A method for synthesizing a single molecular weight of DBCO-PEG45-NHS ester
The heterobifunctional PEG(X-PEG-Y) linker DBCO-PEG45-NHS Ester was prepared by a simplified twelve-step synthetic route, which solved the problems of complex synthesis and high cost in the existing technology, realized efficient and economical drug synthesis, and expanded its application in the biomedical field.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- WUHAN AOFEI TECH CO LTD
- Filing Date
- 2024-12-06
- Publication Date
- 2026-06-09
Smart Images
Figure CN122167720A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of organic synthesis, specifically to a single-molecular-weight heterobifunctional PEG (X-PEG-Y) linker DBCO-PEG45-NHS. Ester(N-{138-[(2,5-dioxoylidetetrahydro-1H-pyrrolo-1-yl)oxy]-138-oxoylide-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132,135-tetradecanoyloxa-13octadecanoyl-1-yl}-4-oxoylide-4-{11-azatricyclo[10.4.0.0]} 4,9 Synthetic method of hexadecene-1(12),4(9),5,7,13,15-hexaen-2-ynyl-11-ylbutyramide. 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] The bioorthogonal chemical reaction of DBCO reagents with hydrophilic polyethylene glycol (PEG) chains and azide (N3) is an important class of click chemistry reactions. Due to its mild reaction conditions, rapid reaction rate, and good biocompatibility, it has been widely used in bioconjugation, materials science, radiochemistry, drug discovery, and proteomics. Figure 3, WO2022056494A1; WO2023173132A1; J.Controll.Relea., 2024,370,302-309; J.Am.Chem.Soc., 2024,146(26), 17728-17737; J.Am.Chem.Soc., 2024,146(40),27382-27391; Sci.Adv., 2024,10(19),eadm9561; Bioconjugate Chem., 2024, 35(4), 465-471; Nat. Commun., 2024, 15, 8695-8708).
[0005] Given the importance of DBCO reagents with hydrophilic polyethylene glycol (PEG) chains 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 single molecular weight heterobifunctional PEG (X-PEG-Y) linker DBCO-PEG45-NHS Ester(N-{138-[(2,5-dioxoylidetetrahydro-1H-pyrrolo-1-yl)oxy]-138-oxoylide-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132,135-tetradecanoyloxa-13octadecanoyl-1-yl}-4-oxoylide-4-{11-azatricyclo[10.4.0.0]} 4,9The preparation method of hexadecene-1(12),4(9),5,7,13,15-hexaden-2-ynyl-11-yl)butyramide is related to the field of organic synthesis. The heterobifunctional PEG(X-PEG-Y) linker DBCO-PEG45-NHS Ester(N-{138-[(2,5-dioxoylidetetrahydro-1H-pyrrolo-1-yl)oxy]-138-oxoylide-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132,135-tetradecanoyloxa-13octadecanoyl-1-yl}-4-oxoylide-4-{11-azatricyclo[10.4.0.0]} 4,9 Hexadecyl-1(12),4(9),5,7,13,15-hexaden-2-yn-11-yl)butyramide has strong application potential in bioconjugation, materials science, radiochemistry, drug discovery, and proteomics. This invention uses 2-[(1-phenyl-2,5,8,11,14,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69-tetraoxaheptadecane-71-yl)oxy]eth-1-ol as the starting material. Through twelve conventional transformations including protection, substitution, addition, condensation, and deprotection, the target product was successfully prepared in a ~20% 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 excellent economic benefits and market prospects. 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 DBCO heterobifunctional molecules with 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, 80 mmol) was dissolved in dichloromethane (1000 mL) at room temperature, followed by the addition of triethylamine (2.0 eq., 17.3 g) and p-toluenesulfonyl chloride (TsCl, 2.0 eq., 32.7 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 (110 g, 97%). No further purification was required, and it was used directly in the next step.
