Somatostatin large fragment coupling process based on spps-lpps hybrid method

Abstract

The application relates to the technical field of polypeptide synthesis, and discloses a somatostatin large fragment coupling process based on an SPPS-LPPS mixed method, which aims to solve the problems that SPPS fragments are prone to folding and LPPS coupling efficiency is low when somatostatin is synthesized by using the existing SPPS-LPPS mixed method. Fragments I of 1-21 and fragments II of 22-31 of somatostatin are synthesized by using SPPS, and the fragment quality is guaranteed by optimizing a solid-phase carrier, performing phased conformation washing and performing differential activation; then, the fragments are connected by realizing conformation regulation, precise activation and gradient temperature coupling in the LPPS stage, and the unreacted fragments are recovered synchronously; the application can inhibit peptide chain folding and activation agent hydrolysis, improve coupling efficiency and fragment utilization rate, guarantee the purity of a coupling intermediate, simultaneously enhance process stability and economy, and adapt to the industrialized production demand of somatostatin.

Description

Technical Field

[0001] This invention relates to the field of peptide synthesis technology, and in particular to a large fragment coupling process for semaglutide based on the SPPS-LPPS hybrid method. Background Technology

[0002] Semaglutide, with its enhanced DPP-4 resistance due to the Aib substitution at position 8 and its high albumin binding due to the 18C-dicarboxylic acid-AEEA-AEEA-γ-Glu modification of the Lys side chain at position 26, is widely used in the treatment of type 2 diabetes and obesity. Its chemical structure is a linear polypeptide containing 31 amino acids, including sterically hindered amino acids such as α-aminoisobutyric acid, tryptophan, and arginine, as well as multiple hydrophobic residues. This presents significant challenges to the total synthesis of semaglutide, such as low coupling efficiency of large fragments and difficulty in controlling impurities. Currently, the synthesis of semaglutide in the industry mainly relies on total... Solid-phase synthesis (SPPS) and liquid-phase synthesis (LPPS) are two main methods of peptide chain construction. SPPS achieves peptide chain elongation by coupling amino acids one by one onto a solid support. LPPS completes the whole chain construction by activating and coupling fragments in solution. However, in the SPPS process, as the peptide chain length increases, the peptide chain is prone to hydrophobic folding and aggregation on the surface of the solid support, resulting in incomplete coupling of sterically hindered amino acids. Although the LPPS process can avoid the folding problem caused by the solid support, the fragments have low solubility in solution, and the activator is prone to reaction with the deprotection reagent and deactivation, making it difficult to meet the purity and yield requirements for industrial mass production.

[0003] To address the aforementioned issues, existing technologies have developed various SPPS-LPPS hybrid methods. For example, CN116693653A describes a method for the large-scale production of semaglutide, which involves separating semaglutide into 3-6 intermediate polypeptide fragments containing 2-15 amino acids, synthesizing each fragment using SPPS, and then sequentially coupling them with LPPS according to the amino acid sequence, thereby achieving large-scale production and improved yield of semaglutide. CN112010961A describes a solid-liquid synthesis method for semaglutide... A method for synthesizing semaglutide, disclosed in Publication No. CN113754753A, involves solid-phase synthesis of fragment I (positions 1-19), liquid-phase synthesis of side-chain monomers, solid-phase synthesis of fragment II (positions 21-31) coupled with the side-chain monomers, and finally liquid-phase condensation of fragments I and II, thereby avoiding precious metal residues and reducing costs.

[0004] The existing SPPS-LPPS hybrid method still has two major technical problems: First, the synergistic optimization of SPPS and LPPS is lacking. The existing scheme simply splits the fragments and uses SPPS and LPPS processes separately, without matching the folding state of the SPPS fragments with the LPPS coupling conditions. As a result, the β-sheet structure carried by the SPPS fragments enters the LPPS stage with insufficient exposure of active sites, which affects the coupling efficiency. Second, there is an imbalance between steric hindrance and activation in LPPS coupling. The semaglutide fragment contains multiple hydrophobic residues and large steric hindrance protecting groups. The existing LPPS stage relies on a single solvent to dissolve the fragment, which easily leads to fragment aggregation and affects coupling selectivity and yield. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the shortcomings of the existing technology. To this end, we propose a large fragment conjugation process for semaglutide based on the SPPS-LPPS hybrid method.

[0006] To achieve the above objectives, this application adopts the following technical solution: a semaglutide large fragment conjugation process based on SPPS-LPPS hybrid method, comprising the following steps: S1: Semaglutide is cleaved into fragment I composed of amino acids 1-21 and fragment II composed of amino acids 22-31, and fragment I and fragment II are synthesized separately using solid-phase synthesis; during SPPS synthesis, the solid-phase support is first swollen and activated, then the initial C-terminal amino acid is loaded and the unreacted active site is blocked, and the peptide chain is extended by a cyclic operation of first removing the N-terminal protecting group and then conjugating amino acids. After each preset round of amino acid conjugation, the peptide chain conformation is regulated and washed, and finally... S1: The resin was cut and purified to obtain pure fragment I and pure fragment II; S2: Fragment I and fragment II were coupled using a liquid-phase synthesis method. First, the conformation of fragment I was regulated, and then the conformationally regulated fragment I was precisely activated by the carboxyl group. Simultaneously, the N-terminal protecting group of fragment II was removed and washed; The deprotected fragment II was added dropwise to the activated fragment I solution at a preset ratio, the pH of the reaction system was adjusted and the coupling reaction was completed by gradient temperature control; After the coupling reaction was terminated, the target coupling product was precipitated and separated, and the filtrate was separated and concentrated to recover unreacted fragment I; S3: The target coupling product was purified and freeze-dried to obtain the semaglutide coupling intermediate.

[0007] Preferably, the solid support in S1 is polyethylene glycol-800 grafted trichlorotriphenylmethyl resin, and the resin substitution degree is 0.35-0.45 mmol / g.

[0008] Preferably, the conformation control washing described in S1 uses a mixture of polar amide solvent and trifluoroethanol, wherein the volume ratio of polar amide solvent to trifluoroethanol in the mixture is 2.5-3.5:1.

[0009] Preferably, the conformational regulation of fragment I in S2 adopts a composite regulation system of hydroxypropyl-β-cyclodextrin and polyethylene glycol-200, wherein the concentration of hydroxypropyl-β-cyclodextrin in the composite regulation system is 3.5%-4.5%, and the mass ratio of hydroxypropyl-β-cyclodextrin to polyethylene glycol-200 is 1.75-2.25:1.

[0010] Preferably, the precise activation of the carboxyl group of fragment I in S2 adopts an activation system of N-hydroxysuccinimide, N,N'-diisopropylcarbodiimide and imidazole, with a molar ratio of N-hydroxysuccinimide, N,N'-diisopropylcarbodiimide and imidazole of 1.1-1.2:1.2:0.4. The precise activation is carried out in an ice bath environment at 0-5℃, and the activation reaction time is 12-18 min.

