A covalent organic framework and a preparation method thereof, and application thereof in protein renaturation

Abstract

The application discloses a kind of covalent organic framework and its preparation method, application in protein renaturation, belong to composite functional material technical field.The covalent organic framework with the pore size of 1.2-6.5nm provided in the application is prepared by Schiff base reaction of aldehyde group monomer and phenanthridine amino monomer.The preparation method provided in the application: under the catalysis of acetic acid, aldehyde group monomer and amino phenanthridine monomer are dissolved in the mixed solution of mesitylene and 1, 4-dioxane and react, separate and collect, obtain the covalent organic framework.The covalent organic framework prepared in the application can limit the degree of freedom of denatured protein, reduce protein misfolding and aggregation, promote protein correct folding, and can stabilize the correct folding intermediate state through hydrophobic interaction and π-π conjugation interaction, and through the precise regulation of confined space and interface interaction, the complete renaturation of protein can be realized, at the same time, the renaturation of most denatured proteins can be realized, with wide universality.

Description

Technical Field

[0001] This application belongs to the field of composite functional materials technology, and in particular relates to a covalent organic framework and its preparation method, and its application in protein refolding. Background Technology

[0002] Proteins occupy a central position in the life sciences, serving not only as the basic molecular structural units of organisms but also ensuring the normal functioning of life processes through participation in various biochemical processes, including enzyme-catalyzed reactions, cytoskeleton construction, and signal transduction. They also have wide applications in biomanufacturing and biomedicine. However, if proteins fail to fold correctly according to their designed three-dimensional structure, misfolded proteins may aggregate into insoluble aggregates. These aggregates form plaques or fibers within cells, negatively impacting cellular function and potentially causing the proteins to lose their normal function or activity. Currently, protein refolding is mainly achieved through dilution, dialysis, gel filtration, and additive-assisted methods. However, these methods suffer from drawbacks such as low refolding efficiency, limitation to certain proteins preventing large-scale industrial application, high cost, and complex operation. Therefore, finding efficient and simple methods to refold a variety of proteins and realize their value in biomanufacturing and biomedicine is an urgent problem to be solved.

[0003] Covalent organic frameworks can be structured and designed to develop two-dimensional (2D) or three-dimensional (3D) structures based on combinations of organic structural units and specific geometries. This structural design can synthesize highly ordered porous structures and fine-tune their physical and chemical properties. Currently, there are no precedents for protein refolding using covalent organic frameworks. Summary of the Invention

[0004] This application discloses a covalent organic framework and its preparation method, as well as its application in protein refolding, aiming to solve the technical problems of low protein refolding rate, limited applicable protein types, high cost, and complex operation of existing materials.

[0005] To achieve the above objectives, the technical solution of this application is:

[0006] The first aspect of this application provides a covalent organic framework prepared via a Schiff base from an aldehyde monomer and a phenanthridine amino monomer.

[0007] The covalent organic framework has a pore size of 1.2-6.5 nm.

[0008] Preferably, in conjunction with the first aspect, the aldehyde monomer is one of pyromellitic pyromellitic aldehyde, 2-hydroxy-1,3,5-phenyltricarboxaldehyde, 2,4-dihydroxy-1,3,5-pyromellitic pyromellitic aldehyde, trialdehyde-resorcinol, 2,4,6-tris(4-aldehydephenyl)-1,3,5-triazine, and 2,4,6-tris-(4-formyl-biphenyl-4-yl)-1,3,5-triazine.

[0009] Preferably, in conjunction with the first aspect, the phenanthridine amino monomer is one of 3,8-diamino-6-phenylphenanthridine, 3,8-diamino-5-ethyl-6-phenylphenanthridine bromide, 3,8-diamino-5-ethyl-6-phenylphenanthridine fluoride, 3,8-diamino-5-ethyl-6-phenylphenanthridine chloride, and 3,8-diamino-5-ethyl-6-phenylphenanthridine iodide.

[0010] The second aspect of this application provides a method for preparing the covalent organic framework described in the first aspect, the method comprising:

[0011] Under acetic acid catalysis, aldehyde monomers and aminophenanthridine monomers are dissolved in a mixed solution of mesitylene and 1,4-dioxane. After reaction under vacuum, the mixture is separated and collected to obtain the covalent organic framework.

[0012] Preferably, in conjunction with the second aspect, the molar ratio of the phenanthrene amino monomer to the aldehyde monomer is 1:(1-3).

[0013] Preferably, in conjunction with the second aspect, the molar amount of acetic acid is 0.5-10 times the molar amount of the aldehyde monomer, and the concentration is 3-18M.

