Method for synthesizing semaglutide by using whole fragment

Abstract

According to the method for synthesizing the semaglutide by using the whole fragment, the 2 peptide is simple to synthesize and low in cost, and is permitted to be used as a starting material in supervision, so that the steps required by solid-phase synthesis are greatly reduced in a manner of completely using the fragment peptide, and the time required by synthesis and the dosage of a solvent are greatly reduced. According to the method, after coupling is completed in sequence from a carbon terminal to a nitrogen terminal, a Dde protecting group is selectively removed, uBtO-Oct-gamma-Glu (OtBu)-AEA-AEA-OH is coupled, full-protection semaglutide is synthesized in a solid phase mode, and semaglutide is obtained through splitting decomposition, and the method greatly reduces synthesis steps and solvent dosage, reduces purification cost and is beneficial to industrial large-scale production.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide drug synthesis, specifically to the technical field of semaglutide synthesis, and more particularly to a method for synthesizing semaglutide using whole fragments, which significantly reduces the steps required for solid-phase synthesis, thereby significantly reducing the time and solvent usage required for synthesis, reducing purification costs, and facilitating industrial scale-up production. Background Art

[0002] Semaglutide, English name: Semaglutide, peptide sequence is:

[0003] H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Oc t-γ-Glu-AEEA-AEEA)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

[0004] Semaglutide is a new long-acting GLP-1 analog developed by Novo Nordisk of Denmark. Its amino acid sequence contains the unnatural amino acid aminoisobutyric acid at position 2, and the Lys side chain amino group at position 20 has been modified with PEG, γ-Glu, and octadecandioic acid. Compared to liraglutide, this modified semaglutide is more hydrophilic, inhibits DPP-4 hydrolysis, prolongs its biological half-life, achieves long-term blood sugar reduction, promotes pancreatic cell regeneration, and prolongs gastric emptying, among other benefits. It is currently widely used worldwide.

[0005] Patent WO2013098191A1 discloses a method for preparing semaglutide, which is a linear 29-mer peptide prepared by biosynthesis.

[0006] H-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH, and then the N-terminus and side chain are modified by chemical methods to finally obtain the finished product of semaglutide. This method requires a combination of biological and chemical methods and is relatively difficult.

[0007] Patent CN106478806A discloses a solid-phase synthesis method for synthesizing semaglutide, which uses 2-Cl-Resin as a starting resin carrier and sequentially connects the corresponding amino acids in the semaglutide sequence through a solid-phase synthesis method, wherein lysine uses Dde-Lys(Fmoc)-OH as a raw material, and after linking the side chain raw material, the Dde protecting group of lysine is removed to perform a continuous condensation reaction of the peptide chain. During the reaction, specific microwave technology is used to obtain semaglutide-2-Cl-Resin; the semaglutide-2-Cl-Resin is subjected to a cutting and sedimentation reaction and freeze-dried to obtain crude semaglutide peptide.

[0008] Patent CN106928343A discloses a method for preparing semaglutide, which involves solid-phase synthesis of linear semaglutide chains, followed by removal of Lys side chain protecting groups and coupling of side chain modifying groups, followed by cleavage to yield the peptide product. This patent utilizes a one-by-one coupling method for amino acids. As the carbon chain grows, the numerous hydrophobic protecting groups interact with the amino acids, making coupling difficult. This process produces defective peptides that are difficult to remove, and increasing the feed and time does not fundamentally address the problem.

[0009] Patent CN108034004A discloses a method for synthesizing semaglutide, in which amino acids 1-4 of semaglutide are used as the first fragment, amino acids 5-31 are coupled, the Lys side chain at position 20 is removed and sequentially coupled as the second fragment, and finally the two fragments are coupled, followed by cleavage and precipitation to obtain semaglutide. This method reduces the difficulty of synthesis and improves the yield to a certain extent, but the effect is limited.