[0011] Preferably, the preset molar ratio of fragment I to fragment II in S2 is 1:1.1-1.5. The fragment II solution is added dropwise to the fragment I activation solution. The gradient temperature control is to first stir in an ice bath at 0-5℃ for 30 min, and then raise the temperature to 22-25℃ and continue stirring for 3-3.2 h. The pH of the reaction system is adjusted by N,N-diisopropylethylamine, and the pH of the system after adjustment is 8.0-8.5.

[0012] Preferably, the amino acid coupling in S1 employs a differentiated activation system. For conventional sterically hindered amino acids, the activation system consists of Fmoc-amino acid, 1,3-dicyclohexylcarbodiimide, and 1-hydroxybenzotriazole, dissolved in a polar amide solvent, activated at room temperature for 5-8 min, and then coupled for 2-2.5 h. For highly sterically hindered amino acids, the activation system consists of Fmoc-amino acid, (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylamino-morpholine-carbonhexafluorophosphate, and 1-hydroxy-7-azobenzotriazole, dissolved in a mixture of a polar amide solvent and dimethyl sulfoxide, activated at 30°C for 8-10 min, and then coupled for 2.5-3 h.

[0013] Preferably, the purity of fragment I in S1 is ≥97.5%, the purity of fragment II is ≥98%, and the content of single impurities in both fragments is ≤0.5%.

[0014] Preferably, the precipitation of the target coupling product in S2 is carried out using isopropyl ether pre-cooled to 10-20°C, the volume of isopropyl ether added is 2-3 times the volume of the coupling termination liquid, the filtrate is separated into layers using n-hexane, the volume ratio of n-hexane to filtrate is 1:1, and the shaking separation time is 15-25 min.

[0015] Preferably, the resin cutting in S1 uses a cutting fluid containing trifluoroacetic acid, trifluoroethanol and ethylenedithiol, wherein the volume percentage of TFA in the cutting fluid is 6%, TFE is 4%, EDT is 0.5%, and the cutting time is 1 hour.

[0016] The technical effects and advantages of this invention are as follows:

[0017] In this invention, a low-resistivity fragment preparation system for SPPS solid-phase synthesis was constructed. By selectively grafting CTC resin with PEG-800 and controlling its substitution degree, combined with staged conformation washing of DMF / TFE mixture, and adopting a differentiated activation strategy based on the differences in amino acid steric hindrance, the problems of easy folding of peptide chains and incomplete coupling of sterically hindered amino acids in the SPPS stage were solved. By inhibiting the hydrophobic aggregation of fragments I and II, the purity of the SPPS-synthesized fragments was ensured to be stable and meet the requirements of subsequent coupling, and low-purity fragments or folded structures were avoided from being introduced into the LPPS stage and causing side reactions.

[0018] In this invention, a synergistic mechanism among conformation, activation, and recovery of LPPS liquid-phase coupling was established. Fragment I was conformationally regulated, precisely activated, and coupled with gradient temperature control through an HP-β-CD and PEG-200 composite system. This not only broke the hydrophobic folds of the fragment to expose the active sites but also reduced the hydrolysis of the activator and fragment aggregation. At the same time, the target product and impurities were separated by isopropyl ether gradient precipitation, and unreacted fragments were recovered from the filtrate. This improved the LPPS coupling efficiency and selectivity, controlled the generation of impurities such as dimers, and also enabled the recycling of unreacted fragments.

[0019] In this invention, a synergistic optimization mechanism for the entire SPPS-LPPS process was constructed, clarifying the matching standard between SPPS fragment purity and LPPS coupling conditions. This enables the low-resistivity fragments in the SPPS stage to adapt to the conformational regulation and activation system of LPPS, avoiding efficiency loss caused by mismatch between fragment quality and coupling conditions, and improving the overall efficiency and stability of the semaglutide large fragment coupling process. Attached Figure Description

[0020] The disclosure of this invention is illustrated with reference to the accompanying drawings. It should be understood that the drawings are for illustrative purposes only and are not intended to limit the scope of protection of this invention. In the drawings, the same reference numerals are used to refer to the same parts:

[0021] Figure 1 This is the HPLC chromatogram of fragment I of the present invention;

[0022] Figure 2 This is the HPLC chromatogram of fragment II of the present invention;

[0023] Figure 3 This is the HPLC chromatogram of the coupling intermediate of the present invention. Detailed Implementation

[0024] It is readily understood that, based on the technical solution of this invention, those skilled in the art can propose various interchangeable structural methods and implementations without altering the essential spirit of the invention. Therefore, the following detailed embodiments and accompanying drawings are merely illustrative examples of the technical solution of this invention and should not be considered as the entirety of the invention or as limitations or restrictions on the technical solution of this invention.

[0025] The meanings of the abbreviations used in this invention are listed in Table 1 below:

[0026] SPPS Solid-phase peptide synthesis LPPS Liquid-phase peptide synthesis CTC Chlortriphenylmethyl PEG polyethylene glycol TFA Trifluoroacetic acid TFE Trifluoroethanol DMF N,N-Dimethylformamide DMSO dimethyl sulfoxide DIC N,N'-Diisopropylcarbodiimide HOBt 1-Hydroxybenzotriazole COMU (1-Cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylamino-morpholine-carbonhexafluorophosphate HOAt 1-Hydroxy-7-azobenzotriazole DIPEA N,N-Diisopropylethylamine HOSu N-hydroxysuccinimide EDT Ethylene dithiol HP-β-CD Hydroxypropyl-β-cyclodextrin UPLC Ultra-high performance liquid chromatography OtBu tert-butyl ester Trt Triphenylmethyl Boc tert-Butoxycarbonyl Pbf 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl Alloc allyloxycarbonyl Fmoc fluorene methyloxycarbonyl

[0027] Table 1

[0028] Example 1: This example provides a large fragment conjugation process for semaglutide based on the SPPS-LPPS hybrid method, including the following steps:

[0029] S1: SPPS solid-phase synthesis fragment.

[0030] The first 21 amino acids of the solid-phase synthesized semaglutide sequence were designated as fragment I, and the second 22-31 amino acids of the solid-phase synthesized semaglutide sequence were designated as fragment II.

[0031] S11: Solid-phase synthesis of fragment I;

[0032] Semaglutide fragment I has an amino acid sequence from position 1 to 21, specifically as follows:

[0033] Fmoc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Lys(Alloc)-Gly-OH;

[0034] S111: Pretreated solid support;

[0035] Take 10g of PEG-800 grafted chlorotriphenylmethyl resin (CTC), with a substitution degree of 0.4mmol / g and a particle size of 100-200 mesh, and place it in a 250mL solid-phase reaction column. Add 100mL of dichloromethane (DCM) and stir at room temperature to swell for 30min to ensure that the resin is fully expanded. Then, drain the swollen solution and wash the resin three times with 50mL of N,N-dimethylformamide (DMF). After each wash, drain the solution to complete the resin activation.

