A polyethylene glycol linker for protein modification and a synthesis method thereof
The simplified chemical synthesis steps for preparing polyethylene glycol linkers solve the problems of complex systems and high costs in existing technologies, enabling the preparation of low-cost and readily available polyethylene glycol linkers for protein modification and modification of nanoliposome materials.
Patent Information
- Application Number
- CN202111169857.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2021-10-08
- Publication Date
- 2024-12-20
- Estimated Expiration
- 2041-10-08
AI Technical Summary
Existing methods for synthesizing polyethylene glycol linkers for protein modification are complex, costly, and require readily available raw materials, and the resulting linkers are inconvenient to use.
Polyethylene glycol linkers were synthesized through a series of chemical reaction steps, including the synthesis of compounds 2, 3, 4, 5, and 6. Polyethylene glycol linkers with one end of a thiol group and the other end of a biotin group were prepared by dichloromethane extraction and water washing.
The synthesis process is simplified, the cost is reduced, the raw materials are readily available, and the prepared linker is easy to modify nanoliposome materials, making it convenient to introduce biotin groups on the surface of proteins or nanoliposome particles.
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Figure CN114085233B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of chemical synthesis, and in particular relates to a polyethylene glycol linker for protein modification and a synthesis method thereof. Background Art
[0002] Polyethylene glycol is an excellent chemical fusion promoter used in protoplast fusion. It is a polymer of ethylene glycol with the molecular formula HOCH2 (CH2O·CH2) n CH2OH. It is a white, slightly yellow waxy solid, soluble in water, and has a slight odor. There are a series of polymers with different relative molecular weights. Those with a relative molecular weight between 200 and 6000 can be used as a melting agent, but those with a relative molecular weight of 1000 are the best (in liquid form). PEG is highly water-soluble. In liquid media, the ether bonds on the surface of its molecules carry a weak negative charge. In Ca 2+ With the participation of ions, positively charged surface proteins or negatively charged glycoproteins can be transported through Ca 2+ In 50% PEG, free water disappears, which can lead to cell dehydration and cause changes in plasma membrane structure and cell fusion.
[0003] Polyethylene glycol linkers can be used to modify various types of biological drugs. By forming formulation materials through cationic lipophilic liposome nanoparticles, it is possible to instantly load targeting molecules and achieve the mounting of any drug, which can provide mature technical support for the establishment of a personalized drug library for cancer patients.
[0004] Most of the existing methods for synthesizing polyethylene glycol linkers for protein modification have complex systems, high costs, and difficult-to-obtain raw materials. In addition, the prepared polyethylene glycol linkers are inconvenient to use. Summary of the invention
[0005] The object of the present invention is to provide a polyethylene glycol linker for protein modification and a synthesis method thereof, so as to solve the problems raised in the above background technology.
[0006] To achieve the above object, the present invention provides the following technical solution: a polyethylene glycol linker for protein modification, having a chemical structure of
[0007] A method for synthesizing a polyethylene glycol linker for protein modification comprises the following steps in chronological order:
[0008] S1: Synthesis of compound 2: at 0°C, methylsulfonyl chloride was added to a dichloromethane solution of compound 1 and triethylamine. After the reaction was completed at room temperature, water was added to the reaction solution, and the mixture was extracted, washed with water, and dried to obtain compound 2;
[0009] S2: Synthesis of compound 3: Potassium thioglycolate was added to the DMF solution of compound 2. After the reaction was completed at room temperature, water was added to the reaction solution, extracted, washed with water, and dried through a column to obtain compound 3;
[0010] S3: Synthesis of compound 4: Compound 3 synthesized in the above step is added to methanol and water, lithium hydroxide monohydrate is added, and after the reaction is completed at room temperature under nitrogen protection, extraction is performed and the compound 4 is obtained by drying through a column;
[0011] S4: Synthesis of compound 5: Add compound 4 synthesized in the above step to HCl for reaction at room temperature, and after the reaction is complete, dry it to obtain compound 5;
[0012] S5: Synthesis of compound 6: dissolve pentafluorophenol activated ester in dichloromethane, add compound 5 synthesized in the above step, and after the reaction is completed at room temperature, add water to the reaction solution, extract, wash with water, and dry through a column to obtain compound 6;
[0013] The chemical structure of compound 1 is The chemical structure of compound 2 is The chemical structure of compound 3 is The chemical structure of compound 4 is The chemical structure of compound 5 is The chemical structure of compound 6 is
[0014] Preferably, in step S1, the water washing is performed three times.
