METHOD FOR PREPARING LIXISENATIDE

Abstract

A method for preparing Lixisenatide. According to a peptide sequence structure of Lixisenatide peptide, specially protected serine dipeptide is used as a raw material and coupled into a peptide sequence. Because of a ring-shaped structure similar to that of proline is formed, the rotation of a peptide bond can be effectively prevented, the contraction of a peptide chain curling agent is suppressed, so that active functional groups are fully exposed, thereby facilitating the coupling of the amino acid, and reducing the occurrence of defects and other side effects.

Claims

1. A method for preparing lixisenatide, comprising: step 1: synthesizing Fmoc-Lys-resin by solid-phase synthesis; step 2: coupling an amino acid or a dipeptide to the Fmoc-Lys-resin according to the peptide sequence of lixisenatide to obtain lixisenatide peptide resin; wherein the dipeptide is selected from the group consisting of -Gly-Thr, -Phe-Thr, -Thr-Ser, -Leu-Ser, -Ser-Ser and -Pro-Ser; and step 3: cleaving the lixisenatide peptide resin to obtain lixisenatide.

2. The method according to claim 1, wherein coupling -Gly-Thr is performed by using Fmoc-Gly-Thr(PSI ME, ME Pro)-OH; coupling -Phe-Thr is performed by using Fmoc-Phe-Thr(PSI ME, ME Pro)-OH; coupling -Thr-Ser is performed by using Fmoc-Thr(tBu)-Ser(PSI ME, ME Pro)-OH; coupling -Leu-Ser is performed by using Fmoc-Leu-Ser(PSI ME, ME Pro)-OH; coupling -Ser-Ser is performed by using Fmoc-Ser(tBu)-Ser(PSI ME, ME Pro)-OH; and coupling -Pro-Ser is performed by using Fmoc-Pro-Ser(PSI ME, ME Pro)-OH,

3. The method according to claim 1, wherein coupling agent for the coupling is a mixture of HOBt and DIC; and wherein the molar ratio of HOBt to DIC is 1:1.

4. The method according to claim 1, wherein cleaving agent for the cleaving comprises TFA and component B; and wherein the component B is selected from the group consisting of PhSMe, PhOMe, EDT, H.sub.2O, TIS and PhOH.

5. The method according to claim 1, after the step 3, further comprising steps of purification and salt conversion.

6. The method according to claim 1, wherein the coupling is in particular performed by coupling sequentially the following: 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 and Boc-His(Trt)-OH.

7. The method according to claim 1, wherein the coupling is in particular performed by coupling sequentially the following: 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 and Boc-His(Trt)-OH.

8. The method according to claim 1, wherein the coupling is in particular performed by coupling sequentially the following: 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 and Boc-His(Trt)-OH.

9. The method according to claim 1, wherein the coupling is in particular performed by coupling sequentially the following: 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 and Boc-His(Trt)-OH.

10. The method according to claim 1, wherein the coupling is in particular performed by coupling sequentially the following: 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 and Boc-His(Trt)-OH.

11. The method according to claim 1, wherein the coupling is in particular performed by coupling sequentially the following: 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 and Boc-His(Trt)-OH.

12. The method according to claim 1, wherein the coupling is in particular performed by coupling sequentially the following: 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 or Boc-His(Trt)-OH.

13. The method according to claim 1, wherein the coupling is in particular performed by coupling sequentially the following: 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 and Boc-His(Trt)-OH.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIG. 1 shows the mass spectrum of the crude peptide prepared in Example 1.

[0067] FIG. 2 shows the mass spectrum of the crude peptide prepared in Comparative Example 1.

DETAILED DESCRIPTION OF EMBODIMENTS

[0068] The present invention provides methods for preparing lixisenatide. Those skilled in the art can learn from the content of this document and appropriately improve the process parameters. In particular, it should be noted that all similar replacements and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is apparent to those skilled in the art that the methods and applications herein may be modified or appropriately changed and combined to implement and use the present invention, without departing from the spirit, scope, and scope of the present invention.

[0069] The reagents or instruments used in the present invention are all common commercial products and are all available in the market.

[0070] Among them, the names and abbreviations of the materials are shown in Table 1:

TABLE-US-00002 TABLE 1 Names and abbreviations of the materials Fmoc 9-fluorenylmethyloxycarbonyl Resin Resin tBu Tert-butyl OtBu Tert-butoxy Trt Triphenylmethyl DCM Dichloromethane DBLK 20% hexahydropyridine/DMF solution DIPEA N,N-diisopropylethylamine HOBt 1-hydroxybenzotriazole HOAt 1-hydroxy-7-azobenzotriazole PyBOP Benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate DMSO Dimethyl Sulfoxide HATU O-(7-azobenzotriazol-1-oxo)-N,N,N,N-tetramethyluronium hexafluorophosphate HPLC High performance liquid chromatography DMF N,N-Dimethylformamide TFA Trifluoroacetic acid HBTU O-benzotriazole- N,N,N,N-tetramethyluronium hexafluorophosphate PyAOP Triazolo[4,5-b]pyridin-3-oxy)tri-1-pyrrolidinyl hexafluorophosphate PhOH Phenol PhOMe Anisole PheSMe Thioanisole EDT 1,2-ethanedithiol TIS Triisopropylsilane

[0071] In the following, the present invention will be further illustrated in combination with examples:

Example 1

[0072] In this example, only dipeptide A, dipeptide C, dipeptide D, dipeptide E, and dipeptide F were introduced to prepare lixisenatide.

