Solid-phase synthesis method of semeglutide

Abstract

The invention provides a solid-phase synthesis method of semeglutide, and belongs to the technical field of synthesis of medical intermediates, and the method specifically comprises the following steps: segmenting a semeglutide main chain into three shorter polypeptide fragments, namely a fragment 4, a fragment 5 and a fragment 2, which are 7-14 sites, 15-22 sites and 23-37 sites; and respectively synthesizing and then assembling fragments to obtain a target product. The method specifically comprises the following steps: a) synthesizing a 7-14 peptide fragment and a 15-22 peptide fragment, and assembling the 7-14 peptide fragment and the 15-22 peptide fragment into a 7-22 peptide fragment, namely a fragment 1; b) synthesizing a 23-37 peptide fragment, selectively removing a protecting group A11oc on Lys26, and connecting with a side chain to form a fragment 3; c) connecting the peptide fragment 7-22 with the peptide fragment 23-37, and removing a protecting group to obtain a crude product; and d) separating and purifying to obtain a finished product. According to the invention, the purpose of high-purity, high-yield and low-cost synthesis of the semeglutide bulk drug can be achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical intermediate synthesis, and specifically relates to a solid-phase synthesis method of semaglutide. Background Art

[0002] Semaglutide is a glucagon-like peptide 1 (GLP-1) receptor agonist, mainly used for blood glucose control in patients with type 2 diabetes and can also reduce cardiovascular risks. It stimulates insulin secretion, suppresses appetite, and delays gastric emptying by mimicking the action of the GLP-1 hormone. Semaglutide is available in two dosage forms: an injection once a week and an oral tablet once a day.

[0003] The polypeptide sequence of semaglutide is as follows:

[0004] H-His 7 -Aib 8 -Glu 9 -Gly 10 -Thr 11 -Phe 12 -Thr 13 -Ser 14 -Asp 15 -Val 16 -Ser 17 -Ser 18 -Tyr 19 -Leu 20 -Glu 21 -Gly 22 -Gln 23 -Ala 24 -Ala 25 -Lys 26 (AEEA-AEEA-γ-Glu-OctadecanedioicAcid)-Glu 27 -Phe 28 -Ile 29 -Ala 30 -Trp 31 -Leu 32 -Val 33 -Arg 34 -Gly 35 -Arg 36 -Gly 37 -OH.

[0005] The invention with the publication number of CN110894227A discloses a solid-phase synthesis method of liraglutide. The method comprises the following steps: according to the amino acid sequence from the N-terminus to the C-terminus of the main chain of liraglutide, solid-phase synthesizing a first resin peptide of a polypeptide fragment of amino acids 1-20; removing the side-chain protecting group of lysine at the 12th position of the first resin peptide of the polypeptide fragment, and then performing a coupling reaction with the compound Pal-Glu(OtBu)-OH to obtain a third resin peptide of the polypeptide fragment; according to the amino acid sequence from the C-terminus to the N-terminus of the main chain of liraglutide, solid-phase synthesizing a second resin peptide of a polypeptide fragment of amino acids 21-31, and then performing a cleavage reaction to remove the resin to obtain the polypeptide fragment two; after removing the terminal protecting group of the third resin peptide of the polypeptide fragment, performing a coupling reaction with the polypeptide fragment two by a solid-phase synthesis method to obtain a liraglutide resin; and then performing cleavage and purification to obtain liraglutide. The method of the invention is simple to operate, the obtained product has a high purity and a low cost, and is beneficial to industrial production.

[0006] The invention patent with the publication number of CN116120427B discloses a synthesis method of semaglutide. The method comprises coupling amino acids or peptide segments from the C-terminus to the N-terminus in sequence by a solid-phase synthesis method according to the amino acid sequence of semaglutide to obtain a fully protected resin of semaglutide, and performing cleavage and purification to obtain semaglutide; the peptide segment comprises a dipeptide S20-S21 formed by amino acids at positions 20-21, and the S20-S21 is R1-Lys(Fmoc)-Glu(OtBu)-OH, wherein R1 is Dde or ivDde, Fmoc is a side-chain protecting group, and R1 is a main-chain protecting group. The total yield of the crude semaglutide produced by the invention reaches 57.07%, the HPLC purity reaches 77.71%, the production of impurities is reduced, the synthesis and purification processes are simple, the selected materials are cheap and easily available, the comprehensive production cost is low, and it is more beneficial to industrial production. The total yield of the crude semaglutide synthesized is only 57.07%. The HPLC purity of the crude semaglutide is only 77.71%.