[0014] In conjunction with the second aspect, preferably, the reaction time under vacuum conditions is 10-180 h and the temperature is 30-180 °C.

[0015] The third aspect of this application provides the application of covalent organic frameworks prepared by the preparation method described in the second aspect in protein refolding.

[0016] The fourth aspect of this application provides for the application described in the third aspect, including:

[0017] After mixing the denatured protein with the covalent organic framework described in the first aspect, the protein is separated and collected to obtain the refolded protein.

[0018] Preferably, in conjunction with the fourth aspect, the denatured protein is one of microperoxidase, lysozyme, nattokinase, trypsin, papain, laccase, or glucose oxidase.

[0019] And / or, when the denatured protein is mixed with the covalent organic framework described in the first aspect, the temperature is 20-50°C.

[0020] Compared with the prior art, the advantages or beneficial effects of the embodiments of this application include at least the following:

[0021] The covalent organic framework with a pore size of 1.2-6.5 nm provided in this application is prepared by Schiff base reaction of aldehyde monomers and phenanthrene amino monomers. Firstly, it not only restricts the degrees of freedom of denatured proteins, reducing protein misfolding and aggregation and promoting correct protein folding, but also stabilizes the correct folding intermediate state through hydrophobic interactions and π-π conjugation interactions, and achieves near-complete protein refolding through precise control of confined space and interface interactions. Secondly, it improves protein refolding while significantly increasing refolding speed and efficiency. Thirdly, experimental verification shows that based on the stability of the covalent organic framework's own structure, the refolding performance remains essentially unchanged after recycling, with refolding rates exceeding 95% for trypsin, nattokinase, glucose oxidase, papain, etc. Fourthly, it can refold most denatured proteins, exhibiting broad applicability. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Powder diffraction patterns of NKCOF-121, NKCOF-121-X, NKCOF-121-1O, NKCOF-121-1O-X, NKCOF-121-2O, and NKCOF-121-20-X prepared for embodiments of this application;

[0024] Among them, (a) NKCOF-121; (b) NKCOF-121-X; (c) NKCOF-121-1O; (d) NKCOF-121-1O-X; (e) NKCOF-121-2O; (f) NKCOF-121-20-X;

[0025] Figure 2 Powder diffraction patterns of NKCOF-121-3O, NKCOF-121--3O-X, NKCOF-122, NKCOF-122-X, NKCOF-123, and NKCOF-123-X prepared for embodiments of this application;

[0026] Among them, (a) NKCOF-121-3O; (b) NKCOF-121--3O-X; (c) NKCOF-122; (d) NKCOF-122-X; (e) NKCOF-123; (f) NKCOF-123-X;

[0027] Figure 3 Pore ​​size distribution diagrams of NKCOF-121, NKCOF-121-1O, NKCOF-121-2O, NKCOF-121-3O, NKCOF-122, and NKCOF-123 prepared for embodiments of this application;

[0028] Among them, (a) NKCOF-121; (b) NKCOF-121-1O; (c) NKCOF-121-2O; (d) NKCOF-121-3O; (e) NKCOF-122; (f) NKCOF-123;

[0029] Figure 4 Pore ​​size distribution diagrams of NKCOF-121-X, NKCOF-121-1O-X, NKCOF-121-2O-X, NKCOF-121-3O-X, NKCOF-122-X, and NKCOF-123-X prepared for embodiments of this application;

[0030] Among them, (a) NKCOF-121-X; (b) NKCOF-121-1O-X; (c) NKCOF-121-2O-X; (d) NKCOF-121-3O-X; (e) NKCOF-122-X; (f) NKCOF-123-X;

[0031] Figure 5 Hydrophobicity diagrams of NKCOF-121, NKCOF-121-1O, NKCOF-121-X, NKCOF-121-2O, NKCOF-121-3O, NKCOF-121-2O-X, NKCOF-122, NKCOF-123, NKCOF-122-X, NKCOF-121-1O-X, NKCOF-121-3O-X, and NKCOF-123-X prepared for embodiments of this application;

[0032] Among them, (a) NKCOF-121; (b) NKCOF-121-1O; (c) NKCOF-121-X; (d) NKCOF-121-2O; (e) NKCOF-121-3O; (f) NKCOF-121-2O -X; (g) NKCOF-122; (h) NKCOF-123; (i) NKCOF-122-X; (j) NKCOF-121-1O-X; (k) NKCOF-121-3O-X; (l) NKCOF-123-X;

