A chiral nanozyme based on copper and L-histidine coordination and its preparation method and application

Abstract

The present invention belongs to the field of chiral catalysis technology, and specifically relates to a chiral nanozyme based on copper and L-histidine coordination, a preparation method thereof, and an application in the selective catalysis of L / D-tyrosinol. Based on the regular structure-activity relationship between nanozymes and natural enzymes, the structure of natural tyrosinase (TYR) is simulated, metallic copper is used as the catalytic active site, and L-histidine is used as a chiral recognition ligand, and the catalytic principle of natural enzymes is combined into the synthetic design of nanozymes. Based on this biomimetic strategy, a chiral nanozyme based on copper and L-histidine coordination is successfully synthesized; at the same time, a preparation method of the chiral nanozyme is provided, and then its application in the selective catalysis of L / D-tyrosinol is also provided.

Description

Technical Field

[0001] The present invention belongs to the technical field of chiral catalysis, and specifically relates to a chiral nanozyme based on the coordination of copper and L-histidine, a preparation method thereof, and an application thereof in the selective catalysis of L / D-tyrosinol. Background Art

[0002] Most biochemical reactions in living organisms rely on the efficient and specific catalytic action of natural enzymes. Natural enzymes can regulate the metabolism of organisms, transmit genetic information, maintain life activity, and catalyze biochemical reactions. Because of their high catalytic efficiency, especially the efficient discrimination of chiral substrates, natural enzymes are widely used in industrial production, medicine, and biochemistry. However, the inherent defects of natural enzymes, such as high preparation and purification costs, poor operating stability, catalytic efficiency dependent on reaction conditions, and difficulty in recycling and reuse, limit their actual production applications. In order to meet the current huge application needs, artificial enzymes with lower costs are gradually being developed and used as substitutes for natural enzymes. Compared with natural enzymes and traditional artificial enzymes, nanozymes are nanomaterials with enzyme-like activity, which have the advantages of stable structure, high efficiency, adjustable activity, low cost, large-scale preparation, and recyclability.

[0003] Although nanozymes have great application prospects, existing nanozyme research still faces many challenges. Compared with natural enzymes, nanozymes have poor selectivity and specificity. In addition, the synthetic design and mechanism research of nanozymes are still very limited, which is not conducive to the further development of chiral nanozymes. Therefore, improving the chiral selective catalytic effect of nanozymes is an urgent problem to be solved. Summary of the invention

[0004] In order to create a nanozyme with high chiral selectivity, this application simulates the structure of natural tyrosinase (TYR) based on the regular structure-activity relationship between nanozymes and natural enzymes, uses metallic copper as the catalytic active site, and L-histidine as the chiral recognition ligand, combining the catalytic principle of natural enzymes into the synthetic design of nanozymes. Based on this biomimetic strategy, a chiral nanozyme based on copper and L-histidine coordination was successfully synthesized; a preparation method of the chiral nanozyme was also provided, and then its application in the selective catalysis of L / D-tyrosinol was also provided.

[0005] The technical solution of the present invention is as follows:

[0006] A chiral nanozyme based on the coordination of copper and L-histidine. The chiral nanozyme is prepared by hydrothermal reaction of copper chloride and L-histidine.

[0007] Furthermore, the copper chloride is copper chloride dihydrate CuCl2·2H2O; the molar ratio of the copper chloride to the required amount of L-histidine is (0.1-10):(1-100).

[0008] Preferably, the copper chloride is copper chloride dihydrate CuCl2·2H2O; and the molar ratio of the copper chloride to the required amount of L-histidine is 1:10.

[0009] Preferably, the hydrothermal reaction conditions are: 150-230° C. for 8 h to 18 h.

[0010] Another object of the present invention is to protect the preparation method of the chiral nanozyme based on copper and L-histidine coordination, comprising the following steps:

[0011] (1) Weigh copper chloride and L-histidine, dissolve them in water or ethanol or N,N-dimethylformamide (DMF), and stir to mix well to obtain a mixed solution;

[0012] (2) transferring the mixed solution obtained in step (1) into a hydrothermal reactor for reaction, and obtaining a suspension after the reaction is completed;

[0013] (3) Cool to room temperature, remove the supernatant by centrifugation, wash the precipitate with water and centrifuge; after drying, a gray-brown powder is obtained, which is the Cu@L-His chiral nanozyme.

