Method for preparing semeglutide by natural chemical connection method

Abstract

The invention discloses a method for preparing semeglutide by using a natural chemical ligation method, which comprises the following steps: (1) synthesizing semeglutide in two segments, namely an N-terminal segment with carboxyl-terminal hydrazide and a C-terminal segment with amino-terminal cysteine; (2) connecting the two fragments through a natural chemical connection method under a buffer salt condition; and (3) performing desulfurization treatment to convert Cys into target amino acid, and performing purification and freeze-drying after complete treatment. According to the method, the semeglutide is divided into the C-terminal polypeptide fragment and the N-terminal polypeptide fragment, the C-terminal polypeptide fragment and the N-terminal polypeptide fragment are connected through natural chemical connection reaction after being prepared respectively, then desulfurization treatment is conducted, the high-purity and high-yield semeglutide is finally obtained, the problem of difficult sequences is solved, the synthesis efficiency and purity of the semeglutide are remarkably improved, and the method is suitable for industrial production. The method is suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of polypeptide synthesis, and more particularly to a method for preparing smegglutinin using a natural chemical linking method. Background Technology

[0002] Smegglutide is a long-acting glucagon-like peptide-1 (GLP-1) receptor agonist widely used to treat metabolic diseases. Its core indications include: type 2 diabetes (by stimulating insulin secretion and delaying gastric emptying to lower blood glucose, approved in 2017), reduction of cardiovascular disease risk (expanded for diabetic patients with heart disease in 2020), and chronic kidney disease (CKD) (approved in 2025 based on the FLOW trial, reducing the risk of kidney disease progression and cardiovascular death by 24%). In addition, high-dose formulations (such as Wegovy) are used for obesity, achieving a 20.7% weight loss over 72 weeks.

[0003] Structurally, semaglutide is a GLP-1 (7-37) chain where Aib replaces Ala at position 8, Arg replaces Lys at position 34, and a 1,18-octadecanoic acid fatty chain is attached to Lys at position 26, along with glutamic acid and a short-chain PEG modification. This side-chain modification not only allows it to bind tightly to albumin, masking the DPP-4 enzyme hydrolysis site, but also reduces renal excretion, prolonging its biological half-life and achieving a long-lasting effect. Its sequence is as follows:

[0004] H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-L ys(AEEAc-AEEAc-γ-Glu-1,18-octadecanedioic acid)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH

[0005] Currently, the most effective chemical synthesis method for peptide drugs is solid-phase synthesis using the Fmoc method. This method starts from the C-terminus of the sequence, attaches the first amino acid to the resin, removes the Fmoc protecting group at the N-terminus of the amino acid, and then couples the next amino acid in sequence, removes the N-terminal protection, and so on, until the coupling of the last N-terminal amino acid is completed. Finally, the peptide is cleaved from the resin.

[0006] Chinese patents CN112250755A and CN106478806B employ a method of coupling smegglutinin amino acid sequences one by one. During the gradual reaction of the peptide chain, intermolecular and intramolecular aggregation and folding significantly affect the deprotection and coupling reactions, resulting in a higher proportion of missing and fragmented peptides in the final product, reducing the synthesis yield and increasing the difficulty of purification. Chinese patent CN110372785A, which uses a fragmentation method to prepare smegglutinin, also faces some problems. For example, the prepared protected fragment is too hydrophobic and has poor solubility, making analysis and purification difficult. Furthermore, the condensation reaction of the protected fragment is challenging and prone to racemic reactions, resulting in the presence of racemic impurities in the final product that are difficult to remove.

[0007] Therefore, this invention proposes a method for preparing smegglutinin using natural chemical ligation. First, a short, high-purity fragment is prepared. The subsequent ligation reaction conditions are mild, efficient, and selective. The resulting crude smegglutinin has high purity, a simple impurity profile, and few racemic and deletion peptide impurities, thus significantly reducing the difficulty of subsequent purification and improving the overall yield. Furthermore, the entire process is relatively simple, easy to operate, and improves synthesis efficiency while reducing waste liquid generation, making it suitable for large-scale production. Summary of the Invention

[0008] In view of this, the present invention provides a method for preparing smegglutinin using a natural chemical linking method.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0010] A method for preparing smegglutinin using a natural chemical linking method includes the following steps:

[0011] (1) The smegglutinin sequence was inserted into Ala 24 -Ala 25 The process involved synthesizing N-terminal and C-terminal fragments separately. The N-terminal fragment had the sequence H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-NH-NH2, while the C-terminal fragment had the sequence H-Cys-Lys(AEEAc-AEEAc-γ-Glu-1,18-octadecanoic acid)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

[0012] (2) The carbon terminus of the N-terminal fragment is converted into a mercaptoester and linked to the C-terminal fragment in a buffer salt solution via a natural chemical linking method to obtain the smegglutinin precursor.

