CN116396497B - Metal organic framework material, ligand structure thereof and application of metal organic framework material in nano enzyme - Google Patents

Abstract

The invention discloses a metal organic framework material, a ligand structure thereof and application thereof in nano-enzyme, and belongs to the technical field of biology. The invention provides nano-enzyme with good sterilization effect based on a metal organic framework material. The invention prepares a carboxylic acid ligand and applies the carboxylic acid ligand to the synthesis of novel metal organic framework materials, the synthesized MOF materials can be used as nano enzymes, have peroxidase activity, show good sterilization capability in vivo and in vitro sterilization tests, are vital to resisting bacterial pathogens and treating infected wounds, and have good application prospects and development potential in the field of antibiosis. In addition, the metal organic framework material synthesis method provided by the invention has the advantages of simple process, mild conditions and the like.

Description

A metal-organic framework material and its ligand structure and its application in nanozymes

Technical field

The invention relates to a metal-organic framework material and its ligand structure and its application in nanozymes, and belongs to the field of biotechnology.

Background technique

The spread of pathogenic bacteria has always been a fatal challenge faced by mankind. It not only seriously threatens human health, but is also one of the main causes of human death worldwide. Although current antibiotics, metal ions and quaternary ammonium salts can effectively combat bacterial attacks, they will also greatly increase microbial resistance. As a substitute for these traditional antibiotics, the rapid development of nanozymes brings hope to antibacterial technology. Because nanozymes have bactericidal activity and nanostructures that produce photothermal effects, they can catalyze the inactivation of drug-resistant bacteria through sustained reactions. They have excellent development prospects in biomedicine and are expected to become a more durable and safe antibacterial disinfection. agent.

Nanozymes are nanomaterials with enzyme-like properties, showing excellent enzymatic catalytic activity, such as peroxidase-like (POD), catalase-like (CAT), superoxide dismutase-like (SOD), glucose-like Catalytic activity of oxidase (GOx) and glutathione peroxidase-like (GPx) classes. Among them, peroxide-like nanozyme (POD) can use physiological concentration (3mmol/L) of hydrogen peroxide to activate its enzyme-like reaction and locally generate a large amount of bactericidal active oxygen. It can avoid denaturation or protease hydrolysis and maintain long-lasting bactericidal activity. This enables disinfection treatment that is sensitive to bacteria and plays a high-level role in pathological sites. However, this antibacterial therapy has been severely challenged due to the lack of rational design of nanozymes with good catalytic reactivity. Therefore, developing a nanozyme with better catalytic reactivity is an important issue facing this field.

Metal Organic Frameworks (MOFs), as a class of porous materials composed of metal ions and organic ligands, can be precisely designed de novo or modified post-synthetically to customize rich and well-defined enzyme-mimetic states. The research area has become a focus of research. Moreover, metal-organic framework materials are highly similar to biological enzymes in physical and chemical properties, and have an ordered spatial structure with metal-centered catalytic sites and sites available for substrate adsorption. Therefore, it is very necessary to provide a metal-organic framework material used in nanozymes and a preparation method thereof.

Contents of the invention

In order to provide a nanozyme with good bactericidal effect, the present invention provides a metal organic framework material and a preparation method thereof, and uses it as a nanozyme.

Technical solution of the present invention:

One of the purposes of the present invention is to provide a metal organic framework material, which is referred to as MOF-ET13 for short and has a chemical formula of [Cu ₄ Ce ₄ (L) ₂ ], in which L is C ₇₂ H ₅₀ O ₈ .

The second object of the present invention is to provide a method for preparing the above-mentioned metal organic framework material. The method is as follows: dissolving Ce(NO ₃ ) ₃ •6H ₂ O and Cu(NO ₃ ) ₂ •3H ₂ O in an ethanol solution, and The ligand was dissolved in the ethanol solution, and then the two solutions were mixed and stirred at room temperature for 2.5 hours. After the reaction was completed, MOF-ET13 was obtained by centrifugation, washing and drying.

It is further limited that the molar ratio of Ce(NO ₃ ) ₃ •6H ₂ O, Cu(NO ₃ ) ₂ •3H ₂ O and ligand is 1:1:1.

It is further limited that the volume ratio of ethanol and water in the ethanol solution is 1:1.

It is further limited that after the reaction is completed, it is washed three times with water and ethanol.

To further qualify, the drying temperature is 333 K.

To further limit, the structure of the ligand is:

.

To further limit, the preparation method of the ligand includes the following steps:

S1, add 3,5-dibromo-1-trimethylsilylbenzene, 4'-(carboxy)biphenyl-4-boronic acid, 1.4 g of palladium on carbon and sodium carbonate to absolute ethanol, and stir under argon Under protection, heat the reaction. After the reaction is completed, slowly cool the reaction system to room temperature, add water to dilute and filter, acidify the filtrate with hydrochloric acid, filter, wash the precipitate, and dry to obtain a white powder, which is Intermediate 1;

S2, heat iodine monochloride until liquid, then transfer to a three-necked flask placed on ice, and add N,N-dimethylformamide, stir evenly, add intermediate 1, heat to room temperature, and the reaction is completed Finally, pour the reaction solution into methylene chloride, filter, collect the solid, and dry to obtain a light yellow powder, which is intermediate 2;

S3, add the intermediate 2, 2,5-dimethyl-1,4-phenylene diborate pinacol ester and tetrakis (triphenylphosphine) palladium to the potassium carbonate aqueous solution, and heat the reaction under argon protection , after the reaction is completed, the reaction system is slowly cooled to room temperature, diluted with water and filtered. The filtrate is acidified with hydrochloric acid, centrifuged, and the precipitate is collected. The precipitate is washed and then added to ethanol. The solvent is removed by rotary evaporation and dried to obtain a tan solid. That is the ligand.

Further limit, the molar volume of 3,5-dibromo-1-trimethylsilylbenzene, 4'-(carboxy)biphenyl-4-boronic acid, 1.4 grams of palladium on carbon, sodium carbonate and absolute ethanol in S1 The ratio is 10mmol:22mmol:80mmol:180mL.

It is further limited that the heating reaction temperature in S1 is 70°C and the time is 30 hours.

To further limit, the filtrate in S1 is acidified to pH 1 with 2 mol/L hydrochloric acid.

To further qualify, water is used to wash the precipitate in S1.

To further limit, the drying process in S1 is: place under vacuum conditions at 50°C for 24 hours.

To further limit, the molar ratio of iodine monochloride to intermediate 1 in S2 is 34:6.2.

To further limit, the reaction time in S2 is 64h.

To further limit, the molar ratio of potassium carbonate, intermediate 2, 2,5-dimethyl-1,4-phenylene diborate pinacol ester and tetrakis(triphenylphosphine)palladium in S3 is 70:5 :2.5:0.25.

To further limit, the reaction temperature in S3 is 80°C and the time is 20h.

To further limit, the filtrate in S3 is acidified using 6 mol/L hydrochloric acid.

To further limit, the centrifugation rate in S3 is 6000 rpm.

To further qualify, the precipitate in S3 was washed first with water and then three times with ethanol.

To further limit, the drying temperature in S3 is 65°C and the time is 24 hours.

The third object of the present invention is to provide the application of the above-mentioned metal organic framework material as a nanoenzyme bactericide.

The fourth object of the present invention is to provide a nanozyme bactericide based on the above-mentioned metal organic framework material. Specifically, the nanozyme bactericide is used to inhibit the growth of Escherichia coli and Staphylococcus aureus.

The invention has the following beneficial effects:

The present invention prepares a carboxylic acid ligand and applies it to the synthesis of new metal-organic framework materials. The MOF material can be used as a nanozyme, has peroxidase activity, and shows good performance in in vivo and in vitro bactericidal tests. Bactericidal ability is crucial for fighting bacterial pathogens and treating infected wounds, and has good application prospects and development potential in the antibacterial field. In addition, the synthesis method of metal organic framework materials provided by the present invention also has the advantages of simple process and mild conditions.

