CN106632655B - Preparation method of exenatide and product thereof - Google Patents

Preparation method of exenatide and product thereof Download PDF

Info

Publication number
CN106632655B
CN106632655B CN201611247180.9A CN201611247180A CN106632655B CN 106632655 B CN106632655 B CN 106632655B CN 201611247180 A CN201611247180 A CN 201611247180A CN 106632655 B CN106632655 B CN 106632655B
Authority
CN
China
Prior art keywords
fmoc
gly
tbu
ser
leu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201611247180.9A
Other languages
Chinese (zh)
Other versions
CN106632655A (en
Inventor
王慧
王惠嘉
郭添
张忠旗
李乾
高长波
苏晨灿
韩广
王万科
赵金礼
杨小琳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shaanxi HuiKang Bio Tech Co Ltd
Original Assignee
Shaanxi HuiKang Bio Tech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shaanxi HuiKang Bio Tech Co Ltd filed Critical Shaanxi HuiKang Bio Tech Co Ltd
Priority to CN201611247180.9A priority Critical patent/CN106632655B/en
Publication of CN106632655A publication Critical patent/CN106632655A/en
Application granted granted Critical
Publication of CN106632655B publication Critical patent/CN106632655B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Endocrinology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Vascular Medicine (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses a preparation method of exenatide, which comprises the steps of Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-CONH2The full-protection fragment of (a) is prepared from the following full-protection fragments of three fragments: Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-COOH, Thr-Ser-Asp-Leu-Ser-Lys-Gln-COOH, His-Gly-Glu-Gly-Thr-Phe-COOH. The invention overcomes the problems of difficult purification and difficult scale of the existing solid-phase synthesis exenatide, improves the synthesis efficiency, reduces the accumulation of impurities and reduces the purification difficulty.