1.1 Preparation of Fmoc-Lys(Boc)-Rink Amide-MBHA Resin

[0073] 380 g of dry Rink Amide-MBHA Resin (substitution degree of 0.45 mmol/g) was weighted and added into a solid-phase reaction column. The resin was first washed twice with DMF, followed by swelling in 2-3 times the resin bed volume of DMF for 30 minutes. The resin washed three times with DMF and then twice with DCM and left for feeding.

[0074] Under the condition of ice bath, 238.9 g of Fmoc-Lys(Boc)-OH (510 mmol) and 72.4 g of HOAt (536 mmol) were dissolved in a mixed solvent of DMF and DCM. After the amino acid was dissolved, 67.5 g (535 mmol) of DIC was slowly added and activated for 3 minutes. The resulting reaction solution was poured into the reaction column, and the reaction was carried out under stirring with air-blowing. The reaction was stopped when ninhydrin test shown that the resin was transparent. The resin was washed 3 times with DMF and shrunk by methanol. The resin was dried under reduced pressure to give 421 g of Fmoc-Lys(Boc)-Rink Amide-MBHA Resin with a substitution degree detected of 0.406 mmol/g.

1.2 Preparation of Lixisenatide Peptide Resin

[0075] 421 g (230 mmol) of Fmoc-Lys(Boc)-Rink Amide-MBHA Resin with a substitution degree of 0.406 mmol/g prepared in method 1.1 was weighted and added into a solid-phase reaction column. The resin was washed twice with DMF, followed by swelling in DMF for 30 minutes. After the deprotection of Fmoc by DBLK, the resin was washed 6 times with DMF. 239.1 g (510 mmol) Fmoc-Lys(Boc)-OH, 72.2 g (536 mmol) HOBt, and 67.7 g (535 mmol) DIC were dissolved in a mixed solution of DCM and DMF (volume ratio of 1:1), and then added into the solid-phase reaction column and allowed to react at room temperature for 2 h (the completion of the reaction was determined by ninhydrin test; if the resin was colorless and transparent, the reaction was complete; if the resin was colored, the reaction was incomplete, and in this case, the coupling reaction was carried out for additional 1 h until the resin detected was transparent).

[0076] The steps of deprotecting Fmoc and coupling of corresponding amino acid added were repeated, and the following coupling was completed according to the order of the sequence by using certain coupling method: 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, and Boc-His(Trt)-OH. After the reaction was completed, the resin was shrunk by methanol and dried under vacuum overnight to give 2018 g of lixisenatide peptide resin.

1.3 Cleaving of Lixisenatide Peptide Resin and Preparation of Refined Peptide

[0077] 700 g of lixisenatide peptide resin obtained in 1.2 was placed in a cleaving reactor and a cleaving agent (TFA:PhSMe:EDT:TIS:H.sub.2O=84:5:5:5:1, (V/V)) was added at an amount of 8 ml/g (cleaving agent/resin). The resulting mixture was stirred for 2.5 h at room temperature and then filtered through a sand core funnel, and the filtrate was collected. The resin was washed twice with a small amount of TFA. The filtrates were combined and concentrated under reduced pressure. Ice-cold anhydrous ether was added to the filtrates for precipitation followed by centrifugation. The cake of the crude peptide was washed three times with anhydrous ether and dried under vacuum to give 341 g white powdery solid of lixisenatide crude peptide. MS (FIG. 1) MALDI-TOF: (M+H).sup.+=4859.216. The yield of the crude peptide was 99.4% and the purity detected by HPLC was 69.1%.

[0078] 44 g of the lixisenatide crude peptide obtained in 1.3 was dissolved and subjected to refining by using NOVASEP RP-HPLC system under conditions: wavelength 220 nm, reversed-phase C18 column. The lixisenatide was purified by using a normal 0.1% TFA/water and acetonitrile mobile phase system and salt conversion was performed. The fractions of the peak of interest were collected, concentrated by rotary evaporation and lyophilized to give 12.6 g of lixisenatide refined peptide. The purity of the refined peptide detected by HPLC was 99.3% and the yield was 28.8%.

Example 2

[0079] In this example, only dipeptide A, dipeptide B, dipeptide D, and dipeptide F were introduced to prepare lixisenatide.

2.1 Preparation of Fmoc-Lys(Boc)-Rink Amide Resin

[0080] 356 g of dry Rink Amide Resin (substitution degree of 0.42 mmol/g) was weighted and added into a solid-phase reaction column. The resin was first washed twice with DMF, followed by swelling in 2-3 times the resin bed volume of DMF for 30 minutes. The resin washed three times with DMF and then twice with DCM and left for feeding.

[0081] Under the condition of ice bath, 210.9 g of Fmoc-Lys(Boc)-OH (450 mmol), 63.8 g of HOBt (473 mmol) and 170.6 g of HBTU (450 mmol) were dissolved in a mixed solvent of DMF and DCM. After the amino acid was dissolved, 87.6 g (675 mmol) of DIPEA was slowly added and activated for 3 minutes. The resulting reaction solution was poured into the reaction column, and the reaction was carried out under stirring with air-blowing. The reaction was stopped when ninhydrin test shown that the resin was transparent. The resin was washed 3 times with DMF and shrunk by methanol. The resin was dried under reduced pressure to give 390 g of Fmoc-Lys (Boc)-Rink Amide Resin with a substitution degree detected of 0.385 mmol/g.

