A synthetic method of semaglutide

Abstract

The invention provides a method for synthesizing semaglutide, wherein Fmoc-N(FMPB)-Gly-OtBu and HBTU react with a swollen AM Resin in the presence of an activator to obtain Fmoc-N(FMPB-AM Resin)-Gly-OtBu; Fmoc-Arg(Pbf)-OH and HOBT are added to a reaction system of a resin and a PIP / DMF solution, and reacted under DIC activation conditions until the ninhydrin test is negative; the above steps of removing Fmoc protection and coupling the corresponding amino acid or polypeptide fragment are repeated, and the peptide resin coupling is completed sequentially from the C-terminus to the N-terminus according to the semaglutide sequence, and the crude semaglutide is obtained by cleavage, filtration, precipitation, and drying. The method adopts the condensation of the modified Gly with the amino resin to obtain the resin, which can be successfully completed at a higher temperature, and the synthesis efficiency and synthesis yield are higher.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide synthesis, and particularly relates to a method for synthesizing semaglutide. Background Art

[0002] The molecular formula of semaglutide is C 187 H 291 N 45 O 59 , with a molecular weight of 4113.64, is a GLP-1 receptor agonist that was originally approved for the treatment of type 2 diabetes. Its trade names include Ozempic and Wegovy. It stimulates the production of insulin and inhibits the secretion of glucagon, thereby reducing appetite and food intake, thereby achieving the effects of lowering blood sugar and reducing weight. The half-life of semaglutide in the body is about one week, so it can be conveniently administered once a week.

[0003] Regarding the synthesis method of semaglutide, existing patents such as the Chinese patent with patent number CN202010393322.2 use Wang resin as a carrier, Boc-His 1 (Trt)-Aib 2 -Glu 3 (OtBu)-OH, Fmoc-Gly 4 -Thr 5 (tBu)-Phe 6 -OH, Fmoc-Lys 20 [Octadecanedioic(OtBu)-γ-Glu(OtBu)-PEG2-PEG2]-OH fragment was used as the starting material for the synthesis of semaglutide resin. However, Wang resin was used as the carrier to couple Gly 31 , in the removal of Gly 31 When Fmoc protecting group is used, [+Gly] impurity will be generated; when Boc-His is used in the synthesis process 1 (Trt)-Aib 2 -Glu 3 (OtBu)-OH fragment will produce racemic impurities of D-Glu; after the synthesis of semaglutide resin is completed, the linker on WangResin will break when the peptide resin is cleaved and connect to the Trp side chain, generating new impurities. In another Chinese patent with patent number CN201810137173.6, CTC resin is used as a carrier, Fmoc-Arg 30 (Pbf)-Gly 31 -OH、Fmoc-Arg 28 (Pbf)-Gly 29 -OH、Fmoc-Trp26 (Boc)-Leu 27 -Val 28 -OH、Fmoc-Glu 22 (OtBu)-Phe 23 -Ile 24 -Ala 25 -OH、Alloc-Lys 20 [Octadecanedioic(OtBu)-γ-Glu(OtBu)-PEG2-PEG2]-OH, Fmoc-Gln 17 (Trt)-Ala 18 -Ala 19 -OH、Fmoc-Glu 15 (OtBu)-Gly 16 -OH、Fmoc-Tyr 13 (tBu)-Leu 14 -OH、Fmoc-Asp 9 (OtBu)-Val 10 -OH、Fmoc-Thr 5 (OtBu)-Phe 6 -OH、Fmoc-Glu 3 (OtBu)-Gly 4 -OH, Fmoc-His 1 (Trt)-Aib 2 -OH fragments are used as starting materials to synthesize semaglutide resin; CTC resin is used as a carrier. Due to the unstable peptide chain of CTC resin at high temperature, the peptide chain will fall off, greatly reducing the yield of the crude product; too many fragments are used, which greatly increases the production cost and difficulty, and is not conducive to large-scale production.

[0004] The above-mentioned record of background technical knowledge is intended to help ordinary technicians in this field understand the existing technologies that are relatively close to the present invention, and at the same time facilitate the understanding of the inventive concept and technical solution of the present invention. It should be clear that in the absence of clear evidence that the above-mentioned content has been disclosed before the filing date of this patent application, the above-mentioned background technology should not be used to evaluate the novelty of the technical solution of this application. Summary of the invention

[0005] Technical issues

[0006] In order to solve the above problems, the purpose of the present invention is to provide a method for synthesizing semaglutide, which uses modified Gly and amino resin to condense to obtain a resin, which can be successfully completed at a higher temperature, with higher synthesis efficiency and yield. Compared with the use of Wang resin, the generation of new impurities during the cracking process is avoided; the use of fragments is reduced, the cost is greatly saved, the generation of impurities is avoided, the purification difficulty and production cost are reduced, and the product yield and product purity are improved.

