Application of Michael addition method based on hydrogen bond activation to rapid preparation of covalent organic framework and immobilized enzyme
The covalent organic framework (COF) can be rapidly prepared under conventional conditions by using a hydrogen bond-activated Michael addition strategy, which solves the problem of long preparation time and achieves efficient enzyme immobilization and improved photothermal catalytic performance.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-30
- Publication Date
- 2026-03-31
AI Technical Summary
The preparation time of existing covalent organic frameworks (COFs) is relatively long and difficult to achieve rapidly under normal conditions, which affects their application efficiency and stability.
A covalent organic framework (COF) was prepared using a hydrogen bond-activated Michael addition strategy under conventional solvent and weak acid catalysis conditions. The rapid synthesis of COFs was achieved by utilizing the intramolecular hydrogen bond activation effect.
Rapid preparation of COFs was achieved, which have high crystallinity, high specific surface area and high stability, and are suitable for enzyme immobilization, significantly improving the catalytic performance and photothermal conversion performance of enzymes.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of porous organic materials, specifically relating to the preparation of a new type of covalent organic framework and its application in immobilized enzymes. Background Technology
[0002] In recent years, covalent organic frameworks (COFs), as a novel type of crystalline porous polymer, have several advantages over traditional porous materials, making them ideal solid carriers for enzyme immobilization. (1) Tunable pore size. By changing the length and geometry of the building blocks, the pore size of COFs can be easily adjusted, thereby promoting enzyme embedding in the pores of the covalent organic framework and providing better protection for enzyme formation. (2) High specific surface area and pore volume. This means that COFs can provide sufficient space to accommodate large enzymes, resulting in high enzyme loading rates. (3) Designable and regular structures. This allows us to precisely install functional groups in COFs to achieve additional functions. For example, photothermal functions can be achieved by introducing functional groups (e.g., azo groups). (4) Easy post-modification. Pre-introduced anchoring sites (such as carboxyl, amino, hydroxyl, etc.) can achieve covalent linkage between enzymes and COFs through post-modification reactions. Functional groups can also be introduced through post-modification to provide a suitable microenvironment for efficient enzyme conversion.
[0003] Covalent organic frameworks (COFs), a class of crystalline porous organic polymers, are constructed by connecting organic components through covalent bonds. In this context, the linkers not only play a crucial role in connecting vertices and linkers to ordered two-dimensional or three-dimensional network structures, but also significantly influence the material's properties. The development of COFs is inseparable from an understanding of fundamental chemistry; expanding new bonding reaction types is an important means of constructing entirely new COFs. The earliest COFs were linked by BO bonds, but their strong reversibility made it impossible to guarantee the overall framework stability. Subsequently, C=N linked COFs were gradually developed, receiving extensive research and undergoing significant progress. β-ketoenamines, as a variant derived from imine bonds, exhibit significantly improved stability due to their significantly reduced reversibility. Although many types of COF linkages have been developed, the preparation time for COFs is currently quite long, generally requiring several days. While some special methods, such as acid-based grinding and microwave synthesis, can shorten the preparation time, they essentially still rely on externally applied complex conditions to facilitate COF formation. Developing a linker bond that allows for the rapid preparation of COFs under conventional conditions is both important and challenging. Summary of the Invention
[0004] The purpose of this invention is to rapidly prepare covalent organic frameworks using a hydrogen bond-activated Michael addition strategy. These materials have advantages such as high crystallinity, high porosity, abundant functional groups, and high stability, and can be easily designed in terms of structure and function.
[0005] Another objective of this invention is to immobilize enzymes for catalytic transformation of chiral substrates, identify COFs rich in azophenyl groups that can achieve good solar energy absorption and photothermal conversion performance, and provide a suitable catalytic microenvironment for enzymes by rapidly heating to 52±3 °C under light conditions after enzyme immobilization.
[0006] Other objects of the present invention will be apparent to those skilled in the art directly from the foregoing and the following description.
[0007] The first aspect of this invention is to propose a hydrogen bond-activated Michael addition strategy for the preparation of COFs, achieving rapid and direct preparation of COFs materials. The key feature is that COFs can be rapidly prepared using conventional solvents under weak acid catalytic conditions.