2.2 Preparation of Lixisenatide Peptide Resin

[0082] 421 g (150 mmol) of Fmoc-Lys(Boc)-Rink Amide Resin with a substitution degree of 0.385 mmol/g prepared in method 2.1 was weighted and added into a solid-phase reaction column. The resin was washed twice with DMF, followed by swelling in DMF for 30 minutes. After the deprotection of Fmoc by DBLK, the resin was washed 6 times with DMF. 211.3 g (450 mmol) Fmoc-Lys(Boc)-OH, 64.8 g (473 mmol) HOBt, and 63.9 g (473 mmol) DIC were dissolved in a mixed solution of DCM and DMF (volume ratio of 1:1), and then added into the solid-phase reaction column and allowed to react at room temperature for 2 h (the completion of the reaction was determined by ninhydrin test; if the resin was colorless and transparent, the reaction was complete; if the resin was colored, the reaction was incomplete, and in this case, the coupling reaction was carried out for additional 1 h until the resin detected was transparent).

[0083] The steps of deprotecting Fmoc and coupling of corresponding amino acid added were repeated, and the following coupling was completed according to the order of the sequence by using certain coupling method: 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, and Boc-His(Trt)-OH. After the reaction was completed, the resin was shrunk by methanol and dried under vacuum overnight to give 1611 g of lixisenatide peptide resin.

2.3 Cleaving of Lixisenatide Peptide Resin and Preparation of Refined Peptide

[0084] 600 g of lixisenatide peptide resin obtained in 2.2 was placed in a cleaving reactor and a cleaving agent (TFA:PhSMe:EDT:H.sub.2O=91:3:3:3, (V/V)) was added in an amount of 10 ml/g (cleaving agent/resin). The resulting mixture was stirred for 2.5 h at room temperature and then filtered through a sand core funnel, and the filtrate was collected. The resin was washed twice with a small amount of TFA. The filtrates were combined and concentrated under reduced pressure. Ice-cold anhydrous ether was added to the filtrates for precipitation followed by centrifugation. The cake of the crude peptide was washed three times with anhydrous ether and dried under vacuum to give 251 g white powdery solid of lixisenatide crude peptide. MS MALDI-TOF: (M+H).sup.+=4859.017. The yield of the crude peptide was 101.2% and the purity detected by HPLC was 66.6%.

[0085] 47 g of the lixisenatide crude peptide obtained in 2.3 was dissolved and subjected to refining by using NOVASEP RP-HPLC system under conditions: wavelength 220 nm, reversed-phase C18 column. The lixisenatide was purified by using a normal 0.1% TFA/water and acetonitrile mobile phase system and salt conversion was performed. The fractions of the peak of interest were collected, concentrated by rotary evaporation and lyophilized to give 12.4 g of lixisenatide refined peptide. The purity of the refined peptide detected by HPLC was 99.1% and the yield was 26.4%.

Example 3

[0086] In this example, only dipeptide A, dipeptide B, dipeptide D, and dipeptide E were introduced to prepare lixisenatide.

3.1 Preparation of Fmoc-Lys(Boc)-Siber Resin

[0087] 155 g of dry Siber Resin (substitution degree of 0.35 mmol/g) was weighted and added into a solid-phase reaction column. The resin was first washed twice with DMF, followed by swelling in 2-3 times the resin bed volume of DMF for 30 minutes. The resin washed three times with DMF and then twice with DCM and left for feeding.

[0088] Under the condition of ice bath, 77.6 g of Fmoc-Lys(Boc)-OH (165 mmol), and 23.5 g of HOBt (173 mmol) were dissolved in a mixed solvent of DMF and DCM. After the amino acid was dissolved, 22.1 g (173 mmol) of DIC was slowly added and activated for 3 minutes. The resulting reaction solution was poured into the reaction column, and the reaction was carried out under stirring with air-blowing. The reaction was stopped when ninhydrin test shown that the resin was transparent. The resin was washed 3 times with DMF and shrunk by methanol. The resin was dried under reduced pressure to give 368 g of Fmoc-Lys (Boc)-Siber Resin with a substitution degree detected of 0.323 mmol/g.

3.2 Preparation of Lixisenatide Peptide Resin

[0089] 368 g (54 mmol) of Fmoc-Lys(Boc)-Siber Resin with a substitution degree of 0.323 mmol/g prepared in method 3.1 was weighted and added into a solid-phase reaction column. The resin was washed twice with DMF, followed by swelling in DMF for 30 minutes. After the deprotection of Fmoc by DBLK, the resin was washed 6 times with DMF. 77.9 g (165 mmol) Fmoc-Lys(Boc)-OH, 23.5 g (173 mmol) HOBt, and 22.4 g (173 mmol) DIC were dissolved in a mixed solution of DCM and DMF (volume ratio of 1:1), and then added into the solid-phase reaction column and allowed to react at room temperature for 2 h (the completion of the reaction was determined by ninhydrin test; if the resin was colorless and transparent, the reaction was complete; if the resin was colored, the reaction was incomplete, and in this case, the coupling reaction was carried out for additional 1 h until the resin detected was transparent).

[0090] The steps of deprotecting Fmoc and coupling of corresponding amino acid added were repeated, and the following coupling was completed according to the order of the sequence by using certain coupling method: 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, and Boc-His(Trt)-OH. After the reaction was completed, the resin was shrunk by methanol and dried under vacuum overnight to give 831 g of lixisenatide peptide resin.

