Method for synthesizing semeglutide through large-fragment SPPS-LPPS mixing method

Abstract

The invention provides a method for synthesizing semeglutide by a large-fragment SPPS-LPPS mixing method, which comprises the following steps: cutting into two polypeptide fragments: the sequences 1-16 are fragments I, and the sequences 17-31 are fragments II; coupling protective amino acid monomers at corresponding sequence positions through SPPS to synthesize a full-protection fragment I and a full-protection fragment II; introducing tert-butyl ester to protect carboxyl at the carbon terminal of the fragment II, and carrying out Fmoc removal / condensation reaction on the fragment I and the fragment II through an LPPS (Liquefied Peptide Peptide) method to obtain fully-protected semeglutide; and removing the protecting group to obtain crude peptide, and purifying to obtain the semeglutide. According to the method, peptide impurity deletion caused by DKP side reaction, M + 106 impurities introduced by cracking and M + 270 impurities introduced by amine ester exchange in one-by-one synthesis of the semeglutide by taking wang resin as a solid-phase carrier are effectively avoided, the total yield is greater than 60%, the purity is greater than 99.0%, the single impurity content is less than 0.1%, the process is simple, and the method is more suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide drug synthesis technology, and relates to a method for synthesizing semaglutide, specifically a method for synthesizing semaglutide using a large fragment SPPS-LPPS mixed method. Background Technology

[0002] Smegglutide is a novel long-acting GLP-1 analog developed by Novo Nordisk of Denmark. From the amino acid sequence, the 2nd position of the peptide sequence is the non-natural amino acid aminoisobutyric acid, and the Lys side chain amino group at the 20th position of the peptide sequence has been modified by AEEA, γ-Glu and octadecanoic acid.

[0003] Chinese name: Sermaglutide; English name: Sermaglutide; Peptide sequence: H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γGlu-Octadecanedioicacid)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg -Gly-OH.

[0004] Compared with liraglutide, the modified smegglutide has enhanced hydrophilicity, inhibits the hydrolysis of DPP-4 enzyme, prolongs the biological half-life, has a long-lasting effect in lowering blood sugar, promotes pancreatic islet cell regeneration, and prolongs gastric emptying, among other effects, and has broad application prospects.

[0005] Patent publication number CN101133082A, published on February 27, 2008, entitled "Invention Patent for Acylated GLP-1 Compound", discloses a semi-synthetic process route, which uses fermented 29 peptide as raw material to react with the active ester of the side chain under alkaline conditions to introduce a side chain at the 20-position Lys, and then introduces the nitrogen-terminated His-Aib dipeptide.

[0006] There are two main strategies for the solid-phase total synthesis of smegraglutide: one is the solid-phase one-to-one coupling method, which uses Wang resin as a solid support to couple fully protected amino acid monomers or short peptide fragments at corresponding sequence positions one by one, followed by cleavage and purification to obtain smegraglutide. The other strategy is the large-fragment SPPS-LPPS hybrid synthesis strategy.

[0007] During the development of the one-to-one coupling process, the applicant discovered (such as) Figure 2(As shown) Because the Wang resin is linked by ester bonds and peptide chains, and ester bonds are less stable than amide bonds, DKP side reactions can occur, resulting in impurities with missing peptides. Additionally, the applicant also discovered the M+270 impurity, which is inferred to have occurred during the removal of the Fmoc protecting group at position 29 of Gly. Figure 2 The amine-ester exchange reaction shown introduces an inserted peptide (inserted Gly-Arg-Gly); in addition, during the cleavage process, the p-methylenephenol carbocation of the Wang resin linker is captured by the peptide chain, thereby introducing the M+106 impurity.

[0008] Patent publication number CN113045641A, published on June 29, 2021, entitled "A Method for Preparing Semaglutide," discloses a fragment synthesis method for semaglutide. The specific process involves first synthesizing a fully protected fragment (His) using 2-CTC resin. 1 -Ala 18 ) and fragment two (Ala 19 -Gly 31 After coupling with R6 (R6 is 4-(hydroxymethyl)phenol) in the liquid phase, smegglutinin is obtained. The disadvantage of this method is that the coupling site is Ala at position 18, which is prone to racemization during the coupling reaction, resulting in more phase-differentiated peptide byproducts.

[0009] Patent publication number CN114249810A, published on March 29, 2022, entitled "A Method for Synthesizing Semaglutide," discloses an isonitrile-mediated large-fragment liquid-phase coupling strategy for the synthesis of semaglutide. Specifically, it involves first using Wang resin as a solid-phase support to synthesize fragment 1-Gln. 17 -Gly 31 Simultaneously, the fully protected fragment Fmoc-His was synthesized using 2-CTC resin. 1 -Gly 16 -OH, after thioesterification and deprotection, yields fragment two Fmoc-His 1 -Gly 16 -SR, then fragment one and fragment two are coupled under isonitrile-mediated coupling, and then the Fmoc protecting group is removed to obtain smegglutinin. The whole process requires two TFA deprotection, which generates a lot of acidic waste and uses thiols with a pungent smell.