[0033] Figure 6 The refolding rate of myoglobin with NKCOF-121, NKCOF-121-1O, NKCOF-121-2O, NKCOF-121-3O, NKCOF-121-X, NKCOF-121-1O-X, NKCOF-121-2O-X, and NKCOF-121-3O-X prepared for the embodiments of this application;

[0034] Figure 7 The refolding rate of parathyroid hormone of NKCOF-121, NKCOF-121-1O, NKCOF-121-2O, NKCOF-121-3O, NKCOF-121-X, NKCOF-121-1O-X, NKCOF-121-2O-X, and NKCOF-121-3O-X prepared for the embodiments of this application;

[0035] Figure 8 The refolding rate of microperoxidase by NKCOF-121, NKCOF-121-1O, NKCOF-121-2O, NKCOF-121-3O, NKCOF-121-X, NKCOF-121-1O-X, NKCOF-121-2O-X, and NKCOF-121-3O-X prepared for the embodiments of this application;

[0036] Figure 9 The refolding rates of NKCOF-122, NKCOF-122-X, NKCOF-123, and NKCOF-123-X prepared for the embodiments of this application to different proteins;

[0037] Figure 10 Cyclic stability graphs of NKCOF-122, NKCOF-122-X, NKCOF-123, and NKCOF-123-X prepared for embodiments of this application against different proteins. Detailed Implementation

[0038] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0039] In the following description of this embodiment, the term "and / or" is used to describe the association relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, B existing alone, and A and B existing simultaneously. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0040] In the following description of this embodiment, the term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0041] Those skilled in the art should understand that, in the following description of the embodiments of this application, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0042] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms "a" and "the" as used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0043] It should be noted that all raw materials and reagents in the embodiments of this application were purchased from the market or prepared according to conventional methods known to those skilled in the art. For example, pyromellitic aldehyde, 2-hydroxy-1,3,5-phenyltricarboxaldehyde, 2,4-dihydroxy-1,3,5-pyromellitic aldehyde, trialdehyde-resorcinol, 2,4,6-tris(4-aldehyde-phenyl)-1,3,5-triazine, 2,4,6-tri-(4-formyl-biphenyl-4-yl)-1,3,5-triazine, 3,8-diamino-6-phenylphenanthridine, 3,8-diamino-5-ethyl-6-phenylphenanthridine bromide, 3,8-diamino-5-ethyl-6-phenylphenanthridine fluoride, 3,8-diamino-5-ethyl-6-phenylphenanthridine chloride, and 3,8-diamino-5-ethyl-6-phenylphenanthridine iodide were all purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0044] In a first aspect, embodiments of this application provide a covalent organic framework, which is prepared by a Schiff base reaction of an aldehyde monomer and a phenanthridine amino monomer.

[0045] The covalent organic framework has a pore size of 1.2-6.5 nm.

[0046] Firstly, it not only restricts the degrees of freedom of denatured proteins, reducing protein misfolding and aggregation and promoting correct protein folding, but also stabilizes the correct folding intermediate state through hydrophobic interactions and π-π conjugation interactions. Furthermore, it enables complete protein refolding through precise regulation of confined space and interface interactions. Secondly, it not only improves protein refolding but also significantly increases refolding speed and efficiency. Thirdly, experimental verification shows that the stability of the covalent organic framework structure itself ensures that refolding performance remains essentially unchanged after recycling, with refolding rates exceeding 95% for trypsin, nattokinase, glucose oxidase, papain, and other proteins. Fourthly, it can refold most denatured proteins, demonstrating broad applicability.

[0047] In the embodiments of this application, the aldehyde monomer is preferably one of pyromellitic pyromellitic aldehyde, 2-hydroxy-1,3,5-phenyltricarboxaldehyde, 2,4-dihydroxy-1,3,5-pyromellitic pyromellitic aldehyde, trialdehyde-resorcinol, 2,4,6-tris(4-aldehydephenyl)-1,3,5-triazine, and 2,4,6-tri-(4-formyl-biphenyl-4-yl)-1,3,5-triazine; the phenanthridine amino monomer is preferably one of 3,8-diamino-6-phenylphenanthridine, 3,8-diamino-5-ethyl-6-phenylphenanthridine bromide, 3,8-diamino-5-ethyl-6-phenylphenanthridine fluoride, 3,8-diamino-5-ethyl-6-phenylphenanthridine chloride, and 3,8-diamino-5-ethyl-6-phenylphenanthridine iodide. The specific chemical structures and properties of these aldehyde and phenanthridine amino monomers enable them to play a crucial role in protein refolding. Trimethylolpropionate (PMP) and its derivatives possess multiple aldehyde functional groups, providing numerous reaction sites. These aldehyde groups can form Schiff base bonds with amino groups in protein molecules, enabling the reconnection and stabilization of folded or denatured protein molecules, restoring them to their correct three-dimensional structure. The phenanthridine structure exhibits planar rigidity and high conjugation, providing non-covalent interactions such as π-π interactions and hydrogen bonds, further stabilizing the structure of refolded proteins and preventing re-denaturation. Therefore, the covalent organic frameworks synthesized from these aldehyde and phenanthridine amino monomers possess the characteristics of both monomers. Through their unique chemical structures and reactivity, they provide multiple binding sites and a stable molecular framework, ultimately achieving protein refolding. The selection and design of these materials are based on their ability to provide the necessary chemical and physical environment to support and promote the correct folding and functional recovery of proteins.

