CN107286234B

CN107286234B - Method for reducing and/or removing default peptide in polypeptide solid phase synthesis

Info

Publication number: CN107286234B
Application number: CN201610200628.5A
Authority: CN
Inventors: 陈友金; 宓鹏程; 陶安进; 袁建成
Original assignee: Hybio Pharmaceutical Co Ltd
Current assignee: Hybio Pharmaceutical Co Ltd
Priority date: 2016-03-31
Filing date: 2016-03-31
Publication date: 2021-06-08
Anticipated expiration: 2036-03-31
Also published as: CN107286234A

Abstract

The invention relates to the field of polypeptide synthesis, in particular to a method for reducing and/or removing a default peptide in polypeptide solid phase synthesis. The method adopts a fully protected peptide fragment containing default amino acids and amino acids (C-N) before the default amino acids to carry out solid phase synthesis of the polypeptide, simultaneously solves the problems of amino acid default caused by incomplete Fmoc removal and incomplete condensation reaction, and can reduce the default peptide impurities to be below the quality control limit in the synthesis stage. Specifically, the peptide can be Ala in liraglutide²Default impurities and Thr⁵The default impurities are respectively reduced from more than 1 percent to undetectable and less than 0.15 percent, and Ser in nesiritide can be reduced¹⁹The default peptide impurities were reduced to undetectable.

Description

Method for reducing and/or removing default peptide in polypeptide solid phase synthesis

Technical Field

The invention relates to the field of polypeptide synthesis, in particular to a method for reducing and/or removing a default peptide in polypeptide solid phase synthesis.

Background

In solid phase polypeptide synthesis, some polypeptides have incomplete reaction due to the existence of difficult sequences, and default peptide (non-target peptide) impurities are easily formed. There are some default peptide impurities that are very close in nature to the product itself, for which existing separation techniques have little separation effect.

In the Fmoc solid phase synthesis method, the default peptide is generated mainly due to aggregation of the peptide chain, incomplete Fmoc removal of the temporary protecting group or incomplete condensation reaction. For incomplete Fmoc removal, complete removal can be achieved with 2% DBU, but with side reactions. The condensation reaction is incomplete, the pseudoproline adopted has a good effect, but the pseudoproline is expensive, can be used only when serine and threonine are needed, and has no universality. In addition, the generation of default peptide impurities is generally a result of incomplete Fmoc removal and incomplete condensation reactions acting simultaneously, and a single method does not completely solve the problem of amino acid defaults.

Two secondary structure areas exist in the GLP-1 compound, intramolecular interaction causes peptide chains to aggregate, reaction is difficult to carry out in the two areas, and various default peptide impurities are easy to generate. Liraglutide as GLP-1 analogue, Ala produced when Fmoc solid phase method was used²Default impurities and Thr⁵Default impurities are very close to the main peak in the chromatogram and cannot be effectively removed by the existing HPLC purification methods.

Disclosure of Invention

In view of the above, the present invention provides a method for reducing and/or removing a default peptide in solid phase synthesis of a polypeptide. The method adopts a fully protected peptide fragment containing default amino acids and amino acids (C-N) before the default amino acids to carry out solid phase synthesis of the polypeptide, simultaneously solves the problems of amino acid default caused by incomplete Fmoc removal and incomplete condensation reaction, and can reduce the default peptide impurities to be below the quality control limit in the synthesis stage. Specifically, the peptide can be Ala in liraglutide²Default impurities and Thr⁵The default impurities are respectively reduced from more than 1 percent to undetectable and less than 0.15 percent, and Ser in nesiritide can be reduced¹⁹The default peptide impurities were reduced to undetectable.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides the use of a peptide stretch consisting of a default amino acid and an amino acid immediately adjacent to the default amino acid and near the C-terminus for reducing and/or eliminating the default peptide in solid phase synthesis of a polypeptide.

In some embodiments of the invention, the number of amino acids immediately adjacent to the default amino acid and near the C-terminus is at least 1.

