A method for synthesizing a single molecular weight bromoacetamide-PEG44-azide

Abstract

The application discloses a synthesis method of a single molecular weight heterobifunctional PEG (X-PEG-Y) connector Bromoacetamide-PEG44-Azide, and relates to the field of organic synthesis. The biological orthogonal chemical reaction of azide reagent with hydrophilic polyethylene glycol chain, alkyne, dibenzocyclooctyne (DBCO) and bicyclo[6,1,0]nonyne (BCN) is widely applied due to mild reaction conditions, fast speed and good biocompatibility. The application successfully prepares a brand-new single molecular weight azide heterobifunctional molecule Bromoacetamide-PEG44-Azide through ten steps of conventional transformation such as protection, bromination and substitution, with a total yield of about 30% and a scale of hundreds of grams. The route is simple in operation and treatment, mild in reaction conditions, is a brand-new process synthesis route, and has good economic benefits and market prospects.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis, specifically to a method for synthesizing a single molecular weight heterobifunctional PEG (X-PEG-Y) linker Bromoacetamide-PEG44-Azide (N-(134-azido-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetraoxa-134-tetraco-1-yl)-2-bromoacetamide). Background Technology

[0002] PEGylation is the process of attaching polymer PEG chains to molecular and macroscopic structures (such as drugs, therapeutic proteins, or vesicles), which can improve the safety and efficacy of many therapeutic methods. The chain is covalently bound to the drug and its steric stability is maintained. Therefore, PEGylated proteins or peptides are non-toxic, non-antigenic, non-immunogenic, and highly water-soluble. Figure 1Furthermore, it further prolongs the half-life of PEGylated drugs and reduces dosing frequency by inhibiting renal excretion. PEGylated products consist of various reagents, including linkers, crosslinking agents, and labels. Different reagents include amine, carboxyl, and carboxyl-based PEGylations. PEGylation has three main advantages: 1) Improved pharmacokinetic and pharmacodynamic properties: Studies have shown that PEGylation significantly alters in vivo pharmacokinetic properties, including prolonging plasma half-life, increasing drug release in vivo, and reducing renal clearance. 2) Enhanced drug stability: After PEGylation, proteins and peptides form a thicker hydration film on their surface, preventing aggregation and precipitation. Modifying the bond between PEG and lipid derivatives (acyl groups, ethers, disulfide bonds, etc.) can also increase liposome stability. In addition, the flexible chains of PEG can create steric hindrance, protecting the modification from protease attack and increasing its stability. 3) Improved drug distribution in vivo: After PEG modification, the molecular weight of the drug increases, which greatly reduces the glomerular filtration rate during systemic administration, thereby reducing its excretion in urine. In addition, PEGylation of drugs improves systemic circulation stability and prolongs retention time, thereby enhancing drug distribution in vivo. It is especially beneficial for the accumulation of macromolecular drugs at tumor and inflammatory sites, and has an enhanced retention effect, thereby prolonging the therapeutic time of drugs in vivo (ACS Nano, 2021, 15(9), 14022-14048; Chem. Rev., 2022, 122(16), 13516-13546; Bioconjugate Chem., 2023, 34(6), 941-960; Org. Process Res. Dev. 2024, 28(4), 860-890). Pegylated drugs are currently used to treat cancer, chronic kidney disease, hepatitis, multiple sclerosis, hemophilia, and gastrointestinal disorders. ADAGEN, from Enzon Pharmaceuticals in the United States, was the first PEGylated protein to receive FDA approval in March 1990. Since ADAGEN's introduction, numerous PEGylated proteins and peptides have followed suit, and many other drugs are currently in clinical trials or development. Among FDA-approved PEGylated drugs, Pegasys and Neulasta both achieved global sales exceeding $5 billion in 2011.

