A method for synthesizing polyethylene glycol carboxyl-activated esters

Abstract

This invention discloses a method for synthesizing polyethylene glycol (PEG) carboxyl-activated esters. This preparation method utilizes centrifugation, taking advantage of the density difference in the solvent to accelerate the extraction process, thus saving production time. Furthermore, purification via membrane treatment in hydrochloric acid solution improves product purity. The use of hydrochloric acid prevents the degradation of the carboxyl-activated ester in water, while the membrane treatment rapidly removes insoluble substances, ensuring purity while preventing deactivation of the carboxyl-activated ester. Both methods employed are highly adaptable to industrial applications. The re-optimized synthesis and purification steps significantly improve the purity and yield of the PEG carboxyl-activated ester. This method for preparing PEG carboxyl-activated esters is simple, yields high purity and high output, and is easily industrialized. It can be used for drug modification, hydrogel preparation, medical sponges, tissue sealants, bio-adhesives, biological scaffolds, fluorescence imaging, and more.

Description

Technical Field

[0001] This invention belongs to the field of polyethylene glycol preparation technology, specifically relating to a method for synthesizing polyethylene glycol carboxyl-activated esters. Background Technology

[0002] Carboxyl-activated ester modified polyethylene glycol (NHS-PEG-NHS) has significant applications in pharmaceuticals, chemical engineering, clinical medicine, and tissue engineering due to its rapid reaction with any amino-containing substance. For example, in the pharmaceutical field, NHS-PEG-NHS can be used to modify drugs and increase their metabolic half-life. Secondly, it can be used as a crosslinking agent to prepare hydrogels and biogels. Furthermore, NHS-PEG-NHS can be used for fluorescein labeling, which has great application value in diagnostics, detection, and imaging. However, commercially available NHS-PEG-NHS is currently expensive, costing between 800-1500 yuan per 100 mg. At the same time, the yield of industrially produced NHS-PEG-NHS is low, the production cycle is long, and the catalyst and byproducts in the final reaction step are difficult to remove, greatly limiting the development of scientific research and other fields. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for synthesizing polyethylene glycol carboxyl-activated esters, so as to solve the problems of low yield and high preparation cost of NHS-PEG-NHS in the prior art.

[0004] To achieve the above objectives, the present invention employs the following technical solution:

[0005] A method for synthesizing polyethylene glycol carboxyl-activated esters includes the following steps:

[0006] Step 1: Dissolve polyethylene glycol, acid anhydride and 4-dimethylaminopyridine in a solvent to obtain product solution A. Mix product solution A with a 0.1-2M hydrochloric acid aqueous solution of saturated sodium chloride and shake. Centrifuge the shaken solution to obtain organic phase centrifuged product B. Dry centrifuged product B with anhydrous magnesium sulfate and evaporate the solvent to obtain evaporated product C. Add petroleum ether to precipitate evaporated product C and rotary evaporate to obtain carboxylated polyethylene glycol.

[0007] Step 2: Dissolve carboxylated polyethylene glycol, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a solvent, mix and react to obtain product solution D. Mix product solution D with 0.1-2M hydrochloric acid aqueous solution of saturated sodium chloride and shake. Centrifuge the shaken solution to obtain organic phase centrifuged product E. Dry centrifuged product E with anhydrous magnesium sulfate and evaporate the solvent to obtain evaporated product F. Add petroleum ether to precipitate evaporated product F and rotary evaporate to obtain carboxyl-activated ester polyethylene glycol.

[0008] Step 3: Dissolve the carboxyl-activated polyethylene glycol in a hydrochloric acid solution with pH = 1.0-6.0, filter through a filter membrane, and then freeze dry using a freeze dryer to obtain the final polyethylene glycol carboxyl-activated ester.

[0009] A further improvement of the present invention is that:

[0010] Preferably, in step 1, the concentration of polyethylene glycol in the solvent is 0.1%-80%, and the mixing equivalent ratio of polyethylene glycol, acid anhydride and 4-dimethylaminopyridine is (0.01-1):(0.01-1):(0.01-1).

[0011] Preferably, the specific process in step 1 is as follows: polyethylene glycol, acid anhydride, and 4-dimethylaminopyridine are dissolved in a solvent according to a set ratio and reacted at 20-39℃ for 4-72h to obtain product solution A; product solution A is mixed with an equal volume of 0.1-2M hydrochloric acid aqueous solution of saturated sodium chloride and shaken; the shaken solution is centrifuged at 500-50000rpm for 1-60min and the supernatant is discarded; an equal volume of 0.1-2M hydrochloric acid aqueous solution is added, mixed, shaken, and centrifuged to obtain the supernatant; the process of adding hydrochloric acid aqueous solution, mixing, shaking, and centrifuging to obtain the supernatant is repeated 1-5 times to obtain centrifuged product B; centrifuged product B is dried with anhydrous magnesium sulfate to remove water, and then the solvent is evaporated to 1-10mL in a vacuum rotary evaporator to obtain evaporated product C; petroleum ether is added to evaporated product C to precipitate, and the mixture is further rotary evaporated to dryness to obtain carboxylated polyethylene glycol.

[0012] Preferably, in steps 1 and 2, the solvent is anhydrous dichloromethane, anhydrous chloroform, anhydrous dichloroethane, anhydrous ethanol, anhydrous methanol, anhydrous DMF, anhydrous acetone, anhydrous methyl ethyl ketone, anhydrous tetrahydrofuran, anhydrous xylene, anhydrous acetonitrile, anhydrous dimethyl sulfoxide, anhydrous pyridine, or anhydrous N,N-dimethylformamide.

[0013] Preferably, in step 1, the polyethylene glycol has the structural formula I, II, III, or IV, where n is 10-10000; specifically:

[0014]

[0015] Preferably, in step 1, the acid anhydride is acetic anhydride, propionic anhydride, succinic anhydride, glutaric anhydride, maleic anhydride, phthalic anhydride, dimethylmaleic acid, pyromellitic anhydride, dodecanoic anhydride, 3-nitro-1,8-naphthalenedicarboxylic anhydride, bicyclo[2.2.2]oct-5-ene-2,3-dicarboxylic anhydride, cis-5-norbornene-ex-2,3-dicarboxylic anhydride, pyridine-3,4-dicarboxylic anhydride, benzothioxanone dicarboxylic anhydride, 2,3- Pyridine dicarboxylic anhydride, creatine anhydride, chlorobridged anhydride, crotonic anhydride, biphenyl anhydride, carnonic anhydride, NA anhydride, indigo anhydride, palmitic anhydride, acrylic anhydride, itaconic anhydride, 4-fluorophthalic anhydride, trimellitic anhydride, trimethylacetic anhydride, acetylsalicylic anhydride, thioglycolic anhydride, methylnadic anhydride, norbornene anhydride, 3-methylitanic anhydride, 2-methacrylic anhydride, hexahydrophthalic anhydride, 3-hydroxyphthalic anhydride, 2,3-pyridinedicarboxylic anhydride Azithiopic anhydride, N-decenylsuccinic anhydride, 1,2-cyclopentadicarboxylic anhydride, S-acetylmercaptosuccinic anhydride, 1,1-cyclohexyldiacetic anhydride, 2,2-dimethylsuccinic anhydride, 3,3-tetramethyleneglutarate anhydride, cyclobutane-1,2-dicarboxylic anhydride, 4,5-dimethoxyphthalic anhydride, 4,4'-oxobisphthalic anhydride, 1,2,4-trimeric anhydride acyl chloride, 1,4,5,8-naphthalenetetracarboxylic anhydride, D-propanediol Acid-N-carboxyl-cyclic anhydride, 1,8-naphthalenedicarboxylic anhydride-4-sulfonate potassium, (+)-diacetyl-L-tartaric anhydride, 4-methacryloyloxyethyltriphenyltrihydride, 4,4'-diphthalic anhydride, 5-(2,5-dioxotetrahydro-3-furanyl)-3-methyl-3-cyclohexene-1,2-dicarboxylic anhydride or (S)-3-acetoxydihydro-2,5-furandione O-acetyl-L-malic anhydride.