[0048] Secondly, embodiments of this application also provide a method for preparing the covalent organic framework described in the first aspect, the method comprising:

[0049] Under acetic acid catalysis, aldehyde monomers and aminophenanthridine monomers are dissolved in a mixed solution of mesitylene and 1,4-dioxane. After reaction under vacuum, the mixture is separated and collected to obtain the covalent organic framework.

[0050] It should be noted that the preparation process of this application involves a Schiff base reaction of an aldehyde monomer and an aminophenanthridine monomer under the catalysis of acetic acid. The process is simple, and the resulting covalent organic framework has high selectivity and stable yield.

[0051] In the embodiments of this application, the molar ratio of the phenanthrene amino monomer to the aldehyde monomer is preferably 1:(1-3), for example, 1:1, 1:2, 1:3 or any ratio within this range. By controlling the molar ratio of the aldehyde monomer to the phenanthrene amino monomer, the reaction rate can be controlled via the imine bond condensation reaction, thereby precisely controlling the structure of the resulting covalent organic framework.

[0052] In this embodiment, the molar amount of acetic acid is 0.5-10 times the molar amount of the aldehyde monomer, and the concentration is preferably 3-18 M. By controlling the amount of acetic acid added as a catalyst, the reaction rate can be increased, the generation of byproducts can be reduced, and thus the purity and yield of the covalent organic framework can be improved.

[0053] In this embodiment, the reaction time under vacuum conditions is preferably 10-180 h, and the temperature is preferably 30-180 °C. By controlling the reaction temperature and time, the reaction rate and reaction equilibrium can be controlled, thereby improving the purity and yield of the covalent organic framework.

[0054] The third aspect of this application provides the application of covalent organic frameworks prepared by the method described in the second aspect in protein refolding methods. Based on the prepared covalent organic frameworks with pore sizes of 1.2-6.5 nm, which possess stability and biocompatibility, the covalent organic frameworks can achieve protein refolding through precise control of hydrophobic interactions, π-π conjugation interactions, and confined space and interface interactions.

[0055] The fourth aspect of this application provides the application described in the third aspect, comprising: mixing denatured protein with the covalent organic framework described in the first aspect, and then separating and collecting the protein to obtain refolded protein.

[0056] It should be noted that this application does not particularly limit the method of protein refolding using covalent organic frameworks, but the preferred method is the solid-phase column chromatography method. The steps include: packing a column with covalent organic framework material; delivering the denatured protein into the chromatography column using a peristaltic pump, and collecting the flow-through liquid at a constant flow rate to obtain the refolded protein. Because the prepared covalent organic frameworks vary in size, they can refold corresponding denatured proteins. Efficient, rapid, and stable refolding can be achieved by controlling the flow rate, temperature, and concentration during the protein refolding process.

[0057] In this embodiment, the denatured protein is preferably one of microperoxidase, lysozyme, nattokinase, trypsin, papain, laccase, or glucose oxidase; the temperature for mixing the denatured protein with the covalent organic framework described in the first aspect is preferably 20-50°C. The resulting covalent organic framework can achieve the refolding of most proteins, and experiments have shown that by controlling the temperature during the refolding process, the refolding rate of proteins is above 95%, demonstrating broad applicability.

[0058] The technical solution of this application will be further described below with reference to specific embodiments.

[0059] Example 1

[0060] This embodiment provides a method for preparing A1-covalent organic framework (NKCOF-121), specifically including:

[0061] Weigh 0.12 mmol of 3,8-diamino-6-phenylphenanthridine and 0.12 mmol of trimesin, add them to a thick-walled heat-resistant glass tube, then add 0.7 mL of trimesin, 0.3 mL of 1,4-dioxane and 0.1 mL of 6M acetic acid aqueous solution. Then rapidly freeze in liquid nitrogen, then evacuate and seal the tube with a hydrogen-oxygen flame. Place it in an oven at 150 °C for 3 days to obtain A1-covalent organic framework (NKCOF-121).