3.3 Cleaving of Lixisenatide Peptide Resin and Preparation of Refined Peptide

[0091] 800 g of lixisenatide peptide resin obtained in 3.2 was placed in a cleaving reactor and a cleaving agent (TFA:PhSMe:EDT:PhOMe=90:5:3:2, (V/V)) was added in an amount of 12 ml/g (cleaving agent/resin). The resulting mixture was stirred for 3 h at room temperature and then filtered through a sand core funnel, and the filtrate was collected. The resin was washed twice with a small amount of TFA. The filtrates were combined and concentrated under reduced pressure. Ice-cold anhydrous ether was added to the filtrates for precipitation followed by centrifugation. The cake of the crude peptide was washed three times with anhydrous ether and dried under vacuum to give 241 g white powdery solid of lixisenatide crude peptide. MS MALDI-TOF: (M+H).sup.+=4859.512. The yield of the crude peptide was 98.9% and the purity detected by HPLC was 68.4%.

[0092] 50 g of the lixisenatide crude peptide obtained in 3.3 was dissolved and subjected to refining by using NOVASEP RP-HPLC system under conditions: wavelength 220 nm, reversed-phase C18 column. The lixisenatide was purified by using a normal 0.1% TFA/water and acetonitrile mobile phase system and salt conversion was performed. The fractions of the peak of interest were collected, concentrated by rotary evaporation and lyophilized to give 13.7 g of lixisenatide refined peptide. The purity of the refined peptide detected by HPLC was 99.2% and the yield was 27.4%.

Example 4

[0093] In this example, only dipeptide B, dipeptide D, and dipeptide E were introduced to prepare lixisenatide.

4.1 Preparation of Fmoc-Lys(Boc)-Rink Amide-MBHA Resin

[0094] 135 g of dry Rink Amide-MBHA Resin (substitution degree of 0.24 mmol/g) was weighted and added into a solid-phase reaction column. The resin was first washed twice with DMF, followed by swelling in 2-3 times the resin bed volume of DMF for 30 minutes. The resin washed three times with DMF and then twice with DCM and left for feeding.

[0095] Under the condition of ice bath, 46.8 g of Fmoc-Lys(Boc)-OH (100 mmol), and 14.2 g of HOBt (105 mmol) were dissolved in a mixed solvent of DMF and DCM. After the amino acid was dissolved, 13.5 g (105 mmol) of DIC was slowly added and activated for 3 minutes. The resulting reaction solution was poured into the reaction column, and the reaction was carried out under stirring with air-blowing. The reaction was stopped when ninhydrin test shown that the resin was transparent. The resin was washed 3 times with DMF and shrunk by methanol. The resin was dried under reduced pressure to give 143 g of Fmoc-Lys (Boc)-Siber Resin with a substitution degree detected of 0.223 mmol/g.

4.2 Preparation of Lixisenatide Peptide Resin

[0096] 143 g (32 mmol) of Fmoc-Lys(Boc)-Rink Amide-MBHA Resin with a substitution degree of 0.223 mmol/g prepared in method 4.1 was weighted and added into a solid-phase reaction column. The resin was washed twice with DMF, followed by swelling in DMF for 30 minutes. After the deprotection of Fmoc by DBLK, the resin was washed 6 times with DMF. 45.6 g (96 mmol) Fmoc-Lys(Boc)-OH, 13.8 g (101 mmol) HOBt, and 12.7 g (101 mmol) DIC were dissolved in a mixed solution of DCM and DMF (volume ratio of 1:1), and then added into the solid-phase reaction column and allowed to react at room temperature for 2 h (the completion of the reaction was determined by ninhydrin test; if the resin was colorless and transparent, the reaction was complete; if the resin was colored, the reaction was incomplete, and in this case, the coupling reaction was carried out for additional 1 h until the resin detected was transparent).

[0097] The steps of deprotecting Fmoc and coupling of corresponding amino acid added were repeated, and the following coupling was completed according to the order of the sequence by using certain coupling method: 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, and Boc-His(Trt)-OH. After the reaction was completed, the resin was shrunk by methanol and dried under vacuum overnight to give 401 g of lixisenatide peptide resin.

4.3 Cleaving of Lixisenatide Peptide Resin and Preparation of Refined Peptide

[0098] 400 g of lixisenatide peptide resin obtained in 4.2 was placed in a cleaving reactor and a cleaving agent (TFA:PhSMe:EDT:PhOH:H.sub.2O=85:5:3:5:2, (V/V)) was added in an amount of 15 ml/g (cleaving agent/resin). The resulting mixture was stirred for 3 h at room temperature and then filtered through a sand core funnel, and the filtrate was collected. The resin was washed twice with a small amount of TFA. The filtrates were combined and concentrated under reduced pressure. Ice-cold anhydrous ether was added to the filtrates for precipitation followed by centrifugation. The cake of the crude peptide was washed three times with anhydrous ether and dried under vacuum to give 156 g white powdery solid of lixisenatide crude peptide. MS MALDI-TOF: (M+H).sup.+=4859.347. The yield of the crude peptide was 102.6% and the purity detected by HPLC was 67.9%.

[0099] 46 g of the lixisenatide crude peptide obtained in 4.3 was dissolved and subjected to refining by using NOVASEP RP-HPLC system under conditions: wavelength 220 nm, reversed-phase C18 column. The lixisenatide was purified by using a normal 0.1% TFA/water and acetonitrile mobile phase system and salt conversion was performed. The fractions of the peak of interest were collected, concentrated by rotary evaporation and lyophilized to give 11.6 g of lixisenatide refined peptide. The purity of the refined peptide detected by HPLC was 99.3% and the yield was 27.1%.

Example 5

[0100] In this example, only dipeptide C, and dipeptide E were introduced to prepare lixisenatide.