[0010] Patent publication number CN114685645A, publication date July 1, 2022, entitled "A Method for Synthesizing Semaglutide," discloses a strategy for constructing semaglutide by naturally chemically linking thiols and Cys. Specifically, it involves first synthesizing fully protected His through 2-CTC resin. 1 -Gln 17 -OH, followed by thioesterification and deprotection to obtain the fragment IHis.1 -Gln 17 -SR, and the fragment IIICys was also obtained through solid-state synthesis. 18 -Gly 31 Fragment I and fragment III are naturally linked under alkaline conditions. Finally, the 18-position Cys is converted into Ala through a desulfurization process to complete the construction of smegglutinin. The entire process route is relatively long. Because the 18-position Cys is introduced as a natural linker, it needs to be converted into Ala through a subsequent desulfurization process. Furthermore, it is easy to oxidize and form disulfide bonds to form dimers during desulfurization. Summary of the Invention

[0011] To overcome the problems of side reactions such as DKP, M+270, and M+106 introduced by the aforementioned step-by-step solid-phase synthesis methods, as well as the various shortcomings of the aforementioned large-fragment methods, this invention provides a simpler and more efficient synthetic strategy.

[0012] This invention provides a method for synthesizing semagrapeptide using a large-fragment SPPS-LPPS hybrid method. This invention cleaves semagrapeptide into two polypeptide fragments of similar length: sequence His... 1 -Gly 16 As fragment I (hexapeptide), the sequence Gln 17 -Gly 31 As fragment II (pentapeptide); the fully protected fragment I / fragment II can be obtained by sequentially coupling corresponding amino acid monomers or fragments with 2-CTC resin as a solid-phase support, followed by cleavage under 1%-5% trifluoroacetic acid dichloromethane solution / hexafluoroisopropanol dichloromethane solution / trifluoroethanol dichloromethane solution or other weakly acidic conditions. Tert-butyl ester protection is introduced at the C-terminus of fragment II. Then, fragments I and II are cleaved via LPPS method through a de-Fmoc / condensation reaction to obtain the fully protected smegglutinin. After removing the protecting group, the crude smegglutinin peptide is obtained, which is then purified to obtain smegglutinin. The solid-phase synthesis of fragment peptides in this invention requires only 2.0 equivalents of coupling monomers per step to achieve complete reaction, resulting in high atom economy and low cost. Furthermore, the synthesis of each fragment peptide can be performed in parallel, significantly shortening batch synthesis time and increasing production efficiency. In the condensation reaction of fragment peptide I and fragment peptide II, the equivalent ratio of fragment peptides is between 1:1 and 1:1.1, ensuring complete reaction. The condensation coupling reaction of the fragment peptides is carried out in a liquid-phase reactor, facilitating large-scale production. Additionally, this invention uses 2-CTC resin as the solid-phase support in the solid-phase synthesis of fragment I and fragment II, effectively avoiding the missing peptide impurities generated by DKP side reactions, effectively avoiding M+106 impurities introduced by Wang resin cleavage, and avoiding the removal of Gly from tripeptides on Wang resin. 3 The M+270 impurity is introduced by amine ester exchange when the Fmoc protecting group is used.

[0013] To achieve the above objectives, the present invention adopts the following technical solution: This invention first provides a method for synthesizing smegglutinin using a large-fragment SPPS-LPPS hybrid method. The method involves: cleaving smegglutinin into two polypeptide fragments: fragment I consisting of sequences 1-16 and fragment II consisting of sequences 17-31; using 2-ChlorotritylChloride resin as a solid-phase support, sequentially coupling protective amino acid monomers at corresponding sequence positions via SPPS; and cleaving under fluoroalcohol or weak acid conditions to obtain fully protected fragment I and fully protected fragment II. A tert-butyl ester was introduced to protect the carboxyl group at the carbon terminus of the fully protected fragment II; under the action of an organic base, the Fmoc protecting group of the nitrogen-terminated amino group was removed to obtain the nitrogen-terminated bare fragment II; the structure of the nitrogen-terminated bare fragment II is shown below: H-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γGlu(α-OtBu)-Octadecanedioicacid(mono-tBu))- Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu. The fully protected fragment I and the nitrogen-terminated naked fragment II react under condensation conditions to obtain the fully protected smegglutinin; the protecting group is removed under acidic conditions to obtain crude smegglutinin, which is then purified to obtain pure smegglutinin.

