Amino acid tags, methods of making the same, and methods of polypeptide synthesis

Abstract

The application provides an amino acid tag, a preparation method thereof and a polypeptide synthesis method. The amino acid tag comprises hydrophobic amino acids; the hydrophobic amino acids comprise phenylalanine and / or proline. The amino acid tag can solve the problem of poor polypeptide synthesis effect in the prior art and is suitable for the field of polypeptide synthesis.

Description

Technical Field

[0001] This invention relates to the field of polypeptide synthesis, and more specifically, to an amino acid tag, its preparation method, and a method for polypeptide synthesis. Background Technology

[0002] In the field of peptide synthesis, solid-phase peptide synthesis (SPPS) is currently the widely used standard method in commercial applications. Its core principle is to keep the growing peptide attached to a solid support, and achieve separation of the target product from other reactants through phase separation. To overcome the many limitations of SPPS, liquid-phase peptide synthesis (LPPS) has emerged. It is carried out under homogeneous reaction conditions and has advantages such as requiring less solvent, raw materials and reagents, faster reaction kinetics, and direct monitoring of the reaction by means of HPLC or LCMS. However, how to efficiently separate the target product from reactants and byproducts has become a key problem restricting its development. Currently, there are two existing technical solutions that are most similar to the application scenario of this invention: one is a hydrophobic soluble linker system, which serves as a label-assisted LPPS support and removes byproducts through precipitation or water washing (see, for example, Takahashi, D. et al. Angewandte Chemie International Edition 129:2017 7911-7915 and US Patent Application Publication No. US 20180215782); the other is a hydrophilic linker system, represented by polyethylene glycol (PEG), in which byproducts can be removed by using environmentally friendly organic solvents to extract hydrophilic label-assisted LPPS supports for liquid-phase peptide synthesis (see, for example, Fischer, PM; Zheleva, DI (2002) Journal of Peptide Science 8:529-542).

[0003] However, existing technologies all have significant shortcomings. First, while SPPS is the industry standard, it requires numerous resin washing steps, which is time-consuming and costly. The large amount of solvent generated is environmentally unfriendly, and the heterogeneous reaction system results in slow reaction kinetics, making it difficult to maximize product conversion efficiency and easily leading to side reactions such as peptide aggregation. Subsequent reaction monitoring and process optimization also present significant challenges. Second, the hydrophobic soluble linker system, a key component of LPPS, suffers from severe solubility problems as the peptide chain lengthens. Furthermore, aqueous washing has limited effectiveness in removing reagents and byproducts, easily interfering with downstream synthesis steps. Residual water in the organic layer may also affect peptide coupling reactions, requiring additional dehydration steps. On the other hand, the PEG derivatives of hydrophilic PEG linker systems are mostly polydisperse with variable molecular weights, increasing the complexity of analyzing and purifying high molecular weight PEG conjugates and making it difficult to synthesize long peptides (greater than or equal to 15 amino acid residues). These shortcomings limit the efficiency, applicability, and industrial application potential of liquid-phase peptide synthesis to varying degrees. Summary of the Invention

[0004] The main objective of this invention is to provide an amino acid tag and its preparation method, as well as a method for polypeptide synthesis, to solve the problems of poor polypeptide synthesis effect, high cost, and difficulty in synthesizing long polypeptides in the prior art.

[0005] To achieve the above objectives, according to a first aspect of the present invention, an amino acid tag is provided, the amino acid tag comprising hydrophobic amino acids; the hydrophobic amino acids comprising phenylalanine and / or proline.

[0006] Furthermore, amino acid tags also include hydrophobic regulatory components and / or linkers.

[0007] Furthermore, the hydrophobic regulating components include sarcosine and / or 2-(2-(2-aminoethoxy)ethoxy)acetyl.

[0008] Furthermore, the label contains 5 to 10 hydrophobic amino acids.

[0009] Furthermore, the label contains 5-7 hydrophobic amino acids and 2-3 hydrophobic regulatory components.

[0010] Furthermore, the hydrophobic amino acids include five phenylalanines.

[0011] Furthermore, the linkage sequence is the amino acid tag from C-terminus to N-terminus, and the amino acid tag includes any one of the following: a) 5 phenylalanines, 2 proline, and a linker; b) 5 phenylalanines, 2 sarcosines, and a linker; c) 5 phenylalanines, 2 2-(2-(2-aminoethoxy)ethoxy)acetyl groups, and a linker.

[0012] To achieve the above objective, according to a second aspect of the present invention, a method for preparing the above-mentioned amino acid tag is provided, the method comprising: coupling a resin with a hydrophobic amino acid in the order of the amino acid tag from the C-terminus to the N-terminus, followed by cleavage to remove the resin and obtain the amino acid tag.

[0013] Furthermore, the preparation method further includes: coupling the resin with a hydrophobic amino acid in the order of the amino acid tag from the C-terminus to the N-terminus to obtain a first product; coupling the first product with a linker to obtain a second product; cleaving the second product to remove the resin and obtain an amino acid tag; preferably, the preparation method further includes: coupling the resin, the hydrophobic amino acid and the hydrophobic regulating component in the order of the amino acid tag from the C-terminus to the N-terminus.

[0014] To achieve the above objectives, according to a third aspect of the present invention, a method for polypeptide synthesis is provided, the method comprising: coupling the above-mentioned amino acid tag with each amino acid to be linked sequentially in the order from the C-terminus to the N-terminus of the target polypeptide, followed by cleavage to remove the amino acid tag and obtain the target polypeptide.