[0062] Its reaction equation is shown in equation (1):

[0063]

[0064] Example 2

[0065] This embodiment provides a method for preparing A2-covalent organic framework (NKCOF-121-1O), specifically including:

[0066] Weigh 0.12 mmol of 3,8-diamino-6-phenylphenanthridine and 0.06 mmol of 2-hydroxy-1,3,5-benzenetrialdehyde, add them to a thick-walled heat-resistant glass tube, then add 0.7 mL of mesitylene, 0.3 mL of 1,4-dioxane and 0.1 mL of 6M acetic acid aqueous solution. Then rapidly freeze in liquid nitrogen, then evacuate, then seal the tube with a hydrogen-oxygen flame, and place it in an oven at 150 °C for 3 days to obtain A2-covalent organic framework (NKCOF-121-1O).

[0067] Its reaction equation is shown in equation (2):

[0068]

[0069] Example 3

[0070] This embodiment provides a method for preparing A3-covalent organic framework (NKCOF-121-2O), specifically including:

[0071] Weigh 0.12 mmol of 3,8-diamino-6-phenylphenanthridine and 0.04 mmol of 2,4-dihydroxy-1,3,5-pyromellitic aldehyde, add them to a thick-walled heat-resistant glass tube, then add 0.7 mL of mesitylene, 0.3 mL of 1,4-dioxane and 0.1 mL of 6M acetic acid aqueous solution. Then rapidly freeze in liquid nitrogen, then evacuate, then seal the tube with a hydrogen-oxygen flame, and place it in an oven at 150 °C for 3 days to obtain A3-covalent organic framework (NKCOF-121-2O).

[0072] Its reaction equation is shown in equation (3):

[0073]

[0074] Example 4

[0075] This embodiment provides a method for preparing A4-covalent organic framework (NKCOF-121-3O), specifically including:

[0076] Weigh 0.12 mmol of 3,8-diamino-6-phenylphenanthridine and 0.08 mmol of trialdehyde phloroglucinol, add them to a thick-walled heat-resistant glass tube, then add 0.7 mL of mesitylene, 0.3 mL of 1,4-dioxane and 0.1 mL of 6M acetic acid aqueous solution. Then rapidly freeze in liquid nitrogen, then evacuate, then seal the tube with a flame using an oxyhydrogen generator, and place it in an oven at 150 °C for 3 days to obtain A4-covalent organic framework (NKCOF-121-3O).

[0077] Its reaction equation is shown in equation (4):

[0078]

[0079] Example 5

[0080] This embodiment provides a method for preparing A5-covalent organic framework (NKCOF-122), specifically including:

[0081] Weigh 0.12 mmol of 3,8-diamino-6-phenylphenanthridine and 0.08 mmol of 2,4,6-tris(4-aldehydephenyl)-1,3,5-triazine, and add them to a thick-walled heat-resistant glass tube. Then add 0.7 mL of mesitylene, 0.3 mL of 1,4-dioxane and 0.1 mL of 6M acetic acid aqueous solution. The tube is then rapidly frozen in liquid nitrogen, evacuated, and sealed with a flame using an oxyhydrogen generator. The tube is then placed in an oven at 150 °C and reacted for 3 days to obtain A5-covalent organic framework (NKCOF-122).

[0082] Its reaction equation is shown in equation (5):

[0083]

[0084] Example 6

[0085] This embodiment provides a method for preparing A6-covalent organic framework (NKCOF-123), specifically including:

[0086] Weigh 0.12 mmol of 3,8-diamino-6-phenylphenanthridine and 0.08 mmol of 2,4,6-tri-(4-formyl-biphenyl-4-yl)-1,3,5-triazine, add them to a thick-walled heat-resistant glass tube, then add 0.7 mL of mesitylene, 0.3 mL of 1,4-dioxane and 0.1 mL of 6M acetic acid aqueous solution, then rapidly freeze in liquid nitrogen, then evacuate, then seal the tube with a hydrogen-oxygen flame, and place it in an oven at 150 °C for 3 days to obtain A6-covalent organic framework (NKCOF-123).

[0087] Its reaction equation is shown in equation (6):

[0088]

[0089] Example 7

[0090] This embodiment provides a method for preparing A7-covalent organic framework (NKCOF-121-X), specifically including:

[0091] Weigh 0.12 mmol of 3,8-diamino-6-phenylphenanthridine halide and 0.08 mmol of trimesin, add them to a thick-walled heat-resistant glass tube, then add 0.7 mL of trimesin, 0.3 mL of 1,4-dioxane and 0.1 mL of 6M acetic acid aqueous solution. Then rapidly freeze in liquid nitrogen, then evacuate, then seal the tube with a hydrogen-oxygen flame, and place it in an oven at 150 °C for 3 days to obtain A7-covalent organic framework (NKCOF-121-X).