[0007] Technical Solution

[0008] In order to achieve the above object, the present invention includes the following solutions.

[0009] Scheme 1, a method for synthesizing a fully protected semaglutide peptide resin, comprising:

[0010] Step 1: Fmoc-N(FMPB)-Gly-OtBu and HBTU are reacted with swollen AM Resin in the presence of an activator to obtain Fmoc-N(FMPB-AM Resin)-Gly-OtBu;

[0011] Step 2: Fmoc-Arg(Pbf)-OH and HOBT are added to the reaction system of resin and PIP / DMF solution, and reacted under DIC activation conditions until the ninhydrin test is negative;

[0012] Step 3: Repeat the above steps of removing Fmoc protection and coupling the corresponding amino acid or polypeptide fragment, and complete the peptide resin coupling from the C-terminus to the N-terminus in sequence according to the semaglutide sequence. Finally, wash the peptide resin at least 3 times with an equal volume of methanol to the resin, and vacuum dry to obtain the semaglutide fully protected peptide resin Boc-His(Trt)-Aib-Glu(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser (tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys[Octadecanedioic(OtBu)-γ-Glu(OtBu)-PEG2 -PEG2]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-N(FMPB-AMResin)-Gly-OtBu.

[0013] Furthermore, in step 1, the Fmoc-N(FMPB)-Gly-OtBu structure is selected from at least one of the following structures (F1)-(F6):

[0014] (F1); (F2);

[0015] (F3); (F4);

[0016] (F5); (F6).

[0017] Furthermore, in step 1, the degree of substitution of the AM Resin is between 0.1-1 mmol / L.

[0018] Furthermore, in step 1, the swelling of AM Resin is specifically performed by swelling the resin with DMF of 2-5 times the volume of AM Resin for 1-2 hours, and then removing the DMF.

[0019] Furthermore, in step 1, the swollen AM Resin needs to be washed with NMM / DMF solution and DMF in sequence before participating in the reaction to remove the detergent.

[0020] Furthermore, the resin is washed with NMM / DMF solution by washing the resin at least twice with 1-6% NMM / DMF solution of 2-5 times the volume of the swollen AM Resin, and then removing the NMM / DMF solution.

[0021] Furthermore, the resin is washed with DMF by washing the resin at least 5 times with DMF having a volume of 2-5 times that of the swollen AM Resin, and then removing the DMF.

[0022] Furthermore, in step 1, the activating agent is NMM with twice the equivalent of Fmoc-N(FMPB)-Gly-OtBu.

[0023] Furthermore, step one specifically includes: adding Fmoc-N(FMPB)-Gly-OtBu and HBTU in a ratio of 3:2.85 into an activation bottle, adding DMF to dissolve, and then adding NMM twice the equivalent of Fmoc-N(FMPB)-Gly-OtBu, and slowly adding it to the reactor containing the AM Resin in step one after activation, and continuously reacting at 35±5°C. After the reaction is completed, the reaction solution is extracted and washed with DMF at least 3 times to obtain Fmoc-N(FMPB-AM Resin)-Gly-OtBu.

[0024] Furthermore, the activation is performed at 0°C for 5-10 min.

[0025] Furthermore, the end of the reaction means that the reaction is completed when the ninhydrin test is negative.

[0026] Furthermore, in step 2, the reaction system of the resin and the PIP / DMF solution comprises: adding an equal volume of the PIP / DMF solution to the resin obtained in step 1 and reacting for 5 minutes, removing the reaction solution and then adding an equal volume of the PIP / DMF solution to the resin and reacting for 15 minutes, and removing the reaction solution and adding an equal volume of DMF to the resin and washing the resin at least 8 times until the ninhydrin test is positive.

[0027] Furthermore, the PIP / DMF solution is a 10-40% PIP / DMF solution.

[0028] Furthermore, step 2 specifically includes: adding Fmoc-Arg(Pbf)-OH and HOBT to DMF solution in a ratio of 3:3, adding DIC equivalent to Fmoc-Arg(Pbf)-OH for activation under ice-water bath conditions, adding the reaction solution to a reactor, continuing the reaction at 35±5°C until the ninhydrin test is negative, removing the reaction solution, and washing at least 3 times with an equal volume of DMF to the resin.

[0029] Furthermore, the activation time is 5-10 minutes.

[0030] Further, the steps of completing the peptide resin coupling from the C-terminus to the N-terminus in step 3 include: using Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys[Octadecanedioic(OtBu)-γ-Glu(OtBu)-PEG2-PEG2]-OH, Fmoc-Ala-OH, Fmoc-A la-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Leu-OH, 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-Thr(tBu)-Phe-OH, Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH complete the peptide resin coupling.