Description

Preparation method of exenatide and product thereof
Technical Field
The invention belongs to the technical field of polypeptide synthesis, and particularly relates to a method for synthesizing exenatide through a fragment method.
Background
Exenatide is an active polypeptide containing 39 amino acids, and the amino acid sequence is as follows: h2N-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH2The exenatide injection is an analogue of GLP-1, can stimulate islet beta cell regeneration, promote insulin secretion, inhibit glucagon release and have the effect of controlling blood sugar, and the exenatide injection can improve blood sugar control by reducing fasting and postprandial blood sugar concentrations of type 2 diabetic patients.
The preparation method of exenatide mainly adopts the traditional classical solid-phase polypeptide synthesis method at present, and the method has low synthesis efficiency and high purification cost.
Therefore, the development of a method for synthesizing exenatide with high efficiency and low cost is a technical problem to be solved urgently in the field.
The synthesis of exenatide is disclosed by Zhang in the Fine chemical engineering, 10 months 2014, the synthesis method divides exenatide into six segments to carry out solid phase synthesis, the traditional scheme of synthesizing carbon-terminal amidation by using polypeptide and using amino Resin such as Rink Amide-AM Resin or Rink Amide-MBHA Re sin with high price is used in the synthesis method, and the cost is high. The number of fragments is large, and the synthesis cost is high.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for synthesizing exenatide by a segmentation method, which has high synthesis efficiency, low purification cost, easy flow and easy scale production, overcomes the defects of low efficiency, low product content and high purification cost of synthesizing long peptide by the traditional classical solid-phase polypeptide synthesis method, and also overcomes the defects of complex process and high purification cost when solid-phase liquid-phase mixing is used.
The method comprises the steps of dividing 39 amino acids of exenatide into 4 sections by using a C-N extension strategy, respectively synthesizing 4 sections of full-protection sections, assembling and connecting the 4 sections of full-protection sections in sequence to obtain exenatide resin, cutting to obtain an exenatide crude product, and purifying the crude product to obtain an exenatide product.
More specifically, the present invention is achieved by the following means.
A preparation method of exenatide, which comprises the steps of Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-CONH2The full-protection fragment of (a) is prepared from the following full-protection fragments of three fragments: Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-COOH, Thr-Ser-Asp-Leu-Ser-Lys-Gln-COOH, His-Gly-Glu-Gly-Thr-Phe-COOH.
Wherein the full protective fragment is Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin, Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-COOH, Fmoc-Thr (tBu) -Ser (tBu) -Asp (Otbu) -Leu-Lys (Ser tBu) -Lys (Boc) -Gln (trt) -COOH, respectively, Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr (tBu) -Phe-COOH.
Wherein, the preparation method comprises the following steps:
(1) fragment synthesis
Synthesizing Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin and 3 full protection fragments;
(2) assembling and connecting the segments and cutting the segments to obtain the product
And (2) performing fragment assembly connection on the 4 total-protection fragments prepared in the step (1) according to the amino acid sequence of the exenatide to obtain exenatide with protected nitrogen end and side chain, then processing by using cutting fluid to obtain crude exenatide, and purifying to obtain an exenatide product.
Wherein, the preparation method of Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin comprises the following steps:
(1) preparation of Fmoc-Ser (tBu) -MBHA Resin
Removing Fmoc from Fmoc-Rink Amide MBHA Resin, adding N, N-dimethylformamide, Fmoc-Ser (tBu) -OH, 1-hydroxy benzotriazole, benzotriazole-N, N, N ', N ' -tetramethylurea tetrafluoroborate and N, N ' -diisopropylethylamine, and reacting under the protection of nitrogen to obtain Fmoc-Ser (tBu) -MBHA Resin;
(1) elongation of amino acid chain
Removing Fmoc from the Fmoc-Ser (tBu) -MBHA Resin obtained in the step (1), adding N, N-dimethylformamide, Fmoc-Pro-OH, 1-hydroxy phenylpropyl triazole, benzotriazole-N, N, N ', N ' -tetramethylurea tetrafluoroborate and N, N ' -diisopropylethylamine, and reacting under the protection of nitrogen to obtain Fmoc-Pro-Ser (tBu) -MBHA Resin; Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Ile-OH, Fmoc-Phe-OH are connected in this order to obtain Fmoc-Phe-Ile-Glu (Ou tb) -Trp-Leu-Lys (Boc) -Asn (trt) -Gly-Gly-Pro-Ser (tBu) -Ser tBu (tBu) -Gly-Ala-Pro-Pro (Ser (tBu) -Ilk-Ile-HA.
Wherein the molar ratio of the 1-hydroxy phenylpropyl triazole, the N, N, N ', N ' -tetramethyluronium tetrafluoroborate, the N, N ' -diisopropylethylamine and the Fmoc-Rink Amide MBHA Resin in the step (1) is (2-4): 2-4: 1;
wherein, the preparation method of the 3 total protection fragments Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-COOH, Fmoc-Thr (tBu) -Ser (tBu) -Asp (Otbu) -Leu-Ser (tBu) -Lys (Boc) -Gln (Trt) -COOH, Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr (tBu) -Phe-COOH comprises the following steps:
(1) connection of amino acids: forming an Fmoc-amino acid-2-chlorotrityl chloride Resin structure by using N-terminal amino acid, and then sequentially connecting hydroxyl amino acids of which the N terminals are protected by Fmoc and the branched chains are also protected to form the 2-chlorotrityl chloride Resin structure of an amino acid chain; the amino acid with the N-terminal protected by Fmoc and the side chain protected is represented as Fmoc-amino acid (protecting group) -OH; and
(2) cutting treatment: and (2) cutting the 2-chlorotrityl chloride Resin structure of the amino acid chain obtained in the step (1) to obtain the full-protection fragment.
Wherein the preparation method of Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-COOH comprises the following steps: Fmoc-Arg (pbf) -OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu (Otbu) -OH and Fmoc-Met-OH are connected to Fmoc-Leu-2-chlorotrityl chloride resin in sequence to obtain Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-2-chlorotrityl chloride resin;
wherein, the Fmoc-Thr (tBu) -Ser (tBu) -Asp (Otbu) -Leu-Ser (tBu) -Lys (Boc) -Gln (Trt) -COOH is prepared by connecting the Fmoc-Gln (Trt) -2-chlorotrityl chloride resin with Fomc-Lys (Boc) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Leu-OH, Fmoc-Asp (Otbu) -OH, Fmoc-Ser (tBu) -OH and Fmoc-Thr (tBu) -OH in sequence to obtain Fmoc-Thr (tBu) -Ser (tBu) -Asp (Otbu) -Leu-Ser (tBu) -Lys (Boc) -Gln (Trt) -2-chlorotrityl chloride resin which is cut to obtain the product;
the preparation method of Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr (tBu) -Phe-COOH comprises the following steps: Fmoc-Thr (tBu) -OH, Fomc-Gly-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Gly-OH and Fmoc-His (Trt) -OH are connected to Fmoc-Phe-2-chlorotrityl chloride resin in sequence to obtain Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr (tBu) -Phe-2-chlorotrityl chloride resin, and the Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr (tBu) is cut to obtain the product.
The preparation method of the Fmoc-amino acid-2-chlorotrityl chloride Resin comprises the following steps: swelling 2-chlorotrityl chloride resin with dichloromethane, and reacting with Fmoc-amino acid-OH and N, N' -diisopropylethylamine to obtain a product; preferably, the ratio of 2-chlorotrityl chloride resin, Fmoc-amino acid-OH and N, N' -diisopropylethylamine is 1:1: 4.
Wherein, the reaction conditions for sequentially connecting the hydroxyl amino acid (the structure is Fmoc-amino acid (protecting group) -OH) with the N end protected by Fmoc and the branched chain protected by Fmoc are as follows: fmoc-amino acid-2-chlorotrityl chloride Resin is subjected to Fmoc removal, and then N, N-dimethylformamide, Fmoc-amino acid (protecting group) -OH to be connected, 1-hydroxy phenylpropyl triazole, benzotriazole-N, N, N ', N ' -tetramethyluronium tetrafluoroborate and N, N ' -diisopropylethylamine are added for reaction to obtain Fmoc-amino acid (protecting group) -2-chlorotrityl chloride Resin.
Adding mixed cutting fluid of trifluoroethanol, acetic acid and dichloromethane in a volume ratio of 2:1:7 into the to-be-cut segment, reacting at normal temperature, then carrying out solid-liquid separation, adjusting the pH of the liquid to be neutral, and separating to obtain the product.
Wherein the molar ratio of each Fmoc-amino acid (protecting group) -OH to be connected to each Fmoc-amino acid-2-chlorotrityl chloride Resin is (2-4): 1, preferably the ratio is 2: 1; the molar ratio of the 1-hydroxy phenylpropyl triazole, the benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate to the 2-chlorotrityl chloride Resin is (2-4): 1, and the preferable molar ratio is 3:3:3: 1.
The Fmoc removal condition is that the Fmoc removal is carried out twice by using a mixed solution of piperidine and N, N-dimethylformamide in a volume ratio of 1: 4.
The method for assembling, connecting and cutting the segments to obtain the product comprises the following steps:
(1) preliminary assembly
Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin is subjected to Fmoc removal, and a mixture of N, N-dimethylformamide and dimethyl sulfoxide, N-methylpyrrolidone and Fmoc-Met-Glu (Otbu) -Ala-Val (Arg pbf) -Leu-COOH, 1-hydroxyphenyltriazole and N, N-diisopropyl carbodiimide are added to react to obtain Fmoc-Met-Glu (Otbu) -Ala-Val-Arg-pbf) -Leu-Phe-Ile-Glu (Otbu) -Trp-Lys -Leu-lys (boc) -asn (trt) -Gly-Pro-ser (tbu) -Gly-Ala-Pro-ser (tbu) -Rink Amide MBHA Resin;
(2) obtaining the product
