A method for preparing a polypeptide containing a Sar linker

Abstract

The present invention provides a method for preparing a polypeptide containing a Sar linker, which belongs to the technical field of polypeptide synthesis. The method for preparing a polypeptide containing a Sar linker comprises: (1) Fmoc- β ‑Asp‑PG‑(Sar) m ‑OH and H‑Sar‑OH undergo condensation reaction to obtain Fmoc‑ β ‑Asp‑PG‑(Sar) n ‑OH; (2) Fmoc‑ β ‑Asp‑PG‑(Sar) n ‑OtBu was deprotected to obtain Fmoc‑ β ‑Asp‑(Sar) n The present invention adopts a full liquid phase synthesis method to prepare a polypeptide containing a Sar linker under mild conditions. The product has a stable yield and high purity, and the post-processing method is simple, which is conducive to industrial scale-up production.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide synthesis, and in particular to a method for preparing a polypeptide containing a Sar linker. Background Art

[0002] The linker containing multiple Sars is a polypeptide composed of 2 to 11 amino acids. Its molecular structure is shown in the figure. Its amino acids are Fmoc- β -Asp-(Sar) n -OH, where n is an integer between 1 and 10.

[0003]

[0004] Following the eras of chemotherapy, targeted therapy, and immunotherapy, ADC-type drugs, combining cytotoxic drugs with targeted monoclonal antibodies, have ushered in the fourth era of oncology drug therapy with remarkable success. Technological advances have also led to the emergence of a variety of new conjugate drugs: RDC, SMDC, PDC, ISAC, FDC, ACC, VDC, AOC, ABC, and others. The connecting component is called a linker.

[0005] However, the solid-phase synthesis method of a linker containing multiple Sars as a linker will inevitably produce piperazine dione impurities during the scale-up process, which in turn causes difficulties in the separation and purification of the missing dipeptide impurities and increases the cost.

[0006] Therefore, it is urgent to develop a safe, efficient and industrially producible method for synthesizing polypeptides containing Sar linkers.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The present invention aims to provide a method for preparing a polypeptide containing a Sar linker. This method utilizes a fully liquid-phase synthesis method, enabling safe, efficient, and industrialized production of polypeptides containing multiple Sar linkers. The method utilizes mild reaction conditions, produces high-quality, multiple Sar linkers in high yield, and simplifies post-processing, facilitating industrial scale-up.

[0009] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0010] The present invention provides a method for preparing a polypeptide containing a Sar linker, the preparation method comprising the following steps:

[0011] (1) Fmoc- β -Asp-PG-(Sar)m -OH and H-(Sar) k -OH undergoes condensation reaction to obtain Fmoc- β -Asp-PG-(Sar) n -OH; the reaction formula is as follows:

[0012] ;

[0013] Wherein, PG represents a protecting group, m is an integer between 0 and 9, k is an integer between 1 and 9, and n is an integer between 1 and 10;

[0014] (2) Fmoc- β -Asp-PG-(Sar) n -OH was deprotected to obtain Fmoc- β -Asp-(Sar) n -OH, the reaction formula is as follows:

[0015] ;

[0016] Wherein, PG represents a protecting group, and n is an integer between 1 and 10.

[0017] Furthermore, in step (1), before the condensation reaction, the preparation method further comprises the following steps: β -Asp-PG-(Sar) m -OH activation treatment and / or raw material H-(Sar) k Protection treatment of -OH.

[0018] Furthermore, the activation treatment comprises the following steps: β -Asp-PG-(Sar) m -OH, the first solvent, the first additive and the first condensing agent are mixed and activated to activate Fmoc- β -Asp-PG-(Sar) m -OH.

[0019] Furthermore, the protection treatment comprises the following steps: k -OH, the second solvent and the silicon protection reagent are mixed, and the silicon protection H-(Sar) is reacted to form a protective layer. k -OH.

[0020] Furthermore, in the activation treatment, the activation reaction temperature is 20-50° C., and the activation reaction time is 2-12 h.

[0021] Furthermore, in the protection treatment, the temperature of the protection reaction is 20-50° C., and the time of the protection reaction is 2-12 h.

[0022] Furthermore, in the activation treatment, the Fmoc- β -Asp-PG-(Sar) m The molar ratio of -OH, the first additive and the first condensing agent is 1:(1-1.5):(1-1.5).

[0023] Furthermore, in the protection process, the H-(Sar) k The molar ratio of -OH to silicon protecting agent is 1:(1.5~3.0).

[0024] Furthermore, the first solvent and the second solvent are each independently selected from any one or a combination of at least two of dichloroethane, dichloromethane or tetrahydrofuran.

[0025] Furthermore, the first additive is selected from N -Hydroxysuccinimide, N,N' - Any one of disuccinimidyl carbonate, pentafluorophenol, 1-hydroxybenzotriazole or ethyl 2-oximecyanoacetate, or a combination of at least two thereof.

[0026] Furthermore, the first condensing agent is selected from N,N' -Dicyclohexylcarbodiimide, N,N' -diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or benzotriazole- N,N,N',N' -Tetramethyluronium hexafluorophosphate, any one or a combination of at least two.

[0027] Furthermore, the silicon protecting agent is selected from trimethylchlorosilane and / or N,O -Bistrimethylsilylacetamide.

[0028] Furthermore, in step (1), the Fmoc- β -Asp-PG-(Sar) m -OH and H-(Sar) k The molar ratio of -OH is (0.8~1.2):(0.8~1.2);

[0029] Furthermore, in step (1), the temperature of the condensation reaction is 20-30° C., and the time of the condensation reaction is 2-20 h.

[0030] Furthermore, in step (1), after the condensation reaction is completed, the following post-treatment steps are also included:

[0031] The reaction solution obtained by the condensation reaction is concentrated, washed, and precipitated in sequence to obtain the Fmoc- β -Asp-PG-(Sar) n -OH.

[0032] Furthermore, in step (1), the washing solvent is selected from citric acid solution and / or water.

[0033] Furthermore, in step (1), the precipitation solvent is selected from petroleum ether and / or n-heptane.

[0034] Furthermore, in steps (1) and (2), the protecting group PG is selected from -OtBu or -OAll.

[0035] Furthermore, in step (2), when the protecting group -OtBu is removed, the reagents used for the deprotection treatment include trifluoroacetic acid and a solvent.

[0036] Furthermore, in step (2), when the protecting group -OtBu is removed, the solvent is selected from dichloromethane, ethyl acetate, 1,4-dioxane, N,N - Any one of dimethylformamide or water or a combination of at least two thereof.

[0037] Furthermore, in step (2), when the protecting group -OAll is removed, the reagents used for the deprotection treatment include bistriphenylphosphine palladium dichloride, N -methylmorpholine and solvent.

[0038] Furthermore, in step (2), when the protecting group -OAll is removed, the solvent includes any one of tetrahydrofuran, N,N-dimethylformamide, dichloromethane or water, or a combination of at least two thereof.

[0039] Furthermore, in step (2), when the protecting group -OtBu is removed, the temperature of the deprotection treatment is 20-30°C, and the time of the deprotection treatment is 0.5-3 h.

[0040] Furthermore, in step (2), when the protecting group -OAll is removed, the temperature of the deprotection treatment is 20-60° C., and the time of the deprotection treatment is 6-18 h.

[0041] Furthermore, in step (2), when the protecting group -OtBu is removed, the Fmoc- β -Asp-PG-(Sar) n The mass ratio of -OtBu and trifluoroacetic acid is 1:(10~20).

[0042] Furthermore, in step (2), when the protecting group -OAll is removed, the Fmoc- β-Asp-PG-(Sar) n The mass ratio of -OtBu, bistriphenylphosphine palladium dichloride and N-methylmorpholine is 1: (0.1~0.2): (10~20).