[0015] Furthermore, the side chains of the amino acids to be linked have protecting groups attached.

[0016] Furthermore, the protecting groups include tBu and / or TTrt.

[0017] Furthermore, lysis involves mixing the peptide fragments obtained after all coupling reactions are completed with the lysis buffer, removing the tags from the peptide fragments, and obtaining the target peptide.

[0018] Furthermore, the lysis buffer includes TFA.

[0019] Furthermore, the side chains of the target peptide's amino acids have no protecting groups, and the TFA concentration is 85-95%.

[0020] Furthermore, the side chains of the target peptide's amino acids are attached with protecting groups, and the concentration of TFA is 1% to 5%.

[0021] Furthermore, the target polypeptide has a length of 5 to 20 amino acid residues.

[0022] By applying the technical solution of this invention and utilizing the hydrophobic amino acid tag in this application for liquid-phase peptide synthesis, the resulting peptide is less prone to degradation, produces fewer byproducts, is less susceptible to water interference, and can be adapted to the synthesis of long peptides, thus improving the overall effect of liquid-phase peptide synthesis. Attached Figure Description

[0023] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0024] Figure 1 The chromatogram of Fmoc-P2F5-Sieber in Example 1 of this application is shown.

[0025] Figure 2 The mass spectrum of Fmoc-P2F5-Sieber in Example 1 of this application is shown.

[0026] Figure 3 The chromatogram of HMPA-P2F5 in Example 1 of this application is shown.

[0027] Figure 4 The mass spectrum of HMPA-P2F5 in Example 1 of this application is shown.

[0028] Figure 5 The chromatogram of HMPB-P2-F5 in Example 2 of this application is shown.

[0029] Figure 6 The chromatogram of Rink-P2-F5 in Example 3 of this application is shown.

[0030] Figure 7 The chromatogram of HMPA-Sar2-F5 in Example 4 of this application is shown.

[0031] Figure 8 The mass spectrum of HMPA-Sar2-F5 in Example 4 of this application is shown.

[0032] Figure 9 The chromatogram of HMPB-Sar2F5-Sieber in Example 5 of this application is shown.

[0033] Figure 10 The chromatogram of Rink-Sar2-F5 in Example 6 of this application is shown.

[0034] Figure 11 The chromatogram of HMPA-AEEA-F5 in Example 7 of this application is shown.

[0035] Figure 12 The mass spectrum of HMPB-AEEA-F5 in Example 8 of this application is shown.

[0036] Figure 13 The chromatogram of HMPB-AEEA-F5 in Example 8 of this application is shown.

[0037] Figure 14 The chromatogram of Rink-AEEA-F5 in Example 9 of this application is shown.

[0038] Figure 15 The chromatogram of the crude oily product Y(tBu)GGFL-HMPA-P2F5 from Example 10 of this application is shown.

[0039] Figure 16 The chromatogram of the C-terminal carboxylic acid compound YGGFL-OH with unprotected side chains from Example 10 of this application is shown.

[0040] Figure 17 The mass spectrum of the C-terminal carboxylic acid compound YGGFL-OH with unprotected side chains from Example 10 of this application is shown.

[0041] Figure 18 The chromatogram of the crude oily product Y(tBu)GGFL-HMPB-P2F5 from Example 11 of this application is shown.

[0042] Figure 19 The chromatogram of the carboxylic acid Y(tBu)GGFL-OH compound with protected side chains from Example 11 of this application is shown.

[0043] Figure 20 The mass spectrum of the carboxylic acid Y(tBu)GGFL-OH compound with protected side chains from Example 11 of this application is shown.

[0044] Figure 21 The chromatogram of the crude oily product Y(tBu)GGFL-Rink-P2F5 from Example 12 of this application is shown.

[0045] Figure 22 The chromatogram of the C-terminal amide YGGFL-NH2 compound with unprotected side chains from Example 12 of this application is shown.

[0046] Figure 23 The mass spectrum of the C-terminal amide YGGFL-NH2 compound with unprotected side chains from Example 12 of this application is shown.

[0047] Figure 24 The chromatogram of the target polypeptide in Example 13 of this application specification is shown.

[0048] Figure 25 The mass spectrum of the target polypeptide in Example 13 of this application specification is shown. Detailed Implementation

[0049] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0050] As mentioned in the background section, the limitations of existing peptide synthesis techniques result in poor synthesis efficiency. Therefore, in this application, the inventors attempt to develop a new tag and apply it to the liquid-phase synthesis of peptides, thus proposing a series of protection schemes.

[0051] In a first typical embodiment of this application, an amino acid tag is provided, the tag comprising hydrophobic amino acids, including phenylalanine and / or proline.

[0052] Based on the shortcomings of the existing technology, the core technical objective of this invention is to provide a novel soluble tag with superior performance that is adapted to the needs of liquid-phase synthesis. It employs a hydrophobic amino acid combination as the core unit of the tag, enabling precise matching of the hydrophobicity control requirements of liquid-phase synthesis for soluble tags. This overcomes the limitations of existing linker systems in terms of hydrophobic compatibility or hydrophilicity defects: firstly, it overcomes the defects of existing hydrophobic linker systems in peptide synthesis, such as decreased solubility with peptide chain elongation, poor byproduct removal, and susceptibility to water interference; secondly, it overcomes the defects of existing hydrophilic PEG linker systems in peptide synthesis, such as inconsistent molecular weight, complex purification and analysis, and difficulty in adapting to the synthesis of long peptides. The hydrophobic amino acid combination of this application forms the core structure, possessing tunable hydrophobicity, and is adaptable to the homogeneous reaction and product separation requirements of liquid-phase peptide synthesis.

