A copper complex of 2,3-diketo-l-gulonic acid and its preparation method and application

Abstract

The application discloses a copper 2,3-diketo-L-gulonic acid complex and a preparation method and application thereof, and belongs to the technical field of metal complexes and bioactive small molecules. The copper 2,3-diketo-L-gulonic acid complex is formed by coordination of 2,3-diketo-L-gulonic acid and copper ions. The complex can have an insertion type interaction with DNA and mediate DNA damage, and realizes multiple bioactivities of antibacteriophage, antibacterial and antitumor by virtue of the core action mechanism, has multiple application scenes, remarkable effects and a wide application range.

Description

Technical Field

[0001] This invention belongs to the field of metal complexes and bioactive small molecules, and particularly relates to a 2,3-dione-L-gulonic acid copper complex, its preparation method and application. Background Technology

[0002] With the increasing prominence of drug-resistant bacteria and the growing demand for cancer treatment, the development of functional molecules with multiple biological activities has become an important direction in the fields of medicine and biotechnology. Current antibacterial drug development faces challenges such as the continuous emergence of drug resistance, limited mechanisms of action, and a lack of novel lead compounds. Meanwhile, antitumor drug development also presents challenges such as complex targets, significant individual variability, and the difficulty in balancing safety and efficacy. Therefore, developing chemical molecules with novel mechanisms of action and multiple biological activities is of great significance for expanding the development pathways for anti-infective and antitumor drugs.

[0003] In recent years, metal complexes have shown great potential in antibacterial and antitumor drug research due to their unique coordination structures, tunable physicochemical properties, and ability to interact with biomolecules such as DNA. Some metal complexes can exert biological effects by inducing DNA damage, interfering with nucleic acid metabolism, or influencing cell proliferation, thus becoming an important research direction for the development of novel bioactive molecules. However, currently available small molecules that simultaneously possess antibacterial and antitumor activities, along with good chemical stability and application development potential, remain relatively limited.

[0004] Furthermore, phage contamination is a significant factor affecting production stability and product quality in fermentation, biopharmaceutical production, and strain application. Current phage control methods largely rely on physical isolation, process management, or host modification, which often suffer from limitations in applicability, high implementation costs, or insufficient versatility. Therefore, if a small chemical molecule possesses antibacterial and antitumor activities while also exhibiting antiphage activity, its application value in the biopharmaceutical and biomanufacturing fields will be further expanded.

[0005] Based on this, developing a small chemical molecule that has a relatively simple preparation process, stable chemical properties, and combines antiphage, antibacterial, and antitumor activities is of great scientific significance and practical application value for enriching the types of multifunctional active molecules and expanding their application scope in related fields. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a 2,3-diketogulonic acid copper complex, its preparation method, and its applications. This complex is a novel complex (2,3-DKG-Cu complex) formed by the coordination of 2,3-diketogulonic acid (2,3-DKG) with copper ions. It can be applied in antiphage, antibacterial, and antitumor applications.

[0007] To achieve the above objectives, the present invention provides the following technical solution: One of the objectives of this invention is to provide a 2,3-dione-L-gulonic acid copper complex, which is formed by coordination of 2,3-dione-L-gulonic acid with copper ions.

[0008] The 2,3-diketone-L-gulonate copper complex was prepared by mixing and incubating 2,3-DKG solution with a soluble copper salt solution. Its coordination complex structure was qualitatively characterized using UV-Vis spectroscopy and NMR. This complex can insert into DNA and mediate DNA damage. Based on this mechanism, it can effectively inhibit the replication process of bacteriophage DNA, exhibiting significant anti-phage activity. Simultaneously, it can damage bacterial DNA, thereby exerting a highly effective antibacterial effect. Furthermore, it can induce DNA strand breaks in tumor cells, triggering cell cycle arrest and apoptosis, thus inhibiting tumor cell proliferation and demonstrating good anti-tumor activity.

[0009] Furthermore, the copper ions include Cu 2+ Or its reduction product Cu in the system + .