[0031] According to the present invention, amino resin, Gly 31The use of Gly after α-NH2 was modified by FMPB and α-COOH was modified by tBu solved the problem of the first amino acid Gly at the C-terminus. 31 The α-NH2 of the molecule was modified with FMPB, which effectively avoided the technical problems of peptide chain falling off from the carrier during high temperature coupling when using CTCResin, and also avoided the problem of Gly 31 The problem of [+Gly] impurities and impurities during cleavage; using Fmoc-Lys 20 [Octadecanedioic(OtBu)-γ-Glu(OtBu)-PEG2-PEG2]-OH fragment, which simplified the coupling of semaglutide and improved the purity and yield of crude peptide; using Boc-His 1 (Trt)-Aib 2 -Glu 3 (OtBu)-Gly 4 -OH、Fmoc-Thr 5 (tBu)-Phe 6 -OH、Fmoc-Glu 15 (OtBu)-Gly 16 The -OH fragment is used as the starting material, which greatly reduces the D-His racemic impurities, [-Thr] and [-Glu] missing impurities, significantly improves the purity of the crude product, is conducive to purification, improves the yield of the product, reduces the production cost, and has good application prospects.

[0032] Scheme 2, the fully protected semaglutide peptide resin prepared by the method described in Scheme 1 above, specifically: Boc-His(Trt)-Aib-Glu(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys[Octadecanedioic(OtBu)-γ-Glu(OtBu)-PEG2-PEG2]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-N(FMPB-AM Resin)-Gly-OtBu.

[0033] Scheme 3, a method for synthesizing semaglutide, comprising adding a cleavage solution to the fully protected semaglutide peptide resin described in Scheme 2 above, collecting the filtrate after the reaction, adding it to cold methyl tert-butyl ether, collecting the precipitate by centrifugation, and drying to obtain a crude semaglutide.

[0034] Furthermore, the lysate is a mixed solution of trifluoroacetic acid, 1,2-ethanedithiol, triisopropylsilane and water in a volume ratio of 88-95:6-3:3-1:3-1.

[0035] Furthermore, the added amount of the lysate is 10-15 mL / g in a volume-to-mass ratio with respect to the semaglutide fully protected peptide resin.

[0036] Furthermore, the reaction time is at least 2 hours.

[0037] Furthermore, the reaction temperature is 25±5°C.

[0038] Furthermore, the filtrate includes the filtrate collected after the reaction and the filtrate obtained by washing the resin with TFA at least three times.

[0039] Furthermore, after collecting the precipitate by centrifugation, the precipitate is washed with cold methyl tert-butyl ether at least 3 times.

[0040] Furthermore, the crude yield of semaglutide is not less than 97.0%, and the HPLC purity is not less than 71.11%.

[0041] Furthermore, the synthesis method of semaglutide also includes: dissolving the crude semaglutide in a phosphate buffer solution of pH 6.8 containing 0.15-0.45 mmol / L hydroxyethyl-β-cyclodextrin, filtering through a 0.22 μm organic filter membrane, and chromatographically purifying to obtain purified semaglutide. The isoelectric point of semaglutide is about 5.4. The use of pH 6.8 phosphate buffer to dissolve crude semaglutide helps to improve the solubility of semaglutide, so that it is fully dissolved, the crude concentration is not less than 55g / L, and the crude peptide solution after dissolution is more stable in nature and less prone to precipitation, thereby avoiding peptide aggregation, adsorption, and separation. The inventors further found that adding a trace amount of hydroxyethyl-β-cyclodextrin to the pH 6.8 phosphate buffer for dissolving crude semaglutide can significantly improve the separation effect, so that after only conventional chromatographic purification, a pure semaglutide with a purity of not less than 99.5% and a maximum single impurity of not more than 0.3% can be obtained, which greatly simplifies the purification steps and is greatly improved compared to the complicated preparation and purification process of the prior art.

[0042] The present invention fundamentally solves the problems of [+Gly] impurities, D-Glu racemic impurities and new impurities generated due to the breakage of the connection on Wang Resin during the cleavage process in patent CN202010393322.2; it also solves the problem of CTC Resin as a carrier, the peptide chain is easy to fall off at high temperature and too many fragments are used for coupling in patent CN201810137173.6.

[0043] Based on the common knowledge in this field, the above-mentioned preferred conditions can be combined with each other to obtain a specific implementation method.

[0044] The raw materials and reagents involved in the present invention are all common commercially available products, and the operations involved are all routine operations in the art unless otherwise specified.

