Short peptide-Zn coordination compound with SOD (superoxide dismutase) activity as well as preparation and application of short peptide-Zn coordination compound

Abstract

The invention discloses an oligopeptide-Zn coordination compound with SOD (superoxide dismutase) activity as well as preparation and application of the oligopeptide-Zn coordination compound, and belongs to the technical field of polypeptides. The specific synthesis steps of the oligopeptide-Zn (DT-Zn) coordination compound are as follows: designing and synthesizing oligopeptide; characterizing the synthesized oligopeptide through mass spectrum and high performance liquid chromatography; respectively dispersing oligopeptide and zinc sulfate in deionized water, and shaking uniformly; and adding a zinc sulfate solution into the oligopeptide solution, oscillating, uniformly mixing, and incubating at the constant temperature of 37 DEG C for 30 minutes to obtain the DT-Zn coordination compound. A coordination compound is obtained through direct coordination of N-terminal acetylated and C-terminal aminated oligopeptide (Ac-FFFFHHHK-NH2) and zinc ions, particulate matter monodispersed in an aqueous solution is self-assembled into spiral fibers, a natural enzyme activity center is formed, the SOD activity is greatly improved, the effect of removing ROS is achieved, healing of multiple bacterial infected wounds is promoted, and the effect of removing the ROS is achieved. And the biological safety is good.

Description

Technical Field

[0001] This invention belongs to the field of peptide nanozyme technology, specifically, it relates to a short peptide-Zn coordination compound with SOD activity and its preparation and application. Background Technology

[0002] Short peptides are short chains composed of several amino acid monomers arranged in a specific sequence and linked by peptide bonds. Compared with proteins, which have large molecular weights and complex structures, short peptides have become an ideal biomedical material due to their unique advantages such as simple structure, easy synthesis, good biocompatibility, non-toxicity, self-degradability, and ease of functionalization and modification.

[0003] Compared to inorganic nanoparticles or metal complexes, peptides and their derivatives are simple yet essential building blocks of life, exhibiting excellent biocompatibility in the construction of supramolecular enzymes. Current research has focused on replicating the catalytic capabilities of enzymes by developing enzyme-like peptides. These peptides, as enzyme mimics, offer advantages over traditional enzymes, including higher stability under extreme conditions, simpler synthesis, lower cost, greater tunability, lower immunogenicity, and enhanced functionality. These properties make them highly promising for applications in biotechnology, medicine, and industry, bringing new hope for human health and sustainable development.

[0004] Superoxide dismutase (SOD) holds great potential in scavenging reactive oxygen species and treating wound infections, but its clinical application faces bottlenecks such as poor stability of natural enzymes, high immunogenicity, and insufficient cell membrane permeability. Existing artificial SOD mimics, such as small molecule metal complexes and nanozymes, generally suffer from poor biocompatibility, easy dissociation of metal ions leading to toxicity, or poor biodegradability, making it difficult to balance efficacy and safety. This severely limits their application in in vivo therapy. Therefore, it is necessary to explore a safer compound that combines SOD-like activity with broad-spectrum antibacterial capabilities. Summary of the Invention

[0005] To overcome the technical problems existing in the background art, this invention proposes a short peptide-Zn coordination compound with SOD activity, its preparation and application. By designing and synthesizing short peptides, zinc ions are introduced as coenzyme factors to coordinate with histidine and lysine in the short peptides, causing the monodisperse particles in aqueous solution to self-assemble into helical fibers, forming natural enzyme active centers, thus greatly enhancing SOD activity, playing a role in scavenging ROS, and promoting the healing of wounds infected with multiple bacteria. This coordination compound can also achieve efficient bacterial killing through a unique physical-mechanical bactericidal mechanism, exhibiting excellent antibacterial properties compared with the biochemical effects of traditional antibiotics.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: A short peptide-Zn coordination compound with SOD activity, wherein the short peptide-Zn coordination compound contains an amino acid sequence that can successfully coordinate with zinc ions, specifically Ac-FFFFHHHK-NH2.