[0010] Therefore, there is an urgent need to provide a method for synthesizing semaglutide using the whole fragment to solve the problems of high impurities, low purity, low yield, high cost, complicated operation steps and excessive waste liquid in the existing semaglutide synthesis process. Summary of the Invention

[0011] In order to address the deficiencies of the prior art, the present invention aims to provide a method for synthesizing semaglutide using full fragments. Since the synthesis of the two peptides is simple, the cost is low, and they are allowed to be used as starting materials under regulation, the present invention uses all fragment peptides to greatly reduce the steps required for solid-phase synthesis, thereby greatly reducing the time and solvent required for synthesis. The fragments used include Fmoc-Gly-Arg(Pbf)-OH, Fmoc-Val-Arg(Pbf)-OH, Fmoc-Trp(Boc)-Leu-OH, Fmoc-Ile-Ala-OH, Fmoc-Glu(OtBu)-Phe-OH, Fmoc-Gln(Trt)-Ala-OH, Fmoc-Ala-Lys(Dde)-OH, uBtO-Oct-γ-Glu(OtBu)-AEEA-AEEA-OH, Fmoc -Glu(OtBu)-Gly-OH, Fmoc-Tyr(tBu)-Leu-OH, Fmoc-Ser(tBu)-Ser(tBu)-OH, Fmoc-Asp(OtBu)-Val-OH, Fmoc-Thr(tBu)-Ser(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH, after the coupling is completed in sequence from the carbon end to the nitrogen end, the Dde protecting group is selectively removed and uBtO-Oct-γ-Glu(OtBu)-AEEA-AEEA-OH is coupled to synthesize fully protected semaglutide in solid phase, and semaglutide crude product is obtained through cracking and precipitation. The method of the present invention greatly reduces the synthesis steps and the amount of solvent used, reduces the purification cost, and is conducive to industrial scale-up production.

[0012] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0013] The present invention provides a method for synthesizing semaglutide using whole fragments. The synthesis method comprises using an amino resin as a starting resin and preparing it by a solid-phase peptide synthesis method, obtaining a semaglutide resin by the peptide solid-phase synthesis method, and then cracking the semaglutide resin to obtain a crude semaglutide product; wherein all fragment peptides are used in the synthesis process of the semaglutide multi-resin.

[0014] As a preferred embodiment of the present invention, the synthesis method is:

[0015] 1) Select amino acids 1-4 of the semaglutide sequence as Boc-His 1 (Trt)-Aib 2 -Glu 3 (OtBu)-Gly 4-OH fragment, with amino acids 19-20 of the semaglutide sequence as the dipeptide Fmoc-Ala 19 -Lys 20 (Dde)-OH fragment; semaglutide side chain as the 4-peptide uBtO-Oct-γ-Glu(OtBu)-AEEA-AEEA-OH fragment;

[0016] 2) After the amino resin is swollen with solvent, the solvent is drained and Fmoc removal reagent is added for deprotection; after draining and washing, the indene test result is positive; activated Fmoc-Gly-Arg(Pbf)-OH is added to react with the condensing agent; after the indene test result is negative, draining and washing are repeated, and the coupling reaction is carried out according to the amino acid sequence;

[0017] 3) Selectively removing the Dde protecting group and performing side chain modification to obtain fully protected semaglutide peptide resin;

[0018] 4) A cleavage reagent is added to the fully protected semaglutide peptide resin, and the crude semaglutide peptide is obtained through precipitation and drying.

[0019] As a preferred embodiment of the present invention, the amino resin is one of Fmoc-Gly-Wang resin and Fmoc-Gly-CTC resin, and the resin substitution degree is 0.3-0.5 mmol / g.

