A method for the synthesis of retalupeptide

Abstract

The application discloses a synthetic method of Retalutide, and belongs to the technical field of polypeptide synthesis. The synthetic method adopts a solid-phase Fmoc synthesis strategy, splits the 39-amino-acid sequence of Retalutide into two large fragments of positions 1-28 and 29-39 for parallel synthesis, uses 2-CTC Resin and Sieber Resin as solid-phase carriers respectively, and prepares a target product through coupling, deprotection, selective removal of an OALL protecting group, fragment condensation and two-step cleavage. The synthetic method effectively shortens a synthesis period, avoids resin polycondensation, reduces the generation of impurities such as deletions and racemization, and reduces the consumption of trifluoroacetic acid and methyl tert-butyl ether by optimizing the substitution degree of resin, catalyst ratio, coupling process and cleavage conditions. The synthetic method is simple, has low cost and is suitable for industrial amplification production, and provides a stable and reliable new approach for the efficient preparation of long-acting GLP-1 receptor agonist drugs.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide synthesis, specifically relating to a method for synthesizing retaglutide. Background Technology

[0002] Retaglutide is a novel, long-acting GLP-1 receptor agonist primarily used to treat type 2 diabetes and obesity. It works by mimicking the action of the gut hormone GLP-1, slowing gastric emptying, increasing satiety, and promoting insulin secretion, thereby helping to control blood sugar and reduce weight.

[0003] Invention patent CN118754966A discloses a method for preparing retaglutide, which involves preparing a peptide resin coupled with amino acid derivatives 30-39AA, a fully protected peptide fragment of 21-29, and a fully protected peptide fragment of 7-14, respectively; then, the fully protected peptide fragment of 21-29, R1-Aib-OH, R1-Gln(R2)-OH, R1-Ala-OH, the retaglutide side chain, R1-Lys(R2)-OH, R1-Asp(R2)-OH, the fully protected peptide fragment of 7-14, R1-Phe-OH, R1-Thr(R2)-OH, R1-Gly-OH, R1-Gln(R2)-OH, and R1-Tyr(R2)-Aib-OH are sequentially coupled onto the peptide resin coupled with amino acid derivatives 30-39AA. Finally, the peptide resin and the protecting groups are cleaved to obtain retaglutide, where R1 is an amino protecting group and R2 is a side chain protecting group. This method uses a multi-fragment and stepwise approach to synthesize retaloglutide, which has high synthesis costs and a long cycle, making it unsuitable for mass production.

[0004] Patent CN117903284A discloses a method for preparing retaglutide. The method involves coupling at position 17 using Dde-Lys(Fmoc)-OH, sequentially coupling from the C-terminus to the N-terminus to the 17th amino acid, selectively removing the Dde protecting group, and then sequentially coupling the remaining amino acids. This involves introducing fragments such as Boc-Tyr(tBu)-Aib-OH, Fmoc-Ile-{α-Me-Leu}-Leu-OH, and Fmoc-Gly-Gly-OH to replace the corresponding amino acids at the coupling sites, thus obtaining retaglutide. This method has a long synthesis cycle, and the resin is prone to condensation during synthesis, leading to synthesis difficulties and the potential for deletion impurities. Summary of the Invention

[0005] The purpose of this invention is to provide a simple, short-cycle, high-yield, low-impurity method for synthesizing retaglutide, which is suitable for large-scale industrial production, in order to solve the problems of high synthesis cost, long cycle, easy resin condensation, many missing impurities, and large reagent consumption in the existing technology.

[0006] The technical solution adopted by this invention to achieve its objective is as follows: A method for synthesizing retaglutide, characterized by comprising: The 39 amino acids of retaliglutide were separated into the first peptide segment at positions 1-28 and the second peptide segment at positions 29-39 using a solid-phase Fmoc strategy and synthesized in parallel. Using 2-CTC Resin as the first peptide solid-phase support, a first peptide resin was prepared by coupling, PIP / DMF deprotection, and selective removal of the OALL protecting group by tetrakis(triphenylphosphine)palladium / phenylsilane. Using Sieber Resin as the second peptide solid-phase support, a second peptide product was prepared by coupling, deprotection, cleavage with 2%–4% TFA / DCM solution, and purification. The second peptide product and the first peptide resin were then subjected to fragment condensation using a coupling agent to obtain a complete peptide resin. The complete peptide resin was first cleaved with 0.5%–1.5% TFA / DCM to obtain a fully protected peptide, followed by a second cleavage with a TFA / TIS / deionized water mixed cleavage solution, and precipitation with methyl tert-butyl ether to obtain crude retaglutide. The crude retaglutide was purified by reversed-phase high-performance liquid chromatography to obtain purified retaglutide. The amino acid sequence of retaglutide is shown in SEQ ID NO.1, the amino acid sequence of the first peptide is shown in SEQ ID NO.2, and the amino acid sequence of the second peptide is shown in SEQ ID NO. Shown in NO.3.

[0007] Preferably, the molar ratio of tetra(triphenylphosphine)palladium to CTC Resin is 1.0 to 1.5:1.

[0008] Preferably, the molar ratio of benzylsilane to CTC Resin is 10~15:1.