[0045] Beneficial Effects

[0046] The scheme of the present invention adopts the condensation of modified Gly and amino resin to obtain resin. Compared with the traditional CTC resin, the connection of peptide chains can be successfully completed at a higher temperature, the synthesis efficiency is higher, and the synthesis yield is improved; compared with the use of Wang resin, the generation of new impurities in the cracking process is avoided; the use of fragments is reduced, and the cost is greatly saved. Due to the characteristics of the structure of Gly itself, it is very easy to generate [+Gly] impurities when using Wang resin carrier. This impurity is similar to the property of the target peptide and is difficult to separate during purification, resulting in a reduced product yield. The use of modified Gly and amino resin to obtain Gly-amino resin avoids the generation of [+Gly] impurities at this site, thereby reducing the difficulty of purification, improving the product yield, and reducing the production cost. Therefore, the scheme of the present invention helps to reduce the increase of impurities, racemic impurities and missing impurities, improve the purity and yield of the crude product, is conducive to purification, improves the yield of the product, reduces the production cost, and is conducive to industrial production.

[0047] The present invention adopts the above technical solution to achieve the above purpose, which makes up for the shortcomings of the prior art and has reasonable design and convenient operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to make the above and / or other purposes, features, advantages and examples of the present invention more obvious and easy to understand, the drawings required for use in the specific implementation of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying creative work.

[0049] Figure 1 It represents the technical roadmap of the synthesis method of semaglutide;

[0050] Figure 2 A schematic diagram showing the yield of crude semaglutide peptide;

[0051] Figure 3 Schematic diagram showing the HPLC purity of crude semaglutide peptide;

[0052] Figure 4 A schematic diagram showing the purity of purified semaglutide;

[0053] Figure 5Schematic diagram showing the maximum single impurity content of purified semaglutide. DETAILED DESCRIPTION

[0054] Those skilled in the art can refer to the content of this article and appropriately replace and / or modify the process parameters to achieve the same. However, it should be particularly noted that all similar replacements and / or modifications are obvious to those skilled in the art and are considered to be included in the present invention. The products and preparation methods described in the present invention have been described through preferred examples. It is obvious that relevant personnel can modify or appropriately change and combine the products and preparation methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0055] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. The present invention uses the methods and materials described herein; however, other suitable methods and materials known in the art may also be used. The materials, methods, and examples described herein are illustrative only and are not intended to be limiting. All publications, patent applications, patents, provisional applications, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification, including definitions, shall prevail.

[0056] Unless otherwise specified, all percentages, parts, ratios, etc. are by weight; otherwise specified, including but not limited to, "%", "wt%", "mass%" means weight percentage, "mol%" means molar percentage, and "vol%" means volume percentage.

[0057] When a quantity, concentration or other numerical value or parameter is given as a range, a preferred range or a series of upper preferred values ​​and lower preferred values, it should be understood that it specifically discloses all ranges formed by any pair of numerical values ​​of any larger range limit or preferred value and any smaller range limit or preferred value, regardless of whether the range is disclosed separately. For example, when describing the range of "1 to 5 (1-5)", the described range should be understood to include the range of "1 to 4 (1-4)", "1 to 3 (1-3)", "1 to 2 (1-2)", "1 to 2 (1-2) and 4 to 5 (4-5)", "1 to 3 (1-3) and 5" etc. Unless otherwise stated, where a numerical range is described herein, the range includes the range end values ​​and all integers and fractions within the range.

[0058] Unless specifically stated, the materials, methods, and examples described herein are illustrative only and not limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are still described herein.

[0059] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. At the same time, the embodiments in this application and the features in the embodiments can be combined with each other without conflict.

[0060] In order to facilitate the understanding of the embodiments of the present invention, the abbreviations and key terms that may be involved in the embodiments of the present invention are first explained or defined. The relevant explanations or definitions do not absolutely limit the meanings of the abbreviations and key terms. For undefined abbreviations or key terms, they are commonly understood by those skilled in the art.

[0061] AM Resin: amino resin;

[0062] HBTU: full name O-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate, benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate, is a condensation agent commonly used in peptide synthesis;

[0063] HOBT: 1-Hydroxybenzotriazole, an amide condensation agent commonly used in peptide synthesis;

[0064] Fmoc: 9-fluorenylmethyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, is an amino protecting group commonly used in organic synthesis;

[0065] PIP: piperidine;

[0066] DMF: N,N-dimethylformamide;

[0067] DIC: 1,3-diisopropylcarbodiimide, N,N'-Diisopropylcarbodiimide;

[0068] NMM: N-Methylmorpholine.

[0069] The present invention is described in detail below

[0070] Embodiment 1:

[0071] Provided is a method for preparing a fully protected semaglutide peptide resin, the specific technical route is as follows Figure 1 As shown, the following steps are included:

[0072] (1) Weigh AM Resin with a substitution degree of 0.5 mmol / g, add DMF twice the volume of the resin to swell the resin for 1.5 h, remove the DMF, add 5% NMM / DMF solution twice the volume of the resin to wash the resin twice, remove the 5% NMM / DMF solution, then wash the resin five times with DMF, and remove the DMF solution.