[0010] A second objective of this invention is to provide a method for preparing the 2,3-dione-L-gulonic acid copper complex as described in claim 1, comprising the following steps: Under anaerobic conditions, L-dehydroascorbic acid was mixed with N,N-dimethylformamide (DMF), and sodium hydroxide solution was added to react. The reaction was terminated with acetic acid to prepare a 2,3-diketone-L-gulonic acid solution. A solution of 2,3-dione-L-gulonic acid and a solution of a soluble copper salt were mixed at room temperature to form the copper complex of 2,3-dione-L-gulonic acid.

[0011] Furthermore, the ratio of L-dehydroascorbic acid, N,N-dimethylformamide, sodium hydroxide solution, and acetic acid is 0.12 mmol: 40 µL: 200 µL: 200 µL.

[0012] Furthermore, the concentration of the sodium hydroxide solution is 0.75M; the concentration of the acetic acid is 1.5M.

[0013] Furthermore, the specific steps for terminating the reaction are as follows: after reacting the raw materials in an ice bath for 30 seconds, acetic acid is immediately added to terminate the reaction.

[0014] Further, the concentration of the 2,3-dione-L-gulonic acid solution is 0.5-2 mM; the concentration of the soluble copper salt solution is 100-400 µM.

[0015] The third objective of this invention is to provide an application of the aforementioned 2,3-dione-L-gulonic acid copper complex in the preparation of antiphage products.

[0016] The fourth objective of this invention is to provide a method for preparing the antiphage product, comprising the following steps: diluting 2,3-dione-L-gulonic acid solution and soluble copper salt solution to 0.5 mM and 100 µM respectively, then mixing them and incubating at room temperature for 10 min.

[0017] The fifth objective of this invention is to provide an application of the aforementioned 2,3-dione-L-gulonic acid copper complex in the preparation of antibacterial products.

[0018] The sixth objective of this invention is to provide a method for preparing an antibacterial product, comprising the following steps: diluting a 2,3-dione-L-gulonic acid solution and a soluble copper salt solution to 2 mM and 400 µM respectively, then mixing them and incubating at room temperature for 10 min.

[0019] The seventh objective of this invention is to provide an application of the aforementioned 2,3-dione-L-gulonic acid copper complex in the preparation of antitumor products.

[0020] The eighth objective of this invention is to provide a method for preparing the antitumor product, comprising the following steps: diluting a 2,3-dione-L-gulonic acid solution and a soluble copper salt solution to 2 mM and 400 µM respectively, then mixing them and incubating at room temperature for 10 min.

[0021] Compared with the prior art, the present invention has the following advantages and technical effects: 1) The raw materials for the preparation of 2,3-DKG-Cu complex are readily available reagents such as L-dehydroascorbic acid, N,N-dimethylformamide, and copper salt. The preparation process is simple, the reaction efficiency is high, and it has the technical feasibility for large-scale production.

[0022] 2) This complex can undergo insertional interactions with DNA and mediate DNA damage. Through this core mechanism of action, it achieves multiple biological activities, including antiphage, antibacterial, and antitumor effects, with diverse applications and a wide range of applicability. In terms of antiphage, it can target and inhibit the DNA replication of bacteriophages, exhibiting potential broad-spectrum antiphage effects. In terms of antibacterial, it can directly cause bacterial DNA damage, with highly efficient and broad-spectrum antibacterial effects. In terms of antitumor, it can exert tumor cell cytotoxicity through multiple target mechanisms, such as regulating cell cycle checkpoints and inhibiting DNA repair, with a clear and efficient antitumor mechanism. Attached Figure Description