[0053] In a preferred embodiment, the amino acid tag further includes a hydrophobic regulatory component and / or a linker.

[0054] In a preferred embodiment, the hydrophobicity regulating component includes sarcosine (hereinafter referred to as Sar) and / or 2-(2-(2-aminoethoxy)ethoxy)acetyl (hereinafter referred to as AEEA).

[0055] In a preferred embodiment, the label contains 5 to 7 hydrophobic amino acids (including but not limited to 5, 6 or 7).

[0056] In a preferred embodiment, the label contains 5 to 7 hydrophobic amino acids and / or 2 to 3 hydrophobic regulatory components.

[0057] In a preferred embodiment, the hydrophobic amino acids include five phenylalanines.

[0058] In a preferred embodiment, the linkage order is the amino acid tag from C-terminus to N-terminus, and the amino acid tag includes any one of the following: a) 5 phenylalanines, 2 proline (SEQ ID NO: 1) and a linker (from C-terminus to N-terminus, i.e., linker-P2F5 hereinafter).

[0059] b) 5 phenylalanines, 2 sarcosines (SEQ ID NO: 2) and a linker (from the C-terminus to the N-terminus, i.e., linker-Sar2-F5 below);

[0060] c) 5 phenylalanines, 2 2-(2-(2-aminoethoxy)ethoxy)acetyl groups (SEQ ID NO: 2) and a linker (from the C-terminus to the N-terminus, i.e., linker-AEEA-F5 below).

[0061] SEQ ID NO: 1: PPFFFFF.

[0062] SEQ ID NO: 2: XXFFFFF, where "X" includes sarcosine or 2-(2-(2-aminoethoxy)ethoxy)acetyl.

[0063] The linkers mentioned above include, but are not limited to, one or more of HMPA, HMPB, Rink, Sieber, Ramage, PAL, HMBA, ICam, or ABHL, or other common linkers suitable for liquid-phase peptide synthesis. Those skilled in the art can flexibly replace them according to actual needs, all of which can meet the diverse liquid-phase synthesis scenarios of C-terminal carboxylic acids and amide substrates with free or protected side chains. This ensures both synthesis efficiency and product purity while improving the flexibility and adaptability of liquid-phase peptide synthesis technology, providing more reliable technical support for long peptide synthesis and industrial applications.

[0064] The amino acid tag of this application can be adjusted according to the properties of the target polypeptide to be synthesized. The hydrophobicity of the tag can be regulated by flexibly selecting the types and combination ratios of hydrophobic amino acids and hydrophobic regulatory components to adapt to different polypeptide linking reactions. Those skilled in the art can make adjustments themselves.

[0065] The linker can be flexibly replaced according to the actual synthesis scenario (such as substrate type and reaction conditions), improving the adaptability of the technology. Based on the flexible selection and replacement of the above-mentioned components, the tag of this application can be adapted to a variety of substrates (including but not limited to unprotected C-terminal amide peptides, unprotected C-terminal carboxylic acid peptides, and fully protected C-terminal carboxylic acid peptides), further broadening the scope of substrate applicability. It can simultaneously meet the liquid phase synthesis requirements of substrates with free or protected C-terminal carboxylic acids or amides, and further solve the problem of single substrate adaptability in existing technologies.

[0066] In a second typical embodiment of this application, a method for preparing the above-mentioned tag is provided, comprising: coupling resin with hydrophobic amino acids in the order of amino acid tag from C-terminus to N-terminus, followed by cleavage to remove resin and obtain amino acid tag.

[0067] In a preferred embodiment, the preparation method further includes: coupling the resin with a hydrophobic amino acid in the order of the amino acid tag from the C-terminus to the N-terminus to obtain a first product; coupling the first product with a linker to obtain a second product; cleaving the second product to remove the resin and obtain an amino acid tag; preferably, the preparation method further includes: coupling the resin, the hydrophobic amino acid and the hydrophobic regulating component in the order of the amino acid tag from the C-terminus to the N-terminus.

[0068] In a preferred embodiment, the resin includes, but is not limited to, amide resins. Preferably, the amide resins include, but are not limited to, Sieber resins. Those skilled in the art can select the resin specifications according to actual needs, and this application does not impose any limitations.

[0069] The chemical formula of the method for synthesizing the label in this application is shown below:

[0070] .

[0071] The tag proposed in this application, through the combination of hydrophobic amino acids and solid-phase synthesis, enables the rapid synthesis and acquisition of soluble tags with different hydrophobicities, meeting the needs of liquid-phase peptide synthesis. The synthesis method is simple, and its properties can be adjusted by modifying the specific amino acids and hydrophobicity control components of the tag according to the properties of the target peptide. Furthermore, the linker can be flexibly selected and replaced according to the properties of the target peptide, adapting to substrates with free or protected C-terminal carboxylic acids or amides. Compared with existing peptide synthesis tags and methods, this approach offers better performance and is more conducive to widespread adoption.

[0072] In a third typical embodiment of this application, a method for polypeptide synthesis is provided, the method comprising: coupling the above-mentioned tag with the amino acid to be linked in the order from the C-terminus to the N-terminus of the target polypeptide, followed by cleavage to remove the tag and obtain the target polypeptide.