[0007] The specific steps of the preparation method are as follows: 1) Design and synthesize the short peptide Ac-FFFFHHHK-NH2; 2) Characterization of the molecular weight of short peptides: The synthesized short peptides were characterized by mass spectrometry and high performance liquid chromatography; 3) Preparation of short peptide and zinc ion solution: Disperse the short peptide and zinc sulfate separately in deionized water and shake well; 4) Preparation of coordination compound: Add zinc sulfate solution to short peptide solution and shake to mix evenly. Incubate at 37 °C for 30 min to obtain short peptide-Zn coordination compound.

[0008] Preferably, in step 3), the concentrations of the short peptide and the zinc sulfate solution are 1 mg / mL.

[0009] Preferably, in step 4), the molecular weight ratio of the short peptide solution to the zinc sulfate solution is 1:1.

[0010] The aforementioned coordination compounds can be used in the treatment of wound inflammation caused by antibacterial and bacterial infections.

[0011] Furthermore, the bacteria that this coordination compound acts on are one or more of Escherichia coli, Salmonella, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The beneficial effects of this invention are:

[0012] (1) In this invention, zinc ions are introduced as coenzyme factors into short peptides with an amino acid series of Ac-FFFFHHHK-NH2, and coordinate with histidine and lysine in the short peptides, so that the particles dispersed in the aqueous solution self-assemble into helical fibers to form natural enzyme active centers. This invention synthesizes short peptide-Zn (DT-Zn) coordination compounds through a specific reaction system, which greatly enhances SOD activity.

[0013] (2) The coordination compound prepared in this invention constructs an enzyme-like catalytic pocket centered on Zn²⁺ by biomimeticly mimicking the active center structure of natural SOD. It achieves the dual-core function of traditional Cu / Zn-SOD with mononuclear Zn²⁺, avoiding the use of toxic metal Cu²⁺. At the same time, it greatly enhances the activity of SOD, solves the problem of easy inactivation and poor permeability of natural SOD in wound application, fully exerts the role of clearing ROS, enhances the anti-inflammatory ability, and promotes the healing of wounds infected with multiple bacteria. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the amino acid sequence structure of a short peptide; Figure 2 This is the mass spectrum (MS) of a short peptide. Figure 3 This is a high-performance liquid chromatogram (HPLC) of a short peptide. Figure 4 This is a schematic diagram of the coordination between a short peptide and zinc ions at a molecular weight ratio of 1:1. Figure 5 Mass spectrometry (MS) of DT-Zn coordination compound (1:1); Figure 6 The UV-Vis absorption spectrum of the DT-Zn coordination compound (1:1). Figure 7 The infrared absorption spectrum (FTIR) of the DT-Zn coordination compound (1:1); Figure 8 Circular dichroism (CD) spectrum of DT-Zn coordination compound (1:1); Figure 9 Transmission electron microscopy (TEM) and dynamic light scattering (DLS) particle size distributions of the DT-Zn coordination compound (1:1) before and after coordination. Figure 10 Schematic diagram of SOD activity of DT-Zn coordination compound (1:1); Figure 11 A schematic diagram illustrating the superoxide anion radical scavenging ability of the DT-Zn coordination compound (1:1); Figure 12 The diagram shows the bactericidal effect of DT-Zn coordination compound (1:1) on different bacteria; Figure 13 The effect of DT-Zn coordination compound (1:1) on inflammatory wounds with bacterial infection. Detailed Implementation

[0015] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the content described. Example 1

[0016] The specific steps for preparing a short peptide-Zn coordination compound with SOD activity are as follows: 1) Design and synthesize the short peptide Ac-FFFFHHHK-NH2, the structural diagram of which is shown below. Figure 1 As shown.