[0003] Bioorthogonal chemistry refers to chemical reactions that can occur in living cells or tissues without interfering with the organism's own biochemical reactions. The essence of bioorthogonality lies in studying biomacromolecules such as proteins and lipids without causing cellular toxicity. A range of chemical linking strategies have been researched and explored to meet the requirements of bioorthogonality, such as click chemistry. Click chemistry describes selective, modular, broad-ranging, and high-yield chemical reactions that can rapidly synthesize new compounds through heteroatom linkages (CXCs). Common reaction structures in click chemistry include azides (N3), alkynes, dibenzocyclooctyne (DBCO), bicyclic [6,1,0]nonyne (BCN), trans-cyclooctene (TCO), and tetrazines. Click chemistry reactions can be classified into three categories: 1) Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC); 2) Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC); and 3) Inverse Electron-Demand Diels–Alder (iEDDA). As a simple and efficient connection method, click chemistry has been widely applied in the biomedical field, such as in fluorescence imaging, targeted therapy, antibody-drug conjugate (ADC) synthesis, and protein degradation-targeting chimeras (PROTACs) synthesis. Figure 2 , Bioconjugate Chem., 2021, 32(12), 2457-2479; Chem. Rev., 2022, 122 (1), 340-384; Chem. Soc. Rev., 2022, 51, 1336-1376; Chem. Soc. Rev., 2023, 52, 7737-7772).

[0004] Bioorthogonal chemical reactions of hydrophilic polyethylene glycol (PEG)-containing azide (N3) reagents with alkynes, dibenzocyclooctyne (DBCO), and bicyclic [6,1,0]nonyne (BCN) constitute an important class of click chemistry reactions. Due to their mild reaction conditions, rapid reaction rate, and good biocompatibility, they have been widely applied in bioconjugation, materials science, radiochemistry, drug discovery, and proteomics. Figure 3 , WO2013127949A1; WO2023196445A1; Bioorg.Med.Chem.Lett., 2013, 23(17), 5006-5010; Chem.Eur.J., 2014, 20(32), 10038-10051; C atal.Commun.,2017,101,5-9;J.Med.Chem.,2021,64(18),13853-13872;J.Mater.Chem.B,2024,12,9296-9311;Biomacromolecules 2024,25(3),1972-1977). However, the relevant compounds are all obtained from commercially available PEG2000 (mixture) as starting material through several conventional conversion steps, and the final products are also mixtures that exhibit a certain normal distribution, and the components are not singular.

[0005] Given the importance of hydrophilic polyethylene glycol (PEG) chains in azide (N3) reagents in bioorthogonal chemical reactions and their potential applications in bioconjugation, materials science, radiochemistry, drug discovery, and proteomics, it is essential to design novel routes and synthesize novel structures to meet the growing market demand. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a single molecular weight heterobifunctional PEG (X-PEG-Y) linker, Bromoacetamide-PEG44-Azide N-(134-azido-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetraoxa-134-tetratetraco-1-yl)-2-bromoacetamide, which relates to the field of organic synthesis. The heterobifunctional PEG (X-PEG-Y) linker Bromoacetamide-PEG44-Azide N-(134-azido-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetraoxa-134-tetraco-1-yl)-2-bromoacetamide has great potential applications in bioconjugation, materials science, radiochemistry, drug discovery, and proteomics. This invention uses 2-[(1-phenyl-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77,80,83,86,89,92,95,98,101,104,107,110,113,116,119,122,125,128,131-tetratetraoxa-133-yl)oxy]ethane-1-ol as the starting material. Through ten conventional transformations including protection, bromination, substitution, and deprotection, the target product was successfully prepared in a ~30% overall yield and on a 100-gram scale. This route is simple to operate and operates under mild reaction conditions, representing a novel synthetic process with significant economic benefits and market potential. Attached Figure Description

[0007] Figure 1 Wide range of applications of polyethylene glycolation

[0008] Figure 2 Main types of bioorthogonal chemistry

[0009] Figure 3 Representative bifunctional azidomolecules with ultralong PEG hydrophilic chains have been reported. Detailed Implementation

[0010] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0011] Example

[0012]

[0013] [Step 1]

[0014] Compound 1 (100 g, 47 mmol) was dissolved in dichloromethane (1000 mL) at room temperature, followed by the addition of triethylamine (2.0 eq., 9.6 g) and methanesulfonyl chloride (MsCl, 2.0 eq., 14.6 g). The mixture was stirred at room temperature for 16 hours. TLC monitoring (MeOH:DCM = 10:1) showed that the starting material reacted completely. The reaction mixture was washed once with water and then with saturated brine. The organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a yellow oily substance 2 (104 g, 100%). No further purification was required, and it was used directly in the next step.