[0015] Preferably in any of the above schemes, in step S2, the number of water washings is 8 times.
[0016] Preferably in any of the above schemes, in step S5, the number of water washings is 2 times.
[0017] In any of the above schemes, preferably, in steps S1, 2, 3, 4, and 5, the extractant used in the extraction is dichloromethane.
[0018] Preferably in any of the above schemes, in step S4, the concentration of HCl is 6 mol / L.
[0019] The technical effects and advantages of the present invention are as follows: the synthetic method of the polyethylene glycol linker for protein modification has readily available raw materials, simple operation and low cost; and the prepared polyethylene glycol linker has a thiol group at one end and a biotin group at the other end, so that the thiol group and the protein linker can be conveniently used to modify the monomers of the nanoliposome preparation material, thereby conveniently introducing the biotin group on the surface of the protein or the nanoliposome particle. DETAILED DESCRIPTION
[0020] The following will be described clearly and completely in conjunction with the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0021] A polyethylene glycol linker for protein modification, the chemical structure is
[0022] Embodiment 1:
[0023] A method for synthesizing a polyethylene glycol linker for protein modification comprises the following steps in chronological order:
[0024] S1: Synthesis of compound 2: at 0°C, methylsulfonyl chloride was added to a dichloromethane solution of compound 1 and triethylamine. After the reaction was completed at room temperature, water was added to the reaction solution, and the mixture was extracted, washed with water, and dried to obtain compound 2;
[0025] S2: Synthesis of compound 3: Potassium thioglycolate was added to the DMF solution of compound 2. After the reaction was completed at room temperature, water was added to the reaction solution, extracted, washed with water, and dried through a column to obtain compound 3;
[0026] S3: Synthesis of compound 4: Compound 3 synthesized in the above step is added to methanol and water, lithium hydroxide monohydrate is added, and after the reaction is completed at room temperature under nitrogen protection, extraction is performed and the compound 4 is obtained by drying through a column;
[0027] S4: Synthesis of compound 5: Add compound 4 synthesized in the above step to HCl for reaction at room temperature, and after the reaction is complete, dry it to obtain compound 5;
[0028] S5: Synthesis of compound 6: dissolve biotin pentafluorophenol activated ester in dichloromethane, add compound 5 synthesized in the above step, and after the reaction is completed at room temperature, add water to the reaction solution, extract, wash with water, and dry through a column to obtain compound 6;
[0029] The chemical structure of compound 1 is The chemical structure of compound 2 is The chemical structure of compound 3 is The chemical structure of compound 4 is The chemical structure of compound 5 is The chemical structure of compound 6 is The polyethylene glycol repeating unit of the polyethylene glycol part in the middle of the molecules of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6 can be 1 to n repeating units.
[0030] Specifically, in step S1, the number of water washings is 3 times.
[0031] Specifically, in step S2, the number of water washings is 8 times.
[0032] Specifically, in step S5, the number of water washings is 2 times.
[0033] Specifically, in steps S1, 2, 3, 4, and 5, the extraction agent used during extraction is dichloromethane.
[0034] Specifically, in step S4, the concentration of HCl is 6 mol / L.
