Method for catalyzing demethylation of N6-methyladenosine

Abstract

The invention relates to preparation of a novel hierarchical pore metal organic framework catalyst loaded with a natural enzyme and application of the catalyst in catalysis of demethylation of N6-methyladenosine. Based on an active oxygen oxidation N6-methyladenosine demethylation mechanism, glucose oxidase and an iron-based metal organic framework are combined to prepare the novel glucose oxidase loaded iron-based metal organic framework catalyst. The iron-based metal organic framework is used for catalyzing glucose oxidase to oxidize hydrogen peroxide generated in situ on glucose to generate active oxygen, and efficient N6-methyladenosine demethylation conversion is achieved. Wherein the iron-based metal organic framework is of a hierarchical pore structure and is constructed through mediation of a surfactant. The hierarchical pore structure overcomes the defect that micropores of the traditional metal organic framework are incompatible with enzyme molecules in size, not only improves the loading capacity of the enzyme, but also protects the enzyme from being inactivated. The novel catalyst-mediated N6-methyladenosine demethylation reaction provided by the invention has the advantages of mild reaction conditions, simple experimental operation steps, avoidance of the use of a high-toxicity oxidant, good biocompatibility, hopeful promotion to RNA chains and even in vivo, and provision of support for epigenetics research of complete transcription gene sequences.

Description

Technical Field

[0001] The present invention relates to the field of nanocomposite material technology and organic chemistry, and in particular to the preparation of a novel multi-level porous metal organic framework catalyst loaded with natural enzymes and the catalytic N 6 -Application of methyladenosine demethylation. Background Art

[0002] Nucleic acids contain a large number of different covalent chemical modifications, and these chemically modified bases play important functions in eukaryotic cells. In particular, nucleic acid methylation and demethylation play a key role in the regulation of gene expression. For example, methylation of gene regulatory regions usually leads to silencing of downstream genes, while removal of methyl groups activates the expression of downstream genes, thereby participating in the corresponding biological processes. 6 -methyladenosine (m 6 A) is the most abundant epigenetic modification in mRNA of plants, animals and viruses, and is involved in regulating mRNA transcription, splicing, nuclear export, translation, stability and other unknown physiological processes, playing a fundamental regulatory role in gene expression. 6 A is formed by the methyltransferase complex WTAP, METTL3 or METTL14, while demethylation occurs under the catalysis of FTO or AlkBH5. 6 A is a dynamic modification, and this reversible methylation phenomenon will seriously affect gene expression. Therefore, exploring the methylation and demethylation of such nucleic acids is an important part of epigenetics, and has strong theoretical significance and practical value for its development.

[0003] Currently, for m 6 Although the catalytic mechanism and function mediated by FTO and AlkBH5 have been widely studied and applied, it is still difficult to regulate this biological process by chemical methods. 6 Biomimetic methods for the chemical demethylation of A are still rare. It has been reported that ammonium bicarbonate-activated hydrogen peroxide can oxidize m 6 A, and through the intermediate N 6 -Hydroxymethyladenosine (hm6A), N 6 -aldehyde adenosine (f 6 A) and N 6 -Peroxymethyladenosine (oxm 6 A) to produce the demethylated product adenosine (A). This discovery breaks the traditional enzyme-catalyzed modification of m 6The limitation of A has promoted the development of chemical methods to regulate nucleic acid modification and gene expression (Chem. Sci. 2015, 6, 3013-3017.). However, this method has a low conversion efficiency (37%) and requires a large excess of hydrogen peroxide (50-100 equivalents). The reaction conditions are relatively harsh, which limits its further application. In view of this, researchers use small molecule photosensitizers (flavin mononucleotide, vitamin B2, 1,4-dicyanobenzene, methylene blue, etc.) to trigger m under ultraviolet, blue or red light. 6 The oxidation of A achieved a relatively efficient demethylation reaction (52% to 79%) (Angew. Chem. Int. Edit. 2019, 131, 5082-5086.). However, this method must provide continuous illumination and be carried out in an oxygen atmosphere. Although it improves the conversion rate and biocompatibility of the reaction to a certain extent, it also increases the complexity of the experimental operation. In addition, the photosensitizer is easy to aggregate during the reaction, which reduces the catalytic activity and is difficult to separate and recover from the reaction system, thus restricting the promotion and application of photocatalysts. Therefore, there is an urgent need to develop a new type of recyclable catalyst to achieve simple and efficient m 6 A chemical transformation to further advance epigenetic demethylation research. Summary of the invention

[0004] The purpose of the present invention is to develop a novel multi-level porous metal organic framework catalyst loaded with glucose oxidase, which is m 6 A demethylation reaction provides a green, convenient and efficient method.