[0015] [Step 2]
[0016] Compound 3 (90 g, 75 mmol) was dissolved in tetrahydrofuran (THF, 1000 mL) at room temperature. The mixture was cooled to 0°C, and sodium hydride (NaH, 2.0 eq., 6.1 g) was added. The mixture was stirred at room temperature for 2 hours. The mixture was then cooled to 0°C, and compound 2 (1.2 eq., 108 g) was added dropwise. The mixture was slowly heated to 25°C and reacted for 16 hours. The reaction was monitored by TLC (MeOH:DCM = 10:1), which showed that the starting material had reacted completely. 1000 mL of water was added to the reaction system, and the reaction solution was directly concentrated under reduced pressure to remove most of the solvent. Ethyl acetate was added to extract impurities, and the mixture was extracted three times. 1000 mL of dichloromethane (DCM) was added to extract three times. The organic phases were combined, washed with saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a yellow oily substance 4 (150 g, 84%). No further purification was required, and it was used directly in the next step.
[0017] [Step 3]
[0018] Compound 4 (150 g, 64 mmol) was dissolved in methanol (MeOH, 1000 mL) at room temperature. 1 mL of hydrochloric acid was added, and the mixture was stirred at room temperature for 2 hours. The reaction was monitored by TLC (MeOH:DCM = 10:1), indicating that the starting material had reacted completely. Most of the methanol was concentrated under reduced pressure. 1 L of dichloromethane and 1 L of water were added, and the mixture was stirred for 10 minutes. The mixture was allowed to stand and separate into layers. The aqueous phase was extracted twice with dichloromethane. The organic phases were combined and washed once with saturated brine and once with sodium bicarbonate solution. The organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a white solid crude product 5 (140 g, 98%). The crude product was dissolved in 500 mL of dichloromethane, and 200 g of 100-200 mesh silica gel powder was added and mixed. The mixture was subjected to column chromatography. Impurities were removed using the mobile phase (PE:EA = 10:1-1:1-0:1), and the white solid product 5 (120 g, 85%) was obtained.
[0019] [Step 4]
[0020] Under a nitrogen atmosphere and at room temperature, compound 5 (120 g, 54 mmol) was dissolved in tetrahydrofuran (THF, 2000 mL), sodium lumps (100 mg) were added, and tert-butyl acrylate 6 (2 eq., 14 g) was added dropwise. The mixture was stirred at room temperature for 12 hours. TLC monitoring (MeOH:DCM = 10:1) showed that the starting material had reacted completely. The mixture was quenched with 100 mL of water, and concentrated under reduced pressure to remove most of the tetrahydrofuran (THF). 1 L of dichloromethane and 1 L of water were added and stirred for 10 minutes. The mixture was allowed to stand and separate into layers. The aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed once with saturated brine and once with sodium bicarbonate solution. The organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a white solid crude product 7 (126 g, 100%). The crude product was dissolved in 500 mL of dichloromethane, and 200 g of sodium bicarbonate was added. Mix the sample with 100-200 mesh silica gel powder, pass it through the column for chromatography, and use the mobile phase (PE:EA = 10:1-1:1-0:1) to remove impurities, and obtain a white solid product 7 (120g, 95%).
[0021] [Step 5]
[0022] Compound 7 (120 g, 50 mmol) was dissolved in methanol (1000 mL) under nitrogen atmosphere and room temperature. The reaction was carried out under 5% Pd / C (5 g) and 15 psi hydrogen atmosphere for 16 hours. TLC monitoring (MeOH:DCM = 10:1) showed complete reaction of the starting material. Palladium on carbon was filtered off, and the mixture was concentrated under reduced pressure to obtain a white solid crude product 8 (115 g, 100%). The crude product was dissolved in 500 mL of dichloromethane (DCM), and 200 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 (PE:EA = 10:1-1:1-0:1), and the white solid product 8 (110 g, 95%) was obtained.
[0023] [Step 6]
[0024] Compound 8 (110 g, 48 mmol) was dissolved in dichloromethane (1000 mL) at room temperature, followed by the addition of triethylamine (2.0 eq., 9.8 g) and p-toluenesulfonyl chloride (TsCl, 2.0 eq., 18.6 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 9 (110 g, 97%). No further purification was required, and it was used directly in the next step.
[0025] [Step 7]
[0026] Compound 9 (110 g, 47 mmol) was dissolved in acetonitrile (1000 mL) at room temperature, and lithium bromide (2.0 eq., 8.4 g) was added. The mixture was heated to 80 °C and stirred for 16 hours. The reaction was monitored by TLC (MeOH:DCM = 10:1), which 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 10 (100 g, 91%). No further purification was required, and it was used directly in the next step.
[0027] [Step 8]
[0028] Compound 10 (100 g, 45 mmol) was dissolved in N,N-dimethylformamide (DMF, 1000 mL) at room temperature. Compound 11 (2.0 eq., 17 g) and cesium carbonate (2.0 eq., 29.5 g) were added. The mixture was heated to 80 °C and stirred for 16 hours. The reaction was monitored by TLC (MeOH:DCM = 10:1), indicating that the starting materials had 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 (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 12 (120 g), crude product. The crude product was dissolved in 500 mL of DCM, and 300 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=20:1-10:1) as the mobile phase to pass the product to obtain 12 white solid products (85g, 82%).
[0029] [Step 9]
[0030] Compound 12 (85 g, 37 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 complete reaction of the starting materials. 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 combined organic phases were washed once with saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a white solid, compound 13 (90 g), crude. The crude product was dissolved in 100 mL of dichloromethane (DCM), and 150 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 a white solid product 13 (70g, 87%).
[0031] [Step 10]
[0032] Compound 13 (70 g, 32 mmol) was dissolved in dichloromethane (DCM, 1000 mL) at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2 eq., 12.5 g) and compound 14 (2 eq., 20 g) were added. The mixture was stirred at room temperature for 2 hours. TLC monitoring (MeOH:DCM = 10:1) showed complete reaction of the starting material. The mixture was extracted once with 500 mL of water, and the aqueous phase was purified using 500 mL of dichloromethane. Extracted twice with chloromethane, 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 15 (95 g) crude product. The crude product was dissolved in 100 mL of DCM, mixed with 150 g of 100-200 mesh silica gel powder, and subjected to column chromatography. The mobile phase was used to remove impurities (PE:EA = 10:1-1:1-0:1) and the product was filtered through (MeOH:DCM = 20:1-8:1) to obtain a yellow solid product 15 (69 g, 87%).
[0033] [Step 11]
[0034] Compound 15 (69 g, 28 mmol) was dissolved in dichloromethane (DCM, 1000 mL) at room temperature, and 300 mL of trifluoroacetic acid (TFA) was added. The mixture was stirred at room temperature for 2 hours. The reaction was monitored by TLC (MeOH:DCM = 10:1), which showed that the starting material reacted completely. The mixture was concentrated under reduced pressure to obtain a white solid compound 16 (90 g) crude product. The crude product was dissolved in 100 mL of dichloromethane (DCM), and 150 g of 100-200 mesh silica gel powder was added and mixed. The mixture was then subjected to column chromatography. The mobile phase was (PE:EA = 10:1-1:1-0:1) to remove impurities, and (MeOH:DCM = 20:1-8:1) to remove the product to obtain a white solid product 16 (40 g, 59%).
[0035] 1 H NMR(400MHz,DMSO-d6,ppm)δ7.70(s,1H),7.62(dd,J=22.6,7.2Hz,2H),7.52-7.22(m,6H),5.00(d,J=14.3Hz,1H),3.58-3.45(m,188 H),3.07-3.03(m,1H),2.59-2.51(m,1H),2.34(t,J=6.5Hz,2H),2.20(dt,J=15.1,7.6Hz,1H),2.04-1.91(m,1H),1.80-1.68(m,1H).
[0036] [Step 12]
[0037] Compound 16 (40 g, 16 mmol) was dissolved in DCM (500 mL) at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 4 eq., 12.3 g) and N-hydroxysuccinimide (HOSu, 2 eq., 5.1 g) were added. The mixture was stirred at room temperature for 2 hours. TLC monitoring (MeOH:DCM = 10:1) showed complete reaction of the starting material. The mixture was extracted once with 500 mL of water, and the aqueous phase was extracted twice 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 give the white solid final product DBCO-PEG45-NHS. Ester(N-{138-[(2,5-dioxoylidetetrahydro-1H-pyrrolo-1-yl)oxy]-138-oxoylide-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132,135-tetradecanoyloxa-13octadecanoyl-1-yl}-4-oxoylide-4-{11-azatricyclo[10.4.0.0]} 4,9 [Hexadecyl-1(12),4(9),5,7,13,15-hexaen-2-yn-11-yl}butyramide (40g, 96%).
[0038] 1 H NMR (400MHz, DMSO-d6, ppm) δ7.70(s,1H),7.65(d,J=7.8Hz,1H),7.59(d,J=7.6Hz,1H),7.49-7.25(m,6H),5.00(d,J=14.1Hz,1H),3.69(t,J=5.9Hz,2H ),3.47(s,183H),2.89(t,J=6.0Hz,2H),2.78(s,4H),2.55(dd,J=16.4,8.0 Hz,1H),2.21(dd,J=15.3,7.5Hz,1H),2.05-1.87(m,1H),1.82-1.62(m,1H).
[0039] This invention uses 2-[(1-phenyl-2,5,8,11,14,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69-tetroxaocitaco-71-yl)oxy]eth-1-ol as the starting material, and through twelve conventional transformation steps including protection, substitution, addition, condensation, and deprotection, successfully prepared the target product DBCO-PEG45-NHS in a ~20% overall yield and on a 100-gram scale. Ester(N-{138-[(2,5-dioxoylidetetrahydro-1H-pyrrolo-1-yl)oxy]-138-oxoylide-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132,135-tetradecanoyloxa-13octadecanoyl-1-yl}-4-oxoylide-4-{11-azatricyclo[10.4.0.0]} 4,9 [Hexadecyl-1(12),4(9),5,7,13,15-hexaen-2-yn-11-yl}butanamide]; This route is simple to operate and has mild reaction conditions. It is a brand-new process synthesis route with good economic benefits and market prospects.
[0040] 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 DBCO-PEG45-NHS Ester(N-{138-[(2,5-dioxoylidetetrahydro-1H-pyrrolo-1-yl)oxy]-138-oxoylide-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132,135-tetradecanoyloxa-13octadecanoyl-1-yl}-4-oxoylide-4-{11-azatricyclo[10.4.0.0]} 4,9 The preparation method of hexadecene-1(12),4(9),5,7,13,15-hexaden-2-yn-11-ylbutyramide is as follows:
2. The heterobifunctional PEG (X-PEG-Y) linker DBCO-PEG45-NHS according to claim 1 Ester(N-{138-[(2,5-dioxoylidetetrahydro-1H-pyrrolo-1-yl)oxy]-138-oxoylide-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132,135-tetradecanoyloxa-13octadecanoyl-1-yl}-4-oxoylide-4-{11-azatricyclo[10.4.0.0]} 4,9 A method for synthesizing hexadecene-1(12),4(9),5,7,13,15-hexaen-2-yn-11-ylbutyramide, 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 4 is as follows: under room temperature conditions, compound 3 is dissolved in tetrahydrofuran, cooled to 0 degrees Celsius, sodium hydride and compound 2 are added, and 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 4, which can be directly used for the next reaction without further purification. The specific method for synthesizing compound 5 is as follows: under room temperature conditions, compound 4 is dissolved in methanol, hydrochloric acid is added, the temperature is raised to react, and after the reaction is completed, the reaction solution is extracted, desolventized, and purified to obtain compound 5. The specific method for synthesizing compound 7 is as follows: under a nitrogen atmosphere and room temperature, compound 5 is dissolved in tetrahydrofuran, compound 6 is added, and the reaction is carried out at room temperature. After the reaction is completed, the reaction solution is quenched, 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 acetonitrile, lithium bromide is added, the temperature is raised and the reaction is carried out. After the reaction is completed, the reaction solution is concentrated, extracted, desolventized, and purified to obtain compound 10. No further purification is required, and it can be directly used for the next reaction. The specific method for synthesizing compound 12 is as follows: under room temperature conditions, compound 10 is dissolved in N,N-dimethylformamide, compound 11 and cesium carbonate are added sequentially, the temperature is raised and the reaction is carried out. After the reaction is completed, the reaction solution is concentrated, extracted, desolventized and purified to obtain compound 