[0014] As a preferred embodiment of the present invention, the steps of the method for synthesizing smegglutinin are as follows: 1) Synthesis of fully protected fragment I and fully protected fragment II: 1) Synthesis of fully protected fragment I: Dichloromethane was added to a solid-phase reactor containing 2-ChlorotritylChloride resin for swelling. The solvent was removed, and a dry dichloromethane solution of Fmoc-Glu(OtBu)-Gly-OH and DIPEA was added. The amino acids at positions 15 and 16 were linked to the resin. DCM / MeOH / DIPEA was added to block unreacted or poorly reacted chlorination sites on the resin. The resin was washed clean with solvent, and a deprotecting agent was added to remove the Fmoc protecting group. An activated Fmoc-Leu-OH / HOBt / DIC mixture was added to perform the coupling reaction of the amino acid monomer at position 14. After a negative indene test result, the solution was removed, and the peptide resin was washed with organic solvent to complete the coupling reaction. The above steps were repeated. The solid-phase synthesis of fragment I was carried out according to the following sequence: Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(OtBu)-Gly-OH, Boc-His(Trt)-Aib-OH. After coupling, the resin was washed with organic solvent and dried under vacuum at room temperature. The peptide resin was then cleaved in fluorinated alcohol solution or 1%-5% TFA dichloromethane solution to obtain fully protected fragment I. 2) Synthesis of fully protected fragment II: Dichloromethane was added to a solid-phase reactor containing 2-ChlorotritylChloride resin for swelling. The solvent was then removed, and a dry dichloromethane solution of Fmoc-Arg(Pbf)-Gly-OH and DIPEA was added. Amino acids at positions 30 and 31 were linked to the resin. Unreacted or poorly reacted chlorination sites on the resin were blocked with DCM / MeOH / DIPEA. The resin was washed clean with solvent, and a deprotecting agent was added to remove the Fmoc protecting group. An activated Fmoc-Arg(Pbf)-Gly-OH / HOBt / DIC mixture was added to couple the amino acid monomers at positions 28 and 29. After a negative indole test result, the solution was removed, and the peptide resin was washed with organic solvent to complete the coupling reaction. The above steps were repeated according to... The following sequences were used for solid-phase synthesis of fragment II: 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(AEEA-AEEA-γGlu(α-OtBu)-Octadecanedioicacid(mono-tBu))-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH. After coupling, the resin was washed with organic solvent and dried under vacuum at room temperature. The peptide resin was then cleaved in fluorinated alcohol solution or 1%-5% TFA dichloromethane solution to obtain fully protected fragment II. (ii) Synthesis of fragment II with exposed nitrogen terminus: The fully protected fragment II was dissolved in DMSO, and 1.0-10.0 equivalents of di-tert-butyl dicarbonate were added. After stirring until homogeneous, 0.1-0.5 equivalents of DMAP were added. The reaction was carried out at room temperature until the reactants were completely reacted. The product was collected by back-electrolysis with water or an ether solvent to obtain the carbon-terminated tert-butylated fully protected fragment II. The fully protected fragment II with carbon-terminal tert-butyl esterification was deprotected by removing the Fmoc protecting group of the nitrogen-terminal amino group in a DMF solution containing 10%-50% by volume of pyrrolidine or piperidine, yielding fragment II with the nitrogen-terminal naked group. (iii) Synthesis of fully protected smegglutinin: The fully protected fragment I was dissolved in DMSO, a condensing agent was added and activated at room temperature for 15 minutes, then the nitrogen-terminated bare fragment II was added and reacted at 25-50℃ until the content of nitrogen-terminated bare fragment II was less than 2%; the product was collected by back-electrolysis with water or ether solvent to obtain the fully protected smegglutinin. (iv) Synthesis of crude smegglutinin: The fully protected smegglutinin obtained in step 3) is further deprotected under lysis conditions, precipitated in an ether solvent, and the solid is collected by centrifugation. The solid is washed twice with an ether solvent and collected by centrifugation to obtain crude smegglutinin. 5) Preparation of pure Smegglutinin: The crude Smegglutinin solution obtained in step 4) was purified by reverse silica gel chromatography under elution conditions to obtain refined Smegglutinin.

[0015] As a preferred embodiment of the present invention, the structure of the fully protected segment I is as follows: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH; The structure of the fully protected segment II is shown below: Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γGlu(α-OtBu)-Octadecanedioicacid(mono-tBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH.

[0016] In a preferred embodiment of the present invention, the deprotecting agent is a mixture of piperidine and an organic solvent or a mixture of pyrrolidine and an organic solvent; when the deprotecting agent is a mixture of piperidine and an organic solvent, the mass percentage of piperidine is 10%-50%; when the deprotecting agent is a mixture of pyrrolidine and an organic solvent, the mass percentage of pyrrolidine is 10%-50%; the organic solvent is one or more of dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone or 2-methyltetrahydrofuran.

[0017] As a preferred embodiment of the present invention, in step one), the amount of protected amino acid monomer is 2-5 times the equivalent of the total molar number of the starting resin.

[0018] As a preferred embodiment of the present invention, in step one), the fluorinated alcohol solution is a 15%-50% hexafluoroisopropanol dichloromethane solution or a 20%-50% trifluoroethanol dichloromethane solution.

