A trinuclear schiff base cobalt complex and hybrid enzyme, and preparation method and application thereof

Abstract

This invention provides a trinuclear Schiff base cobalt complex and a hybrid enzyme, along with their preparation method and applications, relating to the field of cobalt complex technology. The trinuclear Schiff base cobalt complex provided by this invention has a trinuclear cluster structure, where three cobalt ions are bridged by two completely deprotonated Schiff base ligands and one acetate ion to form a unique trinuclear arrangement. This multinuclear configuration can generate a multi-metal synergistic catalytic effect, exhibiting superior superoxide dismutase (SOD) activity. The interaction between the naphthalene ring large π system in the Schiff base ligand and the trinuclear cobalt center endows the trinuclear Schiff base cobalt complex with higher SOD activity. In the hybrid enzyme formed by hybridization with serum protein, serum protein acts as a protein scaffold, providing a suitable chiral microenvironment. Its positively charged amino acid residues promote the enrichment and activation of superoxide anions, thereby achieving a synergistic enhancement effect between the organometallic complex and the serum protein carrier, resulting in a hybrid enzyme with high SOD activity.

Description

Technical Field

[0001] This invention relates to the field of cobalt complex technology, specifically to a trinuclear Schiff base cobalt complex and hybrid enzyme, its preparation method, and its application. Background Technology

[0002] Schiff base metal complexes are organometallic hybrid systems formed by Schiff base ligands and transition metal ions through coordination bonds. The imine nitrogen atom (-C=N-) and adjacent electron-donating atoms (such as phenolic hydroxyl oxygen, pyridine nitrogen, etc.) in the Schiff base ligand serve as coordination sites, chelating with transition metal ions to form stable cyclic structures. Due to their unique structural characteristics and properties, Schiff base metal complexes show broad application prospects in catalysis, biomedicine, and functional materials. Among them, cobalt Schiff base complexes, due to the abundant oxidation states and diverse coordination configurations of cobalt, can mimic the active sites of various metalloenzymes, showing promising applications in catalytic oxidation and superoxide dismutase (SOD) simulation.

[0003] Superoxide dismutase (SOD) catalyzes the dismutation of superoxide anion radicals into oxygen and hydrogen peroxide, playing a crucial role in protecting cells from oxidative damage. Natural SODs suffer from complex extraction processes, poor stability, and high costs; therefore, developing artificial SOD mimics has significant application value. Artificial metalloenzyme systems combine the high activity of metal complexes with the high stability of biological carriers. Hybrid enzymes constructed by anchoring Schiff base cobalt complexes into protein cavities have been successfully used for SOD mimicry. However, most existing Schiff base cobalt complexes have a single-nuclear structure, resulting in relatively low SOD activity (SOD activity, referring to the mimicry activity of the SOD mimic). Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a trinuclear Schiff base cobalt complex and a hybrid enzyme, as well as its preparation method and application. The trinuclear Schiff base cobalt complex provided by this invention exhibits high superoxide dismutase activity.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a trinuclear Schiff base cobalt complex having the structure shown in Formula I: Formula I.

[0006] This invention provides a method for preparing the trinuclear Schiff base cobalt complex described above, comprising the following steps: Cobalt acetate, 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine, triethylamine and anhydrous ethanol were mixed and subjected to a coordination reaction to obtain a trinuclear Schiff base cobalt complex. The mass ratio of cobalt acetate to triethylamine is 1.5~2.5:1; The mass ratio of cobalt acetate to anhydrous ethanol is 1:80~200; The coordination reaction was carried out at a temperature of 80°C for 72 hours.

[0007] Preferably, the molar ratio of cobalt acetate to 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine is 2 to 4:1.

[0008] Preferably, the preparation method of the 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine includes the following steps: 2-hydroxy-1-naphthaldehyde, 1,3-propanediamine and solvent are mixed and subjected to a Schiff base reaction to obtain the 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine.

[0009] Preferably, the molar ratio of 2-hydroxy-1-naphthaldehyde to 1,3-propanediamine is 1.5 to 3:1.

[0010] Preferably, the Schiff base reaction is carried out at a temperature of 60-80°C for 5-8 hours.

[0011] The present invention provides a hybrid enzyme comprising a serum protein and an organometallic complex, wherein the organometallic complex binds to the serum protein via coordination and non-covalent binding. The organometallic complex is the trinuclear Schiff base cobalt complex or the trinuclear Schiff base cobalt complex described in the above technical solution; The serum proteins include bovine serum albumin and / or human serum albumin.

[0012] Preferably, the molar ratio of the serum protein to the organometallic complex is 1:1 to 100.

