Solid-phase synthesis method for glp-1 / gip dual agonist

Abstract

The present invention provides a use of a dipeptide fragment in solid-phase synthesis of a polypeptide compound, and a dipeptide fragment-based solid-phase synthesis method for a polypeptide compound. The dipeptide fragment is selected from the following structures: Gly-Gly, Ile-Aib, Tyr-Aib, Thr-αMePhe and Glu-Gly.

Description

Solid-phase synthesis of GLP-1 / GIP dual agonists Technical Field

[0001] The present invention belongs to the field of solid-phase synthesis of polypeptide compounds. More specifically, the present invention provides the use of a dipeptide fragment in the solid-phase synthesis of a polypeptide compound, and a method for solid-phase synthesis of a dipeptide fragment of a polypeptide compound. The dipeptide fragment is selected from the following structures: Gly-Gly, Ile-Aib, Tyr-Aib, Thr-αMePhe, and Glu-Gly. Background Art

[0002] The solid-phase synthesis of polypeptide compounds uses a solid-phase resin carrier as a synthesis platform. Amino acids are connected to the resin carrier one by one according to a predetermined sequence. The synthesized peptide chain is then cut off with a cleavage agent and further purified to obtain a high-purity polypeptide.

[0003] However, when synthesizing peptides with more than 20 amino acids, the content of oligopeptide byproducts, mainly residue deletions, increases significantly and accumulates continuously. Because these oligopeptide byproducts are very similar to the target peptide in molecular weight, solubility, polarity and structure, they bring difficulties to separation and purification.

[0004] Therefore, it is necessary to continuously improve the synthesis method of long-chain polypeptide compounds in order to increase the synthesis yield and purity of long-chain polypeptide compounds and reduce production costs. Summary of the Invention

[0005] According to one aspect of the present invention, a method for solid-phase synthesis of a dipeptide fragment of a polypeptide compound is provided, wherein the polypeptide compound is a compound of formula AI and has the following structure:

[0006] H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2 formula AI (SEQ ID NO: 12),

[0007] wherein optionally, at any one lysine selected from positions 16, 24, 28 and 40, the side chain ε-amino group is chemically modified via ([2-(2-amino-ethoxy)-ethoxy]-acetyl)a-(γ-Glu)b-CO-(CH2)c-COOH, wherein each a is independently an integer from 0 to 5, each b is independently an integer from 0 to 5, and each c is independently an integer from 10 to 24,

[0008] The dipeptide fragment is selected from the following structures: Gly-Gly, Ile-Aib, Tyr-Aib, Thr-αMePhe and Glu-Gly.

[0009] According to another aspect of the present invention, there is provided a use of a dipeptide fragment in solid-phase synthesis of a polypeptide compound, wherein the dipeptide fragment is selected from the following structures: Gly-Gly, Ile-Aib, Tyr-Aib, Thr-αMePhe and Glu-Gly, and the polypeptide compound is a compound of formula AI and has the following structure:

[0010] H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2 formula AI (SEQ ID NO: 12),

[0011] Optionally, at any one lysine selected from positions 16, 24, 28 and 40, the side chain ε-amino group is chemically modified via ([2-(2-amino-ethoxy)-ethoxy]-acetyl)a-(γ-Glu)b-CO-(CH2)c-COOH, wherein each a is independently an integer from 0 to 5, each b is independently an integer from 0 to 5, and each c is independently an integer from 10 to 24.

[0012] Compared with solid-phase condensation coupling using individual amino acids one by one, the present invention uses specific dipeptide fragments for segmented coupling, circumventing difficult sites with low yields and difficult synthesis, reducing amino acid racemization and side reactions, and improving the synthesis yield, with a final yield of up to about 10-about 32%. At the same time, the purity of the crude product and the purity of the pure product of the polypeptide synthesized according to the method of the present invention are both improved, and the purity of the pure product can reach about 94.9-about 99.57%. Moreover, the method of the present invention can achieve stable scale-up production without sacrificing the final yield. For example, the method of the present invention can stably pass multiple batches of verification such as small test batches, pilot batches, toxicology batches, engineering batches, and GMP batches without sacrificing the final yield and crude product purity. It can also be stably scaled up from a 10 mmol laboratory scale to a 50 mmol-300 mmol large-scale production without sacrificing the final yield and crude product purity.

[0013] Compared to the sequential solid-phase synthesis method, which links amino acids 1 to 40 one by one from the C-terminus, the method of the present invention significantly improves the final yield. Compared to the solid-phase synthesis method, which first synthesizes a large fragment containing amino acids 1 to 14 from the C-terminus and then links amino acids 15 to 40 one by one, the large-fragment synthesis method requires a larger amount of fragment (e.g., 4 molar equivalents). The method of the present invention does not require excessive amounts of individual amino acid and dipeptide fragments, thus reducing production costs and improving the final yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the H NMR spectrum of compound P016.

[0015] Figure 2 is the C NMR spectrum of compound P016. DETAILED DESCRIPTION

[0016] Unless otherwise specified, all scientific and technical terms in the present invention shall have the same meanings as those known to those skilled in the art. In the event of any inconsistency, the definitions provided in the present invention shall prevail.

[0017] It should be understood that all materials, methods, examples and detailed descriptions of figures are for illustrative purposes only and are not to be construed as limiting the present invention unless otherwise expressly stated.

[0018] As used herein, the terms "comprising" and "including" are synonymous, indicating that other ingredients or steps that do not affect the end result may be included. This term encompasses the terms "consisting of" and "consisting essentially of." Products and methods according to the present invention may include, consist of, or consist essentially of the basic technical features and / or limitations of the present invention described herein, as well as any additional and / or optional ingredients, components, steps, or limitations described herein.

[0019] Unless otherwise stated, in the context of the present invention each numerical range is meant to include both endpoints and any number and sub-range within the numerical range.

[0020] Unless otherwise specified, all materials and reagents used in the present invention are commercially available.

[0021] The following specific embodiments are applicable to the solid-phase synthesis method of a dipeptide fragment of a polypeptide compound according to the present invention, and are also applicable to the use of the dipeptide fragment in the solid-phase synthesis of a polypeptide compound.

[0022] [Peptide compounds]

[0023] According to the present invention, the polypeptide compound is a compound of formula AI and has the following structure:

[0024] H-Tyr1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2 formula AI (SEQ ID NO: 12),

[0025] Optionally, at any one lysine selected from positions 16, 24, 28 and 40, the side chain ε-amino group is chemically modified via ([2-(2-amino-ethoxy)-ethoxy]-acetyl)a-(γ-Glu)b-CO-(CH2)c-COOH, wherein each a is independently an integer from 0 to 5, each b is independently an integer from 0 to 5, and each c is independently an integer from 10 to 24.

[0026] In some embodiments, each a is independently an integer from 1 to 5, each b is independently an integer from 1 to 5, and each c is independently an integer from 12 to 22.

[0027] The polypeptide compound of Formula AI comprises 40 amino acid residues. In the context herein, unless otherwise indicated or clearly contradictory, amino acid position numbering is calculated from the N-terminus, the leftmost end of the peptide chain or structural formula of the polypeptide compound. For example, using the polypeptide compound of Formula AI as an example, the N-terminal amino acid is tyrosine (Tyr) at position 1, and the C-terminal amino acid is lysine (Lys) at position 40.

[0028] In the context of this document, unless otherwise specified or clearly contradictory, "polypeptide compound", "polypeptide" and "compound" are used interchangeably to refer to the polypeptide compound of formula AI.

[0029] In some embodiments, the polypeptide compound is a compound of Formula AI and has the following structure:

[0030] H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40-NH2 formula AI (SEQ ID NO: 1),

[0031] The four lysine residues selected from positions 16, 24, 28 and 40 are not chemically modified with the side chain ε-amino group. 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO: 1)

[0032] In some of the embodiments, the polypeptide compound is a compound of formula AI and has the following structure:

[0033] H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2 formula AI (SEQ ID NO: 12),

[0034] Wherein at any one lysine selected from positions 16, 24, 28 and 40, the side chain ε-amino group is chemically modified via ([2-(2-amino-ethoxy)-ethoxy]-acetyl)a-(γ-Glu)b-CO-(CH2)c-COOH, wherein each a is independently an integer from 0 to 5, each b is independently an integer from 0 to 5, and each c is independently an integer from 10 to 24.

