Solid-phase synthesis method of GIP / GLP-1 receptor agonist

Abstract

The invention provides a solid-phase synthesis method of a GIP / GLP-1 receptor agonist. Amidated resin with an Fmoc protected amino group is used as a raw material, a solid-phase polypeptide synthesis method is used for preparing the tilpoitide, amino acid and fragment polypeptide are sequentially coupled according to an amino acid sequence of the tilpoitide, finally, the tilpoitide-resin is cracked to obtain a tilpoitide crude product, and then the tilpoitide pure product is obtained through operations such as purification and the like. Wherein the used polypeptide fragments comprise the following components in parts by weight: 1 to 5 parts of pentapeptide fragments: Boc-Tyr (tBu)-Aib-Glu (OtBu)-Gly-Thr (tBu)-OH, 6 to 7 parts of dipeptide fragments: Fmoc-Phe-Thr (tBu)-OH, 8 to 10 parts of tripeptide fragments: Fmoc-Ser (tBu)-Asp (OtBu)-Tyr (tBu)-OH, 11 to 14 parts of tetrapeptide fragments: Fmoc-Ser (tBu)-Ile-Aib-Leu-OH, and 36 to 38 parts of tripeptide fragments: Fmoc-Pro-Pro-Pro-OH. According to the method, through synthesis and coupling of the intermediate polypeptide, generation of impurities is remarkably reduced, the purity of crude peptide is improved, and a potential synthesis mode is provided for current preparation of telpotide.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a solid-phase synthesis method for a GIP / GLP-1 receptor agonist. Background Technology

[0002] GIP / GLP-1 receptor dual agonists are an emerging and rapidly developing class of drugs, with telpoitin as a benchmark.

[0003] Tirzepatide is a novel incretin-based antidiabetic drug and the world's first dual agonist targeting both the glucose-dependent insulinotropic peptide (GIP) receptor and the glucagon-like peptide-1 (GLP-1) receptor. Its development was driven by the goal of achieving superior glycemic control and weight management by superimposing the GIP pathway synergistic effect on a single GLP-1 target drug. The specific sequence of tirzepatide is Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-(Eic-γ-Glu-AEEA-AEEA-Lys)-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2, with the molecular formula C2. 225 H 348 N 48 O 68 Its molecular weight is 4810.53. The structural formula is shown in Formula I below:

[0004]

[0005] Formula I

[0006] The chemical structure of telpolide contains a linear polypeptide of 39 amino acids (including 2 non-natural amino acids Aib) and C. 20 In solid-phase synthesis, there are some technical difficulties in the coupling synthesis of non-natural amino acids and long amino acid chains for fatty acid side chains.

[0007] Patent application CN119529056A discloses a solid-phase preparation method for telpolide, which sequentially couples amino acids and small peptide fragments. By selecting specific small peptide fragments and specific coupling agents, the number of coupling reactions in the solid-phase synthesis method is greatly reduced and the efficiency of the coupling reaction is improved. The yield of the crude telpolide is 105% and the purity is 75.74%. However, it still has the disadvantages of low purity of crude telpolide and high reagent cost.

[0008] Patent application CN119241683A discloses a method for synthesizing telpoide. The method includes first synthesizing fully protected peptides 1-17 and 18-39, respectively, and then performing liquid-phase splicing and deprotection treatment on the fully protected peptides 1-17 and 18-39 to obtain crude telpoide with a purity of 81.48%. This method has the disadvantages of difficult intermediate purification, difficult purification steps, and difficulty in controlling the reaction process in subsequent liquid-phase reactions, which is not conducive to large-scale production.

[0009] Patent application CN118666986A discloses a method for preparing telpoeptide, comprising preparing a peptide resin coupled with amino acid derivatives 15-39AA, an R1-(3-14AA)-OH fragment, or an R1-(5-14AA)-OH fragment; and then sequentially coupling R1-(3-14AA)-OH and R1-Tyr(R2)-Aib-OH onto the peptide resin coupled with amino acid derivatives 15-39AA using a solid-phase synthesis method. Alternatively, R1-(5-14AA)-OH, R1-Gly-OH, R1-Glu(R2)-OH, and R1-Tyr(R2)-Aib-OH can be sequentially coupled onto a peptide resin coupled with amino acid derivatives 15-39AA using a solid-phase synthesis method. Finally, the peptide resin and protecting group are cleaved to obtain telpoeptide. The purity of the crude telpoeptide prepared by this method is only 63.40%~65.70%, and the yield is 95.56%~103.25%.