[0015] [Step 2]

[0016] Compound 2 (104 g, 47 mmol) was dissolved in acetonitrile (MeCN, 1000 mL) at room temperature, and lithium bromide (2.0 eq., 8.3 g) was added. The mixture was heated to 80 °C and stirred for 16 hours. TLC monitoring (MeOH:DCM = 10:1) showed that the starting material reacted completely. The reaction system was cooled to room temperature, 500 mL of water was added, and most of the acetonitrile was concentrated under reduced pressure. The mixture was extracted three times with 500 mL of dichloromethane, and the organic phases were combined. The organic phase was washed once with saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a white solid compound 3 (95 g, 91%), which could be used directly in the next step without further purification.

[0017] [Step 3]

[0018] Compound 3 (95 g, 45 mmol) was dissolved in N,N-dimethylformamide (DMF, 1000 mL) at room temperature. Compound 4 (2.0 eq., 16.2 g) and cesium carbonate (2.0 eq., 28.5 g) were added. The mixture was heated to 80°C and stirred for 16 hours. TLC monitoring (MeOH:DCM = 10:1) showed that the starting materials reacted completely. The reaction system was cooled to room temperature, 500 mL of water was added, and most of the N,N-dimethylformamide (DMF) was concentrated using an oil pump. Extracted three times with 500 mL of dichloromethane, the organic phases were combined and washed once with saturated brine. The organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a white solid compound 5 (100 g) crude product. The crude product was dissolved in 500 mL of dichloromethane (DCM), and 150 g of 100-200 mesh silica gel powder was added and mixed. The mixture was subjected to column chromatography. The mobile phase was used to remove impurities (PE:EA = 10:1-1:1-0:1) and the product was removed by passing it through a column (MeOH:DCM = 20:1-10:1) to obtain a white solid product 5 (85 g, 87%).

[0019] [Step 4]

[0020] Compound 5 (85 g, 38 mmol) was dissolved in methanol (MeOH, 1000 mL) at room temperature. 10 mL of hydrazine hydrate was added, and the mixture was heated to 80 °C and stirred for 2 hours. TLC monitoring (MeOH:DCM = 10:1) showed that the starting material reacted completely. The reaction system was cooled to room temperature, and most of the methanol (MeOH) was concentrated under reduced pressure. The mixture was extracted three times with 500 mL of water and 500 mL of dichloromethane (DCM). The organic phases were combined, washed once with saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a white solid compound 6 (87 g) crude product. The crude product was dissolved in 100 mL of dichloromethane (DCM), and 120 g of 100-200 mesh silica gel powder was added and mixed. The mixture was subjected to column chromatography. The mobile phase was filtered to remove impurities using (PE:EA = 10:1-1:1-0:1) and to remove the product using (MeOH:DCM = 20:1-8:1) to obtain a white solid product 6 (60 g, 75%).

[0021] [Step 5]

[0022] Compound 6 (60 g, 28 mmol) was dissolved in methanol (MeOH, 600 mL) at room temperature, and di-tert-butyl dicarbonate (Boc₂O, 2 eq., 12.4 g) was added. The mixture was stirred at room temperature for 16 hours. TLC monitoring (MeOH:DCM = 10:1) showed that the starting material reacted completely. Most of the methanol (MeOH) was concentrated under reduced pressure, and 500 mL of water was added and stirred for 10 minutes. The aqueous phase was extracted three times with 500 mL of dichloromethane (DCM). The organic phases were combined, washed once with saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a white solid compound 7 (70 g) crude product. The crude product was dissolved in 100 mL of dichloromethane (DCM), and 120 g of... Mix the sample with 100-200 mesh silica gel powder, pass it through the column for column chromatography, use (PE:EA=10:1-1:1-0:1) as the mobile phase to remove impurities, and use (MeOH:DCM=20:1-8:1) as the mobile phase to pass the product to obtain yellow solid product 7 (55g, 89%).