[0035] In one embodiment, the polypeptide compound of formula AI is a compound selected from the following:

[0036] Compound P001: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2(SEQ ID NO:1)

[0037] Compound P007: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu21 -Phe 22 -Val 23 -Lys 24 (AEEA - AEEA - γGlu - CO - (CH2) 16 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2, that is, Y 1 -Aib 2 -E 3 -G 4 -T 5 -αMePhe 6 -T 7 -S 8 -D 9 -Y 10 -Aib 11 -I 12 -Aib 13 -L 14 -D 15 -K 16 -Q 17 -A 18 -Q 19 -A 20 -E 21 -F 22 -V 23 -K 24 (AEEA - AEEA - γGlu - CO - (CH2) 16 -COOH)-W 25 -L 26 -L 27 -K 28 -G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P 37 -P38 -S 39 -K 40 (SEQ ID NO: 2), the structure is as follows:

[0038] Compound P008: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 (AEEA-AEEA-γGlu-CO-(CH2) 16 -COOH)-Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2, i.e., Y 1 -Aib 2 -E 3 -G 4 -T 5 -αMePhe 6 -T 7 -S 8 -D 9 -Y 10 -Aib11 -I 12 -Aib 13 -L 14 -D 15 -K 16 -Q 17 -A 18 -Q 19 -A 20 -HAVE BEEN 21 -F 22 -V 23 -K 24 -W 25 -L 26 -L 27 -K 28 (AEEA-AEEA-γGlu-CO-(CH2) 16 -COOH)-G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P 37 -P 38 -S 39 -K 40 (SEQ ID NO:3),the structure is as follows:

[0039] Compound P013: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 (AEEA-AEEA-γGlu-CO-(CH2)18 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2, that is, Y 1 -Aib 2 -E 3 -G 4 -T 5 -αMePhe 6 -T 7 -S 8 -D 9 -Y 10 -Aib 11 -I 12 -Aib 13 -L 14 -D 15 -K 16 -Q 17 -A 18 -Q 19 -A 20 -E 21 -F 22 -V 23 -K 24 (AEEA-AEEA-γGlu-CO-(CH2) 18 -COOH)-W 25 -L 26 -L 27 -K 28 -G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P 37 -P 38 -S 39 -K 40 (SEQ ID NO:4), the structure is as follows:

[0040] Compound P014: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 (AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2, i.e., Y 1 -Aib 2 -E 3 -G 4 -T 5 -αMePhe 6 -T 7 -S 8 -D 9 -Y 10 -Aib 11 -I 12 -Aib 13 -L 14 -D 15 -K 16-Q 17 -A 18 -Q 19 -A 20 -HAVE BEEN 21 -F 22 -V 23 -K 24 (AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-W 25 -L 26 -L 27 -K 28 -G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P 37 -P 38 -S 39 -K 40 (SEQ ID NO:5),the structure is as follows:

[0041] Compound P015: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 (AEEA-AEEA-γGlu-γGlu-γGlu-CO-(CH2) 16 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28-Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2, that is, Y 1 -Aib 2 -E 3 -G 4 -T 5 -αMePhe 6 -T 7 [[ID=3s]]-S 8 -D 9 -Y 10 -Aib 11 -I 12 -Aib 13 / / -L 14 -D 15 -K 16 -Q 17 -A 18 -Q 19 -A 20 -E 21 -F 22 -V 23 / / -K 24 (AEEA - AEEA - γGlu - γGlu - γGlu - CO - (CH2) 16 -COOH)-W 25 -L 26 -L 27 -K 28 -G 29 -G 30 -P 31 -S 32 -S<F 33 -G 34 -A 35 -P 36 -P 37 -P 38 -S 39 -K 40 (SEQ ID NO:6), the structure is as follows:

[0042] Compound P016: H - Tyr 1 -Aib 2 -Glu 3 -Gly4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 (AEEA - AEEA - AEEA - γGlu - CO - (CH2) 20 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2, that is, Y 1 -Aib 2 -E 3 -G 4 -T 5 -αMePhe 6 -T 7 -S 8 -D 9 -Y 10 -Aib 11 -I 12 -Aib 13 -L 14 -D 15 -K 16 -Q 17 -A 18 -Q 19 -A 20 -E21 -F 22 -V 23 -K 24 (AEEA-AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-W 25 -L 26 -L 27 -K 28 -G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P 37 -P 38 -S 39 -K 40 (SEQ ID NO:7),the structure is as follows:

[0043] Compound P017:H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 (AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-Gly 29 -Gly 30 - Pro 31 - Ser 32-Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2, i.e., Y 1 -Aib 2 -E 3 -G 4 -T 5 -αMePhe 6 -T 7 -S 8 -D 9 -Y 10 -Aib 11 -I 12 -Aib 13 -L 14 -D 15 -K 16 -Q 17 -A 18 -Q 19 -A 20 -E 21 -F 22 -V 23 -K 24 -W 25 -L 26 -L 27 -K 28 (AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P 37 -P 38 -S 39 -K 40 (SEQ ID NO:8), the structure is as follows:

[0044] Compound P018: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8-Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 (AEEA - AEEA - γGlu - γGlu - γGlu - CO - (CH2) 16 -COOH)-Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2, that is, Y 1 -Aib 2 -E 3 -G 4 -T 5 -αMePhe 6 -T 7 -S 8 -D 9 -Y 10 -Aib 11 -I 12 -Aib 13 -L 14 -D 15 -K 16 -Q 17 -A 18 -Q 19 -A 20 -E 21 -F 22 -V 23 -K 24 -W 25 -L 26-L 27 -K 28 (AEEA-AEEA-γGlu-γGlu-γGlu-CO-(CH2) 16 -COOH)-G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P 37 -P 38 -S 39 -K 40 (SEQ ID NO:9),the structure is as follows:

[0045] Compound P019: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 (AEEA-AEEA-AEEA-γGlu-CO-(CH2) 16 -COOH)-Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro37 -Pro 38- Ser 39 -Lys 40 -NH2, i.e., Y 1 -Aib 2 -E 3 -G 4 -T 5 -αMePhe 6 -T 7 -S 8 -D 9 -Y 10 -Aib 11 -I 12 -Aib 13 -L 14 -D 15 -K 16 -Q 17 -A 18 -Q 19 -A 20 -E 21 -F 22 -V 23 -K 24 -W 25 -L 26 -L 27 -K 28 (AEEA-AEEA-AEEA-γGlu-CO-(CH2) 16 -COOH)-G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P 37 -P 38 -S 39 -K 40 (SEQ ID NO: 10), the structure is as follows:

[0046] and

[0047] Compound P020: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 (AEEA - AEEA - AEEA - γGlu - CO - (CH2) 20 -COOH)-Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2, that is, Y 1 -Aib 2 -E 3 -G 4 -T 5 -αMePhe 6 -T 7 -S 8 -D 9 -Y 10 -Aib 11 -I 12 -Aib 13 -L 14 -D 15 -K 16 -Q 17 -A 18 -Q 19 -A 20 -E 21 -F 22 -V 23 -K 24 -W 25 -L 26 -L 27 -K 28(AEEA-AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P 37 -P 38 -S 39 -K 40 (SEQ ID NO: 11), the structure is as follows:

[0048] wherein AEEA represents [2-(2-amino-ethoxy)-ethoxy]-acetyl.

[0049] In a specific embodiment, the polypeptide compound is compound P016: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 (AEEA-AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34-Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2, i.e., Y 1 -Aib 2 -E 3 -G 4 -T 5 -αMePhe 6 -T 7 -S 8 -D 9 -Y 10 -Aib 11 -I 12 -Aib 13 -L 14 -D 15 -K 16 -Q 17 -A 18 -Q 19 -A 20 -E 21 -F 22 -V 23 -K 24 (AEEA-AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-W 25 -L 26 -L 27 -K 28 -G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P 37 -P 38 -S 39 -K 40 (SEQ ID NO: 7), molecular formula C 243 H 382 N 52 O 73 , the structure is as follows:

[0050] The main chain of compound P016 contains 40 amino acid residues, and the side chain ε-amino group of lysine at position 24 is connected to the following five parts in sequence: AEEA, AEEA, AEEA, γ-Glu and -CO-(CH2) 20 -COOH.