[0020] As a preferred embodiment of the present invention, in step 2), the amino acid sequence is:

[0021] Fmoc-Val-Arg(Pbf)-OH, Fmoc-Trp(Boc)-Leu-OH, Fmoc-Ile-Ala-OH, Fmoc-Glu(OtBu)-Phe-OH, Fmoc-Gln(Trt)-Ala-OH, Fmoc-Ala-Lys(Dde)-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmo c-Tyr(tBu)-Leu-OH, Fmoc-Ser(tBu)-Ser(tBu)-OH, Fmoc-Asp(OtBu)-Val-OH, Fmoc-Thr(tBu)-Ser(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH.

[0022] As a preferred embodiment of the present invention, in step 2), the amount of the Fmoc-protected amino acid or protected amino acid fragment is 1.5-2.5 times the total molar number of the resin fed.

[0023] As a preferred embodiment of the present invention, in step 2), the solvent is one or more combinations of DCM, DMF, NMP, and DMSO.

[0024] As a preferred embodiment of the present invention, the condensing agent used is selected from one of DIC / HOBt, HBTU / HOBT / DIEA, and PyBop / HOBT / DIEA.

[0025] As a preferred embodiment of the present invention, in step 2), the Fmoc removal reagent is a mixed solution of piperidine and DMF, which is composed of 20% by volume of piperidine and 80% by volume of DMF.

[0026] As a preferred embodiment of the present invention, in step 4), the lysis reagent is a mixed reagent of TFA, EDT and TIS in a volume ratio of 90:5:5.

[0027] As a preferred embodiment of the present invention, step 3) is specifically as follows: after the coupling is completed, hydrazine hydrate / DMF solution is added to remove Dde for side chain modification, methanol / DCM are alternately washed, and vacuum dried to obtain a fully protected semaglutide peptide resin.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1) The present invention reduces the steps of solid-phase synthesis of fully protected semaglutide from the original 70 steps to 27 steps by using a large number of fragment peptides. Almost all the original sites (except Aib, Gly, Oct, AEEA) may undergo deletion and racemization, but the number of sites that may undergo racemization or deletion is reduced to less than half. After cleavage and precipitation, crude semaglutide is obtained.

[0030] 2) The present invention significantly reduces the steps required for solid-phase synthesis by using only fragment peptides, thereby significantly reducing the time required for synthesis and the amount of solvent used.

[0031] 3) The method of the present invention greatly reduces the number of synthesis steps and the amount of solvent used, reduces the purification cost, and is conducive to industrial scale-up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0033] Figure 1 This is a chromatogram of the crude peptide obtained in Example 1 of the present invention.

[0034] Figure 2 This is the chromatogram of the crude peptide obtained in Comparative Example 1.

[0035] Figure 3 This is the chromatogram of the crude peptide obtained in Comparative Example 2. DETAILED DESCRIPTION

[0036] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0037] The present invention provides a method for synthesizing semaglutide using all fragments. By using all fragment peptides, the steps required for solid-phase synthesis are greatly reduced, thereby greatly reducing the time and solvent usage required for synthesis. The fragments used include Fmoc-Gly-Arg(Pbf)-OH, Fmoc-Val-Arg(Pbf)-OH, Fmoc-Trp(Boc)-Leu-OH, Fmoc-Ile-Ala-OH, Fmoc-Glu(OtBu)-Phe-OH, Fmoc-Gln(Trt)-Ala-OH, Fmoc-Ala-Lys(Dde)-OH, uBtO-Oct-γ-Glu(OtBu)-AEEA-AEEA-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-T After the coupling is completed in sequence from the carbon end to the nitrogen end, yr(tBu)-Leu-OH, Fmoc-Ser(tBu)-Ser(tBu)-OH, Fmoc-Asp(OtBu)-Val-OH, Fmoc-Thr(tBu)-Ser(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, and Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH are selectively removed from the Dde protecting group and coupled with uBtO-Oct-γ-Glu(OtBu)-AEEA-AEEA-OH to synthesize fully protected semaglutide in solid phase, and semaglutide crude product is obtained through cracking and precipitation. The method of the present invention greatly reduces the synthesis steps and the amount of solvent used, reduces the purification cost, and is conducive to industrial scale-up production.