[0007] Patent WO2009083549A1 prepares semaglutide by a process of gene recombination-bio-fermentation in series with chemical synthesis. The process uses Fmoc-His-Aib-OSu and Lys 26The main-chain 29-peptide with the side chain attached reacts to obtain Fmoc-semaglutide, and then the Fmoc group is removed with piperidine to obtain the crude product of semaglutide. However, the product produced by this method contains 12-17% of disubstituted impurities (33-peptide), namely His-Aib-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH. Due to partial removal of the Fmoc group on the main chain, disubstituted impurities are formed, resulting in too low overall yield. Summary of the Invention

[0008] The object of the present invention is to provide a solid-phase synthesis method of semaglutide, which can achieve the synthesis purpose of high purity, high yield and low cost of semaglutide API.

[0009] To solve the above technical problems, the technical solution adopted by the present invention is:

[0010] A solid-phase synthesis method of semaglutide, the specific method is as follows:

[0011] The semaglutide main chain is divided into three shorter polypeptide fragments at positions 7-14, 15-22, and 23-37, specifically: fragment 4, fragment 5, and fragment 2;

[0012] After separate synthesis, the target product is obtained through fragment assembly;

[0013] Specifically, it includes the following steps:

[0014] a) Synthesize the 7-14 peptide segment and the 15-22 peptide segment, and assemble the two into a 7-22 peptide segment, namely: fragment 1;

[0015] b) After synthesizing the 23-37 peptide segment, selectively remove the protecting group A11oc on Lys 26 and then connect it with the side chain to form fragment 3;

[0016] c) Connect the 7-22 peptide segment with the 23-37 peptide segment, remove the protecting group, and obtain the crude product;

[0017] d) Through separation and purification, the finished product is obtained.

[0018] Furthermore, the sequence of fragment 1 is:

[0019] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH。

[0020] Furthermore, the sequence of Fragment 2 is as follows:

[0021] Fmoc-Gln(Trt)-Ala-Ala-Lys(alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH, where the alloc protecting group is selectively removed and formed after side chain connection.

[0022] Furthermore, the sequence of Fragment 3 is as follows:

[0023] Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH.

[0024] Furthermore, the sequence of Fragment 4 is as follows:

[0025] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-OH.

[0026] Furthermore, the sequence of Fragment 5 is as follows:

[0027] Fmoc-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH.

[0028] Furthermore, the said Fragment 1 is obtained by the assembly of Fragment 4 and Fragment 5, and the specific steps include:

[0029] a) Synthesize Fragment 4 and Fragment 5 successively on the solid-phase synthesis resin;

[0030] b) Connect Fragment 4 and Fragment 5 through a condensation reaction to form Fragment 1.

[0031] Furthermore, the said Fragment 2 is obtained by selectively removing Lys 26After the A11oc protecting group on it, it is connected to the side chain to form Fragment 3. The specific steps include:

[0032] a) Selectively remove the A11oc protecting group; b) The deprotected Lys 26 is connected to the side chain AEEA - AEEA - γ - Glu - OctadecanedioicAcid - OtBu to form Fragment 3.

[0033] Furthermore, the connection between Fragment 1 and Fragment 3 is carried out by solid - phase synthesis method. The specific steps include:

[0034] a) Condense Fragment 1 and Fragment 3 under the action of a condensing agent;

[0035] b) Remove all protecting groups to obtain a crude product.