In some embodiments of the invention, the default peptide comprises no more than 0.15% by weight of the polypeptide.

In some embodiments of the invention, the polypeptide is liraglutide and the default amino acid is Ala²、Thr⁵。

In some embodiments of the invention, the peptide fragment is a fully protected peptide fragment.

The invention also provides a method for reducing and/or removing the default peptide in the solid-phase synthesis of the polypeptide, wherein the solid-phase synthesis is carried out by adopting the method which comprises the default amino acid and the amino acid which is adjacent to the default amino acid and close to the C end.

In some embodiments of the invention, the number of amino acids immediately adjacent to the default amino acid and near the C-terminus in the solid phase synthesis method is at least 1.

In some embodiments of the invention, the default peptide comprises no more than 0.15% by weight of the polypeptide in the solid phase synthesis method.

In some embodiments of the invention, the polypeptide is liraglutide and the default amino acid is Ala in a solid phase synthesis method²、Thr⁵；

Or the polypeptide is nesiritide, and the default amino acid is Ser¹⁹；

The peptide segment is a full-protection peptide segment.

The invention also provides a method for reducing and/or removing default peptides in solid phase synthesis of polypeptides,

the polypeptide is liraglutide, and comprises the following steps:

step 1: preparing Fmoc-Gly-Wang Resin by reacting Wang Resin with Fmoc-Gly-OH;

step 2: Fmoc-Arg (pbf) -OH, Fmoc-Gly-OH, Fmoc-Arg (pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (alloc) -OH, Fmoc-Ala-OH, Fmoc-Gln (trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, (Fmoc-Val-OH, Fmoc-Asp-OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -Phe-OH, Fmoc-Gly-OH, Fmoc-Ala-Glu (OtBu) -OH, Boc-His (trt) -OH, Fmoc-Glu-OtBu and palmitic acid to obtain a liraglutide peptide resin;

and step 3: cracking the liraglutide peptide resin to obtain a crude liraglutide product;

and 4, step 4: purifying the crude liraglutide by HPLC to obtain a pure liraglutide product;

the polypeptide is nesiritide, and comprises the following steps:

step 1: preparing Fmoc-His (trt) -2-CTC Resin by reacting 2-CTC Resin with Fmoc-His (trt) -OH;

step 2: Fmoc-Arg (pbf) -OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys (Boc) -OH, Fmoc-Cys (trt) -OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Ser (tBu) -Gly-OH and Fmoc-Ile-OH, Fmoc-Arg (pbf) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Met-OH, Fmoc-Lys (Boc) -OH, Fmoc-Arg (pbf) -OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys-trt-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Gly-OH, Fmoc-Gln (trt) -OH, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-Lys (Boc) -OH, Fmoc-Pro-OH and Fmoc-Ser (tBu) -OH to obtain nesiritide peptide resin;

and step 3: cracking the nesiritide peptide resin to obtain a crude nesiritide product;

and 4, step 4: and purifying the nesiritide crude product by HPLC to obtain a nesiritide pure product.

The invention provides the use of a peptide stretch consisting of a default amino acid and an amino acid immediately adjacent to the default amino acid and near the C-terminus for reducing and/or eliminating the default peptide in solid phase synthesis of a polypeptide. The invention adopts the full-protection peptide fragment containing the default amino acid and the previous amino acid (C-N) to carry out the solid phase synthesis of the polypeptide, simultaneously solves the problems of amino acid default caused by incomplete Fmoc removal and incomplete condensation reaction, and can reduce the default peptide impurities to be below the quality control limit in the synthesis stage. Specifically, the peptide can be Ala in liraglutide²Default impurities and Thr⁵The default impurities are respectively reduced from more than 1 percent to undetectable and less than 0.15 percent, and Ser in nesiritide can be reduced¹⁹The default peptide impurities were reduced to undetectable.