[0010] CN115160429A discloses a solid-phase synthesis method for amino acid fragments containing Aib, wherein Example 33 involves the use of amino acid fragments Fmoc-Ile-Aib-Leu-OH and Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-OH, but the purity of the crude thiopeptide prepared by this method is only 75.3% at most, and the yield is 58.9%.

[0011] In the aforementioned prior art, when preparing telpoide using the conventional Fmoc solid-phase synthesis strategy, the synthesis process on the resin often faces problems such as increased side reactions and accumulation of deleted sequences due to the long target polypeptide sequence, the presence of multiple hydrophobic amino acids, and fragments that easily form secondary structures. This results in generally low purity of the crude peptide obtained from cleavage, typically only 63.4%~81.48%. The low purity of the crude peptide severely increases the burden on subsequent purification processes, significantly reduces the final yield, and increases production costs.

[0012] Given the aforementioned technical problems with existing technologies, there is an urgent need for a more efficient solid-phase synthesis method for peptides that yields crude peptides with higher purity. Summary of the Invention

[0013] The purpose of this invention is to overcome the above-mentioned shortcomings in the prior art and provide a new solid-phase synthesis method for GIP / GLP-1 receptor agonists, thereby achieving higher synthesis efficiency, higher purity of crude peptides, fewer impurities, and greater suitability for industrial production.

[0014] This invention employs a multi-fragment synthesis method, using pentapeptide fragments (1-5), dipeptide fragments (6-7), tripeptide fragments (8-10), tetrapeptide fragments (11-14), and tripeptide fragments (36-38) to couple amino acids that are later in the sequence. The use of amino acid fragments addresses the low reaction efficiency of non-natural amino acids (Aib) during coupling. For example, the solid-phase synthesis strategy using pentapeptide fragments (1-5: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-OH) offers higher reaction efficiency compared to the previously used tetrapeptide fragment strategy (Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH). Furthermore, the combination of terminal 1-14 peptide fragments effectively reduces the number of Fmoc removal operations, lowers the mismatch rate, improves the reaction environment kinetically, and significantly increases the purity of the crude peptide.

[0015] This invention provides the following technical solution: a solid-phase synthesis method for a GIP / GLP-1 receptor agonist, comprising the following steps:

[0016] Step 1: Following the Fmoc solid-phase synthesis method, using an amidated resin with Fmoc-protected amino groups as the solid-phase synthesis support, after deprotection with a deprotecting agent, the corresponding Fmoc-protected amino acid and peptide fragment are added to the coupling solvent for coupling reaction. After the reaction is complete, Fmoc is removed with a deprotecting agent, and then coupled with the next Fmoc-protected amino acid and peptide fragment in the following sequence to obtain telpoeptide-resin: sequentially with Fmoc-Ser(tBu)-OH, Fmoc-Pro-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, and Fmoc-Ser(tBu)-OH, Fmoc-Pro-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, and Fmoc-Ser(tBu)-OH, Fmoc-Pro-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, and Fmoc-Ala-Pro-OH, respectively. c-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH,, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmo c-Trp-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu))-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys-OH, Fmoc-Asp-OH, Fmoc-Ser(tBu)-Ile-Aib-L eu-OH, Fmoc-Ser(tBu)-Asp(OtBu)-Tyr(tBu), Fmoc-Phe-Thr(tBu)-OH, Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-OH coupling reaction.

[0017] Step 2: Add the telpoeptide-resin obtained in Step 1 to the lysis buffer for lysis, add the precipitating solvent to the lysis solution to induce crystallization, filter and dry to obtain crude telpoeptide.

[0018] In one embodiment of the present invention, in step one, the amidated resin with Fmoc protected amino groups is selected from one or more of Fmoc-Rink Amide-AM Resin resin, Fmoc-Sieber Amide Resin resin, Fmoc-Knorr Amide Resin resin, or Fmoc-PAL-PEG Resin resin.