[0036] Furthermore, when carrying out the condensation reaction, the condensing agent is one of the following combinations: DIC / HOBT, DIC / HOAT, TBTU / HOBT / DIPEA, HBTU / HOBT / DIPEA, HATU / HOAT / DIPEA.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] For the industrial large - scale production of semaglutide bulk drug, using the conventional solid - phase synthesis method has great synthetic challenges, which are mainly reflected in that there are a large number of hydrophobic amino acids between the sequences His 7 and Lys 26 making the hydrogen bonds between peptide chains stable, resulting in serious β - sheet formation, enhancing the interaction between peptide chains, and causing resin polycondensation. In the present invention, the main chain of maruglutide is divided into 3 shorter polypeptide fragments at positions 7 - 14, 15 - 22, and 23 - 37 and synthesized simultaneously. The 7 - 14 peptide segment (Fragment 4) and the 15 - 22 peptide segment (Fragment 5) are assembled into a 7 - 22 peptide segment (Fragment 1). The 14 - th amino acid Ser and the 22 - nd amino acid Gly are both amino acids with relatively small steric hindrance, and the racemization risk of Ser is relatively small, while Gly cannot undergo racemization. Therefore, the connection method selected in the present invention has a small racemization risk and the prepared finished product has a high purity. On the other hand, after assembling the 23 - 37 peptide segment (Fragment 2), the protecting group A11oc on Lys 26 is selectively removed. Then it is connected to the side chain to form Fragment 3. Then the 7 - 22 peptide segment (Fragment 1) and the 23 - 37 peptide segment (Fragment 3) are connected, and then the protecting groups are removed. The yield and purity of the crude semaglutide prepared by the present invention are both relatively high. Through conventional purification means, semaglutide fine peptide with a purity of more than 99.1% and a single impurity of less than 0.1% can be obtained with a high yield. Brief Description of the Drawings

[0039] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as a limitation of the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0040] Figure 1 Flow chart for the preparation of semaglutide. Specific embodiments

[0041] In the following text, only some exemplary embodiments are simply described. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of the embodiments of the present invention. Therefore, the drawings and descriptions are considered to be exemplary in nature rather than restrictive. The embodiments of the present invention will be described in detail below with reference to the drawings.

[0042] Refer to Figure 1 , this embodiment discloses a solid-phase synthesis method for semaglutide, and the specific method is as follows:

[0043] The semaglutide backbone is divided into three shorter polypeptide fragments at positions 7 - 14, 15 - 22, and 23 - 37, specifically: fragment 4, fragment 5, and fragment 2;

[0044] After separate synthesis, the target product is obtained through fragment assembly;

[0045] Specifically, it includes the following steps:

[0046] a) Synthesize the 7 - 14 peptide segment and the 15 - 22 peptide segment, and assemble the two into a 7 - 22 peptide segment, namely: fragment 1;

[0047] b) After synthesizing the 23 - 37 peptide segment, selectively remove the protecting group A11oc on 26 Lys, and form fragment 3 by connecting with the side chain;

[0048] c) Connect the 7 - 22 peptide segment with the 23 - 37 peptide segment, remove the protecting group, and obtain the crude product;

[0049] d) Through separation and purification, the finished product is obtained.

[0050] Furthermore, the sequence of fragment 1 is:

[0051] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH。

[0052] Furthermore, the sequence of Fragment 2 is as follows:

[0053] Fmoc-Gln(Trt)-Ala-Ala-Lys(alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH, where the alloc protecting group is formed after being selectively removed and the side chain is connected.

[0054] Furthermore, the sequence of Fragment 3 is as follows:

[0055] Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH.

[0056] Furthermore, the sequence of Fragment 4 is as follows:

[0057] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-OH.

[0058] Furthermore, the sequence of Fragment 5 is as follows:

[0059] Fmoc-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH.

[0060] Furthermore, the said Fragment 1 is obtained by the assembly of Fragment 4 and Fragment 5, and the specific steps include:

[0061] a) Synthesize Fragment 4 and Fragment 5 on the solid-phase synthesis resin in sequence;

[0062] b) Connect Fragment 4 and Fragment 5 through a condensation reaction to form Fragment 1.

[0063] Furthermore, the said Fragment 2 is obtained by selectively removing Lys 26After the A11oc protecting group on it, it is connected to the side chain to form fragment 3. The specific steps include:

[0064] a) Selectively remove the A11oc protecting group; b) The deprotected Lys 26 is connected to the side chain AEEA - AEEA - γ - Glu - OctadecanedioicAcid - OtBu to form fragment 3.

[0065] Furthermore, the connection between fragment 1 and fragment 3 is carried out by solid - phase synthesis method. The specific steps include:

[0066] a) Condense fragment 1 and fragment 3 under the action of a condensing agent;

[0067] b) Remove all protecting groups to obtain the crude product.

[0068] Furthermore, when carrying out the condensation reaction, the condensing agent is one of the following combinations: DIC / HOBT, DIC / HOAT, TBTU / HOBT / DIPEA, HBTU / HOBT / DIPEA, HATU / HOAT / DIPEA.