According to the assembly and ligation method of step (1), Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -RinkAmide MBHA Resin is sequentially assembled and ligated with Fmoc-Thr tBu) -Ser (Asp (Ou) -Leu-Ser tbu) -Lys (Boc) -Gln (trt) -COOH, Fmoc-His (trt) -Gly-Glu OtBu (tbu) -Gly-Thr (Phe-COOH), and removing Fmoc to obtain exenatide resin, adding the exenatide resin into the cutting liquid for reaction, separating to obtain an exenatide crude product, purifying by reverse phase chromatography, and freeze-drying to obtain an exenatide product.
Wherein the cutting fluid in the step of assembling, connecting and cutting the fragments to obtain the product is a mixed solution which comprises, by mass, 83% of trifluoroacetic acid, 5% of phenol, 4% of thioanisole, 3% of water and 5% of triisopropylsilane.
Wherein the molar ratio of Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-COOH, Fmoc-Thr (tBu) -Ser (tBu) -Asp (Otbu) -Leu-Ser (tBu) -Lys (Boc) -Gln (Trt) -COOH, Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr (tBu) -Phe-COOH to Fmoc-Rink Amide MBHA Resin is (1-3): 1-3).
Wherein in the preliminary assembly step, the molar ratio of 1-hydroxy phenylpropyl triazole, N-diisopropyl carbodiimide to Fmoc-Rink Amide MBHA Resin is (2-6) to 1; the volume ratio of the N, N-dimethylformamide to the dimethyl sulfoxide to the N-methylpyrrolidone in the mixed solution is (0.5-1): 1-2): 1.
The invention also provides an exenatide product prepared by the preparation method, and the purity of the exenatide product is higher than 98%.
More specifically, the invention provides a solid-phase fragment synthesis method of exenatide, which comprises the following steps:
1. synthesis of Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin
(1) Synthesis of Fmoc-Ser (tBu) -MBHA Resin
Swelling Fmoc-Rink Amide MBHA Resin with N, N-dimethylformamide, removing Fmoc-twice with a mixed solution of piperidine and N, N-dimethylformamide in a volume ratio of 1:4, adding N, N-dimethylformamide, Fmoc-Ser (tBu) -OH, 1-hydroxy phenyl triazole, benzotriazole-N, N, N ', N ' -tetramethylurea tetrafluoroboric acid and N, N ' -diisopropylethylamine, and stirring at normal temperature for 1-3 hours under the protection of nitrogen to obtain Fmoc-Ser (tBu) -MBHA Resin;
(2) synthesis of Fmoc-Pro-Ser (tBu) -MBHA Resin
Removing Fmoc-twice from Fmoc-Ser (tBu) -MBHA Resin by using a mixed solution of piperidine and N, N-dimethylformamide in a volume ratio of 1:4, adding N, N-dimethylformamide, Fmoc-Pro-OH, 1-hydroxy phenylpropyl triazole, benzotriazole-N, N, N ', N ' -tetramethylurea tetrafluoroborate and N, N ' -diisopropylethylamine, and stirring at normal temperature for 1-3 hours under the protection of nitrogen to obtain Fmoc-Pro-Ser (tBu) -MB HA Resin;
(3) synthesis of Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin
Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Ile-OH, Fmoc-Phe-OH are connected to Fmoc-Pro-Ser (tBu) -MBU in this order according to the method of step (2) to obtain Fmoc-Phe-Ile-Glu- Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin;
2. synthesis of Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-COOH
(1) Synthesis of Fmoc-Leu-2-chlorotrityl chloride resin
Swelling 2-chlorotrityl chloride resin with dichloromethane, adding dichloromethane, Fmoc-Leu-OH and N, N' -diisopropylethylamine, and stirring at normal temperature for 1-3 hours under the protection of nitrogen to obtain Fmoc-Leu-2-chlorotrityl chloride resin;
(2) synthesis of Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-2-chlorotrityl chloride resin
Connecting Fmoc-Arg (pbf) -OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Met-OH to Fmoc-Leu-2-chlorotrityl chloride resin in this order according to the method of step 1 (2) to Fmoc-Leu-2-chlorotrityl chloride resin to obtain Fmoc-Met-Glu (Otbu) -Glu (Glu tbOu) -Ala-Val-Arg (pbf) -Leu-2-chlorotrityl chloride resin;
(3) cutting process
To Fmoc-Met-Glu (Otbu)Adding cutting fluid into Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-2-chlorotrityl chloride resin, stirring for 1-3 hours at normal temperature, filtering, and slowly adding 2-20% NaHCO into the filtrate3The solution is neutral, and precipitates are separated out to obtain Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-COOH; the cutting fluid is a mixed solution of trifluoroethanol, acetic acid and dichloromethane in a volume ratio of 2:1: 7;
3. synthesis of Fmoc-Thr (tBu) -Ser (tBu) -Asp (Otbu) -Leu-Ser (tBu) -Lys (Boc) -Gln (Trt) -COOH
Attaching Fmoc-Gln (Trt) -OH to the 2-chlorotrityl chloride resin according to the method of step (1) in step 2 to obtain Fmoc-Gln (Trt) -2-chlorotrityl chloride resin, and attaching Fomc-Lys (Boc) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Leu-OH, Fmoc-Asp (Otbu) -OH, Fmoc-ThrtBu (Ser) (ThrtBu) -Ser (Thr) (Leu) (Boc-Gln) (Trt) -COOH, Fmoc-ThrtBu) -Ser (Thr-Thr) to the Fmoc-Thr) chloride resin in sequence according to the method of step 2 to obtain Fmoc-Gln (Trt) -OH, Fmoc-Gln (Trt) -2-chlorotrityl chloride resin, and attaching Fomc-Lys (Boc) -Boc) -OH to the Fmoc-chlorotrityl chloride resin in sequence according to the method of step 2 Performing a cleavage treatment according to the method in step 2 (3) to obtain Fmoc-Thr (tBu) -Ser (tBu) -Asp (Otbu) -Leu-Ser (tBu) -Lys (Boc) -Gln (Trt) -COOH;
4. synthesis of Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr (tBu) -Phe-COOH
Attaching Fomc-Phe-OH to 2-chlorotrienyl chloride Resin according to the method of step (1) in step 2 to obtain Fmoc-Phe-2-chlorotrienyl chloride Resin, and further attaching Fmoc-Thr (tBu) -OH, Fomc-Gly-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Gly-OH, Fmoc-His Trt) -OH to Fmoc-Phe-2-chlorotrienyl chloride Resin in sequence according to the method of step (2) in step 2 to obtain Fmoc-His Trt- (Gly) -Glu (Otbu) -Gly-Thr (tBu) -Phe-2-chlorotrienyl chloride Resin, Fmoc-His-Glu (Gly-Glu) (Gly-Thru) -Phe-2-chlorotrienyl chloride Resin, Fmoc-His-Glu (Tru) (Gly-Thru) -Phe-2-chlorotrienyl chloride Resin, and then performing cleavage according to the method of step 3 in step 2-chlorotrienyl chloride Resin, obtaining Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr (tBu) -Phe-COOH;
5. synthesis of Exenatide
(1) Assembly and linkage of Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin
Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -Rink Amide MB HA Resin obtained in the step 1 is subjected to Fmoc-removing twice by using a mixed solution with piperidine and N, N-dimethylformamide in the volume ratio of 1:4, and added with a mixed solution of N, N-dimethylformamide, dimethyl sulfoxide and N-methylpyrrolidone in the volume ratio of (0.5-1) to (1-2) a mixed solution of 1 and Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-COOH, 1-hydroxy phenylpropyl triazole, 1-Leu-Lys (Boc, N-Met-Glu (Otbu) -Ala, Stirring N, N-diisopropyl carbodiimide at normal temperature for 3-4 hours under the protection of nitrogen to obtain Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin;
(2) assemble and connect exenatide
According to the assembly and ligation method of step (1), Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin is sequentially assembled and ligated with Fmoc-Thr-tBu) -Ser (Asp (Otbu) -Leu-Ser (tBu) -Lys (Boc) -Gln (Trt) -COOH, and Fmoc-Trt) -Gly-Glu (Otbu) -Gly-Thr (Phe-COOH, removing Fmoc-twice by using a mixed solution of piperidine and N, N-dimethylformamide in a volume ratio of 1:4 to obtain exenatide resin, stirring at room temperature for 1-3 hours by using a cutting solution consisting of 83% by mass of trifluoroacetic acid, 5% by mass of phenol, 4% by mass of phenylmethylsulfide, 3% by mass of water and 5% by mass of triisopropylsilane, filtering, separating out a precipitate by using cold ethyl ether to obtain an exenatide crude product, and purifying and freeze-drying the exenatide crude product by using reverse phase chromatography to obtain exenatide;
in the above step 1, the molar ratio of Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Asn (trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Glu (otbu) -OH, Fmoc-Ile-OH, Fmoc-Phe-OH and Fmoc-Rink Amide MBHA Resin is (2 to 4):1, Fmoc-Rink Amide MBHA Resin and 1-hydroxyphenyltriazole, benzotriazol-N, the molar ratio of N, N ', N ' -tetramethylurea tetrafluoroboric acid to N, N ' -diisopropylethylamine is 1 (2-4) to (2-4).
In the step 2 (1), the molar ratio of the 2-chlorotrityl chloride resin to Fmoc-Leu-OH and N, N '-diisopropylethylamine is 1:1:4, and in the step 2 (2), the molar ratio of Fmoc-Arg (pbf) -OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu (otbu) -OH, Fmoc-Met-OH and 2-chlorotrityl chloride resin is 2-4: 1, 2-chlorotrityl chloride resin to 1-hydroxy-phenyl triazole, benzotriazole-N, N, N', N '-tetramethyluronium tetrafluoroborate and N, N' -diisopropylethylamine is 1: 2-4 (2-4).
The molar ratio of the 1-hydroxy phenylpropyl triazole, the N, N-diisopropyl carbodiimide and the Fmoc-Rink AmidemBHA Resin is (2-6): 1.
Wherein the four fragments of exenatide are respectively: (1) Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin, (2) Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-COOH, (3) Fmoc-Thr (tBu) -Ser tBu) -Asp (tbOu) -Leu-Ser tBu (Lys) (Boc) -Gln (Trt) -COOH, (4) Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr tBu-Phe-COOH and (tBu-COOH).
Wherein the cutting processing method of the steps (2), (3) and (4) comprises the following steps: adding a mixed cutting fluid of trifluoroethanol, acetic acid and dichloromethane in a volume ratio of 2:1:7 into each fragment of resin peptide, stirring for 1-3 hours at normal temperature, filtering, and slowly adding 2-20% NaHCO into the filtrate3The aqueous solution is neutral, and precipitates are separated out.
In the step 5, the molar ratio of Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-COOH, Fmoc-Thr (tBu) -Ser (tBu) -Asp (Otbu) -Leu-Ser (tBu) -Lys (Boc) -Gln (Trt) -COOH, Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr (Ftbu) -Phe-COOH to Fmoc-Rink Amide MBHA Resin is (1-3): 1-3);
in the step 5, the molar ratio of the 1-hydroxy phenylpropyl triazole, the N, N-diisopropyl carbodiimide to the Fmoc-Rink Amide MBHA Resin is (2-6): 1.
The invention has the following beneficial effects:
1. according to the method, the exenatide is synthesized by a fragment synthesis method, and the carbon chains of all fragments are shorter than those of a one-by-one extension method, so that the impurity accumulation is reduced, and the purification difficulty is reduced.
2. The fragment is synthesized by a solid phase method, the synthesis operation is simple, the complicated product treatment process of a liquid phase method is omitted, the yield is stable, and the purity is high.
3. The invention reduces the number of fragments to the maximum extent, reduces the usage amount of the 2-chlorotrityl chloride resin as much as possible to reduce the cost, and simultaneously considers the solubility of the fragments. And the synthetic steps are reduced to the maximum extent on the basis.
4. The invention overcomes the problems of difficult purification and difficult scale of the existing solid-phase synthesis of the exenatide, improves the synthesis efficiency, and provides a new method for effectively solving the problem of large scale synthesis of the exenatide, reducing the purification difficulty and improving the product purity.
Drawings
FIG. 1 is a mass spectrum of exenatide synthesized in example 1.
FIG. 2 is a liquid chromatogram of exenatide synthesized in example 1.
Detailed Description
The present invention will be described in further detail with reference to the following drawings and examples, but the present invention is not limited to these examples.
The following is a brief description of the specification:
2-chlorotrityl chloride Resin, which is named 2-chlorotrityl chloride Resin;
Fmoc-Rink Amide MBHA Resin, its name is 4- (2 ', 4' -dimethoxyphenyl-fluorenylmethoxycarbonyl-aminomethyl) -phenoxyacetamido-methylbenzhydrylamine Resin
Fmoc: fmoc group
pbf, tbu, Otbu, Trt, Boc are protecting groups with the names 2,2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl, tert-butyl, tert-butoxy, trityl, tert-butoxycarbonyl, respectively.
The 'full-protection fragment' refers to a fragment with a protection group obtained by protecting a group which influences synthesis in amino acid.
The invention provides a preparation method of exenatide, which comprises the steps of Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-CONH2The full-protection fragment of (a) is prepared from the following full-protection fragments of three fragments: Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-COOH, Thr-Ser-Asp-Leu-Ser-Lys-Gln-COOH, His-Gly-Glu-Gly-Thr-Phe-COOH. This is the result obtained by the inventors analyzing the sequence and combining the properties of the respective amino acids while taking into consideration the cost of synthesis, selecting and experimenting between the respective factors, and resolving the 39 amino acid positions into four fragments as described above. The selection is the result of creative labor of the inventor, so that the cost is effectively reduced;
wherein the full protective fragment is Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin, Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (Fpbf) -Leu-COOH, Fmoc-Thr (tBu) -Ser tBu) -Asp (Otbu) -Leu-Lys (Boc) -Gln (Trt) -COOH, Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr tBu) -Phe-COOH, respectively. The protecting group selected for each amino acid in the three sequences may be any protecting group as long as the desired technical effect of protection can be achieved, but the inventors prefer the best effect of the substituent. The Fmoc-Leu-OH, Fmoc-Gln (trt) -OH and Fmoc-Phe-OH are selected as the carbon ends of the three full-protection fragments, the Fmoc-His (trt) -OH, Fmoc-Thr (tBu) -OH and the Fmoc-Met-OH are selected as the nitrogen ends of the full-protection fragments, so that the reaction activities of the amino acid as the carbon end and the nitrogen end are considered to be favorable for the assembly reaction of the fragments, and the selection of breakpoints is considered to balance the length, the solubility and the number of the full-protection fragments, so that the synthesis steps can be effectively reduced, the efficiency is improved, and the cost is reduced; the use of Boc-His (Trt) -OH at the nitrogen terminus can reduce processing steps after assembly is complete.
The preparation method comprises the following steps:
(1) fragment synthesis
Synthesizing Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin and 3 full protection fragments;
in one embodiment, Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin is prepared as follows:
(1) preparation of Fmoc-Ser (tBu) -MBHA Resin
Removing Fmoc from Fmoc-Rink Amide MBHA Resin, adding N, N-dimethylformamide, Fmoc-Ser (tBu) -OH, 1-hydroxy benzotriazole, benzotriazole-N, N, N ', N ' -tetramethylurea tetrafluoroborate and N, N ' -diisopropylethylamine, and reacting under the protection of nitrogen to obtain Fmoc-Ser (tBu) -MBHA Resin; the raw material used in the step is Fmoc-Rink Amide MBHA Resin which is formed by connecting MBHA Resin with Fmoc protection modified Rink Amide Linker. The substitution degree of the compound is 0.34mmol/g, but other substitution degrees can achieve the same or similar technical effects and are also within the protection scope of the invention. The degree of substitution 0.34mmol/g used in the present invention is an optimum value in terms of the balance of factors such as yield of the synthesized fragment, purity, and utilization rate of the resin.
(2) Elongation of amino acid chain
Removing Fmoc times from Fmoc-Ser (tBu) -MBHA Resin obtained in the step (1), adding N, N-dimethylformamide, Fmoc-Pro-OH, 1-hydroxy phenylpropyl triazole, benzotriazole-N, N, N ', N ' -tetramethylurea tetrafluoroborate and N, N ' -diisopropylethylamine, and reacting under the protection of nitrogen to obtain Fmoc-Pro-Ser (tBu) -MBHA Resin; then Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Ile-OH, Fmoc-Phe-OH are connected in sequence according to the method to obtain Fmoc-Phe-Ile-Glu (Ou tb) -Trp-Leu-Lys (Boc) -Asn (trt) -Gly-Gly-Pro-Ser (tBu) -Ser tBu (tBu) -Gly-Ala-Pro-Pro (Ser tBu) -Ile-HA;
preferably, the molar ratio of the 1-hydroxy phenylpropyl triazole, the N, N, N ', N ' -tetramethyluronium tetrafluoroborate, the N, N ' -diisopropylethylamine and the Fmoc-Rink Amide MBHA Resin in the step (1) is (2-4): 2-4: 1; the proportion of each substance in the preparation process can be properly adjusted, and the obtained product can still meet the requirements, so that the proper adjustment of the proportion of each substance in the preparation process is also within the protection scope of the invention.
(2) Assembling and connecting the segments and cutting the segments to obtain the product
And (2) performing fragment assembly connection on the 4 total-protection fragments prepared in the step (1) according to the amino acid sequence of the exenatide to obtain exenatide with protected nitrogen end and side chain, then processing by using cutting fluid to obtain crude exenatide, and purifying to obtain an exenatide product.
Wherein the 3 total-protection fragments Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-COOH, Fmoc-Thr (tBu) -Ser (tBu) -Asp (Otbu) -Leu-Ser (tBu) -Lys (Boc) -Gln (Trt) -COOH, Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr (tBu) -Phe-COOH were prepared by a method comprising the steps of:
(1) connection of amino acids: forming an Fmoc-amino acid-2-chlorotrityl chloride Resin structure by using N-terminal amino acid, and then sequentially connecting hydroxyl amino acids of which the N terminals are protected by Fmoc and the branched chains are also protected to form the 2-chlorotrityl chloride Resin structure of an amino acid chain; the amino acid with the N-terminal protected by Fmoc and the side chain protected is represented as Fmoc-amino acid (protecting group) -OH; and
(2) cutting treatment: and (2) cutting the 2-chlorotrityl chloride Resin structure of the amino acid chain obtained in the step (1) to obtain the full-protection fragment.
The step is a step of chain extension of amino acids, and the Fmoc-amino acid-2-chlorotrietyl chloride Resin structure in the present invention is Fmoc-Leu-2-chlorotrietyl chloride Resin, Fmoc-Gln (Trt) -2-chlorotrietyl chloride Resin and Fmoc-Phe-2-chlorotrietyl chloride Resin, and the N-terminal of the sequential linkage is Fmoc-protected, and the branched chain is also protected, that is, the structure represented as Fmoc-amino acid (protecting group) -OH may be Fmoc-Arg (pbf) -OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Glu (Ou) tbOH, Fmoc-Glu (Otbu) -OH, Fmoc-Met-OH, Fomc-Lys (Lys), Fmoc-OH, Fmoc-tBu-OH) and Fmoc-Phe-amino acid (protecting group) -OH, Fmoc-Leu-OH, Fmoc-Asp (Otbu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fomc-Gly-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Gly-OH, Fmoc-His (Trt) -OH.
The preparation method of the Fmoc-amino acid-2-chlorotrityl chloride Resin comprises the following steps: swelling 2-chlorotrityl chloride resin with dichloromethane, and reacting with Fmoc-amino acid-OH and N, N' -diisopropylethylamine to obtain the product.
The starting material used in the above method for preparing Fmoc-amino acid-2-chlorotrityl chloride Resin is 2-chlorotrityl chloride Resin, which is relatively inexpensive, thereby reducing the cost on the starting material. The 2-chlorotrityl chloride Resin is named as 2-chlorotrityl chloride Resin, the substitution degree is 1.74mmol/g, but the 2-chlorotrityl chloride Resin with other substitution degrees can achieve the same or similar technical effects and is also in the protection scope of the invention. The substitution degree of the 2-chlorotrityl chloride Resin can be all the substitution degrees commercialized at present, and the substitution degree of 1.74mmol/g used in the invention is an optimal value balanced from factors such as yield, purity, Resin utilization rate and the like of a synthetic fragment.
