Solid-phase synthesis method of semeglutide with high coupling efficiency

Abstract

The invention discloses a solid-phase synthesis method of semeglutide with high coupling efficiency, and belongs to the technical field of medicine synthesis. According to the method, Sieber Linker-AM resin is selected and combined with DMF swelling to reduce peptide chain aggregation, and Fmoc-N (HMB)-Gly-OH is adopted as the first amino acid of a carbon terminal to avoid the problem of instability of the carbon terminal. Aiming at high-steric-hindrance amino acids Ile23, Leu26 and Val27 and a special amino acid Aib, a high-steric-hindrance adaptive activation system is respectively introduced, and a low-temperature and double-activator condensation strategy is adopted for the Aib, so that the condensation efficiency is effectively improved. In addition, a Lys20 solid-phase acylation and fragment recombination auxiliary synthesis strategy is adopted, and the long-chain synthesis pressure is shared. According to the method, the condensation efficiency, the crude product purity and the recovery rate of the semeglutide are remarkably improved, the synthesis period is shortened, the cost is reduced, and an efficient, green and stable technical scheme is provided for industrial production of the high-quality semeglutide.

Description

Technical Field

[0001] This invention relates to the field of drug synthesis technology, specifically to a solid-phase synthesis method for semaglutide with high coupling efficiency. Background Technology

[0002] Smegglutinin is a GLP-1 receptor agonist prepared via solid-phase peptide synthesis (SPPS) or recombinant expression technology, and is widely used in the treatment of type 2 diabetes and obesity. Its core is a linear polypeptide chain containing 31 amino acids, with a high concentration of lysine (Lys...). 20 The structure is complex due to fatty acid side chain modification at the ) site.

[0003] Currently, the preparation of smegraglutide mainly relies on solid-phase peptide synthesis strategies. However, traditional solid-phase synthesis faces many challenges when dealing with complex peptide chains like smegraglutide. Firstly, the peptide chain contains isoleucine (Ileucine)... 23 ), Leucine (Leu 26 ) and valine (Val 27 These are all sterically hindered amino acids, with large steric hindrance leading to low coupling efficiency between the active amino acid ester and the free amino group on the resin. This easily results in incomplete condensation products, i.e., deleted sequence impurities, severely affecting the purity of the crude peptide and the final yield. Secondly, 2-aminoisobutyric acid (Aib), as an α-methyl amino acid, has a unique spatial structure that makes its condensation equally difficult and prone to polymerization during activation, further reducing condensation efficiency and product quality. Furthermore, the condensation of the first amino acid at the C-terminus, glycine (Gly), typically suffers from poor stability and low activation efficiency, easily triggering side reactions. As the peptide chain elongates, especially with sequences containing hydrophobic amino acids, the peptide chain easily aggregates on the solid support surface or in solution, forming secondary structures that make the active site difficult to access, further exacerbating the condensation difficulty. Finally, smegglutinin in Lys... 20 The position requires modification of fatty acid side chains. If liquid-phase acylation is performed in the later stage of long-chain synthesis or after cleavage, hydrolysis byproducts are easily generated and the reaction efficiency is difficult to control, which increases the difficulty and cost of purification.

[0004] Existing patent CN120173087A discloses a solid-phase synthesis method for smegglutinin, the core of which is to break down the complete peptide chain into shorter peptide fragments that are easier to synthesize. However, it fails to effectively solve the problems of instability and poor sequence fit of the first amino acid glycine at the C-terminus, as well as the difficulty of condensing sterically hindered amino acids. Its overall yield and process stability are difficult to meet the requirements of high-standard industrial production.

[0005] In summary, the current solid-phase synthesis process of smegglutide still has many shortcomings, and there is an urgent need to develop a new method that can effectively improve its condensation efficiency, crude product purity and yield, shorten the synthesis cycle and reduce costs, so as to meet the high standards required for industrial drug production. Summary of the Invention

[0006] The purpose of this invention is to provide a high-coupling-efficiency solid-phase synthesis method for semaglutide, aiming to overcome the instability of the first C-terminal amino acid glycine, the difficulty in condensing highly hindered amino acids and special amino acids such as Aib, and the challenges faced by existing solid-phase synthesis methods for semaglutide, such as the instability of glycine, a high-steric amino acid, and the difficulty in condensing Lys. 20 This invention addresses issues such as the complexity of side-chain acylation. Through a series of innovative technological inventions, it significantly improves the condensation efficiency, crude product purity, and yield of smegglutinin, shortens the synthesis cycle, reduces production costs, and enhances process robustness, providing a highly efficient, stable, economical, and environmentally friendly new technological solution for the industrial production of smegglutinin.

[0007] The specific technical solution is as follows: A solid-phase synthesis method for smegglutinin with high coupling efficiency includes fragment 1-resin synthesis, fragment 2 synthesis, fragment 3 synthesis, and fragment coupling and recombination. The method is characterized in that fragment 1-resin synthesis includes: the first amino acid condensation using Fmoc-N(HMB)-Gly-OH; and the high-steric-hindrance amino acid condensation using a PyBOP / DIPEA system. Fragment 2 synthesis includes: Lys... 20 Solid-phase acylation modification of the side chain; the synthesis of fragment 3 includes: Aib amino acid condensation using a dual activator strategy combined with low-temperature condensation and ultrasonic-assisted treatment; fragment coupling and recombination includes: coupling fragment 1-resin with fragment 2, then coupling with fragment 3, and finally global deprotection and purification to obtain smegglutinin.

[0008] Furthermore, the resin fragment 1 is: Fmoc-Phe 22 -Ile 23 -Ala 24 -Trp 25 (Boc)-Leu 26 -Val 27 -Arg 28 (Pbf)-Gly 29 -Arg 30 (Pbf)-Gly 31 - Resin; The second segment is: Fmoc-Gln 17 (Trt)-Ala 18 -Ala 19 -Lys 20(acyl)-Glu 21 (OtBu)-OH; The third segment is: Fmoc-His 1 (Trt)-Aib-Glu 3 (OtBu)-Gly 4 -Thr 5 (tBu)-Phe 6 -Thr 7 (tBu)-Ser 8 (tBu)-Asp 9 (OtBu)-Val 10 -Ser 11 (tBu)-Ser 12 (tBu)-Tyr 13 (tBu)-Leu 14 -Glu 15 (OtBu)-Gly 16 -OH.