[0073] (2) Weigh Fmoc-N(FMPB)-Gly-OtBu and HBTU in a ratio of Fmoc-N(FMPB)-Gly-OtBu:HBTU = 3:2.85 and add them to an activation bottle. Then add DMF to dissolve, and then add NMM twice the equivalent of Fmoc-N(FMPB)-Gly-OtBu, and activate at 0°C for 10 minutes. Finally, slowly add it to AM Resin, and continue the reaction at 35±2°C until the ninhydrin test is negative. Draw off the reaction solution and wash it with DMF 3 times to obtain Fmoc-N(FMPB-AM Resin)-Gly-OtBu. (3) Add 1.0 times the volume of the resin to the reactor. 20% PIP / DMF solution was reacted for 5 minutes. The reaction solution was removed. Then, 1.0 times the volume of the resin was added to the reactor. 20% PIP / DMF solution was reacted for 15 minutes. The reaction solution was removed. Then, the resin was washed 8 times with 1.0 times the volume of the resin. The ninhydrin test was positive. Fmoc-Arg(Pbf)-OH and HOBT were weighed in a ratio of Fmoc-Arg(Pbf)-OH:HOBT=3:3. Then, DMF solution was added. In an ice-water bath, DIC equivalent to Fmoc-Arg(Pbf)-OH was added for activation for 10 minutes. The activated reaction solution was added to the reactor. The reaction was continued at 35±2℃ until the ninhydrin test was negative. After the reaction was completed, the reaction solution was removed. Then, the resin was washed 3 times with 1.0 times the volume of the resin.

[0074] (4) Repeat the above steps of removing Fmoc protection and coupling the corresponding amino acid or polypeptide fragment, and complete the peptide resin coupling from the C-terminus to the N-terminus according to the semaglutide sequence, wash the resin three times with 1.0 times the resin volume of methanol, and vacuum dry the peptide resin to obtain the semaglutide fully protected peptide resin Boc-His(Trt)-Aib-Glu(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-S er(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys[Octadecanedioic(OtBu)-γ-Glu(OtBu)-PEG 2-PEG2]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-N(FMPB-AMResin)-Gly-OtBu.

[0075] Wherein, in step (4), the peptide resin coupling is completed in sequence from the C-terminus to the N-terminus using Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys[Octadecanedioic(OtBu)-γ-Glu(OtBu)-PEG2-PEG2]-OH, Fmoc-Ala-OH, Fmoc-Ala -OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-O H, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH complete the peptide resin coupling.

[0076] Embodiment 2:

[0077] Provided is a method for preparing semaglutide. The method comprises the following steps: on the basis of Example 1, placing the obtained semaglutide fully protected peptide resin in a cleavage reactor; mixing trifluoroacetic acid, 1,2-ethanedithiol, triisopropylsilane and water in a volume ratio of 90:5:2.5:2.5 to prepare a cleavage solution, adding 15 mL / g of resin into the cleavage solution, and continuously reacting for 4.0 h at 25±2° C.; filtering the reaction mixture with a sand core funnel, collecting the filtrate, washing the resin with a small amount of TFA for 3 times, adding the combined filtrate into precooled methyl tert-butyl ether, collecting a precipitate by centrifugation, washing the precipitate with cold methyl tert-butyl ether for 3 times, and vacuum drying to obtain a solid, i.e., a crude semaglutide peptide, wherein the crude product yield is 97.0% and the HPLC purity is 71.11%.

[0078] Embodiment 3:

[0079] Provided is a method for preparing semaglutide. The method comprises the following steps: on the basis of Example 1, placing the obtained semaglutide fully protected peptide resin in a cleavage reactor; mixing trifluoroacetic acid, 1,2-ethanedithiol, triisopropylsilane and water in a volume ratio of 93:3:2:2 to prepare a cleavage solution, adding the mixture into the cleavage solution at a ratio of 10 mL / g of resin, and continuously reacting for 3 hours at 25±2°C; filtering the reaction mixture with a sand core funnel, collecting the filtrate, washing the resin with a small amount of TFA for 3 times, adding the combined filtrate into pre-cooled methyl tert-butyl ether, collecting a precipitate by centrifugation, washing the precipitate with cold methyl tert-butyl ether for 3 times, and vacuum drying to obtain a solid, i.e., a crude semaglutide peptide.