The invention adopts the relative full-protection peptide fragment of default amino acid to carry out solid phase synthesis, not only has cheap and easily obtained raw materials, but also can effectively reduce the impurities of the default peptide, can be applied to the synthesis of all polypeptides, and has wide industrial production value.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows an HPLC chromatogram of crude liraglutide prepared in example 3;

FIG. 2 shows an HPLC chromatogram of a crude liraglutide prepared by a comparative example;

FIG. 3 shows the HPLC chromatogram of a pure liraglutide prepared in example 4;

FIG. 4 shows an HPLC chromatogram of a pure nesiritide product obtained in example 9;

fig. 5 shows the HPLC profile of a pure liraglutide prepared by the comparative example.

Detailed Description

The invention discloses a method for reducing and/or removing a default peptide in polypeptide solid phase synthesis, and a person skilled in the art can use the content for reference and appropriately modify the process parameters to realize the purpose. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention provides a solution for default of amino acid in polypeptide synthesis, in particular to a GLP-1 compound, in particular to liraglutide, which comprises the following detailed steps:

step 1: preparing Fmoc-Gly-Wang Resin by reacting Wang Resin with Fmoc-Gly-OH;

and 4, step 4: and purifying the crude liraglutide by HPLC to obtain a pure liraglutide product.

The specific steps of nesiritide are as follows:

The invention provides the use of a peptide stretch consisting of a default amino acid and an amino acid immediately adjacent to the default amino acid and near the C-terminus for reducing and/or eliminating the default peptide in solid phase synthesis of a polypeptide. The invention adopts the full-protection peptide fragment containing the default amino acid and the previous amino acid (C-N) to carry out the solid phase synthesis of the polypeptide, simultaneously solves the problems of amino acid default caused by incomplete Fmoc removal and incomplete condensation reaction, and can reduce the default peptide impurities to be below the quality control limit in the synthesis stage. Specifically, the peptide can be Ala in liraglutide²Default impurities and Thr⁵Default impurities are respectivelyThe content of Ser in nesiritide can be reduced from 1% to undetectable and below 0.15%¹⁹The default peptide impurities were reduced to undetectable.

1. The invention is intended to protect a solution to the amino acid default first, specifically described as using a solid phase synthesis of a fully protected peptide fragment containing the default amino acid and its preceding amino acid;

2. the method is mainly applied to Ala in liraglutide²And Thr⁵The removal of default impurities, the present invention intends to protect all preparation methods against these two impurities;

or Ser in nesiritide¹⁹Removal of default peptide impurities.

3. The method has universality, and particularly the application of the method in the liraglutide and nesiritide.

The invention provides a method for reducing and/or removing default peptides in polypeptide solid phase synthesis, wherein raw materials and reagents used in the method are all available on the market.

The abbreviations and English meanings referred to in the present invention are shown in Table 1:

TABLE 1 abbreviations and English meanings to which the invention relates

Abbreviations and English	Means of
		DIC	N, N' -diisopropylcarbodiimide
DCM	Methylene dichloride
		Et₂O	Anhydrous diethyl ether
MeOH	Methanol
		DMAP	4-dimethylaminopyridine
HOBt	1-hydroxybenzotriazoles
		TFA	Trifluoroacetic acid
EDT
		1, 2-ethanedithiol
DMF			N, N-dimethylformamide
	Phenol	Phenol and its preparation
20％DBLK			20% hexahydropyridine (v)/N, N-dimethylformamide (v)
	DBU	1, 8-diazabicyclo [5.4.0 ]]Undec-7-enes

The invention is further illustrated by the following examples:

EXAMPLE 1 preparation of Fmoc-Gly-Wang Resin

Weighing 40g of Wang Resin with the substitution degree of 0.75mmol/g, adding the Wang Resin into a solid phase reaction column, washing with DMF for 2 times, swelling with DMF for 30min, washing with DMF for 2 times, dissolving Fmoc-Gly-OH (13.38g, 45mmol) and HOBt (6.38g, 47.3mmol) in DMF, adding DIC (5.95g, 47.3mmol) after activating for 3-5 min after ice bath for 10min, adding the reaction column, simultaneously adding DMAP (0.55g, 4.5mmol), stirring with nitrogen for 2h, draining the reaction liquid, washing with DMF for 4 times, washing with DCM for 3 times, adding acetic anhydride (61.2g, 600mmol) and pyridine (47.5g, 600mmol) solution, sealing for 8h, draining the sealing liquid, washing with DMF for 6 times, washing with DCM for 2 times, shrinking with MeOH, drying to obtain 43.6g of Fmoc-Gly-Wang Resin with the substitution degree of 0.32 mmol/g.

