WO2017114192A1 - 一种利西拉来的制备方法 - Google Patents

一种利西拉来的制备方法 Download PDF

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WO2017114192A1
WO2017114192A1 PCT/CN2016/110374 CN2016110374W WO2017114192A1 WO 2017114192 A1 WO2017114192 A1 WO 2017114192A1 CN 2016110374 W CN2016110374 W CN 2016110374W WO 2017114192 A1 WO2017114192 A1 WO 2017114192A1
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fmoc
boc
lys
pro
ser
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PCT/CN2016/110374
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English (en)
French (fr)
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陈新亮
宓鹏程
陶安进
袁建成
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深圳翰宇药业股份有限公司
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Priority to US16/067,067 priority Critical patent/US20200277328A1/en
Priority to EP16880982.0A priority patent/EP3398959B1/en
Publication of WO2017114192A1 publication Critical patent/WO2017114192A1/zh

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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
    • C07K1/06—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
    • C07K1/061—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 protecting groups
    • C07K1/062—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 protecting groups for alpha- or omega-carboxy functions
    • 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
    • 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
    • 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
    • C07K1/10—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using coupling 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
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • 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
    • C07K1/061—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 protecting groups

Definitions

  • the invention relates to the technical field of polypeptide drug synthesis, in particular to a preparation method of lixisenatide.
  • Lixisenatide is a glucagon-like peptide-1 (GLP-1) receptor agonist based on the C-terminal 6 lysine residues of the GLP-1 analogue Exendin-4 with hypoglycemic activity.
  • GLP-1 glucagon-like peptide-1
  • the novel GLP-1 analog synthesized by the modification of the base can tolerate the degradation of dipeptidyl peptidase IV (DPP4) in vivo.
  • DPP4 dipeptidyl peptidase IV
  • lixisenatide is indicated for the use of metformin, sulfonylureas, thiazolidinediones, metformin and sulfonylureas, metformin and thiazolidinediones in combination with type II diabetes.
  • adjuvant therapy for patients who cannot effectively control blood sugar to improve control of blood sugar is indicated.
  • a conventional method for synthesizing lixisenatide is a solid phase stepwise synthesis (SPPS) method, which has the characteristics of simple operation and low equipment requirements.
  • SPPS solid phase stepwise synthesis
  • the yield of the solid phase stepwise synthesis method is low, and the peptide tends to bend the secondary structure on the synthetic resin, causing the reaction site to be wrapped, and the resulting lixisenatide is coarse.
  • the peptide has low purity and complicated impurities, which makes it difficult to obtain purification of the product, and it is difficult to obtain a higher purity lixisenatide.
  • fragment synthesis is a commonly used method in solid phase synthesis.
  • Fragment synthesis method is to prepare a fully-protected peptide fragment by directly coupling the sequence to the designated peptide resin according to the amino acid sequence, thereby avoiding the amino acid site which is difficult to couple, and avoiding the coupling of amino acids one by one.
  • a side reaction such as a defect can obtain a crude peptide having a relatively high purity and reduce the difficulty of purification.
  • the fragment synthesis method uses a conventional resin, and a large amount of the expensive 2-CTC resin is required to prepare a fragment peptide resin, and the 2-CTC resin has high acid sensitivity, and the peptide is easily detached from the resin when coupled, thereby causing The yield of the prepared fragments is not high, the operation steps are relatively cumbersome, and the production cost is also greatly improved.
  • the technical problem to be solved by the present invention is to provide a preparation method of lixisenatide, which is simple in operation, high in total yield, and high in purity of the obtained product.
  • the invention provides a preparation method of lixisenatide, comprising:
  • Step 1 Solid phase synthesis of Fmoc-Lys-resin
  • Step 2 coupling a amino acid or a dipeptide to the Fmoc-Lys-resin according to a peptide sequence of lixisenatide to prepare a lixisenatide resin;
  • the dipeptide is selected from the group consisting of -Gly-Thr, -Phe-Thr , -Thr-Ser, -Leu-Ser, -Ser-Ser or -Pro-Ser;
  • Step 3 The lixisenatide resin is cracked to obtain lixisenatide.
  • the method provided by the present invention will be the 4th to 5th positions of the lixisenatide peptide sequence (-Gly-Thr, denoted as dipeptide A), and the 6th to 7th position (-Phe-Thr, denoted as dipeptide B), 7 to 8 (-Thr-Ser, denoted as dipeptide C), 10 to 11 (-Leu-Ser, denoted as dipeptide D), 32 to 33 (-Ser-Ser, denoted as dipeptide E) or the amino acids of positions 31 to 32 and 37 to 37 (-Pro-Ser, denoted as dipeptide F) are coupled with a dipeptide as a raw material, and the selected dipeptide raw material is dipeptide A to F.
  • One or more sites that do not use a dipeptide as a raw material are coupled using a single amino acid as a raw material.
  • the method can effectively prevent the rotation of the peptide bond, inhibit the shrinkage of the peptide chain curling agent, and fully expose the active functional group (primary amine), thereby facilitating the coupling of amino acids and reducing the occurrence of side reactions such as defects.
  • Coupling-Pro-Ser uses Fmoc-Pro-Ser (PSI ME, ME Pro)-OH.
  • Fmoc-Gly-Thr (PSI ME, ME Pro)-OH is as in Formula II-a;
  • Fmoc-Phe-Thr (PSI ME, ME Pro)-OH has the structure of formula II-b;
  • Fmoc-Leu-Ser PSI ME, ME Pro
  • Fmoc-Ser(tBu)-Ser(PSI ME, ME Pro)-OH is as in Formula II-e;
  • Fmoc-Pro-Ser PSI ME, ME Pro
  • Formula II-f The structure of Fmoc-Pro-Ser (PSI ME, ME Pro)-OH is as in Formula II-f.
  • the resin of the Fmoc-Lys-resin is Rink Amide resin, Rink Amide-MBHA resin, Rink Amide-AM resin or Siber resin.
  • the protective group of Lys is Boc.
  • the degree of substitution of the Fmoc-Lys(Boc)-resin is from 0.1 mmol/g to 0.6 mmol/g.
  • the degree of substitution ranges from 0.2 mmol/g to 0.4 mmol/g.
  • the coupled coupling agent is a mixture of HOBt and DIC; wherein the molar ratio of HOBt to DIC is 1:1.
  • the coupling step comprises: after removing the Fmoc protection, the coupling reaction is carried out by mixing the mixture of DCM and DMF as a solvent with a coupling agent.
  • the molar ratio of the deprotected resin to the amino acid to be coupled is 1:3.
  • the coupling step is repeated until the entire peptide chain is synthesized.
  • the conditions of the coupling reaction are room temperature reaction for 2 h.
  • the room temperature is from 10 ° C to 30 ° C.
  • the coupling reaction is continued for 1 h if the coupling reaction is incomplete.
  • the mixture was cooled with methanol, and the resin was vacuum dried overnight.
  • the cleavage cleavage agent is cleavable with TFA cleavage agent comprising Component B;
  • B component is selected from the PhSMe, PhOMe, EDT, H 2 O, TIS , or PhOH.
  • the volume ratio of TFA, PhSMe, PhOMe, EDT, H 2 O, TIS, PhOH in the lysing agent is (80 to 90): (0 to 5): (0 to 3): (0 to 5): ( 0 to 5): (0 to 2): (0 to 5).
  • the volume ratio of TFA, PhSMe, EDT, TIS, H 2 O in the lysate is 84:5:5:5:1.
  • the volume ratio of TFA, PhSMe, EDT, H 2 O in the lysing agent is 91:3:3:3.
  • the volume ratio of TFA, PhSMe, EDT, PhOMe in the lysing agent is 90:5:3:2.
  • the volume ratio of TFA, EDT, H 2 O in the lysing agent is 90:5:5.
  • the volume ratio of TFA, PhSMe, PhOH, EDT, H 2 O in the lysing agent is 85:5:3:5:2.
  • the step of lysing comprises: mixing the lixisenatide peptide resin with the lysate, and lysing at room temperature for 2.5 h to 3 h, washing the resin with TFA, and precipitating with anhydrous diethyl ether to obtain a linear crude peptide of lixisenatide.
  • the temperature of the precipitate of anhydrous diethyl ether is from 0 to 4 °C.
  • the mass-to-volume ratio of the lixisenatide resin to the lysate is from 1:8 to 15.
  • step 3 further comprises the steps of purifying and salt turning.
  • the purified chromatogram uses NOVASEP RP-HPLC system with a detection wavelength of 220 nm, the column is a reverse phase C18 column, the mobile phase A phase is a 0.1% by volume aqueous solution of TFA, and the mobile phase B phase is acetonitrile.
  • the elution gradient was 18% acetonitrile-48% acetonitrile for 45 min, 48% isocratic for 15 min: after purification, the salt was transferred by salt chromatography.
  • the specific salt is converted to acetate, hydrochloride, citrate, phosphate, trifluoroacetate, sodium, potassium or ammonium.