[0043] Furthermore, in step (2), when the protecting group -OtBu is removed, the deprotection treatment further includes the following post-treatment steps:

[0044] The reaction solution obtained by deprotection treatment was concentrated and added dropwise to methyl tert-butyl ether to precipitate a solid, which was then centrifuged and washed to obtain Fmoc- β -Asp-(Sar) n -OH.

[0045] Furthermore, the washing solvent is selected from methyl tert-butyl ether.

[0046] Furthermore, in step (2), when the protecting group -OAll is removed, the deprotection treatment further includes the following post-treatment steps:

[0047] The reaction solution obtained by deprotection treatment was concentrated, dissolved in dichloromethane, washed and concentrated, and added dropwise to petroleum ether to precipitate solids, which were then centrifuged and dried to obtain Fmoc- β -Asp-(Sar) n -OH.

[0048] Furthermore, the washing solvent is selected from citric acid solution and / or saturated sodium chloride solution.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] The liquid-phase synthesis method of a multi-Sar linker of the present invention uses simple and inexpensive starting materials to produce high-quality multi-Sar linkers in a high yield under mild reaction conditions. The post-processing method is simple, thereby facilitating industrial scale-up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0052] Figure 1 The product Fmoc- β HPLC spectrum of -Asp-(Sar)4-OH.

[0053] Figure 2 The product Fmoc- β LC-MS spectrum of -Asp-(Sar)4-OH.

[0054] Figure 3 The intermediate Fmoc- β HPLC spectrum of -Asp-OAll-(Sar)4-OH.

[0055] Figure 4 The intermediate Fmoc- β LC-MS spectrum of -Asp-OAll-(Sar)4-OH.

[0056] Figure 5 The product Fmoc- β HPLC spectrum of -Asp-(Sar)7-OH.

[0057] Figure 6 The product Fmoc- β LC-MS spectrum of -Asp-(Sar)7-OH.

[0058] Figure 7 The intermediate Fmoc- β HPLC spectrum of -Asp-OtBu-(Sar)3-OH.

[0059] Figure 8 The intermediate Fmoc- β LC-MS spectrum of -Asp-OtBu-(Sar)3-OH. DETAILED DESCRIPTION

[0060] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including" and other forms is non-limiting.

[0061] It should be noted that the following description sets forth specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in a variety of other ways than those described herein, and those skilled in the art may make similar generalizations without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0062] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0063] The present invention provides a method for preparing a polypeptide containing a Sar linker, the preparation method comprising the following steps:

[0064] (1) Fmoc- β -Asp-PG-(Sar) m -OH and H-(Sar) k -OH undergoes condensation reaction to obtain Fmoc- β -Asp-PG-(Sar) n -OH; the reaction formula is as follows:

[0065] ;

[0066] Wherein, PG represents a protecting group, m is an integer between 0 and 9, k is an integer between 1 and 9, and n is an integer between 1 and 10;

[0067] (2) Fmoc- β -Asp-PG-(Sar) n -OH was deprotected to obtain Fmoc- β -Asp-(Sar) n -OH, the reaction formula is as follows:

[0068] ;

[0069] Wherein, PG represents a protecting group, and n is an integer between 1 and 10.

[0070] As an optional implementation, m is an integer between 0 and 9, for example, it can be 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9.

[0071] As an optional implementation, k is an integer between 1 and 9, for example, it can be 1, 2, 3, 4, 5, 6, 7, 8, or 9.

[0072] As an optional implementation, n is an integer between 1 and 10, for example, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0073] As an optional embodiment, when n is an integer between 1 and 10, the Fmoc- β -Asp-PG-(Sar) m-OH and H-(Sar) k The condensation reaction of H-(Sar)4-OH is repeated multiple times until a polypeptide chain with the desired number of Sar linkers is obtained. For example, when the final product n=4 and k=1, the condensation reaction needs to be performed three times. Alternatively, an intermediate fragment can be selected for condensation to obtain a polypeptide chain with the desired number of Sar linkers. For example, when the final product n=7, an intermediate with m=3 and a H-(Sar)4-OH starting material with k=4 can be selected for condensation.

[0074] As an optional embodiment, in step (1), before the condensation reaction, the preparation method further comprises the following steps: β -Asp-PG-(Sar) m -OH activation treatment and / or raw material H-(Sar) k Protection treatment of -OH.

[0075] It should be noted that, in the present invention, when performing the condensation reaction, the strategy of first activating the amino acid providing the carboxyl group and then condensing it with the amino group of the silicon-protected amino acid is adopted, which can omit the deprotection step in the synthesis and avoid racemization.

[0076] As an optional embodiment, in step (1), the activation treatment includes the following steps:

[0077] The raw material Fmoc- β -Asp-PG-(Sar) m -OH, the first solvent, the first additive and the first condensing agent are mixed and reacted to activate Fmoc- β -Asp-PG-(Sar) m -OH.

[0078] As an optional implementation, in step (1), the protection process includes the following steps:

[0079] The raw material H-(Sar) k -OH, the second solvent and the silicon protecting agent are mixed and reacted to protect H-(Sar) with silicon. k -OH.

[0080] It should be noted that the present invention utilizes silyl groups to replace the active hydrogen in the carboxyl group to obtain an intermediate with high chemical stability. After the reaction is completed, the silyl groups are removed through post-treatment hydrolysis to restore the protected active hydrogen. Existing research has confirmed that there is no loss of enantiomeric purity under silylation conditions. Silyl protection also addresses solubility and amino group nucleophilicity issues, converting zwitterionic amino acids into soluble silyl esters and releasing nucleophilic free amino groups. Excess silyl groups can also bind to free amino groups to increase their nucleophilicity.

[0081] As an optional embodiment, in step (1), in the activation treatment, the temperature of the activation reaction is 20-50°C, for example, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, etc., and the time of the activation reaction is 2-12h, for example, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, etc.

[0082] As an optional embodiment, in step (1), in the protection treatment, the temperature of the protection reaction is 20-50°C, for example, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, etc., and the time of the protection reaction is 2-12 h, for example, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, etc.

[0083] As an optional embodiment, in step (1), during the activation treatment, the Fmoc- β -Asp-PG-(Sar) m The molar ratio of -OH, the first additive and the first condensing agent is 1:(1~1.5):(1~1.5), for example, it can be 1:1:1, 1:1:1.1.2, 1:1:1.4, 1:1:1.5, 1:1.2:1, 1:1.2:1.1.2, 1:1.2:1.4, 1:1.2:1.5, 1:1.4:1, 1:1.4:1.1.2, 1:1.4:1.4, 1:1.4:1.5, 1:1.5:1.5, etc.

[0084] As an optional embodiment, in step (1), during the activation treatment, the H-(Sar) k The molar ratio of -OH to the silicon protecting agent is 1:(1.5-3.0), for example, it can be 1:1.5, 1:1.6, 1:1.8, 1:2, 1:2.2, 1:2.4, 1:2.5, 1:2.6, 1:2.8, 1:3.0, etc.

[0085] As an optional embodiment, in step (1), the first solvent is selected from any one of dichloroethane, dichloromethane or tetrahydrofuran, or a combination of at least two thereof.

[0086] As an optional embodiment, in step (1), the second solvent is selected from any one of dichloroethane, dichloromethane or tetrahydrofuran, or a combination of at least two thereof.

[0087] As an optional embodiment, in step (1), the first additive is selected from N -Hydroxysuccinimide (HOSu), N,N' - Any one of disuccinimidyl carbonate (DSC), pentafluorophenol (PfpOH), 1-hydroxybenzotriazole (HOBt) or ethyl 2-oximecyanoacetate (Oxymapure) or a combination of at least two thereof.

