Covalent organic framework, preparation method thereof and application of covalent organic framework in protein renaturation

Abstract

The invention discloses a covalent organic framework as well as a preparation method and application thereof in protein renaturation, and belongs to the technical field of composite functional materials. The covalent organic framework with the pore diameter of 1.2-6.5 nm is prepared from an aldehyde group monomer and a phenanthridine amino monomer through a Schiff base reaction. The preparation method provided by the invention comprises the following steps: under the catalytic action of acetic acid, dissolving an aldehyde group monomer and an amino phenanthridine monomer in a mixed solution of mesitylene and 1, 4-dioxane to react, separating and collecting to obtain the covalent organic framework. The covalent organic framework prepared by the invention can limit the degree of freedom of denatured protein, reduce wrong folding and aggregation of the protein, promote correct folding of the protein, and stabilize a correct folding intermediate state through hydrophobic interaction and pi-pi conjugate interaction, so that the covalent organic framework has a good application prospect. And moreover, complete renaturation of the protein can be realized through accurate regulation and control of interaction between the confinement space and the interface, and meanwhile, renaturation of most denatured proteins can be realized, so that the method has wide universality.

Description

Technical Field

[0001] The present application belongs to the technical field of composite functional materials, and in particular relates to a covalent organic framework and a preparation method thereof, and an application thereof in protein renaturation. Background Art

[0002] Proteins occupy a core position in the field of life sciences. They are not only the basic molecular structural units of organisms, but also ensure the normal progress of life activities by participating in various biochemical processes, including enzyme catalysis, cytoskeleton construction and signal transmission. They are also widely used in biomanufacturing and biomedicine. However, if proteins fail to fold correctly according to their designed three-dimensional structures, misfolded proteins may aggregate into insoluble aggregates, which form plaques or fibers in cells, have a negative impact on cell function, and may cause proteins to lose their normal functions or activities. At present, protein refolding is mainly achieved through dilution, dialysis, gel filtration, and additive-assisted methods. However, these methods have the disadvantages of low refolding efficiency, only for some proteins, and cannot be applied on a large scale in industrial applications, high cost and complex operation. Therefore, how to efficiently and simply achieve the refolding of multiple proteins and give full play to their value in the fields of 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 the combination of organic building blocks and specific geometric shapes. The structural design can synthesize highly ordered pore structures and fine-tune their physical and chemical properties. There is currently no precedent for the refolding of proteins through covalent organic frameworks. Summary of the invention

[0004] The present application discloses a covalent organic framework and a preparation method thereof, and application in protein renaturation, aiming to solve the technical problems of low protein renaturation rate of existing materials, few types of applicable proteins, high cost and complex operation.

[0005] In order to achieve the above purpose, the technical solution of this application is:

[0006] The first aspect of the present application provides a covalent organic framework, which is prepared by using an aldehyde monomer and a phenanthridine amino monomer via a Schiff base;

[0007] The pore size of the covalent organic framework is 1.2-6.5 nm.

[0008] In combination with the first aspect, preferably, the aldehyde monomer is one of trimesaldehyde, 2-hydroxy-1,3,5-benzenetricarbaldehyde, 2,4-dihydroxy-1,3,5-benzenetricarbaldehyde, trialdehyde pyroglutamic acid, 2,4,6-tris(4-formylphenyl)-1,3,5-triazine, and 2,4,6-tris-(4-formyl-biphenyl-4-yl)-1,3,5-triazine.

[0009] In combination with the first aspect, preferably, 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 the present application provides a method for preparing the covalent organic framework described in the first aspect, the method comprising:

[0011] Under the catalysis of acetic acid, the aldehyde monomer and the aminophenanthridine monomer are dissolved in a mixed solution of mesitylene and 1,4-dioxane, reacted under vacuum conditions, and then separated and collected to obtain the covalent organic framework.

[0012] In combination with the second aspect, preferably, the molar ratio of the phenanthridine amino monomer to the aldehyde monomer is 1:(1-3).