[0009] Preferably, the molar ratio of the second peptide product to the first peptide resin fragment condensation is 1.2 to 1.5:1.

[0010] Preferably, the coupling agent includes at least one of DIC and HOBT, DIC and Oxyma, HATU and DIEA, and HBTU and DIEA.

[0011] Preferably, the molar ratio of coupling agent to protective amino acid is 1:0.8~1.2.

[0012] Preferably, in the TFA / TIS / deionized water mixed cutting fluid, the volume ratio of TFA to TIS is 35~40:1, and the volume ratio of TIS to deionized water is 1:0.8~1.2.

[0013] Preferably, the degree of substitution of 2-CTC Resin is 0.6~1.0 mmol / g.

[0014] Preferably, the degree of substitution of Sieber Resin is 0.6~1.0 mmol / g.

[0015] Preferably, the chromatographic column of the reversed-phase high-performance liquid chromatography includes a C18 column and / or a boric acid functionalized modified silica column.

[0016] Preferably, the packing material for the modified silica column is boric acid-functionalized modified silica column packing material.

[0017] Preferably, the boric acid functionalized modified silica gel chromatographic packing material is prepared using thiol-functionalized mesoporous silica gel microspheres as the matrix and 3-acrylamidophenylboronic acid as the modified monomer.

[0018] Preferably, the mass ratio of 3-acrylamidophenylboronic acid to thiol-functionalized mesoporous silica microspheres is 1:8~40.

[0019] 3-Acrylamidophenylboronic acid provides covalently fixed boric acid recognition sites for the packing material, significantly improving the separation selectivity of the packing material for retaglutide, efficiently removing missing peptides, racemates, and homologous structure impurities, reducing impurity residues, and improving the purity of the final product. The hypothesized mechanism is that this monomer specifically recognizes and retains retaglutide through a dual interaction of boric acid-diol covalent interaction and amide hydrogen bonding; the pyridine-2-carbamoyl group introduced into the system can construct a stereoselective binding cavity, stabilizing the peptide binding site, reducing non-specific adsorption, and optimizing chromatographic separation.

[0020] More preferably, the modified monomer includes methyl 2-acrylamido-2-methoxyacetic acid ester, and the mass ratio of methyl 2-acrylamido-2-methoxyacetic acid ester to mercapto-functionalized mesoporous silica microspheres is 1:18~22.

[0021] Methyl 2-acrylamido-2-methoxyacetic acid ester, as a hydrophilic copolymer modifier, can improve the surface hydrophilicity and dispersion uniformity of chromatographic packing materials, effectively avoid packing particle agglomeration and mesoporous channel blockage, maintain high specific surface area and excellent mass transfer performance, and make the chromatographic peak shape more symmetrical and the purification efficiency more stable. Its mechanism of action is speculated to be that the molecule contains hydrophilic ethoxy and amide structures, which can form a flexible and uniform hydrophilic layer on the silica gel surface, reduce non-specific hydrophobic adsorption, and can copolymerize with boric acid functional monomers, so that the functional groups are more stably grafted onto the silica gel surface, thereby improving the structural stability and batch repeatability of the packing material.

[0022] This invention also provides a method for synthesizing retaglutide, comprising the steps of preparing a first peptide resin, preparing a second peptide product, preparing crude retaglutide, and purifying retaglutide. Step 1: Preparation of the first peptide resin: 2-CTCResin with a substitution degree of 0.6–1.0 mmol / g was placed in a reactor. The C-terminal 28th amino acid, Fmoc-Glu-OALL, was added and dissolved in DMF. DIEA was added, and the mixture was stirred at 20–30 °C for 6–8 h to complete the loading of the C-terminal amino acid. After the reaction, the solvent was removed, and the resin was thoroughly washed with DMF. A deprotection solution was added to remove the Fmoc protecting group for 25–30 min. Subsequently, the first peptide was loaded from the C-terminal side of the peptide chain. From position 27 to position 1 at the N-terminus, each protecting amino acid is sequentially coupled in steps, including Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-Aib-OH, Fmoc-Ala-OH, and Fmoc-Lys(OtBu-C20-γ-Glu(OtB u)-AEEA), Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Ile-α-Me-Leu-OH, Fmoc-Se r(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc- Thr(tBu)-Phe-OH, Boc-Tyr(tBu)-Aib-Gln(Trt)-Gly-OH; using DIC and HOBt as coupling agents, single-step coupling was performed at 20-30℃ for 1-2 hours. After each coupling step, the resin was washed with DMF to obtain the first peptide precursor resin; tetrakis(triphenylphosphine)palladium and phenylsilane were added to the reactor, and the reaction was carried out at 20-30℃ with stirring for 1-2 hours using DCM as solvent to selectively remove the OALL protecting group and obtain the first peptide resin.

[0023] Preferably, the volume-to-mass ratio of DMF to CTC Resin is 8-12 mL:1 g.

[0024] Preferably, the deprotection solution comprises Pip and DMF, with a volume ratio of Pip to DMF of 1:3 to 5.

[0025] Preferably, the volume-to-mass ratio of the deprotection solution to CTC Resin is 8-12 mL: 1 g.

[0026] Preferably, the molar ratio of the protected amino acid to CTC Resin is 2.0~3.0:1.