[0080] Embodiment 4:

[0081] Provided is a preparation method of semaglutide. The method comprises the following steps: on the basis of Example 1, placing the obtained semaglutide fully protected peptide resin in a cleavage reactor; mixing trifluoroacetic acid, 1,2-ethanedithiol, triisopropylsilane and water in a volume ratio of 88:6:3:3 to prepare a cleavage solution, adding the mixture into the cleavage solution in a ratio of 12 mL / g of resin, and continuously reacting for 2 hours at 25±2°C; filtering the reaction mixture with a sand core funnel, collecting the filtrate, washing the resin with a small amount of TFA for 3 times, adding the combined filtrate into pre-cooled methyl tert-butyl ether, collecting a precipitate by centrifugation, washing the precipitate with cold methyl tert-butyl ether for 3 times, and vacuum drying to obtain a solid, i.e., a crude semaglutide peptide.

[0082] Embodiment 5:

[0083] Provided is a method for preparing semaglutide. The method comprises the following steps: on the basis of Example 1, placing the obtained semaglutide fully protected peptide resin in a cleavage reactor; mixing trifluoroacetic acid, 1,2-ethanedithiol, 4-methyl-5-acetylthiazole, triisopropylsilane and water in a volume ratio of 90:3:2:2.5:2.5 to prepare a cleavage solution, adding the mixture into the cleavage solution at a ratio of 15 mL / g of resin, and continuously reacting for 4.0 h at 25±2° C.; filtering the reaction mixture with a sand core funnel, collecting the filtrate, washing the resin with a small amount of TFA for 3 times, adding the combined filtrate into precooled methyl tert-butyl ether, collecting the precipitate by centrifugation, washing the precipitate with cold methyl tert-butyl ether for 3 times, and vacuum drying to obtain a solid, i.e., a crude semaglutide peptide.

[0084] Embodiment 6:

[0085] Provided is a method for preparing semaglutide. The method comprises the following steps: on the basis of Example 1, placing the obtained semaglutide fully protected peptide resin in a cleavage reactor; mixing trifluoroacetic acid, 1,2-ethanedithiol, 4-methyl-5-acetylthiazole, triisopropylsilane and water in a volume ratio of 93:1.5:1.5:2:2 to prepare a cleavage solution, adding the mixture into the cleavage solution at a ratio of 10 mL / g of resin, and continuously reacting for 3 hours at 25±2°C; filtering the reaction mixture with a sand core funnel, collecting the filtrate, washing the resin with a small amount of TFA for 3 times, adding the combined filtrate into pre-cooled methyl tert-butyl ether, collecting a precipitate by centrifugation, washing the precipitate with cold methyl tert-butyl ether for 3 times, and vacuum drying to obtain a solid, i.e., a crude semaglutide peptide.

[0086] Embodiment 7:

[0087] Provided is a method for preparing semaglutide. The method comprises the following steps: on the basis of Example 1, placing the obtained semaglutide fully protected peptide resin in a cleavage reactor; mixing trifluoroacetic acid, 1,2-ethanedithiol, 4-methyl-5-acetylthiazole, triisopropylsilane and water in a volume ratio of 88:4:2:3:3 to prepare a cleavage solution, adding the mixture into the cleavage solution at a ratio of 12 mL / g of resin, and continuously reacting for 2 hours at 25±2° C.; filtering the reaction mixture with a sand core funnel, collecting the filtrate, washing the resin with a small amount of TFA for 3 times, adding the combined filtrate into precooled methyl tert-butyl ether, collecting a precipitate by centrifugation, washing the precipitate with cold methyl tert-butyl ether for 3 times, and vacuum drying to obtain a solid, i.e., a crude semaglutide peptide.

[0088] Experimental Example 1:

[0089] The yield and HPLC purity of crude semaglutide peptide obtained in Examples 2 to 7 were respectively calculated, and the results were shown as follows: Figure 2 and Figure 3 As shown, Figure 2 The yield of crude semaglutide peptide is shown. Figure 3 The HPLC purity of crude semaglutide peptide is shown. It can be seen that in the scheme of the present invention, trifluoroacetic acid, 1,2-ethanedithiol, triisopropylsilane and water are mixed in a specific volume ratio to form a cleavage solution, and then the semaglutide fully protected peptide resin is subjected to a cleavage reaction and filtered, washed, precipitated and dried to obtain a crude peptide, with a yield of not less than 96.5% and an HPLC purity of not less than 71%. The method for preparing semaglutide is simple, and the yield and purity can meet general requirements. Further studies have found that replacing 5-10% of 1,2-ethanedithiol in the cleavage solution with 4-methyl-5-acetylthiazole can further improve the cleavage efficiency on the basis of the original cleavage solution, more efficiently break the peptide bonds on the resin, and more thoroughly release the polypeptide, with the yield being increased to not less than 97.9% and the HPLC purity being increased to not less than 77%.