[0013] This invention provides a method for preparing the hybrid enzyme described in the above technical solution, comprising the following steps: Serum protein and buffer solution are mixed to obtain serum protein buffer solution; The serum protein buffer, organometallic complex, and solvent were mixed and subjected to a hybridization reaction to obtain a hybrid enzyme.

[0014] This invention provides the application of the trinuclear Schiff base cobalt complex described in the above-described technical solution, the trinuclear Schiff base cobalt complex described in the above-described technical solution, the hybrid enzyme described in the above-described technical solution, or the hybrid enzyme prepared by the above-described technical solution in catalyzing the disproportionation of superoxide anion free radicals into oxygen and hydrogen peroxide.

[0015] This invention provides a trinuclear Schiff base cobalt complex with the structure shown in Formula I. The trinuclear Schiff base cobalt complex provided by this invention has a trinuclear cluster structure. One molecule of the trinuclear Schiff base cobalt complex has three cobalt atoms. The three cobalt ions are bridged by two completely deprotonated Schiff base ligands and one acetate ion to form a unique trinuclear arrangement. This multinuclear configuration can produce a multi-metal synergistic catalytic effect different from mono / binuclear systems. The three metal centers can jointly participate in substrate activation, electron transfer, and intermediate stabilization processes, exhibiting superior superoxide dismutase activity. Moreover, the ligand in the trinuclear Schiff base cobalt complex provided by this invention is 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine. Compared with conventional Schiff base ligands (based on benzene-containing salicylaldehyde), the naphthalene ring significantly expands the π-conjugated system of the ligand, affecting the electronic structure, coordination ability, and optical, electrical, and magnetic properties of the complex. The interaction between the large π-system of the naphthalene ring and the trinuclear cobalt center endows the trinuclear Schiff base cobalt complex with higher superoxide dismutase activity. Moreover, 1,3-propanediamine in 2-hydroxy-1-naphthaldehyde acetal has a longer carbon chain (three methylene groups) than the common ethylenediamine, which provides greater flexibility and spatial adjustment capability for the ligand and is conducive to the formation of special polynuclear structures.

[0016] This invention provides a method for preparing the trinuclear Schiff base cobalt complex described in the above technical solution, comprising the following steps: mixing cobalt acetate, 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine, triethylamine, and anhydrous ethanol, and carrying out a coordination reaction to obtain the trinuclear Schiff base cobalt complex; the mass ratio of cobalt acetate to triethylamine is 1.5~2.5:1; the mass ratio of cobalt acetate to anhydrous ethanol is 1:80~200; the temperature of the coordination reaction is 80℃, and the time is 72h. This invention, by controlling the amounts of the solvent anhydrous ethanol and the coordination catalyst triethylamine, as well as the temperature and time of the coordination reaction, enables the phenolic oxygen in the ligand 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine to connect two or more cobalt centers via a bridging coordination mechanism. Furthermore, the spatial configuration of the ligand, the flexibility of the 1,3-propanediamine chain segment in the ligand, and the rigidity and steric hindrance brought by the naphthalene ring collectively regulate the distance and arrangement between cobalt atoms, which is beneficial to the self-assembly of the trinuclear Schiff base cobalt complex. Moreover, the preparation method provided by this invention is simple to operate and suitable for industrial production.

[0017] This invention provides a hybrid enzyme comprising a serum protein and an organometallic complex. The organometallic complex binds to the serum protein via coordination and non-covalent binding. The organometallic complex is a trinuclear Schiff base cobalt complex as described in the above-mentioned technical solutions. The serum protein includes bovine serum albumin (BSA) and / or human serum albumin (HSA). The hybrid enzyme provided by this invention uses serum protein as a protein scaffold, providing a suitable chiral microenvironment that enhances the antioxidant activity of the trinuclear Schiff base cobalt complex. The positively charged amino acid residues on its surface promote the enrichment and activation of superoxide anions, causing redox reactions in the system's ground-state electron transitions. This achieves a synergistic enhancement effect between the organometallic complex and the serum protein carrier, resulting in high superoxide dismutase activity in the hybrid enzyme. Attached Figure Description

[0018] Figure 1 This is a structural diagram of a trinuclear Schiff base cobalt complex; Figure 2 Single-crystal simulation and PXRD spectra of trinuclear Schiff base cobalt complexes; Figure 3 The UV-Vis absorption spectrum of the trinuclear Schiff base cobalt complex; Figure 4 The UV-Vis absorption spectra of bovine serum albumin, trinuclear Schiff base cobalt complex, and hybrid enzymes are shown. Figure 5 The absorbance of trinuclear Schiff base cobalt complexes at different concentrations at 550 nm varies with time. Figure 6 The absorbance of the hybrid enzyme at different concentrations at 550 nm changes over time. Figure 7 The concentration-relative rate of increase curves for trinuclear Schiff base cobalt complexes and hybrid enzymes are shown. Detailed Implementation

[0019] This invention provides a trinuclear Schiff base cobalt complex having the structure shown in Formula I: Formula I.