[0008] Preferably, the synthesis method 1 involves directly adding p-phenylenediamine monomer, a tri-linked monomer containing β-ketene dimethylamine, and a catalyst to a reactor, sealing the reactor under normal pressure, and obtaining COFs powder material through Michael addition, followed by cleaning and purification.
[0009] Preferably, monomer 1 is a di- or tri-linked amino monomer.
[0010] More preferably, monomer 2 is a tri-linked monomer containing β-ketoene dimethylamine.
[0011] In a preferred embodiment of the present invention, the amino monomer is any one of p-phenylenediamine, 2-sulfonic p-phenylenediamine, 2,5-dihydroxyp-phenylenediamine, 4,4'-biphenylenediamine, p-diaminoazobenzene, p-diaminoynylbenzene, 1,3,5-tris(4-aminophenyl)amine, 1,3,5-tris(4-aminophenyl)benzene, 1,3,5-tris(4-aminophenyl)triazine, and 1,3,5-tris(4-(4-aminophenyl)phenyl)amine.
[0012] In a preferred embodiment of the present invention, the monomer containing β-ketoenyldimethylamine is 1,3,5-triβ-ketoenyldimethylamine-2,4,6-phloroglucinol or 1,4-diβ-ketoenyldimethylamine-2,5-hydroquinone.
[0013] Preferably, the catalyst is a compound containing acidic functional groups.
[0014] More preferably, the catalyst is one of acetic acid or lactic acid.
[0015] Preferably, the obtained COFs gel is a β-enoneamine-linked COFs powder material.
[0016] Preferably, the pore size of the COFs material is 2.2-3.3 nm.
[0017] Preferably, the specific surface area of the COFs material is 1271-1478 m². 2 / g.
[0018] Preferably, the molar ratio of organic monomer 1 to organic monomer 2 is 3:2.
[0019] Preferably, the amount of catalyst acid used in the reaction system is 100 μL.
[0020] Preferably, the amount of organic solvent used in the reaction system is 1 mL.
[0021] Preferably, the reaction system temperature is 120-180 ℃.
[0022] Preferably, the solvent in the reaction system is o-dichlorobenzene or n-butanol.
[0023] Preferably, the reaction time is 1-120 minutes, more preferably 60 minutes.
[0024] Preferably, the sealed reaction vessel is a type of high-pressure steel reactor with a polytetrafluoroethylene lining.
[0025] The synthesis method of this invention constructs imine-based COFs gel materials through a group protection strategy. The general formula can be expressed as follows:
[0026] In the general formula: Organic monomer 1 mainly includes any one of p-phenylenediamine, 2-sulfonic p-phenylenediamine, 2,5-dihydroxyp-phenylenediamine, 4,4'-biphenylenediamine, p-diaminoazobenzene, p-diaminoynylbenzene, 1,3,5-tris(4-aminophenyl)amine, 1,3,5-tris(4-aminophenyl)benzene, 1,3,5-tris(4-aminophenyl)triazine, and 1,3,5-tris(4-(4-aminophenyl)phenyl)amine.
[0027] Organic monomer 2 mainly includes: 1,3,5-trisβ-ketoenyldimethylamine-2,4,6-phloroglucinol and 1,4-diβ-ketoenyldimethylamine-2,5-hydroquinone.
[0028] The monomers 1 and 2 mentioned above can be combined in any way [3+2] or [3+3], and the target COFs can be prepared under any of the following conditions: catalyst conditions.
[0029] The catalyst is either acetic acid or lactic acid.
[0030] Preferably, the synthesis rate is controlled at 120 °C or 180 °C, and the selected reaction time is 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 60 minutes, or 120 minutes.
[0031] Preferably, the solvents used in the chemical stability test include water, aqueous solutions with pH=0 and pH=12, and different organic solvents, including DMF, CH2Cl2, CHCl3, THF, n-hexane, toluene, and acetonitrile.
[0032] Preferably, the photothermal conversion performance of the lipase-COF composite material is equivalent to that of simulated solar light intensity (1 kW / m²). 2 It was carried out under irradiation.
[0033] Further preferred, the lipase and COF composite material dispersed in solution can locally raise the temperature of the system to 50±3 °C within one hour under light conditions.