[0016] Preferably, in step 2, the mass fraction of the carboxylated polyethylene glycol in the solvent is 0.1%-80%.

[0017] Preferably, in step 2, the mixing ratio of carboxylated polyethylene glycol, EDC and NHS is (0.01-1):(0.01-1):(0.01-1).

[0018] Preferably, step 2 involves mixing carboxylated polyethylene glycol, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide in a specific ratio, reacting at 20-39°C for 4-72 hours to obtain product solution D. Product solution D is then mixed with an equal volume of 0.1-2M hydrochloric acid aqueous solution containing saturated sodium chloride, shaken for 1-60 minutes, and centrifuged at 500-50000 rpm for 1-60 minutes. The supernatant is discarded, and the process of adding saturated sodium chloride aqueous solution, shaking, centrifuging, and discarding the supernatant is repeated 1-5 times to obtain centrifuged product E. This product E is dried with anhydrous magnesium sulfate to remove water, and then evaporated to 1-10 mL using a vacuum rotary evaporator to obtain evaporated product F. 20-100 mL of petroleum ether is added to the evaporated product to precipitate it, and the mixture is further evaporated to dryness to obtain carboxyl-activated polyethylene glycol.

[0019] Preferably, polyethylene glycol carboxyl activated ester is dissolved in an aqueous hydrochloric acid solution with pH = 1.0-6.0, filtered through a 0.01-0.45 μm filter membrane, and then freeze-dried using a freeze dryer to obtain polyethylene glycol carboxyl activated ester.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention discloses a method for synthesizing polyethylene glycol carboxyl-activated esters. This preparation method utilizes centrifugation, taking advantage of the difference in solvent density to accelerate the extraction speed, thus saving production time. In the final step of the carboxyl-activated ester synthesis, the catalyst and byproducts are often difficult to remove completely, resulting in turbidity when dissolved in water. This invention improves product purity by purifying the product through a membrane in hydrochloric acid solution. Using hydrochloric acid avoids the degradation of the carboxyl-activated ester in water, while the membrane rapidly removes insoluble substances, ensuring purity while preventing deactivation of the carboxyl-activated ester. Both methods employed have strong industrial adaptability, and the re-optimized synthesis and purification steps significantly improve the purity and yield of the polyethylene glycol carboxyl-activated ester. This method for preparing polyethylene glycol carboxyl-activated esters is simple, high in purity and yield, and easy to industrialize. It can be used for drug modification, hydrogel preparation, medical sponges, tissue sealants, bio-glues, biological scaffolds, fluorescence imaging, etc. Attached Figure Description

[0022] Figure 1 The NHS-PEG-NHS proton NMR spectrum of Example 1 of this invention;

[0023] Figure 2 The carbon spectrum of NHS-PEG-NHS in Example 1 of this invention;

[0024] Figure 3 The NHS-PEG-NHS proton NMR spectrum of Example 1 of this invention;

[0025] Figure 4 This is the carbon spectrum of NHS-PEG-NHS in Example 1 of the present invention. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to the specific preparation process:

[0027] This invention further provides a method for preparing polyethylene glycol carboxyl-activated esters by hydroxyl synthesis of polyethylene glycol, comprising the following steps:

[0028] (1) Synthesize polyethylene glycol with carboxyl end groups;

[0029] In step (1), in the polyethylene glycol carboxyl-activated ester reaction, the mass fraction of polyethylene glycol in the solvent can be 0.1%-80%, preferably 1%-50%, and more preferably 10%. Polyethylene glycol, acid anhydride, and 4-dimethylaminopyridine (DMAP) are dissolved in the solvent at an equivalent ratio of 0.01-1:0.01-1:0.01-1 and reacted at 20-39°C for 1-24 hours. More preferably, the equivalent ratio is 1:2:5. After the reaction, product solution A is obtained. Product solution A is mixed with an equal volume of 0.1-2M hydrochloric acid aqueous solution of saturated sodium chloride and shaken. Washing with saturated sodium chloride solution is crucial during this process, as it removes impurities and unreacted acid anhydride, DMAP, etc. After washing with saturated sodium chloride, the traditional method is to let the solution stand and wait for separation. However, this waiting time is long, sometimes exceeding one day, greatly slowing down production efficiency. The present invention then employs centrifugation parameters of 500-50000 rpm for 1-60 min on a centrifuge. Preferably, the centrifugation speed is 3000 rpm and the centrifugation time is 5 min. The supernatant is discarded, and this process is repeated 1-5 times. The centrifuged product is dried with anhydrous magnesium sulfate to remove water, and then the solvent is evaporated to 1-10 mL on a vacuum rotary evaporator. Petroleum ether is added to precipitate the product, and rotary evaporation is continued to obtain the product, which is carboxylated polyethylene glycol with the molecular formula HOOC-PEG-COOH.

[0030] The solvent can be any solvent that can dissolve polyethylene glycol, such as anhydrous dichloromethane, anhydrous chloroform, anhydrous dichloroethane, anhydrous ethanol, anhydrous methanol, anhydrous DMF, anhydrous acetone, anhydrous methyl ethyl ketone, anhydrous tetrahydrofuran, anhydrous xylene, anhydrous acetonitrile, anhydrous dimethyl sulfoxide, anhydrous pyridine, anhydrous N,N-dimethylformamide, etc.

[0031] (2) Synthesize polyethylene glycol with carboxyl-activated esters at the end groups;

[0032] In step (2), during the reaction of the polyethylene glycol carboxyl activated ester, the mass fraction of the carboxylated polyethylene glycol can be 0.01%-80%. The carboxylated polyethylene glycol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) are dissolved in a solvent and reacted. The ratio of carboxylated polyethylene glycol: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC): N-hydroxysuccinimide (NHS) is a:b:c (equivalent ratio), where a, b, and c can be 0.01 to 1, and more preferably 1:2:5.

[0033] The specific reaction process is as follows: HOOC-PEG-COOH, EDC and NHS are mixed in proportion and reacted at 20-39℃ for 4-72h. After the reaction solution is mixed with an equal volume of 0.1-2M hydrochloric acid aqueous solution of saturated sodium chloride, the mixture is shaken for 1-60min and centrifuged at 500-50000rpm for 1-60min. The supernatant is discarded. This process is repeated 1-5 times. After drying with anhydrous magnesium sulfate, the solvent is evaporated to about 1-10mL in a vacuum rotary evaporator. Petroleum ether is added to precipitate the product, and rotary evaporation is continued to obtain the product, which is polyethylene glycol carboxyl activated ester.

[0034] The solvent used to dissolve the carboxylated polyethylene glycol by mass fraction can be anhydrous dichloromethane, anhydrous chloroform, anhydrous dichloroethane, anhydrous ethanol, anhydrous methanol, anhydrous DMF, anhydrous acetone, anhydrous methyl ethyl ketone, anhydrous tetrahydrofuran, anhydrous xylene, anhydrous acetonitrile, anhydrous dimethyl sulfoxide, anhydrous pyridine, anhydrous N,N-dimethylformamide, etc.