[0020] This invention provides a method for preparing the trinuclear Schiff base cobalt complex described above, comprising the following steps: Cobalt acetate, 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine, triethylamine and anhydrous ethanol were mixed and subjected to a coordination reaction to obtain a trinuclear Schiff base cobalt complex. The mass ratio of cobalt acetate to triethylamine is 1.5~2.5:1; The mass ratio of cobalt acetate to anhydrous ethanol is 1:80~200; The coordination reaction was carried out at a temperature of 80°C for 72 hours.

[0021] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0022] In this invention, the preparation method of 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine may include the following steps: 2-hydroxy-1-naphthaldehyde, 1,3-propanediamine, and a solvent are mixed and subjected to a Schiff base reaction to obtain the 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine. This invention condenses 2-hydroxy-1-naphthaldehyde with 1,3-propanediamine to obtain a Schiff base ligand, 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine, containing two imine nitrogen atoms and two phenolic hydroxyl oxygen atoms. This ligand, after deprotonation, can provide multiple N / O coordination sites.

[0023] In this invention, the synthetic route for 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine is shown in Route 1: Route 1.

[0024] In this invention, the molar ratio of 2-hydroxy-1-naphthaldehyde to 1,3-propanediamine can be 1.5 to 3:1, specifically 1.8:1, 2:1, 2.2:1, or 2.5:1; the solvent can include ethanol, specifically anhydrous ethanol; the mass ratio of 2-hydroxy-1-naphthaldehyde to the solvent can be 1:10 to 50, specifically 1:20, 1:30, or 1:40. In this invention, the temperature of the Schiff base reaction can be 60 to 80°C, specifically 65°C, 70°C, or 75°C; the time of the Schiff base reaction can be 5 to 8 hours, specifically 6 hours or 7 hours. In this invention, the Schiff base reaction can be carried out under reflux conditions.

[0025] After completing the Schiff base reaction, the present invention may further include cooling the amine-aldehyde condensation solution obtained from the Schiff base reaction to room temperature and then separating the solid and liquid, washing the obtained solid, and obtaining the 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine.

[0026] In this invention, the solid-liquid separation can specifically be filtration; the filtration can be performed at room temperature; the washing can be ethanol washing, specifically anhydrous ethanol washing; the number of washings can be 2-3 times; the molar ratio of the salicylaldehyde to the volume of anhydrous ethanol used in a single washing can be 10 mmol: 5-10 mL, specifically 10 mmol: 6 mL or 10 mmol: 8 mL. In this invention, during each washing process, the solid and anhydrous ethanol are mixed and filtered; this invention does not have a specific limitation on the time of a single washing, but only the time of a single filtration, which can be 1-5 min, specifically 2 min or 4 min.

[0027] In this invention, the cobalt acetate can specifically be cobalt acetate tetrahydrate (C4H6CoO4∙4H2O); the molar ratio of cobalt acetate to 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine can be 2~4:1, specifically 2.5:1, 3:1 or 3.5:1; the mass ratio of cobalt acetate to triethylamine is 1.5~2.5:1, specifically 1.8:1, 2:1 or 2.3:1; the mass ratio of cobalt acetate to anhydrous ethanol is 1:80~200, specifically 1:100, 1:126.7 or 1:150. In this invention, the coordination reaction temperature is 80℃; the coordination reaction time is 72h.

[0028] The present invention provides a hybrid enzyme comprising a serum protein and an organometallic complex, wherein the organometallic complex binds to the serum protein via coordination and non-covalent binding. The organometallic complex is the trinuclear Schiff base cobalt complex or the trinuclear Schiff base cobalt complex described in the above technical solution; The serum proteins include bovine serum albumin and / or human serum albumin.

[0029] In this invention, the molar ratio of serum protein to organometallic complex can be 1:1 to 100, specifically 1:5, 1:10, 1:30 or 1:50.

[0030] This invention provides a method for preparing the hybrid enzyme described in the above technical solution, comprising the following steps: Serum protein and buffer solution are mixed to obtain serum protein buffer solution; The serum protein buffer, organometallic complex, and solvent were mixed and subjected to a hybridization reaction to obtain a hybrid enzyme.