[0009] Furthermore, the solid-phase synthesis method for smegglutinin with high coupling efficiency includes the following steps: S1: Sieber Linker-AM resin with a substitution degree of 0.43 mmol / g was swollen in DMF at room temperature for 30 minutes and washed three times with DMF, 15 mL / g of resin each time; 4.0 mol equivalents of Fmoc-N(HMB)-Gly-OH were dissolved in DMF to prepare a 0.2 mol / L solution, and 6.0 mol equivalents of DIC and 6.0 mol equivalents of Oxyma were added, and the solution was activated at 5 °C for 15 minutes to generate activated amino acids; the activated amino acids were added to the swollen Sieber Linker-AM resin and condensed at 30 °C for 2.5 hours to complete the loading of the first amino acid; the remaining amino acids were then condensed sequentially until Fmoc-Phe 22 After condensation, the peptide chain remains on the resin without cleavage, washing, and drying to obtain fragment 1-resin. S2: CTC resin with a substitution degree of 0.43 mmol / g was swollen at room temperature and washed three times with DMF, 15 mL / g of resin each time. 4.0 mol equivalents of Fmoc-Glu(OtBu)-OH and 4.0 mol equivalents of DIPEA were mixed in N,N-dimethylformamide, and the swollen CTC resin was added. Condensation was carried out at room temperature for 3 hours to complete the loading. Subsequently, Fmoc-Lys(Alloc)-OH was activated in the DIC / Oxyma system at 5°C for 15 minutes, and then added to the CTC resin. The condensation reaction was carried out at 30°C for 2.5 hours. The remaining amino acids were then condensed sequentially until Fmoc-Gln...17 (Trt); then Lys 20 Solid-phase acylation modification of side chains: The modified resin was reacted with lysis buffer in an ice bath for 3 hours. The filtrate was collected and precipitated with MTBE at 0℃. After centrifugation, washing, drying and purification were performed to obtain fragment 2. S3: CTC resin with a substitution degree of 0.25 mmol / g was swollen at room temperature and washed three times with DMF, 15 mL / g of resin each time; the first amino acid, Fmoc-Gly-OH, was activated in a DIC / Oxyma system at 5 °C for 15 minutes, followed by condensation with CTC resin for 2.5 hours; subsequently, the remaining amino acids were condensed sequentially until Fmoc-His... 1 (Trt); After condensation, the peptide was gently lysed with lysis buffer for 3 hours to remove the fully protected peptide from the resin. The filtrate was collected, precipitated with MTBE at 0°C, washed by centrifugation, dried, and purified to obtain fragment 3. S4: First, the resin of fragment 1 is coupled with the resin of fragment 2 to obtain the intermediate resin. The intermediate resin is condensed with fragment 3 to obtain the full-length protected smegglutinin resin. It is placed in the lysis buffer and subjected to global deprotection in an ice bath for 3 hours. The solid peptide is precipitated with MTBE at 0°C, washed by centrifugation, dried, purified, and finally lyophilized to obtain the smegglutinin product.

[0010] Furthermore, the remaining amino acids in step S1 include conventional amino acids and sterically hindered amino acids; the sterically hindered amino acids include: Ile 23 Leu 26 and Val 27 The activation process uses a PyBOP / DIPEA system. The specific steps are as follows: 4.0 molar equivalents of Fmoc-sterically hindered amino acids are dissolved in DMF, 6.0 molar equivalents of PyBOP are added, and then 6.0 molar equivalents of DIPEA are added. The mixture is then activated at 5°C for 15 minutes.

[0011] Furthermore, in step S2, Lys 20 Solid-phase acylation modification of side chains includes: on the solid phase, firstly, cytosyl... 20 Selective de-Alloc protection was performed, followed by acylation with fatty acid N-succinimide ester; the lysis buffer was TFA / DCM / TIS, with a volume ratio of TFA:DCM:TIS=0.5:94.5:5.

[0012] Furthermore, the method for acylation of the fatty acid N-succinimide ester is as follows: 3.5 molar equivalents of fatty acid N-succinimide ester and 1.0 molar equivalent of DIPEA are dissolved in a DCM / DMF (1:1, v / v) solution and stirred at room temperature for 2 hours; the fatty acid N-succinimide ester is C18-diacid-γ-Glu-(AEEA)2-NHS.

[0013] Furthermore, the remaining amino acids in step S3 include conventional amino acids and Aib. The condensation of Aib employs a dual-activator strategy combined with low-temperature condensation and ultrasonic-assisted treatment. The specific steps include: dissolving 4.0 mol equivalents of Fmoc-Aib-OH in DMF to prepare a 0.2 mol / L solution; adding 4.8 mol equivalents of DIC and 4.8 mol equivalents of Oxyma for pre-activation for 5 minutes; then adding 6.0 mol equivalents of PyBOP and 6.0 mol equivalents of DIPEA for activation for 10 minutes; and adding 0.5 mol equivalents of TIS to suppress side reactions. After the reaction proceeds for 1 hour, ultrasonic-assisted treatment is performed for 2 minutes to break up aggregation, and then the reaction continues for 2 hours. The lysis buffer is TFA / DCM / TIS, with a volume ratio of TFA:DCM:TIS = 1:94:5.

[0014] Further, the specific steps for the condensation of fragment 1-resin and fragment 2 in step S4 include: taking fragment 1-resin prepared in step S1, removing the nitrogen-terminated Fmoc protecting group, and thoroughly washing to obtain Fmoc-free fragment 1-resin; dissolving 3.0 molar equivalents of fragment 2 prepared in step S2 in DMF:DMSO=3:2 (v / v), adding 3.0 molar equivalents of HATU and 6.0 molar equivalents of DIPEA for pre-activation, and then adding it to the Fmoc-free fragment 1-resin, and reacting at room temperature for 12 hours.

[0015] Furthermore, the specific steps for coupling the intermediate resin and fragment 3 in step S4 include: first, removing the Fmoc protecting group from the nitrogen end of the intermediate resin, washing it to obtain the Fmoc-free intermediate resin; dissolving 3.0 molar equivalents of fragment 3 prepared in step S3 in DMF:DMSO=3:2 (v / v), adding 3.0 molar equivalents of HATU and 6.0 molar equivalents of DIPEA for pre-activation, and then adding it to the Fmoc-free intermediate resin, reacting at room temperature for 24 hours.