Description of the drawings

Figure 1 shows the synthetic route for preparing ligands for metal organic framework materials;

Figure 2 is the ¹ H-NMR spectrum of intermediate 1 prepared in Example 1;

Figure 3 is the ¹³ C-NMR spectrum of intermediate 1 prepared in Example 1;

Figure 4 is the mass spectrum of Intermediate 1 prepared in Example 1;

Figure 5 is the ¹ H-NMR spectrum of intermediate 2 prepared in Example 1;

Figure 6 is the ¹³ C-NMR spectrum of intermediate 2 prepared in Example 1;

Figure 7 is a mass spectrum of intermediate 2 prepared in Example 1;

Figure 8 is a ¹ H-NMR spectrum of the ligand prepared in Example 1;

Figure 9 is a ¹³ C-NMR spectrum of the ligand prepared in Example 1;

Figure 10 is a mass spectrum of the ligand prepared in Example 1;

Figure 11 is an X-ray structural characterization diagram of the metal organic framework material MOF-ET13 prepared in Example 1;

Figure 12 shows the test results of the antibacterial activity of the metal organic framework material MOF-ET13 prepared in Example 1 against E. coli;

Figure 13 is a test of the antibacterial activity of the metal organic framework material MOF-ET13 prepared in Example 1 against Staphylococcus aureus;

Figure 14 is the in vivo antibacterial activity test results of the metal organic framework material MOF-ET13 prepared in Example 1.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention and are not intended to limit the present invention. The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

In addition, the technical features involved in different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in this field unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.

3,5-dibromo-1-trimethylsilylbenzene (CAS: 17878-23-8) and 4'-(carboxy)biphenyl-4-boronic acid (CAS: 872341-95- 2) and 2,5-dimethyl-1,4-phenylenediboronic acid pinacol ester (CAS: 303006-89-5) were purchased directly from Sigma-Aldrich.

The elemental analysis of the following examples was carried out using the German Elementar UNICUBE elemental analyzer.

Example 1:

The process of preparing the metal organic framework material MOF-ET13 in this embodiment is as follows:

(1) As shown in Figure 1, synthesize the ligand:

①Synthetic intermediate 1:

Add 180 ml of absolute ethanol, 3,5-dibromo-1-trimethylsilylbenzene (raw material 1, 3.08 g, 10 mmol), and 4'-(carboxy)biphenyl-4-boronic acid to the three-necked flask. (raw material 2, 5.32 g, 22 mmol), 1.4 g of palladium on carbon and sodium carbonate (8.48 g, 80 mmol). The mixture was evacuated and then backfilled with argon three times, heated and stirred at 70°C. 30 hours. After the reaction is completed, slowly cool the reaction system to 25°C, dilute with 500 ml of water, filter, then acidify the filtrate with 2 mol/L hydrochloric acid to pH=1, filter the precipitate, wash with water, dry, and finally at 50° C was placed under vacuum conditions for 24 hours to obtain 4.34 grams of white powder, which was Intermediate 1, with a yield of 80%.

Structural characterization of the obtained intermediate 1:

<1>NMR characterization results:

Hydrogen spectrum: ¹ H NMR (400 MHz, DMSO): δ 8.05 (m, 7 H), 7.86 (d, 4 H), 7.38 (m, 8H), 0.35 (s, 9 H), as shown in Figure 2 .

Carbon spectrum: ¹³ C NMR (100 MHz, DMSO): δ 167.69, 144.13, 140.90, 140.62, 139.21,138.55, 132.28, 130.23, 129.63, 127.88, 127.50, 126.96, 126 .71, 1.43, as shown in Figure 3.

<2>Mass spectrometry characterization results:

ESI(m/z): [M+H] ⁺ theoretical calculation C ₃₅ H ₃₀ O ₄ Si, 542.71; actual measurement 543.29. As shown in Figure 4.

<3>Elemental analysis test results:

Theoretical calculation of C ₃₅ H ₃₀ O ₄ Si, C, 77.46, H, 5.57, O, 11.79; actual measurement of C, 78.23, H, 6.03, O, 12.13.

In summary, it can be seen that the structure of the obtained intermediate 1 is as follows:

.