[0034] Preferably, the lipase and COFs composite material is kept under light throughout the actual chiral catalytic conversion process, and a water cooling system is used to keep the external ambient temperature at room temperature to avoid errors caused by instrument overheating.
[0035] Preferably, the wavelength range is 320 nm to 2500 nm.
[0036] Preferably, a collimated beam with a diameter of 40mm is used.
[0037] The synthesis method used in this invention can achieve gram-scale preparation by expanding the reaction vessel.
[0038] Preferably, 4.5 mmol of organic monomer 1 and 3 mmol of organic monomer 2 are ground and mixed.
[0039] Preferably, a 100mL reaction vessel is used.
[0040] Preferably, 25 mL of o-dichlorobenzene, 25 mL of n-butanol and 5 mL of 6M lactic acid aqueous solution are added.
[0041] Preferably, the reaction temperature is 120 °C.
[0042] Preferably, the reaction time is 60 minutes.
[0043] Compared with existing inventions, this invention has the following innovations: 1. A class of β-ketoenamine COFs based on intramolecular hydrogen bond-activated Michael addition reactions were designed and synthesized. Due to the intramolecular hydrogen bond activation effect, highly crystallinity and high specific surface area covalent organic frameworks can be prepared within minutes. Furthermore, the synthesis of COFs can be easily extended to the gram scale with very high spacetime yield (511 kg / m³ / day).
[0044] 2. A novel photothermal enhancement strategy was devised to significantly improve the catalytic performance of the enzyme. Under light irradiation, the biocatalyst exhibited enhanced catalytic activity for eight racemic substrates (ee value > 99%, conversion rate close to 50% of theoretical yield), nearly three times higher than the catalytic activity without light irradiation and with the free enzyme.
[0045] Figure 1 This invention relates to the monomer structure and COF structure of a strategy for constructing intramolecular hydrogen bond activated covalent organic framework materials.
[0046] Figure 2 : A schematic diagram of the synthetic route for the covalent organic framework material prepared in this invention.
[0047] Figure 3 The mechanism and model molecule preparation route of the covalent organic framework material prepared in this invention.
[0048] Figure 4 X-ray powder diffraction and structural simulation diagram of the representative covalent organic framework material prepared by this invention.
[0049] Figure 5 Infrared spectrum of a representative covalent organic framework material prepared by this invention.
[0050] Figure 6 Solid-state NMR spectra of representative covalent organic framework materials prepared in this invention.
[0051] Figure 7 : 77K nitrogen isothermal adsorption-desorption curve of the representative covalent organic framework material prepared by this invention.
[0052] Figure 8 X-ray powder diffraction patterns of representative covalent organic framework materials prepared in this invention at different reaction times.
[0053] Figure 9 Photographs showing the gram-scale preparation process of the representative covalent organic framework material prepared by this invention.
[0054] Figure 10 X-ray powder diffraction pattern of the representative covalent organic framework material prepared by this invention at the gram scale.
[0055] Figure 11Infrared spectra of representative covalent organic framework materials prepared by this invention at the gram scale.
[0056] Figure 12 X-ray powder diffraction patterns of representative covalent organic framework materials prepared by this invention at different synthesis times under high temperature conditions.
[0057] Figure 13 Infrared spectra of representative covalent organic framework materials prepared by this invention at different synthesis times under high temperature conditions.
[0058] Figure 14 X-ray powder diffraction patterns of representative covalent organic framework materials prepared in this invention after treatment in different solvents.
[0059] Figure 15 Scanning electron microscope image of a representative covalent organic framework material prepared in this invention.
[0060] Figure 16 High-resolution transmission electron microscopy images of representative covalent organic framework materials prepared in this invention.
[0061] Figure 17 Water contact angle diagram of a representative covalent organic framework hydrogel material prepared in this invention.
[0062] Figure 18 X-ray powder diffraction pattern of a representative covalently immobilized enzyme within a covalent organic framework prepared in this invention.
[0063] Figure 19 The 77K nitrogen isothermal adsorption-desorption curve and pore size distribution of the representative covalent organic framework enzyme prepared in this invention.
[0064] Figure 20 Confocal microscopy images of enzymes covalently immobilized in the representative covalent organic framework prepared in this invention.