[0019] In one embodiment of the present invention, the degree of substitution of the resin is 0.4-0.8 mmol / g.

[0020] In one embodiment of the present invention, in step one, the deprotection agent includes an aprotic organic solvent containing an organic base; in one embodiment, the deprotection agent can be any one of the following: a DCM solution containing 15-30% piperidine (PIP) by volume, a DMF solution containing 15-30% piperidine, or an NMP solution containing 15-30% piperidine, more specifically, a DMF solution containing 20% ​​piperidine.

[0021] In one embodiment of the present invention, in step one, for the coupling method of the non-natural amino acid Aib, the pentapeptide fragments 1-5: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-OH and the tetrapeptide fragments 11-14: Fmoc-Ser(tBu)-Ile-Aib-Leu-OH are directly used. The defect of low coupling efficiency of non-natural amino acids is solved by directly coupling the peptide fragments protected by Fmoc, thereby improving the reaction yield and purity.

[0022] In one specific embodiment of the present invention, the Fmoc-protected amino acids and polypeptide fragments in step one are pre-activated before the coupling reaction. Pre-activation can significantly reduce the occurrence of anisotropic peptides and deletion peptides in the product, reduce amino acid racemization, reduce product isomer impurities, facilitate preparation and purification, and improve product yield and purity.

[0023] As a specific embodiment of the present invention, the activating reagent used for pre-activation before the coupling reaction described in step one is any one of HOBT / DIC, HOAT / DIC, HBTU / DIC, HATU / DIC, HOBT / DIEA, HOAT / DIEA, HBTU / DIEA, HATU / DIEA, TBTU / DIC, CDI / DIEA, and EEDQ / DIEA.

[0024] As a specific embodiment of the present invention, before the coupling reaction described in step one, the molar ratio of the activating reagent used for pre-activation to the amidated resin with Fmoc protected amino groups is 4~6:1.

[0025] As a specific embodiment of the present invention, the coupling reaction temperature in each step of step one is 15~30℃.

[0026] In one embodiment of the present invention, the coupling solvent in step one is an organic solvent, which is selected from one or more of NMP, DMSO, acetonitrile, ethanol, tetrahydrofuran, and DMF.

[0027] In one specific embodiment of the present invention, the lysis buffer in step two is a combination of TFA, TIS, and EDT. The lysis buffer described in this invention does not contain added water, which significantly improves the product properties and prevents stickiness.

[0028] In one specific embodiment of the present invention, the mass ratio of the lysis solution in step two is TFA:TIS:EDT=90-95:1-5:1-5, preferably TFA:TIS:EDT=95:3:2.

[0029] In one embodiment of the present invention, the precipitation solvent in step two is an ether solvent, which includes one or more of petroleum ether, diethyl ether, dipropyl ether, isopropyl ether, methyl tert-butyl ether, and ethylene glycol dimethyl ether. In a specific embodiment of the present invention, in step two, the mass-to-volume ratio (g / mL) of the telpopeptide-resin to the lysis buffer is 1:5 to 1:0, or more specifically 1:10.

[0030] In one specific embodiment of the present invention, in step two, the pyrolysis temperature is 15-35℃ and the pyrolysis time is 0.5-2 h.

[0031] As a specific embodiment of the present invention, the crude telpoeptide is dried in a vacuum drying oven containing desiccants (phosphorus pentoxide, calcium chloride, etc.), which can reduce impurities generated by hydrolysis, increase the yield of crude product, and reduce costs.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] This invention employs a multi-fragment synthesis method, using pentapeptide fragments (1-5), dipeptide fragments (6-7), tripeptide fragments (8-10), tetrapeptide fragments (11-14), and tripeptide fragments (36-38) to couple amino acids in the later sequence. The use of amino acid fragments solves the problem of low reaction efficiency during the coupling of non-natural amino acids (Aib). For example, the solid-phase synthesis strategy using the 1-5 pentapeptide fragment (Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-OH) has higher reaction efficiency compared to the previously commonly used tetrapeptide fragment strategy (Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH). The combination of terminal 1-14 peptide fragments effectively reduces the number of Fmoc removal operations in the reaction, lowers the mismatch rate, improves the reaction environment kinetically, and significantly increases the purity of the crude peptide. Compared with the prior art, the purity of crude peptide of telpoeptide is greatly increased from 81.48% to 93.36%~97.62%, with an absolute increase of at least about 12% and a relative increase of more than 14.5%.