[0014] Another object of the present invention is to protect the use of the chiral nanozyme prepared by the above method or the chiral nanozyme described in claim 1 in the selective catalysis of L / D-tyrosinol.

[0015] Principle of the invention: Inspired by the structure of the active center site of natural tyrosinase (TYR), the present invention uses metallic copper as the catalytic active site and L-histidine as the chiral recognition ligand to construct an artificial peroxidase (Cu@L-His) with high enantiomeric selectivity. The core innovation is that Cu@L-His shows higher selectivity for L-Tyrosinol, with biased affinity and catalytic activity. The nanozyme synthesized by the present invention has a particle size of less than 200 nm, has excellent biocompatibility, low cytotoxicity, and also shows high selectivity for L-tyrosinol in a complex macrophage environment.

[0016] Beneficial Effects of the Invention

[0017] (1) The present invention is based on the rational simulation of the structure of natural enzymes, utilizes the unique properties of nanomaterials, combines the selectivity of natural enzymes and the stability of nanozymes, constructs active sites and selective sites in the nanosystem, and forms a structure similar to the catalytic pocket of natural enzymes, thereby constructing an artificial peroxidase with high enantiomeric selectivity and high catalytic activity.

[0018] (2) Compared with the complicated synthesis method of chiral discriminating nanozymes in existing inventions, the present invention does not require multi-step synthesis. It only uses metallic copper as the catalytic site and L-histidine as the chiral recognition ligand. By utilizing the simple coordination between the two, a chiral discriminating nanozyme with high enantiomeric selectivity and high catalytic activity can be synthesized in one step.

[0019] (3) Cu@L-His has a peroxidase activity comparable to that of natural horseradish peroxidase (HRP), which greatly improves the efficiency of chiral selective catalysis. Cu@L-His also shows higher selectivity for the L-isomer, with biased affinity and catalytic activity.

[0020] (4) This excellent enantioselective catalytic ability can be detected by a variety of methods such as ultraviolet absorption spectroscopy and fluorescence spectroscopy. It has detection stability and is less affected by the external environment.

[0021] (5) The present invention has biocompatibility and low cytotoxicity. Its chiral selective catalytic ability can be realized in a complex cellular environment. Cu@L-His also exhibits enantioselective catalytic effect in macrophages and can selectively catalyze more L-isomers.

[0022] (6) The preparation method of the present invention is simple and rapid, and does not require expensive instruments and equipment; the materials are green and environmentally friendly, and the raw materials for preparation are non-biotoxic. During the preparation process, it is not necessary to add organic surfactants and other reagents that have an impact on the environment; the preparation cost is low, and the raw material cost is low and easily available; it is suitable for industrial production.

[0023] (7) The Cu@L-His chiral nanozyme prepared by the present invention has a good morphology and nanoscale size, and a uniform particle size distribution. No common stabilizers such as polyvinyl pyrrolidone need to be added during the synthesis process.

[0024] (8) This work will provide new insights into the design and synthesis of chiral-selective nanozymes and has very broad application prospects in the field of chiral discrimination. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a transmission electron microscopy image of the Cu@L-His chiral nanozyme prepared in Example 1;

[0026] Figure 2 The hydrated particle size test results of the Cu@L-His chiral nanozyme prepared in Example 1;

[0027] Figure 3 Zeta potential test results of Cu@L-His chiral nanozyme prepared in Example 1;

[0028] Figure 4 This is the XRD spectrum of the Cu@L-His chiral nanozyme prepared in Example 1;

[0029] Figure 5 This is the infrared spectrum of the Cu@L-His chiral nanozyme prepared in Example 1;

[0030] Figure 6 The test results of distinguishing enantiomers of the Cu@L-His chiral nanozyme prepared in Example 1 by fluorescence spectroscopy;

[0031] Figure 7 The test results of distinguishing enantiomers of the Cu@L-His chiral nanozyme prepared in Example 1 by ultraviolet absorption spectroscopy;

[0032] Figure 8 This is the test result of enantiomer differentiation of Cu@L-His chiral nanozyme prepared in Example 1 in macrophages.