[0016] Furthermore, in step S4, the pyrolysis solution is TFA / H2O / TIS, with a volume ratio of TFA / H2O / TIS = 95:2.5:2.5.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) Significantly improved condensation efficiency: By using Sieber Linker-AM resin combined with DMF swelling, peptide chain aggregation can be effectively reduced, and the coupling difficulty of sterically hindered amino acids can be lowered. Furthermore, the optimized activation condensation strategy for sterically hindered amino acids can further reduce the generation of impurities and improve condensation efficiency. At the same time, the low-temperature, dual-activator coupling strategy and ultrasonic assistance of Aib effectively inhibit the polymerization tendency of Aib, and its coupling yield is significantly improved. These improvements work synergistically to ensure the thoroughness of condensation during peptide chain elongation and greatly reduce the generation of deletion sequence impurities.

[0018] (2) Significantly improved purity and stability of crude product: The first amino acid at the C-terminus, glycine, was treated with the Fmoc-N(HMB)-Gly-OH specific reagent, effectively avoiding C-terminal instability and side reactions. Its hydrolysis products were easily washed away, significantly improving the purity of the C-terminal crude peptide. Furthermore, Lys 20 Solid-phase acylation avoids the hydrolysis byproducts that may result from liquid-phase acylation. The fragment recombination-assisted synthesis strategy distributes the pressure of long-chain synthesis to short peptide fragments, effectively solving the problem of long-chain peptide aggregation and significantly improving the overall synthesis success rate, recovery rate, and purity of the final product.

[0019] (3) Cost optimization: The fragment recombination strategy effectively shortens the overall synthesis cycle and significantly improves production efficiency. While ensuring condensation efficiency, it effectively avoids the waste of excessive reagents, thereby reducing raw material costs and making it suitable for large-scale industrial production. Attached Figure Description

[0020] Figure 1 This is a simplified flowchart illustrating a solid-phase synthesis method for smegglutinin with high coupling efficiency according to the present invention.

[0021] Figure 2 The purity and yield of the crude smegglutinin prepared for the examples and comparative examples. Detailed Implementation

[0022] The following embodiments further explain and illustrate the technical solutions of the present invention. It should be specifically noted that each specific embodiment is a concretization and explanation of the technical solution and should not be considered as a limitation on the scope of protection of the present invention. Those skilled in the art still have the right to modify the technical solutions of these embodiments and make equivalent substitutions for some or all of the technical features, and these modifications or substitutions do not change the essence of the corresponding technical solutions, nor do they cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions described in the present invention.

[0023] This invention segments the smegglutinin sequence into three short peptide fragments, including fragment 1-resin, containing Lys 20Fragments 2 and 3 at the site were synthesized separately on a solid phase, and then the fragments were recombined to synthesize the complete smegru peptide chain. (See attached image.) Figure 1 The diagram shown is a simplified flowchart of the solid-phase synthesis method for smegglutinin according to the present invention. The target product smegglutinin is synthesized through the following steps.

[0024] HO-Gly 31 -Arg 30 -Gly 29 -Arg 28 -Val 27 -Leu 26 -Trp 25 -Ala 24 -Ile 23 -Phe 22 -Lys 20 (acyl)-Glu 21 -Ala 19 -Ala 18 -Gln 17 -Gly 16 -Glu 15 -Leu 14 -Tyr 13 -Ser 12 -Ser 11 -Val 10 -Asp 9 -Ser 8 -Thr 7 -Phe 6 -Thr 5 -Gly 4 -Glu 3 -Aib-His 1 .

[0025] Among them, Ile 23 Leu 26 and Val 27 It belongs to a highly sterically hindered amino acid and is located in fragment 1; Lys 20 The amino acid with side chain modification is located in fragment 2, and its modifying group acyl is C18-diacid-γ-Glu-(AEEA)2; Aib is an amino acid unique to smegglutinin and is located in fragment 3; the remaining amino acids are conventional amino acids.

[0026] A universal solid-phase synthesis cycle for amino acid condensation: Resin swelling and washing: The resin was fully swollen with N,N-dimethylformamide (DMF) at room temperature for 30 min, and then washed repeatedly with DMF 3 times, each time at a volume of 15 mL / g of resin, to remove low molecular weight impurities, optimize the solvation environment of the resin-peptide chain, minimize the aggregation of peptide chains during the elongation process, and thus improve the condensation efficiency of subsequent amino acids and the stability of peptide chain linkage.

[0027] Conventional amino acid activation: Activation was performed using an N,N'-diisopropylcarbodiimide (DIC) / ethyl 2-oxime cyanoacetate (Oxyma) system. Feed ratio: Fmoc-amino acid:DIC:Oxyma = 4.0 eq:4.8 eq:4.8 eq. Solvent: DMF. Activation temperature: 5℃, activation time: 10 minutes.

[0028] Amino acid condensation reaction: The activated amino acid intermediate was added to the resin, bubbled with nitrogen, and the temperature was increased to 30°C for 2.5 hours. The reaction was complete when a negative result was detected with ninhydrin. The resin was washed 6 times with DMF for 5 minutes each time.

[0029] Fmoc deprotection: Add 10 mL / g of deprotection solution (20% piperidine / DMF, v / v) to the resin, react for 5 minutes, drain, and then add an equal volume of deprotection solution again, reacting for 15 minutes. The free amino group is positive when tested with ninhydrin. After the reaction is complete, wash the resin 6 times with DMF, 5 minutes each time.

[0030] 1. Fragment 1 - Preparation of Resin Fragment sequence: Fmoc-Phe 22 -Ile 23 -Ala 24 -Trp 25 (Boc)-Leu 26 -Val 27 -Arg 28 (Pbf)-Gly 29 -Arg 30 (Pbf)-Gly 31 - Resin.

[0031] Resin: A Sieber Linker-AM resin with a degree of substitution of 0.43 mmol / g was selected as the solid-phase support. The Sieber Linker structure can be cleaved under mild acidic conditions to retain other protecting groups.

[0032] Synthesis process: (1) Loading of the first amino acid: 4.0 eq Fmoc-glycine-N-hydroxymethylbenzotriazole ester (Fmoc-N(HMB)-Gly-OH) was dissolved in DMF and added to a 6.0 eq DIC / 6.0 eq Oxyma system for activation condensation. The activation was carried out at 5°C for 15 minutes. Then the activated amino acid solution was added to the swollen resin and reacted at 30°C for 2.5 hours to complete the loading of the first amino acid.