[0092] Its reaction equation is shown in equation (7):

[0093]

[0094]

[0095] Example 8

[0096] This embodiment provides a method for preparing A8-covalent organic framework (NKCOF-121-1O-X), specifically including:

[0097] Weigh 0.12 mmol of 3,8-diamino-6-phenylphenanthridine halide and 0.08 mmol of 2-hydroxy-1,3,5-benzenetrialdehyde, add them to a thick-walled heat-resistant glass tube, then add 0.7 mL of mesitylene, 0.3 mL of 1,4-dioxane and 0.1 mL of 6M acetic acid aqueous solution, then rapidly freeze in liquid nitrogen, then evacuate, then seal the tube with a hydrogen-oxygen flame, and place it in an oven at 150 °C for 3 days to obtain A8-covalent organic framework (NKCOF-121-1O-X).

[0098] Its reaction equation is shown in equation (8):

[0099]

[0100] Example 9

[0101] This embodiment provides a method for preparing A9-covalent organic framework (NKCOF-121-2O-X), specifically including:

[0102] Weigh 0.12 mmol of 3,8-diamino-6-phenylphenanthridine halide and 0.08 mmol of 2,4-dihydroxy-1,3,5-pyromellitic aldehyde, add them to a thick-walled heat-resistant glass tube, then add 0.7 mL of mesitylene, 0.3 mL of 1,4-dioxane and 0.1 mL of 6M acetic acid aqueous solution, then rapidly freeze in liquid nitrogen, then evacuate, then seal the tube with a hydrogen-oxygen flame, and place it in an oven at 150 °C for 3 days to obtain A9-covalent organic framework (NKCOF-121-2O-X).

[0103] Its reaction equation is shown in equation (9):

[0104]

[0105] Example 10

[0106] This embodiment provides a method for preparing A10-covalent organic framework (NKCOF-121-3O-X), specifically including:

[0107] Weigh 0.12 mmol of 3,8-diamino-6-phenylphenanthridine halide and 0.08 mmol of trialdehyde phloroglucinol, add them to a thick-walled heat-resistant glass tube, then add 0.7 mL of mesitylene, 0.3 mL of 1,4-dioxane and 0.1 mL of 6M acetic acid aqueous solution, then rapidly freeze in liquid nitrogen, then evacuate, then seal the tube with a hydrogen-oxygen flame, and place it in an oven at 150 °C for 3 days to obtain A10-covalent organic framework (NKCOF-121-3O-X).

[0108] Its reaction equation is shown in equation (10):

[0109]

[0110] Example 11

[0111] This embodiment provides a method for preparing A11-covalent organic framework (NKCOF-122-X), specifically including:

[0112] Weigh 0.12 mmol of 3,8-diamino-6-phenylphenanthridine halide and 0.08 mmol of 2,4,6-tris(4-aldehydephenyl)-1,3,5-triazine, and add them to a thick-walled heat-resistant glass tube. Then add 0.7 mL of mesitylene, 0.3 mL of 1,4-dioxane, and 0.1 mL of 6M acetic acid aqueous solution. The tube is then rapidly frozen in liquid nitrogen, evacuated, and sealed with a flame using an oxyhydrogen generator. The tube is then placed in an oven at 150 °C and reacted for 3 days to obtain A11-covalent organic framework (NKCOF-122-X).

[0113] Its reaction equation is shown in equation (11):

[0114]

[0115] Example 12

[0116] This embodiment provides a method for preparing A12-covalent organic framework (NKCOF-123-X), specifically including:

[0117] Weigh 0.12 mmol of 3,8-diamino-6-phenylphenanthridine halide and 0.08 mmol of 2,4,6-tris-(4-formyl-biphenyl-4-yl)-1,3,5-triazine, add them to a thick-walled heat-resistant glass tube, then add 0.7 mL of mesitylene, 0.3 mL of 1,4-dioxane and 0.1 mL of 6M acetic acid aqueous solution, then rapidly freeze in liquid nitrogen, then evacuate, then seal the tube with a hydrogen-oxygen flame, and place it in an oven at 150 °C for 3 days to obtain A12-covalent organic framework (NKCOF-123-X).

[0118] Its reaction equation is shown in equation (12):

[0119]

[0120] To verify the conjugated system of the covalent organic framework prepared in the examples, the adsorption rate was calculated by measuring the fluorescence values ​​of amino acids before and after adsorption, thereby verifying the conjugated system of the material.

