Preparation method of semeglutide

Abstract

The invention discloses a preparation method of semeglutide, which comprises the following steps: respectively using 8-bit and 12-bit single pseudo-proline serine in a combined manner, using 1-2-bit, 3-4-bit, 5-6-bit and 29-30-bit dipeptide fragments in a combined manner, using 27-29-bit and 14-16-bit 2, 4-dimethoxy benzyl modified glycine tripeptide fragments in a combined manner, and further comprising a 20-bit lysine fragment with a side chain group, the method is used for synthesizing semeglutide resin, and the semeglutide resin is cracked and purified to obtain the semeglutide. The preparation method combining the fragments is beneficial to completing solid phase coupling of the semeglutide, greatly improves the substitution value of the semeglutide peptide resin and the purity and yield of a crude product, remarkably reduces the purification difficulty of the crude product, finally reduces the synthesis cost, and is beneficial to industrial mass production.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a preparation method of semaglutide. Background Art

[0002] Semaglutide is a new long-acting GLP-1 analogue developed by Novo Nordisk. Its sequence is:

[0003] Molecular formula: C 187 H 291 N 45 O 59 . It is used to treat type 2 diabetes, reduce overweight, and reduce major adverse cardiovascular events in patients with established cardiovascular disease.

[0004] Patents CN104356224A, CN105753964A, CN106928343A, CN106478806A, CN108203462A, CN108359006A, CN108676087A, and CN109021092A utilize a solid-phase strategy using Fmoc (9-fluorenylmethoxycarbonyl), an amino protecting group widely used in peptide synthesis. Its core function is to enable efficient deprotection under mild alkaline conditions while avoiding side reactions. This method involves long synthesis cycles for sequential amino acid coupling. During stepwise coupling, the resin shrinks severely, resulting in incomplete reactions and defective peptides. The selected solid-phase support has limited substitution values, resulting in low overall yields, high levels of impurities, and difficult purification.

[0005] Patents CN108059666A, CN109627317A and CN113045641A use a solid-phase fragment condensation method for synthesis. Each fragment input in the solid-phase fragment condensation is 2-5 times excessive, which seriously wastes the peptide fragments and causes high synthesis costs. At the same time, the resin substitution value of the solid-phase fragment condensation is limited, the material throughput is reduced, the solvent is wasted, and a large amount of waste liquid is generated.

[0006] Patent CN115677845A prepares semaglutide by using dipeptides as feedstock at specific sites, avoiding racemization at six sites during solid-phase synthesis. CN106749613A and CN108034004A use prefabricated fragments. However, this synthesis method still fails to address the aggregation phenomenon during backbone synthesis, resulting in limited substitution values ​​for solid-phase synthesis resins and reduced material throughput.

[0007] In the main chain modification method to prevent aggregation, Fmoc was used in CN113880935A. N(Dmb) Gly-OH (N-[(2,4-dimethoxybenzyl]-N-[9H-fluoren-9-yl]methoxycarbonyl]glycine), but the subsequent Glu (glutamic acid) coupling is very difficult, and Fmoc N(Dmb) Gly-OH is expensive. In CN109456402A, Val at position 10 and Ser at position 11 are synthesized by using Fmoc-Val10-Ser11 Ser11 (Psi Me,Me Pro)-OH.

[0008] Since the sequence of semaglutide is long and belongs to a medium-length peptide, the main chain is the sequence of semaglutide excluding the Oct-γ-Glu-AEEA-AEEA fragment connected to the Lys side chain at position 20 of the side chain. The main chain synthesis has serious aggregation phenomenon at high substitution values. Therefore, those skilled in the art are still looking forward to methods for obtaining products with good quality with high product yield and low synthesis cost, especially new methods for reducing costs and reducing waste liquid generation, which are very necessary and important for large-scale and industrial production. Summary of the Invention

[0009] The present invention provides a preparation method of semaglutide, which is conducive to completing the solid-phase coupling of semaglutide through the combination of preparation methods of certain specific fragments, greatly improving the substitution value of semaglutide peptide resin, the purity and yield of crude products, significantly reducing the difficulty of crude product purification, and ultimately reducing the synthesis cost, which is conducive to large-scale industrial production.

[0010] In order to solve the above technical problems, a technical solution adopted by the present invention is: the present invention provides a method for preparing semaglutide, comprising the following steps: Step S1, preparing a semaglutide resin as a carrier for solid phase synthesis; Step S2, solid-phase synthesis of semaglutide: using monopseudoproline at positions 8 and 12 in combination, dipeptide fragments at positions 1-2, 3-4, 5-6, and 29-30, and 2,4-dimethoxybenzyl-modified glycine tripeptide fragments at positions 27-29 and 14-16, and also including a lysine fragment with a side chain group at position 20, to synthesize semaglutide resin; Step S3, cleaving the fully protected semaglutide and removing the protecting groups to obtain crude semaglutide peptide; Step S4: Purify the crude semaglutide peptide and exchange the salt to obtain semaglutide.