[0033] (2) Peptide chain elongation: The remaining amino acids are condensed onto the peptide chain using the universal solid-phase synthesis cycle until Fmoc-Phe 22 The sterically hindered amino acids were activated using a 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) / N,N-diisopropylethylamine (DIPEA) system. The feed ratio was: sterically hindered amino acid:PyBOP:DIPEA = 4.0 eq:6.0 eq:6.0 eq; activation temperature: 5℃; activation time: 15 minutes; continuous, slow, and uniform stirring. After condensation, the peptide chains remained on the resin without cleavage. They were washed with DMF, dichloromethane (DCM), and methanol, and dried to obtain fragment 1-resin.

[0034] The Fmoc-N(HMB)-Gly-OH exhibits higher activation efficiency and stability, with fewer side reactions, effectively avoiding the carbon-terminal instability and poor sequence fit issues that may exist with conventional Gly-OH. Its hydrolysis product, HMB-OH, is highly polar and easily removed by DMF washing, reducing interference with subsequent purification processes and contributing to improved crude product purity. The DIC / Oxyma system is environmentally friendly and produces fewer byproducts, which helps improve reaction yield and reduce byproduct generation.

[0035] The PyBOP / DIPEA system exhibits stronger condensation activity for sterically hindered amino acids, effectively overcoming steric hindrance, inhibiting peptide chain aggregation, and improving peptide chain solubility, thereby significantly enhancing the condensation completion rate and final crude product purity of sterically hindered amino acids.

[0036] 2. Synthesis of Fragment 2 and Lys 20 Side-chain solid-phase acylation modification Fragment sequence: Fmoc-Gln 17 (Trt)-Ala 18 -Ala 19 -Lys 20 (acyl)-Glu 21 (OtBu)-OH; Resin: CTC resin with a degree of selective substitution of 0.43 mmol / g was used. This resin allows peptide cleavage under extremely weak acidic conditions without removing conventional side-chain protecting groups.

[0037] Synthesis process: (1) Loading of the first amino acid: 4.0 eq of Fmoc-Glu(OtBu)-OH and 4.0 eq of DIPEA were dissolved in DMF, and the swollen CTC resin was added. The reaction was carried out at room temperature for 3 hours to complete the loading of the first amino acid and obtain Fmoc-Glu(OtBu)-CTC resin.

[0038] (2) Peptide chain elongation: The amino acids are sequentially condensed onto the peptide chain using the universal solid-phase synthesis cycle until Fmoc-Gln 17 (Trt). Among them, Lys 20 The condensation was performed using Fmoc-Lys(Alloc)-OH to obtain crude peptide resin.

[0039] (3) Selective removal of Alloc groups: The crude peptide resin was sequentially placed in a deprotection system of 0.1 eq tetrakis(triphenylphosphine)palladium (Pd(PPh3)4) and 10 eq phenylsilane (PhSiH3) in DCM for 5 minutes each time. After each treatment, it was immediately washed thoroughly with DCM and a washing solution containing 0.5% DIPEA (v / v) in a DCM:DMF ratio of 1:1 (v / v) to remove metal residues. The above treatment was repeated until the Alloc groups were completely removed.

[0040] (4) Acylation modification: 3.5 eq of C18-diacid-γ-Glu-(AEEA)2-NHS and 1.0 eq of DIPEA were dissolved in DCM:DMF = 1:1 (v / v), added to the resin, and stirred at room temperature for 2 hours. After the reaction, the resin was washed 10 times with DMF and then 5 times with DCM to obtain fragment 2-CTC resin. The NHS ester in C18-diacid-γ-Glu-(AEEA)2-NHS is an N-hydroxysuccinimide ester, which is an active ester that can directly react with exposed Lys 20 The side chain ε-NH2 reacts to form a stable amide bond. This solid-phase acylation strategy effectively avoids the hydrolysis byproduct problem that may occur during liquid-phase acylation, improving modification efficiency and product purity. The DIPEA is used to neutralize the acid released during the reaction.

[0041] (5) Lysis: Under ice bath conditions, 15 mL / g of pre-cooled lysis buffer was added to fragment 2-CTC resin, and the reaction was carried out at room temperature for 3 hours. The lysis buffer was filtered and the filtrate was collected. The crude peptide was precipitated with methyl tert-butyl ether (MTBE) at 0°C, centrifuged, washed with MTBE, and dried to obtain fragment 2 crude peptide. The lysis buffer was prepared in a volume ratio of trifluoroacetic acid (TFA):DCM:triisopropylsilane (TIS) = 0.5:94.5:5.

[0042] (6) Purification: Fragment 2 was purified by preparative reversed-phase high-performance liquid chromatography, and the target product was collected to obtain high-purity fragment 2.

[0043] 3. Synthesis of Fragment 3 Fragment sequence: Fmoc-His 1 (Trt)-Aib-Glu 3 (OtBu)-Gly 4 -Thr 5 (tBu)-Phe 6 -Thr 7 (tBu)-Ser 8 (tBu)-Asp 9 (OtBu)-Val 10 -Ser 11 (tBu)-Ser 12 (tBu)-Tyr 13 (tBu)-Leu 14 -Glu 15 (OtBu)-Gly 16 -OH.

[0044] Resin: CTC resin with a degree of selective substitution of 0.25 mmol / g was used.

[0045] Synthesis process: (1) Loading of the first amino acid: 4.0 eq of Fmoc-Gly-OH was dissolved in DMF, and 4.8 eq of DIC and 4.8 eq of Oxyma were added for activation condensation at 5 °C for 15 minutes. Then the activated amino acid solution was added to the swollen resin and condensed at 30 °C for 2.5 hours to complete the loading of the first amino acid.

[0046] (2) Peptide chain elongation: The activated remaining amino acids are condensed onto the peptide chain using the universal solid-phase synthesis cycle until Fmoc-His 1(Trt). To suppress the polymerization tendency of Aib during activation and improve condensation efficiency, a low-temperature condensation condition of -5℃ was adopted, and a dual-activator strategy was used in the activation system. Specific steps: 4.0 eq of Fmoc-Aib-OH was dissolved in DMF, and 4.8 eq of DIC / 4.8 eq of Oxyma was added. Pre-activation was performed at -5℃ for 5 minutes, maintaining the temperature. Then, 6.0 eq of PyBOP / 6.0 eq of DIPEA was added, and activation was carried out for 10 minutes. 0.5 equivalents of triisopropylsilane were introduced to suppress side reactions. After the Aib condensation reaction proceeded for 1 hour, ultrasonic treatment was used for 2 minutes to break up aggregation, and then the reaction continued for another 2 hours. After condensation, crude peptide resin was obtained.