[0019] As a preferred embodiment of the present invention, in step three), the condensing agent is HATU+DIPEA.

[0020] As a preferred embodiment of the present invention, in step four), the lysis buffer is a combination of trifluoroacetic acid and several of the following: phenol, water, ethylene dithiol, triisopropylsilane, dimethyl sulfide, anisole, dithiothreitol, or 3,6-dioxo-1,8-octanedithiol. The volume percentage of trifluoroacetic acid in the lysis buffer is not less than 80%. The mass ratio of the lysis buffer to the fully protected smegglutinin is 5:1-10:1, the reaction temperature is 10℃-30℃, and the reaction time is 1.5-5h. The ether solvent is one or a mixture of isopropyl ether, methyl tert-butyl ether, or diethyl ether.

[0021] As a preferred embodiment of the present invention, in step one), the molar ratio of resin, protected amino acid monomer, HOBt and DIC in the resin / protected amino acid monomer / HOBt / DIC mixture is 1:2-5:2-5:2-5.

[0022] As a preferred embodiment of the present invention, in step five), the eluent is an aqueous phase of 0.05-0.2% TFA aqueous solution and an organic phase of acetonitrile; or an aqueous phase of 10-50 mmol / L potassium dihydrogen phosphate aqueous solution and an organic phase of acetonitrile; or an aqueous phase of 10-50 mmol / L sodium dihydrogen phosphate aqueous solution and an organic phase of acetonitrile.

[0023] Compared with the prior art, the present invention has the following beneficial effects: 1) This invention achieves the synthesis of smegglutinin using a large-fragment solid-liquid phase mixing method. First, fully protected fragment I (hexapeptide) and fully protected fragment II (pentadecapeptide) are synthesized using a solid-phase synthesis method. Tert-butyl ester protection is introduced at the C-terminus of the fully protected fragment II. Then, under the action of an organic base, the Fmoc protecting group of the N-terminal amino group is removed to obtain the N-terminal naked fragment II. The fully protected fragment I and the N-terminal naked fragment II react under condensation conditions to obtain the fully protected smegglutinin. Subsequently, the protecting group is removed under acidic conditions to obtain crude smegglutinin peptide, which is then purified to obtain pure smegglutinin.

[0024] 2) The solid-phase synthesis of the fragment peptides of the present invention requires only 2.0 equivalents of coupling monomers for each step to achieve complete reaction, resulting in high atom economy and low cost. In addition, the synthesis of each fragment peptide can be carried out in parallel, greatly shortening the batch synthesis time and increasing production efficiency. The coupling docking reaction of the fragment peptides can be completed with an equivalent ratio of fragment peptides between 1:1 and 1:1.1. The coupling docking reaction of the fragment peptides is carried out in a liquid-phase reactor, which makes it easier to scale up production.

[0025] 3) In addition, the present invention uses 2-CTC resin as a solid-phase support in the solid-phase synthesis of fragment I and fragment II, which effectively avoids the deletion peptide impurities generated by the DKP side reaction, effectively avoids the M+106 impurity introduced by the cleavage of Wang resin, and the M+270 impurity introduced by the amino ester exchange of the tripeptide on Wang resin. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This relates to the synthesis strategy.

[0028] Figure 2 This is a side reaction introduced during the synthesis of smegglutinin using Wang resin.

[0029] Figure 3 It is a solid-phase synthesis of fully protected fragment I (hexapeptide).

[0030] Figure 4 It is a solid-phase synthesis of fully protected fragment II (pentapeptide).

[0031] Figure 5 It is the synthesis of fragment II with the nitrogen-terminated naked end.

[0032] Figure 6 It involves the coupling of fragment I and fragment II and the synthesis of smegglutinin.

[0033] Figure 7 This is the HPLC chromatogram of the prepared pure smegglutinin. Detailed Implementation

[0034] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0035] See Figure 1This invention provides a method for synthesizing smegglutinin using a large-fragment SPPS-LPPS hybrid method, comprising: cleaving smegglutinin into two polypeptide fragments with similar peptide sequence lengths: sequence 1-16 as fragment I and sequence 17-31 as fragment II; using 2-CTC resin as a solid-phase support, SPPS is used to sequentially couple protective amino acid monomers at corresponding sequence positions to synthesize fully protected fragment I and fragment II; tert-butyl ester is introduced to protect the carbon terminus of fragment II; then fragment I and fragment II are coupled via LPPS through a de-Fmoc / condensation reaction to obtain fully protected smegglutinin; subsequently, the protecting group is removed to obtain crude smegglutinin peptide, which is then purified to obtain smegglutinin.

