Protein-modified probes containing α,β-unsaturated aldehyde structures, their preparation methods, and applications

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of protein modification probe preparation technology, and in particular to protein modification probes containing α,β-unsaturated aldehyde structures, their preparation methods, and applications. Background Technology

[0002] During food processing, especially thermal processing, lipids can release large amounts of α,β-unsaturated aldehydes, such as acrolein and 4-hydroxynonenal, 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 can lead to protein oxidation, carbonylation, cross-linking, and aggregation, causing protein quality deterioration and ultimately reducing food quality. Therefore, it is necessary to pay attention to the modifying effects of α,β-unsaturated aldehydes on food proteins during food processing, and to assess the potential impact of α,β-unsaturated aldehydes on proteins during various food processing processes.

[0003] However, traditional analytical techniques, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and standard proteomics methods, cannot directly visualize the degree of protein modification during food processing, nor can they provide a comprehensive analysis of the modified components. Summary of the Invention

[0004] The purpose of this invention is to solve the problems existing in the prior art, and to propose a method for preparing a protein-modified probe containing an α,β-unsaturated aldehyde structure.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A protein-modified probe containing an α,β-unsaturated aldehyde structure is shown in the following structural formula:

[0007] .

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

[0009] S1, in an inert solvent, 6-heptyneic acid and 2-buten-1,4-diol are esterified in the presence of a condensing agent to obtain an intermediate product;

[0010] 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.

[0011] The synthetic chemical formula is as follows:

[0012] .

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

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

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

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

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

[0018] S102, wash with saturated salt water;

[0019] S103, retain the organic phase and dry with anhydrous sodium sulfate;

[0020] S104, silica gel column chromatography was used to separate the intermediate product.

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

[0022] The intermediate product was dissolved in dichloromethane, and Dess-Martin oxidant was added to carry out the oxidation reaction at -10℃ to 50℃ for 2 to 48 hours.

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

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

[0025] Dilute the reaction solution with a haloalkanes solvent;

[0026] Quenching is performed by adding a mixed solution of sodium thiosulfate and sodium bicarbonate;

[0027] After separation, the organic phase was washed sequentially with sodium bicarbonate solution and saturated saline solution.

[0028] After drying with anhydrous sodium sulfate, it was purified by silica gel column chromatography.

[0029] The use of a protein modification probe containing an α,β-unsaturated aldehyde structure in assessing the degree of α,β-unsaturated aldehyde modification in proteins.

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

[0031] The present invention provides a protein modification probe containing an α,β-unsaturated aldehyde structure, which can modify food proteins under various food processing conditions, directly visualize the degree of protein modification during food processing, and facilitate comprehensive analysis of the modified components. Attached Figure Description

[0032] Figure 1 The present invention provides a hydrogen NMR spectrum of the probe.

[0033] Figure 2 The carbon NMR spectrum of the probe is provided for this invention;

[0034] Figure 3 This is a schematic diagram comparing the electrophilicity of the probe proposed in this invention with that of acrolein and 4-hydroxynonenal;

[0035] Figure 4 This is a schematic diagram showing the degree of modification (fluorescence intensity) of the protein (cod protein);

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

[0037] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0038] A method for preparing a protein-modified probe containing an α,β-unsaturated aldehyde structure, the synthetic chemical formula of which is as follows:

[0039] ;

[0040] The specific steps are as follows:

[0041] 5 mmol of 6-heptyneic acid was dissolved in 10 mL of dichloromethane. Under ice bath conditions, 2 mmol of 4-dimethylaminopyridine and 6 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added sequentially. After stirring for 30 min, the solution was added dropwise to a dichloromethane solution containing 10 mmol of 2-buten-1,4-diol, and the reaction was continued at room temperature for 24 h. After the reaction was complete, the reaction solution was washed three times with 1M HCl and once with saturated brine. The organic phase was dried over anhydrous sodium sulfate and separated by silica gel column chromatography to obtain the intermediate product. 500 mg of the intermediate product was dissolved in 10 mL of dichloromethane. Under ice bath conditions, 1550 mg of Dess-Martin oxidant was added, and the reaction was magnetically stirred for 3 h. After the reaction was complete, the reaction solution was diluted with 40 mL of dichloromethane, and 50 mL of a saturated sodium thiosulfate-saturated sodium bicarbonate (1:1) mixed solution was added. The mixture was stirred at room temperature for 15 min. After separation, the organic phase was washed with sodium bicarbonate and saturated saline solution. After drying the organic phase with anhydrous sodium sulfate, the target probe was obtained by silica gel column chromatography.