[0088] As a preferred embodiment, in step (1), the first additive is N- Hydroxysuccinimide (HOSu).

[0089] It should be noted that the preferred first additive of the present invention is N- Hydroxysuccinimide (HOSu) can further improve the conversion rate of the condensation reaction.

[0090] As an optional embodiment, in step (1), the first condensing agent is selected from N,N' -Dicyclohexylcarbodiimide (DCC), N,N' -Diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) or benzotriazole- N,N,N',N' -Tetramethyluronium hexafluorophosphate (HBTU) or a combination of at least two thereof.

[0091] As a preferred embodiment, in step (1), the first condensing agent is selected from N,N' -Dicyclohexylcarbodiimide (DCC).

[0092] It should be noted that the preferred first condensing agent of the present invention is selected from N,N' -Dicyclohexylcarbodiimide (DCC) can further improve the conversion rate of the condensation reaction.

[0093] As an optional embodiment, in step (1), the silicon protecting agent is selected from trimethylchlorosilane and / or N,O-bistrimethylsilylacetamide (BSA).

[0094] As a preferred embodiment, in step (1), the silicon protection agent is selected from N,O -Bistrimethylsilylacetamide (BSA).

[0095] It should be noted that, in the present invention, the preferred silicon protecting agent is N,O-bis(trimethylsilyl)acetamide (BSA), which can further improve the conversion rate of the condensation reaction.

[0096] As shown above, in step (1), Fmoc- β -Asp-PG-(Sar) m -OH and H-(Sar) k -OH proceeds according to the following reaction pathway:

[0097] .

[0098] As an optional embodiment, in step (1), the Fmoc- β -Asp-PG-(Sar) m -OH and H-(Sar) k The molar ratio of -OH is (0.8~1.2):(0.8~1.2), for example, it can be 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1:0.8, 1:0.9, 1:1.1, 1:1.2, etc.

[0099] As an optional embodiment, in step (1), in the Fmoc- β -Asp-PG-(Sar) m -OH and H-(Sar) k During the condensation reaction of -OH, the temperature of the condensation reaction is 20~30°C, for example, it can be 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, etc., and the time of the condensation reaction is 2~20 h, for example, it can be 2 h, 4 h, 5 h, 6 h, 8 h, 10 h, 12 h, 14 h, 15 h, 16 h, 18 h, 20 h, etc.

[0100] As an optional embodiment, in step (1), in the Fmoc- β -Asp-PG-(Sar) m -OH and H-(Sar) k After the condensation reaction of -OH is completed, the following post-treatment steps are also included:

[0101] The reaction solution obtained by the condensation reaction is concentrated, washed, and precipitated in sequence to obtain the Fmoc- β -Asp-PG-(Sar) n -OH.

[0102] As an optional embodiment, in step (1), the washing solvent is selected from citric acid solution and / or water.

[0103] As an optional embodiment, in step (1), the precipitation solvent is selected from petroleum ether.

[0104] As a preferred embodiment, in step (1), in the Fmoc- β -Asp-PG-(Sar) m -OH and H-(Sar) k After the condensation reaction of -OH is completed, the following post-treatment steps are also included:

[0105] The reaction solution obtained by the condensation reaction was concentrated, washed with citric acid solution and water, and then petroleum ether was added thereto for precipitation to obtain the Fmoc- β -Asp-PG-(Sar) n -OH.

[0106] As an optional embodiment, in steps (1) and (2), the protecting group PG is selected from -OtBu or -OAll.

[0107] As an optional embodiment, when n is an integer between 1 and 10, and the protecting group PG is selected from -OtBu, the preparation method of the polypeptide containing the Sar linker is carried out according to the following reaction path:

[0108] .

[0109] As an optional embodiment, when n is an integer between 1 and 10, and the protecting group PG is selected from -OAll, the method for preparing the polypeptide containing the Sar linker is carried out according to the reaction path shown below:

[0110] .

[0111] As an optional embodiment, in step (2), when the protecting group -OtBu is removed, the reagents used for the deprotection treatment include trifluoroacetic acid and a solvent.

[0112] As an optional embodiment, in step (2), when the protecting group -OtBu is removed, the solvent is selected from dichloromethane, ethyl acetate, 1,4-dioxane, N,N - any one of dimethylformamide or water or a combination of at least two;

[0113] As an optional embodiment, in step (2), when the protecting group -OAll is removed, the reagents used for the deprotection treatment include bistriphenylphosphine palladium dichloride, N -methylmorpholine and solvent.

[0114] As an optional embodiment, in step (2), when the protecting group -OAll is removed, the solvent includes any one of tetrahydrofuran, N,N-dimethylformamide, dichloromethane or water, or a combination of at least two thereof.

[0115] As an optional embodiment, in step (2), when the protecting group -OtBu is removed, the temperature of the deprotection treatment is 20-30°C, for example, 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, etc., and the time of the deprotection treatment is 0.5-3 h, for example, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, etc.

[0116] As an optional embodiment, in step (2), when the protecting group -OAll is removed, the temperature of the deprotection treatment is 20-60°C, for example, 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, etc., and the time of the deprotection treatment is 6-18 h, for example, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, etc.

[0117] As an optional embodiment, in step (2), when the protecting group -OtBu is removed, the Fmoc- β -Asp-PG-(Sar) n The mass ratio of -OH to trifluoroacetic acid is 1:(10-20), for example, it can be 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, etc.

[0118] As an optional embodiment, in step (2), when the protecting group -OAll is removed, the Fmoc- β -Asp-PG-(Sar) n -OH, bistriphenylphosphine palladium dichloride and N The mass ratio of -methylmorpholine is 1:(0.1~0.2):(10~20), for example, it can be 1:0.1:10, 1:0.11:11, 1:0.12:12, 1:0.13:13, 1:0.14:14, 1:0.15:15, 1:0.16:16, 1:0.17:17, 1:0.18:18, 1:0.19:19, 1:0.2:20, etc.

[0119] As an optional embodiment, in step (2), when the protecting group -OtBu is removed, the deprotection treatment further includes the following post-treatment steps:

[0120] The reaction solution obtained by deprotection treatment was concentrated and added dropwise to methyl tert-butyl ether to precipitate a solid, which was then centrifuged and washed to obtain Fmoc- β -Asp-(Sar) n -OH.

[0121] As an optional embodiment, the washing solvent is selected from methyl tert-butyl ether.

[0122] As an optional embodiment, in step (3), when the protecting group -OAll is removed, the deprotection treatment further includes the following post-treatment steps: concentrating the reaction solution obtained by the deprotection treatment, dissolving it in dichloromethane, washing it and concentrating it, adding it dropwise to petroleum ether, precipitating a solid, and then centrifuging and drying it to obtain Fmoc- β -Asp-(Sar) n -OH;

[0123] As an optional embodiment, the washing solvent is selected from citric acid solution and / or saturated sodium chloride solution.

[0124] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0125] In the reactions of the steps of the following examples, the reactions were detected by HPLC. The specific method of HPLC was:

[0126] Column: ACQUITY UPLC BEH C18 Column, 130 Å, 1.7 μm, 2.1 mm x 100 mm

[0127] Column temperature: 35°C;

[0128] Flow rate: 1.0 mL / min;

[0129] Detector: UV (215 nm);

[0130] Mobile phase: A. Chromatographic grade water containing 0.1% trifluoroacetic acid;

[0131] B. Chromatographic grade acetonitrile containing 0.1% trifluoroacetic acid;

[0132] The gradient program looks like this:

[0133] 0 min 98% A and 2% B

[0134] 1 min 98% A and 2% B

[0135] 2 min 70% A and 30% B

[0136] 22 min20%A and 80%B

[0137] 24 min20%A and 80%B

[0138] 25 min98%A and 2%B.