  • dipeptide A dipeptide A, dipeptide C, dipeptide D, dipeptide E, dipeptide F are introduced to prepare lixisenatide.
  • the coupling is specifically coupled in sequence: Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-Ser( PSI ME, ME Pro)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-Ser(PSI ME, ME Pro)-OH, Fmoc-Pro-OH , Fmoc-Gly-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu
  • dipeptide A dipeptide A, dipeptide B, dipeptide D, dipeptide F are introduced to prepare lixisenatide.
  • the coupling is specifically coupled in sequence: Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc) -OH, Fmoc-Pro-Ser (PSI ME, ME Pro)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-Ser (PSI ME, ME Pro)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp ( Boc)-OH, Fmoc-
  • dipeptide A dipeptide A, dipeptide B, dipeptide D, dipeptide E are introduced to prepare lixisenatide.
  • the coupling is specifically coupled in sequence: Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc) -OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-Ser (PSI ME, ME Pro ) -OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-
  • dipeptide B only dipeptide B, dipeptide D, dipeptide E are introduced to prepare lixisenatide.
  • the coupling is specifically coupled in sequence: Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc) -OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-Ser (PSI ME, ME Pro ) -OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp ( Boc)-OH,
  • dipeptide C dipeptide E is introduced to prepare lixisenatide.
  • the coupling is specifically coupled in sequence: Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc) -OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-Ser (PSI ME, ME Pro ) -OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp ( Boc)-OH,
  • only dipeptide E is introduced to prepare lixisenatide.
  • the coupling is specifically coupled in sequence: Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc) -OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-Ser (PSI ME, ME Pro ) -OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp ( Boc)-OH,
  • only dipeptide D is introduced to prepare lixisenatide.
  • the coupling is specifically coupled in sequence: Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc) -OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu ) -OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp ( Boc)
  • dipeptide A dipeptide A, dipeptide B, dipeptide D, dipeptide E, dipeptide F are introduced to prepare lixisenatide.
  • the coupling is specifically coupled in sequence: Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc) -OH, Fmoc-Pro-Ser (PSI ME, ME Pro)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-Ser (PSI ME, ME Pro ) -OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp ( Boc)-OH, Fmo
  • the present invention is coupled to the peptide sequence by using a specific protected serine dipeptide as a raw material, and the peptide structure can be effectively prevented from rotating due to the formation of a proline-like cyclic structure.
  • the contraction of the peptide chain curling agent is inhibited, and the active functional group (primary amine) is sufficiently exposed, thereby facilitating the coupling of amino acids and reducing the occurrence of side reactions such as defects.
  • the yield of the sperm peptide of the present invention can be as high as 25.8% to 28.8%, which is superior to the sequential coupling method (about 15.4%). Meanwhile, the purity of the sperm peptide of the present invention was 99.3%. The yield is guaranteed while ensuring purity.
  • Figure 1 shows the mass spectrum of the crude peptide prepared in Example 1
  • Figure 2 shows the mass spectrum of the crude peptide prepared in Comparative Example 1.
  • the invention provides a preparation method of lixisenatide, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application of the present invention may be modified or combined and modified to achieve and apply the present invention without departing from the scope of the present invention. Invention technology.
  • the reagents or instruments used in the present invention are all commercially available, and are commercially available.
  • the Fmoc-Lys(Boc)-Rink Amide-MBHA Resin 421g (230mmol) with a substitution degree of 0.406mmol/g prepared by the method of 1.1 was added to the solid phase reaction column, washed twice with DMF, and swelled with DMF for 30 minutes. The Fmoc protection was removed with DBLK and then washed 6 times with DMF.
  • the crude peptide weight yield was 99.4% and the HPLC purity was 69.1%.
  • lixisenatide crude peptide obtained in 1.3 and purify it. After dissolving, it adopts NOVASEP RP-HPLC system with wavelength of 220nm.
  • the column is reversed phase C18 column. It is purified by conventional 0.1% TFA/water and acetonitrile mobile phase system. Peak fraction, concentrated by rotary evaporation, lyophilized
  • the lixisenatide sperm peptide was 12.6 g, the HPLC purity was 99.3%, and the phage yield was 28.8%.
  • the Fmoc-Lys(Boc)-Rink Amide Resin 421g (150mmol) prepared by the method of 2.1 was replaced by a solid phase reaction column, washed twice with DMF, and swollen with DMF for 30 minutes. DBLK was removed from Fmoc protection and then washed 6 times with DMF. 211.3 g (450 mmol) of Fmoc-Lys(Boc)-OH, 64.8 g (473 mmol) of HOBt, and 63.9 g (473 mmol) of DIC were dissolved in a mixed solution of DCM and DMF in a volume ratio of 1:1, and added to a solid phase reaction column.
  • the reaction was carried out at room temperature for 2 h (the end point of the reaction was determined by the ninhydrin method. If the resin was colorless and transparent, the reaction was complete, and the resin developed color, indicating that the reaction was incomplete, and the coupling reaction was further carried out for 1 h until the resin was detected to be transparent).
  • the crude peptide weight yield was 101.2% and the HPLC purity was 66.6%.
  • lixisenatide crude peptide obtained in 2.3 and purify it. After dissolving, it adopts NOVASEP RP-HPLC system with wavelength of 220nm. The column is reversed C18 column. It is purified by conventional 0.1% TFA/water and acetonitrile mobile phase system. The peak fraction was concentrated by rotary evaporation, and lyophilized to obtain 12.4 g of lixisenatide sperm peptide, HPLC purity was 99.1%, and the yield of phage was 26.4%.
  • the reaction was stopped when the reaction of the resin was transparent, and the DMF was washed 3 times, and the methanol was shrunk and dried under reduced pressure to obtain 368 g of Fmoc-Lys(Boc)-Siber Resin, and the degree of substitution of the resin was 0.323 mmol/g.
  • the Fmoc-Lys(Boc)-Siber Resin 368g (54mmol) with a degree of substitution of 0.323mmol/g prepared by the method of 3.1 was added to the solid phase reaction column, washed twice with DMF, and swollen with DMF for 30 minutes, then DBLK was used. The Fmoc protection was removed and then washed 6 times with DMF.
  • lixisenatide crude peptide obtained in 3.3 and purify it. After dissolving, it adopts NOVASEP RP-HPLC system with wavelength of 220nm.
  • the column is reversed phase C18 column. It is purified by conventional 0.1% TFA/water and acetonitrile mobile phase system.
  • the peak fraction was concentrated by rotary evaporation, and lyophilized to obtain 13.7 g of lixisenatide quercetin, the purity of HPLC was 99.2%, and the yield of phage was 27.4%.
  • the Fmoc-Lys(Boc)-Rink Amide-MBHA Resin 143g (32mmol) prepared by the method of 4.1 was replaced by a solid phase reaction column, washed twice with DMF, and swollen with DMF for 30 minutes. , use DBLK to remove Fmoc protection, and then use 6 times of DMF washing, 45.6g (96mmol) Fmoc-Lys(Boc)-OH, 13.8g (101mmol) HOBt, 12.7g (101mmol) DIC was dissolved in a mixture of DCM and DMF in a volume ratio of 1:1, and added to the solid.
  • reaction column react at room temperature for 2 h (the end point of the reaction is determined by the ninhydrin method. If the resin is colorless and transparent, the reaction is complete, the resin develops color, indicating that the reaction is incomplete, and the coupling reaction is required for 1 h until the resin is transparent) .
  • the crude peptide weight yield was 102.6% and the HPLC purity was 67.9%.
  • the Fmoc-Lys(Boc)-Rink Amide-AM Resin 207g (105mmol) prepared by the method of 5.1 was replaced by 0.58 mmol/g, added to the solid phase reaction column, washed twice with DMF, and swollen with DMF for 30 minutes. The Fmoc protection was removed with DBLK and then washed 6 times with DMF. 147.6 g (315 mmol) of Fmoc-Lys(Boc)-OH, 44.8 g (330 mmol) of HOBt, 41.6 g (330 mmol) of DIC were dissolved in a mixed solution of DCM and DMF in a volume ratio of 1:1, and added to a solid phase reaction column.
  • the reaction was carried out at room temperature for 2 h (the end point of the reaction was determined by the ninhydrin method. If the resin was colorless and transparent, the reaction was complete, and the resin developed color, indicating that the reaction was incomplete, and the coupling reaction was further carried out for 1 h until the resin was detected to be transparent).
  • lixisenatide crude peptide obtained in 5.3 and purify it. After dissolving, it adopts NOVASEP RP-HPLC system with wavelength of 220nm. The column is reversed C18 column. It is purified by conventional 0.1% TFA/water and acetonitrile mobile phase system. The peak fraction was concentrated by rotary evaporation, and lyophilized to obtain 11.9 g of lixisenatide quercetin, the purity of HPLC was 99.3%, and the yield of phage was 28.1%.
