A method for solid-phase synthesis of polypeptides

Abstract

This invention relates to a method for solid-phase synthesis of peptides, comprising the following steps: (A) removing temporary protecting groups of amino acids from a solid-phase resin; (B) sequentially soaking or rinsing the solid-phase resin with an acidic solution and an alkaline solution; (C) sequentially coupling amino acids according to a target sequence via a one-to-one condensation method to synthesize a peptide having the target sequence; and (D) cleaving the peptide having the target sequence from the solid-phase resin to obtain the peptide product. This invention effectively improves the purity of peptide products in solid-phase synthesis of peptides, especially when synthesizing peptide sequences with long sequences that easily form secondary structures.

Description

Technical Field

[0001] This invention relates to a method for solid-phase synthesis of polypeptides. Background Technology

[0002] Polypeptides are compounds consisting of 10 to 100 amino acids linked by peptide bonds, similar to proteins. They are naturally occurring bioactive substances found in living organisms and have been known for over a century. Tens of thousands of polypeptides have been discovered in living organisms, exhibiting broad and important biological activities, acting extensively on the endocrine, immune, digestive, cardiovascular, hematopoietic, and musculoskeletal systems. Polypeptides have been used as pharmaceuticals for over 70 years, attracting increasing attention due to their unique advantages such as low toxicity, high specificity, and small molecular weight. To date, over 100 polypeptide drugs have been approved globally, including blockbuster drugs such as glatiramer, octreotide, liraglutide, dulaglutide, and semaglutide, all with annual sales in the billions of dollars.

[0003] Currently, peptide products on the market are mainly produced through three methods: animal tissue extraction, gene recombination expression, and chemical synthesis. Peptide drugs extracted from animal tissues will be gradually phased out. While gene engineering methods, primarily based on DNA recombination technology, have solved the problem of peptide production difficulties to some extent, some sequences have long production cycles, low expression efficiency, and the obtained products are even difficult to purify. Furthermore, some complex peptide molecules, especially those containing non-natural amino acids or modified peptides, cannot be realized through biological expression. Chemical synthesis is clearly the most direct method for synthesizing peptide drugs. It allows for convenient and rapid modification of peptide structures, such as introducing non-natural amino acids to improve stability, and chemically modifying them to adjust their hydrophilicity / hydrophobicity and affinity for certain targets, thereby enhancing the drug-likeness of the peptide molecules. Chemical synthesis methods mainly include liquid-phase synthesis and solid-phase synthesis. The former is generally used for the synthesis of shorter peptide sequences, while the structural complexity of peptide drugs is increasing, and solid-phase synthesis technology is now widely used for their synthesis.

[0004] Solid-phase synthesis of peptides was invented in 1963 by American chemist Professor R. Bruce Merrifield, marking a milestone in peptide chemistry. Its basic principles are as follows: Figure 1As shown, this method begins with the first amino acid attached to an insoluble carrier. Through a cyclical process of activation, coupling, and removal of temporary protecting groups, the protecting amino acids (semi-permanent protecting groups protecting active side chains) are "assembled" one by one to form the target polypeptide molecule. Finally, the polypeptide is cleaved from the insoluble carrier while simultaneously removing the semi-permanent protecting groups to obtain the polypeptide product. The significant advantages of this method are: it simplifies and accelerates multi-step synthesis, avoiding losses caused by manual operation and repeated material transfer; it enables rapid separation of unreacted substances through filtration and washing, avoiding the significant losses caused by lengthy intermediate separation and purification steps (recrystallization or silica gel column purification) in liquid-phase peptide synthesis; it promotes the complete completion of individual difficult reactions, achieving a high yield of the final product; the use of a solid-phase carrier can alleviate peptide chain self-aggregation, facilitating the smooth progress of the synthesis reaction; and all reactions can be carried out in a single reaction vessel, facilitating automation. Particularly noteworthy is the high versatility and short initial process development time of solid-phase synthesis, making it highly suitable for the development of polypeptide drugs. With the development and optimization of this solid-phase synthesis method, a large number of polypeptides, hormones and some proteins with important biological activities have been synthesized in a short time, which has greatly promoted the development of life science research. For this, Professor Merrifield was awarded the Nobel Prize in Chemistry in 1984.

