Synthesis method of retaglutide and retaglutide

Abstract

The invention relates to the technical field of polypeptide synthesis, in particular to a synthesis method of retaglutide and the retaglutide prepared by the method. Comprising the following steps: synthesizing retaglutide side chain peptide resin through an Fmoc / tBu solid phase method; removing a Lys17 main chain protecting group Dde or Alloc, and sequentially coupling main chain protected amino acids or protected amino acid fragments to obtain retaglutide peptide resin; cracking the peptide resin by adopting an acidic mixed solution to obtain crude peptide; and performing HPLC (High Performance Liquid Chromatography) purification, salt conversion, concentration and freeze-drying on the crude peptide to obtain refined retaglutide. According to the retaglutide synthesis method provided by the invention, the purity of the obtained retaglutide product is greater than 99.0%, and the purity of a single impurity is less than 0.10%. The synthesis method is simple to operate, high in crude peptide purity, easy to purify and prepare, low in comprehensive synthesis cost and very suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide synthesis, and in particular to a synthesis method of retaglutide and retaglutide. Background Art

[0002] Diabetes is a disorder of sugar, fat and protein metabolism caused by insufficient insulin secretion and insulin resistance. It involves all systems in the body and can even induce many fatal complications, seriously endangering human health. Among Chinese diabetics, type 2 diabetes accounts for more than 90%, type 1 diabetes accounts for about 5%, and gestational diabetes and other types of diabetes account for about 5% in total.

[0003] As the number of diabetic patients increases, my country's diabetes-related medical expenditures are also on the rise, ranking second in the world. Although the scale of diabetic patients and medical expenditures in my country are large, the overall diagnosis rate and per capita medical expenditures of patients are both low.

[0004] The successful research results of dual receptor agonists and the clinical effectiveness of single receptor agonists provide support for GLP-1R, GIPR, and GCGR triple receptor agonists. Theoretically, the effects of triple receptor agonists are based on a "three-pronged" approach. GLP-1R agonists provide weight loss and insulin secretion-promoting effects; GCGR agonists activate independent synergistic weight loss mechanisms; GIPR agonists further buffer glucagon-mediated hepatic glucose production by enhancing insulin secretion. Balanced triple receptor agonists are similar in length to endogenous glucagon and close in sequence to the GLP-1 analog exendin-4.

[0005] Retatrutide is a triple agonist peptide for glucagon receptor (GCGR), glucose-dependent insulinotropic polypeptide receptor (GIPR) and glucagon-like peptide-1 receptor (GLP-1R) developed by Eli Lilly and Company. Retatrutide participates in GCGR in vivo and can improve glucose tolerance in ipGTT through GIP or GLP-1 receptors, leading to significant weight loss and increased energy expenditure through glucagon receptor activation.

[0006] Retaglutide peptide sequence: H-Tyr 1 -Aib 2 -Gln 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10- Ser 11 -Ile 12 -αMeLeu 13 -Leu 14 -Asp 15 -Lys 16 -Lys 17 (AEEA-γ-Glu-Eicosanedioic acid)-Ala 18 -Gln 19 -Aib 20 - Wake up 21 - Phew 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 - Wake up 35 - Pro 36 - Pro 37 - Pro 38 - Ser 39 -NH2。

[0007] At present, there have been some research reports on the synthesis method of retaglutide. For example, in the Chinese patent application with publication number CN117024528B, multiple fully protected fragments Fmoc-Pro-Pro-Pro-OH, Fmoc-Ser (tBu) -Gly-OH, Fmoc-Gly-Gly-OH, Fmoc-Leu-Leu-OH, Fmoc-Gln (Trt) -Aib-OH, Fmoc-Lys (AEEA-γGlu (OtBu) - Eicosanedioic acid (OtBu)) -OH Fmoc-Ile-αMeLeu-OH, Fmoc-Thr (tBu) -Phe-OH, Boc-Tyr (tBu) -Aib-Gln (Trt) -Gly-OH are selected for the synthesis of retaglutide. The quality control of the intermediates is difficult, the cost is high, and it is not conducive to large-scale production. For another example, in the Chinese patent application with publication number CN 117903284 A, Dde-Lys(Fmoc)-OH is used at position 17 to synthesize the side chain first and then the main chain, and Boc-Tyr(tBu)-Aib-OH, Fmoc-Gly-Gly-OH, and Fmoc-Ile-{α-Me-Leu}-Leu-OH fragments are used for coupling in protecting amino acids, among which Fmoc-Ile-{α-Me-Leu}-Leu-OH has a low degree of industrialization, is expensive, has a high synthesis cost, and is not easy to industrialize. In addition, the crude peptide obtained by this method has a low purity and is not purified and refined, so the degree of industrialization is still unclear. Summary of the invention