  • the Fmoc-Lys(Boc)-Rink Amide-AM Resin 137g (70mmol) prepared by the method of 6.1 was replaced by 0.508mmol/g, added to the solid phase reaction column, washed twice with DMF, and swollen with DMF for 30 minutes. The Fmoc protection was removed with DBLK and then washed 6 times with DMF. 98.5 g (210 mmol) of Fmoc-Lys(Boc)-OH, 30.6 g (220 mmol) of HOBt, 27.9 g (220 mmol) of DIC were dissolved in a mixed solution of DCM and DMF in a volume ratio of 1:1, and added to a solid phase reaction column.
  • the reaction was carried out at room temperature for 2 h (the end point of the reaction was determined by the ninhydrin method. If the resin was colorless and transparent, the reaction was complete, and the resin developed color, indicating that the reaction was incomplete, and the coupling reaction was further carried out for 1 h until the resin was detected to be transparent).
  • the crude peptide weight yield was 99.6% and the HPLC purity was 61.8%.
  • This example only introduces dipeptide D to prepare lixisenatide.
  • Fmoc-Lys(Boc)-Rink prepared by the method of 7.1 with an alternative degree of 0.508 mmol/g Amide-AM Resin 137 g (70 mmol) was added to a solid phase reaction column, washed twice with DMF, and the resin was swollen with DMF for 30 minutes, then Fmoc protected with DBLK, and then washed 6 times with DMF.
  • lixisenatide crude peptide obtained in 7.3 and purify it. After dissolving, it adopts NOVASEP RP-HPLC system with wavelength of 220nm.
  • the column is reversed phase C18 column. It is purified by conventional 0.1% TFA/water and acetonitrile mobile phase system. The peak fraction, concentrated by rotary evaporation, and lyophilized to obtain 12.8 g of lixisenatide sperm peptide, HPLC purity 99.1%, and phage yield 26.6%.
  • the F0oc-Lys(Boc)-Rink Amide-AM Resin 136g (70mmol) prepared by the method of 8.1 was replaced by 0.508mmol/g, added to the solid phase reaction column, washed twice with DMF, and swollen with DMF for 30 minutes. The Fmoc protection was removed with DBLK and then washed 6 times with DMF. 98.5 g (210 mmol) of Fmoc-Lys(Boc)-OH, 31.6 g (220 mmol) of HOAt, 109.5 g (210 mmol) of PyAOP, 54.3 g (420 mmol) of DIPEA were dissolved in a mixture of DCM and DMF in a volume ratio of 1:1.
  • lixisenatide crude peptide obtained in 8.3 and purify it. After dissolving, it adopts NOVASEP RP-HPLC system with wavelength of 220nm.
  • the column is reversed phase C18 column. It is purified by conventional 0.1% TFA/water and acetonitrile mobile phase system.
  • the peak fraction was concentrated by rotary evaporation, and lyophilized to obtain 14.5 g of lixisenatide quercetin, the purity of HPLC was 99.2%, and the yield of phage was 27.9%.
  • the Fmoc-Lys(Boc)-Rink Amide-AM Resin 462g (230mmol) prepared by the D1.1 method was replaced by 0.51mmol/g, added to the solid phase reaction column, washed twice with DMF, and swollen with DMF 30. After a minute, Fmoc protection was removed with DBLK and then washed 6 times with DMF.
  • 323.6 g (690 mmol) of Fmoc-Lys(Boc)-OH, 98.1 g (725 mmol) of HOBt, 92.4 g (724 mmol) of DIC were dissolved in a mixed solution of DCM and DMF in a volume ratio of 1:1, and added to a solid phase reaction column.
  • the reaction was carried out at room temperature for 2 h (the end point of the reaction was determined by the ninhydrin method. If the resin was colorless and transparent, the reaction was complete, and the resin developed color, indicating that the reaction was incomplete, and the coupling reaction was further carried out for 1 h until the resin was detected to be transparent).
  • the crude lixisenatide peptide obtained in D1.3 was purified by 49g, dissolved in NOVASEP RP-HPLC system, the wavelength was 220nm, the column was reversed C18 column, and the conventional 0.1% TFA/water and acetonitrile mobile phase system was purified and then transferred to salt.
  • the peak fraction of the target was collected, concentrated by rotary evaporation, and lyophilized to obtain 7.5 g of lixisenatide quercetin, HPLC purity 98.6%, and phage yield 15.4%.
  • Example 7 4859.318 100.8% 59.6% 26.6% 99.1%
  • Example 8 4859.478 97.6% 68.9% 27.9% 99.2%
  • Comparative example 1 4859.288 96.8% 44.5% 15.4% 98.6%

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Abstract

一种利西拉来的制备方法。根据利西拉来肽的肽序结构，采用特定的保护的丝氨酸二肽作为原料偶联到肽序中，由于形成了类似脯氨酸的环状结构，能有效的防止肽键旋转，抑制肽链卷曲剂收缩，使活性官能团充分暴露，从而有利于氨基酸的偶联，减少缺损等副反应的发生。

Description

一种利西拉来的制备方法
本申请要求于2015年12月31日提交中国专利局、申请号为201511032042.4、发明名称为“一种利西拉来的制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及多肽药物合成技术领域，尤其涉及一种利西拉来的制备方法。
背景技术
利西拉来(Lixisenatide)是胰高血糖素样肽-1(GLP-1)受体激动剂，是基于具降糖活性的GLP-1类似物Exendin-4的C末端6个赖氨酸残基的修饰而合成的新型GLP-1类似物，能耐受体内二肽基肽酶Ⅳ(DPP4)的降解作用。利西拉来的结构如式I所示：
Figure PCTCN2016110374-appb-000001
其肽序为：
H-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-Ser-Lys-Lys-Lys-Lys-Lys-Lys-NH2
作为GLP-1类似物的一种，利西拉来适用于服用二甲双胍、磺脲类、噻唑烷二酮类、二甲双胍和磺脲类联用、二甲双胍和噻唑烷二酮类联用的II型糖尿病患者中，不能有效控制血糖的患者的辅助治疗，以改善对血糖的控制。