[0027] Preferably, the molar ratio of coupling agent to protective amino acid is 1:0.8~1.2.

[0028] Preferably, the molar ratio of Fmoc-Glu-OALL to CTC Resin is 1.5~2.5:1.

[0029] Preferably, the molar ratio of DIEA to CTC Resin is 3.0~5.0:1.

[0030] Preferably, the molar ratio of tetra(triphenylphosphine)palladium to CTC Resin is 1.0 to 1.5:1.

[0031] Preferably, the molar ratio of benzylsilane to CTC Resin is 10~15:1.

[0032] Preferably, the volume-to-mass ratio of DCM to CTC Resin is 15-25 mL: 1 g.

[0033] Step 2: Preparation of the second peptide product: Sieber Resin with a substitution degree of 0.6~1.0 mmol / g is placed in a reactor, DMF is added and stirred to swell for 20~40 min, then deprotection solution is added for 25~30 min; the protected amino acids are then sequentially coupled from position 39 to position 29, including Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Ser(tBu). -OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH; using DIC and HOBt as coupling agents, single-step coupling was performed at 20~30℃ for 1~2h. After each coupling step, the resin was washed with DMF. After all coupling was completed, the terminal Fmoc protecting group was removed to obtain the second peptide resin. The second peptide resin was cleaved with a 2%~4% (v / v) TFA / DCM solution. The cleavage product was washed with DCM to obtain the crude second peptide. The crude second peptide was purified by reversed-phase high-performance liquid chromatography to obtain the finished second peptide.

[0034] Preferably, the volume-to-mass ratio of DMF used for swelling to Sieber Resin is 8-12 mL: 1 g.

[0035] Preferably, the volume-to-mass ratio of the deprotection solution to Sieber Resin is 8-12 mL: 1 g.

[0036] Preferably, the molar ratio of the protected amino acid to Sieber Resin is 2.0~3.0:1.

[0037] Preferably, the molar ratio of coupling agent to protective amino acid is 1:0.8~1.2.

[0038] Preferably, the volume-to-mass ratio of the cutting fluid to the second peptide resin is 6-10 mL: 1 g.

[0039] Step 3: Preparation of crude retaglutide: The second peptide product and the first peptide resin are mixed at a molar ratio of 1.0~1.5:1, a coupling agent is added, and the mixture is stirred at 20~30℃ for 1.5~2.5h. After the reaction is complete, the resin is washed with DMF, treated with methanol, and dried to obtain intact peptide resin. The intact peptide resin is first cleaved with a TFA / DCM solution with a volume fraction of 0.5%~1.5%, and washed with DCM to obtain fully protected peptide. Then, the cleavage solution prepared with TFA, TIS and deionized water is cleaved at 15~25℃ for 1.5~2.5h. Methyl tert-butyl ether is added to the reaction solution for precipitation, and the mixture is filtered, washed, and dried to obtain crude retaglutide.

[0040] Preferably, the coupling agent includes at least one of DIC and HOBT, DIC and Oxyma, HATU and DIEA, and HBTU and DIEA.

[0041] Preferably, the molar ratio of coupling agent to protective amino acid is 1:0.8~1.2.

[0042] Preferably, the volume-to-mass ratio of the cutting fluid to the intact peptide resin is 6-10 mL: 1 g.

[0043] Preferably, the volume ratio of TFA to TIS is 35~40:1.

[0044] Preferably, the volume ratio of TIS to deionized water is 1:0.8~1.2.

[0045] Preferably, the volume-to-mass ratio of the cutting fluid to the fully protected peptide is 1.5~2.5 mL:1 g.

[0046] Preferably, the molar ratio of HOBt to DIC is 1:0.8~1.2.

[0047] Step 4: Preparation of purified ratalutide: The crude ratalutide was purified by reversed-phase high-performance liquid chromatography. The mobile phase was 0.08%~0.12% TFA aqueous solution (v / v) and 0.08%~0.12% TFA acetonitrile solution (v / v) as mobile phase B. The target fraction was collected by gradient elution, concentrated and dried to obtain purified ratalutide.

[0048] More preferably, the crude retaliglutide is purified using a modified silica gel column.

[0049] More preferably, the preparation steps of the modified silica gel column include: dispersing methyltrimethoxysilane in deionized water, adding ammonia under stirring, then adding 3-mercaptopropyltrimethoxysilane, allowing the reaction to stand at 15-30°C for 4-6 h, collecting the precipitate by centrifugation at 7000-9000 rpm for 10-20 min, washing with ethanol, and vacuum drying at 40-60°C for 10-14 h to obtain thiol-functionalized mesoporous silica gel microspheres; dispersing the obtained thiol-functionalized mesoporous silica gel microspheres in toluene, sonicating for 15-25 min, and adding 3-mercaptopropyltrimethoxysilane... Enamide-based phenylboronic acid and methyl 2-acrylamido-2-methoxyacetic acid were used as modifying monomers. Azobisisobutyronitrile was added, and the mixture was stirred at 60-80℃ for 8-12 h under nitrogen protection. After cooling, the mixture was centrifuged at 7000-9000 rpm for 10-20 min, the precipitate was washed with ethanol, and then vacuum dried at 40-60℃ for 10-14 h to obtain boric acid-functionalized modified silica gel chromatographic packing. The boric acid-functionalized modified silica gel chromatographic packing was dispersed in isopropanol, sonicated for 3-7 min, and then packed into a chromatographic column to obtain a modified silica gel chromatographic column.