[0017] 2) The molecular weight of the synthesized short peptides was characterized by mass spectrometry (MS) and high-performance liquid chromatography (HPLC); the mass spectra are shown below. Figure 2As shown, the high-performance liquid chromatogram is as follows: Figure 3 As shown, characterization revealed that when carrying three charges, the molecular weight was 396.7, consistent with the theoretical mass of the molecule. High-resolution mass spectrometry characterization showed that the purity was over 90%.

[0018] 3) Disperse the short peptide and zinc sulfate separately in deionized water to prepare a solution with a concentration of 1 mg / mL, and shake well.

[0019] 4) Add zinc sulfate solution to the short peptide solution at a molecular weight ratio of 1:1, shake to mix thoroughly, and then incubate at 37 ℃ for 30 min to obtain the DT-Zn coordination compound. Its structural schematic diagram is shown below. Figure 4 As shown.

[0020] The synthesized coordination compounds were characterized using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MA / FAST), and the mass spectra are shown below. Figure 5 As shown, the molecular weight is 1249.5, which is consistent with the theoretical mass of this coordination compound. Example 2

[0021] The application of the DT-Zn coordination compound (1:1) prepared in Example 1 in in vitro sterilization includes the following steps: 1) Activation and culture of bacteria: Escherichia coli, Salmonella, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii were cultured separately in liquid culture medium at 37 °C and 150 rpm for 12 h until their concentration reached 1 × 10⁻⁶. 8 ~10 9 CFU / mL, bacterial suspension at 9700 g Collect bacterial cells by centrifugation for 5 min, wash 2-3 times with sterile deionized water, and then dilute the bacteria to a concentration of 1×10⁻⁶. 6 ~10 7 CFU / mL; 2) Test the bactericidal performance of DT-Zn: Transfer 1 mL of diluted bacterial solution to a 1.5 mL centrifuge tube, slowly add 0.2 mL of DT-Zn coordination compound (1:1) solution, incubate in a shaker at 37 ℃ for 30 min and then measure the bacterial concentration; at the same time, compare the bactericidal ability of DT and Zn²⁺, and other operation steps and reaction parameters are the same as DT-Zn.

[0022] 3) Colony count: Take 0.2 mL of bacterial culture before and after DT, Zn²⁺, and DT-Zn treatments, and dilute with sterile deionized water at a concentration of 10 μL. 5 Take 0.2 mL of the diluted liquid and spread it on a solid culture medium. Incubate the medium on a shaker at 37 °C for 18 h and count the number of colonies on the culture dish.

[0023] The effects of DT, Zn²⁺ and DT-Zn on different bacteria are as follows: Figure 12 As shown, compared with the bacterial groups treated with DT and Zn²⁺, the bacterial group treated with DT-Zn exhibits highly efficient bactericidal ability and broad-spectrum antibacterial properties. Example 3

[0024] The application of the DT-Zn coordination compound (1:1) prepared in Example 1 in in vivo anti-inflammatory activity is described in the following steps: 1) Establishing a mouse wound model: First, mice were divided into three groups. After shaving the hair on their backs, circular skin wounds with a diameter of 8 mm were created. One group served as the control group, receiving 50 µL of physiological saline; the other two groups received 50 µL of (10) [unclear text - possibly a specific type of saline wound]. 8 The wound was infected with a bacterial solution of Escherichia coli (CFU / mL). On the second day, 50 µL of normal saline was dripped into the wound of the uninfected group; the other two infected groups were treated with either 50 µL of normal saline or 50 µL of DT-Zn (200 µg / mL) coordination compound solution.

[0025] 2) Record the wound healing status of the three groups of mice within 15 days of treatment.

