WO2023279323A1 - Method for synthesizing glp-1 analog - Google Patents

Method for synthesizing glp-1 analog Download PDF

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WO2023279323A1
WO2023279323A1 PCT/CN2021/105192 CN2021105192W WO2023279323A1 WO 2023279323 A1 WO2023279323 A1 WO 2023279323A1 CN 2021105192 W CN2021105192 W CN 2021105192W WO 2023279323 A1 WO2023279323 A1 WO 2023279323A1
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fmoc
glu
fragment
otbu
resin
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PCT/CN2021/105192
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French (fr)
Chinese (zh)
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汪伟
黄嘉诚
尹传龙
唐洋明
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深圳翰宇药业股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/08—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to the technical field of polypeptide synthesis, in particular to a method for synthesizing GLP-1 analogues.
  • GLP-1 glucagon-like peptide-1
  • GLP-1 receptor binds to the GLP-1 receptor and acts on pancreatic ⁇ cells to increase the biosynthesis and secretion of insulin; at the same time Increase the number of islet ⁇ cells, inhibit the secretion of glucagon; inhibit appetite and food intake, delay the emptying of gastric contents, etc.
  • GLP-1 analogues are clinically developed for the treatment of type II diabetes and obesity, including liraglutide (Liraglutide), semaglutide (Semaglutide), etc.
  • the peptide sequence of liraglutide is:
  • the peptide sequence of semaglutide is:
  • the existing methods for preparing GLP-1 analogues can be roughly divided into two categories: genetic recombination methods and chemical synthesis methods.
  • Genetic recombination method Firstly, construct a recombinant expression vector, ferment, and purify the main chain fragment of the peptide, and then chemically connect the side chain fragment to the main chain fragment to obtain the crude peptide of the target product, such as patents CN1271086, CN105154498, etc.
  • the development cycle of this method is long, the technology is difficult, and the impurities are not easy to control.
  • Chemical synthesis methods including the following:
  • Fmoc/tBu strategy is used for solid-phase synthesis, temporary protecting groups such as Mtt, Aloc or ivDde are selected for lysine side chains, and the main chains are coupled one by one from the C-terminus to the N-terminus according to the peptide sequence, and then removed
  • the side chain amino acids are coupled in sequence according to the peptide sequence, and finally the target product crude peptide is obtained by trifluoroacetic acid cleavage, such as patents CN102286092, CN106928343, CN109369798, etc.
  • Patent CN106749613 first synthesizes the following three fragments by solid phase or liquid phase method: [1-16], [17-22] and [23-31], then condenses the fragments in the liquid phase to obtain a fully protected peptide, and finally trifluoroacetic acid Cleavage to obtain crude semaglutide peptide.
  • Patent CN109456401 first synthesizes six fragments by liquid phase or solid phase method: [1-4], [5-9], [10-16], [17-22], [23-27], [28-31], Then the fragments were coupled to a resin to obtain a peptide resin, and finally trifluoroacetic acid was cleaved to obtain a crude semaglutide peptide.
  • Patent CN11037278 firstly synthesized Oct- ⁇ -Glu(tBu)-AEEA-AEEA-OH side chain fragment and Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)- Val-OH hexapeptide fragments, and then these two fragments and other amino acids are coupled to the resin to obtain a peptide resin, and finally trifluoroacetic acid is cleaved to obtain a crude semaglutide peptide.
  • Fragment [17-22] selected by patent CN106749613 in fragment condensation, fragment [5-9], [17-22] and [23-27] selected by patent CN109456401, fragment Fmoc-Thr(tBu)-Phe selected by patent CN11037278 -Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-OH will racemize the C-terminal amino acid during coupling, resulting in more racemic impurities, and the purity of the crude peptide is low. At the same time, it is cumbersome to use liquid phase to synthesize fragments. Therefore, it is necessary to provide a synthetic method of GLP-1 analogs with high purity and yield of crude peptide and less Des-Thr 5 impurities to adapt to industrial production.
  • the purpose of the present invention is to provide a method for synthesizing GLP-1 analogues, the purity and yield of the crude peptide obtained by the method are high, the Des-Thr 5 impurity is less, and the operation is simple, which is suitable for industrial production.
  • the invention provides a method for synthesizing GLP-1 analogues, comprising the following steps:
  • Step 1 Synthesizing a fragment 1 coupled with a protecting group at the N-terminal of the amino acid sequence shown in SEQ ID NO: 1, on the His side chain, and on the Glu side chain;
  • Step 2 Synthesizing Fragment 2 with protective groups coupled to the N-terminal of the amino acid sequence shown in SEQ ID NO: 2, on the Thr side chain, on the Ser side chain, on the Asp side chain, on the Tyr side chain, and on the Glu side chain;
  • Step 3 sequentially coupling the amino acid shown in SEQ ID NO:3, Fragment 2 and Fragment 1 on the solid phase carrier in the order from the C-terminal to the N-terminal to obtain a peptide resin;
  • Step 4 Cleavage the peptide resin to obtain crude peptide.
  • the present invention firstly synthesizes the fragment 1 to 4 of the N-terminal of the GLP-1 analogue, the fragment 2 of the 5th to 12th position, and then sequentially couples SEQ ID NO:
  • the amino acid shown in 2 Fragment 2 and Fragment 1 are obtained from peptide resin, and cleaved to obtain crude peptide.
  • the crude peptide obtained by the synthesis method of the invention has high purity, high yield and less Des-Thr5 impurities.
  • the purity of the crude peptide is 80% to 82%, the yield is 38% to 40%, and the impurity of Des-Thr 5 is about 0.07 to 0.08%. .
  • the step 1 is specifically: sequentially coupling Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-X-OH and Boc-His(Trt)-OH, piperidine removes the Fmoc protecting group, and fragment 1 is obtained by weak acid cleavage.
  • the peptide sequence of Fragment 1 is Boc-His(Trt)-X-Glu(OtBu)-Gly-OH.
  • X is Ala or Aib.
  • the step 2 is specifically: according to the sequence from the C-terminus to the N-terminus of the amino acid sequence shown in SEQ ID NO: 2, sequentially coupling Fmoc-Gly-OH, Fmoc-Glu(OtBu)- OH, Fmoc-Leu-OH, Fmoc-Tyr(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-Ser(psiMe, Mepro), Fmoc-Asp(OtBu)-OH, Fmoc-Ser( tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH and Fmoc-Thr(tBu)-OH, piperidine removes the Fmoc protecting group, and weak acid cleavage to obtain Fragment 2.
  • the weak acid used in the weak acid cracking is one or more of trifluoroethanol, hexafluoroisopropanol, acetic acid and trifluoroacetic acid.
  • step 3 of the present invention the coupling of the amino acids shown in SEQ ID NO:3 is specifically to couple the protected amino acids one by one in the order from the C-terminal to the N-terminal of the amino acid sequence shown in SEQ ID NO:3; the protected amino acid is the protected group protected amino acids.
  • the protected amino acid is Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, 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(Y)-OH, Fmoc-Ala-OH , Fmoc-Ala-OH and Fmoc-Gln(Trt)-OH.
  • Y is Pal-Glu-OtBu or Oct(OtBu)-Glu-OtBu-AEEA-AEEA.
  • the peptide sequence of the GLP-1 analog is (N-terminal to C-terminal): H-His-X-Glu-Gly-Thr 5 -Phe-Thr-Ser-Asp-Val 10 -Ser-Ser-Tyr -Leu-Glu 15 -Gly-Gln-Ala-Ala-Lys 20 (Y)-Glu-Phe-Ile-Ala-Trp 25 -Leu-Val-Arg-Gly-Arg 30 -Gly-OH.
  • X is Ala
  • Y is Pal-Glu-OtBu
  • the synthetic GLP-1 analogue is liraglutide, and its peptide sequence is:
  • X is Aib
  • Y is Oct(OtBu)-Glu-OtBu-AEEA-AEEA
  • the synthetic GLP-1 analog is semaglutide, and its peptide sequence is:
  • the type of solid resin is not particularly limited, and the types commonly used in the field are sufficient.
  • the solid resin used in steps 1-3 is 2-chlorotrityl chloride resin or Wang resin.
  • the removal agent used to remove the N-terminal Fmoc protecting group in the present invention is preferably piperidine solution. Including but not limited to this.
  • the coupling agent for coupling is DIPCDI/HOBt, PyBop/HOBt/DIPEA, HBTU/HOBt/DIPEA, DIPCDI/HOAt, HATU/HOAt/DIPEA and PyAop/HOAt/DIPEA one or several.
  • the lysate of the lysis is a trifluoroacetic acid solution containing a capture agent, and the capture agent is one or more of PhSMe, PhOH, H 2 O, TIS, PhOMe, and EDT.
  • the lysate is a combination of TFA, H 2 O and PhOH, wherein the volume ratio of TFA, H 2 O and PhOH is 90:5:5.
  • the present invention firstly synthesizes the fragment 1 of the 1st to 4th position of the N-terminal of the GLP-1 analogue, and the fragment 2 of the 5th to 12th position of the N-terminal, and then sequentially couples the SEQ ID NO on the solid phase carrier according to the order from the C-terminal to the N-terminal : amino acid shown in 3, fragment 2 and fragment 1, obtain peptide resin, crack, obtain crude peptide.
  • the crude peptide obtained by the synthesis method of the invention has high purity and high total yield.
  • Fig. 1 shows the preparation process flowchart of GLP-1 analogue
  • Figure 2 shows a typical chromatogram of liraglutide fragment one
  • Figure 3 shows a typical chromatogram of liraglutide crude peptide
  • Figure 4 shows a typical chromatogram of semaglutide fragment one
  • Fig. 5 shows the typical chromatogram of semaglutide crude peptide
  • Figure 6 shows a typical chromatogram of a refined peptide.
