A biomimetic metal enzyme with a polyamide derivative as a core, and its preparation method and application

Abstract

The present invention belongs to the field of biomedicine and relates to biomimetic metalloenzymes, and in particular refers to a biomimetic metalloenzyme with a polyamide derivative as a core, and its preparation method and application. The present application prepares a nucleic acid-polyamide polymer catalyst with a core-shell structure having targeting, high biosafety and low immunogenicity. In this system, the catalytic active center is protected by a dense outer nucleic acid shell, while the molecular structure and hydrophobic properties determined by the hyperbranched polymer polyamide are utilized to create a highly "pure" catalytic environment inside, avoiding the dilemma of biomimetic enzyme inactivation in a complex system, and also avoiding the hidden danger of metal ion leakage in the body. By utilizing the three-dimensional structure of the material, it can enter the cell autonomously through recognition, avoiding the use of highly toxic cationic transfection agents. Some of the more major problems currently faced by biomimetic metalloenzymes are solved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a biomimetic metal enzyme, in particular to a biomimetic metal enzyme with a polyamide derivative as a core, and a preparation method and application thereof. Background Art

[0002] Intracellular catalysis has attracted increasing attention in recent years because it offers a simple and efficient approach for directly controlling biochemical processes or producing molecules of interest in cells. To achieve enhanced functionality and realize the usefulness of catalysis in cells, it would be desirable to expand the range of "catalyzable" reactions using these intracellular catalysts, thereby enabling more abiotic transformations. However, most metal complexes with catalytic properties exhibit reduced activity or even complete inactivation under biologically relevant conditions because the coordinated biomolecules are poisoned in such environments. A widely adopted solution is to introduce artificial scaffolds, such as single-chain nanoparticles (SCNPs), dense-shell nanoparticles (DSNPs), proteins, and inorganic nanomaterials, for these catalytic metal complexes. These intracellular catalytic strategies have successfully brought a wider range of catalytic species into cells; however, accompanying issues such as cytotoxicity, low cellular uptake, difficult metal scaffold assembly, and limited protection of the metal center still often hinder their application.

[0003] Intracellular biomimetic metalloenzymes show great potential in biocatalysis and medical applications, but their practical application still faces multiple challenges. The inventors of this application previously explored the possibility of intracellular catalysis using SNAs, but the catalytic effect in animal models was unsatisfactory (Molecularly pure miktoarm spherical nucleic acids: preparation and usage as a scaffold for abiotic intracellular catalysis). Further summarizing their experience, the inventors discovered that biomimetic metalloenzymes still have the following problems:

[0004] First, the complex biological environment within the cell significantly affects the stability of biomimetic metalloenzymes, causing them to become inactivated. In particular, metal ions within the cell may be interfered with by other competing ligands, leading to decreased enzyme activity or even inactivation.

[0005] Second: Intracellular biomimetic metalloenzymes need to overcome the barrier of biological membranes when entering the cell.

[0006] Third: The catalytic efficiency of intracellular biomimetic metalloenzymes is often limited in complex biological systems.

[0007] Fourth, the potential toxicity of metal ions from biomimetic metalloenzymes to cells is also a key consideration. Excessive or overactive free heavy metal ions in cells can cause cytotoxic reactions, including oxidative stress, protein dysfunction, and membrane damage.

[0008] Fifth: The immunogenic reaction that may be caused by long-term exposure is also a key point that needs attention. Summary of the Invention

[0009] In order to solve the above technical problems, the present invention proposes a bionic metalloenzyme with a polyamide derivative as the core, and a preparation method and application thereof.

[0010] The technical solution of the present invention is achieved as follows:

[0011] A method for preparing a biomimetic metalloenzyme with a polyamide derivative as a core comprises the following steps:

[0012] (1) Synthesis of Alkyl-PAMAM Core

[0013]

[0014] Using propiolic acid as a raw material, hydroxysuccinimide is used to activate the carboxyl group in a solvent of dichloromethane or DMF under the catalysis of triethylamine and dicyclohexylcarbodiimide to obtain an activated ester of succinimide (compound 1), and the equivalent ratio of propiolic acid, hydroxysuccinimide, dicyclohexylcarbodiimide and triethylamine is 1:1:1.5:1.5. Then, an excess amount of compound 1 (100 equiv.) is used to react with PAMAM-NH2 (1.0 equiv.) with a third-generation outer layer containing 16 amino arms in an ultra-dry methanol system or an ultra-dry ethanol system for 3 to 7 days under the catalysis of triethylamine to obtain a terminal alkyne-modified hyperbranched polyamide derivative Alkyl-PAMAM (compound 2) as the core molecule.

[0015] The CAS number of PAMAM-NH2 is 153891-46-4.