[0005] In solid-phase synthesis, the benzyloxycarbonyl (Z) group was initially used as a temporary protecting group (protecting group of the α-amino group). Its removal requires strong acidolysis conditions. Therefore, the tert-butyloxycarbonyl (Boc) protecting group, which can be removed under trifluoroacetic acid (TFA) conditions, was later adopted. This method offers relatively mild removal conditions and minimizes racemization during the removal process. However, this method is still unsuitable for the synthesis of peptides containing tryptophan and other acid-labile peptides. Furthermore, the final cleavage often employs highly corrosive hydrofluoric acid or trifluoromethanesulfonic acid methods, placing extremely stringent demands on the equipment.

[0006] Later, Carpino et al. developed fluorene methoxycarbonyl (Fmoc) as an α-amino protecting group for amino acid raw materials in solid-phase peptide synthesis. It can be removed by secondary amine molecules such as piperidine, but it is relatively stable to acids, so it can be used in combination with semi-permanent protecting groups such as tert-butyl (tBu) and triphenylmethyl (Trt). In this way, the final cleavage can be achieved in TFA, the process is relatively mild, and the requirements for equipment are much lower. In recent years, solid-phase synthesis of peptides based on the Fmoc / tBu method has been widely used. The specific synthesis consists of the following four steps: 1) Deprotection: The resin is treated with an alkaline solvent (such as piperidine) to remove the protecting group Fmoc of the amino group to release the α-amino group; 2) Activation and coupling: The carboxyl group of the next amino acid to be coupled is activated by an activator and then couples with the released α-amino group to form a peptide bond; 3) Steps 1) and 2) are repeated until the synthesis is complete; 4) Cleavage: The peptide is cleaved from the resin, and the semi-permanent protecting groups of the amino acid side chains are also removed. Finally, the cleavage solution is added to diethyl ether or methyl tert-butyl ether for crystallization to obtain the peptide product.

[0007] Peptide solid-phase synthesis technology based on the Fmoc / tBu method has many advantages. However, because it is a continuous production process, errors in the final peptide product are accumulated due to side reactions such as fragmentation, deletion, isomerization, oxidation, reduction, and hydrolysis at each step. This poses a significant challenge to subsequent peptide purification processes (mainly high-performance liquid chromatography purification), especially when preparing longer peptide sequences or peptide sequences that are prone to secondary structure formation. Problems become particularly severe, such as low purity of crude peptides (i.e., cleaved peptide products), unsatisfactory synthesis yields, and numerous impurities affecting purification. This is, in fact, the main reason limiting the application of peptide solid-phase synthesis technology to longer peptide sequences and even protein synthesis. Therefore, synthesizing high-purity peptide products is undoubtedly one of the most important directions for improving the efficiency (synthesis efficiency and purification efficiency) of solid-phase peptide production processes. Summary of the Invention

[0008] The purpose of this invention is to provide a method for solid-phase synthesis of peptides that effectively improves the purity of peptide products, especially when synthesizing peptide sequences that are long and prone to forming secondary structures.

[0009] The present invention adopts the following technical solution:

[0010] A method for solid-phase synthesis of polypeptides, comprising the following steps:

[0011] (A) Removing temporary amino acid protecting groups from the solid resin;

[0012] (B) The solid resin is soaked or rinsed sequentially with acidic and alkaline solutions;

[0013] (C) A polypeptide with the target sequence is synthesized by sequentially coupling amino acids according to the target sequence through one-to-one condensation.

[0014] (D) The polypeptide having the target sequence is cleaved from the solid resin to obtain the polypeptide product.

[0015] Furthermore, the polypeptide is a polypeptide having 10-100 amino acids, preferably a polypeptide having 20-100 amino acids, and more preferably a polypeptide having 40-100 amino acids.

[0016] Furthermore, in step (A), the temporary protecting group of the amino acid is Fmoc.

[0017] Further, in step (A), the solid resin is a king resin, a Rink resin, an HMPA-PEGA resin, an FMPBAM resin, or a DHP HM resin. King resin and Rink resin are preferred.

[0018] Furthermore, in step (A), the solution for removing the temporary protecting group of the amino acid is an alkaline solution. Specifically, it can be a solution of a secondary amine molecule. More specifically, the alkaline solution can be a DMF solution of piperidine, a DMF solution of 4-methylpiperidine, a solution of N-methylpyrrolidone of piperidine, a solution of N-methylpyrrolidone of 4-methylpiperidine, or a combination thereof, wherein the alkaline solution contains 15 vol% to 25 vol% of piperidine or 4-methylpiperidine.

[0019] Furthermore, in step (B), the solvents for the acidic and alkaline solutions are dichloromethane, N-methylpyrrolidone, dimethylformamide, dimethyl sulfoxide, or water.