[0073] The chemical formula of the polypeptide synthesized in this application is shown below:

[0074] .

[0075] In a preferred embodiment, a protecting group is attached to the side chain of the amino acid to be linked.

[0076] As is known to those skilled in the art, amino acid side chains, being active functional groups, all have protecting groups. Protecting groups are common raw materials in peptide synthesis, and their purpose is to ensure that each coupling reaction only reacts with the carboxyl or amino group of the main chain. Therefore, the protecting groups on the amino acid side chains of this application can be any kind of protecting group known to those skilled in the art to protect the amino acid. This application does not impose any restrictions.

[0077] In a preferred embodiment, the protecting group includes, but is not limited to, one or more of tBu, Trt, or Boc.

[0078] In a preferred embodiment, lysis includes mixing the polypeptide fragments obtained after all the coupling reactions are completed with a lysis buffer, removing the tag from the polypeptide fragments, and obtaining the target polypeptide.

[0079] In a preferred embodiment, the lysis buffer comprises TFA.

[0080] In a preferred embodiment, the side chains of the amino acids of the target polypeptide have no protecting groups, and the concentration of TFA is 85-95%.

[0081] In a preferred embodiment, the side chains of the amino acids of the target polypeptide are connected with protecting groups, and the concentration of TFA is 1% to 5%.

[0082] The peptide synthesis method of this application allows for adjustment of the lysis buffer concentration based on whether the target peptide's side chains require protecting groups. If the final target peptide does not require protecting groups, the TFA concentration in the lysis buffer is controlled at 85-95%, and the tag and protecting groups are lysed together. If the final target peptide still needs further ligation, the lysis buffer concentration is controlled at 1-5%, ensuring that protecting groups remain attached to the target peptide, allowing the ligation reaction to continue. Those skilled in the art can adjust and select the lysis buffer concentration according to actual production needs to obtain the target peptide.

[0083] In a preferred embodiment, the target polypeptide has a length of 5 to 20 amino acid residues.

[0084] Unless otherwise specified, the reagents used in the embodiments of this application are all commercially available products.

[0085] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0086] Example 1

[0087] Preparation of HMPA-P2F5:

[0088] The chemical formula of HMPA-P2F5 (SEQ ID NO: 1) is as follows:

[0089] .

[0090] Starting with Fmoc-Sieber amide resin (substitution degree 0.7 mmol / g, styrene 1% DVB, 100-200 mesh), HMPA-P2F5 was prepared via solid-phase synthesis in a solid-phase reactor using the Fmoc strategy, as follows:

[0091] Coupled P2F5 units on Sieber resin: The preparation of P2F5 on Fmoc-Sieber amide resin was carried out on a 7.0 mmol scale (10 g starting resin).

[0092] Step 1: Swelling. Use DMF (60 mL, 6V, 20 min, repeated 3 times) to swell, and then drain the solution.

[0093] Step 2: Deprotection. Use 20% piperidine DMF solution (60 mL, 6V, drain after 15 min, repeat twice) to deprotect, and wash with DMF (60 mL, 6V, drain after 3 min, repeat 8 times).

[0094] Step 3: Coupling. Under nitrogen protection, DMF (60 mL, 6V), Fmoc-AA-OH (14.0 mmol, 2.0 eq, AA=Pro, Phe), and Oxyma (14.0 mmol, 2.0 eq) were added sequentially to a four-necked flask and stirred until dissolved. After cooling to 5±5℃, DIC (15.4 mmol, 2.2 eq) was added dropwise. After activation for 10±5 min, the mixture was added to a solid-phase flask. After reacting at room temperature for 3 h, Kaiser analysis was performed. Once the resin was colorless and the reaction was complete, the atmosphere was purged.

[0095] Wash with DMF (60 mL, 6V, drain after 3 min, repeat 6 times).

[0096] The deprotection and coupling cycles were repeated a total of seven times to obtain Fmoc-P2F5-Sieber peptide resin.

[0097] After the final DMF wash, the resin was washed with DCM (60 mL, 6 V, 3 min, repeated twice), MTBE (60 mL, 6 V, 3 min, repeated twice), and then washed with DCM (60 mL, 6 V, 3 min, repeated twice).

[0098] Dry in a nitrogen stream for 4 hours.

[0099] The peptide resin Fmoc-P2F5-Sieber (i.e., the first product) was obtained with a weight gain of 99.5%, a purity of 93% for the lysate sample, and a QTOF of m / z of 947.4765 (M). + H + ). Fmoc-P2F5-Sieber (the method for synthesizing the peptide resin (first product) in subsequent Examples 2-9 is consistent with the method and steps in this Example).

[0100] The chromatogram of Fmoc-P2F5-NH2 is as follows: Figure 1 As shown, the mass spectrum is as follows Figure 2 As shown. The chemical formula of Fmoc-P2F5-NH2 is as follows:

[0101] .

[0102] HMPA groups were coupled to Fmoc-P2F5-Sieber on peptide resin: A portion (1.2 g, 0.7 mmol) of Fmoc-P2F5-Sieber on peptide resin was swollen with DMF (7.2 mL, 6 V, for 20 min, then drained, repeated 3 times), deprotected with DMF solution of 20% piperidine (7.2 mL, 6 V, for 15 min, then drained, repeated 2 times), and washed with DMF (7.2 mL, 6 V, for 3 min, then drained, repeated 8 times).