[0069] The specific method is as follows:

[0070] a: Use Wang resin solid - phase carrier. Fragment 2 and fragment 5 are coupled with Fmoc - G1y - OH to prepare Fmoc - Gly - Wang resin; Fragment 4 is coupled with Fmoc - Ser(tBu) - OH to prepare Fmoc - Ser(tBu) - Wang. Then, the protected amino acids are sequentially coupled in sequence to obtain the target peptide fragments 2, fragment 4 and fragment 5 respectively.

[0071] b: Cut fragment 4 from the resin, and then fragment 4 and fragment 5 which is connected to the resin and has its Fmoc protecting group removed:

[0072] (Asp(OtBu) - Val - Ser(tBu) - Ser(tBu) - Tyr(tBu) - Leu - Glu(OtBu) - Gly - Wang) are connected to form fragment 1 connected to the resin: Boc - His(Trt) - Aib - Glu(OtBu) - Gly - Thr(tBu) - Phe - Thr(Trt) - Ser(tBu) - Asp(OtBu) - Val - Ser(tBu) - Ser(tBu) - Tyr(tBu) - Leu - Glu(OtBu) - Gly - Wang.

[0073] Subsequently, Fragment 1 was cut off from the resin to form Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH.

[0074] c: For Fragment 2 attached to the resin:

[0075] The protecting group Alloc on (Fmoc-Gln(Trt)-Ala-Ala-Lys(alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang) was removed to form Fmoc-Gln(Trt)-Ala-Ala-Lys-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH, which was then linked to the side chain to obtain Fragment 3 attached to the resin:

[0076] Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang.

[0077] d: Fragment 1 was coupled with Fragment 3 attached to the resin (Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang) to obtain the fully protected semaglutide attached to the resin.

[0078] e: The fully protected semaglutide attached to the resin was cleaved, purified, and lyophilized to obtain the finished product of semaglutide.

[0079] To facilitate further understanding of the present invention by those skilled in the art, the present invention will be further elaborated below in conjunction with specific operation steps.

[0080] 1. In step a, the solid support is Wang resin with a substitution degree of 0.1 - 0.6 mmol / g. After connecting the amino acid to the resin, after removing the Fmoc protection, the corresponding Fmoc - protected amino acid is added at a feeding ratio of 2 - 5 times for the coupling reaction. Each coupling reaction is a solid - phase peptide - coupling reaction carried out in the presence of a condensing agent. The end - point of each step of the coupling reaction is detected by Kaiser reagent. After the reaction is completed, the Fmoc is removed with a de - protecting reagent and then coupled with the next Fmoc - protected amino acid; the operation is repeated until the fully protected fragments 2, fragment 4, and fragment 5 are synthesized respectively. The de - protecting reagent is preferably a 20% piperidine in DMF solution (volume ratio); the condensing agent used in the coupling reaction is one of the following combinations: DIC / HOBT, DIC / HOAT, TBTU / HOBT / DIPEA, HBTU / HOBT / DIPEA, HATU / HOAT / DIPEA.

[0081] 2. In step b, the method for cleaving the peptide chain from the resin is: reacting the peptide chain attached to the resin with a mixture of DCM and hexafluoroisopropanol (HFIP) at a ratio of 1 - 50:1. The preferred ratio is 5:1. The mixture is stirred at room temperature for 2 - 3 hours, and the preferred ratio is 5:1. After the mixture is stirred at room temperature for 2 - 3 hours, the resin is filtered off. After the filtrate is dried by suction, it is purified and used in step d.

[0082] 3. In step c, the method for removing the protecting group A11oc on Lys 26 is to use Pd(PPh3)4 at 0.1 - 0.4 times the synthetic scale amount and phenylsilane (or morpholine) at 10 - 30 times the synthetic scale amount to remove it under solid - phase conditions for 10 - 90 minutes.

[0083] 4. In steps b, c, and d, the condensing agent used in the coupling reaction is one of the following combinations: DIC / HOBT, DIC / HOAT, TBTU / HOBT / DIPEA, HBTU / HOBT / DIPEA, HATU / HOAT / DIPEA.

[0084] 5. In step e, the cleavage reagent is a TFA solution added with a scavenger at a volume ratio of 1 - 5%, and the scavenger is one or more of anisole, benzyl mercaptan, ethylene dithiol, mercaptoethanol, phenol, water, and TIS.

[0085] 6. The preferred cleavage reagent ratio is: TFA / benzyl mercaptan / water

[0086] / TIS = 90 / 2.5 / 5.0 / 2.5.