[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 For the detection of different concentrations of 2,3-DKG and Cu by agarose gel electrophoresis 2+ And the degradation effect of the 2,3-DKG-Cu complex on plasmid pcDNA4.0; Figure 2 2,3-DKG, Cu 2+ Atomic force microscopy (AFM) imaging of calf thymus DNA (CT-DNA) after treatment with 2,3-DKG-Cu complex; Figure 3 For comet experiments to detect 2,3-DKG and Cu 2+ And the damaging effect of 2,3-DKG-Cu complex on intracellular DNA (the left side of each group is a global view, and the right side is a magnified view of the local area). Figure 4 Quantitative analysis results of comet experiments; Figure 5 The inhibitory effect of antiphages on the replication of different phages; Figure 6 The inhibitory effect of antibacterial products on bacterial proliferation; Figure 7 The anti-tumor formula inhibits tumor cell proliferation and apoptosis. Figure 8 Representative histograms of cell cycle distribution after different treatments were generated by flow cytometry (PI staining); where A: PBS control group; B: 2,3-DKG treatment group (2mM); C: Cu 2+ Individual treatment group (400 μM); D: DAT treatment group; Figure 9This is a statistical representation of cell cycle proportions; where A, B, and C are the proportions of cells after different treatments in G0 / G1, S, and G2 / M phases, respectively, and D is the proportion of cells in different cycles after different treatments. Figure 10 The distribution map of apoptosis induced by 2,3-DKG-Cu complex was detected by flow cytometry; where A: control group; B: DAT treatment group; C: Cu 2+ Treatment alone (400µM); D: 2,3-DKG treatment alone (2mM), Q1: percentage of necrotic cells; Q2: percentage of cells in the intermediate and late stages of apoptosis; Q3: percentage of normal cells; Q4: percentage of cells in the early stages of apoptosis; Figure 11 The results show the statistical results of cell apoptosis rate; where A: total apoptosis rate, B: early apoptosis rate. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] This invention provides a 2,3-dione-L-gulonic acid copper complex and its preparation method, specifically including the following steps: I. Preparation steps of the core product: 2,3-dione-L-gulonate copper complex 1) Preparation of 2,3-diketo-L-gulonic acid (2,3-DKG) solution: Under anaerobic conditions, 0.12 mmol of L-dehydroascorbic acid ((L)-DHA) was thoroughly mixed with 40 µL of N,N-dimethylformamide, and 200 µL of 0.75 M sodium hydroxide solution was added. The mixture was placed in an ice bath and reacted for 30 s. Immediately afterwards, 200 µL of 1.5 M acetic acid was added to terminate the reaction, yielding the 2,3-DKG solution. All the above procedures were performed in a glove box purged with high-purity nitrogen under anaerobic conditions.

[0030] 2) Complex synthesis: The 2,3-DKG solution (concentration adjusted to 0.5-2 mM) prepared above was thoroughly mixed with a soluble copper salt solution (concentration adjusted to 100-400 µM) at room temperature and incubated to form a 2,3-diketone-L-gulonic acid copper complex; the copper ion in this complex is Cu. 2+ Or its reduction product Cu in the system + The concentration of the soluble copper salt solution is adjusted using deionized water.

[0031] II. Application This invention provides 2,3-DKG-Cu complex stock solutions of different concentrations according to application scenarios, as shown in the examples below. The "stock solution" can be prepared fresh by diluting a high-concentration 2,3-DKG and CuCl2 stock solution in a certain proportion, or it can be pre-prepared and stored for a short period of time under inert gas protection.

[0032] 1. Preparation steps of antiphage products 1) Prepare a 2,3-DKG solution according to the above core product preparation steps, and at the same time prepare a soluble copper salt solution; 2) Dilute the 2,3-DKG solution to 0.5 mM and the soluble copper salt solution to 100 µM; 3) Mix the two diluted solutions at room temperature and incubate for 10 minutes to obtain the 2,3-dione-L-gulonic acid copper complex product for antiphage.

[0033] The 2,3-DKG-Cu complex can inhibit phage DNA replication by interacting with and causing DNA breaks in phage genomic DNA, thereby reducing phage proliferation and protecting host bacteria. It can be used for phage contamination control and strain preservation in fermentation processes.

[0034] 2. Preparation steps of antibacterial products 1) Prepare a 2,3-DKG solution according to the above core product preparation steps, and at the same time prepare a soluble copper salt solution; 2) Dilute the 2,3-DKG solution to 2 mM and the soluble copper salt solution to 400 µM; 3) Mix the two diluted solutions at room temperature and incubate for 10 minutes to obtain the antibacterial 2,3-dione-L-gulonic acid copper complex product.

[0035] When using, dilute this antibacterial product to the working concentration in LB or other culture media. The dosage of 2,3-DKG and Cu can be adjusted according to the sensitivity of different bacterial strains. 2+ Final concentration. For example, Escherichia coli MG1655, Salmonella ATCC14025, or Klebsiella pneumoniae KP1.

[0036] 2,3-DKG-Cu complexes can bind to DNA through insertion, causing damage to bacterial DNA and thus inhibiting bacterial proliferation. They can be used for disinfection, sterilization, and pathogen control.