5.1 Preparation of Fmoc-Lys(Boc)-Rink Amide-AM Resin

[0101] 547 g of dry Rink Amide-MBHA Resin (substitution degree of 0.56 mmol/g) was weighted and added into a solid-phase reaction column. The resin was first washed twice with DMF, followed by swelling in 2-3 times the resin bed volume of DMF for 30 minutes. The resin washed three times with DMF and then twice with DCM and left for feeding.

[0102] Under the condition of ice bath, 431.2 g of Fmoc-Lys(Boc)-OH (918 mmol), and 130.5 g of HOBt (964 mmol) were dissolved in a mixed solvent of DMF and DCM. After the amino acid was dissolved, 121.5 g (964 mmol) of DIC was slowly added and activated for 3 minutes. The resulting reaction solution was poured into the reaction column, and the reaction was carried out under stirring with air-blowing. The reaction was stopped when ninhydrin test shown that the resin was transparent. The resin was washed 3 times with DMF and shrunk by methanol. The resin was dried under reduced pressure to give 621 g of Fmoc-Lys(Boc)-Rink Amide-AM Resin with a substitution degree detected of 0.508 mmol/g.

5.2 Preparation of Lixisenatide Peptide Resin

[0103] 207 g (105 mmol) of Fmoc-Lys(Boc)-Rink Amide-AM Resin with a substitution degree of 0.508 mmol/g prepared in method 5.1 was weighted and added into a solid-phase reaction column. The resin was washed twice with DMF, followed by swelling in DMF for 30 minutes. After the deprotection of Fmoc by DBLK, the resin was washed 6 times with DMF. 147.6 g (315 mmol) Fmoc-Lys(Boc)-OH, 44.8 g (330 mmol) HOBt, and 41.6 g (330 mmol) DIC were dissolved in a mixed solution of DCM and DMF (volume ratio of 1:1), and then added into the solid-phase reaction column and allowed to react at room temperature for 2 h (the completion of the reaction was determined by ninhydrin test; if the resin was colorless and transparent, the reaction was complete; if the resin was colored, the reaction was incomplete, and in this case, the coupling reaction was carried out for additional 1 h until the resin detected was transparent).

[0104] The steps of deprotecting Fmoc and coupling of corresponding amino acid added were repeated, and the following coupling was completed according to the order of the sequence by using certain coupling method: 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, and Boc-His(Trt)-OH. After the reaction was completed, the resin was shrunk by methanol and dried under vacuum overnight to give 1046 g of lixisenatide peptide resin.

5.3 Cleaving of Lixisenatide Peptide Resin and Preparation of Refined Peptide

[0105] 1000 g of lixisenatide peptide resin obtained in 5.2 was placed in a cleaving reactor and a cleaving agent (TFA:EDT:H.sub.2O=90:5:5, (V/V)) was added in an amount of 10 ml/g (cleaving agent/resin). The mixture was stirred for 3 h at room temperature and then filtered through a sand core funnel, and the filtrate was collected. The resin was washed twice with a small amount of TFA. The filtrates were combined and concentrated under reduced pressure. Ice-cold anhydrous ether was added to the filtrates for precipitation followed by centrifugation. The cake of the crude peptide was washed three times with anhydrous ether and dried under vacuum to give 487 g white powdery solid of lixisenatide crude peptide. MS MALDI-TOF: (M+H).sup.+=4859.109. The yield of the crude peptide was 100.6% and the purity detected by HPLC was 67.1%.

[0106] 43 g of the lixisenatide crude peptide obtained in 5.3 was dissolved and subjected to refining by using NOVASEP RP-HPLC system under conditions: wavelength 220 nm, reversed-phase C18 column. The lixisenatide was purified by using a normal 0.1% TFA/water and acetonitrile mobile phase system and salt conversion was performed. The fractions of the peak of interest were collected, concentrated by rotary evaporation and lyophilized to give 11.9 g of lixisenatide refined peptide. The purity of the refined peptide detected by HPLC was 99.3% and the yield was 28.1%.

Example 6

[0107] In this example, only dipeptide E was introduced to prepare lixisenatide.

6.1 Preparation of Fmoc-Lys(Boc)-Rink Amide-AM Resin

[0108] 547 g of dry Rink Amide-AM Resin (substitution degree of 0.56 mmol/g) was weighted and added into a solid-phase reaction column. The resin was first washed twice with DMF, followed by swelling in 2-3 times the resin bed volume of DMF for 30 minutes. The resin washed three times with DMF and then twice with DCM and left for feeding.

[0109] Under the condition of ice bath, 431.2 g of Fmoc-Lys(Boc)-OH (918 mmol), and 130.5 g of HOBt (964 mmol) were dissolved in a mixed solvent of DMF and DCM. After the amino acid was dissolved, 121.5 g (964 mmol) of DIC was slowly added and activated for 3 minutes. The resulting reaction solution was poured into the reaction column, and the reaction was carried out under stirring with air-blowing. The reaction was stopped when ninhydrin test shown that the resin was transparent. The resin was washed 3 times with DMF and shrunk by methanol. The resin was dried under reduced pressure to give 621 g of Fmoc-Lys(Boc)-Rink Amide-AM Resin with a substitution degree detected of 0.508 mmol/g.

