzyme integrated catalyst, in which the chemical catalyst uses silicon powder as a template to make holes in the preparation of COF materials. The specific preparation is as follows:

[0084] Take 475.5 mg PTSA (2.5 mmol) and 83.8 mg Bpy (0.45 mmol), grind them with a mortar for 15 minutes, add 630 mg silicon powder (SiO2), and continue grinding for 15 minutes to form a uniform yellow powder; then add 63 mg Tp (0.3 mmol) and continue grinding and mixing until the color changes from yellow to orange; then drop 100 μL water and continue grinding for 15 minutes, and the color changes from orange to dark red; finally, transfer the mixture to a pressure tube, heat at 120 ° C for 16 hours to produce a dark red powder, then cool to room temperature, wash the powder with 100 ° C water to remove PTSA, and extract with N, N-dimethylacetamide (DMAC) for 24 hours, then wash with DMAC, water and acetone to remove the residue, and finally dry under reduced pressure to obtain SiO2@TpBpy. SiO2@TpBpy was heated to 800°C in an Ar atmosphere at a heating rate of 5°C / min and pyrolyzed for 2 h to obtain C-SiO2@TpBpy. After cooling to room temperature, it was immersed in 300 mL of NaOH (2M) solution for 24 h to remove the SiO2 template. The product was collected by filtration, washed with water and ethanol, and then dried in a vacuum oven at 60°C for 12 h to obtain SiO2-COF.

[0085] 50 mg SiO2-COF was added to 50 mL methanol suspension containing 17 mg [Rh(Cp*)Cl2]2, and after ultrasonic dispersion for 5 to 10 min, it was placed on a shaker for 24 h, filtered, washed with a large amount of deionized water, and vacuum dried at 60 °C for 12 h to obtain Rh-SiO2-COF.

[0086] Mix 6 mg of Rh-SiO2-COF with 1 mL of AaeUPO enzyme solution (90 U / mL), add 1 mL of 80% (NH4)2SO4 solution at 0°C for precipitation for 1 hour, then add 2.5% glutaraldehyde solution for cross-linking for 35 minutes, wash with PBS buffer (0.05 M, pH = 5.0) and centrifuge to obtain a chemical-enzyme integrated catalyst.

[0087] Comparative Example 1

[0088] System of free enzyme AaeUPO enzyme solution and chemical catalyst Rh-macro-COF (the preparation of Rh-macro-COF is the same as in Example 1).

[0089] Comparative Example 2

[0090] The free enzyme was subjected to cross-linking treatment to obtain a cross-linked enzyme CLEAs-AaeUPO (which is more convenient to recover than the free enzyme) and a chemical catalyst Rh-macro-COF (the preparation of Rh-macro-COF is the same as in Example 1).

[0091] The specific preparation of the cross-linking enzyme is as follows: 1 mL of AaeUPO enzyme solution (90 U / mL) is added to 1 mL of 80% (NH4)2SO4 solution at 0°C for precipitation for 1 hour, and then a 2.5% volume concentration glutaraldehyde solution is added for cross-linking for 35 minutes, and then washed with PBS buffer (0.05 M, pH = 5.0) and centrifuged to obtain 4 mg of cross-linking enzyme.

[0092] The catalytic effects of the chemical enzyme integrated catalysts prepared in Examples 1 to 11 and Comparative Example 3 and the free enzyme chemical catalyst systems in Comparative Examples 1 and 2 and Reference Examples 1 and 2 on the oxygen functionalization reaction of inert CH bonds were verified.

[0093] (I) Catalytic in-situ generation of hydrogen peroxide

[0094] The Rh-COF series chemical catalysts prepared in Examples 1 to 11 and Comparative Examples 1 to 3 (the chemical catalysts of Comparative Examples 1 and 2 are the same as those of Example 1, so the experiment is not repeated) are used to generate hydrogen peroxide in situ, respectively, and the specific operation is as follows: COF-based chemical catalyst (14 groups of experiments are respectively 6 mg of Rh-macro-COF in Examples 1 to 9, 3 mg of Rh-macro-COF in Example 10, 9 mg of Rh-macro-COF in Example 11, 6 mg of Rh-TpBpy-COF in Comparative Example 1, Rh-NT-COF in Comparative Example 2, and Rh-SiO2-COF in Comparative Example 3), 0.0408 g of sodium formate, 2 mL of PBS (0.05 M, pH = 5.0), room temperature 25°C, 800 rpm stirring for 4 hours. The yield of catalytic H2O2 in situ synthesis is shown in Table 1 below.