[0023] [Step 6]

[0024] Compound 7 (55 g, 24 mmol) was dissolved in methanol (MeOH, 500 mL) at room temperature, and reacted with 5% Pd / C (5 g) under a 15 psi hydrogen atmosphere for 16 hours. TLC monitoring (MeOH:DCM = 10:1) showed that the starting material reacted completely. Pd / C was filtered off, and the mixture was concentrated under reduced pressure to obtain a white solid crude product 8 (50 g, 100%). The crude product was dissolved in 100 mL of dichloromethane (DCM), and 60 g of 100-200 mesh silica gel powder was added and mixed. The mixture was then subjected to column chromatography. Impurities were filtered through the mobile phase (PE:EA = 10:1-1:1-0:1), and the product was filtered through the mobile phase (MeOH:DCM = 10:1) to obtain a white solid product 8 (40 g, 77%).

[0025] [Step 7]

[0026] Compound 8 (40 g, 19 mmol) was dissolved in dichloromethane (DCM, 400 mL) at room temperature, followed by the addition of triethylamine (2.0 eq., 3.8 g) and methanesulfonyl chloride (MsCl, 2.0 eq., 2.0 g). The mixture was stirred at room temperature for 16 hours. TLC monitoring (MeOH:DCM = 10:1) showed that the starting material reacted completely. The reaction mixture was washed once with water and then with saturated brine. The organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a yellow oily substance 9 (41 g, 100%). No further purification was required, and it was used directly in the next step.

[0027] [Step 8]

[0028] Compound 9 (41 g, 19 mmol) was dissolved in N,N-dimethylformamide (DMF, 400 mL) at room temperature. Potassium azide (KN3, 2.0 eq., 3 g) was added, and the mixture was heated to 80°C and stirred for 16 hours. TLC monitoring (MeOH:DCM = 10:1) showed that the starting material reacted completely. The reaction system was cooled to room temperature, and 500 mL of water was added. Most of the N,N-dimethylformamide (DMF) was concentrated using an oil pump. The mixture was extracted three times with 500 mL of dichloromethane. The organic phases were combined, washed once with saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a yellow oily substance 10 (50 g) crude product. The crude product was dissolved in 100 mL of dichloromethane (DCM), and 80 g of... Mix 100-200 mesh silica gel powder with the sample, pass through column chromatography, use (PE:EA=10:1-1:1-0:1) as the mobile phase to remove impurities, and use (MeOH:DCM=20:1-10:1) as the mobile phase to pass through the product to obtain 10 (40g, 100%) of white solid product.

[0029] [Step 9]

[0030] Compound 10 (40 g, 19 mmol) was dissolved in 6N hydrochloric acid (HCl, 200 mL) at room temperature and stirred for 16 hours. TLC monitoring (MeOH:DCM = 10:1) showed that the starting material reacted completely. The mixture was concentrated under reduced pressure to obtain a yellow oily crude product 11 (40 g). The crude product was dissolved in 100 mL of dichloromethane (DCM), and 80 g of 100-200 mesh silica gel powder was added and mixed. The mixture was then subjected to column chromatography. Impurities were removed using a mobile phase of (PE:EA = 10:1-1:1-0:1), and the product was removed using a mobile phase of (MeOH:DCM = 20:1-10:1) to obtain a white solid product 11 (35 g, 89%).

[0031] [Step 10]

[0032] Compound 11 (35 g, 17 mmol) was dissolved in dichloromethane (DCM, 400 mL) at room temperature, followed by the addition of triethylamine (2.0 eq., 3.4 g) and compound 12 (2.0 eq., 6.9 g). The mixture was stirred at room temperature for 16 hours. TLC monitoring (MeOH:DCM = 10:1) showed that the starting material reacted completely. The reaction mixture was washed once with water and then with saturated brine. The organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a yellow oily crude product, Bromoacetamide-PEG44-Azide (40 g). The crude product was dissolved in 100 mL of dichloromethane (DCM), and 60 g of... Mix the sample with 100-200 mesh silica gel powder, pass through column chromatography, and use the mobile phase (PE:EA = 10:1-1:1-0:1) to remove impurities, and (MeOH:DCM = 20:1-10:1) to pass through the product to obtain a white solid product Bromoacetamide-PEG44-Azide (30g, 81%).

[0033] 1 H NMR (400MHz, CDCl) 3， ppm)δ3.88(s,2H),3.84–3.76(m,1H),3.69–3.55(m,178H),3.48–3.45(m,2H),3.38(t,J=5.1Hz,2H).