  • the present invention provides a method for synthesizing GLP-1 analogues, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to realize it.
  • all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method and application herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention Invent technology.
  • the reagents and instruments used in the present invention are all common commercial products and can be purchased in the market.
  • the invention provides a method for synthesizing GLP-1 analogues, comprising the following steps:
  • Step 1 Synthesizing a fragment 1 coupled with a protecting group at the N-terminal of the amino acid sequence shown in SEQ ID NO: 1, on the His side chain, and on the Glu side chain;
  • Step 2 Synthesizing Fragment 2 with protective groups coupled to the N-terminal of the amino acid sequence shown in SEQ ID NO: 2, on the Thr side chain, on the Ser side chain, on the Asp side chain, on the Tyr side chain, and on the Glu side chain;
  • Step 3 sequentially coupling the amino acid shown in SEQ ID NO:3, Fragment 2 and Fragment 1 on the solid phase carrier in the order from the C-terminal to the N-terminal to obtain a peptide resin;
  • Step 4 Cleavage the peptide resin to obtain crude peptide.
  • the peptide sequence of the GLP-1 analog is (N-terminal to C-terminal): H-His-X-Glu-Gly-Thr 5 -Phe-Thr-Ser-Asp-Val 10 -Ser-Ser-Tyr -Leu-Glu 15 -Gly-Gln-Ala-Ala-Lys 20 (Y)-Glu-Phe-Ile-Ala-Trp 25 -Leu-Val-Arg-Gly-Arg 30 -Gly-OH (Formula I).
  • the synthesis strategy provided by the present invention is: the main chain peptide sequence of the above-mentioned GLP-1 analog is divided into three parts, fragment one (SEQ ID NO:1), fragment two (SEQ ID NO:2) and the remaining amino acids (SEQ ID NO: 3).
  • SEQ ID NO:1 fragment one
  • SEQ ID NO:2 fragment two
  • SEQ ID NO: 3 the remaining amino acids
  • the amino acid sequence shown in SEQ ID NO: 1 N terminal ⁇ C terminal
  • the specific sequence is: His-X-Glu-Gly, wherein, X is Ala or Aib.
  • amino acid sequence shown in SEQ ID NO: 2 is (N-terminal ⁇ C-terminal), which is the sequence numbered 5-16 in the peptide sequence shown in formula I, and the specific sequence is: Thr-Phe-Thr-Ser-Asp- Val-Ser-Ser-Tyr-Leu-Glu-Gly.
  • amino acid sequence (N-terminal ⁇ C-terminal) shown in SEQ ID NO:3 is the sequence numbered 17th to 31st in the peptide sequence shown in formula I, and the specific sequence is: Gln-Ala-Ala-Lys(Y)- Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly.
  • Y is Pal-Glu-OtBu or Oct(OtBu)-Glu-OtBu-AEEA-AEEA.
  • Fragment 1 The peptide sequence of Fragment 1 is Boc-His(Trt)-X-Glu(OtBu)-Gly-OH, wherein X is Ala or Aib. Using 2-chlorotrityl chloride resin as a carrier, according to the polypeptide solid-phase synthesis method, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-X-OH and Boc-His(Trt)-OH.
  • Fragment 2 The peptide sequence of Fragment 2 is Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(psiMe,Mepro)-Ser(tBu) -Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH.
  • 2-chlorotrityl chloride resin as a carrier, according to the polypeptide solid-phase synthesis method, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Tyr(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-Ser(psiMe, Mepro), Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Phe-OH and Fmoc-Thr(tBu)-OH.
  • Synthesis of peptide resin use 2-chlorotrityl chloride resin or Wang resin as the carrier, according to the peptide solid-phase synthesis method, according to the peptide sequence, sequentially couple Fmoc-Gly-OH, Fmoc from C-terminal to N-terminal -Arg(Pbf)-OH, 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(Y)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH , fragment two and fragment one, wherein Y is Pal-Glu
  • Embodiment 2 the synthesis of fragment two
  • Embodiment 6 the synthesis of semaglutide peptide resin
  • Embodiment 7 the synthesis of semaglutide
  • the crude peptide obtained in Example 4 or 7 was prepared and purified by high performance liquid chromatography, with tetraalkylsilane bonded silica gel as the stationary phase, 0.2% acetic acid solution as the mobile phase A, and acetonitrile as the mobile phase B , monitoring wavelength 280nm, gradient elution to collect target peak fractions, concentrated, freeze-dried to obtain liraglutide or semaglutide fine peptide, purity 99.7%, (chromatogram shown in Figure 6).
  • the purity of the obtained crude peptide was 58%, and the total yield was about 15%.

Abstract

The present invention relates to the technical field of polypeptide synthesis, in particular to a method for synthesizing a GLP-1 analog. In the present invention, fragment 1 and fragment 2 respectively at positions 1-4 and positions 5-12 of an N terminus of a GLP-1 analog are firstly synthesized, and then the remaining amino acids, fragment 2 and fragment 1 are sequentially coupled on a solid-phase carrier in a sequence from a C terminus to the N terminus to obtain a peptide resin which is cleaved to obtain a crude peptide. The GLP-1 analogs, namely liraglutide and semaglutide, are synthesized using a specific synthesis strategy of the present invention, so that the purity and the total yield of the crude peptide are high, the synthesis process is simple, and the method is suitable for large-scale production.

Description

一种合成GLP-1类似物的方法A method for synthesizing GLP-1 analogs 技术领域technical field
本发明涉及多肽合成技术领域，尤其涉及一种合成GLP-1类似物的方法。The invention relates to the technical field of polypeptide synthesis, in particular to a method for synthesizing GLP-1 analogues.
背景技术Background technique
GLP-1(胰高血糖素样肽-1)是由人体肠道细胞分泌的一种肽类激素，与GLP-1受体结合后作用于胰岛β细胞，增加胰岛素的生物合成和分泌；同时增加胰岛β细胞数量，抑制胰高血糖素的分泌；抑制食欲及摄食，延缓胃内容物排空速度等。针对这些特点，临床上开发GLP-1类似物用于II型糖尿病和肥胖症的治疗，包括利拉鲁肽(Liraglutide)、索马鲁肽(Semaglutide)等。GLP-1 (glucagon-like peptide-1) is a peptide hormone secreted by human intestinal cells, which binds to the GLP-1 receptor and acts on pancreatic β cells to increase the biosynthesis and secretion of insulin; at the same time Increase the number of islet β cells, inhibit the secretion of glucagon; inhibit appetite and food intake, delay the emptying of gastric contents, etc. In view of these characteristics, GLP-1 analogues are clinically developed for the treatment of type II diabetes and obesity, including liraglutide (Liraglutide), semaglutide (Semaglutide), etc.
利拉鲁肽的肽序为：The peptide sequence of liraglutide is:
H-His-Ala-Glu-Gly-Thr 5-Phe-Thr-Ser-Asp-Val 10-Ser-Ser-Tyr-Leu-Glu 15-Gly-Gln-Ala-Ala-Lys 20(γ-Glu-Pal)-Glu-Phe-Ile-Ala-Trp 25-Leu-Val-Arg-Gly-Arg 30-Gly-OH。 H-His-Ala-Glu-Gly-Thr 5 -Phe-Thr-Ser-Asp-Val 10 -Ser-Ser-Tyr-Leu-Glu 15 -Gly-Gln-Ala-Ala-Lys 20 (γ-Glu- Pal)-Glu-Phe-Ile-Ala- Trp25 -Leu-Val-Arg-Gly- Arg30 -Gly-OH.
索马鲁肽的肽序为：The peptide sequence of semaglutide is:
H-His-Aib-Glu-Gly-Thr 5-Phe-Thr-Ser-Asp-Val 10-Ser-Ser-Tyr-Leu-Glu 15-Gly-Gln-Ala-Ala-Lys 20(AEEA-AEEA-γ-Glu-OctadecanedioicAcid)-Glu-Phe-Ile-Ala-Trp 25-Leu-Val-Arg-Gly-Arg 30-Gly-OH。 H-His-Aib-Glu-Gly-Thr 5 -Phe-Thr-Ser-Asp-Val 10 -Ser-Ser-Tyr-Leu-Glu 15 -Gly-Gln-Ala-Ala-Lys 20 (AEEA-AEEA- γ-Glu-Octadecanedioic Acid)-Glu-Phe-Ile-Ala-Trp 25 -Leu-Val-Arg-Gly-Arg 30 -Gly-OH.
现有制备GLP-1类似物的方法大致可分为基因重组法和化学合成法两类。基因重组法：首先通过构建重组表达载体、发酵、纯化得到肽主链片段，然后化学方法将侧链片段连接至主链片段得到目标产品粗肽，如专利CN1271086、CN105154498等。该方法开发周期长，技术难度大，杂质不易控制。化学合成法，包括以下几种：The existing methods for preparing GLP-1 analogues can be roughly divided into two categories: genetic recombination methods and chemical synthesis methods. Genetic recombination method: Firstly, construct a recombinant expression vector, ferment, and purify the main chain fragment of the peptide, and then chemically connect the side chain fragment to the main chain fragment to obtain the crude peptide of the target product, such as patents CN1271086, CN105154498, etc. The development cycle of this method is long, the technology is difficult, and the impurities are not easy to control. Chemical synthesis methods, including the following:
(1)氨基酸逐个偶联：采用Fmoc/tBu策略固相合成，赖氨酸侧链选择Mtt、Aloc或ivDde等临时保护基，按照肽序从C端至N端逐个偶联主链，然后脱除赖氨酸侧链保护基，按照肽序依次偶联侧链氨基酸，最后 三氟乙酸裂解得到目标产品粗肽，如专利CN102286092、CN106928343、CN109369798等。(1) Coupling of amino acids one by one: Fmoc/tBu strategy is used for solid-phase synthesis, temporary protecting groups such as Mtt, Aloc or ivDde are selected for lysine side chains, and the main chains are coupled one by one from the C-terminus to the N-terminus according to the peptide sequence, and then removed In addition to the lysine side chain protecting group, the side chain amino acids are coupled in sequence according to the peptide sequence, and finally the target product crude peptide is obtained by trifluoroacetic acid cleavage, such as patents CN102286092, CN106928343, CN109369798, etc.