[0013] (3) The smegglutinin precursor was desulfurized to convert Cys to Ala. After complete desulfurization, it was purified and lyophilized to obtain smegglutinin.

[0014] In one embodiment of the present invention, in step (1), a C-terminal fragment is prepared using a solid-phase peptide synthesis method, and the peptide sequence of the C-terminal fragment is sequentially coupled onto a solid-phase resin. After drying the peptide resin, it is added to a lysis buffer to lyse the peptide from the solid-phase resin, thereby removing the side chain protecting groups. The peptide is then crystallized and dried to obtain the C-terminal fragment. The lysis buffer can be a TFA / TIS / H2O mixture of 95 / 2.5 / 2.5 (v / v / v).

[0015] Preferably, the solid resin used to synthesize the C-terminal fragment is selected from royal resin and 2-CTC resin.

[0016] Preferably, in step (1), the method for synthesizing the C-terminal fragment is as follows:

[0017] Weigh 2 g of 2-CTC resin with a substitution degree of 0.8 mmol / g and add it to the reactor. Swell twice with DMF for 10 minutes each time. Weigh 1 mmol of Fmoc-Gly-OH and dissolve it in 10 mL of DMF. Add the dissolved reaction solution to the reactor. Add 2 mmol of DIPEA to the resin reaction solution and react at room temperature for 1 hour. After the reaction is complete, add methanol to the reaction solution to block unreacted active sites in the resin and react for 30 minutes. After the reaction is complete, drain the solution and wash the resin with DMF. Soak the resin twice with PIPE / DMF (1 / 4, v / v) solution for 20 minutes each time. Dissolve 2 mmol of Fmoc-Arg(Pbf)-OH and 2 mmol of TBTU in an appropriate amount of DMF, then add 4 mmol of DIPEA and mix well. Add this mixture to the resin and react at room temperature with stirring for 2 hours.

[0018] The coupling was completed sequentially according to the sequence; after the peptide resin was dried, it was added to the lysis buffer to lyse the peptide from the solid resin, while removing the side chain protecting groups. Then, it was crystallized and dried to obtain the C-terminal fragment.

[0019] In one embodiment of the present invention, in step (1), an N-terminal fragment is prepared using a solid-phase peptide synthesis method, and is sequentially coupled onto a 2-CTC resin according to the peptide sequence of the N-terminal fragment. After drying the peptide resin, it is added to a lysis buffer to cleave the peptide from the 2-CTC resin, thereby removing the side chain protecting groups. The peptide is then crystallized and dried to obtain the N-terminal fragment. The lysis buffer can be a TFA / TIS / H2O mixture of 95 / 2.5 / 2.5 (v / v / v).

[0020] Preferably, in step (1), the N-terminal fragment synthesis method is as follows:

[0021] Weigh 2 g of 2-CTC resin with a substitution degree of 0.8 mmol / g and add it to the reactor. Swell twice with DMF for 10 minutes each time. Then cool to 0°C, and slowly add a mixture of 1 mmol DIPEA and 1 mmol hydrazine hydrate to the DMF. React at room temperature for 1 hour. After the reaction is complete, methanol is added to the reaction solution to block unreacted active sites in the resin, and the reaction is carried out for 30 minutes. Finally, the resin is washed with DMF. Dissolve 3 mmol Fmoc-Ala-OH and 3 mmol HATU in an appropriate amount of DMF, then add 6 mmol DIPEA and mix well. Add this mixture to the resin and react at room temperature with stirring for 2 hours.

[0022] The coupling was completed sequentially according to the sequence; the peptide resin was dried and then added to the lysis buffer to lyse the peptide from the solid resin, while the side chain protecting groups were removed and crystallized and dried to obtain the N-terminal fragment.