[0011] Furthermore, in step S2, the following fragments are used in combination in the solid phase synthesis of semaglutide: f) In the coupling of His1-Aib2-Glu3-Gly4, Boc-His(Trt)-Aib-OH is used in the synthesis of His1-Aib2, and Fmoc-Glu(OtBu)-Gly-OH is introduced in the synthesis of Glu3-Gly4; or the commercially available tetrapeptide Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH is used for direct coupling; g) introducing commercially available Fmoc-Thr(tBu)-Phe-OH at the Thr5-Phe6 position; h) Fmoc-Ser (Psi) was introduced at Ser8 and Ser12 Me,Me Pro)-OH; Fmoc-Leu-Glu(OtBu)-N(Dmb)Gly-OH was used at Leu14-Glu15-Gly16; i) using Fmoc-L-Lys[Oct-(OtBu)-Glu-(OtBu)-AEEA-AEEA]-OH at Lys20; Alternatively, Fmoc-Lys(Mtt)-OH, Fmoc-Lys(Alloc)-OH, or Fmoc-Lys(ivDde)-OH was used at position 20, and the side chain protecting group was selectively removed, followed by coupling with homemade Oct-(OtBu)-Glu-(OtBu)-AEEA-AEEA-OH; j) Fmoc-Val-Arg(Pbf)-N(Dmb)Gly-OH was used at Val27-Arg28-Gly29; alternatively, Fmoc-Val-OH alone was used at position 27, Fmoc-Arg(Pbf)-OH alone was used at position 28, and commercially available Fmoc-Gly-Arg(Pbf)-OH was used at positions Gly29-Arg30.

[0012] The present invention improves the production efficiency of semaglutide through convergent polypeptide fragment condensation and uses two in situ peptide backbone modification methods to prevent aggregation during semaglutide synthesis. The combination of the two methods addresses the aforementioned difficulties currently faced in semaglutide synthesis, resulting in a fully protected semaglutide resin. The protecting groups are then cleaved and removed to obtain crude semaglutide peptide, which is then purified and salted to obtain semaglutide. Leveraging technological advantages to achieve cost advantages, the present invention establishes a leading process platform for semaglutide API.

[0013] The use of Boc-His(Trt)-Aib-OH at positions 1-2 reduces the byproducts of histidine racemization; the use of Fmoc-Glu(OtBu)-Gly-OH at positions 3-4 reduces the byproducts of poly-Gly at this position; the use of Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH reduces the byproducts of histidine racemization and poly-Gly; the use of Fmoc-Thr(tBu)-Phe-OH at positions 5-6 reduces the defective products of less Thr; the use of Fmoc-L-Lys[Oct-(OtBu)-Glu-(OtBu)-AEEA-AEEA]-OH or Oct-(OtBu)-Glu-(OtBu)-AEEA-AEEA-OH at position 20 can reduce the byproducts of incomplete side chain coupling; the use of commercially available Fmoc-Gly-Arg(Pbf)-OH at positions Gly29-Arg30 can reduce the byproducts of poly-Gly at this position. The combined use of these commercially available and synthetic fragments can significantly reduce the appearance of by-products and ease the difficulty of purification.

[0014] Modification of the polypeptide backbone inhibits the formation of the polypeptide β-sheet structure. Me,Me Pro)-OH and introduced pseudo-proline structures at Ser8 and Ser12 to achieve in situ peptide backbone modification instead of introducing the dipeptide Fmoc-Val-Ser(Psi Me,Me Pro)-OH and Fmoc-Phe-Thr(Psi Me,Me Pro)-OH expensive unit; at the same time, newly constructed Fmoc-Leu-Glu(OtBu)-N(Dmb)Gly-OH and Fmoc-Val-Arg(Pbf)-N(Dmb)Gly-OH as peptide backbone modification structural units are introduced into the Leu14-Glu15-Gly16 and Val27-Arg28-Gly29 sites, respectively. In this way, the N atoms in the peptide chain have no hydrogen, which greatly reduces the formation of hydrogen bonds between the peptide chains and destroys the β-pleated aggregation structure between the peptide chains. In the embodiment, the substitution value of the solid phase resin can be greatly improved, the synthesis yield and the purity of the crude peptide can be increased, and the cost of the synthesis and subsequent purification of the semaglutide raw material can be reduced.