[0034] Meanwhile, due to the increased purity, the proportion of the target main peak increases significantly, while the number of impurity peaks decreases. In the HPLC chromatogram of the product, the impurities with relative retention times between 0.9 and 1.1 are greatly reduced from 3.92% to 0.46% to 0.55%. In subsequent purification steps, this results in a reduction in the difficulty of separating the target product from structurally similar impurities (such as deleted sequences). This helps to obtain active pharmaceutical ingredients that meet higher pharmacopoeia standards, with lower levels of related impurities, and fundamentally ensures product quality and batch consistency.

[0035] The aforementioned increase in purity and decrease in impurity content are significant and unexpected in the field of long-chain complex peptide synthesis, representing a key breakthrough in the optimization of the entire production process. Attached Figure Description

[0036] Figure 1 This is the HPLC chromatogram of the crude telpoeptide obtained in Example 1.

[0037] Figure 2 This is the mass spectrum of the crude telpoeptide obtained in Example 1.

[0038] Figure 3 This is the HPLC chromatogram of the crude telpoeptide obtained in Example 2. Detailed Implementation

[0039] The present invention will be further described below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0040] To facilitate understanding of the present invention by those skilled in the art, the technical solution of the present invention will be further described below in conjunction with specific embodiments, but the following content should not limit the scope of protection claimed in the claims of the present invention in any way.

[0041] The amino acids and resins used in this invention were all purchased from Jier Biochemical Co., Ltd.

[0042] The English abbreviations and their Chinese names appearing in this invention are as follows:

[0043]

[0044] Example 1

[0045] (1) Preparation of telpoeptide-resin

[0046] Weigh 10 g (6.1 mmol, degree of substitution 0.61 mmol / g) of Fmoc-Rink-Amide-AM-Resin resin and add it to a solid-phase peptide synthesizer. Add 60 mL of DCM and purge the resin with nitrogen for 5 minutes to swell it. Then, dry the resin under vacuum. Add 60 mL of the prepared deprotection reagent, 15% PIP / DMF solution, three times for 10 minutes each time. Wash the resin six times with 60 mL of DMSO each time. Weigh 9.35 g (24.4 mmol, 4 eq) of Fmoc-Ser(tBu)-OH, 3.31 g (24.4 mmol, 4 eq) of HOAT, and 60 mL of DMSO into a 100 mL single-necked flask and dissolve completely. Incubate on ice (0-5℃) for 10 minutes. Add 3.8 mL of DIC (24.4 mmol, 4 eq) and pre-activate for 20 minutes. Add the solution to the resin, purge with nitrogen, and couple the reaction at 15℃ for 60 minutes. Take a small amount of resin and test it with Kaiser's reagent to determine the reaction endpoint. If the resin is colorless and transparent, the reaction is complete. Continue the reaction for another 60 minutes (if the resin is colored, the reaction is incomplete; test every 30 minutes until the resin is colorless and transparent, then continue the reaction for another 60 minutes). After the reaction reaches the endpoint, remove the solvent and wash the resin 6 times with 80 mL of DMSO each time. Repeat the above steps, condensing the telpopeptide sequence sequentially from C-terminus to N-terminus, completing the condensation with Fmoc-Pro-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, and Fmoc-A la-OH, Fmoc-Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioicacid(mon-tBu))-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys-OH , Fmoc-Asp-OH, Fmoc-Ser(tBu)-Ile-Aib-Leu-OH, Fmoc-Ser(tBu)-Asp(OtBu)-Tyr(tBu), Fmoc-Phe-Thr(tBu)-OH, Boc-Tyr(tBu)-Aib-Glu(OtBu)

[0047] The coupling reaction of -Gly-Thr(tBu)-OH was carried out. Finally, the following thiopeptide-resin was obtained: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln(Trt)-Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioicacid(mon-tBu))-Ala-Phe-Val-Gln(Trt)-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-35.64 g.