[0005] The technical solution to achieve the purpose of the present invention is: based on the active oxygen oxidation m 6 A demethylation mechanism, glucose oxidase and iron-based metal organic framework were assembled to construct a new chemical-enzyme composite nanocatalyst (HP-MOF / Fe@GOx), which was used to catalyze glucose oxidase to oxidize glucose in situ to generate hydrogen peroxide to produce reactive oxygen species, triggering m 6 A demethylation reaction. The iron-based metal organic framework is a multi-level porous structure.

[0006] The method comprises the following steps:

[0007] (1) Preparation of a hierarchical porous iron-based metal organic framework (HP-MOF / Fe): a surfactant and a ferrous ion salt are dissolved in a suitable solvent to form a surfactant and metal salt solution; 2-aminoterephthalic acid is dissolved in a suitable solvent to form an organic ligand solution; the organic ligand solution is added to the surfactant and metal salt solution to form a mixed solution; the mixed solution is refluxed and heated under nitrogen protection for a period of time, centrifuged, washed, and dried to obtain HP-MOF / Fe.

[0008] (2) Hierarchical porous iron-based metal organic framework loaded with glucose oxidase (HP-MOF / Fe@GOx): Glucose oxidase was dissolved in water to form a glucose oxidase aqueous solution, to which hierarchical porous MOF-Fe was added, ultrasonically dispersed, stirred at room temperature for a period of time, centrifuged, washed, and freeze-dried to obtain HP-MOF / Fe@GOx catalyst, and the enzyme loading was detected.

[0009] (3) HP-MOF / Fe@GOx catalysis 6 A demethylation: In aqueous solution of glucose, m 6 A is used as raw material, and the catalyst HP-MOF / Fe@GOx is added. The reaction is carried out at a certain temperature for a period of time to obtain the demethylated product A, and the catalyst is recovered by centrifugation. The reaction formula is as follows:

[0010]

[0011] The ferrous ion salts in step (1) include ferrous chloride and ferrous acetate, and the molar ratio of the ferrous ion salts to the organic ligand 2-aminoterephthalic acid is 1:1; the surfactants include polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG-400), and cetyltrimethylammonium bromide (CTAB), and the ratio of the ferrous ion salts to the metal ions and the organic ligands is 0 to 300 g / mol; the solvents include N,N-dimethylformamide (DMF), methanol and water, and the ratio of the ferrous ion salts to the metal ions and the organic ligands is 20 L / mol; the reaction temperatures are 100 to 150° C. (the solvent is DMF), 80 to 100° C. (the solvent is water) and 50 to 70° C. (the solvent is methanol); and the reaction time is 2 to 12 h.

[0012] In step (2), the feed ratio of glucose oxidase to HP-MOF / Fe is 0.1-1.1 g / g; the stirring time is 0.5-4 h; and the enzyme loading detection method includes Bradford method and BCA method.

[0013] The catalyst HP-MOF / Fe@GOx and the substrate m in step (3) 6 The feed ratio of A is 0.05-0.5 g / g; the glucose concentration is 3-9 mM; the reaction temperature is 25-50° C.; and the reaction time is 2-6 h.

[0014] The present invention has the following advantages:

[0015] 1) This method constructs a multi-level porous metal organic framework catalyst loaded with glucose oxidase, organically connects the glucose oxidation catalyzed by glucose oxidase and the Fenton reaction mediated by the iron-based metal organic framework, and rapidly converts the hydrogen peroxide generated in situ into reactive oxygen species, achieving efficient m 6 A demethylation reaction. This method has mild reaction conditions, simple experimental steps, and the catalyst can be recycled and reused, avoiding the use of highly toxic oxidants. It has good biocompatibility and is expected to be extended to RNA chains and even living bodies, providing support for epigenetic research on the entire transcribed gene sequence.

[0016] 2) Surfactants as soft templates mediate the preparation of hierarchical porous metal-organic frameworks, which not only has the characteristics of easy reaction conditions and simple post-processing, but also can effectively avoid the network penetration and collapse of the material structure.