[0015] Furthermore, the step S2 also includes, before step S21, the removal of a temporary protecting group, wherein the temporary protecting group is an amino protecting group, and the temporary protecting group removal reagent used is a DMF solution (containing 0.1 M HOBt) with a volume percentage of 15% to 25% piperidine or a DMF solution (containing 0.1 M HOBt) with a volume percentage of 1 to 2% DBU.

[0016] Furthermore, the resin raw material of the semaglutide resin in step S1 is Wang resin or acid-sensitive resin.

[0017] In step S1, the specific solid phase synthesis method is a conventional technical means for those skilled in the art.

[0018] Furthermore, the coupling agent used in step S2 is a combination of DIC and HOBt in a molar ratio of 1:1, a combination of DIC and Oxyma in a molar ratio of 1:1, or a combination of HBTU, HOBt and DIEA in a molar ratio of 1:1:2, or a combination of PyBOP, HOBt and DIEA in a molar ratio of 1:1:2.

[0019] Furthermore, in step S3, an amino deprotecting agent is used to remove the protecting group, and the amino deprotecting agent used is a DMF solution (containing 0.1 M HOBt) with a volume percentage of 15% to 25% piperidine or a DMF solution (containing 0.1 M HOBt) with a volume percentage of 1 to 2% DBU.

[0020] Preferably, the amino deprotection reagent used is a DMF solution with a volume percentage of 20% piperidine (containing 0.1M HOBt) or a DMF solution with a volume percentage of 1% DBU (containing 0.1M HOBt); preferably, a DMF solution with a volume percentage of 20% piperidine (containing 0.1M HOBt)

[0021] Furthermore, the cleavage solution for the cleavage of the fully protected semaglutide in step S3 is a mixed solution of TFA and H2O in a volume ratio of 95:5, or a mixed solution of TFA, EDT, TIS, PhOH, and H2O in a volume ratio of 80:5:5:5:5, or a mixed solution of TFA, EDT, TIS, and H2O in a volume ratio of 92.5:2.5:2.5:2.5. Preferably, a mixed solution of TFA, EDT, TIS, and H2O in a volume ratio of 92.5:2.5:2.5:2.5.

[0022] Furthermore, the purification in step S4 is performed by reverse-phase high performance liquid chromatography; the chromatographic column is a C18 column; the mobile phases include A and B as follows: A: 0.1% TFA acetonitrile: water = 4:1 (v:v, containing 0.1% TFA); B: 0.1% TFA / water solution.

[0023] Furthermore, in step S4, the salt exchange is specifically performed by using reverse phase high performance liquid chromatography, a C18 column, and a mobile phase consisting of A: 0.5% AcOH acetonitrile: water = 4:1 (v:v, containing 0.5% AcOH); B: 0.5% AcOH / water; and C: 100 mM ammonium acetate solution.

[0024] The present invention has the following characteristics:

[0025] Given the current demand for semaglutide APIs, significantly improving its production efficiency and changing the synthesis scheme are all things that all practitioners can think of. In the synthesis of semaglutide, we tried to introduce a scheme that introduced cheap peptide backbone modifications and combined the use of different fragments. Surprisingly, we found that the purity of the crude peptide reached more than 80%, which greatly reduced the difficulty of the final liquid chromatography purification.

[0026] The present invention uses a peptide backbone to modify amino acids, resulting in a high substitution value for solid-phase synthesis resins, low amino acid input, and low material costs. This improves the substitution value of the resin in solid-phase synthesis, increases material throughput, and allows for the production of more product using the same volume of reaction vessel and solvent, while reducing waste generation. The crude peptide purity reaches over 80%, significantly reducing the difficulty of final liquid chromatography purification. Therefore, the present invention is characterized by high throughput, low cost, low waste, high efficiency, and easy purification, making it very suitable for large-scale, industrialized production.

[0027] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is an HPLC analysis chart of the crude semaglutide peptide of Example 1 of the present invention; Figure 2 This is an HPLC analysis chart of pure semaglutide peptide according to Example 1 of the present invention; Figure 3 This is an HPLC analysis chart of crude semaglutide peptide of Example 2 of the present invention; Figure 4 This is an HPLC analysis chart of pure semaglutide peptide according to Example 2 of the present invention; Figure 5 This is an HPLC analysis chart of crude semaglutide peptide of Example 3 of the present invention; Figure 6 This is an HPLC analysis chart of pure semaglutide peptide according to Example 3 of the present invention. DETAILED DESCRIPTION

[0029] The following describes the specific embodiments of the present invention through specific examples. Those skilled in the art will readily understand the advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented in various other forms, i.e., various modifications and variations are possible without departing from the scope of the present invention.

[0030] If there is a conflict between the Chinese name and the structural formula of the compound in the present invention, the structural formula shall prevail, unless there is an obvious error in the structural formula.

[0031] The meanings of the material abbreviations used in the present invention are shown in Table 1.