[0095] The H2O2 concentration was determined by potassium iodide method: 400 μL of the reaction solution was added with 300 μL of pre-prepared iodine detection reagent (containing 0.4 M potassium iodide, 0.05 M sodium hydroxide, 10 -4 300 μL of 0.5 M ammonium molybdate) and 300 μL of 0.5 M potassium hydrogen phthalate solution were mixed evenly, and detected by ultraviolet spectrophotometer to read the absorbance at 351 nm.

[0096] Table 1 The yield of in-situ H2O2 produced by catalysts

[0097]

[0098] As can be seen from the data in Table 1, COF-based chemical catalysts prepared with different amounts of [Rh(Cp)Cl2]2 have a significant effect on the catalytic effect of in-situ generation of H2O2. The catalytic effect of COF-based chemical catalysts is not directly positively correlated with the content of [Rh(Cp)Cl2]2, indicating that the catalytic activity depends not only on the amount of its components, but may also be affected by other factors such as catalyst structure and surface properties. Therefore, when screening catalysts, it is necessary to comprehensively consider the yield of H2O2 and the content of [Rh(Cp)Cl2]2.

[0099] (II) Catalytic conversion of EBA into HEBA

[0100] The chemical-enzyme catalysts prepared in Examples 1 to 11 and Comparative Example 3 and the enzyme and catalyst systems in Comparative Examples 1 and 2 and Reference Examples 1 and 2 were used in the reaction of converting EBA into HEBA, and the specific method is as follows.

[0101] Examples 1 to 11, Comparative Example 3: 10 mg of chemical-enzyme catalyst, 0.0408 g of sodium formate, 1.7 mL of PBS (0.05 M, pH = 5.0), 0.3 mL of ACN as a cosolvent, 10 mM EBA, room temperature 25 ° C, stirring at 800 rpm, react for 9 hours, and then centrifuge to separate the catalyst after the reaction is completed. The reaction solution is filtered with a 0.45 μm organic filter membrane and the yield of HEBA is determined by liquid chromatography.

[0102] Comparative Example 1, Comparative Example 2, Control Example 1: 6 mg Rh-COF chemical catalyst, 1 mL AaeUPO enzyme solution (90 U / mL), 0.0408 g sodium formate, 0.7 mL PBS (0.05 M, pH = 5.0), co-solvent 0.3 mL ACN, 10 mM EBA, room temperature 25 ° C, stirring at 800 rpm, react for 9 hours, after the reaction is completed, the catalyst is centrifuged and the reaction solution is filtered with a 0.45 μm organic filter membrane and the yield of HEBA is determined by liquid chromatography.

[0103] Control Example 2: 6 mg Rh-macro-COF catalyst, 4 mg cross-linking enzyme, 0.0408 g sodium formate, 1.7 mL PBS (0.05 M, pH = 5.0), cosolvent 0.3 mL ACN, 10 mM EBA, room temperature 25 ° C, stirring at 800 rpm, react for 9 hours, and then centrifuge to separate the catalyst after the reaction is completed. The reaction solution is filtered with a 0.45 μm organic filter membrane and the yield of HEBA is determined by liquid chromatography.

[0104] The liquid chromatography detection conditions are as follows: HPLC-20AD XR HP high performance liquid chromatography system (Shimadzu Corporation, Japan) was used to determine the concentrations of EBA and HEBA. Chromatographic column: C18-RP; detection temperature: 25°C; flow rate: 0.5 mL min -1 ; Mobile phase: A mixture of a 0.1% trifluoroacetic acid solution (Solution A) and a 0.095% trifluoroacetic acid acetonitrile solution (Solution B) was used for gradient elution, wherein the volume fraction of Solution B was as follows: 0 min: 35%, to 7 min: 80%, to 9 min: 35%, to 10 min: 35%. EBA and HEBA were detected using a UV-visible detector (237 nm).

[0105] Liquid chromatography test sample: Take 200 μL of the reaction solution sample after filtering through the organic filter membrane, mix it with 800 μL of solution A, and centrifuge it at 13000 rpm for 4 minutes at 4°C. Transfer the supernatant to a sample bottle for testing.

[0106] Use the standards to prepare standard solutions with concentrations of 2 mM, 4 mM, 6 mM, 8 mM, 10 mM and 12 mM to draw a standard curve.

[0107] Attached photos Fig.10 A is the HPLC of the AaeUPO@Rh-macro-COF catalytic reaction system of Example 8, which clearly shows the peak positions of the substrate and product of the EBA to HEBA conversion reaction on the liquid phase diagram, and is consistent with the attached figure Fig.10 Substrate standards and attached diagram for B Fig.10 The peak positions of the C product standards on the liquid phase diagram are consistent.