[0037] 3. Preparation steps of anti-tumor products 1) Prepare a 2,3-DKG solution according to the above core product preparation steps, and at the same time prepare a soluble copper salt solution; 2) Dilute the 2,3-DKG solution to 2 mM and the soluble copper salt solution to 400 µM; 3) Mix the two diluted solutions at room temperature and incubate for 10 minutes to obtain the antitumor 2,3-dione-L-gulonic acid copper complex product.

[0038] When using this anti-tumor product, dilute it to the working concentration in cell culture medium such as DMEM. It is recommended to prepare and use it immediately. Alternatively, it can be stored at 4°C in the dark for a short period of time, not exceeding 2 weeks.

[0039] The 2,3-DKG-Cu complex can induce DNA strand breaks and disrupt the cell cycle in tumor cells, triggering apoptosis and thus inhibiting tumor cell proliferation; it can be prepared into pharmaceutically acceptable formulations for use in tumor treatment-related fields.

[0040] The core of the preparation of each product in this invention is the coordination binding of 2,3-DKG with copper ions. For products with different uses, only the dilution concentration of the 2,3-DKG solution and the soluble copper salt solution needs to be adjusted. The remaining operating parameters are consistent with the general requirements for the preparation of the core product.

[0041] Unless otherwise specified, "room temperature" in this invention refers to 37°C.

[0042] All raw materials used in this invention were purchased commercially. Unless otherwise specified, experimental materials, reagents, and methods can be applied using conventional techniques in the field.

[0043] The DAP, DAB, and DAT in the following embodiments of the present invention are merely example formulations. Those skilled in the art can adjust the formulation concentration, solvent, and buffer system without departing from the spirit of the present invention.

[0044] The 2,3-DKG-Cu complex of the present invention can be prepared as: an aqueous additive for fermentation systems, a spray or soaking solution for disinfection / sterilization, or an injection / oral preparation / topical drug delivery preparation for antitumor purposes; it can also be combined with conventional excipients (such as PBS, physiological saline, buffer systems, lyophilization protectants, polyethylene glycol, etc.) to form compound or sustained-release preparations.

[0045] The technical solution of the present invention will be further illustrated by the following embodiments.

[0046] Example 1: Preparation and characterization of 2,3-DKG-Cu complex 1) Preparation of 2,3-diketo-L-gulonic acid (2,3-DKG) stock solution: In an oxygen-free, nitrogen-blown glove box, 0.12 mmol of L-dehydroascorbic acid and 40 µL of N,N-dimethylformamide were thoroughly mixed at room temperature. 200 µL of 0.75 M NaOH solution was added, and the mixture was placed in an ice bath and reacted for 30 s. Immediately afterward, 200 µL of 1.5 M acetic acid was added to terminate the reaction, resulting in 600 µL of 200 mM 2,3-DKG solution, which was stored at -80 °C.

[0047] 2) Preparation of CuCl2 stock solution: Prepare a 20mM CuCl2 stock solution with deionized water at room temperature and store at 4℃.

[0048] UV-Vis spectroscopy characterization: Using Tris-HCl as the detection buffer system, the characteristic absorption peak of 2,3-DKG at 288 nm was recorded; Cu was added. 2+ The absorption peak then red-shifted and the absorbance increased; after adding EDTA, the red-shifted peak returned to its initial position and the absorbance decreased, indicating that the chelation formed a complex and that the process was reversible.

[0049] Nuclear magnetic resonance characterization: The complex was prepared to a suitable concentration in deuterated water, and then... 1 HNMR, 13 CNMR, 1H-1HCOSY, and 1H-13CHSQC spectroscopic analyses were used to confirm the occurrence and binding mode of coordination complexation.

[0050] Example 2: The insertion, binding, and cleavage of DNA by the complex This invention verifies the DNA damage capability of the 2,3-DKG-Cu complex from four dimensions: circular DNA (plasmid), linear DNA (phage genome), atomic force microscopy (AFM) observation, and intracellular comet experiment.