Example 2 Synthesis of liraglutide peptide resin

Weighing 31.3g of Fmoc-Gly-Wang Resin with the substitution degree of 0.32mmol/g, adding the Fmoc-Gly-Wang Resin into a solid phase reaction column, washing with DMF for 2 times, swelling with DMF for 30min, washing with DMF for 2 times, dissolving Fmoc-Arg (pbf) -OH (19.46g, 30mmol) and HOBt (4.26g, 31.5mmol) in DMF, carrying out ice bath for 10min, adding DIC (3.97g, 31.5mmol) for activation for 3-5 min, adding the mixture into the solid phase reaction column, stirring with nitrogen for reaction for 2h, completely detecting ninhydrin, pumping out reaction liquid, washing with DMF for 3 times, deprotecting 20% DBLK (5+7min), and washing with DMF for 6 times;

dissolving Fmoc-Gly-OH (8.92g, 30mmol) and HOBt (4.26g, 31.5mmol) in DMF, carrying out ice bath for 10min, adding DIC (3.97g, 31.5mmol) for activation for 3-5 min, adding into a solid phase reaction column, stirring with nitrogen for reaction for 2h, completely detecting ninhydrin, pumping out reaction liquid, washing with DMF for 3 times, deprotecting 20% DBLK (5+7min), and washing with DMF for 6 times;

Fmoc-Arg (pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (alloc) -OH, Fmoc-Ala-OH, Fmoc-Gln (trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr-ThrBu) -OH, Fmoc-Leu-OH, Fmoc-Gly-Ala-OH, Fmoc-Ala, Fmoc-Thr (tBu) -Phe-OH, Fmoc-Gly-OH, Fmoc-Ala-Glu (OtBu) -OH, Boc-His (trt) -OH, Fmoc-Glu-OtBu and palmitic acid, to give 86.2g of liraglutide peptide resin.

Example 3 cleavage of liraglutide peptide resin

86.2g of liraglutide peptide resin was transferred to a 1000ml single neck round bottom flask and 860m was addedl freezing the lysate for 2H (TFA: Phenol: H)₂O: EDT 87.5: 5: 5: 2.5), stirring and reacting for 2h at room temperature, filtering, adding the filtrate into 8.6L of frozen anhydrous ether, centrifuging, washing and drying to obtain 37.3g of crude peptide, wherein the yield is as follows: 99.5%, purity: 53.79%, see FIG. 1.

Example 4 preparation of pure liraglutide

1. Sample treatment: the solid crude peptide is treated with 10% acetonitrile/90% water (V/V), the sample is completely dissolved by ultrasonic treatment, and then the solid crude peptide is filtered by a filter membrane, and the filtrate is collected for later use.

HPLC purification:

and (3) purification conditions: a chromatographic column: the chromatographic column using the octaalkylsilane bonded silica as a stationary phase has the following diameters and lengths: 50mm by 250 mm. Mobile phase: phase A: 0.1% trifluoroacetic acid 85% water/15% isopropanol solution in water; phase B: acetonitrile of 0.1% trifluoroacetic acid, flow rate: 50-80ml/min, gradient: 40% B-60% B, detection wavelength: 275 nm. The amount of the sample was 3 g.

And (3) purification process: washing the chromatographic column with acetonitrile over 50%, and balancing the sample loading amount of 1.5-3 g. Eluting with linear gradient for 40min, collecting target peak to obtain fraction with purity higher than 95%, concentrating the collected target peak fraction to about 10-30mg/ml under reduced pressure at water temperature not higher than 35 deg.C, and purifying with the second step.