[0023] Preferably, in step (2), the natural chemical bonding uses a buffer salt solution comprising guanidine hydrochloride, Na₂HPO₄, MPAA, and TCEP, with a pH of 6.5-7.5. In a preferred embodiment, the buffer salt solution comprises guanidine hydrochloride at a concentration of 5-7 M and Na₂HPO₄ at a concentration of 0-200 mM.

[0024] Preferably, in step (2), the ligation via natural chemical linkage includes: first preparing a solution containing 6 M guanidine hydrochloride and 0.2 M Na₂HPO₄ at pH=3, and dissolving the N-terminal fragment in this solution: at -10 ± 5 o Add 0.2 M NaNO2 solution at C. After 30 min, add MPAA solution and slowly adjust the pH to 6.5 with 2 M NaOH solution. Then add the C-terminal fragment and TCEP solution and stir at room temperature for 3 h. After the reaction is complete, adjust the pH to 4.9 and a large amount of white solid precipitates. After filtration, the smegglutinin precursor is obtained.

[0025] Preferably, in step (3), the desulfurization process uses 2-mercaptoethanol or t-BuSH with VA-044 to convert Cys to Ala.

[0026] Preferably, in step (3), the desulfurization treatment includes:

[0027] The precursor of smegglutinin was dissolved in a solution containing 6 M guanidine hydrochloride, 0.1 M Na2HPO4, 20 mM TCEP, and pH=7. Then, t-BuSH and VA-044 solutions were added and the reaction was carried out under nitrogen protection. After 5 h of reaction, the product was purified and lyophilized to obtain smegglutinin.

[0028] The present invention achieves the following technical effects compared to the prior art:

[0029] (1) The method of the present invention prepares smegglutinin through natural chemical linkage, which effectively improves the purity, reduces the difficulty of subsequent purification, and improves the overall efficiency of smegglutinin synthesis.

[0030] (2) The method of the present invention is simple and easy to implement, and has high practical value in the laboratory-scale synthesis and industrial production of smegglutinin. Attached Figure Description

[0031] Figure 1 The reaction equation for the preparation of smegglutinin using natural chemical methods.

[0032] Figure 2 The HPLC chromatogram of smegglutinin prepared by conventional methods.

[0033] Figure 3 This is the HPLC chromatogram of the C-terminal fragment of the present invention;

[0034] Figure 4 This is the HPLC chromatogram of the N-terminal fragment of this invention. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] This invention discloses a method for preparing smegglutinin using a natural chemical linking method, comprising the following steps:

[0037] (1) The synthesis of smegglutinin is divided into the synthesis of the N-terminal fragment and the synthesis of the C-terminal fragment. The sequence of the N-terminal fragment is H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-NH-NH2; the sequence of the C-terminal fragment is H-Cys-Lys(AEEAc-AEEAc-γ-Glu-1,18-octadecanoic acid)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH;

[0038] The method for synthesizing C-end fragments is as follows:

[0039] Weigh 2-CTC resin with a substitution degree of 0.8 mmol / g and add it to the reactor. Swell twice with DMF for 10 minutes each time. Weigh 1 mmol of Fmoc-Gly-OH and dissolve it in 10 mL of DMF. Add the dissolved reaction solution to the reactor. Add 1 mmol of DIPEA to the resin reaction solution and react at room temperature for 1 hour. Continue to add methanol to the reaction solution to block unreacted active sites in the resin and react for 30 minutes. After the reaction is complete, drain the solution and wash the resin with DMF. Soak the resin twice with PIPE / DMF (1 / 4, v / v) solution for 20 minutes each time. Dissolve 2 mmol of Fmoc-Gly-OH and 2 mmol of TBTU in an appropriate amount of DMF, then add 4 mmol of DIPEA and mix well. Add this mixture to the resin and react at room temperature with stirring for 2 hours.

[0040] The peptides were coupled sequentially according to their sequences. After the peptide resin was dried, it was added to the lysis buffer to lyse the peptides from the solid resin, while removing the side chain protecting groups. The C-terminal fragments were then obtained by crystallization and drying.