[0016] The structure of Alkyl-PAMAM is:

[0017]

[0018] (2) Synthesis of PAMAM-SNA

[0019]

[0020] 19.8-90.2 mg of compound 2 (5-21 μmol, 1.0 equiv.) was weighed and dissolved in 5-10 mL of DMF and added to a 25 mL round-bottom flask equipped with a magnetic rod. 108.8-456.2 mg (31-130 μmol, 6.2 equiv.) of azido-tris (triazole, azido-TTA) was dissolved in 2-5 mL of DMF and added to the above system after complete dissolution, and Ar gas was passed through. 5.85-24.6 mg (32.5-136.5 μmol, 6.5 equiv.) of anhydrous CuSO4 and 17.0-72.4 mg (32.5-136.5 μmol, 6.5 equiv.) of TBTA ligand (Cas) were weighed and added to the above system. No.:510758-28-8) were mixed and dissolved in 1-5 mL of DMF. After mixing, the mixture was allowed to stand for 5 minutes. The solution was then transferred to a flask system protected by Ar atmosphere and stirred at moderate speed at room temperature for two days. After two days, CPG glass beads loaded with N3-DNA chains (the amount of loaded DNA was 77.5-325.5 μmol, 15.5 equiv.) were added to the system. 10.4-43.5 mg (57.5-241.5 μmol, 1 Anhydrous CuSO₄ (1.5 equiv.) and 30.5-128.1 mg (11.5 equiv.) of TBTA ligand were dissolved in 2-10 mL of DMF. After mixing, the mixture was allowed to stand for 5 minutes before being added to the above reaction system. The system was maintained under an argon atmosphere and stirred at high speed at room temperature for 7 days. After completion of the reaction, the reaction system was filtered to retain the residue (CPG glass beads). The CPG glass beads were then rinsed with acetonitrile 5-7 times under suction, the liquid was drained, and then blown dry with nitrogen.

[0021] Wherein: the sequence of the N3-DNA chain is shown in SEQ ID No.1:

[0022] 5'-N3-TTTTTTTTTTTTTTTTTTTTGGGTGGTGGTGGTTGTGGTGGTGGTGGT-3'.

[0023] (3) Purification of PAMAM-SNA

[0024] Place the CPG glass beads obtained in step (2) into a brown sample bottle, add 1-5 mL of ammonia water, seal the bottle, and allow to stand at room temperature for 24 hours. Excess ammonia is then blown away with nitrogen. The mixture of unreacted DNA and fragments removed and the SNA target product is separated and purified using RP-HPLC to obtain pure SNA. The solution is then lyophilized to obtain SNA lyophilized powder, which can be stored for long periods (>2 years) at -20°C.

[0025] The biomimetic metalloenzyme prepared in this application has the following general structural formula:

[0026]

[0027] Where:

[0028] R is a complex of TBTA, and its structural formula is: For the N3-DNA strand: 5'-N3-TTTTTTTTTTTTTTTTTTGGGTGGTGGTGGTTGTGGTGGTGGTGGT-3'.

[0029] Application of the above-mentioned biomimetic metalloenzyme in the preparation of intracellular catalysts.

[0030] Application of the above-mentioned biomimetic metalloenzyme in preparing reagents for catalyzing click reactions.

[0031] The present invention has the following beneficial effects:

[0032] 1. The present application prepares a nucleic acid-polyamide polymer catalyst with a core-shell structure that is targeted, highly biosafe, and low immunogenic. In this system, the catalytic active center is protected by a dense outer nucleic acid shell. At the same time, the molecular structure and hydrophobic properties of the hyperbranched polymer polyamide are used to create a highly "pure" catalytic environment inside, avoiding the dilemma of biomimetic enzyme inactivation in complex systems and the hidden danger of metal ion leakage in the body. By utilizing the three-dimensional structure of the material, it can enter cells autonomously through recognition, avoiding the use of highly toxic cationic transfection agents. Some of the more major problems currently faced by biomimetic metalloenzymes are solved.

[0033] 2. The biomimetic metal enzyme of the present application has a three-dimensional structure of the multivalent nucleic acid conjugate, which can wrap the catalytic active center well inside the material to ensure its catalytic activity in a complex environment; the multivalent nucleic acid of a specific sequence is modified in the outer layer of the material as a dense shell to form a spherical nucleic acid nanostructure with a special three-dimensional structure. Both polyamide and nucleic acid materials have high biocompatibility. At the same time, because the outer layer is modified with a dense negatively charged nucleic acid outer layer, the protective effect of the invented material on the copper ions in the inner core is very significant. While maintaining its good catalytic activity in the body, it also solves the risk of metal ion leakage. By utilizing the recognition effect of the outer nucleic acid, it can enter the target cells efficiently and highly targeted, and the outer nucleic acid sequence can be customized. By simply replacing the sequence of the nucleic acid chain (J.Am.Chem.Soc.2020,142,31,13350-13355. or Adv.Sci.2024,11,2308924.), it is possible to achieve the purpose of targeting different cells and delivering enzyme-mimicking catalysts to them. The method is simple and reliable.

[0034] 3. This application utilizes a core-shell structure to "bind" internal metal ions, significantly improving the biosafety of the material. The single-stranded nucleic acid, the primary component of the material, is also highly biocompatible. Therefore, this polyvalent nucleic acid conjugate with a polyamide derivative as its core is capable of efficiently catalyzing click reactions in the complex biochemical environment of the body, representing a promising intracellular biomimetic metalloenzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 MALDI-ToF mass spectrum of the 16-arm polyamide alkyne derivative core.

[0037] Figure 2 HPLC curves and agarose gel images of PAMAM-SNA and DNA; the left picture is the HPLC curve of PAMAM-SNA (SNA) (red) and DNA raw material (black), and the right picture is the agarose gel image of the two.

[0038] Figure 3 This is the MALDI-ToF mass spectrum of PAMAM-SNA (SNA).

[0039] Figure 4 Schematic diagram of the PAMAM-SNA (SNA) structure (in order to clearly show the inner core, the size ratio of the DNA single chain to the inner core is not the actual ratio).