(2)片段缩合：(2) Fragment condensation:
专利CN106749613首先固相或液相法合成以下三个片段：[1-16]、[17-22]和[23-31]，然后在液相中将片段缩合得到全保护肽，最后三氟乙酸裂解得到索马鲁肽粗肽。Patent CN106749613 first synthesizes the following three fragments by solid phase or liquid phase method: [1-16], [17-22] and [23-31], then condenses the fragments in the liquid phase to obtain a fully protected peptide, and finally trifluoroacetic acid Cleavage to obtain crude semaglutide peptide.
专利CN109456401首先液相或固相法合成六个片段：[1-4]、[5-9]、[10-16]、[17-22]、[23-27]、[28-31]，然后将片段偶联至树脂得到肽树脂，最后三氟乙酸裂解得到索马鲁肽粗肽。Patent CN109456401 first synthesizes six fragments by liquid phase or solid phase method: [1-4], [5-9], [10-16], [17-22], [23-27], [28-31], Then the fragments were coupled to a resin to obtain a peptide resin, and finally trifluoroacetic acid was cleaved to obtain a crude semaglutide peptide.
专利CN11037278首先通过固相法合成Oct-γ-Glu(tBu)-AEEA-AEEA-OH侧链片段和Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-OH六肽片段，然后将这两个片段和其它氨基酸偶联至树脂得到肽树脂，最后三氟乙酸裂解得到索马鲁肽粗肽。Patent CN11037278 firstly synthesized Oct-γ-Glu(tBu)-AEEA-AEEA-OH side chain fragment and Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)- Val-OH hexapeptide fragments, and then these two fragments and other amino acids are coupled to the resin to obtain a peptide resin, and finally trifluoroacetic acid is cleaved to obtain a crude semaglutide peptide.
以上化学合成法中，氨基酸逐个偶联由于分子内或分子间的氢键缔合导致偶联困难，产生大量的缺损氨基酸杂质，这些杂质与产品的理化性质相近，最终导致纯化困难收率低。片段缩合中专利CN106749613选择的片段[17-22]、专利CN109456401选择的片段[5-9]、[17-22]和[23-27]、专利CN11037278选择的片段Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-OH在偶联时C端氨基酸会发生消旋，产生较多的消旋杂质，粗肽纯度低。同时采用液相合成片段操作繁琐。因此，提供一种粗肽纯度和收率高，Des-Thr 5杂质较少的GLP-1类似物合成方法以适应工业化生产十分必要。 In the above chemical synthesis methods, the coupling of amino acids one by one is difficult due to intramolecular or intermolecular hydrogen bond associations, resulting in a large number of defective amino acid impurities, which are similar to the physical and chemical properties of the product, resulting in difficulty in purification and low yield. Fragment [17-22] selected by patent CN106749613 in fragment condensation, fragment [5-9], [17-22] and [23-27] selected by patent CN109456401, fragment Fmoc-Thr(tBu)-Phe selected by patent CN11037278 -Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-OH will racemize the C-terminal amino acid during coupling, resulting in more racemic impurities, and the purity of the crude peptide is low. At the same time, it is cumbersome to use liquid phase to synthesize fragments. Therefore, it is necessary to provide a synthetic method of GLP-1 analogs with high purity and yield of crude peptide and less Des-Thr 5 impurities to adapt to industrial production.
发明内容Contents of the invention
有鉴于此，本发明目的在于提供一种合成GLP-1类似物的方法，该方法获得的粗肽纯度和收率较高，Des-Thr 5杂质较少，且操作简单，适合工业化生产。 In view of this, the purpose of the present invention is to provide a method for synthesizing GLP-1 analogues, the purity and yield of the crude peptide obtained by the method are high, the Des-Thr 5 impurity is less, and the operation is simple, which is suitable for industrial production.
本发明提供一种合成GLP-1类似物的方法，包括以下步骤：The invention provides a method for synthesizing GLP-1 analogues, comprising the following steps:
步骤1：合成在SEQ ID NO:1所示氨基酸序列N端、His侧链上、Glu侧链上偶联有保护基的片段一；Step 1: Synthesizing a fragment 1 coupled with a protecting group at the N-terminal of the amino acid sequence shown in SEQ ID NO: 1, on the His side chain, and on the Glu side chain;
步骤2：合成在SEQ ID NO:2所示氨基酸序列N端、Thr侧链上、Ser侧链上、Asp侧链上、Tyr侧链上、Glu侧链上偶联有保护基的片段二；Step 2: Synthesizing Fragment 2 with protective groups coupled to the N-terminal of the amino acid sequence shown in SEQ ID NO: 2, on the Thr side chain, on the Ser side chain, on the Asp side chain, on the Tyr side chain, and on the Glu side chain;
步骤3：在固相载体上按照从C端到N端的顺序，依次偶联SEQ ID NO:3所示的氨基酸、片段二和片段一，获得肽树脂；Step 3: sequentially coupling the amino acid shown in SEQ ID NO:3, Fragment 2 and Fragment 1 on the solid phase carrier in the order from the C-terminal to the N-terminal to obtain a peptide resin;
步骤4：将所述肽树脂裂解，获得粗肽。Step 4: Cleavage the peptide resin to obtain crude peptide.
本发明首先合成GLP-1类似物N端第1～4位的片段一第5～12位的片段二，然后在固相载体上按照从C端到N端的顺序，依次偶联SEQ ID NO:2所示的氨基酸、片段二和片段一，获得肽树脂，裂解，获得粗肽。本发明合成方法得到的粗肽纯度高，收率高，Des-Thr5杂质少。实验表明，采用本发明特定的合成策略合成利拉鲁肽和索马鲁肽，粗肽纯度为80％～82％，收率为38％～40％，Des-Thr 5杂质约0.07～0.08％。 The present invention firstly synthesizes the fragment 1 to 4 of the N-terminal of the GLP-1 analogue, the fragment 2 of the 5th to 12th position, and then sequentially couples SEQ ID NO: The amino acid shown in 2, Fragment 2 and Fragment 1 are obtained from peptide resin, and cleaved to obtain crude peptide. The crude peptide obtained by the synthesis method of the invention has high purity, high yield and less Des-Thr5 impurities. Experiments show that liraglutide and semaglutide are synthesized using the specific synthesis strategy of the present invention, the purity of the crude peptide is 80% to 82%, the yield is 38% to 40%, and the impurity of Des-Thr 5 is about 0.07 to 0.08%. .
一些实施方案中，所述步骤1具体为：在固相载体上按照从C端到N端的顺序，依次偶联Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-X-OH和Boc-His(Trt)-OH，哌啶脱除Fmoc保护基，弱酸裂解，获得片段一。In some embodiments, the step 1 is specifically: sequentially coupling Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-X-OH and Boc-His(Trt)-OH, piperidine removes the Fmoc protecting group, and fragment 1 is obtained by weak acid cleavage.
具体地，片段一的肽序为Boc-His(Trt)-X-Glu(OtBu)-Gly-OH。其中，X为Ala或Aib。Specifically, the peptide sequence of Fragment 1 is Boc-His(Trt)-X-Glu(OtBu)-Gly-OH. Wherein, X is Ala or Aib.
一些实施方案中，所述步骤2具体为：按照SEQ ID NO:2所示氨基酸序列C端到N端的顺序，在固相载体上依次偶联Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-Ser(psiMe,Mepro)、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH和Fmoc-Thr(tBu)-OH，哌啶脱除Fmoc保护基，弱酸裂解，获得片段二。In some embodiments, the step 2 is specifically: according to the sequence from the C-terminus to the N-terminus of the amino acid sequence shown in SEQ ID NO: 2, sequentially coupling Fmoc-Gly-OH, Fmoc-Glu(OtBu)- OH, Fmoc-Leu-OH, Fmoc-Tyr(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-Ser(psiMe, Mepro), Fmoc-Asp(OtBu)-OH, Fmoc-Ser( tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH and Fmoc-Thr(tBu)-OH, piperidine removes the Fmoc protecting group, and weak acid cleavage to obtain Fragment 2.
本发明步骤1和步骤2中，所述弱酸裂解采用的弱酸为三氟乙醇、六氟异丙醇、醋酸和三氟乙酸中的一种或几种。In step 1 and step 2 of the present invention, the weak acid used in the weak acid cracking is one or more of trifluoroethanol, hexafluoroisopropanol, acetic acid and trifluoroacetic acid.
本发明步骤3中，所述偶联SEQ ID NO:3所示的氨基酸具体为按照SEQ ID NO:3所示的氨基酸序列C端到N端的顺序逐一偶联保护氨基酸；保护氨基酸即被保护基保护的氨基酸。具体地，所述保护氨基酸为 Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、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(Y)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH和Fmoc-Gln(Trt)-OH。In step 3 of the present invention, the coupling of the amino acids shown in SEQ ID NO:3 is specifically to couple the protected amino acids one by one in the order from the C-terminal to the N-terminal of the amino acid sequence shown in SEQ ID NO:3; the protected amino acid is the protected group protected amino acids. Specifically, the protected amino acid is Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, 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(Y)-OH, Fmoc-Ala-OH , Fmoc-Ala-OH and Fmoc-Gln(Trt)-OH.