[0036] Example 1 This embodiment provides the solid-phase synthesis of fully protected fragment I (hexapeptide) (e.g. Figure 3 (as shown) Synthesis of peptide resins: Weigh 20.00 g of 2-ChlorotritylChloride resin (resin substitution degree is 0.76 mmol / g) and add it to a solid-phase reactor. Add 200 mL of dichloromethane to swell the resin for 30 minutes, and then remove the solvent. Dissolve Fmoc-Glu(OtBu)-Gly-OH (14.67 g, 30.40 mmol) and diisopropylethylamine (7.86 g, 60.80 mmol) in 300 mL of dry dichloromethane. Add the above solution to the solid-phase reactor and react at 20℃-30℃ for 3 hours. Remove the solvent and wash the resin 5 times with DMF, 200 mL each time. Add 300 mL of a mixture of DCM / MeOH / DIPEA (volume ratio 80:15:5) and react at room temperature for 30 minutes (to block unreacted chlorinated groups on the resin). The solvent was removed, and the resin was washed 5 times with DMF, 200 mL each time. 250 mL of 20% piperidine DMF solution (v / v, piperidine:DMF=1:4) was added, and the mixture was reacted at room temperature for 30 minutes. The solution was removed, and the resin was washed 5 times with DMF, 200 mL each time. The indole test result was positive.

[0037] Weigh out Fmoc-Leu-OH (10.74 g, 30.40 mmol) and HOBt (4.11 g, 30.40 mmol), dissolve them in 300 mL of DMF, cool to 10-15 °C, add DIC (3.84 g, 30.40 mmol), and continue activation at 10-15 °C for 15 minutes. Add the resulting activated solution to a solid-phase reactor and react at 20-30 °C. Take a small amount of resin every hour for indene testing until the indene test is negative. Drain the solution and wash the resin 5 times with DMF, 200 mL each time. Add 250 mL of 20% piperidine DMF solution (v / v, piperidine:DMF = 1:4). The reaction was carried out at 20℃-30℃ for 30 minutes, then dried. The resin was washed 5 times with DMF, 200mL each time. The indene test result was positive. (It should be noted that the above values ​​are only one example. As long as the molar ratio of 2-ChlorotritylChloride resin / protected amino acid monomer / HOBt / DIC is 1:2-5:2-5:2-5, it is applicable to this invention. Piperidine in the above operation is an Fmoc protecting group removal agent. Other deprotection agents can also be used, such as 20% pyrrolidine DMF solution, 1% DBUDM FMF solution or 50% diethylamine DMF solution, to remove Fmoc.)

[0038] Repeat the steps described above to perform the solid-phase synthesis reaction of fragment I according to the following sequence: Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(OtBu)-Gly-OH, Boc-His(Trt)-Aib-OH; after coupling, wash the resin three times with 300 mL of dichloromethane each time; then wash the resin three times with 300 mL of methanol each time; dry under vacuum at room temperature to obtain the peptide resin of fragment I, the structure of which is shown below: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-CTCResin.

[0039] Pyrolysis: Prepare 300 mL of lysis buffer with a ratio of hexafluoroisopropanol to dichloromethane of 1:4 (volume ratio). Add the peptide resin containing fragment I under ice bath conditions, stir for 30 min under ice bath conditions, and then continue the reaction at room temperature for 2 hours. After the reaction is complete, filter off the resin under reduced pressure, and concentrate the filtrate under reduced pressure at 25-35 °C to obtain fully protected fragment I (hexapeptide). Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (29.57g, yield 76.0%).

[0040] Example 2 This embodiment provides the solid-phase synthesis of fully protected fragment II (pentapeptide) (e.g. Figure 4 (as shown) Synthesis of peptide resins: Weigh 20.00 g of 2-ChlorotritylChloride resin (resin substitution degree is 0.76 mmol / g) and add it to a solid-phase reactor. Add 200 mL of dichloromethane to swell the resin for 30 minutes, and then remove the solvent. Dissolve Fmoc-Arg(Pbf)-Gly-OH (21.46 g, 30.40 mmol) and diisopropylethylamine (7.86 g, 60.80 mmol) in 300 mL of dry dichloromethane. Add the above solution to the solid-phase reactor and react at 20℃-30℃ for 3 hours. Remove the solvent and wash the resin 5 times with DMF, 200 mL each time. Add 300 mL of a mixture of DCM / MeOH / DIPEA (volume ratio 80:15:5) and react at room temperature for 30 minutes (to block unreacted chlorinated groups on the resin). The resin was dried and washed 5 times with DMF, 200 mL each time. 250 mL of 20% piperidine DMF solution (v / v, piperidine:DMF=1:4) was added and reacted at room temperature for 30 minutes. The resin was dried and washed 5 times with DMF, 200 mL each time. The indole test result was positive.