[0051] [Application of dipeptide fragments in solid-phase synthesis of polypeptide compounds]

[0052] According to one aspect of the present invention, there is provided a use of a dipeptide fragment in solid phase synthesis of a polypeptide compound, wherein the dipeptide fragment is selected from the following structures: Gly-Gly, Ile-Aib, Tyr-Aib, Thr-αMePhe and Glu-Gly, and the polypeptide compound is a compound of formula AI and has the following structure: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2 formula AI (SEQ ID NO: 12),

[0053] Optionally, at any one lysine selected from positions 16, 24, 28 and 40, the side chain ε-amino group is chemically modified via ([2-(2-amino-ethoxy)-ethoxy]-acetyl)a-(γ-Glu)b-CO-(CH2)c-COOH, wherein each a is independently an integer from 0 to 5, each b is independently an integer from 0 to 5, and each c is independently an integer from 10 to 24.

[0054] Unless clearly contradictory or explicitly stated otherwise, the following description of the solid-phase synthesis method of dipeptide fragments is applicable to the use of dipeptide fragments in the solid-phase synthesis of polypeptide compounds.

[0055] In some embodiments, each a is independently an integer from 1 to 5, each b is independently an integer from 1 to 5, and each c is independently an integer from 12 to 22.

[0056] [Dipeptide fragment solid phase synthesis method]

[0057] According to one aspect of the present invention, a method for solid phase synthesis of a dipeptide fragment of a polypeptide compound is provided, wherein the polypeptide compound is a compound of formula AI and has the following structure: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33-Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2 formula AI (SEQ ID NO: 12),

[0058] wherein optionally, at any one lysine selected from positions 16, 24, 28 and 40, the side chain ε-amino group is chemically modified via ([2-(2-amino-ethoxy)-ethoxy]-acetyl)a-(γ-Glu)b-CO-(CH2)c-COOH, wherein each a is independently an integer from 0 to 5, each b is independently an integer from 0 to 5, and each c is independently an integer from 10 to 24,

[0059] The dipeptide fragment is selected from the following structures: Gly-Gly, Ile-Aib, Tyr-Aib, Thr-αMePhe, and Glu-Gly.

[0060] In the context of the present invention, "dipeptide fragment solid phase synthesis," "fragment solid phase synthesis," and "segmented coupling" are used interchangeably to refer to the stepwise condensation coupling of dipeptide fragments and individual amino acids using solid phase synthesis. "Condensation" and "coupling" are used interchangeably to refer to the dehydration condensation coupling of the amino group of the amino acid being linked with the carboxyl group of the amino acid to be linked, forming an amide bond or a peptide bond.

[0061] In some embodiments, each a is independently an integer from 1 to 5, each b is independently an integer from 1 to 5, and each c is independently an integer from 12 to 22.

[0062] In some embodiments, the method for solid phase synthesis of a polypeptide compound using a dipeptide fragment comprises:

[0063] The peptide chain of the formula AI polypeptide compound is prepared by a dipeptide fragment solid phase synthesis method, and

[0064] Optionally, the side chain ε-amino group is chemically modified at any one lysine selected from positions 16, 24, 28 and 40 via ([2-(2-amino-ethoxy)-ethoxy]-acetyl)a-(γ-Glu)b-CO-(CH2)c-COOH, wherein each a is independently an integer from 0 to 5, each b is independently an integer from 0 to 5, and each c is independently an integer from 10 to 24.

[0065] In some embodiments, the method for solid phase synthesis of a polypeptide compound using a dipeptide fragment comprises:

[0066] The peptide chain of the formula AI polypeptide compound was prepared by a dipeptide fragment solid phase synthesis method without chemical modification of the ε-amino group of the lysine side chain, thereby obtaining the polypeptide compound P001.

[0067] In some embodiments, the method for solid phase synthesis of a polypeptide compound using a dipeptide fragment comprises:

[0068] The peptide chain of the formula AI polypeptide compound is prepared by a dipeptide fragment solid phase synthesis method, and

[0069] Then, at any one lysine selected from positions 16, 24, 28 and 40, the side chain ε-amino group is chemically modified via ([2-(2-amino-ethoxy)-ethoxy]-acetyl)a-(γ-Glu)b-CO-(CH2)c-COOH, wherein each a is independently an integer from 0 to 5, each b is independently an integer from 0 to 5, and each c is independently an integer from 10 to 24.

[0070] According to one aspect of the present invention, a method for solid-phase synthesis of a dipeptide fragment of a polypeptide compound is provided, wherein the polypeptide compound of formula AI is a compound selected from the following:

[0071] Compound P001: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2(SEQ ID NO:1)

[0072] Compound P007: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 (AEEA-AEEA-γGlu-CO-(CH2) 16 -COOH)-Trp[[ID=七十二]] 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- [[ID=9九百九十九]]Ser 39 -Lys 40 -NH2, that is, Y 1 -Aib 2 -E 3 -G4 -T 5 -αMePhe 6 -T 7 -S 8 -D 9 -Y 10 -Aib 11 -I 12 -Aib 13 -L 14 -D 15 -K 16 -Q 17 -A 18 -Q 19 -A 20 -E 21 -F 22 -V 23 -K 24 (AEEA-AEEA-γGlu-CO-(CH2) 16 -COOH)-W 25 -L 26 -L 27 -K 28 -G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P 37 -P 38 -S 39 -K 40 (SEQ ID NO: 2), the structure is as follows:

[0073] Compound P008: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala20 -Glu 21 -Phe 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 (AEEA - AEEA - γGlu - CO - (CH2) 16 -COOH)-Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2, that is, Y 1 -Aib 2 -E 3 -G 4 -T 5 -αMePhe 6 -T 7 -S 8 -D 9 -Y 10 -Aib 11 -I 12 -Aib 13 -L 14 -D 15 -K 16 -Q 17 -A 18 -Q 19 -A 20 -E 21 -F 22 -V 23 -K 24 -W 25 -L 26 -L 27 -K 28 (AEEA - AEEA - γGlu - CO - (CH2) 16 -COOH)-G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P37 -P 38 -S 39 -K 40 (SEQ ID NO: 3), the structure is as follows:

[0074] Compound P013: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 (AEEA-AEEA-γGlu-CO-(CH2) 18 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2, i.e., Y 1 -Aib 2 -E 3 -G 4 -T 5 -αMePhe 6 -T 7 -S 8 -D 9 -Y10 -Aib 11 -I 12 -Aib 13 -L 14 -D 15 -K 16 -Q 17 -A 18 -Q 19 -A 20 -E 21 -F 22 -V 23 -K 24 (AEEA-AEEA-γGlu-CO-(CH2) 18 -COOH)-W 25 -L 26 -L 27 -K 28 -G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P 37 -P 38 -S 39 -K 40 (SEQ ID NO: 4), the structure is as follows:

[0075] Compound P014: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24(AEEA - AEEA - γGlu - CO - (CH2) 20 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2, that is, Y 1 -Aib 2 -E 3 -G 4 -T 5 -αMePhe 6 -T 7 -S 8 -D 9 -Y 10 -Aib 11 -I 12 -Aib 13 -L 14 -D 15 -K 16 -Q[[ID=6​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​

[0076] Compound P015: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 (AEEA-AEEA-γGlu-γGlu-γGlu-CO-(CH2) 16 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2, that is, Y 1 -Aib 2 -E 3 -G 4 -T 5 -αMePhe 6 -T 7 -S 8 -D 9 -Y 10 -Aib<了 11 -I 12 -Aib 13 -L It should be noted that there seems to be a misspelling in the original text where "了0001687" appears. It is assumed to be a typo and has been translated as "0001687" in the above translation. If this is not the case, the translation needs to be adjusted according to the correct content.14 -D 15 -K 16 -Q 17 -A 18 -Q 19 -A 20 -HAVE BEEN 21 -F 22 -V 23 -K 24 (AEEA-AEEA-γGlu-γGlu-γGlu-CO-(CH2) 16 -COOH)-W 25 -L 26 -L 27 -K 28 -G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P 37 -P 38 -S 39 -K 40 (SEQ ID NO:6),the structure is as follows:

[0077] Compound P016: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 (AEEA-AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-Trp 25-Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2, that is, Y 1 -Aib 2 -E 3 -G 4 -T 5 -αMePhe 6 -T 7 -S 8 -D 9 -Y 10 -Aib 11 -I 12 -Aib 13 -L 14 -D 15 -K 16 -Q 17 -A 18 -Q 19 -A 20 -E 21 -F 22 -V 23 -K 24 (AEEA - AEEA - AEEA - γGlu - CO - (CH2) 20 -COOH)-W 25 -L 26 -L 27 -K 28 -G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P 37 -P 38 -S 39 -K 40 (SEQ ID NO:7), the structure is as follows:

[0078] Compound P017: H - Tyr1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 (AEEA - AEEA - γGlu - CO - (CH2) 20 -COOH)-Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2, that is, Y 1 -Aib 2 -E 3 -G 4 -T 5 -αMePhe 6 -T 7 -S 8 -D 9 -Y 10 -Aib 11 -I 12 -Aib 13 -L 14 -D 15 -K 16 -Q 17 -A18 -Q 19 -A 20 -HAVE BEEN 21 -F 22 -V 23 -K 24 -W 25 -L 26 -L 27 -K 28 (AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P 37 -P 38 -S 39 -K 40 (SEQ ID NO:8),the structure is as follows:

[0079] Compound P018:H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 (AEEA-AEEA-γGlu-γGlu-γGlu-CO-(CH2) 16 -COOH)-Gly 29-Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2, i.e., Y 1 -Aib 2 -E 3 -G 4 -T 5 -αMePhe 6 -T 7 -S 8 -D 9 -Y 10 -Aib 11 -I 12 -Aib 13 -L 14 -D 15 -K 16 -Q 17 -A 18 -Q 19 -A 20 -E 21 -F 22 -V 23 -K 24 -W 25 -L 26 -L 27 -K 28 (AEEA-AEEA-γGlu-γGlu-γGlu-CO-(CH2) 16 -COOH)-G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P 37 -P 38 -S 39 -K 40 (SEQ ID NO:9), the structure is as follows:

[0080] Compound P019: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 (AEEA - AEEA - AEEA - γGlu - CO - (CH2) 16 -COOH)-Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2, that is, Y 1 -Aib 2 -E 3 -G 4 -T 5 -αMePhe 6 -T 7 -S 8 -D 9 -Y 10 -Aib 11 -I 12 -Aib 13 -L 14 -D 15 -K 16 -Q 17 -A 18 -Q 19 -A 20 -E 21 -F 22-V 23 -K 24 -W 25 -L 26 -L 27 -K 28 (AEEA - AEEA - AEEA - γGlu - CO - (CH2) 16 -COOH)-G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P 37 -P 38 -S 39 -K 40 (SEQ ID NO:10), the structure is as follows:

[0081] and

[0082] Compound P020: H - Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 (AEEA - AEEA - AEEA - γGlu - CO - (CH2) 20 -COOH)-Gly 29 -Gly 30 -Pro 31 -Ser32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2, i.e., Y 1 -Aib 2 -E 3 -G 4 -T 5 -αMePhe 6 -T 7 -S 8 -D 9 -Y 10 -Aib 11 -I 12 -Aib 13 -L 14 -D 15 -K 16 -Q 17 -A 18 -Q 19 -A 20 -E 21 -F 22 -V 23 -K 24 -W 25 -L 26 -L 27 -K 28 (AEEA-AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P 37 -P 38 -S 39 -K 40 (SEQ ID NO: 11), the structure is as follows:

[0083] wherein AEEA represents [2-(2-amino-ethoxy)-ethoxy]-acetyl.

[0084] According to one aspect of the present invention, a method for solid phase synthesis of a dipeptide fragment of a polypeptide compound is provided, wherein the polypeptide compound is compound P016: H-Tyr 1-Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 (AEEA - AEEA - AEEA - γGlu - CO - (CH2) 20 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​-Q 19 -A 20 -E 21 -F 22 -V 23 -K 24 (AEEA-AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-W 25 -L 26 -L 27 -K 28 -G 29 -G 30 -P 31 -S 32 -S 33 -G 34 -A 35 -P 36 -P 37 -P 38 -S 39 -K 40 (SEQ ID NO: 7), molecular formula C 243 H 382 N 52 O 73 , the structure is as follows:

[0085] The dipeptide fragment is selected from the following structures: Gly-Gly, Ile-Aib, Tyr-Aib, Thr-αMePhe and Glu-Gly.

[0086] In some embodiments, the method for solid phase synthesis of a polypeptide compound using a dipeptide fragment comprises:

[0087] The peptide chain of the formula AI polypeptide compound is prepared by a dipeptide fragment solid phase synthesis method, and

[0088] Then, at lysine 24, the acetyl group was synthesized via ([2-(2-amino-ethoxy)-ethoxy]-acetyl)3-(γ-Glu)1-CO-(CH2) 20 -COOH was chemically modified with the side chain ε-amino group to obtain polypeptide compound P016.

[0089] [Synthesis of peptide chains of polypeptide compounds]

[0090] Solid-phase synthesis methods commonly used to synthesize polypeptide compounds can be used to prepare the polypeptide compounds of Formula AI of the present invention. In the polypeptide solid-phase synthesis method, single amino acids are used as building blocks to construct polypeptide compounds. The C-terminal amino acid of the peptide chain of the polypeptide compound to be connected is pre-amino-protected. The carboxyl group of the amino-protected C amino acid is connected to an insoluble resin support via an appropriate linker. The amino-protecting group is then removed and the peptide is condensed and coupled with an excess of the second amino acid from the C-terminus that has its carboxyl group activated and amino-protected to form a peptide bond (i.e., an amide bond). The condensation coupling of amino acids is repeated in this manner until the desired length of the peptide chain of the polypeptide compound to be synthesized is achieved. An appropriate reagent is then selected to cleave the product from the support resin. The product is then separated and purified by filtration and washing to obtain the peptide chain of the polypeptide compound of Formula AI.

[0091] [Protective Group]

[0092] To achieve controlled synthesis of peptide compounds, it is necessary to protect functional groups on amino acids that are not expected to undergo condensation coupling, such as the free amino group (-NH2) of the amino acid to be linked and polar groups on the side chain (e.g., amino-NH2, carboxyl-COOH, hydroxyl-OH). Free amino groups refer to nitrogen atoms that are not forming amide bonds, including primary amino groups (-NH2) and secondary amino groups (-NH-).

[0093] Protective groups commonly used in solid-phase synthesis of polypeptides can be used in the present invention, as long as they do not affect the stepwise coupling solid-phase synthesis method of the present invention.

[0094] Amino-protecting groups useful in the present invention include hydrocarbonoxycarbonyl groups, such as alkoxycarbonyl groups (e.g., tert-butyloxycarbonyl (Boc)), alkenyloxycarbonyl groups (e.g., allyloxycarbonyl (Alloc)), aralkyloxycarbonyl groups (e.g., benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc)), silyloxycarbonyl groups (e.g., trimethoxysilylethoxycarbonyl (Teoc)); acyl groups, including carbonyl groups (e.g., phthalimido (Pht), 5,5-dimethyl-cyclohexane-1,3-dione-2-ethylidene (Dde)), sulfonyl groups (e.g., p-toluenesulfonyl (Tos)), haloalkylacyl groups (e.g., trifluoroacetyl (Tfa)); and alkyl groups, including aralkyl groups, such as trityl (Trt), 2,4-dimethoxybenzyl (Dmb), p-methoxybenzyl (Pmb), and benzyl (Bn).

[0095] Hydroxyl protecting groups useful in the present invention include t-butyl tBu.

[0096] Carboxyl protecting groups useful in the present invention include tert-butyloxy OtBu.

[0097] Amide protecting groups useful in the present invention include trityl Trt.

[0098] In some embodiments, lysine Lys uses Boc, Mtt, Alloc, or Dde as a side chain ε-amino protecting group, tryptophan Trp uses Boc as a side chain amino protecting group, serine Ser uses tBu as a side chain hydroxyl protecting group, tyrosine Tyr uses tBu as a side chain hydroxyl protecting group, threonine Thr uses tBu as a side chain hydroxyl protecting group, aspartic acid Asp uses tert-butyloxy OtBu as a side chain carboxyl protecting group, glutamic acid Glu uses tert-butyloxy OtBu as a side chain carboxyl protecting group, and / or glutamine Gln uses Trt as a side chain amide protecting group. Glu(γ-OtBu) indicates that a tert-butyloxy group is attached to the γ-carboxyl group of glutamic acid and is used in the peptide chain synthesis of the polypeptide compound of formula AI; Glu(α-OtBu) indicates that a tert-butyloxy group is attached to the α-carboxyl group of glutamic acid and is used to modify the side chain ε-amino group of lysine.

[0099] In some embodiments, for the lysine at positions 16, 24, 28, and 40, the lysine to be chemically modified with the side chain ε-amino group uses Mtt, Alloc, or Dde as the side chain ε-amino protecting group, and the lysine not to be chemically modified with the side chain ε-amino group uses Boc as the side chain ε-amino protecting group.

