Method for preparing two-arm PEG modifier

Abstract

The invention provides a synthesis method of a two-arm PEG modifier. The synthesis method comprises the step of reacting mPEG-SC with lysine in a mixed solvent system of an organic solvent and water. Compared with an existing preparation process, the method has the advantages that the reaction coupling efficiency and the yield of a final product, namely, the two-arm PEG lysine are greatly improved, the applicability of the process to mPEG-SC with different molecular weights is expanded, and the method is suitable for industrial production of the two-arm PEG lysine.

Description

[0001] This application claims priority to the prior art of the applicant’s earlier application filed with the China National Intellectual Property Administration on September 26, 2025, with patent application number 202511396815.0 and entitled “Method for preparing a two-arm PEG modifier”; the entire contents of the earlier application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of organic synthesis, specifically relating to a method for preparing two-arm PEG modifiers using lysine or its salts. Background Technology

[0003] Polyethylene glycol (PEG) is a linear, highly hydrophilic polymer that is non-toxic to humans, has good biocompatibility, and low immunogenicity. When linked to drugs, it can impart the excellent properties of the polymer itself to the drug molecules.

[0004] Polyethylene glycol modification technology has been developed for decades, and various branched PEG modifiers have been developed, such as two-armed PEG modifiers. Compared with single-armed PEG modifiers, their molecular weight increases several times, thereby increasing the size of the conjugate molecule, improving the selectivity for modification sites, thus prolonging the in vivo retention time and reducing immunogenicity.

[0005] Methods for preparing two-arm PEG modifiers using lysine have been reported in the literature, mainly including:

[0006] 1) US Patent US20130177961 discloses a method for synthesizing an aqueous system. It uses a borax buffer solution with a pH of 8.0-8.3 as the reaction solvent. After reacting monooxy polyethylene glycol succinimide carbonate (mPEG-SC) with lysine at room temperature for 24 hours, the corresponding mPEG2-lysine (10 kDa) is obtained through acidification, extraction, crystallization and DEAE column chromatography purification. The final product yield is about 50%, and the reaction time is as long as 24 hours.

[0007] 2) Chinese patent CN114716663A discloses an organic synthesis method that uses a mixed solvent of anhydrous ethanol and dichloromethane as the reaction solvent. Lysine is added during the reaction. After the reaction, the crude mPEG2-lysine is obtained by MTBE precipitation. The crude product is then purified by DEAE column chromatography and other purification steps to obtain the final product mPEG2-lysine (10 kDa). The yield of the final product is not high (about 71%). Moreover, the method uses the method of adding lysine, which makes it difficult to accurately control the amount of reagent added and easily generates monosubstituted impurities.

[0008] Therefore, it is particularly necessary to develop new methods for synthesizing mPEG2-Lysine with high coupling efficiency and suitable for industrial production. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides a method for synthesizing a two-arm PEG modifier, aiming to improve reaction coupling efficiency and thereby increase the yield of the final product, the two-arm PEG modifier.

[0010] According to an embodiment of the present invention, the two-arm PEG modifier prepared by the present invention is a two-arm PEG lysine, and its preparation method includes reacting mPEG-SC of formula (I) with lysine or its salt in a mixed system of organic solvent A and water under alkaline conditions.

[0011]

[0012] In equations (I) and (II), n is the same, and is an integer between 4 and 500.

[0013] According to an embodiment of the present invention, n is an integer between 40 and 400.

[0014] According to an embodiment of the present invention, n is an integer between 300 and 500.

[0015] According to an embodiment of the present invention, n is an integer of 450, i.e., the molecular weight of mPEG-SC is 20 kDa.

[0016] According to an embodiment of the present invention, the molecular weight of the two-arm PEGylated lysine prepared by the present invention is 40 kDa.

[0017] According to an embodiment of the present invention, the lysine salt is lysine dihydrochloride.