[0034] This invention uses a single component, 2-[(1-phenyl-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77,80,83,86,89,92,95,98,101,104,107,110,113,116,119,122,125,128,131-tetratetraoxa-133-yl)oxy]eth-1-ol, as the starting material. Through ten conventional transformation steps including protection, bromination, substitution, and deprotection, the target product Bromoa was successfully prepared in a ~30% overall yield and on a 100-gram scale. cetamide-PEG44-Azide(N-(134-azido-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetraoxa-134-tetraco-1-yl)-2-bromoacetamide); This route is simple to operate and has mild reaction conditions. It is a completely new synthetic route with good economic benefits and market prospects.

[0035] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A heterobifunctional PEG (X-PEG-Y) linker, Bromoacetamide-PEG44-Azide N-(134-azido-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetraoxa-134-tetratetraco-1-yl)-2-bromoacetamide, prepared by the following method:

2. The method for synthesizing the heterobifunctional PEG(X-PEG-Y) linker Bromoacetamide-PEG44-Azide N-(134-azido-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetraoxatritratetradecane-1-yl)-2-bromoacetamide according to claim 1, characterized in that: The specific method for synthesizing compound 2 is as follows: compound 1 is dissolved in dichloromethane, and triethylamine and p-toluenesulfonyl chloride are added sequentially. The reaction is carried out at room temperature. After the reaction is completed, the reaction solution is extracted and the solvent is removed to obtain compound 2. No further purification is required, and it can be directly used for the next reaction. The specific method for synthesizing compound 3 is as follows: under room temperature conditions, compound 2 is dissolved in acetonitrile, lithium bromide is added, the temperature is raised to react, and after the reaction is completed, the temperature is lowered to room temperature. The reaction solution is diluted, concentrated, extracted, and desolventized to obtain compound 3, which does not require further purification and can be directly used in the next reaction. The specific method for synthesizing compound 5 is as follows: under room temperature conditions, compound 3 is dissolved in N,N-dimethylformamide, compound 4 and cesium carbonate are added, the temperature is raised to react, and after the reaction is completed, the temperature is lowered to room temperature. The reaction solution is diluted, concentrated, extracted, desolventized, and purified to obtain compound 5. The specific method for synthesizing compound 6 is as follows: under room temperature conditions, compound 5 is dissolved in methanol, hydrazine hydrate is added, the temperature is raised to react, and after the reaction is completed, the temperature is lowered to room temperature. The reaction solution is concentrated, diluted, extracted, desolventized, and purified to obtain compound 6. The specific method for synthesizing compound 7 is as follows: under room temperature conditions, compound 6 is dissolved in methanol, di-tert-butyl dicarbonate is added, and the reaction is carried out at room temperature. After the reaction is completed, the temperature is lowered to room temperature, and the reaction solution is concentrated, diluted, extracted, desolventized, and purified to obtain compound 7. The specific method for synthesizing compound 8 is as follows: under a nitrogen atmosphere and room temperature, compound 7 is dissolved in methanol, 5% palladium on carbon is added, hydrogen is used to replace the gas three times, the reaction is stirred under a hydrogen atmosphere, and after the reaction is completed, the palladium on carbon is filtered off the reaction solution, and the filtrate is purified by solvent removal and separation to obtain compound 8. The specific method for synthesizing compound 9 is as follows: compound 8 is dissolved in dichloromethane, and triethylamine and p-toluenesulfonyl chloride are added sequentially. The reaction is carried out at room temperature. After the reaction is completed, the reaction solution is extracted and the solvent is removed to obtain compound 9. No further purification is required, and it can be directly used for the next reaction. The specific method for synthesizing compound 10 is as follows: under room temperature conditions, compound 9 is dissolved in N,N-dimethylformamide, potassium azide is added, the temperature is raised to react, and after the reaction is completed, the temperature is lowered to room temperature. The reaction solution is diluted, concentrated, extracted, desolventized, and purified to obtain compound 10. The specific method for synthesizing compound 11 is as follows: under room temperature conditions, compound 10 is dissolved in hydrochloric acid and reacted at room temperature. After the reaction is completed, the reaction solution is concentrated, solvent removed, and purified to obtain compound 11. The final product, Bromoacetamide-PEG44-Azide(N-(134-azido-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111, The specific method for synthesizing 114,117,120,123,126,129,132-tetratetraoxa-134-tetracosyl-2-bromoacetamide is as follows: under room temperature conditions, compound 11 is dissolved in dichloromethane, and triethylamine and compound 12 are added sequentially. The reaction is carried out at room temperature. After the reaction is completed, the reaction solution is extracted, desolventized, and purified to obtain the final product Bromoacetamide-PEG44-Azide.