[0036] S112: Initial C-terminal amino acid Gly loading and blocking;

[0037] Fmoc-Gly-OH and N,N-diisopropylethylamine (DIPEA) were added to the activated resin and dissolved in 80 mL of DMF. The reaction was carried out at room temperature with shaking for 2 h. The fixed molar equivalents of Fmoc-Gly-OH and DIPEA were 2.0 eq, and the DMF concentration was 0.2 mmol / mL. After the reaction was completed, the reaction solution was dried under vacuum, and the resin was washed three times with 50 mL of DMF solution. 100 mL of methanol / DMF mixture (1:1 volume ratio) was added, and the mixture was shaken at room temperature for 30 min to inactivate unreacted resin active sites. A small amount of resin was taken, and ninhydrin reagent was added. The mixture was heated in a boiling water bath for 30 s. If the resin turned colorless, it indicated that Gly... 21 If the resin is fully loaded and turns blue, then methanol / DMF mixture needs to be added to extend the sealing time to 45-60 minutes until the test is qualified. Then, wash the resin three times alternately with 50 mL of DMF solution and DCM solution, and dry it for later use.

[0038] S113: Removal of Fmoc protecting group;

[0039] Add 50 mL of 20% piperidine / DMF solution to the resin, shake and react at room temperature for 5 min, then dry it. Add another 50 mL of piperidine / DMF solution of the same concentration and continue shaking and reacting for 15 min. After deprotection, wash the resin three times alternately with 50 mL of DMF solution and DCM solution, and then dry it for later use.

[0040] S114: Amino acid activation and coupling;

[0041] This step requires the use of two activation systems based on the steric hindrance differences of the amino acid side chains. The specific operation is as follows:

[0042] For conventional sterically hindered amino acids, such as Gly, Ala, Val, Leu, and Glu (OtBu): Fmoc-target amino acid, 1,3-dicyclohexylcarbodiimide (DIC), and 1-hydroxybenzotriazole (HOBt) were dissolved in 50 mL of DMF solution. After activation at room temperature for 5 min, DIPEA was added to adjust the pH of the system to 8.0-8.5. The activated solution was then added to the reaction column and reacted with shaking at room temperature for 2 h. The fixed molar equivalents of the target amino acid were 1.5 eq, the fixed molar equivalents of DIC were 1.2 eq, the fixed molar equivalents of HOBt were 1.1 eq, the fixed molar equivalents of DIPEA were 2.0 eq, and the concentration of DMF solution was 0.3 mmol / mL.

[0043] For sterically hindered amino acids, such as Aib 2Thr(tBu), Ser(tBu), etc.: Fmoc-target amino acid, (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylamino-morpholine-carbonhexafluorophosphate (COMU), 1-hydroxy-7-azobenzotriazole (HOAt) and DIPEA were dissolved in a DMF / dimethyl sulfoxide (DMSO) mixture at a volume ratio of 7:3. After activation at 30℃ for 8 min, the activated solution was added to the reaction column and reacted at 30℃ with shaking for 2.5 h. The fixed molar equivalents of the target amino acid were 1.8 eq, COMU was 1.2 eq, HOAt was 1.1 eq, and DIPEA was 2.0 eq.

[0044] After each coupling cycle, a small amount of resin is tested for ninhydrin. If it is colorless, proceed to the subsequent washing step. If it is light blue, the reaction can be extended to 2.5 hours for conventional amino acids and to 3 hours for sterically hindered amino acids. If it is dark blue, 0.5 eq of activator DIC or COMU and 0.5 eq of auxiliary reagent HOBt or HOAt need to be added.

[0045] S115: Conformation-controlled washing;

[0046] After each of the four amino acid couplings, i.e., after the 4th, 8th, 12th, 16th and 20th coupling rounds, the resin was washed three times with a 50 mL DMF / trifluoroethanol (TFE) mixture at a volume ratio of 3:1, with each wash followed by 5 min of shaking to disrupt the early hydrophobic folding of the peptide chain. The volume ratio of the mixture can be finely adjusted within the range of 2.5-3.5:1 to ensure that the TFE concentration is within the range of 25%-40% to exert its conformational regulation effect.

[0047] S116: Resin cutting and crude peptide preparation;

[0048] Complete N-end His 1 After coupling, 50 mL of 20% piperidine / DMF solution was added to the resin, and the mixture was shaken at room temperature for 5 min before being dried. Then, another 50 mL of piperidine / DMF solution of the same concentration was added and the mixture was shaken for 15 min. After deprotection, the resin was washed three times alternately with 50 mL of DMF solution and DCM solution. After drying, the resin was transferred to a vacuum dryer and dried under vacuum at 40 °C for 4 h to obtain the resin-loaded fragment I precursor.

[0049] Add 80 mL of cutting solution to the dry resin and react with shaking at room temperature for 1 h. The cutting solution is a mixture of 6% trifluoroacetic acid (TFA) / DCM, 4% TFE, and 0.5% ethylenedithiol (EDT). TFE can enhance peptide chain solubility, and EDT can inhibit His side chain oxidation.

[0050] The filtrate was collected by filtration. The resin was washed three times with 10 mL of DCM. The filtrates were combined and then slowly added with 5 times the volume of pre-cooled n-hexane / ethyl acetate mixture at a volume ratio of 3:1 while stirring. The mixture was allowed to stand at 4°C for 30 min to allow the crude peptide to precipitate fully. After centrifugation at 4000 r / min for 10 min, the supernatant was discarded. The precipitate was washed three times with pre-cooled n-hexane and dried under vacuum to obtain fragment I crude peptide.

[0051] S117: Purification and characterization of fragment I;

[0052] The crude peptide was dissolved in a 20% acetonitrile / water solution containing 0.1% TFA, and the concentration was adjusted to 20 mg / mL. After filtration through a 0.45 μm filter, the solution was loaded onto a reversed-phase C18 column and eluted using a gradient of acetonitrile / water solution, with the acetonitrile concentration increasing from 20% to 45% at a flow rate of 10 mL / min and a column temperature of 30 °C. The column was monitored by a UV detector, and the main peak fraction was collected. The main peak fractions were combined, concentrated under reduced pressure to remove acetonitrile, and freeze-dried for 12 h to obtain pure fragment I. The detection results are shown in Table 2. Figure 1 As shown.

[0053]

[0054] Table 2

[0055] S12: Solid-phase synthesis of fragment II;

[0056] Semaglutide fragment II has amino acid sequences from positions 22 to 31, specifically:

[0057] Fmoc-Ala-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-OH;

[0058] S121: Pretreated solid support;

[0059] Take 5g of CTC resin with a substitution degree of 0.4mmol / g and a particle size of 100-200 mesh, place it in a 1000mL solid-phase reaction column, add 50mL of DCM, stir and swell at room temperature for 30min, then dry the swollen solution, wash the resin three times with 50mL of DMF, and dry it after each wash to complete the resin activation.