[0026] The anti-inflammatory effects of DT-Zn coordination compounds are as follows: Figure 13 As shown, compared with the control group treated with saline, mice in the bacterial infection group exhibited significant skin inflammation on the second day. After treatment with both saline and DT-Zn, the DT-Zn-treated mice showed better wound healing. This indicates that DT-Zn coordination compounds can scavenge excess free radicals at the wound site through their SOD activity, reducing oxidative stress damage and inflammatory responses, creating a favorable microenvironment for tissue repair, effectively treating bacterial infection wounds, and promoting wound healing. Experimental Analysis I. Spectral Data Validation of DT-Zn Coordination Compounds

[0027] The absorption spectra of the raw material short peptide (DT), zinc ions (Zn²⁺), and short peptide-zinc coordination compound (DT-Zn) were detected by UV-Vis spectrophotometry. The detection results are as follows: Figure 6 As shown, the absorption spectrum of the DT-Zn coordination compound changed significantly, with a new characteristic absorption peak appearing near 220 nm. This indicates that the imidazole group of histidine in DT coordinated with Zn²⁺.

[0028] The infrared spectra of DT and DT-Zn were detected using a Fourier transform infrared spectrometer. The detection results are as follows: Figure 7 As shown, from Figure 7The peaks at 2816 cm⁻¹ and 2730 cm⁻¹ are characteristic peaks generated by the Fermi resonance of the free -NH₂ group at the end of the lysine (K) side chain in DT. These peaks are significantly weakened in DT-Zn, providing evidence of direct coordination between the amino group of the lysine (K) residue side chain in DT and Zn²⁺. The stretching vibration peak at 1600-1650 cm⁻¹ is the C=N / C=C stretching vibration peak in DT, which red-shifts after coordination with Zn²⁺, a key indicator of histidine coordination. The peak at 620 cm⁻¹ in DT-Zn is stronger than that in DT, which is due to the weak out-of-plane deformation vibration of the aromatic ring in DT, while the newly formed Zn-N stretching vibration has strong infrared activity. These data indicate that Zn²⁺ coordinates with the imidazole group in DT.

[0029] The circular dichroism (CD) spectra of DT and DT-Zn solutions were detected by circular dichroism spectroscopy (CD), and the results are as follows: Figure 8 As shown, the results indicate that coordination of Zn²⁺ induces DT to form a more stable and ordered secondary structure, which is an important structural basis for its biological functions.

[0030] The morphology and particle size of DT and DT-Zn were detected by transmission electron microscopy and dynamic light scattering particle size analyzer. The results are as follows: Figure 9 As shown, DT initially appears as small, round nanoparticles. After coordinating with Zn²⁺ to obtain DT-Zn coordination compounds, they self-assemble into larger nanofibers. II. Verification of SOD activity of DT-Zn coordination compounds

[0031] The SOD activities of DT, Zn²⁺, and DT-Zn were detected and compared using a total SOD activity assay kit (WST-8 method). WST-8 reacts with superoxide anions catalyzed by xanthine oxidase to produce water-soluble formazan dye. Since SOD can catalyze the disproportionation of superoxide anions, this reaction step can be inhibited by SOD. Therefore, SOD activity is negatively correlated with the amount of formazan dye produced. The enzyme activity of SOD was calculated by colorimetric analysis of the WST-8 products.

[0032] The specific operating steps are as follows: Take 20 μL of DT-Zn coordination compound solution, add 160 μL of prepared WST-8 / enzyme working solution and 20 μL of reaction starter solution, incubate in a shaker at 37 ℃ for 30 min, record the absorbance value at 450 nm wavelength using a microplate reader, and calculate the inhibition rate of SOD on the WST-8 reaction. Compare the SOD activities of DT and Zn²⁺, with other operating steps and reaction parameters the same as for DT-Zn.

[0033] like Figure 10As shown, the absorbance value of the DT-Zn group was significantly lower than that of the DT group and the Zn²⁺ group, and the inhibition rate of the WST-8 reaction was significantly improved.