3. The heterobifunctional PEG(X-PEG-Y) linker Bromoacetamide-PEG44-Azide (N-(134-azido-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetraoxytetratetracarbazide-1-yl)-2-bromoacetamide) according to claim 2, characterized in that: The organic base used in steps 1, 7, and 10 is one of triethylamine, N,N-diisopropylethylamine, imidazole, and pyridine.

4. A heterobifunctional PEG (X-PEG-Y) linker according to claim 2 A method for synthesizing Bromoacetamide-PEG44-Azide (N-(134-azido-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetraoxa-134-tetratetraco-1-yl)-2-bromoacetamide), characterized in that: The inorganic base used in step 3 is one of potassium phosphate, potassium monohydrogen phosphate, sodium phosphate, sodium monohydrogen phosphate, potassium carbonate, potassium bicarbonate, sodium carbonate, sodium bicarbonate, cesium carbonate, potassium hydroxide, sodium hydroxide, and lithium hydroxide.

5. A heterobifunctional PEG (X-PEG-Y) linker according to claim 2 A method for synthesizing Bromoacetamide-PEG44-Azide (N-(134-azido-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetraoxa-134-tetratetraco-1-yl)-2-bromoacetamide), characterized in that: The solvent used in steps 4, 5 and 6 is one of methanol, ethanol, isopropanol and tert-butanol.

6. A heterobifunctional PEG (X-PEG-Y) linker according to claim 2 A method for synthesizing Bromoacetamide-PEG44-Azide (N-(134-azido-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetraoxa-134-tetratetraco-1-yl)-2-bromoacetamide), characterized in that: The highly polar aprotic solvent used in steps 3 and 8 is one of N,N-dimethylformamide (DMF), dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), and dimethyl sulfoxide (DMSO).

7. A heterobifunctional PEG (X-PEG-Y) linker according to claim 2 A method for synthesizing Bromoacetamide-PEG44-Azide (N-(134-azido-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetraoxa-134-tetratetraco-1-yl)-2-bromoacetamide), characterized in that: The chlorinated solvent used in steps 1, 7, and 10 is one of dichloromethane (DCM), chloroform (CHCl3), carbon tetrachloride, o-dichlorobenzene, chlorobenzene, and dibromomethane.

8. The method for synthesizing the heterobifunctional PEG(X-PEG-Y) linker Bromoacetamide-PEG44-Azide (N-(134-azido-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetradecano-1-yl)-2-bromoacetamide) according to claim 2, characterized in that: The protic acid used in step 9 is one of hydrochloric acid, sulfuric acid, trifluoroacetic acid, and methanesulfonic acid.

9. A method for synthesizing a heterobifunctional PEG(X-PEG-Y) linker Bromoacetamide-PEG44-Azide (N-(134-azido-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetradecano-1-yl)-2-bromoacetamide) according to claim 2, characterized in that: The brominating reagent used in step 2 is one of lithium bromide, sodium bromide, potassium bromide, phosphorus tribromide, and N-bromosuccinimide (NBS).

10. A heterobifunctional PEG (X-PEG-Y) linker according to claim 2 A method for synthesizing Bromoacetamide-PEG44-Azide (N-(134-azido-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetraoxa-134-tetratetraco-1-yl)-2-bromoacetamide), characterized in that: The reaction temperature in steps 2, 3, 4 and 8 is one of 40-150℃.

11. A heterobifunctional PEG (X-PEG-Y) linker according to claim 2 A method for synthesizing Bromoacetamide-PEG44-Azide (N-(134-azido-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetraoxa-134-tetratetraco-1-yl)-2-bromoacetamide), characterized in that: The reaction temperature in steps 1, 5, 6, 7, 9, and 10 is one of 0-30℃.

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