[0020] Furthermore, in step (B), the solute in the acidic solution is one or more of trifluoroacetic acid, trifluoroethanol, and hydrochloric acid; its volume fraction in the acidic solution is 0.1% to 20%, preferably 1% to 15%, and more preferably 5% to 10%.

[0021] Furthermore, in step (B), the solute in the alkaline solution is one or more of N,N-diisopropylethylamine, triethylamine, pyridine, and ammonia; its volume fraction in the alkaline solution is 1% to 10%, preferably 2% to 8%, and more preferably 4% to 6%.

[0022] Furthermore, in step (B), when the solid resin is treated with acidic and alkaline solutions in sequence, the specific method can be soaking or continuous flow rinsing, wherein oscillation, stirring, bubbling, and combinations thereof can be performed simultaneously during soaking.

[0023] Furthermore, in step (C), the condensing agent used in the coupling of the amino acids is selected from one or more of 1-hydroxybenzotriazole monohydrate (HOBT), N,N-dicyclohexylcarbodiimide (DCC), N,N-diisopropylcarbodiimide (DIC), O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), and 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazine-4-one (DEPBT).

[0024] Furthermore, in step (D), the lysis buffer is a mixture of trifluoroacetic acid, triethylsilane, anisole and phenol in a volume ratio of 85:5:5:5.

[0025] Furthermore, the polypeptide is an exenatide analogue, which includes the following steps:

[0026] (1) Weigh 2g of solid resin with a bonding amount of 0.5mmol / g, i.e., a total bonding amount of 1mmol; add a solution of secondary amine molecules to it, stir for 20 minutes to remove the Fmoc group; after the Fmoc removal reaction is completed, wash the solid resin alternately with DMF and IPA; at this time, add 2mmol of the amino acid Fmoc-Lys(Dde)-OH with a protecting group, 2mmol of TBTU and 4mmol of DIPEA to the solid resin, stir for 2 hours at room temperature to couple the amino acid Lys40; after the coupling reaction is completed, wash the solid resin alternately with DMF and IPA; repeat the above Fmoc removal and amino acid linkage process according to the peptide sequence of the exenatide analog until the synthesis ends at Gly29;

[0027] (2) Starting from the synthesis of Asn28, a solution of secondary amine molecules was first added and the reaction was stirred for 20 minutes to remove the Fmoc group. After the Fmoc removal reaction was completed, the solid resin was washed alternately with DMF and IPA. After washing, the solid resin was washed with an acidic solution, then with an alkaline solution, and finally with DMF. Then, the amino acid Asn28 was linked. At this time, 2 mmol of the amino acid Fmoc-Asn(Trt)-OH with a protecting group, 2 mmol of TBTU and 4 mmol of DIPEA were added to the solid resin and the reaction was stirred at room temperature for 2 hours. After the linking reaction was completed, the solid resin was washed alternately with DMF and IPA. The above Fmoc removal and amino acid linking process was repeated according to the peptide sequence of the exenatide analog until Leu10 was synthesized.

[0028] (3) Starting from Asp9, repeat the Fmoc removal process and amino acid linkage process in step (1). Link different amino acids one by one to the solid resin according to the peptide sequence of the exenatide analog. The main chain synthesis ends at His1. Then, treat the solid resin twice with 2% hydrazine hydrate / DMF for 10 minutes each time to remove the Dde protecting group. The subsequent coupling conditions of AEEAc and MPA are: 2 mmol AEEAc / MPA, 2 mmol HOBT and 2 mmol DIC. Stir the reaction at room temperature for 2 hours.

[0029] (4) After drying the solid resin obtained in step (3), add it to the lysis buffer to lyse the peptide from the solid resin. At the same time, remove the side chain protecting groups and then obtain the crude peptide of exenatide analogue by crystallization and drying.

[0030] The beneficial effects of this invention are as follows:

[0031] The method of the present invention effectively improves the purity of peptide products by treating the solid-phase resin with acidic and alkaline solutions in sequence, thereby reducing the difficulty of subsequent purification and improving the overall efficiency of peptide solid-phase synthesis.

[0032] In particular, for some polypeptide sequences that are long and prone to forming secondary structures, the method of the present invention can significantly improve the purity of such polypeptide products synthesized in solid phase.

[0033] The number and content of potential impurities in peptide products prepared by this method are reduced accordingly, thereby enhancing the biological activity of the sample and improving the safety and efficacy of pharmaceutical peptides.

[0034] The method of this invention is simple and easy to implement, and has high practical value in both laboratory-scale synthesis and industrial production of peptides. Attached Figure Description

[0035] Figure 1 This is a basic schematic diagram of peptide solid-phase synthesis technology.