6.2 Preparation of Lixisenatide Peptide Resin

[0110] 137 g (70 mmol) of Fmoc-Lys(Boc)-Rink Amide-AM Resin with a substitution degree of 0.508 mmol/g prepared in method 6.1 was weighted and added into a solid-phase reaction column. The resin was washed twice with DMF, followed by swelling in DMF for 30 minutes. After the deprotection of Fmoc by DBLK, the resin was washed 6 times with DMF. 98.5 g (210 mmol) Fmoc-Lys(Boc)-OH, 30.6 g (220 mmol) HOBt, and 27.9 g (220 mmol) DIC were dissolved in a mixed solution of DCM and DMF (volume ratio of 1:1), and then added into the solid-phase reaction column and allowed to react at room temperature for 2 h (the completion of the reaction was determined by ninhydrin test; if the resin was colorless and transparent, the reaction was complete; if the resin was colored, the reaction was incomplete, and in this case, the coupling reaction was carried out for additional 1 h until the resin detected was transparent).

[0111] The steps of deprotecting Fmoc and coupling of corresponding amino acid added were repeated, and the following coupling was completed according to the order of the sequence by using certain coupling method: 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, and Boc-His(Trt)-OH. After the reaction was completed, the resin was shrunk by methanol and dried under vacuum overnight to give 728 g of lixisenatide peptide resin.

6.3 Cleaving of Lixisenatide Peptide Resin and Preparation of Refined Peptide

[0112] 728 g of lixisenatide peptide resin obtained in 6.2 was placed in a cleaving reactor and a cleaving agent (TFA:EDT:H.sub.2O=90:5:5, (V/V)) was added in an amount of 10 ml/g (cleaving agent/resin). The mixture was stirred for 3 h at room temperature and then filtered through a sand core funnel, and the filtrate was collected. The resin was washed twice with a small amount of TFA. The filtrates were combined and concentrated under reduced pressure. Ice-cold anhydrous ether was added to the filtrates for precipitation followed by centrifugation. The cake of the crude peptide was washed three times with anhydrous ether and dried under vacuum to give 338.9 g white powdery solid of lixisenatide crude peptide. MS MALDI-TOF: (M+H).sup.+=4859.205. The yield of the crude peptide was 99.6% and the purity detected by HPLC was 61.8%.

[0113] 58 g of the lipisatetide crude peptide obtained in 6.3 was dissolved and subjected to refining by using NOVASEP RP-HPLC system under conditions: wavelength 220 nm, reversed-phase C18 column. The lixisenatide was purified by using a normal 0.1% TFA/water and acetonitrile mobile phase system and salt conversion was performed. The fractions of the peak of interest were collected, concentrated by rotary evaporation and lyophilized to give 14.9 g of lixisenatide refined peptide. The purity of the refined peptide detected by HPLC was 99.2% and the yield was 25.8%.

Example 7

[0114] In this example, only dipeptide D was introduced to prepare lixisenatide.

7.1 Preparation of Fmoc-Lys(Boc)-Rink Amide-AM Resin

[0115] 547 g of dry Rink Amide-AM Resin (substitution degree of 0.56 mmol/g) was weighted and added into a solid-phase reaction column. The resin was first washed twice with DMF, followed by swelling in 2-3 times the resin bed volume of DMF for 30 minutes. The resin washed three times with DMF and then twice with DCM and left for feeding.

[0116] Under the condition of ice bath, 431.2 g of Fmoc-Lys(Boc)-OH (918 mmol), and 130.5 g of HOBt (964 mmol) were dissolved in a mixed solvent of DMF and DCM. After the amino acid was dissolved, 121.5 g (964 mmol) of DIC was slowly added and activated for 3 minutes. The resulting reaction solution was poured into the reaction column, and the reaction was carried out under stirring with air-blowing. The reaction was stopped when ninhydrin test shown that the resin was transparent. The resin was washed 3 times with DMF and shrunk by methanol. The resin was dried under reduced pressure to give 621 g of Fmoc-Lys(Boc)-Rink Amide-AM Resin with a substitution degree detected of 0.508 mmol/g.

7.2 Preparation of Lixisenatide Peptide Resin

[0117] 137 g (70 mmol) of Fmoc-Lys(Boc)-Rink Amide-AM Resin with a substitution degree of 0.508 mmol/g prepared in method 7.1 was weighted and added into a solid-phase reaction column. The resin was washed twice with DMF, followed by swelling in DMF for 30 minutes. After the deprotection of Fmoc by DBLK, the resin was washed 6 times with DMF. 98.5 g (210 mmol) Fmoc-Lys(Boc)-OH, 31.6 g (220 mmol) HOAt, and 27.9 g (220 mmol) DIC were dissolved in a mixed solution of DCM and DMF (volume ratio of 1:1), and then added into the solid-phase reaction column and allowed to react at room temperature for 2 h (the completion of the reaction was determined by ninhydrin test; if the resin was colorless and transparent, the reaction was complete; if the resin was colored, the reaction was incomplete, and in this case, the coupling reaction was carried out for additional 1 h until the resin detected was transparent).

[0118] The steps of deprotecting Fmoc and coupling of corresponding amino acid added were repeated, and the following coupling was completed according to the order of the sequence by using certain coupling method: 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, and Boc-His(Trt)-OH. After the reaction was completed, the resin was shrunk by methanol and dried under vacuum overnight to give 742 g of lixisenatide peptide resin.