②Synthetic intermediate 2:

Iodine monochloride (5.5 g, 34 mmol) was heated to liquid and transferred by pipette to a three-necked flask on ice. N,N-dimethylformamide was added and the solution was stirred for 5 minutes, then Intermediate 1 (3.36 g, 6.2 mmol) was added. The mixture was heated to 25°C, stirred for 64 hours, poured into methylene chloride, filtered, and the white solid was collected. After drying, 3.07 g of light yellow powder was obtained, which was Intermediate 2, with a yield of 83%.

Structural characterization of the obtained intermediate 2:

<1>NMR characterization results:

Hydrogen spectrum: ¹ H NMR (400 MHz, DMSO): δ 8.17 (d, 2 H), 8.11(d, 4 H), 8.04(d, 1H), 7.88 (d, 4 H), 7.39(m, 8 H), as shown in Figure 5.

Carbon spectrum: ¹³ C NMR (100 MHz, DMSO): δ 167.69, 144.13, 141.58, 140.90, 138.49,136.17, 130.23, 129.63, 127.89, 127.72, 126.85, 126.71, 96. 83, as shown in Figure 6.

<2>Mass spectrometry characterization results:

ESI(m/z): [M+H] ⁺ theoretical calculation C ₃₂ H ₂₁ IO ₄ , 596.42; actual measurement 597.23. As shown in Figure 7.

<3>Elemental analysis test results:

Theoretical calculation C ₃₂ H ₂₁ IO ₄ , C, 64.44, H, 3.55, O, 10.73; actual measurement C, 65.23, H, 4.03, O, 11.46.

In summary, it can be seen that the structure of the obtained intermediate 2 is as follows:

.

③Synthetic ligands:

Dissolve potassium carbonate (9.7 g, 70 mmol) in 30 ml of water, then transfer the solution to a 250 ml three-necked flask, and then add intermediate 2 (2.98 g, 5 mmol) and 2 to the three-necked flask in sequence. , 5-Dimethyl-1,4-phenylene diboronate pinacol ester (raw material 3, 0.829 g, 2.5 mmol) and tetrakis (triphenylphosphine) palladium (0.29 g, 0.25 mmol), will The mixture was evacuated and then backfilled with argon three times, then heated and stirred at 80°C for 20 hours. After the reaction, the reaction system was slowly cooled to 25°C, the mixture was diluted with water, filtered, acidified with 6 mol/L hydrochloric acid, and then centrifuged at 6000 rpm to collect the precipitate. The precipitate was then washed first with water and then with ethanol three times, 50 ml each time. Finally, it was suspended in ethanol, the solvent was removed by rotary evaporation, and finally dried at 65°C for 24 hours to obtain 0.83 g of tan solid, which was the ligand, with a yield of 92%.

Structural characterization of the obtained ligands:

<1>NMR characterization results:

Hydrogen spectrum: ¹ H NMR (400 MHz, DMSO): δ 8.13(m, 14 H), 7.96(m, 10 H), 7.41(m, 16H), 2.60 (s, 6 H), as shown in Figure 8 .

Carbon spectrum: ¹³ C NMR (100 MHz, DMSO): δ 167.69, 144.13, 140.90, 139.87, 139.35,139.15, 139.02, 134.85, 130.23, 129.63, 127.89, 127.71, 127 .17, 126.94,126.71, 126.62, 21.45, as shown in the figure 9 shown.

<2>Mass spectrometry characterization results:

ESI(m/z): [M+H] ⁺ theoretical calculation C ₇₂ H ₅₀ O ₈ , 1042.18; actual measurement 1043.01. As shown in Figure 10.

<3>Elemental analysis test results:

Theoretical calculation of C ₇₂ H ₅₀ O ₈ , C, 82.90, H,4.83, O, 12.27; actual measurement of C, 83.14, H,5.68, O, 12.99.

In summary, it can be seen that the structure of the obtained ligand 1 is as follows:

.