[0048] (2) Preparation of crude telpoeptide

[0049] A lysis buffer (TFA:TIS:EDT = 90:5:5, mass ratio, same below) was prepared to lyse telpoeptide-resin (1 g resin = 10 mL lysis buffer) at 25℃ for 1.5 h. The resin was filtered off, and isopropyl ether was added to crystallize (lysis reagent to isopropyl ether volume ratio 1:3). After centrifugation, the solid was washed twice with isopropyl ether, centrifuged again, and dried in a vacuum drying oven to obtain 3.78 g of crude telpoeptide white powder. The yield was 3.78 / (4*0.521*1568.84 / 1000) = 115.6%, and the purity was 93.36% (HPLC chromatogram as shown). Figure 1 As shown, the mass spectrum is as follows Figure 2 (As shown).

[0050] Example 2

[0051] (1) Preparation of telpoeptide-resin

[0052] Weigh 10 g (6.1 mmol, degree of substitution 0.61 mmol / g) of Fmoc-Rink-Amide-AM-Resin resin and add it to a solid-phase peptide synthesizer. Add 60 mL of DCM, purge the resin with nitrogen for 5 minutes to swell, and then dry it under vacuum. Add 60 mL of the prepared deprotection reagent 20% PIP / DMF solution three times for 10 minutes each time. Wash the resin six times with 60 mL of DMF each time. Weigh 11.69 g (30.5 mmol, 5 eq) of Fmoc-Ser(tBu)-OH, 4.14 g (30.5 mmol, 5 eq) of HOBT, and 60 mL of DMF into a 100 mL single-necked flask and dissolve completely. Incubate on ice (0-5℃) for 10 minutes. Add 4.75 mL of DIC (30.5 mmol, 5 eq) and pre-activate for 20 minutes. Add the solution to the resin, purge with nitrogen, and couple the reaction at 25℃ for 60 minutes. Take a small amount of resin and test it with Kaiser's reagent to determine the reaction endpoint. If the resin is colorless and transparent, the reaction is complete. Continue the reaction for another 60 minutes (if the resin is colored, the reaction is incomplete; test every 30 minutes until the resin is colorless and transparent, then continue the reaction for another 60 minutes). After the reaction reaches the endpoint, remove the solvent and wash the resin 6 times with 80 mL of DMF each time. Repeat the above steps, condensing the telpopeptide sequence sequentially from C-terminus to N-terminus, completing the condensation with Fmoc-Pro-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, and Fmoc- Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioicacid(mon-tBu))-OH、Fmoc-Gln(Trt)-OH、Fmoc-Ala-OH、Fmoc-Ile-OH、Fmoc-Lys-OH、Fmoc-Asp-OH、Fmoc -Coupling reactions of Ser(tBu)-Ile-Aib-Leu-OH, Fmoc-Ser(tBu)-Asp(OtBu)-Tyr(tBu), Fmoc-Phe-Thr(tBu)-OH, Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-OH.Finally, the following thiopeptide-resin was obtained: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln(Trt)-Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioicacid(mon-tBu))-Ala-Phe-Val-Gln(Trt)-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-35.80g.

[0053] (2) Preparation of crude telpoeptide

[0054] A lysis buffer (TFA:TIS:EDT = 95:3:2, mass ratio, same below) was prepared to lyse the resin peptide (1g resin = 5 mL lysis buffer) at 25℃ for 1.5 h. The resin was filtered off, and methyl tert-butyl ether (MTBE) was added to crystallize the peptide (lysis buffer to MTBE volume ratio = 1:10). The solid was centrifuged, washed twice with MTBE, centrifuged again, and dried in a vacuum drying oven to obtain 3.80 g of crude white powder of telpopeptide. The yield was 3.80 / (4*0.521*1568.84 / 1000) = 116.2%, and the purity was 97.62%. Figure 3 (As shown).