[0035] (3) The obtained polyethylene glycol carboxyl activated ester was subjected to membrane treatment;

[0036] In step (3), the initially obtained polyethylene glycol carboxyl-activated ester is dissolved in hydrochloric acid aqueous solution with pH = 1.0-6.0, filtered through a 0.1-0.45 μm filter membrane, and then freeze-dried to obtain polyethylene glycol carboxyl-activated ester with relatively high purity. How to remove the byproducts acylurea produced after the reaction of EDC, NHS, and polyethylene glycol carboxyl groups has always been a problem in industrial production. Most commercially available carboxyl-activated esters contain incompletely removed byproducts, causing turbidity upon redissolution in water, greatly affecting their further applications, especially in biomedical applications. This process can solve this problem. On the one hand, the use of hydrochloric acid solution effectively inhibits the hydrolysis of polyethylene glycol carboxyl-activated ester in a short time, ensuring the product's effectiveness. On the other hand, the membrane filtration method can remove the byproducts produced in the reaction, greatly improving product purity and facilitating industrial implementation.

[0037] The yield of the polyethylene glycol carboxyl activated ester is 50-70%, and its purity is above 99%.

[0038] The carboxyl-activated ester-modified polyethylene glycol provided by this invention is composed of polyethylene glycol, acetic anhydride, propionic anhydride, succinic anhydride, glutaric anhydride, maleic anhydride, phthalic anhydride, dimethylmaleic acid, pyromellitic anhydride, dodecanoic anhydride, 3-nitro-1,8-naphthalenedicarboxylic anhydride, bicyclo[2.2.2]oct-5-ene-2,3-dicarboxylic anhydride, cis-5-norbornene-ex-2,3-dicarboxylic anhydride, pyridine-3,4-dicarboxylic anhydride, and benzothioxanthate dicarboxylic anhydride. 2,3-Pyridinedicarboxylic anhydride, creatine anhydride, chlorobridged anhydride, crotonic anhydride, biphenyl anhydride, carronic anhydride, Na anhydride, indigo anhydride, palmitic anhydride, acrylic anhydride, itaconic anhydride, 4-fluorophthalic anhydride, trimellitic anhydride, trimethylacetic anhydride, acetylsalicylic anhydride, thioglycolic anhydride, methylnadic anhydride, norbornene anhydride, 3-methylitanic anhydride, 2-methacrylic anhydride, hexahydrophthalic anhydride, 3-hydroxyphthalic anhydride, 2,3-pyrazinic acid Anhydride, N-decenylsuccinic anhydride, 1,2-cyclopentadicarboxylic anhydride, S-acetylmercaptosuccinic anhydride, 1,1-cyclohexyldiacetic anhydride, 2,2-dimethylsuccinic anhydride, 3,3-tetramethyleneglutarate anhydride, cyclobutane-1,2-dicarboxylic anhydride, 4,5-dimethoxyphthalic anhydride, 4,4'-oxobisphthalic anhydride, 1,2,4-trimeric anhydride acyl chloride, 1,4,5,8-naphthalenetetracarboxylic anhydride, D-alanine-N-carboxyl-cyclic anhydride, Potassium 1,8-naphthalenedicarboxylic anhydride-4-sulfonate, (+)-diacetyl-L-tartaric anhydride, 4-methacryloyloxyethyl trimellitic anhydride, 4,4'-diphthalic anhydride, 5-(2,5-dioxotetrahydro-3-furanyl)-3-methyl-3-cyclohexene-1,2-dicarboxylic anhydride, (S)-3-acetoxydihydro-2,5-furandione O-acetyl-L-malic anhydride, DMAP, EDC, and NHS are prepared by reaction.

[0039] In the polyethylene glycol of the aforementioned carboxyl-activated ester, the polyethylene glycol has structural formulas I, II, III, and IV, where n can specifically be 10-1000000, etc.

[0040]

[0041]

[0042] In the polyethylene glycol carboxyl-activated ester, the carboxylation of the polyethylene glycol terminus is performed using acid anhydrides. Specific acid anhydrides include acetic anhydride, propionic anhydride, succinic anhydride, glutaric anhydride, maleic anhydride, phthalic anhydride, dimethylmaleic acid, pyromellitic anhydride, dodecanoic anhydride, 3-nitro-1,8-naphthalenedicarboxylic anhydride, bicyclo[2.2.2]oct-5-ene-2,3-dicarboxylic anhydride, cis-5-norbornene-ex-2,3-dicarboxylic anhydride, pyridine-3,4-dicarboxylic anhydride, and benzene. Thioxanol dicarboxylic anhydride, 2,3-pyridine dicarboxylic anhydride, creatine anhydride, chlorobridged anhydride, crotonic anhydride, biphenyl anhydride, caronic anhydride, norbornene anhydride, indigo anhydride, palmitic anhydride, acrylic anhydride, itaconic anhydride, 4-fluorophthalic anhydride, trimellitic anhydride, trimethylacetic anhydride, acetylsalicylic anhydride, thioglycolic anhydride, methylnadic anhydride, norbornene anhydride, 3-methylitanic anhydride, 2-methacrylic anhydride, hexahydrophthalic anhydride, 3-hydroxyphthalic anhydride, 2,3-pyrazine dicarboxylic anhydride Anhydrides, N-decenylsuccinic anhydride, 1,2-cyclopentadicarboxylic anhydride, S-acetylmercaptosuccinic anhydride, 1,1-cyclohexyldiacetic anhydride, 2,2-dimethylsuccinic anhydride, 3,3-tetramethyleneglutarate anhydride, cyclobutane-1,2-dicarboxylic anhydride, 4,5-dimethoxyphthalic anhydride, 4,4'-oxobisphthalic anhydride, 1,2,4-trimethicone acyl chloride, 1,4,5,8-naphthalenetetracarboxylic anhydride, D-alanine-N-carboxyl-cyclic anhydride, 1,8-naphthalenedicarboxylic acid Anhydrides such as potassium 4-sulfonate, (+)-diacetyl-L-tartaric anhydride, 4-methacryloyloxyethyl trimellitic anhydride, 4,4'-diphthalic anhydride, 5-(2,5-dioxotetrahydro-3-furanyl)-3-methyl-3-cyclohexene-1,2-dicarboxylic anhydride, (S)-3-acetoxydihydro-2,5-furandione O-acetyl-L-malic anhydride, etc., among which the structural formulas of several commonly used anhydrides are V, VI, VII, VIII, IX, X, and XI.

[0043]

[0044]

[0045] In the polyethylene glycol carboxyl-activated ester, polyethylene glycol can be converted into carboxylated polyethylene glycol through a chemical reaction with acid anhydride. Taking the reaction of succinic anhydride as an example, the specific structural formulas can be VI, VII, VIII, and IX.

[0046]

[0047]

[0048]

[0049] In the polyethylene glycol carboxyl-activated ester, the carboxylated polyethylene glycol can be further used to obtain the polyethylene glycol carboxyl-activated ester. Taking the carboxyl end grafted with succinic anhydride as an example, the specific structural formula can be X, XI, XII, or XIII.