[0008] In view of the above technical problems, the purpose of the present invention is to provide a method for synthesizing retaglutide, wherein the purity of the obtained retaglutide product is greater than 99.0%, and the single impurity is less than 0.10%. The synthesis method is simple to operate, the crude peptide has high purity, is easy to purify and prepare, and has low comprehensive synthesis cost, and is very suitable for industrial production.

[0009] In a first aspect, the present application provides a method for synthesizing retaglutide, comprising the following steps: (1) Synthesis of retaglutide side chain peptide resin by Fmoc / tBu solid phase method: Using amino resin as the starting resin, sequentially couple protected amino acids or protected amino acid fragments from C-terminus to N-terminus. 19 and Aib 20 , Gly 29 and Gly 30 The materials Fmoc-Gln(Trt)-Aib-OH and Fmoc-Gly-Gly-OH were used for coupling respectively; Lys 17The site was coupled using the material R-Lys(Fmoc)-OH (R = Dde or Alloc); (2) Removal of Lys 17 The main chain protecting group Dde or Alloc is sequentially coupled with the main chain protecting amino acid or protecting amino acid fragment to obtain the retaglutide peptide resin. 12 αMeLeu 13 Fmoc-Ile-OH and Fmoc-αMeLeu-OH were used for coupling, and Tyr 1 and Aib 2 Coupling was performed using Boc-Tyr(tBu)-Aib-OH; (3) using an acidic mixed solution to cleave the peptide resin to obtain a crude peptide; (4) The crude peptide is purified by HPLC, converted to salt, concentrated, and freeze-dried to obtain refined retaglutide peptide.

[0010] The sequence of retaglutide is H-Tyr 1 -Aib 2 -Gln 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Ile 12 -αMeLeu 13 -Leu 14 -Asp 15 -Lys 16 -Lys 17 (AEEA-γ-Glu-Eicosanedioic acid)-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 -NH2, wherein two adjacent Gly are coupled sequentially using Fmoc-Gly-OH and Fmoc-Gly-OH, which is very easy to introduce Gly impurities, causing great difficulties for purification. Therefore, in the present invention, Fmoc-Gly-Gly-OH is used to replace the two couplings of Fmoc-Gly-OH, which can completely avoid the insertion of Gly impurities.

[0011] In some embodiments, the amino resin is an aminomethyl resin having an active amino group: ,in represents a solid phase support, such as polystyrene resin.

[0012] According to a preferred embodiment of the present invention, the step (1) specifically comprises: synthesizing the side chain peptide resin of retaglutide by the Fmoc / tBu solid phase method, using amino resin as the starting resin, and sequentially connecting Fmoc-Linker, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH.H2O, Fmoc-Pro-OH.H2O, Fmoc-Pro-OH.H2O, Fmoc-Ala-OH.H2O, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)- -OH, Fmoc-Pro-OH.H2O, Fmoc-Gly-Gly-OH, Fmoc-Glu(OtBu)-OH.H2O, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu (OtBu)-OH.H2O, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Ala-OH.H2O, Fmoc-Gln(Trt)-Aib-OH, Fmoc-Ala-OH.H2O, R-Lys(Fmoc)-OH(R= Dde or Alloc), Fmoc-AEEA-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosanedioate to obtain R-Lys(AEEA-γ-Glu-(OtBu)-Eicosanedioic acid-OtBu)-Ala-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-Leu-Glu(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Linker-AM Resin, wherein R = Dde or Alloc.

[0013] According to a preferred embodiment of the present invention, in the step (1), the degree of substitution of the amino resin is 0.4 to 0.8 mmol / g, more preferably, the degree of substitution of the amino resin is 0.4 to 0.6 mmol / g.

[0014] According to a preferred embodiment of the present invention, in step (1), the condensation reagent used for coupling is one or a combination of DIC / HOBT, Oxyma / DIC, TBTU / DIEA, PyBOP / DIEA.

