Microwave synthesis method of Angiopep-2
The synthesis process of Angiopep-2 was optimized by microwave synthesis technology, which solved the problem of low efficiency of traditional solid-phase synthesis method, and realized efficient and simple synthesis and large-scale production of Angiopep-2, improving yield and purity.
Patent Information
- Application Number
- CN202511201338.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-26
- Publication Date
- 2025-11-28
AI Technical Summary
Traditional solid-phase synthesis methods for Angiopep-2 are inefficient, require large amounts of solvent, and are difficult to scale up, making them unsuitable for synthesizing complex secondary peptides.
Microwave synthesis technology was employed to synthesize Angiopep-2 by optimizing microwave power, reaction temperature, and coupling reagent combinations, combined with a microwave synthesizer. This included the preparation of protected amino acid tyrosine resin, fully protected peptide resin, crude peptide, and refined peptide. Specific resins and deprotection reagents were used to shorten reaction time and improve synthesis efficiency.
It significantly improves the yield and purity of Angiopep-2, shortens the reaction time, is suitable for efficient synthesis and large-scale application, and is simple to operate with good reproducibility.
Smart Images
Figure CN121021644A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of peptide synthesis technology, specifically relating to a microwave synthesis method for Angiopep-2. Background Technology
[0002] Angiopep-2 is a polypeptide composed of 19 amino acids, with the molecular formula C2. 104 H 149 N 29 O 31 Its peptide sequence is TFFYGGSRGKRNNFKTEEY. By specifically binding to low-density lipoprotein receptor-related protein 1 (LRP-1), it can efficiently penetrate the blood-brain barrier and target tumor tissue, demonstrating significant application value in drug delivery systems, treatment of neurodegenerative diseases, and targeted therapy for brain tumors. It can also serve as a molecular imaging probe for precise lesion visualization. However, its complex secondary structure (such as β-sheets) and long peptide chain characteristics place high demands on the synthesis process.
[0003] Traditional solid-phase peptide synthesis (SPPS) suffers from problems such as long reaction times (several hours per step), low reaction efficiency, large solvent consumption, and difficulty in scaling up. In contrast, microwave synthesis technology, through molecular-level high-frequency heating, shortens the single-step reaction to minutes, significantly improving synthesis efficiency while reducing solvent consumption and supporting precise temperature and pressure control, making it particularly suitable for the synthesis of long peptides with complex structures.
[0004] This invention utilizes a microwave peptide synthesizer and, by optimizing microwave power, reaction temperature, and coupling reagent combinations, successfully improved the yield and purity of Angiopep-2. This process, characterized by high efficiency, provides an effective solution for the efficient synthesis and large-scale application of Angiopep-2. Summary of the Invention
[0005] To address the shortcomings of existing technologies, the present invention aims to provide a microwave synthesis method for Angiopep-2.
[0006] This invention describes a microwave synthesis method for Angiopep-2, comprising the following steps:
[0007] (1) Preparation of resin for protecting amino acid tyrosine:
[0008] The resin solid support was coupled with Fmoc-Tyr(tBu)-OH to obtain Fmoc-Tyr(tBu)-resin;
[0009] (2) Preparation of Angiopep-2 fully protected peptide resin:
[0010] On the Fmoc-Tyr(tBu)-resin obtained in step (1), Fmoc-protected amino acids were sequentially coupled from the C-terminus to the N-terminus of the Angiopep-2 peptide sequence using a microwave synthesizer and a solid-phase synthesis method to obtain the following fully protected Angiopep-2 peptide resin:
[0011] Thr(tBu)-Phe-Phe-Tyr(tBu)-Gly-Gly-Ser(tBu)-Arg(pbf)-Gly-Lys(Boc)-Arg(pbf)-Asn(Trt)-Asn(Trt)-Phe-Lys(Boc)-Thr(tBu)-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-resin;
[0012] (3) Preparation of crude Angiopep-2 peptide:
[0013] The Angiopep-2 fully protected peptide resin obtained in step (2) was lysed with a lysis buffer to obtain crude Angiopep-2 peptide;
[0014] (4) Preparation of Angiopep-2 priapeptide:
[0015] Angiopep-2 crude peptide was purified by reverse preparative liquid chromatography to obtain Angiopep-2 refined peptide.