[0050]

[0051]

[0052] Example 1

[0053] Weigh 200 mg of polyethylene glycol (molecular weight 8000), 16.7 mg of succinic anhydride, and 8.14 mg of DMAP and dissolve them in 200 mL of anhydrous dichloromethane. React at 20 °C for 72 h. Mix the reaction product with an equal volume of 0.1 M hydrochloric acid solution containing saturated sodium chloride and shake. Centrifuge at 500 rpm for 60 min, discard the supernatant, and add an equal volume of 0.1 M hydrochloric acid solution containing saturated sodium chloride. Centrifuge again under the same conditions, repeating this process 5 times. Add anhydrous magnesium sulfate to dry the mixture. Then, evaporate the dichloromethane to 1 mL using a rotary evaporator, add 20 mL of petroleum ether to precipitate the precipitate, and continue rotary evaporation until dry. Weigh the product.

[0054] The obtained HOOC-PEG-COOH was reacted with 1.07 mg EDC and 1.61 mg NHS in anhydrous dichloromethane for 72 hours at 20 °C. The resulting reaction solution was mixed with an equal volume of 0.1 M hydrochloric acid solution saturated with sodium chloride, shaken for 30 min, and then centrifuged at 500 rpm for 60 min. The supernatant was discarded, and an equal volume of 0.1 M hydrochloric acid solution saturated with sodium chloride was added again. This process was repeated five times. Anhydrous magnesium sulfate was added to remove water, and the dichloromethane was evaporated to 1 mL using a rotary evaporator. 20 mL of petroleum ether was added to precipitate the precipitate, and the mixture was further evaporated to dryness to obtain polyethylene glycol carboxyl-activated ester. The polyethylene glycol carboxyl-activated ester was dissolved in hydrochloric acid solution at pH 1.0 and filtered through a 0.45 μm filter membrane to obtain the final product. The NMR spectrum of the final product (…) Figure 1 and Figure 2 ).

[0055] Example 2

[0056] Weigh 16g of polyethylene glycol (molecular weight 8000), 1.5216g of glutaric anhydride, and 651.2mg of DMAP and dissolve them in 200mL of anhydrous dichloromethane. React at 39℃ for 4h. Mix the reaction product with an equal volume of 2M hydrochloric acid aqueous solution of saturated sodium chloride and shake. Then centrifuge at 50000rpm for 1min, discard the supernatant, and add an equal volume of 2M hydrochloric acid aqueous solution of saturated sodium chloride. Centrifuge again under the same conditions. Repeat this centrifugation once more. Add anhydrous magnesium sulfate to dry and remove water. Then evaporate the dichloromethane to 10mL using a rotary evaporator. Add 100mL of petroleum ether to precipitate the precipitate and continue rotary evaporation until the final volume is reached. Weigh the product.

[0057] The obtained HOOC-PEG-COOH was reacted with 191.70 mg EDC and 115.09 mg NHS at 25 °C for 12 h. The resulting reaction solution was mixed with an equal volume of 2M hydrochloric acid solution saturated with sodium chloride and shaken. The mixture was then centrifuged at 50,000 rpm for 1 min, the supernatant was discarded, and another equal volume of 2M hydrochloric acid solution saturated with sodium chloride was added. This process was repeated once more. Anhydrous magnesium sulfate was added to remove water, and dichloromethane was evaporated to 10 mL using a rotary evaporator. 100 mL of petroleum ether was added to precipitate the precipitate, and the mixture was further evaporated to obtain polyethylene glycol carboxyl-activated ester. The polyethylene glycol carboxyl-activated ester was dissolved in hydrochloric acid solution at pH 6.0 and filtered through a 0.01 μm membrane to obtain the final product. The NMR spectrum of the final product (…) Figure 3 and Figure 4 ).

[0058] Example 3

[0059] Take 20g of tetra-arm polyethylene glycol (molecular weight 10000), 2.0g of succinic anhydride, and 977mg of DMAP and dissolve them in 200mL of dichloromethane. React at 30℃ for 4h. Mix the reaction product with an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride and shake. Then centrifuge at 3000rpm for 10min, discard the supernatant, add an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride and mix. Centrifuge again under the same conditions. Repeat the centrifugation once. Add anhydrous magnesium sulfate to dry and remove water. Then evaporate the dichloromethane to 10mL on a rotary evaporator, add 100mL of petroleum ether to precipitate, and continue to evaporate to dryness. Weigh the product.

[0060] The obtained HOOC-PEG-COOH was reacted with 383.4 mg EDC and 230.18 mg NHS in anhydrous dichloromethane at 25 °C for 6 hours. The resulting reaction solution was mixed with an equal volume of 1M hydrochloric acid solution saturated with sodium chloride and shaken. The mixture was then centrifuged at 3000 rpm for 1 min, the supernatant was discarded, and another equal volume of 1M hydrochloric acid solution saturated with sodium chloride was added. This process was repeated three times. Anhydrous magnesium sulfate was added to remove water, and the dichloromethane was evaporated to 10 mL using a rotary evaporator. 50 mL of petroleum ether was added to precipitate the product, and the mixture was further evaporated to dryness to obtain polyethylene glycol carboxyl-activated ester. The polyethylene glycol carboxyl-activated ester was dissolved in hydrochloric acid solution at pH 4.0 and filtered through a 0.45 μm membrane to obtain the final product. The NMR spectrum of the final product was then analyzed.

[0061] Example 4

[0062] Weigh 20g of six-arm polyethylene glycol (molecular weight 10000), 4.0g of succinic anhydride, and 1.47g of DMAP and dissolve them in 200mL of anhydrous dichloromethane. React at 30℃ for 24h. Mix the reaction product with an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride and shake. Then centrifuge at 3000rpm for 15min, discard the supernatant, and add an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride. Centrifuge again under the same conditions. Repeat this centrifugation process 3 times. Add anhydrous magnesium sulfate to dry and remove water. Then evaporate the dichloromethane to 10mL using a rotary evaporator, add 50mL of petroleum ether to precipitate, and continue to evaporate to dryness. Weigh the product to obtain approximately 16g of HOOC-PEG-COOH.

[0063] 0.16 g of HOOC-PEG-COOH was dissolved in anhydrous dichloromethane at a ratio of 0.01:0.01:1 with 5.75 mg EDC and 345.27 mg NHS. The mixture was reacted at 25 °C for 6 hours. The resulting reaction solution was mixed with an equal volume of 1 M hydrochloric acid solution containing saturated sodium chloride and shaken. The mixture was then centrifuged at 3000 rpm for 20 min. The supernatant was discarded, and an equal volume of 1 M hydrochloric acid solution containing saturated sodium chloride was added and mixed again. This process was repeated three times. Anhydrous magnesium sulfate was added to remove water. The dichloromethane was evaporated to 10 mL using a rotary evaporator. 50 mL of petroleum ether was added to precipitate the product, and the mixture was further evaporated to dryness to obtain polyethylene glycol carboxyl activated ester. The polyethylene glycol carboxyl activated ester was dissolved in hydrochloric acid solution at pH 4.0 and filtered through a 0.25 μm filter membrane to obtain the final product.

[0064] Example 5

[0065] Weigh 20g of octagonal polyethylene glycol (molecular weight 8000), 3.0g of succinic anhydride, and 1.95g of DMAP and dissolve them in 200mL of anhydrous dichloromethane. React at 10℃ for 24h. Mix the reaction product with an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride and shake. Then centrifuge at 3000rpm for 30min, discard the supernatant, and add an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride. Centrifuge again under the same conditions. Repeat this centrifugation process 3 times. Add anhydrous magnesium sulfate to dry and remove water. Then evaporate the dichloromethane to 10mL on a rotary evaporator, add 50mL of petroleum ether to precipitate, and continue to evaporate to dryness. Weigh the product. Approximately 16g of HOOC-PEG-COOH can be obtained.