[0100] In some embodiments, for the lysine at positions 16, 24, 28, and 40, the lysine to be chemically modified with the side chain ε-amino group uses Mtt as the side chain ε-amino protecting group, and the lysine not to be chemically modified with the side chain ε-amino group uses Boc as the side chain ε-amino protecting group.

[0101] In some embodiments, Mtt is used as the side chain ε-amino protecting group for lysine at position 24, and Boc is used as the side chain ε-amino protecting group for lysine at positions 16, 28, and 40.

[0102] Table 1 Protective groups that can be used in the present invention

[0103] Note: The dot “·” in the protecting group structural formula represents the connection site between the protecting group and the protected group of the amino acid.

[0104] The Boc solid phase synthesis method in which the α-amino group is protected with a tert-butyloxycarbonyl (Boc) group, the Fmoc solid phase synthesis method in which the α-amino group is protected with a 9-fluorenylmethyloxycarbonyl (Fmoc) group, and a combination of the Boc solid phase synthesis method and the Fmoc solid phase synthesis method can all be used in the present invention.

[0105] Taking the Fmoc solid-phase synthesis method as an example, the peptide chain of the formula AI polypeptide compound can be prepared as shown in the reaction scheme below.

[0106] As shown above, according to the Fmoc solid-phase synthesis method, a C-terminal amino acid with an Fmoc-protected α-amino group is coupled to a resin support via a linker, and the Fmoc protecting group of the C-terminal amino acid attached to the resin is removed. Starting from the rightmost C-terminus of the peptide chain of the polypeptide compound and moving toward the leftmost N-terminus, single amino acids and dipeptide fragments are sequentially connected, wherein the carboxyl group of the amino acid to be connected is condensed with the amino group of the amino acid to be connected, wherein the amino acid to be connected has a carboxyl group to be coupled and an Fmoc-protected amino group.

[0107] In some embodiments, when preparing the peptide chain of the polypeptide compound of formula AI, the C-terminal amino acid (i.e., lysine K at position 40) protected by Fmoc is 40 ) is coupled with the resin support through a linker to obtain Fmoc-K 40 (ε amino protecting group)-linker-resin. Then, K 40 The α-amino group of K was removed by Fmoc protection to obtain 40 (ε amino protecting group)-linker-resin. The second amino acid from the C-terminus (i.e., the amino acid to be connected, Serine S at position 39) with α-amino protected by Fmoc was added. 39 ) carboxyl group and K 40 (εamino protecting group)-linker-resin K 40 (i.e., the amino group of the amino acid to be connected) is coupled to obtain Fmoc-S 39 (Hydroxy Protecting Group)-K 40 (ε amino protecting group)-linker-resin. In this way, starting from the rightmost C-terminus of the peptide chain of the polypeptide compound and moving toward the leftmost N-terminus, i.e., along the direction from amino acid position 40 to amino acid position 1 (C-40→N-01), single amino acids and dipeptide fragments are sequentially condensed to obtain the peptide chain of the polypeptide compound of formula AI.

[0108] Formula AI (SEQ ID NO: 1).

[0109] In the Boc solid phase synthesis method, the reaction route is similar, and the main difference is that the Fmoc protecting group used in the Fmoc solid phase synthesis method is replaced by a Boc protecting group.

[0110] [Dipeptide fragment]

[0111] The polypeptide compound of Formula AI comprises 40 amino acid residues. In some embodiments, during the step of preparing the peptide chain of the polypeptide compound of Formula AI, the dipeptide fragment is selected from any of the following structures: Gly-Gly, Ile-Aib, Tyr-Aib, Thr-αMePhe, and Glu-Gly. Each dipeptide fragment may occur one or more times.

[0112] In some embodiments, the dipeptide fragment is a combination of Gly-Gly, Ile-Aib and Tyr-Aib. Each dipeptide fragment can appear once or multiple times.

[0113] In some embodiments, the dipeptide fragment is a combination of Gly-Gly, Ile-Aib, Tyr-Aib, Thr-αMePhe and Glu-Gly. Each dipeptide fragment can appear once or multiple times.

[0114] In some embodiments, according to the dipeptide fragment solid-phase synthesis method of the present invention, the polypeptide compound of Formula AI can be divided into six dipeptide fragments and then coupled with 28 single amino acid fragments for a 6+28-fragment coupling. Specifically, amino acids 1-14 are divided into nine fragments, and amino acids 15-40 are divided into 25 fragments. Each dipeptide fragment is sequentially linked to the single amino acid fragment from the C-terminus to the N-terminus. Compared to the 4+32-fragment coupling described below, the 6+28-fragment coupling can achieve superior final yield and crude product purity.

[0115] In some embodiments, the amino acids 1-14 of the formula AI polypeptide compound can be divided into 9 fragments for coupling, specifically the dipeptide fragments Tyr-Aib, Glu-Gly, Thr-αMePhe, Tyr-Aib and Ile-Aib, and 4 single amino acid fragments other than these 5 dipeptide fragments; and the amino acids 15-40 can be divided into 25 fragments for coupling, specifically the dipeptide fragment Gly-Gly, and 24 single amino acids other than this 1 dipeptide fragment, and each dipeptide fragment is coupled one by one with each single amino acid fragment in the order from C-terminus to N-terminus.

[0116] In some embodiments, according to the dipeptide fragment solid-phase synthesis method of the present invention, the polypeptide compound of Formula AI can be divided into four dipeptide fragments and then coupled with 32 single amino acid fragments for 4+32 fragment coupling. Specifically, amino acids 1-14 are divided into 11 fragments, and amino acids 15-40 are divided into 25 fragments. Each dipeptide fragment is sequentially linked to the single amino acid fragment from the C-terminus to the N-terminus.

[0117] In some embodiments, the amino acids 1-14 of the formula AI polypeptide compound can be divided into 11 fragments for coupling, specifically the dipeptide fragments Tyr-Aib, Tyr-Aib, Ile-Aib, and 8 single amino acid fragments other than these 3 dipeptide fragments; and the amino acids 15-40 can be divided into 25 fragments for coupling, specifically the dipeptide fragment Gly-Gly, and 24 single amino acids other than this 1 dipeptide fragment, and each dipeptide fragment is coupled one by one with the single amino acid fragment in the order from C-terminus to N-terminus.

[0118] In some embodiments, in the step of preparing the peptide chain of the polypeptide compound of formula AI, the peptide chain is divided into the following 6 fragments: Tyr 1 -Aib 2 Glu 3 -Gly 4 、Thr 5 -αMePhe 6 、Tyr 10 -Aib 11 、Ile 12 -Aib 13 、Gly 29 -Gly 30 , and the following 28 sites except these 6 fragments are coupled with single amino acids: Thr 7 、Ser 8 、Asp 9 、Leu 14 、Asp 15 、Lys 16 、Gln 17 、Ala 18 、Gln 19 、Ala 20 Glu 21 、Phe 22 、Val 23 、Lys 24 Trp 25 、Leu 26 、Leu 27 、Lys 28 、Pro 31 、Ser 32 、Ser 33 、Gly 34 、Ala 35 、Pro 36 、Pro 37 、Pro 38 、Ser 39 and Lys 40 .

[0119] In some embodiments, in the step of preparing the peptide chain of the polypeptide compound of formula AI, the peptide chain is divided into the following four fragments: Tyr 1 -Aib 2 、Tyr 10 -Aib 11 、Ile 12 -Aib 13 、Gly 29 -Gly 30 , and coupled with single amino acids at the following 32 sites except these 4 fragments: Glu 3 、Gly 4 、Thr 5 、αMePhe 6 、Thr 7 、Ser 8 、Asp 9 、Leu 14 、Asp 15 、Lys 16 、Gln 17 、Ala 18 、Gln 19 、Ala 20 Glu 21 、Phe 22 、Val 23 、Lys 24 Trp 25 、Leu 26 、Leu 27 、Lys 28 、Pro 31 、Ser 32 、Ser 33 、Gly 34 、Ala 35 、Pro 36 、Pro 37 、Pro 38 、Ser 39 and Lys 40 .

[0120] In some embodiments, the free amino group of the dipeptide fragment, in particular the free α-amino group, has an Fmoc protecting group, or has a Boc protecting group, or has both an Fmoc protecting group and a Boc protecting group. The free α-amino group of the dipeptide fragment refers to the α-position nitrogen atom of the dipeptide fragment that does not form an amide bond, including primary amino groups -NH2 and secondary amino groups -NH-.