[0042] The chemical structure of the target probe is as follows:

[0043] .

[0044] To verify the effectiveness of the target probe, food protein modification and visualization detection were performed:

[0045] The target probe was dissolved in dimethyl sulfoxide to prepare a 10 mM stock solution, which was then added to a food protein solution to modify the food protein under various food processing conditions. Subsequently, a copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) click chemistry method was used to fluorescently label the modified protein with 6-carboxyfluorescein azide (FAM-Azide). The specific procedures are as follows:

[0046] Take 10 μL of food protein solution and 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). Incubate the mixture at 37°C for 2 hours. Perform fluorescence imaging analysis of the modified protein on a 10% SDS-PAGE gel using the ChemiDoc XRS+ system, and verify the protein bands by Coomassie brilliant blue staining.

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

[0048] Design and synthesize corresponding clickable probes, and co-incubate them with food systems to modify target proteins;

[0049] Couple labeled proteins with reporter tags via click-through chemical reactions;

[0050] Analyze the effect of editing using report tags;

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

[0052] Obtain the NMR spectra (H1N and C1N spectra) of the probe, such as Figure 1 and Figure 2 As shown, the NMR spectrum confirms that the prepared probe contains an α,β-unsaturated aldehyde structure and an alkynyl group that can be used for click chemistry reactions;

[0053] The probe exhibits electrophilicity and protein modification capabilities similar to those of typical α,β-unsaturated aldehydes in food.

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

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

[0056] like Figure 5 As shown, the probe is suitable for assessing the degree of α,β-unsaturated aldehyde modification in proteins under various processing conditions:

[0057] At three different processing temperatures (25°C, 90°C, and 180°C), the degree of modification of cod protein by α,β-unsaturated aldehydes can be visually shown from the fluorescence imaging, while samples without added probes cannot be fluorescence imaged.

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

[0059] The present invention provides a protein modification probe containing an α,β-unsaturated aldehyde structure, which can modify food proteins under various food processing conditions, directly visualize the degree of protein modification during food processing, and facilitate comprehensive analysis of the modified components.

[0060] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A protein-modified probe containing an α,β-unsaturated aldehyde structure, characterized in that: The structural formula is as follows: 。 2. A method for preparing a protein modification probe having an α,β-unsaturated aldehyde structure, which comprises preparing a protein modification probe having an α,β-unsaturated aldehyde structure as claimed in claim 1, characterized by: It comprises the following steps: S1, in an inert solvent, 6-heptyne acid is subjected to esterification reaction with 2-buten-1, 4-diol in the presence of a condensing agent system to obtain an intermediate product; S2, the intermediate product obtained in step S1 is subjected to oxidation reaction, and after treatment, a protein modification probe containing an α, β-unsaturated aldehyde structure is obtained.

3. The method for preparing a protein-modified probe containing an α,β-unsaturated aldehyde structure according to claim 2, characterized in that: In the step S1, the condensing agent system comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, the molar ratio of the 6-heptyne 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 method for preparing a protein-modified probe containing an α,β-unsaturated aldehyde structure according to claim 2, characterized in that: The esterification reaction is carried out at-10℃-50℃, and the reaction time is 2-48 hours, and the inert solvent is selected from at least one of dichloromethane, chloroform or tetrahydrofuran.

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

5.

6. The method for preparing a protein-modified probe containing an α,β-unsaturated aldehyde structure according to claim 2, characterized in that: After the completion of the reaction of the step S1, the following treatment process is used to obtain the intermediate product: S101, the reaction solution is washed with 1M hydrochloric acid solution for 2-4 times; S102, washed with saturated brine; S103, the organic phase is dried with anhydrous sodium sulfate; S104, the intermediate product is obtained by silica gel column chromatography separation.

7. The method for preparing a protein-modified probe containing an α,β-unsaturated aldehyde structure according to claim 2, characterized in that, In the step S2, the oxidation reaction specifically comprises: The intermediate product is dissolved in dichloromethane, Dess-Martin oxidant is added for oxidation reaction, which is carried out at-10℃-50℃, and the reaction time is 2-48 hours.

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

1.

9. The method for preparing a protein-modified probe containing an α,β-unsaturated aldehyde structure according to claim 8, characterized in that: After the completion of the reaction in the step S2: The reaction solution is diluted with a halogenated hydrocarbon solvent; A mixed solution of sodium thiosulfate and sodium bicarbonate is added for quenching; After separation, the organic phase is washed with sodium bicarbonate solution and saturated brine in sequence; After drying with anhydrous sodium sulfate, it is purified by silica gel column chromatography.

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

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