[0036] Figure 2 The image shows the HPLC chromatogram of the crude peptide of the exenatide analog prepared by the conventional solid-phase synthesis method of peptides according to the comparative example.

[0037] Figure 3 The image shows the HPLC chromatogram of the crude peptide of the exenatide analog prepared by the synthesis method of Example 1 of the present invention.

[0038] Figure 4 The image shows the HPLC chromatogram of the crude peptide of the exenatide analog prepared by the synthesis method of Example 2 of the present invention.

[0039] Figure 5The HPLC chromatogram is shown for the crude peptide of the exenatide analog prepared by the synthesis method of Example 3 of the present invention.

[0040] Figure 6 The HPLC chromatogram is shown for the crude peptide of the exenatide analog prepared by the synthesis method of Example 4 of the present invention.

[0041] Figure 7 The HPLC chromatogram is shown for the crude peptide of the exenatide analog prepared by the synthesis method of Example 5 of the present invention.

[0042] Figure 8 The HPLC chromatogram is shown for the crude peptide of the exenatide analog prepared by the synthesis method of Example 6 of the present invention.

[0043] Figure 9 The HPLC chromatogram is shown for the crude peptide of the exenatide analog prepared by the synthesis method of Example 7 of the present invention. Detailed Implementation

[0044] The present invention will be further described below through specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0045] This invention provides a method for solid-phase synthesis of polypeptides, mainly comprising the following steps: 1) removing temporary amino acid protecting groups from a solid resin; 2) treating the solid resin sequentially with an acidic solution and an alkaline solution; 3) coupling amino acids sequentially according to the target sequence by a one-to-one condensation method to synthesize a polypeptide having the target sequence; 4) cleaving the polypeptide having the target sequence from the solid resin to obtain the polypeptide product.

[0046] The "peptide product" or "crude peptide" mentioned in this invention refers to a product obtained directly through solid-phase synthesis of peptides without further purification. This peptide product can be further purified using techniques such as high-performance liquid chromatography to obtain the final peptide product.

[0047] The polypeptide product of the present invention may be a polypeptide having 10 to 100 amino acids, preferably a polypeptide having 20 to 100 amino acids, and more preferably a polypeptide having 40 to 100 amino acids.

[0048] The solid resin used in step 1) of the method of the present invention is linked to an amino acid containing a temporary protecting group. In one specific embodiment of the present invention, the temporary protecting group of the amino acid is Fmoc.

[0049] The acidic solution used in step 2) of the method of the present invention comprises an acid and a solvent, wherein the acidic solution comprises 0.1 vol% to 20 vol% of acid, preferably 1 vol% to 15 vol% of acid, and more preferably 5 vol% to 10 vol% of acid. When the acid content is less than 0.1 vol%, the acidic solution is too weak to function effectively in the treatment of the solid-phase resin; when the acid content is greater than 20 vol%, the acidity is too strong and may pose a risk of cleaving the polypeptide chains off the solid-phase resin. The acid may be selected from trifluoroacetic acid, trifluoroethanol, hydrochloric acid, and combinations thereof, and the solvent may be selected from dichloromethane, N-methylpyrrolidone, dimethylformamide, dimethyl sulfoxide, and water.

[0050] The alkaline solution used in step 2) of the method of the present invention comprises an alkali and a solvent, wherein the alkaline solution comprises 1 vol% to 10 vol% alkali, preferably 2 vol% to 8 vol% alkali, and more preferably 4 vol% to 6 vol% alkali. When the alkali content is less than 1 vol%, the alkaline solution is too weak to function effectively in the treatment of the solid resin; when the alkali content is greater than 10 vol%, excessive alkalinity may cause problems such as racemic and aspartic imide side reactions. The alkali may be selected from N,N-diisopropylethylamine, triethylamine, pyridine, ammonia, and combinations thereof, and the solvent may be selected from dichloromethane, N-methylpyrrolidone, dimethylformamide, dimethyl sulfoxide, and water.

[0051] Based on current laboratory observations and related research, we speculate that the reason why acid / base treatment can improve product purity may be that acid / base treatment can remove some intermediate products generated during Fmoc removal, converting them to the expected free amino groups to the greatest extent, thus improving the removal efficiency of Fmoc removal and reducing the generation of related impurity peptides.

[0052] In step 4) of the method of the present invention, the peptide resin having the target sequence is added to the lysis buffer (a mixture of TFA, triethylsilane, anisole sulfide and phenol in a volume ratio of 85:5:5:5) and reacted for 3 hours to obtain the peptide product.