[0055] Example 3

[0056] (1) Preparation of telpoeptide-resin

[0057] Weigh 10 g (6.1 mmol, degree of substitution 0.61 mmol / g) of Fmoc-Rink-Amide-AM-Resin resin and add it to a solid-phase peptide synthesizer. Add 60 mL of DCM and purge the resin with nitrogen for 5 minutes to swell it. Then, dry the resin under vacuum. Add 60 mL of the prepared deprotection reagent, 30% PIP / DMF solution, three times for 10 minutes each time. Wash the resin six times with 60 mL of tetrahydrofuran each time. Weigh 14.03 g (36.6 mmol, 6 eq) of Fmoc-Ser(tBu)-OH, 6.21 g (36.6 mmol, 6 eq) of HOBT, and 60 mL of tetrahydrofuran into a 100 mL single-necked flask and dissolve completely. Incubate on ice (0-5℃) for 10 minutes, then add 5.7 mL of DIC (36.6 mmol, 6 eq) for pre-activation for 20 minutes. Add the solution to the resin, purge with nitrogen, and couple the reaction at 30℃ for 60 minutes. Take a small amount of resin and test it with Kaiser's reagent to determine the reaction endpoint. If the resin is colorless and transparent, the reaction is complete. Continue the reaction for another 60 minutes (if the resin is colored, the reaction is incomplete; test every 30 minutes until the resin is colorless and transparent, then continue the reaction for another 60 minutes). After the reaction reaches the endpoint, remove the solvent and wash the resin six times with 80 mL of tetrahydrofuran each time. Repeat the above steps, condensing the telposide sequence sequentially from C-terminus to N-terminus, completing the condensation with Fmoc-Pro-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, and Fmoc-Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu))-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys-OH, Fmoc-Asp-OH, Fmoc-Ser(tBu)-Ile-Aib-Le Coupling reactions of u-OH, Fmoc-Ser(tBu)-Asp(OtBu)-Tyr(tBu), Fmoc-Phe-Thr(tBu)-OH, Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-OH.Finally, the following thiopeptide-resin was obtained: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln(Trt)-Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioicacid(mon-tBu))-Ala-Phe-Val-Gln(Trt)-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-35.52 g.

[0058] (2) Preparation of crude telpoeptide

[0059] A lysis buffer (TFA:TIS:EDT = 94:4:4, mass ratio) was prepared to lyse the resin peptide (1g resin = 20mL lysis buffer) at 25℃ for 1.5 h. The resin was filtered off, and petroleum ether was added to crystallize the peptide (lysis buffer to methyl tert-butyl ether volume ratio = 1:20). The peptide was centrifuged, and the solid was washed twice with petroleum ether, centrifuged again, and dried in a vacuum drying oven to obtain 3.75 g of crude telpopeptide white powder. The yield was 3.75 / (4*0.521*1568.84 / 1000) = 114.7%, and the purity was 95.36%.

[0060] Comparative Example 1

[0061] Referring to the synthesis method disclosed in patent application CN119241683A, fully protected peptides 1-17 and 18-39 were first synthesized separately. Then, the fully protected peptides 1-17 and 18-39 were subjected to liquid phase splicing and deprotection treatment to obtain crude telpoeptide with a purity of 81.48%.

[0062] The comparison between Examples 1-3 and Comparative Example 1 is shown in Table 1 below. Compared with the prior art, the purity of crude peptide of telpoeptide is greatly increased from 81.48% to 93.36%-97.62%, with an absolute increase of at least about 12% and a relative increase of more than 14.5%.

[0063] Meanwhile, due to the increased purity, the proportion of the target main peak increases significantly, while the number of impurity peaks decreases. In the HPLC chromatogram of the product, the impurities with relative retention times between 0.9 and 1.1 are greatly reduced from 3.92% to 0.46%-0.55%. In subsequent purification steps, this results in a reduction in the difficulty of separating the target product from structurally similar impurities (such as deleted sequences). This helps to obtain active pharmaceutical ingredients that meet higher pharmacopoeia standards, with lower levels of related impurities, and fundamentally ensures product quality and batch consistency.

[0064] The aforementioned increase in purity and decrease in impurity content are significant and unexpected in the field of long-chain complex peptide synthesis, representing a key breakthrough in the optimization of the entire production process.

[0065] The improved purity of the crude telpoeptide of the present invention is mainly due to the synthesis strategy of the present invention, which effectively suppresses the main side reaction pathways, can greatly reduce the mismatch rate in the amino acid coupling process, and reduce the shedding of commonly used protecting groups of amino acids. This process mainly reduces the proportion of deletion peptides, the main impurity.