[0066] 16g of HOOC-PEG-COOH was dissolved in anhydrous dichloromethane at a ratio of 1:0.01:0.01 and reacted at 25°C for 18 hours. The resulting reaction solution was mixed with an equal volume of 1M hydrochloric acid solution containing saturated sodium chloride, and then centrifuged at 3000 rpm for 10 min. The supernatant was discarded, and an equal volume of 1M hydrochloric acid solution containing saturated sodium chloride was added and mixed again. This process was repeated three times. Anhydrous magnesium sulfate was added to dry the mixture, and the dichloromethane was evaporated to 10 mL using a rotary evaporator. 50 mL of petroleum ether was added to precipitate the product, and the mixture was further evaporated to dryness to obtain polyethylene glycol carboxyl activated ester. The polyethylene glycol carboxyl activated ester was dissolved in hydrochloric acid solution at pH 3.0 and filtered through a 0.25 μm filter membrane to obtain the final product.

[0067] Example 6

[0068] Polyethylene glycol (20g), phthalic anhydride, and DMAP were dissolved in anhydrous dichloroethane at an equivalent ratio of 1:1:1, with polyethylene glycol having a mass fraction of 0.1%. The mixture was reacted at 39°C for 5 hours. The reaction product was then mixed with an equal volume of 1M hydrochloric acid solution containing saturated sodium chloride and shaken. The mixture was then centrifuged at 1000 rpm for 20 minutes. The supernatant was discarded, and an equal volume of 1M hydrochloric acid solution containing saturated sodium chloride was added and mixed. The mixture was then centrifuged again under the same conditions. This process was repeated 5 times. Anhydrous magnesium sulfate was added to remove water. Subsequently, dichloromethane was evaporated to 10 mL using a rotary evaporator. 50 mL of petroleum ether was added to precipitate the product, and the mixture was further evaporated to dryness to obtain 16g of carboxylated polyethylene glycol.

[0069] The obtained HOOC-PEG-COOH (0.16) was dissolved in anhydrous dichloroethane at an equivalent ratio of 0.01:1:0.01 with EDC (479 mg) and NHS (4.60 mg), wherein the mass fraction of carboxylated polyethylene glycol was 0.1%. The reaction was carried out at 39 °C for 4 h. The resulting reaction solution was mixed with an equal volume of 1M hydrochloric acid aqueous solution with saturated sodium chloride, and then centrifuged at 5000 rpm for 1 min. The supernatant was discarded, and an equal volume of 1M hydrochloric acid aqueous solution with saturated sodium chloride was added and mixed again. This process was repeated 5 times. Anhydrous magnesium sulfate was added to dry the solution and remove water. The dichloromethane was evaporated to 10 mL on a rotary evaporator, and 50 mL of petroleum ether was added to precipitate the product. The product was then evaporated to dryness to obtain polyethylene glycol carboxyl activated ester. The polyethylene glycol carboxyl activated ester was dissolved in hydrochloric acid aqueous solution with pH = 6.0 and filtered through a 0.45 μm filter membrane to obtain the final product.

[0070] Example 7

[0071] Polyethylene glycol (20g), lauric anhydride, and DMAP were dissolved in anhydrous ethanol at an equivalent ratio of 1:0.2:0.5, with polyethylene glycol accounting for 1% by mass. The reaction was carried out at 35°C for 8 hours. The reaction product was mixed with an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride and shaken. The mixture was then centrifuged at 2000 rpm for 15 minutes. The supernatant was discarded, and an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride was added and mixed. The mixture was centrifuged again under the same conditions. This process was repeated 5 times. Anhydrous magnesium sulfate was added to dry the mixture. Then, anhydrous ethanol was evaporated to 10 mL using a rotary evaporator. 50 mL of petroleum ether was added to precipitate the precipitate, and the mixture was further evaporated to dryness to obtain carboxylated polyethylene glycol.

[0072] The obtained HOOC-PEG-COOH was dissolved in anhydrous ethanol with EDC and NHS at an equivalent ratio of 1:0.2:0.5, wherein the mass fraction of carboxylated polyethylene glycol was 1%. The reaction was carried out at 35°C for 10 h. The resulting reaction solution was mixed with an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride, and then centrifuged at 1000 rpm for 10 min. The supernatant was discarded, and an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride was added and mixed. The mixture was centrifuged again under the same conditions, and this process was repeated 5 times. Anhydrous magnesium sulfate was added and dried to remove water. The anhydrous ethanol was evaporated to dryness again on a rotary evaporator to obtain polyethylene glycol carboxyl activated ester. The polyethylene glycol carboxyl activated ester was dissolved in hydrochloric acid aqueous solution of pH=3.0 to 10 mL, and 50 mL of petroleum ether was added to precipitate. The mixture was then evaporated to dryness and filtered through a 0.45 μm filter membrane to obtain the final product.

[0073] Example 8

[0074] Polyethylene glycol (20g), 3-nitro-1,8-naphthalenedicarboxylic anhydride, and DMAP were dissolved in anhydrous methanol at an equivalent ratio of 5:8:8, with polyethylene glycol accounting for 10% by mass. The mixture was reacted at 30°C for 5 hours. The reaction product was then mixed with an equal volume of 1M hydrochloric acid solution containing saturated sodium chloride and shaken. The mixture was then centrifuged at 5000 rpm for 20 minutes. The supernatant was discarded, and an equal volume of 1M hydrochloric acid solution containing saturated sodium chloride was added and mixed. The mixture was centrifuged again under the same conditions, and this process was repeated 5 times. Anhydrous magnesium sulfate was added to remove water. Subsequently, the anhydrous methanol was evaporated to 10 mL using a rotary evaporator. 50 mL of petroleum ether was added to precipitate the product, and the mixture was further evaporated to dryness to obtain carboxylated polyethylene glycol.

[0075] The obtained HOOC-PEG-COOH was dissolved in anhydrous methanol with EDC and NHS in an equivalent ratio of 5:8:8, wherein the mass fraction of carboxylated polyethylene glycol was 10%. The reaction was carried out at 30°C for 20 h. The resulting reaction solution was mixed with an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride, and then centrifuged at 1000 rpm for 10 min each time. The supernatant was discarded, and an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride was added and mixed again. The same centrifugation was repeated 5 times. Anhydrous magnesium sulfate was added to dry the solution and remove water. The anhydrous methanol was evaporated to 10 mL on a rotary evaporator, and 50 mL of petroleum ether was added to precipitate the product. The product was then evaporated to dryness to obtain polyethylene glycol carboxyl activated ester. The polyethylene glycol carboxyl activated ester was dissolved in hydrochloric acid aqueous solution at pH 5.0 and filtered through a 0.45 μm filter membrane to obtain the final product.

[0076] Example 9

[0077] Polyethylene glycol (20g), 3-nitro-1,8-naphthalenedicarboxylic anhydride, and DMAP were dissolved in anhydrous DMF at an equivalent ratio of 6:5:10, with polyethylene glycol accounting for 20% by mass. The reaction was carried out at 28°C for 10 hours. The reaction product was mixed with an equal volume of 1M hydrochloric acid solution containing saturated sodium chloride and shaken. The mixture was then centrifuged at 10,000 rpm for 20 minutes. The supernatant was discarded, and an equal volume of 1M hydrochloric acid solution containing saturated sodium chloride was added and mixed. The mixture was centrifuged again under the same conditions. This process was repeated 4 times. Anhydrous magnesium sulfate was added to dry the mixture. The anhydrous DMF was then evaporated to 10 mL using a rotary evaporator. 50 mL of petroleum ether was added to precipitate the product, and the mixture was further evaporated to dryness to obtain carboxylated polyethylene glycol.