[0087] The abbreviations used in the specification and claims have the following meanings:

[0088] Fmoc 9-Fluorenylmethyloxycarbonyl Wang Resins Wang Resin tBu tert-Butyl Pbf 2,2,4,6,7-Pentamethylbenzofuran-5-sulfonyl Trt Triphenylmethyl Alloc (2-Propenyloxy)carbonyl Boc tert-Butyloxycarbonyl Aib 2-Methylalanine

[0089] The specific synthesis is as follows:

[0090] Example 1: Synthesis of Fmoc-Gly-Wang;

[0091] Place 500.0 g of the carrier Wang resin (sub = 0.42 mmol / g) in a synthesis column, wash it twice with 2400 mL of DMF, add 2400 mL of DCM to swell for 30 min; after filtering off the DCM by suction, add a mixed DCM solution of Fmoc-Gly-OH / DIC / HOBT [weigh 282.4 g (400 mmol) of Fmoc-Gly-OH and 64.8 g (480 mmol) of HOBT and place them in an amino acid activation bottle, add 2000 mL of a mixed solution of DMF and DCM with a volume ratio of 1:1, stir to dissolve, add 76.4 mL (480 mmol) of DIC at 0 °C, activate for 5 minutes, after reacting for 10 min, add 4.8 g (4 mmol) of DMAP; react for 2 h, filter off the reaction solution, wash it twice with 2400 mL of DMF, add 2400 mL of the capping reagent (480 mL of acetic anhydride and 408 mL of pyridine dissolved in 1512 mL of DMF) and react for 2 h, filter off the reaction solution by suction, wash it twice with DMF, DCM, and methanol respectively, and obtain 609.3 g of Fmoc-Gly-Wang after vacuum drying: Take a sample to measure the substitution degree as 0.26 mmol / g.

[0092] Example 2: Synthesis of Fmoc-Ser(tBu)-Wang;

[0093] Place 500.0 g of the support Wang resin (sub = 0.42 mmol / g) in a synthesis column, wash it twice with 2400 mL of DMF, add 2400 mL of DCM to swell for 30 min; after filtering off the DCM by suction, add a mixed DCM solution of Fmoc-Ser(tBu)-OH / DIC / HOBT [weigh 312.6 g (400 mmol) of Fmoc-Ser(tBu)-OH and 64.8 g (480 mmol) of HOBT and place them in an amino acid activation bottle, add 2000 mL of a mixed solution of DMF and DCM with a volume ratio of 1:1 and stir to dissolve, add 76.4 ml (480 mmol) of DIC at 0 °C, activate for 5 minutes, after reacting for 10 min, add 4.8 g (4 mmol) of DMAP: react for 2 h, filter off the reaction solution, wash it twice with 2400 ml of DMF, add 2400 mL of the capping reagent (dissolve 480 ml of acetic anhydride and 408 ml of pyridine in 1512 ml of DMF) and react for 2 h, filter off the reaction solution by suction, wash it twice with DMF, DCM, and methanol respectively, and obtain 682.3 g of Fmoc-Ser(tBu)-Wang after vacuum drying: take a sample to measure the substitution degree as 0.30 mmol / g.

[0094] Example 3: Preparation of the peptide chain fragment 2 resin;

[0095] Accurately weigh 69.23 g (synthesis scale 18 mmol) of Fmoc-Gly-Wang resin with a substitution degree of 0.26 mmol / g in Example 1 and place it in a synthesis column. Add 1000 ml of DCM to swell for 30 min. After suction filtration to remove DCM, wash with 800 ml of DMF twice. Add 1000 ml of 20% piperidine / DMP solution for deprotection twice, reacting for 10 min and 10 min respectively. Then wash with 800 ml of DMF, DCM, and DMF twice respectively. Add 500 ml of a DMF solution containing 25.41 g (36 mmol) of Fmoc-Arg(Pbf)-OH, 5.35 g (39.6 mmol) of HOBT, and 6.3 ml (39.6 mmol) of DIC, stir the reaction under nitrogen bubbling for 2 h. The end point of the reaction is determined by the Kaiser reagent test result. After the reaction reaches the end point, suck out the reaction solution and wash with 800 ml of DMF, DCM, and DMF twice respectively. Subsequently, deprotect again. Repeat this cyclic operation. According to the sequence of peptide chain fragment 2, couple with protected amino acids one by one. The sequentially connected protected amino acids are: 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-Lys(Alloc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH. Obtain the peptide chain fragment 2 resin with side chains protected: Fmoc-Gln(Trt)-Ala-Ala-Lys(alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang.