[0103] HMPA linker 4-(hydroxymethyl)phenoxyacetic acid (1.5 mmol, 2.0 equivalent), Oxyma (1.5 mmol, 2.0 equivalent), and DIC (2.1 mmol, 3.0 equivalent) in DMF (7.2 mL, 6 V) were added to the reaction vessel and mixed by bubbling with nitrogen for 3 h. The reaction vessel was drained and washed with DMF (7.2 mL, 6 V, 3 min, then evacuated, repeated 6 times). After the last DMF wash, the resin was washed with DCM (60 mL, 6 V, 3 min, repeated twice), MTBE (60 mL, 6 V, 3 min, repeated twice), and then with DCM (60 mL, 6 V, 3 min, repeated twice). The resin was dried in a nitrogen stream for 4 h. The peptide resin HMPA-P2F5-Sieber showed a weight gain of 100.5%.

[0104] HMPA-P2F5-NH2 cleavage from Sieber resin: HMPA-P2F5-Sieber on the peptide resin was mixed with a 3% TFA solution in DCM (12 mL, 10 V) for 30 min, filtered, and washed with another DCM solution. The filtrate was neutralized with pyridine and concentrated under reduced pressure. The resulting oily substance was dissolved in DMF (2 mL), and purified water (20 mL) was added, causing the product to precipitate. The precipitate was filtered, washed with purified water, and dried in a nitrogen stream for 24 h to obtain HMPA-P2F5. The chromatogram is shown below. Figure 3 As shown, the mass spectrum is as follows Figure 4 As shown, the product purity is 91.27%, QTOF: m / z 1111.6354 (M + H + ).

[0105] Example 2

[0106] Preparation of HMPB-P2-F5:

[0107] The chemical formula of HMPB-P2-F5 is shown below:

[0108] .

[0109] HMPB groups were coupled to Fmoc-P2F5-Sieber on peptide resin: A portion (1.2 g, 0.7 mmol) of Fmoc-P2F5-Sieber on the peptide resin was swollen with DMF (7.2 mL, 6 V, evacuated after 20 min, repeated 3 times), deprotected with DMF solution of 20% piperidine (7.2 mL, 6 V, evacuated after 15 min, repeated 2 times), and washed with DMF (7.2 mL, 6 V, evacuated after 3 min, repeated 8 times). The HMPB linker 4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid (1.5 mmol, 2.0 equivalent), Oxyma (1.5 mmol, 2.0 equivalent), and DIC (2.1 mmol, 3.0 equivalent) were added to the reaction vessel in a solution of DMF (7.2 mL, 6 V) and mixed by bubbling with nitrogen for 4 h. The reaction vessel was drained and washed with DMF (7.2 mL, 6V, 3 min, then drained, repeated 6 times). After the final DMF wash, the resin was washed with DCM (60 mL, 6V, 3 min, repeated twice), then with MTBE (60 mL, 6V, 3 min, repeated twice), and then with DCM (60 mL, 6V, 3 min, repeated twice). The resin was dried in a nitrogen stream for 4 h.

[0110] The yield of peptide resin HMPB-P2F5-Sieber was 98.5%.

[0111] Cleavage of HMPB-P2F5-NH2 from Sieber resin: HMPB-P2F5-Sieber on peptide resin was mixed with 1% TFA in DCM solution (12 mL, 10 V) for 15 min, filtered, and the filtrate was neutralized with pyridine. The cleavage was repeated 4 times. The filtrates were combined and concentrated under reduced pressure. The resulting oily substance was dissolved in DMF (2 mL), and purified water (20 mL) was added, causing the product to precipitate. The product was filtered, the filter cake was washed with purified water, and dried in a nitrogen stream for 24 h to obtain HMPB-P2-F5 with a purity of 90.35% and QTOF: m / z 1169.6933 (M + H + ), chromatogram as Figure 5 As shown.

[0112] Example 3

[0113] Preparation of Rink-P2-F5:

[0114] The chemical formula of Rink-P2-F5 is shown below:

[0115] .

[0116] To couple the Rink group to the peptide resin with Fmoc-P2F5-Sieber: A portion (1.2 g, 0.7 mmol) of Fmoc-P2F5-Sieber on the peptide resin was swollen with DMF (7.2 mL, 6 V, for 20 min, then drained, repeated 3 times), deprotected with DMF solution of 20% piperidine (7.2 mL, 6 V, for 15 min, then drained, repeated 2 times), and washed with DMF (7.2 mL, 6 V, for 3 min, then drained, repeated 8 times). Fmoc-Rink linker (p-[a-[1-(9H-fluorene-9-yl)-methoxyformamido]-2,4-dimethoxybenzyl]-phenoxyacetic acid (1.5 mmol, 2.0 equivalent), Oxyma (1.5 mmol, 2.0 equivalent), and DIC (2.1 mmol, 3.0 equivalent) in DMF (7.2 mL, 6 V) was added to the reaction vessel and mixed by bubbling with nitrogen for 4 h. The reaction vessel was drained and washed with DMF (7.2 mL, 6 V, evacuation after 3 min, repeated 6 times). Deprotection was performed using a 20% piperidine DMF solution (7.2 mL, 6 V, evacuation after 15 min, repeated 2 times), followed by washing with DMF (7.2 mL, 6 V, evacuation after 3 min, repeated 8 times).

