GLP-1 derivative and preparation method thereof

Abstract

The invention belongs to the field of medicine synthesis, and particularly relates to a series of GLP-1 derivatives and a preparation method thereof. The GLP-1 derivative comprises a polypeptide part, a linker part and a side chain part, the amino acid sequence of the polypeptide part is as follows: H-X-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F-I-A-W-L-V-R-G-R-G; the amino acid sequence of the polypeptide part is Wherein X is alpha-aminoisobutyric acid; the structure of a linker part of the GLP-1 derivative is oligomeric ethylene glycol-oligomeric ethylene glycol-glutamic acid; the side chain part of the GLP-1 derivative is composed of two sections of short-chain fatty acids linked by 2, 6-diaminocaproic acid. The GLP-1 derivative prepared by the invention can be combined with a GLP-1 receptor and activate the receptor, so that the compound has extremely high application potential in treatment of metabolic diseases such as type 2 diabetes mellitus and obesity.

Description

Technical Field

[0001] The present invention belongs to the field of drug synthesis, and specifically relates to a series of GLP-1 derivatives and preparation methods thereof. Background Art

[0002] Peptide hormone compounds such as angiotensin, adrenocorticotropic hormone (ACTH), calcitonin, insulin, glucagon, glucagon-like peptide-1 (GLP-1), and glucagon-like peptide-2 have been widely used in clinical practice. These peptide drugs play an important role in regulating the body's physiological processes. However, in clinical treatment, natural peptides and peptide derivatives generally have the problem of high clearance rate, which limits their wide application. For many treatment plans, it is necessary to maintain a high blood concentration of the drug in the human body, otherwise the drug cannot be maintained for a long time in the human body, which will bring a series of undesirable consequences. For example, natural peptides are rapidly metabolized and quickly cleared from the body, resulting in a short effective period of the drug. Patients need to take drugs frequently, which increases the complexity of treatment and the burden on patients.

[0003] In order to overcome this problem, the art proposes to optimize the composition of the drug to delay the release rate of the peptide and adjust its in vivo release curve. Although this strategy is effective in some cases, its scope of application is limited, especially for peptide compounds with high clearance rates, which often fail to achieve the expected effect.

[0004] Taking gastrointestinal hormones as an example, its key hormones include glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). GIP has excellent hypoglycemic effect, but it is not effective in treating non-insulin-dependent diabetes mellitus (NIDDM), which limits its application in clinical treatment. GLP-1, on the other hand, has shown excellent efficacy in promoting insulin secretion and inhibiting glucagon secretion in NIDDM patients, significantly reducing blood sugar levels in patients, and has become an important candidate drug for the treatment of diabetes. GLP-1 is a peptide hormone derived from proglucagon, which can effectively lower blood sugar by regulating insulin secretion and inhibiting glucagon. However, the half-life of GLP-1 in the body is extremely short, only about 2 minutes. This means that the biological activity of GLP-1 is very limited, which seriously affects the effectiveness of its clinical treatment.

[0005] Although existing GLP-1 receptor agonists (GLP-1 derivatives) have demonstrated significant therapeutic effects in the treatment of type 2 diabetes and obesity, they still face several key challenges and limitations: 1) Short half-life: Natural GLP-1 and its early derivatives are metabolized rapidly in the body and are usually rapidly degraded within a few minutes to an hour, which requires patients to inject the drug frequently, increasing the inconvenience and burden during treatment and reducing patient compliance with treatment; 2) Easy to be enzymatically hydrolyzed: GLP-1 derivatives are easily decomposed by enzymes (such as DPP-4 enzyme) in the body, which leads to the rapid loss of their activity, shortening the effective action time of the drug, reducing the therapeutic effect, and also placing higher requirements on drug dosage management.

[0006] Although GLP-1 and its derivatives show great potential in the treatment of type 2 diabetes, their rapid clearance rate limits their application, and this problem urgently needs further research and improvement. Summary of the invention

[0007] Based on the above, in order to solve the problem that GLP-1 derivatives have a short half-life, resulting in a short therapeutic effect and easy inactivation, the present invention proposes a series of GLP-1 derivatives and preparation methods thereof.

[0008] The objects of the present invention are: 1. To provide a method for preparing a GLP-1 derivative to reduce the preparation cost; Second, prolong the duration of action of GLP-1 derivatives in the body and reduce their clearance rate; 3. Improve the therapeutic effect of GLP-1 derivatives.