[0051] 1.1) Circular DNA (plasmid) fragmentation: Using pcDNA4.0 supercoiled closed circular DNA as a model (100 ng), different concentrations of 2,3-DKG-Cu complexes (groups A, B, C, D, E, F, G, H, and I) and corresponding 2,3-DKG (the 2,3-DKG stock solution prepared in Example 1 was diluted to 200 μM, 150 μM, 100 μM, 50 μM, 2 μM, 1.5 μM, 1 μM, 0.5 μM, and 0.4 μM, respectively) and Cu were added to Tris-HCl buffer (pH=7.2). 2+ (The CuCl2 stock solution prepared in Example 1 was diluted to 4 μM, 10 μM, 20 μM, 30 μM, and 40 μM, respectively), and then incubated at 37 °C before agarose gel electrophoresis (see [link to example]). Figure 1 (A and B in the text).

[0052] The groups for different concentrations of the 2,3-DKG-Cu complex were defined as follows: Group A: 5 μL of 2,3-DKG at a concentration of 50 μM was mixed with 5 μL of CuCl2 at a concentration of 10 μM; Group B: 5 μL of 100 μM 2,3-DKG was mixed with 5 μL of 20 μM CuCl2. Group C: 5 μL of 150 μM 2,3-DKG was mixed with 5 μL of 30 μM CuCl2; Group D: 5 μL of 2,3-DKG at a concentration of 200 μM was mixed with 5 μL of CuCl2 at a concentration of 50 μM.

[0053] Group E: 5 μL of 0.4 mM 2,3-DKG was mixed with 5 μL of 4 μM CuCl2. Group F: 5 μL of 0.5 mM 2,3-DKG was mixed with 5 μL of 4 μM CuCl2; Group G: 5 μL of 1 mM 2,3-DKG was mixed with 5 μL of 4 μM CuCl2; Group H: 5 μL of 1.5 mM 2,3-DKG was mixed with 5 μL of 4 μM CuCl2.

[0054] Group I: 5 μL of 2,3-DKG at a concentration of 2 mM was mixed with 5 μL of CuCl2 at a concentration of 4 μM.

[0055] Figure 1 For the detection of different concentrations of 2,3-DKG and Cu by agarose gel electrophoresis 2+ The graph shows the degradation effect of the 2,3-DKG-Cu complex on plasmid pcDNA4.0. It can be seen that the 2,3-DKG-Cu complex is effective when the 2,3-DKG concentration is between 50-200 mM and the Cu concentration is high. 2+ At concentrations in the range of 50-10 μM, the complex can transform supercoiled DNA (SC) into a nicked circular / linear form (NC). However, adding the same concentration of 2,3-DKG or Cu alone will not produce the same result. 2+ The plasmid remained in the SC state (Figure A). When the concentration of 2,3-DKG was increased to the range of 2-0.4 mM, the complex at this concentration exhibited stronger fragmentation and degradation of the plasmid. At this concentration, 2,3-DKG alone had a certain ring-opening effect on the plasmid, but no significant degradation effect (Figure B).

[0056] 2. AFM observation of CT-DNA morphology: Calf thymus DNA (CT-DNA) from different treatment groups was adsorbed onto mica sheets, and the DNA conformation was observed using AFM. The specific method is as follows: Sample preparation for AFM imaging: Prepare a DNA (500 ng / μL) stock solution and a deposition buffer containing 1 mM MgCl2, 10 mM HEPES (pH 7.5); take 50 ng of CT-DNA, 50 µM 2,3-DKG, 10 µM CuCl2 and the deposition buffer and mix to obtain 50 µl, incubate at 25°C for 30 minutes; at the same time, set 50 µM 2,3-DKG, 10 µM CuCl2 and Control.

[0057] AFM imaging: 2-5 µL of sample was deposited and dried on a mica sheet at room temperature. The sample was then scanned and imaged using an MFP-3D atomic force microscope at a scanning speed of approximately 2 Hz under a high voltage field of ±220 V. The imaging was analyzed using Oxford Instruments Asylum Research AFM software.

[0058] Experimental results showed that the DNA conformation of the 2,3-DKG and CuCl2 treatment groups was similar to that of the control group, while the DNA morphology of the 2,3-DKG-Cu complex group changed significantly: the linear length of the DNA was significantly shortened, which is presumably due to the complex molecules cutting or degrading the DNA into smaller fragments, resulting in DNA damage.