[0038] In the present invention, amino acids 1 to 4 of the semaglutide sequence are selected as Boc-His 1 (Trt)-Aib 2 -Glu 3 (OtBu)-Gly 4-OH fragment, with amino acids 19-20 of the semaglutide sequence as the dipeptide Fmoc-Ala 19 -Lys 20 (Dde)-OH fragment; semaglutide side chain as the 4-peptide uBtO-Oct-γ-Glu(OtBu)-AEEA-AEEA-OH fragment.

[0039] Example 1

[0040] This example uses the preparation of the whole-fragment synthetic semaglutide:

[0041] 1) Select amino acids 1-4 of the semaglutide sequence as Boc-His 1 (Trt)-Aib 2 -Glu 3 (OtBu)-Gly 4 -OH fragment, with amino acids 19-20 of the semaglutide sequence as the dipeptide Fmoc-Ala 19 -Lys 20 (Dde)-OH fragment; semaglutide side chain as the 4-peptide uBtO-Oct-γ-Glu(OtBu)-AEEA-AEEA-OH fragment.

[0042] 2) After the Fmoc-Gly-Wang resin is swollen with DCM, the solvent is drained and the Fmoc removal reagent is added for deprotection; after draining and washing, the indene test result is positive; a mixture of activated Fmoc-Gly-Arg(Pbf)-OH, HOBT, and DIC is added for reaction; if the indene test result is negative, the resin is drained and washed;

[0043] According to the amino acid sequence Fmoc-Val-Arg(Pbf)-OH, Fmoc-Trp(Boc)-Leu-OH, Fmoc-Ile-Ala-OH, Fmoc-Glu(OtBu)-Phe-OH, Fmoc-Gln(Trt)-Ala-OH, Fmoc-Ala-Lys(Dde)-OH, Fmoc-Glu(OtBu)-Gly-OH , Fmoc-Tyr(tBu)-Leu-OH, Fmoc-Ser(tBu)-Ser(tBu)-OH, Fmoc-Asp(OtBu)-Val-OH, Fmoc-Thr(tBu)-Ser(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH.

[0044] 3) After the coupling is completed, 200 mL of 2% hydrazine hydrate / DMF solution is added to remove Dde for side chain modification, and the mixture is washed alternately with methanol / DCM three times, 200 mL each time, and vacuum dried to obtain fully protected semaglutide peptide resin.

[0045] 4) Cleavage: Prepare 250 mL of a 90:5:5 volume ratio of TFA, EDT, and TIS as a cleavage reagent. Add the fully protected semaglutide peptide resin under ice-cooling conditions. After 0.5 h, return to room temperature and continue the reaction for 2 h. After the reaction is complete, add anhydrous isopropyl ether to precipitate. Centrifuge the precipitate four times, adding 300 mL of anhydrous isopropyl ether each time. After drying, the resulting product is crude semaglutide, yielding 20.54 g of crude peptide.

[0046] Chromatographic detection was performed using the following method:

[0047] Mobile phase: Phase A: 50 mmol / L KH2PO4, H3PO4, pH = 2.5; Phase B: 80% acetonitrile.

[0048] Chromatographic column: Aglient Pursuit C18 3μm 4.6×250mm.

[0049] Column temperature: 45°C; detection wavelength: 215 nm; flow rate: 1 mL / min; injection volume: 5 μL.

[0050] The elution gradient is shown in Table 1.

[0051] Table 1. Elution gradient

[0052] step Time min B％ Flow rate mL / min 1 0 52 1 2 15 62 1 3 30 100 1 4 34 100 1 5 35 52 1 6 40 52 1

[0053] The crude semaglutide peptide prepared by the method of the present invention has a chromatographic result as shown in FIG. Figure 1 As shown in Table 2, the crude peptide was quantified as 18.44 g by reference, with a total yield of 89.63% and a purity of 91.59%.