[0033] Specific implementation measures

[0034] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0035] Example 1

[0036] A method for preparing a chiral nanozyme based on copper and L-histidine coordination, specifically comprising the following steps:

[0037] (1) Weigh 0.0340 g of cupric chloride dihydrate (CuCl2·2H2O) and 0.3103 g of L-histidine and dissolve them in 35 mL of deionized water. Stir and mix well to obtain a dark blue mixed solution.

[0038] (2) The mixed solution was transferred into a polytetrafluoroethylene hydrothermal reactor and reacted at 180°C for 8 h-18 h. After the reaction, a brown-grey suspension was obtained. The mixture was naturally cooled to room temperature and the supernatant was removed by centrifugation. The precipitate was repeatedly washed with deionized water and centrifuged at a speed of 7000-9000 r / min and the centrifugation time was 10-20 min each time until the supernatant was basically colorless. The solid obtained by centrifugation was dried in an oven at 60-70°C to obtain a grey-brown powder, which was the Cu@L-His chiral nanozyme. The obtained Cu@L-His chiral nanozyme was stored at 4°C in the dark. Figure 1 As shown in the figure, the synthesized Cu@L-His chiral nanozyme has a regular morphology and uniform size. Figure 2 As shown in Figure 2, the hydrated particle size of the synthesized Cu@L-His chiral nanozyme is about 122 nm. Figure 3 As shown in Figure 2, the Zeta potential of the synthesized Cu@L-His chiral nanozyme is +39 mV. Figure 4 , Figure 5 As shown, in the infrared spectrum and XRD spectrum, the characteristic peaks of the synthesized Cu@L-His chiral nanozyme are consistent with those of the raw material L-histidine, proving that L-histidine is successfully incorporated into the chiral nanozyme and the structure is not destroyed.

[0039] Example 2

[0040] The performance of the Cu@L-His chiral nanozyme synthesized in Example 1 in selectively catalyzing L / D-tyrosinol was further tested below.

[0041] 1. Characterization of chiral discrimination performance by fluorescence spectroscopy

[0042] The ability of the Cu@L-His chiral nanozyme synthesized in Example 1 to selectively catalyze chiral tyrosinol isomers in the presence of H2O2 was tested by fluorescence spectroscopy. Figure 6 shown.

[0043] Specific operation method: (1) Using deionized water as solvent, prepare a Cu@L-His chiral nanozyme solution with a concentration of 500 μg / mL synthesized in Example 1; and an L-tyrosinol solution and a D-tyrosinol solution with a concentration of 500 μg / mL.

[0044] (3) Set the parameters of the fluorescence spectrometer as follows: excitation wavelength 310 nm, emission wavelength 350 nm-500 nm, excitation voltage 900 V, slit 5 nm, and scanning speed 2400 nm / min.

[0045] (4) After 40 μL of Cu@L-His chiral nanozyme solution was mixed with 2 mL of L-tyrosinol solution and D-tyrosinol solution in the dark for 15 min, 4 μL of H2O2 (30%) was added. Starting from the 0th minute, the fluorescence emission spectra of L-tyrosinol solution and D-tyrosinol solution were detected every 20 min.

[0046] Results: By Figure 6It can be seen from the fluorescence spectrum that the fluorescence intensity of L-tyrosinol solution and D-tyrosinol solution cannot completely distinguish the two in the initial stage. As the reaction time with Cu@L-His and H2O2 increases, the difference in fluorescence intensity between the two gradually increases, and the emission peak of L-tyrosinol solution at 350-500 nm is significantly higher than that of D-tyrosinol. This is because Cu@L-His chiral nanozyme has chiral recognition ligands, which can selectively bind more L-isomers and then oxidize more L-tyrosinol, thus having a clear distinction in the fluorescence spectrum.

[0047] 2. Characterization of chiral discrimination performance by UV absorption spectroscopy

[0048] The ability of the Cu@L-His chiral nanozyme synthesized in Example 1 to selectively catalyze L / D-tyrosinol in the presence of H2O2 was tested by ultraviolet absorption spectroscopy. Figure 7 shown.

[0049] Specific operation method:

[0050] (1) Using deionized water as solvent, prepare a 500 μg / mL solution of the Cu@L-His chiral nanozyme synthesized in Example 1.

[0051] (2) Using deionized water as the solvent, prepare L-tyrosinol solutions and D-tyrosinol solutions with concentrations of 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL, respectively.

[0052] (3) The ELISA reader was set at 25°C, the reaction time was 150 min, the measurement interval was 5 min, and the detection wavelength was 320 nm.