[0139] Example 1

[0140] This example provides a polypeptide containing a Sar linker (Fmoc-β -Asp-(Sar)4-OH) preparation method, the Fmoc- β The preparation method of -Asp-(Sar)4-OH comprises the following steps:

[0141] S1, Fmoc- β Activation of -Asp-OtBu and protection of H-Sar-OH

[0142] S1-1, to a 250 mL reaction flask, 100 mL of dichloroethane, 10 g of Fmoc- β -Asp-OtBu, 3.36 g of HOSu, stirred at 20-25 ° C to clarify. Then cooled to 0 ° C, 6.02 g of DCC was slowly added in batches. After the addition was complete, the reaction system was slowly heated to 20-25 ° C and stirred for 2 hours. After the reaction was completed, the suspension was filtered to remove the by-product DCU. The filtrate obtained was Fmoc- β -Asp-OtBu activation solution, which is directly used in the subsequent condensation reaction.

[0143] S1-2. To a 100 mL reaction flask, 40 mL of dichloroethane, 3.24 g of H-Sar-OH, and 17.78 mL of BSA were added in sequence. The mixture was stirred at 20-25°C for 2 h. The silicon protective solution was directly used in the subsequent condensation reaction.

[0144] S2, Fmoc- β Preparation of -Asp-OtBu-Sar-OH

[0145]

[0146] The two solutions of S1-1 and S1-2 were mixed and stirred at 20-25 °C for 16 h. After the reaction was complete by HPLC, 300 mL of water was added to quench the reaction. The pH was then adjusted to about 2 with 10 wt% citric acid and stirred for 5 min. The aqueous phase was extracted twice with DCM. The organic phases were combined and washed three times with saturated NaCl solution, concentrated, and slowly added dropwise to PE. A large amount of solid precipitated. After centrifugation and drying, 10.7 g of white solid Fmoc- β -Asp-OtBu-Sar-OH, purity 96.32%, yield 91%.

[0147] S3, Fmoc- β Activation of -Asp-OtBu-Sar-OH and protection of H-Sar-OH

[0148] S3-1, to a 100 mL reaction flask, 50 mL of dichloroethane, 6.4 g of Fmoc-β -Asp-OtBu-Sar-OH, 1.83 g of HOSu, stirred at 20-25 ° C to clarify. Then cooled to 0 ° C, 3.28 g of DCC was slowly added in batches. After the addition was complete, the reaction system was slowly heated to 20-25 ° C and stirred for 2 hours. After the reaction was completed, the suspension was filtered to remove the by-product DCU. The filtrate obtained was Fmoc- β -Asp-OtBu-Sar-OH activation solution, which is directly used in the subsequent condensation reaction.

[0149] S3-2. Add 15 mL of dichloroethane, 1.36 g of H-Sar-OH, and 7.45 mL of BSA to a 50 mL reaction flask in sequence and stir at 20-25°C for 2 h. The silicon protective solution is directly used in the subsequent condensation reaction.

[0150] S4, Fmoc- β Preparation of -Asp-OtBu-(Sar)2-OH

[0151]

[0152] The two solutions of S3-1 and S3-2 were mixed and stirred at 20-25 °C for 16 h. After the reaction was complete after HPLC detection, 100 mL of water was added to quench the reaction. The pH was then adjusted to about 2 with 10 wt% citric acid and stirred for 5 min. The organic phase was concentrated and dissolved with EA, and 1 wt% NaHCO3 was added and stirred for 10 min. The organic phase was discarded, and 10 wt% citric acid was added to the aqueous phase to adjust the pH to about 2. The organic phase was washed three times with saturated NaCl solution and concentrated. It was slowly added dropwise to PE. A large amount of solid precipitated. After centrifugation and drying, 4.8 g of white solid Fmoc- β -Asp-OtBu-(Sar)2-OH, purity 97.15%, yield 85%.

[0153] S5, Fmoc- β Activation of -Asp-OtBu-(Sar)2-OH and Protection of H-Sar-OH

[0154] S5-1, 30 mL of dichloroethane, 3.18 g of Fmoc- β -Asp-OtBu-(Sar)2-OH, 0.73 g of HOSu, stirred at 20-25 ° C to clarify. Then cooled to 0 ° C, 1.30 g of DCC was slowly added in batches. After the addition was complete, the reaction system was slowly heated to 20-25 ° C and stirred for 2 hours. After the reaction was completed, the suspension was filtered to remove the by-product DCU. The filtrate obtained was Fmoc-β -Asp-OtBu-(Sar)2-OH activation solution, which is directly used in the subsequent condensation reaction.

[0155] S5-2. Add 10 mL of dichloroethane, 0.77 g of H-Sar-OH, and 4.07 mL of BSA to a 50 mL reaction flask in sequence and stir at 20-25°C for 2 h. The silicon protective solution is directly used in the subsequent condensation reaction.

[0156] S6, Fmoc- β Preparation of -Asp-OtBu-(Sar)3-OH

[0157]

[0158] The two solutions of S5-1 and S5-2 were mixed and stirred at 20-25 °C for 16 h. After the reaction was complete by HPLC, 100 mL of water was added to quench the reaction. The pH was then adjusted to about 2 with 10 wt% citric acid and stirred for 5 min. The aqueous phase was extracted twice with DCM. The organic phases were combined, washed three times with saturated NaCl solution, and concentrated. The organic phases were slowly added dropwise to PE. A large amount of solid precipitated. After centrifugation and drying, 3 g of white solid Fmoc- β -Asp-OtBu-(Sar)3-OH, purity 94.72%, yield 83%.

[0159] S7, Fmoc- β Activation of -Asp-OtBu-(Sar)3-OH and Protection of H-Sar-OH

[0160] S7-1, 170 mL of dichloroethane, 16.29 g of Fmoc- β -Asp-OtBu-(Sar)3-OH, 3.31 g of HOSu, stirred at 20-25 ° C to clarify. Then cooled to 0 ° C, 5.93 g of DCC was slowly added in batches. After the addition was complete, the reaction system was slowly heated to 20-25 ° C and stirred for 2 hours. After the reaction was completed, the suspension was filtered to remove the by-product DCU. The filtrate obtained was Fmoc- β -Asp-OtBu-(Sar)3-OH activation solution, which is directly used in the subsequent condensation reaction.

[0161] S7-2. To a 100 mL reaction flask, add 40 mL of dichloroethane, 3.485 g of H-Sar-OH, and 19.11 mL of BSA in sequence and stir at 20-25°C for 2 h. The silicon protective solution is directly used in the subsequent condensation reaction.

[0162] S8, Fmoc- β Preparation of -Asp-OtBu-(Sar)4-OH

[0163]

[0164] The two solutions of S7-1 and S7-2 were mixed and stirred at 20-25 °C for 16 h. After the reaction was complete by HPLC, 200 mL of water was added to quench the reaction. The pH was then adjusted to about 2 with 10 wt% citric acid and stirred for 5 min. The aqueous phase was extracted twice with DCM. The organic phases were combined, washed three times with saturated NaCl solution, and concentrated. The organic phases were slowly added dropwise to PE. A large amount of solid precipitated. After centrifugation and drying, 3 g of white solid Fmoc- β -Asp-OtBu-(Sar)4-OH, purity 95.38%, yield 78%.