[0054] Table 2. Chromatographic results

[0055]

[0056]

[0057] Comparative Example 1

[0058] This comparative example was prepared by the following method:

[0059] Fmoc-Lys 20 Preparation of (Dde)-OH Semaglutide Peptide Resin:

[0060] Step 1: The 20th amino acid of the semaglutide sequence is used as the Fmoc-Lys(Dde)-OH fragment.

[0061] Step 2: Weigh 13.6g of Fmoc-Gly-Wang resin (Sub = 0.37mmol / g) and add it to a solid phase reactor. Add 80mL of DCM to swell the resin for 0.5h. Drain the solvent and add 80mL of a 20% piperidine / DMF solution (i.e., a DMF solution containing 20% piperidine by volume). Deprotection reaction for 10+20min. Drain and wash six times with 100mL of DMF. The indene test result is positive. Weigh 9.73g of Fmoc-Arg(Pbf)-OH, 2.5g of HOBT, 3.0mL of DIC, and 60mL of DMF solution. Activate in an ice bath for 10 minutes at a temperature not exceeding 10°C. Add the activated solution to the reactor and react for 1h. After the indene test result is negative, drain. Add DMF and wash three times, 100mL each time.

[0062] Repeat the above steps to prepare the following amino acid sequences: Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc -Glu(OtBu)-OH, Fmoc-Leu-OH, Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu )-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH, Boc-His(Trt)-OH for coupling reaction.

[0063] After the coupling is completed, 200 mL of 2% hydrazine hydrate / DMF solution is added to remove Dde for side chain modification:

[0064] Repeat the above steps and perform coupling reaction according to the amino acid sequence: Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-γ-Glu(OtBu)-OH, uBtO-Oct-OH;

[0065] The mixture was washed alternately with methanol / DCM three times, 200 mL each time, and dried under vacuum to obtain fully protected semaglutide peptide resin.

[0066] Step 3, cleavage: Prepare 250mL of a mixture of TFA, EDT, and TIS in a volume ratio of 90:5:5 as a cleavage reagent. Add the fully protected semaglutide peptide resin under ice bath conditions. After 0.5h, return to room temperature and continue the reaction for 2h. After the reaction is completed, add anhydrous isopropyl ether to precipitate. Centrifuge the precipitate 4 times, adding 300mL of isopropyl ether each time. The product obtained after drying is the crude semaglutide peptide, and the crude peptide yield is 17.45g.

[0067] Chromatographic detection was performed using the following method:

[0068] Mobile phase: Phase A: 50 mmol / L KH2PO4, H3PO4, pH = 2.5; Phase B: 80% acetonitrile.

[0069] Chromatographic column: Aglient Pursuit C18 3μm 4.6×250mm.

[0070] Column temperature: 45°C; detection wavelength: 215 nm; flow rate: 1 mL / min; injection volume: 5 μL.

[0071] The elution gradient is shown in Table 3.

[0072] Table 3. Elution gradient

[0073] step Time min B％ Flow rate mL / min 1 0 52 1 2 15 62 1 3 30 100 1 4 34 100 1 5 35 52 1 6 40 52 1

[0074] The crude semaglutide peptide prepared by the method of Comparative Example 1 was subjected to chromatographic detection and the chromatographic results were as follows: Figure 2 As shown in Table 4, the crude peptide was quantified as 14.45 g by reference, with a total yield of 70.25% and a purity of 83.04%.

[0075] Table 4. Chromatographic results

[0076]

[0077]

[0078] Comparative Example 2

[0079] This comparative example uses Fmoc-Lys 20(Dde)-OH fragment, uBtO-Oct-γ-Glu(OtBu)-AEEA-AEEA-OH fragment and Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH fragment are used in the preparation of semaglutide peptide resin:

[0080] In step 1, the 20th amino acid of the semaglutide sequence is used as the Fmoc-Lys(Dde)-OH fragment, and the semaglutide side chain is used as a whole uBtO-Oct-γ-Glu(OtBu)-AEEA-AEEA-OH fragment; the 1st to 4th amino acids of the semaglutide sequence are used as the Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH fragment.