[0117] After the final DMF wash, the resin was washed with DCM (60 mL, 6V, 3 min, repeated twice), then with MTBE (60 mL, 6V, 3 min, repeated twice), and finally with DCM (60 mL, 6V, 3 min, repeated twice). It was dried in a nitrogen stream for 4 h to obtain the peptide resin Rink-P2F5-Sieber, with a yield of 99.8%.

[0118] Rink-P2F5-NH2 cleavage from Sieber resin: Rink-P2F5-Sieber on peptide resin was mixed with a lysis buffer solution of 95% TFA / 5% TIPS (12 mL, 10 V) for 90 min, filtered, and the filter cake was washed with a small amount of TFA. The filtrate was concentrated under reduced pressure. The resulting concentrate was slowly added dropwise to MTBE (24 mL, 20 V), and the product precipitated. The precipitate was filtered, the filter cake was washed with MTBE, and the filter cake was dried in a nitrogen stream for 24 h to obtain the product Rink-P2-F5 with a purity of 86.35%. The chromatogram is shown below. Figure 6 As shown, QTOF: m / z 1246.5990 (M + H + ).

[0119] Example 4

[0120] Preparation of HMPA-Sar2-F5:

[0121] The chemical formula of HMPA-Sar2-F5 is as follows:

[0122] .

[0123] The only difference between this embodiment and Example 1 is that Fmoc-Pro-OH is replaced with Fmoc-Sar-OH for coupling, and the yield of peptide resin HMPA-Sar2F5-Sieber is 101.3%.

[0124] The peptide resin HMPA-Sar2F5-Sieber was then cleaved in a 3% TFA DCM solution, neutralized with pyridine, and crystallized in a DMF / H2O system to obtain HMPA-Sar2-F5 with a purity of 85.11%. The chromatogram is shown below. Figure 7 As shown, the mass spectrum is as follows Figure 8 As shown, QTOF: m / z 1059.5065 (M + H + ).

[0125] Example 5

[0126] Preparation of HMPB-Sar2-F5:

[0127] The chemical formula of HMPB-Sar2-F5 is as follows:

[0128] .

[0129] The only difference from Example 2 is that Fmoc-Pro-OH was replaced with Fmoc-Sar-OH for coupling. The yield of the peptide resin HMPB-Sar2F5-Sieber was 101.3%. The peptide resin HMPB-Sar2F5-Sieber was then cleaved in a 1% TFA solution in DCM, neutralized with pyridine, and crystallized in a DMF / H2O system to obtain HMPB-Sar2-F5 with a purity of 81.40%. The chromatogram is shown below. Figure 9 As shown, QTOF: m / z 1117.6733 (M + H + ).

[0130] Example 6

[0131] Preparation of Rink-Sar2-F5:

[0132] The chemical formula of Rink-Sar2-F5 is as follows:

[0133] .

[0134] The only difference from Example 3 is that Fmoc-Pro-OH was replaced with Fmoc-Sar-OH for coupling. The yield of the peptide resin Rink-Sar2F5-Sieber was 98.3%. The peptide resin Rink-Sar2F5-Sieber was then cleaved in a 95% TFA solution, followed by MTBE crystallization to obtain Rink-Sar2-F5 with a purity of 77.87%. The chromatogram is shown below. Figure 10 As shown, QTOF: m / z1194.6654 (M + H + ).

[0135] Example 7

[0136] Preparation of HMPA-AEEA-F5:

[0137] The chemical formula of HMPA-AEEA-F5 is as follows:

[0138] .

[0139] The only difference from Example 1 is that Fmoc-Pro-OH was replaced with Fmoc-AEEA-OH for coupling, resulting in a 100.0% weight gain yield of the peptide resin HMPA-AEEA-F5-Sieber. Subsequently, the peptide resin HMPA-AEEA-F5-Sieber was cleaved in a 3% TFA DCM solution, neutralized with pyridine, and crystallized in a DMF / H2O system to obtain HMPA-AEEA-F5 with a purity of 91.53%. The chromatogram is shown below. Figure 11 As shown, QTOF: m / z 1062.5879 (M + H + ).

[0140] Example 8

[0141] Preparation of HMPB-AEEA-F5:

[0142] The chemical formula of HMPB-AEEA-F5 is shown below:

[0143] .

[0144] The only difference from Example 2 is that Fmoc-Pro-OH was replaced with Fmoc-AEEA-OH for coupling, resulting in a 99.0% weight gain yield of the peptide resin HMPB-AEEA-F5-Sieber. Subsequently, the peptide resin HMPB-AEEA-F5-Sieber was cleaved in a 1% TFA DCM solution, neutralized with pyridine, and crystallized in a DMF / H2O system to obtain HMPB-AEEA-F5 with a purity of 96.70%. The mass spectrum is shown below. Figure 12 As shown, the chromatogram is as follows Figure 13 As shown, QTOF: m / z 1102.5291 (M-H2O) + H + ).

[0145] Example 9

[0146] Preparation of Rink-AEEA-F5:

[0147] The chemical formula of Rink-AEEA-F5 is as follows:

[0148] .

[0149] The only difference from Example 2 is that Fmoc-Pro-OH was replaced with Fmoc-AEEA-OH for coupling. The yield of the peptide resin Rink-AEEA-F5-Sieber was 99.5%. The peptide resin Rink-AEEA-F5-Sieber was then cleaved in a 95% TFA solution, followed by MTBE crystallization to obtain Rink-AEEA-F5 with a purity of 89.64%. The chromatogram is shown below. Figure 14 As shown, QTOF: m / z1197.6656 (M + H + ).