[0121] In some embodiments, the dipeptide fragment having an Fmoc protecting group is selected from the group consisting of: Fmoc-Gly-Gly-OH, Fmoc-Ile-Aib-OH, Fmoc-Tyr(tBu)-Aib-OH, Fmoc-Thr(tBu)-αMePhe-OH and Fmoc-Glu(OtBu)-Gly-OH.

[0122] In some embodiments, the dipeptide fragment having a Boc protecting group is selected from the group consisting of: Boc-Gly-Gly-OH, Boc-Ile-Aib-OH, Boc-Tyr(tBu)-Aib-OH, Boc-Thr(tBu)-αMePhe-OH and Boc-Glu(OtBu)-Gly-OH.

[0123] In some embodiments, the dipeptide fragment having both an Fmoc protecting group and a Boc protecting group is selected from the group consisting of: Fmoc-Gly-Gly-OH, Fmoc-Ile-Aib-OH, Fmoc-Tyr(tBu)-Aib-OH, Fmoc-Thr(tBu)-αMePhe-OH, Fmoc-Glu(OtBu)-Gly-OH and Boc-Tyr(tBu)-Aib-OH.

[0124] In some embodiments, the dipeptide fragment having both an Fmoc protecting group and a Boc protecting group is a combination of Fmoc-Gly-Gly-OH, Fmoc-Ile-Aib-OH, Fmoc-Tyr(tBu)-Aib-OH, Fmoc-Thr(tBu)-αMePhe-OH, Fmoc-Glu(OtBu)-Gly-OH and Boc-Tyr(tBu)-Aib-OH.

[0125] A dipeptide fragment having both an Fmoc protecting group and a Boc protecting group means that in the same embodiment, a dipeptide fragment having an Fmoc protecting group and another dipeptide fragment having a Boc protecting group coexist, rather than the same dipeptide fragment containing both an Fmoc protecting group and a Boc protecting group.

[0126] In some embodiments, in the step of preparing the peptide chain of the polypeptide compound of formula AI, the dipeptide fragment having both Fmoc protecting group and Boc protecting group is Boc-Tyr 1 (tBu)-Aib 2 -OH, Fmoc-Glu 3 (OtBu)-Gly 4 -OH, Fmoc-Thr 5 (tBu)-αMePhe 6 -OH, Fmoc-Gly29 -Gly 30 -OH, Fmoc-Tyr 10 (tBu)-Aib 11 -OH and Fmoc-Ile 12 -Aib 13 -OH. The last amino acid to be added is Tyr 1 The Boc protecting group is connected to the amino group -NH2 of the peptide, which increases the stability of the peptide chain of the polypeptide compound of formula AI compared to the case of connecting Fmoc.

[0127] According to the present invention, when preparing the peptide chain of the formula AI polypeptide compound, starting from the rightmost C-terminus of the peptide chain and toward the leftmost N-terminus, that is, along the direction from the 40th amino acid to the 1st amino acid (C-40→N-01), the following dipeptide fragments and single amino acids are sequentially condensed (6+28 fragment coupling): Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH.H2O, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc -Lys(Mtt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Glu(γ-OtBu)-OH, Fmoc-Ala-OH.H2O, Fmoc-Gln (Trt)-OH, Fmoc-Ala-OH.H2O, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc- Leu-OH, Fmoc-Ile-Aib-OH, Fmoc-Tyr(tBu)-Aib-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmo c-Thr(tBu)-OH, Fmoc-Thr(tBu)-αMePhe-OH, Fmoc-Glu(γ-OtBu)-Gly-OH, Boc-Tyr(tBu)-Aib-OH.

[0128] According to the present invention, when preparing the peptide chain of the formula AI polypeptide compound, starting from the rightmost C-terminus of the peptide chain and moving toward the leftmost N-terminus, that is, along the direction from the 40th amino acid to the 1st amino acid (C-40→N-01), the following dipeptide fragments and single amino acids are sequentially condensed (4+32 fragment coupling): Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc -Pro-OH, Fmoc-Ala-OH.H2O, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fm oc-Gly-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Mtt )-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Glu(γ-OtBu)-OH, Fmoc-Ala-OH.H2O, Fmoc-Gln(Trt)-OH, F moc-Ala-OH.H2O, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc- Ile-Aib-OH, Fmoc-Tyr(tBu)-Aib-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-αMePhe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(γ-OtBu)-OH, Boc-Tyr(tBu)-Aib-OH.

[0129] [Lysine side chain ε-amino modification]

[0130] In some embodiments, in addition to preparing the peptide chain of the polypeptide compound of Formula AI by dipeptide fragment solid phase synthesis, the side chain ε-amino group of lysine is also modified.

[0131] In some embodiments, after the peptide chain of the polypeptide compound of Formula AI is prepared by a dipeptide fragment solid phase synthesis method, the side chain ε-amino group of lysine is further modified.

[0132] In some embodiments, the side chain ε-amino modification of lysine comprises chemical modification of the side chain ε-amino group at any one lysine selected from positions 16, 24, 28 and 40 via ([2-(2-amino-ethoxy)-ethoxy]-acetyl)a-(γ-Glu)b-CO-(CH2)c-COOH, wherein each a is independently an integer from 0 to 5, each b is independently an integer from 0 to 5, and each c is independently an integer from 10 to 24.

[0133] In some embodiments, the modification of the ε-amino group of the lysine side chain comprises:

[0134] For lysine using Mtt, Alloc, or Dde as a side chain ε-amino protecting group, deprotection is performed, and a 2-(2-amino-ethoxy)-ethoxy]-acetyl group, b γ-Glu, and 1 -CO-(CH2)c-COOH are sequentially connected to the deprotected lysine ε-amino group. Preferably, Mtt is used as the protecting group for the ε-amino group of the lysine side chain because, compared with the case where Alloc or Dde is used as the lysine side chain ε-amino protecting group, Mtt as the protecting group for the lysine side chain ε-amino group can increase the final yield of the polypeptide compound of formula AI while maintaining a crude product purity comparable to that when Alloc or Dde is used.

[0135] In some embodiments, 2-(2-amino-ethoxy)-ethoxy]-acetyl is linked by using Fmoc-AEEA-OH, γ-Glu is linked by using Fmoc-Glu(α-OtBu)-OH, and -OC-(CH2)c-COOtBu is linked by using HOOC-(CH2)c-COOtBu, which is converted to -OC-(CH2)c-COOtBu by post-treatment.

[0136] In some embodiments, the method for solid phase synthesis of a polypeptide compound using a dipeptide fragment comprises:

[0137] The peptide chain of the formula AI polypeptide compound is prepared by a dipeptide fragment solid phase synthesis method,

[0138] For lysine using Mtt, Alloc or Dde as the side chain ε-amino protecting group, deprotection is performed, and

[0139] At the side chain ε-amino group of the deprotected lysine, 2-(2-amino-ethoxy)-ethoxy]-acetyl is attached by using Fmoc-AEEA-OH, γ-Glu is attached by using Fmoc-Glu(α-OtBu)-OH, and -OC-(CH2)c-COOtBu is attached by using HOOC-(CH2)c-COOtBu; and

[0140] After post-treatment, a polypeptide compound of formula AI is obtained.

[0141] In some embodiments, the method for solid phase synthesis of a polypeptide compound using a dipeptide fragment comprises:

[0142] The peptide chain of the polypeptide compound of formula AI is prepared by a dipeptide fragment solid phase synthesis method, wherein the 24th lysine has Mtt as a side chain ε-amino protecting group, and the 16th, 28th and 40th lysines have Boc as side chain ε-amino protecting groups;

[0143] Deprotecting the side chain ε-amino group of lysine at position 24; and

[0144] At the deprotected lysine 24, the ligand was synthesized via ([2-(2-amino-ethoxy)-ethoxy]-acetyl)3-(γ-Glu)1-CO-(CH2) 20 -COOH was chemically modified with the side chain ε-amino group to obtain polypeptide compound P016.