[0018] According to embodiments of the present invention, the organic solvent A includes, but is not limited to, one or more of alcohol solvents, ketone solvents, ether solvents, ester solvents, sulfone or sulfoxide solvents, halogenated hydrocarbon solvents, and nitrile solvents. The alcohol solvents include, but are not limited to, methanol, ethanol, propanol, and isopropanol. The ketone solvents include, but are not limited to, acetone. The ether solvents include, but are not limited to, diethyl ether and tetrahydrofuran. The ester solvents include, but are not limited to, ethyl formate and ethyl acetate. The sulfone or sulfoxide solvents include, but are not limited to, dimethyl sulfoxide (DMSO). The halogenated hydrocarbon solvents include, but are not limited to, dichloromethane. The nitrile solvents include, but are not limited to, acetonitrile (CH3CN).

[0019] According to an embodiment of the present invention, the organic solvent A is selected from one or more of alcohol solvents, ketone solvents, ether solvents, ester solvents, sulfoxide solvents, halogenated hydrocarbon solvents, and nitrile solvents.

[0020] According to an embodiment of the present invention, the organic solvent A is selected from one or more of methanol, ethanol, propanol, isopropanol, acetone, tetrahydrofuran, ethyl acetate, dimethyl sulfoxide, dichloromethane, and acetonitrile.

[0021] According to an embodiment of the present invention, organic solvent A is selected from one or more of isopropanol, acetone, tetrahydrofuran, acetonitrile, methanol, dimethyl sulfoxide, and ethanol.

[0022] According to an embodiment of the present invention, organic solvent A is selected from acetonitrile or dimethyl sulfoxide.

[0023] According to an embodiment of the present invention, the volume ratio of the organic solvent A to water is (0.2-50):1; preferably (1-20):1; more preferably (1-15):1.

[0024] According to an embodiment of the present invention, the alkaline conditions are provided by adding one or more alkaline reagents, which include organic bases and inorganic bases. The organic or inorganic base is selected from triethylamine, pyridine, DIPEA, DBU, imidazole, sodium hydroxide, potassium hydroxide, sodium tert-butoxide, potassium tert-butoxide, potassium bicarbonate, sodium carbonate, sodium bicarbonate, cesium carbonate, and potassium carbonate.

[0025] According to an embodiment of the present invention, the molar ratio of mPEG-SC to alkaline reagent is 1:(1-10), preferably 1:(1-5); more preferably 1:(1-2.5).

[0026] According to an embodiment of the present invention, the molar ratio of mPEG-SC to alkaline reagent is 1:2.2.

[0027] According to an embodiment of the present invention, the molar ratio of mPEG-SC to lysine or its salt is 1:(0.4-1), for example 1:(0.45-0.7) or 1:(0.5-0.7).

[0028] According to an embodiment of the present invention, the molar ratio of mPEG-SC to lysine or its salt is 1:0.55.

[0029] According to an embodiment of the present invention, the reaction is carried out at a temperature of 0–60°C, for example at a temperature of 30–60°C.

[0030] According to an embodiment of the present invention, the reaction is carried out at 30±5°C.

[0031] According to an embodiment of the present invention, the reaction time is 0.1 to 10 h, preferably 1 to 5 h, more preferably 1 to 2 h; wherein the reaction time is the duration of the reaction after all the solution of lysine or its salt has been added to the reaction system.

[0032] According to an embodiment of the present invention, the preparation method includes: Step 1, dissolving mPEG-SC in organic solvent A, stirring to dissolve, and adding an alkaline reagent; Step 2, dissolving lysine or its salt in water, and adding the aqueous solution of lysine or its salt to the reaction system of Step 1.

[0033] Beneficial effects

[0034] This invention employs an organic solvent / water two-phase system, which not only ensures the dissolution of the substrate mPEG-SC and lysine, but also effectively avoids the hydrolysis of mPEG-SC, greatly improving the reaction coupling efficiency and the yield of the final product, two-arm PEG lysine. This expands the applicability of the process to mPEG-SC with different molecular weights and is suitable for the industrial production of two-arm PEG lysine. Detailed Implementation

[0035] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0036] The terms “two-armed PEG lysine,” “branched acid,” and “branched carboxylic acid” can be used interchangeably in this article, referring to the product formed by a lysine molecule being linked to a PEG molecule via two amino groups through amide bonds.