目前一种常规合成利西拉来的方法是固相逐步合成(SPPS)法，该方法具有操作简单、设备要求低等特点。但是因为利西拉来由44个氨基酸组成，固相逐步合成法的产品收率低，肽在合成树脂上容易发生二级结构的弯曲，导致反应位点被包裹，得到的利西拉来粗肽纯度低，杂质繁杂，给产品纯化带来困难，不易得到较高纯度的利西拉来。
对于解决偶联困难的氨基酸问题，片段合成法是固相合成方法中一种常用的手段。片段合成法是先将偶联困难的序列制备成全保护肽片段，按照氨基酸序列将其作为整体直接偶联到指定肽树脂上，从而避开偶联困难的氨基酸位点，避免逐个氨基酸偶联产生的缺损等副反应，能够获得纯度相对高的粗肽，降低纯化难度。但片段合成法采用了常规的树脂外，还需使用大量价格较昂贵的2-CTC树脂制备片段肽树脂，且2-CTC树脂酸敏感性高，偶联时肽容易从树脂上脱落，从而导致制备的片段收率不高，操作步骤相对繁琐，生产成本也大幅度提高。
发明内容
有鉴于此，本发明要解决的技术问题在于提供一种利西拉来的制备方法，该方法操作简便，总收率高，所得产物纯度较高。
本发明提供了一种利西拉来的制备方法，包括：
步骤1：固相合成Fmoc-Lys-树脂；
步骤2：根据利西拉来的肽序在所述Fmoc-Lys-树脂上偶联氨基酸或二肽制得利西拉来肽树脂；所述二肽选自-Gly-Thr、-Phe-Thr、-Thr-Ser、-Leu-Ser、-Ser-Ser或-Pro-Ser；
步骤3：所述利西拉来肽树脂经裂解制得利西拉来。
本发明提供的方法将利西拉来肽序中第4～5位(-Gly-Thr，记为二肽A)、第6～7位(-Phe-Thr，记为二肽B)、第7～8位(-Thr-Ser，记为二肽C)、第10～11位(-Leu-Ser，记为二肽D)、第32～33位(-Ser-Ser，记为二肽E)或第31～32位、第37～37位(-Pro-Ser，记为二肽F)的氨基酸以二肽为原料进行偶联，选用的二肽原料为二肽A～F中任一种或多种，不采用二肽为原料的位点则采用单个氨基酸为原料进行偶联。该方法能够有效防止肽键旋转，抑制肽链卷曲剂收缩，使活性官能团(伯胺)充分暴露，从而有利于氨基酸的偶联，减少缺损等副反应的发生。
在本发明的实施例中，
偶联-Gly-Thr采用Fmoc-Gly-Thr(PSI ME,ME Pro)-OH；
偶联-Phe-Thr采用Fmoc-Phe-Thr(PSI ME,ME Pro)-OH；
偶联-Thr-Ser采用Fmoc-Thr(tBu)-Ser(PSI ME,ME Pro)-OH；
偶联-Leu-Ser采用Fmoc-Leu-Ser(PSI ME,ME Pro)-OH；
偶联-Ser-Ser采用Fmoc-Ser(tBu)-Ser(PSI ME,ME Pro)-OH；
偶联-Pro-Ser采用Fmoc-Pro-Ser(PSI ME,ME Pro)-OH。
其中，Fmoc-Gly-Thr(PSI ME,ME Pro)-OH的结构如式II-a；
Fmoc-Phe-Thr(PSI ME,ME Pro)-OH的结构如式II-b；
Fmoc-Thr(tBu)-Ser(PSI ME,ME Pro)-OH的结构如式II-c；
Fmoc-Leu-Ser(PSI ME,ME Pro)-OH的结构如式II-d；
Fmoc-Ser(tBu)-Ser(PSI ME,ME Pro)-OH的结构如式II-e；
Fmoc-Pro-Ser(PSI ME,ME Pro)-OH的结构如式II-f。
Figure PCTCN2016110374-appb-000002
Figure PCTCN2016110374-appb-000003
在本发明中，Fmoc-Lys-树脂的树脂为Rink Amide树脂、Rink Amide-MBHA树脂、Rink Amide-AM树脂或Siber树脂。
其中，Lys的保护基为Boc。
作为优选，Fmoc-Lys(Boc)-树脂的替代度为0.1mmol/g～0.6mmol/g。优选的，替代度范围为0.2mmol/g～0.4mmol/g。
在本发明的实施例中，偶联的偶联剂为HOBt与DIC的混合物；其中HOBt与DIC的摩尔比为1:1。
在本发明中，所述偶联的步骤包括：脱除Fmoc保护后，以DCM和DMF的混合液为溶剂，与偶联剂混合后进行偶联反应。
作为优选，脱除保护的树脂与待偶联氨基酸的摩尔比为1:3。
重复偶联的步骤直至合成整个肽链。
在本发明中，偶联反应的条件为室温反应2h。所述室温为10℃～30℃。
作为优选，如偶联反应不完全则继续偶联反应1h。
在本发明中，偶联反应结束后以甲醇收缩，树脂真空干燥过夜。
在本发明的实施例中，裂解的裂解剂为裂解的裂解剂包括TFA与B 组分；所述B组分选自PhSMe、PhOMe、EDT、H2O、TIS或PhOH。
作为优选，裂解剂中TFA、PhSMe、PhOMe、EDT、H2O、TIS、PhOH的体积比为(80～90)：(0～5):(0～3):(0～5):(0～5)：(0～2)：(0～5)。
优选的，裂解液中TFA、PhSMe、EDT、TIS、H2O的体积比为84:5:5:5:1。
优选的，裂解剂中TFA、PhSMe、EDT、H2O的体积比为91:3:3:3。
优选的，裂解剂中TFA、PhSMe、EDT、PhOMe的体积比为90:5:3:2。
优选的，裂解剂中TFA、EDT、H2O的体积比为90:5:5。
优选的，裂解剂中TFA、PhSMe、PhOH、EDT、H2O的体积比为85:5:3:5:2。
在本发明中，裂解的步骤包括：将利西拉来肽树脂与裂解液混合，室温裂解2.5h～3h后，以TFA洗涤树脂、以无水乙醚沉淀，获得利西拉来线性粗肽。
作为优选，无水乙醚沉淀的温度为0～4℃。
作为优选，利西拉来肽树脂与裂解液的质量-体积比为1:8～15。
在本发明的实施例中，步骤3后还包括纯化、转盐的步骤。
具体的，纯化的色谱采用NOVASEP RP-HPLC系统，检测波长220nm，色谱柱为反相C18柱，流动相A相为体积分数为0.1％的TFA水溶液，流动相B相为乙腈。洗脱梯度为18％乙腈-48％乙腈45min，48％等度15min：纯化后经转盐色谱，转盐。
具体的转盐为转化为醋酸盐，盐酸盐，枸橼酸盐，磷酸盐，三氟乙酸盐，钠盐，钾盐或铵盐。
在一些实施例中，仅引入二肽A，二肽C、二肽D、二肽E、二肽F，制备利西拉来。
偶联具体为依次偶联：Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Pro-Ser(PSI ME，ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-Ser(PSI ME，ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Gly-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、 Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-Ser(PSI ME，ME Pro)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Thr(tBu)-Ser(PSI ME，ME Pro)-OH、Fmoc-Phe-OH、Fmoc-Gly-Thr(PSI ME，ME Pro)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH和Boc-His(Trt)-OH。
在一些实施例中，仅引入二肽A，二肽B、二肽D、二肽F，制备利西拉来。
偶联具体为依次偶联：Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Pro-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-Ser(PSI ME,ME Pro)-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-Ser(PSI ME,ME Pro)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Phe-Thr(PSI ME,ME Pro)-OH、Fmoc-Gly-Thr(PSI ME,ME Pro)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH和Boc-His(Trt)-OH。
在一些实施例中，仅引入二肽A，二肽B、二肽D、二肽E，制备利西拉来。
偶联具体为依次偶联：Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、 Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-Ser(PSI ME,ME Pro)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Phe-Thr(PSI ME,ME Pro)-OH、Fmoc-Gly-Thr(PSI ME,ME Pro)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH和Boc-His(Trt)-OH。
在一些实施例中，仅引入二肽B、二肽D、二肽E，制备利西拉来。
偶联具体为依次偶联：Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-Ser(PSI ME,ME Pro)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Phe-Thr(PSI ME,ME Pro)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH和Boc-His(Trt)-OH。
在一些实施例中，仅引入二肽C、二肽E，制备利西拉来。
偶联具体为依次偶联：Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、 Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Leu-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Thr(tBu)-Ser(PSI ME,ME Pro)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH和Boc-His(Trt)-OH。
在一些实施例中，仅引入二肽E，制备利西拉来。
偶联具体为依次偶联：Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Leu-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-Gly-OH和Boc-His(Trt)-OH。
在一些实施例中，仅引入二肽D，制备利西拉来。
偶联具体为依次偶联：Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、 Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-Ser(PSI ME,ME Pro)-OH、Fmoc-Leu-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-Gly-OH或Boc-His(Trt)-OH。
在一些实施例中，仅引入二肽A，二肽B，二肽D，二肽E，二肽F，制备利西拉来。