[0059] Figure 22,3-DKG, Cu 2+ AFM imaging of CT-DNA after treatment with 2,3-DKG-Cu complex; the results showed that 2,3-DKG or Cu... 2+ The DNA morphology of the treatment group alone was similar to that of the blank control, while the DNA treated with the 2,3-DKG-Cu complex showed obvious breaks / fragments, suggesting that the complex has a stronger damaging effect on DNA.

[0060] 3. Comet assay for intracellular DNA damage: Log-phase HeLa cells were seeded into 6-well plates to form a monolayer. PBS blank control, 2 mM 2,3-DKG control (1 mL dissolved in PBS), 400 μM CuCl2 control (1 mL dissolved in PBS), and a treatment group containing 2,3-DKG-Cu complex (1 mL dissolved in PBS) were added. After 24 h of culture, an alkaline comet assay was used to detect DNA damage such as single-strand and double-strand breaks.

[0061] Figure 3 For comet experiments to detect 2,3-DKG and Cu 2+ The images show the damaging effects of the 2,3-DKG-Cu complex on intracellular DNA (the left side of each group is a global view, and the right side is a magnified view of a local area). Under a fluorescence microscope, it can be observed that the 2,3-DKG-Cu treatment group shows obvious tailing, while the control group does not show obvious tailing.

[0062] Tail DNA% and Olive Tail Moment were quantitatively analyzed using software such as ImageJ / OpenComet. Intracellular DNA damage was detected using the comet assay: Log-phase HeLa cells were seeded into 6-well plates to form a monolayer. PBS blank control, 2 μM 2,3-DKG control (1 mL dissolved in PBS), 400 μM CuCl2 control (1 mL dissolved in PBS), and a treatment group containing 2,3-DKG-Cu complex (1 mL dissolved in PBS) as described in "Example 3, Preparation Steps of Antitumor Products" were added. After 24 h of culture, the basic comet assay was used to detect DNA damage such as single-strand and double-strand breaks. Figure 4The results of the comet experiment are presented in the figure. As can be seen from the figure, the average Tail DNA percentage in the Control group was 19.28%, and the average Olive Tail Moment was 7.53; the average Olive Tail DNA percentage in the 2,3-DKG treatment group was 23.66%, and the average Olive Tail Moment was 6.50; the average Tail DNA percentage in the CuCl2 treatment group was 20.38%, and the average Olive Tail Moment was 4.19; and the average Tail DNA percentage in the 2,3-DKG-Cu complex treatment group was 59.48%, and the average Olive Tail Moment was 62.62. Statistical analysis showed that compared with the Control group, the 2,3-DKG treatment group, and the CuCl2 treatment group, the Tail DNA % and Olive Tail Momen of the 2,3-DKG-Cu complex treatment group were significantly increased. However, compared with the Control group, there were no significant changes in Tail DNA % and Olive Tail Momen in the 2,3-DKG treatment group and the CuCl2 treatment group, indicating that the 2,3-DKG-Cu complex can induce DNA damage in cells.

[0063] Application Example 1 The preparation of an antiphage product includes the following steps: Under anaerobic nitrogen protection, the 2,3-DKG stock solution with a concentration of 200 mM prepared in Example 1 was diluted to 0.5 mM with deionized water, and the CuCl2 stock solution with a concentration of 20 mM prepared in Example 1 was diluted to 100 μM. The two were mixed at a volume ratio of 500 μL:500 μL and incubated at room temperature for 10 min (during which the mixture was slowly inverted to mix) to obtain the 2,3-diketone-L-gulonate copper complex product for phage resistance (DAP, 2,3-DKG-Cu-AntiPhage).

[0064] Application Example 2 The preparation of an antibacterial product includes the following steps: Under anaerobic nitrogen protection, the 2,3-DKG stock solution with a concentration of 200 mM prepared in Example 1 was diluted to 2 mM with deionized water, and the CuCl2 stock solution with a concentration of 20 mM prepared in Example 1 was diluted to 400 μM. The two were mixed and incubated at room temperature for 10 min (during which they were slowly inverted to mix) to obtain the antibacterial 2,3-diketone-L-gulonate copper complex product (DAB, 2,3-DKG-Cu-AntiBac).