其中，在保护氨基酸Fmoc-Lys(Y)-OH中，Y为Pal-Glu-OtBu或Oct(OtBu)-Glu-OtBu-AEEA-AEEA。Wherein, in the protected amino acid Fmoc-Lys(Y)-OH, Y is Pal-Glu-OtBu or Oct(OtBu)-Glu-OtBu-AEEA-AEEA.
本发明中，GLP-1类似物的肽序为(N端到C端)：H-His-X-Glu-Gly-Thr 5-Phe-Thr-Ser-Asp-Val 10-Ser-Ser-Tyr-Leu-Glu 15-Gly-Gln-Ala-Ala-Lys 20(Y)-Glu-Phe-Ile-Ala-Trp 25-Leu-Val-Arg-Gly-Arg 30-Gly-OH。 In the present invention, the peptide sequence of the GLP-1 analog is (N-terminal to C-terminal): H-His-X-Glu-Gly-Thr 5 -Phe-Thr-Ser-Asp-Val 10 -Ser-Ser-Tyr -Leu-Glu 15 -Gly-Gln-Ala-Ala-Lys 20 (Y)-Glu-Phe-Ile-Ala-Trp 25 -Leu-Val-Arg-Gly-Arg 30 -Gly-OH.
一些实施方案中，X为Ala，Y为Pal-Glu-OtBu，合成的GLP-1类似物为利拉鲁肽，其肽序为：In some embodiments, X is Ala, Y is Pal-Glu-OtBu, and the synthetic GLP-1 analogue is liraglutide, and its peptide sequence is:
H-His-Ala-Glu-Gly-Thr 5-Phe-Thr-Ser-Asp-Val 10-Ser-Ser-Tyr-Leu-Glu 15-Gly-Gln-Ala-Ala-Lys 20(γ-Glu-Pal)-Glu-Phe-Ile-Ala-Trp 25-Leu-Val-Arg-Gly-Arg 30-Gly-OH。 H-His-Ala-Glu-Gly-Thr 5 -Phe-Thr-Ser-Asp-Val 10 -Ser-Ser-Tyr-Leu-Glu 15 -Gly-Gln-Ala-Ala-Lys 20 (γ-Glu- Pal)-Glu-Phe-Ile-Ala- Trp25 -Leu-Val-Arg-Gly- Arg30 -Gly-OH.
一些实施方案中，X为Aib，Y为Oct(OtBu)-Glu-OtBu-AEEA-AEEA，合成的GLP-1类似物为索马鲁肽，其肽序为：In some embodiments, X is Aib, Y is Oct(OtBu)-Glu-OtBu-AEEA-AEEA, and the synthetic GLP-1 analog is semaglutide, and its peptide sequence is:
H-His-Aib-Glu-Gly-Thr 5-Phe-Thr-Ser-Asp-Val 10-Ser-Ser-Tyr-Leu-Glu 15-Gly-Gln-Ala-Ala-Lys 20(AEEA-AEEA-γ-Glu-OctadecanedioicAcid)-Glu-Phe-Ile-Ala-Trp 25-Leu-Val-Arg-Gly-Arg 30-Gly-OH。 H-His-Aib-Glu-Gly-Thr 5 -Phe-Thr-Ser-Asp-Val 10 -Ser-Ser-Tyr-Leu-Glu 15 -Gly-Gln-Ala-Ala-Lys 20 (AEEA-AEEA- γ-Glu-Octadecanedioic Acid)-Glu-Phe-Ile-Ala-Trp 25 -Leu-Val-Arg-Gly-Arg 30 -Gly-OH.
本发明对固体树脂的种类没有特殊限定，本领域常用的种类即可。在一些具体实施例中，步骤1～3中所采用的固体树脂为2-氯三苯甲基氯树脂或Wang树脂。In the present invention, the type of solid resin is not particularly limited, and the types commonly used in the field are sufficient. In some specific embodiments, the solid resin used in steps 1-3 is 2-chlorotrityl chloride resin or Wang resin.
在固体树脂上偶联氨基酸时，由于每个氨基酸都有保护基，需要脱除N端保护基再进行偶联，本发明脱除N端Fmoc保护基所采用的脱除剂优选哌啶溶液，包括但不仅限于此。When coupling amino acids on a solid resin, since each amino acid has a protecting group, it is necessary to remove the N-terminal protecting group before coupling. The removal agent used to remove the N-terminal Fmoc protecting group in the present invention is preferably piperidine solution. Including but not limited to this.
本发明步骤1～3中，所述偶联的偶联剂为DIPCDI/HOBt、PyBop/HOBt/DIPEA、HBTU/HOBt/DIPEA、DIPCDI/HOAt、HATU/HOAt/DIPEA和PyAop/HOAt/DIPEA中的一种或几种。In steps 1 to 3 of the present invention, the coupling agent for coupling is DIPCDI/HOBt, PyBop/HOBt/DIPEA, HBTU/HOBt/DIPEA, DIPCDI/HOAt, HATU/HOAt/DIPEA and PyAop/HOAt/DIPEA one or several.
本发明步骤4中，所述裂解的裂解液为含捕捉剂的三氟乙酸溶液，所 述捕捉剂为PhSMe、PhOH、H 2O、TIS、PhOMe、EDT中的一种或几种。一些具体实施例中，裂解液为TFA、H 2O和PhOH的组合，其中TFA、H 2O和PhOH的体积比为90:5:5。 In step 4 of the present invention, the lysate of the lysis is a trifluoroacetic acid solution containing a capture agent, and the capture agent is one or more of PhSMe, PhOH, H 2 O, TIS, PhOMe, and EDT. In some specific embodiments, the lysate is a combination of TFA, H 2 O and PhOH, wherein the volume ratio of TFA, H 2 O and PhOH is 90:5:5.
本发明首先合成GLP-1类似物N端第1～4位的片段一、第5～12位的片段二，然后在固相载体上按照从C端到N端的顺序，依次偶联SEQ ID NO:3所示的氨基酸、片段二和片段一，获得肽树脂，裂解，获得粗肽。本发明合成方法得到的粗肽纯度高，总收率高。实验表明，采用本发明特定的合成策略合成利拉鲁肽和索马鲁肽，粗肽纯度为80％～82％，产品纯度高达99.75％，总收率为38～40％。The present invention firstly synthesizes the fragment 1 of the 1st to 4th position of the N-terminal of the GLP-1 analogue, and the fragment 2 of the 5th to 12th position of the N-terminal, and then sequentially couples the SEQ ID NO on the solid phase carrier according to the order from the C-terminal to the N-terminal : amino acid shown in 3, fragment 2 and fragment 1, obtain peptide resin, crack, obtain crude peptide. The crude peptide obtained by the synthesis method of the invention has high purity and high total yield. Experiments show that by using the specific synthesis strategy of the present invention to synthesize liraglutide and semaglutide, the purity of the crude peptide is 80%-82%, the product purity is as high as 99.75%, and the total yield is 38-40%.
附图说明Description of drawings
图1示GLP-1类似物的制备工艺流程图；Fig. 1 shows the preparation process flowchart of GLP-1 analogue;
图2示利拉鲁肽片段一的典型色谱图；Figure 2 shows a typical chromatogram of liraglutide fragment one;
图3示利拉鲁肽粗肽的典型色谱图；Figure 3 shows a typical chromatogram of liraglutide crude peptide;
图4示索马鲁肽片段一的典型色谱图；Figure 4 shows a typical chromatogram of semaglutide fragment one;
图5示索马鲁肽粗肽的典型色谱图；Fig. 5 shows the typical chromatogram of semaglutide crude peptide;
图6示精肽的典型色谱图。Figure 6 shows a typical chromatogram of a refined peptide.
具体实施方式detailed description
本发明提供了一种合成GLP-1类似物的方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。The present invention provides a method for synthesizing GLP-1 analogues, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method and application herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention Invent technology.
本发明采用的试剂、仪器皆为普通市售品，皆可于市场购得。The reagents and instruments used in the present invention are all common commercial products and can be purchased in the market.
本发明中，英文缩写的含义如表1所示。In the present invention, the meanings of English abbreviations are shown in Table 1.
表1Table 1
缩写及英文Abbreviation and English 含义meaning
HOAtHOAt 1-羟基-7-偶氮苯并三氮唑1-Hydroxy-7-azobenzotriazole
DIPCDIDIPCDI 二异丙基碳二亚胺Diisopropylcarbodiimide
DCCDCC 二环己基碳二亚胺Dicyclohexylcarbodiimide
HOBtHOB 1-羟基苯并三唑1-Hydroxybenzotriazole
HATUHATU 2-(7-偶氮苯并三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯2-(7-Azobenzotriazole)-N,N,N’,N’-tetramethyluronium hexafluorophosphate
HBTUHBTU 苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸盐Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate
DIPEADIPEA N,N-二异丙基乙胺N,N-Diisopropylethylamine
PyBOPPyBOP 苯并三唑-1-基-氧基三吡咯烷基六氟磷酸盐Benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate
DMFDMF N,N-二甲基甲酰胺N,N-Dimethylformamide
THETHE 三氟乙醇Trifluoroethanol
DCMDCM 二氯甲烷Dichloromethane
TFATFA 三氟乙酸Trifluoroacetate
PhSMePhSMe 苯甲硫醚Thioanisole
PhOHPhOH 苯酚phenol
PhOMePhOMe 苯甲醚Anisole
EDTEDT 乙二硫醇Ethanedithiol
TISTIS 三异丙基硅烷Triisopropylsilane
PalPal 棕榈酸Palmitic acid
OctOctober 十八烷二酸octadecanedioic acid
AEEAAEEA 2-(2-(2-氨基乙氧基)乙氧基)乙酸2-(2-(2-Aminoethoxy)ethoxy)acetic acid
本发明提供一种合成GLP-1类似物的方法，包括以下步骤：The invention provides a method for synthesizing GLP-1 analogues, comprising the following steps:
步骤1：合成在SEQ ID NO:1所示氨基酸序列N端、His侧链上、Glu侧链上偶联有保护基的片段一；Step 1: Synthesizing a fragment 1 coupled with a protecting group at the N-terminal of the amino acid sequence shown in SEQ ID NO: 1, on the His side chain, and on the Glu side chain;
步骤2：合成在SEQ ID NO:2所示氨基酸序列N端、Thr侧链上、Ser侧链上、Asp侧链上、Tyr侧链上、Glu侧链上偶联有保护基的片段二；Step 2: Synthesizing Fragment 2 with protective groups coupled to the N-terminal of the amino acid sequence shown in SEQ ID NO: 2, on the Thr side chain, on the Ser side chain, on the Asp side chain, on the Tyr side chain, and on the Glu side chain;
步骤3：在固相载体上按照从C端到N端的顺序，依次偶联SEQ ID NO:3所示的氨基酸、片段二和片段一，获得肽树脂；Step 3: sequentially coupling the amino acid shown in SEQ ID NO:3, Fragment 2 and Fragment 1 on the solid phase carrier in the order from the C-terminal to the N-terminal to obtain a peptide resin;
步骤4：将所述肽树脂裂解，获得粗肽。Step 4: Cleavage the peptide resin to obtain crude peptide.