[0009] To achieve the above objectives, the present invention adopts the following technical solutions.

[0010] A GLP-1 derivative, The GLP-1 derivative comprises a polypeptide portion, a linker portion and a side chain portion; The amino acid sequence of the polypeptide portion of the GLP-1 derivative is: HXEGTFTSDVSSYLEGQAAKE-FIAWLVRGRG; Wherein X is α-aminoisobutyric acid; The linker part structure of the GLP-1 derivative is oligoethylene glycol-oligoethylene glycol-glutamic acid; The side chain of the GLP-1 derivative is composed of two short-chain fatty acids linked by 2,6-diaminohexanoic acid.

[0011] As a preference, The structural formula of the GLP-1 derivative is shown below: .

[0012] A method for preparing a GLP-1 derivative, The method comprises: 1) Calculate the weight of each raw material based on the polypeptide sequence of the GLP-1 derivative; 2) Pre-treat the resin and drain it for later use; 3) Take the first amino acid at the C-terminus, add a solvent and a protective agent, mix them, place them in a reactor, and then place the reactor in a shaker to react and obtain polypeptide chain A; 4) Mix methanol, solvent and protective agent to form a blocking solution, use the blocking solution to block polypeptide chain A, wash it and drain it; 5) Add a cleavage reagent to the reactor to remove the protecting group, wash and dry the resin after removing the protecting group, and test it by ninhydrin method. If the resin has color, it means that the condensation is incomplete, and the reaction time is extended. If the resin is colorless, it means that the reaction is complete; 6) After the reaction is complete, the second amino acid at the C-terminus, the solvent and the protective agent are mixed and added to the reactor, and the reactor is then placed in a shaker for reaction. After the reaction is completed, the resin obtained in step 2) is tested by the ninhydrin method. If the resin has color, it means that the condensation is incomplete, and the reaction time is extended. If the resin is colorless, it means that the reaction is complete. After the reaction is complete, a cleavage reagent is added to the reactor to remove the protective group. After the protective group is removed, it is washed and dried. The resin obtained in step 2) is tested by the ninhydrin method. If the resin has color, it means that the deprotection is successful; 7) Repeat steps 5) to 6) to sequentially connect the remaining amino acids of the polypeptide, wherein: Fmoc-Lys(dde)-OH was used for amino acid 20, and Boc-His(trt)-OH was used for N-terminal His]; 8) Add a cleavage reagent to the reactor to remove the protecting group (DDE) of the 20th amino acid and connect the side chain amino acids in sequence according to the method of steps 5) to 6), then use the cleavage reagent to completely remove the polypeptide protecting group and the resin, and then precipitate to obtain a crude polypeptide; 9) Separate the target peptide and impurities by HPLC, and freeze-dry the target peptide to obtain the target product GLP-1 derivative.

[0013] As a preference, Step 2) The resin is a dichloro resin, and the pretreatment is to soak it in a sufficient amount of DCM and drain it, which is repeated at least 3 times.

[0014] As a preference, The cleavage reagents in step 5), step 6) and step 7) are all 20% piperidine solution.

[0015] As a preference, Step 8) the cleavage reagent for removing the protecting group (DDE) of the 20th amino acid is hydrazine hydrate; In step 8), the cleavage reagent for completely removing the polypeptide protecting groups and the resin is a 95% TFA solution.

[0016] The present invention provides a GLP-1 derivative that has been subjected to specific chemical modification or has introduced a disulfide bond. The key point of the present invention lies in the composition and combination of the linker part and the side chain part in the amino acid sequence of the GLP-1 derivative. The present invention optimizes the molecular structure of the GLP-1 derivative in depth, makes key adjustments in the amino acid sequence, and introduces unique side chain modifications to significantly improve the affinity with albumin. This design enhances the stability of the molecule and makes it have stronger resistance to degradation in the body. The function obtained by replacing, deleting or adding one or more amino acids in the polypeptide is similar to the polypeptides that are identical or similar to the existing GLP-1 derivatives, and the polypeptides that have at least 90% homology in the amino acid sequence.