[0108] The catalytic results (i.e., the yield of catalyzing the conversion of EBA to HEBA) of the chemical-enzyme catalysts prepared in Examples 1 to 11 and Comparative Example 3 and the enzyme and catalyst systems of Comparative Examples 1, 2 and Reference Examples 1, 2 are shown in Table 2 below.

[0109] Table 2 The yield of EBA converted to HEBA catalyzed by the catalyst

[0110]

[0111] As can be seen from the data in Table 2, from the yield of HEBA, the catalytic effect of the chemical-enzyme integrated catalyst is not directly positively correlated with the H2O2 yield of the Rh-COF series chemical catalyst. For example, in Examples 4, 5, and 6, the pore size of the macropore is too small to effectively fix the enzyme, and the pore size is too large, which may cause the structure to collapse, which is also not conducive to the fixation of the enzyme, resulting in a decrease in the HEBA yield; in Examples 7, 8, and 9, the amount of rhodium complex added is too low to ensure the supply of H2O2, and too much addition may cause the catalyst and the enzyme to be mutually inactivated, which will also cause a decrease in the HEBA yield. In Comparative Examples 1, 2, and 3, the pore structure of TpBpy-COF synthesized by different methods is not suitable for enzyme immobilization, which will also cause a decrease in the HEBA yield. This shows that in the chemical-enzyme cascade reaction, the catalytic activity of the integrated catalyst is not only related to the H2O2 yield of the Rh-COF series chemical catalyst, but also closely related to the synergistic match between it and the AaeUPO enzyme catalyst.

[0112] In summary, when screening catalysts, multiple factors such as H2O2 yield, HEBA yield, and [Rh(Cp)Cl2]2 content should be considered comprehensively. By comparing the performance of different catalysts on these indicators, their catalytic performance can be more comprehensively evaluated, so as to select the catalyst most suitable for a specific reaction.

[0113] In addition, by comparing the control examples 1 and 2 with Example 1, that is, comparing the catalytic conversion efficiency of EBA by the catalytic system in which the AaeUPO free enzyme and the CLEAs-AaeUPO cross-linked enzyme were simply mixed with the Rh-macro-COF catalyst and the AaeUPO@Rh-macro-COF chemical-enzyme integrated catalyst, as shown in FIG. Fig.12 As shown in the figure, the conversion rate of EBA by the catalytic system (free AaeUPO+Rh-macro-COF) is only 32%, while the conversion rate of EBA by the catalytic system (CLEAs-AaeUPO+Rh-macro-COF) is increased to 51%, and the conversion rate of the AaeUPO@Rh-macro-COF chemical-enzyme integrated catalyst of the present invention is as high as 99%. This significant improvement is attributed to (1) the CLEAs-AaeUPO fixed in the macropores of macro-COF has higher stability than free AaeUPO and unfixed CLEAs-AaeUPO; (2) the integration of the chemical catalytic unit and the enzyme catalytic unit shortens the mass transfer distance of the substrate and improves the catalytic efficiency.

[0114] In summary, the chemical-enzyme integrated catalyst of the present invention uses a covalent organic framework COF material with a macroporous structure obtained by making holes with polystyrene balls as a hard template as a carrier, anchors the rhodium (III) complex through the bipyridine unit in the COF material ligand, and fixes AaeUPO in the macroporous structure through cross-linking, thereby achieving simultaneous fixation of the metal site catalyzing oxygen reduction and the non-specific peroxygenase AaeUPO catalyzing the oxygen functionalization of the CH bond, not only achieving in situ production of hydrogen peroxide to drive the efficient conversion of the inert CH bond oxygen functionalization reaction catalyzed by AaeUPO, but also effectively inhibiting the destruction of the active center of AaeUPO by high concentration of hydrogen peroxide, and the obtained chemical-enzyme integrated catalyst has both high catalytic activity and high stability and reusability.

Claims

1. A chemical-enzyme integrated catalyst for oxygen functionalization of inert CH bonds, characterized in that: The chemical-enzyme integrated catalyst is obtained by simultaneously fixing a rhodium (III) complex and a non-specific peroxygenase on a covalent organic framework COF material having a macroporous structure inside. The covalent organic framework (COF) material with a macroporous structure was prepared by using trialdehyde pyrogallol and 5,5'-diamino-2,2'-bipyridine as ligands and polystyrene spheres as templates to prepare pores, which was denoted as macro-COF.

2. The chemical-enzyme integrated catalyst for oxygen functionalization reaction of inert CH bonds according to claim 1, characterized in that: The macro-COF material anchors the rhodium (III) complex [Rh(Cp*)Cl2]2 through the coordination of the bipyridine unit and fixes the nonspecific peroxygenase AaeUPO within the macroporous structure through cross-linking.