[0041] The method for synthesizing N-terminal fragments is as follows:

[0042] Weigh 2 g of 2-CTC resin with a substitution degree of 0.8 mmol / g and add it to the reactor. Swell twice with DMF for 10 minutes each time. Then cool to 0°C, and slowly add a mixture of 1 mmol DIPEA and 1 mmol hydrazine hydrate to the DMF. React at room temperature for 1 hour. After the reaction is complete, methanol is added to the reaction solution to block unreacted active sites in the resin, and the reaction is carried out for 30 minutes. Finally, the resin is washed with DMF. Dissolve 3 mmol Fmoc-Ala-OH and 3 mmol HATU in an appropriate amount of DMF, then add 6 mmol DIPEA and mix well. Add this mixture to the resin and react at room temperature with stirring for 2 hours.

[0043] The peptides were coupled sequentially according to their sequences. After drying, the peptide resin was added to the lysis buffer to lyse the peptides from the solid resin. At the same time, the side chain protecting groups were removed, and the peptides were crystallized and dried to obtain the N-terminal fragments.

[0044] (2) First, prepare a solution containing 6 M guanidine hydrochloride and 0.2 M Na₂HPO₄ with pH=3. Dissolve the N-terminal fragment in this solution: at -10 ± 5 o Add 0.2 M NaNO2 solution at C. After 30 min, add MPAA solution to treat the mixture and slowly adjust the pH to 6.5 with 2 M NaOH solution. Then add the C-terminal fragment and TCEP solution and stir at room temperature for 3 h. After the reaction is complete, adjust the pH to 4.9 and a large amount of white solid precipitates. After filtration, the smegglutinin precursor is obtained for subsequent desulfurization treatment.

[0045] The buffer salts used in the natural chemical linkage include guanidine hydrochloride, Na2HPO4, MPAA and TCEP. The pH of the buffer salt solution is 6.5-7.5. For example, the pH of the buffer salt solution can be 6.5, 7.0, 7.5, or in the range of 6.5-7.0 or 7.0-7.5.

[0046] (3) The smegglutinin precursor was desulfurized to convert Cys into the target amino acid. After complete desulfurization, it was purified and lyophilized to obtain smegglutinin.

[0047] The desulfurization process uses 2-mercaptoethanol or t-BuSH with VA-044 to convert Cys to Ala;

[0048] The desulfurization process is as follows: Smegglutide precursor is dissolved in a solution containing 6 M guanidine hydrochloride, 0.1 M Na2HPO4, 20 mM TCEP and pH=7, then t-BuSH and VA-044 solutions are added and the reaction is carried out under nitrogen protection. After 5 h of reaction, it is purified and lyophilized to obtain smegglutide.

[0049] The meanings of the abbreviations used in this invention are listed in Table 1 below.

[0050] Table 1: Abbreviation Name Comparison Table

[0051] abbreviation Chinese name TFA Trifluoroacetic acid TIS Triisopropylsilane DIPEA N,N-Diisopropylethylamine DMF N,N-Dimethylformamide DCC N,N-Dicyclohexylcarbodiimide DIC N,N-Diisopropylcarbodiimide <![CDATA[HOBt·H2O]]> 1-Hydroxybenzotriazole monohydrate TBTU O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate HATU 2-(7-Azobenzotriazole)-N,N,N',N'-Tetramethylurea hexafluorophosphate HPLC High performance liquid chromatography AEEAc 2-[2-(2-aminoethoxy)ethoxy]acetic acid IPA Isopropanol Dde 1-(4,4-Dimethyl-2,6-dioxocyclohexylene)ethyl DCM dichloromethane MPAA 4-Mercaptophenylacetic acid TCEP Tris(2-carboxyethyl)phosphine t-BuSH tert-Butylthiol VA-044 Azobisisobutyrazoline hydrochloride

[0052] The sources of materials and instruments used in the following comparative examples / exemplary cases are as follows:

[0053] Polypeptide solid-phase synthesis resins: amino resins (CTC resin, Wang resin) were purchased from Xi'an Lanxiao, and the protected amino acids used were purchased from Bachem; TFA was purchased from Aladdin; triisopropylsilane was purchased from Sinopharm Group; HATU, TBTU, DIPEA, HOBt·H2O and DIC were all purchased from Suzhou Haofan; DMF and IPA were purchased from Xilong Scientific.

[0054] Analytical high-performance liquid chromatography (HPLC): Thermo U3000, analytical software: Chromeleon, column: Kromasil C8, flow rate: 1.0 mL / min, column temperature: 35 ˚C, detector wavelength: 220 nm, injection volume: 20 μL. Mobile phase A: 0.1 TFA / H₂O, mobile phase B: 0.1 TFA / ACN, fluidity gradient: mobile phase B changed by 15% - 45% within 15 min.