[0040] Figure 5 AFM characterization of PAMAM-SNA (SNA).

[0041] Figure 6 DLS characterization of PAMAM-SNA (SNA) in 0.5 M PBS.

[0042] Figure 7 Cell uptake experiment of Cy3-modified PAMAM-SNA using MCF7 cells; scale bar, 20 μm.

[0043] Figure 8 The intracellular biocompatibility of PAMAM-SNA catalyst (cell line is HL7702 cells).

[0044] Figure 9Toxicity test of PAMAM-SNA in living zebrafish.

[0045] Figure 10 Nuclease stability test of DNA and PAMAM-SNA.

[0046] Figure 11 This is the catalytic experiment of TBTA and PAMAM-SNA in PBS.

[0047] Figure 12 Catalytic experiments were performed in DMEM medium containing FBS.

[0048] Figure 13 This is the reaction mechanism of PAMAM-SNA and sodium ascorbate.

[0049] Figure 14 This is the intracellular catalytic experiment of PAMAM-SNA (the cell line is MCF-7). DETAILED DESCRIPTION

[0050] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0052] The sequence of the nucleic acid chain N3-DNA chain used in this application is shown in SEQ ID No. 1 (provided by Sangon Biotech (Shanghai) Co., Ltd.):

[0053] 5'-N3-TTTTTTTTTTTTTTTTTTTTGGGTGGTGGTGGTTGTGGTGGTGGTGGT-3'.

[0054] Example 1

[0055] A method for preparing a biomimetic metalloenzyme with a polyamide derivative as a core comprises the following steps:

[0056] (1) Synthesis of Alkyl-PAMAM Core

[0057]

[0058] Using propiolic acid as the raw material, hydroxysuccinimide is used to activate the carboxyl group in the solvent of dichloromethane or DMF under the catalysis of triethylamine and dicyclohexylcarbodiimide to obtain the activated ester of succinimide (compound 1), wherein the equivalent ratio of propiolic acid, hydroxysuccinimide, dicyclohexylcarbodiimide and triethylamine is 1:1:1.5:1.5; then, an excess of compound 1 (100 equiv.) is used to react with PAMAM-NH2 (1.0 equiv.) with a third-generation outer layer containing 16 amino arms in an ultra-dry methanol system under the catalysis of triethylamine for 3 to 7 days to obtain a terminal alkyne-modified hyperbranched polyamide derivative Alkyl-PAMAM (compound 2) as the core molecule.

[0059] The CAS number of PAMAM-NH2 is 153891-46-4.

[0060] The structure of Alkyl-PAMAM is:

[0061]

[0062] (2) Synthesis of PAMAM-SNA

[0063]

[0064] 19.8-90.2 mg of compound 2 (5 μmol, 1.0 equiv.) was dissolved in 5-10 mL of DMF and added to a 25 mL round-bottom flask equipped with a magnetic rod. 108.8-456.2 mg (31 μmol, 6.2 equiv.) of azido-tris (triazole, azido-TTA) was dissolved in 2-5 mL of DMF and added to the above system after complete dissolution, and Ar gas was passed through. 5.85 mg (32.5 μmol, 6.5 equiv.) of anhydrous CuSO4 and 17.0 mg (32.5 μmol, 6.5 equiv.) of TBTA ligand (CasNo.: 510758-28-8) were mixed and dissolved in 1-5 mL of DMF. After mixing, the mixture was allowed to stand for 5 minutes, and then the solution was transferred to the flask system protected by Ar atmosphere and stirred at room temperature at a medium speed. The reaction was stirred for two days. After two days, CPG glass beads loaded with N3-DNA chains (77.5 μmol, 15.5 equiv) were added to the system. 10.4 mg (57.5 μmol, 11.5 equiv) of anhydrous CuSO4 and 30.5 mg (11.5 equiv) of TBTA ligand were mixed and dissolved in 2 mL of DMF. After mixing, the mixture was allowed to stand for 5 minutes before being added to the reaction system. The system was maintained under an argon atmosphere and stirred at high speed at room temperature for 7 days. After completion of the reaction, the reaction system was filtered to retain the residue (CPG glass beads). The CPG glass beads were then rinsed with acetonitrile 5-7 times under suction, the liquid was drained, and then blown dry with nitrogen.

[0065] (3) Purification of PAMAM-SNA

[0066] The CPG glass beads obtained in step (2) were placed in a brown sample bottle, 1 mL of ammonia water was added, the bottle was sealed, and the bottle was left at room temperature for 24 hours. Excess ammonia was then blown away with nitrogen gas. The excised DNA and PAMAM-SNA mixture were separated and purified by HPLC to obtain pure PAMAM-SNA. The solution was then lyophilized to obtain PAMAM-SNA lyophilized powder (abbreviated as SNA), which can be stored for long periods of time (>2 years) at -20°C.

[0067] Maldi-ToF mass spectrometry was performed on the Alkyl-PAMAM core and PAMAM-SNA prepared in this example:

[0068] The chemical structure and time-of-flight mass spectrometry characterization of the Alkyl-PAMAM core are as follows Figure 1 As shown: According to the software, the calculated value of the core molecular weight is 4310.4585, and the measured value is 4333.4483, which is the mass of one molecule of sodium ion. The tested mass is highly consistent with the predicted mass, proving the successful synthesis of the core material polyamide alkyne derivative.