[0053] The following specific implementation scheme further explains or illustrates the content of the present invention, but these examples should not be construed as limiting the scope of protection of the present invention.

[0054] In this specific embodiment, the polypeptide product may be an exenatide analog [Lys40(ε-AEEAc-MPA)-NH2]-Exendin-4 (hereinafter referred to as CJC-1134, whose polypeptide sequence is: 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-Pro-Pro-Ser-Lys(AEEAc-MPA)-NH2).

[0055] The meanings of the abbreviations used in this invention are listed in Table 1 below.

[0056] Table 1. Abbreviation Name Comparison Table

[0057] .

[0058] The sources of materials and instruments used in the following comparative examples / exemplary cases are as follows:

[0059] Solid-phase resins: Amino resins (Ramage resin, NovaSyn TGA resin) and the protected amino acids used were purchased from Bachem; TFA was purchased from Aladdin; triethylsilane, anisole, and phenol were purchased from Sinopharm Group; TBTU, DIPEA, HOB, and DIC were all purchased from Suzhou Haofan; DMF and IPA were purchased from Xilong Scientific; and DCM was purchased from Shandong Jinling.

[0060] High performance liquid chromatography: Thermo U3000, analysis software: Chromeleon, column: Kromasil C8, flow rate: 1.0 mL / min, column temperature: 35˚C, detector wavelength: 220 nm, injection volume: 20 μl.

[0061] The HPLC gradient is as follows:

[0062] .

[0063] Crude peptide CJC-1134 was prepared using conventional solid-phase peptide synthesis methods in the comparative example.

[0064] The specific steps of conventional solid-phase peptide synthesis methods are as follows:

[0065] (1) Weigh 2 g of solid-phase resin (amino resin, Ramage Resin) with a bonding amount of 0.5 mmol / g, i.e., a total bonding amount of 1 mmol. Add a 20% (v / v) DMF solution of piperidine to it and stir for 20 minutes to remove the Fmoc group. After the Fmoc removal reaction is completed, wash the solid-phase resin alternately with DMF and IPA. At this time, add 2 mmol Fmoc-Lys(Dde)-OH, 2 mmol TBTU and 4 mmol DIPEA to the solid-phase resin and stir for 2 hours at room temperature to couple the amino acid Lys40. After the coupling reaction is completed, wash the solid-phase resin alternately with DMF and IPA. Repeat the above Fmoc removal and amino acid linkage process according to the peptide sequence of CJC-1134, and the main chain synthesis ends at His1. The solid resin was then treated twice with 2% hydrazine hydrate / DMF for 10 minutes each time to remove the Dde protecting group. The subsequent coupling conditions for AEEAc and MPA were 2 mmol AEEAc / MPA, 2 mmol HOBT and 2 mmol DIC, and the reaction was stirred at room temperature for 2 hours.

[0066] (2) After drying the solid resin in step (1), add it to the lysis buffer (a mixture of TFA, triethylsilane, anisole sulfide and phenol in a volume ratio of 85:5:5:5) to cleave the peptide from the solid resin. At the same time, remove the side chain protecting groups and then obtain the crude peptide CJC-1134 by crystallization and drying.

[0067] The HPLC chromatogram of the crude peptide CJC-1134 prepared by conventional solid-phase peptide synthesis method is shown below. Figure 2 As shown, the integral results of the HPLC curves are presented in Table 2 below. From the integral results in Table 2, it can be seen that the crude CJC-1134 peptide prepared in the comparative example contains impurities (pre-impurities and post-impurities) and CJC-1134. The peak area of ​​CJC-1134 accounts for 26.55% of the total peak area, meaning that the purity of the crude CJC-1134 peptide prepared by the conventional method is 26.55%.

[0068] Table 2. HPLC curve integration results of the crude CJC-1134 peptide prepared in the comparative example.

[0069] .

[0070] Example 1: Preparation of crude CJC-1134 peptide by the method of the present invention

[0071] The steps for preparing crude CJC-1134 peptide according to the method for improving the purity of solid-phase synthesized polypeptide products of the present invention are as follows:

[0072] (1) Weigh 2 g of solid-phase resin (amino resin, Ramage resin) with a bonding amount of 0.5 mmol / g, i.e., a total bonding amount of 1 mmol. Add a 20% (v / v) DMF solution of piperidine to it and stir for 20 minutes to remove the Fmoc group. After the Fmoc removal reaction is completed, wash the solid-phase resin alternately with DMF and IPA. At this time, add 2 mmol of the amino acid Fmoc-Lys(Dde)-OH with a protecting group, 2 mmol of TBTU and 4 mmol of DIPEA to the solid-phase resin and stir for 2 hours at room temperature to couple the amino acid Lys40. After the coupling reaction is completed, wash the solid-phase resin alternately with DMF and IPA. Repeat the above Fmoc removal and amino acid linkage process according to the peptide sequence of CJC-1134 until Gly29 is reached.