[0017] Through these improvements, the half-life of the drug can be extended, so that it can maintain its effectiveness in the body for a longer period of time. By extending the half-life of the drug, the drug has made a breakthrough in stability and can improve treatment compliance. At the same time, through the optimization of the molecular structure, GLP-1 derivatives have stronger resistance to DPP-4 enzymatic hydrolysis and can maintain activity for a longer period of time. This not only improves the therapeutic effect of the drug, but also reduces the patient's dosage and treatment costs.

[0018] These innovative peptide drugs will provide more efficient and lasting treatments for diabetic patients, reducing the need for frequent dosing. Not only will patients benefit from longer-term blood sugar control, but they will also significantly improve their quality of life and reduce the inconvenience and treatment costs caused by frequent medication. With the introduction of these improved drugs, the treatment of diabetes will enter a new stage, solving the high clearance problem faced by existing peptide drugs and laying a solid foundation for future clinical applications.

[0019] The beneficial effects of the present invention are as follows: The present invention provides a more effective and widely applicable treatment scheme, which improves the efficacy and stability of GLP-1 derivatives by optimizing the molecular structure; The GLP-1 derivative prepared by the present invention can bind to and activate the GLP-1 receptor. This activation value makes the compound have extremely high application potential in the treatment of metabolic diseases such as type 2 diabetes and obesity, and can provide patients with more sustained and safe treatment effects, expanding its application prospects in the medical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the mass spectrum of Example 1; Figure 2 The analytical liquid phase of Example 1; Figure 3 is the mass spectrum of Example 2; Figure 4 is the analytical liquid phase of Example 2; Figure 5 is the mass spectrum of Example 3; Figure 6 The analytical liquid phase of Example 3; DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with specific embodiments and the accompanying drawings. A person of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention.

[0022] Unless otherwise specified, the raw materials used in the examples of the present invention are all commercially available or available to those skilled in the art; unless otherwise specified, the methods used in the examples of the present invention are all methods known to those skilled in the art.

[0023] Example 1: A series of GLP-1 derivatives and a method for preparing the same, the method comprising: 1) Calculate the weight of each raw material according to the polypeptide sequence: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(-OEG-OEG-Glu-glutaric acid-Lys-adipic acid)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly; The raw materials are as follows:

[0024] 2) Place the dichlororesin in a 150 mL reactor, add 50 mL DCM and soak for 2 h, wash the resin with DMF and drain, repeat the operation 4 times to drain the resin; 3) Weigh 0.3 mmol Fmoc-Gly-OH (the first amino acid at the C-terminus), add 80 mL DCM and 4 mL DIEA into the reactor, and then place the reactor in a shaker at 30 °C for 2 h to obtain polypeptide chain A; 4) Methanol, DIEA and DCM were mixed in a volume ratio of 1:1:2 to obtain a solution, and polypeptide chain A was blocked with the solution for 0.5 h, washed with DMF 4 times, and dried; 5) Add 20% piperidine solution (piperidine / DMF=1:4) to the reactor to remove the Fmoc protecting group. After the protection is removed, wash it four times with DMF and drain it. Take a small amount of resin for ninhydrin test. If the resin has color, it means that the condensation is incomplete and the reaction time should be extended. If the resin is colorless, it means that the reaction is complete. 6) Weigh 0.9 mmol Fmoc-Arg (Pbf) -OH (the second amino acid at the C-terminus), 60 mg HOBT, and 0.5 mL DIC into the reactor, place the reactor in a shaker at 30 °C for 1 h, take a small amount of resin for testing, and use the ninhydrin method to test. If the resin has color, it means that the condensation is incomplete, and the reaction time should be extended. If the resin is colorless, it means that the reaction is complete. After the reaction is complete, wash the resin four times with DMF and drain it; 7) Add piperidine solution (piperidine / DMF=1:4) to the reactor, shake on a decolorizing shaker for 20 min to remove the Fmoc protecting group on the resin, wash four times with DMF, drain, and check whether the protecting group is completely removed. Take a small amount of resin and test it with the ninhydrin method. If the resin has color, it means that the deprotection is successful; 8) Follow steps 5) and 6) to sequentially connect the remaining amino acids [use Fmoc-Lys(dde)-OH for the 20th amino acid and Boc-His(trt)-OH for the N-terminal His]; 9) Add hydrazine hydrate to remove DDE, connect the side chain amino acids in sequence according to steps 5) and 6), react with a cleavage reagent 95% TFA solution for 2 h, remove all the peptide protecting groups and resin, and use ice ether to precipitate the crude peptide; 10) Separate the target peptide from impurities by high performance liquid chromatography (HPLC) and send it for freeze drying. After the target peptide is freeze dried into powder, it is subjected to QC quality inspection; The quality inspection conditions are: The column model was C18, the mobile phase was Pump A: 0.1% Trifluoroacetic in 100% water, Pump B was 0.1% Trifluoroacetic in 100% acetonitrile, the UV wavelength was 214 nm or 220 nm, the column temperature was 40°C, and the flow rate was 1% / min.