[0032] Table 1 Abbreviations of materials used in the present invention

[0033] Example: A method for preparing semaglutide, comprising the following steps: Step S1: Preparation of Fmoc-Gly-Wang resin: Weigh 137.1 g of resin with a substitution value of 0.85 mmol / g into a reaction kettle. Add DCM to swell the resin and wash the resin three times with DMF. Weigh 1.4 eq (equivalent, meaning the amount equivalent to a specific or conventional value) of Fmoc-Gly-OH, 0.1 eq of DMAP, and 2.1 eq of DIC into the reaction kettle and react for 1 hour. Wash the resin three times with DMF and dry it with methanol. The substitution value is measured to be 0.57 mmol / g.

[0034] Step S21: Removal of temporary protecting group: Removal of Fmoc protecting group: After each coupling step is completed and the coupling is confirmed to be complete, add 20% hexahydropyridine in 0.1 mol / L HOBt DMF solution to remove the Fmoc protecting group, wash the resin with DMF, and check with indene.

[0035] Removal of the side chain Mtt protecting group of Fmoc-Lys(Mtt)-OH (Fmoc-N'-methyltrityl-L-lysine): Wash the resin with DCM, then add 1% TFA / DCM solution to remove the side chain Mtt protecting group. After removal, wash the resin with DCM several times, and then wash the resin with DMF.

[0036] Removal of the Alloc protecting group on the side chain of Fmoc-Lys(Alloc)-OH (N-[(9H-fluorene-9-methoxy)carbonyl]-N'-[(2-propyleneoxy)carbonyl]-L-lysine): Wash the resin with anhydrous DCM, then weigh 0.3 eq of tetrakis(triphenylphosphine)palladium and dissolve it in DCM. Measure 30 eq of phenylsilane and prepare a DCM solution. First, add the phenylsilane solution in DCM, then dropwise add the tetrakis(triphenylphosphine)palladium solution in DCM to remove the Alloc protecting group. Wash the resin several times with DCM, then rinse with DMF.

[0037] Removal of the ivDde protecting group of the Fmoc-Lys(ivDde)-OH side chain: Add 5% (80%) hydrazine hydrate DMF solution to the reactor to remove the ivDde protecting group. After removal, wash the resin with DMF.

[0038] Step S2 includes steps S22 and S23, wherein step S22: coupling of protected amino acids: Weigh 1.5 eq of amino acids (all Fmoc-protected amino acids in the semaglutide sequence excluding the reference fragment and the introduced Fmoc-protected single pseudo-proline), 1.5 eq of HOBt, and 1.5 eq of DIC, dissolve them, and transfer them into a peptide synthesis reactor. React at 0°C for 1 h, then at room temperature for 8 h, and wash with DMF. Repeat the above steps to couple the individual amino acids in the sequence in sequence according to the selected synthetic route, including: Fmoc-Arg(Pbf)-OH 30, Fmoc-Arg(Pbf)-OH 28, Fmoc-Val-OH 27, Fmoc-Leu-OH 26, Fmoc-Trp(Boc)-OH 25, Fmoc-Ala-OH 24, Fmoc-Ile-OH23, Fmoc-Phe-OH 22, Fmoc-Glu(OtBu)-OH 21, Fmoc-Lys(Mtt)-OH 20, Fmoc-Lys(Alloc)-OH20, Fmoc-Lys(ivDde)-OH 20, Fmoc-Ala-OH 19, Fmoc-Ala-OH 18, Fmoc-Gln(Trt)-OH 17, Fmoc-Gly-OH 16. Fmoc-Glu(OtBu)-OH 15. Fmoc-Leu-OH 14. Fmoc-Tyr(tBu)-OH 13. Fmoc-Ser(Psi Me,Me Pro)-OH (monopseudoproserine) 12, Fmoc-Sre(tBu)-OH 11, Fmoc-Val-OH 10, Fmoc-Asp(OtBu)-OH 9, Fmoc-Ser(Psi Me,MePro)-OH (monopseudoproserine) 8, Fmoc-Thr(tBu)-OH 7, Fmoc-Arg(Pbf)-OH 30, Fmoc-Leu-OH 26, Fmoc-Trp(Boc)-OH 25, Fmoc-Ala-OH 24, Fmoc-Ile-OH 23, Fmoc-Phe-OH 22. Fmoc-Glu(OtBu)-OH 21. Fmoc-Ala-OH 19. Fmoc-Ala-OH 18. Fmoc-Gln(Trt)-OH 17. Fmoc-Gly-OH 16. Fmoc-Glu(OtBu)-OH 15. Fmoc-Leu-OH 14. Fmoc-Tyr(tBu)-OH 13. Fmoc-Ser(Psi Me,Me Pro)-OH 12 (monopseudoproline) Fmoc-Ser(tBu)-OH 12, Fmoc-Sre(tBu)-OH 11, Fmoc-Val-OH 10, Fmoc-Asp(OtBu)-OH 9, Fmoc-Ser(Psi Me,Me Pro)-OH 8 (single pseudo-proline) or Fmoc-Ser(tBu)-OH 8, Fmoc-Thr(tBu)-OH 7, Fmoc-Phe-OH 6, Fmoc-Thr(tBu)-OH 5; until the coupling of all amino acids in the polypeptide chain is completed.