[0060] S122: C-terminal amino acid Gly 31 Load and enclosure;

[0061] Add Fmoc-Gly-OH and DIPEA to the activated resin, dissolve in 80 mL of DMF, and react with shaking at room temperature for 2 h. The fixed molar equivalents of Fmoc-Gly-OH and DIPEA are 2.0 eq, and the DMF concentration is 0.2 mmol / mL. After the reaction, dry the reaction mixture, wash the resin three times with 30 mL of DMF solution, add 50 mL of methanol / DMF mixture (1:1 volume ratio), and seal with shaking at room temperature for 30 min. Take a small amount of resin, add ninhydrin reagent, and heat in a boiling water bath for 30 s. If the resin becomes colorless, it indicates that Gly... 31 If the resin is fully loaded and turns blue, then methanol / DMF mixture needs to be added to extend the sealing time to 45-60 minutes until the test is qualified. Then, wash the resin three times alternately with 50 mL of DMF solution and DCM solution, and dry it for later use.

[0062] S123: Removal of Fmoc protecting group;

[0063] Add 30 mL of 20% piperidine / DMF solution to the resin, shake and react at room temperature for 5 min, then dry it. Add another 30 mL of piperidine / DMF solution of the same concentration and continue shaking and reacting for 15 min. After deprotection, wash the resin three times alternately with 50 mL of DMF solution and DCM solution, and then dry it for later use.

[0064] S124: Amino acid activation and coupling;

[0065] This step requires the use of two activation systems based on the steric hindrance differences of the amino acid side chains. The specific operation is as follows:

[0066] For conventional sterically hindered amino acids, such as Ala 22 Ala 27 Val 29 Ile 26 Phe 25 Glu(OtBu) 24 The following steps were performed: Fmoc-target amino acid, DIC, and HOBt were dissolved in 50 mL of DMF solution and activated at room temperature for 5 min. Then, DIPEA was added to adjust the pH of the system to 8.0-8.5. The activated solution was added to the reaction column and the reaction was carried out at room temperature with shaking for 2 h. The fixed molar equivalents of the target amino acid were 1.5 eq, the fixed molar equivalents of DIC were 1.2 eq, the fixed molar equivalents of HOBt were 1.1 eq, the fixed molar equivalents of DIPEA were 2.0 eq, and the concentration of DMF solution was 0.3 mmol / mL.

[0067] For sterically hindered amino acids, such as Trp(Boc) 28 Arg(Pbf) 30The target amino acid, COMU, HOAt, and DIPEA were dissolved in a DMF / DMSO mixture at a volume ratio of 7:3. After activation at 30°C for 8 min, the activated solution was added to the reaction column and reacted at 30°C with shaking for 2.5 h. The fixed molar equivalents of the target amino acid were 1.8 eq, COMU was 1.2 eq, HOAt was 1.1 eq, and DIPEA was 2.0 eq.

[0068] Coupled Trp(Boc) 28 At that time, 0.2% DTT was added to the activation solution under light-protected conditions to prevent oxidation; Arg(Pbf) was coupled. 30 If necessary, extend the reaction time to 2 hours;

[0069] After each coupling cycle, a small amount of resin is tested for ninhydrin. If it is colorless, proceed to the subsequent washing step. If it is light blue, the reaction can be extended to 2.5 hours for conventional amino acids and to 3 hours for sterically hindered amino acids. If it is dark blue, 0.5 eq of activator DIC or COMU and 0.5 eq of auxiliary reagent HOBt or HOAt need to be added.

[0070] S125: Conformation-controlled washing;

[0071] After each of the four amino acid couplings is completed, i.e. after the fourth and eighth rounds of coupling, the resin is washed three times with 30 mL of DMF / TFE mixture at a volume ratio of 3:1, with each wash and shaking for 5 min. The volume ratio of the mixture can be finely adjusted within the range of 2.5-3.5:1 to ensure that the TFE concentration is within the range of 25%-40%.

[0072] S126: Resin cutting and crude peptide preparation;

[0073] Complete N-end Ala 22 After coupling, 30 mL of 20% piperidine / DMF solution was added to the resin, and the mixture was shaken at room temperature for 5 min before being dried. Then, another 30 mL of piperidine / DMF solution of the same concentration was added and the mixture was shaken for 15 min. After deprotection, the resin was washed three times alternately with 50 mL of DMF solution and DCM solution. After drying, the resin was transferred to a vacuum dryer and dried under vacuum at 40 °C for 4 h to obtain the resin-loaded fragment II precursor.

[0074] Add 40 mL of cleavage solution to the dry resin and react with shaking at room temperature for 1 h. The cleavage solution is a mixture of 6% TFA / DCM, 4% TFE and 0.5% EDT. TFE can enhance the solubility of peptide chains and EDT can protect the Trp side chains.

[0075] The filtrate was collected by filtration. The resin was washed three times with 5 mL of DCM. The filtrates were combined and then slowly added with 5 times the volume of pre-cooled n-hexane / ethyl acetate mixture at a volume ratio of 3:1 while stirring. The mixture was allowed to stand at 4°C for 30 min to allow the crude peptide to precipitate fully. After centrifugation at 4000 r / min for 10 min, the supernatant was discarded. The precipitate was washed three times with pre-cooled n-hexane and dried under vacuum to obtain fragment II crude peptide.

[0076] S127: Purification and characterization of fragment II;

[0077] The crude peptide was dissolved in a 20% acetonitrile / water solution containing 0.1% TFA, and the concentration was adjusted to 20 mg / mL. After filtration through a 0.45 μm filter, the sample was loaded onto a reversed-phase C18 column and eluted using a gradient of acetonitrile / water solution, with the acetonitrile concentration increasing from 30% to 50%, a flow rate of 7 mL / min, and a column temperature of 30 °C. The column was monitored by a UV detector, and the main peak fraction was collected. The main peak fractions were combined, concentrated under reduced pressure to remove acetonitrile, and freeze-dried for 12 h to obtain pure fragment II. The detection results are shown in Table 3. Figure 2 As shown.

[0078]

[0079] Table 3

[0080] S2: LPPS liquid-phase coupling and purification.

[0081] S21: Pretreatment before liquid-phase synthesis;

[0082] Take 1.0 mmol of fragment I and place it in a 250 mL three-necked flask. Add 30 mL of DMF / DMSO / TFE compound solvent with a volume ratio of 5:3:2. Stir at 300 r / min for 10 min at room temperature until the solution is clear and transparent. Use a UV spectrophotometer to detect the absorbance of the solution. If the absorbance fluctuation is ≤0.02 AU, it indicates that the solution is uniformly dissolved. If a small precipitate appears, add 1-2 mL of TFE and continue stirring for 5 min until the precipitate is completely dissolved.