[0096] Example 4: Preparation of peptide chain fragment 4 resin;

[0097] Accurately weigh 75.26 g (synthesis scale: 18 mmol) of Fmoc-Ser(tBu)-Wang resin with a substitution degree of 0.30 mmol / g in Example 1 and place it in a synthesis column. Add 1000 ml of DCM to swell for 30 min. After suction filtration to remove DCM, wash it twice with 800 ml of DMF. Add 1000 ml of 20% piperidine / DMP solution for deprotection twice, reacting for 10 min and 10 min respectively. Then wash it twice with 800 ml of DMF, DCM, and DMF respectively. Add 500 ml of a DMF solution containing 14.61 g (36 mmol) of Fmoc-Thr(Trt)-OH, 5.35 g (39.6 mmol) of HOBT, and 6.3 ml (39.6 mmol) of DIC, and stir the reaction under nitrogen for 2 h. The end point of the reaction is determined by the Kaiser reagent test result. After the reaction reaches the end point, suction off the reaction solution and wash it twice with 800 ml of DMF, DCM, and DMF respectively. Subsequently, deprotect again. Repeat this cyclic operation, and couple with protected amino acids one by one according to the sequence of peptide chain fragment 2. The protected amino acids connected in sequence are: Fmoc-Thr(Trt)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH, Fmoc-His(Trt)-OH. Obtain peptide chain fragment 4 resin:

[0098] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Wang。

[0099] Example 5: Preparation of peptide chain fragment 5 resin;

[0100] Accurately weigh 69.23 g (synthesis scale 18 mmol) of Fmoc-Gly-Wang resin with a substitution degree of 0.26 mmol / g in Example 1 and place it in a synthesis column. Add 1000 ml of DCM to swell for 30 min. After suction filtration to remove DCM, wash with 800 ml of DMF twice. Add 1000 ml of 20% piperidine / DMP solution to deprotect twice, reacting for 10 min and 10 min respectively. Then wash with 800 ml of DMF, DCM, and DMF twice respectively. Add 500 ml of a DMF solution containing 19.42 g (36 mmol) of Fmoc-Glu(OtBu)-OH, 5.35 g (39.6 mmol) of HOBT, and 6.3 ml (39.6 mmol) of DIC, stir the reaction under nitrogen for 2 h, and determine the end point of the reaction based on the detection result of Kaiser reagent. After the reaction reaches the end point, suction off the reaction solution and wash with 800 ml of DMF, DCM, and DMF twice respectively. Subsequently, deprotect again. Repeat the above cyclic operation, and couple with protected amino acids one by one according to the sequence of peptide chain fragment 2. The protected amino acids connected in sequence are:

[0101] Fmoc-Glu(OtBu)-OH,

[0102] Fmoc-Leu-OH,

[0103] Fmoc-Tyr(tBu)-OH,

[0104] Fmoc-Ser(tBu)-OH,

[0105] Fmoc-Ser(tBu)-OH,

[0106] Fmoc-Val-OH,

[0107] Fmoc-Asp(OtBu)-OH.

[0108] Obtain peptide chain fragment 5 resin:

[0109] Fmoc-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Wang.

[0110] Example 6: Method for cleaving the fully protected peptide chain fragment 4 from the resin;

[0111] Put the peptide chain fragment 4 connected to the resin into a mixed solvent of DCM and hexafluoroisopropanol (HFIP). The ratio of the mixed solvent of DCM and hexafluoroisopropanol (HFIP) is 1 - 50:1. The preferred ratio is 5:1. After stirring the mixture at room temperature for 2 - 3 h, filter to remove the resin. After the filtrate is dried by suction, peptide chain fragment 4 is obtained:

[0112] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-OH. It was used in Example 7 after purification.

[0113] Example 7: Method for connecting peptide chain fragment 4 and fragment 5 on resin;

[0114] Take the peptide chain fragment 5 resin prepared in Example 5:

[0115] Add 1000 ml of 20% piperidine / DMP solution to Fmoc-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Wang110g for deprotection twice, reacting for 10 min and 10 min respectively: Then wash it twice with 800 ml of DMF, DCM, and DMF respectively, and add the peptide chain fragment 4 prepared in Example 6:

[0116] Add 500 ml of DMF solution of Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-OH, 5.35 g (39.6 mmol) of HOBT, and 6.3 ml (39.6 mmol) of DIC, stir and react for 2 h under nitrogen bubbling. The end point of the reaction is determined by the Kaiser reagent test result. After the reaction reaches the end point, draw off the reaction solution and wash it twice with 800 ml of DMF, DCM, and DMF respectively. Obtain the peptide chain fragment 1 connected to the resin:

[0117] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Wang.