[0165] S9, Fmoc- β Preparation of -Asp-(Sar)4-OH

[0166]

[0167] 4 mL of trifluoroacetic acid and 8 mL of dichloromethane were added to a 50 mL reaction bottle, and 1 g of Fmoc- β -Asp-OtBu-(Sar)4-OH, react at room temperature for 2 h. After the reaction is complete, the reaction solution is concentrated and slowly added dropwise to MTBE. A large amount of solid precipitates. Centrifuge, wash three times with MTBE, and dry to obtain 0.531 g of white solid Fmoc- β -Asp-(Sar)4-OH.

[0168] The obtained product Fmoc- β -Asp-(Sar)4-OH was detected by HPLC, and the HPLC spectrum was as follows Figure 1 As shown, the product purity was measured to be 96.06% and the yield was 58%.

[0169] in, Figure 1 The integration results are shown in Table 1 below:

[0170] Table 1

[0171]

[0172] The obtained product was detected by LC-MS, and the theoretical relative molecular mass was 639.66, and ESI-MS m / z: found [M+H] + 640.3, the result is as follows Figure 2 shown.

[0173] Example 2

[0174] This example provides a polypeptide containing a Sar linker (Fmoc- β -Asp-(Sar)4-OH) preparation method, the Fmoc- β The preparation method of -Asp-(Sar)4-OH comprises the following steps:

[0175] S1, Fmoc- β -Activation of Asp-OAll and protection of H-Sar-OH

[0176] S1-1, to a 250 mL reaction flask, 50 mL of dichloroethane, 5 g of Fmoc- β -Asp-OAll, 1.74g HOSu, stirred at 20-25℃ to clarify. Then cooled to 0℃, 3.13g DCC was slowly added in batches. After the addition was complete, the reaction system was slowly heated to 20-25℃ and stirred for 2 hours. After the reaction was completed, the suspension was filtered to remove the by-product DCU. The filtrate obtained was Fmoc- β -Asp-OAll activation solution, which is directly used in the subsequent condensation reaction.

[0177] S1-2. Add 20 mL of dichloroethane, 1.68 g of H-Sar-OH, and 9.27 mL of BSA to a 100 mL reaction flask in sequence and stir at 20-25°C for 2 h. The silicon protective solution is directly used in the subsequent condensation reaction.

[0178] S2, Fmoc- β Preparation of -Asp-OAll-Sar-OH

[0179]

[0180] The two solutions of S1-1 and S1-2 were mixed and stirred at 20-25 °C for 16 h. After the reaction was complete after HPLC detection, 150 mL of water was added to quench the reaction. The pH was then adjusted to about 2 with 10 wt% citric acid and stirred for 5 min. The aqueous phase was extracted twice with DCM. The organic phases were combined, washed three times with saturated NaCl solution, and concentrated. The organic phases were slowly added dropwise to PE. A large amount of solid precipitated. After centrifugation and drying, 5.15 g of white solid Fmoc- β -Asp-OAll-Sar-OH, purity 98.35%, yield 87%.

[0181] S3, Fmoc- β Activation of -Asp-OAll-Sar-OH and protection of H-Sar-OH

[0182] S3-1, to a 100 mL reaction flask, 30 mL of dichloroethane, 2.98 g of Fmoc- β -OAll-OtBu-Sar-OH, 0.81 g of HOSu, stirred at 20-25 ° C to clarify. Then cooled to 0 ° C, 1.45 g of DCC was slowly added in batches. After the addition was complete, the reaction system was slowly heated to 20-25 ° C and stirred for 2 h. After the reaction was completed, the resulting suspension was filtered to remove the by-product DCU. The filtrate obtained was Fmoc- β -Asp-OAll-Sar-OH activation solution, which is directly used in the subsequent condensation reaction.

[0183] S3-2. Add 15 mL of dichloroethane, 0.85 g of H-Sar-OH, and 4.68 mL of BSA to a 50 mL reaction flask in sequence and stir at 20-25°C for 2 h. The silicon protective solution is directly used in the subsequent condensation reaction.

[0184] S4, Fmoc- β Preparation of -Asp-OAll-(Sar)2-OH

[0185]

[0186] The two solutions of S3-1 and S3-2 were mixed and stirred at 20-25°C for 16 h. After the reaction was complete as determined by HPLC, 50 mL of water was added to quench the reaction. The pH was then adjusted to approximately 2 with 10 wt% citric acid and stirred for 5 min. The organic phase was concentrated and dissolved with EA. 1 wt% NaHCO3 was added and stirred for 10 min. The organic phase was discarded and 10% citric acid was added to the aqueous phase to adjust the pH to approximately 2. The organic phase was washed three times with saturated NaCl solution and concentrated. The product was slowly added dropwise to PE. A large amount of solid precipitated. The product was centrifuged and dried to obtain 2.819 g of white solid Fmoc- β -Asp-OAll-(Sar)2-OH, purity 97.03%, yield 82%.

[0187] S5, Fmoc- β Activation of -Asp-OAll-(Sar)2-OH and Protection of H-Sar-OH

[0188] S5-1, to a 50 mL reaction flask, 15 mL of dichloroethane, 1.41 g of Fmoc- β-Asp-OAll-(Sar)2-OH, 0.332 g of HOSu, and stirring at 20-25°C until clarified. Then, cool to 0°C and slowly add 0.595 g of DCC in batches. After addition, slowly warm the reaction system to 20-25°C and stir for 2 hours. After the reaction is complete, filter the resulting suspension to remove the byproduct DCU. The resulting filtrate is the activated solution of Fmoc-β-Asp-OAll-Sar-Sar-OH, which is used directly in the subsequent condensation reaction.

[0189] S5-2. Add 5 mL of dichloroethane, 0.351 g of H-Sar-OH, and 1.92 mL of BSA to a 25 mL reaction flask in sequence and stir at 20-25°C for 2 h. The silicon protective solution is directly used in the subsequent condensation reaction.

[0190] S6, Fmoc- β Preparation of -Asp-OAll-(Sar)3-OH

[0191]

[0192] The two solutions of S5-1 and S5-2 were mixed and stirred at 20-25 °C for 16 h. After the reaction was complete after HPLC detection, 20 mL of water was added to quench the reaction. The pH was then adjusted to about 2 with 10 wt% citric acid and stirred for 5 min. The aqueous phase was extracted twice with DCM. The organic phases were combined, washed three times with saturated NaCl solution, and concentrated. The organic phases were slowly added dropwise to PE. A large amount of solid precipitated. After centrifugation and drying, 1.406 g of white solid Fmoc- β -Asp-OAll-(Sar)3-OH, purity 95%, yield 88%.

[0193] S7, Fmoc- β Activation of -Asp-OAll-(Sar)3-OH and Protection of H-Sar-OH

[0194] S7-1, to a 50 mL reaction flask, 15 mL of dichloroethane, 1.41 g of Fmoc- β -Asp-OAll-(Sar)3-OH, 0.293 g of HOSu, stirred at 20-25 ° C to clarify. Then cooled to 0 ° C, 0.526 g of DCC was slowly added in batches. After the addition was complete, the reaction system was slowly heated to 20-25 ° C and stirred for 2 hours. After the reaction was completed, the suspension was filtered to remove the by-product DCU. The filtrate obtained was Fmoc- β -Asp-OAll-(Sar)3-OH activation solution, which is directly used in the subsequent condensation reaction.

[0195] S7-2. Add 5 mL of dichloroethane, 0.31 g of H-Sar-OH, and 1.75 mL of BSA to a 25 mL reaction flask in sequence and stir at 20-25°C for 2 h. The silicon protective solution is directly used in the subsequent condensation reaction.