[0031] This invention mixes serum protein and a buffer solution to obtain a serum protein buffer solution.

[0032] In this invention, the buffer solution may include a phosphate buffer solution (PB); the pH value of the phosphate buffer solution may be 7; the phosphate in the phosphate buffer solution may be sodium dihydrogen phosphate and disodium hydrogen phosphate; the total phosphate concentration (total concentration of sodium dihydrogen phosphate and disodium hydrogen phosphate) of the phosphate buffer solution may be 0.01 mol / L. In this invention, the concentration of serum protein in the serum protein buffer may be 1 × 10⁻⁶. -3 mol / L.

[0033] After obtaining the serum protein buffer, the present invention mixes the serum protein buffer, the organometallic complex and the solvent to carry out a hybridization reaction to obtain a hybrid enzyme.

[0034] In this invention, the solvent may include dimethyl sulfoxide (DMSO); the organometallic complex and the solvent may be used in the form of an organometallic complex solution; the concentration of the organometallic complex solution may be 1 × 10⁻⁶. - 3 The concentration is mol / L; the volume ratio of the serum protein buffer to the organometallic complex solution can be 1:1. In this invention, the temperature of the hybridization reaction can be 4°C; the time of the hybridization reaction can be 12 h; the hybridization reaction can be carried out under stirring conditions.

[0035] After completing the hybridization reaction, the present invention may further include dialysis of the hybrid feed solution obtained from the hybridization reaction to obtain a dialysis product; centrifugation of the dialysis product, concentration of the supernatant, and separation by gel column chromatography to obtain an eluent; centrifugation of the eluent, concentration of the supernatant, and obtaining the hybrid enzyme.

[0036] In this invention, the dialysis solution can be a phosphate buffer solution; the dialysis can include continuous first dialysis, second dialysis, and third dialysis; the first dialysis time can be 3 hours; the second dialysis time can be 4 hours; and the third dialysis time can be 5 hours. This invention removes unhybridized organometallic complexes and the solvent DMSO by dialysis of a hybrid feed solution using a phosphate buffer solution.

[0037] In this invention, the conditions for gel column chromatography may include: a column chromatography temperature of 4-10°C, specifically 6°C or 8°C; a Sephadex G-75 column as the gel column; a phosphate buffer solution as the eluent; a pH value of the phosphate buffer solution of 6.8-7.4, specifically 7 or 7.2; a total phosphate concentration of the phosphate buffer solution of 0.005-0.05 mol / L, specifically 0.01 mol / L, 0.02 mol / L, or 0.04 mol / L; an elution flow rate of 0.1-1.0 mL / min, specifically 0.2 mL / min, 0.5 mL / min, or 0.8 mL / min; and a collection volume of 0.5-2 mL per tube, specifically 1 mL or 1.5 mL. This invention removes unhybridized serum proteins through gel column chromatography; moreover, this invention employs a simple non-covalent hybridization method to prepare high-purity hybrid enzymes through a purification method combining dialysis and gel column chromatography.

[0038] This invention provides the application of the trinuclear Schiff base cobalt complex described in the above-described technical solution, the trinuclear Schiff base cobalt complex described in the above-described technical solution, the hybrid enzyme described in the above-described technical solution, or the hybrid enzyme prepared by the above-described technical solution in catalyzing the disproportionation of superoxide anion free radicals into oxygen and hydrogen peroxide.

[0039] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] In all embodiments of the present invention, 1,3-propanediamine, ethylenediamine, anhydrous ethanol, cobalt acetate tetrahydrate, dimethyl sulfoxide, sodium monohydrogen phosphate, and sodium dihydrogen phosphate were all purchased from Xilong Chemical Co., Ltd.; 2-hydroxy-1-naphthaldehyde and bovine serum albumin were all purchased from Aladdin Reagent Co., Ltd. (Shanghai). Except for bovine serum albumin, all the above reagents were of analytical grade.

[0041] Example 1 10 mmol of 2-hydroxy-1-naphthaldehyde and 5 mmol of 1,3-propanediamine were dissolved in 50 mL of anhydrous ethanol and subjected to a Schiff base reaction at 80 °C under reflux for 5 h. After the reaction was completed, the resulting amine-aldehyde condensate was cooled to room temperature and filtered. The resulting solid was washed three times with anhydrous ethanol at room temperature. After each wash, the solid and liquid were separated by vacuum filtration. The volume of anhydrous ethanol used in each wash was 10 mL. 2-hydroxy-1-naphthaldehyde condensate 1,3-propanediamine, denoted as H2L (i.e., a ligand with two H coordinates), was obtained as a yellow powder solid with a yield of 79%.