[0037] "Branched acid content" refers to the mass percentage of cladinated acids, determined by size exclusion chromatography. After the reaction is complete, the cladinated acid content is determined by size exclusion chromatography, and this percentage is the reaction coupling efficiency in this text.

[0038] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0039] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0040] Example 1: Solvent Screening Experiment for mPEG-SC and Lysine Dihydrochloride

[0041] Using mPEG-SC with a molecular weight of 20 kDa as the raw material, the molar ratio of mPEG-SC: lysine dihydrochloride: base was 1:0.55:2.2. 10 g of mPEG-SC was weighed using an analytical balance and added to a 100 mL round-bottom flask. A stir bar was added, followed by 30 mL of reaction solvent A. The mixture was stirred and dissolved in an oil bath at 30 ± 5 °C. 191.6 μL of DIPEA was then added. 60.25 mg of lysine dihydrochloride was weighed and added to solvent B, stirred until completely dissolved, and then added to the above reaction system. The mixture was stirred and reacted for 1–2 h. The branched acid content was determined by size exclusion chromatography.

[0042] Table 1

[0043]

[0044] Experimental Conclusions: By investigating the effect of different solvent systems on coupling efficiency, the results show that the coupling efficiency can reach over 87.5% when the reaction solvent is a mixture of propanol / water, acetone / water, tetrahydrofuran / water, acetonitrile / water, methanol / water, dimethyl sulfoxide / water, and ethanol / water. Among these, the coupling efficiency can reach over 95% when acetonitrile / water and dimethyl sulfoxide / water are used as the reaction solvents. However, when lysine dihydrochloride is dissolved in dichloromethane, ethyl acetate, methanol, or acetonitrile before being added to the reaction system, the coupling efficiency is generally lower. The experimental results indicate that water is the preferred solvent for lysine dihydrochloride.

[0045] Example 2: Screening Experiment with Different Bases

[0046] Using mPEG-SC with a molecular weight of 20 kDa as the raw material, the molar ratio of mPEG-SC: lysine dihydrochloride: base was 1:0.55:2.2. 10 g of mPEG-SC was weighed using an analytical balance and added to a 100 mL round-bottom flask. A stir bar was added, followed by 30 mL of dimethyl sulfoxide. The mixture was stirred and dissolved in an oil bath at 30 ± 5 °C, and then the base was added. 60.25 mg of lysine dihydrochloride and 10 mL of water were weighed, completely dissolved, and added to the above reaction system. The mixture was stirred and reacted at 30 ± 5 °C for 1–2 h. The branched acid content was determined by size exclusion chromatography.

[0047] Table 2

[0048] Group Alkali (2.2 eq) Branched acid content 3-1 Triethylamine 93.6％ 3-2 Pyridine 93.5％ 3-3 DIPEA 95.6％ 3-4 DBU 81.5％ 3-5 imidazole 87.4％ 3-6 Sodium hydroxide 80.5％ 3-7 potassium hydroxide 81.7％ 3-8 Sodium tert-butoxide 80.4％ 3-9 Potassium tert-butoxide 80.4％ 3-10 Potassium bicarbonate 92.5％ 3-11 Sodium carbonate 94.0％ 3-12 Sodium bicarbonate 93.9％ 3-13 cesium carbonate 89.9％ 3-14 Potassium carbonate 94.3％

[0049] Experimental conclusion: By investigating the effect of different bases on the content of branched acid, the results showed that the content of branched acid could reach more than 80% under both organic and inorganic base conditions. Among them, when the bases were triethylamine, pyridine, DIPEA, potassium bicarbonate, sodium carbonate, sodium bicarbonate, and potassium carbonate, the content of branched acid was more than 92%.