偶联具体为依次偶联：Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Pro-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-Ser(PSI ME,ME Pro)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Phe-Thr(PSI ME,ME Pro)-OH、Fmoc-Gly-Thr(PSI ME,ME Pro)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH和Boc-His(Trt)-OH。
本发明根据利西拉来肽的肽序结构，采用特定的保护的丝氨酸二肽作为原料偶联到肽序中，由于形成了类似脯氨酸的环状结构，能有效的防止肽键旋转，抑制肽链卷曲剂收缩，使活性官能团(伯胺)充分暴露，从而有利于氨基酸的偶联，减少缺损等副反应的发生。根据实验，本发明精肽收率可高达25.8％～28.8％，优于顺序逐个偶联方法(约15.4％)。同时，本发明精肽的纯度为99.3％。在保证了纯度的同时保证了收率。
附图说明
图1示实施例1制得制得粗肽的质谱图；
图2示对比例1制得制得粗肽的质谱图。
具体实施方式
本发明提供了一种利西拉来的制备方法，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。
本发明采用的试剂或仪器皆为普通市售品，皆可于市场购得。
其中，各材料的名称及缩写如表1：
表1各材料的名称及缩写
Fmoc 9-芴甲氧羰基
Resin 树脂
tBu 叔丁基
OtBu 叔丁氧基
Trt 三苯甲基
DCM 二氯甲烷
DBLK 20％六氢吡啶/DMF溶液
DIPEA N,N-二异丙基乙胺
HOBt 1-羟基苯并三唑
HOAt 1-羟基-7-偶氮苯并三氮唑
PyBOP 六氟磷酸苯并三唑-1-基-氧基三吡咯烷基
DMSO 二甲亚砜
HATU O-(7-偶氮苯并三氮唑-1-氧)-N,N,N’,N’-四甲基脲鎓六氟磷酸盐
HPLC 高效液相色谱
DMF N,N-二甲基甲酰胺
Figure PCTCN2016110374-appb-000004
下面结合实施例，进一步阐述本发明：
实施例1
本实施例仅引入二肽A，二肽C、二肽D、二肽E、二肽F，制备利西拉来。
1.1 Fmoc-Lys(Boc)-Rink Amide-MBHA Resin的制备
称取干燥Rink Amide-MBHA Resin 380g(替代度为0.45mmol/g)加入到固相反应柱中，首先DMF洗涤树脂2遍，再用2～3倍树脂床层体积DMF溶胀树脂30分钟，DMF洗涤3次，DCM洗涤2次，等待投料。
在冰浴冷却的条件下，将238.9g Fmoc-Lys(Boc)-OH(510mmol)、72.4g HOAt(536mmol)溶于DMF和DCM的混合溶剂中，待氨基酸溶解后，慢慢加入DIC 67.5g(535mmol)，活化3min后将反应液倒入反应柱中，鼓气搅拌反应；采用茚三酮检测树脂反应透明时停止反应，DMF洗涤3遍，甲醇收缩、减压干燥得Fmoc-Lys(Boc)-Rink Amide-MBHA Resin 421克，检测树脂替代度为0.406mmol/g。
1.2 lixisenatide肽树脂的制备
取1.1方法制备的替代度为0.406mmol/g的Fmoc-Lys(Boc)-Rink Amide-MBHA Resin 421g(230mmol)，加入固相反应柱中，用DMF洗涤2次，用DMF溶胀树脂30分钟后，用DBLK脱除Fmoc保护，然后用DMF洗涤6次，。将239.1g(510mmol)Fmoc-Lys(Boc)-OH，72.2g(536mmol)HOBt，67.7g(535mmol)DIC溶于体积比为1:1的DCM和DMF混合溶 液，加入固相反应柱中，室温反应2h(反应终点以茚三酮法检测为准，如果树脂无色透明，则反应完全，树脂显色，表示反应不完全，需再偶联反应1h至树脂检测透明)。
重复上述脱除Fmoc保护和加入相应氨基酸偶联的步骤，按照片段的顺序，采用一定的偶联方法，依次完成Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Pro-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Gly-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-Ser(PSI ME,ME Pro)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Thr(tBu)-Ser(PSI ME,ME Pro)-OH、Fmoc-Phe-OH、Fmoc-Gly-Thr(PSI ME,ME Pro)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH、Boc-His(Trt)-OH的偶联。反应结束后用甲醇收缩，树脂真空干燥过夜，得到2018克lixisenatide肽树脂。
1.3 lixisenatide肽树脂的裂解和精肽制备
取1.2得到的lixisenatide肽树脂700g置于裂解反应器中，以8ml/g(裂解液/树脂)的裂解液用量入裂解试剂(TFA：PhSMe：EDT：TIS：H2O＝84:5:5:5:1，(V/V))，室温搅拌2.5h。反应物用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤2次，合并滤液后减压浓缩。加入冰冻的无水乙醚沉淀、离心，无水乙醚洗涤粗肽滤饼3次，真空干燥得到白色粉末固体，lixisenatide粗肽341g，质谱(图1)MALDI-TOF：(M+H)+＝4859.216。粗肽重量收率为99.4％，HPLC纯度为69.1％。
取1.3中得到的lixisenatide粗肽44g进行精制，溶解后采用NOVASEP RP-HPLC系统，波长220nm，色谱柱为反相C18柱，常规0.1％TFA/水、乙腈流动相体系纯化后转盐，收集目的峰馏分，旋转蒸发浓缩，冻干得到 lixisenatide精肽12.6g，HPLC纯度99.3％，精肽收率28.8％。
实施例2
本实施例仅引入二肽A，二肽B、二肽D、二肽F，制备利西拉来。
2.1 Fmoc-Lys(Boc)-Rink Amide Resin的制备
称取干燥Rink Amide Resin356g(替代度为0.42mmol/g)加入到固相反应柱中，首先DMF洗涤树脂2遍，再用2～3倍树脂床层体积DMF溶胀树脂30分钟，DMF洗涤3次，DCM洗涤2次，等待投料。
在冰浴冷却的条件下，将210.9g Fmoc-Lys(Boc)-OH(450mmol)、63.8g HOBt(473mmol)、170.6g HBTU(450mmol)溶于DMF和DCM的混合溶剂中，待氨基酸溶解后，慢慢加入DIPEA 87.6g(675mmol)，活化3min后将反应液倒入反应柱中，鼓气搅拌反应；采用茚三酮检测树脂反应透明时停止反应，DMF洗涤3遍，甲醇收缩、减压干燥得Fmoc-Lys(Boc)-Rink Amide Resin 390克，检测树脂替代度为0.385mmol/g。
2.2lixisenatide肽树脂的制备
取2.1方法制备的替代度为0.385mmol/g的Fmoc-Lys(Boc)-Rink Amide Resin 421g(150mmol)，加入固相反应柱中，用DMF洗涤2次，用DMF溶胀树脂30分钟后，用DBLK脱除Fmoc保护，然后用DMF洗涤6次，。将211.3g(450mmol)Fmoc-Lys(Boc)-OH，64.8g(473mmol)HOBt，63.9g(473mmol)DIC溶于体积比为1:1的DCM和DMF混合溶液，加入固相反应柱中，室温反应2h(反应终点以茚三酮法检测为准，如果树脂无色透明，则反应完全，树脂显色，表示反应不完全，需再偶联反应1h至树脂检测透明)。
重复上述脱除Fmoc保护和加入相应氨基酸偶联的步骤，按照片段的顺序，采用一定的偶联方法，依次完成Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Pro-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-Ser(PSI ME,ME Pro)-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、 Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-Ser(PSI ME,ME Pro)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Phe-Thr(PSI ME,ME Pro)-OH、Fmoc-Gly-Thr(PSI ME,ME Pro)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH、Boc-His(Trt)-OH的偶联。反应结束后用甲醇收缩，树脂真空干燥过夜，得到1611克lixisenatide肽树脂。
2.3 lixisenatide肽树脂的裂解和精肽制备
取2.2得到的lixisenatide肽树脂600g置于裂解反应器中，以10ml/g(裂解液/树脂)的裂解液用量入裂解试剂(TFA：PhSMe：EDT：H2O＝91:3:3:3，(V/V))，室温搅拌2.5h。反应物用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤2次，合并滤液后减压浓缩。加入冰冻的无水乙醚沉淀、离心，无水乙醚洗涤粗肽滤饼3次，真空干燥得到白色粉末固体，lixisenatide粗肽251g，质谱MALDI-TOF：(M+H)+＝4859.017。粗肽重量收率为101.2％，HPLC纯度为66.6％。
取2.3中得到的lixisenatide粗肽47g进行精制，溶解后采用NOVASEP RP-HPLC系统，波长220nm，色谱柱为反相C18柱，常规0.1％TFA/水、乙腈流动相体系纯化后转盐，收集目的峰馏分，旋转蒸发浓缩，冻干得到lixisenatide精肽12.4g，HPLC纯度99.1％，精肽收率26.4％。
实施例3
本实施例仅引入二肽A，二肽B、二肽D、二肽E，制备利西拉来。
3.1 Fmoc-Lys(Boc)-Siber Resin的制备
称取干燥Siber Resin 155g(替代度为0.35mmol/g)加入到固相反应柱中，首先DMF洗涤树脂2遍，再用2～3倍树脂床层体积DMF溶胀树脂30分钟，DMF洗涤3次，DCM洗涤2次，等待投料。
在冰浴冷却的条件下，将77.6g Fmoc-Lys(Boc)-OH(165mmol)、23.5g  HOBt(173mmol)溶于DMF和DCM的混合溶剂中，待氨基酸溶解后，慢慢加入DIC 22.1g(173mmol)，活化3min后将反应液倒入反应柱中，鼓气搅拌反应；采用茚三酮检测树脂反应透明时停止反应，DMF洗涤3遍，甲醇收缩、减压干燥得Fmoc-Lys(Boc)-Siber Resin 368克，检测树脂替代度为0.323mmol/g。
3.2 lixisenatide肽树脂的制备
取3.1方法制备的替代度为0.323mmol/g的Fmoc-Lys(Boc)-Siber Resin 368g(54mmol)，加入固相反应柱中，用DMF洗涤2次，用DMF溶胀树脂30分钟后，用DBLK脱除Fmoc保护，然后用DMF洗涤6次，。将77.9g(165mmol)Fmoc-Lys(Boc)-OH，23.5g(173mmol)HOBt，22.4g(173mmol)DIC溶于体积比为1:1的DCM和DMF混合溶液，加入固相反应柱中，室温反应2h(反应终点以茚三酮法检测为准，如果树脂无色透明，则反应完全，树脂显色，表示反应不完全，需再偶联反应1h至树脂检测透明)。