[0145] In some embodiments, the method for solid phase synthesis of a polypeptide compound using a dipeptide fragment comprises:

[0146] The peptide chain of the polypeptide compound of formula AI is prepared by a dipeptide fragment solid phase synthesis method, wherein the 24th lysine has Mtt as a side chain ε-amino protecting group, and the 16th, 28th and 40th lysines have Boc as side chain ε-amino protecting groups;

[0147] Deprotect the side chain ε-amino group of lysine at position 24;

[0148] At the deprotected lysine 24, 2-(2-amino-ethoxy)-ethoxy]-acetyl was attached by using Fmoc-AEEA-OH, γ-Glu was attached by using Fmoc-Glu(α-OtBu)-OH, and HOOC-(CH2) 20 -COOtBu is linked to -OC-(CH2)c-COO-tBu, thereby via ([2-(2-amino-ethoxy)-ethoxy]-acetyl)3-(γ-Glu)1-CO-(CH2) 20 -COO-tBu to chemically modify the side chain ε-amino group of lysine 24; and

[0149] After post-treatment, polypeptide compound P016 was obtained.

[0150] [Reaction conditions for solid phase synthesis]

[0151] Before starting to prepare the peptide chain, the C-terminal amino acid is first linked to a resin as a support via its carbonyl group. The resin that can be used in the present invention is not particularly limited, as long as it has the ability to bind to reactive carbonyl groups and is insensitive to the reagents and reaction conditions of the stepwise condensation coupling and deprotection reactions. Examples of such resins include amino-type resins, such as MBHA resin and Sieber resin; halomethyl resins, such as chloromethyl resins, especially chloromethyl-polystyrene-divinylbenzene polymers, and bromomethyl resins; hydroxymethyl resins; phenol resins; tert-butoxy-carbonyl hydrazide resins; and cross-linked poly-N-acryloyl-pyridine resins.

[0152] In some embodiments, the resin is an amino-type resin, such as MBHA resin and Sieber Resin.

[0153] In some embodiments, the resin has a low substitution value, preferably a substitution value of less than about 5 mmol / g, more preferably a substitution value of less than about 2 mmol / g, even more preferably a substitution value of less than about 1 mmol / g, still more preferably a substitution value of less than about 0.6 mmol / g, or more preferably a substitution value of about 0.3 to about 0.65 mmol / g.

[0154] In some embodiments, swelling of the resin is performed prior to attachment of the C-terminal amino acid to the resin support.

[0155] The swollen resin has a larger network space, allowing the reagent molecules to enter the resin to react with the functional groups and accommodate the growing peptide chain. Optional swelling agents include the following organic solvents: dichloromethane (DCM), LDCM, N-methylpyrrolidone (NMP), 2-isopropylethylamine (DIEA), methyl tert-butyl ether (MTBE), trifluoroacetic acid (TFA), or any combination thereof, such as DIEA / DCM.

[0156] In some embodiments, optional coupling agents (i.e., condensing agents) include: N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azobenzotriazole (HOAt), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 6-chloro-1-hydroxybenzotriazole (Cl-HOBt), or any combination thereof.

[0157] In some embodiments, the coupling agent is optionally used in combination with a suitable solvent such as N,N-dimethylformamide (DMF), for example, in the form of a combination of DIC / HOBt, HOBt / DMF, DIC / HOAt, HBTU / Cl-HOBt, DMF / DIC / HOBt, DMF / DCM / DIC / HOBt.

[0158] In some embodiments, the condensation temperature is 5±5°C to 28±5°C, preferably 10±5°C to 25±5, for example 0-33°C, including both endpoints and any value and subrange within the range of 0-33°C, for example 0°C, 5°C, 10°C, 15°C, 20°C, 23°C, 30°C or 33°C.

[0159] In some embodiments, the condensing agent is used in an amount of about 2.0 to about 4.0 molar equivalents relative to the amount of each amino acid or dipeptide to be linked.

[0160] In some embodiments, the deprotecting agent comprises piperidine (PIP), hexafluoroisopropanol (HFIP), or a combination thereof.

[0161] In some of the described protocols, the deprotecting agent is optionally used in combination with a suitable solvent (eg, DMF or DCM), for example, in the form of a combination of PIP / DMF or HFIP / DCM.

[0162] In some embodiments, after modification of the ε-amino group of the side chain of lysine, post-treatment is further performed, including resin cleavage, product purification and lyophilization, to obtain a pure polypeptide compound.

[0163] In some embodiments, the cracking agent includes trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT), triisopropylsilane (TIS), DODT, phenol (ArOH), MPR, ArSCH3, or any combination thereof; preferably, a cracking system based on TFA, wherein the cracking system based on TFA means that the volume ratio of TFA is about 80-about 95 vol%, and the volume ratio of EDT, TIS, DODT, ArOH, MPR and / or ArSCH3 is about 1-about 10 vol%, with H2O making up the balance. For example,

[0164] TFA:H2O:EDT:TIS=approximately 94.0:2.0:2.0:2.0(V:V:V:V)

[0165] TFA:H2O:DODT:TIS=approximately 94.0:2.0:2.0:2.0(V:V:V:V)

[0166] TFA:H2O:EDT:TIS:ArOH=about 92.0:2.0:2.0:2.0:2.0(V:V:V:V:V)

[0167] TFA:H2O:TIS:EDT:ArOH:ArSCH3=approximately 81.5:5:1:2.5:5:5(V:V:V:V:V:V)

[0168] TFA:TIS:H2O:MPR=approximately 94:2.5:2.5:1(V:V:V:V)

[0169] TFA:H2O:EDT:TIS:ArOH=about 86.5:5:2.5:1:5(V:V:V:V:V);

[0170] Preferably, for example,

[0171] TFA:H2O:EDT:TIS:ArOH=about 92.0:2.0:2.0:2.0:2.0(V:V:V:V:V)

[0172] TFA:H2O:TIS:EDT:ArOH:ArSCH3=about 81.5:5:1:2.5:5:5(V:V:V:V:V:V);

[0173] TFA:H2O:TIS:ArOH:ArSCH3=about 84:5:1:5:5(V:V:V:V:V),

[0174] Where V represents the volume ratio.

[0175] In some embodiments, the dried polypeptide compound-resin is cut with a TFA cracking system, then filtered, concentrated, precipitated, and filtered to obtain a cracked intermediate of the polypeptide compound AI with the resin carrier removed; then purified and separated by HPLC, the components collected, classified, salt exchanged, and concentrated to obtain a purified intermediate of the polypeptide compound AI, which was then freeze-dried to obtain the polypeptide compound AI.

[0176] Those skilled in the art will appreciate that the reaction reagents, such as swelling agents, condensing agents, deprotecting agents, cleavage agents, and solvents, can be matched, mixed, and diluted according to methods commonly used in the art.

[0177] Example

[0178] Example 1: Segmented Coupling of Peptide Compound P016 (6+28 Fragment Coupling)

[0179] [Reaction materials: amino acids, dipeptide fragments and resin]

[0180] Table 2 Amino acid fragments and resins

[0181] R stands for resin, A stands for amino acid, and side stands for side chain.

[0182] [Synthesis of Fmoc-Rink Linker-Nle-MBHA Resin]

[0183] 1. Resin swelling and condensation

[0184] Add 102.0 g of the starting MBHA resin to 0.7 L of DCM and allow to swell with stirring for 30 minutes. Drain the solution and repeat the reaction several times. Ninhydrin colorimetric analysis indicates that the swollen resin is colorless and transparent, passing the colorimetric analysis. Next, add the Fmoc-Nle-OH solution to the reaction flask containing the swollen resin. DIC (condensing agent) may be added during the reaction to complete the condensation reaction. After condensation is complete, drain the solution and pump in DMF. Repeat the washing and draining steps several times to obtain the Fmoc-Nle-MBHA resin.

[0185] 2. Resin Deprotection

[0186] Prepare a PIP / DMF solution (1:4 by volume) (deprotecting agent) in a piperidine flask and add it to the reaction flask. Stir and react with Fmoc-Nle-MBHA Resin for 30 minutes. Drain the solution and repeat several times. Develop the resin with ninhydrin. The resin will turn purple, yielding the deprotected resin, NH2-Nle-MBHA-Resin.

[0187] 3. Resin condensation

[0188] Add the Fmoc-Rink Linker solution to the reaction flask containing the deprotected NH2-Nle-MBHA-Resin resin. Ninhydrin colorimetry indicates that the resin will be colorless and transparent, indicating complete condensation. Drain the solution as much as possible and pump in DMF. Repeat the washing and draining steps 1-2 times to obtain the Fmoc-Rink Linker-Nle-MBHA Resin.

[0189] [Preparation of the peptide chain of crude peptide compound P016 by segmented coupling]

[0190] 1. Resin deprotection

[0191] Take 93.8g of Fmoc-Lys(Boc)-OH and 31.0g of condensing agent HOBt, add them to a dissolution bottle containing DMF / DCM, cool and stir until completely dissolved, cool in a cold bath temperature of ≤-5°C for more than 30 minutes, then slowly add DIC / DCM solution, stir, and obtain Fmoc-Lys(Boc)-OH solution for use.

