Sulfonated thia-bridged azacalixarene and its complex with manganese in scavenging reactive oxygen species

Abstract

This invention belongs to the field of nanomaterials technology, and relates to the application of sulfonated thiobridged azocalixarene and its complexes with manganese in scavenging reactive oxygen species. The sulfonated thiobridged azocalixarene has the building unit TSAC4A and the chemical formula C2. 48 H 28 N8Na4O 16 S8, with the following structural formula, shows that manganese ions, acting as coordinating metals, form a stable metal complex with TSAC4A in water. This invention utilizes Mn... 2+ -TSAC4A exhibits excellent scavenging ability for superoxide anions, with a scavenging efficiency far exceeding that of TSAC4A and Mn at the same concentration. 2+ The summation of TSAC4A and Mn indicates that TSAC4A and Mn 2+ It exhibits a good synergistic effect in the scavenging of superoxide anions.

Description

Technical Field

[0001] This invention belongs to the field of nano-supramolecular materials technology, and in particular relates to the application of sulfonated thio-bridged azocalixarene and its complexes with manganese in the scavenging of reactive oxygen species (ROS). Background Technology

[0002] In living organisms, reactive oxygen species (ROS) are natural byproducts of normal oxygen metabolism and play a crucial role in fundamental physiological activities such as cell signaling and the regulation of cell proliferation, differentiation, apoptosis, and immune responses. However, excessive ROS is a significant pathophysiological characteristic of many inflammatory diseases. Excessive ROS accumulation leads to the oxidation of lipids, proteins, and nucleic acids, further exacerbating local tissue damage and causing chronic inflammatory diseases such as wound sepsis, inflammatory bowel disease, acute liver / kidney injury, and liver fibrosis. Therefore, efficient ROS scavenging can reduce oxidative stress-induced cellular damage and is of great significance for the treatment of inflammatory diseases.

[0003] Therefore, we urgently need to develop new materials for the treatment of ROS-related diseases. In previous research, the inventors discovered that calixarenes can significantly enhance their ROS scavenging ability by binding with metal ions. However, many calixarenes, such as sulfonated calix-4-aryl (SC4A) and sulfonated azo calix-4-aryl (SAC4A), currently exhibit poor ROS scavenging effects on their own and also show limited ability to scavenge ROS by binding with metal ions such as Mn. 2+ Their binding ability is not strong. Therefore, it is of great significance to develop a calixarene with good self-scavenging ability of ROS and strong binding ability with metal ions. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a supramolecular material that can efficiently remove ROS, thereby effectively improving the ability to remove ROS.

[0005] The first aspect of the present invention provides the application of sulfonated thiobridged azocalixarene in scavenging reactive oxygen species, said sulfonated thiobridged azocalixarene having the building unit TSAC4A and the chemical formula C 48 H 28 N8Na4O 16 S8, the structural formula is as follows:

[0006]

[0007] A second aspect of the invention provides the application of sulfonated sulfur-bridged azocalixarene complexes with manganese in the scavenging of reactive oxygen species.

[0008] A third aspect of the present invention provides a method for preparing sulfonated thiobridged azocalixarenes, comprising the following steps:

[0009] Step 1: Dissolve p-tert-butylphenol, S8, and sodium hydroxide in diphenyl ether at a molar ratio of 2:2:1; gradually raise the temperature to 150-180℃ with stirring, maintain for 1-2 hours, and purge with nitrogen for protection; gradually raise the temperature to 200-250℃ over 2-3 hours, maintain for 2-4 hours; gradually cool to room temperature, dilute with toluene, and wash with 0.5-1 mol / L sulfuric acid solution; add a large amount of ethanol to precipitate the solid, filter, and recrystallize with dichloromethane and ethanol to obtain a white solid, namely tert-butylsulfide bridged calix 4-aromatic hydrocarbon t-buTC4A;

[0010] Step 2: Dissolve t-buTC4A, phenol, and aluminum trichloride in toluene at a molar ratio of 1:0.5:10; stir at 60-90℃ for 1.5-3 hours; after cooling, wash with water 2-4 times, separate the liquids, and rotary evaporate to obtain a pale yellow product; stir the pale yellow product in methanol and reflux for 20-50 minutes; cool and filter to obtain a white solid, which is thiobridged calix 4 aromatic hydrocarbon TC4A;