重复上述脱除Fmoc保护和加入相应氨基酸偶联的步骤，按照片段的顺序，采用一定的偶联方法，依次完成Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-Ser(PSI ME,ME Pro)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Phe-Thr(PSI ME,ME Pro)-OH、Fmoc-Gly-Thr(PSI ME,ME Pro)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH、Boc-His(Trt)-OH的偶联。反应结束后用甲醇收缩，树脂真空干燥过夜，得到831克lixisenatide肽树脂。
3.3 lixisenatide肽树脂的裂解和精肽制备
取3.2得到的lixisenatide肽树脂800g置于裂解反应器中，以12ml/g(裂解液/树脂)的裂解液用量入裂解试剂(TFA：PhSMe：EDT：PhOMe＝90:5:3:2，(V/V))，室温搅拌3h。反应物用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤2次，合并滤液后减压浓缩。加入冰冻的无水乙醚沉淀、离心，无水乙醚洗涤粗肽滤饼3次，真空干燥得到白色粉末固体，lixisenatide粗肽241g，质谱MALDI-TOF：(M+H)+＝4859.512。粗肽重量收率为98.9％，HPLC纯度为68.4％。
取3.3中得到的lixisenatide粗肽50g进行精制，溶解后采用NOVASEP RP-HPLC系统，波长220nm，色谱柱为反相C18柱，常规0.1％TFA/水、乙腈流动相体系纯化后转盐，收集目的峰馏分，旋转蒸发浓缩，冻干得到lixisenatide精肽13.7g，HPLC纯度99.2％，精肽收率27.4％。
实施例4
本实施例仅引入二肽B、二肽D、二肽E，制备利西拉来。
4.1 Fmoc-Lys(Boc)-Rink Amide-MBHA Resin的制备
称取干燥Rink Amide-MBHA Resin 135g(替代度为0.24mmol/g)加入到固相反应柱中，首先DMF洗涤树脂2遍，再用2～3倍树脂床层体积DMF溶胀树脂30分钟，DMF洗涤3次，DCM洗涤2次，等待投料。
在冰浴冷却的条件下，将46.8g Fmoc-Lys(Boc)-OH(100mmol)、14.2g HOBt(105mmol)溶于DMF和DCM的混合溶剂中，待氨基酸溶解后，慢慢加入DIC 13.5g(105mmol)，活化3min后将反应液倒入反应柱中，鼓气搅拌反应；采用茚三酮检测树脂反应透明时停止反应，DMF洗涤3遍，甲醇收缩、减压干燥得Fmoc-Lys(Boc)-Siber Resin 143克，检测树脂替代度为0.223mmol/g。
4.2 lixisenatide肽树脂的制备
取4.1方法制备的替代度为0.223mmol/g的Fmoc-Lys(Boc)-Rink Amide-MBHA Resin 143g(32mmol)，加入固相反应柱中，用DMF洗涤2次，用DMF溶胀树脂30分钟后，用DBLK脱除Fmoc保护，然后用 DMF洗涤6次，将45.6g(96mmol)Fmoc-Lys(Boc)-OH，13.8g(101mmol)HOBt，12.7g(101mmol)DIC溶于体积比为1:1的DCM和DMF混合溶液，加入固相反应柱中，室温反应2h(反应终点以茚三酮法检测为准，如果树脂无色透明，则反应完全，树脂显色，表示反应不完全，需再偶联反应1h至树脂检测透明)。
重复上述脱除Fmoc保护和加入相应氨基酸偶联的步骤，按照片段的顺序，采用一定的偶联方法，依次完成Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-Ser(PSI ME,ME Pro)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Phe-Thr(PSI ME,ME Pro)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH、Boc-His(Trt)-OH的偶联。反应结束后用甲醇收缩，树脂真空干燥过夜，得到401克lixisenatide肽树脂。
4.3 lixisenatide肽树脂的裂解和精肽制备
取4.2得到的lixisenatide肽树脂400g置于裂解反应器中，以15ml/g(裂解液/树脂)的裂解液用量入裂解试剂(TFA：PhSMe：EDT：PhOH：H2O＝85:5:3:5:2，(V/V))，室温搅拌3h。反应物用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤2次，合并滤液后减压浓缩。加入冰冻的无水乙醚沉淀、离心，无水乙醚洗涤粗肽滤饼3次，真空干燥得到白色粉末固体，lixisenatide粗肽156g，质谱MALDI-TOF：(M+H)+＝4859.347。粗肽重量收率为102.6％，HPLC纯度为67.9％。
取4.3中得到的lixisenatide粗肽46g进行精制，溶解后采用NOVASEP  RP-HPLC系统，波长220nm，色谱柱为反相C18柱，常规0.1％TFA/水、乙腈流动相体系纯化后转盐，收集目的峰馏分，旋转蒸发浓缩，冻干得到lixisenatide精肽11.6g，HPLC纯度99.3％，精肽收率27.1％。
实施例5
本实施例仅引入二肽C、二肽E，制备利西拉来。
5.1 Fmoc-Lys(Boc)-Rink Amide-AM Resin的制备
称取干燥Rink Amide-AM Resin 547g(替代度为0.56mmol/g)加入到固相反应柱中，首先DMF洗涤树脂2遍，再用2～3倍树脂床层体积DMF溶胀树脂30分钟，DMF洗涤3次，DCM洗涤2次，等待投料。
在冰浴冷却的条件下，将431.2g Fmoc-Lys(Boc)-OH(918mmol)、130.5g HOBt(964mmol)溶于DMF和DCM的混合溶剂中，待氨基酸溶解后，慢慢加入DIC 121.5g(964mmol)，活化3min后将反应液倒入反应柱中，鼓气搅拌反应；采用茚三酮检测树脂反应透明时停止反应，DMF洗涤3遍，甲醇收缩、减压干燥得Fmoc-Lys(Boc)-Rink Amide-AM Resin 621克，检测树脂替代度为0.508mmol/g。
5.2 lixisenatide肽树脂的制备
取5.1方法制备的替代度为0.508mmol/g的Fmoc-Lys(Boc)-Rink Amide-AM Resin 207g(105mmol)，加入固相反应柱中，用DMF洗涤2次，用DMF溶胀树脂30分钟后，用DBLK脱除Fmoc保护，然后用DMF洗涤6次，。将147.6g(315mmol)Fmoc-Lys(Boc)-OH，44.8g(330mmol)HOBt，41.6g(330mmol)DIC溶于体积比为1:1的DCM和DMF混合溶液，加入固相反应柱中，室温反应2h(反应终点以茚三酮法检测为准，如果树脂无色透明，则反应完全，树脂显色，表示反应不完全，需再偶联反应1h至树脂检测透明)。
重复上述脱除Fmoc保护和加入相应氨基酸偶联的步骤，按照片段的顺序，采用一定的偶联方法，依次完成Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、 Fmoc-Gly-OH、Fmoc-Ser(tBu)-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Leu-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Thr(tBu)-Ser(PSI ME,ME Pro)-OH、Fmoc-Phe--OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH、Boc-His(Trt)-OH的偶联。反应结束后用甲醇收缩，树脂真空干燥过夜，得到1046克lixisenatide肽树脂。
5.3 lixisenatide肽树脂的裂解和精肽制备
取5.2得到的lixisenatide肽树脂1000g置于裂解反应器中，以10ml/g(裂解液/树脂)的裂解液用量入裂解试剂(TFA：EDT：H2O＝90:5:5，(V/V))，室温搅拌3h。反应物用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤2次，合并滤液后减压浓缩。加入冰冻的无水乙醚沉淀、离心，无水乙醚洗涤粗肽滤饼3次，真空干燥得到白色粉末固体，lixisenatide粗肽487g，质谱MALDI-TOF：(M+H)+＝4859.109。粗肽重量收率为100.6％，HPLC纯度为67.1％。
取5.3中得到的lixisenatide粗肽43g进行精制，溶解后采用NOVASEP RP-HPLC系统，波长220nm，色谱柱为反相C18柱，常规0.1％TFA/水、乙腈流动相体系纯化后转盐，收集目的峰馏分，旋转蒸发浓缩，冻干得到lixisenatide精肽11.9g，HPLC纯度99.3％，精肽收率28.1％。
实施例6
本实施例仅引入二肽E，制备利西拉来。
6.1 Fmoc-Lys(Boc)-Rink Amide-AM Resin的制备
称取干燥Rink Amide-AM Resin 547g(替代度为0.56mmol/g)加入到固相反应柱中，首先DMF洗涤树脂2遍，再用2～3倍树脂床层体积 DMF溶胀树脂30分钟，DMF洗涤3次，DCM洗涤2次，等待投料。
在冰浴冷却的条件下，将431.2g Fmoc-Lys(Boc)-OH(918mmol)、130.5g HOBt(964mmol)溶于DMF和DCM的混合溶剂中，待氨基酸溶解后，慢慢加入DIC 121.5g(964mmol)，活化3min后将反应液倒入反应柱中，鼓气搅拌反应；采用茚三酮检测树脂反应透明时停止反应，DMF洗涤3遍，甲醇收缩、减压干燥得Fmoc-Lys(Boc)-Rink Amide-AM Resin 621克，检测树脂替代度为0.508mmol/g。
6.2 lixisenatide肽树脂的制备
取6.1方法制备的替代度为0.508mmol/g的Fmoc-Lys(Boc)-Rink Amide-AM Resin 137g(70mmol)，加入固相反应柱中，用DMF洗涤2次，用DMF溶胀树脂30分钟后，用DBLK脱除Fmoc保护，然后用DMF洗涤6次。将98.5g(210mmol)Fmoc-Lys(Boc)-OH，30.6g(220mmol)HOBt，27.9g(220mmol)DIC溶于体积比为1:1的DCM和DMF混合溶液，加入固相反应柱中，室温反应2h(反应终点以茚三酮法检测为准，如果树脂无色透明，则反应完全，树脂显色，表示反应不完全，需再偶联反应1h至树脂检测透明)。
重复上述脱除Fmoc保护和加入相应氨基酸偶联的步骤，按照片段的顺序，采用一定的偶联方法，依次完成Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Leu-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-Gly-OH、Boc-His(Trt)-OH的偶联。反应结束后用甲醇收缩，树脂真空干燥过夜，得到728克lixisenatide肽树脂。
6.3 lixisenatide肽树脂的裂解和精肽制备