[0039] Step S23: Coupling of fragments: Depending on the synthetic route, different fragments are introduced into the solid phase synthesis of semaglutide.

[0040] Weigh 1.5 eq of the peptide fragment (the fragment referenced in the synthetic route selected in the semaglutide sequence), 1.5 eq of PyBop, and 1.5 eq of DIEA, dissolve them, and transfer them to a peptide synthesis reactor. React at 0°C for 1 h, then at room temperature for 8-15 h. Wash with DMF. Depending on the selected synthetic route, selectively couple Fmoc-Gly-Arg(pbf)-OH or Fmoc-Val-Arg(Pbf)-N(Dmb)Gly-OH at positions 29-30; Fmoc-Lys[Octadecanedioic acid mono-tert-butyl ester-Glu(AEEA-AEEA)-OtBu]-OH (i.e., Fmoc-L-Lys[Oct-(OtBu)-Glu-(OtBu)-AEEA-AEEA]-OH) at position 20, or couple Octadecanedioic acid to the side chain at position 20. mono-tert-butylester-γ-Glu(OtBu)AEEA-AEEA-OH (corresponding to "using Fmoc-Lys(Mtt)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Lys(ivDde)-OH at position 20, selectively removing the side chain protecting group, and coupling the homemade Oct-(OtBu)-Glu-(OtBu)-AEEA-AEEA-OH"); Couple Fmoc-Leu-Glu(OtBu)-N(Dmb)Gly-OH; couple Fmoc-Thr(tBu)-Phe-OH at positions 5-6; couple Fmoc-Glu(OtBu)-Gly-OH at positions 3-4; couple Boc-His(Trt)-Aib-OH at positions 1-2 or Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH at positions 1-4 until all amino acids in the polypeptide chain are coupled.

[0041] Steps S22 and S23 correspond to step S2 in claim 1, namely, the solid-phase synthesis of semaglutide is accomplished by sequentially coupling a single protected amino acid and a fragment. Step S22 couples a single protected amino acid in solid-phase synthesis, coupling one site at a time; step S23 couples a polypeptide fragment intermediate in solid-phase synthesis, coupling multiple consecutive sites at a time. The order of these two steps completely follows the amino acid sequence of semaglutide from carbon to nitrogen terminus in solid-phase synthesis.

[0042] Step S3: Cleavage (cleavage) of the polypeptide: The fully protected semaglutide is cleaved and the protecting groups are removed to obtain the crude semaglutide peptide, specifically: Method 1: Weigh the dried peptide resin and prepare a cutting solution (2.5% water, 2.5% triisopropylsilane, 2.5% 1,4-phenylenedimethanethiol, 92.5% trifluoroacetic acid, v:v:v:v:v). Add the peptide resin and cutting solution sequentially to a glass reactor and stir for 3 hours. After the reaction, collect the cutting solution, rotary evaporate, precipitate with methyl tert-butyl ether, filter, and dry to obtain a dry crude product. The crude product was dissolved in deionized water and acetonitrile. HPLC analysis revealed a purity of 72.37%.

[0043] Method 2: Weigh the dried peptide resin and prepare a cutting solution, Regent K (5% water, 5% methyl phenyl sulfide, 5% phenol, 2.5% 1,4-ethanedithiol, 82.5% trifluoroacetic acid, v:v:v:v:v). Add the peptide resin and cutting solution sequentially to a glass reactor and stir for 3 hours. After the reaction, collect the cutting solution, rotary evaporate, precipitate with methyl tert-butyl ether, filter, and dry to obtain a dry crude product.

[0044] Step S4: Purification and purity detection of target polypeptide: The crude semaglutide peptide is purified and salt-exchanged to obtain semaglutide, specifically: Dissolve the crude product from step S3 in deionized water and ammonia as a cosolvent. Purify the crude product using reversed-phase HPLC with the following mobile phases: A: acetonitrile:water = 4:1 (v:v, containing 0.1% TFA); B: 0.1% TFA / water. Dissolve the purified product from the previous step in deionized water and acetic acid as a cosolvent. Salt exchange the purified product using reversed-phase HPLC with the following mobile phases: A: acetonitrile:water = 4:1 (v:v, containing 0.5% AcOH); B: 0.5% AcOH / water; C: 100 mM ammonium acetate solution.