[0078] The obtained HOOC-PEG-COOH was dissolved in anhydrous DMF with EDC and NHS at an equivalent ratio of 6:5:10, wherein the mass fraction of carboxylated polyethylene glycol was 1%. The reaction was carried out at 28°C for 30 h. The resulting reaction solution was mixed with an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride, and then centrifuged at 5000 rpm for 15 min. The supernatant was discarded, and an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride was added and mixed. The mixture was centrifuged again under the same conditions, and this process was repeated 5 times. Anhydrous magnesium sulfate was added to dry the solution and remove water. The anhydrous DMF was evaporated to 10 mL using a rotary evaporator. 50 mL of petroleum ether was added to precipitate the product, and the mixture was evaporated to dryness to obtain polyethylene glycol carboxyl activated ester. The polyethylene glycol carboxyl activated ester was dissolved in hydrochloric acid aqueous solution at pH 3.0 and filtered through a 0.25 μm filter membrane to obtain the final product.

[0079] Example 10

[0080] Polyethylene glycol (20g), chlorobrines, and DMAP were dissolved in anhydrous acetone at an equivalent ratio of 0.6:1:0.5, with polyethylene glycol accounting for 30% by mass. The reaction was carried out at 27°C for 12 hours. The reaction product was mixed with an equal volume of 1M hydrochloric acid solution containing saturated sodium chloride and shaken. The mixture was then centrifuged at 5000 rpm for 20 minutes. The supernatant was discarded, and an equal volume of 1M hydrochloric acid solution containing saturated sodium chloride was added and mixed. The mixture was centrifuged again under the same conditions. This process was repeated four times. Anhydrous magnesium sulfate was added to remove water. Then, anhydrous acetone was evaporated to 10 mL using a rotary evaporator. 50 mL of petroleum ether was added to precipitate the product, and the mixture was further evaporated to dryness to obtain carboxylated polyethylene glycol.

[0081] The obtained HOOC-PEG-COOH was dissolved in anhydrous acetone with EDC and NHS in an equivalent ratio of 0.6:1:0.5, wherein the mass fraction of carboxylated polyethylene glycol was 10%. The reaction was carried out at 27°C for 40 h. The resulting reaction solution was mixed with an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride, and then centrifuged at 1000 rpm for 20 min. The supernatant was discarded, and an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride was added and mixed again. This process was repeated 5 times. Anhydrous magnesium sulfate was added to dry the mixture, and the anhydrous acetone was evaporated to 10 mL on a rotary evaporator. 50 mL of petroleum ether was added to precipitate the product, and the mixture was evaporated to dryness to obtain polyethylene glycol carboxyl activated ester. The polyethylene glycol carboxyl activated ester was dissolved in hydrochloric acid aqueous solution at pH 5.0 and filtered through a 0.25 μm filter membrane to obtain the final product.

[0082] Example 11

[0083] Polyethylene glycol (20g), biphenyl anhydride, and DMAP were dissolved in anhydrous acetonitrile at an equivalent ratio of 1:0.1:0.2, with polyethylene glycol accounting for 40% by mass. The reaction was carried out at 26°C for 1 hour. The reaction product was mixed with an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride and shaken. The mixture was then centrifuged at 2000 rpm for 40 minutes. The supernatant was discarded, and an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride was added and mixed. The mixture was centrifuged again under the same conditions. This process was repeated 4 times. Anhydrous magnesium sulfate was added to dry the mixture. Then, anhydrous methyl ethyl ketone was evaporated to 10 mL using a rotary evaporator. 50 mL of petroleum ether was added to precipitate the product, and the mixture was further evaporated to dryness to obtain carboxylated polyethylene glycol.

[0084] The obtained HOOC-PEG-COOH was dissolved in anhydrous acetonitrile with EDC and NHS at an equivalent ratio of 1:0.1:0.2, wherein the mass fraction of carboxylated polyethylene glycol was 20%. The reaction was carried out at 26°C for 45 h. The resulting reaction solution was mixed with an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride, and then centrifuged at 500 rpm for 25 min. The supernatant was discarded, and an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride was added and mixed. The mixture was centrifuged again under the same conditions, and this process was repeated 5 times. Anhydrous magnesium sulfate was added to dry the mixture, and the anhydrous acetonitrile was evaporated to 10 mL on a rotary evaporator. 50 mL of petroleum ether was added to precipitate the product, and the mixture was evaporated to dryness to obtain polyethylene glycol carboxyl activated ester. The polyethylene glycol carboxyl activated ester was dissolved in hydrochloric acid aqueous solution at pH 5.0 and filtered through a 0.15 μm filter membrane to obtain the final product.

[0085] Example 12

[0086] Polyethylene glycol (20g), norborneol adienoic anhydride, and DMAP were dissolved in anhydrous tetrahydrofuran at an equivalent ratio of 1:1:1, with polyethylene glycol accounting for 50% by mass. The reaction was carried out at 24°C for 12 hours. The reaction product was mixed with an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride and shaken. The mixture was then centrifuged at 2500 rpm for 20 minutes. The supernatant was discarded, and an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride was added and mixed. The mixture was centrifuged again under the same conditions. This process was repeated three times. Anhydrous magnesium sulfate was added to dry the mixture. Then, the anhydrous tetrahydrofuran was evaporated to 10 mL using a rotary evaporator. 50 mL of petroleum ether was added to precipitate the product, and the mixture was further evaporated to dryness to obtain carboxylated polyethylene glycol.

[0087] The obtained HOOC-PEG-COOH was dissolved in anhydrous tetrahydrofuran at an equivalent ratio of 1:1:1, with the carboxylated polyethylene glycol having a mass fraction of 40%. The reaction was carried out at 24°C for 20 h. The resulting reaction solution was mixed with an equal volume of 1M hydrochloric acid aqueous solution containing saturated sodium chloride. The mixture was then centrifuged at 1000 rpm for 30 min, and the supernatant was discarded. An equal volume of 1M hydrochloric acid aqueous solution containing saturated sodium chloride was added and mixed again. This process was repeated 5 times. Anhydrous magnesium sulfate was added to remove water, and the anhydrous tetrahydrofuran was evaporated to dryness again using a rotary evaporator to obtain polyethylene glycol carboxyl activated ester. The polyethylene glycol carboxyl activated ester was dissolved in hydrochloric acid aqueous solution at pH 5.0 and filtered through a 0.45 μm filter membrane to obtain the final product.

[0088] Example 13

[0089] Polyethylene glycol (20g), trimethylacetic anhydride, and DMAP were dissolved in anhydrous xylene at an equivalent ratio of 4:5:6, with polyethylene glycol accounting for 40% by mass. The reaction was carried out at 20°C for 22 hours. The reaction product was mixed with an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride and shaken. The mixture was then centrifuged at 3000 rpm for 20 minutes. The supernatant was discarded, and an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride was added and mixed. The mixture was centrifuged again under the same conditions. This process was repeated 5 times. Anhydrous magnesium sulfate was added to dry the mixture. Then, the anhydrous xylene was evaporated to 10 mL using a rotary evaporator. 50 mL of petroleum ether was added to precipitate the product, and the mixture was further evaporated to dryness to obtain carboxylated polyethylene glycol.