[0150] Example 10

[0151] Y(tBu)GGFL-HMPA-P2F5, liquid-phase peptide synthesis using HMPA-P2F5 and strong cleavage from the linker:

[0152] The chemical formula of Y(tBu)GGFL (SEQ ID NO: 2)-HMPA-P2F5 is as follows:

[0153] .

[0154] Amino acid chain elongation on peptide resin HMPA-P2F5-NH2: DCM (10V) was added to a four-necked flask, and the temperature was lowered to 0-10℃. Fmoc-Leu-OH (2.0 eq), DMAP (0.10 eq), and EDCI (4.0 eq) were added sequentially, and the mixture was stirred until dissolved. Then HMPA-P2F5-NH2 (1.0 eq) was added, and the mixture was allowed to return to room temperature (20-30℃) for 3 hours. The mixture was then extracted and washed twice using a 20% NaCl aqueous solution (10V). The system was then cooled to 0-10℃, and mercaptosuccinic acid (3.0 eq) was added, followed by dropwise addition of DBU (8 eq). The mixture was reacted at 0-10℃ for 2 hours. After the reaction was complete, the mixture was extracted and washed twice using a 5% sodium carbonate aqueous solution (10V), and then extracted and washed once more using a 20% NaCl aqueous solution (10V).

[0155] Repeat the amino acid coupling and deprotection steps to couple the Fmoc-protected amino acids (with tBu-protected side chain -OH) sequentially from the C-terminus to the N-terminus. After all coupling is complete, concentrate the system to obtain an oily substance with the following chemical formula and a purity of 83.20% (RT24.5 is the amino acid raw material). The chromatogram is shown below. Figure 15 As shown, QTOF: m / z 555.3367 (M + H + ).

[0156] .

[0157] Strong cleavage of the unprotected C-terminal carboxylic acid YGGFL (SEQ ID NO: 3)-OH from the linker: The crude oily product Y(tBu)GGFL-HMPA-P2F5 was mixed with a cleavage buffer solution (12 mL, 10 V) of TFA:triisopropylsilane:1,2-ethylenedithiol:water (85:5:5:5 v / v ratio) for 90 min. The mixture was filtered, and the filter cake was washed with a small amount of TFA. The filtrate was concentrated under reduced pressure. The resulting concentrate was slowly added dropwise to MTBE (24 mL, 20 V), and the product precipitated. The precipitate was filtered, the filter cake was washed with MTBE, and dried in a nitrogen stream for 24 h. The unprotected C-terminal carboxylic acid YGGFL (SEQ ID NO: 3)-OH compound was obtained with a purity of 89.98%. The chromatogram is shown below. Figure 16 As shown, QTOF: m / z 555.2897 (M + H + Mass spectrum as shown Figure 17 As shown.

[0158] Example 11

[0159] Y(tBu)GGFL-HMPB-P2F5, liquid-phase peptide synthesis and soft cleavage from the linker using HMPB-P2F5:

[0160] The chemical formula of Y(tBu)GGFL-HMPB-P2F5 is as follows:

[0161] .

[0162] The preparation method was the same as in Example 10, using liquid-phase synthesis. The process involved liquid-phase coupling, aqueous extraction and washing, liquid-phase deprotection, and aqueous washing, with coupling performed in order from the C-terminus to the N-terminus. After all coupling was complete, the system was concentrated to obtain an oily substance with the following chemical formula and a purity of 68%. The chromatogram is shown below. Figure 18 As shown, QTOF: m / z 881.9533 (M + 2H + ).

[0163] .

[0164] Soft cleavage of the protected carboxylic acid Y(tBu)GGFL-OH from the linker: The crude oily product Y(tBu)GGFL-HMPB-P2F5 was mixed with a 1% TFA solution in DCM (12 mL, 10V) for 15 min, filtered, and the filtrate was neutralized with pyridine. The cleavage was repeated four times. The filtrates were combined and concentrated under reduced pressure to about 5V. The mixture was washed four times with saturated brine (10V, extract and separate), and the organic phase was dried over anhydrous sodium sulfate. The organic phase was then added dropwise to pre-cooled n-heptane (30V), and the product precipitated. The mixture was filtered, the filter cake was washed with n-heptane, and the filter cake was dried under a nitrogen stream for 24 h. The protected carboxylic acid Y(tBu)GGFL-OH compound was obtained with a purity of 95.57%, and the chromatogram is shown below. Figure 19 As shown, the mass spectrum is as follows Figure 20 As shown, QTOF: m / z633.3343 (M + Na + ).

[0165] Example 12

[0166] Y(tBu)GGFL-Rink-P2F5, liquid-phase peptide synthesis using Rink-P2F5 and strong cleavage from the linker:

[0167] The chemical formula of Y(tBu)GGFL-Rink-P2F5 is as follows:

[0168] .

[0169] The preparation method was the same as in Example 10, using liquid-phase synthesis. The process involved liquid-phase coupling, aqueous extraction and washing, liquid-phase deprotection, and aqueous washing, with coupling performed in order from the C-terminus to the N-terminus. After all coupling was complete, the system was concentrated to obtain an oily substance with the following chemical formula and a purity of 73.53%. The chromatogram is shown below. Figure 21 As shown, QTOF: m / z 920.5566 (M + H + ).

[0170] .