[0090] Embodiment 8:

[0091] Provided is a method for preparing semaglutide, which is further purified on the basis of Example 2, specifically, dissolving a crude semaglutide in a phosphate buffer solution at pH 6.8, and passing through a 0.22 μm organic filter membrane; selecting a C18 column with a diameter and a length of 5 cm×25 cm, a phase A of 0.1% TFA aqueous solution, and a phase B of 0.1% TFA acetonitrile solution; a flow rate of 75 mL / min, a gradient of 30-50% for the phase B, an elution time of 60 min, a detection wavelength of 230 nm, and chromatographic purification to obtain purified semaglutide.

[0092] Embodiment 9:

[0093] Provided is a method for preparing semaglutide, which is further purified on the basis of Example 2, specifically, the crude semaglutide is dissolved in a phosphate buffer solution of pH 6.8 containing 0.15 mmol / L hydroxyethyl-β-cyclodextrin, and passed through a 0.22 μm organic filter membrane; a C18 column is selected, with a diameter and a length of 5 cm×25 cm, phase A is a 0.1% TFA aqueous solution, and phase B is a 0.1% TFA acetonitrile solution; the flow rate is 75 mL / min, the gradient of phase B is 30-50%, the elution time is 60 min, the detection wavelength is 230 nm, and the purified semaglutide is obtained by chromatography purification.

[0094] Embodiment 10:

[0095] Provided is a method for preparing semaglutide, which is further purified on the basis of Example 2, specifically, dissolving a crude semaglutide in a phosphate buffer solution of pH 6.8 containing 0.45 mmol / L hydroxyethyl-β-cyclodextrin, and passing through a 0.22 μm organic filter membrane; selecting a C18 column with a diameter and a length of 5 cm×25 cm, a phase A of 0.1% TFA aqueous solution, and a phase B of 0.1% TFA acetonitrile solution; a flow rate of 75 mL / min, a gradient of 30-50% for the phase B, an elution time of 60 min, a detection wavelength of 230 nm, and chromatographic purification to obtain purified semaglutide.

[0096] Embodiment 11:

[0097] Provided is a method for preparing semaglutide, which is further purified on the basis of Example 2, specifically, dissolving a crude semaglutide in a phosphate buffer solution of pH 6.8 containing 0.25 mmol / L hydroxyethyl-β-cyclodextrin, and passing through a 0.22 μm organic filter membrane; selecting a C18 column with a diameter and a length of 5 cm×25 cm, a phase A of 0.1% TFA aqueous solution, and a phase B of 0.1% TFA acetonitrile solution; a flow rate of 75 mL / min, a gradient of 30-50% for the phase B, an elution time of 60 min, a detection wavelength of 230 nm, and chromatographic purification to obtain purified semaglutide.

[0098] Embodiment 12:

[0099] Provided is a method for preparing semaglutide, which is further purified on the basis of Example 2, specifically, the crude semaglutide is dissolved in a phosphate buffer solution of pH 6.8 containing 0.4 mmol / L hydroxyethyl-β-cyclodextrin, and passed through a 0.22 μm organic filter membrane; a C18 column is selected, with a diameter and a length of 5 cm×25 cm, a phase A of 0.1% TFA aqueous solution, and a phase B of 0.1% TFA acetonitrile solution; a flow rate of 75 mL / min, a gradient of 30-50% for the phase B, an elution time of 60 min, a detection wavelength of 230 nm, and chromatographic purification to obtain purified semaglutide.

[0100] Experimental Example 2:

[0101] The purity and maximum single impurity of the purified semaglutide obtained in Examples 8-12 were statistically analyzed, and the statistical results were as follows: Figure 4 and Figure 5 As shown, Figure 4The purity of the purified semaglutide obtained in each example is shown. It can be seen that the purity of the purified semaglutide can be increased to 99.2% by using a phosphate buffer of pH 6.8 to dissolve the crude semaglutide and purifying it by conventional chromatography. Further, adding a trace amount of hydroxyethyl-β-cyclodextrin to the phosphate buffer can significantly improve the separation effect, so that the purity can be obtained after only conventional chromatography purification. Not less than 99.5%; Figure 5 The maximum single impurity of purified semaglutide obtained in each embodiment is shown. It can be seen that the addition of a trace amount of hydroxyethyl-β-cyclodextrin can reduce the maximum single impurity of chromatographically purified semaglutide to a level not higher than 0.3%. Only simple operations are required to greatly simplify the purification steps and significantly improve the purity of purified semaglutide, which is a great improvement over the complicated preparation and purification processes of the prior art.

[0102] The conventional techniques in the above embodiments are prior arts known to those skilled in the art, and thus will not be described in detail here.

[0103] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

[0104] Although the present invention has been described in detail and some specific embodiments have been cited, it is obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention.