[0090] The obtained HOOC-PEG-COOH was dissolved in anhydrous xylene solution with EDC and NHS in an equivalent ratio of 4:5:6, wherein the mass fraction of carboxylated polyethylene glycol was 40%. The reaction was carried out at 20℃ for 30 h. The resulting reaction solution was mixed with an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride, shaken, and then centrifuged at 1000 rpm for 30 min. The supernatant was discarded, and an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride was added and mixed again. The same centrifugation was repeated 5 times. Anhydrous magnesium sulfate was added to dry the solution and remove water. The anhydrous xylene was evaporated to 10 mL on a rotary evaporator. 50 mL of petroleum ether was added to precipitate the product, and the mixture was evaporated to dryness to obtain polyethylene glycol carboxyl activated ester. The polyethylene glycol carboxyl activated ester was dissolved in hydrochloric acid aqueous solution at pH 4.0 and filtered through a 0.25 μm filter membrane to obtain the final product.

[0091] Example 14

[0092] Polyethylene glycol (20g), N-decenyl succinic anhydride, and DMAP were dissolved in anhydrous acetonitrile at an equivalent ratio of 3:1:1, with polyethylene glycol accounting for 30% by mass. The reaction was carried out at 22°C for 20 hours. The reaction product was mixed with an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride and shaken. The mixture was then centrifuged at 4000 rpm for 10 minutes. The supernatant was discarded, and an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride was added and mixed. The mixture was centrifuged again under the same conditions. This process was repeated 5 times. Anhydrous magnesium sulfate was added to dry the mixture. Then, anhydrous acetonitrile was evaporated to 10 mL using a rotary evaporator. 50 mL of petroleum ether was added to precipitate the product, and the mixture was further evaporated to dryness to obtain carboxylated polyethylene glycol.

[0093] The obtained HOOC-PEG-COOH was dissolved in anhydrous acetonitrile with EDC and NHS in an equivalent ratio of 3:1:1, wherein the mass fraction of carboxylated polyethylene glycol was 30%. The reaction was carried out at 22℃ for 20 h. The resulting reaction solution was mixed with an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride and shaken. Then, it was centrifuged at 5000 rpm for 5 min, and the supernatant was discarded. An equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride was added and mixed. The mixture was centrifuged again under the same conditions. This centrifugation was repeated 5 times. Anhydrous magnesium sulfate was added to dry the mixture. The anhydrous acetonitrile was evaporated to 10 mL on a rotary evaporator. 50 mL of petroleum ether was added to precipitate the product, and the mixture was evaporated to dryness to obtain polyethylene glycol carboxyl activated ester. The polyethylene glycol carboxyl activated ester was dissolved in hydrochloric acid aqueous solution at pH 5.0 and filtered through a 0.01 μm filter membrane to obtain the final product.

[0094] Example 15

[0095] Polyethylene glycol (20g), 2,2-dimethylsuccinic anhydride, and DMAP were dissolved in anhydrous dimethyl sulfoxide in an equivalent ratio of 2:1:1, with polyethylene glycol accounting for 40% by mass. The reaction was carried out at 33°C for 18 hours. The reaction product was mixed with an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride and shaken. The mixture was then centrifuged at 5000 rpm for 5 minutes. The supernatant was discarded, and an equal volume of 1M hydrochloric acid aqueous solution of saturated sodium chloride was added and mixed. The mixture was centrifuged again under the same conditions. This process was repeated 5 times. Anhydrous magnesium sulfate was added to dry the mixture. Then, the anhydrous dimethyl sulfoxide was evaporated to 10 mL using a rotary evaporator. 50 mL of petroleum ether was added to precipitate the product, and the mixture was further evaporated to dryness to obtain carboxylated polyethylene glycol.

[0096] The obtained HOOC-PEG-COOH was dissolved in anhydrous dimethyl sulfoxide at an equivalent ratio of 2:1:1, with the carboxylated polyethylene glycol having a mass fraction of 40%. The reaction was carried out at 33°C for 20 h. The resulting reaction solution was mixed with an equal volume of 1M hydrochloric acid aqueous solution containing saturated sodium chloride. The mixture was then centrifuged at 1000 rpm for 20 min, and the supernatant was discarded. An equal volume of 1M hydrochloric acid aqueous solution containing saturated sodium chloride was added and mixed again. This process was repeated 5 times. Anhydrous magnesium sulfate was added to remove water. The anhydrous dimethyl sulfoxide was evaporated to 10 mL using a rotary evaporator. 50 mL of petroleum ether was added to precipitate the product, and the mixture was further evaporated to dryness to obtain polyethylene glycol carboxyl activated ester. The polyethylene glycol carboxyl activated ester was dissolved in hydrochloric acid aqueous solution at pH 6.0 and filtered through a 0.45 μm filter membrane to obtain the final product.

[0097] In the above embodiments, the solvent can be replaced with anhydrous dichloromethane, anhydrous chloroform, anhydrous dichloroethane, anhydrous ethanol, anhydrous methanol, anhydrous DMF, anhydrous acetone, anhydrous methyl ethyl ketone, anhydrous tetrahydrofuran, anhydrous xylene, anhydrous acetonitrile, anhydrous dimethyl sulfoxide, anhydrous pyridine, or anhydrous N,N-dimethylformamide.

[0098] Acid anhydrides can include acetic anhydride, propionic anhydride, succinic anhydride, glutaric anhydride, maleic anhydride, phthalic anhydride, dimethylmaleic anhydride, pyromellitic anhydride, dodecanoic anhydride, 3-nitro-1,8-naphthalenedicarboxylic anhydride, bicyclo[2.2.2]oct-5-ene-2,3-dicarboxylic anhydride, cis-5-norbornene-ex-2,3-dicarboxylic anhydride, pyridine-3,4-dicarboxylic anhydride, benzothioxanone dicarboxylic anhydride, and 2,3-pyridinedicarboxylic acid. Anhydride, creatine anhydride, chlorobridged anhydride, crotonic anhydride, biphenyl anhydride, carronic anhydride, NA anhydride, indigo anhydride, palmitic anhydride, acrylic anhydride, itaconic anhydride, 4-fluorophthalic anhydride, trimellitic anhydride, trimethylacetic anhydride, acetylsalicylic anhydride, thioglycolic anhydride, methylnadic anhydride, norbornene anhydride, 3-methylitanic anhydride, 2-methacrylic anhydride, hexahydrophthalic anhydride, 3-hydroxyphthalic anhydride, 2,3-pyrazinic acid Anhydride, N-decenylsuccinic anhydride, 1,2-cyclopentadicarboxylic anhydride, S-acetylmercaptosuccinic anhydride, 1,1-cyclohexyldiacetic anhydride, 2,2-dimethylsuccinic anhydride, 3,3-tetramethyleneglutarate anhydride, cyclobutane-1,2-dicarboxylic anhydride, 4,5-dimethoxyphthalic anhydride, 4,4'-oxobisphthalic anhydride, 1,2,4-trimeric anhydride acyl chloride, 1,4,5,8-naphthalenetetracarboxylic anhydride, D-alanine- N-Carboxyl-cyclic anhydride, 1,8-naphthalenedicarboxylic anhydride-4-sulfonate potassium, (+)-diacetyl-L-tartaric anhydride, 4-methacryloyloxyethyltriphenyltrihydride, 4,4'-diphthalic anhydride, 5-(2,5-dioxotetrahydro-3-furanyl)-3-methyl-3-cyclohexene-1,2-dicarboxylic anhydride or (S)-3-acetoxydihydro-2,5-furandione O-acetyl-L-malic anhydride.