[0121] The test procedure was as follows: For tetramethylrhodamine (TAMRA), the excitation and emission wavelengths were set to 550 nm and 580 nm, respectively. Using an excitation wavelength of 550 nm, the emission spectrum from 555 to 650 nm was collected, and the fluorescence intensity at 580 nm was used for analysis. Detection was performed at room temperature using a 1 cm path length cell and buffer solutions (50 mM Tris-HCl, pH = 8.0 and 5 mM MgCl2). The interaction was achieved by vibrating 62 μM NKCOFs with 50 nM of the fluorescent dye FAM and TAMRA at 200 rpm for 3 hours at 25 °C. The adsorption rate was then calculated by subtracting the fluorescence value after adsorption from the fluorescence value before adsorption and then dividing by the fluorescence value before adsorption. For tryptophan, phenylalanine, or tyrosine, the interaction was achieved by vibrating 1 mg / mL of the amino acid with 1 mg of NKCOFs at 200 rpm for 3 hours at 25 °C. The concentrations of amino acids before and after adsorption were detected at a wavelength of 280 nm using a spectrophotometer. The amount of tryptophan, phenylalanine, or tyrosine adsorbed by NKCOFs was calculated, and the results are shown in Table 1.

[0122] Table 1 Adsorption of aromatic amino acids by covalent organic frameworks

[0123]

[0124]

[0125] Therefore, the adsorption of the conjugated dye tetramethylrhodamine by the covalent organic framework demonstrates that NKCOFs themselves are strongly conjugated systems. Subsequently, since proteins themselves contain many aromatic amino acids, and these amino acids can form conjugated interactions with NKCOFs, the adsorption of aromatic amino acids by NKCOFs demonstrates the π-π conjugated interaction between NKCOFs and proteins.

[0126] To verify the structural properties of the covalent organic frameworks prepared in the embodiments of this application, powder diffraction and pore size distribution tests were performed on the covalent organic frameworks prepared in the embodiments of this application. The results are as follows: Figures 1 to 4 As shown.

[0127] according to Figure 1 and Figure 2 As shown, the peak positions of the covalent organic frameworks prepared in the examples are all before 5°, indicating their success, and they also have good crystallinity.

[0128] according to Figure 3 and Figure 4 As shown, by measuring nitrogen adsorption in covalent organic frameworks and fitting the data using the Non-Local Density Functional Theory (NLDFT) model, the micropore or mesopore distribution of the covalent organic frameworks was obtained, and the pore size was calculated. The NKCOF-121 series consisted entirely of micropores with pore sizes between 1.6 and 2.0 nm; the NKCOF-122 series had pore sizes between 3.8 and 4.4 nm; and the NKCOF-123 series had pore sizes between 5.1 and 5.6 nm, indicating that the prepared covalent organic frameworks all possessed nanopore size distributions.

[0129] To verify the overall performance of the covalent organic frameworks prepared in the embodiments of this application, the hydrophobic properties and refolding rates of different proteins were tested. The results are as follows: Figures 5 to 9 As shown.

[0130] according to Figure 5 It is known that a certain amount of NKCOFs are pressed into thin sheets using a tablet press, and their hydrophilicity and hydrophobicity are measured using a water contact angle meter. The size of the hydrophobic angle characterizes the hydrophobicity of the covalent organic framework. The stronger the hydrophobicity, the stronger the hydrophobic interaction with the protein, and these covalent organic frameworks possess a certain degree of hydrophobicity.

[0131] The refolding experiment was performed by diluting the denatured protein into a refolding buffer. A refolding solution was prepared by dispersing NKCOFs in ultrapure water (3 mg / mL NKCOFs). To perform protein refolding, 100 μL of the denatured protein solution was added to 900 μL of the refolding buffer and incubated at 25°C for 4 hours. The refolding rate of the denatured protein was assessed by measuring protein activity, such as… Figure 6-9 As shown.

[0132] according to Figure 6-9 As is known, myoglobin, parathyroid hormone, and microperoxidase are approximately 2 nm in size. The NKCOF-121 series exhibits a refolding rate of over 88% for these three proteins. Furthermore, stronger hydrophobic interactions result in better refolding performance; therefore, NKCOF-121 achieves the highest refolding rate, exceeding 95%. Lysozyme, nattokinase, trypsin, and papain are approximately 4 nm in size, and the NKCOF-122 series shows a refolding rate of over 90% for all four proteins. Glucose oxidase, laccase, and peroxidase are approximately 5 nm in size, and the NKCOF-123 series shows a refolding rate of over 90% for all three proteins.