[0042] The characterization results of 2-hydroxy-1-naphthaldehyde acetalized with 1,3-propanediamine are as follows: Elemental analysis: Calcd. (%) for C 25 H 22 N2O2(382.17): C, 78.51; H, 5.80; N, 7.32. Found (%): C, 78.08; H, 5.76; N, 7.28; Infrared analysis: FT-IR (KBr, cm -1 ): 3413 (s), 1638 (s), 1543 (s), 1500 (w), 1446 (w), 1325 (w), 1207 (m), 1104 (w), 1009 (w), 887 (w), 748 (w); Nuclear magnetic resonance hydrogen spectrum analysis 1 H NMR (400 MHz, DMSO): δ 14.18 (s, 3H), 9.17 (d,4H), 8.07 (d, 4H), 7.74 (d, 4H), 7.64 (dd, 4H), 7.39 (ddd, 4H), 7.19 (dd,2H), 7.19 (s, 2H), 6.75 (d, 4H), 3.77 (q, 9H), 3.01 (s, 1H), 2.13 (p, 4H).

[0043] 0.2 mmol of C4H6CoO4∙4H2O was dissolved in 3 mL of ethanol, and 3 drops of triethylamine were added dropwise. Then, 5 mL of an ethanol solution of 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine (with a molar amount of 0.1 mmol) was added dropwise to the resulting solution. The coordination reaction was carried out at 80 °C for 72 h. After the reaction was complete, the resulting coordination solution was filtered, and the resulting solid was washed with ethanol to obtain a trinuclear Schiff base cobalt complex, a yellow blocky crystal, with a yield of 39%.

[0044] The characterization results of the trinuclear Schiff base cobalt complex are as follows: Elemental analysis: Calcd. (%) for C 54 H 46 Co3N4O8(1055.13): C, 61.43; H, 4.39; N, 5.31. Found (%): C, 61.39; H, 4.38; N, 5.30; Infrared analysis: FT-IR (KBr, cm -1): 3440 (s), 2919 (w), 1617 (s), 1569 (m), 1545 (w), 1460 (w), 1436 (m), 1342 (m), 1191 (w), 826 (w), 746 (w).

[0045] Test Example 1 The main infrared spectral data of the 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine and the trinuclear Schiff base cobalt complex prepared in Example 1 were compared, and the results are shown in Table 1.

[0046] Table 1. Main infrared spectral data (KBr, cm⁻¹) of 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine and trinuclear Schiff base cobalt complexes -1 )

[0047] As shown in Table 1, the range is 3413~3445 cm. -1 The broad peak can be attributed to intermolecular hydrogen bonding, and is the absorption peak of the phenol OH group; 1608~1639 cm⁻¹ -1 It is a characteristic absorption peak of C=N Schiff bases; 1430~1497 cm⁻¹ -1 It is an in-plane OH bending vibration; 1142~1208 cm -1 It is the CO stretching vibration. The peak position of the trinuclear Schiff base cobalt complex relative to the 1,3-propanediamine acetal ligand of 2-hydroxy-1-naphthaldehyde will be slightly shifted.

[0048] The crystal structure of the trinuclear Schiff base cobalt complex prepared in Example 1 was determined. The crystals of the trinuclear Schiff base cobalt complex were tested using a single-crystal diffractometer from Agilent Technologies, with MoKα radiation and the conditions set as λ=0.71073Å and T=293(2)K. The structure was solved by a direct method, followed by mixed hydrogenation, where anisotropic thermal parameters were used for non-hydrogen atoms and isotropic thermal parameters were used for hydrogen atoms. The obtained crystallographic data and structural refinement parameters are shown in Table 2.

[0049] Table 2 Crystallographic data and structural refinement parameters of trinuclear Schiff base cobalt complexes

[0050] Based on the crystallographic data and structural refinement parameters of the trinuclear Schiff base cobalt complexes in Table 2, the structural diagrams of the trinuclear Schiff base cobalt complexes are obtained, as follows: Figure 1 As shown in Table 3, some bond lengths and bond angles in the trinuclear Schiff base cobalt complex are shown in Table 3, where the symmetry code is: (A) -x+1, -y+1, -z.