[0055] The conventional method for preparing smegglutinin is as follows:

[0056] (1) Weigh 2 g of solid-phase resin (Fmoc-Gly-Wang Resin) with a bonding amount of 0.5 mmol / g, i.e., a total bonding amount of 1 mmol. Add a 20% (v / v) DMF solution of piperidine to it and stir for 20 minutes to remove the Fmoc group. After washing the solid-phase resin with DMF, add 2 mmol Fmoc-Arg(Pbf)-OH, 2 mmol TBTU and 4 mmol DIPEA to the solid-phase resin and stir for 2 hours at room temperature to couple the amino acid Arg. 36 After the coupling reaction was completed, the solid resin was washed with DMF. The Fmoc removal and amino acid linkage process was repeated according to the peptide sequence of smegglutinin, and the main chain synthesis ended at His. Then, the solid resin was treated twice with 2% hydrazine hydrate / DMF for 10 minutes each time to remove the Dde protecting group at position 26 of Lys. The subsequent coupling conditions for AEEAc and γ-Glu, octadecyl diacid were 2 mmol AEEAc, 2 mmol HOBt·H2O, and 2 mmol DIC, and the reaction was stirred at room temperature for 2 hours.

[0057] (2) After drying the solid resin in step (1), add it to the 5 ± 5 ℃ lysis buffer (a mixture of TFA, triisopropylsilane and water in a volume ratio of 95:2.5:2.5). Then, under nitrogen protection, control the reaction at room temperature for 2 h to cleave the peptide from the solid resin and remove the side chain protecting groups. Then, obtain 2.53 g of crude smegglutinin by crystallization and drying.

[0058] Purification conditions for smegglutinin: Column: Kromasil-C8-10 μm-50 × 250 mm, detection wavelength: 220 nm, primary purification mobile phase: A: 0.05 M sodium acetate buffer pH 7.5, B: 75% ACN / 25% A, secondary purification mobile phase: A: 0.1% TFA / H2O, B: 0.1% TFA / ACN.

[0059] Sample purification: Crude smegglutinin was dissolved in 0.1% TFA / H2O, and after system equilibration, a first purification injection was initiated. After injection, a gradient elution was performed at a flow rate of 60 mL / min, with mobile phase B increasing from 40% to 50% (mobile phase A from 60% to 50%) over 90 min. The qualified purified fractions were collected, combined, and a second purification injection was initiated. After injection, a gradient elution was performed at a flow rate of 60 mL / min, with mobile phase B increasing from 30% to 50% (mobile phase A from 70% to 50%) over 60 min. The qualified purified fractions were collected, combined, and lyophilized to obtain the final product. The total yield of smegglutinin was 15.8%, and the purity was 99.30%. The HPLC chromatogram of smegglutinin prepared using conventional solid-phase peptide synthesis methods is shown below. Figure 2 As shown.

[0060] Examples of the preparation of smegglutinin using the natural chemical linking method of the present invention are as follows:

[0061] Example 1:

[0062] C-terminal fragment synthesis: 20 g of 2-CTC resin with a substitution degree of 0.8 mmol / g was weighed and added to the reactor, and swollen twice with DMF for 10 minutes each time. Fmoc-Gly-OH (2.97 g, 10 mmol) was weighed and dissolved in DMF, and the dissolved reaction solution was added to the reactor. DIPEA (3.49 mL, 20 mmol) was added to the resin reaction solution, and the reaction was carried out at 25°C for 1 hour. After the reaction was complete, 10 mL of methanol was added to the reaction solution to block unreacted active sites in the resin, and the reaction was carried out for 30 minutes. After the reaction was complete, the solution was drained, and the resin was washed with DMF. The resin was soaked twice with a PIPE / DMF (1 / 4, v / v) solution for 20 minutes each time. Dissolve approximately two equivalents of Fmoc-Arg(Pbf)-OH (12.98 g, 20 mmol) and two equivalents of TBTU (6.10 g, 20 mmol) in an appropriate amount of DMF. Then add approximately four equivalents of DIPEA and mix thoroughly. Add the mixture to the resin to carry out the coupling reaction. After the reaction has proceeded for at least two hours, drain the solution and wash the resin with DMF.

