Protein modified probe containing alpha, beta-unsaturated aldehyde structure as well as preparation method and application of protein modified probe

Abstract

The invention relates to the technical field of preparation of protein modified probes, and discloses a preparation method of a protein modified probe containing an alpha, beta-unsaturated aldehyde structure, and the preparation method of the protein modified probe containing the alpha, beta-unsaturated aldehyde structure comprises the following steps: S1, in an inert solvent, enabling 6-heptynoic acid to react with 2-butene-1, 2, 4-triazole-1, 2, 4-triazole-1, 2-triazole-1, 2-triazole-1, 2-triazole-1, 2-triazole-1, 2-triazole-1, 2- carrying out esterification reaction on 1, 4-diol in the presence of a condensing agent system to obtain an intermediate product; and S2, carrying out oxidation reaction on the intermediate product obtained in the step S1, and carrying out post-treatment to obtain the probe compound containing the alpha, beta-unsaturated aldehyde structure. The protein modification probe containing the alpha, beta-unsaturated aldehyde structure prepared by the invention can modify food protein under various food processing conditions, can directly visualize the protein modification degree in the food processing process, and is easy to comprehensively analyze modified components.

Description

Technical Field

[0001] The present invention relates to the technical field of protein modification probe preparation, and particularly to a protein modification probe containing an α,β-unsaturated aldehyde structure, its preparation method and application. Background Art

[0002] During the processing of lipids in various foods, especially during thermal processing, a large amount of α,β-unsaturated aldehyde compounds such as acrolein and 4-hydroxynonenal can be released due to lipid peroxidation. These α,β-unsaturated aldehydes have strong electrophilic properties and can covalently modify cysteine, histidine, and lysine residues in proteins. Such modifications may lead to phenomena such as protein oxidation, carbonylation, cross-linking, and aggregation, causing deterioration of protein quality and ultimately resulting in a decline in food quality. Therefore, it is necessary to pay attention to the modification of food proteins by α,β-unsaturated aldehydes during food processing to evaluate the potential impact of α,β-unsaturated aldehydes on proteins in various food processing processes.

[0003] However, traditional analytical techniques, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and standard proteomics methods, can neither directly visualize the degree of protein modification during food processing nor comprehensively analyze the modified components. Summary of the Invention

[0004] The purpose of the present invention is to solve the problems existing in the prior art, and a preparation method of a protein modification probe containing an α,β-unsaturated aldehyde structure is proposed.

[0005] To achieve the above purpose, the present invention adopts the following technical scheme:

[0006] A protein modification probe containing an α,β-unsaturated aldehyde structure has the following structural formula:

[0007]

[0008] A preparation method of a protein modification probe containing an α,β-unsaturated aldehyde structure, for preparing a protein modification probe containing an α,β-unsaturated aldehyde structure, includes the following steps:

[0009] S1, in an inert solvent, reacting 6-heptynoic acid with 2-butene-1,4-diol in the presence of a condensing agent system to obtain an intermediate product;

[0010] S2, performing an oxidation reaction on the intermediate product obtained in step S1, and obtaining a probe compound containing an α,β-unsaturated aldehyde structure after post-treatment;

[0011] The synthesis chemical formula is as follows:

[0012]

[0013] Preferably, in the step S1, the condensing agent system comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine. The molar ratio of 6-heptynoic acid to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:(1.0 - 1.5), and the molar ratio to 4-dimethylaminopyridine is 1:(0.3 - 0.6).

[0014] Preferably, the esterification reaction is carried out under the conditions of -10°C to 50°C, and the reaction time is 2 to 48 hours. The inert solvent is selected from at least one of dichloromethane, chloroform or tetrahydrofuran.

[0015] Preferably, in the step S1, the molar ratio of 6-heptynoic acid to 2-butene-1,4-diol is 1:1.5 - 2.5.

[0016] Preferably, after the reaction in the step S1 is completed, the intermediate product is obtained through the following treatment process:

[0017] S101, washing the reaction solution with 1M hydrochloric acid solution 2 - 4 times;

[0018] S102, washing with saturated brine;

[0019] S103, retaining the organic phase and drying it with anhydrous sodium sulfate;

[0020] S104, separating by silica gel column chromatography to obtain the intermediate product.

[0021] Preferably, in the step S2, the oxidation reaction specifically includes:

[0022] Dissolving the intermediate product in dichloromethane, adding Dess-Martin oxidant for oxidation reaction, which is carried out under the conditions of -10°C to 50°C, and the reaction time is 2 to 48 hours.

[0023] Preferably, in the step S2, the hypervalent iodine reagent is Dess-Martin oxidant, and its mass ratio to the intermediate compound is (2.5 - 4.0):1.