[0050] Example 3: Experiment to investigate the reaction temperature of the system

[0051] Using mPEG-SC with a molecular weight of 20 kDa as the raw material, the molar ratio of mPEG-SC: lysine dihydrochloride: base was 1:0.55:2.2. 10 g of mPEG-SC was weighed using an analytical balance and added to a 100 mL round-bottom flask. A stir bar was added, followed by 30 mL of dimethyl sulfoxide. The mixture was stirred and dissolved in an oil bath, with the internal temperature controlled at A. 191.6 μL of DIPEA was added. 60.25 mg of lysine dihydrochloride was weighed, dissolved completely in 10 mL of water, and then added to the above reaction system. The mixture was stirred and reacted at temperature A for 1–2 h. The branched acid content was determined by size exclusion chromatography.

[0052] Table 3

[0053] Group 4-1 4-2 4-3 4-4 4-5 4-6 4-7 Temperature A 0℃ 10℃ 20℃ 30℃ 40℃ 50℃ 60℃ Branched acid content 91.4％ 93.8％ 95.6％ 95.7％ 95.3％ 95.5％ 95.5％

[0054] Experimental conclusion: By investigating the effect of different reaction temperatures on the content of branched acid, the results showed that the content of branched acid was above 91% in the reaction temperature range of 0 to 60℃, and the content of branched acid was slightly higher in the reaction temperature range of 30℃ to 60℃.

[0055] Example 4: Experiment on the dosage of lysine dihydrochloride

[0056] Using mPEG-SC with a molecular weight of 20 kDa as the raw material, the molar ratio of mPEG-SC: lysine dihydrochloride: base was 1:X:2.2. 10 g of mPEG-SC was weighed using an analytical balance and added to a 100 mL round-bottom flask. A stir bar was added, followed by 30 mL of dimethyl sulfoxide. The mixture was stirred and dissolved in an oil bath at 30 ± 5 °C, and then 191.6 μL of LDPEA was added. Lysine dihydrochloride was weighed, dissolved in 10 mL of water, and then added to the above reaction system. The mixture was stirred and reacted at 30 ± 5 °C for 1–2 h. The content of branched acid was determined by size exclusion chromatography.

[0057] Table 4

[0058] Group Dosage of lysine dihydrochloride X Branched acid content 5-1 0.4eq 76.0％ 5-2 0.45eq 85.3％ 5-3 0.50eq 91.7％ 5-4 0.55eq 95.5％ 5-5 0.60eq 95.4％ 5-6 0.70eq 95.3％

[0059] Experimental conclusion: By investigating the effect of lysine dihydrochloride dosage on the reaction coupling efficiency, the results showed that when the dosage of lysine dihydrochloride was 0.4 eq, the reaction coupling efficiency was only 76%. As the dosage increased to 0.55 eq, the reaction coupling efficiency could be increased to 95.5%. Further increasing the dosage of lysine dihydrochloride (e.g., 0.6-0.7 eq) did not significantly change the coupling efficiency.

[0060] Example 5, Scale-up Experiment

[0061] Using mPEG-SC with a molecular weight of 20 kDa as the raw material, the molar ratio of mPEG-SAC: lysine dihydrochloride: alkali was 1:0.55:2.2. 298 g of mPEG-SC was weighed using a balance and added to a 5 L glass reactor. 894 mL of dimethyl sulfoxide was added, and the reactor temperature was maintained at 30 ± 5 °C while stirring to dissolve the solution. 57.1 mL of DIPEA was then added. Weigh 1.832 g of lysine dihydrochloride and add it to 89.4 mL of water. After complete dissolution, add the dissolved lysine to the above reaction system and stir at 30±5℃ for 1–2 h. Dilute the system dropwise with water 15–30 times, and perform ultrafiltration using a PES 5K membrane. After ultrafiltration, load the sample onto a DEAE ion exchange column, wash with purified water for 1–2 column volumes, and then elute with a high-concentration sodium chloride solution. Collect the eluent, adjust the pH to approximately 3 with HCl, extract with DCM, dry to anhydrous sodium sulfate, filter, concentrate under reduced pressure to remove the solvent, precipitate with 6000 mL of isopropanol at 0℃, filter, and vacuum dry to obtain 253.5 g of branched carboxylic acid, with a yield of 85.1%. The content of branched carboxylic acid in the final product was almost 100% as determined by size exclusion chromatography.