[0047] (3) Mild lysis: 15 mL / g of pre-cooled lysis buffer was added to the obtained crude peptide resin. After reacting at room temperature for 3 hours, the lysis buffer was filtered and the filtrate was collected. The crude peptide was precipitated with MTBE at 0℃, centrifuged, washed with MTBE, and dried to obtain fragment 3 crude peptide. The lysis buffer had a volume ratio of TFA:DCM:TIS = 1:94:5. This lysis condition can cleave the linkage between the peptide and the resin without affecting the protecting groups of the amino acid side chains on the peptide chain.

[0048] (4) Purification: The crude peptide of fragment 3 was purified by preparative reversed-phase high-performance liquid chromatography, and the target product was collected to obtain high-purity fragment 3.

[0049] 4. Fragment condensation, recombination, and purification (1) Coupling of Fragment 1-resin and Fragment 2: Fragment 1-resin was deprotected by removing the nitrogen-terminal Fmoc protecting group with a 20% piperidine / DMF solution and washed thoroughly to obtain Fmoc-deprotected Fragment 1-resin; 3.0 eq of Fragment 2 was dissolved in DMF:DMSO=3:2 (v / v), and 3.0 eq of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) / 6.0 eq of DIPEA was added for preactivation, and then added to the Fmoc-deprotected Fragment 1-resin and reacted at room temperature for 12 hours; after the reaction was completed, the resin was filtered and washed thoroughly to obtain intermediate resin.

[0050] (2) Coupling of intermediate resin with fragment 3: First, remove the Fmoc protecting group from the nitrogen end of the intermediate resin and wash it to obtain the Fmoc-free intermediate resin; Dissolve 3.0 eq fragment 3 in DMF:DMSO=3:2 (v / v), add 3.0 eq HATU / 6.0 eq DIPEA for pre-activation, and then add it to the Fmoc-free intermediate resin. React at room temperature for 24 hours; After the reaction is completed, filter and wash the resin thoroughly to obtain the full-length protected smegglutinin resin.

[0051] (3) Cleavage and global deprotection: The fully protected smegglutinin resin was placed in a lysis buffer and reacted in an ice bath for 3 hours. Then, it was precipitated with ice-cold MTBE, the solid was collected by centrifugation, washed several times, and then vacuum dried to obtain the final crude smegglutinin peptide. The lysis mixture used for lysis was TFA / H2O / TIS with a volume ratio of 95:2.5:2.5.

[0052] (4) Purification and freeze-drying: The final fine purification was performed by preparative reversed-phase high-performance liquid chromatography, and the target product was collected. The purified peptide solution was freeze-dried to obtain a high-purity smegglutinin white powder product.

[0053] In the following examples and comparative examples, each amino acid was protected with Fmoc, and the side chain protecting groups were selected according to the standard Fmoc-SPPS strategy.

[0054] Table 1. Reagents used in the following examples and comparative examples.

[0055] Example 1. Fragment 1 - Preparation of Resin Weigh 46.5 g of Sieber Linker-AM resin (20 mmol) with a degree of substitution of 0.43 mmol / g into a solid-phase synthesis reaction column, add 600 mL of DMF to swell at room temperature for 30 minutes, drain the solvent, and then wash three times with DMF.

[0056] (1) First amino acid loading: Weigh 35.7 g of Fmoc-N(HMB)-Gly-OH (80 mmol, 4.0 eq) and 16.3 g of Oxyma (120 mmol, 6.0 eq), dissolve them in 400 mL of DMF, and cool to 5 °C in an ice-water bath. Slowly add 18.9 mL of LDIC (120 mmol, 6.0 eq), and activate the mixture by stirring at 5 °C for 15 minutes. Add this activated solution to the above resin, heat to 30 °C, and react with nitrogen bubbling and stirring for 2.5 hours. After the reaction is complete, vent the reaction solution and wash the resin 6 times with DMF.

[0057] (2) Elongation of peptide chains: Fmoc deprotection: Add 200 mL of 20% piperidine / DMF solution (v / v) and react for 5 minutes. After emptying the container, add another 200 mL and react for 15 minutes. After confirming a positive ninhydrin test, wash 6 times with DMF.

[0058] Conventional amino acid condensation: Weigh the corresponding Fmoc-conventional amino acid (80 mmol, 4.0 eq) and Oxyma (96 mmol, 4.8 eq) and dissolve them in DMF. After activating with DIC (96 mmol, 4.8 eq) at 5 °C for 10 minutes, add it to the reaction column and react at 30 °C for 2.5 hours.

[0059] Condensation of high-position β-branching amino acids: Weigh the corresponding Fmoc-high-position β-branching amino acid (80 mmol, 4.0 eq) and 62.5 g PyBOP (120 mmol, 6.0 eq) and dissolve them in DMF. Then add 20.9 mL DIPEA (120 mmol, 6.0 eq), activate at 5 °C for 15 minutes, add to the reaction column, and react at 30 °C for 2.5 hours.

[0060] Repeat the above deprotection and condensation cycle until the full sequence of fragment 1 is synthesized. After the reaction is complete, wash with DMF, DCM, and methanol, and dry to obtain fragment 1-resin for later use.

[0061] 2. Preparation and purification of fragment 2 Weigh 46.5 g (20 mmol) of CTC resin with a degree of substitution of 0.43 mmol / g into the reaction column and swell and wash it according to the method in step 1.

[0062] (1) Loading of the first amino acid: Weigh 34.0 g of Fmoc-Glu(OtBu)-OH (80 mmol, 4.0 eq) and dissolve it in 400 mL of DMF. Add 13.9 mL of DIPEA (80 mmol, 4.0 eq), mix well, and then add it to the resin. React at room temperature for 3 hours. Drain the reaction solution and wash 6 times with DMF.

[0063] (2) Elongation of peptide chains: Amino acid condensation: The conventional amino acid condensation method in step 1 was used for each amino acid condensation, and the coupling was performed sequentially until Fmoc-Gln was reached. 17 (Trt). Lys 20 The condensation is performed using Fmoc-Lys(Alloc)-OH.

[0064] (3) Lys 20 Modification Selective removal of alloc groups: The crude peptide resin was sequentially treated in a deprotection system of 2.31 g Pd(PPh3)4 (2.0 mmol, 0.1 eq) and 21.6 g PhSiH3 (200 mmol, 10 eq) in DCM. After each treatment for 5 minutes, the resin was immediately washed thoroughly with 200 mL of DCM and 200 mL of a DCM:DMF = 1:1 (v / v) washing solution containing 0.5% DIPEA (v / v) to remove metal residues. The above treatment was repeated until alloc groups were completely removed.