[0024] Preferably, after the reaction in the step S2:

[0025] Diluting the reaction solution with a halogenated hydrocarbon solvent;

[0026] Adding a mixed solution of sodium thiosulfate and sodium bicarbonate for quenching;

[0027] After liquid separation, washing the organic phase with sodium bicarbonate solution and saturated brine in sequence;

[0028] Purified by silica gel column chromatography after drying with anhydrous sodium sulfate.

[0029] Use of a protein modification probe containing an α,β-unsaturated aldehyde structure for evaluating the degree of α,β-unsaturated aldehyde-modified protein.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] A protein modification probe containing an α,β-unsaturated aldehyde structure prepared by the present invention can modify food proteins under various food processing conditions, can directly visualize the degree of protein modification during food processing, and is easy to comprehensively analyze the modified components. Brief Description of the Drawings

[0032] Figure 1 1H NMR spectrum of the probe proposed by the present invention;

[0033] Figure 2 13C NMR spectrum of the probe proposed by the present invention;

[0034] Figure 3 Schematic diagram comparing the electrophilic ability of the probe proposed by the present invention with acrolein and 4-hydroxynonenal;

[0035] Figure 4 Schematic diagram of the degree of protein (cod protein) modification (fluorescence intensity);

[0036] Figure 5 Fluorescence imaging diagrams showing the degree of modification of cod protein by α,β-unsaturated aldehyde at three different processing temperatures (25 °C, 90 °C, 180 °C). Detailed Embodiments

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0038] A preparation method of a protein modification probe containing an α,β-unsaturated aldehyde structure, and the synthesis chemical formula is as follows:

[0039]

[0040] Specifically, the following steps are carried out:;

[0041] Dissolve 5 mmol of 6-heptynoic acid in 10 mL of dichloromethane. Under ice bath conditions, sequentially add 2 mmol of 4-dimethylaminopyridine and 6 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to the solution. After stirring the solution for 30 min, dropwise add it into a dichloromethane solution containing 10 mmol of 2-butene-1,4-diol, and continue to react at room temperature for 24 h. After the reaction is completed, wash the reaction solution three times with 1M HCl, wash it once with saturated brine, retain the organic phase, dry it with anhydrous sodium sulfate, and separate it by silica gel column chromatography to obtain the intermediate product. Dissolve 500 mg of the intermediate product in 10 mL of dichloromethane, add 1550 mg of Dess-Martin oxidant under ice bath conditions, and stir magnetically for 3 h. After the reaction is completed, dilute the reaction solution with 40 mL of dichloromethane, add 50 mL of a mixed solution of saturated sodium thiosulfate-saturated sodium bicarbonate (1:1), and stir at room temperature for 15 min. After liquid separation, wash the organic phase with sodium bicarbonate and saturated brine. After drying the organic phase with anhydrous sodium sulfate, separate it by silica gel column chromatography to obtain the target probe.

[0042] The chemical structural formula of the obtained target probe is as follows:

[0043]

[0044] To verify the effect of the target probe, food protein modification and visualization detection are now carried out:

[0045] Dissolve the target probe in dimethyl sulfoxide to prepare a 10 mM mother liquor, and add it to the food protein solution to modify the food protein under various food processing conditions. Subsequently, the copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) click chemistry method is used to fluorescently label the modified protein with 6-carboxyfluorescein azide (FAM-Azide). The specific operation is as follows:

[0046] Take 10 μL of the food protein solution, mix it with 18 μL of phosphate buffer (PB, 0.2 M, pH 7.4), 4 μL of FAM-Azide solution (2.5 mM), 2.5 μL of tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) solution (10 mM), 2.5 μL of CuSO4 solution (20 mM), and 2.5 μL of sodium ascorbate (NaVc) solution (300 mM). Place the mixed system at 37 °C and react for 2 hours. Use the ChemiDoc XRS+ system to perform fluorescence imaging analysis on the modified protein on a 10% SDS-PAGE gel, and verify the protein bands by Coomassie Brilliant Blue staining.

[0047] It should be noted that click chemistry, as a bioorthogonal method, can achieve efficient and specific labeling of protein modifications. Given the complex composition of both food systems and biological environments, click chemistry labeling can effectively reduce interference from other components when applied to food systems. Therefore, click chemistry is used in this example, and its application in food protein modification research includes the following steps:

[0048] (1) Design and synthesize the corresponding clickable probes and co-incubate them with the food system to modify the target protein;

[0049] (2) coupling the labeled protein to the reporter tag via click chemistry;

[0050] (3) Analyze modification effects using reporter tags;

[0051] By introducing clickable probes, analysis of protein modifications in complex food systems can be achieved under minimal interference conditions.