[0062] Comparative Example 1

[0063] Using mPEG-SC with a molecular weight of 20 kDa as the raw material, the molar ratio of mPEG-SC: lysine dihydrochloride: triethylamine was 1:0.5:2.0. 10 g of mPEG-SC was weighed using an analytical balance and added to a 100 mL round-bottom flask. A stir bar was added, followed by 40 mL of dichloromethane. The mixture was stirred and dissolved in a 30°C oil bath, and then 101 mg of triethylamine was added. 54.7 mg of lysine dihydrochloride was weighed, dissolved in 4 mL of ethanol, and added to the above reaction system. The mixture was stirred and reacted at 30°C for 1 h. The branched acid content was determined to be 46.5% by size exclusion chromatography.

Claims

1. A method for preparing two-arm PEG-lysine of formula (II), comprising reacting mPEG-SC of formula (I) with lysine or a salt thereof in a mixture of organic solvent A and water under alkaline conditions. in, n is an integer between 4 and 500.

2. The method of claim 1, wherein n is an integer between 200 and 500; Preferably, n is an integer between 300 and 500, for example, n is an integer of 450.

3. The method according to any one of claims 1-2, wherein the lysine salt is lysine dihydrochloride.

4. The method according to any one of claims 1-3, wherein the organic solvent A includes, but is not limited to, one or more of alcohol solvents, ketone solvents, ether solvents, ester solvents, sulfone or sulfoxide solvents, halogenated hydrocarbon solvents, and nitrile solvents; Preferably, the organic solvent A is selected from one or more of alcohol solvents, ketone solvents, ether solvents, ester solvents, sulfoxide solvents, halogenated hydrocarbon solvents, and nitrile solvents; Preferably, the organic solvent A is selected from one or more of methanol, ethanol, propanol, isopropanol, acetone, tetrahydrofuran, ethyl acetate, dimethyl sulfoxide, dichloromethane, and acetonitrile; Preferably, according to an embodiment of the present invention, organic solvent A is selected from one or more of isopropanol, acetone, tetrahydrofuran, acetonitrile, methanol, dimethyl sulfoxide, and ethanol; More preferably, organic solvent A is selected from acetonitrile or dimethyl sulfoxide.

5. The method according to any one of claims 1-4, wherein the volume ratio of the organic solvent A to water is (0.2-50):1; Preferably, the volume ratio of the organic solvent A to water is (1-20):1; More preferably, the volume ratio of the organic solvent A to water is (1-15):

1.

6. The method according to any one of claims 1-5, wherein the alkaline conditions are provided by adding one or more alkaline reagents, the alkaline reagents including organic bases and inorganic bases; Preferably, the organic or inorganic base is selected from triethylamine, pyridine, DIPEA, DBU, imidazole, sodium hydroxide, potassium hydroxide, sodium tert-butoxide, potassium tert-butoxide, potassium bicarbonate, sodium carbonate, sodium bicarbonate, cesium carbonate, and potassium carbonate. Preferably, the molar ratio of mPEG-SC to alkaline reagent is 1:(1-10); More preferably, the molar ratio of mPEG-SC to alkaline reagent is 1:(1-5).

7. The method according to any one of claims 1-6, wherein the molar ratio of mPEG-SC to lysine or its salt is 1:(0.4-1); Preferably, the molar ratio of mPEG-SC to lysine or its salt is 1:(0.45-0.7); Preferably, the molar ratio of mPEG-SC to lysine or its salt is 1:(0.5-0.7); Preferably, the molar ratio of mPEG-SC to lysine or its salt is 1:0.

55.

8. The method according to any one of claims 1-7, wherein the reaction is carried out at 0-60°C; Preferably, the reaction is carried out at 30–60°C; Preferably, the reaction is carried out at 30±5°C.

9. The method of any one of claims 1-8, wherein the method comprises: Step 1: Dissolve mPEG-SC in organic solvent A, stir to dissolve, and add alkaline reagent; Step 2: Dissolve lysine or its salt in water, and add the aqueous solution of lysine or its salt to the reaction system of Step 1.

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