[0053] (4) Add 40 μL of Cu@L-His chiral nanozyme solution and 200 μL of L-tyrosinol solution and D-tyrosinol solution of different concentrations to a 96-well plate, mix for 15 min in the dark, add 4 μL of H2O2 (30%), and place the plate in an ELISA instrument for detection and recording of experimental data.

[0054] Results: By Figure 7 From the change of the absorbance at 320 nm in the UV absorption spectrum of (a), it can be seen that the initial absorption of L-type and D-type at 320 nm cannot completely distinguish the two. As the reaction time with Cu@L-His and H2O2 increases, the difference in absorption at 320 nm between the two gradually increases, and finally the two can be clearly distinguished. Figure 7 As can be seen in (b), as the substrate concentration increases, the difference in UV absorption also gradually increases.

[0055] Example 3

[0056] The ability of the Cu@L-His chiral nanozyme synthesized in Example 1 to selectively catalyze L / D-tyrosinol in the macrophage environment is characterized below. Figure 8 shown.

[0057] Specific operation method:

[0058] (1) Select normal mouse mononuclear macrophage leukemia cells RAW264.7 cells. The cell culture method can be any method known to those skilled in the art without any particular limitation. In the present invention, the cells are cultured in an incubator at 37°C containing 5% (v / v) CO2, and the culture medium used is DMEM culture medium containing 10% inactivated fetal bovine serum. First, the cells are cultured at 2×10 5 The cells were inoculated at a density of 100 μg / mL in a 6-well plate, and 2 mL of culture medium (DMEM culture medium containing 10% inactivated fetal bovine serum) was added to each well. The 6-well plate was placed in an incubator at 37°C containing 5% (v / v) CO2 for 48 h. Fresh culture medium was replaced with 2 mL per well every 24 h. After RAW264.7 cells were cultured in the 6-well plate until they adhered to the wall, the 6-well plate was removed for later use.

[0059] (2) Using DMEM culture medium as solvent, prepare a 150 μg / mL Cu@L-His chiral nanozyme solution synthesized in Example 1; a 1 mg / mL L-tyrosinol solution and a 1 mg / mL D-tyrosinol solution; and a 30 mM H2O2 solution.

[0060] (3) First, 2 mL of L-tyrosinol solution and D-tyrosinol solution prepared in (2) were incubated with RAW264.7 cells for 0.5 h, respectively, to allow them to fully enter the cells through endocytosis. Then, the culture medium was discarded, and 2 mL of Cu@L-His chiral nanozyme solution prepared in (2) and 200 μL of H2O2 solution prepared in (2) were added and incubated for 4 h.

[0061] (4) After 4 h of co-incubation, the changes in fluorescence intensity were observed by two-photon laser confocal microscopy.

[0062] Results: By Figure 8 It can be seen that through the endocytosis function of RAW264.7 cells, L / D-tyrosinol and Cu@L-His chiral nanozymes entered the complex environment inside the cells. After 4 hours, the confocal fluorescence intensity after the reaction of L-tyrosinol and Cu@L-His chiral nanozymes was significantly higher than that of D-tyrosinol, which was consistent with the change of fluorescence intensity in the solution environment.

Claims

1. A Cu@L-His chiral nanozyme, characterized in that: The chiral nanozyme is prepared by the following method: (1) Weigh 0.0340 g of cupric chloride dihydrate and 0.3103 g of L-histidine, dissolve them in deionized water, and stir to mix well to obtain a mixed solution; (2) transferring the mixed solution obtained in step (1) into a hydrothermal reactor and reacting at 180° C. for 8-18 hours to obtain a suspension after the reaction is completed; (3) Cool to room temperature, remove the supernatant by centrifugation, wash the precipitate with water and centrifuge; after drying, a gray-brown powder is obtained, which is the Cu@L-His chiral nanozyme.

2. The Cu@L-His chiral nanozyme according to claim 1, characterized in that The centrifugal speed is 7000-9000r / min, and the centrifugal time is 10-20min.

3. The Cu@L-His chiral nanozyme according to claim 1, characterized in that The drying temperature is 60-70℃.

4. Use of the Cu@L-His chiral nanozyme according to any one of claims 1 to 3 in the selective catalytic oxidation of L-tyrosinol for non-therapeutic purposes.

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