[0015] According to a preferred embodiment of the present invention, in step (1), the reaction temperature is 25-35°C.

[0016] According to a preferred embodiment of the present invention, in step (1), the amount of Fmoc-protected amino acid or protected short peptide fragment is 2 to 6 times the total molar number of the resin fed.

[0017] According to a preferred embodiment of the present invention, step (2) is specifically as follows: when R = Dde, the Dde protecting group is removed by 2-3% hydrazine hydrate / DMF solution; when R = Alloc, when removing the Alloc protecting group, it is removed by a mixed solution of tetrakistriphenylphosphine palladium / DCM; then Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-αMeLeu-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Boc-Tyr(tBu)-Aib-OH are coupled in sequence to obtain retaglutide peptide resin.

[0018] According to a preferred embodiment of the present invention, in step (2), the reaction solvent is one or a combination of dichloromethane, N,N-dimethylformamide, and N-methylpyrrolidone.

[0019] According to a preferred embodiment of the present invention, in step (2), the reaction temperature is 25-35°C.

[0020] According to a preferred embodiment of the present invention, the acidic mixed solution in step (3) is a combination of trifluoroacetic acid (TFA) and one or more of phenol, water, 1,2-ethanedithiol, triisopropylsilane, thioanisole, anisole, dithiothreitol, 3,6-dioxo-1 or 8-octanedithiol, wherein the volume ratio of trifluoroacetic acid is not less than 85%.

[0021] According to a preferred embodiment of the present invention, the acidic mixed solution in step (3) is 6 to 10 mL / g of the weight of the resin.

[0022] According to a preferred embodiment of the present invention, in step (3), the lysis time is 2.5 to 3.5 hours.

[0023] According to a preferred embodiment of the present invention, in step (3), the cracking temperature is 15 to 30°C.

[0024] According to a preferred embodiment of the present invention, the step (4) is specifically as follows: adding water to the crude retaglutide peptide and stirring, adjusting the pH to 8.0-9.0 with aqueous ammonia to completely dissolve the peptide, filtering the solution with a 0.45 μm microporous filter membrane, purifying the solution by reversed-phase preparative chromatography, and collecting the retaglutide fraction; then performing salt conversion by reversed-phase preparative chromatography, collecting the retaglutide fraction, concentrating under reduced pressure to obtain a retaglutide aqueous solution, and freeze-drying the solution to obtain refined retaglutide peptide.

[0025] In a second aspect, the present application provides a retaglutide prepared according to the method described in the first aspect. In some embodiments, the retaglutide product has a purity greater than 99.0%, and a single impurity is less than 0.10%.

[0026] Compared with the prior art, the present invention has the following beneficial effects: 1. The retaglutide synthesis method of the present invention can obtain a retaglutide product with a purity greater than 99.0%, and a single impurity less than 0.10%. The synthesis method is simple to operate, has a high purity of crude peptide, is easy to purify and prepare, has a low comprehensive synthesis cost, and is very suitable for the industrial production of retaglutide.

[0027] 2. The two adjacent Gly in the retaglutide sequence are easily introduced into the Gly impurity by coupling with Fmoc-Gly-OH and Fmoc-Gly-OH in sequence, which brings great difficulties to purification. In the retaglutide synthesis method of the present invention, the two couplings of Fmoc-Gly-Gly-OH instead of Fmoc-Gly-OH can completely avoid the insertion of Gly impurities and improve the purity of the product.

[0028] 3. In the prior art, Fmoc-Ile-αMeLeu-OH or Fmoc-Ile-αMeLeu-Leu-OH fragment peptide is used to couple Ile in the retaglutide sequence. 12 and αMeLeu 13 However, the material is expensive, resulting in low industrialization, great coupling difficulty, and incomplete coupling. In the synthesis method of retaglutide of the present invention, a step-by-step coupling method is adopted to sequentially couple Fmoc-αMeLeu-OH and Fmoc-Ile-OH, which greatly improves the coupling efficiency, and the purity and yield of the crude peptide are significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a process flow chart of a method for synthesizing retaglutide according to one embodiment of the present invention; Figure 2 is the HPLC spectrum of the crude retaglutide peptide in Example 1 of the present invention; Figure 3 is the HPLC spectrum of the refined retaglutide peptide in Example 1 of the present invention; Figure 4 is the MS graph of refined retaglutide peptide in Example 1 of the present invention; Figure 5 is the HPLC spectrum of the crude retaglutide peptide in Example 2 of the present invention; Figure 6 is the HPLC spectrum of the refined retaglutide peptide in Example 2 of the present invention; Figure 7 This is the MS graph of the refined retaglutide peptide in Example 2 of the present invention. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods, but does not constitute any limitation to the present invention.