[0065] Application Example 3 The preparation of an anti-tumor product includes the following steps: Under anaerobic nitrogen protection, the 2,3-DKG stock solution with a concentration of 200 mM prepared in Example 1 was diluted to 2 mM with deionized water, and the CuCl2 stock solution with a concentration of 20 mM prepared in Example 1 was diluted to 400 μM. The two were mixed and incubated at room temperature for 10 min to obtain the antitumor 2,3-diketone-L-gulonate copper complex product (DAT, 2,3-DKG-Cu-AntiTumor).

[0066] Example 3: Validation of the use of antiphages (inhibition of phage replication) (1) Host bacteria pretreatment: In order to reduce the influence of intracellular copper ion background on the effect of complex, Escherichia coli MG1655, Salmonella ATCC14025 or Klebsiella pneumoniae KP1 were continuously passaged in M9 basal medium to obtain strains with relatively low intracellular copper content. (2) Infection and treatment: After centrifugation, the logarithmic phase bacterial culture was resuspended in LB medium containing DAP (using the anti-phage formulation prepared in Example 1) or LB control medium without complex; different phages were added at MOI=0.1 and cultured at 37℃ with shaking for 90 min; (3) Detection indicators: Phage titer (Log PFU / mL) was measured by sampling 4 hours after infection; (4) Detection results: After DAP treatment, the titer of Escherichia coli phage T1 decreased by about 4 log, the titer of T7 decreased by about 3 log, the titer of Salmonella phage PhiSal decreased by about 4 log, and the titer of Klebsiella pneumoniae phage PhiKP decreased by about 3 log (see [link to DAP treatment]). Figure 5 ).

[0067] Example 4: Validation of antibacterial use (inhibition of bacterial proliferation) (1) Pretreatment of strains: In order to reduce the influence of intracellular copper ion background on the effect of complex, Escherichia coli MG1655, Salmonella ATCC14025 or Klebsiella pneumoniae KP1 were continuously passaged in M9 basal medium to obtain strains with relatively low intracellular copper content. (2) Treatment and culture: After centrifugation, the logarithmic phase bacterial culture was resuspended in M9 basal medium containing DAB (the antibacterial formula prepared in Example 2) and M9 control medium without complex. 2,3-DKG (2mM) or CuCl2 (400μM) were used as control groups. All groups were cultured at 37℃ with shaking for 24h. (3) Detection indicators: OD600 was measured and a growth curve was plotted; (4) Detection results: After DAB treatment, the growth of strain MG1655 was significantly inhibited for the first 12 hours, the growth of ATCC14025 was significantly inhibited for the first 8 hours, and the growth of KP1 was significantly inhibited for the first 7 hours (see [link]). Figure 6 ).

[0068] Example 5: Validation of anti-tumor applications (inhibition of tumor cell proliferation, induction of apoptosis) (1) CCK-8 cell viability assay: A549 and Hep G2 tumor cell lines were selected and treated with DAT (using the antitumor formulation prepared in Example 3) for 24 h. Cell viability was assessed using CCK-8. The results showed that the survival rate of A549 cells decreased to about 40.73% and the survival rate of Hep G2 cells decreased to about 44.84% after DAT treatment (see [link to CCK-8 assay]). Figure 7 ); (2) Cell cycle arrest: Cell cycle distribution was detected by PI staining flow cytometry.

[0069] Figure 8 Representative histograms (PI staining) for cell cycle distribution after different treatments were generated by flow cytometry. A: PBS control group; B: 2,3-DKG alone treatment group (2 mM); C: Cu 2+ Individual treatment group (400 μM); D: DAT treatment group. Figure 9 The cell cycle proportions were statistically analyzed, where A, B, and C represent the proportions of cells in G0 / G1, S, and G2 / M phases after different treatments, respectively, and D represents the proportions of cells in different phases after different treatments. The results showed that, compared with the PBS control group, the DAT treatment group ( Figure 8 D in Figure 9 A decrease in the G0 / G1 phase ratio and an increase in the S and / or G2 / M phase ratio indicate cell cycle arrest, suggesting that DAT treatment interfered with a key step in cell division, leading to DNA damage. In contrast, the 2,3-DKG-treated group (…) Figure 8 B in Figure 9 ) and Cu 2+ Individual processing group ( Figure 8 C in Figure 9 The cell cycle distribution was consistent with that of the PBS control group.