[0083] Take 1.1 mmol of fragment II and place it in a 100 mL Erlenmeyer flask. Add 20 mL of anhydrous DMF and stir at room temperature for 5 min until completely dissolved to form a clear solution.

[0084] The purity of fragment I and fragment II after processing was detected by UPLC. The purity of fragment I must be ≥97.5% and the purity of fragment II must be ≥98%, and the content of single impurities must be ≤0.5%, which meets the conditions for subsequent operation.

[0085] S22: Conformational regulation:

[0086] Based on the amino acid composition of fragment I, which contains 7 hydrophobic residues and 3 amidation sites, this scheme adopts a composite system of cyclodextrin and polyether, as detailed below:

[0087] Preparation of the conditioning system: Take 1.2 g of hydroxypropyl-β-cyclodextrin (HP-β-CD), dissolve it in 10 mL of anhydrous DMF, and stir magnetically until completely dissolved to prepare a 4% HP-β-CD solution; take 0.6 g of polyethylene glycol-200 (PEG-200), with an average molecular weight of 190-210, and add it directly to the above HP-β-CD solution, stir for 5 min to form a homogeneous mixture, with a mass ratio of HP-β-CD to PEG-200 of 2:1;

[0088] The above-mentioned regulator mixture was slowly added dropwise to the pretreated fragment I solution at a rate of 1 mL / min. During the addition process, the rate was maintained at 300 r / min to avoid excessive local concentration that could lead to fragment aggregation. The reaction system temperature was then controlled at 22 °C and stirred for 30 min. This allowed HP-β-CD to encapsulate the hydrophobic residues of fragment I through hydrophobic interactions, and PEG-200 to inhibit refolding through steric hindrance.

[0089] Take 2 mL of the regulated solution and detect it using a circular dichroism chromatograph. Scan 190-250 nm and record the intensity of the β-sheet characteristic peak at 217 nm. The intensity of the β-sheet characteristic peak should be ≤3000 a.u., and the intensity of the α-helix characteristic peak at 208 nm should be ≥1500 a.u. Detect the fluorescence intensity of the C-terminal carboxyl group of fragment I using a fluorescent probe method. The intensity should be greater than 85% of the initial value to prove that the active site is fully exposed. Detect the particle size distribution of the solution using a dynamic light scattering instrument. More than 90% of the particles should be ≤5 nm in size, and there should be no aggregated particles larger than 10 nm to avoid increased steric hindrance due to aggregation during coupling.

[0090] S23: Precise activation;

[0091] To efficiently convert the C-terminal carboxyl group of conformationally regulated fragment I into an active ester, while avoiding inactivation by the activator reacting with the deprotecting agent of fragment II, the specific procedures are as follows:

[0092] Preparation of activation system: Take 1.1 mmol of N-hydroxysuccinimide (HOSu), place it in a 50 mL beaker, add 5 mL of anhydrous DMF, and stir magnetically until completely dissolved; add 1.2 mmol of N,N'-diisopropylcarbodiimide (DIC) and 0.4 mmol of imidazole to the above solution in sequence, and stir at room temperature for 5 min to form a homogeneous activation solution;

[0093] The above-mentioned activating solution was slowly added dropwise to the conformationally regulated fragment I solution at a rate of 0.5 mL / min. During the addition process, an ice bath was maintained and the mixture was magnetically stirred at a rate of 300 r / min to avoid local overheating that could lead to the decomposition of the active ester. The reaction was then maintained at 0 °C for 15 min to suppress the hydrolysis side reaction of DIC and promote the acylation reaction of carboxyl groups with the activator.

[0094] While activating fragment I, the N-terminal Fmoc deprotection of fragment II was simultaneously performed. 5 mL of 20% piperidine / DMF solution was added to the DMF solution containing the qualified fragment II, and the mixture was stirred at room temperature for 40 min. The deprotection was detected using ninhydrin reagent. After the deprotection was completed, the fragment II solution was transferred to a centrifuge tube, 10 mL of DMF was added, and the mixture was centrifuged at 4000 r / min for 5 min. The supernatant was discarded, and the mixture was washed three times. Fragment II was then redissolved in 10 mL of DMF for later use.

[0095] S24: Liquid-phase coupling reaction;

[0096] Take the precisely activated fragment I solution and place it in a 250 mL three-necked flask. Slowly add the deprotected and washed fragment II solution using a constant pressure dropping funnel. The molar ratio of fragment I to fragment II is 1:1.1, and the dropping rate is controlled at 1 mL / min to avoid excessively high local concentrations that could cause the amino group of fragment II to react with multiple active esters to form dimers. It is important to note that the fragment II solution must be added dropwise to the activated fragment I solution; the reverse operation is not allowed, as it will cause excessive aggregation of the active esters in fragment I, increasing self-coupling side reactions. Immediately after the addition is complete, add 3 mmol of DIPEA to the mixed solution. The molar ratio of DIPEA to fragment I is 3:1. Mix the system uniformly by magnetic stirring, while neutralizing the carboxylic acid generated in the reaction to maintain the pH of the system at 8.0-8.5. This pH range maximizes the nucleophilicity of the amino group and inhibits the hydrolysis of the active esters.

[0097] Next, a gradient temperature-controlled coupling reaction was carried out. First, the mixture was placed in an ice bath and stirred at 300 rpm for 30 min. The low temperature suppressed the hydrolysis side reaction of the active ester of fragment I and promoted the N-terminal amino group Ala of fragment II. 22 Nucleophilic attack on the C-terminal active ester of fragment I; then remove the ice bath, slowly raise the system temperature to 22-25℃, maintain the stirring speed, and continue the reaction for 3h. Strictly control the temperature to avoid excessive temperature leading to the removal of the protecting group or degradation of the peptide chain.

[0098] 10 μL samples were taken at 0.5 h, 1 h, 2 h, and 3 h of the reaction, filtered through a 0.22 μm filter membrane, and then analyzed by UPLC. When the residual peak areas of fragment I and fragment II were both ≤5%, the peak area of ​​the target product was ≥95%, and the peak area of ​​the dimer was ≤1.5%, 0.2 mL of glacial acetic acid was added, and the mixture was stirred for 5 min. The pH of the system was adjusted to 6.0-6.5, and the reaction was terminated by protonation of the amino group, while avoiding excessive acidity that could lead to the removal of the protecting group.