[0171] Strong cleavage of the unprotected C-terminal amide YGGFL (SEQ ID NO: 3)-NH2 from the linker: The crude oily product Y(tBu)GGFL-Rink-P2F5 was mixed with a cleavage buffer solution (12 mL, 10 V) of TFA:triisopropylsilane:1,2-ethylenedithiol:water (85:5:5:5 v / v ratio) for 90 min. The mixture was filtered, and the filter cake was washed with a small amount of TFA. The filtrate was concentrated under reduced pressure. The resulting concentrate was slowly added dropwise to MTBE (24 mL, 20 V), and the product precipitated. The precipitate was filtered, the filter cake was washed with MTBE, and dried in a nitrogen stream for 24 h to obtain the unprotected C-terminal amide YGGFL (SEQ ID NO: 3)-NH2 compound with a purity of 96.82%. The chromatogram is shown below. Figure 22 As shown, the mass spectrum is as follows Figure 23 As shown, MS: m / z 556.4 (M + H + ).

[0172] Example 13

[0173] Fmoc-W(Boc)LIAGGPS(tBu)S(tBu)GAPPPS(tBu)(SEQ ID NO: 4)-Rink-P2F5, using Rink-P2F5 for liquid-phase peptide synthesis and strong cleavage from the linker.

[0174] The polypeptide synthesized in this application is Fmoc-WLIAGGPSSGAPPPS-NH2, with a length of 15 peptides. It was synthesized using liquid-phase methods consistent with those in Example 12, involving liquid-phase coupling, aqueous extraction and washing, liquid-phase deprotection, and aqueous washing, with coupling performed in the order from C-terminus to N-terminus. After coupling, it was cleaved under the strong cleavage conditions of Example 12, ultimately yielding a C-terminal amide 15-peptide compound without side chain protection, with a purity of 87.21%. The chromatogram is shown below. Figure 24 As shown, the mass spectrum is as follows Figure 25 As shown, MS: m / z 807.9038 (M+2H) + ).

[0175] .

[0176] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: This application, by utilizing a combination of hydrophobic amino acids and employing solid-phase synthesis, can rapidly obtain soluble tags with different hydrophobicities. The tags of this application can meet the needs of liquid-phase peptide synthesis. The tag synthesis of this application is simple, and the elements of the tag can be flexibly selected according to the different properties of the target peptide. The linker can be flexibly replaced to adapt to substrates with free or protected C-terminal carboxylic acids or amides, making it suitable for the synthesis of long peptides. Compared with existing tags, the synthesis cost is low, the effect is better when applied to peptide synthesis, and it is more suitable for industrial synthesis and promotion.

[0177] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An amino acid tag, characterized in that, The amino acid tag consists of hydrophobic amino acids, hydrophobic regulatory components, and linkers; The hydrophobic amino acids include phenylalanine and / or proline.

2. The label according to claim 1, characterized in that, The hydrophobic regulating components include sarcosine and / or 2-(2-(2-aminoethoxy)ethoxy)acetyl.

3. The amino acid tag according to claim 1, characterized in that, The amino acid tag contains 5 to 10 of the hydrophobic amino acids.

4. The amino acid tag according to claim 1, characterized in that, The amino acid tag contains 5 to 7 of the hydrophobic amino acids and 2 to 3 hydrophobic regulatory components.

5. The amino acid tag according to any one of claims 1-4, characterized in that, The hydrophobic amino acids include five phenylalanines.

6. The amino acid tag according to claim 5, characterized in that, The connection order is from the C-terminus to the N-terminus of the amino acid tag, and the amino acid tag includes any of the following: a) 5 phenylalanines, 2 prolines, and one linker; b) 5 phenylalanines, 2 sarcosines, and one linker; c) 5 phenylalanines, 2 2-(2-(2-aminoethoxy)ethoxy)acetyl groups, and one linker.

7. A method for preparing an amino acid tag according to any one of claims 1 to 6, characterized in that, The preparation method includes: The resin, the hydrophobic amino acid, and the hydrophobic regulating component are coupled in the order of the amino acid tag from C-terminus to N-terminus to obtain the first product. The first product is coupled to the linker to obtain the second product; The second product is cleaved to remove the resin and obtain the amino acid tag.

8. A method for synthesizing polypeptides, characterized in that, The method includes: coupling the amino acid tag of any one of claims 1 to 6 with each amino acid to be linked in sequence from the C-terminus to the N-terminus of the target polypeptide, followed by cleavage to remove the amino acid tag and obtain the target polypeptide.

9. The method according to claim 8, characterized in that, The side chain of the amino acid to be joined has a protecting group attached.

10. The method according to claim 9, characterized in that, The protecting groups include tBu and / or Trt.

11. The method according to claim 8, characterized in that, The lysis process involves mixing the polypeptide fragments obtained after all the coupling reactions are completed with a lysis buffer, removing the tag from the polypeptide fragments, and obtaining the target polypeptide.

12. The method according to claim 11, characterized in that, The lysis solution includes TFA.

13. The method according to claim 12, characterized in that, The target polypeptide has no protecting groups on the side chains of its amino acids, and the concentration of TFA is 85-95%.

14. The method according to claim 12, characterized in that, The target polypeptide has a protecting group attached to the side chain of an amino acid, and the concentration of TFA is 1% to 5%.

15. The method according to claim 8, characterized in that, The target polypeptide has a length of 5 to 20 amino acid residues.

Images