[0031] In the specific implementation manner of the present invention, the Chinese meanings of the English abbreviations used in the application documents are shown in Table 1.

[0032] Table 1

[0033] The present invention is further described below with reference to examples.

[0034] Retaglutide peptide resin: Boc-Tyr(tBu)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr( tBu)-Ser(tBu)-Ile-α-MeLeu-Leu-Asp(OtBu)-Lys(Boc)-Lys(AEEA-γ-Glu-(OtBu)-Eicosanedioic acid-OtBu)-Ala-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-Leu-Glu(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Linker-AM Resin.

[0035] Using amino resin as the starting resin, through activation and coupling reaction, the amino acid resin Fmoc-Linker-AM Resin loaded with the first site is obtained, and then the side chain peptide resin of retaglutide is obtained by removing Fmoc protection, protecting amino acid activation and coupling, and then removing Lys 17 The main chain Dde or Alloc protecting group is sequentially coupled with the protected amino acids shown in Table 2 to prepare the Retaglutide peptide resin. The protected amino acids or fragments corresponding to the protected amino acids used in this example are shown below: Table 2

[0036] Example 1: Preparation of Retaglutide Side Chain Peptide Resin like Figure 1 As shown, 750 mmol AM Resin (substitution degree is 0.55 mmol / g) is taken, and after swelling and washing, the protected amino acid Fmoc-Linker (1.5 eq), condensation reagent DIEA (3 eq), and TBTU (1.5 eq) are added into the reaction kettle, dissolved with DMF, and coupled for 120 to 300 minutes at 25 to 35 ° C under stirring, filtered and washed to obtain Fmoc-Linker-AM Resin.

[0037] After removing Fmoc protection with 20% Pip / DMF solution, add protected amino acid Fmoc-Ser(tBu)-OH (3eq) and HOBt (3eq), dissolve with appropriate amount of DMF and cool to 0-15°C. Take DIC (3eq) and slowly add it to the protected amino acid DMF solution under stirring, stir and react at 0-15°C for 5-15 minutes to obtain the activated protected amino acid solution.

[0038] The activated protected amino acid solution is added to the resin to be reacted, and the coupling reaction is carried out at 25-35°C for 120-300 minutes, and then filtered and washed to obtain Fmoc-Ser(tBu)-Linker-AM Resin.

[0039] Using the same method as above, the protected amino acids or fully protected amino acid fragment peptides in Table 2 were sequentially connected: Fmoc-Pro-OH.H2O, Fmoc-Pro-OH.H2O, Fmoc-Pro-OH.H2O, Fmoc-Ala-OH.H2O, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH.H2O, Fmoc-Gly-Gly-OH, Fmoc-Glu(OtBu)-OH.H2O, Fmoc-Leu-OH, Fmoc-L Dde-Lys(AEEA-γ-Glu-(OtBu)-Eicosanedioic acid mono-tert-butyl ester was obtained by mixing with 2-hydroxy-1,2-dioic acid and 1,2-dioic acid with 1,2-dioic acid and 1,2-dioic acid. acid-OtBu)-Ala-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-Leu-Glu(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Linker-AM Resin.

[0040] Preparation of Retaglutide Peptide Resin Lys removal 17 After adding the main chain protecting group Dde, each main chain site was coupled in turn, and the Dde protecting group was removed using 2-3% hydrazine hydrate / DMF solution. The reaction was carried out at 5-35°C for 10 min, and this process was repeated 3 times. After washing the peptide resin, the following sites of the main chain were connected in sequence: Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-αMeLeu-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Boc-Tyr(tBu)-Aib-OH, and 6500.3 g of retaglutide peptide resin was obtained.

[0041] Preparation of crude retaglutide peptide To the prepared retaglutide peptide resin, an acidic mixed solution was added, the mixed solution ratio was TFA:EDT / Tis / phenol / H2O=90:2.5:2.5:2.5:2.5, and the amount was 8 mL / g resin; the reaction was stirred at 15-30°C for 2-4 hours to obtain a reaction mixture, which was filtered using a sand core funnel, the filtrate was collected, and the resin was washed 3 times with a small amount of TFA, the filtrate was combined, MTBE was added to precipitate, and the precipitate was washed 3 times with MTBE, and 3536.3 g of off-white powder was obtained, i.e., crude retaglutide, and the purity of the crude product was 81.81% ( Figure 2 ).