(2) Synthesis of metal organic framework material MOF-ET13:

Dissolve 1 mmol Ce(NO ₃ ) ₃ •6H ₂ O and 1 mmol Cu(NO ₃ ) ₂ •3H ₂ O in 20 ml water/ethanol (volume ratio 1:1) solution, and then add 1 mmol moles of ligand were dissolved in 5 ml of water/ethanol (volume ratio 1:1) solution. The two solutions were then mixed and magnetically stirred at 25°C for 2.5 hours. After the reaction, the product was collected by centrifugation and washed three times with water and ethanol, 50 ml each time. After vacuum drying at 333 K, MOF-ET13 was finally obtained.

Structural characterization of the obtained MOF-ET13:

<1> The synthesized MOF-ET13 crystal was placed in a glass capillary tube, and the crystal structure was tested using single crystal X-rays. The instrument was a Bruker-Apex II CCD detector, which was collected with a Cu Kα (λ=1.54178Å) X-ray source. Data are corrected for absorption by the SADABS program and not for extinction or decay. Use the SHELXTL software package to directly solve the problem, and the test results are shown in Figure 11.

Antimicrobial activity testing;

Coat the entire plate with E. coli and Staphylococcus aureus using a sterile coating stick, then place the plate in an incubator and incubate at 36 ± 1°C for 1 hour. Then set up five groups: <1> The control group is NaAc-HAc buffer; <2>H ₂ O ₂ ; <3> MOF-ET13 + H ₂ O ₂ ; <4> H ₂ O ₂ + light; <5> MOF-ET13+H ₂ O ₂ + light. Among them, the light irradiation time is 15 minutes, and the MOF-ET13 concentration is 50 μg/ml. The plate counting method was then used to determine the in vitro bactericidal effect of 50 μg/ml MOF-ET13 nanozyme on Escherichia coli and Staphylococcus aureus under weak acid conditions.

The test results are shown in Figures 12 and 13. Compared with the control group NaAc-HAc, pH=4.0, MOF-ET13 in the presence of 500 microliters of 3 mmol/L H ₂ O ₂ has better effects on E. coli or Staphylococcus aureus all showed excellent antibacterial effects. Then, when it coexists with 500 microliters of 3 mmol/L H ₂ O ₂ under light conditions, it has a significant inhibitory effect on the growth of Escherichia coli and Staphylococcus aureus, with sterilization rates of 99.87% and 99.95% respectively.

(4) Use the metal organic framework material MOF-ET13 prepared above as a nanozyme, and conduct an in vivo antibacterial activity test on it;

Twelve mice with average weight gain were randomly divided into 3 groups, and the mice were anesthetized, and then the wounds of the mice were treated with 100 μl of 1×10 ⁸ CFU/mL Staphylococcus aureus suspension and infected. 12 hours, <1> the control group is NaAc-HAc buffer; <2> MOF-ET13+H ₂ O ₂ ; <3> MOF-ET13 + H ₂ O ₂ + light. Among them, the light irradiation time is 15 minutes, and the MOF-ET13 concentration is 50 μg/ml.

The test results are shown in Figure 14. In the change curve of the wound area in each group over time, compared with the NaAc-HAc buffer blank control group, the MOF-ET13+H ₂ O ₂ group can significantly reduce the wound area in the same time. area, which shows that the addition of MOF-ET13 can significantly improve the antibacterial activity in vivo.

Obviously, the above-mentioned embodiments are only examples for clear explanation and are not intended to limit the implementation. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. An exhaustive list of all implementations is neither necessary nor possible. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims (4)

1. A metal organic framework material, characterized in that the material is referred to as MOF-ET13 for short, and its chemical formula is [Cu ₄ Ce ₄ (L) ₂ ], in which L is C ₇₂ H ₅₀ O ₈ ; The ligand structure for preparing the metal organic framework material is: 2. An application of the metal organic framework material according to claim 1, characterized in that it is used to prepare nanozyme bactericides. 3. The application of the metal-organic framework material as claimed in claim 1 as a nanozyme bactericide, characterized in that the metal-organic framework material is used in combination with H ₂ O ₂ . 4. A nanoenzyme bactericide, characterized in that the nanoenzyme bactericide is the metal organic framework material according to claim 1 and is used to inhibit the growth of Escherichia coli and Staphylococcus aureus.