7.3 Cleaving of Lixisenatide Peptide Resin and Preparation of Refined Peptide

[0119] 742 g of lixisenatide peptide resin obtained in 7.2 was placed in a cleaving reactor and a cleaving agent (TFA:PhSMe:PhOH:EDT:H.sub.2O=85:5:3:5:2, (V/V)) was added in an amount of 10 ml/g (cleaving agent/resin). The resulting mixture was stirred for 3 h at room temperature and then filtered through a sand core funnel, and the filtrate was collected. The resin was washed twice with a small amount of TFA. The filtrates were combined and concentrated under reduced pressure. Ice-cold anhydrous ether was added to the filtrates for precipitation followed by centrifugation. The cake of the crude peptide was washed three times with anhydrous ether and dried under vacuum to give 341.3 g white powdery solid of lixisenatide crude peptide. MS MALDI-TOF: (M+H).sup.+=4859.318. The yield of the crude peptide was 100.8% and the purity detected by HPLC was 59.6%.

[0120] 48 g of the lipisatetide crude peptide obtained in 7.3 was dissolved and subjected to refining by using NOVASEP RP-HPLC system under conditions: wavelength 220 nm, reversed-phase C18 column. The lixisenatide was purified by using a normal 0.1% TFA/water and acetonitrile mobile phase system and salt conversion was performed. The fractions of the peak of interest were collected, concentrated by rotary evaporation and lyophilized to give 12.8 g of lixisenatide refined peptide. The purity of the refined peptide detected by HPLC was 99.1% and the yield was 26.6%.

Example 8

[0121] In this example, only dipeptide A, dipeptide B, dipeptide D, dipeptide E, and dipeptide F were introduced to prepare lixisenatide.

8.1 Preparation of Fmoc-Lys(Boc)-Rink Amide-AM Resin

[0122] 547 g of dry Rink Amide-AM Resin (substitution degree of 0.56 mmol/g) was weighted and added into a solid-phase reaction column. The resin was first washed twice with DMF, followed by swelling in 2-3 times the resin bed volume of DMF for 30 minutes. The resin washed three times with DMF and then twice with DCM and left for feeding.

[0123] Under the condition of ice bath, 431.2 g of Fmoc-Lys(Boc)-OH (918 mmol), and 130.5 g of HOBt (964 mmol) were dissolved in a mixed solvent of DMF and DCM. After the amino acid was dissolved, 121.5 g (964 mmol) of DIC was slowly added and activated for 3 minutes. The resulting reaction solution was poured into the reaction column, and the reaction was carried out under stirring with air-blowing. The reaction was stopped when ninhydrin test shown that the resin was transparent. The resin was washed 3 times with DMF and shrunk by methanol. The resin was dried under reduced pressure to give 621 g of Fmoc-Lys(Boc)-Rink Amide-AM Resin with a substitution degree detected of 0.508 mmol/g.

8.2 Preparation of Lixisenatide Peptide Resin

[0124] 136 g (70 mmol) of Fmoc-Lys(Boc)-Rink Amide-AM Resin with a substitution degree of 0.508 mmol/g prepared in method 8.1 was weighted and added into a solid-phase reaction column. The resin was washed twice with DMF, followed by swelling in DMF for 30 minutes. After the deprotection of Fmoc by DBLK, the resin was washed 6 times with DMF. 98.5 g (210 mmol) Fmoc-Lys(Boc)-OH, 31.6 g (220 mmol) HOAt, 109.5 g (210 mmol) of PyAOP, and 54.3 g (420 mmol)) of DIPEA were dissolved in a mixture of DCM and DMF (volume ratio of 1:1), and then added into the solid-phase reaction column and allowed to react at room temperature for 2 h (the completion of the reaction was determined by ninhydrin test; if the resin was colorless and transparent, the reaction was complete; if the resin was colored, the reaction was incomplete, and in this case, the coupling reaction was carried out for additional 1 h until the resin detected was transparent).

[0125] The steps of deprotecting Fmoc and coupling of corresponding amino acid added were repeated, and the following coupling was completed according to the order of the sequence by using certain coupling method: 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, and Boc-His(Trt)-OH. After the reaction was completed, the resin was shrunk by methanol and dried under vacuum overnight to give 751 g of lixisenatide peptide resin.

8.3 Cleaving of Lixisenatide Peptide Resin and Preparation of Refined Peptide

[0126] 742 g of lixisenatide peptide resin obtained in 8.2 was placed in a cleaving reactor and a cleaving reagent (TFA:PhSMe:PhOH:EDT:H.sub.2O=85:5:3:5:2, (V/V)) was added in an amount of 10 ml/g (cleaving reagent/resin). The resulting mixture was stirred for 3 h at room temperature and then filtered through a sand core funnel, and the filtrate was collected. The resin was washed twice with a small amount of TFA. The filtrates were combined and concentrated under reduced pressure. Ice-cold anhydrous ether was added to the filtrates for precipitation followed by centrifugation. The cake of the crude peptide was washed three times with anhydrous ether and dried under vacuum to give 335.6 g white powdery solid of lixisenatide crude peptide. MS MALDI-TOF: (M+H).sup.+=4859.478. The yield of the crude peptide was 97.6% and the purity detected by HPLC was 68.9%.

[0127] 52 g of the lipisatetide crude peptide obtained in 8.3 was dissolved and subjected to refining by using NOVASEP RP-HPLC system under conditions: wavelength 220 nm, reversed-phase C18 column. The lixisenatide was purified by using a normal 0.1% TFA/water and acetonitrile mobile phase system and salt conversion was performed. The fractions of the peak of interest were collected, concentrated by rotary evaporation and lyophilized to give 14.5 g of lixisenatide refined peptide. The purity of the refined peptide detected by HPLC was 99.2% and the yield was 27.9%.