[0041] Weigh out Fmoc-Arg(Pbf)-Gly-OH (21.46 g, 30.40 mmol) and HOBt (4.11 g, 30.40 mmol), dissolve them in 300 mL of DMF, cool to 10-15 °C, add DIC (3.84 g, 30.40 mmol), and continue activation at 10-15 °C for 15 minutes. Add the resulting activated solution to a solid-phase reactor and react at 20-30 °C. Take a small amount of resin every hour for indene testing until the indene test is negative. Dry the resin and wash it 5 times with DMF, 200 mL each time. Add 250 mL of 20% piperidine DMF solution (v / v, piperidine:DMF = ... (1:4), react at 20℃-30℃ for 30 minutes, dry under vacuum, wash the resin 5 times with DMF, 200mL each time, and the indene test result is positive. (It should be noted that the above values ​​are only one example. As long as the molar ratio of 2-ChlorotritylChloride resin / protected amino acid monomer / HOBt / DIC is 1:2-5:2-5:2-5, it is applicable to this invention. Piperidine in the above operation is an Fmoc protecting group removal agent. Other deprotection agents can also be used, such as 20% pyrrolidine DMF solution, 1% DBUDM FMF solution, or 50% diethylamine DMF solution, to remove Fmoc.)

[0042] Repeat the steps described above to perform the solid-phase synthesis of fragment II (fifteen-peptide) according to the following sequence: 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(AEEA-AEEA-γGlu(α-OtBu)-Octadecanedioicacid(mono- tBu))-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH; after coupling, the resin was washed three times with dichloromethane, 300 mL each time; then washed three times with methanol, 300 mL each time; dried under vacuum at room temperature to obtain peptide resin of fragment II (pentapeptide): Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γGlu(α-OtBu)-Octadecanedioicacid(mono -tBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-CTCResin.

[0043] Pyrolysis: Prepare 300 mL of lysis buffer with a ratio of hexafluoroisopropanol to dichloromethane of 1:4 (volume ratio). Add the peptide resin containing fragment II under ice bath conditions, stir for 30 min under ice bath conditions, and then continue the reaction at room temperature for 2 hours. After the reaction is complete, filter off the resin under reduced pressure, and concentrate the filtrate under reduced pressure at 25-35 °C to obtain the fully protected fragment II. Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γGlu(α-OtBu)-Octadecanedioicacid(mono- tBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH (41.64g, yield 75%).

[0044] Example 3 This embodiment provides the synthesis of fragment II with a nitrogen-terminated naked end (e.g., Figure 5 (as shown) Weigh 41.64 g (11.4 mmol) of the fully protected fragment II obtained in Example 2 into a 1 L round-bottom flask, add 500 mL of DMSO to dissolve it, then cool in an ice bath for 10 minutes, add di-tert-butyl dicarbonate (7.46 g, 34.20 mmol), stir to dissolve, then slowly add DMAP (0.42 g, 3.42 mmol), remove the ice bath, and react at room temperature until fragment II reacts completely as monitored by HPLC. Pour the reaction solution into ice water to crystallize, filter to collect the solid, dry it, and use it directly in the next step.

[0045] The crude product was added to a 1L round-bottom flask, and 500 mL of 20% pyrrolidine DMF solution (volume ratio 1:4) was added under ice bath. After 10 min, the ice bath was removed, and the mixture was reacted at room temperature for 1 hour. The mixture was then concentrated under reduced pressure, and then slurried twice with 100 mL of isopropyl ether each time. The mixture was filtered, and the filter cake was washed with a small amount of n-heptane. The filter cake was collected and dried under reduced pressure to obtain crude fragment II with exposed nitrogen terminus, which was directly used in the next step of the reaction.

[0046] Example 4 This embodiment provides the synthesis of crude smegglutinin peptide (e.g., Figure 6 (As shown) Weigh 26.86 g (10.5 mmol) of the fully protected fragment I obtained in Example 1 into a 1 L round-bottom flask, add 500 mL of DMSO to dissolve, then cool in an ice bath for 10 min. Add DIPEA (1.55 g, 12.00 mmol) and HATU (4.00 g, 10.5 mmol) and continue activation in an ice bath for 15 min. Add the nitrogen-terminated naked fragment II (34.86 g, 10.0 mmol) obtained in Example 3 in portions, remove the ice bath, and react at room temperature until the reaction of fragment II is complete as monitored by TLC. Add the reaction solution dropwise to 1.5 L of ice water to crystallize, filter and collect the solid. The solid is slurried with 200 mL of 5% sodium bicarbonate aqueous solution, filtered under reduced pressure and collected to obtain the fully protected smegglutinin.

[0047] Fully protected semaglutide is completely deprotected in one step: Prepare 500 mL of lysis buffer with TFA / EDT / TIS / H2O = 87.5:5:2.5:5 (V / V), cool in an ice bath for 30 min, add the cooled lysis buffer to the reaction flask containing the fully protected semaglutide, and react naturally at elevated temperature for 2-3 hours. Concentrate the reaction solution under reduced pressure to 200 mL at 20-30 °C, and slowly add the resulting concentrate dropwise to 1.5 L of pre-cooled isopropyl ether to crystallize. Collect the solid by centrifugation, wash the solid twice with fresh pre-cooled isopropyl ether (500 mL each time), collect the solid by centrifugation, and dry under reduced pressure at room temperature for 1 hour to obtain crude semaglutide.