[0099] S25: Gradient purification and SPPS fragment recovery;

[0100] Take the reaction solution after liquid-phase coupling termination and place it in a 500mL Erlenmeyer flask. Slowly add 2.5 times the volume of pre-cooled isopropyl ether at 200r / min, with the isopropyl ether temperature in the range of 10-20℃ and the dropping rate controlled at 3mL / min to avoid local supersaturation leading to co-precipitation of impurities. Isopropyl ether is a weakly polar solvent, which can reduce the solubility of the target coupling product and make it precipitate preferentially, while unreacted small molecule fragments and sterically hindered dimer impurities remain soluble in the mixed solvent phase, achieving preliminary separation. After the addition is complete, let it stand at 15℃ for 30min to allow the precipitate to settle fully. Filter under reduced pressure using a Buchner funnel, collect the filter cake, and wash the filter cake twice with 10mL of pre-cooled isopropyl ether to remove residual impurities and solvent on the surface. After vacuum drying the filter cake for 1h, dissolve it in 5mL of acetonitrile / water (volume ratio 1:1). UPLC analysis shows a purity ≥85% and sterically hindered dimer impurities ≤3%.

[0101] The filtrate was transferred to a 500 mL separatory funnel, and an equal volume of n-hexane was added. After shaking for 3 min, the mixture was allowed to stand for 15 min to separate into three phases, forming an upper n-hexane phase, a middle mixed solvent phase, and a lower trace aqueous phase. The middle mixed solvent phase was collected, and 5 g of anhydrous sodium sulfate was added to dry it for 30 min. After filtering to remove water, the mixture was concentrated by vacuum distillation at 40 °C. Five times the volume of -20 °C pre-cooled ice-cold diethyl ether was added dropwise to the concentrate. After stirring for 5 min, the mixture was allowed to stand for 30 min, and then centrifuged at 4000 r / min for 10 min. The precipitate was collected to obtain recovered fragment I. UPLC analysis showed that the purity of recovered fragment I was ≥95%. The upper n-hexane phase and the lower aqueous phase were combined, and the solvent was recovered by distillation. The residue was treated according to hazardous waste regulations.

[0102] The above filter cake was ultrasonically dissolved in an acetonitrile / water mixture for 5 min, and then filtered through a 0.22 μm organic phase filter membrane to remove insoluble particles, resulting in a purified sample solution, which was then refrigerated at 4°C for later use.

[0103] S26: Preparation of coupling intermediates;

[0104] The purified sample solution was dissolved in a 20% acetonitrile / water mixture containing 0.1% TFA, with the concentration adjusted to 20 mg / mL. After filtration through a 0.22 μm organic phase filter membrane, the sample was loaded onto a reversed-phase C18 column and eluted using an acetonitrile / water gradient. The acetonitrile concentration was increased from 30% to 50%, the flow rate was controlled at 7 mL / min, and the column temperature was 30℃. The main peak fraction with a retention time of 18.2–19.0 ​​min was collected. The combined target fraction was concentrated to one-tenth of its original volume under reduced pressure at 40℃, and then placed in a freeze dryer. The fraction was pre-frozen at -50℃ for 4 h, sublimated at -30℃ for 12 h, and desorbed at 25℃ for 4 h to obtain a white, loose, powdery coupling intermediate. The detection results are shown in Table 4. Figure 3 As shown.

[0105]

[0106] Table 4

[0107] Example 2 describes a large fragment conjugation process for semaglutide based on a SPPS-LPPS hybrid method. The difference from Example 1 is that the substitution rate of the solid support used in the SPPS solid-phase synthesis stage is 0.35 mmol / g. Specific details are as follows:

[0108] Take 10g of PEG-800 grafted CTC resin with a substitution degree of 0.35mmol / g and a particle size of 100-200 mesh, place it in a 250mL solid-phase reaction column, add 100mL of DCM, stir and swell at room temperature for 30min, dry it under vacuum, and wash it three times with 50mL of DMF; for the synthesis of fragment II, take 5g of resin of the same specification and treat it according to the same swelling and washing steps.

[0109] Due to the reduced resin substitution degree, Fmoc-Gly-OH corresponds to the C-terminal Gly of fragment I. 21 C-terminal Gly of fragment II 31 The loading reaction time needs to be extended to 2.2 hours to ensure a loading rate ≥98%; for the coupling of other amino acids, the reaction time for conventional sterically hindered amino acids is maintained at 2 hours, while for highly sterically hindered amino acids such as Aib... 2 Trp(Boc) 28 The reaction time was maintained at 2.5 h, and the resin was colorless as detected by ninhydrin, indicating complete coupling.

[0110] After resin cutting, the crude product yield of fragment I was ≥91%, and the purity after reverse-phase C18 purification was ≥97.5%; the crude product yield of fragment II was ≥92%, and the purity after purification was ≥98%; circular dichroism spectroscopy showed that the β-sheet content of fragment I was ≤42%, which met the low-resistance requirement for subsequent LPPS coupling.

[0111] Everything else remains the same as in Example 1.

[0112] Example 3 describes a large fragment conjugation process for semaglutide based on a SPPS-LPPS hybrid method. The difference from Example 1 is that the substitution rate of the solid support used in the SPPS solid-phase synthesis stage is 0.45 mmol / g. Specific details are as follows:

[0113] Take 10g of PEG-800 grafted CTC resin with a substitution degree of 0.45mmol / g and a particle size of 100-200 mesh, place it in a 250mL solid-phase reaction column, add 120mL of DCM and stir at room temperature for 35min to ensure that the resin is fully expanded to relieve peptide chain crowding; after drying, wash 3 times with 50mL of DMF. When synthesizing fragment II, take 5g of the same resin and process it in the same way.

[0114] After every 4 amino acid coupling steps, the resin was washed 4 times with 50 mL of DMF / TFE mixture (volume ratio 2.5:1), with shaking for 6 min each time. By increasing the TFE concentration, the over-folding of the peptide chain was broken. The content of fragment I β-sheet was ≤43% as detected by circular dichroism chromatography.

[0115] After resin cleavage, the crude yields of fragments I and II were 92% and 93%, respectively. After purification, the purity of fragment I was ≥97.5% and the purity of fragment II was ≥98%. The sterically hindered amino acid Arg(Pbf) was also tested. 30 The coupling rate is ≥97%, and there are no significant missing peptide impurities.

[0116] Everything else remains the same as in Example 1.

[0117] Example 4 describes a large fragment conjugation process for semaglutide based on a SPPS-LPPS hybrid method. The difference from Example 1 is that the substitution rate of the solid support used in the SPPS solid-phase synthesis stage is 0.42 mmol / g. Specific details are as follows:

[0118] Take 10g of PEG-800 grafted CTC resin, with a substitution degree of 0.42mmol / g and a particle size of 100-200 mesh, swell it in 110mL of DCM at room temperature for 32min, dry it under vacuum, and wash it three times with 50mL of DMF; the Fmoc-Gly-OH loading reaction time is 2h, the coupling efficiency is ≥99%, and no reaction extension is required;

[0119] After every 4 coupling steps, wash 3 times with 50 mL of LMF / TFE mixture (3:1 volume ratio), shaking for 5 min each time, ensuring fragment I β-sheet content ≤36%; for sterically hindered amino acid coupling, such as Aib 2 Trp(Boc) 28 The amount of DIPEA in the activation solution was increased to 2.2 eq, and the coupling rate was ≥99%.