[0073] (2) Starting with the synthesis of Asn28, a 20% (v / v) DMF solution of piperidine was first added, and the mixture was stirred for 20 minutes to remove the Fmoc group. After the Fmoc removal reaction was completed, the solid resin was washed alternately with DMF and IPA. After washing, the solid resin was washed with a 0.1% (v / v) TFA DCM solution, then washed twice with a 5% (v / v) DIPEA DCM solution, and finally washed with DMF. Then, the amino acid Asn28 was ligated. At this time, 2 mmol of the amino acid Fmoc-Asn(Trt)-OH with a protecting group, 2 mmol of TBTU, and 4 mmol of DIPEA were added to the solid resin, and the mixture was stirred at room temperature for 2 hours. After the ligation reaction was completed, the solid resin was washed alternately with DMF and IPA. The above Fmoc removal and amino acid ligation process was repeated according to the peptide sequence of CJC-1134 until Leu10 was reached.

[0074] (3) Starting from Asp9, repeat the Fmoc removal and amino acid linkage process in step (1), and link different amino acids one by one to the solid resin according to the peptide sequence of CJC-1134. The main chain synthesis ends at His1. Then, treat the solid resin twice with 2% hydrazine hydrate / DMF for 10 minutes each time to remove the Dde protecting group. The subsequent coupling conditions of AEEAc and MPA are: 2 mmol AEEAc / MPA, 2 mmol HOBT and 2 mmol DIC, and stir the reaction at room temperature for 2 hours.

[0075] (4) After drying the solid resin in step (3), add it to the lysis buffer (a mixture of TFA, triethylsilane, anisole and phenol in a volume ratio of 85:5:5:5) to cleave the peptide from the solid resin. At the same time, remove the side chain protecting groups and then obtain the crude peptide CJC-1134 by crystallization and drying.

[0076] The HPLC chromatogram of the crude CJC-1134 peptide prepared in Example 1 is shown below. Figure 3 As shown, the integral results of the HPLC curves are presented in Table 3 below.

[0077] Table 3. HPLC curve integration results of the crude CJC-1134 peptide prepared in Example 1

[0078] .

[0079] As shown in the integral results in Table 3, the crude CJC-1134 peptide prepared in Example 1 contains impurities (pre-impurities and post-impurities) and CJC-1134, with the peak area of ​​CJC-1134 accounting for 34.42% of the total peak area. This indicates that the purity of the crude CJC-1134 peptide prepared by the method in Example 1 is 34.42%. Compared with the conventional method of the comparative example, the method in Example 1 improved the purity of the crude CJC-1134 peptide by 30%.

[0080] Example 2: Preparation of crude CJC-1134 peptide by the method of the present invention

[0081] The method steps of Example 2 are similar to those of Example 1, except that in steps (1) and (2), a DMF solution with a concentration of 20% by volume of 4-methylpiperidine is used to remove the Fmoc group, and in step (2), a DCM solution with a concentration of 10% by volume of TFE is used to clean the solid resin, and then the solid resin is cleaned twice with a DCM solution with a concentration of 5% by volume of TEA.

[0082] The HPLC chromatogram of the crude CJC-1134 peptide prepared in Example 2 is shown below. Figure 4 As shown, the integral results of the HPLC curves are presented in Table 4 below.

[0083] Table 4. HPLC curve integration results of the crude CJC-1134 peptide prepared in Example 2

[0084] .

[0085] As shown in the integral results in Table 4, the crude CJC-1134 peptide prepared in Example 2 contains impurities (pre-impurities and post-impurities) and CJC-1134, with the peak area of ​​CJC-1134 accounting for 34.12% of the total peak area. This indicates that the purity of the crude CJC-1134 peptide prepared by the method in Example 2 is 34.12%. Compared with the conventional method of the comparative example, the method in Example 2 improved the purity of the crude CJC-1134 peptide by 29%.

[0086] Example 3: Preparation of crude CJC-1134 peptide by the method of the present invention

[0087] The method steps of Example 3 are similar to those of Example 1, except that in step (2), the solid resin is cleaned with a DCM solution of 1 volume% TFA and then cleaned twice with a DCM solution of 10 volume% DIPEA.