[0048] Preparation and purification of crude smegglutinin: The aqueous solution of crude semaglutide obtained in the previous step was filtered through a mixed microporous membrane with a pore size of 0.45 μm. Purification was performed using high-performance liquid chromatography (HPLC). The semaglutide fraction was collected according to the position of the reference standard. The fraction was concentrated under reduced pressure and freeze-dried to obtain 25.50 g of pure semaglutide (purity 99.8%, total yield 62%).

[0049] The HPLC chromatogram of the obtained pure smegglutinin is shown in [reference needed]. Figure 7 See Table 1.

[0050] Table 1. HPLC chromatographic results No. Retention time / min Peak area / mAU*MIN Peak height / mAU relative area / % Relative retention time Separation / USP 1 19.742 0.016 0.045 0.01 na 2.16 2 20.742 173.906 597.589 99.89 na na 3 21.608 0.123 0.473 0.07 na na 4 22.008 0.056 0.131 0.03 na na na na na na na na na Total 174.102 598.238 100 0.00 The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A process for the synthesis of semaglutide by large fragment SPPS-LPPS hybrid method, characterized in that, The method is: cutting semaglutide into two polypeptide fragments: sequence 1-sequence 16 is fragment I, and sequence 17-sequence 31 is fragment II; coupling protected amino acid monomers corresponding to the sequence position one by one by SPPS with 2-ChlorotritylChloride resin as a solid phase carrier, and cleaving under fluorinated alcohol or weak acid conditions to obtain fully protected fragment I and fully protected fragment II; Introducing t-butyl ester to protect the carboxyl group at the carbon end of the fully protected fragment II; removing the Fmoc protecting group of the amino group at the nitrogen end under the action of an organic base to obtain the fragment II with the naked nitrogen end; the structure of the fragment II with the naked nitrogen end is as follows: H-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-gammaGlu(alpha-OtBu)-Octadecanedioic acid(mono-tBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu; The fully protected fragment I and the fragment II with the naked nitrogen end are reacted under the condition of a condensing agent to obtain fully protected semaglutide; the protecting group is removed under acidic conditions to obtain crude semaglutide peptide, and pure semaglutide is obtained after purification.

2. The process for synthesis of semaglutide by large fragment SPPS-LPPS hybrid method as claimed in claim 1 wherein, The steps of the method for synthesizing semaglutide are: I) Synthesis of fully protected fragment I and fully protected fragment II: 1) Synthesis of fully protected fragment I: adding dichloromethane to 2-ChlorotritylChloride resin for swelling, drying the solvent, adding a dry dichloromethane solution containing Fmoc-Glu(OtBu)-Gly-OH and DIPEA, connecting the amino acids at the 15th and 16th positions to the resin, adding DCM / MeOH / DIPEA to close the unreacted or difficultly reacted chloro sites on the resin; washing the resin, adding a deprotection reagent to remove the Fmoc protecting group, adding an activated Fmoc-Leu-OH / HOBt / DIC mixture to perform the coupling reaction of the amino acid monomer at the 14th position, and the indene test result is negative; after drying the solution, the coupling reaction is completed after washing the peptide resin with an organic solvent; repeating the above steps, and performing the solid-phase synthesis reaction of fragment I according to the following sequence: Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(OtBu)-Gly-OH, Boc-His(Trt)-Aib-OH; after the coupling is completed, the resin is washed; vacuum drying at room temperature; and cleaving the peptide resin under the condition of a fluorinated alcohol solution or a 1%-5% TFA dichloromethane solution to obtain fully protected fragment I; 2) Synthesis of the fully protected fragment II: The 2-Chlorotrityl Chloride resin was swelled in dichloromethane, and then Fmoc-Arg(Pbf)-Gly-OH and DIPEA in dry dichloromethane were added to the resin to couple the amino acids at the 30th and 31st positions. DCM / MeOH / DIPEA was added to block the unreactive or difficultly reactive chloro sites on the resin. After the resin was washed, a deprotection reagent was added to remove the Fmoc protecting group. Then, a mixture of activated Fmoc-Arg(Pbf)-Gly-OH / HOBt / DIC was added to couple the amino acids at the 28th and 29th positions. After the indenyl test was negative, the solution was removed by suction, and the peptide resin was washed with organic solvent to complete the coupling reaction. The above steps were repeated to synthesize the fragment II according to the following sequence: 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(AEEA-AEEA-γGlu(α-OtBu)-Octadecanedioic acid(mono-tBu))-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH. After the coupling was completed, the resin was washed, and then dried in vacuum at room temperature. The peptide resin was cleaved in a fluorinated alcohol solution or a 1%-5% TFA dichloromethane solution to obtain the fully protected fragment II; II. Synthesis of the fragment II with a naked nitrogen end: The fully protected fragment II was dissolved in DMSO, and then 1.0-10.0 equivalents of di-tert-butyl dicarbonate and 0.1-0.5 equivalents of DMAP were added. After stirring until the raw material was completely reacted, the product was collected by water or ether solvent to obtain the fully protected fragment II with a tert-butylated carbon end; The fully protected fragment II with a tert-butylated carbon end was treated with a 5%-30% pyrrolidine or piperidine DMF solution to remove the Fmoc protecting group of the nitrogen end amino group, thereby obtaining the fragment II with a naked nitrogen end; III. Synthesis of the fully protected semaglutide: The fully protected fragment I was dissolved in DMSO, and then a condensing agent was added and activated at room temperature for 15 minutes. The fragment II with a naked nitrogen end was added, and the reaction was carried out at 25-50°C until the content of the fragment II with a naked nitrogen end was less than 2%. The product was collected by water or ether solvent to obtain the fully protected semaglutide; IV. Synthesis of the semaglutide crude peptide: The fully protected semaglutide obtained in step III was treated under the cleavage solution conditions to remove all the protecting groups at one time. The solid was precipitated in an ether solvent, centrifuged to collect the solid, washed, and centrifuged again to collect the solid, thereby obtaining the semaglutide crude peptide; V. Preparation of the semaglutide pure product: The semaglutide crude peptide obtained in step IV was purified by a reverse silica gel chromatographic column under the eluent conditions to obtain the semaglutide pure product.