[0120] The yields of fragments I and II after purification were 94% and 95%, respectively. The subsequent LPPS conjugation completion rate was ≥98.5%, and the purity of the conjugation intermediate was ≥99.7%.

[0121] Everything else remains the same as in Example 1.

[0122] Example 5: A large fragment conjugation process for semaglutide based on the SPPS-LPPS hybrid method, which differs from Example 1 in that the concentration of HP-β-CD used in the LPPS conformation regulation stage is 3.5%, as detailed below:

[0123] Dissolve 1.05g of HP-β-CD in 10mL of anhydrous DMF to prepare a 3.5% HP-β-CD solution; add 0.6g of PEG-200 and stir for 6min to form a homogeneous mixture. The mass ratio of HP-β-CD to PEG-200 is 1.75:1.

[0124] When adding the regulator mixture to fragment I solution, the dropping rate was reduced to 0.8 mL / min. After dropping, the solution was stirred at room temperature for 35 min to ensure that HP-β-CD fully encapsulates the hydrophobic residues. The exposure rate of active sites was ≥80% as detected by fluorescent probe method, and the fragment particle size was ≤6 nm as detected by dynamic light scattering instrument.

[0125] During fragment I activation, the amount of HOSu in the activation solution was increased to 1.2 eq to compensate for the slight decrease in the exposure rate of active sites, and the active ester formation rate was ≥95%.

[0126] Everything else remains the same as in Example 1.

[0127] Example 6: A large fragment conjugation process for semaglutide based on SPPS-LPPS hybrid method, which differs from Example 1 in that the concentration of HP-β-CD used in the LPPS conformation regulation stage is 4.5%, as detailed below:

[0128] Dissolve 1.35g of HP-β-CD in 10mL of anhydrous DMF to prepare a 4.5% HP-β-CD solution; add 0.6g of PEG-200 and stir for 8min to ensure uniform dissolution. The mass ratio of HP-β-CD to PEG-200 is 2.25:1.

[0129] When the regulator mixture was added dropwise to the fragment I solution, 0.5 mL of DMSO was added simultaneously to reduce the risk of intermolecular aggregation of HP-β-CD molecules. After addition, the mixture was stirred at room temperature for 30 min. Dynamic light scattering analysis showed that 90% of the particles had a diameter ≤8 nm and the active site exposure rate was ≥87%.

[0130] When LPPS is coupled, the amount of DIPEA is reduced to 2.8 eq to avoid side reactions caused by the synergistic effect of high concentration of HP-β-CD and organic base, and the dimer impurities are ≤2.0%.

[0131] Everything else remains the same as in Example 1.

[0132] Example 1: A semaglutide large fragment conjugation process based on SPPS-LPPS hybrid method, differing from Example 1 in that the HP-β-CD concentration used in the LPPS conformation regulation stage is 3.8%, as detailed below:

[0133] Dissolve 1.14g HP-β-CD in 10mL anhydrous DMF to prepare a 3.8% HP-β-CD solution; add 0.6g PEG-200 and stir for 6min to form a homogeneous mixture. The mass ratio of HP-β-CD to PEG-200 is 1.9:1, which takes into account both hydrophobic inclusion and anti-aggregation effects.

[0134] The dropwise addition rate of the regulator mixture was 1 mL / min, and the mixture was stirred at 22℃ for 30 min after addition. The content of fragment I β-sheet detected by circular dichroism chromatography was ≤10%, and the exposure rate of active site detected by fluorescent probe method was ≥86%.

[0135] When fragment I is activated, the active ester formation rate is ≥99%, the LPPS coupling completion rate is ≥99%, the purity of the coupling intermediate is ≥99.8%, and the dimer impurity is ≤1.2%.

[0136] Everything else remains the same as in Example 1.

[0137] Example 8 illustrates a large-fragment conjugation process for semaglutide based on a SPPS-LPPS hybrid method. The difference from Example 1 is that the SPPS-purified fragments achieve the minimum acceptable purity, with fragment I having a purity of 97.5% and fragment II having a purity of 98%. Specific details are as follows:

[0138] For fragment I purification, the elution gradient on a reverse-phase C18 column was 0-70 min, with acetonitrile at 20%-43%, and the main peak fraction with a retention time of 17.8-18.5 min was collected, controlling the content of oxidized peptide impurities to 0.5%. For fragment II purification, the elution gradient was 0-70 min, with acetonitrile at 30%-48%, and the content of single impurities was 0.4%.

[0139] During coupling, the molar ratio of fragment I to fragment II was increased to 1:1.15 to compensate for the loss of active sites in the low-purity fragment; the room temperature ripening reaction time was extended to 3.2 h to ensure a coupling completion rate of ≥95%.

[0140] The coupling intermediate was purified to a purity of 99.5%, with an overall yield of 58% and no new impurities.

[0141] Everything else remains the same as in Example 1.

[0142] Example 9 illustrates a large-fragment conjugation process for semaglutide based on a SPPS-LPPS hybrid method. Unlike Example 1, the SPPS-purified fragments achieve conventional optimized purity, with fragment I having a purity of 98.8% and fragment II having a purity of 99.2%. Specific details are as follows:

[0143] For fragment I purification, the elution gradient was 0-70 min, with acetonitrile at 20%-45%, and the main peak fraction with a retention time of 17.6-18.6 min was collected, with a single impurity content of 0.2% for the deleted peptide. For fragment II purification, the elution gradient was 0-70 min, with acetonitrile at 30%-50%, and a single impurity content of 0.15%, balancing purity and purification efficiency.

[0144] During fragment I conformation regulation, the HP-β-CD concentration was maintained at 4%, the stirring time was shortened to 28 min, and the active site exposure rate was ≥85%; during coupling, the fragment molar ratio was 1:1.1, the aging reaction time at room temperature was 3 h, and the coupling completion rate was ≥98.5%.

[0145] The SPPS purification time was increased compared to Example 8, the overall yield was 60.5%, and the purity of the coupling intermediate was 99.7%.

[0146] Everything else remains the same as in Example 1.

[0147] Example 10 describes a large fragment conjugation process for semaglutide based on a SPPS-LPPS hybrid method. Unlike Example 1, the SPPS-purified fragments exhibit high purity, with fragment I having a purity of 99.6% and fragment II having a purity of 99.7%. Specific details are as follows:

[0148] For fragment I purification, a slow gradient elution was used: 20% acetonitrile for 0-5 min; 20%-46% acetonitrile for 5-75 min. The narrow-range main peak fraction with a retention time of 18.0-18.4 min was collected, with a single impurity content of 0.1%. For fragment II purification, the elution gradient was: 30% acetonitrile for 0-5 min; 30%-51% acetonitrile for 5-75 min, with a single impurity content of 0.08%.