[0133] To verify the cycling stability of the covalent organic framework prepared in the embodiments of this application, a cycling stability test was performed on the covalent organic framework prepared in the embodiments of this application, and the results are as follows. Figure 10 As shown.

[0134] A covalent organic framework material (50-100 mg) was packed into the column. Denatured protein (5-10 mg / mL, 10 mL) was then delivered into the column using a peristaltic pump at a constant flow rate (0.1-0.5 mL / min). The flow-through was collected to obtain the refolded protein. The solid-phase column was washed with 50-100 mL of ultrapure water, and the refolding process was repeated. The activity of the refolded protein was measured each time, and the cycling performance of the solid-phase column was calculated.

[0135] according to Figure 10 To date, NKCOFs were prepared into solid-phase columns, and their cycling performance was measured. When refolding proteins with similar pore sizes, the refolding rate of NKCOFs did not decrease significantly after 30 cycles, remaining above 80%. This demonstrates their structural stability and good cycling performance.

[0136] Therefore, the covalent organic framework with a pore size of 1.2-6.5 nm provided in this application is prepared by a Schiff base reaction of an aldehyde monomer and a phenanthrene amino monomer. The preparation method provided in this application involves dissolving the aldehyde monomer and the aminophenanthrene monomer in a mixed solution of mesitylene and 1,4-dioxane under acetic acid catalysis, reacting the solutions, separating and collecting the contents to obtain the covalent organic framework. The covalent organic framework prepared in this application can restrict the degrees of freedom of denatured proteins, reduce protein misfolding and aggregation, promote correct protein folding, and stabilize the correct folding intermediate state through hydrophobic interactions and π-π conjugation interactions. Furthermore, it can achieve complete protein renaturation through precise control of confined space and interface interactions, and can simultaneously achieve renaturation of most denatured proteins, exhibiting broad applicability.

[0137] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

[0138] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of this application.

Claims

1. The application of a covalent organic framework in protein refolding, wherein the application is for non-disease diagnosis or treatment purposes, characterized in that, The covalent organic framework is prepared by Schiff base reaction of an aldehyde monomer and a phenanthreneamine amino monomer; the pore size of the covalent organic framework is 1.2-6.5 nm. The aldehyde monomer is one of the following: pyromellitic pyromellitic aldehyde, 2-hydroxy-1,3,5-phenyltricarboxaldehyde, 2,4-dihydroxy-1,3,5-pyromellitic pyromellitic aldehyde, trialdehyde-resorcinol, 2,4,6-tris(4-aldehydephenyl)-1,3,5-triazine, and 2,4,6-tris-(4-formyl-biphenyl-4-yl)-1,3,5-triazine; The phenanthridine amino monomer is one of 3,8-diamino-6-phenylphenanthridine, 3,8-diamino-5-ethyl-6-phenylphenanthridine bromide, 3,8-diamino-5-ethyl-6-phenylphenanthridine fluoride, 3,8-diamino-5-ethyl-6-phenylphenanthridine chloride, and 3,8-diamino-5-ethyl-6-phenylphenanthridine iodide.

2. The application of the covalent organic framework according to claim 1 in protein refolding, characterized in that, The method for preparing the covalent organic framework includes: Under acetic acid catalysis, aldehyde monomers and aminophenanthridine monomers are dissolved in a mixed solution of mesitylene and 1,4-dioxane. After reaction under vacuum, the mixture is separated and collected to obtain the covalent organic framework.

3. The application of the covalent organic framework according to claim 2 in protein refolding, characterized in that, The molar ratio of the phenanthridine amino monomer to the aldehyde monomer is 1:(1-3).

4. The application of the covalent organic framework according to claim 2 in protein refolding, characterized in that, The molar amount of acetic acid is 0.5-10 times the molar amount of the aldehyde monomer, and the concentration is 3-18 M.

5. The application of the covalent organic framework according to claim 2 in protein refolding, characterized in that, The reaction time under vacuum conditions is 10-180 h, and the temperature is 30-180 ℃.

6. The application of the covalent organic framework according to claim 1 in protein refolding, characterized in that, include: After mixing the denatured protein with the covalent organic framework, the protein is separated and collected to obtain the refolded protein.

7. The application of the covalent organic framework according to claim 6 in protein refolding, characterized in that, The denatured protein is one of microperoxidase, lysozyme, nattokinase, trypsin, papain, laccase, and glucose oxidase. And / or, the temperature is 20-50 °C when the denatured protein is mixed with the covalent organic framework.

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