The reaction conditions for sequentially connecting the hydroxyl amino acid (the structure is Fmoc-amino acid (protecting group) -OH) with the N end protected by Fmoc and the branched chain protected by Fmoc are as follows: removing Fmoc from Fmoc-amino acid-2-chloro rityl chloride Resin, adding N, N-dimethylformamide, Fmoc-amino acid (side chain protecting group) -OH to be connected, 1-hydroxy phenylpropyl triazole, benzotriazole-N, N, N ', N ' -tetramethylurea tetrafluoro, boric acid and N, N ' -diisopropylethylamine to react to obtain Fmoc-amino acid (protecting group) -2-chloro chloride Resin;
wherein the molar ratio of each Fmoc-amino acid (protecting group) -OH to be connected to each Fmoc-amino acid-2-chlorotrityl chloride Resin is (2-4): 1, preferably the ratio is 2: 1; the molar ratio of the 1-hydroxy phenylpropyl triazole, the benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate to the 2-chlorotrityl chloride Resin is (2-4): 1, and the preferable molar ratio is 3:3:3: 1.
Preferably, the Fmoc removal condition is that the Fmoc removal is carried out by using a mixed solution of piperidine and N, N-dimethylformamide in a volume ratio of 1: 4. The feeding amount of each substance in the step is taken as a reference in the preparation of the Fmoc-amino acid-2-chlorotrietyl chloride Resin structure, and the feeding amount is effectively controlled by the control, so that the product quality is maintained, and the cost is controlled.
(2) Cutting treatment: and (2) cutting the 2-chlorotrityl chloride Resin structure of the amino acid chain obtained in the step (1) to obtain the full-protection fragment.
Specifically, the synthesis methods of the three fragments are as follows:
the method for assembling, connecting and cutting the segments to obtain the product comprises the following steps:
(1) preliminary assembly
Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin is subjected to Fmoc removal, and a mixture of N, N-dimethylformamide and dimethyl sulfoxide, N-methylpyrrolidone and Fmoc-Met-Glu (Otbu) -Ala-Val (Arg pbf) -Leu-COOH, 1-hydroxyphenyltriazole and N, N-diisopropyl carbodiimide are added to react to obtain Fmoc-Met-Glu (Otbu) -Ala-Val-Arg-pbf) -Leu-Phe-Ile-Glu (Otbu) -Trp-Lys -Leu-lys (boc) -asn (trt) -Gly-Pro-ser (tbu) -Gly-Ala-Pro-ser (tbu) -Rink Amide MBHA Resin;
(2) obtaining the product
According to the assembly and ligation method of step (1), Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -RinkAmide MBHA Resin is sequentially assembled and ligated with Fmoc-Thr tBu) -Ser (Asp (Ou) -Leu-Ser tbu) -Lys (Boc) -Gln (trt) -COOH, Fmoc-His (trt) -Gly-Glu OtBu (tbu) -Gly-Thr (Phe-COOH), and removing Fmoc to obtain exenatide resin, adding the exenatide resin into the cutting liquid for reaction, separating to obtain an exenatide crude product, purifying by reverse phase chromatography, and freeze-drying to obtain an exenatide product.
Preferably, the molar ratio of Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-COOH, Fmoc-Thr (tBu) -Ser (tBu) -Asp (Otbu) -Leu-Ser (tBu) -Lys (Boc) -Gln (Trt) -COOH, Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr (tBu) -Phe-COOH to Fmoc-Rink Amide MBHA Resin is (1-3): 1-3; the proportion ensures that the cost of synthesis is minimized, and simultaneously, the purity and the process meet the requirements.
In a specific embodiment, the molar ratio of the 1-hydroxyphenyltriazole, the N, N-diisopropylcarbodiimide and the Fmoc-Rink Amide MBHA Resin in the preliminary assembly step is (2-6): 1; the volume ratio of the N, N-dimethylformamide to the dimethyl sulfoxide to the N-methylpyrrolidone in the mixed solution is (0.5-1): 1-2): 1.
The proportion of each substance in the preparation process can be properly adjusted, and the obtained product can still meet the requirements, so that the proper adjustment of the proportion of each substance in the preparation process and the numerical value of the parameters of the reaction conditions are also within the protection scope of the invention. The ingredient ratio of each substance in the preparation steps is controlled to be within the range, which is the result of the consideration of a plurality of factors on the ingredient ratio of the preparation method of each step, and the technical effect can still be achieved by floating up and down 10% within the protection range.
In addition, the invention also provides an exenatide product prepared by the preparation method, and the purity of the exenatide product is higher than 98%.
Examples
In the following, the sources of the respective substances used in the examples are explained, and if not specifically stated, the raw materials and instruments used are commercially available, and the instruments and raw materials are conventionally used in the art as long as they meet the experimental requirements.
The substitution degree of Fmoc-Rink Amide MBHA Resin is 0.34mmol/g, and the substitution degree of 2-chlorotrityl chloride Resin is 1.74mmol/g, which are all produced by Tianjin Nankai and science and technology Limited.
The solid phase polypeptide synthesis reactor is a general polypeptide synthesis reactor, and is purchased from CS936X CSBio Peptide Synthesizer of Dus Nuo import and export trade Co.
The analytical high performance liquid chromatograph is Hitachi full-automatic L2000.
The preparative high performance liquid chromatograph is Innovative Hengtong LC3000, the C18 analytical chromatographic column is Dalian physical chemistry research institute 4.6mm multiplied by 250mm, and the C18 preparative chromatographic column is Chengdu science popularization biology limited company 40.1mm multiplied by 450 mm.
The amino acids used were obtained from Gill Biochemical Co.Ltd, Shanghai.
The LTQ-XL electrospray ionization mass spectrometer is manufactured by Thermo Finnigan, USA.
Piperidine, N, N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, dichloromethane, trifluoroacetic acid, phenol, thioanisole, triisopropylsilane, 1-hydroxyphenyltriazole, benzotriazole-N, N, N ', N ' -tetramethyluronium tetrafluoroborate, N, N ' -diisopropylethylamine, N, N-diisopropylcarbodiimide and other reagents are all made of domestic AR or CP.
Example 1
1. Synthesis of Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin
(1) Synthesis of Fmoc-Ser (tBu) -MBHA Resin
Adding 50g of Fmoc-Rink Amide MBHA Resin into a reactor, adding 500mLN, N-dimethylformamide to soak the Resin for 30 minutes to fully swell the Resin, removing N, N-dimethylformamide by suction filtration, adding 500mL of mixed solution of piperidine and N, N-dimethylformamide with the volume ratio of 1:4 into the reactor, reacting for 5 minutes, removing the mixed solution by suction filtration, adding 500mL of mixed solution of piperidine and N, N-dimethylformamide with the volume ratio of 1:4, reacting for 20 minutes, suction filtration, washing the Resin with isopropanol for 2 times, washing the Resin with N, N-dimethylformamide for 3 times, each time for 500mL, completing Fmoc-Rink Amide MBHA Resin removal twice, adding 500mL of N, N-dimethylformamide, 13.04g of Fmoc-Ser (tbu) -OH, adding, 4.59g of 1-hydroxy phenylpropyl triazole, 12.9g of benzotriazole-N, N, N ', N ' -tetramethyluronium tetrafluoroborate and 5.9mL of N, N ' -diisopropylethylamine are stirred for 1.5 hours at normal temperature under the protection of nitrogen, and the mixture is subjected to suction filtration, isopropanol and N, N-dimethylformamide are used for washing the Resin for 2 times, 500mL each time, and the Resin is subjected to suction filtration to obtain Fmoc-Ser (tBu) -MBHA Resin.
(2) Synthesis of Fmoc-Pro-Ser (tBu) -MBHA Resin
The Fmoc-Ser (tBu) -MBHA Resin obtained is Fmoc-removed twice by using a mixed solution of piperidine and N, N-dimethylformamide with the volume ratio of 1:4 according to the method of the step (1), 500mL of N, N-dimethylformamide, 11.47g of Fmoc-Pro-OH, 4.59g of 1-hydroxy benzotriazole, 12.90g of benzotriazole-N, N, N ', N ' -tetramethylurea tetrafluoroboric acid and 5.9mL of N, N ' -diisopropylethylamine are added, the mixture is stirred for 1.5 hours at normal temperature under the protection of nitrogen, and the Resin is washed 2 times and 500mL each time by using isopropanol and N, N-dimethylformamide to obtain Fmoc-Pro-Ser (tBu) -MBHA Resin.
(3) Synthesis of Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin
The Fmoc-Pro-OH, 11.47g of Fmoc-Pro-OH, 10.58g of Fmoc-Ala-OH, 10.11g of Fmoc-Gly-OH, 13.04g of Fmoc-Ser (tBu) -OH, 11.47g of Fmoc-Pro-OH, 10.11g of Fmoc-Gly-OH, 20.29g of Fmoc-Glu (Asn Trt) -OH, 15.93g of Fmoc-Lys (Boc) -OH, 12.02g of Fmoc-Leu-OH, 14.50g of Fmoc-Trp-OH, 14.47g of Fmoc-Glu (tBu) -OH, 12.02g of Fmoc-E-OH, 13.17g of Fmoc-Leu-Ala-Lys, 14.50g of Fmoc-Trp-OH, 14.47g of Fmoc-Glu (Ty-25 g of Fmoc-Phe-Lys-Gly-OH, 13.25 g of Fmoc-Glu-Ty-2, and the same amount of other reagents as used in Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin.
The molar ratio of Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Glu (Ou tb) -OH, Fmoc-Ile-OH, Fmoc-Phe-OH and Fmoc-Rink Amide MBHA Resin is 2:1, the molar ratio of Fmoc-Rink Amide HA Resin to 1-hydroxy phenylpropyl triazole, benzotriazol-N, the molar ratio of N ', N ' -tetramethylurea tetrafluoroboric acid to N, N ' -diisopropylethylamine is 1:2:2: 2.
2. Synthesis of Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-COOH
(1) Synthesis of Fmoc-Leu-2-chlorotrityl chloride resin
Adding 50g of 2-chlorotrityl chloride resin into a reactor, adding 500mL of dichloromethane to soak the resin for 30 minutes to fully swell the resin, performing suction filtration to remove the dichloromethane, adding 500mL of dichloromethane, 30.75g of Fmoc-Leu-OH, 60.2mL of N, N '-diisopropylethylamine into the reactor, adding the molar ratio of the 2-chlorotrityl chloride resin to the Fmoc-Leu-OH and the N, N' -diisopropylethylamine into the reactor to be 1:1:4, stirring for 1.5 hours at normal temperature under the protection of nitrogen, performing suction filtration, washing the resin with isopropanol and N, N-dimethylformamide respectively for 2 times, 500mL each time, and obtaining the Fmoc-Gly-2-chlorotrityl chloride resin.
(2) Synthesis of Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-COOH
