A method for producing hydroxyl radicals by oxidase-phthalocyanine supramolecular near-infrared enzyme cascade catalysis

Abstract

The application discloses a method for producing hydroxyl radicals by using an oxidase-phthalocyanine supramolecular near-infrared light enzyme cascade catalysis, and belongs to the technical field of near-infrared light catalysis. The oxidase-phthalocyanine supramolecular light enzyme is constructed by non-covalent interaction, and is used for producing hydroxyl radicals under the irradiation of near-infrared light. The supramolecular near-infrared light enzyme is prepared, so that the hydroxyl radicals can be generated under a lower concentration of hydrogen peroxide and a lower loading of a photocatalyst. The confinement effect of the supramolecular light enzyme enables the hydrogen peroxide generated by oxygen removal to be quickly and in-situ utilized by the phthalocyanine, greatly improves the efficiency of the generation of the hydroxyl radicals, and reduces the amount of the phthalocyanine. Finally, the generation rate of the hydroxyl radicals can be controlled by controlling the concentrations of the enzyme, the substrate and the phthalocyanine, which is of great significance to specific application scenarios such as the preparation of ultra-high molecular weight polymers. The application can greatly promote the application of the hydroxyl radicals in the fields of controllable radical polymerization, photodynamic therapy and 3D printing.

Description

Technical Field

[0001] This invention belongs to the field of near-infrared photocatalysis technology, specifically relating to a method for the production of hydroxyl radicals via an oxidase-phthalocyanine supramolecular near-infrared photocatalytic cascade. Background Technology

[0002] Hydroxyl radicals are highly reactive free radicals widely used in environmental remediation, pollution control, virus inactivation, and tumor killing. Furthermore, hydroxyl radicals are used to initiate free radical polymerization, yielding a variety of high-performance polymer materials. Currently, the main technologies for generating hydroxyl radicals include the Fenton reaction, electrochemical oxidation, and photochemical oxidation. However, these methods generally require large amounts of oxidants / catalysts or rely on high-energy short-wavelength light (such as ultraviolet light), making them not only cumbersome and demanding in terms of conditions but also often requiring complex reaction apparatus. In recent years, with the growing awareness of sustainable development, society has gradually recognized the importance of environmental protection and urgently needs to develop a simple, green method to generate hydroxyl radicals in a gentle manner.

[0003] Near-infrared light accounts for more than half of the solar energy spectrum, and its rational use is beneficial for maximizing solar energy utilization. Compared to shorter wavelengths of light, the biggest advantage of near-infrared light is its strong penetrating power, making it widely used in fields such as industrial material drying, deep tissue repair, photodynamic therapy, and photothermal therapy. Enzymes, known as natural catalysts, can efficiently and specifically catalyze specific biochemical reactions under mild conditions. If enzyme catalysis and near-infrared photocatalysis can be cascaded to form a supramolecular near-infrared photoenzyme to generate hydroxyl radicals, the biocompatibility of the reaction system will be greatly increased. Furthermore, by controlling the amount of each component in the cascaded catalytic system, the generation rate and concentration of hydroxyl radicals can be controlled, which will have a profound impact on specific applications such as controlled radical polymerization, the preparation of ultra-high molecular weight polymers, and photodynamic therapy. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for the production of hydroxyl radicals by an oxidase-phthalocyanine supramolecular near-infrared photoenzyme cascade.

[0005] The method for producing hydroxyl radicals by an oxidase-phthalocyanine supramolecular near-infrared photoenzyme cascade catalysis according to the present invention comprises the following steps:

[0006] (1) Oxidase and phthalocyanine are added to buffer solution or water in sequence to make their concentrations 1-1000 U / mL and 0.1-1000 μM, respectively, and stirred evenly to prepare a solution containing supramolecular near-infrared photoenzyme.

[0007] (2) Add the substrate of oxidase to the supramolecular near-infrared photoenzyme solution obtained in step (1) to make its concentration 1-500 mM; then, let it stand for 0-100 minutes at 0-60°C to generate hydrogen peroxide.