[0099] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for synthesizing polyethylene glycol carboxyl-activated esters, characterized in that, Includes the following steps: Step 1: Dissolve polyethylene glycol, acid anhydride and 4-dimethylaminopyridine in a solvent to obtain product solution A. Mix product solution A with a 0.1-2 M hydrochloric acid aqueous solution of saturated sodium chloride and shake. Centrifuge the shaken solution to obtain organic phase centrifuged product B. Dry centrifuged product B with anhydrous magnesium sulfate and evaporate the solvent to obtain evaporated product C. Add petroleum ether to precipitate evaporated product C and rotary evaporate to obtain carboxylated polyethylene glycol. The concentration of polyethylene glycol in the solvent is 0.1%-80%, and the mixing equivalent ratio of polyethylene glycol, acid anhydride and 4-dimethylaminopyridine is (0.01~1):(0.01~1):(0.01~1). Step 2: Dissolve carboxylated polyethylene glycol, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a solvent, mix and react to obtain product solution D. Mix product solution D with 0.1-2 M hydrochloric acid aqueous solution of saturated sodium chloride and shake. Centrifuge the shaken solution to obtain organic phase centrifuged product E. Dry centrifuged product E with anhydrous magnesium sulfate and evaporate the solvent to obtain evaporated product F. Add petroleum ether to precipitate evaporated product F and rotary evaporate to obtain carboxyl-activated ester polyethylene glycol. In step 2, the mixing ratio of carboxylated polyethylene glycol, EDC, and NHS is (0.01-1):(0.01-1):(0.01-1). Step 2 involves mixing carboxylated polyethylene glycol, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide in a specific ratio and reacting them at 20-39°C for 4-72 hours to obtain product solution D. Product solution D is then mixed with an equal volume of 0.1-2 M hydrochloric acid aqueous solution containing saturated sodium chloride and shaken for 1-60 minutes. The mixture is then centrifuged at 500-50000 rpm for 1-60 minutes, and the supernatant is discarded. This process of adding saturated sodium chloride aqueous solution, shaking, centrifuging, and discarding the supernatant is repeated 1-5 times to obtain centrifuged product E. This product E is dried with anhydrous magnesium sulfate and then evaporated to 1-10 mL using a vacuum rotary evaporator to obtain evaporated product F. 20-100 mL of petroleum ether is added to the evaporated product to precipitate it, and the mixture is further evaporated to dryness to obtain carboxyl-activated polyethylene glycol. In step 2, the carboxylated polyethylene glycol has a mass fraction of 0.1%-80% in the solvent; Step 3: Dissolve polyethylene glycol carboxyl activated ester in hydrochloric acid aqueous solution with pH=1.0-6.0, filter through a 0.01-0.45μm filter membrane, and then freeze dry using a freeze dryer to obtain polyethylene glycol carboxyl activated ester.

2. The method for synthesizing polyethylene glycol carboxyl-activated ester according to claim 1, characterized in that, The specific process in step 1 is as follows: Polyethylene glycol, acid anhydride, and 4-dimethylaminopyridine are dissolved in a solvent according to a set ratio and reacted at 20-39℃ for 4-72 h to obtain product solution A. Product solution A is mixed with an equal volume of 0.1-2 M hydrochloric acid aqueous solution of saturated sodium chloride and shaken. The shaken solution is centrifuged at 500-50000 rpm for 1-60 min and the supernatant is discarded. An equal volume of 0.1-2 M hydrochloric acid aqueous solution is added, mixed, shaken, and centrifuged to obtain the supernatant. This process of adding hydrochloric acid aqueous solution, mixing, shaking, and centrifuging is repeated 1-5 times to obtain centrifuged product B. Centrifuged product B is dried with anhydrous magnesium sulfate to remove water and then placed in a vacuum rotary evaporator to evaporate the solvent to 1-10 mL to obtain evaporated product C. Petroleum ether is added to evaporated product C to precipitate it, and the mixture is further rotary evaporated to dryness to obtain carboxylated polyethylene glycol.

3. The method for synthesizing polyethylene glycol carboxyl-activated ester according to claim 1, characterized in that, In steps 1 and 2, the solvent is anhydrous dichloromethane, anhydrous chloroform, anhydrous dichloroethane, anhydrous ethanol, anhydrous methanol, anhydrous DMF, anhydrous acetone, anhydrous methyl ethyl ketone, anhydrous tetrahydrofuran, anhydrous xylene, anhydrous acetonitrile, anhydrous dimethyl sulfoxide, anhydrous pyridine, or anhydrous N,N-dimethylformamide.

4. The method for synthesizing polyethylene glycol carboxyl-activated ester according to claim 1, characterized in that, In step 1, the polyethylene glycol has the structural formula I, II, III, or IV, where n is 10-10000; specifically: I II III IV.

5. The method for synthesizing polyethylene glycol carboxyl-activated ester according to claim 1, characterized in that, In step 1, the acid anhydrides are acetic anhydride, propionic anhydride, succinic anhydride, glutaric anhydride, maleic anhydride, phthalic anhydride, dimethylmaleic acid, pyromellitic anhydride, dodecanoic anhydride, 3-nitro-1,8-naphthalenedicarboxylic anhydride, bicyclo[2.2.2]oct-5-ene-2,3-dicarboxylic anhydride, cis-5-norbornene-ex-2,3-dicarboxylic anhydride, pyridine-3,4-dicarboxylic anhydride, benzothioxanone dicarboxylic anhydride, and 2,3-pyridine dicarboxylic anhydride. Carboxylic anhydride, creatine anhydride, chlorobridged anhydride, crotonic anhydride, biphenyl anhydride, carnelian anhydride, NA anhydride, indigo anhydride, palmitic anhydride, acrylic anhydride, itaconic anhydride, 4-fluorophthalic anhydride, trimellitic anhydride, trimethylacetic anhydride, acetylsalicylic anhydride, thioglycolic anhydride, methylnadic anhydride, norbornene anhydride, 3-methylitanic anhydride, 2-methacrylic anhydride, hexahydrophthalic anhydride, 3-hydroxyphthalic anhydride, 2,3-pyrazine Anhydrides, N-decenylsuccinic anhydride, 1,2-cyclopentadicarboxylic anhydride, S-acetylmercaptosuccinic anhydride, 1,1-cyclohexyldiacetic anhydride, 2,2-dimethylsuccinic anhydride, 3,3-tetramethyleneglutarate anhydride, cyclobutane-1,2-dicarboxylic anhydride, 4,5-dimethoxyphthalic anhydride, 4,4'-oxobisphthalic anhydride, 1,2,4-trimeric anhydride acyl chloride, 1,4,5,8-naphthalenetetracarboxylic anhydride, D-alanine -N-Carboxyl-cyclic anhydride, 1,8-naphthalenedicarboxylic anhydride-4-sulfonate potassium, (+)-diacetyl-L-tartaric anhydride, 4-methacryloyloxyethyltriphenyltrihydride, 4,4'-diphthalic anhydride, 5-(2,5-dioxotetrahydro-3-furanyl)-3-methyl-3-cyclohexene-1,2-dicarboxylic anhydride or (S)-3-acetoxydihydro-2,5-furandione O-acetyl-L-malic anhydride.

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