[0065] Acylation modification: Weigh 58.6 g (70 mmol, 3.5 eq) of activated C18-diacid-γ-Glu-(AEEA)2-NHS ester with the side chain, dissolve it in 400 mL of a mixed solvent of DCM:DMF = 1:1 (v / v), add 3.5 mL of DIPEA (20 mmol, 1.0 eq), and add it to the above resin. Stir the mixture at room temperature for 2 hours. After the reaction is complete, wash thoroughly with DMF and DCM to obtain fragment 2-CTC resin.

[0066] (4) Lysis and purification: Under ice bath conditions, 700 mL of pre-cooled lysis buffer TFA:DCM:TIS = 0.5:94.5:5 (v / v / v) was added to the peptide resin, and the reaction was carried out at room temperature for 3 hours. The resin was filtered, and the filtrate was collected. The filtrate was slowly added dropwise to 6 L of MTBE pre-cooled to 0 °C with stirring, and a large amount of white solid precipitated. After standing for 30 minutes, the precipitate was collected by centrifugation, washed three times with MTBE, and dried under vacuum to obtain the crude product. The crude product was purified by preparative reversed-phase high-performance liquid chromatography, and the target peak was collected to finally obtain 14.0 g of high-purity fragment 2.

[0067] 3. Preparation and purification of fragment 3 Weigh 23.3 g (10 mmol) of CTC resin with a degree of substitution of 0.25 mmol / g into the reaction column and swell and wash it according to the method in step 1.

[0068] (1) Loading of the first amino acid: Weigh 11.9g Fmoc-Gly-OH (40mmol, 4.0eq), activate it using the DIC / Oxyma system, add it to the resin, and condense at 30℃ for 2.5 hours.

[0069] (2) Elongation of peptide chains: Conventional amino acid condensation: The conventional amino acid condensation method in step 1 was used for each coupling sequentially.

[0070] The specific condensation of Aib: The reaction column was cooled to -5°C. 13.0 g of Fmoc-Aib-OH (40 mmol, 4.0 eq) was weighed and dissolved in 200 mL of DMF. 6.8 g of Oxyma (48 mmol, 4.8 eq) and 7.5 mL of DIC (48 mmol, 4.8 eq) were added, and the mixture was pre-activated at -5°C for 5 minutes. Then, 31.2 g of PyBOP (60 mmol, 6.0 eq) and 10.5 mL of DIPEA (60 mmol, 6.0 eq) were added, and activation continued for 10 minutes. This activated solution was added to the resin, and after reacting for 1 hour, sonication was performed for 2 minutes, followed by another 2 hours of reaction.

[0071] (3) Cleavage and purification: The crude peptide resin was transferred to a reaction flask, and 700 mL of pre-cooled lysis buffer TFA:DCM:TIS=1:94:5 (v / v / v) was added. The mixture was stirred at room temperature for 3 hours. The resin was filtered, and the filtrate was collected. The filtrate was slowly added dropwise to 7 L of MTBE pre-cooled to 0 °C with stirring, and a large amount of white solid precipitated. After standing for 30 minutes, the precipitate was collected by centrifugation, washed three times with MTBE, and dried under vacuum to obtain the crude product. The crude product was purified by preparative reversed-phase high-performance liquid chromatography, the target peak was collected, and lyophilized to obtain 16.0 g of high-purity fragment 3.

[0072] 4. Preparation of Smegglutinin (1) Coupling of Fragment 1-Resin with Fragment 2: The Fragment 1-resin obtained in step 1 was placed in a reaction vessel, and the nitrogen-terminated Fmoc protecting group was removed with 20% piperidine / DMF, followed by thorough washing. 93 g (60 mmol, 3.0 eq) of Fragment 2 obtained in step 2 was weighed and dissolved in a mixed solution of 600 mL DMF and 400 mL DMSO. 22.8 g HATU (60 mmol, 3.0 eq) and 10.4 mL LDPEA (120 mmol, 6.0 eq) were added, and the mixture was pre-activated at room temperature for 15 minutes. The activated solution was added to the resin in the reaction vessel, and the reaction was carried out at room temperature for 12 hours. After the reaction was completed, the reaction solution was filtered, and the resin was thoroughly washed with DMF and DCM to obtain the intermediate resin.

[0073] (2) Coupling of intermediate resin with fragment 3: The intermediate resin obtained in the previous step was washed again with 20% piperidine / DMF to remove the nitrogen-terminated Fmoc protecting group. 192 g (60 mmol, 3.0 eq) of fragment 3 obtained in step 3 was weighed, and the activation solution was added to the resin using the same HATU / DIPEA activation system and feed amount as in the previous step. The reaction was carried out at room temperature for 24 hours. After the reaction was completed, the reaction solution was filtered, and the resin was thoroughly washed with solvents such as DMF, DCM, and methanol. After vacuum drying, the full-length protected smegglutinin resin was obtained.

[0074] (3) Cleavage and global deprotection: The fully protected peptide resin obtained in the previous step was placed in a reaction flask and slowly added to 150 mL of lysis buffer TFA:H2O:TIS = 95:2.5:2.5 (v / v / v) under ice bath cooling. The reaction was carried out for 3 hours under ice bath cooling. After the reaction was completed, the resin was removed by filtration and the filtrate was collected. The filtrate was added dropwise to 1.5 L of ice-cold MTBE to precipitate, the solid was collected by centrifugation, washed several times and dried under vacuum to obtain crude smegglutinin peptide.

[0075] (4) Purification and freeze-drying: The obtained crude peptide was dissolved in an appropriate amount of aqueous solution containing acetonitrile and purified by preparative reversed-phase high-performance liquid chromatography. The target product fraction with qualified purity was collected, combined, and freeze-dried to obtain pure smegglutinin.

[0076] Comparative Example 1 Traditional solid-phase total synthesis methods Wang resin with a substitution degree of 0.5 mmol / g was selected and pre-swollen with DCM. The first Gly was prepared using Fmoc-Gly-OH, with DIC / HOBT as the activator at a molar ratio of 1:1.2:1.2. The condensation temperature was 25℃, and the condensation time was 1 hour. Subsequent amino acids were protected with Fmoc and activated with DIC / HOBT at a molar ratio of 1:1.2:1.2. The condensation temperature was 25℃, and the condensation time was 1 hour. If the ninhydrin test was positive, the condensation was repeated once. Lys 20 After cleavage of the whole peptide chain, liquid-phase acylation modification was performed. Cleavage and purification: A standard TFA cleavage mixture was used. The crude peptide was purified by preparative reversed-phase high-performance liquid chromatography and then lyophilized to obtain the pure product.