3. Second step HPLC purification:

and (3) purification conditions: a chromatographic column: the chromatographic column using the octaalkylsilane bonded silica as a stationary phase has the following diameters and lengths: 50mm by 250 mm. Mobile phase: 0.15% perchloric acid in water as phase A, 0.15% perchloric acid in acetonitrile as phase B, gradient: 40% B-70% B, detection wavelength: 275 nm. The amount of sample was 1.8 g.

And (3) purification process: after the chromatographic column was washed clean and equilibrated with 50% or more acetonitrile, the first-step purified fraction was loaded in an amount of 1.9 g. Eluting with linear gradient for 40min, collecting target peak to obtain fraction with purity higher than 97%, concentrating the collected target peak fraction to about 15-25mg/ml under reduced pressure rotary evaporation at water temperature not higher than 35 deg.C, and desalting to purify sample.

4. Third step, HPLC desalination purification: a chromatographic column: the chromatographic column using the octaalkylsilane bonded silica as a stationary phase has the following diameters and lengths: 50mm by 250 mm. Taking 0.01% ammonia water solution as phase A, taking chromatographic pure acetonitrile as phase B, and gradient: 30% B-60% B, detection wavelength: 275 nm. The amount of sample was 1.2 g.

And (3) purification process: after the chromatographic column is washed clean and balanced by more than 50% acetonitrile, the second-step purified fraction is loaded, and the loading amount is 1.3 g. Eluting with linear gradient for 30min, collecting target peak to obtain fraction with purity higher than 98%, concentrating the collected target peak fraction to about 65mg/ml under reduced pressure and rotary evaporation at water temperature not higher than 35 deg.C, and freeze drying to obtain 0.76g of liraglutide with purity of 99.65%, with total yield of 25.3%, as shown in FIG. 3.

Example 5 preparation of Fmoc-His (trt) -2-CTC Resin

Weighing 60g of 2-CTC Resin with the substitution degree of 0.5mmol/g, adding the 2-CTC Resin into a solid phase reaction column, washing with DMF for 2 times, swelling with DMF for 30min, washing with DMF for 2 times, dissolving Fmoc-His (trt) -OH (74.4g, 120mmol) in DMF, adding DIPEA (30.96g, 240mmol) after ice bath for 5min, activating for 3-5 min, adding the mixture into the reaction column, stirring with nitrogen for reaction for 1h, adding MeOH (19.2g, 600mmol) into the reaction liquid, continuing stirring with nitrogen for reaction for 20min, draining the reaction liquid, washing with DMF for 4 times, washing with DCM for 3 times, shrinking the MeOH, and drying to obtain 66g of Fmoc-His (trt) -2-CTC Resin with the substitution degree of 0.28 mmol/g.

Example 6 Synthesis of nesiritide peptide resin

Weighing 34.7g of Fmoc-His (trt) -2-CTC Resin with the substitution degree of 0.28mmol/g, adding the Fmoc-His (trt) -2-CTC Resin into a solid-phase reaction column, washing with DMF for 2 times, swelling with DMF for 30min, washing with DMF for 2 times, dissolving Fmoc-Arg (pbf) -OH (19.46g, 30mmol), PyBOP (15.6g, 30mmol) and HOBt (4.26g, 31.5mmol) in DMF, carrying out ice bath for 10min, adding DIPEA (7.74g, 60mmol) for activation for 3-5 min, adding the mixture into the solid-phase reaction column, stirring with nitrogen for 2h to complete ninhydrin detection reaction, pumping out reaction liquid, washing with DMF for 3 times, deprotecting 20% DBLK (5+7min), and washing with DMF for 6 times;

dissolving Fmoc-Arg (pbf) -OH (19.46g, 30mmol), PyBOP (15.6g, 30mmol) and HOBt (4.26g, 31.5mmol) in DMF, carrying out ice bath for 10min, adding DIPEA (7.74g, 60mmol) for activation for 3-5 min, adding into a solid phase reaction column, stirring with nitrogen for reaction for 2h, completely detecting ninhydrin, pumping out reaction liquid, washing with DMF for 3 times, deprotecting 20% DBLK (5+7min), and washing with DMF for 6 times;

Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys (Boc) -OH, Fmoc-Cys (trt) -OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Ser (tBu) -Gly-OH and Fmoc-Ile-OH, Fmoc-Arg (pbf) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Met-OH, Fmoc-Lys (Boc) -OH, Fmoc-Arg (pbf) -OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-trt) -OH, Fmoc-Gly-OH, Fmoc-Ser tBu) -OH, (Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Leu-Lys (tBu) -OH, Fmoc-Gly-, Fmoc-Gln (trt) -OH, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-Lys (Boc) -OH, Fmoc-Pro-OH and Fmoc-Ser (tBu) -OH to obtain 61g of nesiritide peptide resin.

Example 7 Nexillin peptide resin cleavage

61g of liraglutide peptide resin was transferred to a 1000ml single-neck round-bottom flask, and 610ml of frozen lysate (TFA: Phenol: PhSMe: H) for 2H was added₂O: EDT 82.5: 5: 5: 5: 2.5), stirring and reacting for 2h at room temperature, filtering, adding the filtrate into 6.1L of frozen anhydrous ether, centrifuging, washing and drying to obtain 25.3g of linear peptide, wherein the yield is as follows: 99.5%, purity: 73.1 percent.

Example 8 Nexilic peptide Linear peptide Oxidation

25.3g of a linear peptide was weighed and prepared at a ratio of 8 to 10mg/ml as a reaction solution [ a mixed solution of purified water and acetonitrile (1:1 ═ v: v) ]. And (3) after fully grinding the linear peptide, slowly adding the linear peptide into the reaction solution while stirring until the linear peptide is completely dissolved, adjusting the pH value of the reaction solution to 8.1-8.4 by using 25% ammonia water, introducing oxygen, stirring for reacting for 4 hours, stopping the reaction when the HPLC shows complete oxidation, and adjusting the pH value to 3.0-3.5 by using TFA.

EXAMPLE 9 preparation of pure Nexillin

1. Sample treatment: the oxidized crude peptide solution in example 8 is concentrated by rotary evaporation under reduced pressure to remove most of the acetonitrile in the solution, i.e. the volume after rotary evaporation is 50-60% of the volume before rotary evaporation. Filtering with 0.45 μm microporous membrane, and mixing filtrates.

HPLC purification:

and (3) purification conditions: a chromatographic column: the chromatographic column using octadecylsilane chemically bonded silica as a stationary phase has the following diameter and length: 50mm by 250 mm. Mobile phase: phase A: adjusting the pH value to 3.0 by 0.2 percent sulfuric acid solution and ammonia water; phase B: acetonitrile, flow rate: 50-80ml/min, gradient: 10% B-30% B, detection wavelength: 230 nm. The amount of the sample was 3 g.

And (3) purification process: washing the chromatographic column with acetonitrile over 50%, and balancing the sample loading amount of 1.5-3 g. Eluting with linear gradient for 60min, collecting target peak to obtain fraction with purity higher than 95%, concentrating the collected target peak fraction to about 10-30mg/ml under reduced pressure under water temperature not higher than 32 deg.C, and purifying with the second step.

3. Second step HPLC purification:

and (3) purification conditions: a chromatographic column: the chromatographic column using octadecylsilane chemically bonded silica as a stationary phase has the following diameter and length: 50mm by 250 mm. Mobile phase: 0.2% phosphoric acid solution (pH adjusted to 6.5 with ammonia) as phase a, acetonitrile solution as phase B, gradient: 10% B-30% B, detection wavelength: 230 nm. The amount of sample was 1.8 g.

And (3) purification process: after the chromatographic column was washed clean and equilibrated with 50% or more acetonitrile, the first-step purified fraction was loaded in an amount of 1.9 g. Eluting with linear gradient for 60min, collecting target peak to obtain fraction with purity higher than 97%, concentrating the collected target peak fraction to about 15-25mg/ml under reduced pressure under water temperature not higher than 32 deg.C, and desalting to purify sample.