[0196] S8, Fmoc- β Preparation of -Asp-OAll-(Sar)4-OH

[0197]

[0198] The two solutions of S7-1 and S7-2 were mixed and stirred at 20-25 °C for 16 h. After the reaction was complete after HPLC detection, 20 mL of water was added to quench the reaction. The pH was then adjusted to about 2 with 10 wt% citric acid and stirred for 5 min. The aqueous phase was extracted twice with DCM. The organic phases were combined, washed three times with saturated NaCl solution, and concentrated. The organic phases were slowly added dropwise to PE. A large amount of solid precipitated. After centrifugation and drying, 1.26 g of white solid Fmoc- β -Asp-OAll-(Sar)4-OH.

[0199] The obtained intermediate was tested by HPLC, and the HPLC spectrum was as follows: Figure 3 As shown, the product purity was measured to be 99.17% and the yield was 80%.

[0200] in, Figure 3 The integration results are shown in Table 2 below:

[0201] Table 2

[0202]

[0203] The obtained product was detected by LC-MS, and the theoretical relative molecular mass was 679.72, and ESI-MS m / z: found [M+H] + 679.27, the result is Figure 4 shown.

[0204] S9, Fmoc- β Preparation of -Asp-(Sar)4-OH

[0205]

[0206] To a 50 mL reaction bottle, 12 mL THF, 1.187 g Fmoc- β-Asp-OAll-(Sar)4-OH, 0.202 g PdCl2(PPh3)2, 1.523 g redistilled N-methylmorpholine, react at 20-25 ° C for 6 h. After the reaction is completed, the reaction solution is concentrated, dissolved with DCM, washed with citric acid solution and saturated NaCl solution three times, and then concentrated. It is slowly added dropwise to PE, and a large amount of solid is precipitated. Centrifugal drying gives 0.84 g of white solid Fmoc- β -Asp-(Sar)4-OH, purity 95.42%, yield 75%.

[0207] Example 3

[0208] This example provides a polypeptide containing a Sar linker (Fmoc- β -Asp-(Sar)7-OH) preparation method, the Fmoc- β The preparation method of -Asp-(Sar)7-OH comprises the following steps:

[0209] S1 to S8 are completely consistent with Example 1.

[0210] S9, Fmoc- β Activation of -Asp-OtBu-(Sar)4-OH and Protection of H-Sar-OH

[0211] S9-1, 150 mL of dichloroethane, 14.29 g of Fmoc- β -Asp-OtBu-(Sar)4-OH, 2.60 g of HOSu, stirred at 20-25 ° C to clarify. Then cooled to 0 ° C, 4.67 g of DCC was slowly added in batches. After the addition was complete, the reaction system was slowly heated to 20-25 ° C and stirred for 2 hours. After the reaction was completed, the suspension was filtered to remove the by-product DCU. The filtrate obtained was Fmoc- β -Asp-OtBu-(Sar)4-OH activation solution, which is directly used in the subsequent condensation reaction.

[0212] S9-2. To a 100 mL reaction flask, add 40 mL of dichloroethane, 2.745 g of H-Sar-OH, and 15.05 mL of BSA in sequence and stir at 20-25°C for 2 h. The silicon protective solution was directly used in the subsequent condensation reaction.

[0213] S10, Fmoc- β Preparation of -Asp-OtBu-(Sar)5-OH

[0214]

[0215] The two solutions of S9-1 and S9-2 were mixed and stirred at 20-25 °C for 16 h. After the reaction was complete by HPLC, 200 mL of water was added to quench the reaction. The pH was then adjusted to about 2 with 10 wt% citric acid and stirred for 5 min. The aqueous phase was extracted twice with DCM. The organic phases were combined, washed three times with saturated NaCl solution, and concentrated. The organic phases were slowly added dropwise to PE. A large amount of solid precipitated. After centrifugation and drying, 13.92 g of white solid Fmoc- β -Asp-OtBu-(Sar)5-OH, purity 93.24%, yield 88%.

[0216] S11, Fmoc- β Activation of -Asp-OtBu-(Sar)5-OH and Protection of H-Sar-OH

[0217] S11-1. To a 500 mL reaction flask, 100 mL of dichloroethane, 8.08 g of Fmoc- β -Asp-OtBu-(Sar)5-OH, 1.34 g of HOSu, stirred at 20-25 ° C to clarify. Then cooled to 0 ° C, 2.39 g of DCC was slowly added in batches. After the addition was complete, the reaction system was slowly heated to 20-25 ° C and stirred for 2 hours. After the reaction was completed, the suspension was filtered to remove the by-product DCU. The filtrate obtained was Fmoc- β -Asp-OtBu-(Sar)5-OH activation solution, which is directly used in the subsequent condensation reaction.

[0218] S11-2. To a 100 mL reaction flask, add 20 mL of dichloroethane, 1.41 g of H-Sar-OH, and 7.72 mL of BSA in sequence and stir at 20-25°C for 2 h. The silicon protective solution is directly used in the subsequent condensation reaction.

[0219] S12, Fmoc- β Preparation of -Asp-OtBu-(Sar)6-OH

[0220]

[0221] The two solutions of S11-1 and S11-2 were mixed and stirred at 20-25 °C for 16 h. After the reaction was complete by HPLC, 100 mL of water was added to quench the reaction. The pH was then adjusted to about 2 with 10 wt% citric acid and stirred for 5 min. The aqueous phase was extracted twice with DCM. The organic phases were combined, washed three times with saturated NaCl solution, and concentrated. The organic phases were slowly added dropwise to PE. A large amount of solid precipitated. After centrifugation and drying, 6.55 g of white solid Fmoc- β-Asp-OtBu-(Sar)6-OH, purity 93.02%, yield 74%.

[0222] S13, Fmoc- β Activation of -Asp-OtBu-(Sar)6-OH and Protection of H-Sar-OH

[0223] S13-1. To a 500 mL reaction flask, 100 mL of dichloroethane, 4.62 g of Fmoc- β -Asp-OtBu-(Sar)6-OH, 0.70 g of HOSu, stirred at 20-25 ° C to clarify. Then cooled to 0 ° C, 1.25 g of DCC was slowly added in batches. After the addition was complete, the reaction system was slowly heated to 20-25 ° C and stirred for 2 hours. After the reaction was completed, the suspension was filtered to remove the by-product DCU. The filtrate obtained was Fmoc- β -Asp-OtBu-(Sar)6-OH activation solution, which is directly used in the subsequent condensation reaction.

[0224] S13-2. Add 10 mL of dichloroethane, 0.74 g of H-Sar-OH, and 4.04 mL of BSA to a 50 mL reaction flask in sequence and stir at 20-25°C for 2 h. The silicon protective solution is directly used in the subsequent condensation reaction.

[0225] S14, Fmoc- β Preparation of -Asp-OtBu-(Sar)7-OH

[0226]

[0227] The two solutions of S11-1 and S11-2 were mixed and stirred at 20-25 °C for 16 h. After the reaction was complete by HPLC, 100 mL of water was added to quench the reaction. The pH was then adjusted to about 2 with 10 wt% citric acid and stirred for 5 min. The aqueous phase was extracted twice with DCM. The organic phases were combined, washed three times with saturated NaCl solution, and concentrated. The organic phases were slowly added dropwise to PE. A large amount of solid precipitated. After centrifugation and drying, 4.15 g of white solid Fmoc- β -Asp-OtBu-(Sar)7-OH, purity 91.87%, yield 83%.

[0228] S15, Fmoc- β Preparation of -Asp-(Sar)7-OH

[0229]

[0230] 6 mL of trifluoroacetic acid and 12 mL of dichloromethane were added to a 50 mL reaction bottle, and 1.5 g of Fmoc- β -Asp-OtBu-(Sar)7-OH, react at room temperature for 2 h. After the reaction is complete, the reaction solution is concentrated and slowly added dropwise to MTBE. A large amount of solid precipitates, which is filtered, washed three times with MTBE, and dried to obtain 0.679 g of a white solid Fmoc- β -Asp-(Sar)7-OH.