[0065] Figure 21 Near-infrared-ultraviolet spectra of enzymes covalently immobilized by the representative covalent organic framework prepared in this invention.
[0066] Figure 22 The temperature rise curve under light after covalent immobilization of the representative covalent organic framework prepared in this invention is compared with that of the enzyme solution and the blank solution.
[0067] Figure 23 : A schematic diagram of the chiral substrate catalytic transformation under light irradiation after the enzyme is covalently immobilized in a representative covalent organic framework prepared in this invention.
[0068] Figure 24The graph shows a comparison of the chiral substrate catalytic conversion rate under light irradiation with that of free enzymes and non-covalently immobilized enzymes, based on the representative covalent organic framework enzymes prepared in this invention.
[0069] Figure 25 The relative activity of the representative covalently immobilized enzyme prepared by this invention under light irradiation in a catalytic cycling experiment. Detailed Implementation
[0070] Unless otherwise stated in the context of this application, the technical terms and abbreviations used herein have their conventional meanings as known to those skilled in the art; unless otherwise stated, the raw material compounds used in the following examples are all commercially available. Example 1 is a method for synthesizing the material, Example 2 is a solvent exchange test of the material, Example 3 is a freeze resistance test of the material, and Example 4 is a test of the representative material on solar thermal seawater evaporation.
[0071] The specific implementation method for the general rapid synthesis of a class of covalent organic framework materials and their performance characterization in immobilized enzymes and catalysis, as mentioned in this invention, is as follows. Conversely, the following examples are only for further explanation and illustration of this invention and should not be considered as limiting the scope of the invention.
[0072] Example 1: According to the above general formula, p-phenylenediamine (0.075 mmol, 8.1 mg) and 1,3,5-trisβ-ketoenyldimethylamine-2,4,6-phloroglucinol (0.05 mmol, 20.9 mg) were weighed, added to a mortar and ground until uniformly mixed. The mixture was then transferred to a reactor, and 0.5 mL of o-dichlorobenzene, 0.5 mL of n-butanol and 0.1 mL of 6 mol / L lactic acid aqueous solution were added. The reactor was sealed at room temperature and pressure and placed in an oven at 120 °C for 1-120 minutes. After the reaction, the mixture was cooled to room temperature, filtered, and thoroughly washed with N,N-dimethylformamide and methanol to obtain a deep red covalent organic framework powder material.
[0073] like Figure 4 As shown, the powder X-ray diffraction pattern indicates that the synthesized COFs material has high crystallinity. Figure 4 Infrared spectroscopy further confirmed that this material is a hydrogen-bonded COF material, with 2915 cm⁻¹ being a particularly rich source of hydrogen bonds. -1 The CH stretching vibration peak of the methyl group in dimethylamine and the 3092-3409 cm⁻¹ -1 The disappearance of the NH stretching vibration peak of the amino group indicates that the Michael addition-elimination reaction has occurred and COF has been generated. Figure 7 The nitrogen isotherm adsorption-desorption curves of representative covalent organic framework materials at 77 K are shown, with NKCOF-72 having a BET surface area of 11478 m².2 / g, with a pore size of 2.2 nm.
[0074] Example 2: These types of covalent organic framework materials can enable gram-scale experiments, as illustrated in the diagram. Figure 9 As shown, p-phenylenediamine (4.5 mmol, 0.49 g) and 1,3,5-trisβ-enone dimethylamine-2,4,6-phloroglucinol (3 mmol, 1.26 g) were weighed, added to a mortar and ground until uniformly mixed. The mixture was then transferred to a 100 mL reaction vessel, and 25 mL of o-dichlorobenzene, 25 mL of n-butanol and 5 mL of 6 mol / L lactic acid aqueous solution were added. The reaction vessel was sealed at room temperature and pressure and placed in an oven at 120 ℃ for 60 minutes. After the reaction, the mixture was cooled to room temperature, filtered, and thoroughly washed with N,N-dimethylformamide and methanol to obtain 1.17 g of deep red covalent organic framework powder material.