112.89g of Fmoc-Arg (pbf) -OH, 59.06g of Fmoc-Val-OH, 54.17g of Fmoc-Ala-OH, 74.04g of Fmoc-Glu (Otbu) -OH, 74.04g of Fmoc-Glu (Otbu) -OH, 74.04g of Fmoc-Glu (Otbu) -OH, 64.64g of Fmoc-Met-OH were sequentially linked to Fmoc-Leu-2-chlorotrityl chloride resin according to the method of step 1 (2): 23.51g of 1-hydroxy phenylpropyl triazole, 66.0g of benzotriazole-N, N, N ', N ' -tetramethyluronium tetrafluoroborate and 30.1mL of N, N ' -diisopropylethylamine, and the resin is washed 2 times with 500mL of isopropanol and N, N-dimethylformamide respectively to obtain Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-2-chlorotrityl chloride resin.
The molar ratio of the Fmoc-Arg (pbf) -OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Met-OH and 2-chlorotrityl chloride resin is 2:1, 2-chlorotrityl chloride resin, 1-hydroxy benzotriazole, benzotriazole-N, N, N ', N ' -tetramethyluronium tetrafluoroborate and N, N ' -diisopropylethylamine is 1:2:2: 2.
(3) Cutting process
Adding 500mL of cutting fluid into Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-2-chlorotrityl chloride resin, wherein the volume ratio of the cutting fluid to the cutting fluid is 2:1:7, stirring for 1.5 hours at normal temperature, filtering, and slowly adding 20% NaHCO into the filtrate3The aqueous solution is neutralized, precipitates are separated out, the precipitates are collected and dried in vacuum at normal temperature for 1 hour, and 142.31g of Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-COOH are obtained.
3. Synthesis of Fmoc-Thr (tBu) -Ser (tBu) -Asp (Otbu) -Leu-Ser (tBu) -Lys (Boc) -Gln (Trt) -COOH
Attaching 53.13g of Fmoc-Gln (Trt) -OH to 50g of 2-chlorotrityl chloride resin according to the method of step 2 (1) to obtain Fmoc-Gln (Trt) -2-chlorotrityl chloride resin, and attaching 40.76g of Fomc-Lys (Boc) -OH, 66.71g of Fmoc-Ser (tBu) -OH, 61.49g of Fmoc-Leu-OH, 71.60g of Fmoc-Asp (Otbu) -OH, 66.71g of Fmoc-Ser (tBu) -OH, 69.17g of Fmoc-ThrBu (ThrBu) -OH to obtain Fmoc (Ser) (Asp-tBu) -Thru) and (Thr) -Boc-Lys-T-Boc-Lys-Thru) -2-chlorotrityl chloride resin in the order of step 2 (2) to obtain Fmoc-Leu-2-chlorotrityl chloride resin (Lys) (40.76 g of Fmoc-Boc) -2) to obtain Fmoc-Thru-Ser (Thr) -OH, 61. -chlorotrityl chloride resin was cleaved according to the method of (3) in step 2 to obtain 135.42gFmoc-Thr (tBu) -Ser (tBu) -Asp (Otbu) -Leu-Ser (tBu) -Lys (Boc) -Gln (Trt) -COOH.
4. Synthesis of Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr (tBu) -Phe-COOH
After connecting 33.70g of Fomc-Phe-OH to 50g of 2-chlorotrityl chloride Resin according to the method of step 2 (1), Fmoc-Phe-2-chlorotrityl chloride Resin was obtained, and then connecting 69.17g of Fomc-Thr (tBu) -OH, 51.73g of Fmoc-Gly-OH, 7404g of Fmoc-Glu (otBu) tbOH, 51.73g of Fmoc-Gly-OH, 107.83g of Fomc-His (Trt) -OH to Fmoc-Phe-2-chlorotrityl chloride Resin in this order according to the method of step 2 (2), Fmoc-Gly-OH, 107.83g of Fomc-His (Trt) -OH were obtained, Fmoc-Gly-Glu (Otbu) -Gly-Thr (tBu) -Phe-2-chlorotrityl chloride Resin (Fmoc-Phe-2-chlorotrityl chloride Resin, Trust-His- (Thr) -Gly-Thr) was subjected to the method of step 2 (2) to cleavage by the method of step 2 (2) of Fmoc-Phe-Thr-Phe-Glu-2-chlorotrityl chloride Resin (2), 95.01g of Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr (tBu) -Phe-COOH were obtained.
5. Assemble and connect to synthesize exenatide
(1) Assembly and linkage of Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin
According to the method of step 1 (1), 45.23g of Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin was Fmoc-twice from a mixed solution of piperidine and N, N-dimethylformamide in a volume ratio of 1:4, 600mL of a mixed solution of N, N-dimethylformamide, dimethylsulfoxide and N-methylpyrrolidone in a volume ratio of 1:1:1 and 27.53g of Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-COOH, 23.51g of 1-hydroxyphenyltriazole, 1-hydroxyphenyltriazole, 5.3mL of N, N-diisopropylcarbodiimide, under nitrogen protection, stirred at room temperature for 3 hours, and washed with isopropanol and N, N-dimethylformamide for 2 times, each 600mL of 2 times, to obtain Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (trt) -Gly-Gly-Pro-Ser (tBu) -Ser tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin;
(2) assemble and connect exenatide
According to the assembly and ligation method of step (1), 26.63g of Fmoc-Thr (tBu) -Ser (tBu) -Asp (Otbu) -Leu-Lys (Boc) -Ala-Val-Arg (pbf) -Leu-Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin was sequentially assembled and ligated to Fmoc-Met-Glu (Otbu) -Asp (Otbu) -Leu-Ser (tBu) -Lys (Boc) -Gln (Trt) -COOH, 20.49g of Fmoc-His-Trt) -Gly-Glu Otbu- (Gly-Thr) (tBu) -Phe-COOH, removing Fmoc twice by using 600ml of mixed solution with the volume ratio of piperidine to N, N-dimethylformamide being 1:4 to obtain exenatide resin, stirring at room temperature for 2 hours by using cutting fluid consisting of 83 mass percent of trifluoroacetic acid, 5 mass percent of phenol, 4 mass percent of phenylmethylsulfide, 3 mass percent of water and 5 mass percent of triisopropylsilane, filtering, separating out precipitate by using cold anhydrous ether to obtain crude exenatide, purifying the crude exenatide by reverse phase chromatography, and freeze-drying to obtain 22.92g of exenatide, wherein the yield is 32.2%;
Fmoc-Lys (Boc) -Asn (Trt) -Gly-COOH, Fmoc-Val-Arg (pbf) -Leu-Phe-Ile-Glu (Otbu) -Trp-Leu-COOH, Fmoc-Met-Glu (Otbu) -Ala-COOH, Fmoc-Thr (tBu) -Ser (tBu) -Asp (Otbu) -Leu-Ser (tBu) -Lys (Boc) -Gln (Trt) -COOH, Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr (tBu) -Phe-COOH and Fmoc-Rink Amide MBHA Resin are in a molar ratio of 1:1, 1-hydroxyphenyltriazole, N-diisopropylcarbodiimide to Fmoc-Rink Amide MBHA Resin of 2:2: 1.
The structure of the synthesized product is characterized by a mass spectrometer, and the result is shown in figure 1, and as can be seen from the figure, the molecular weight and the molecular ion peak of the synthesized product are consistent with those of exenatide, which indicates that the synthesized product is exenatide. The purity of the synthesized exenatide is determined by a liquid chromatograph, a liquid chromatogram is shown in figure 2, and the purity of the exenatide is more than 98% as can be seen from figure 2.
Example 2
In step 1 of this embodiment, the molar ratio of Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Ile-OH, Fmoc-Phe-OH and Fmoc-Rink Amide MBHA Resin is 3:1, Fmoc-Rink Amide MBHA Resin and 1-hydroxy phenylpropyl triazole, benzotriazole-N, the molar ratio of N, N ', N ' -tetramethyluronium tetrafluoroborate to N, N ' -diisopropylethylamine was 1:3:3:3, and the other steps of this step were the same as in example 1.
In step 2 of this example, the molar ratio of Fmoc-Arg (pbf) -OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Met-OH and 2-chlorotrityl chloride resin was 3:1, and the molar ratio of 2-chlorotrityl chloride resin to 1-hydroxyphenyltriazole, benzotriazol-N, N, N ', N ' -tetramethyluronium tetrafluoroborate and N, N ' -diisopropylethylamine was 1:3:3, and the other steps of this example were the same as in example 1.
In steps 3 to 4 of this example, the molar ratio of 2-chlorotrityl chloride resin to Fmoc-amino acid, 1-hydroxy benzotriazole, benzotriazole-N, N '-tetramethyluronium tetrafluoroborate, N' -diisopropylethylamine was the same as in step 2 of this example, and the synthesis method was the same as in example 1. In step 5 of this embodiment, the molar ratio of Fmoc-Lys (Boc) -Asn (Trt) -Gly-COOH, Fmoc-Val-Arg (pbf) -Leu-Phe-Ile-Glu (Otbu) -Trp-Leu-COOH, Fmoc-Met-Glu (Otbu) -Ala-COOH, Fmoc-Thr (tBu) -Ser (tBu) -Asp (Otbu) -Leu-Ser (tBu) -Lys (Boc) -Gln (Trt) -COOH, Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr (tBu) -Phe-COOH to Fmoc-Rink Amide MBHA Resin is 2:1, 1-hydroxy phenylpropyl triazole, N-diisopropyl carbodiimide to Fmoc-Rink MBamide HA Resin is 4:1, 24.91g of exenatide was obtained with a yield of 35%.
The structure of the synthesized product was characterized by a mass spectrometer using the same conditions as in example 1, and the molecular weight and molecular ion peak of the synthesized product were consistent with those of exenatide. And (3) adopting a liquid chromatograph to measure the purity of the synthesized exenatide, wherein the purity is more than 98%.
Example 3
In step 1 of this embodiment, the molar ratio of Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Ile-OH, Fmoc-Phe-OH and Fmoc-Rink Amide MBHA Resin is 4:1, Fmoc-Rink Amide MBHA Resin and 1-hydroxy phenylpropyl triazole, benzotriazole-N, the molar ratio of N, N ', N ' -tetramethyluronium tetrafluoroborate to N, N ' -diisopropylethylamine was 1:4:4:4, and the other steps of this step were the same as in example 1.
In step 2 of this example, the molar ratio of Fmoc-Arg (pbf) -OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Met-OH and 2-chlorotrityl chloride resin was 4:1, and the molar ratio of 2-chlorotrityl chloride resin to 1-hydroxyphenyltriazole, benzotriazol-N, N, N ', N ' -tetramethyluronium tetrafluoroborate and N, N ' -diisopropylethylamine was 1:4:4, and the other steps of this example were the same as in example 1.
In steps 3 to 4 of this example, the molar ratio of 2-chlorotrityl chloride resin to Fmoc-amino acid, 1-hydroxy benzotriazole, benzotriazole-N, N '-tetramethyluronium tetrafluoroborate, N' -diisopropylethylamine was the same as in step 2 of this example, and the synthesis method was the same as in example 1. In step 5 of this example, the molar ratio of Fmoc-Lys (Boc) -Asn (Trt) -Gly-COOH, Fmoc-Val-Arg (pbf) -Leu-Phe-Ile-Glu (Otbu) -Trp-Leu-COOH, Fmoc-Met-Glu (Otbu) -Ala-COOH, Fmoc-Thr (tBu) -Ser (tBu) -Asp (Otbu) -Leu-Ser (tBu) -Lys (Boc) -Gln (Trt) -COOH, Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr (tBu) -Phe-COOH to Fmoc-Rink Amide MBHA Resin was 3:1, 1-hydroxy phenylpropyl triazole, N-diisopropyl carbodiimide to Fmoc-Rink Amide HA: 6:1, 21.78g of exenatide was obtained with a yield of 30.6%.
The structure of the synthesized product was characterized by a mass spectrometer using the same conditions as in example 1, and the molecular weight and molecular ion peak of the synthesized product were consistent with those of exenatide. And (3) adopting a liquid chromatograph to measure the purity of the synthesized exenatide, wherein the purity is more than 98%.