[0231] The obtained product Fmoc- β -Asp-(Sar)7-OH was detected by HPLC, and the HPLC spectrum was as follows Figure 5 As shown, the product purity was measured to be 96.55% and the yield was 48%.

[0232] in, Figure 5 The integration results are shown in Table 3 below:

[0233] Table 3

[0234]

[0235] The obtained product was detected by LC-MS, and the theoretical relative molecular mass was 852.37, and ESI-MS m / z: found [M+H] + 853.37, the result is as follows Figure 6 shown.

[0236] Example 4

[0237] This example provides a polypeptide containing a Sar linker (Fmoc- β -Asp-(Sar)7-OH) is prepared in the following way, which is different from Example 3 in that the intermediate with m=3 is condensed with the raw material H-(Sar)4-OH with k=4, and the Fmoc- β The preparation method of -Asp-(Sar)7-OH comprises the following steps:

[0238] S1 to S6 are completely consistent with Example 1.

[0239] The intermediate Fmoc- β -Asp-OtBu-(Sar)3-OH was detected by HPLC, and the HPLC spectrum was as follows Figure 7 As shown, the product purity was measured to be 97.82% and the yield was 48%.

[0240] in, Figure 7 The integration results are shown in Table 4 below:

[0241] Table 4

[0242]

[0243] The obtained product was detected by LC-MS, and the theoretical relative molecular mass was 624.28, and ESI-MS m / z: found [M+H] + 625.28, the result is Figure 8 shown.

[0244] S7, Fmoc- β Activation of -Asp-OtBu-(Sar)3-OH and Protection of H-(Sar)4-OH

[0245] S7-1. To a 50 mL reaction flask, 10 mL of dichloroethane, 0.83 g of Fmoc- β -Asp- OtBu -(Sar)3-OH, 0.17 g of HOSu, stirred at 20-25 ° C to clarify. Then cooled to 0 ° C, 0.30 g of DCC was slowly added in batches. After the addition was complete, the reaction system was slowly heated to 20-25 ° C and stirred for 2 hours. After the reaction was completed, the suspension was filtered to remove the by-product DCU. The filtrate obtained was Fmoc- β -Asp- OtBu -(Sar) 3-OH activation solution, which is directly used in the subsequent condensation reaction.

[0246] S7-2. To a 50 mL reaction flask, add 10 mL of dichloroethane, 0.60 g of H-(Sar)4-OH, 0.97 mL of BSA, and 0.69 mL of DIPEA in sequence and stir at 20-25°C for 2 h. The silicon protective solution was directly used in the subsequent condensation reaction.

[0247] S8, Fmoc- β Preparation of -Asp- OtBu -(Sar)7-OH

[0248]

[0249] The two solutions of S7-1 and S7-2 were mixed and stirred at 35-40 °C for 16 h. After the reaction was complete after HPLC detection, 20 mL of water was added to quench the reaction. The pH was then adjusted to about 2 with 10 wt% citric acid and stirred for 5 min. The aqueous phase was extracted twice with DCM. The organic phases were combined, washed three times with saturated NaCl solution, and concentrated. The organic phases were slowly added dropwise to PE. A large amount of solid precipitated. After centrifugation and drying, 1.26 g of white solid Fmoc- β -Asp- OtBu -(Sar)7-OH, purity 95.37%, yield 95%.

[0250] S9, Fmoc- β Preparation of -Asp-(Sar)7-OH

[0251]

[0252] 3 mL of trifluoroacetic acid and 6 mL of dichloromethane were added to a 25 mL reaction bottle, and 1.2 g of Fmoc- β -Asp-OtBu-(Sar)7-OH, react at room temperature for 2 h. After the reaction is complete, the reaction solution is concentrated and slowly added dropwise to MTBE. A large amount of solid precipitates, which is filtered, washed three times with MTBE, and dried to obtain 0.532 g of a white solid Fmoc- β -Asp-(Sar)7-OH, purity 98.76%, yield 97%.

[0253] Example 5

[0254] This example provides a polypeptide containing a Sar linker (Fmoc- β -Asp-Sar-OH) is prepared in the following manner, which differs from Example 1 only in that:

[0255] The additive HOSu in S1, S3, and S5 was replaced by an equal molar amount of HOBt, and the condensing agent DCC was replaced by an equal molar amount of EDCI;

[0256] The Fmoc- β -Asp-OtBu-Sar-OH purity was 89.64% and the yield was 93%;

[0257] The Fmoc- β -Asp-OtBu-(Sar)2-OH had a purity of 71.15% and a yield of 89%;

[0258] The Fmoc- β The purity of -Asp-OtBu-(Sar)3-OH was 68.06%, and the yield was 86%.

[0259] Example 6

[0260] This example provides a polypeptide containing a Sar linker (Fmoc- β -Asp-Sar-OH) is prepared in the following manner, which differs from Example 1 only in that:

[0261] The additive HOSu in S1, S3, and S5 was replaced by an equal molar amount of DSC, and the condensing agent DCC was replaced by an equal molar amount of HBTU;

[0262] The Fmoc- β -Asp-OtBu-Sar-OH purity was 0.79% and the yield was 93%;

[0263] The Fmoc- β -Asp-OtBu-(Sar)2-OH purity was 11.44% and the yield was 88%;

[0264] The Fmoc- β The purity of -Asp-OtBu-(Sar)3-OH was 8.65%, and the yield was 81%.

[0265] Example 7

[0266] This example provides a polypeptide containing a Sar linker (Fmoc- β -Asp-Sar-OH) is prepared in the following manner, which differs from Example 1 only in that:

[0267] The protecting group BSA of S1, S3, and S5 was replaced with an equimolar amount of trimethylsilyl chloride;

[0268] The Fmoc- β -Asp-OtBu-Sar-OH purity was 88.02% and the yield was 92%;

[0269] The Fmoc- β -Asp-OtBu-(Sar)2-OH had a purity of 70.54% and a yield of 82%;

[0270] The Fmoc- β The purity of -Asp-OtBu-(Sar)3-OH was 79.79%, and the yield was 78%.

[0271] Example 8

[0272] This example provides a polypeptide containing a Sar linker (Fmoc- β -Asp-Sar-OH) is prepared in the following manner, which differs from Example 1 only in that:

[0273] The activation reaction steps of S1, S3, and S5 were not performed, and the corresponding amounts of HOSu and DCC of S1, S3, and S5 of Example 1 were directly added during the condensation process of S2, S4, and S6;

[0274] The Fmoc- β -Asp-OtBu-Sar-OH purity was 7.34% and the yield was 98%;

[0275] The Fmoc- β -Asp-OtBu-(Sar)2-OH purity was 4.11% and the yield was 87%;

[0276] The Fmoc- β The purity of -Asp-OtBu-(Sar)3-OH was 2.74%, and the yield was 83%.

[0277] Example 9

[0278] This example provides a polypeptide containing a Sar linker (Fmoc- β -Asp-Sar-OH) is prepared in the following manner, which differs from Example 1 only in that:

[0279] The silicon protection steps of S1, S3, and S5 were not performed, and the corresponding amount of BSA of S1, S3, and S5 of Example 1 was directly added during the condensation process of S2, S4, and S6;

[0280] The Fmoc- β -Asp-OtBu-Sar-OH purity was 88.83% and the yield was 94%;

[0281] The Fmoc- β -Asp-OtBu-(Sar)2-OH had a purity of 27.63% and a yield of 88%;

[0282] The Fmoc- β The purity of -Asp-OtBu-(Sar)3-OH was 48.02%, and the yield was 82%.