[0075] like Figure 10 As shown, the powder X-ray diffraction pattern indicates that the COFs material synthesized at the gram scale has high crystallinity. Figure 11 Infrared spectroscopy further confirmed that the chemical composition of the gram-scale synthesized COFs materials is consistent with that of the small-scale synthesized COFs.
[0076] Example 3: To effectively immobilize lipase, the covalent organic framework material (COF) needs to be pre-activated. First, the COF was activated in PB buffer (50 mM, pH = 7.5) until no guest molecules effluxed from the material. The activated COF (30 mg) was then introduced into a lipase solution (2.0 mL, 6.0 mg / mL), using 50 mM PB buffer as the solvent. The mixture was shaken at 100 rpm and 37°C. The material was then washed with water to remove loosely adsorbed lipase, yielding composite materials labeled lipase@@NKCOF-73 and lipase@@NKCOF-73. After washing, the supernatant from each operation was collected, and protein concentration was quantified according to Brandford's standard protocol, followed by fitting a calibration curve for the lipase.
[0077] Example 4: The chiral resolution performance of the immobilized lipase was tested using (R,S)-1-phenylethanol as an example. In the enzyme activity assay, the lipase preferentially catalyzed the transesterification between (R)-phenylethanol and vinyl acetate to produce (R)-phenylacetate and acetaldehyde. Finally, unreacted (S)-phenylethanol and the generated (R)-phenylacetate were obtained in the reaction system. Therefore, the chiral decomposition of 1-phenylethanol was achieved. Typically, 1-phenylethanol (0.25 mmol, 30.4 mg) and vinyl acetate (1.0 mmol, 84.1 mg) were added to 5.0 mL of n-hexane. Then, the immobilized catalyst (containing 1.0 mg of lipase) was added to the reaction system and irradiated under solar light intensity (1 kW / m²). 2 The mixture was stirred for 20 hours under circulating condensate while maintaining an ambient temperature of 25 °C. The reaction mixture was analyzed using high-performance liquid chromatography (HPLC). For cyclic catalytic experiments, Lipase-COFs were separated by centrifugation and directly added to the next reaction step.
[0078] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any non-innovative changes and modifications made by those skilled in the art to the technical solution of the present invention based on the above content without departing from the scope of the technical solution of the present invention, such as changing only the ratio of raw materials and reagents, reaction time, and operating procedures, should be included within the protection scope of the present invention.
Claims
1. A method for rapid preparation of covalent organic frameworks based on intramolecular hydrogen bond activation strategy for Michael addition, characterized in that, The di- and tri-connected amino monomers, b-ketoenamine monomers and catalysts are directly added into the reactor, and a COFs material is obtained by heating under normal pressure in a closed state; The amino monomer is an amino-containing organic molecule; the b-ketoenamine monomer is a carbonyl, carbon-carbon double bond and amine-containing organic molecule; and the catalyst is a compound containing an acidic functional group.
2. The method of claim 1, wherein, The amino monomer is selected from any one of p-phenylenediamine, 4,4'-diphenyl diamine, p-diaminoazobenzene, p-diaminoalkynylbenzene, 1,3,5-tris(4-aminophenyl)benzene, and 1,3,5-tris(4-aminophenyl)triazine.
3. The method of claim 1, wherein, The carbonyl, carbon-carbon double bond and amine-containing organic molecule is 1,3,5-tri-b-ketoenamine dimethylamine-2,4,6-mellitic acid.
4. The method of claim 1, wherein, The catalyst is selected from one of acetic acid or lactic acid.
5. The method of claim 1, wherein, The reaction solvent is selected from one or more of o-dichlorobenzene or n-butanol.
6. The method of claim 1, wherein, The step of washing the COFs material with an organic solvent and drying is also included.
7. The covalent organic framework material prepared according to the method of any one of claims 1 to 6, characterized in that, The material is a b-ketoenamine connected covalent organic framework material obtained by Michael addition.
8. The material of claim 7, wherein, The pore size of the COFs is 2.2-3.2 nm.
9. The covalent organic framework of claim 7, wherein the structure of the repeat unit is wherein the dotted line indicates a repeating unit.
10. Use of a covalent organic framework according to any one of claims 7-9, characterized in that, The material is used for solar absorption, photo-thermal conversion, biological macromolecule loading or biological composite preparation catalysis.