[0088] The HPLC chromatogram of the crude CJC-1134 peptide prepared in Example 3 is shown below. Figure 5 As shown, the integral results of the HPLC curves are presented in Table 5 below.

[0089] Table 5. HPLC curve integration results of the crude CJC-1134 peptide prepared in Example 3.

[0090] .

[0091] As shown in the integral results in Table 5, the purity of the crude CJC-1134 peptide prepared by the method in Example 3 is 32.80%. Compared with the conventional method of the comparative example, the method in Example 3 increased the purity of the crude CJC-1134 peptide by 24%.

[0092] Example 4: Preparation of crude CJC-1134 peptide by the method of the present invention

[0093] The method steps of Example 4 are similar to those of Example 1, except that in step (2), the solid resin is cleaned with a DCM solution of 5 volume% TFE, and then the solid resin is cleaned twice with a DCM solution of 2 volume% TEA.

[0094] The HPLC chromatogram of the crude CJC-1134 peptide prepared in Example 4 is shown below. Figure 6 As shown, the integral results of the HPLC curves are presented in Table 6 below.

[0095] Table 6. HPLC curve integration results of the crude CJC-1134 peptide prepared in Example 4

[0096] .

[0097] As shown in the integral results in Table 6, the purity of the crude CJC-1134 peptide prepared by the method in Example 4 is 34.86%. Compared with the conventional method of the comparative example, the method of Example 4 improved the purity of the crude CJC-1134 peptide by 31%.

[0098] Example 5: Preparation of crude CJC-1134 peptide by the method of the present invention

[0099] The method steps of Example 5 are similar to those of Example 1, except that in step (2), the solid resin is cleaned with a DCM solution of 20 volume% TFE, and then the solid resin is cleaned twice with a DCM solution of 10 volume% TEA.

[0100] The HPLC chromatogram of the crude CJC-1134 peptide prepared in Example 5 is shown below. Figure 7 As shown, the integral results of the HPLC curves are presented in Table 7 below.

[0101] Table 7. HPLC curve integration results of the crude CJC-1134 peptide prepared in Example 5.

[0102] .

[0103] As shown in the integral results in Table 7, the purity of the crude CJC-1134 peptide prepared by the method in Example 5 is 34.93%. Compared with the conventional method of the comparative example, the method in Example 5 increased the purity of the crude CJC-1134 peptide by 32%.

[0104] Example 6 Preparation of crude CJC-1134 peptide by the method of the present invention

[0105] The method steps of Example 6 are similar to those of Example 1, except that in step (2), the solid resin is cleaned with a DCM solution of 25 volume% TFE, and then the solid resin is cleaned twice with a DCM solution of 15 volume% TEA.

[0106] The HPLC chromatogram of the crude CJC-1134 peptide prepared in Example 6 is shown below. Figure 8 As shown, the integral results of the HPLC curves are presented in Table 8 below.

[0107] Table 8. HPLC curve integration results of the crude CJC-1134 peptide prepared in Example 6

[0108] .

[0109] As shown in the integral results in Table 8, the purity of the crude CJC-1134 peptide prepared by the method in Example 6 is 27.96%. Compared with the conventional method of the comparative example, the method of Example 6 improved the purity of the crude CJC-1134 peptide by 5%.

[0110] Example 7 Preparation of crude CJC-1134 peptide by the method of the present invention

[0111] The method steps of Example 7 are similar to those of Example 1, except that the solid resin used in step (1) is NovaSyn TGA resin, and the solid resin is washed with an aqueous solution of 0.1% hydrochloric acid in step (2), and then washed twice with an aqueous solution of 0.2% ammonia.

[0112] The HPLC chromatogram of the crude CJC-1134 peptide prepared in Example 7 is shown below. Figure 9 As shown, the integral results of the HPLC curves are presented in Table 9 below.

[0113] Table 9. HPLC curve integration results of the crude CJC-1134 peptide prepared in Example 7

[0114] .

[0115] As shown in the integral results in Table 9, the purity of the crude CJC-1134 peptide prepared by the method in Example 7 is 27.96%. Compared with the conventional method of the comparative example, the method of Example 7 improved the purity of the crude CJC-1134 peptide by 5%.

[0116] Based on the above embodiments, Table 10 below shows the solutes and solvents of the acidic and alkaline solutions used in step (2) of each embodiment.

[0117] Table 10. Acidic and alkaline solutions used in step (2) of each embodiment.

[0118] .