[0283] Example 10

[0284] This example provides a polypeptide containing a Sar linker (Fmoc- β -Asp-Sar-OH) is prepared in the following manner, which differs from Example 1 only in that:

[0285] The activation reaction and silicon protection step of S1, S3, and S5 were not performed. HOSu and DCC corresponding to the amounts of S1, S3, and S5 in Example 1 were directly added to the condensation process of S2, S4, and S6 without adding BSA.

[0286] The Fmoc- β -Asp-OtBu-Sar-OH purity was 61.72% and the yield was 90%;

[0287] The purity of Fmoc-β-Asp-OtBu-(Sar)2-OH prepared by S4 was 77.74% and the yield was 91%;

[0288] The purity of Fmoc-β-Asp-OtBu-(Sar)3-OH prepared by S6 was 43.05% and the yield was 83%.

[0289] In summary, the method of the present invention demonstrated stable yields and high product purity in 10-gram-scale experiments. This invention provides a novel liquid-phase synthesis method for multiple Sar linkers. Using simple, inexpensive starting materials, the method produces high-quality multiple Sar linkers in high yield under mild reaction conditions. The post-processing method is simple, facilitating industrial scale-up.

[0290] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a polypeptide containing a Sar linker, characterized in that: The preparation method comprises the following steps: (1) Fmoc- β -Asp-PG-(Sar) m -OH and H-(Sar) k -OH undergoes condensation reaction to obtain Fmoc- β -Asp-PG-(Sar) n -OH; the reaction formula is as follows: ； Wherein, PG represents a protecting group, the protecting group PG is selected from -OtBu or -OAll, m is an integer between 0 and 9, k is an integer between 1 and 9, and n is an integer between 1 and 10; (2) Fmoc- β -Asp-PG-(Sar) n -OH was deprotected to obtain Fmoc- β -Asp-(Sar) n -OH, the reaction formula is as follows: ； Wherein, PG represents a protecting group, the protecting group PG is selected from -OtBu or -OAll, and n is an integer between 1 and 10; In step (1), before the condensation reaction, the preparation method further comprises the following steps: β -Asp-PG-(Sar) m Activation treatment of -OH and raw material H-(Sar) k -OH protection treatment; Wherein, the activation treatment comprises the following steps: β -Asp-PG-(Sar) m -OH, the first solvent, the first additive and the first condensing agent are mixed and activated to activate Fmoc- β -Asp-PG-(Sar) m -OH; the first additive is selected from N -Hydroxysuccinimide, N,N' - Any one of disuccinimidyl carbonate, pentafluorophenol, 1-hydroxybenzotriazole or ethyl 2-oximecyanoacetate, or a combination of at least two thereof; Wherein, the protection treatment comprises the following steps: k -OH, the second solvent and the silicon protection reagent are mixed, and the silicon protection H-(Sar) is reacted to form a protective layer. k -OH.

2. The method for preparing a polypeptide containing a Sar linker according to claim 1, wherein In the activation treatment, the activation reaction temperature is 20-50° C., and the activation reaction time is 2-12 h; In the protection treatment, the protection reaction temperature is 20-50° C., and the protection reaction time is 2-12 h.

3. The method for preparing a polypeptide containing a Sar linker according to claim 1, wherein In the activation treatment, the Fmoc- β -Asp-PG-(Sar) m The molar ratio of -OH, the first additive and the first condensing agent is 1:(1-1.5):(1-1.5); In the protection treatment, the H-(Sar) k The molar ratio of -OH to silicon protecting agent is 1:(1.5~3.0).

4. The method for preparing a polypeptide containing a Sar linker according to claim 1, wherein The first solvent and the second solvent are each independently selected from any one or a combination of at least two of dichloroethane, dichloromethane or tetrahydrofuran; And / or, the first condensing agent is selected from N,N' -Dicyclohexylcarbodiimide, N,N' -diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or benzotriazole- N,N,N',N' -Any one or a combination of at least two of tetramethyluronium hexafluorophosphate; And / or, the silicon protecting agent is selected from trimethylchlorosilane and / or N,O -Bistrimethylsilylacetamide.

5. The method for preparing a polypeptide containing a Sar linker according to claim 1, wherein In step (1), the Fmoc- β -Asp-PG-(Sar) m -OH and H-(Sar) k The molar ratio of -OH is (0.8~1.2):(0.8~1.2).

6. The method for preparing a polypeptide containing a Sar linker according to claim 1, wherein In step (1), the temperature of the condensation reaction is 20-30° C., and the time of the condensation reaction is 2-20 h.

7. The method for preparing a polypeptide containing a Sar linker according to claim 1, wherein In step (1), after the condensation reaction is completed, the following post-treatment steps are also included: The reaction solution obtained by the condensation reaction is concentrated, washed, and precipitated in sequence to obtain the Fmoc- β -Asp-PG-(Sar) n -OH; and / or, the washing solvent is selected from citric acid solution and / or water; And / or, the precipitation solvent is selected from petroleum ether and / or n-heptane.

8. The method for preparing a polypeptide containing a Sar linker according to claim 1, wherein In step (2), when the protecting group -OtBu is removed, the reagents used for the deprotection treatment include trifluoroacetic acid and a solvent; In step (2), when the protecting group -OAll is removed, the reagents used for the deprotection treatment include bistriphenylphosphine palladium dichloride, N -methylmorpholine and solvent.

9. The method for preparing a polypeptide containing a Sar linker according to claim 8, wherein In step (2), when the protecting group -OtBu is removed, the solvent is selected from dichloromethane, ethyl acetate, 1,4-dioxane, N,N - any one of dimethylformamide or water or a combination of at least two; In step (2), when the protecting group -OAll is removed, the solvent includes tetrahydrofuran, N,N - Any one of dimethylformamide, dichloromethane or water, or a combination of at least two thereof.

10. The method for preparing a polypeptide containing a Sar linker according to claim 8, wherein In step (2), when the protecting group -OtBu is removed, the temperature of the deprotection treatment is 20-30°C, and the time of the deprotection treatment is 0.5-3 h; In step (2), when the protecting group -OAll is removed, the temperature of the deprotection treatment is 20-60° C., and the time of the deprotection treatment is 6-18 h.

11. The method for preparing a polypeptide containing a Sar linker according to claim 8, wherein: In step (2), when the protecting group -OtBu is removed, the Fmoc- β -Asp-PG-(Sar) n The mass ratio of -OtBu and trifluoroacetic acid is 1:(10~20); In step (2), when the protecting group -OAll is removed, the Fmoc- β -Asp-PG-(Sar) n -OtBu, bistriphenylphosphine palladium dichloride and N The mass ratio of -methylmorpholine is 1:(0.1~0.2):(10~20).

12. The method for preparing a polypeptide containing a Sar linker according to claim 8, wherein In step (2), when the protecting group -OtBu is removed, the deprotection treatment further includes the following post-treatment steps: The reaction solution obtained by deprotection treatment was concentrated and added dropwise to methyl tert-butyl ether to precipitate a solid, which was then centrifuged and washed to obtain Fmoc- β -Asp-(Sar) n -OH; and / or, the washing solvent is selected from methyl tert-butyl ether; In step (2), when the protecting group -OAll is removed, the deprotection treatment further includes the following post-treatment steps: the reaction solution obtained by the deprotection treatment is concentrated, dissolved in dichloromethane, washed and concentrated, and then added dropwise to petroleum ether to precipitate a solid, which is then centrifuged and dried to obtain Fmoc- β -Asp-(Sar) n -OH; And / or, the washing solvent is selected from citric acid solution and / or saturated sodium chloride solution.

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