[0017] 3) The high specific surface area and porosity of the metal organic framework facilitate the diffusion and transport of reactants, products and reaction intermediates, greatly improving the mass transfer efficiency of the reaction. In addition, the construction of the multi-level pore structure overcomes the shortcomings of the incompatibility between the micropores of the traditional metal organic framework and the size of the enzyme molecules, which increases the enzyme loading capacity on the one hand and protects them from inactivation on the other. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The morphology of the multi-level porous iron-based metal organic framework under a scanning electron microscope;

[0019] Figure 2 The morphology of the multi-level porous iron-based metal organic framework under a transmission electron microscope;

[0020] Figure 3 This is the morphology of a traditional iron-based metal-organic framework under a scanning electron microscope;

[0021] Figure 4 This is the morphology of a traditional iron-based metal-organic framework under a transmission electron microscope;

[0022] Figure 5 The nitrogen adsorption-desorption curves of hierarchical iron-based metal-organic frameworks and traditional iron-based metal-organic frameworks;

[0023] Figure 6 The pore size distribution curves of the hierarchical pore Fe-based metal-organic framework and the traditional Fe-based metal-organic framework;

[0024] Figure 7 N 6 -Methyladenosine demethylation reaction product 1 H-NMR;

[0025] Figure 8 N 6-Methyladenosine demethylation reaction product 13 C-NMR;

[0026] Fig. 9 The catalyst recycling performance. DETAILED DESCRIPTION

[0027] The following examples are provided to help further understand the present invention, but the present invention is not limited thereto.

[0028] Example 1 Preparation of multi-level porous iron-based metal organic framework

[0029] Ferrous chloride tetrahydrate (0.198 g, 1 mmol) and PVP (0.150 g) were dissolved in DMF (20 mL) to form a DMF solution of metal ions and surfactants, and 2-aminoterephthalic acid (0.181 g, 1 mmol) was dissolved in DMF (20 mL) to form a DMF solution of organic ligands, which was then added to the DMF solution of ferric chloride and PVP. After ultrasonic treatment for 5 min, the mixture was refluxed at 120 ° C for 4 h under nitrogen protection, and then naturally cooled to room temperature. The precipitate was collected by centrifugation, washed twice with DMF, washed three times with deionized water, and vacuum dried to obtain 0.092 g of a brown solid. The scanning electron microscope, transmission electron microscope, and nitrogen adsorption and desorption experimental results of the product are shown in Figure 2. Figure 1 , 2 , 5 and 6.

[0030] Example 2 Preparation of traditional iron-based metal organic framework

[0031] Dissolve ferrous chloride tetrahydrate (0.198 g, 1 mmol) in DMF (20 mL) to form a DMF solution of metal ions, then dissolve 2-aminoterephthalic acid (0.181 g, 1 mmol) in DMF (20 mL) to form a DMF solution of organic ligands, then add it to the DMF solution of ferric chloride, sonicate for 5 min, reflux at 120 ° C for 4 h under nitrogen protection, then cool naturally to room temperature, collect the precipitate by centrifugation, wash twice with DMF, wash three times with deionized water, and vacuum dry to obtain 0.122 g of brown solid. The scanning electron microscope of the product and the results of nitrogen adsorption and desorption experiments are shown in Figure 2. Figure 3 , 4 , 5, 6.

[0032] Example 3 Preparation of a multi-level porous iron-based metal organic framework catalyst loaded with glucose oxidase

[0033] 0.05 g of the hierarchical porous iron-based metal-organic framework obtained in Example 1 was ultrasonically dispersed in a glucose oxidase aqueous solution (10 mL, 5 mM), stirred at room temperature for 2 h, centrifuged to collect the precipitate, washed with deionized water until there was no free enzyme in the supernatant, and freeze-dried to obtain a new type of glucose oxidase-loaded hierarchical porous iron-based metal-organic framework catalyst. The enzyme loading rate was measured by the BCA method and was 15.4%.