[0069] The theoretical calculated molecular weight of PAMAM-SNA is 157215.8947Da (~157.22kDa), and the measured value is close to the theoretical value. At the same time, since the material is a multivalent nucleic acid conjugate, it can carry multiple charges. The mass-to-charge ratios of the material with 2 and 3 charges can also be observed in the figure, which are approximately ~78.61kDa and ~52.44kDa, respectively. The test results are highly consistent with the theoretical calculated values. It is speculated that the structural schematic diagram of the PAMAM-SNA material in the present invention is as follows Figure 4 shown.

[0070] HPLC and agarose gel characterization of the N3-DNA chain raw material and PAMAM-SNA (SNA) used in this example:

[0071] like Figure 2 The left figure shows the HPLC curves of the raw material N3-DNA (black curve) and the product SNA (red curve). Since SNA is a polyvalent nucleic acid, its hydrophilicity is enhanced, resulting in a shortened retention time on HPLC. The right figure shows the agarose gel electrophoresis analysis of the two materials. The gel electrophoresis experiment shows that due to the larger size of SNA, its migration rate on the gel is slower than that of the raw DNA, resulting in a closer distance to the upper gel well.

[0072] Dry particle size of PAMAM-SNA (characterized by AFM)

[0073] Through AFM test, such as Figure 5 As shown: it can be seen that the flat size of the material in the dry state is about 30-40nm (due to the collapse of nucleic acids in the vertical direction, the flat particle size of the material is larger to a certain extent); in the vertical direction, since the outer layer of DNA is a flexible chain, it will collapse to a large extent in the dry state, so the dry height of SNA is measured to be 1.5-2.5nm, which is basically the particle size of the inner core.

[0074] Dynamic hydration particle size of PAMAM-SNA in aqueous solution (DLS characterization)

[0075] The dynamic hydration particle size test (DLS) experiment of SNA in 0.5M PBS was carried out. Figure 6 As shown in the figure, the dynamic hydration particle size of SNA in a higher concentration salt solution is 60-80nm, and the distribution width is narrow, indicating that the SNA material is still well dispersed in the high concentration salt solution, basically in a dispersed state, and the size is relatively uniform.

[0076] Furthermore, by adjusting the raw materials and reaction conditions in this example, it was found that the same product as in Example 1 could still be prepared. The adjusted data are shown in Examples 2-5:

[0077] Example 2

[0078] A method for preparing a biomimetic metalloenzyme with a polyamide derivative as a core comprises the following steps:

[0079] (1) Synthesis of Alkyl-PAMAM Core

[0080]

[0081] Using propiolic acid as the raw material, the carboxyl group is activated using hydroxysuccinimide in the solvent of dichloromethane or DMF under the catalysis of triethylamine and dicyclohexylcarbodiimide to obtain the activated ester of succinimide (compound 1), wherein the equivalent ratio of propiolic acid, hydroxysuccinimide, dicyclohexylcarbodiimide and triethylamine is 1:1:1.5:1.5; then, an excess of compound 1 (100 equiv.) and PAMAM-NH2 (1.0 equiv.) with a third-generation outer layer containing 16 amino arms are reacted in an ultra-dry ethanol system under the catalysis of triethylamine for 7 days to obtain a terminal alkyne-modified hyperbranched polyamide derivative Alkyl-PAMAM (compound 2) as the core molecule.

[0082] The CAS number of PAMAM-NH2 is 153891-46-4.

[0083] The structure of Alkyl-PAMAM is shown in Example 1.

[0084] (2) Synthesis of PAMAM-SNA

[0085]

[0086] Dissolve compound 2 (21 μmol, 1.0 equiv.) in 10 mL of DMF and add it to a 25 mL round-bottom flask equipped with a magnetic separator. Dissolve the azido-triazole ligand (130.2 μmol, 6.2 equiv.) in 5 mL of DMF. Once dissolved, add the above system and flow through with Ar gas. Weigh anhydrous CuSO4 (136.5 μmol, 6.5 equiv.) and TBTA ligand (Cas). No.: 510758-28-8) were mixed and dissolved in 5 mL of DMF. After mixing, the mixture was allowed to stand for 5 minutes. The solution was then transferred to a flask under an Ar atmosphere and stirred at moderate speed at room temperature for two days. After two days, CPG glass beads loaded with N3-DNA chains (325.5 μmol, 15.5 equiv. of DNA) were added to the system. Anhydrous CuSO4 (241.5 μmol, 11.5 equiv.) and TBTA ligand (11.5 equiv.) were mixed and dissolved in 10 mL of DMF. After mixing, the mixture was allowed to stand for 5 minutes before being transferred to the above reaction system. The system was maintained under an Ar atmosphere and stirred at high speed at room temperature for 7 days. After completion of the reaction, the reaction system was filtered to retain the residue (CPG glass beads). The CPG glass beads were rinsed 5-7 times with acetonitrile under suction, the liquid was drained, and then blown dry with nitrogen.

[0087] Wherein: the sequence of the N3-DNA chain is shown as SEQ ID No.1.

[0088] (3) Purification of PAMAM-SNA

[0089] The CPG glass beads obtained in step (2) were placed in a brown sample bottle, 5 mL of ammonia water was added, the bottle was sealed, and the bottle was left at room temperature for 24 hours. Excess ammonia was then blown away with nitrogen gas. The excised DNA and PAMAM-SNA mixture were separated and purified by HPLC to obtain pure PAMAM-SNA. The solution was then lyophilized to obtain PAMAM-SNA lyophilized powder, which can be stored for long periods of time (>2 years) at -20°C.

[0090] Example 3

[0091] A method for preparing a biomimetic metalloenzyme with a polyamide derivative as a core comprises the following steps:

[0092] (1) Synthesis of Alkyl-PAMAM Core

[0093]

[0094] Using propiolic acid as the raw material, hydroxysuccinimide is used to activate the carboxyl group in the solvent of dichloromethane or DMF under the catalysis of triethylamine and dicyclohexylcarbodiimide to obtain the activated ester of succinimide (compound 1); the equivalent ratio of propiolic acid, hydroxysuccinimide, dicyclohexylcarbodiimide and triethylamine is 1:1:1.5:1.5; then, an excess of compound 1 (100 equiv.) and PAMAM-NH2 (1.0 equiv.) with a third-generation outer layer containing 16 amino arms are reacted in an ultra-dry methanol system or an ultra-dry ethanol system for 3 to 7 days under the catalysis of triethylamine to obtain a terminal alkyne-modified hyperbranched polyamide derivative Alkyl-PAMAM (compound 2) as the core molecule.

[0095] The CAS number of PAMAM-NH2 is 153891-46-4.

[0096] The structure of Alkyl-PAMAM is shown in Example 1.

[0097] (2) Synthesis of PAMAM-SNA

[0098]

[0099] Dissolve compound 2 (10 μmol, 1.0 equiv.) in 5-10 mL of DMF and add it to a 25 mL round-bottom flask equipped with a magnetic separator. Dissolve azido-tris (triazole, azido-TTA) (62 μmol, 6.2 equiv.) in 3 mL of DMF. After complete dissolution, add it to the above system and pass Ar gas. Weigh anhydrous CuSO4 (65 μmol, 6.5 equiv.) and TBTA (Cas). No.: 510758-28-8) were mixed and dissolved in 3 mL of DMF. After mixing, the mixture was allowed to stand for 5 minutes. The solution was then transferred to a flask under an Ar atmosphere and stirred at moderate speed at room temperature for two days. After two days, CPG glass beads loaded with N3-DNA chains (155 μmol, 15.5 equiv. of DNA) were added to the system. Anhydrous CuSO4 (115 μmol, 11.5 equiv.) and TBTA ligand (115 μmol, 11.5 equiv.) were mixed and dissolved in 4 mL of DMF. After mixing, the mixture was allowed to stand for 5 minutes before being transferred to the above reaction system. The system was maintained under an Ar atmosphere and stirred at high speed at room temperature for 7 days. After completion of the reaction, the reaction system was filtered to retain the residue (CPG glass beads). The CPG glass beads were rinsed 5-7 times with acetonitrile under vacuum, the liquid was drained, and then blown dry with nitrogen.

[0100] Wherein: the sequence of the N3-DNA chain is shown as SEQ ID No.1.

[0101] (3) Purification of PAMAM-SNA

[0102] The CPG glass beads obtained in step (2) were placed in a brown sample bottle, 3 mL of ammonia water was added, the bottle was sealed, and the bottle was left at room temperature for 24 hours. Excess ammonia was then blown away with nitrogen gas. The excised DNA and PAMAM-SNA mixture was separated and purified by HPLC to obtain pure PAMAM-SNA. The solution was then lyophilized to obtain PAMAM-SNA lyophilized powder, which can be stored for long periods of time (>2 years) at -20°C.

[0103] Example 4

[0104] A method for preparing a biomimetic metalloenzyme with a polyamide derivative as a core comprises the following steps:

[0105] (1) Synthesis of Alkyl-PAMAM Core

[0106]

[0107] Using propiolic acid as the raw material, the carboxyl group is activated using hydroxysuccinimide in the solvent of dichloromethane or DMF under the catalysis of triethylamine and dicyclohexylcarbodiimide to obtain the activated ester of succinimide (compound 1), and the equivalent ratio of propiolic acid, hydroxysuccinimide, dicyclohexylcarbodiimide and triethylamine is 1:1:1.5:1.5; then, an excess of compound 1 (100 equiv.) and PAMAM-NH2 (1.0 equiv.) with a third-generation outer layer containing 16 amino arms are reacted in an ultra-dry methanol system or an ultra-dry ethanol system for 7 days under the catalysis of triethylamine to obtain a terminal alkyne-modified hyperbranched polyamide derivative Alkyl-PAMAM (compound 2) as the core molecule.

[0108] The CAS number of PAMAM-NH2 is 153891-46-4.

[0109] The structure of Alkyl-PAMAM is shown in Example 1.

[0110] (2) Synthesis of PAMAM-SNA

[0111]

[0112] Compound 2 (15 μmol, 1.0 equiv.) was weighed and dissolved in 8 mL of DMF and added to a 25 mL round-bottom flask equipped with a magnetic separator. Azido-tris (triazole, azido-TTA) (93 μmol, 6.2 equiv.) was dissolved in 4 mL of DMF and added to the above system after complete dissolution. Ar gas was then introduced. Anhydrous CuSO4 (97.5 μmol, 6.5 equiv.) and TBTA (Cas) (97.5 μmol, 6.5 equiv.) were weighed and added. No.: 510758-28-8) were mixed and dissolved in 4 mL of DMF. After mixing, the mixture was allowed to stand for 5 minutes. The solution was then transferred to a flask under an Ar atmosphere and stirred at moderate speed at room temperature for two days. Two days later, CPG glass beads loaded with N3-DNA chains (232.5 μmol, 15.5 equiv. of DNA) were added to the system. Anhydrous CuSO4 (172.5 μmol, 11.5 equiv.) and TBTA ligand (172.5 μmol, 11.5 equiv.) were mixed and dissolved in 8 mL of DMF. After mixing, the mixture was allowed to stand for 5 minutes before being transferred to the above reaction system. The system was maintained under an Ar atmosphere and stirred at high speed at room temperature for 7 days. After completion of the reaction, the reaction system was filtered to retain the residue (CPG glass beads). The CPG glass beads were then rinsed with acetonitrile six times under suction, the liquid was drained, and then blown dry with nitrogen.

[0113] Wherein: the sequence of the N3-DNA chain is shown as SEQ ID No.1.

[0114] (3) Purification of PAMAM-SNA

[0115] Place the CPG glass beads obtained in step (2) into a brown sample bottle, add 1-5 mL of ammonia water, seal the bottle, and leave it at room temperature for 24 hours. Then, blow off the excess ammonia with nitrogen gas. The excised DNA and PAMAM-SNA mixture are separated and purified by HPLC to obtain pure PAMAM-SNA. The solution is then lyophilized to obtain PAMAM-SNA lyophilized powder, which can be stored for long periods of time (>2 years) at -20°C.

[0116] Example 5

[0117] A method for preparing a biomimetic metalloenzyme with a polyamide derivative as a core comprises the following steps:

[0118] (1) Synthesis of Alkyl-PAMAM Core

[0119]

[0120] Using propiolic acid as the raw material, the carboxyl group is activated using hydroxysuccinimide in the solvent of dichloromethane or DMF under the catalysis of triethylamine and dicyclohexylcarbodiimide to obtain the activated ester of succinimide (compound 1), and the equivalent ratio of propiolic acid, hydroxysuccinimide, dicyclohexylcarbodiimide and triethylamine is 1:1:1.5:1.5; then, an excess of compound 1 (100 equiv.) and PAMAM-NH2 (1.0 equiv.) with a third-generation outer layer containing 16 amino arms are reacted in an ultra-dry methanol system or an ultra-dry ethanol system for 6 days under the catalysis of triethylamine to obtain a terminal alkyne-modified hyperbranched polyamide derivative Alkyl-PAMAM (compound 2) as the core molecule.

[0121] The CAS number of PAMAM-NH2 is 153891-46-4.

[0122] The structure of Alkyl-PAMAM is shown in Example 1.

[0123] (2) Synthesis of PAMAM-SNA

[0124]

[0125] 19.8-90.2 mg of compound 2 (8 μmol, 1.0 equiv.) was weighed and dissolved in 6 mL of DMF and added to a 25 mL round-bottom flask equipped with a magnetic rod. 49.6 μmol, 6.2 equiv. of azido-tris (triazole, azido-TTA) was dissolved in 3 mL of DMF and added to the above system after complete dissolution, and Ar gas was passed through. 52 μmol, 6.5 equiv. of anhydrous CuSO4 and 52 μmol, 6.5 equiv. of TBTA ligand (Cas) were weighed and added. No.: 510758-28-8) were dissolved in 2 mL of DMF, allowed to stand for 5 minutes, and then the solution was transferred to a flask under an Ar atmosphere and stirred at moderate speed at room temperature for two days. Two days later, CPG glass beads loaded with N3-DNA chains (124 μmol, 15.5 equiv. of DNA) were added to the system. Anhydrous CuSO4 (92 μmol, 11.5 equiv.) and TBTA ligand (92 μmol, 11.5 equiv.) were dissolved in 2-10 mL of DMF, mixed, and allowed to stand for 5 minutes before being transferred to the above reaction system. The system was maintained under an Ar atmosphere and stirred at high speed at room temperature for 7 days. After completion of the reaction, the reaction system was filtered to retain the residue (CPG glass beads). The CPG glass beads were rinsed six times with acetonitrile under vacuum, the liquid was drained, and then blown dry with nitrogen.

[0126] Wherein: the sequence of the N3-DNA chain is shown as SEQ ID No.1.

[0127] (3) Purification of PAMAM-SNA

[0128] The CPG glass beads obtained in step (2) were placed in a brown sample bottle, and 4 mL of ammonia water was added, followed by sealing the bottle with a cap and leaving it at room temperature for 24 hours. Excess ammonia was then blown away with nitrogen gas, and the excised DNA and PAMAM-SNA mixture was separated and purified by HPLC to obtain pure PAMAM-SNA. The solution was then lyophilized to obtain PAMAM-SNA lyophilized powder, which can be stored for long periods (>2 years) at -20°C.

[0129] Implementation effect examples

[0130] 1. Cellular uptake experiment of PAMAM-SNA (characterization by confocal microscopy)

[0131] Cy3-PAMAM-SNA was prepared using Cy3-labeled nucleic acid chains for cell uptake experiments. The N3-DNA-Cy3 used here is:

[0132] 5'-N3-TTTTTTTTTTTTTTTTTTTTGGGTGGTGGTGGTTGTGGTGGTGGTGGT-Cy3-3';

[0133] The preparation steps of Cy3-PAMAM-SNA are as follows: Compound 2 (15 μmol, 1.0 equiv.) was weighed and dissolved in 8 mL of DMF and added to a 25 mL round-bottom flask equipped with a magnetic rod. Azido-tris (triazole, azido-TTA) (93 μmol, 6.2 equiv.) was dissolved in 4 mL of DMF and added to the above system after complete dissolution, and Ar gas was passed through. Anhydrous CuSO4 (97.5 μmol, 6.5 equiv.) and TBTA ligand (Cas No.: 510758-28-8) (97.5 μmol, 6.5 equiv.) were weighed and dissolved in 4 mL of DMF. After mixing, the mixture was allowed to stand for 5 minutes, and then the solution was poured into the flask system protected by Ar atmosphere and stirred at moderate speed at room temperature for two days. After two days, N3 loaded was added to the system. CPG glass beads loaded with a DNA-Cy3 chain (DNA loading: 232.5 μmol, 15.5 equiv). Anhydrous CuSO₄ (172.5 μmol, 11.5 equiv) and TBTA ligand (172.5 μmol, 11.5 equiv) were mixed and dissolved in 8 mL of DMF. After mixing, the mixture was allowed to stand for 5 minutes before being added to the above reaction system. The system was maintained under an argon atmosphere and stirred at high speed at room temperature for 7 days. After completion of the reaction, the reaction system was filtered to retain the residue (CPG glass beads). The CPG glass beads were then rinsed six times with acetonitrile under vacuum, the liquid was drained, and then blown dry with nitrogen to obtain Cy3-PAMAM-SNA.

[0134] The prepared Cy3-PAMAM-SNA was subjected to cell uptake experiments. According to the experimental results Figure 7 It can be seen that Cy3-PAMAM-SNA has a very high cell uptake efficiency and can efficiently enter cells without the assistance of a transfection agent.

[0135] 2. Biocompatibility testing of PAMAM-SNA

[0136] The test steps refer to Chem.Sci., 2021, 12, 15843-15848. The cytotoxicity of PAMAM-SNA was evaluated using the classic MTT cytotoxicity assay. RAW264.7 cells were treated with DMEM containing PAMAM-SNA in a range of 0.625 to 5 μM (final concentration) and further incubated for 24 hours. Cells without SNA were used as controls. After incubation, the culture medium was removed and the cells were washed with PBS, and then the cell culture medium was replaced with 120 μL of fresh MTT solution (0.5 mg / mL in DMEM) and incubated for another 1.5 hours. The culture medium was replaced with DMSO (100 μL) and the absorbance of the solution at 595 nm was measured with a microplate reader, and the cell viability was calculated by comparison.

[0137] According to the biocompatibility test results Figure 8 It can be seen that the biocompatibility of PAMAM-SNA is very excellent. Even when the concentration is 10 times higher than the working concentration (0.5 μM), the survival rate of normal somatic cells still reaches more than 95%, indicating that the material has high biosafety.

[0138] 3. Toxicity testing of PAMAM-SNA in living zebrafish

[0139] The test experiment refers to Chem.Sci., 2021, 12, 15843-15848. The injection concentrations of 20nM PAMAM-SNA+120nM copper sulfate, 120nM TBTA catalyst+120nM copper sulfate, 120μM azidotriazole ligand+120nM copper sulfate, and 120nM copper sulfate were respectively calculated based on the CuSO4 concentration. The four groups of solutions were injected into the zebrafish through the abdominal cavity with an injection volume of 2μL / fish, and the survival of the zebrafish was observed.

[0140] The results are as follows Figure 9 As shown in the figure, the survival curve shows that the PAMAM-SNA catalyst has a better survival curve than the traditional TBTA ligand chelated copper catalyst or the copper ion catalyst without ligand, indicating that it is safer for living organisms.

[0141] 4. PAMAM-SNA and anti-enzymatic stability test

[0142] The assay procedure involved enzymatic digestion of SNA material and ssDNA using the manufacturer's recommended conditions. Digestion progress was monitored by reversed-phase HPLC using rhodamine dye as an internal standard. The extent of digestion was calculated using normalized integral values ​​for the corresponding material at different time points. The HPLC conditions used were: 0.05 M TEAA / ACN, volume ratio from 0.95 / 0.05 to 0.05 / 0.95 over 50 minutes, at a flow rate of 1 mL / min.

[0143] Through stability test data Figure 10 It can be seen that PAMAM-SNA material has higher stability and resistance to enzyme cleavage than free DNA.

[0144] 5. In vitro catalytic experiments of PAMAM-SNA

[0145] The test steps refer to Chem. Sci., 2021, 12, 15843-15848. The specific steps are as follows: (1) PBS buffer system: 5 μL of a catalyst solution containing 1 μM PAMAM-SNA and 5 μM CuSO4, and 5 μL of a catalytic solution containing 5 μM TBTA catalyst and 5 μM CuSO4, respectively, were added to 500 μL of a 1× PBS solution containing two catalytic substrates, namely 20 μM 3-hydroxy-7-azidocoumarin and 20 μM p-methylphenylacetylene, based on the CuSO4 concentration. The fluorescence change of the system was measured by a fluorescence spectrophotometer to obtain the catalytic yield. (2) Serum-culture medium system: 5 μL of catalyst solution I containing 0.5 μM PAMAM-SNA and 3 μM CuSO4 and 5 μL of catalyst solution II containing 20 μM TBTA and 20 μM CuSO4 were prepared. The two solutions were added to 500 μL of DMEM culture medium containing two catalytic substrates, namely 20 μM 3-hydroxy-7-azidocoumarin and 20 μM p-methylphenylacetylene, and the culture medium contained 8% fetal bovine serum. The fluorescence changes of the system were measured by a fluorescence spectrophotometer to obtain the catalytic yield.