[0011] Step 3: Dissolve p-aminobenzenesulfonic acid and sodium carbonate in water at a molar ratio of 10:5, and sonicate or heat at 50-55℃ until completely dissolved to obtain the first mixture, wherein the concentration of p-aminobenzenesulfonic acid is 300-400 mol / L; cool to below 30℃, dissolve sodium nitrite in water, and add it to the first mixture, wherein the concentration of sodium nitrite is 150-200 mol / L; cool to 0-5℃ in an ice-salt bath to obtain the second mixture; add concentrated hydrochloric acid dropwise to the second mixture, wherein the volume ratio of concentrated hydrochloric acid to the second mixture is 1:5 to 1:10; cool to 0-5℃ to obtain the third mixture; take TC4A and sodium acetate trihydrate into a container, wherein the molar ratio of TC4A to sodium acetate trihydrate is 2-4:25-50, and add methanol and N,N-dimethyl... Formamide, wherein the volume ratio of methanol to N,N-dimethylformamide is 5:8, and the molar ratio of methanol to TC4A is 3:10 to 5:10, is cooled to 0-5℃ to obtain a fourth mixture; the third mixture is slowly added to the fourth mixture, and the color is observed to change from orange to orange-red and then to brick-red; the mixture is placed in an ice bath for 1-2 hours, and then gradually heated to room temperature for 1-2 hours; the pH is adjusted to 2, and the mixture is heated to 50-70℃ for 30-50 minutes; the heating is stopped, and methanol, N,N-dimethylformamide, and water are removed by rotary evaporation; N,N-dimethylformamide is added to the solid to dissolve the product, and the mixture is filtered to remove the salt, and the filtrate is evaporated to dryness; methanol is added to dissolve the product, ethyl acetate is added to precipitate the product, and the solid product, sulfonated thiobridged azocalix 4-aromatic hydrocarbon TSAC4A, is obtained by filtration.

[0012] The advantages and beneficial effects of this invention are:

[0013] 1. The present invention Mn 2+-TSAC4A exhibits excellent scavenging ability for superoxide anions, etc., and its scavenging efficiency is higher than that of Mn at the same concentration. 2+ More than twice that of SAC4A and TSAC4A, and far greater than that of TSAC4A and Mn. 2+ The summation of TSAC4A and Mn indicates that TSAC4A and Mn 2+ It exhibits a good synergistic effect in the scavenging of superoxide anions.

[0014] 2. The TSAC4A of this invention has a well-defined chemical structure, good batch-to-batch reproducibility, and high chemical and thermal stability. TSAC4A and Mn 2+ Metal complexes formed in water can effectively remove ROS and have the potential to treat inflammatory diseases. Attached Figure Description

[0015] Figure 1 NMR spectrum of sulfobridged calix 4-aromatic hydrocarbon (TC4A);

[0016] Figure 2 NMR spectrum of sulfonated sulfobridged azocalix 4-aromatic hydrocarbon (TSAC4A);

[0017] Figure 3 This is the mass spectrum of sulfonated sulfur-bridged azocalix 4-aromatic hydrocarbon (TSAC4A);

[0018] Figure 4 Scavenging O2 at a concentration of 60 μM for sulfonated calix-4 aromatic monomers (SPhOH), sulfonated calix-4 aromatic hydrocarbons (SC4A), sulfonated azocalix-4 aromatic monomers (SAPhOH), sulfonated azocalix-4 aromatic hydrocarbons (SAC4A), and sulfonated sulfobridged azocalix-4 aromatic hydrocarbons (TSAC4A). - Before and after comparison images;

[0019] Figure 5 (a) in the figure represents a manganese ion and a sulfonated thio-bridged azocalix 4 aromatic metal complex (Mn). 2+ -TSAC4A), manganese ions and sulfonated azocalix 4 aromatic metal complexes (Mn 2+ -SAC4A), sulfonated sulfobridged azo calix 4 aromatics (TSAC4A), sulfonated azo calix 4 aromatics (SAC4A), manganese ions (Mn 2+ ) Scavenging O2 at a concentration of 60 μM - Before and after comparison images;

[0020] Figure 5 (b) is a manganese ion and sulfonated thio-bridged azocalix 4 aromatic metal complex (Mn) 2+ -TSAC4A), manganese ions and sulfonated azocalix 4 aromatic metal complexes (Mn 2+-SAC4A), sulfonated sulfobridged azo calix 4 aromatics (TSAC4A), sulfonated azo calix 4 aromatics (SAC4A), manganese ions (Mn 2+ ) Scavenging O2 at a concentration of 60 μM - Before and after comparison images;

[0021] Figure 6 (a) represents the addition of different concentrations of Mn when the concentration of sulfonated thio-bridged azocalix 4-aromatic hydrocarbon (TSAC4A) is 60 μM. 2 + Its removal of O2 - Efficiency variation graph;

[0022] Figure 6 (b) shows the addition of different concentrations of Mn when the concentration of sulfonated azocalix-4 aromatic hydrocarbon (SAC4A) is 60 μM. 2+ Its removal of O2 - Efficiency variation graph;

[0023] Figure 7 Manganese ions (Mn) 2+ UV-Vis titration plots of 500 μM for sulfonated thio-bridged azo calix 4 aromatic hydrocarbon (TSAC4A) and sulfonated thio-bridged azo calix 4 aromatic hydrocarbon (TSAC4A).

[0024] Figure 8 Direct UV titration of Mn in 20 μM sulfonated thiobridged azo calixarene (TSAC4A) 2+ The graph shows the changes in ultraviolet absorption of TSAC4A. Detailed Implementation

[0025] The present invention will be further described in detail below through specific embodiments. The following embodiments are merely descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0026] The preparation steps of sulfonated sulfobridged azocalix 4-aromatic hydrocarbon (TSAC4A) are as follows:

[0027] Step 1: Add 30g of p-tert-butylphenol, 51.2g of S8, and 4g of sodium hydroxide to the reaction flask. Add 40ml of diphenyl ether to dissolve the reactants. Gradually raise the temperature to 160℃ with stirring, maintain for 1h, and purge with nitrogen. Gradually raise the temperature to 230℃ over 3h, maintain for 3h, and purge with nitrogen. Gradually cool to room temperature, dilute with toluene, and wash with 0.5 mol / L sulfuric acid solution. Add a large amount of ethanol to precipitate the solid, filter, and recrystallize with dichloromethane and ethanol to obtain about 20g of white solid, namely tert-butylsulfide bridged calix 4-aromatic (t-buTC4A).

[0028] Step 2: Add 5g t-buTC4A, 0.35g phenol, and 9.73g aluminum trichloride to the reaction flask, and add 200ml toluene to dissolve the reactants; heat to 80℃ and stir for 2 hours. After cooling, wash twice with water, separate the contents, and rotary evaporate to obtain a pale yellow product. Stir the pale yellow product in methanol and reflux for 30 minutes. Cool and filter to obtain approximately 2.64g of a white solid, namely thiobridged calix 4-aromatic hydrocarbon (TC4A). Figure 1 The NMR spectrum of TC4A (a sulfur-bridged cup 4-aromatic) shows that the assignment of H in the NMR spectrum is correct and the integration is accurate.

[0029] Step 3: Dissolve 300 mg of p-aminobenzenesulfonic acid and 91 mg of sodium carbonate in water, and sonicate or heat at 50-55°C until completely dissolved to obtain the first mixture; cool to below 30°C. Dissolve 118 mg of sodium nitrite in water and add it to the first mixture, cooling to 0°C in an ice-salt bath to obtain the second mixture; add 1 ml of concentrated hydrochloric acid dropwise to the second mixture to obtain the third mixture, and cool to 0-5°C. Take 200 mg of TC4A and 697 mg of sodium acetate trihydrate into a round-bottom flask, add 1 ml of methanol and 1.6 ml of N,N-dimethylformamide (methanol:N,N-dimethylformamide = 5:8), and cool to 0-5°C to obtain the fourth mixture. Slowly add the third mixture to the fourth mixture; the color will change from orange to orange-red and then to brick-red. Incubate in an ice bath for 1 hour, then gradually heat to room temperature for 1 hour. Adjust the pH to approximately 2, heat to 60°C, and continue heating for 30 minutes. Stop heating and remove methanol, N,N-dimethylformamide, and water by rotary evaporation.

[0030] The product was dissolved in N,N-dimethylformamide and filtered to remove the salt. The filtrate was then evaporated to dryness. The product was dissolved in methanol and precipitated by adding ethyl acetate. The precipitate was obtained by filtration and the solid product was obtained, which was sulfonated thiobridged azocalix 4-aromatic (TSAC4A).

[0031] Figure 2 The NMR spectrum of sulfonated thio-bridged azocalix 4-aromatic hydrocarbon (TSAC4A) shows that the assignment of H in the NMR spectrum is correct and the integration is accurate.

[0032] Figure 3 The mass spectrum of sulfonated thiobridged azocalix 4-aromatic hydrocarbon (TSAC4A) shows a mass-to-nucleus ratio of 1230.96, which is correct.

[0033] Determination of superoxide anion scavenging:

[0034] (1) Sample preparation: The following samples were prepared: sulfonated calix 4 aromatic monomers (SPhOH; CAS: 98-67-9), sulfonated calix 4 aromatics (SC4A; CAS: 112269-92-8), sulfonated azo calix 4 aromatic monomers (SAPhOH; CAS: 2918-83-4), sulfonated azo calix 4 aromatics (SAC4A; CAS: 221181-37-9), and sulfonated sulfur-bridged azo calix 4 aromatics (TSAC4A) were prepared with manganese ions (Mn). 2+ Prepare 1 mM solutions separately.

[0035] (2) Preparation of WST-8 / enzyme working solution: Prepare an appropriate amount of WST-8 / enzyme working solution according to a volume of 160 μl for each reaction. Mix 151 μl of SOD detection buffer, 8 μl of WST-8 and 1 μl of enzyme solution evenly to prepare 160 μl of WST-8 / enzyme working solution.

[0036] (3) Preparation of reaction start-up working solution: Dissolve and mix the reaction start-up solution (40X) in the kit, and dilute it by adding 39 μl of SOD detection buffer to every 1 μl of reaction start-up solution (40X). Mix well to obtain the reaction start-up working solution. Prepare an appropriate amount of reaction start-up working solution according to the number of samples to be tested (including standards).

[0037] (4) Refer to Table 1 to set up the sample wells and various blank control wells using a 96-well plate. Add the test samples and other solutions in the order shown in the table below. Mix thoroughly after adding the reaction start-up working solution.

[0038] Scavenging efficiency = [(A blank - A blank control) - (A sample - A sample control)] / (A blank - A blank control) × 100%

[0039] Table 1

[0040] sample Sample control blank Blank control Sample to be tested 20μl 20μl － － SOD detection buffer － 20μl 20μl 40μl WST-8 / Enzyme Working Solution 160μl 160μl 160μl 160μl Reaction start-up working fluid 20μl － 20μl －

[0041] The specific operation is as follows: Add the sample to be tested, water, WST-8 / enzyme working solution and starter solution to the centrifuge tubes in the order shown in Table 2, for a total of 6 groups of 12 centrifuge tubes. Place the centrifuge tubes in a water bath and incubate at 37°C for 30 minutes. Then add the liquid in the centrifuge tubes to a 96-well plate and measure the absorbance at 450 nm.

[0042] Table 2

[0043]

[0044] As shown in Table 2, sulfonated calix-4 aromatic monomers (SPhOH), sulfonated calix-4 aromatic hydrocarbons (SC4A), sulfonated azocalix-4 aromatic monomers (SAPhOH), sulfonated azocalix-4 aromatic hydrocarbons (SAC4A), and sulfonated sulfobridged azocalix-4 aromatic hydrocarbons (TSAC4A) were used to remove O2 at a concentration of 60 μM. - The results of the comparison are shown in the figure. Figure 4 It can be seen that at this concentration, the scavenging effect is: TSAC4A > SAC4A > SC4A > SAPhOH > SPhOH.

[0045] Refer to Table 1 to set up the sample wells and various blank control wells using a 96-well plate. Add the test samples and other solutions in the order shown in Table 3. After adding the reaction start-up working solution, mix thoroughly.

[0046] The specific operation is as follows: Add the sample to be tested, water, WST-8 / enzyme working solution and starter solution to the centrifuge tubes in the order shown in Table 3, for a total of 6 groups of 12 centrifuge tubes. Place the centrifuge tubes in a water bath and incubate at 37°C for 30 minutes. Then add the liquid in the centrifuge tubes to a 96-well plate and measure the absorbance at 450 nm.

[0047] Table 3

[0048]

[0049] As shown in Table 3, manganese ions and sulfonated sulfur-bridged azocalix 4 aromatic metal complexes (Mn) 2+ -TSAC4A), manganese ions and sulfonated azocalix 4 aromatic metal complexes (Mn 2+ -SAC4A), sulfonated sulfobridged azo calix 4 aromatics (TSAC4A), sulfonated azo calix 4 aromatics (SAC4A), manganese ions (Mn 2+ ) Scavenging O2 at a concentration of 60 μM - Comparing the effects, the Mn in the complex 2+ The concentration was 60 μM. Results are shown below. Figure 5 In (a), we can see the scavenging effect at this concentration: Mn 2+ -TSAC4A>Mn 2+ -SAC4A>TSAC4A>Mn 2+ .

[0050] Refer to Table 1 to set up the sample wells and various blank control wells using a 96-well plate. Add the test samples and other solutions in the order listed in the table. After adding the reaction start-up working solution, mix thoroughly.

[0051] The specific operation is as follows: Add the sample to be tested, water, WST-8 / enzyme working solution and starter solution to the centrifuge tubes in the order shown in Table 4, for a total of 6 groups of 12 centrifuge tubes. Place the centrifuge tubes in a water bath and incubate at 37°C for 30 minutes. Then add the liquid in the centrifuge tubes to a 96-well plate and measure the absorbance at 450 nm.

[0052] Table 4

[0053]

[0054] As shown in Table 4, manganese ions and sulfonated sulfur-bridged azocalix 4 aromatic metal complexes (Mn) 2+ -TSAC4A), manganese ions and sulfonated azocalix 4 aromatic metal complexes (Mn 2+ -SAC4A), sulfonated sulfobridged azo calix 4 aromatics (TSAC4A), sulfonated azo calix 4 aromatics (SAC4A), manganese ions (Mn 2+ ) Scavenging O2 at a concentration of 60 μM - The effects were compared. Among them, Mn in the coordination compound... 2 + The concentration was 300 μM. Results are shown below. Figure 5 In (b), it can be seen that at this concentration, the scavenging effect is: Mn 2+ -TSAC4A>Mn 2+ -SAC4A>TSAC4A>Mn 2+ .

[0055] Figure 5 (b) and Figure 5 Compared to (a) in the middle, it can be seen that increasing Mn 2+ Concentration, metal complex scavenging O2 - The effect is significantly enhanced.

[0056] When the concentration of sulfonated thio-bridged azo calix 4 aromatic hydrocarbon (TSAC4A) is 60 μM, different concentrations of Mn are added. 2+ Its removal of O2 - Changes in efficiency, such as Figure 6 As shown in (a) above, it can be seen that as Mn 2+ Increased concentration leads to the removal of O2 by metal complexes. - The effect also improves accordingly, reaching its maximum at around 200uM.

[0057] When the concentration of sulfonated azocalix-4 aromatic hydrocarbon (SAC4A) is 60 μM, different concentrations of Mn are added. 2+ Its removal of O2 - Changes in efficiency, such as Figure 6 As shown in (b) above, it can be seen that as Mn... 2+ Increased concentration leads to the removal of O2 by metal complexes.- The effect also improves accordingly, reaching its maximum at around 200uM.

[0058] contrast Figure 6 (a) Figure 6 As can be seen from (b) in the figure, Mn 2+ -SAC4A Clears O2 - The effect is not as good as Mn 2+ -TSAC4A.

[0059] Refer to Table 5 below for monitoring Mn 2+ The change in UV absorption of TSAC4A at 365 nm when the concentration of TSAC4A is 500 μM.

[0060] Table 5

[0061] Concentration / μM 50 100 150 200 250 300 350 400 450 <![CDATA[Mn 2+ / 10mM]]> 1.5 3 4.5 6 7.5 9 10.5 12 13.5 Concentration / μM 450 400 350 300 250 200 150 100 50 TSAC4A / 1mM 135 120 105 90 75 60 45 30 15 water 165 180 195 210 225 240 255 270 285

[0062] Figure 7 Manganese ions (Mn) 2+ The UV-Vis titration plots of Mn and sulfonated thioazo-bridged azocalix 4-aromatic hydrocarbon (TSAC4A) at a combined concentration of 500 μM were used to monitor the UV absorption change of TSAC4A at 365 nm. It can be seen that the peak is 0.5. 2+ The coordination mode with TSAC4A is n:n.

[0063] Figure 8 To achieve a TSAC4A concentration of 20 μM, 20 μM, 40 μM, 80 μM, 160 μM, 320 μM, 640 μM, and 1000 μM were added. 2+ The UV absorption changes of TSAC4A are shown. The absorption of calixarene decreases, and there is an isoabsorption point around 400 nm, indicating coordination between the two in water, indirectly representing the formation of a complex.

[0064] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several modifications and improvements can be made without departing from the inventive concept, and these all fall within the protection scope of the present invention.

Claims

1. A process for the preparation of a sulfonated thia-bridged azacalixarene, characterized in that, The sulfonated sulfur-bridged azacalixarene, a building unit TSAC4A, has a chemical formula of C 48 H 28 N8Na4O 16 S8, and a structural formula as follows: The steps are as follows: Step 1: Dissolve p-tert-butylphenol, S8, and sodium hydroxide in diphenyl ether at a molar ratio of 2:2:1; gradually heat to 150-180℃ with stirring, maintain for 1-2 hours, and purge with nitrogen; gradually heat to 200-250℃ over 2-3 hours, maintain for 2-4 hours; gradually cool to room temperature, dilute with toluene, and wash with 0.5-1 mol / L sulfuric acid solution; add a large amount of ethanol to precipitate, filter, and recrystallize with dichloromethane and ethanol to obtain a white solid, namely tert-butylsulfide bridged calix 4 aromatic hydrocarbon. t- buTC4A; Step 2, t- buTC4A, phenol, and aluminum trichloride were dissolved in toluene at a molar ratio of 1:0.5:10; the mixture was stirred at 60-90℃ for 1.5-3 hours, cooled, washed with water 2-4 times, separated, and rotary evaporated to obtain a pale yellow product; the pale yellow product was stirred in methanol and refluxed for 20-50 minutes. Cool and filter to obtain a white solid, which is TC4A, a sulfur-bridged calix aromatic hydrocarbon. Step 3: Dissolve p-aminobenzenesulfonic acid and sodium carbonate in water at a molar ratio of 10:5, and sonicate or heat at 50-55℃ until completely dissolved to obtain the first mixture, wherein the concentration of p-aminobenzenesulfonic acid is 300-400 mol / L; cool to below 30℃, dissolve sodium nitrite in water, and add it to the first mixture, wherein the concentration of sodium nitrite is 150-200 mol / L; cool to 0-5℃ in an ice-salt bath to obtain the second mixture; add concentrated hydrochloric acid dropwise to the second mixture, wherein the volume ratio of concentrated hydrochloric acid to the second mixture is 1:5~1:10; cool to 0-5℃ to obtain the third mixture; take TC4A and sodium acetate trihydrate into a container, wherein the molar ratio of TC4A to sodium acetate trihydrate is 2-4:25-50, and add methanol and N,N-dimethyl... Formamide, wherein the volume ratio of methanol to N,N-dimethylformamide is 5:8, and the molar ratio of methanol to TC4A is 3:10~5:10, is cooled to 0-5℃ to obtain a fourth mixture; the third mixture is slowly added to the fourth mixture, and the color is observed to change from orange to orange-red and then to brick red; the mixture is placed in an ice bath for 1-2 hours, and then gradually heated to room temperature for 1-2 hours; the pH is adjusted to 2, and the mixture is heated to 50-70℃ for 30-50 minutes; the heating is stopped, and methanol, N,N-dimethylformamide, and water are removed by rotary evaporation; N,N-dimethylformamide is added to the solid to dissolve the product, and the mixture is filtered to remove the salt, and the filtrate is evaporated to dryness; methanol is added to dissolve the product, ethyl acetate is added to precipitate the product, and the solid product, sulfonated thiobridged azocalix 4-aromatic hydrocarbon TSAC4A, is obtained by filtration.

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