[0149] The SPPS purification time was increased compared to Example 9, but the LPPS conjugation completion rate was only improved to 99.2%; the purity of the conjugation intermediate was 99.9%, and the overall yield of the whole process was 61%.

[0150] Everything else remains the same as in Example 1.

[0151] Comparative Example 1: Fragment I was prepared according to the method in CN112010961A, with a crude product yield of 82%, a purified product yield of 78%, a purified purity of 95.2%, and a single impurity content of 0.8%; Fragment II was prepared with a crude product yield of 83%, a purified product yield of 80%, a purified purity of 96.5%, and a single impurity content of 0.9%; the peak area of ​​the coupling intermediate was 3.2%, and the peak area of ​​the oxidized impurity was 1.1%. Only fragment I was recovered, with a purity of 88%, and the overall yield of the entire process was 45%.

[0152] In the solid-phase synthesis stage, Comparative Example 1 used ordinary ungrafted PEG-800 CTC resin and did not perform DMF / TFE stage conformation washing, resulting in severe hydrophobic folding of peptide chains and incomplete coupling of sterically hindered amino acids. The fragment yield and purity were significantly lower than those of the present invention.

[0153] In the liquid-phase coupling stage, Comparative Example 1 lacked the HP-β-CD-PEG-200 composite conformation control system, resulting in poor solubility of the fragments in the liquid phase, insufficient exposure of active sites, low coupling efficiency, and high content of impurities such as dimers. At the same time, no efficient fragment recovery process was established, and the utilization rate of unreacted fragments was low.

[0154] The overall yield of Comparative Example 1 was 45%, which is lower than that of the present invention. It also had a high content of single impurities and high solvent consumption, which does not meet the requirements of industrial mass production for yield, purity and cost control. The present invention, through SPPS-LPPS synergistic optimization of the entire process, achieves a double improvement in yield and purity, while reducing impurities and solvent consumption, which is more suitable for the industrial production needs of semaglutide.

[0155] The technical scope of this invention is not limited to the content described above. Those skilled in the art can make various modifications and variations to the above embodiments without departing from the technical concept of this invention, and all such modifications and variations should fall within the protection scope of this invention.

Claims

1. A semaglutide large fragment conjugation process based on SPPS-LPPS hybrid method, characterized in that, Includes the following steps: S1: Semaglutide was cleaved into fragment I consisting of amino acids 1-21 and fragment II consisting of amino acids 22-31. Fragment I and fragment II were synthesized separately using solid-phase synthesis. In the solid-phase synthesis process, polyethylene glycol-800 grafted trichlorotriphenylmethyl resin with a substitution degree of 0.35-0.45 mmol / g was selected as the solid-phase support. The solid-phase support was first swollen and activated, then the initial C-terminal amino acid was loaded and the unreacted active site was blocked. The peptide chain was extended by first removing the N-terminal protecting group and then using a differentiated activation system for amino acid coupling in a cyclic operation. The differentiated activation system includes, for conventional sterically hindered amino acids, an activation system composed of Fmoc-amino acid, 1,3-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole, dissolved in N,N-dimethylformamide; and for highly sterically hindered amino acids, an activation system composed of Fmoc-amino acid, (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylamino-morpholine-carbonhexafluorophosphate and 1-hydroxy-7-azobenzotriazole, dissolved in a mixture of N,N-dimethylformamide and dimethyl sulfoxide. After each preset round of amino acid coupling, the peptide chain conformation is controlled by washing with a mixture of N,N-dimethylformamide and trifluoroethanol, wherein the volume ratio of N,N-dimethylformamide to trifluoroethanol in the mixture is 2.5-3.5:

1. Finally, the peptide chain is cut and purified with resin to obtain fragment I with a purity ≥97.5% and a single impurity content ≤0.5% and fragment II with a purity ≥98% and a single impurity content ≤0.5%. S2: Fragment I and fragment II were coupled using a liquid-phase synthesis method. First, the conformation of fragment I was controlled using a composite control system of hydroxypropyl-β-cyclodextrin and polyethylene glycol-200. The concentration of hydroxypropyl-β-cyclodextrin in the composite control system was 3.5%-4.5%, and the mass ratio of hydroxypropyl-β-cyclodextrin to polyethylene glycol-200 was 1.75-2.25:

1. Then, the conformationally controlled fragment I was precisely activated for carboxyl groups using an activation system of N-hydroxysuccinimide, N,N'-diisopropylcarbodiimide, and imidazole. The molar ratio of N-hydroxysuccinimide, N,N'-diisopropylcarbodiimide, and imidazole was 1.1-1.2:1.2:0.

4. The precise activation was carried out in an ice bath environment at 0-5℃ for 12-18 min. Simultaneously, the N-terminal protecting group of fragment II was removed and washed. The deprotected fragment II was added dropwise to the activated fragment I solution at a molar ratio of 1:1.1-1.

5. The pH of the reaction system was adjusted to 8.0-8.5 using N,N-diisopropylethylamine. The mixture was first stirred in an ice bath at 0-5℃ for 30 min, and then heated to 22-25℃ and stirred for 3-3.2 h to complete the coupling reaction. After the coupling reaction was terminated, the target coupling product was separated by isopropyl ether precipitation, and the filtrate was treated with n-hexane for separation and concentration to recover unreacted fragment I. S3: The target conjugated product is purified and freeze-dried to obtain the semaglutide conjugated intermediate.

2. The semaglutide large fragment conjugation process based on the SPPS-LPPS hybrid method according to claim 1, characterized in that: The activation system of conventional sterically hindered amino acids described in S1 is activated at room temperature for 5-8 min and then coupled for 2-2.5 h.

3. The semaglutide large fragment conjugation process based on the SPPS-LPPS hybrid method according to claim 1, characterized in that: The sterically hindered amino acid activation system described in S1 is activated at 30℃ for 8-10 min and then coupled for 2.5-3 h.

4. The semaglutide large fragment conjugation process based on the SPPS-LPPS hybrid method according to claim 1, characterized in that: The isopropyl ether in S2 is pre-cooled to 10-20°C, the volume of the isopropyl ether added is 2-3 times the volume of the coupling termination liquid, the volume ratio of n-hexane to filtrate is 1:1, and the shaking and layering time is 15-25 min.

5. The semaglutide large fragment conjugation process based on the SPPS-LPPS hybrid method according to claim 1, characterized in that: The resin cutting described in S1 uses a cutting fluid containing trifluoroacetic acid, trifluoroethanol and ethylenedithiol. The volume percentage of TFA in the cutting fluid is 6%, TFE is 4%, EDT is 0.5%, and the cutting time is 1 hour.

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