[0063] Following the peptide sequence, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-phe-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Lys(Dde)-OH, and Boc-Cys(Trt)-OH were coupled sequentially. After the reaction was completed, the Dde protecting group was removed using 2% N2H4·H2O / DMF, and then Fmoc-AEEAc-OH, Fmoc-AEEAc-OH, Fmoc-Glu-OtBu, and Octadecanedioicacid mono-tert-butyl ester were coupled.

[0064] The lysis reaction was carried out using 6-7 mL of lysis reagent (TFA / TIS / H2O = 95 / 2.5 / 2.5, v / v / v) per gram of peptide derivative resin. The peptide derivative resin was completely added to the lysis reagent, and the reaction was carried out under nitrogen bubbling and stirring, with the temperature controlled at 10 ± 5 °C. o After approximately 30 minutes of reaction, the reaction solution was heated to room temperature and the reaction continued for another 3.0 ± 0.5 h. After the reaction was complete, solid-liquid separation was performed, and the liquid was transferred to cold MTBE to precipitate. The precipitate was repeatedly centrifuged, washed with MTBE, and then vacuum dried at room temperature to obtain 21.76 g of the C-terminal fragment peptide with a purity of 92.34%.

[0065] Example 2:

[0066] Synthesis of the N-terminal fragment His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-NH-NH2: 20 g of 2-CTC resin with a substitution degree of 0.8 mmol / g was weighed and added to a reactor, and swollen twice with DMF for 10 minutes each time. The mixture was then cooled to 0 °C, and a mixture of DIPEA (1.74 mL, 10 mmol) and hydrazine hydrate (0.485 mL, 10 mmol) was slowly added to the DMF, initiating the reaction at room temperature. After 2 h of reaction, methanol was added and stirred for 15 min. Finally, the resin was washed with DMF. Dissolve approximately 4 equivalents of Fmoc-Ala-OH·H2O (13.18 g, 40 mmol) and 4 equivalents of HATU (15.21 g, 40 mmol) in an appropriate amount of DMF. Then add approximately 8 equivalents of DIPEA and mix thoroughly. Add the mixture to the resin to carry out the coupling reaction. After the reaction has proceeded for at least two hours, drain the solution and wash the resin with DMF.

[0067] Following the polypeptide sequence, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH·H2O, and Boc-Aib-His(Trt)-OH were sequentially coupled. After the reaction was completed, the resin was washed three times with DCM, with 6-7 ppm of DCM per gram of polypeptide derivative resin. The lysis reaction was carried out using mL of lysis reagent (TFA / TIS / H2O = 95 / 2.5 / 2.5, v / v / v). The peptide derivative resin was completely added to the lysis reagent, and the reaction was carried out under nitrogen bubbling and stirring, with the temperature controlled at 10 ± 5 °C. o C. After approximately 30 minutes of reaction, the reaction solution was heated to room temperature and the reaction continued for another 3.0 ± 0.5 h. After the reaction was complete, solid-liquid separation was performed, and the liquid was transferred to cold MTBE to precipitate. The precipitate was repeatedly centrifuged and washed with MTBE, then vacuum dried at room temperature to obtain 22.93 g of N-terminal fragment peptide with a purity of 90.04%.

[0068] Example 3:

[0069] First, prepare a buffer solution (6 M guanidine hydrochloride, 0.2 M Na2HPO4, pH=3). Dissolve 5.0 g of the N-terminal fragment in 500 mL of the above buffer solution: at -10 ± 5... o Add 42 mL of 0.2 M NaNO2 solution at C. After 30 min, add 42 mL of MPAA solution to treat the mixture, and slowly adjust the pH to 6.5 with 2 M NaOH solution. Then add 5.2 g of the C-terminal fragment and TCEP solution, and stir at room temperature for 3 h. After the reaction is complete, adjust the pH to 4.9, and a large amount of white solid precipitates. After filtration, the smegglutinin precursor is obtained and directly subjected to desulfurization treatment.

[0070] Example 4:

[0071] Desulfurization treatment: The smegglutinin precursor was dissolved in a buffer solution (500 mL) containing 6 M guanidine hydrochloride, 0.1 M Na2HPO4, 20 mM TCEP, and pH=7. Then, 30 mL of t-BuSH and 300 mL of 0.1 M VA-044 solution were added, and the reaction was carried out under nitrogen protection. After 5 h of reaction, purification was performed.