[0051] Table 3. Partial bond lengths and bond angles in trinuclear Schiff base cobalt complexes

[0052] The trinuclear Schiff base cobalt complex prepared in Example 1 was subjected to powder X-ray diffraction (PXRD) tests on a Rigaku D / max-2550 diffractometer, and the diffraction data obtained were compared with the diffraction data obtained by simulation using crystal data. Figure 2 Single-crystal simulation and PXRD spectra of trinuclear Schiff base cobalt complexes, by Figure 2 It can be seen that the PXRD test results of the trinuclear Schiff base cobalt complex prepared in Example 1 are in good agreement with the single crystal simulation, proving that the prepared trinuclear Schiff base cobalt complex has good phase purity.

[0053] At room temperature, the trinuclear Schiff base cobalt complex obtained in Example 1 was prepared into a complex solution using a phosphate buffer solution with pH = 7 and a total phosphate concentration of 1 × 10⁻⁶. -6 The concentration was mol / L, with 3 mL of phosphate buffer solution as a reference solution. UV-Vis spectra were measured at 0 h, 24 h and 48 h after the preparation of the complex solution, and data were collected in the range of 200-600 nm.

[0054] Figure 3 The UV-Vis absorption spectrum of the trinuclear Schiff base cobalt complex is shown below. Figure 3 It can be seen that as time goes on, the peak of the complex does not undergo a blue shift, nor does it undergo a red shift, and no new absorption peaks appear, indicating that the trinuclear Schiff base cobalt complex is stable in phosphate buffer solution at room temperature.

[0055] Example 2 Sodium dihydrogen phosphate and disodium hydrogen phosphate were dissolved separately in deionized water and mixed to prepare a phosphate buffer solution with pH=7 and a total phosphate concentration of 0.01 mol / L.

[0056] Bovine serum albumin was dissolved in phosphate buffer solution to prepare a solution with a concentration of 1×10⁻⁶. -3 Bovine serum albumin buffer (BSA) was prepared by dissolving the trinuclear Schiff base cobalt complex obtained in Example 1 in dimethyl sulfoxide (DMSO) to obtain a concentration of 1 × 10⁻⁶ mol / L. -3A mol / L complex solution was prepared. The obtained bovine serum albumin buffer and the complex solution were mixed at a 1:1 volume ratio and the mixture was stirred at 4°C for 12 h to induce hybridization. After the hybridization reaction was complete, the mixture was dialyzed three times with phosphate buffer, with dialysis fluid replacement times of 3 h, 4 h, and 5 h respectively. After dialysis, the mixture was centrifuged, and the supernatant was concentrated. The concentrate was then subjected to gel column chromatography. The gel column chromatography conditions were: column temperature 4°C, Sephadex G-75 column, phosphate buffer solution with pH=7 and a total phosphate concentration of 0.01 mol / L as eluent, elution flow rate 0.5 mL / min, and a collection volume of 1 mL per tube. The eluent was centrifuged at high speed, and the supernatant was concentrated to obtain the hybrid enzyme.

[0057] Test Example 2 At room temperature, the hybrid enzyme and bovine serum albumin prepared in Example 2 were prepared into hybrid enzyme solution and bovine serum albumin solution respectively using the phosphate buffer solution in Test Example 1. Using 3 mL of phosphate buffer solution as a reference solution, ultraviolet-visible spectroscopy was performed to collect data from 200 to 600 nm.

[0058] Figure 4 This is the UV-Vis absorption spectrum of bovine serum albumin, the trinuclear Schiff base cobalt complex, and the hybrid enzyme. The black line represents bovine serum albumin, the red line represents the trinuclear Schiff base cobalt complex, and the blue line represents the hybrid enzyme. Figure 3 The starting point of the horizontal axis is 200nm. Figure 4 The starting point of the horizontal axis is 250nm, therefore Figure 3 The peak at 250 nm of the trinuclear Schiff base cobalt complex is located at Figure 4 The starting position of the horizontal axis). Figure 4 It is known that the aromatic amino acids of tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) in bovine serum albumin undergo π-π reactions. The transition process causes a characteristic absorption peak in the UV-Vis absorption spectrum around 279 nm. The UV absorption peak in the 200–300 nm region of trinuclear Schiff base cobalt complexes is mainly due to the n-π transitions of the benzene or naphthalene rings in the complex molecule. The absorption in the 330–380 nm region is mainly caused by the transition, while the absorption in the 330–380 nm region is mainly due to the C=N bonds in the trinuclear Schiff base cobalt complex. π-π The transition caused the hybrid enzyme to exhibit not only the characteristic peaks of the trinuclear Schiff base cobalt complex but also those of bovine serum albumin (BSAP). This indicates that BSAP binds to the trinuclear Schiff base cobalt complex to form the hybrid enzyme.