4. Third step, HPLC desalination purification: a chromatographic column: the chromatographic column using octadecylsilane chemically bonded silica as a stationary phase has the following diameter and length: 50mm by 250 mm. Taking a 0.2% ammonium acetate solution as a phase A, taking chromatographic pure acetonitrile as a phase B, and performing gradient: 30% B-60% B, detection wavelength: 275 nm. The amount of sample was 1.2 g.

And (3) purification process: after the chromatographic column is washed clean and balanced by more than 50% acetonitrile, the second-step purified fraction is loaded, and the loading amount is 1.3 g. Eluting with linear gradient for 30min, collecting target peak to obtain fraction with purity higher than 98%, concentrating the collected target peak fraction to about 65mg/ml under reduced pressure and rotary evaporation at water temperature not higher than 35 deg.C, and freeze drying to obtain 0.76g active drug nesiritide with purity higher than 98.5%, with total yield of 25.3%, as shown in FIG. 4.

Comparative example liraglutide Synthesis

Weighing 6.25g of Fmoc-Gly-Wang Resin with the substitution degree of 0.32mmol/g, adding the Fmoc-Arg (pbf) -OH (3.89g, 6mmol) and HOBt (0.85g, 6.3mmol) into a solid phase reaction column, washing with DMF for 2 times, swelling with DMF for 30min, washing with DMF for 2 times, dissolving Fmoc-Arg (pbf) -OH (3.89g, 6.3mmol) in DMF, carrying out ice bath for 10min, adding DIC (0.79g, 6.3mmol) into the solid phase reaction column after activation for 3-5 min, stirring with nitrogen for reaction for 2h, completely detecting ninhydrin, pumping out reaction liquid, washing with DMF for 3 times, deprotecting 20% DBLK (5+7min), and washing with DMF for 6 times;

dissolving Fmoc-Gly-OH (1.78g, 6mmol) and HOBt (0.85g, 6.3mmol) in DMF, carrying out ice bath for 10min, adding DIC (0.79g, 6.3mmol) for activation for 3-5 min, adding into a solid phase reaction column, stirring with nitrogen for reaction for 2h, completely detecting ninhydrin, pumping out reaction liquid, washing with DMF for 3 times, deprotecting 20% DBLK (5+7min), and washing with DMF for 6 times;

Fmoc-Arg (pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (alloc) -OH, Fmoc-Ala-OH, Fmoc-Gln (trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr-ThrBu) -OH, Fmoc-Leu-OH, Fmoc-Gly-Ala-OH, Fmoc-Ala, Fmoc-Thr (tBu) -Phe-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH, Boc-His (Trt) OH, Fmoc-Glu-OtBu and palmitic acid, to give 16.9g of liraglutide peptide resin. The liraglutide peptide resin was cleaved as in example 3 to give 7.3g of crude liraglutide peptide, yield: 97.3%, purity: 50.08%, the spectrum is shown in FIG. 2.

The crude liraglutide was purified as in example 4 to give 0.70g of fine liraglutide with 97.74% purity in a total yield of 23.3%, as shown in fig. 5.

The results are shown in table 2, in comparison with the liraglutide prepared in example 3 and example 4:

TABLE 2 comparison of liraglutide prepared in comparative example with that prepared in example

Example 3 compared to the comparative example, the crude peptide obtained by the process of the present invention was significantly superior (P < 0.05) to the comparative example in both purity and default peptide impurities, and the purified peptide obtained by the same process was purified, and the purified peptide obtained by example 4 was significantly superior to the purified peptide obtained by the comparative example in both purity and default peptide impurities.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A method for reducing and/or removing a default peptide in solid phase synthesis of a polypeptide,

the polypeptide is liraglutide, and comprises the following steps:

step 1: preparing Fmoc-Gly-Wang Resin by reacting Wang Resin with Fmoc-Gly-OH;