[0077] Comparative Example 2 Similar to the previous example, except that Wang resin was used for the synthesis of fragment 1, which was swollen with DMF, and the first Gly was activated and coupled using the Fmoc-Gly-OH / DIC / Oxyma system.

[0078] Comparative Example 3 Similar to the previous example, except that the condensation of the sterically hindered amino acid and Aib was performed using the traditional DIC / HOBT system.

[0079] Product testing: High-performance liquid chromatography (HPLC) (1) Sample preparation and injection: Take 1 mg of sample and dilute and dissolve it with 10 mL of pure water. Injection volume: 1 μL.

[0080] (2) Chromatographic column and mobile phase system: reversed-phase column C18; mobile phase: phase A is pure water, phase B is acetonitrile, elution gradient from 95%A:5%B to 5%A:95%B by volume percentage. Flow rate 1.0 mL / min.

[0081] (3) The absorption peak was detected at 220 nm.

[0082] Table 2 Comparison of crude semaglutide synthesis results between the examples and comparative examples

[0083] Analyze the results in Table 2: (1) Example: The crude peptide purity reached 93.2%, the crude peptide yield was 96.0%, and the synthesis cycle was shortened to 5 days. This demonstrates the innovative technical inventions proposed in this invention, including Sieber Linker-AM resin swelling, Fmoc-N(HMB)-Gly-OH first-order condensation, targeted optimization of sterically hindered amino acid and Aib condensation, and Lys 20 Solid-phase acylation and fragment recombination strategies, working synergistically, significantly improved the synthesis efficiency, purity, and yield of smegglutinin, and substantially shortened the synthesis cycle, as shown in the attached figure. Figure 2 As shown.

[0084] (2) Comparative Example 1: Using traditional solid-phase total synthesis, the crude peptide purity was only 70.0%, the yield was 65.0%, and the synthesis cycle was as long as 9 days. This fully exposes the many problems that traditional solid-phase synthesis faces when dealing with complex peptide chains, such as condensation difficulties, aggregation, many impurities, and long synthesis time.

[0085] (3) Comparative Example 2: With only optimization of the activator system and feed ratio, the purity of the synthesized crude peptide was 87.2%, the yield was 91.4%, and the synthesis cycle was 5 days. Although the optimization of the activator system and the increase of the feed ratio of high-steric amino acids slightly improved the synthesis, they did not fully solve the problems of Aib condensation, carbon-terminal stability, and peptide chain aggregation, and the overall improvement was limited. This shows that optimization of a single activator is not enough to fully solve the complex problems of smegglutinin synthesis.

[0086] (4) Comparative Example 3: Using only fragment recombination without optimizing condensation, the synthesized crude peptide had a purity of 84.2%, a yield of 87.2%, and a synthesis cycle of 6 days. The fragment recombination strategy did alleviate the problems caused by long chain aggregation to some extent, thus improving the purity and yield, and slightly shortening the synthesis time. However, due to the lack of targeted condensation optimization for key difficulties such as sterically hindered amino acids and Aib, the problem of incomplete local condensation still exists, resulting in a significant difference in performance compared to the examples.

[0087] In summary, the method of this invention significantly improves the purity and yield of crude smegglutinin and drastically shortens the synthesis cycle. This fully demonstrates the significant technological advancements and beneficial effects of this invention in improving condensation efficiency, ensuring high product purity and yield, reducing production costs, and enhancing the environmental friendliness of the process. This invention provides an efficient, stable, economical, and highly valuable technical solution for the large-scale industrial production of high-quality smegglutinin, as well as for the synthesis of other similar complex peptides in the future.

Claims

1. A solid-phase synthesis method for smegglutinin with high coupling efficiency, comprising fragment 1-resin synthesis, fragment 2 synthesis, fragment 3 synthesis, and fragment coupling and recombination, characterized in that, The synthesis of fragment 1-resin includes: the first amino acid condensation using Fmoc-N(HMB)-Gly-OH; the high-steric hindrance amino acid condensation using the PyBOP / DIPEA system; the synthesis of fragment 2 includes: Lys 20 Solid-phase acylation modification of the side chain; the synthesis of fragment 3 includes: Aib amino acid condensation using a dual activator strategy combined with low-temperature condensation and ultrasonic-assisted treatment; fragment coupling and recombination includes: coupling fragment 1-resin with fragment 2, then coupling with fragment 3, and finally global deprotection and purification to obtain smegglutinin.

2. The solid-phase synthesis method for semaglutide with high coupling efficiency as described in claim 1, characterized in that, The resin fragment 1 is: Fmoc-Phe 22 -Ile 23 -Ala 24 -Trp 25 (Boc)-Leu 26 -Val 27 -Arg 28 (Pbf)-Gly 29 -Arg 30 (Pbf)-Gly 31 - Resin; The second segment is: Fmoc-Gln 17 (Trt)-Ala 18 -Ala 19 -Lys 20 (acyl)-Glu 21 (OtBu)-OH; The third segment is: Fmoc-His 1 (Trt)-Aib-Glu 3 (OtBu)-Gly 4 -Thr 5 (tBu)-Phe 6 -Thr 7 (tBu)-Ser 8 (tBu)-Asp 9 (OtBu)-Val 10 -Ser 11 (tBu)-Ser 12 (tBu)-Tyr 13 (tBu)-Leu 14 -Glu 15 (OtBu)-Gly 16 -OH。 3. The solid-phase synthesis method for semaglutide with high coupling efficiency as described in claim 1, characterized in that, Includes the following steps: S1: Sieber Linker-AM resin with a substitution degree of 0.43 mmol / g was swollen in DMF at room temperature for 30 minutes and washed three times with DMF, 15 mL / g of resin each time; 4.0 mol equivalents of Fmoc-N(HMB)-Gly-OH were dissolved in DMF to prepare a 0.2 mol / L solution, and 6.0 mol equivalents of DIC and 6.0 mol equivalents of Oxyma were added, and the solution was activated at 5 °C for 15 minutes to generate activated amino acids; the activated amino acids were added to the swollen Sieber Linker-AM resin and condensed at 30 °C for 2.5 hours to complete the loading of the first amino acid; the remaining amino acids were then condensed sequentially until Fmoc-Phe 22 After condensation, the peptide chain remains on the resin without cleavage, washing, and drying to obtain fragment 1-resin. S2: CTC resin with a substitution degree of 0.43 mmol / g was swollen at room temperature and washed three times with DMF, 15 mL / g of resin each time. 4.0 mol equivalents of Fmoc-Glu(OtBu)-OH and 4.0 mol equivalents of DIPEA were mixed in N,N-dimethylformamide, and the swollen CTC resin was added. Condensation was carried out at room temperature for 3 hours to complete the loading. Subsequently, Fmoc-Lys(Alloc)-OH was activated in the DIC / Oxyma system at 5°C for 15 minutes, and then added to the CTC resin. The condensation reaction was carried out at 30°C for 2.5 hours. The remaining amino acids were then condensed sequentially until Fmoc-Gln... 17 (Trt); then Lys 20 Solid-phase acylation modification of side chains: The modified resin was reacted with lysis buffer in an ice bath for 3 hours. The filtrate was collected and precipitated with MTBE at 0℃. After centrifugation, washing, drying and purification were performed to obtain fragment 2. S3: CTC resin with a degree of substitution of 0.25 mmol / g was swollen at room temperature and washed three times with DMF, 15 mL / g of resin each time; the first amino acid Fmoc-Gly-OH was activated in the DIC / Oxyma system at 5 °C for 15 minutes, and then CTC resin was added for condensation for 2.5 hours. The remaining amino acids were then condensed sequentially until Fmoc-His was obtained. 1 (Trt); After condensation, the peptide was gently lysed with lysis buffer for 3 hours to remove the fully protected peptide from the resin. The filtrate was collected, precipitated with MTBE at 0°C, washed by centrifugation, dried, and purified to obtain fragment 3. S4: First, the resin of fragment 1 is coupled with the resin of fragment 2 to obtain the intermediate resin. The intermediate resin is condensed with fragment 3 to obtain the full-length protected smegglutinin resin. It is placed in the lysis buffer and subjected to global deprotection in an ice bath for 3 hours. The solid peptide is precipitated with MTBE at 0°C, washed by centrifugation, dried, purified, and finally lyophilized to obtain the smegglutinin product.

4. The solid-phase synthesis method for smegglutinin with high coupling efficiency as described in claim 3, characterized in that, The remaining amino acids in step S1 include conventional amino acids and sterically hindered amino acids; the sterically hindered amino acids include: Ile 23 Leu 26 and Val 27 The activation process uses a PyBOP / DIPEA system. The specific steps are as follows: 4.0 molar equivalents of Fmoc-sterically hindered amino acids are dissolved in DMF, 6.0 molar equivalents of PyBOP are added, and then 6.0 molar equivalents of DIPEA are added. The mixture is then activated at 5°C for 15 minutes.

5. The solid-phase synthesis method for smegglutinin with high coupling efficiency as described in claim 3, characterized in that, Lys in step S2 20 Solid-phase acylation modification of side chains includes: on the solid phase, firstly, cytosyl... 20 Selective de-Alloc protection was performed, followed by acylation with fatty acid N-succinimide ester; the lysis buffer was TFA / DCM / TIS, with a volume ratio of TFA:DCM:TIS=0.5:94.5:

5.

6. The solid-phase synthesis method for smegglutinin with high coupling efficiency as described in claim 5, characterized in that, The method for acylation of the fatty acid N-succinimide ester is as follows: 3.5 molar equivalents of fatty acid N-succinimide ester and 1.0 molar equivalent of DIPEA are dissolved in a DCM / DMF (1:1, v / v) solution and stirred at room temperature for 2 hours; the fatty acid N-succinimide ester is C18-diacid-γ-Glu-(AEEA)2-NHS.

7. The solid-phase synthesis method for smegglutinin with high coupling efficiency as described in claim 3, characterized in that, The remaining amino acids in step S3 include conventional amino acids and Aib. The condensation of Aib employs a dual-activator strategy combined with low-temperature condensation and ultrasonic-assisted treatment. The specific steps include: dissolving 4.0 mol equivalents of Fmoc-Aib-OH in DMF to prepare a 0.2 mol / L solution; adding 4.8 mol equivalents of DIC and 4.8 mol equivalents of Oxyma for pre-activation for 5 minutes; then adding 6.0 mol equivalents of PyBOP and 6.0 mol equivalents of DIPEA for activation for 10 minutes; and adding 0.5 mol equivalents of TIS to suppress side reactions. After the reaction proceeds for 1 hour, ultrasonic-assisted treatment is performed for 2 minutes to break up aggregation, and then the reaction continues for 2 hours. The lysis buffer is TFA / DCM / TIS, with a volume ratio of TFA:DCM:TIS = 1:94:

5.

8. The solid-phase synthesis method for smegglutinin with high coupling efficiency as described in claim 3, characterized in that, The specific steps for the condensation of fragment 1-resin and fragment 2 in step S4 include: taking fragment 1-resin prepared in step S1, removing the nitrogen-terminated Fmoc protecting group, and washing thoroughly to obtain Fmoc-free fragment 1-resin; dissolving 3.0 molar equivalents of fragment 2 prepared in step S2 in DMF:DMSO=3:2 (v / v), adding 3.0 molar equivalents of HATU and 6.0 molar equivalents of DIPEA for pre-activation, and then adding it to the Fmoc-free fragment 1-resin, and reacting at room temperature for 12 hours.

9. The solid-phase synthesis method for smegglutinin with high coupling efficiency as described in claim 3, characterized in that, The specific steps for coupling the intermediate resin and fragment 3 in step S4 include: first, removing the Fmoc protecting group from the nitrogen end of the intermediate resin, washing it to obtain the Fmoc-free intermediate resin; dissolving 3.0 molar equivalents of fragment 3 prepared in step S3 in DMF:DMSO=3:2 (v / v), adding 3.0 molar equivalents of HATU and 6.0 molar equivalents of DIPEA for pre-activation, and then adding it to the Fmoc-free intermediate resin, reacting at room temperature for 24 hours.

10. The solid-phase synthesis method for smegglutinin with high coupling efficiency as described in claim 3, characterized in that, In step S4, the pyrolysis solution is TFA / H2O / TIS, with a volume ratio of TFA / H2O / TIS = 95:2.5:2.5.

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