[0081] Step 2: Weigh 13.6g of Fmoc-Gly-Wang resin (Sub = 0.37mmol / g) and add it to a solid phase reactor. Add 80mL of DCM to swell the resin for 0.5h. Drain the solvent and add 80mL of a 20% piperidine / DMF solution (i.e., a DMF solution containing 20% piperidine by volume). Deprotection reaction for 10+20min. Drain and wash six times with 100mL of DMF. The indene test result is positive. Weigh 9.73g of Fmoc-Arg(Pbf)-OH, 2.5g of HOBT, 3.0mL of DIC, and 60mL of DMF solution. Activate in an ice bath for 10 minutes at a temperature not exceeding 10°C. Add the activated solution to the reactor and react for 1h. After the indene test result is negative, drain. Add DMF and wash three times, 100mL each time.

[0082] Repeat the above steps to prepare the following amino acid sequences: Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Ala-OH, Fmoc -Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH were coupled. After the coupling was completed, 200 mL of 2% hydrazine hydrate / DMF solution was added to remove Dde for side chain modification. Finally, the mixture was washed alternately with methanol / DCM three times, 200 mL each time. Vacuum drying was performed to obtain fully protected semaglutide peptide resin.

[0083] Step 3, cleavage: Prepare 250mL of a mixture of TFA, EDT, and TIS in a volume ratio of 90:5:5 as a cleavage reagent. Add the fully protected semaglutide peptide resin under ice bath conditions. After 0.5h, return to room temperature and continue the reaction for 2h. After the reaction is completed, add anhydrous isopropyl ether to precipitate. Centrifuge the precipitate 4 times, adding 300mL of isopropyl ether each time. The product obtained after drying is the crude semaglutide peptide, and the crude peptide yield is 20.96g.

[0084] Chromatographic detection was performed using the following method:

[0085] Mobile phase: Phase A: 50 mmol / L KH2PO4, H3PO4, pH = 2.5; Phase B: 80% acetonitrile.

[0086] Chromatographic column: Aglient Pursuit C18 3μm 4.6×250mm.

[0087] Column temperature: 45°C; detection wavelength: 215 nm; flow rate: 1 mL / min; injection volume: 5 μL.

[0088] The elution gradient is shown in Table 5.

[0089] Table 5. Elution gradient

[0090] step Time min B％ Flow rate mL / min 1 0 52 1 2 15 62 1 3 30 100 1 4 34 100 1 5 35 52 1 6 40 52 1

[0091] The crude semaglutide peptide prepared by the method of comparative example 2 was subjected to chromatographic detection and the chromatographic results were as follows: Figure 3 As shown in Table 6, the crude peptide was quantified as 17.61 g by reference, with a total yield of 85.63% and a purity of 85.33%.

[0092] Table 6. Chromatographic results

[0093]

[0094]

[0095] It can be seen that the present invention synthesizes semaglutide by using a large number of fragment peptides, and after the fragment peptides are coupled in sequence from the carbon end to the nitrogen end, a 2% hydrazine hydrate / DMF solution is used to selectively remove the Dde protecting group, and the remaining side chain fragments are coupled in sequence. The fully protected semaglutide is synthesized in solid phase, and the crude semaglutide peptide is obtained after cleavage and precipitation, reducing the original 70 steps of the solid phase synthesis of fully protected semaglutide to 27 steps, and almost all the original sites (except Aib, Gly, Oct, AEEA) may be missing and racemized, and the number of sites that are racemized or missing is reduced to less than half. The method of the present invention greatly reduces the reaction steps, reduces the solvent cost, improves the purity of the crude peptide, greatly reduces the purification cost, and is conducive to industrial scale-up production.