[0016] Furthermore, in step (2), the microwave synthesizer also includes: a nitrogen metering pump, an amino acid and condensing agent solvent bottle, a DMF solvent bottle, a deprotection solvent bottle, a reactor, and a microwave heating device; wherein, the amino acid and condensing agent solvent, DMF solvent, and deprotection solvent are added to the reactor through the nitrogen metering pump; the microwave heating device can set the microwave temperature and power according to the experimental content and requirements, and control the microwave reaction time.
[0017] Further, in step (2), the steps of synthesizing peptide sequences using the microwave synthesizer include S1, resin swelling; S2, resin deprotection; S3, coupling; wherein step S3 includes amino acid microwave coupling, three resin washings, amino acid microwave deprotection, and six resin washings.
[0018] The resin synthesized by microwave is any one of Rink Amide-AM resin, Wang resin, and 2-chlorotriphenylmethylchloro(2-CTC) resin; the deprotection reagent used is any one of 30% (v / v) piperidine / DMF, 25% (v / v) piperidine / DMF, 20% (v / v) piperidine / DMF, and 10% (v / v) piperazine / DMF.
[0019] In step (2), the steps of synthesizing peptide sequences using the microwave synthesizer include S1, resin swelling; S2, resin deprotection; S3, coupling; wherein step S3 includes microwave coupling of amino acids, three washings of the resin, microwave deprotection of amino acids, and six washings of the resin.
[0020] The resin synthesized by microwave is any one of Rink Amide-AM resin, Wang resin, and 2-chlorotriphenylmethylchloro(2-CTC) resin; the deprotection reagent used is any one of 30% (v / v) piperidine / DMF, 25% (v / v) piperidine / DMF, 20% (v / v) piperidine / DMF, and 10% (v / v) piperazine / DMF.
[0021] Further, in step (2), the coupling is carried out in the presence of a condensing agent selected from any one or more combinations of N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), benzotriazine-1-yl-oxytripyrrolylphosphine hexafluorophosphate (PyBop), (7-azabenzotriazine-1-oxy)tripyrrolylphosphine hexafluorophosphate (PyAOP), 2-(7-oxobenzotriazine)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), O-benzotriazine-tetramethylurea hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBT), 1-hydroxy-7-azabenzotriazole (HOAT), 4-dimethylaminopyridine (DMAP), ethyl 2-oxime cyanoacetate (Oxyma), N,N-diisopropylethylamine (DIEA), or N-methylmorpholine (NMM).
[0022] Furthermore, in step (2), the added amino acids and condensing agents are mixed with the solvent to form a mixture, wherein the solvent is selected from any one or any mixture of N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF) in any proportion.
[0023] Further, the preferred molar ratio of the condensing agent to the amino acid is 1:1 or 2:1; the amino acid concentration is 0.2M-0.5M; the concentration of the condensing agent is 0.2-1.5M; preferably, the amino acid concentration is 0.3M-0.4M and the concentration of the condensing agent is 0.3M-1.2M.
[0024] Furthermore, in step (2), the reaction temperature for the microwave coupling of amino acids is 30-100℃, the reaction power is 50-300W, and the reaction time is 120s-600s.
[0025] Furthermore, step (2) specifically includes the following steps:
[0026] a. After adding amino acid resin to the reactor of the microwave synthesizer, continue to add DMF for washing and swelling, then add deprotection reagent, set reaction conditions, and turn on the microwave heating device to carry out microwave deprotection reaction;
[0027] b. After the deprotection reaction is completed, add the mixture of amino acid and solvent and the mixture of condensing agent and solvent into the reactor, set the reaction conditions, turn on the microwave heating device to carry out the microwave coupling reaction, then add the deprotection reagent and turn on the microwave heating device to carry out the microwave deprotection reaction.
[0028] c. After the amino acid deprotection reaction is complete, repeat step b, and sequentially couple Fmoc-protected amino acids according to the peptide sequence from C-terminus to N-terminus of Angiopep-2: Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Arg(pbf)-OH, Fmoc-S er(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, to obtain Thr(tBu)-Phe-Phe-Tyr(tBu) -Gly-Gly-Ser(tBu)-Arg(pbf)-Gly-Lys(Boc)-Arg(pbf)-Asn(Trt)-Asn(Trt)-Phe-Lys(Boc)-Thr(tBu)-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-resin;
[0029] d. The resin obtained in step c
[0030] The resin containing Thr(tBu)-Phe-Phe-Tyr(tBu)-Gly-Gly-Ser(tBu)-Arg(pbf)-Gly-Lys(Boc)-Arg(pbf)-Asn(Trt)-Asn(Trt)-Phe-Lys(Boc)-Thr(tBu)-Glu(OtBu)-Glu(OtBu)-Tyr(tBu) is washed and dried to obtain the Angiopep-2 fully protected peptide resin.