12. The specific method for synthesizing compound 13 is as follows: under room temperature conditions, compound 12 is dissolved in methanol, hydrazine hydrate is added, the temperature is raised to react, and after the reaction is completed, the reaction solution is concentrated, extracted, desolventized, and purified to obtain compound 13. The specific method for synthesizing compound 15 is as follows: under room temperature conditions, compound 13 is dissolved in dichloromethane, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and compound 14 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 compound 15. The specific method for synthesizing compound 16 is as follows: under room temperature conditions, compound 15 is dissolved in dichloromethane, trifluoroacetic acid is added, and the reaction is carried out at room temperature. After the reaction is completed, the reaction solution is concentrated, separated and purified to obtain compound 16. The final product, DBCO-PEG45-NHS Ester(N-{138-[(2,5-dioxanetetrahydro-1H-pyrrolo-1-yl)oxy]-138-oxonyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132,135-tetradecanoyloxa-13octadecanoyl-1-yl}-4-oxonyl-4-{11-azatricyclo[10.4.0.0]} 4,9 The specific method for synthesizing hexadecene-1(12),4(9),5,7,13,15-hexaen-2-yn-11-yl}butanamide is as follows: under room temperature conditions, compound 16 is dissolved in dichloromethane, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide 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 DBCO-PEG45-NHS Ester.
3. The heterobifunctional PEG (X-PEG-Y) linker DBCO-PEG45-NHS according to claim 2 Ester(N-{138-[(2,5-dioxoylidetetrahydro-1H-pyrrolo-1-yl)oxy]-138-oxoylide-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132,135-tetradecanoyloxa-13octadecanoyl-1-yl}-4-oxoylide-4-{11-azatricyclo[10.4.0.0]} 4,9 A method for synthesizing hexadecene-1(12),4(9),5,7,13,15-hexaen-2-yn-11-ylbutyramide, characterized in that: The organic base used in steps 1 and 6 is one of triethylamine, N,N-diisopropylethylamine, imidazole, and pyridine.
4. The heterobifunctional PEG (X-PEG-Y) linker DBCO-PEG45-NHS according to claim 2 Ester(N-{138-[(2,5-dioxoylidetetrahydro-1H-pyrrolo-1-yl)oxy]-138-oxoylide-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132,135-tetradecanoyloxa-13octadecanoyl-1-yl}-4-oxoylide-4-{11-azatricyclo[10.4.0.0]} 4,9 A method for synthesizing hexadecene-1(12),4(9),5,7,13,15-hexaen-2-yn-11-ylbutyramide, characterized in that: The inorganic base used in step 8 is one of potassium phosphate, sodium phosphate, potassium carbonate, sodium carbonate, and cesium carbonate.
5. The heterobifunctional PEG (X-PEG-Y) linker DBCO-PEG45-NHS according to claim 2 Ester(N-{138-[(2,5-dioxoylidetetrahydro-1H-pyrrolo-1-yl)oxy]-138-oxoylide-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132,135-tetradecanoyloxa-13octadecanoyl-1-yl}-4-oxoylide-4-{11-azatricyclo[10.4.0.0]} 4,9 A method for synthesizing hexadecene-1(12),4(9),5,7,13,15-hexaen-2-yn-11-ylbutyramide, characterized in that: The solvent used in steps 3 and 5 is one of methanol, ethanol, isopropanol, and tert-butanol.
6. The heterobifunctional PEG (X-PEG-Y) linker DBCO-PEG45-NHS according to claim 2 Ester(N-{138-[(2,5-dioxoylidetetrahydro-1H-pyrrolo-1-yl)oxy]-138-oxoylide-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132,135-tetradecanoyloxa-13octadecanoyl-1-yl}-4-oxoylide-4-{11-azatricyclo[10.4.0.0]} 4,9 A method for synthesizing hexadecene-1(12),4(9),5,7,13,15-hexaen-2-yn-11-ylbutyramide, characterized in that: The protic acid used in step 11 is one of hydrochloric acid, sulfuric acid, trifluoroacetic acid, and methanesulfonic acid.