[0096] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form or substance. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for synthesizing semaglutide using whole fragments, characterized in that: The method comprises: using amino resin as a starting resin, obtaining semaglutide resin by a polypeptide solid phase synthesis method, and then cracking to obtain a crude semaglutide product; wherein fragment peptides are used entirely in the synthesis process of the semaglutide multi-resin.

2. A method for synthesizing semaglutide using full fragments according to claim 1, characterized in that: The method described is: 1) Select amino acids 1-4 of the semaglutide sequence as Boc-His 1 (Trt)-Aib 2 -Glu 3 (OtBu)-Gly 4 -OH fragment, with amino acids 19-20 of the semaglutide sequence as the dipeptide Fmoc-Ala 19 -Lys 20 (Dde)-OH fragment; semaglutide side chain as the 4-peptide uBtO-Oct-γ-Glu(OtBu)-AEEA-AEEA-OH fragment; 2) After the amino resin is swollen with solvent, the solvent is drained and Fmoc removal reagent is added for deprotection; after draining and washing, the indene test result is positive; activated Fmoc-Gly-Arg(Pbf)-OH is added to react with the condensing agent, and the indene test result is negative, and then draining and washing are performed; Repeat and carry out coupling reaction according to the amino acid sequence; 3) Selectively removing the Dde protecting group and performing side chain modification to obtain fully protected semaglutide peptide resin; 4) A cleavage reagent is added to the fully protected semaglutide peptide resin, and the crude semaglutide peptide is obtained through precipitation and drying.

3. A method for synthesizing semaglutide using full fragments according to claim 1 or 2, characterized in that: The amino resin is one of Fmoc-Gly-Wang resin and Fmoc-Gly-CTC resin, and the resin substitution degree is 0.3-0.5 mmol / g.

4. The method for synthesizing semaglutide using the whole fragment according to claim 2, characterized in that: In step 2), the amino acid sequence is: Fmoc-Val-Arg(Pbf)-OH, Fmoc-Trp(Boc)-Leu-OH, Fmoc-Ile-Ala-OH, Fmoc-Glu(OtBu)-Phe-OH, Fmoc-Gln(Trt)-Ala-OH, Fmoc-Ala-Lys(Dde)-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmo c-Tyr(tBu)-Leu-OH, Fmoc-Ser(tBu)-Ser(tBu)-OH, Fmoc-Asp(OtBu)-Val-OH, Fmoc-Thr(tBu)-Ser(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH.

5. The method for synthesizing semaglutide using whole fragments according to claim 2, characterized in that: In step 2), the amount of the Fmoc-protected amino acid or protected amino acid fragment is 1.5-2.5 times the total molar number of the resin fed.

6. The method for synthesizing semaglutide using whole fragments according to claim 2, characterized in that: In step 2), the solvent is one or more combinations of DCM, DMF, NMP, and DMSO.

7. The method for synthesizing semaglutide using whole fragments according to claim 2, characterized in that: The condensing agent used is selected from one of DIC / HOBt, HBTU / HOBT / DIEA and PyBop / HOBT / DIEA.

8. The method for synthesizing semaglutide using whole fragments according to claim 2, characterized in that: In step 2), the Fmoc removal reagent is a mixed solution of piperidine and DMF, which is composed of 20% piperidine by volume and 80% DMF by volume.

9. The method for synthesizing semaglutide using whole fragments according to claim 2, characterized in that: In step 4), the lysis reagent is a mixed reagent of TFA, EDT and TIS in a volume ratio of 90:5:

5.

10. A method for synthesizing semaglutide using whole fragments according to any one of claims 2 to 9, characterized in that: Step 3) is specifically as follows: after the coupling is completed, hydrazine hydrate / DMF solution is added to remove Dde for side chain modification, methanol / DCM are alternately washed, and vacuum dried to obtain fully protected semaglutide peptide resin.

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