[0045] Example 1: Synthesis Route 1 of Semaglutide

[0046] Fmoc-Gly-Arg(pbf)-OH was introduced at positions 29-30 of semaglutide; Fmoc-Lys[Octadecanedioic acid mono-tert-butyl ester-Glu(AEEA-AEEA)-OtBu]-OH was introduced at position 20; Fmoc-Thr(tBu)-Phe-OH was introduced at positions 5-6; and Boc-His(Trt)-Aib-Glu (OtBu)-Gly-OH was introduced at positions 1-4.

[0047] Weigh 54.5 g of 0.57 mmol / L Fmoc-Gly-Wang resin synthesized in step S1, swell the resin with DCM, wash the resin three times with DMF, and repeat the deprotection and amino acid coupling steps according to the methods in steps S21 and S22 to couple Fmoc-Val-OH 27, Fmoc-Leu-OH 26, Fmoc-Trp(Boc)-OH 25, Fmoc-Ala-OH 24, Fmoc-Ile-OH 23, Fmoc-Phe-OH 22, Fmoc-Glu(OtBu)-OH 21, Fmoc-Ala-OH 19, Fmoc-Ala-OH18, Fmoc-Gln(Trt)-OH 17, Fmoc-Gly-OH 16, Fmoc-Glu(OtBu)-OH 15, Fmoc-Leu-OH 14. Fmoc-Tyr(tBu)-OH 13, Fmoc-Ser(tBu)-OH 12, Fmoc-Sre(tBu)-OH 11, Fmoc-Val-OH 10, Fmoc-Asp(OtBu)-OH 9, Fmoc-Ser(tBu)-OH 8, Fmoc-Thr(tBu)-OH 7.

[0048] During the synthesis process, according to the method in step S23, Fmoc-Gly-Arg(pbf)-OH 29-30, Fmoc-Lys[Octadecanedioic acid mono-tert-butyl ester-Glu(AEEA-AEEA)-OtBu]-OH20, Fmoc-Thr(tBu)-Phe-OH 5-6, and Boc-His(Trt)-Aib-Glu (OtBu)-Gly-OH 1-4 were coupled in sequence to complete the solid phase synthesis of semaglutide.

[0049] According to the method 1 in step S3, the peptide resin was cleaved and deionized water and acetonitrile were used as solvents to dissolve the resulting crude product. The purity was determined by HPLC to be 78.46%. The sample was then purified according to the method in step S4. The purity met the standard requirements and was 99.49%. The final lyophilized product weighed 47.71 g, and the total yield was 37.46%. Figure 1 and Figure 2 .

[0050] Figure 1This is the HPLC analysis chart of semaglutide synthesized via synthetic route 1. The upper part is the peak shape diagram. In the lower part of the table, the third column is the peak time, the fourth column is the peak area, the fifth column is the peak width (min), the sixth column is the half-peak width (min), the seventh column is the peak height (mAu), and the eighth column is the area percentage (%). Each row represents a different peak in the peak shape diagram. Among them, the peak at the peak time of 20.870 is the main peak of the target product. The higher the peak area percentage here, the higher the purity of the crude peptide synthesized this time, and the better the synthesis effect.

[0051] Figure 2 This is the HPLC analysis chart of the final product obtained after the crude peptide is purified and salted. The upper part is also a peak shape chart, and the table below is the data description of the peak shape chart above. Figure 1 Similarly, the higher the area percentage of the main peak with an elution time of 24.717, the higher the purity of the purified peptide, and the better the synthesis and purification effect. At the same time, the lower the area percentage of other peaks excluding the main peak, the lower their relative content in the pure peptide, and the better the effect.

[0052] Example 2: Synthesis Route 2 of Semaglutide

[0053] Fmoc-Val-Arg(Pbf)-N(Dmb)Gly-OH was introduced at positions 27-29 of semaglutide; Fmoc-Lys[Octadecanedioic acid mono-tert-butyl ester-Glu(AEEA-AEEA)-OtBu]-OH was introduced at position 20; and Fmoc-Ser(Psi Me,Me Pro)-OH (monopseudoproline); Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH was introduced at positions 1-4.

[0054] Weigh 54.1 g of 0.57 mmol / L Fmoc-Gly-Wang resin synthesized in step S1, swell the resin with DCM, wash the resin three times with DMF, and repeat the deprotection and amino acid coupling steps according to the methods in steps S21 and S22 to couple Fmoc-Arg(Pbf)-OH 30, Fmoc-Leu-OH 26, Fmoc-Trp(Boc)-OH 25, Fmoc-Ala-OH24, Fmoc-Ile-OH 23, Fmoc-Phe-OH 22, Fmoc-Glu(OtBu)-OH 21, Fmoc-Ala-OH 19, Fmoc-Ala-OH 18, Fmoc-Gln(Trt)-OH 17, Fmoc-Gly-OH 16, Fmoc-Glu(OtBu)-OH 15. Fmoc-Leu-OH14, Fmoc-Tyr(tBu)-OH 13. Fmoc-Ser(Psi Me,Me Pro)-OH (monopseudoproserine) 12, Fmoc-Sre(tBu)-OH 11, Fmoc-Val-OH 10, Fmoc-Asp(OtBu)-OH 9, Fmoc-Ser(Psi Me,Me Pro)-OH (monopseudoproline) 8, Fmoc-Thr(tBu)-OH 7, Fmoc-Phe-OH 6, Fmoc-Thr(tBu)-OH 5.

[0055] During the synthesis process, according to the method in step S23, Fmoc-Val-Arg(Pbf)-N(Dmb)Gly-OH 27-29, Fmoc-Lys[Octadecanedioic acid mono-tert-butyl ester-Glu(AEEA-AEEA)-OtBu]-OH 20, and Boc-His(Trt)-Aib-Glu (OtBu)-Gly-OH 1-4 were coupled in sequence to complete the solid phase synthesis of semaglutide.

[0056] According to the method 1 in step S3, the peptide resin was cleaved and deionized water and acetonitrile were used as solvents to dissolve the resulting crude product. The purity was determined by HPLC to be 78.325%. The sample was then purified according to the method in step S4. The purity met the standard requirements and was 99.88%. The final lyophilized product weighed 46.54 g, and the total yield was 36.54%. Figure 3 and Figure 4 .

[0057] Example 3: Semaglutide Synthesis Route 3

[0058] Fmoc-Val-Arg(Pbf)-N(Dmb)Gly-OH was introduced at positions 27-29 of semaglutide; Fmoc-Lys[Octadecanedioic acid mono-tert-butyl ester-Glu(AEEA-AEEA)-OtBu]-OH was introduced at position 20; Fmoc-Leu-Glu(OtBu)-N(Dmb)Gly-OH was introduced at positions 14-16; and Fmoc-Ser(Psi)-H was introduced at positions 12 and 8. Me,Me Fmoc-Glu (OtBu)-Gly-OH was introduced at the 3-4 position of Pro)-OH (monopseudoproline); Boc-His(Trt)-Aib-OH was introduced at the 1-2 position.

[0059] Weigh 54.8 g of 0.57 mmol / L Fmoc-Gly-Wang resin synthesized in step S1, swell the resin with DCM, wash the resin three times with DMF, and repeat the deprotection and amino acid coupling steps according to the methods in steps S21 and S22 to couple Fmoc-Arg(Pbf)-OH 30, Fmoc-Leu-OH 26, Fmoc-Trp(Boc)-OH 25, Fmoc-Ala-OH24, Fmoc-Ile-OH 23, Fmoc-Phe-OH 22, Fmoc-Glu(OtBu)-OH 21, Fmoc-Lys(Mtt)-OH 20, Fmoc-Ala-OH 19, Fmoc-Ala-OH 18, Fmoc-Gln(Trt)-OH 17, Fmoc-Tyr(tBu)-OH 13. Fmoc-Ser(Psi Me,Me Pro)-OH (monopseudoproserine) 12, Fmoc-Sre(tBu)-OH 11, Fmoc-Val-OH 10, Fmoc-Asp(OtBu)-OH 9, Fmoc-Ser(Psi Me,Me Pro)-OH (monopseudoproline) 8, Fmoc-Thr(tBu)-OH 7, Fmoc-Phe-OH 6, Fmoc-Thr(tBu)-OH 5.

[0060] During the synthesis process, according to the method in step S23, Fmoc-Val-Arg(Pbf)-N(Dmb)Gly-OH 27-29, Octadecanedioic acid mono-tert-butyl ester-γ-Glu(OtBu)AEEA-AEEA-OH20 side chain, Fmoc-Glu (OtBu)-Gly-OH 3-4, and Boc-His(Trt)-Aib-OH 1-2 were coupled in sequence to complete the solid phase synthesis of semaglutide.

[0061] According to the method 1 in step S3, the peptide resin was cleaved and deionized water and acetonitrile were used as solvents to dissolve the crude product. The purity of the product was 80.685% by HPLC. The sample was then purified according to the method in step S4. The purity met the standard requirements and was 99.60%. The final lyophilized product weighed 49.69 g and the total yield was 39.01%. Figure 5 and Figure 6 .

[0062] And, through Figures 1-6 This indicates that the crude semaglutide peptide synthesized according to the third synthetic route of Example 3 has the best purity and the method is optimal.

[0063] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structures made using the contents of the present invention's description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing semaglutide, characterized in that: The steps include: Step S1, preparing a semaglutide resin as a carrier for solid phase synthesis; Step S2, solid-phase synthesis of semaglutide: using monopseudoproline at positions 8 and 12 in combination, dipeptide fragments at positions 1-2, 3-4, 5-6, and 29-30, and 2,4-dimethoxybenzyl-modified glycine tripeptide fragments at positions 27-29 and 14-16, and also including a lysine fragment with a side chain group at position 20, to synthesize semaglutide resin; Step S3, cleaving the fully protected semaglutide and removing the protecting groups to obtain crude semaglutide peptide; Step S4: Purify the crude semaglutide peptide and exchange the salt to obtain semaglutide.

2. The method for preparing semaglutide according to claim 1, wherein: In step S2, the following fragments are used in combination in the solid phase synthesis of semaglutide: a) In the coupling of His1-Aib2-Glu3-Gly4, Boc-His(Trt)-Aib-OH is used in the synthesis of His1-Aib2, and Fmoc-Glu(OtBu)-Gly-OH is introduced in the synthesis of Glu3-Gly4; or the commercially available tetrapeptide Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH is used for direct coupling; b) introducing commercially available Fmoc-Thr(tBu)-Phe-OH at the Thr5-Phe6 position; c) Fmoc-Ser (Psi) was introduced at Ser8 and Ser12 Me,Me Pro)-OH; Fmoc-Leu-Glu(OtBu)-N(Dmb)Gly-OH was used at Leu14-Glu15-Gly16; d) using Fmoc-L-Lys[Oct-(OtBu)-Glu-(OtBu)-AEEA-AEEA]-OH at Lys20; Alternatively, Fmoc-Lys(Mtt)-OH, Fmoc-Lys(Alloc)-OH, or Fmoc-Lys(ivDde)-OH was used at position 20, and the side chain protecting group was selectively removed, followed by coupling with homemade Oct-(OtBu)-Glu-(OtBu)-AEEA-AEEA-OH; e) Fmoc-Val-Arg(Pbf)-N(Dmb)Gly-OH was used at Val27-Arg28-Gly29; alternatively, Fmoc-Val-OH alone was used at position 27, Fmoc-Arg(Pbf)-OH alone was used at position 28, and commercially available Fmoc-Gly-Arg(Pbf)-OH was used at positions Gly29-Arg30.

3. The method for preparing semaglutide according to claim 1, wherein: Before step S2, the method further includes step S21 of removing a temporary protecting group, wherein the temporary protecting group is an amino protecting group, and the temporary protecting group removing reagent used is a DMF solution with a volume percentage of 15% to 25% piperidine or a DMF solution with a volume percentage of 1 to 2% DBU.

4. The method for preparing semaglutide according to claim 1, wherein: The resin raw material of the semaglutide resin in step S1 is Wang resin or acid-sensitive resin.

5. The method for preparing semaglutide according to claim 1, wherein: The coupling agent used in step S2 is a combination of DIC and HOBt in a molar ratio of 1:1, a combination of DIC and Oxyma in a molar ratio of 1:1, or a combination of HBTU, HOBt and DIEA in a molar ratio of 1:1:2, or a combination of PyBOP, HOBt and DIEA in a molar ratio of 1:1:

2.

6. The method for preparing semaglutide according to claim 1, wherein: In step S3, an amino deprotection reagent is used to remove the protecting group. The amino deprotection reagent used is a DMF solution with a volume percentage of 15% to 25% piperidine or a DMF solution with a volume percentage of 1 to 2% DBU.

7. The method for preparing semaglutide according to claim 1, wherein: The cleavage solution for the cleavage of the fully protected semaglutide in step S3 is a mixed solution of TFA and H2O in a volume ratio of 95:5, or a mixed solution of TFA, EDT, TIS, PhOH, and H2O in a volume ratio of 80:5:5:5:5, or a mixed solution of TFA, EDT, TIS, and H2O in a volume ratio of 92.5:2.5:2.5:2.

5.

8. The method for preparing semaglutide according to claim 1, wherein: The purification in step S4 is performed by reverse-phase high performance liquid chromatography; the chromatographic column is a C18 column; the mobile phase comprises A and B as follows: A: 0.1% TFA acetonitrile: water = 4:1; B: 0.1% TFA / water solution.

9. The method for preparing semaglutide according to claim 1, wherein: In step S4, the salt exchange is specifically performed by using reverse phase high performance liquid chromatography, a C18 column, and a mobile phase consisting of A: 0.5% AcOH acetonitrile: water = 4:1; B: 0.5% AcOH / water; and C: 100 mM ammonium acetate solution.

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