[0052] The probe's nuclear magnetic spectrum (hydrogen spectrum and carbon spectrum) is obtained, such as Figure 1 and Figure 2 As shown, the NMR spectrum confirmed that the prepared probe contained an α, β-unsaturated aldehyde structure and an alkyne group that can be used for click chemistry reactions;

[0053] The probe has similar electrophilicity and protein modification ability to typical α,β-unsaturated aldehydes in food:

[0054] like Figure 3 As shown in the figure, the probe has electrophilicity, and the electrophilicity of the probe is similar to that of typical α,β-unsaturated aldehydes (acrolein, 4-hydroxynonenal) in food. The larger the number in the figure, the stronger the electrophilicity of the site.

[0055] like Figure 4 As shown, as the probe concentration increases, the modification degree (fluorescence intensity) of the protein (cod protein) gradually increases;

[0056] like Figure 5 As shown, the probe is suitable for evaluating the extent of α,β-unsaturated aldehyde-modified proteins under various processing conditions:

[0057] At three different processing temperatures (25°C, 90°C, and 180°C), the fluorescence imaging can intuitively show the degree of modification of cod protein by α,β-unsaturated aldehydes, while fluorescence imaging cannot be performed on samples without the addition of probes.

[0058] Therefore, the following conclusions can be drawn:

[0059] A protein modification probe containing an α,β-unsaturated aldehyde structure prepared by the present invention can modify food proteins under various food processing conditions, can directly visualize the degree of protein modification during food processing, and is convenient for a comprehensive analysis of the modified components.

[0060] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, should be covered within the protection scope of the present invention.

Claims

1. A protein modification probe containing an α,β-unsaturated aldehyde structure, characterized in that: Its structural formula is as follows:

2. A method for preparing a protein modification probe containing an α,β-unsaturated aldehyde structure, for preparing a protein modification probe containing an α,β-unsaturated aldehyde structure as shown in claim 1, characterized in that: It includes the following steps: S1. In an inert solvent, 6-heptynoic acid and 2-butene-1,4-diol are subjected to an esterification reaction in the presence of a condensing agent system to obtain an intermediate product; S2. The intermediate product obtained in step S1 is subjected to an oxidation reaction, and after post-treatment, a probe compound containing an α,β-unsaturated aldehyde structure is obtained.

3. The preparation method of a protein modification probe containing an α,β-unsaturated aldehyde structure according to claim 1, characterized in that: In step S1, the condensing agent system includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine. The molar ratio of 6-heptynoic acid to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:(1.0 - 1.5), and the molar ratio to 4-dimethylaminopyridine is 1:(0.3 - 0.6).

4. The preparation method of a protein modification probe containing an α,β-unsaturated aldehyde structure according to claim 2, characterized in that: The esterification reaction is carried out at -10°C to 50°C for 2 to 48 hours. The inert solvent is selected from at least one of dichloromethane, chloroform or tetrahydrofuran.

5. The preparation method of a protein modification probe containing an α,β-unsaturated aldehyde structure according to claim 2, characterized in that: In step S1, the molar ratio of 6-heptynoic acid to 2-butene-1,4-diol is 1:1.5 - 2.

5.

6. The preparation method of a protein modification probe containing an α,β-unsaturated aldehyde structure according to claim 1, characterized in that: After the reaction in step S1 is completed, the following treatment process is carried out to obtain the intermediate product: S101. Wash the reaction solution with 1M hydrochloric acid solution 2 - 4 times; S102. Wash with saturated brine; S103. Retain the organic phase and dry it with anhydrous sodium sulfate; S104. Separate by silica gel column chromatography to obtain the intermediate product.

7. The preparation method of a protein modification probe containing an α,β-unsaturated aldehyde structure according to claim 1, wherein, In step S2, the oxidation reaction specifically includes: Dissolve the intermediate product in dichloromethane, add Dess-Martin oxidant to carry out the oxidation reaction, which is carried out at -10°C to 50°C for 2 to 48 hours.

8. The preparation method of a protein modification probe containing an α,β-unsaturated aldehyde structure according to claim 7, characterized in that: In step S2, the hypervalent iodine reagent is Dess-Martin oxidant, and its mass ratio to the intermediate compound is (2.5 - 4.0):

1.

9. The preparation method of a protein modification probe containing an α,β-unsaturated aldehyde structure according to claim 8, characterized in that: In step S2, after the reaction ends: Dilute the reaction solution with a halogenated hydrocarbon solvent; Add a mixed solution of sodium thiosulfate and sodium bicarbonate to quench; After liquid separation, wash the organic phase with sodium bicarbonate solution and saturated brine in sequence; Purify by silica gel column chromatography after drying with anhydrous sodium sulfate.

10. Use of a protein modification probe containing an α,β-unsaturated aldehyde structure as described in claim 1 in the evaluation of the degree of α,β-unsaturated aldehyde-modified protein.

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