[0008] (3) Place the solution after the reaction in step (2) under near-infrared light to irradiate the phthalocyanine under near-infrared light to sensitize hydrogen peroxide and produce hydroxyl radicals.

[0009] Oxidases have two functions: first, to produce hydrogen peroxide by consuming substrates and oxygen; and second, to increase the ability and efficiency of photosensitizing hydrogen peroxide to produce hydroxyl radicals by confining phthalocyanines through non-covalent interactions.

[0010] Further, in step (1), the oxidase is an oxidase from the glucose-methanol-choline (GMC) oxidoreductase family and other enzymes that can remove oxygen to produce hydrogen peroxide; preferably, the oxidase is one or more of glucose oxidase, choline oxidase, pyranose oxidase, formic acid oxidase, lactate oxidase, oxalate oxidase and cholesterol oxidase, and their corresponding substrates are glucose, choline, glucose, formic acid, lactic acid, oxalic acid and cholesterol, respectively.

[0011] Phthalocyanine is a phthalocyanine or naphthol containing a coordinating metal (aluminum, zinc, copper, magnesium, iron, nickel, lithium, manganese, etc.); a phthalocyanine or naphthol without a coordinating metal; or a phthalocyanine containing a substituent (carboxyl group, sulfonic acid group, carboxyl group with 1 to 10 carbons, ethylene glycol substituent); preferably, the phthalocyanine is one or more of tetrasulfonic acid zinc phthalocyanine, tetracarboxylate zinc phthalocyanine, tetrasulfonic acid aluminum phthalocyanine, tetracarboxylate aluminum phthalocyanine, zinc phthalocyanine, and aluminum phthalocyanine.

[0012] The buffer solution is one or more of the following: phosphate buffer, citrate phosphate buffer, carbonate phosphate buffer, acetate buffer, barbiturate phosphate buffer, and tris(hydroxymethyl)aminomethane phosphate buffer;

[0013] In buffer solutions or water, the concentrations of oxidase and phthalocyanine are 20–300 U / mL and 1–50 μM, respectively.

[0014] In step (2), the concentration of the oxidase substrate is 1–50 mM; and it is allowed to stand for 5–30 minutes at 10–30°C.

[0015] In step (3), the near-infrared light source used for the near-infrared catalytic production of hydroxyl radicals has a central emission wavelength of 600–900 nm and an energy density of 10–1000 mW / cm². -2 Preferably, the center emission wavelength of the near-infrared light source is 700–750 nm, and the energy density of the light source is 20–140 mW / cm². -2 .

[0016] The method of this invention has the following advantages: First, the production of hydroxyl radicals by cascading oxidase deoxygenation with near-infrared photocatalysis is a first in this invention, innovative and simple to operate, generating hydroxyl radicals under mild conditions. Second, compared to traditional methods of generating hydroxyl radicals using photosensitized hydrogen peroxide, this invention, through the preparation of supramolecular near-infrared photoenzymes, can generate hydroxyl radicals at lower hydrogen peroxide concentrations and photocatalyst loadings. The confinement effect of the supramolecular photoenzyme allows the hydrogen peroxide produced by enzyme deoxygenation to be rapidly and in situ utilized by phthalocyanine, significantly improving the efficiency of hydroxyl radical generation and reducing the amount of phthalocyanine used. Finally, the generation rate of hydroxyl radicals can be controlled by adjusting the concentrations of enzyme, substrate, and phthalocyanine, which is of great significance for specific applications such as the preparation of ultra-high molecular weight polymers. This invention can greatly promote the application of hydroxyl radicals in controlled radical polymerization, photodynamic therapy, and 3D printing. Attached Figure Description