Comparative Example 1

D1.1 Preparation of Fmoc-Lys(Boc)-Rink Amide-AM Resin

[0128] 410 g of dry Rink Amide-AM Resin (substitution degree of 0.56 mmol/g) was weighted and added into a solid-phase reaction column. The resin was first washed twice with DMF, followed by swelling in 2-3 times the resin bed volume of DMF for 30 minutes. The resin washed three times with DMF and then twice with DCM and left for feeding.

[0129] Under the condition of ice bath, 323.5 g of Fmoc-Lys(Boc)-OH (690 mmol), and 97.9 g of HOBt (725 mmol) were dissolved in a mixed solvent of DMF and DCM. After the amino acid was dissolved, 92.4 g (725 mmol) of DIC was slowly added and activated for 3 minutes. The resulting reaction solution was poured into the reaction column, and the reaction was carried out under stirring with air-blowing. The reaction was stopped when ninhydrin test shown that the resin was transparent. The resin was washed 3 times with DMF and shrunk by methanol. The resin was dried under reduced pressure to give 462 g of Fmoc-Lys(Boc)-Rink Amide-AM Resin with a substitution degree detected of 0.51 mmol/g.

D1.2 Preparation of Lixisenatide Peptide Resin Via Sequential Coupling

[0130] 462 g (230 mmol) of Fmoc-Lys(Boc)-Rink Amide-AM Resin with a substitution degree of 0.51 mmol/g prepared in method D1.1 was weighted and added into a solid-phase reaction column. The resin was washed twice with DMF, followed by swelling in DMF for 30 minutes. After the deprotection of Fmoc by DBLK, the resin was washed 6 times with DMF. 323.6 g (690 mmol) Fmoc-Lys(Boc)-OH, 98.1 g (725 mmol) HOBt, and 92.4 g (724 mmol) DIC were dissolved in a mixture of DCM and DMF (volume ratio of 1:1), and then added into the solid-phase reaction column and allowed to react at room temperature for 2 h (the completion of the reaction was determined by ninhydrin test; if the resin was colorless and transparent, the reaction was complete; if the resin was colored, the reaction was incomplete, and in this case, the coupling reaction was carried out for additional 1 h until the resin detected was transparent).

[0131] The steps of deprotecting Fmoc and coupling of corresponding amino acid added were repeated, and the following coupling was completed according to the order of the sequence by using certain coupling method: 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, and Boc-His(Trt)-OH. After the reaction was completed, the resin was shrunk by methanol and dried under vacuum overnight to give 2350 g of lixisenatide peptide resin.

D1.3 Cleaving of Lixisenatide Peptide Resin and Preparation of Refined Peptide

[0132] 800 g of lixisenatide peptide resin obtained in D1.2 was placed in a cleaving reactor and a cleaving agent (TFA:PhSMe:PhOH:EDT:H.sub.2O=85:5:3:5:2, (V/V)) was added in an amount of 12 ml/g (cleaving agent/resin). The resulting mixture was stirred for 3 h at room temperature and then filtered through a sand core funnel, and the filtrate was collected. The resin was washed twice with a small amount of TFA. The filtrates were combined and concentrated under reduced pressure. Ice-cold anhydrous ether was added to the filtrates for precipitation followed by centrifugation. The cake of the crude peptide was washed three times with anhydrous ether and dried under vacuum to give 370.6 g white powdery solid of lixisenatide crude peptide. MS (FIG. 2) MALDI-TOF: (M+H).sup.+=4859.288. The yield of the crude peptide was 96.8% and the purity detected by HPLC was 44.5%.

[0133] 49 g of the lipisatetide crude peptide obtained in D1.3 was dissolved and subjected to refining by using NOVASEP RP-HPLC system under conditions: wavelength 220 nm, reversed-phase C18 column. The lixisenatide was purified by using a normal 0.1% TFA/water and acetonitrile mobile phase system and salt conversion was performed. The fractions of the peak of interest were collected, concentrated by rotary evaporation and lyophilized to give 7.5 g of lixisenatide refined peptide. The purity of the refined peptide detected by HPLC was 98.6% and the yield was 15.4%.

Example 9

[0134] The refined peptides prepared in examples 1 to 8 and Comparative Example 1 were tested. The mass spectrum of the crude peptide obtained in Example 1 is shown in FIG. 1, and the mass spectrum of the crude peptide obtained in Comparative Example 1 is shown in FIG. 2. The mass spectra of the products obtained in the other examples are similar. The purity and yield of the polypeptide obtained in each example are shown in Table 2:

TABLE-US-00003 TABLE 2 Purity and yield of polypeptide MS MALDI-TOF: Yield of Purity of Yield of Refined Purity of Refined (M + H).sup.+ Crude Peptide Crude Peptide Peptide Peptide Example 1 4859.216 99.4% 69.1% 28.8% 99.3% Example 2 4859.017 101.2% 66.6% 26.4% 99.1% Example 3 4859.512 98.9% 68.4% 27.4% 99.2% Example 4 4859.347 102.6% 67.9% 27.1% 99.3% Example 5 4859.109 100.6% 67.1% 28.1% 99.3% Example 6 4859.205 99.6% 61.8% 25.8% 99.2% 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 4859.288 96.8% 44.5% 15.4% 98.6% Example 1

[0135] The results show that the technical solution of introducing dipeptide segment to the synthesis method is better than the method of comparative example in terms of the purity of the crude peptide, the purity of the refined peptide, and the yield of the refined peptide.

[0136] The above are merely preferred embodiments of the present invention, and it should be pointed out that those of ordinary skill in the art can also make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications should also be regarded as in the scope of protection of the present invention.