[0146] The PAMAM-SNA catalyst prepared by the present invention was used to conduct in vitro catalytic experiments to preliminarily verify the catalytic performance of the material. Figure 11 and Figure 12 As shown: Compared with the commercial catalyst TBTA, in the presence of PBS, it can be seen that TBTA and PAMAM-SNA both have catalytic activity ( Figure 11 When the environment is more complex (i.e., in a culture medium with fetal bovine serum), the catalytic performance of the commercially available catalyst is greatly affected, while the PAMAM-SNA catalyst of the present invention can still exhibit high catalytic performance ( Figure 12 ).

[0147] The reaction catalyzed by the present invention is a click reaction between 3-hydroxy-7-azidocoumarin and p-methylphenylacetylene. The coumarin derivative after the reaction is fluorescent. Cu(I) is a highly efficient ligand catalyst obtained by reacting PAMAM-SNA, the precursor of the intracellular artificial metalloenzyme prepared by the present invention, with sodium ascorbate. The reaction mechanism is as follows: Figure 13 shown.

[0148] VI. Intracellular catalytic experiments of PAMAM-SNA (characterization by confocal microscopy)

[0149] The specific experimental steps were to incubate 300,000 RAW 264.7 cells for 16 hours to attach to the plate, and then culture the cells with three mixed catalyst solutions of TBTA 20μM + CuSO4 20μM, PAMAM-SNA 0.5μM + CuSO4 3μM, and PAMAM-SNA 1μM + CuSO4 6μM in an incubator for 6 hours, then add two substrates (50μM) and sodium L-ascorbate (1mM) in new DMEM solution and culture for 2 hours. After washing with PBS three times, 500μL of PBS was added to the cells and observed using a confocal microscope.

[0150] Results from intracellular catalysis experiments Figure 14 It can be seen that compared with the commercially available catalyst TBTA, the PAMAM-SNA of the present invention can effectively achieve the catalysis of the substrate in the target cells (the catalytic product has green fluorescence), and can still maintain catalytic activity at a lower working concentration.

[0151] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a biomimetic metalloenzyme with a polyamide derivative as a core, characterized in that: The steps are: (1) Propiolic acid, hydroxysuccinimide, dicyclohexylcarbodiimide and triethylamine are activated in an organic solvent I to obtain an activated ester of succinimide, namely compound 1; compound 1, PAMAM-NH2 and triethylamine are further reacted in an organic solvent II to obtain compound 2, namely Alkyl-PAMAM; (2) Alkyl-PAMAM and azidotriazole ligands from step (1) were dissolved in DMF, and anhydrous CuSO4 and TBTA ligand DMF solution was added in an inert gas atmosphere. After stirring at room temperature, CPG glass beads loaded with N3-DNA chains were added, and the reaction was continued with high-speed stirring in an inert gas atmosphere. After the reaction was completed, the CPG glass beads were collected by filtration, acetonitrile elution, and nitrogen drying; (3) The CPG glass beads treated in step (2) were placed in a brown bottle, and after adding ammonia water, the bottle was sealed and placed. Then, the excess ammonia was blown away with nitrogen gas, and the liquid in the brown bottle was collected and separated and purified by HPLC to obtain a biomimetic metalloenzyme containing a polyamide derivative as the core, namely PAMAM-SNA; The structural formula of the TBTA ligand is: ； The sequence of the N3-DNA chain in step (2) is shown in SEQ ID No. 1, and an N3- group is connected to its 5' end.

2. The method for preparing a biomimetic metalloenzyme with a polyamide derivative as a core according to claim 1, characterized in that: In the step (1), the equivalent ratio of propiolic acid, hydroxysuccinimide, dicyclohexylcarbodiimide and triethylamine is 1:1:1.5:1.5; the equivalent ratio of compound 1, PAMAM-NH2 and triethylamine is 80-120:1:20; the organic solvent I is dichloromethane or DMF; and the organic solvent II is ultra-dry methanol or ultra-dry ethanol.

3. The method for preparing a biomimetic metalloenzyme with a polyamide derivative as a core according to claim 2, characterized in that: The activation reaction temperature is 0-25° C., the time is 4-8 hours, and the reaction time is 3-7 days.

4. The method for preparing a biomimetic metalloenzyme with a polyamide derivative as a core according to claim 3, characterized in that: The stirring time at room temperature is 2-3 days, and the high-speed stirring reaction time is 5-7 days.

5. The method for preparing a biomimetic metalloenzyme with a polyamide derivative as a core according to claim 4, characterized in that: The sealing time in step (3) is 24 hours.

6. A biomimetic metalloenzyme prepared by the method according to any one of claims 1 to 5, having the following general structural formula: Where: R is a complex of TBTA, and its structural formula is: ; It is an N3-DNA chain, the nucleotide sequence of which is shown in SEQ ID No. 1, and N3 is connected to its 5' end.

7. Use of the biomimetic metalloenzyme according to claim 6 in preparing intracellular catalysts.

8. Use of the biomimetic metalloenzyme according to claim 7 in preparing a reagent for catalyzing click reactions.

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