取6.2得到的lixisenatide肽树脂728g置于裂解反应器中，以10ml/g(裂解液/树脂)的裂解液用量入裂解试剂(TFA：EDT：H2O＝90:5:5，(V/V))，室温搅拌3h。反应物用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤2次，合并滤液后减压浓缩。加入冰冻的无水乙醚沉淀、离心，无水乙醚洗涤粗肽滤饼3次，真空干燥得到白色粉末固体，lixisenatide粗肽338.9g，质谱MALDI-TOF：(M+H)+＝4859.205。粗肽重量收率为99.6％，HPLC纯度为61.8％。
取6.3中得到的lixisenatide粗肽58g进行精制，溶解后采用NOVASEP RP-HPLC系统，波长220nm，色谱柱为反相C18柱，常规0.1％TFA/水、乙腈流动相体系纯化后转盐，收集目的峰馏分，旋转蒸发浓缩，冻干得到lixisenatide精肽14.9g，HPLC纯度99.2％，精肽收率25.8％。
实施例7
本实施例仅引入二肽D，制备利西拉来。
7.1 Fmoc-Lys(Boc)-Rink Amide-AM Resin的制备
称取干燥Rink Amide-AM Resin 547g(替代度为0.56mmol/g)加入到固相反应柱中，首先DMF洗涤树脂2遍，再用2～3倍树脂床层体积DMF溶胀树脂30分钟，DMF洗涤3次，DCM洗涤2次，等待投料。
在冰浴冷却的条件下，将431.2g Fmoc-Lys(Boc)-OH(918mmol)、130.5g HOBt(964mmol)溶于DMF和DCM的混合溶剂中，待氨基酸溶解后，慢慢加入DIC 121.5g(964mmol)，活化3min后将反应液倒入反应柱中，鼓气搅拌反应；采用茚三酮检测树脂反应透明时停止反应，DMF洗涤3遍，甲醇收缩、减压干燥得Fmoc-Lys(Boc)-Rink Amide-AM Resin 621克，检测树脂替代度为0.508mmol/g。
7.2 lixisenatide肽树脂的制备
取7.1方法制备的替代度为0.508mmol/g的Fmoc-Lys(Boc)-Rink  Amide-AM Resin 137g(70mmol)，加入固相反应柱中，用DMF洗涤2次，用DMF溶胀树脂30分钟后，用DBLK脱除Fmoc保护，然后用DMF洗涤6次。将98.5g(210mmol)Fmoc-Lys(Boc)-OH，31.6g(220mmol)HOAt，27.9g(220mmol)DIC溶于体积比为1:1的DCM和DMF混合溶液，加入固相反应柱中，室温反应2h(反应终点以茚三酮法检测为准，如果树脂无色透明，则反应完全，树脂显色，表示反应不完全，需再偶联反应1h至树脂检测透明)。
重复上述脱除Fmoc保护和加入相应氨基酸偶联的步骤，按照片段的顺序，采用一定的偶联方法，依次完成Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-Ser(PSI ME,ME Pro)-OH、Fmoc-Leu-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-Gly-OH、Boc-His(Trt)-OH的偶联。反应结束后用甲醇收缩，树脂真空干燥过夜，得到742克lixisenatide肽树脂。
7.3 lixisenatide肽树脂的裂解和精肽制备
取7.2得到的lixisenatide肽树脂742g置于裂解反应器中，以10ml/g(裂解液/树脂)的裂解液用量入裂解试剂(TFA：PhSMe：PhOH：EDT：H2O＝85:5:3:5:2，(V/V))，室温搅拌3h。反应物用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤2次，合并滤液后减压浓缩。加入冰冻的无水乙醚沉淀、离心，无水乙醚洗涤粗肽滤饼3次，真空干燥得到白色粉末固体，lixisenatide粗肽341.3g，质谱MALDI-TOF：(M+H)+＝4859.318。 粗肽重量收率为100.8％，HPLC纯度为59.6％。
取7.3中得到的lixisenatide粗肽48g进行精制，溶解后采用NOVASEP RP-HPLC系统，波长220nm，色谱柱为反相C18柱，常规0.1％TFA/水、乙腈流动相体系纯化后转盐，收集目的峰馏分，旋转蒸发浓缩，冻干得到lixisenatide精肽12.8g，HPLC纯度99.1％，精肽收率26.6％。
实施例8
本实施例仅引入二肽A，二肽B，二肽D，二肽E，二肽F，制备利西拉来。
8.1 Fmoc-Lys(Boc)-Rink Amide-AM Resin的制备
称取干燥Rink Amide-AM Resin 547g(替代度为0.56mmol/g)加入到固相反应柱中，首先DMF洗涤树脂2遍，再用2～3倍树脂床层体积DMF溶胀树脂30分钟，DMF洗涤3次，DCM洗涤2次，等待投料。
在冰浴冷却的条件下，将431.2g Fmoc-Lys(Boc)-OH(918mmol)、130.5g HOBt(964mmol)溶于DMF和DCM的混合溶剂中，待氨基酸溶解后，慢慢加入DIC 121.5g(964mmol)，活化3min后将反应液倒入反应柱中，鼓气搅拌反应；采用茚三酮检测树脂反应透明时停止反应，DMF洗涤3遍，甲醇收缩、减压干燥得Fmoc-Lys(Boc)-Rink Amide-AM Resin 621克，检测树脂替代度为0.508mmol/g。
8.2 lixisenatide肽树脂的制备
取8.1方法制备的替代度为0.508mmol/g的Fmoc-Lys(Boc)-Rink Amide-AM Resin 136g(70mmol)，加入固相反应柱中，用DMF洗涤2次，用DMF溶胀树脂30分钟后，用DBLK脱除Fmoc保护，然后用DMF洗涤6次。将98.5g(210mmol)Fmoc-Lys(Boc)-OH，31.6g(220mmol)HOAt，109.5g(210mmol)PyAOP，54.3g(420mmol)DIPEA溶于体积比为1:1的DCM和DMF混合溶液，加入固相反应柱中，室温反应2h(反应终点以茚三酮法检测为准，如果树脂无色透明，则反应完全，树脂显色，表示反应不完全，需再偶联反应1h至树脂检测透明)。
重复上述脱除Fmoc保护和加入相应氨基酸偶联的步骤，按照片段的 顺序，采用一定的偶联方法，依次完成Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Pro-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-Ser(PSI ME,ME Pro)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Phe-Thr(PSI ME,ME Pro)-OH、Fmoc-Gly-Thr(PSI ME,ME Pro)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH、Boc-His(Trt)-OH的偶联。反应结束后用甲醇收缩，树脂真空干燥过夜，得到751克lixisenatide肽树脂。
8.3 lixisenatide肽树脂的裂解和精肽制备
取8.2得到的lixisenatide肽树脂742g置于裂解反应器中，以10ml/g(裂解液/树脂)的裂解液用量入裂解试剂(TFA：PhSMe：PhOH：EDT：H2O＝85:5:3:5:2，(V/V))，室温搅拌3h。反应物用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤2次，合并滤液后减压浓缩。加入冰冻的无水乙醚沉淀、离心，无水乙醚洗涤粗肽滤饼3次，真空干燥得到白色粉末固体，lixisenatide粗肽335.6g，质谱MALDI-TOF：(M+H)+＝4859.478。粗肽重量收率为97.6％，HPLC纯度为68.9％。
取8.3中得到的lixisenatide粗肽52g进行精制，溶解后采用NOVASEP RP-HPLC系统，波长220nm，色谱柱为反相C18柱，常规0.1％TFA/水、乙腈流动相体系纯化后转盐，收集目的峰馏分，旋转蒸发浓缩，冻干得到lixisenatide精肽14.5g，HPLC纯度99.2％，精肽收率27.9％。
对比例1
D1.1 Fmoc-Lys(Boc)-Rink Amide-AM Resin的制备
称取干燥Rink Amide-AM Resin 410g(替代度为0.56mmol/g)加入到固相反应柱中，首先DMF洗涤树脂2遍，再用2～3倍树脂床层体积DMF溶胀树脂30分钟，DMF洗涤3次，DCM洗涤2次，等待投料。
在冰浴冷却的条件下，将323.5g Fmoc-Lys(Boc)-OH(690mmol)、97.9g HOBt(725mmol)溶于DMF和DCM的混合溶剂中，待氨基酸溶解后，慢慢加入DIC 92.4g(725mmol)，活化3min后将反应液倒入反应柱中，鼓气搅拌反应；采用茚三酮检测树脂反应透明时停止反应，DMF洗涤3遍，甲醇收缩、减压干燥得Fmoc-Lys(Boc)-Rink Amide-AM Resin 462克，检测树脂替代度为0.51mmol/g。
D1.2顺序偶联制备lixisenatide肽树脂
取D1.1方法制备的替代度为0.51mmol/g的Fmoc-Lys(Boc)-Rink Amide-AM Resin 462g(230mmol)，加入固相反应柱中，用DMF洗涤2次，用DMF溶胀树脂30分钟后，用DBLK脱除Fmoc保护，然后用DMF洗涤6次，。将323.6g(690mmol)Fmoc-Lys(Boc)-OH，98.1g(725mmol)HOBt，92.4g(724mmol)DIC溶于体积比为1:1的DCM和DMF混合溶液，加入固相反应柱中，室温反应2h(反应终点以茚三酮法检测为准，如果树脂无色透明，则反应完全，树脂显色，表示反应不完全，需再偶联反应1h至树脂检测透明)。
重复上述脱除Fmoc保护和加入相应氨基酸偶联的步骤，按照片段的顺序，采用一定的偶联方法，依次完成Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Leu-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-Gly-OH、Boc-His(Trt)-OH的偶联。反应结束后用甲醇收缩，树脂真空干燥过夜，得到2350克lixisenatide肽树脂。
D1.3 lixisenatide肽树脂的裂解和精肽制备
称取D1.2得到的lixisenatide肽树脂800克置于裂解反应器中，以12ml/g(裂解液/树脂)的裂解液用量入裂解试剂(TFA：PhSMe：PhOH：EDT：H2O＝85:5:3:5:2，(V/V))，室温搅拌3h。反应物用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤2次，合并滤液后减压浓缩。加入冰冻的无水乙醚沉淀、离心，无水乙醚洗涤粗肽滤饼3次，真空干燥得到白色粉末固体，lixisenatide粗肽370.6g，质谱(图2)MALDI-TOF：(M+H)+＝4859.288。粗肽重量收率为96.8％，HPLC纯度为44.5％。
取D1.3中得到的lixisenatide粗肽49g进行精制，溶解后采用NOVASEP RP-HPLC系统，波长220nm，色谱柱为反相C18柱，常规0.1％TFA/水、乙腈流动相体系纯化后转盐，收集目的峰馏分，旋转蒸发浓缩，冻干得到lixisenatide精肽7.5g，HPLC纯度98.6％，精肽收率15.4％。
实施例9
检测实施例1～8和对比例1制得的精肽。其中，对实施例1制得粗肽的质谱图如图1，对对比例1制得粗肽的质谱图如图2。其他各实施例制得产品的质谱图与此相似。各实施例获得多肽纯度和收率如表2：