[0013] In combination with the second aspect, preferably, 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 combination with the second aspect, preferably, the reaction time under vacuum conditions is 10-180 hours and the temperature is 30-180°C.

[0015] The third aspect of the present application provides the use of the covalent organic framework prepared by the preparation method described in the second aspect in protein renaturation.

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

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

[0018] In combination with the fourth aspect, preferably, the denatured protein is one of microperoxidase, lysozyme, nattokinase, trypsin, papain, laccase, and 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 the present application include at least:

[0021] The covalent organic framework with a pore size of 1.2-6.5nm provided in the present application is prepared by Schiff base reaction of aldehyde monomer and phenanthridine amino monomer. On the one hand, it can not only limit the degree of freedom of denatured protein, reduce protein misfolding and aggregation, and promote correct protein folding, but also stabilize the correct folding intermediate state through hydrophobic interaction and π-π conjugated interaction, and can achieve nearly complete refolding of protein through precise regulation of confined space and interface interaction; on the second hand, it can improve protein refolding while significantly improving refolding speed and efficiency; on the third hand, it has been experimentally verified that based on the stability of the covalent organic framework's own structure, the refolding performance is basically not reduced after recycling, and the refolding rates of trypsin, nattokinase, glucose oxidase, papain, etc. are all above 95%; on the fourth hand, it can achieve refolding of most denatured proteins, with wide universality. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[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 in the examples of the present 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 in the examples of the present 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 The pore size distribution diagrams of NKCOF-121, NKCOF-121-1O, NKCOF-121-2O, NKCOF-121-3O, NKCOF-122, and NKCOF-123 prepared in the examples of the present 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 The pore size distribution diagram of NKCOF-121-X, NKCOF-121-1O-X, NKCOF-121-2O-X, NKCOF-121-3O-X, NKCOF-122-X, and NKCOF-123-X prepared in the examples of the present 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 graph 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 in the examples of the present 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 renaturation rates 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 in the examples of the present application to myoglobin;

[0034] Figure 7 The renaturation rates 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 in the examples of the present application for parathyroid hormone;

[0035] Figure 8 The renaturation rates 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 in the examples of the present application to microperoxidase;

[0036] Fig. 9 The renaturation rates of NKCOF-122, NKCOF-122-X, NKCOF-123, and NKCOF-123-X prepared in the examples of the present application to different proteins;

[0037] Fig.10 This is a graph showing the cyclic stability of NKCOF-122, NKCOF-122-X, NKCOF-123, and NKCOF-123-X prepared in the examples of the present application to different proteins. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work 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 of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, B exists alone, and A and B exist at the same time. A and B can be singular or plural. The character " / " generally indicates that the associated objects before and after are in an "or" relationship.

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

[0041] Those skilled in the art should understand that in the following description of the embodiments of the present application, the order of serial numbers does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and 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 the present application.

[0042] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0043] It should be noted that all raw materials and reagents in the embodiments of the present application are purchased on the market or prepared according to conventional methods well known to those skilled in the art. For example, trimesic acid, 2-hydroxy-1,3,5-benzene trimesic acid, 2,4-dihydroxy-1,3,5-trisic acid, trialdehyde phloroglucinol, 2,4,6-tris (4-formylphenyl) -1,3,5-triazine, 2,4,6-tris-(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, 3,8-diamino-5-ethyl-6-phenylphenanthridine iodide, etc. are all purchased on the market or prepared according to conventional methods well known to those skilled in the art.

[0044] In a first aspect, an embodiment of the present application provides a covalent organic framework, which is prepared by reacting an aldehyde monomer and a phenanthridine amino monomer via a Schiff base;

[0045] The pore size of the covalent organic framework is 1.2-6.5 nm.