3. The chemical-enzyme integrated catalyst for oxygen functionalization reaction of inert CH bonds according to claim 2, characterized in that: Macro-COF and [Rh(Cp*)Cl2]2 are mixed in a mass ratio of 50:(4-24) to prepare Rh-macro-COF; Rh-macro-COF and AaeUPO enzyme solution with a concentration of 50-100 U / mL are mixed in a dosage ratio of (1-10) mg:1 mL to prepare AaeUPO@Rh-macro-COF.

4. The chemical-enzyme integrated catalyst for oxygen functionalization reaction of inert CH bonds according to claim 1, characterized in that: The pore size of macro-COF materials is between 100 and 400 nm.

5. The chemical-enzyme integrated catalyst for oxygen functionalization reaction of inert CH bonds according to claim 1, characterized in that: The molar ratio of trialdehyde phloroglucinol to 5,5'-diamino-2,2'-bipyridine is 0.3 mmol:(0.1-1) mmol.

6. A method for preparing a chemical-enzyme integrated catalyst for oxygen functionalization reaction of inert CH bonds as claimed in any one of claims 1 to 5, characterized in that: The method comprises the following preparation steps: a. Obtain polystyrene spheres used as templates, denoted as colloidal PS spheres; b. Evenly mix colloidal PS spheres, p-toluenesulfonic acid monohydrate and 5,5'-diamino-2,2'-bipyridine, then add trialdehyde phloroglucinol, continue stirring to complete the reaction, fully dry the reaction solution, then wash with water, and then use tetrahydrofuran to perform Soxhlet extraction to remove the colloidal PS spheres to obtain macro-COF; Preferably, the dosage ratio of colloidal PS spheres, p-toluenesulfonic acid monohydrate, 5,5'-diamino-2,2'-bipyridine, and trialdehyde phloroglucinol is (100-1000) mg: (300-700) mg: (0.1-1) mmol: 0.3 mmol; c. Add macro-COF to a methanol suspension of [Rh(Cp*)Cl2]2, disperse by ultrasonication, fully shake the reaction, filter, wash with water, and dry to obtain Rh-macro-COF; Preferably, the ratio of macro-COF, [Rh(Cp*)Cl2]2, and methanol is 50 mg: (4-24) mg: (30-70) mL; d. Rh-macro-COF was mixed with AaeUPO enzyme solution, and ammonium sulfate solution was added for precipitation treatment, and glutaraldehyde solution was added for cross-linking, and finally, it was washed with potassium phosphate buffer and centrifuged to obtain AaeUPO@Rh-macro-COF; Preferably, the concentration of AaeUPO enzyme solution is 50-100 U / mL, and the dosage ratio of Rh-macro-COF to AaeUPO enzyme solution is (1-10) mg:1 mL; Preferably, the volume concentration of the ammonium sulfate solution is (50-100)%.

7. The method for preparing the chemical-enzyme integrated catalyst for oxygen functionalization reaction of inert CH bonds according to claim 6, characterized in that: The preparation of colloidal PS spheres in step a is as follows: Polyvinyl pyrrolidone is dispersed in water, and nitrogen is continuously bubbled for 10 to 30 minutes, and then styrene from which the stabilizer has been washed is added, and the mixture is fully stirred and mixed, and refluxed at 60 to 95° C. for 10 to 60 minutes, and then potassium persulfate and water are added to initiate polymerization of styrene. The polymerization process is continuously stirred and reacted at 60 to 95° C. at a speed lower than 500 rpm, and the obtained product is poured into a culture dish and dried at 80° C. for 12 hours to obtain colloidal PS spheres with a solid content of 100 g / L; Preferably, the dosage ratio of styrene, polyvinyl pyrrolidone and potassium persulfate is 25-30 g: 1-2 g: 0-1 g.

8. The method for preparing the chemical-enzyme integrated catalyst for oxygen functionalization reaction of inert CH bonds according to claim 6, characterized in that: The diameter of the colloidal PS spheres is adjusted by changing the dosage of potassium persulfate and / or the polymerization reaction temperature, thereby adjusting the pore size of the macro-COF material.

9. Use of a chemical-enzyme integrated catalyst for oxygen functionalization of inert CH bonds as claimed in any one of claims 1 to 5, characterized in that: The direct oxygen functionalization reaction of inert CH bonds catalyzed by the chemical-enzyme integrated catalyst is completed by a one-pot process.

10. Use of the chemical-enzyme integrated catalyst for oxygen functionalization reaction of inert CH bonds according to claim 9, characterized in that: The chemical-enzyme integrated catalyst is used to catalyze the conversion of 4-ethylbenzoic acid into 4-(1-hydroxyethyl)benzoic acid, and the yield reaches more than 99% after 9 hours of reaction.

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