[0066] Table 1 Comparison of results between Examples 1-3 and Comparative Example 1

[0067]

Claims

1. A solid-phase synthesis method for a GIP / GLP-1 receptor agonist, characterized in that, Includes the following steps: Step 1: Following the Fmoc solid-phase synthesis method, using an amidated resin with Fmoc-protected amino groups as the solid-phase synthesis support, after deprotection with a deprotecting agent, the corresponding Fmoc-protected amino acid and peptide fragment are added to the coupling solvent for coupling reaction. After the reaction is complete, Fmoc is removed with a deprotecting agent, and then coupled with the next Fmoc-protected amino acid and peptide fragment in the following sequence to obtain telpoeptide-resin: sequentially with Fmoc-Ser(tBu)-OH, Fmoc-Pro-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, and Fmoc-Ser(tBu)-OH, Fmoc-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, and Fmoc-Ser(tBu)-OH, Fmoc-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, and Fmoc-Ala-OH, Fmoc-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, and Fmoc-Ala-OH, Fmoc-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Pro-Pro-OH, Fmoc-Ala ... c-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH,, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmo c-Trp-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu))-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys-OH, Fmoc-Asp-OH, Fmoc-Ser(tBu)-Ile-Aib-L eu-OH, Fmoc-Ser(tBu)-Asp(OtBu)-Tyr(tBu), Fmoc-Phe-Thr(tBu)-OH, Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-OH coupling reaction; Step 2: Add the telpoeptide-resin obtained in Step 1 to the lysis buffer for lysis, add the precipitation solvent to the lysis solution to induce crystallization, filter and dry to obtain crude telpoeptide.

2. The solid-phase synthesis method of a GIP / GLP-1 receptor agonist according to claim 1, characterized in that, In step one: the amidated resin with Fmoc protected amino groups is selected from one or more of Fmoc-Rink Amide-AM Resin resin, Fmoc-Sieber Amide Resin resin, Fmoc-Knorr Amide Resin resin or Fmoc-PAL-PEG Resin resin.

3. The solid-phase synthesis method of a GIP / GLP-1 receptor agonist according to claim 1, characterized in that, In step one, the deprotection agent includes an aprotic organic solvent containing an organic base, including any one of the following: a DCM solution containing 15-30% piperidine by volume, a DMF solution containing 15-30% piperidine, and an NMP solution containing 15-30% piperidine.

4. The solid-phase synthesis method of a GIP / GLP-1 receptor agonist according to claim 1, characterized in that, In step one: the amino acids and polypeptide fragments protected by Fmoc are pre-activated before the coupling reaction. The pre-activation reagent is one or more of HOBT / DIC, HOAT / DIC, HBTU / DIC, HATU / DIC, HOBT / DIEA, HOAT / DIEA, HBTU / DIEA, HATU / DIEA, TBTU / DIC, CDI / DIEA, and EEDQ / DIEA.

5. The solid-phase synthesis method of a GIP / GLP-1 receptor agonist according to claim 4, characterized in that: The molar ratio of the preactivated reagent to the amidated resin with Fmoc protected amino groups is 4~6:

1.

6. The solid-phase synthesis method of a GIP / GLP-1 receptor agonist according to claim 1, characterized in that, The coupling reaction temperature in step one is 15~30℃.

7. The solid-phase synthesis method of a GIP / GLP-1 receptor agonist according to claim 1, characterized in that, In step one, the coupling solvent is an organic solvent, which is selected from one or more of NMP, DMSO, acetonitrile, ethanol, tetrahydrofuran, and DMF.

8. The solid-phase synthesis method of a GIP / GLP-1 receptor agonist according to claim 1, characterized in that, The mass ratio of the lysis solution in step two is TFA:TIS:EDT = 90-95:1~5:1~5, preferably TFA:TIS:EDT = 95:3:

2.

9. The solid-phase synthesis method of a GIP / GLP-1 receptor agonist according to claim 1, characterized in that, The precipitation solvent in step two is an ether solvent, which includes one or more of petroleum ether, diethyl ether, dipropyl ether, isopropyl ether, methyl tert-butyl ether, and ethylene glycol dimethyl ether.

10. A solid-phase synthesis method for a GIP / GLP-1 receptor agonist according to any one of claims 1 to 9, characterized in that, The mass-to-volume ratio of the telpopeptide-resin to the lysis buffer is 1:5 to 1:20 (g / mL).

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