[0031] After the microwave deprotection reaction and microwave coupling reaction are completed, washing is required before proceeding to the next reaction step.
[0032] The washing process involves washing with DMF or NMP 2-8 times, preferably 3-6 times.
[0033] Further, in step (3), the lysis solution of the present invention is a mixture of trifluoroacetic acid (TFA) and triisopropylsilane (TIS), 3-mercaptopropionic acid (MPA), phenol, anisole sulfide and water; in the mixed solution, the volume ratio of TFA is 85%-97%, preferably 90%-95%; the volume ratio of TIS is 1%-7%, preferably 2%-5%; the volume ratio of MPA is 1%-7%, preferably 2%-5%; the volume ratio of phenol is 1%-5%, preferably 2%-4%; the volume ratio of anisole sulfide is 2%-10%, preferably 3%-7%; and the volume ratio of water is 0.5%-5%, preferably 1%-3%. The most preferred combination is TFA:TIS:MPA:water = 93:3:3:1.
[0034] Furthermore, in step (4), the purification conditions described in this invention refer to reverse preparative liquid chromatography using a C18 10 μm column. A 20*250mm mobile phase was used. Mobile phase A consisted of 100% acetonitrile or 0.1% TFA acetonitrile, and mobile phase B consisted of 0.05% TFA water or 0.1% TFA water. Gradient elution conditions were set: A phase varied from 5% to 60%, and B phase varied from 95% to 40%. The run time was 60-90 min, the flow rate was 10-20 mL / min, and the detection wavelength was 214 nm or 220 nm. The optimal conditions were: mobile phase A was 100% acetonitrile, and mobile phase B was 0.1% TFA water; gradient elution conditions were: A phase varied from 15% to 50%, and B phase varied from 85% to 50%; run time was 80 min; flow rate was 13 mL / min; and detection wavelength was 214 nm.
[0035] The present invention has the following advantages over the prior art:
[0036] 1. This invention provides a microwave synthesis method for Angiopep-2. The yield of crude Angiopep-2 peptide obtained by this invention is 95.94% and the purity is 77.87%, while the yield of crude Angiopep-2 peptide obtained by solid-phase manual synthesis is 91.27% and the purity is 55.67%. In comparison, the microwave synthesis method provided by this invention improves the efficiency of Angiopep-2 synthesis.
[0037] 2. The present invention greatly shortens the coupling reaction time. The reaction time of the present invention for coupling amino acids is 120s to 600s, while the reaction time for solid-phase manual synthesis of coupled amino acids is 2.5h. In comparison, the reaction time of microwave synthesis of coupled amino acids is greatly shortened.
[0038] 3. This invention uses a microwave synthesizer to synthesize peptides, which is simple to operate, has a high reproducibility rate, and is suitable for process optimization and large-scale production. Attached Figure Description
[0039] Figure 1 Here is the HPLC chromatogram of the crude Angiopep-2 peptide obtained in Example 3;
[0040] Figure 2 The mass spectrum of the crude Angiopep-2 peptide obtained in Example 3;
[0041] Figure 3 The image shows the HPLC chromatogram of the Angiopep-2 spermatin obtained in Example 4. Detailed Implementation
[0042] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods.
[0043] The chemical names corresponding to some of the abbreviations used in this invention are as follows:
[0044] Fmoc: fluorenylmethoxycarbonyl, fluorenylmethoxycarbonyl; Glu: glutamic acid; Thr: threonine; Lys: lysine; Phe: phenylalanine; Asn: asparagine; Arg: arginine; Gly: glycine; Ser: serine; Tyr: tyrosine; DMF: N,N-dimethylformamide; DCC: N,N'-dicyclohexylcarbodiimide; DIC: N,N'-diisopropylcarbodiimide; PyAOP: (7-azabenzotriazol-1-oxo)tripyrrolephos hexafluorophosphate; PyBop: benzotriazol-1-yl-oxytripyrrolephosphosiphosphate; HATU: 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate; HBTU: O-benzotriazole-tetramethylurea hexafluorophosphate; HOAT: 1-hydroxy-7-azabenzotriazole; HOBT: 1-hydroxybenzotriazole; Oxyma: ethyl 2-oxime cyanoacetate; DMAP: 4-dimethylaminopyridine; DIEA: N,N-diisopropylethylamine; TFA: trifluoroacetic acid; MeOH: methanol; DODT: 3,6-dioxo-1,8-octanedithiol; TIS: triisopropylsilane; MPA: 3-mercaptopropionic acid.
[0045] Example 1: Preparation of amino acid resin
[0046] 5 g of Wang resin with a substitution degree of 1.24 mmol / g was weighed into a solid-phase reaction column, DMF was added, and the column was bubbled with nitrogen for 30 minutes to swell. 3.87 g of Fmoc-Tyr(tBu)-OH, 0.68 g of HOBt, and 0.02 g of DMAP were weighed and dissolved in DMF. 1 mL of DIEA was added at room temperature, and the reaction was carried out for 4 hours. After washing twice, 2 mL of acetic anhydride and 1.5 mL of pyridine were added, and the mixture was mixed and blocked for 4 hours. The column was washed twice with DMF and three times with DCM. After shrinkage with methanol, the resin was dried to obtain 6.14 g of Fmoc-Tyr(tBu)-Wang Resin, with a substitution degree of 0.45 mmol / g.
[0047] Example 2: Preparation of Angiopep-2 peptide resin
[0048] 2.1 Resin swelling
[0049] Weigh 0.5g of Fmoc-Tyr(tBu)-Wang Resin and add it to the reactor. First, add DMF to wash the resin using a nitrogen metering pump, then add DMF to allow it to swell for 30 minutes. After swelling is complete, add DMF to the reactor to wash it three times.
[0050] 2.2 Resin Deprotection
[0051] An appropriate amount of pre-prepared deprotection reagent (25% (v / v) piperidine / DMF mixed solution, i.e., the volume percentage of piperidine in the mixed solution of piperidine and DMF is 25%) is introduced through a nitrogen metering pump. At the same time, the microwave heating device is turned on to carry out the microwave deprotection reaction. The microwave power gradient is adjusted to 200W~120s, the temperature is 75℃, and the deprotection reaction residence time is 120s. After the deprotection operation is carried out, DMF is added and the mixture is washed 6 times.
[0052] 2.3 Feeding and Reaction
[0053] Weigh 2.54g of Fmoc-Glu(OtBu)-OH and other amino acids in advance and prepare a 0.35M DMF solution. Then weigh 3.28g of HOBT, 13.10g of PyBop, and 8mL of DIEA and prepare a 0.75M DMF solution. Add the prepared amino acid and condensing agent solutions to the amino acid and condensing agent solvent bottles in the microwave synthesizer. Add the amino acid and condensing agent solutions to the reactor using a nitrogen metering pump. Simultaneously turn on the microwave heating device to initiate the microwave coupling reaction. Adjust the microwave power gradient to 200W–300s, the temperature to 75℃, and the coupling reaction residence time to 300s. Perform the coupling operation, then wash three times with DMF. Deprotection of amino acids is performed in step 2.2.
[0054] 2.4 Sequential coupling of amino acids
[0055] Repeat step 2.3, sequentially coupling Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Arg(pbf)-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Gly-OH to the Angiopep-2 peptide sequence. c-Gly-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, to obtain Thr(tBu)-Phe-Phe-Tyr(tBu)-Gly-Gl y-Ser(tBu)-Arg(pbf)-Gly-Lys(Boc)-Arg(pbf)-Asn(Trt)-Asn(Trt)-Phe-Lys(Boc)-Thr(tBu)-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-resin;
[0056] 2.5 Shrink-dried resin
[0057] The resin obtained in step 2.4 was washed twice with DMF (10 mL), twice with DCM (0 mL), and finally shrunk twice with methanol (10 mL). The resin was dried at room temperature or in an oven to constant weight to obtain 1.12 g of Angiopep-2 fully protected peptide resin.
[0058] Example 3: Preparation of crude Angiopep-2 peptide
[0059] Prepare 20 mL of lysis buffer with the following volume ratio: trifluoroacetic acid (TFA): 3-mercaptopropionic acid (MPA): triisopropylsilane (TIS): water = 93%:3%:3%:1%. Add 1.12 g of the fully protected Angiopep-2 resin obtained in Example 2 to 10 mL of lysis buffer, react in an ice-water bath for 2.5 h, filter, wash the peptide resin twice with a small amount of lysis buffer, combine the two washes, filter again, add to cold ether (120 mL), allow to settle, centrifuge, wash the solid three times with ether (100 mL), and obtain 0.496 g of crude Angiopep-2 peptide, with a yield of 95.94% and a purity of 77.87%.
[0060] Figure 1 Here is the HPLC chromatogram of the crude Angiopep-2 peptide obtained in Example 3; Figure 2 The mass spectrum of the crude Angiopep-2 peptide obtained in Example 3; from Figure 1 As can be seen, the peak elution time of the crude Angiopep-2 peptide was 10.666 min, and the purity was 77.87%. Figure 2 As can be seen, the molecular weight of the crude Angiopep-2 peptide is 2300.0958, while the precise molecular weight of Angiopep-2 is 2300.10, indicating that the mass spectrometry results of Angiopep-2 are correct.
[0061] Example 4: Preparation of Angiopep-2 Breast Peptide
[0062] 50 mg of the crude Angiopep-2 peptide obtained in Example 3 was dissolved in 3 mL of water and filtered through a 0.45 μm filter membrane before it could be used as a sample solution for preparation. The filtrate was then passed through a C18 10 μm column. Purification was performed using a 20*250 mm mobile phase. The mobile phase consisted of 100% acetonitrile (A) and 0.1% TFA water. The gradient was 0-80 min, with A% ranging from 15% to 50%. The flow rate was 13 mL / min, and the detection wavelength was 214 nm. The required fractions were collected, and qualified sample peaks were combined. The yield of Angiopep-2 peptide obtained in this step was 30%, with a purity of 90.49%.
[0063] Figure 3The image shows the HPLC chromatogram of the Angiopep-2 spermatin obtained in Example 4. Figure 3 As can be seen, the peak elution time of Angiopep-2 peptide was 10.858 min, and the purity was 90.49%; moreover, the peak elution time of the peptide was basically the same as that of the crude peptide.
[0064] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.
Claims
1. A method for microwave synthesis of Angiopep-2, characterized by: The method comprises the following steps: (1) preparing a protected amino acid tyrosine resin: coupling the resin solid carrier and Fmoc-Tyr(tBu)-OH to obtain Fmoc-Tyr(tBu)-resin; (2) preparing an Angiopep-2 fully protected peptide resin: taking the Fmoc-Tyr(tBu)-resin obtained in step (1), using a microwave synthesizer, and sequentially coupling Fmoc-protected amino acids according to the peptide sequence of Angiopep-2 from the C-terminal to the N-terminal by a solid-phase synthesis method to obtain the following sequence of peptide resin: Thr(tBu)-Phe-Phe-Tyr(tBu)-Gly-Gly-Ser(tBu)-Arg(pbf)-Gly-Lys(Boc)-Arg(pbf)-Asn(Trt)-Asn(Trt)-Phe-Lys(Boc)-Thr(tBu)-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-resin; (3) preparing an Angiopep-2 crude peptide: taking the peptide resin obtained in step (2), and subjecting it to cleavage to obtain an Angiopep-2 crude peptide; (4) preparing an Angiopep-2 refined peptide: subjecting the Angiopep-2 crude peptide to reverse phase chromatography to obtain an Angiopep-2 refined peptide.
2. The microwave synthesis method for Angiopep-2 according to claim 1, characterized in that: In step (2), the steps of synthesizing the peptide sequence by the microwave synthesizer include S1, resin swelling; S2, resin deprotection; S3, coupling; wherein step S3 includes microwave coupling of amino acids, three times of washing the resin, microwave deprotection of amino acids, and six times of washing the resin; The resin synthesized by the microwave is any one of Rink Amide-AM resin, Wang resin, and 2-chlorotrityl chloride (2-CTC) resin; and the deprotection reagent used for deprotection is any one of 30% (v / v) piperidine / DMF, 25% (v / v) piperidine / DMF, 20% (v / v) piperidine / DMF, and 10% (v / v) piperazine / DMF.
3. The microwave synthesis method of Angiopep-2 according to claim 1, characterized in that: In step (2), the coupling is performed in the presence of a condensing agent, and the condensing agent is selected from any one or a combination of N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), benzotriazole-1-yl-oxytrispyrrolidino phosphonium hexafluorophosphate (PyBop), (7-azabenzotriazol-1-yl-oxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), 2-(7-oxabenzotriazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-benzotriazol-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBT), 1-hydroxy-7-azabenzotriazole (HOAT), 4-dimethylaminopyridine (DMAP), 2-oxyma, N,N-diisopropylethylamine (DIEA), or N-methylmorpholine (NMM).
4. The microwave synthesis method for Angiopep-2 according to claim 3, characterized in that: In step (2), the amino acid and the condensing agent are mixed with a solvent to form a mixture, and the solvent is selected from N-methyl pyrrolidone (NMP), dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), or a mixture of any of them in any proportion.
5. The microwave synthesis method of Angiopep-2 according to claim 4, wherein: The preferred molar ratio of the condensing agent to the amino acid is 1:1 or 2:1; the concentration of the amino acid is 0.2M-0.5M; and the concentration of the condensing agent is 0.2-1.5M. 6.The method of claim 1, wherein the Angiopep-2 is synthesized by microwave. In step (2), the reaction temperature for microwave coupling of the amino acid is 30-100℃, the reaction power is 50-300W, and the reaction time is 120s-600s.
7. The microwave synthesis method of Angiopep-2 according to claim 2, wherein: The reaction temperature for resin deprotection and microwave deprotection of the amino acid is 30-100℃, the reaction power is 50-300W, and the reaction time is 60s-300s.
8. The microwave synthesis method of Angiopep-2 according to any one of claims 1-7, characterized in that: Step (2) specifically comprises the following steps: a. After adding Fmoc-Tyr(tBu)-resin into the reactor of the microwave synthesizer, continue to wash and swell with DMF, then add a deprotection reagent, set the reaction conditions, and turn on the heating device of the microwave synthesizer to perform microwave deprotection reaction; b. After the deprotection reaction is completed, add the mixture of the amino acid and the solvent and the mixture of the condensing agent and the solvent into the reactor, set the reaction conditions, and turn on the heating device of the microwave synthesizer to perform microwave coupling reaction, then add a deprotection reagent and turn on the heating device of the microwave synthesizer to perform microwave deprotection reaction; c. After the deprotection reaction of the amino acid is completed, repeat step b, and sequentially couple Fmoc-protected amino acids according to the peptide sequence of Angiopep-2 from C-terminal to N-terminal: Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Arg(pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, to obtain Thr(tBu)-Phe-Phe-Tyr(tBu)-Gly-Gly-Ser(tBu)-Arg(pbf)-Gly-Lys(Boc)-Arg(pbf)-Asn(Trt)-Asn(Trt)-Phe-Lys(Boc)-Thr(tBu)-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-resin; d. The resin obtained in step c is subjected to cleavage reaction to obtain Angiopep-2. Thr(tBu)-Phe-Phe-Tyr(tBu)-Gly-Gly-Ser(tBu)-Arg(pbf)-Gly-Lys(Boc)-Arg(pbf)-Asn(Trt)-Asn(Trt)-Phe-Lys(Boc)-Thr(tBu)-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-resin, after washing and drying, to obtain the Angiopep-2 full-protected peptide resin.
9. A microwave synthesis method for Angiopep-2 according to claim 1, characterized in that: In step (3), the cleavage solution is a mixed solution of trifluoroacetic acid (TFA) and triisopropylsilane (TIS), 3-mercaptopropionic acid (MPA), phenol, anisole and water; In the mixed solution, the volume ratio of TFA is 85%-97%; the volume ratio of TIS is 1%-7%; the volume ratio of MPA is 1%-7%; the volume ratio of phenol is 1%-5%; the volume ratio of anisole is 2%-10%; the volume ratio of water is 0.5%-5%, preferably 1%-3%; and the volume ratio of TFA:TIS:MPA:water is preferably 93:3:3:
1.
10. The method of claim 1, wherein the Angiopep-2 is synthesized by microwave. In step (4), the purification condition refers to reverse phase preparative liquid chromatography, using a column with the specification C18 10μm 20*250mm, mobile phase A is 100% acetonitrile or 0.1% TFA acetonitrile, B is 0.05% TFA water or 0.1% TFA water, set gradient elution conditions for A from 5% to 60%, B from 95% to 40%, running time is 60-90min, flow rate is 10mL / min-20mL / min, detection wavelength is 214nm or 220nm, for separation and purification.