本发明中，GLP-1类似物的肽序为(N端到C端)：H-His-X-Glu-Gly-Thr 5-Phe-Thr-Ser-Asp-Val 10-Ser-Ser-Tyr-Leu-Glu 15-Gly-Gln-Ala-Ala-Lys 20(Y)-Glu-Phe-Ile-Ala-Trp 25-Leu-Val-Arg-Gly-Arg 30-Gly-OH (式I)。 In the present invention, the peptide sequence of the GLP-1 analog is (N-terminal to C-terminal): H-His-X-Glu-Gly-Thr 5 -Phe-Thr-Ser-Asp-Val 10 -Ser-Ser-Tyr -Leu-Glu 15 -Gly-Gln-Ala-Ala-Lys 20 (Y)-Glu-Phe-Ile-Ala-Trp 25 -Leu-Val-Arg-Gly-Arg 30 -Gly-OH (Formula I).
本发明提供的合成策略是：将上述GLP-1类似物的主链肽序分为三部分，片段一(SEQ ID NO:1)、片段二(SEQ ID NO:2)和其余氨基酸(SEQ ID NO:3)。其中，SEQ ID NO:1所示的氨基酸序列(N端→C端)即为式I所示肽序中编号1～4的氨基酸序列，具体序列为：His-X-Glu-Gly，其中，X为Ala或Aib。The synthesis strategy provided by the present invention is: the main chain peptide sequence of the above-mentioned GLP-1 analog is divided into three parts, fragment one (SEQ ID NO:1), fragment two (SEQ ID NO:2) and the remaining amino acids (SEQ ID NO: 3). Wherein, the amino acid sequence shown in SEQ ID NO: 1 (N terminal → C terminal) is the amino acid sequence numbered 1 to 4 in the peptide sequence shown in formula I, and the specific sequence is: His-X-Glu-Gly, wherein, X is Ala or Aib.
SEQ ID NO:2所示的氨基酸序列为(N端→C端)即为式I所示肽序中编号第5～16位的序列，具体序列为：Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly。The amino acid sequence shown in SEQ ID NO: 2 is (N-terminal → C-terminal), which is the sequence numbered 5-16 in the peptide sequence shown in formula I, and the specific sequence is: Thr-Phe-Thr-Ser-Asp- Val-Ser-Ser-Tyr-Leu-Glu-Gly.
SEQ ID NO:3所示的氨基酸序列(N端→C端)即为式I所示肽序中编号第17～31位的序列，具体序列为：Gln-Ala-Ala-Lys(Y)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly。The amino acid sequence (N-terminal→C-terminal) shown in SEQ ID NO:3 is the sequence numbered 17th to 31st in the peptide sequence shown in formula I, and the specific sequence is: Gln-Ala-Ala-Lys(Y)- Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly.
其中，Y为Pal-Glu-OtBu或Oct(OtBu)-Glu-OtBu-AEEA-AEEA。Wherein, Y is Pal-Glu-OtBu or Oct(OtBu)-Glu-OtBu-AEEA-AEEA.
进一步的，本发明GLP-1类似物的合成路线如图1所示，具体包括如下步骤：Further, the synthesis route of the GLP-1 analogs of the present invention is shown in Figure 1, which specifically includes the following steps:
1、片段一的合成：片段一的肽序为Boc-His(Trt)-X-Glu(OtBu)-Gly-OH，其中X是Ala或Aib。以2-氯三苯甲基氯树脂为载体，根据多肽固相合成法，从C端至N端依次偶联Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-X-OH和Boc-His(Trt)-OH。1. Synthesis of Fragment 1: The peptide sequence of Fragment 1 is Boc-His(Trt)-X-Glu(OtBu)-Gly-OH, wherein X is Ala or Aib. Using 2-chlorotrityl chloride resin as a carrier, according to the polypeptide solid-phase synthesis method, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-X-OH and Boc-His(Trt)-OH.
2、片段二的合成：片段二的肽序为Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(psiMe,Mepro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH。以2-氯三苯甲基氯树脂为载体，根据多肽固相合成法，从C端至N端依次偶联Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-Ser(psiMe,Mepro)、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH和Fmoc-Thr(tBu)-OH。2. Synthesis of Fragment 2: The peptide sequence of Fragment 2 is Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(psiMe,Mepro)-Ser(tBu) -Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH. Using 2-chlorotrityl chloride resin as a carrier, according to the polypeptide solid-phase synthesis method, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Tyr(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-Ser(psiMe, Mepro), Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Phe-OH and Fmoc-Thr(tBu)-OH.
3、肽树脂的合成：以2-氯三苯甲基氯树脂或Wang树脂为载体，根据多肽固相合成法，按照肽序，从C端至N端依次偶联Fmoc-Gly-OH、 Fmoc-Arg(Pbf)-OH、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(Y)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、片段二和片段一，其中Y是Pal-Glu-OtBu或Oct(OtBu)-Glu-OtBu-AEEA-AEEA。3. Synthesis of peptide resin: use 2-chlorotrityl chloride resin or Wang resin as the carrier, according to the peptide solid-phase synthesis method, according to the peptide sequence, sequentially couple Fmoc-Gly-OH, Fmoc from C-terminal to N-terminal -Arg(Pbf)-OH, 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(Y)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH , fragment two and fragment one, wherein Y is Pal-Glu-OtBu or Oct(OtBu)-Glu-OtBu-AEEA-AEEA.
下面结合实施例，进一步阐述本发明：Below in conjunction with embodiment, further set forth the present invention:
实施例1：利拉鲁肽片段一的合成Example 1: Synthesis of Liraglutide Fragment One
称取2-氯三苯甲基氯树脂16.7g(替代度为1.2mmol/g，规模20mmo l)，DMF溶胀树脂30分钟，抽干树脂，DMF洗涤2次。另称取Fmoc-Gly-OH 11.89g(40mmol)、DIPEA 10.34g(80mmol)和50mlDMF加入至树脂中，室温反应3小时，抽干树脂，用DMF洗涤3次。加入20％哌啶/DMF溶液用于脱除Fmoc保护基，室温搅拌反应10分钟，抽干树脂，重复操作一次，然后用DMF洗涤6次。另称取Fmoc-Glu(OtBu)-OH 17.02g(40mmol)、HOBT 6.48g(48mmol)、DIPCDI 6.05g(48mmol)和50mlDMF，加入至树脂中，室温反应2小时，以kaiser法检测判断反应是否完全，如树脂无色透明，则表示反应完全；如树脂显色，则表示反应不完全，需要重复上述操作进行二投。此判断标准适用于后续内容中以kaiser法检测判断反应终点。重复上述脱除Fmoc保护基和相应氨基酸偶联的步骤，依次偶联Fmoc-Ala-OH和Boc-His(Trt)-OH，最后DMF洗涤3次，二氯甲烷洗涤3次。Weigh 16.7 g of 2-chlorotrityl chloride resin (the degree of substitution is 1.2 mmol/g, the scale is 20 mmol), swell the resin with DMF for 30 minutes, drain the resin, and wash twice with DMF. Weigh 11.89g (40mmol) of Fmoc-Gly-OH, 10.34g (80mmol) of DIPEA and 50ml of DMF into the resin, react at room temperature for 3 hours, drain the resin, and wash 3 times with DMF. Add 20% piperidine/DMF solution to remove the Fmoc protecting group, stir the reaction at room temperature for 10 minutes, drain the resin, repeat the operation once, and then wash with DMF 6 times. In addition, weigh 17.02g (40mmol) of Fmoc-Glu(OtBu)-OH, 6.48g (48mmol) of HOBT, 6.05g (48mmol) of DIPCDI and 50ml of DMF, add them to the resin, react at room temperature for 2 hours, and use the Kaiser method to detect whether the reaction is Complete, if the resin is colorless and transparent, it means that the reaction is complete; if the resin is colored, it means that the reaction is not complete, and the above operation needs to be repeated for two injections. This judgment standard is applicable to the detection and judgment of the end point of the reaction by the Kaiser method in the subsequent content. Repeat the steps of removing the Fmoc protecting group and coupling the corresponding amino acid, sequentially coupling Fmoc-Ala-OH and Boc-His(Trt)-OH, and finally washing with DMF for 3 times and dichloromethane for 3 times.