[0105] Although the above-mentioned specific embodiments have been shown, described and pointed out the novel features applied to various embodiments, it should be understood that various omissions, substitutions and changes can be made to the form and details of the described device or method without departing from the spirit of the present disclosure. In addition, the above-mentioned various features and methods can be used independently of each other, or can be combined in various ways. All possible combinations and sub-combinations are intended to fall within the scope of the present disclosure. Many of the above-mentioned embodiments include similar components, and therefore, these similar components are interchangeable in different embodiments. Although the present invention has been disclosed in the context of certain embodiments and examples, it should be understood by those skilled in the art that the present invention can extend beyond the specifically disclosed embodiments to other alternative embodiments and / or applications and obvious modifications and equivalents thereof. Therefore, the present invention is not intended to be limited by the specific disclosure of the preferred embodiments herein.

[0106] Matters not covered in the present invention are all known technologies.

Claims

1. A method for synthesizing semaglutide, characterized in that: The cleavage solution is added to the fully protected peptide resin of semaglutide, and the filtrate is collected after the reaction and added to cold methyl tert-butyl ether, and the precipitate is collected by centrifugation and dried to obtain a crude semaglutide; the crude semaglutide is dissolved in a pH 6.8 phosphate buffer containing 0.15-0.45 mmol / L hydroxyethyl-β-cyclodextrin, and filtered through a 0.22 μm organic filter membrane, and purified by chromatography to obtain purified semaglutide; The fully protected semaglutide peptide resin is: Boc-His(Trt)-Aib-Glu(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys[Octadecanedioic(OtBu)-γ-Glu(OtBu)-PEG2-PEG2]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-N(FMPB-AM Resin)-Gly-OtBu; The lysate is a mixed solution of trifluoroacetic acid, 1,2-ethanedithiol, triisopropylsilane and water in a volume ratio of 88-95:6-3:3-1:3-1; 5-10% of 1,2-ethanedithiol in the lysate is replaced by 4-methyl-5-acetylthiazole.

2. The method according to claim 1, characterized in that: The synthesis method of the semaglutide fully protected peptide resin comprises: Step 1: Fmoc-N(FMPB)-Gly-OtBu and HBTU are reacted with swollen AM Resin in the presence of an activator to obtain Fmoc-N(FMPB-AM Resin)-Gly-OtBu; Step 2: Fmoc-Arg(Pbf)-OH and HOBT are added to the reaction system of resin and PIP / DMF solution, and reacted under DIC activation conditions until the ninhydrin test is negative; Step 3: Repeat the steps of removing Fmoc protection and coupling the corresponding amino acid or polypeptide fragment. According to the semaglutide sequence, from C-terminus to N-terminus, use Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys[Octadeca nedioic(OtBu)-γ-Glu(OtBu)-PEG2-PEG2]-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-G ly-OH, Fmoc-Leu-OH, 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-Thr(tBu)-Phe-OH, Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH to complete the peptide resin coupling, and finally wash the peptide resin at least 3 times with an equal volume of methanol to the resin, and vacuum dry to obtain the semaglutide fully protected peptide resin Boc-His(Trt)-Aib-Glu(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)- Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys[Octadecanedioic(O tBu)-γ-Glu(OtBu)-PEG2-PEG2]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-N(FMPB-AM Resin)-Gly-OtBu; In step 1, the Fmoc-N(FMPB)-Gly-OtBu structure is selected from at least one of the following structures (F1)-(F6): <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (F1);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (F2); (F3); (F4); (F5); (F6).

3. The method according to claim 2, characterized in that: In step 1, the substitution degree of the AM Resin is between 0.1-1 mmol / L.

4. The method according to claim 2 or 3, characterized in that: Step one specifically includes: adding Fmoc-N(FMPB)-Gly-OtBu and HBTU in a ratio of 3:2.85 into an activation bottle, adding DMF to dissolve, then adding NMM twice the equivalent of Fmoc-N(FMPB)-Gly-OtBu, and slowly adding it to the reactor containing AMResin in step one after activation, and continuing the reaction at 35±5°C. After the reaction is completed, the reaction solution is extracted and washed with DMF at least 3 times to obtain Fmoc-N(FMPB-AM Resin)-Gly-OtBu.

5. The method according to claim 2, characterized in that: Step 2 specifically includes: adding Fmoc-Arg(Pbf)-OH and HOBT to DMF solution in a ratio of 3:3, adding DIC equivalent to Fmoc-Arg(Pbf)-OH for activation under ice-water bath conditions, adding the reaction solution to a reactor, and continuing the reaction at 35±5° C. until the ninhydrin test is negative, removing the reaction solution, and washing at least 3 times with an equal volume of DMF to the resin.

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