[0034] The scavenging abilities of DT, Zn²⁺, and DT-Zn coordination compounds on superoxide anion radicals in a photochemical system were directly detected and compared using electron paramagnetic resonance (EPR) technology. The classic riboflavin-methionine-nitroblue tetrazolium (NBT) photoreduction reaction system was used as the superoxide anion radical generation source. In this system, riboflavin is excited under light irradiation, transferring electrons to dissolved oxygen to generate superoxide anion radicals. If the analyte exhibits SOD-like activity, catalyzing the disproportionation reaction of superoxide anion radicals, the intensity of the characteristic signal of superoxide anion radicals detected by EPR will be significantly reduced.

[0035] The specific operating procedures were as follows: the blank control group consisted of a superoxide anion radical generation system containing only superoxide anion radicals (riboflavin / L-methionine / NBT concentrations were all 10 mM); the DT-Zn group consisted of a generation system to which 20 μL of DT-Zn coordination compound solution was added. The reaction system was irradiated under a light source of equal intensity for 10 min to fully generate superoxide anion radicals, and then immediately scanned using an EPR spectrometer under the same conditions to capture the characteristic signal of the superoxide anion radicals. Simultaneously, the characteristic signals of the superoxide anion radicals in the DT group and the Zn²⁺ group were compared; other operating procedures and reaction parameters were the same as in the DT-Zn group.

[0036] like Figure 11 As shown, the DT-Zn coordination compound group exhibited the weakest superoxide anion radical characteristic signal intensity, significantly lower than the blank control group. While the signal intensities of the DT and Zn²⁺ groups were slightly reduced, they were still far weaker than the scavenging effect of the DT-Zn group. This demonstrates that the DT-Zn coordination compounds form an enzyme mimicking highly efficient SOD-like activity, effectively scavenging superoxide anion radicals. This characteristic is one of the core mechanisms of its anti-inflammatory and wound-healing effects, enabling it to eliminate a large number of free radicals generated during inflammation and reduce tissue damage.

[0037] This invention designs and synthesizes short peptides, introduces zinc ions as coenzyme factors, and coordinates them with histidine and lysine in the short peptides. This causes the monodisperse particles in the aqueous solution to self-assemble into helical fibers, forming natural enzyme active centers. This significantly enhances SOD activity, enabling the removal of ROS and promoting the healing of wounds infected with multiple bacteria. The coordination compound also achieves highly efficient bacterial killing through a unique physical-mechanical bactericidal mechanism, exhibiting superior antibacterial properties compared to the biochemical effects of traditional antibiotics.

[0038] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A short peptide-Zn coordination compound with SOD activity, characterized in that: The short peptide-Zn coordination compound contains an amino acid sequence capable of successfully coordinating with zinc ions, specifically Ac-FFFFHHHK-NH2.

2. The method for preparing a short peptide-Zn complex compound having SOD activity according to claim 1, characterized in that: The specific steps are as follows: 1) Design and synthesis of short peptide Ac-FFFFHHHK-NH2; 2) Characterization of the molecular weight of the short peptide: the synthesized short peptide is characterized by mass spectrometry and high performance liquid chromatography; 3) Preparation of short peptide and zinc ion solution: the short peptide and zinc sulfate are dispersed in deionized water respectively and shaken uniformly; 4) Preparation of coordination compound: zinc sulfate solution is added to the short peptide solution and shaken, and after uniform mixing, incubated at 37℃ for 30 min to obtain the short peptide-Zn coordination compound.

3. The method for preparing a short peptide-Zn complex compound with SOD activity according to claim 2, characterized in that: In step 3), the concentration of the short peptide and zinc sulfate solution is 1 mg / mL respectively.

4. The method for preparing a short peptide-Zn complex compound with SOD activity according to claim 2, characterized in that: In step 4), the molecular weight ratio of the short peptide solution and the zinc sulfate solution is 1:

1.

5. The short peptide-Zn coordination compound according to any one of claims 1-4 for use in the treatment of antibacterial and bacterial infection wound inflammation.

6. Use according to claim 5, characterized in that: The bacteria are one or more of Escherichia coli, Salmonella, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii.

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