[0034] Example 4 Catalytic N 6 -Demethylation of methyladenosine

[0035] N 6 -Methyladenosine (0.1mmol, 28.1mg) was dissolved in an aqueous solution of glucose (4mmol, 5mL), and a multi-level porous iron-based metal organic framework catalyst (3mg) loaded with glucose oxidase was added thereto. The reaction was stirred at 37°C for 4h, and the solid catalyst was recovered by centrifugation. The reaction solution was poured out, rotary evaporated, and separated by column chromatography to obtain 21.6mg of demethyladenosine with a yield of 81.1%. 1 H-NMR (DMSO-d6, 500MHz) δ = 8.36 (s, 1H), 8.15 (s, 1H), 7.37 (s, 2H), 5.89 (d, J = 6.2Hz, 1H), 5.47 (d, J = 6.3Hz, 1H), 5.46-5.44 ( m, 1H), 5.21 (d, J = 4.6Hz, 1H), 4.64-4.61 (m, 1H), 4.17-4.15 (m, 1H), 3.99-3.97 (m, 1H), 3.70-3.67 (m, 1H), 3.59-3.55 (m, 1H). 13 C-NMR (DMSO-d6, 126MHz) δ = 156.64, 152.86, 149.52, 140.42, 119.83, 88.38, 86.38, 73.91, 71.15, 62.15.

[0036] Example 5 Catalyst recycling

[0037] The catalyst in Example 4 was recovered by centrifugal separation, washed with deionized water and freeze-dried, and then used to catalyze N 6 -Demethylation reaction of methyladenosine, operating conditions and steps of copper example 4, the results are shown in Figure 7 After 4 cycles, the yield can still reach 52.8%.

Claims

1. A catalytic N 6 - A method for demethylating methyladenosine, characterized in that: Assemble hierarchical iron-based metal-organic frameworks and glucose oxidase to construct a new type of glucose oxidase-loaded hierarchical iron-based metal-organic framework catalyst. Use the hierarchical iron-based metal-organic framework to catalyze glucose oxidase to oxidize glucose in situ to generate hydrogen peroxide to produce reactive oxygen, achieving efficient N 6 -Methyladenosine demethylation conversion.

2. The novel glucose oxidase-loaded multi-level porous iron-based metal organic framework catalyst according to claim 1, characterized in that The catalyst is prepared by the following steps: (1) Preparation of a hierarchical porous iron-based metal organic framework: a surfactant and a ferrous ion salt are dissolved in a suitable solvent to form a surfactant and metal salt solution; 2-aminoterephthalic acid is dissolved in a suitable solvent to form an organic ligand solution; the organic ligand solution is added to the surfactant and metal salt solution to form a mixed solution; the mixed solution is refluxed and heated under nitrogen protection for a period of time to obtain a hierarchical porous iron-based metal organic framework. (2) Hierarchical porous iron-based metal-organic framework loaded with glucose oxidase: Glucose oxidase is dissolved in water to form a glucose oxidase aqueous solution, to which a hierarchical porous iron-based metal-organic framework is added, ultrasonically dispersed, and stirred at room temperature for a period of time to obtain a novel hierarchical porous iron-based metal-organic framework catalyst loaded with glucose oxidase.

3. The novel glucose oxidase-loaded multi-level porous iron-based metal organic framework catalyst according to claim 1 or 2, characterized in that The ferrous ion salt described in step (1) includes ferrous chloride and ferrous acetate, and the molar ratio of the ferrous ion salt to the organic ligand 2-aminoterephthalic acid is 1:1; the surfactant includes polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG-400), and cetyltrimethylammonium bromide (CTAB), and the ratio of the ferrous ion salt to the metal ion and the organic ligand is 0-300 g / mol; the solvent includes N,N-dimethylformamide (DMF), methanol and water, and the ratio of the ferrous ion salt to the metal ion and the organic ligand is 20 L / mol; the reaction temperature is 100-150° C. (the solvent is DMF), 80-100° C. (the solvent is water) and 50-70° C. (the solvent is methanol); and the reaction time is 2-12 h. In step (2), the feed ratio of glucose oxidase to the multi-level porous iron-based metal organic framework is 0.1-1.1 g / g; and the stirring time is 0.5-4 h.

4. The glucose oxidase-supported multi-level porous iron-based metal organic framework catalyst catalyzes N 6 -Methyladenosine demethylation reaction characterized by N 6 -methyladenosine is used as a raw material, glucose oxidase is added to load a hierarchical porous iron-based metal organic framework catalyst, and the reaction is carried out at a certain temperature for a period of time to obtain demethyladenosine.

5. The glucose oxidase-supported multi-level porous iron-based metal organic framework catalyst catalyzes N 6 -Methyladenosine demethylation reaction, characterized by The feed ratio of the catalyst to the substrate is 0.05-0.5 g / g; the glucose concentration is 3-9 mM; the reaction temperature is 25-50° C.; and the reaction time is 2-6 h.

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