[0072] Purification conditions for smegglutinin: Column: Kromasil-C8-10 μm-50×250 mm, detection wavelength: 220 mm, purification mobile phase: A: 0.1% TFA / H2O, B: 0.1% TFA / ACN.

[0073] Sample purification: After equilibration of the system, injection was initiated. Following injection, a gradient elution was performed at a flow rate of 60 mL / min, with mobile phase B increasing from 30% to 50% (mobile phase A from 70% to 50%) over 60 min. The purified fractions were collected, combined, and lyophilized to obtain the final product: smegglutinin, with a total yield of 55.3% and a purity of 99.87%.

[0074] Compared with traditional methods for preparing semaglutide, the method of this invention uses a natural chemical linking method to prepare semaglutide, which is more efficient and yields better purity than traditional methods. At the same time, the purification steps are reduced and the operation is simpler, thus improving the production efficiency of semaglutide.

[0075] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing smegglutinin using a natural chemical linking method, characterized in that, Includes the following steps: (1) The smegglutinin sequence was inserted into Ala 24 -Ala 25 The process involves synthesizing N-terminal and C-terminal fragments separately. The N-terminal fragment has the sequence H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-NH-NH2, and the C-terminal fragment has the sequence H-Cys-Lys(AEEAc-AEEAc-γ-Glu-1,18-octadecanoic acid)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH. (2) The carbon terminus of the N-terminal fragment is converted into a mercaptoester and linked to the C-terminal fragment in a buffer salt solution via a natural chemical linking method to obtain the smegglutinin precursor; (3) The smegglutinin precursor is desulfurized to convert Cys to Ala. After complete desulfurization, it is purified and lyophilized to obtain smegglutinin.

2. The method according to claim 1, characterized in that, In step (1), the C-terminal fragment is prepared using a solid-phase peptide synthesis method and coupled sequentially on a solid-phase resin according to the peptide sequence of the C-terminal fragment. The peptide resin is dried and then added to the lysis buffer to lyse the peptide from the solid resin, while removing the side chain protecting groups. The peptide is then crystallized and dried to obtain the C-terminal fragment.

3. The method according to claim 2, characterized in that, The solid resin is selected from royal resin and 2-CTC resin.

4. The method according to claim 2, characterized in that, The pyrolysis solution is a mixture of trifluoroacetic acid, triisopropylsilane, and water in a ratio of 95:2.5:2.

5. 0.5-2.0% (mass / volume) of H-Met-OH or indole is added to the pyrolysis solution.

5. The method according to claim 1, characterized in that, In step (1), the N-terminal fragment is prepared by solid-phase peptide synthesis and is sequentially coupled on 2-CTC resin according to the peptide sequence of the N-terminal fragment. The peptide resin is dried and then added to the lysis buffer to cleave the peptide from the 2-CTC resin, while removing the side chain protecting groups. The peptide is then crystallized and dried to obtain the N-terminal fragment.

6. The method according to claim 5, characterized in that, The pyrolysis solution is a mixture of trifluoroacetic acid, triisopropylsilane, and water in a ratio of 95:2.5:2.

5. 0.5-2.0% (mass / volume) of H-Met-OH or indole is added to the pyrolysis solution.

7. The method according to claim 1, characterized in that, In step (2), the buffer salt contains guanidine hydrochloride, Na2HPO4, MPAA and TCEP, and the pH of the buffer salt solution is 6.5-7.

5.

8. The method according to claim 7, characterized in that, The buffer salt solution contains guanidine hydrochloride at a concentration of 5-7 M and Na2HPO4 at a concentration of 0-200 mM.

9. The method according to claim 1, characterized in that, In step (2), the ligation via natural chemical linkage includes: first, preparing a solution containing 6 M guanidine hydrochloride and 0.2 M Na₂HPO₄ with pH=3, and dissolving the N-terminal fragment in this solution: at -10 ± 5 o Add 0.2 M NaNO2 solution at C; after 30 min, add MPAA solution and slowly adjust the pH to 6.5 with 2 M NaOH solution; then add the C-terminal fragment and TCEP solution, and stir at room temperature for 3 h; after the reaction is complete, adjust the pH to 4.9, and a white solid precipitates. After filtration, the smegglutinin precursor is obtained.

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