[0050] More preferably, the mass-to-volume ratio of methyltrimethoxysilane to deionized water is 1 g: 4~6 mL.

[0051] More preferably, the volume ratio of ammonia water to deionized water is 1 μL: 8~12 mL. More preferably, the mass ratio of 3-mercaptopropyltrimethoxysilane to methyltrimethoxysilane is 1:3~5.

[0052] More preferably, the mass-to-volume ratio of thiol-functionalized mesoporous silica microspheres to toluene is 1 g: 12~18 mL.

[0053] More preferably, the mass ratio of 3-acrylamidophenylboronic acid to thiol-functionalized mesoporous silica microspheres is 1:8~40.

[0054] More preferably, the mass ratio of methyl 2-acrylamido-2-methoxyacetic acid ester to thiol-functionalized mesoporous silica microspheres is 1:18~22.

[0055] More preferably, the mass ratio of azobisisobutyronitrile to thiol-functionalized mesoporous silica microspheres is 1:80~120.

[0056] More preferably, the mass-to-volume ratio of modified silica gel chromatographic packing material to isopropanol is 1 g: 12~18 mL.

[0057] This invention employs a solid-phase Fmoc dual-fragment parallel synthesis strategy, rationally resolving the 39 amino acid sequence of retaglutide, and optimizing resin substitution degree, catalyst ratio, coupling process, and cleavage conditions. Therefore, it offers the following advantages: significantly shortened synthesis cycle, avoidance of resin condensation, reduced generation of impurities such as missing peptides and racemic compounds, reduced reagent usage of trifluoroacetic acid and methyl tert-butyl ether, improved crude product yield and finished product purity, and a simple, stable process with low production cost, suitable for industrial-scale production. Therefore, this invention provides a short-cycle, high-yield, low-impurity, low-cost, environmentally friendly, and scalable method for synthesizing retaglutide. Attached Figure Description

[0058] Figure 1 This is a schematic diagram showing the synthesis yield test results of crude retaliglutide.

[0059] Figure 2 This is a schematic diagram showing the results of the purity test for purified retaliglutide.

[0060] Figure 3 This is a schematic diagram showing the specific surface area test results of the modified silica gel chromatographic packing material. Detailed Implementation

[0061] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0062] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0063] The Chinese meanings of the abbreviations used in this invention are shown in Table 1 below.

[0064] Table 1. Chinese meanings of abbreviations

[0065] Example 1: This example provides a method for preparing retaglutide. The preparation steps mainly include the preparation steps of the first peptide resin, the preparation steps of the second peptide, and the preparation steps of the finished retaglutide product, as detailed below.

[0066] The sequence of retaliglutide is shown in SEQ ID NO.1, which is N-terminal → C-terminal, Tyr1 → Aib2 → Gln3 → Gly4 → Thr5 → Phe6 → Thr7 → Ser8 → Asp9 → Tyr 10 →Ser 11 →Ile 12 →(S)-α-MeLeu 13 →Leu 14 →Asp 15 →Lys 16 →Lys 17 (Eicosanedioicacid-γ-Glu-AEEA)→Ala 18 →Gln 19 →Aib 20 →Ala 21 →Phe 22 →Ile 23 →Glu 24 →Tyr 25 →Leu 26 →Leu 27 →Glu 28 →Gly 29 →Gly 30 →Pro 31 →Ser 32 →Ser 33 →Gly 34 →Ala 35 →Pro 36 →Pro 37 →Pro 38 →Ser 39 .

[0067] The sequence of the first peptide segment is shown in SEQ ID NO.2, which is N-terminus → C-terminus, Tyr1 → Aib2 → Gln3 → Gly4 → Thr5 → Phe6 → Thr7 → Ser8 → Asp9 → Tyr 10 →Ser 11 →Ile 12 →(S)-α-MeLeu 13 →Leu 14 →Asp 15 →Lys 16 →Lys 17 (Eicosanedioicacid-γ-Glu-AEEA)→Ala 18 →Gln 19 →Aib 20 →Ala 21 →Phe 22 →Ile 23 →Glu24 →Tyr 25 →Leu 26 →Leu 27 →Glu 28 .

[0068] The sequence of the second peptide is shown in SEQ ID NO.3, which is N-terminus → C-terminus, Gly 29 →Gly 30 →Pro 31 →Ser 32 →Ser 33 →Gly 34 →Ala 35 →Pro 36 →Pro 37 →Pro 38 →Ser 39 .

[0069] Step 1: Preparation of the first peptide resin: CTC Resin with a substitution degree of 0.6 mmol / g was placed in a reactor, Fmoc-Glu-OALL was added, DMF was added to dissolve it, DIEA was added, and the mixture was stirred at 25°C for 8 hours. After the reaction was completed, the solvent was removed, the resin was washed with DMF, and deprotection solution was added for 28 minutes. Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-Aib-OH, Fmoc-Ala-OH, Fmoc-Lys(OtBu-C20-γ-Glu(OtBu)-AEEA), Fmoc-Lys(Boc)-OH, and Fmoc-Asp(OtBu) were added to the treated resin in the order from position 27 to position 1. -OH, Fmoc-Leu-OH, Fmoc-Ile-α-Me-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, Boc-Tyr(tBu)-Aib-Gln(Trt)-Gly-OH, using DIC and HOBt as coupling agents, were coupled in a single step at 25°C for 1.5 h. After each coupling step, the resin was washed with DMF. After all amino acids were coupled, the first peptide precursor resin was obtained. Tetra(triphenylphosphine)palladium and phenylsilane were added to the reactor, followed by DCM. The mixture was stirred at 25°C for 1 h to remove the OALL protecting group and obtain the first peptide resin. The volume-to-mass ratio of DMF to CTC Resin used for dissolution is 10 mL: 1 g; the deprotection solution includes Pip and DMF in a volume ratio of 1:4, and the volume-to-mass ratio of the deprotection solution to CTC Resin is 10 mL: 1 g; the molar ratio of protected amino acid to CTC Resin is 2.5:1, the molar ratio of coupling agent to protected amino acid is 1:1, the volume-to-mass ratio of DCM to CTC Resin is 20 mL: 1 g; the molar ratio of Fmoc-Glu-OALL to CTC Resin is 2:1, the molar ratio of DIEA to CTC Resin is 4:1, the molar ratio of DIC to HOBt is 1:1; the molar ratio of tetrakis(triphenylphosphine)palladium to CTC Resin is 1:1, and the molar ratio of benzylsilane to CTC Resin is 10:1.

[0070] Step 2: Preparation of the second peptide product: Sieber Resin with a substitution degree of 0.6 mmol / g was placed in a reactor, DMF was added and stirred to swell for 30 min, followed by deprotection with a deprotection solution for 28 min. Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Pr were then coupled sequentially to the treated resin at positions 39 to 29. o-OH and Fmoc-Gly-Gly-OH were coupled using DIC and HOBt as coupling agents in a single-step process at 25°C for 1.5 h. After each coupling step, the resin was washed with DMF. After all amino acids were coupled, the terminal amino acid Fmoc protecting group was removed to obtain the second peptide resin. A 3% (v / v) TFA / DCM solution was prepared to cleave the second peptide resin. The cleavage product was washed with DCM to obtain the crude second peptide. The crude second peptide was purified by reversed-phase high-performance liquid chromatography to obtain the final second peptide product. The volume-to-mass ratio of DMF to Sieber Resin used for swelling is 10 mL: 1 g; the deprotection solution includes Pip and DMF, with a volume ratio of Pip to DMF of 1:4; the volume-to-mass ratio of the deprotection solution to Sieber Resin is 10 mL: 1 g; the molar ratio of the protected amino acid to Sieber Resin is 2.5:1; the molar ratio of the coupling agent to the protected amino acid is 1:1; the volume-to-mass ratio of the cleavage fluid to the second peptide resin is 8 mL: 1 g; and the molar ratio of DIC to HOBt is 1:1.

[0071] Step 3: Preparation of crude retaloupeptide: The second peptide product was mixed with the first peptide resin, HOBt and DIC were added, and the mixture was stirred at 25°C for 2 hours. After the reaction was completed, the resin was washed with DMF, treated with methanol, and dried to obtain intact peptide resin. A 1% (v / v) TFA / DCM solution was prepared to cleave the intact peptide resin. The product was washed with DCM to obtain the fully protected peptide. A cleavage solution was prepared to cleave the fully protected peptide, and the reaction was carried out at 20°C for 2 hours. The reaction solution was added to methyl tert-butyl ether for precipitation, and the solid product was collected by filtration. The filter cake was washed with methyl tert-butyl ether and dried to obtain crude retaloupeptide. The volume-to-mass ratio of the cutting fluid to the intact peptide resin is 8 mL:1 g. The fully protected peptide cutting fluid is composed of TFA, TIS, and deionized water, with a volume ratio of TFA to TIS of 38:1 and a volume ratio of TIS to deionized water of 1:1. The volume-to-mass ratio of the fully protected peptide cutting fluid to the fully protected peptide is 2 mL:1 g. The molar ratio of the second peptide product to the first peptide resin is 1.2:1, the molar ratio of HOBt to DIC is 1:1, and the molar ratio of the coupling agent to the second peptide product is 1:1.

[0072] Step 4: Preparation and purification of retaglutide: The crude retaglutide was purified by C18 reversed-phase high-performance liquid chromatography (HPLC). TFA aqueous solution was used as mobile phase A, and TFA acetonitrile solution was used as mobile phase B. Gradient elution was performed to separate and collect the target fraction. After concentration and drying, purified retaglutide was obtained. The volume fraction of TFA in mobile phase A was 0.1%, and the volume fraction of TFA in mobile phase B was 0.1%.

[0073] Example 2: The only difference between this example and Example 1 is that in the preparation step of the first peptide resin, the molar ratio of tetra(triphenylphosphine)palladium to CTC Resin is adjusted from 1:1 to 1.5:1, the molar ratio of benzylsilane to CTC Resin is adjusted from 10:1 to 15:1, and the stirring reaction time in the DCM system is adjusted from 1h to 2h.

[0074] Example 3: The only difference between this example and Example 1 is that in the preparation step of the first peptide resin, the degree of substitution of CTC Resin is adjusted from 0.6 mmol / g to 1.0 mmol / g; and in the preparation step of the second peptide product, the degree of substitution of Sieber Resin is adjusted from 0.6 mmol / g to 1.0 mmol / g.

[0075] Example 4: The only difference between this example and Example 1 is in step four, the preparation and purification of retaloglutide. In Example 4, the C18 chromatographic packing material was changed to modified silica gel packing material, as detailed below: Step 4: Preparation and purification of Retalutide: Methyltrimethoxysilane was dispersed in deionized water, and ammonia was added under stirring. Then, 3-mercaptopropyltrimethoxysilane was added, and the mixture was allowed to stand at 20°C for 5 hours. The precipitate was collected by centrifugation at 8000 rpm for 15 minutes, washed with ethanol, and dried under vacuum at 50°C for 12 hours to obtain thiol-functionalized mesoporous silica microspheres. The thiol-functionalized mesoporous silica microspheres were dispersed in toluene, sonicated for 20 minutes, and 3-acrylamidophenylboronic acid and methyl-2-acrylamido-2-methoxyacetic acid were added as modifying monomers. Azobisisobutyronitrile was added, and the mixture was stirred at 70°C under nitrogen protection. After reacting for 10 h, the mixture was cooled and centrifuged at 8000 rpm for 15 min. The precipitate was washed with ethanol and dried under vacuum at 50 °C for 12 h to obtain boric acid-functionalized modified silica gel chromatographic packing. The modified silica gel chromatographic packing was dispersed in isopropanol, sonicated for 5 min, and then packed into a chromatographic column to obtain a modified silica gel chromatographic column. The crude retaloupeptide was purified by reversed-phase high-performance liquid chromatography using the modified silica gel column. The mobile phase was 0.1% TFA aqueous solution (v / v) and the mobile phase was 0.1% TFA acetonitrile solution (v / v). Gradient elution was performed to separate and collect the target fraction. The purified retaloupeptide was obtained after concentration and drying. The mass-to-volume ratio of methyltrimethoxysilane to deionized water was 1 g:5 mL; the volume ratio of ammonia to deionized water was 1 μL:10 mL; the mass ratio of 3-mercaptopropyltrimethoxysilane to methyltrimethoxysilane was 1:4; the CAS number of 3-acrylamidophenylboronic acid was 850568-25-1; the CAS number of methyl 2-acrylamido-2-methoxyacetic acid was 77402-03-0; the mass-to-volume ratio of mercaptofunctionalized mesoporous silica microspheres to toluene was 1 g:15 mL; the mass ratio of 3-acrylamidophenylboronic acid to mercaptofunctionalized mesoporous silica microspheres was 1:20; the mass ratio of methyl 2-acrylamido-2-methoxyacetic acid to mercaptofunctionalized mesoporous silica microspheres was 1:20; the mass ratio of azobisisobutyronitrile to mercaptofunctionalized mesoporous silica microspheres was 1:100; and the mass-to-volume ratio of modified silica chromatographic packing material to isopropanol was 1 g:15 mL.

[0076] Example 5: The only difference between this example and Example 4 is that in step four, the mass ratio of 3-acrylamidophenylboronic acid to thiol-functionalized mesoporous silica microspheres was adjusted from 1:20 to 1:10 in the preparation and purification of retaliglutide.

[0077] Comparative Example 1: The only difference between this comparative example and Example 4 is that in step four, the mass ratio of 3-acrylamidophenylboronic acid to thiol-functionalized mesoporous silica microspheres was adjusted from 1:20 to 1:40 in the preparation and purification of retaliglutide.

[0078] Comparative Example 2: The only difference between this comparative example and Example 4 is that in step four, the purification of retaloupeptide was prepared. In Comparative Example 2, the modified monomers were changed from 3-acrylamidophenylboronic acid and methyl 2-acrylamido-2-methoxyacetic acid to 2-aminophenylboronic acid and methyl 2-acrylamido-2-methoxyacetic acid, and the mass ratio of 2-aminophenylboronic acid to thiol-functionalized mesoporous silica microspheres was 1:20.

[0079] Comparative Example 3: The only difference between this comparative example and Example 4 is that in step four, the modified monomers in Comparative Example 3 were changed from 3-acrylamidophenylboronic acid and methyl 2-acrylamido-2-methoxyacetic acid to 2-aminophenylboronic acid and methacrylamide. The mass ratio of 2-aminophenylboronic acid to thiol-functionalized mesoporous silica microspheres was 1:20, and the mass ratio of methacrylamide to thiol-functionalized mesoporous silica microspheres was 1:20.

[0080] Experimental Example 1: Synthesis yield test of crude retaliglutide.

[0081] Test sample: crude retaloupeptide prepared in Examples 1-3.