Claims (9)

1. The preparation method of the exenatide is characterized in that Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-CONH2The full-protection fragment of (a) is prepared from the following full-protection fragments of three fragments: Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-COOH, Thr-Ser-Asp-Leu-Ser-Lys-Gln-COOH, His-Gly-Glu-Gly-Thr-Phe-COOH;
wherein the full protective fragment is Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin, Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (Fpbf) -Leu-COOH, Fmoc-Thr (tBu) -Ser tBu) -Asp (Otbu) -Leu-Lys (Boc) -Gln (Trt) -COOH, Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr tBu) -Phe-COOH, respectively;
wherein the preparation method comprises the following steps:
(1) synthesis of Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin
Synthesizing Fmoc-Ser (tBu) -MBHA Resin
Removing Fmoc from Fmoc-Rink Amide MBHA Resin, adding N, N-dimethylformamide, Fmoc-Ser (tBu) -OH, 1-hydroxyphenyltriazole, benzotriazole-N, N, N '-tetramethyluronium tetrafluoroborate and N, N' -diisopropylethylamine, and reacting under the protection of nitrogen to obtain Fmoc-Ser (tBu) -MBHA Resin;
② Fmoc-Pro-Ser (tBu) -MBHA Resin
Removing Fmoc from the obtained Fmoc-Ser (tBu) -MBHA Resin by using a mixed solution of piperidine and N, N-dimethylformamide according to the method in the step (i), adding N, N-dimethylformamide, Fmoc-Pro-OH, 1-hydroxyphenyltriazole, benzotriazole-N, N, N '-tetramethyluronium tetrafluoroborate and N, N' -diisopropylethylamine, and reacting under the protection of nitrogen to obtain Fmoc-Pro-Ser (tBu) -MBHA Resin;
③ Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin
Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Ile-OH, Fmoc-Phe-OH according to the method of step (II) to obtain Fmoc-Phe-Ile-Glu (Ou) -Trp-Leu-Boc-Asn (Trt) -Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Lys (Trp-Leu) -Asn (Boc) -OH -Pro-Ser (tBu) -Rink Amide MBHA Resin;
(2) synthesis of Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-COOH
Synthesizing Fmoc-Leu-2-chlorotrityl chloride resin
Swelling 2-chlorotrityl chloride resin with dichloromethane, adding Fmoc-Leu-OH and N, N' -diisopropylethylamine, and reacting under the protection of nitrogen to obtain Fmoc-Leu-2-chlorotrityl chloride resin;
② Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-2-chlorotrityl chloride resin
Connecting Fmoc-Arg (pbf) -OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Met-OH to Fmoc-Leu-2-chlorotrityl chloride resin in this order according to the method of step (1) to obtain Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-2-chlorotrityl chloride resin;
cutting treatment
Adding a cutting fluid into Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-2-chlorotrityl chloride resin to react to obtain Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-COOH;
(3) synthesis of Fmoc-Thr (tBu) -Ser (tBu) -Asp (Otbu) -Leu-Ser (tBu) -Lys (Boc) -Gln (Trt) -COOH
Attaching Fmoc-Gln (Trt) -OH to the 2-chlorotrityl chloride resin according to the method of step (2) to obtain Fmoc-Gln (Trt) -OH, and attaching Fmoc-Lys (Boc) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Leu-OH, Fmoc-Asp (Otbu) -OH, Fmoc-Thr- (Ser tBu) -OH, Fmoc-Thr- (tBu) -OH to the Fmoc-Gln (Trt) -2-chlorotrityl chloride resin according to the method of step (2) to obtain Fmoc-Thr- (Thr) -Ser (tBu) -Asp (Otbu) -Leu-Ser (Leu) -Leu- (Lys (Boc) -Boc-Trn- (Trt) -OH, and Fmoc-Thr-Thru) -Leu- (Ser (Leu) -Leu- (Thr) -Leu- (Thr-Ser (Boc-Leu-Asp (Asp-Leu-Gln- (Thr) -Ser (Boc Cutting chlorotrityl chloride resin according to the method of the third step in the step (2) to obtain Fmoc-Thr (tBu) -Ser (tBu) -Asp (Otbu) -Leu-Ser (tBu) -Lys (Boc) -Gln (Trt) -COOH;
(4) synthesis of Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr (tBu) -Phe-COOH
Attaching Fmoc-Phe-OH to 2-chlorotrienyl chloride Resin according to the method of step (2) to obtain Fmoc-Phe-2-chlorotrienyl chloride Resin, and attaching Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Gly-OH, Fmoc-His Trt) -OH to Fmoc-Phe-2-chlorotrienyl chloride Resin in the order of step (2) to obtain Fmoc-His Trt (Gly) -Glu (Otbu) -Gly-Thr (tBu) -Phe-2-chlorotrienyl chloride Resin, Fmoc-His-Glu (Trou) -Gly-Thr) (Fmoc) -Phe-2-chlorotrienyl chloride Resin, Fmoc-His-Glu (Tru) (Gly-Thru) -Phe-2-chlorotrienyl chloride Resin, Fmoc-Phe-2-chlorotrienyl chloride Resin, Fmoc Resin, and (Tru) to cut the step (2-Thr) according to the, obtaining Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr (tBu) -Phe-COOH;
(5) assemble and connect to synthesize exenatide
Assembling and connecting Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin
Fmoc-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Pro-Ser (tBu) -Rink Amide MBHA Resin is subjected to Fmoc removal by using a mixed solution of piperidine and N, N-dimethylformamide, a mixed solution of N, N-dimethylformamide, dimethyl sulfoxide and N-methylpyrrolidone and Fmoc-Met-Glu (Otbu) -Glu (tbOu) -Ala-Val-Arg (pbf) -Leu-COOH, 1-hydroxyphenyltriazole and N, N-diisopropylcarbodiimide are added, and the mixture is reacted under the protection of nitrogen to obtain Fmoc-Met-Glu (Otbu) -Ala-Val) -Leu-Phe-Ile-glu (otbu) -Trp-Leu-lys (boc) -asn (trt) -Gly-Pro-ser (tbu) -Gly-Ala-Pro-ser (tbu) -Rink Amide MBHA Resin;
② assembling and connecting exenatide
According to the assembly and ligation method of step (r), Fmoc-Met-Glu (Otbu) -Ala-Val-Arg (pbf) -Leu-Phe-Ile-Glu (Otbu) -Trp-Leu-Lys (Boc) -Asn (Trt) -Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -Rink Amide HA Resin is sequentially assembled and ligated with Fmoc-Thr (tBu) -Ser (tBu) -Asp (Otbu) -Leu-Lys (Ser tBu) -Lys (Boc) -Gln (t) -COOH, Fmoc-His (Trt) -Gly-Glu (Otbu) -Gly-Thr-COOH (Phe-COOH), removing Fmoc from the mixed solution of piperidine and N, N-dimethylformamide to obtain exenatide resin, stirring the resin with cutting fluid at room temperature, filtering, and precipitating with cold anhydrous ether to obtain crude exenatide.
2. The method for producing exenatide as claimed in claim 1, wherein, in (1), a molar ratio of 1-hydroxyphenyltriazole, benzotriazole-N, N '-tetramethyluronium tetrafluoroborate, N' -diisopropylethylamine to Fmoc-Rink Amide MBHA Resin is (2-4): (2-4): 1.
3. The method for producing exenatide as claimed in claim 1, wherein the mixture of piperidine and N, N-dimethylformamide is a mixture of piperidine and N, N-dimethylformamide in a volume ratio of 1: 4.
4. The method for preparing exenatide as claimed in claim 1, wherein the molar ratio of Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Glu (Ou) -OH, Fmoc-Ile-OH, Fmoc-Phe-OH and Fmoc-Rink Amide MBResin of step (1) is 2:1, the molar ratio of the Fmoc-Rink Amide MBHA Resin to 1-hydroxyphenyltriazole, benzotriazole-N, N, N, N '-tetramethyluronium tetrafluoroborate and N, N' -diisopropylethylamine is 1:2:2: 2.
5. The method for producing exenatide as claimed in claim 1, wherein, in (1) of step (2), the molar ratio of 2-chlorotrityl chloride resin to Fmoc-Leu-OH, N' -diisopropylethylamine is 1:1: 4.
6. The method for producing exenatide peptide according to claim 1, wherein, in step (2), the molar ratio of Fmoc-arg (pbf) -OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-glu (otbu) -OH, Fmoc-Met-OH, and 2-chlorotrityl chloride resin is 2:1, 2-chlorotrityl chloride resin to 1-hydroxyphenyltriazole, benzotriazole-N, -tetramethyluronium borate, N-diisopropylethylamine is 1:2:2:2, respectively.
7. The method for preparing exenatide as claimed in claim 1, wherein the cleavage solution used in the third step of step (2) is a mixture of trifluoroethanol, acetic acid and dichloromethane in a volume ratio of 2:1: 7.
8. The process for producing exenatide as claimed in claim 1, wherein, in (5), the volume ratio of N, N-dimethylformamide to dimethylsulfoxide and N-methylpyrrolidone is 1:1: 1.
9. The method of producing exenatide as claimed in claim 1, wherein the cleavage solution in step (5) is a cleavage solution comprising, by mass, 83% trifluoroacetic acid, 5% phenol, 4% thioanisole, 3% water, and 5% triisopropylsilane.
CN201611247180.9A 2016-12-29 2016-12-29 Preparation method of exenatide and product thereof Active CN106632655B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201611247180.9A CN106632655B (en) 2016-12-29 2016-12-29 Preparation method of exenatide and product thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201611247180.9A CN106632655B (en) 2016-12-29 2016-12-29 Preparation method of exenatide and product thereof