[0046] Among them, on the one hand, it can not only limit the freedom of denatured proteins, reduce protein misfolding and aggregation, and promote correct protein folding, but also stabilize the correct folding intermediate state through hydrophobic interaction and π-π conjugated interaction, and at the same time, through the precise regulation of confined space and interface interaction, it can achieve complete protein renaturation; on the second hand, it can improve protein renaturation while significantly improving the renaturation speed and efficiency; on the third hand, it has been experimentally verified that based on the stability of the covalent organic framework's own structure, the renaturation performance is basically unchanged after cyclic use, and the renaturation rates of trypsin, nattokinase, glucose oxidase, papain, etc. are all above 95%; on the fourth hand, it can achieve the renaturation of most denatured proteins and has wide applicability.

[0047] In the embodiment of the present application, the aldehyde monomer is preferably one of trimesic acid aldehyde, 2-hydroxy-1,3,5-benzene trimesic acid aldehyde, 2,4-dihydroxy-1,3,5-benzene trimesic acid aldehyde, trialdehyde phloroglucinol, 2,4,6-tris(4-aldehyde phenyl)-1,3,5-triazine, and 2,4,6-tris-(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. Among them, the selection of these aldehyde monomers and phenanthridine amino monomers has a specific chemical structure and characteristics, which enable these materials to play an important role in the protein refolding process. Trimethylenediamine and its derivatives have multiple aldehyde functional groups and can provide multiple reaction sites. These aldehyde groups can form Schiff base bonds with the amine groups in protein molecules, and can reconnect and stabilize the folded or denatured protein molecules to restore them to the correct three-dimensional structure. The phenanthridine structure has planar rigidity and high conjugation, and the non-covalent interactions such as π-π interactions and hydrogen bonds provided can further stabilize the structure of the refolded protein to prevent it from being denatured again. Therefore, the covalent organic framework synthesized by the reaction of these aldehyde monomers and phenanthridine amino monomers has the characteristics of two monomers, and through its unique chemical structure and reactivity, it can provide multi-point binding sites and a stable molecular framework, and finally realize the refolding of protein molecules. 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] In a second aspect, the present application also provides a method for preparing the covalent organic framework described in the first aspect, the method comprising:

[0049] Under the catalysis of acetic acid, the aldehyde monomer and the aminophenanthridine monomer are dissolved in a mixed solution of mesitylene and 1,4-dioxane, reacted under vacuum conditions, and then separated and collected to obtain the covalent organic framework.

[0050] It should be noted that the preparation process of the present application is simple, and the prepared covalent organic framework has high selectivity and stable yield by reacting aldehyde monomers and aminophenanthridine monomers under the catalysis of acetic acid through a Schiff base reaction.

[0051] In the embodiment of the present application, the molar ratio of the phenanthridine amino monomer to the aldehyde monomer is preferably 1:(1-3), for example, 1:1, 1:2, 1:3 or any ratio within the range. Among them, by controlling the molar ratio of the aldehyde monomer to the phenanthridine amino monomer, the reaction rate can be controlled through the imine bond condensation reaction, thereby accurately controlling the structure of the obtained covalent organic framework.

[0052] In the embodiment of the present application, 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. Among them, by controlling the amount of catalyst acetic acid added, the reaction rate can be increased, the generation of by-products can be reduced, and the purity and yield of the covalent organic framework can be improved.

[0053] In the embodiment of the present application, the reaction time under vacuum conditions is preferably 10-180 hours, and the temperature is preferably 30-180° C. Among them, by controlling the temperature and time of the reaction, the reaction rate and reaction balance can be controlled, thereby improving the purity and yield of the covalent organic framework.

[0054] The third aspect of the present application provides the use of the covalent organic framework prepared by the preparation method described in the second aspect in a protein renaturation method. Based on the prepared covalent organic framework with a pore size of 1.2-6.5 nm, it has stability and biocompatibility, and the covalent organic framework can achieve protein renaturation through hydrophobic interaction and π-π conjugated interaction, as well as precise regulation of confined space and interface interaction.