[0017] Figure 1 The fluorescence spectrum (a) used to determine the fluorescence quenching mode of zinc tetrasulfonate phthalocyanine on glucose oxidase in Example 1 and the double logarithmic fitting curve (b) used to determine the binding ratio and binding constant. Figure 1 (b) The x-axis is the logarithm of the concentration of zinc tetrasulfonate phthalocyanine; Figure 1 (b) The vertical axis is the logarithm of the change in luminescence intensity of the solution after the addition of glucose oxidase and zinc tetrasulfonate phthalocyanine of different concentrations to the ratio of luminescence intensity. F0 represents the luminescence intensity of glucose oxidase without the addition of zinc tetrasulfonate phthalocyanine, and F represents the luminescence intensity of glucose oxidase with the addition of zinc tetrasulfonate phthalocyanine. Figure 1 (b) is based on Figure 1 (a) The calculated luminescence intensity at 334 nm indicates that zinc tetrasulfonate phthalocyanine quenches glucose oxidase via static quenching, suggesting that glucose oxidase and zinc tetrasulfonate phthalocyanine form a complex. The binding ratio of glucose oxidase to zinc tetrasulfonate phthalocyanine is 1:1, with a binding constant of 7.41 × 10⁻⁶. 5 .

[0018] Figure 2 The output diagram shows the results of determining the interaction between zinc tetrasulfonate phthalocyanine and glucose oxidase through molecular docking (a) and quantum mechanical calculations (b) in Example 1.

[0019] Figure 3 This is a fluorescence test pattern used in Example 1 to determine the generation of hydroxyl radicals;

[0020] Figure 4 This is a fluorescence test chromatogram used in Example 2 to determine the rate of hydroxyl radical generation;

[0021] Figure 5The 1H NMR spectrum of the polymer solution obtained in Example 3;

[0022] Figure 6 The gel permeation chromatography elution curve of the ultra-high molecular weight polymer prepared in Example 3;

[0023] Figure 7 The 1H NMR spectrum of the polymer solution obtained in Example 4; Detailed Implementation

[0024] The specific embodiments of the present invention will be described in further detail below with reference to the examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0025] Example 1

[0026] The supramolecular near-infrared photoenzyme cascade catalyzing the generation of hydroxyl radicals from glucose oxidase and zinc tetrasulfonate phthalocyanine: Glucose oxidase (2.1 mg, 160 U / mL) and zinc tetrasulfonate phthalocyanine (0.02 mg, 12 μM) were added to a cuvette containing 2 mL of phosphate buffer solution (0.2 M, pH 5.7). The mixture was stirred for 1 minute to ensure complete dissolution and homogeneity, preparing a solution containing supramolecular near-infrared photoenzyme. Then, glucose (7.2 mg, 20 mM) was added to the cuvette, and the mixture was incubated at 25 °C for 10 minutes to generate hydrogen peroxide. Next, coumarin-3-carboxylic acid (0.4 mg, 1 mM), a fluorescent probe for hydroxyl radicals (this probe does not affect the generation process or amount of hydroxyl radicals), was added, and near-infrared light with a center wavelength of 730 nm was turned on. The cuvette was placed under near-infrared light at room temperature for irradiation. The distance between the light source and the cuvette was adjusted to ensure that the energy density of the light received by the cuvette was 70 mW / cm². -2 Fluorescence spectroscopy was analyzed every half hour to determine the generation of hydroxyl radicals. The effect of adding zinc tetrasulfonate phthalocyanine on the fluorescence intensity of glucose oxidase was measured to determine the quenching mechanism, binding constant, and binding ratio of phthalocyanine on the enzyme's fluorescence. Molecular docking and quantum mechanical calculations were used to determine the binding mode and action site between the enzyme and phthalocyanine.

[0027] Experimental test analysis: The fluorescence intensity of glucose oxidase gradually decreased with increasing concentration of zinc tetrasulfonate phthalocyanine (see [reference]). Figure 1 (a)) Through fitting, it was found that zinc tetrasulfonate phthalocyanine quenches glucose oxidase in a static manner (see [reference]). Figure 1 (b) indicates that glucose oxidase and zinc tetrasulfonate phthalocyanine form a complex, which we term supramolecular near-infrared photoenzyme. The binding ratio of glucose oxidase to zinc tetrasulfonate phthalocyanine is 1:1, and the binding constant is 7.41 × 10⁻⁶. 5 (see Figure 1(b) Molecular docking and quantum mechanical calculations show that zinc tetrasulfonate phthalocyanine binds to glucose oxidase through non-covalent interactions (including hydrogen bonding, π-π interactions, ionic interactions, and coordination interactions); the main amino acids involved are arginine, tyrosine, tryptophan, and phenylalanine (see [link to relevant documentation]). Figure 2 Coumarin-3-carboxylic acid is a specific fluorescent probe for hydroxyl radicals, as can be seen from fluorescence assays (see [link]). Figure 3 As the illumination time increased, the fluorescence intensity continuously increased, indicating that the glucose oxidase-tetrasulfonic acid zinc phthalocyanine supramolecular near-infrared photoenzyme cascade catalytic system can produce hydroxyl radicals.

[0028] Example 2

[0029] Control of hydroxyl radical generation rate: Glucose oxidase (2.1 mg, 160 U / mL) and zinc tetrasulfonate phthalocyanine (0.02 mg, 12 μM) were added to a cuvette containing 2 mL of phosphate buffer solution (0.2 M, pH 5.7). The mixture was stirred for 1 minute to ensure complete dissolution and homogeneity, preparing a solution containing supramolecular near-infrared photoenzyme. Then, glucose (7.2 mg, 20 mM) was added to the cuvette, and the mixture was allowed to stand at 25°C for 10 minutes to generate hydrogen peroxide. Next, coumarin-3-carboxylic acid (0.4 mg, 1 mM), a fluorescent probe for hydroxyl radicals (this probe does not affect the generation process or quantity of hydroxyl radicals), was added, and near-infrared light with a center wavelength of 730 nm was turned on. The cuvette was irradiated under near-infrared light at room temperature, and the energy density of the light received by the cuvette was adjusted to 70 mW / cm². -2 (Reaction flask #1). Similarly, the reaction solution was prepared in a second cuvette, the only difference being that the amount of glucose oxidase added was reduced to 1.5 mg (114 U / mL), and the energy density of the light received by the cuvette was adjusted to 30 mW / cm² by adjusting the distance between the light source and the cuvette. -2 (Reaction flask #2). Every half hour, both reaction flasks were analyzed using fluorescence spectroscopy (excitation wavelength 370 nm) to determine the generation of hydroxyl radicals. See [link to relevant documentation]. Figure 4 .

[0030] Experimental test analysis: Fluorescence testing revealed (see...) Figure 4 As the illumination time increased, the fluorescence intensity of both reaction flasks continuously increased, indicating that hydroxyl radicals were generated in both flasks. The fluorescence change rate of reaction flask #2 at 445 nm was less than that of reaction flask #1, indicating that the rate of hydroxyl radical production by supramolecular near-infrared photoenzyme can be controlled by adjusting the enzyme concentration and the intensity of the light source.

[0031] Example 3

[0032] Hydroxyl radicals generated by a supramolecular near-infrared photocatalytic cascade of glucose oxidase and zinc tetrasulfonate phthalocyanine are used for the controlled polymerization production of ultra-high molecular weight polymers: In a round-bottom flask, 4-((((2-carboxyethyl)thio)thiocarbonyl)thio)-4-cyanopentanoic acid (6.2 mg, 0.2 mM), N,N-dimethylacrylamide (30 g, 3 M), glucose oxidase (75 mg, 160 U / mL), and zinc tetrasulfonate phthalocyanine (1 mg, 12 μM) are added. Phosphate buffer (0.2 M, pH 5.7) is then added to bring the total reaction volume to 100 mL. Immediately afterwards, glucose (54 mg, 20 mM) is added, and the round-bottom flask is sealed and placed under near-infrared light (emission spectrum center wavelength 730 nm; energy density 30 mW / cm²). -2 Under these conditions, the stirring speed was set to 300 rpm. After 15 hours, the light source was turned off, the reaction flask was opened to expose the viscous polymer to air, and hydroquinone polymerization inhibitor was added to quench the reaction. A small sample was taken and analyzed using 1H NMR spectroscopy and gel permeation chromatography to obtain the monomer conversion, absolute molecular weight of the polymer, and dispersion. See [link to relevant documentation]. Figure 5 and Figure 6 .

[0033] Experimental testing and analysis: The results were obtained through 1H NMR spectroscopy (see [reference]). Figure 5 After reacting for 15 hours, 77% of the N,N-dimethylacrylamide monomer was converted into poly(N,N-dimethylacrylamide) polymer. This indicates that the hydroxyl radicals generated by the glucose oxidase-tetrasulfonic acid zinc phthalocyanine supramolecular near-infrared photoenzyme cascade can be used to initiate controlled radical polymerization to generate polymers. The absolute molecular weight of the obtained polymer was tested using a gel permeation chromatography system equipped with a differential refractive index and multi-angle light scattering laser detector. The results showed that the actual molecular weight of the obtained polymer (1096.3 kg / mol) was close to the theoretical molecular weight (1145.3 kg / mol); even though the molecular weight exceeded 1 million g / mol, the dispersion of the obtained ultra-high molecular weight polymer was only 1.18 (see...). Figure 6 ).

[0034] Example 4

[0035] The hydroxyl radicals generated by the supramolecular near-infrared photoenzyme cascade of choline oxidase-tetrasulfonic acid zinc phthalocyanine were used for controlled polymerization to produce polymers: In a 96-well plate with a single-well volume of 340 μL, 4-((((2-carboxyethyl)thio)thiocarbonyl)thio)-4-cyanopentanoic acid (0.4 mg, 6 mM), N,N-dimethylacrylamide (60 mg, 3 M), choline oxidase (20 U / mL), and tetrasulfonic acid zinc phthalocyanine (0.003 mg, 12 μM) were added, and finally, phosphate buffer (0.2 M, pH 7) was added to bring the total reaction volume to 300 μL. Immediately afterwards, choline chloride (0.8 mg, 20 mM) was added, and the plate was sealed with a sealing film and placed under near-infrared light (emission spectrum center wavelength 730 nm; energy density 70 mW / cm²). -2 The reaction temperature was set to 30℃. After 12 hours, the light source was turned off, the sealing film was opened to expose the polymer solution to air, and hydroquinone polymerization inhibitor was added to quench the reaction. A small sample was taken and subjected to 1H NMR spectroscopy to obtain the monomer conversion rate. (See [reference needed]). Figure 7 .

[0036] Experimental testing and analysis: The results were obtained through 1H NMR spectroscopy (see [reference]). Figure 7 After 12 hours of reaction, 41% of the N,N-dimethylacrylamide monomer was converted into poly(N,N-dimethylacrylamide) polymer. This indicates that the hydroxyl radicals generated by the choline oxidase-tetrasulfonic acid zinc phthalocyanine supramolecular near-infrared photoenzyme cascade can be used to initiate controlled radical polymerization to generate polymers.

[0037] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for the production of hydroxyl radicals via an oxidase-phthalocyanine supramolecular near-infrared photoenzyme cascade, comprising the following steps: (1) Oxidase and phthalocyanine were added to the buffer solution in sequence to make their concentrations 20~160 U / mL and 12~50 μM, respectively, and stirred evenly to prepare a solution containing supramolecular near-infrared photoenzyme. (2) Add the substrate of oxidase to the supramolecular near-infrared photoenzyme solution obtained in step (1) to make its concentration 20~50mM; then, let it stand for 10~30 minutes at 25~30℃ to generate hydrogen peroxide. (3) Place the solution after the reaction in step (2) under near-infrared light to irradiate it, so that phthalocyanine photosensitizes hydrogen peroxide to produce hydroxyl radicals under near-infrared light irradiation. In step (1), the oxidase is glucose oxidase or choline oxidase, the phthalocyanine is zinc tetrasulfonate phthalocyanine, and the buffer solution is phosphate buffer. In step (2), when the oxidase used is glucose oxidase, the substrate of the oxidase is glucose; when the oxidase used is choline oxidase, the substrate of the oxidase is choline chloride. In step (3), the center emission wavelength of the near-infrared light is 700~750 nm, and the energy density of the light source is 30~70 mW / cm². -2 .

Images