[0118] Example 8: Lys of peptide chain fragment 5 26 Selective deprotection of the protecting group on the side chain;

[0119] The peptide chain fragment 2 resin prepared in Example 3 was added to 1000 ml of DCM and washed twice, then 1000 ml of DCM was added. While stirring the resin evenly, 27 ml of phenylsilane was slowly added. After reacting for 3 min, 5.64 g of Pd(PPh3)4 was added, and the reaction was carried out at room temperature for 50 min. The reaction solution was dried by suction. After treating the above resin 3 times with a hydrazine hydrate / DMF (1 / 15) mixed solution, it was washed 6 times with 1000 ml of DCM: Sampling was carried out for Kaiser reagent detection, and the resin showed blue. Lys was obtained. 26 Peptide chain fragment 5 with side chain deprotected and attached to the resin:

[0120] Fmoc-Gln(Trt)-Ala-Ala-Lys 26 -Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang.

[0121] Example 9: Lys of peptide chain fragment 5 26 Fatty acidation of the side chain;

[0122] Weigh 86.96 g of tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu and 8.02 g (59.4 mol) of HOBT into a dissolution flask, add 500 ml of DMF to dissolve. After complete dissolution, place it in an ice-water bath and let it stand for 10 min. Add 9.2 ml (59.4 mmol) of DIC and mix evenly, then activate it in an ice-water bath for 10 min; Add the activated solution to the Fmoc-Gln(Trt)-Ala-Ala-Lys 26 -Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resin obtained in Example 8, and stir the reaction evenly: Kaiser reagent was used to detect the reaction process. After the resin was detected to be colorless, the coupling reaction was terminated: After the peptide resin was prepared, it was washed twice with 1000 ml of DMF, DCM, and methanol respectively, and dried in vacuo to obtain the peptide resin:

[0123] Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang, weighing 262.6 g.

[0124] Example 10: Method for cleaving the fully protected peptide chain fragment 1 from the resin;

[0125] The peptide chain fragment 1 linked to the resin prepared in Example 7 was put into a mixed solvent of DCM and hexafluoroisopropanol (HFIP). The ratio of the mixed solvent of DCM and hexafluoroisopropanol (HFIP) was 1 - 50:1. The preferred ratio was 5:1. After stirring the mixture at room temperature for 2 - 3 hours, the resin was removed by filtration. After the filtrate was dried by suction, the peptide chain fragment 1 was obtained:

[0126] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH. After purification, it was used in Example 12.

[0127] Example 11: Linking the peptide chain fragment 1 and the peptide chain fragment 3;

[0128] Weighed the peptide resin of the peptide chain fragment 3 prepared in Example 9:

[0129] Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic Acid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang 50 g. Added 500 ml of 20% piperidine / DMP solution for deprotection twice, reacting for 10 min and 10 min respectively: Then washed twice with 400 ml of DMF, DCM, and DMF respectively, and added the peptide chain fragment 4 prepared in Example 10:

[0130] A DMF solution of 500 ml containing Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-OH, 5.35 g (39.6 mmol) of HOBT, and 6.3 ml (39.6 mmol) of DIC was stirred under nitrogen for 2 h. The end point of the reaction was determined by the Kaiser reagent. After the reaction reached the end point, the reaction solution was removed by suction, and washed twice with 800 ml of DMF, DCM, and DMF respectively. The fully protected semaglutide linked to the resin was obtained.

[0131] Example 12: Cleavage of the fully protected semaglutide linked to the resin;

[0132] The semaglutide peptide resin obtained in Example 11 was added to 1600 ml of frozen lysis solution (volume ratio of TFA / thioanisole / TIS / 120 = 92.5 / 2.5 / 2.5 / 2.5), and the reaction was stirred at room temperature for 4 h. After the lysis reaction was completed, the resin was filtered, and the resin was washed twice with 100 ml of TFA. The filtrate and the washings were combined, concentrated by rotary evaporation to 1000 ml, and poured into 10 L of frozen methyl tert-butyl ether to precipitate a white solid. After standing for 30 min, it was filtered and washed 6 times with methyl tert-butyl ether, and then dried in vacuo to obtain 71.8 g of crude semaglutide, with a crude peptide yield of 96.8% and a purity of 93.8%.