[0035] Example 2: Synthesis of Compound 2
[0036] The polyethylene glycol repeating units in the polyethylene glycol part of the compound 1 molecule in this example are 5. At 0°C, methylsulfonyl chloride (5.5g, 48mmol) was slowly added to a dichloromethane (150ml) solution of compound 1 (this compound was purchased from Wuhan Boren Kairun Pharmaceutical Co., Ltd., please refer to www.borenpharm.com for specific information) (15.43g, 45.73mmol) and triethylamine (5.08g, 50.30mmol). After adding, it was kept at room temperature for 1h. TLC showed that the reaction was complete. 200ml of water was added to the reaction solution, and dichloromethane was extracted, washed with water 3 times, and dried to obtain 18.82g of yellow oil, which is compound 2, with a yield of 100%. The chemical structure of compound 1 is The chemical structure of compound 2 is
[0037] Example 3: Synthesis of Compound 3
[0038] At room temperature, potassium thioglycolate (7.67 g, 58.9 mmol) was slowly added to a DMF (270 ml) solution of compound 2 (18.82 g, 45.3 mmol), and the mixture was left overnight at room temperature. TLC showed that the reaction was complete. 200 ml of water was added to the reaction solution, and the mixture was extracted with dichloromethane, washed with water 8 times, and the column was dried to obtain 16 g of butter, which was compound 3, with a yield of 89.3%. The chemical formula of compound 3 is
[0039] Example 4: Synthesis of Compound 4
[0040] At room temperature, compound 3 (13 g, 32.87 mmol) was dissolved in 100 ml methanol and 100 ml water, and lithium hydroxide monohydrate (1.44 g, 34.52 mmol) was added. The mixture was incubated at room temperature for 1 h under nitrogen protection. TLC showed that the reaction was complete. The reaction solution was directly extracted with dichloromethane and dried through a column to obtain 10.2 g of a light red oily substance, which was compound 4, with a yield of 87.8%. The chemical structure of compound 4 is
[0041] Example 5: Synthesis of Compound 5
[0042] At room temperature, compound 4 (10.2 g, 28.86 mmol) was dissolved in 6 mol / L HCl (50 ml). After TLC showed that the reaction was complete, 9.187 g of butter was directly dried to obtain compound 5, with a yield of 110%. A small amount of hydrochloric acid was not dried. The chemical structure of compound 5 is
[0043] Example 6: Synthesis of Compound 6
[0044] At room temperature, biotin pentafluorophenol activated ester (12.7 g, 30.94 mmol) was dissolved in dichloromethane (170 ml), and compound 5 (8.373 g, 28.84 mmol) and triethylamine (12.5 g, 123.76 mmol) were added, and the mixture was kept at room temperature overnight. After TLC showed that the reaction was complete, the reaction solution was acidified with water, and then directly extracted with dichloromethane, washed with water twice, and dried over a column to obtain 8.8 g of a white solid with a yield of 63.1%. The white solid was compound 6 (polyethylene glycol linker). The chemical structure of compound 6 is
[0045] Finally, it should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for synthesizing a polyethylene glycol linker for protein modification, characterized in that: The following steps are involved: S1: Synthesis of compound 2: at 0°C, methylsulfonyl chloride was added to a dichloromethane solution of compound 1 and triethylamine. After the reaction was completed at room temperature, water was added to the reaction solution, and the mixture was extracted, washed with water, and dried to obtain compound 2; S2: Synthesis of compound 3: Potassium thioglycolate was added to the DMF solution of compound 2. After the reaction was completed at room temperature, water was added to the reaction solution, extracted, washed with water, and dried through a column to obtain compound 3; S3: Synthesis of compound 4: Compound 3 synthesized in the above step is added to methanol and water, lithium hydroxide monohydrate is added, and after the reaction is completed at room temperature under nitrogen protection, extraction is performed and the compound 4 is obtained by drying through a column; S4: Synthesis of compound 5: Add compound 4 synthesized in the above step to HCl for reaction at room temperature, and after the reaction is complete, dry it to obtain compound 5; S5: Synthesis of compound 6: dissolve pentafluorophenol activated ester in dichloromethane, add compound 5 synthesized in the above step and triethylamine, after the reaction is completed at room temperature, add water to the reaction solution, extract, wash with water, and dry through a column to obtain compound 6; The chemical structure of compound 1 is The chemical structure of compound 2 is The chemical structure of compound 3 is The chemical structure of compound 4 is The chemical structure of compound 5 is The chemical structure of compound 6 is 2. The method for synthesizing a polyethylene glycol linker for protein modification according to claim 1, characterized in that: In step S1, the water washing is performed 3 times.
3. The method for synthesizing a polyethylene glycol linker for protein modification according to claim 1, characterized in that: In step S2, the water washing times are 8 times.
4. The method for synthesizing a polyethylene glycol linker for protein modification according to claim 1, characterized in that: In step S5, the water washing is performed twice.
5. The method for synthesizing a polyethylene glycol linker for protein modification according to claim 1, characterized in that: In steps S1, S2, S3, S4 and S5, the extractant used in the extraction is dichloromethane.
6. The method for synthesizing a polyethylene glycol linker for protein modification according to claim 1, characterized in that: In step S4, the concentration of HCl is 6 mol / L.