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

[0056] It should be noted that the present application does not specifically limit the method of using a covalent organic framework to renature proteins, and preferably a solid phase column method, the steps of which include: loading the covalent organic framework material into a column; delivering the denatured protein to a chromatography column by a peristaltic pump, and collecting the flow-through of the chromatography column at a constant flow rate to obtain a renatured protein. Since the sizes of the prepared covalent organic frameworks are different, they can renature the corresponding denatured proteins. Efficient, rapid, and stable renaturation can be achieved by controlling the flow rate, temperature, concentration, etc. during the protein renaturation process.

[0057] In the embodiment of the present application, the denatured protein is preferably one of microperoxidase, lysozyme, nattokinase, trypsin, papain, laccase, and glucose oxidase; when the denatured protein is mixed with the covalent organic framework described in the first aspect, the temperature is preferably 20-50°C. Among them, the prepared covalent organic framework can realize the renaturation of most proteins, and experiments have proved that by controlling the temperature during the renaturation process, the renaturation rate of proteins is above 95%, which has wide universality.

[0058] The technical solution of the present application will be further described below in conjunction with specific embodiments.

[0059] Example 1

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

[0061] Weigh 3,8-diamino-6-phenylphenanthridine (0.12 mmol) and trimesic acid (0.12 mmol), add them into 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 6 M acetic acid aqueous solution, then quickly freeze in liquid nitrogen, evacuate and seal the tube with a hydrogen-oxygen machine flame, put it in an oven at 150 ° C for 3 days to obtain A1-covalent organic framework (NKCOF-121).

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

[0063]

[0064] Example 2

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

[0066] Weigh 3,8-diamino-6-phenylphenanthridine (0.12 mmol) and 2-hydroxy-1,3,5-benzenetricarboxaldehyde (0.06 mmol), add them into 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 6 M acetic acid aqueous solution, then quickly freeze in liquid nitrogen, evacuate, and then seal the tube with a hydrogen and oxygen machine flame, put it in an oven at 150 ° C for 3 days to obtain A2-covalent organic framework (NKCOF-121-1O).

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

[0068]

[0069] Example 3

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

[0071] Weigh 3,8-diamino-6-phenylphenanthridine (0.12 mmol) and 2,4-dihydroxy-1,3,5-benzenetricarboxaldehyde (0.04 mmol), add them into 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 quickly freeze in liquid nitrogen, evacuate, and then seal the tube with a hydrogen-oxygen machine flame, put it in an oven at 150 ° C for 3 days to obtain A3-covalent organic framework (NKCOF-121-2O).

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

[0073]

[0074] Example 4

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

[0076] Weigh 3,8-diamino-6-phenylphenanthridine (0.12 mmol) and trialdehyde phloroglucinol (0.08 mmol), add them into 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 6 M acetic acid aqueous solution, then quickly freeze in liquid nitrogen, evacuate, and then seal the tube with a hydrogen-oxygen machine flame, put it in an oven at 150 ° C for 3 days to obtain A4-covalent organic framework (NKCOF-121-3O).

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

[0078]

[0079] Example 5

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

[0081] 3,8-Diamino-6-phenylphenanthridine (0.12 mmol) and 2,4,6-tris(4-formylphenyl)-1,3,5-triazine (0.08 mmol) were weighed and added to a thick-walled heat-resistant glass tube, and then 0.7 mL of mesitylene, 0.3 mL of 1,4-dioxane and 0.1 ml of 6 M acetic acid aqueous solution were added, and then quickly frozen in liquid nitrogen, and then vacuumed, and then the tube was sealed with a hydrogen and oxygen machine flame, and placed in an oven at 150 ° C for reaction for 3 days to obtain A5-covalent organic framework (NKCOF-122).

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

[0083]

[0084] Example 6

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

[0086] 3,8-Diamino-6-phenylphenanthridine (0.12 mmol) and 2,4,6-tris-(4-formyl-biphenyl-4-yl)-1,3,5-triazine (0.08 mmol) were weighed and added to a thick-walled heat-resistant glass tube, and then 0.7 mL of mesitylene, 0.3 mL of 1,4-dioxane and 0.1 ml of 6 M acetic acid aqueous solution were added, and then quickly frozen in liquid nitrogen, and then vacuumed, and then the tube was sealed with a hydrogen and oxygen machine flame, and placed in an oven at 150 ° C for reaction for 3 days to obtain A6-covalent organic framework (NKCOF-123).

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

[0088]

[0089] Example 7

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

[0091] Weigh 3,8-diamino-6-phenylphenanthridine halide (0.12 mmol) and trimesic acid (0.08 mmol), add them into 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 6 M acetic acid aqueous solution, then quickly freeze in liquid nitrogen, evacuate, and then seal the tube with a hydrogen-oxygen machine flame, put it in an oven at 150 ° C for 3 days to obtain A7-covalent organic framework (NKCOF-121-X).

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

[0093]

[0094]

[0095] Example 8

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

[0097] Weigh 3,8-diamino-6-phenylphenanthridine halide (0.12 mmol) and 2-hydroxy-1,3,5-benzenetricarboxaldehyde (0.08 mmol), add them into 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 6 M acetic acid aqueous solution, then quickly freeze in liquid nitrogen, evacuate, and then seal the tube with a hydrogen and oxygen machine flame, put it in an oven at 150 ° C for 3 days to obtain A8-covalent organic framework (NKCOF-121-1O-X).

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

[0099]

[0100] Example 9

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

[0102] Weigh 3,8-diamino-6-phenylphenanthridine halide (0.12 mmol) and 2,4-dihydroxy-1,3,5-benzenetricarboxaldehyde (0.08 mmol), add them into 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 6 M acetic acid aqueous solution, then quickly freeze in liquid nitrogen, evacuate, and then seal the tube with a hydrogen and oxygen machine flame, put it in an oven at 150 ° C for 3 days to obtain A9-covalent organic framework (NKCOF-121-2O-X).

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

[0104]

[0105] Example 10

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

[0107] Weigh 3,8-diamino-6-phenylphenanthridine halide (0.12 mmol) and trialdehyde phloroglucinol (0.08 mmol), add them into 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 6 M acetic acid aqueous solution, then quickly freeze in liquid nitrogen, evacuate, and then seal the tube with a hydrogen-oxygen machine flame, put it in an oven at 150 ° C for 3 days to obtain A10-covalent organic framework (NKCOF-121-3O-X).

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

[0109]

[0110] Embodiment 11

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

[0112] Weigh 3,8-diamino-6-phenylphenanthridine halide (0.12 mmol) and 2,4,6-tris(4-formylphenyl)-1,3,5-triazine (0.08 mmol), add them into 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 6 M acetic acid aqueous solution, then quickly freeze in liquid nitrogen, evacuate, and then seal the tube with a hydrogen-oxygen machine flame, put it in an oven at 150 ° C for 3 days to obtain A11-covalent organic framework (NKCOF-122-X).

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

[0114]

[0115] Example 12

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

[0117] Weigh 3,8-diamino-6-phenylphenanthridine halide (0.12 mmol) and 2,4,6-tris-(4-formyl-biphenyl-4-yl)-1,3,5-triazine (0.08 mmol), add them into 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 6 M acetic acid aqueous solution, then quickly freeze in liquid nitrogen, evacuate, and then seal the tube with a hydrogen-oxygen machine flame, put it in an oven at 150 ° C for 3 days to obtain A12-covalent organic framework (NKCOF-123-X).

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

[0119]

[0120] In order to verify the conjugated system of the covalent organic framework prepared in the example, the adsorption rate was calculated by the fluorescence value of the amino acid before and after adsorption, thereby verifying the conjugated system of the material.

[0121] The test steps are as follows: For tetramethylrhodamine (TAMRA), the excitation and emission wavelengths were set to 550nm and 580nm, respectively. Using an excitation wavelength of 550nm, the emission spectrum from 555 to 650nm was collected, and the fluorescence intensity at 580nm was used for analysis. The detection was carried out at room temperature using a cell with a 1cm optical path and a buffer solution (50mM Tris-HCl, pH=8.0 and 5mM MgCl2). 50nM of the fluorescent dyes FAM and TAMRA interacted with 62μM NKCOFs after shaking at 200rpm for 3 hours at 25°C. Subsequently, the adsorption rate was calculated by subtracting the fluorescence value after adsorption from the fluorescence value before adsorption and then dividing it by the fluorescence value before adsorption. For tryptophan, phenylalanine or tyrosine, 1mg / mL of the amino acid interacted with 1mg NKCOFs after shaking at 200rpm for 3 hours at 25°C. The concentration of amino acids before and after adsorption was detected by using a spectrophotometer at a wavelength of 280 nm, and the amount of tryptophan, phenylalanine or tyrosine adsorbed by NKCOFs was calculated. 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 conjugated dye tetramethylrhodamine on the covalent organic framework proved that NKCOFs itself is a strong conjugated system. Subsequently, since there are many aromatic amino acids in the protein itself, and these amino acids will produce conjugated interactions with NKCOFs. Therefore, the adsorption of aromatic amino acids by NKCOFs proved the π-π conjugated interaction between NKCOFs and proteins.

[0126] In order to verify the structural properties of the covalent organic framework prepared in the embodiment of the present application, the covalent organic framework prepared in the embodiment of the present application was subjected to powder diffraction and pore size distribution tests, and the results are as follows: Figures 1 to 4 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 that they are successful and have good crystallinity.

[0128] according to Figure 3 and Figure 4 As shown, by measuring the nitrogen adsorption of the covalent organic framework, the Non-Local Density Functional Theory (NLDFT) model was used for fitting to obtain the micropore or mesopore distribution of the covalent organic framework, and the pore size was calculated. The NKCOF-121 series are all micropores with a pore size between 1.6-2.0nm; the pore size of the NKCOF-122 series is between 3.8-4.4nn; the pore size of the NKCOF-123 series is between 5.1-5.6nm, indicating that the prepared covalent organic frameworks all have a nanopore size distribution.

[0129] In order to verify the comprehensive performance of the covalent organic framework prepared in the examples of the present application, the hydrophobicity and renaturation rate of different proteins of the covalent organic framework prepared in the examples of the present application were tested, and the results are as follows: Figures 5 to 9 shown.

[0130] according to Figure 5 As known, a certain amount of NKCOFs is pressed into thin sheets by a tablet press, and its hydrophilicity is measured by a water contact angle meter. The hydrophobicity of the covalent organic framework is characterized by the size of the hydrophobic angle. The stronger the hydrophobicity, the stronger the hydrophobic interaction with the protein, and these covalent organic frameworks have a certain hydrophobicity.

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

[0132] according to Figure 6-9 As far as we know, the size of myoglobin, parathyroid hormone, and microperoxidase is about 2nm, and the renaturation rate of NKCOF-121 series for these three proteins is above 88%. At the same time, the stronger the hydrophobic effect, the better the renaturation effect, so the renaturation rate of NKCOF-121 is the highest, which is above 95%. The size of lysozyme, nattokinase, trypsin, and papain is about 4nm, and the renaturation rate of NKCOF-122 series for these four proteins is above 90%. The size of glucose oxidase, laccase, and peroxidase is about 5nm, and the renaturation rate of NKCOF-123 series for these three proteins is above 90%.

[0133] In order to verify the cyclic stability of the covalent organic framework prepared in the embodiment of the present application, the cyclic stability test of the covalent organic framework prepared in the embodiment of the present application was carried out, and the results are as follows: Fig.10 shown.