[0025] Test results such as Figure 1 Mass spectrometry, such as Figure 2 Analyze the liquid phase.

[0026] Example 2: A series of GLP-1 derivatives and a method for preparing the same, the method comprising: 1) The weight of each raw material is calculated according to the polypeptide sequence of His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(-OEG-OEG-Glu-glutaric acid-Lys-glutaric acid)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly; the raw materials are as follows:

[0027] 2) Place the dichlororesin in a 150 mL reactor, add 50 mL DCM and soak for 2 h, wash the resin with DMF and drain, repeat the operation 4 times to drain the resin; 3) Weigh 0.3 mmol Fmoc-Gly-OH (the first amino acid at the C-terminus), add 80 mL DCM and 4 mL DIEA into the reactor, and then place the reactor in a shaker at 30 °C for 2 h to obtain polypeptide chain A; 4) Methanol, DIEA and DCM were mixed in a volume ratio of 1:1:2 to obtain a solution, and polypeptide chain A was blocked with the solution for 0.5 h, washed with DMF 4 times, and dried; 5) Add 20% piperidine solution (piperidine / DMF=1:4) to the reactor to remove the Fmoc protecting group. After the protection is removed, wash it four times with DMF and drain it. Take a small amount of resin for ninhydrin test. If the resin has color, it means that the condensation is incomplete and the reaction time should be extended. If the resin is colorless, it means that the reaction is complete. 6) Weigh 0.9 mmol Fmoc-Arg (Pbf) -OH (the second amino acid at the C-terminus), 60 mg HOBT, and 0.5 mL DIC into the reactor, place the reactor in a shaker at 30 °C for 1 h, take a small amount of resin for testing, and use the ninhydrin method to test. If the resin has color, it means that the condensation is incomplete, and the reaction time should be extended. If the resin is colorless, it means that the reaction is complete. After the reaction is complete, wash the resin four times with DMF and drain it; 7) Add piperidine solution (piperidine / DMF=1:4) to the reactor, shake on a decolorizing shaker for 20 min to remove the Fmoc protecting group on the resin, wash four times with DMF, drain, and check whether the protecting group is completely removed. Take a small amount of resin and test it with the ninhydrin method. If the resin has color, it means that the deprotection is successful; 8) Follow steps 5) and 6) to sequentially connect the remaining amino acids [use Fmoc-Lys(dde)-OH for the 20th amino acid and Boc-His(trt)-OH for the N-terminal His]; 9) Add hydrazine hydrate to remove DDE, connect the side chain amino acids in sequence according to steps 5) and 6), react with a cleavage reagent 95% TFA solution for 2 h, remove all the peptide protecting groups and resin, and use ice ether to precipitate the crude peptide; 10) Separate the target peptide from impurities by high performance liquid chromatography (HPLC) and send it for freeze drying. After the target peptide is freeze dried into powder, it is subjected to QC quality inspection; The quality inspection conditions are: The column model was C18, the mobile phase was Pump A: 0.1% Trifluoroacetic in 100% water, Pump B was 0.1% Trifluoroacetic in 100% acetonitrile, the UV wavelength was 214 nm or 220 nm, the column temperature was 40°C, and the flow rate was 1% / min.

[0028] Test results such as Figure 3 Mass spectrometry, such as Figure 4 Analyze the liquid phase.