3. The method of claim 1 or 2 for the synthesis of semaglutide by the large fragment SPPS-LPPS hybrid method, characterized in that, The structure of the fully protected fragment I is shown as follows: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH; The structure of the fully protected fragment II is shown as follows: Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γGlu(α-OtBu)-Octadecanedioicacid(mono-tBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH.

4. The fragment II with the N-terminal exposed: H-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γGlu(α-OtBu)-Octadecanedioicacid(mono-tBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu. The deprotection reagent is a mixture of piperidine and an organic solvent or a mixture of pyrrolidine and an organic solvent; when the deprotection reagent is a mixture of piperidine and an organic solvent, the mass percentage of piperidine is 10%-50%; when the deprotection reagent is a mixture of pyrrolidine and an organic solvent, the mass percentage of pyrrolidine is 10%-50%; the organic solvent is one or more of dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone or 2-methyltetrahydrofuran. In step one), the amount of the protected amino acid monomer is 2-5 times the total moles of the starting resin.

5. The process for synthesis of semaglutide by large fragment SPPS-LPPS hybrid method as claimed in claim 2 wherein, In step one), the fluorinated alcohol solution is a 15%-50% (volume fraction) hexafluoroisopropanol dichloromethane solution or a 20%-50% (volume fraction) trifluoroethanol dichloromethane solution.

6. The process for synthesis of semaglutide by large fragment SPPS-LPPS hybrid method as claimed in claim 2 wherein, In step three), the condensing agent is HATU+DIPEA.

7. The process for synthesis of semaglutide by large fragment SPPS-LPPS hybrid method as claimed in claim 2, wherein, In step four), the cleavage solution is a combination of trifluoroacetic acid and several of phenol, water, ethanedithiol, triisopropylsilane, dimethyl sulfide, benzyl sulfide, anisole, dithiothreitol or 3,6-dioxo-1,8-octanedithiol; the volume percentage of trifluoroacetic acid in the cleavage solution is not less than 80%; the mass ratio of the cleavage solution to the fully protected semaglutide is 5:1-10:1, the reaction temperature is 10-30℃, and the reaction time is 1.5-5h.

8. The process for synthesis of semaglutide by large fragment SPPS-LPPS hybrid method as claimed in claim 2, wherein, In step one), in the resin / protected amino acid monomer / HOBt / DIC mixture, the molar ratio of resin, protected amino acid monomer, HOBt and DIC is 1:2-5:2-5:2-5.

9. The process for synthesis of semaglutide by large fragment SPPS-LPPS hybrid method as claimed in claim 2, wherein, ​ 10. The process for synthesis of semaglutide by large fragment SPPS-LPPS hybrid method as claimed in claim 2 wherein, ​ 11. The process for synthesis of semaglutide by large fragment SPPS-LPPS hybrid method as claimed in claim 1 wherein, In step five), the eluent is water phase 0.05-0.2% TFA aqueous solution and organic phase acetonitrile; or water phase 10-50 mmol / L potassium dihydrogen phosphate aqueous solution and organic phase acetonitrile; or water phase 10-50 mmol / L sodium dihydrogen phosphate aqueous solution and organic phase acetonitrile.

12. Use of the N-terminally naked fragment II according to claim 1 for the synthesis of semaglutide, characterized in that, The structure of the nitrogen terminal exposed fragment II is: H-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γGlu(α-OtBu)-Octadecanedioicacid(mono-tBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu. The application of the nitrogen terminal exposed fragment II in the synthesis of semaglutide.

Images