表2多肽纯度和收率
  质谱MALDI-TOF：(M+H)+ 粗肽收率 粗肽纯度 精肽收率 精肽纯度
实施例1 4859.216 99.4％ 69.1％ 28.8％ 99.3％
实施例2 4859.017 101.2％ 66.6％ 26.4％ 99.1％
实施例3 4859.512 98.9％ 68.4％ 27.4％ 99.2％
实施例4 4859.347 102.6％ 67.9％ 27.1％ 99.3％
实施例5 4859.109 100.6％ 67.1％ 28.1％ 99.3％
实施例6 4859.205 99.6％ 61.8％ 25.8％ 99.2％
实施例7 4859.318 100.8％ 59.6％ 26.6％ 99.1％
实施例8 4859.478 97.6％ 68.9％ 27.9％ 99.2％
对比例1 4859.288 96.8％ 44.5％ 15.4％ 98.6％
结果显示，引入二肽片段的技术方案无论从粗肽纯度、精肽纯度还是精肽收率上都高于对比例方案。
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。

Claims (13)

  1. 一种利西拉来的制备方法，其特征在于，包括：
    步骤1：固相合成Fmoc-Lys-树脂；
    步骤2：根据利西拉来的肽序在所述Fmoc-Lys-树脂上偶联氨基酸或二肽制得利西拉来肽树脂；所述二肽选自-Gly-Thr、-Phe-Thr、-Thr-Ser、-Leu-Ser、-Ser-Ser或-Pro-Ser；
    步骤3：所述利西拉来肽树脂经裂解制得利西拉来。
  2. 根据权利要求1所述的制备方法，其特征在于，
    偶联-Gly-Thr采用Fmoc-Gly-Thr(PSI ME,ME Pro)-OH；
    偶联-Phe-Thr采用Fmoc-Phe-Thr(PSI ME,ME Pro)-OH；
    偶联-Thr-Ser采用Fmoc-Thr(tBu)-Ser(PSI ME,ME Pro)-OH；
    偶联-Leu-Ser采用Fmoc-Leu-Ser(PSI ME,ME Pro)-OH；
    偶联-Ser-Ser采用Fmoc-Ser(tBu)-Ser(PSI ME,ME Pro)-OH；
    偶联-Pro-Ser采用Fmoc-Pro-Ser(PSI ME,ME Pro)-OH。
  3. 根据权利要求1所述的制备方法，其特征在于，所述偶联的偶联剂为HOBt与DIC的混合物；其中HOBt与DIC的摩尔比为1:1。
  4. 根据权利要求1所述的制备方法，其特征在于，所述裂解的裂解剂包括TFA与B组分；所述B组分选自PhSMe、PhOMe、EDT、H2O、TIS或PhOH。
  5. 根据权利要求1所述的制备方法，其特征在于，所述步骤3后还包括纯化、转盐的步骤。
  6. 根据权利要求1所述的制备方法，其特征在于，所述偶联具体为依次偶联：Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Pro-Ser(PSI ME，ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-Ser(PSI ME，ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Gly-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、 Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-Ser(PSI ME，ME Pro)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Thr(tBu)-Ser(PSI ME，ME Pro)-OH、Fmoc-Phe-OH、Fmoc-Gly-Thr(PSI ME，ME Pro)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH和Boc-His(Trt)-OH。
  7. 根据权利要求1所述的制备方法，其特征在于，所述偶联具体为依次偶联：Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Pro-Ser(PSI ME,MEPro)-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-Ser(PSI ME,ME Pro)-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-Ser(PSI ME,ME Pro)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Phe-Thr(PSI ME,ME Pro)-OH、Fmoc-Gly-Thr(PSI ME,ME Pro)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH和Boc-His(Trt)-OH。
  8. 根据权利要求1所述的制备方法，其特征在于，所述偶联具体为依次偶联：Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-Ser(PSI  ME,ME Pro)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Phe-Thr(PSI ME,ME Pro)-OH、Fmoc-Gly-Thr(PSI ME,ME Pro)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH和Boc-His(Trt)-OH。
  9. 根据权利要求1所述的制备方法，其特征在于，所述偶联具体为依次偶联：Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-Ser(PSI ME,ME Pro)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Phe-Thr(PSI ME,ME Pro)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH和Boc-His(Trt)-OH。
  10. 根据权利要求1所述的制备方法，其特征在于，所述偶联具体为依次偶联：Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Leu-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Thr(tBu)-Ser(PSI ME,ME Pro)-OH、Fmoc-Phe--OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH 和Boc-His(Trt)-OH。
  11. 根据权利要求1所述的制备方法，其特征在于，所述偶联具体为依次偶联：Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Leu-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-Gly-OH和Boc-His(Trt)-OH。
  12. 根据权利要求1所述的制备方法，其特征在于，所述偶联具体为依次偶联：Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-Ser(PSI ME,ME Pro)-OH、Fmoc-Leu-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-Gly-OH或Boc-His(Trt)-OH。
  13. 根据权利要求1所述的制备方法，其特征在于，所述偶联具体为依次偶联：Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Pro-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-Ser(PSI ME,ME Pro)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-Ser(PSI ME,ME Pro)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Phe-Thr(PSI ME,ME Pro)-OH、Fmoc-Gly-Thr(PSI ME,ME Pro)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH和Boc-His(Trt)-OH。
PCT/CN2016/110374 2015-12-31 2016-12-16 一种利西拉来的制备方法 WO2017114192A1 (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102558338A (zh) * 2012-02-10 2012-07-11 深圳翰宇药业股份有限公司 一种合成利西拉来的方法
CN103709243A (zh) * 2013-09-10 2014-04-09 深圳翰宇药业股份有限公司 一种制备利西拉来的方法
CN103819553A (zh) * 2013-12-27 2014-05-28 苏贤斌 一种使用固相和液相组合技术制备利西拉来的方法
CN104211801A (zh) * 2014-07-25 2014-12-17 杭州诺泰制药技术有限公司 一种制备利西拉来的方法
CN104844706A (zh) * 2015-06-10 2015-08-19 成都圣诺生物科技股份有限公司 一种合成利西拉来的方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2643515C2 (ru) * 2011-12-29 2018-02-02 Ново Нордиск А/С Дипептид, содержащий непротеиногенную аминокислоту
CA2907454C (en) * 2013-03-21 2021-05-04 Sanofi-Aventis Deutschland Gmbh Synthesis of hydantoin containing peptide products

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102558338A (zh) * 2012-02-10 2012-07-11 深圳翰宇药业股份有限公司 一种合成利西拉来的方法
CN103709243A (zh) * 2013-09-10 2014-04-09 深圳翰宇药业股份有限公司 一种制备利西拉来的方法
CN103819553A (zh) * 2013-12-27 2014-05-28 苏贤斌 一种使用固相和液相组合技术制备利西拉来的方法
CN104211801A (zh) * 2014-07-25 2014-12-17 杭州诺泰制药技术有限公司 一种制备利西拉来的方法
CN104844706A (zh) * 2015-06-10 2015-08-19 成都圣诺生物科技股份有限公司 一种合成利西拉来的方法

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