[0133] Example 13: Purification of semaglutide;

[0134] 50.0 g of the crude peptide prepared in Example 12 was dissolved in 200 ml of acetic acid. After complete dissolution, it was diluted with water to 4000 ml. The solution was filtered through a 0.45-μm filter membrane for standby. Purification method: a C18 preparative column with a diameter of 150 mm, the mobile phase was a 0.1% acetic acid / water - 0.1% acetic acid / acetonitrile system, the sample loading amount was 25 g / time, the flow rate was 300 ml / min, and gradient elution was performed; sample injection was carried out in a cycle before and after the peak, and a refined peptide solution with qualified purity in the in-process analysis was obtained. After desalting, it was freeze-dried to obtain 23.9 g of refined peptide, with a purity of more than 99.1% and a single impurity of less than 0.1%.

[0135] The crude semaglutide prepared by this synthesis method has both high yield and high purity. Through conventional purification means, semaglutide refined peptide with a purity of more than 99.1% and a single impurity of less than 0.1% can be obtained with a high yield. The synthesis of semaglutide raw material medicine with high purity, high yield and low cost is realized.

[0136] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0137] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be noted that any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. A solid phase synthesis method of semaglutide, characterized in that: The specific method is as follows: The main chain of semaglutide is divided into three shorter polypeptide fragments at positions 7-14, 15-22, and 23-37, specifically: fragment 4, fragment 5, and fragment 2; After being synthesized separately, the target product is obtained by assembling the fragments; The specific steps include: a) synthesizing the 7-14 peptide segment and the 15-22 peptide segment, and assembling the two into the 7-22 peptide segment, i.e., fragment 1; b) Selective removal of Lys after synthesis of peptide 23-37 26 After the protecting group A11oc is added, it is connected to the side chain to form fragment 3; c) connecting the 7-22 peptide segment with the 23-37 peptide segment, removing the protecting group, and obtaining a crude product; d) obtaining the finished product through separation and purification.

2. The solid phase synthesis method of semaglutide according to claim 1, characterized in that: The sequence of fragment 1 is: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-As p(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH.

3. The solid phase synthesis method of semaglutide according to claim 1, characterized in that: The sequence of fragment 2 is: Fmoc-Gln(Trt)-Ala-Ala-Lys(alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH, in which the alloc protecting group is selectively removed and the side chain is connected to form.

4. The solid phase synthesis method of semaglutide according to claim 1, characterized in that: The sequence of fragment 3 is: Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH.

5. The solid phase synthesis method of semaglutide according to claim 1, characterized in that: The sequence of fragment 4 is: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-OH.

6. The solid phase synthesis method of semaglutide according to claim 1, characterized in that: The sequence of fragment 5 is: Fmoc-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH.

7. The solid phase synthesis method of semaglutide according to claim 1, characterized in that: The fragment 1 is obtained by assembling the fragment 4 and the fragment 5, and the specific steps include: a) synthesizing fragment 4 and fragment 5 sequentially on a solid phase synthesis resin; b) Fragment 4 and Fragment 5 are connected by a condensation reaction to form Fragment 1.

8. The solid phase synthesis method of semaglutide according to claim 1, characterized in that: The fragment 2 is obtained by selectively removing Lys 26 After the A11oc protecting group is removed, it is connected to the side chain to form fragment 3. The specific steps include: a) selectively removing the A11oc protecting group; b) connecting the deprotected Lys26 to the side chain AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu to form fragment 3.

9. The solid phase synthesis method of semaglutide according to claim 1, characterized in that: The connection between fragment 1 and fragment 3 is carried out by solid phase synthesis, and the specific steps include: a) subjecting fragment 1 and fragment 3 to a condensation reaction under the action of a condensation agent; b) Remove all protecting groups to obtain a crude product.

10. The solid phase synthesis method of semaglutide according to claim 9, characterized in that: During the condensation reaction, the condensing agent is one of the following combinations: DIC / HOBT, DIC / HOAT, TBTU / HOBT / DIPEA, HBTU / HOBT / DIPEA, and HATU / HOAT / DIPEA.

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