[0042] Preparation of Retaglutide Take the crude product of retaglutide, add water and stir, adjust the pH to 9.0-9.5 with ammonia water until it is completely dissolved, and filter the resulting solution with a 0.45 μm mixed microporous filter membrane; Reverse phase preparative chromatography was used for purification and preparation, and the retaglutide fractions were collected; the fractions were combined, converted to sodium salt, and concentrated under reduced pressure to obtain a retaglutide aqueous solution; freeze-dried to obtain 1963.38.68 g of retaglutide refined peptide with a purity of 99.51% and a maximum single impurity of 0.10% ( Figure 3 ), the total yield was 55.33%, and the molecular weight was 4730.76 ( Figure 4 ).

[0043] Example 2: Preparation of Retaglutide Side Chain Peptide Resin like Figure 1 As shown, 750 mmol AM Resin (substitution degree 0.55 mmol / g) was taken and swollen and washed, and then the protected amino acid Fmoc-Linker (1.5 eq), condensation reagent DIEA (3 eq), and TBTU (1.5 eq) were added to the reactor, dissolved with an appropriate amount of DMF, and coupled for 120 to 300 minutes at 25 to 35 ° C under stirring, filtered and washed to obtain Fmoc-Linker-AM Resin.

[0044] Use 20% Pip / DMF solution to remove the Fmoc protecting group, protected amino acid Fmoc-Ser(tBu)-OH (3eq) and HOBt (3eq), dissolve with appropriate amount of DMF and cool to 0-15°C; take DIC (3eq) and slowly add it to the protected amino acid DMF solution with stirring, stir and react at 0-15°C for 5-15 minutes to obtain the activated protected amino acid solution.

[0045] The activated protected amino acid solution is added to the resin to be reacted, and the coupling reaction is carried out at 25-35°C for 120-300 minutes, and then filtered and washed to obtain Fmoc-Ser(tBu)-Linker-AM Resin.

[0046] Using the same method as above, the protected amino acids or fully protected amino acid fragment peptides in Table 2 were sequentially connected: Fmoc-Pro-OH.H2O, Fmoc-Pro-OH.H2O, Fmoc-Pro-OH.H2O, Fmoc-Ala-OH.H2O, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH.H2O, Fmoc-Gly-Gly-OH, Fmoc-Glu(OtBu)-OH.H2O, Fmoc-Leu-OH, Fmoc-Leu -OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH.H2O, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Ala-OH.H2O, Fmoc-Gln(Trt)-Aib-OH, Fmoc-Ala-OH.H2O, Alloc-Lys(Fmoc)-OH, Fmoc-AEEA-OH, Fmoc-Glu-OtBu, and eicosanedioic acid mono-tert-butyl ester were obtained. acid-OtBu)-Ala-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-Leu-Glu(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Linker-AM Resin.

[0047] Preparation of Retaglutide Peptide Resin After removing the main chain protecting group Alloc at Lys17, each main chain site was coupled in sequence. A mixed solution of 0.1% tetrakistriphenylphosphine palladium / DCM was used to remove the Alloc protecting group. The peptide resin was washed and the following sites of the main chain were connected in sequence: Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-αMeLeu-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Boc-Tyr(tBu)-Aib-OH, and 6692.8 g of retaglutide peptide resin were obtained.

[0048] Preparation of crude retaglutide peptide An acidic mixed solution was added to the prepared retaglutide peptide resin, the ratio of the mixed solution was TFA:EDT / Tis / phenol / H2O=90:2.5:2.5:2.5:2.5, and the amount was 8 mL / g resin; the reaction was stirred at 15-30°C for 2-4 hours to obtain a reaction mixture, which was filtered using a sand core funnel, the filtrate was collected, and the resin was washed 3 times with a small amount of TFA, the filtrate was combined, MTBE was added to precipitate, and the precipitate was washed 3 times with MTBE, and 3721.7 g of off-white powder was obtained, i.e., crude retaglutide, and the purity of the crude product was 79.37% ( Figure 5 ).

[0049] Preparation of Retaglutide Take crude retaglutide, add water and stir, adjust pH to 9.0-9.5 with aqueous ammonia until completely dissolved, and filter the resulting solution with a 0.45 μm mixed microporous membrane; Reverse phase preparative chromatography was used for purification and preparation, and qualified fractions were collected; the fractions were combined, converted to sodium salt, and concentrated under reduced pressure to obtain a retaglutide aqueous solution; freeze-dried to obtain 1590.79 g of retaglutide refined peptide with a purity of 99.60% and a maximum single impurity of 0.10% ( Figure 6 ), the total yield was 44.83%, and the molecular weight was 4731.61 ( Figure 7 ).

[0050] The above examples show that the purity of the crude product obtained by the synthesis method provided by the present invention has been greatly improved, the purity of the refined retaglutide peptide product is greater than 99.0%, and the single impurity is less than 0.10%. At the same time, the method provided by the present invention also improves the quality of the final product, the process is simple and easy to control, the degree of industrialization is high, and it has broad practical value and application prospects.

[0051] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for synthesizing retaglutide, characterized in that: The following steps are involved: (1) Synthesis of retaglutide side chain peptide resin by Fmoc / tBu solid phase method: Using amino resin as the starting resin, sequentially couple protected amino acids or protected amino acid fragments from C-terminus to N-terminus. 19 and Aib 20 , Gly 29 and Gly 30 The materials Fmoc-Gln(Trt)-Aib-OH and Fmoc-Gly-Gly-OH were used for coupling respectively; Lys 17 The site was coupled using the material R-Lys(Fmoc)-OH (R = Dde or Alloc); (2) Removal of Lys 17 The main chain protecting group Dde or Alloc is sequentially coupled with the main chain protecting amino acid or protecting amino acid fragment to obtain the retaglutide peptide resin. 12 αMeLeu 13 Fmoc-Ile-OH and Fmoc-αMeLeu-OH were used for coupling, and Tyr 1 and Aib 2 Coupling was performed using Boc-Tyr(tBu)-Aib-OH; (3) using an acidic mixed solution to cleave the peptide resin to obtain a crude peptide; (4) The crude peptide is purified by HPLC, converted to salt, concentrated, and freeze-dried to obtain refined retaglutide peptide.

2. The method for synthesizing retaglutide according to claim 1, characterized in that: The degree of substitution of the amino resin in step (1) is 0.4 to 0.8 mmol / g.

3. The method for synthesizing retaglutide according to claim 1 or 2, characterized in that: In the step (1), the condensation reagent used for coupling is one or a combination of DIC / HOBT, Oxyma / DIC, TBTU / DIEA, PyBOP / DIEA.

4. The method for synthesizing retaglutide according to claim 3, characterized in that: In the step (1), the reaction temperature is 25 to 35°C.

5. The method for synthesizing retaglutide according to claim 1, characterized in that: In the step (1), the amount of Fmoc-protected amino acid or protected short peptide fragment is 2 to 6 times the total molar number of the resin fed.

6. The method for synthesizing retaglutide according to claim 1, characterized in that: In the step (2), when R = Dde, the Dde protecting group is removed by using a 2-3% hydrazine hydrate / DMF solution; when R = Alloc, the Alloc protecting group is removed by using a mixed solution of tetrakistriphenylphosphine palladium / DCM.

7. The method for synthesizing retaglutide according to claim 1, characterized in that: The reaction solvent is one or a combination of dichloromethane, N,N-dimethylformamide and N-methylpyrrolidone.

8. The method for synthesizing retaglutide according to claim 1, characterized in that: The acidic mixed solution described in step (3) is a combination of trifluoroacetic acid TFA and one or more of phenol, water, 1,2-ethanedithiol, triisopropylsilane, thioanisole, anisole, dithiothreitol, 3,6-dioxo-1 or 8-octanedithiol, wherein the volume ratio of trifluoroacetic acid is not less than 85%.

9. The method for synthesizing retaglutide according to claim 1 or 8, characterized in that: In step (3), the acidic mixed solution is 6 to 10 mL / g of the resin weight, the pyrolysis time is 2.5 to 3.5 h, and the pyrolysis temperature is 15 to 30 °C.

10. Retaglutide prepared by the method according to any one of claims 1 to 9, characterized in that: The purity is greater than 99.0%, and each single impurity is less than 0.10%.

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