[0059] Test Example 3 The trinuclear Schiff base cobalt complex prepared in Example 1 and the hybrid enzyme prepared in Example 2 were used to simulate active SOD enzymes, and the SOD activity of the simulated enzymes was determined by the xanthine oxidase-NBT reduction method.

[0060] The trinuclear Schiff base cobalt complex was dissolved in DMSO and prepared to a concentration of 1.0 × 10⁻⁶. -3 A 0.01 mol / L phosphate buffer solution was prepared using NaH₂PO₄ and Na₂HPO₄ to achieve a pH of 7.4. Using this phosphate buffer solution, NBT (nitroblue tetrazolium, used for colorimetric / detection of superanions), XO (xanthine), XOD (xanthine oxidase), and hybrid enzyme solutions were prepared with concentrations of 2.5 × 10⁻⁶ mol / L. -3 mol / L, 1.1×10 -2 mol / L, 4.4×10 -4 mol / L, 1.0×10 -3 The prepared XOD and NBT solutions were prepared at mol / L and used within two days. XO, catalyzed by XOD, generates superoxide anion radicals, which reduce NBT to formazan. Formazan exhibits a maximum UV absorption peak at 550 nm, and the solution color gradually changes from pale yellow to bluish-purple. When a superoxide dismutase (SOD) mimic is added to the system, it competes with NBT for superoxide anion radicals, thus reducing the NBT reduction rate. The reaction rate constant of the trinuclear Schiff base cobalt complex and the hybrid enzyme in the catalytic superoxide anion radical dismutation process can be determined using a spectrophotometer. The activity of SOD was indirectly determined based on the NBT reduction rate before and after the addition of the SOD mimic.

[0061] 100 μL of prepared NBT solution, 200 μL of prepared XO solution, and 1700 μL of prepared phosphate buffer solution were mixed thoroughly. The reduction rate of NBT at 550 nm was determined by spectrophotometry using a UV-Vis spectrometer set to λ=550 nm, and the absorbance values ​​were recorded within a certain time range. XOD was added until ΔA (change in absorbance) ≈ 0.024 min. -1 The absorbance of the trinuclear Schiff base cobalt complex and its hybrid enzyme was measured at 550 nm to determine SOD activity. All experiments were performed at 298 K and pH 7.4 using quantitative analysis. The methods for determining the trinuclear Schiff base cobalt complex and hybrid enzyme were consistent with those described above, with six concentrations and one blank solution tested for each sample.

[0062] Figure 5 The absorbance of trinuclear Schiff base cobalt complexes at different concentrations at 550 nm varies with time. Figure 6The graph shows the change in absorbance of the hybrid enzyme at different concentrations at 550 nm over time. Figure 5 and Figure 6 The concentrations corresponding to a~g are 0.0 μmol / L, 2.0 μmol / L, 4.0 μmol / L, 6.0 μmol / L, 8.0 μmol / L, 10.0 μmol / L, and 12.0 μmol / L, respectively. The smaller the slope of the linear expression, the greater the SOD activity. Figure 5 It can be seen that simply adding the complex or increasing the concentration of the trinuclear Schiff base cobalt complex does not significantly change the slope of the linear curve; Figure 6 It can be seen that the slope of the line decreased significantly after the addition of the hybrid enzyme, and the slope of the line decreased further after increasing the concentration, which proves that the hybrid enzyme has high SOD activity.

[0063] To further determine the SOD activity of the trinuclear Schiff base cobalt complex and hybrid enzyme, the apparent rate constant was calculated using Equation 1. K cat ).

[0064] Formula 1.

[0065] in, V 0 The initial NBT reduction rate (NBT reduction rate without the addition of SOD analogue, blank control); V cat The NBT reduction rate after the addition of a catalyst (a trinuclear Schiff base cobalt complex or a hybrid enzyme); [detector] It indicates the concentration of the molecules; K cat It is the apparent rate constant; K detector The value is approximately 5.88 × 10. 4 M -1 ·s -1 ; [cat] This refers to the concentration of the complex or hybrid enzyme. Because SOD mimics scavenge superoxide anions and compete with NBT, V cat Usually less than V 0 .pass [cat] and V 0 / V cat Plotting -1 (the increase in relative rate) and fitting a straight line, as shown... Figure 7 As shown, the slope of the line is obtained. K cat / K detector [detector]The value of . As can be seen from Equation 1, the slope of the straight line is related to the apparent rate constant of the trinuclear Schiff base cobalt complex or hybrid enzyme. K cat The correlation is positive, therefore it can be preliminarily determined that the trinuclear Schiff base cobalt complex has SOD activity. Figure 7 This is a concentration-relative rate increase curve of SOD-like activity for the trinuclear Schiff base cobalt complex and the hybrid enzyme in the Fridovich assay (NBT = 250 μmol / L), where the red line represents the hybrid enzyme and the black line represents the trinuclear Schiff base cobalt complex. Figure 7 It can be seen that the slope of the linear curve corresponding to the hybrid enzyme is greater than that of the linear curve corresponding to the trinuclear Schiff base cobalt complex, and it is preliminarily inferred that the SOD activity of the hybrid enzyme is greater than that of the SOD activity of the trinuclear Schiff base cobalt complex.