[0029] Example 3: A series of GLP-1 derivatives and a method for preparing the same, the method comprising: 1) Based on: The weight of each raw material was calculated using the polypeptide sequence of His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(-OEG-OEG-Glu-glutaric acid-Lys-succinic acid)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly; The raw materials are as follows:

[0030] 2) Place the dichlororesin in a 150 mL reactor, add 50 mL DCM and soak for 2 h, wash the resin with DMF and drain, repeat the operation 4 times to drain the resin; 3) Weigh 0.3 mmol Fmoc-Gly-OH (the first amino acid at the C-terminus), add 80 mL DCM and 4 mL DIEA into the reactor, and then place the reactor in a shaker at 30 °C for 2 h to obtain polypeptide chain A; 4) Methanol, DIEA and DCM were mixed in a volume ratio of 1:1:2 to obtain a solution, and polypeptide chain A was blocked with the solution for 0.5 h, washed with DMF 4 times, and dried; 5) Add 20% piperidine solution (piperidine / DMF=1:4) to the reactor to remove the Fmoc protecting group. After the protection is removed, wash it four times with DMF and drain it. Take a small amount of resin for ninhydrin test. If the resin has color, it means that the condensation is incomplete and the reaction time should be extended. If the resin is colorless, it means that the reaction is complete. 6) Weigh 0.9 mmol Fmoc-Arg (Pbf) -OH (the second amino acid at the C-terminus), 60 mg HOBT, and 0.5 mL DIC into the reactor, place the reactor in a shaker at 30 °C for 1 h, take a small amount of resin for testing, and use the ninhydrin method to test. If the resin has color, it means that the condensation is incomplete, and the reaction time should be extended. If the resin is colorless, it means that the reaction is complete. After the reaction is complete, wash the resin four times with DMF and drain it; 7) Add piperidine solution (piperidine / DMF=1:4) to the reactor, shake on a decolorizing shaker for 20 min to remove the Fmoc protecting group on the resin, wash four times with DMF, drain, and check whether the protecting group is completely removed. Take a small amount of resin and test it with the ninhydrin method. If the resin has color, it means that the deprotection is successful; 8) Follow steps 5) and 6) to sequentially connect the remaining amino acids [use Fmoc-Lys(dde)-OH for the 20th amino acid and Boc-His(trt)-OH for the N-terminal His]; 9) Add hydrazine hydrate to remove DDE, connect the side chain amino acids in sequence according to steps 5) and 6), react with a cleavage reagent 95% TFA solution for 2 h, remove all the peptide protecting groups and resin, and use ice ether to precipitate the crude peptide; 10) Separate the target peptide from impurities by high performance liquid chromatography (HPLC) and send it for freeze drying. After the target peptide is freeze dried into powder, it is subjected to QC quality inspection; The quality inspection conditions are: The column model was C18, the mobile phase was Pump A: 0.1% Trifluoroacetic in 100% water, Pump B was 0.1% Trifluoroacetic in 100% acetonitrile, the UV wavelength was 214 nm or 220 nm, the column temperature was 40°C, and the flow rate was 1% / min.

[0031] Test results such as Figure 5 Mass spectrometry, such as Figure 6 Analyze the liquid phase.

[0032] The lyophilized powders of Examples 1 to 3 were used to perform a GLP-1R target agonist cell activity detection experiment, and the specific method is as follows.

[0033] 1. Preparation of test culture medium and diluent: Take 450 mL of 90% DMEM and 50 mL of 5% FBS and mix them evenly to prepare the plating medium. The shelf life is 6 months when stored at 2-8°C. 2. Preparation of subculture medium: Take DMEM as solvent, add 10% FBS, 300 μg / mL G418, and 50 μg / mL Hygromycin B and mix well; 3. Preparation of test solution: Using the Bright-Lite Luciferase Assay System, add 100 mL of the staining solution to the substrate and mix well to obtain the detection solution. 4. Preparation of sample gradient solution: Weigh a certain amount of sample and control standard and dissolve them in sterile water to prepare a 1 mM stock solution and prepare gradient dilutions; 5. Cell inoculation and drug treatment: The cells were passaged and expanded in advance. When the cell density reached 80%, the cells were washed 1-2 times with PBS, and the cells were digested with 0.25% trypsin. The digestion was terminated when the cells became round under an inverted microscope (OLYMPUS, IX71). The cells with a cell viability greater than 95% were taken for trypan blue staining and prepared into a cell suspension with a density of 6x105 cells / mL. 100 μL of the cell suspension was inoculated in each well of a 96-well plate. The next day, 20 μL of the gradient diluted sample was added to each well, and the plates were transferred to a 37°C cell culture incubator (Thermo, 3111) and incubated for 3 h. 6. Detection fluid treatment: After the incubation, the cell plate was removed and placed at room temperature for 15 min to allow it to equilibrate to room temperature. Then, the detection solution equilibrated to room temperature was added, 80 μL per well, and the culture plate was placed on a microplate constant temperature oscillator (Haimen Qilin Bell Instrument Co., Ltd., MH-2) and shaken at 500 rpm for 3 min. Finally, the data was collected using a desktop microplate reader (Molecular Devices, SpectraMax M5).