加入裂解液400ml(TFE:DCM＝1:4)，室温搅拌反应2小时，过滤，滤液减压浓缩剩余1/5，倒入甲基叔丁基醚中沉淀，离心，甲基叔丁基醚洗涤3次，真空干燥得到片段一13.0g，收率80％，纯度92％(色谱图如图2所示)。Add 400ml of lysate (TFE:DCM=1:4), stir at room temperature for 2 hours, filter, concentrate the filtrate under reduced pressure and the remaining 1/5, pour into methyl tert-butyl ether for precipitation, centrifuge, methyl tert-butyl ether Washed 3 times and vacuum dried to obtain 13.0 g of Fragment 1 with a yield of 80% and a purity of 92% (the chromatogram is shown in Figure 2).
实施例2：片段二的合成Embodiment 2: the synthesis of fragment two
称取2-氯三苯甲基氯树脂16.7g(替代度为1.2mmol/g，规模20mmol)，DMF溶胀树脂30分钟，抽干树脂，DMF洗涤2次。另称取Fmoc-Gly-OH  11.89g(40mmol)、DIPEA 10.34g(80mmol)和50mlDMF加入至树脂中，室温反应3小时，抽干树脂，用DMF洗涤3次。加入20％哌啶/DMF溶液用于脱除Fmoc保护基，室温搅拌反应10分钟，抽干树脂，重复操作一次，然后用DMF洗涤6次。另称取Fmoc-Glu(OtBu)-OH 17.02g(40mmol)、HOBT 6.48g(48mmol)、DIPCDI 6.05g(48mmol)和50mlDMF，加入至树脂中，室温反应2小时，以kaiser法检测判断反应是否完全，如树脂无色透明，则表示反应完全；如树脂显色，则表示反应不完全，需要重复上述操作进行二投。此判断标准适用于后续内容中以kaiser法检测判断反应终点。重复上述脱除Fmoc保护基和相应氨基酸偶联的步骤，依次偶联Fmoc-Leu-OH、Fmoc-Tyr(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-Ser(psiMe,Mepro)、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH和Fmoc-Thr(tBu)-OH，最后DMF洗涤3次，二氯甲烷洗涤3次。Weigh 16.7 g of 2-chlorotrityl chloride resin (degree of substitution: 1.2 mmol/g, scale: 20 mmol), swell the resin with DMF for 30 minutes, drain the resin, and wash twice with DMF. Weigh 11.89g (40mmol) of Fmoc-Gly-OH, 10.34g (80mmol) of DIPEA and 50ml of DMF into the resin, react at room temperature for 3 hours, drain the resin, and wash 3 times with DMF. Add 20% piperidine/DMF solution to remove the Fmoc protecting group, stir the reaction at room temperature for 10 minutes, drain the resin, repeat the operation once, and then wash with DMF 6 times. In addition, weigh 17.02g (40mmol) of Fmoc-Glu(OtBu)-OH, 6.48g (48mmol) of HOBT, 6.05g (48mmol) of DIPCDI and 50ml of DMF, add them to the resin, react at room temperature for 2 hours, and use the Kaiser method to detect whether the reaction is Complete, if the resin is colorless and transparent, it means that the reaction is complete; if the resin is colored, it means that the reaction is not complete, and the above operation needs to be repeated for two injections. This judgment standard is applicable to the detection and judgment of the end point of the reaction by the Kaiser method in the subsequent content. Repeat the steps of removing the Fmoc protecting group and coupling the corresponding amino acid, and sequentially couple Fmoc-Leu-OH, Fmoc-Tyr(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-Ser(psiMe, Mepro), Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH and Fmoc-Thr(tBu)-OH, finally washed 3 times with DMF , washed three times with dichloromethane.
加入裂解液400ml(TFE:DCM＝1:4)，室温搅拌反应2小时，过滤，滤液减压浓缩剩余1/5，倒入甲基叔丁基醚中沉淀，离心，甲基叔丁基醚洗涤3次，真空干燥得到片段一27.1g，收率77％，纯度95％。Add 400ml of lysate (TFE:DCM=1:4), stir at room temperature for 2 hours, filter, concentrate the filtrate under reduced pressure and the remaining 1/5, pour into methyl tert-butyl ether for precipitation, centrifuge, methyl tert-butyl ether After washing 3 times and vacuum drying, 27.1 g of Fragment 1 was obtained, with a yield of 77% and a purity of 95%.
实施例3：利拉鲁肽肽树脂的合成Example 3: Synthesis of Liraglutide Peptide Resin
称取Wang树脂33.33g(替代度为0.6mmol/g，规模20mmol)，DMF溶胀树脂30分钟，抽干树脂，DMF洗涤2次。另称取Fmoc-Gly-OH 17.84g(60mmol)、HOBt 9.72g(72mmol)、DIPCDI 9.07g(72mmol)、DM AP 2.20g(18mmol)和100mlDMF加入至树脂中，室温反应4小时，抽干树脂，用DMF洗涤6次，甲醇洗涤3次，真空干燥得到Fmoc-Gly-Wang Resin 40.4g，替代度为0.47mmol/g。加入20％哌啶/DMF溶液用于脱除Fmoc保护基，室温搅拌反应10分钟，抽干树脂，重复操作一次，然后用DMF洗涤6次。另称取Fmoc-Arg(Pbf)-OH 37.0g(57mmol)、HOBT 9.23g(68.4mmol)、DIPCDI 8.62g(68.4mmol)和100mlDMF，加入至树脂中，室温反应2小时，以kaiser法检测判断反应是否完全，如树脂无色透明，则表示反应完全；如树脂显色，则表示反应不完全，需要重复 上述操作进行二投。此判断标准适用于后续内容中以kaiser法检测判断反应终点。重复上述脱除Fmoc保护基和相应氨基酸偶联的步骤，依次偶联Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、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(Pal-Glu-OtBu)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、实施例2中的片段二和实施例1中的片段一，最后DMF洗涤3次，二氯甲烷洗涤3次，甲醇洗涤3次，真空干燥得到利拉鲁肽肽树脂。Weigh 33.33g of Wang resin (the degree of substitution is 0.6mmol/g, the scale is 20mmol), swell the resin with DMF for 30 minutes, drain the resin, and wash twice with DMF. Separately weigh 17.84g (60mmol) of Fmoc-Gly-OH, 9.72g (72mmol) of HOBt, 9.07g (72mmol) of DIPCDI, 2.20g (18mmol) of DMAP and 100ml of DMF and add them to the resin, react at room temperature for 4 hours, and drain the resin , washed 6 times with DMF, washed 3 times with methanol, and vacuum-dried to obtain 40.4g of Fmoc-Gly-Wang Resin, with a substitution degree of 0.47mmol/g. Add 20% piperidine/DMF solution to remove the Fmoc protecting group, stir the reaction at room temperature for 10 minutes, drain the resin, repeat the operation once, and then wash with DMF 6 times. Separately weigh 37.0g (57mmol) of Fmoc-Arg(Pbf)-OH, 9.23g (68.4mmol) of HOBT, 8.62g (68.4mmol) of DIPCDI and 100ml of DMF, add them to the resin, react at room temperature for 2 hours, and use the Kaiser method to detect and judge Whether the reaction is complete, if the resin is colorless and transparent, it means that the reaction is complete; if the resin is colored, it means that the reaction is not complete, and the above operation needs to be repeated for two injections. This judgment standard is applicable to the detection and judgment of the end point of the reaction by the Kaiser method in the subsequent content. Repeat the steps of removing the Fmoc protecting group and coupling the corresponding amino acid, and sequentially couple Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, 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( Pal-Glu-OtBu)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, fragment two in Example 2 and fragment one in Example 1, and finally washed with DMF for 3 times, washed 3 times with dichloromethane and 3 times with methanol, and dried in vacuum to obtain liraglutide peptide resin.
实施例4：利拉鲁肽的合成Example 4: Synthesis of Liraglutide
取实施例3中的肽树脂以10ml/g的比例加入裂解液(TFA:H 2O:PhOH＝90:5:5)，室温搅拌反应2小时，过滤，滤液倒入甲基叔丁基醚中沉淀，离心，甲基叔丁基醚洗涤3次，真空干燥得到利拉鲁肽粗肽72g，纯度80％(色谱图如图3所示)，总收率38％。 Take the peptide resin in Example 3 and add it to the lysate (TFA:H 2 O:PhOH=90:5:5) at a ratio of 10ml/g, stir and react at room temperature for 2 hours, filter, and pour the filtrate into methyl tert-butyl ether Precipitate in medium, centrifuge, wash 3 times with methyl tert-butyl ether, and dry in vacuo to obtain 72 g of liraglutide crude peptide with a purity of 80% (as shown in the chromatogram in Figure 3) and a total yield of 38%.
实施例5：索马鲁肽片段一的合成Example 5: Synthesis of Semaglutide Fragment One
称取2-氯三苯甲基氯树脂16.7g(替代度为1.2mmol/g，规模20mmol)，DMF溶胀树脂30分钟，抽干树脂，DMF洗涤2次。另称取Fmoc-Gly-OH 11.89g(40mmol)、DIPEA 10.34g(80mmol)和50mlDMF加入至树脂中，室温反应3小时，抽干树脂，用DMF洗涤3次。加入20％哌啶/DMF溶液用于脱除Fmoc保护基，室温搅拌反应10分钟，抽干树脂，重复操作一次，然后用DMF洗涤6次。另称取Fmoc-Glu(OtBu)-OH 17.02g(40mmol)、HOBT 6.48g(48mmol)、DIPCDI 6.05g(48mmol)和50mlDMF，加入至树脂中，室温反应2小时，以kaiser法检测判断反应是否完全，如树脂无色透明，则表示反应完全；如树脂显色，则表示反应不完全，需要重复上述操作进行二投。此判断标准适用于后续内容中以kaiser法检测判断反应终点。重复上述脱除Fmoc保护基和相应氨基酸偶联的步骤，依次偶联Fmoc-Aib-OH和Boc-His(Trt)-OH，最后DMF洗涤3次，二氯甲烷洗涤3次。Weigh 16.7 g of 2-chlorotrityl chloride resin (degree of substitution: 1.2 mmol/g, scale: 20 mmol), swell the resin with DMF for 30 minutes, drain the resin, and wash twice with DMF. Weigh 11.89g (40mmol) of Fmoc-Gly-OH, 10.34g (80mmol) of DIPEA and 50ml of DMF into the resin, react at room temperature for 3 hours, drain the resin, and wash 3 times with DMF. Add 20% piperidine/DMF solution to remove the Fmoc protecting group, stir the reaction at room temperature for 10 minutes, drain the resin, repeat the operation once, and then wash with DMF 6 times. In addition, weigh 17.02g (40mmol) of Fmoc-Glu(OtBu)-OH, 6.48g (48mmol) of HOBT, 6.05g (48mmol) of DIPCDI and 50ml of DMF, add them to the resin, react at room temperature for 2 hours, and use the Kaiser method to detect whether the reaction is Complete, if the resin is colorless and transparent, it means that the reaction is complete; if the resin is colored, it means that the reaction is not complete, and the above operation needs to be repeated for two injections. This judgment standard is applicable to the detection and judgment of the end point of the reaction by the Kaiser method in the subsequent content. Repeat the above steps of removing the Fmoc protecting group and coupling the corresponding amino acid, sequentially coupling Fmoc-Aib-OH and Boc-His(Trt)-OH, and finally washing with DMF for 3 times and dichloromethane for 3 times.
加入裂解液400ml(TFA:DCM＝1:100)，室温搅拌反应1小时，过滤，滤液中加入20mlDIPEA，减压浓缩至干，加入少量甲醇溶解，倒入水中沉淀，过滤，水洗涤3次，鼓风干燥得到片段一13.7g，收率83％，纯度94％(色谱图如图4所示)。Add 400ml of lysate (TFA:DCM=1:100), stir at room temperature for 1 hour, filter, add 20ml of DIPEA to the filtrate, concentrate to dryness under reduced pressure, add a small amount of methanol to dissolve, pour into water to precipitate, filter, wash with water 3 times, Blast drying gave Fragment 1 13.7g with a yield of 83% and a purity of 94% (the chromatogram is shown in Figure 4).
实施例6：索马鲁肽肽树脂的合成Embodiment 6: the synthesis of semaglutide peptide resin
称取Wang树脂40g(替代度为0.5mmol/g，规模20mmol)，DMF溶胀树脂30分钟，抽干树脂，DMF洗涤2次。另称取Fmoc-Gly-OH 17.84g(60mmol)、HOBt 9.72g(72mmol)、DIPCDI 9.07g(72mmol)、DMAP 2.20g(18mmol)和150mlDMF加入至树脂中，室温反应4小时，抽干树脂，用DMF洗涤6次，甲醇洗涤3次，真空干燥得到Fmoc-Gly-Wang Resin 44.2g，替代度为0.43mmol/g。加入20％哌啶/DMF溶液用于脱除Fmoc保护基，室温搅拌反应10分钟，抽干树脂，重复操作一次，然后用DMF洗涤6次。另称取Fmoc-Arg(Pbf)-OH 37.0g(57mmol)、HOBT 9.23g(68.4mmol)、DIPCDI 8.62g(68.4mmol)和150mlDMF，加入至树脂中，室温反应2小时，以kaiser法检测判断反应是否完全，如树脂无色透明，则表示反应完全；如树脂显色，则表示反应不完全，需要重复上述操作进行二投。此判断标准适用于后续内容中以kaiser法检测判断反应终点。重复上述脱除Fmoc保护基和相应氨基酸偶联的步骤，依次偶联Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、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[Oct(OtBu)-Glu-OtBu-AEEA-AEEA]-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、实施例2中的片段二和实施例5中的片段一，最后DMF洗涤3次，二氯甲烷洗涤3次，甲醇洗涤3次，真空干燥得到索马鲁肽肽树脂。Weigh 40 g of Wang resin (degree of substitution: 0.5 mmol/g, scale: 20 mmol), swell the resin with DMF for 30 minutes, drain the resin, and wash twice with DMF. In addition, Fmoc-Gly-OH 17.84g (60mmol), HOBt 9.72g (72mmol), DIPCDI 9.07g (72mmol), DMAP 2.20g (18mmol) and 150ml DMF were weighed and added to the resin, reacted at room temperature for 4 hours, and dried the resin. Washed 6 times with DMF, washed 3 times with methanol, and dried in vacuum to obtain 44.2 g of Fmoc-Gly-Wang Resin with a substitution degree of 0.43 mmol/g. Add 20% piperidine/DMF solution to remove the Fmoc protecting group, stir the reaction at room temperature for 10 minutes, drain the resin, repeat the operation once, and then wash with DMF 6 times. Separately weigh 37.0g (57mmol) of Fmoc-Arg(Pbf)-OH, 9.23g (68.4mmol) of HOBT, 8.62g (68.4mmol) of DIPCDI and 150ml of DMF, add them to the resin, react at room temperature for 2 hours, and use the Kaiser method to detect and judge Whether the reaction is complete, if the resin is colorless and transparent, it means that the reaction is complete; if the resin is colored, it means that the reaction is not complete, and the above operation needs to be repeated for two injections. This judgment standard is applicable to the detection and judgment of the end point of the reaction by the Kaiser method in the subsequent content. Repeat the steps of removing the Fmoc protecting group and coupling the corresponding amino acid, and sequentially couple Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, 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[ Oct(OtBu)-Glu-OtBu-AEEA-AEEA]-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, fragment two in embodiment 2 and in embodiment 5 Fragment 1, finally washed 3 times with DMF, 3 times with dichloromethane, 3 times with methanol, and vacuum-dried to obtain semaglutide peptide resin.
实施例7：索马鲁肽的合成Embodiment 7: the synthesis of semaglutide
取实施例6中的肽树脂以10ml/g的比例加入裂解液(TFA:H 2O:PhOH＝90:5:5)，室温搅拌反应2小时，过滤，滤液倒入甲基 叔丁基醚中沉淀，离心，甲基叔丁基醚洗涤3次，真空干燥得到利拉鲁肽粗肽80g，纯度82％(色谱图如图5所示)，总收率40％。 Take the peptide resin in Example 6 and add it to the lysate (TFA:H 2 O:PhOH=90:5:5) at a ratio of 10ml/g, stir and react at room temperature for 2 hours, filter, and pour the filtrate into methyl tert-butyl ether Precipitate in medium, centrifuge, wash 3 times with methyl tert-butyl ether, and vacuum dry to obtain 80 g of liraglutide crude peptide, with a purity of 82% (chromatogram shown in Figure 5), and a total yield of 40%.
实施例8：利拉鲁肽或索马鲁肽精肽的制备Example 8: Preparation of liraglutide or semaglutide fine peptide
取实施例4或7中得到的粗肽，采用高效液相色谱仪制备纯化，以四烷基硅烷键合硅胶为固定相，以0.2％醋酸溶液为流动相A相，乙腈为流动相B相，监测波长280nm，梯度洗脱收集目的峰馏分，浓缩、冻干得到利拉鲁肽或索马鲁肽精肽，纯度99.7％，(色谱图如图6所示)。The crude peptide obtained in Example 4 or 7 was prepared and purified by high performance liquid chromatography, with tetraalkylsilane bonded silica gel as the stationary phase, 0.2% acetic acid solution as the mobile phase A, and acetonitrile as the mobile phase B , monitoring wavelength 280nm, gradient elution to collect target peak fractions, concentrated, freeze-dried to obtain liraglutide or semaglutide fine peptide, purity 99.7%, (chromatogram shown in Figure 6).
对比例1：Comparative example 1:
参考专利CN102286092实施例2和3中的操作，首先选取Wang树脂，依次偶联Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、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(Alloc)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Ser(tBu)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ala-OH和Boc-His(Trt)-OH。四三苯基膦钯/苯硅烷脱去赖氨酸侧链Alloc保护基，再偶联Pal-Glu-OtBu，最后裂解液(TFA:PhSMe:PhOMe:EDT＝90:5:3:2)裂解得到粗肽纯度58％，总收率约15％。With reference to the operations in Examples 2 and 3 of the patent CN102286092, first select Wang resin, and sequentially couple Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, 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(Alloc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc -Tyr(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH and Boc-His(Trt )-OH. Tetraphenylphosphine palladium/phenylsilane removes the Alloc protecting group of the lysine side chain, then couples Pal-Glu-OtBu, and finally lyses with the lysate (TFA:PhSMe:PhOMe:EDT=90:5:3:2) The purity of the obtained crude peptide was 58%, and the total yield was about 15%.
对比例2：Comparative example 2:
参考专利CN106749613实施例2和3中的操作：Referring to the operation in the patent CN106749613 embodiment 2 and 3:
(1)首先选取2-氯三苯甲基氯树脂，依次偶联Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Ser(tBu)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu) -OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Aib-OH和Boc-His(Trt)-OH。20％TFE/DCM溶液裂解得到[1-16]片段。(1) First select 2-chlorotrityl chloride resin, and sequentially couple Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(Boc)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc - Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH and Boc-His(Trt)-OH. The [1-16] fragment was obtained by cleavage with 20% TFE/DCM solution.
(2)首先选取2-氯三苯甲基氯树脂，依次偶联Fmoc-Phe-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Lys[Oct(OtBu)-Glu-OtBu-AEEA-AEEA]-OH、Fmoc-Ala-OH、Fmoc-Ala-OH和Fmoc-Gln(Trt)-OH。20％TFE/DCM溶液裂解得到[17-22]片段。(2) First select 2-chlorotrityl chloride resin, and sequentially couple Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys[Oct(OtBu)-Glu-OtBu-AEEA-AEEA] -OH, Fmoc-Ala-OH, Fmoc-Ala-OH and Fmoc-Gln(Trt)-OH. The [17-22] fragment was obtained by cleavage with 20% TFE/DCM solution.
(3)液相法合成[23-31]片段，DCC/HOBt为偶联剂，哌啶液相法脱除Fmoc保护基。(3) The [23-31] fragment was synthesized by a liquid phase method, DCC/HOBt was used as a coupling agent, and the Fmoc protecting group was removed by a piperidine liquid phase method.
(4)液相法将[17-22]片段、[1-16]片段依次与[23-31]片段偶联得到全保护肽，DCC/HOBt为偶联剂，最后三氟乙酸裂解得到索马鲁肽粗肽，纯度约50％，总收率32％。(4) Liquid-phase method, the [17-22] fragment, [1-16] fragment were sequentially coupled with the [23-31] fragment to obtain a fully protected peptide, DCC/HOBt was used as a coupling agent, and finally trifluoroacetic acid was cleaved to obtain the peptide Marutide crude peptide, the purity is about 50%, and the total yield is 32%.
比较本发明和对比例1～2的结果，具体见表2。Compare the results of the present invention and Comparative Examples 1-2, see Table 2 for details.
表2Table 2
组别group 粗肽纯度Crude peptide purity 操作方式Operation method 总收率total yield
实施例4利拉鲁肽Embodiment 4 Liraglutide 82％82% 简单Simple 38％38%
实施例7索马鲁肽Embodiment 7 semaglutide 82％82% 简单Simple 40％40%
对比例1Comparative example 1 58％58% 简单Simple 约15％about 15%
对比例2Comparative example 2 约50％about 50% 繁琐cumbersome 32％32%
由表1结果可知，本发明以特定的片段一、片段二和逐一合成法合成利拉鲁肽和索马鲁肽，粗肽纯度和产品总收率均明显高于对比例1～2，且工艺更加简单。It can be seen from the results in Table 1 that the present invention synthesizes liraglutide and semaglutide with specific fragment one, fragment two and one-by-one synthesis methods, and the purity of the crude peptide and the total yield of the product are significantly higher than those of comparative examples 1-2, and The process is simpler.
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above are only preferred embodiments of the present invention, and it should be pointed out that for those of ordinary skill in the art, some improvements and modifications can also be made without departing from the principle of the present invention, and these improvements and modifications should also be considered Be the protection scope of the present invention.

Claims (10)

  1. 一种合成GLP-1类似物的方法，其特征在于，包括以下步骤：A method for synthesizing GLP-1 analogues, comprising the following steps:
    步骤1：合成在SEQ ID NO:1所示氨基酸序列N端、His侧链上、Glu侧链上偶联有保护基的片段一；Step 1: Synthesizing a fragment 1 coupled with a protecting group at the N-terminal of the amino acid sequence shown in SEQ ID NO: 1, on the His side chain, and on the Glu side chain;
    步骤2：合成在SEQ ID NO:2所示氨基酸序列N端、Thr侧链上、Ser侧链上、Asp侧链上、Tyr侧链上、Glu侧链上偶联有保护基的片段二；Step 2: Synthesizing Fragment 2 with protective groups coupled to the N-terminal of the amino acid sequence shown in SEQ ID NO: 2, on the Thr side chain, on the Ser side chain, on the Asp side chain, on the Tyr side chain, and on the Glu side chain;
    步骤3：在固相载体上按照从C端到N端的顺序，依次偶联SEQ ID NO:3所示的氨基酸、片段二和片段一，获得肽树脂；Step 3: sequentially coupling the amino acid shown in SEQ ID NO:3, Fragment 2 and Fragment 1 on the solid phase carrier in the order from the C-terminal to the N-terminal to obtain a peptide resin;
    步骤4：将所述肽树脂裂解，获得粗肽。Step 4: Cleavage the peptide resin to obtain crude peptide.
  2. 根据权利要求1所述的方法，其特征在于，所述步骤1具体为：按照SEQ ID NO:1所示氨基酸序列C端到N端的顺序，在固相载体上依次偶联Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-X-OH和Boc-His(Trt)-OH，哌啶脱除Fmoc保护基，弱酸裂解，获得片段一；The method according to claim 1, characterized in that, said step 1 is specifically: sequentially coupling Fmoc-Gly-OH on a solid-phase carrier according to the sequence from the C-terminus to the N-terminus of the amino acid sequence shown in SEQ ID NO:1 , Fmoc-Glu(OtBu)-OH, Fmoc-X-OH and Boc-His(Trt)-OH, piperidine removed Fmoc protecting group, weak acid cleavage, obtained fragment one;
    其中，X为Ala或Aib。Wherein, X is Ala or Aib.
  3. 根据权利要求1所述的方法，其特征在于，所述步骤2具体为：按照SEQ ID NO:2所示氨基酸序列C端到N端的顺序，在固相载体上依次偶联Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-Ser(psiMe,Mepro)、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH和Fmoc-Thr(tBu)-OH，哌啶脱除Fmoc保护基，弱酸裂解，获得片段二。The method according to claim 1, characterized in that, said step 2 is specifically: sequentially coupling Fmoc-Gly-OH on a solid-phase carrier according to the sequence from the C-terminus to the N-terminus of the amino acid sequence shown in SEQ ID NO:2 , Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-Ser(psiMe, Mepro), Fmoc-Asp(OtBu )-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH and Fmoc-Thr(tBu)-OH, piperidine removes the Fmoc protecting group, weak acid cleavage, and obtains fragments two.
  4. 根据权利要求2或3所述的方法，其特征在于，所述弱酸为三氟乙醇、六氟异丙醇、醋酸和三氟乙酸中的一种或几种。The method according to claim 2 or 3, wherein the weak acid is one or more of trifluoroethanol, hexafluoroisopropanol, acetic acid and trifluoroacetic acid.
  5. 根据权利要求1所述的方法，其特征在于，步骤3中，所述偶联SEQ ID NO:3所示的氨基酸具体为按照SEQ ID NO:3所示的氨基酸序列C端到N端的顺序逐一偶联保护氨基酸；所述保护氨基酸为Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、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(Y)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH和Fmoc-Gln(Trt)-OH；The method according to claim 1, characterized in that, in step 3, the coupling of the amino acids shown in SEQ ID NO:3 is specifically in accordance with the sequence from the C-terminal to the N-terminal of the amino acid sequence shown in SEQ ID NO:3. Coupling protected amino acids; the protected amino acids are Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, 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(Y)-OH, Fmoc-Ala -OH, Fmoc-Ala-OH and Fmoc-Gln(Trt)-OH;
    其中，Y为Pal-Glu-OtBu或Oct(OtBu)-Glu-OtBu-AEEA-AEEA。Wherein, Y is Pal-Glu-OtBu or Oct(OtBu)-Glu-OtBu-AEEA-AEEA.
  6. 根据权利要求1～5任一项所述的方法，其特征在于，所述固相载体为2-氯三苯甲基氯树脂或Wang树脂。The method according to any one of claims 1-5, characterized in that the solid phase carrier is 2-chlorotrityl chloride resin or Wang resin.
  7. 根据权利要求5所述的方法，其特征在于，所述Fmoc保护基采用哌啶脱除。The method according to claim 5, wherein the Fmoc protecting group is removed by piperidine.
  8. 根据权利要求1所述的方法，其特征在于，步骤1～3中，所述偶联的偶联剂为DIPCDI/HOBt、PyBop/HOBt/DIPEA、HBTU/HOBt/DIPEA、DIPCDI/HOAt、HATU/HOAt/DIPEA和PyAop/HOAt/DIPEA中的一种或几种。The method according to claim 1, characterized in that, in steps 1 to 3, the coupling agent for coupling is DIPCDI/HOBt, PyBop/HOBt/DIPEA, HBTU/HOBt/DIPEA, DIPCDI/HOAt, HATU/ One or more of HOAt/DIPEA and PyAop/HOAt/DIPEA.
  9. 根据权利要求1所述的方法，其特征在于，步骤4中，所述裂解的裂解液为含捕捉剂的三氟乙酸溶液，所述捕捉剂为PhSMe、PhOH、H 2O、TIS、PhOMe、EDT中的一种或几种。 The method according to claim 1, wherein in step 4, the lysate of the cracking is a trifluoroacetic acid solution containing a capture agent, and the capture agent is PhSMe, PhOH, H 2 O, TIS, PhOMe, One or more of EDT.
  10. 根据权利要求1～9任一项所述的方法，其特征在于，在所述步骤4获得粗肽之后还包括将所述粗肽进行HPLC纯化、浓缩、冻干获得精肽的步骤，所述HPLC纯化的色谱条件为：以四烷基硅烷键合硅胶为固定相，以0.2％醋酸溶液为流动相A相，乙腈为流动相B相，监测波长280nm，梯度洗脱。The method according to any one of claims 1 to 9, characterized in that, after obtaining the crude peptide in step 4, it also includes the steps of performing HPLC purification, concentration, and freeze-drying of the crude peptide to obtain a refined peptide, the The chromatographic conditions for HPLC purification are as follows: tetraalkylsilane bonded silica gel is used as the stationary phase, 0.2% acetic acid solution is used as the mobile phase A, acetonitrile is used as the mobile phase B, the monitoring wavelength is 280 nm, and gradient elution is performed.
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