[0066] IC 50 The value represents the concentration of antioxidants lost when the inhibition rate of NBT reduction reaches 50%. IC 50 The magnitude of the value is typically used to quantitatively characterize the activity of SOD. When V 0 / V cat When -1=1, the reciprocal of the slope of the line is... IC 50 The value reveals that the slope of the linear curve representing the increase in concentration-relative rate is related to... IC 50 The values ​​are negatively correlated.

[0067] The apparent rate constants of the trinuclear Schiff base cobalt complex and the hybrid enzyme were calculated according to Equation 2. K cat , IC 50 Value and K cat The results are shown in Table 4.

[0068] K cat = K detector [detector] / IC 50 Formula 2.

[0069] Table 4. Trinuclear Schiff base cobalt complexes and hybrid enzymes IC 50 Value and K cat result

[0070] Table 4 shows that the trinuclear Schiff base cobalt complex IC50 The SOD activity was 48.64 μM, exhibiting better SOD activity compared to other metal complexes. The SOD activity changed after the trinuclear Schiff base cobalt complex formed a hybrid enzyme, indicating that different complexes have different promoting effects on large protein molecules. For the trinuclear Schiff base cobalt, the formation of the hybrid enzyme significantly increased SOD activity, with its apparent rate constant increasing by 12-fold. IC 50 The value is 4.04 μM, indicating good SOD activity.

[0071] The above description is only a preferred embodiment of 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 principle 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 trinuclear Schiff base cobalt complex, characterized in that, It has the structure shown in Equation I: Equation I.

2. The method for preparing the trinuclear Schiff base cobalt complex according to claim 1, characterized in that, Includes the following steps: Cobalt acetate, 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine, triethylamine and anhydrous ethanol were mixed and subjected to a coordination reaction to obtain a trinuclear Schiff base cobalt complex. The mass ratio of cobalt acetate to triethylamine is 1.5~2.5:1; The mass ratio of cobalt acetate to anhydrous ethanol is 1:80~200; The coordination reaction was carried out at a temperature of 80°C for 72 hours.

3. The preparation method according to claim 2, characterized in that, The molar ratio of cobalt acetate to 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine is 2~4:

1.

4. The preparation method according to claim 2, characterized in that, The preparation method of the 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine includes the following steps: 2-hydroxy-1-naphthaldehyde, 1,3-propanediamine and solvent are mixed and subjected to a Schiff base reaction to obtain the 2-hydroxy-1-naphthaldehyde acetal 1,3-propanediamine.

5. The preparation method according to claim 4, characterized in that, The molar ratio of 2-hydroxy-1-naphthaldehyde to 1,3-propanediamine is 1.5 to 3:

1.

6. The preparation method according to claim 4 or 5, characterized in that, The Schiff base reaction is carried out at a temperature of 60-80°C for 5-8 hours.

7. A hybrid enzyme comprising a serum protein and an organometallic complex, wherein the organometallic complex binds to the serum protein via coordination and non-covalent binding. The organometallic complex is the trinuclear Schiff base cobalt complex according to claim 1 or the trinuclear Schiff base cobalt complex according to any one of claims 2 to 6; The serum proteins include bovine serum albumin and / or human serum albumin.

8. The hybrid enzyme according to claim 7, characterized in that, The molar ratio of serum protein to organometallic complex is 1:1 to 100.

9. The method for preparing the hybrid enzyme according to claim 7 or 8, characterized in that, Includes the following steps: Serum protein and buffer solution are mixed to obtain serum protein buffer solution; The serum protein buffer, organometallic complex, and solvent were mixed and subjected to a hybridization reaction to obtain a hybrid enzyme.

10. The application of the trinuclear Schiff base cobalt complex of claim 1, the trinuclear Schiff base cobalt complex of any one of claims 2 to 6, the hybrid enzyme of any one of claims 7 to 8, or the hybrid enzyme prepared by the method of claim 9 in catalyzing the disproportionation of superoxide anion radicals into oxygen and hydrogen peroxide.

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