[0034] The experimental results are shown in the following table:

[0035] It can be seen from the data in the above table that the GLP-1 derivatives of the present invention exhibit the ability to activate the GLP-1 receptor.

Claims

1. A GLP-1 derivative, characterized in that: The GLP-1 derivative comprises a polypeptide portion, a linker portion and a side chain portion; The amino acid sequence of the polypeptide portion of the GLP-1 derivative is: HXEGTFTSDVSSYLEGQAAKE-FIAWLVRGRG; Wherein X is α-aminoisobutyric acid; The linker part structure of the GLP-1 derivative is oligoethylene glycol-oligoethylene glycol-glutamic acid; The side chain of the GLP-1 derivative is composed of two short-chain fatty acids linked by 2,6-diaminohexanoic acid.

2. A GLP-1 derivative according to claim 1, characterized in that: The structural formula of the GLP-1 derivative is shown below: .

3. A method for preparing the GLP-1 derivative according to claim 1 or 2, characterized in that: The method comprises: 1) Calculate the weight of each raw material based on the polypeptide sequence of the GLP-1 derivative; 2) Pre-treat the resin and drain it for later use; 3) Take the first amino acid at the C-terminus, add a solvent and a protective agent, mix them, place them in a reactor, and then place the reactor in a shaker to react and obtain polypeptide chain A; 4) Mix methanol, solvent and protective agent to form a blocking solution, use the blocking solution to block polypeptide chain A, wash it and drain it; 5) Add a cleavage reagent to the reactor to remove the protecting group, wash and dry the resin after removing the protecting group, and test it by ninhydrin method. If the resin has color, it means that the condensation is incomplete, and the reaction time is extended. If the resin is colorless, it means that the reaction is complete; 6) After the reaction is complete, the second amino acid at the C-terminus, the solvent and the protective agent are mixed and added to the reactor, and the reactor is then placed in a shaker for reaction. After the reaction is completed, the resin obtained in step 2) is tested by the ninhydrin method. If the resin has color, it means that the condensation is incomplete, and the reaction time is extended. If the resin is colorless, it means that the reaction is complete. After the reaction is complete, a cleavage reagent is added to the reactor to remove the protective group. After the protective group is removed, it is washed and dried. The resin obtained in step 2) is tested by the ninhydrin method. If the resin has color, it means that the deprotection is successful; 7) Repeat steps 5) to 6) to sequentially connect the remaining amino acids of the polypeptide, wherein: Fmoc-Lys(dde)-OH was used for amino acid 20, and Boc-His(trt)-OH was used for N-terminal His]; 8) Add a cleavage reagent to the reactor to remove the protecting group (DDE) of the 20th amino acid and connect the side chain amino acids in sequence according to the method of steps 5) to 6), then use the cleavage reagent to completely remove the polypeptide protecting group and the resin, and then precipitate to obtain a crude polypeptide; 9) Separate the target peptide and impurities by HPLC, and freeze-dry the target peptide to obtain the target product GLP-1 derivative.

4. The method for preparing a GLP-1 derivative according to claim 3, characterized in that: Step 2) The resin is a dichloro resin, and the pretreatment is to soak it in a sufficient amount of DCM and drain it, which is repeated at least 3 times.

5. The method for preparing a GLP-1 derivative according to claim 3, characterized in that: The cleavage reagents in step 5), step 6) and step 7) are all 20% piperidine solution.

6. The method for preparing a GLP-1 derivative according to claim 3, characterized in that: Step 8) the cleavage reagent for removing the protecting group (DDE) of the 20th amino acid is hydrazine hydrate; In step 8), the cleavage reagent for completely removing the polypeptide protecting groups and the resin is a 95% TFA solution.

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