[0119] The HPLC curve analysis results of the CJC-1134 crude peptides prepared in Examples 1-7 are summarized in Table 11 below. From the integral results of the HPLC curves, it can be seen that, similar to Examples 1 and 2, the purity of the CJC-1134 crude peptides prepared in Examples 3-5 is approximately 34%, indicating that the method of the present invention significantly increases the purity of the polypeptide product (crude peptide) from 26.55% using conventional methods to approximately 34%, an increase of about 30%, demonstrating a very significant effect on improving the purity of the polypeptide product. In Example 6, the excessive concentration of acidic and alkaline solutions may have led to other side reactions, resulting in only a slight increase in the purity of the polypeptide product compared to the conventional method. In Example 7, although the increase in crude peptide purity was not as high as in Examples 1-5, it still reached 15%.

[0120] Table 11. Integrated results of HPLC curves of the crude CJC-1134 peptide prepared in each example.

[0121] .

[0122] The method of this invention significantly improves the purity of solid-phase synthesized peptide products. This level of purity improvement greatly facilitates subsequent peptide purification steps and lays the foundation for improving the overall production efficiency of peptides.

Claims

1. A method for solid-phase synthesis of polypeptides, characterized in that, The polypeptide is an exenatide analog Lys40(ε-AEEAc-MPA)-NH2]-Exendin-4, which includes the following steps: (1) Weigh 2g of solid resin with a bonding amount of 0.5mmol / g, i.e., a total bonding amount of 1mmol; add a solution of secondary amine molecules to it, stir for 20 minutes to remove the Fmoc group; after the Fmoc removal reaction is completed, wash the solid resin alternately with DMF and IPA; at this time, add 2mmol of the amino acid Fmoc-Lys(Dde)-OH with a protecting group, 2mmol of TBTU and 4mmol of DIPEA to the solid resin, stir for 2 hours at room temperature to couple the amino acid Lys40; after the coupling reaction is completed, wash the solid resin alternately with DMF and IPA; repeat the above Fmoc removal and amino acid linkage process according to the peptide sequence of the exenatide analog until the synthesis ends at Gly29; (2) Starting from the synthesis of Asn28, a solution of secondary amine molecules was first added and the reaction was stirred for 20 minutes to remove the Fmoc group. After the Fmoc removal reaction was completed, the solid resin was washed alternately with DMF and IPA. After washing, the solid resin was washed with an acidic solution, then with an alkaline solution, and finally with DMF. Then, the amino acid Asn28 was linked. At this time, 2 mmol of the amino acid Fmoc-Asn(Trt)-OH with a protecting group, 2 mmol of TBTU and 4 mmol of DIPEA were added to the solid resin and the reaction was stirred at room temperature for 2 hours. After the linking reaction was completed, the solid resin was washed alternately with DMF and IPA. The above Fmoc removal and amino acid linking process was repeated according to the peptide sequence of the exenatide analog until Leu10 was synthesized. (3) Starting from Asp9, repeat the Fmoc removal process and amino acid linkage process in step (1). Link different amino acids one by one to the solid resin according to the peptide sequence of the exenatide analog. The main chain synthesis ends at His1. Then, treat the solid resin twice with 2% hydrazine hydrate / DMF for 10 minutes each time to remove the Dde protecting group. The subsequent coupling conditions of AEEAc and MPA are: 2 mmol AEEAc / MPA, 2 mmol HOBT and 2 mmol DIC. Stir the reaction at room temperature for 2 hours. (4) After drying the solid resin obtained in step (3), add it to the lysis buffer to lyse the peptide from the solid resin. At the same time, remove the side chain protecting groups and then obtain the crude peptide of exenatide analogue by crystallization and drying. The solution of the secondary amine molecule in steps (1) and (2) is a DMF solution of piperidine or a DMF solution of 4-methylpiperidine.

2. The method for solid-phase synthesis of polypeptides according to claim 1, characterized in that, The solution of the secondary amine molecule in steps (1) and (2) is a DMF solution of piperidine with a volume concentration of 20% or a DMF solution of 4-methylpiperidine with a volume concentration of 20%.

3. The method for solid-phase synthesis of polypeptides according to claim 1, characterized in that, In step (2), the acidic solution is a DCM solution with a volume concentration of 0.1-1% TFA, a DCM solution with a volume concentration of 5-25% TFE, or hydrochloric acid with a volume concentration of 0.1%. The alkaline solution is a DCM solution with a volume concentration of 5-10% DIPEA, a DCM solution with a volume concentration of 5-15% TEA, or ammonia.

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