[0070] (3) Apoptosis: Apoptosis was detected by Annexin V-FITC / PI double staining flow cytometry.

[0071] Figure 10 The distribution map of apoptosis induced by 2,3-DKG-Cu complex was obtained by flow cytometry. In the diagram, A: control group; B: DAT treatment group; C: Cu... 2+Treatment alone (400 μM); D: 2,3-DKG treatment alone (2 mM), Q1: percentage of necrotic cells; Q2: percentage of cells in the intermediate and late stages of apoptosis; Q3: percentage of normal cells; Q4: percentage of cells in the early stages of apoptosis. Figure 11 The results show the statistical results of cell apoptosis rates, where A represents the total apoptosis rate and B represents the early apoptosis rate. The results showed that the average apoptosis rate in the control group was 4.75%, and the average early apoptosis rate was 3.71%; the average apoptosis rate in the 2,3-DKG-Cu complex treatment group was 22.02%, and the average early apoptosis rate was 14.99%; the average apoptosis rate in the 2,3-DKG treatment group was 5.81%, and the average early apoptosis rate was 4.17%; Cu 2+ The average apoptosis rate in the treatment groups was 6.67%, and the average early apoptosis rate was 4.90%. Statistical analysis showed that the total apoptosis rate and early apoptosis rate in the 2,3-DKG-Cu complex treatment group were significantly higher than those in the control group, the 2,3-DKG treatment group, and the Cu treatment group. 2+ The treatment group showed that the 2,3-DKG-Cu complex significantly increased the apoptosis rate, indicating that the complex DAT can induce apoptosis.

[0072] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A 2,3-dione-L-gulonic acid copper complex, characterized in that, It is formed by coordination of 2,3-diketo-L-gulonic acid with copper ions.

2. A method for preparing the 2,3-dione-L-gulonate copper complex as described in claim 1, characterized in that, Includes the following steps: Under anaerobic conditions, L-dehydroascorbic acid was mixed with N,N-dimethylformamide, and sodium hydroxide solution was added to react. The reaction was terminated with acetic acid to prepare 2,3-dione-L-gulonic acid solution. A solution of 2,3-dione-L-gulonic acid and a solution of a soluble copper salt were mixed at room temperature to form the copper complex of 2,3-dione-L-gulonic acid.

3. The method for preparing the 2,3-dione-L-gulonate copper complex according to claim 2, characterized in that, The ratio of L-dehydroascorbic acid, N,N-dimethylformamide, sodium hydroxide solution and acetic acid is 0.12 mmol: 40 µL: 200 µL: 200 µL; The concentration of the sodium hydroxide solution is 0.75M; the concentration of the acetic acid is 1.5M. The specific steps for terminating the reaction are as follows: after adding sodium hydroxide solution and reacting in an ice bath for 30 seconds, acetic acid is immediately added to terminate the reaction.

4. The method for preparing the 2,3-dione-L-gulonate copper complex according to claim 2, characterized in that, The concentration of the 2,3-diketone-L-gulonic acid solution is 0.5-2 mM; the concentration of the soluble copper salt solution is 100-400 µM.

5. The use of the 2,3-dione-L-gulonate copper complex as described in claim 1 in the preparation of antiphage products.

6. A method for preparing the antiphage product as described in claim 5, characterized in that, Includes the following steps: The 2,3-dione-L-gulonic acid solution and the soluble copper salt solution were diluted to 0.5 mM and 100 µM respectively, then mixed and incubated at room temperature.

7. The use of the 2,3-dione-L-gulonic acid copper complex as described in claim 1 in the preparation of antibacterial products.

8. A method for preparing an antibacterial product as described in claim 7, characterized in that, Includes the following steps: The 2,3-dione-L-gulonic acid solution and the soluble copper salt solution were diluted to 2 mM and 400 µM respectively, then mixed and incubated at room temperature.

9. The use of the 2,3-dione-L-gulonic acid copper complex as described in claim 1 in the preparation of antitumor products.

10. A method for preparing an antitumor product as described in claim 9, characterized in that, Includes the following steps: The 2,3-dione-L-gulonic acid solution and the soluble copper salt solution were diluted to 2 mM and 400 µM respectively, then mixed and incubated at room temperature.

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