[0134] The covalent organic framework material (50-100 mg) is loaded into the column, and then the denatured protein (5-10 mg / mL, 10 mL) is delivered to the chromatography column by a peristaltic pump, and a constant flow rate (0.1-0.5 mL / min) is maintained. The flow-through of the chromatography column is collected to obtain the refolded protein; 50-100 mL of ultrapure water is used to rinse the solid phase column, and then the above refolding steps are repeated. The activity of the refolded protein obtained each time is measured, and the circulation performance of the solid phase column is calculated.

[0135] according to Fig.10 As we know, NKCOFs were prepared into solid phase columns and the cycling performance was measured. After 30 cycles, the renaturation rate of NKCOFs, which was similar to the pore size of proteins, did not decrease significantly and was still above 80%, proving its structural stability and good cycling performance.

[0136] Therefore, the covalent organic framework with a pore size of 1.2-6.5nm provided in the present application is prepared by Schiff base reaction of aldehyde monomers and phenanthridine amino monomers. The preparation method provided in the present application is as follows: under the catalysis of acetic acid, the aldehyde monomers and aminophenanthridine monomers are dissolved in a mixed solution of mesitylene and 1,4-dioxane for reaction, and separated and collected to obtain the covalent organic framework. The covalent organic framework prepared in the present application can limit the degree of freedom of denatured proteins, reduce protein misfolding and aggregation, promote correct protein folding, and can stabilize the correct folding intermediate state through hydrophobic interactions and π-π conjugated interactions, and can achieve complete protein renaturation through precise regulation of confined space and interface interactions. At the same time, the renaturation of most denatured proteins can be achieved, and it has wide universality.

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

[0138] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit the present application. Although the present application has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that the technical solutions recorded in the aforementioned embodiments may still be modified, or some or all of the technical features therein may be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the present application.

Claims

1. A covalent organic framework, characterized in that It is prepared by reacting an aldehyde monomer and a phenanthridinium amino monomer via a Schiff base; The pore size of the covalent organic framework is 1.2-6.5 nm.

2. The covalent organic framework according to claim 1, characterized in that The aldehyde monomer is one of trimesaldehyde, 2-hydroxy-1,3,5-benzenetricarboxaldehyde, 2,4-dihydroxy-1,3,5-pyromelliticarboxaldehyde, trialdehyde phloroglucinol, 2,4,6-tris(4-formylphenyl)-1,3,5-triazine, and 2,4,6-tris-(4-formyl-biphenyl-4-yl)-1,3,5-triazine.

3. The covalent organic framework according to claim 1, characterized in that 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.

4. A method for preparing a covalent organic framework according to any one of claims 1 to 3, characterized in that: The method comprises: Under the catalysis of acetic acid, the aldehyde monomer and the aminophenanthridine monomer are dissolved in a mixed solution of mesitylene and 1,4-dioxane, reacted under vacuum conditions, and then separated and collected to obtain the covalent organic framework.

5. The method for preparing a covalent organic framework according to claim 4, wherein The molar ratio of the phenanthridine amino monomer to the aldehyde monomer is 1:(1-3).

6. The method for preparing a covalent organic framework according to claim 4, wherein The molar amount of the acetic acid is 0.5-10 times the molar amount of the aldehyde monomer, and the concentration is 3-18M.

7. The method for preparing a covalent organic framework according to claim 4, wherein: The reaction time under vacuum conditions is 10-180 hours, and the temperature is 30-180°C.

8. Use of a covalent organic framework prepared according to the preparation method according to any one of claims 4 to 7 in protein renaturation.

9. A use according to claim 8, characterized in that include: After the denatured protein is mixed with the covalent organic framework according to claim 1, the protein is separated and collected to obtain the renatured protein.

10. The use according to claim 9, characterized in that The denatured protein is one of microperoxidase, lysozyme, nattokinase, trypsin, papain, laccase, and glucose oxidase; And / or, when the denatured protein is mixed with the covalent organic framework according to claim 1, the temperature is 20-50°C.

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