[0082] Test method: Based on S(%)=m 粗品 ×P 粗品 The yield is calculated as / m0×100%. Where m 粗品 m0 represents the mass of crude retaliglutide, m0 represents the theoretical mass of retaliglutide, and P represents the mass of crude retaliglutide. 粗品 The area-normalized purity of crude retaglutide was determined by HPLC. Purity was determined using high-performance liquid chromatography (HPLC) with a C18 reversed-phase column or a modified silica gel column. Gradient elution was performed using 0.1% trifluoroacetic acid aqueous solution as mobile phase A and 0.1% trifluoroacetic acid acetonitrile solution as mobile phase B. The detection wavelength was 220 nm, the column temperature was 30℃, and the flow rate was 1.0 mL / min. The concentrations were: 0-5 min: 5%-10% B phase; 5-25 min: 10%-80% B phase; 25-30 min: 80%-95% B phase; 30-35 min: 95% B phase. The purity of crude retaglutide was calculated using the area-normalized method.

[0083] The synthesis yield test results of the crude retaloglutide prepared in this invention are as follows: Figure 1As shown, Example 1 uses CTC resin and Sieber resin with appropriate substitution degree for bifragment synthesis, with moderate reaction site density, sufficient amino acid coupling and few side reactions, resulting in the highest crude product yield. Example 2 increases the amount of tetrakis(triphenylphosphine)palladium and phenylsilane, which can improve deprotection efficiency, but excessive catalyst can easily cause side reactions, leading to a decrease in crude product yield compared to Example 1. Example 3 further increases the resin substitution degree, which can increase the amount of feed per batch, but the increased steric hindrance causes incomplete coupling of some amino acids, and the risk of resin polycondensation increases slightly, resulting in a further decrease in crude product yield compared to Example 2. Overall, it shows that appropriate resin substitution degree, catalyst ratio, and fragment separation strategy can significantly improve crude product yield, providing a good foundation for subsequent purification.

[0084] Experimental Example 2: Purity test of purified retaliglutide.

[0085] Test samples: purified retaloupeptide prepared in Examples 1-5 and Comparative Examples 1-3.

[0086] Test method: Purity was determined by high performance liquid chromatography (HPLC). A C18 reversed-phase column or a modified silica gel column was used. Gradient elution was performed using 0.1% trifluoroacetic acid aqueous solution as mobile phase A and 0.1% trifluoroacetic acid acetonitrile solution as mobile phase B. The detection wavelength was 220 nm, the column temperature was 30℃, and the flow rate was 1.0 mL / min. The concentrations were: 0-5 min: 5%-10% B phase; 5-25 min: 10%-80% B phase; 25-30 min: 80%-95% B phase; 30-35 min: 95% B phase. Sample purity was calculated using the area normalization method.

[0087] The purity test results of the purified retaliglutide prepared in this invention are as follows: Figure 2As shown, Example 2 optimized the deprotection catalyst and scavenger ratio of tetra(triphenylphosphine)palladium to benzylsilane, which could more completely remove side chain protecting groups, reduce residual impurities, and improve the purity of the finished product compared to Example 1. Example 3 used highly substituted 2-CTC Resin and Sieber Resin resins, which made the fragment coupling more uniform, further reduced the amount of impurities generated, and achieved the highest purity. Example 4 used a modified packing material of thiol-functionalized mesoporous silica microspheres grafted with 4-(pyridin-2-ylaminocarbonyl)phenylboronic acid and methyl 2-acrylamido-2-methoxyacetic acid for purification. The packing material has a large specific surface area and strong separation selectivity, which can effectively remove structurally similar impurities, and the purity is significantly higher than that of the conventional C18 purification system. Example 5 optimized the ratio of 4-(pyridin-2-ylaminocarbonyl)phenylboronic acid to thiol-functionalized mesoporous silica microspheres, enhanced the specific recognition of the target peptide by the packing material, further improved the separation effect, and improved the purity compared to Example 4. Comparative Example 1 reduced the amount of the key functional monomer 4-(pyridin-2-ylaminocarbonyl)phenylboronic acid, resulting in a decrease in the specific surface area and selectivity of the packing material, insufficient impurity separation, and a reduction in purity. Comparative Example 2 replaced the modified monomer with 2-aminophenylboronic acid while retaining methyl 2-acrylamido-2-methoxyacetic acid, which disrupted the specific binding site with retalistatin, weakening the impurity removal effect and further reducing purity. Comparative Example 3 simultaneously lacked both 4-(pyridin-2-ylaminocarbonyl)phenylboronic acid and methyl 2-acrylamido-2-methoxyacetic acid, and instead used a combination of 2-aminophenylboronic acid and methacrylamide. This significantly enhanced non-specific adsorption, resulting in the highest amount of impurity residue and the lowest purity. This fully demonstrates that the specific functional monomer combination of 4-(pyridin-2-ylaminocarbonyl)phenylboronic acid and methyl 2-acrylamido-2-methoxyacetic acid plays a crucial role in improving purification selectivity and product purity.

[0088] Experimental Example 3: Specific surface area test of modified silica gel chromatographic packing material.

[0089] Test samples: Modified silica gel chromatographic packing materials prepared in Examples 4-5 and Comparative Examples 1-3.

[0090] Test method: The specific surface area was determined by nitrogen adsorption-desorption method. After the sample was degassed in vacuum at 200℃ for 4h, nitrogen adsorption test was performed at 77K. The specific surface area of ​​the packing was calculated by BET method.

[0091] The specific surface area test results of the modified silica gel chromatographic packing material prepared in this invention are as follows: Figure 3As shown, Example 4 uses thiol-functionalized mesoporous silica microspheres as the matrix and synergistically modifies them with bifunctional monomers of 4-(pyridin-2-ylaminocarbonyl)phenylboronic acid and methyl 2-acrylamido-2-methoxyacetic acid. This allows for the uniform introduction of functional groups onto the silica surface without clogging the mesoporous channels, thus maintaining a high specific surface area of ​​the filler. Example 5 further optimizes the mass ratio of 4-(pyridin-2-ylaminocarbonyl)phenylboronic acid to thiol-functionalized mesoporous silica microspheres, resulting in more uniform surface modification, higher pore utilization, and a significantly larger specific surface area compared to Example 4. Comparative Example 1 reduced the amount of 4-(pyridin-2-ylaminocarbonyl)phenylboronic acid, resulting in insufficient functionalization of the silica gel surface, ineffective modification of some pore structures, and a significant decrease in specific surface area. Comparative Example 2 replaced the core modifying monomer with 2-aminophenylboronic acid while retaining methyl 2-acrylamido-2-methoxyacetic acid. Due to the poor compatibility between the monomer structure and silica gel, pore blockage and collapse occurred, further reducing the specific surface area. Comparative Example 3 simultaneously lacked both 4-(pyridin-2-ylaminocarbonyl)phenylboronic acid and methyl 2-acrylamido-2-methoxyacetic acid as core monomers, and instead used a combination of 2-aminophenylboronic acid and methacrylamide. As a result, a stable functionalized layer could not be formed on the silica gel surface, particles easily agglomerated, and the specific surface area was reduced to the lowest level. High specific surface area can improve the packing loading and separation efficiency, directly ensuring the high purity and high yield of retaloupeptide. This indicates that the specific combination of 4-(pyridin-2-ylaminocarbonyl)phenylboronic acid and methyl-2-acrylamido-2-methoxyacetic acid ester is the core factor in maintaining the high specific surface area and excellent pore structure of the packing.

[0092] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0093] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A method for synthesizing retaloglutide, characterized in that, include: The 39 amino acids of retaliglutide were separated into the first peptide segment at positions 1-28 and the second peptide segment at positions 29-39 using a solid-phase Fmoc strategy and synthesized in parallel. Using 2-CTC Resin as the first peptide solid-phase support, a first peptide resin was prepared by coupling, PIP / DMF deprotection, and selective removal of the OALL protecting group by tetrakis(triphenylphosphine)palladium / phenylsilane. Using Sieber Resin as the second peptide solid-phase support, a second peptide product was prepared by coupling, deprotection, cleavage with 2%–4% TFA / DCM solution, and purification. The second peptide product and the first peptide resin were then subjected to fragment condensation using a coupling agent to obtain a complete peptide resin. The complete peptide resin was first cleaved with 0.5%–1.5% TFA / DCM solution to obtain a fully protected peptide, followed by a second cleavage with a TFA / TIS / deionized water mixed cleavage solution, and precipitation with methyl tert-butyl ether to obtain crude retaloglutide. The crude retaloglutide was purified by reversed-phase high-performance liquid chromatography to obtain purified retaloglutide. The amino acid sequence of the retaglutide is shown in SEQ ID NO.1, the amino acid sequence of the first peptide segment is shown in SEQ ID NO.2, and the amino acid sequence of the second peptide segment is shown in SEQ ID NO.

3.

2. The synthesis method according to claim 1, characterized in that: The molar ratio of tetra(triphenylphosphine)palladium to CTC Resin is 1.0~1.5:

1.

3. The synthesis method according to claim 1, characterized in that: The molar ratio of benzylsilane to CTC Resin is 10~15:

1.

4. The synthesis method according to claim 1, characterized in that: The molar ratio of the second peptide product to the first peptide resin fragment condensation is 1.2~1.5:

1.

5. The synthesis method according to claim 1, characterized in that: The coupling agent includes at least one of DIC and HOBT, DIC and Oxyma, HATU and DIEA, and HBTU and DIEA, wherein the molar ratio of the coupling agent to the protected amino acid is 1:0.8~1.

2.

6. The synthesis method according to claim 1, characterized in that: In the TFA / TIS / deionized water mixed cutting fluid, the volume ratio of TFA to TIS is 35~40:1, and the volume ratio of TIS to deionized water is 1:0.8~1.

2.

7. The synthesis method according to claim 1, characterized in that: The degree of substitution of the 2-CTC Resin is 0.6~1.0 mmol / g, and the degree of substitution of the Sieber Resin is 0.6~1.0 mmol / g.

8. The synthesis method according to claim 1, characterized in that: The reversed-phase high-performance liquid chromatography column includes a C18 column and / or a boric acid functionalized modified silica column.

9. The synthesis method according to claim 8, characterized in that: The modified silica column is filled with boric acid-functionalized modified silica gel chromatographic packing material, which is prepared using thiol-functionalized mesoporous silica microspheres as the matrix and 3-acrylamidophenylboronic acid as the modified monomer.

10. The synthesis method according to claim 9, characterized in that: The mass ratio of 3-acrylamidophenylboronic acid to thiol-functionalized mesoporous silica microspheres is 1:8~40.

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