Publications (2)

Publication Number Publication Date
CN106632655A CN106632655A (en) 2017-05-10
CN106632655B true CN106632655B (en) 2021-02-02

Family

ID=58836365

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201611247180.9A Active CN106632655B (en) 2016-12-29 2016-12-29 Preparation method of exenatide and product thereof

Country Status (1)

Country Link
CN (1) CN106632655B (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107857796B (en) * 2017-11-29 2021-05-04 陕西慧康生物科技有限责任公司 Synthesis method of trifluoroacetyl tripeptide-2
CN107903305B (en) * 2017-12-25 2020-11-13 陕西慧康生物科技有限责任公司 Method for synthesizing dessorrel by solid-liquid combination
EP3986919A1 (en) * 2019-06-18 2022-04-27 Fresenius Kabi iPSUM S.r.l. Process for the manufacture of glucagon
CN110372784A (en) * 2019-07-29 2019-10-25 深圳佳肽生物科技有限公司 The synthetic method and application of Exenatide

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103333237B (en) * 2013-05-07 2015-03-11 海南双成药业股份有限公司 Synthesis of exenatide through solid phase fragment method
CN103613656B (en) * 2013-11-20 2015-03-04 陕西东大生化科技有限责任公司 Solid-phase fragment synthetic method of exenatide

Also Published As

Publication number Publication date
CN106632655A (en) 2017-05-10

Similar Documents

Publication Publication Date Title
US11518794B2 (en) Synthesis method for liraglutide with low racemate impurity
EP2757107B1 (en) Method for solid phase synthesis of liraglutide
CN106699871B (en) Preparation method of liraglutide
CN109180801B (en) Method for synthesizing Somalutide
CN111670194B (en) Preparation of glucagon peptides
CN110372785B (en) Synthesis method of Somalutide
CN106632655B (en) Preparation method of exenatide and product thereof
CN110294800B (en) Preparation method of somaglutide
US20130289241A1 (en) Method for preparing exenatide
CN103613656B (en) Solid-phase fragment synthetic method of exenatide
CN107056927B (en) Preparation method of liraglutide
US20110046349A1 (en) Process for the production of exenatide and of an exenatide analogue
CN106478805B (en) Preparation method of GLP-1 derivative
CN113801197B (en) Preparation method of procalcitonin
WO2023089594A1 (en) Process for the preparation of tirzepatide or pharmaceutically acceptable salt thereof
CN113754753B (en) Synthetic method of somalupeptide
WO2021249564A1 (en) Semaglutide derivative, and preparation method therefor and application thereof
EP4166573A1 (en) Insulin degludec derivative, preparation method therefor, and application thereof
KR20210102362A (en) Improved process for making plecanatide
CN117106055A (en) Synthesis method of telipopeptide
CN106749611B (en) Preparation method of exenatide and product thereof
CN106749610A (en) A kind of preparation method of Exenatide and products thereof
CN103992401B (en) Method for preparing exenatide
EP3398959B1 (en) Method for preparing lixisenatide
WO2014032257A1 (en) Method for preparing bivalirudin

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant