A method for the synthesis of semaglutide

Abstract

This invention belongs to the field of peptide drug synthesis, specifically relating to a method for synthesizing smegglutinin. This method uses a modified amino resin as the solid-phase resin, which can significantly improve the yield and purity of the peptide, and the raw materials are inexpensive and the method is simple.

Description

Technical Field

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

[0002] Smegglutide is a glucagon-like peptide-1 (GLP-1) receptor agonist with 94% amino acid sequence homology to human GLP-1. It promotes insulin secretion and inhibits glucagon secretion, lowering blood glucose levels in patients with type 2 diabetes and reducing the incidence of hypoglycemia. Simultaneously, it slows gastric emptying and suppresses appetite, thus achieving a weight-loss effect. Developed by Novo Nordisk, smegglutide is one of the most watched hypoglycemic and weight-loss drugs globally, with a promising market prospect.

[0003] There are many publicly disclosed synthetic routes for semaglutide in the prior art, with the most common being a chemical solid-phase synthesis method. This method uses Wang resin as the starting resin for solid-phase synthesis, followed by coupling according to the semaglutide amino acid sequence, as seen in patents CN112250755A and CN111217901A. However, the inventors unexpectedly discovered that when using this coupling method, a difficult-to-remove impurity, presumably Trp, exists at approximately 0.88 RRT for semaglutide. This impurity is generated during lysis and has a molecular weight and chemical properties similar to semaglutide, making it difficult to separate effectively on a chromatographic column. Repeated elution can easily affect the yield and purity of the final product. Currently, commonly used impurity removal methods in this field include shortening the lysis time, reducing the acid concentration in the lysis buffer, stepwise lysis, and adding a large amount of cleavage reagent. However, it is still difficult to completely avoid the generation of this impurity, and incomplete deprotection or the addition of a large amount of cleavage reagent can easily introduce other impurities.

[0004] Existing technologies disclose the use of CTC resin as a solid-phase resin for coupling, such as patents CN106928343A and CN110372785A. However, CTC resin is expensive and sensitive to acids and water. During long-chain coupling, peptide chains are easily detached from the resin, resulting in the removal of peptide fragments, leading to low yields and high impurity levels. Other literature discloses synthetic methods using amino resins as solid-phase resins for coupling, such as patent CN114031680A. However, these synthetic routes often involve complex first amino acid structures, making synthesis difficult, with high development costs and the need for customization, hindering industrial-scale mass production. Furthermore, subsequent synthetic steps are also quite cumbersome.

[0005] Therefore, it is necessary to develop a method for synthesizing smegglutinin with high yield and purity, simple steps, and low cost. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a novel method for synthesizing smegglutinin. This method uses a modified amino resin as the solid-phase resin, which can significantly improve the yield and purity of the peptide, and the raw materials are inexpensive and the method is simple.

[0007] A method for synthesizing smegglutinin, the method employing a solid-phase synthesis approach, comprising the following steps:

[0008] (1) After deprotection of the amino resin, it is reacted with the linker and activating agent to obtain the modified resin.

[0009] (2) The first amino acid Fmoc-Gly-OH was activated by an activating reagent, and then DAMP was added to catalyze and couple the modified resin.

[0010] (3) After removing the protection from the resin that has been coupled with amino acids, the second amino acid is coupled by activating the resin with an activating agent.

[0011] (4) According to the smegglutinin sequence, repeat step (3) to couple the amino acids from the C-terminus to the N-terminus to the peptide resin to obtain smegglutinin resin.

[0012] (5) Cleavage the smegglutinin resin and purify it to obtain smegglutinin.

[0013] The degree of substitution of the amino resin is 0.1-1 mmol / g, and the linker is a compound having the structure of Formula I:

[0014]

[0015] Wherein, R1 and R2 are selected from H or C1-C4 alkoxy groups; n is an integer selected from 1-20; the carboxyl group of the linker is connected to the amino group of the amino resin.

[0016] As a preferred embodiment of the present invention, in step (1), the feed ratio of the linker:activator:resin, calculated by resin equivalent, is (1-10):(1-10):1, more preferably (1-5):(1-5):1, and even more preferably (1-3):(1-3):1. More preferably, the equivalent ratio of the linker:activator is 1:1.

[0017] Preferably, in the linker, R1 and R2 are selected from groups H, methoxy, or ethoxy. In some preferred embodiments, R1 and R2 are both H or both methoxy; in other preferred embodiments, R1 is H and R2 is methoxy.

[0018] Preferably, in the linker, n is an integer selected from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; more preferably, n is an integer selected from 1 to 5.

[0019] Preferably, the linker is selected from 4-(hydroxymethyl)phenylacetic acid, 4-(hydroxymethyl)phenylpropionic acid, 4-(hydroxymethyl)phenylbutyric acid, 4-(hydroxymethyl)phenylvaleric acid, 4-(hydroxymethyl)phenylhexanoic acid, 3-(4-(hydroxymethyl)-3-methoxyphenyl)propionic acid, and 3-(4-(hydroxymethyl)-3,5-dimethoxyphenyl)propionic acid.

[0020] Preferably, the activating agent is one or more selected from the combination of HOBT / DIC, Oxyma / DIC, TBTU / DIEA, HBTU / DIEA, PyBOP / DIEA, and HATU / DIEA, more preferably HOBT / DIC and Pybop / DIEA, with an equivalent ratio of 1:1. Further, the activating agents used in steps (1)-(4) can be the same or different.

[0021] Preferably, the amino resin is selected from Am resin, MBHA resin, Rink Amide AM resin, Rink Amide MBHA resin, and Sieber resin.

[0022] Preferably, the degree of substitution of the amino resin is 0.2-0.6 mmol / g, more preferably 0.2-0.5 mmol / g.

[0023] Preferably, in step (4), the coupling method follows the smegglutinin sequence and can be performed by sequential coupling of single amino acids or by fragment coupling. More preferably, in step (4), the amino acids Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH·H2O, and Fmoc-I are coupled sequentially according to the smegglutinin sequence. le-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-L-Lys[Oct-(OtBu)-Glu-(OtBu)-AEEA-AEEA]-OH, Fmoc-Ala -OH·H2O, Fmoc-Ala-OH·H2O, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Leu-OH, Fmoc-Tyr( 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, Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH or amino acids with equivalent structures.

[0024] Preferably, in step (5), the pyrolysis reagent used is trifluoroacetic acid or a combination of trifluoroacetic acid and a capture reagent. The capture reagent is one or more of anisole, ethylenedithiol, anisole, phenol, triisopropylsilane, and water, mixed with trifluoroacetic acid in different volume ratios. More preferably, the pyrolysis reagent, calculated by volume ratio, is 80-100% trifluoroacetic acid, 0-10% anisole, 0-10% ethylenedithiol, 0-10% anisole, 0-10% phenol, 0-10% triisopropylsilane, and 0-10% water, mixed in different volume ratios. In some preferred embodiments, the pyrolysis reagent is trifluoroacetic acid; in other preferred embodiments, the pyrolysis reagent is trifluoroacetic acid: anisole: water: phenol: ethylenedithiol, with a volume ratio of 82.5:5:5:5:2.5.

[0025] Furthermore, the solid-phase synthesis method includes the following steps:

[0026] (1) Add amino resin to a solid-phase reaction column, swell it, then add DBLK solution to remove the protecting group, add 4-(hydroxymethyl)phenylpropionic acid, HOBt / DIC and react with amino resin to obtain the modified resin;

[0027] (2) The first amino acid Fmoc-Gly-OH was activated by HOBt / DIC activating reagent, and then DAMP was added to catalyze and couple with the modified resin.

[0028] (3) After removing the protection from the resin after coupling the amino acids, the second amino acid Fmoc-Arg(Pbf)-OH was activated by the HOBt / DIC activating agent.

[0029] (4) Repeat step (3), sequentially add the amino acids Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH·H2O, Fmoc-I le-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-L-Lys[Oct-(OtBu)-Glu-(OtBu)-AEEA-AEEA]-OH, Fmoc-Ala- OH·H2O, Fmoc-Ala-OH·H2O, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Leu-OH, Fmoc-Tyr(tB The following peptides were coupled together to obtain Smeglucopyrene resin: (u)-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, and Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH.

[0030] (5) Cleavage the smegglutinin resin and purify it to obtain smegglutinin.

[0031] More preferably, in any of the steps (1)-(4), the reaction temperature is 15-30℃, more preferably 20-25℃; and the reaction time is ≥0.5h, more preferably ≥1h.

[0032] In step (5), the pyrolysis reagent used is preferably trifluoroacetic acid: benzyl sulfide: water: phenol: ethylene dithiol, with a volume ratio preferably of 82.5:5:5:5:2.5. The pyrolysis temperature is preferably 15-30℃, more preferably 20-25℃.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] (1) The present invention uses amino resin modified with compound of general formula I as solid phase resin, which not only effectively solves the problem that wang resin is prone to generating Trp impurities and is difficult to remove in the prior art, but also greatly improves the yield of smegglutinin and the production efficiency is higher.

[0035] (2) The materials used in the synthesis route of this invention are cheap and readily available, the synthesis steps are simple and the cost is low, which is more conducive to commercial mass production. Detailed Implementation

[0036] The present invention is further illustrated below by way of embodiments, but these embodiments are not intended to limit the invention to the scope of the embodiments described. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0037] The term "alkoxy" as used in this invention refers to alkyl-O-, preferably C1-C4 alkoxy, such as methoxy, ethoxy, etc.

[0038] The specific meanings of the English abbreviations used in this invention are detailed in the table below.

[0039]

[0040]

[0041] Unless otherwise stated, the material information for the following embodiments and comparative examples is all commercially available.

[0042] Example 1

[0043] (1) Amino resin modification

[0044] Weigh 4.0 g of AM resin with a substitution degree of 0.50 mmol / g and add it to a solid-phase reaction column. After adding DCM to swell the resin for 30 minutes, wash three times with DMF. Add DBLK solution to the reaction column, react for 5 minutes, filter, wash once with DMF, add DBLK solution again, react for 10 minutes, and the Kaiser test is positive. Filter and remove the DBLK solution with DMF.

[0045] 4-(hydroxymethyl)phenylpropionic acid, HOBt, and DMF were added to the reaction column, and the mixture was stirred under purging. DIC was then added, and the reaction was allowed to proceed for 2 hours. After the reaction was complete, the column was washed with DMF and dried under vacuum.

[0046] (2) Peptide resin coupling

[0047] First amino acid coupling: Weigh Fmoc-Gly-OH and HOBt into an activation flask, add DMF, stir to dissolve, and cool to 0-10℃. Add DIC and stir for 5 minutes. Add the activation solution to the resin for reaction, weigh out DMAP, and stir for 4-10 hours. After the reaction, dry the liquid under vacuum, add DMF, wash, and dry again. Add DMF, acetic anhydride, and pyridine to the synthesis column and stir for 2 hours. After the reaction, dry the liquid under vacuum, add DMF, wash, and dry again.

[0048] The second amino acid coupling: DBLK solution was added to the reaction column, reacted for 5 minutes, filtered, washed once with DMF, and then DBLK solution was added again, reacted for 10 minutes, and the Kaiser test was positive. The DBLK solution was then filtered and purified with DMF. The weighed Fmoc-Arg(Pbf)-OH and HOBt were dissolved in DMF and cooled to 0-10℃. The condensation reagent DIC was added, and the reaction was stirred for 5 minutes. The activating solution was added to the resin and stirred for 2 hours. After the reaction, the liquid was dried under vacuum, washed with DMF, and then dried again.

[0049] Repeat the steps of removing Fmoc protection from the second amino acid and adding the corresponding protecting amino acid or fragment for coupling, following the order of the fragments, coupling the esmegglutinin amino acid sequence as follows: Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH·H2O, Fmoc-I le-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-L-Lys[Oct-(OtBu)-Glu-(OtBu)-AEEA-AEEA]-OH, Fmoc-Ala -OH·H2O, Fmoc-Ala-OH·H2O, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Leu-OH, Fmoc-Tyr( 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, Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH, to obtain Smeglucopyrepeptide resin.

[0050] (3) Crude peptide cleavage

[0051] The smegglutinin peptide resin obtained in the above steps was placed in a pyrolysis reactor, and a pyrolysis reagent was added at a ratio of 10 ml / g resin. The pyrolysis reagent was trifluoroacetic acid: benzyl sulfide: water: phenol: ethylene dithiol = 82.5:5:5:5:2.5 (V / V). The mixture was stirred and pyrolyzed at room temperature for 2 hours. The reaction mixture was filtered through a sintered glass funnel, and the filtrate was collected. The resin was washed three times with a small amount of TFA, and the filtrates were combined and concentrated under reduced pressure. Cooled methyl tert-butyl ether was added to precipitate the resin, which was then washed five times with methyl tert-butyl ether and dried under vacuum to obtain crude smegglutinin peptide.

[0052] (4) Purification of arginine peptides

[0053] The crude semaglutide peptide was added to water at a concentration of approximately 5 g / L, and the pH was adjusted to 7.0-8.0 with ammonia. The solution was then filtered to obtain a crude peptide solution. The crude semaglutide peptide solution was purified using a preparative liquid chromatography system (HPLC) through multiple steps. The first step of purification used C18 as the stationary phase and phosphate solution-acetonitrile as the mobile phase, yielding a sample solution with a purity greater than 98%. The second step of purification used C18 as the stationary phase and phosphate solution-acetonitrile as the mobile phase, yielding a sample solution with a purity greater than or equal to 99%. Sodium semaglutide was obtained by salt conversion, followed by vacuum concentration and lyophilization to obtain refined semaglutide peptide.

[0054] Comparative Example 1

[0055] (1) Resin swelling: Weigh 3.77g of Wang resin with a substitution degree of 0.53mmol / g and add it to the solid-phase reaction column. After adding DCM to swell the resin for 30 minutes, wash it three times with DMF.

[0056] Following the synthesis method of steps (2)-(4) in Example 1, smegglutinin peptide was synthesized.

[0057] Based on the synthetic routes of Example 1 and Comparative Example 1, the purity, yield, and impurity content at approximately 0.88 of the crude peptide in step (3) and the refined peptide in step (4) were calculated, and the results are shown in Tables 1-2 below.

[0058] Table 1. Purity and Yield of Example 1 and Comparative Example 1

[0059]

[0060] Table 2 Impurity content of Example 1 and Comparative Example 1

[0061]

[0062]

[0063] The data in the table above shows that, compared to the crude smegglutinin peptide obtained by coupling with ordinary WANG resin, the crude smegglutinin peptide obtained by modifying amino resin has a higher purity. Only 0.5% impurity was detected at approximately 0.88 RRT, far lower than in Comparative Example 1. After purification, the impurities in Example 1 were completely removed, with a purification yield as high as 72%. In contrast, Comparative Example 1 had a low peptide purification yield, and the impurity at approximately 0.88 RRT was not completely removed, leaving 0.4% Trp impurity.

Claims

1. A method for synthesizing smegglutinin, wherein the synthesis method employs a solid-phase synthesis method, comprising the following steps: (1) After deprotection of the amino resin, it is reacted with the linker and activating agent to obtain the modified resin. (2) The first amino acid Fmoc-Gly-OH was activated by an activating reagent, and then DAMP was added to catalyze and couple the modified resin. (3) After removing the protection from the resin after coupling the amino acids, the second amino acid Fmoc-Arg(Pbf)-OH was activated by an activating reagent. (4) According to the smegglutinin sequence, repeat step (3) to couple the amino acids from the C-terminus to the N-terminus to the peptide resin to obtain smegglutinin resin. (5) Cleavage the smegglutinin resin and purify it to obtain smegglutinin. The degree of substitution of the amino resin is 0.1-1 mmol / g, and the linker is a compound having the structure of Formula I: Wherein, R1 and R2 are selected from H groups or C1-C4 alkoxy groups; and n is an integer selected from 1 to 20.

2. The synthesis method according to claim 1, characterized in that, In step (1), the feed ratio of the linker: activator: resin is (1-10): (1-10): 1, more preferably (1-5): (1-5): 1, calculated by resin equivalent.

3. The synthesis method according to claim 1, characterized in that, In the linker, R1 and R2 are selected from groups H, methoxy, or ethoxy; n is an integer selected from 1 to 10.

4. The synthesis method according to claim 3, characterized in that, The linker is selected from 4-(hydroxymethyl)phenylacetic acid, 4-(hydroxymethyl)phenylpropionic acid, 4-(hydroxymethyl)phenylbutyric acid, 4-(hydroxymethyl)phenylvaleric acid, 4-(hydroxymethyl)phenylhexanoic acid, 3-(4-(hydroxymethyl)-3-methoxyphenyl)propionic acid, and 3-(4-(hydroxymethyl)-3,5-dimethoxyphenyl)propionic acid.

5. The synthesis method according to any one of claims 1-4, characterized in that, The activating agent is one or more selected from the combination of HOBT / DIC, Oxyma / DIC, TBTU / DIEA, HBTU / DIEA, PyBOP / DIEA, and HATU / DIEA.

6. The synthesis method according to any one of claims 1-4, characterized in that, The amino resin is selected from Am resin, MBHA resin, Rink Amide AM resin, Rink Amide MBHA resin, and Sieber resin; preferably, the degree of substitution of the amino resin is 0.2-0.6 mmol / g.

7. The synthesis method according to any one of claims 1-6, characterized in that, In step (4), the coupling method is based on the smegglutinin sequence, which can be performed by coupling single amino acid sequences or by using a fragment method.

8. The synthesis method according to claim 7, characterized in that, The coupling method follows the smegglutinin sequence, sequentially coupling the amino acids Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH·H2O, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-L-Lys[Oct-(OtBu)-Glu-(OtBu)-AEEA-AEEA]-OH, Fmoc-Ala-OH·H2O, Fmoc-Ala-OH·H2O, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH·H2O, Fmoc-Leu-OH, Fmoc-Tyr( 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, Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH or amino acids with equivalent structures.

9. The synthesis method according to any one of claims 1-8, characterized in that, In step (5), the pyrolysis reagent used is trifluoroacetic acid or a combination of trifluoroacetic acid and a capture reagent. The capture reagent is one or more of the following: benzyl sulfide, ethylene dithiol, benzyl ether, phenol, triisopropylsilane, and water, which are combined with trifluoroacetic acid in different volume ratios.

10. The synthesis method according to claim 9, characterized in that, The cleavage reagent, calculated by volume ratio, consists of 80-100% trifluoroacetic acid, 0-10% anisole sulfide, 0-10% ethylenedithiol, 0-10% anisole, 0-10% phenol, 0-10% triisopropylsilane, and 0-10% water, mixed in different volume ratios.

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