[0192] Prepare a PIP / DMF solution (1:4 by volume) in a piperidine flask and add it to the drained Fmoc-Rink Linker-Nle-MBHA Resin. Stir for 30 minutes and drain the solution. Wash and drain several times. Ninhydrin color development indicates the resin will turn purple, yielding the deprotected NH2-Rink Linker-Nle-MBHA Resin.

[0193] 2. Connect the C-terminal amino acid (from the leftmost N-terminal of the peptide chain, the 40th amino acid K 40 )

[0194] Add the Fmoc-Lys(Boc)-OH solution to a reaction flask containing the deprotected resin NH2-Rink Linker-Nle-MBHA Resin. Dilute acetic anhydride with DCM solution, add DIEA, and stir thoroughly. Condensation reaction is carried out at a temperature of 10±5°C for 30-60 minutes. Ninhydrin is used for colorimetric detection. If the resin is colorless or pale yellow after ninhydrin, it indicates the absence of free amino groups and complete condensation. Drain the solution as much as possible, pump in DMF, and stir for 2 minutes. Drain the solution, and repeat the washing and draining steps several times to obtain Fmoc-Lys(Boc)-Rink Linker-Nle-MBHA Resin.

[0195] Then, according to the peptide connection sequence in Table 3 below, repeat the above steps to sequentially couple the remaining 39 amino acids to Fmoc-Lys(Boc)-Rink Linker-Nle-MBHA Resin until the coupling is completed to obtain Boc-Tyr 1 (tBu)-Aib 2 -Glu 3 (OtBu)-Gly 4 -Thr 5 (tBu)-αMePhe 6 -Thr 7 (tBu)-Ser 8 (tBu)-Asp 9 (OtBu)-Tyr 10 (tBu)-Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 (OtBu)-Lys 16 (Boc)-Gln 17 (Trt)-Ala 18 -Gln 19 (Trt)-Ala20 -Glu 21 (OtBu)-Phe 22 -Val 23 -Lys 24 (Mtt)-Trp 25 (Boc)-Leu 26 -Leu 27 -Lys 28 (Boc)-Gly 29 -Gly 30 -Pro 31 -Ser 32 (tBu)-Ser 33 (tBu)-Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 (tBu)-Lys 40 (Boc)-Rink Linker-Nle-MBHA Resin (SEQ ID NO: 1).

[0196] Table 3 Peptide sequence

[0197] [Side chain modification]

[0198] 1. Lysine 24 24 Deprotection of the side chain ε-amino group

[0199] Take 57.9g of Fmoc-AEEA-OH and 23.3g of HOBt, add them to a dissolution bottle containing DMF, cool and stir until completely dissolved, and continue to cool in a cold bath temperature of ≤-5°C for more than 30 minutes. Then, slowly add the DIC / DMF solution to the Fmoc-AEEA-OH / HOBt / DMF solution, stir to obtain the Fmoc-AEEA-OH / HOBt / DIC / DMF solution for use.

[0200] Add the prepared HFIP / DCM (volume ratio 1:1) solution (deprotection agent) to Boc-Tyr 1 (tBu)-Aib 2 -Glu 3 (OtBu)-Gly 4 -Thr 5 (tBu)-αMePhe 6 -Thr 7 (tBu)-Ser8 (tBu)-Asp 9 (OtBu)-Tyr 10 (tBu)-Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 (OtBu)-Lys 16 (Boc)-Gln 17 (Trt)-Ala 18 -Gln 19 (Trt)-Ala 20 -Glu 21 (OtBu)-Phe 22 -Val 23 -Lys 24 (Mtt)-Trp 25 (Boc)-Leu 26 -Leu 27 -Lys 28 (Boc)-Gly 29 -Gly 30 -Pro 31 -Ser 32 (tBu)-Ser 33 (tBu)-Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 (tBu)-Lys 40 In the reaction bottle of (Boc)-Rink Linker-Nle-MBHA Resin, stir and react for 30 minutes, drain the solution, and repeat the washing and draining several times. The resin turns purple when detected by ninhydrin color development, indicating that Lysine at position 24 is 24 The side chain ε-amino group is deprotected by Mtt to obtain a peptide chain with deprotected side chain ε-amino group.

[0201] 2. Lysine 24 24 The side chain ε-amino group is connected to 3 AEEA

[0202] Add the Fmoc-AEEA-OH / HOBt / DIC / DMF solution to the reaction bottle containing the deprotected peptide chain, and let the reaction proceed at 10±5°C for 2 hours. Ninhydrin colorimetric detection indicates that the resin is colorless or light yellow, indicating that the condensation is complete. Repeat the washing and draining several times to obtain Lys 24. 24The peptide compound Boc-Tyr with one AEEA attached to the side chain ε-amino group 1 (tBu)-Aib 2 -Glu 3 (OtBu)-Gly 4 -Thr 5 (tBu)-αMePhe 6 -Thr 7 (tBu)-Ser 8 (tBu)-Asp 9 (OtBu)-Tyr 10 (tBu)-Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 (OtBu)-Lys 16 (Boc)-Gln 17 (Trt)-Ala 18 -Gln 19 (Trt)-Ala 20 -Glu 21 (OtBu)-Phe 22 -Val 23 -Lys 24 (Fmoc-AEEA)-Trp 25 (Boc)-Leu 26 -Leu 27 -Lys 28 (Boc)-Gly 29 -Gly 30 -Pro 31 -Ser 32 (tBu)-Ser 33 (tBu)-Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 (tBu)-Lys 40 (Boc)-Rink Linker-Nle-MBHA Resin.

[0203] Repeat the deprotection and AEEA connection twice to obtain lysine 24. 24 The peptide compound Boc-Tyr with three AEEA groups attached to the side chain ε-amino group 1 (tBu)-Aib 2 -Glu 3 (OtBu)-Gly4 -Thr 5 (tBu)-αMePhe 6 -Thr 7 (tBu)-Ser 8 (tBu)-Asp 9 (OtBu)-Tyr 10 (tBu)-Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 (OtBu)-Lys 16 (Boc)-Gln 17 (Trt)-Ala 18 -Gln 19 (Trt)-Ala 20 -Glu 21 (OtBu)-Phe 22 -Val 23 -Lys 24 (Fmoc-AEEA-AEEA-AEEA)-Trp 25 (Boc)-Leu 26 -Leu 27 -Lys 28 (Boc)-Gly 29 -Gly 30 -Pro 31 -Ser 32 (tBu)-Ser 33 (tBu)-Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 (tBu)-Lys 40 (Boc)-Rink Linker-Nle-MBHA Resin.

[0204] 3. Lysine 24 24 Deprotection of ε-amino side chain modification group (Fmoc-AEEA-AEEA-AEEA)

[0205] Deprotection reaction formula:

[0206] Take 63.9g of Fmoc-Glu(α-OtBu)-OH and 23.3g of HOBt, add them to a bottle containing DMF, cool and stir until completely dissolved, continue to cool in a cold bath temperature of ≤-5°C for more than 30 minutes, then slowly add the DIC / DMF solution to the Fmoc-Glu(α-OtBu)-OH / HOBt / DMF solution, stir to obtain Fmoc-Glu(α-OtBu)-OH / HOBt / DIC / DMF for use.

[0207] Add the prepared PIP / DMF solution (volume ratio 1:4) (deprotecting agent) to the 24-position lysine Lys 24 The peptide compound Boc-Tyr with three AEEA groups attached to the side chain ε-amino group 1 (tBu)-Aib 2 -Glu 3 (OtBu)-Gly 4 -Thr 5 (tBu)-αMePhe 6 -Thr 7 (tBu)-Ser 8 (tBu)-Asp 9 (OtBu)-Tyr 10 (tBu)-Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 (OtBu)-Lys 16 (Boc)-Gln 17 (Trt)-Ala 18 -Gln 19 (Trt)-Ala 20 -Glu 21 (OtBu)-Phe 22 -Val 23 -Lys 24 (Fmoc-AEEA-AEEA-AEEA)-Trp 25 (Boc)-Leu 26 -Leu 27 -Lys 28 (Boc)-Gly 29 -Gly 30 -Pro 31 -Ser 32 (tBu)-Ser 33 (tBu)-Gly 34 -Ala 35 -Pro 36 -Pro37 -Pro 38- Ser 39 (tBu)-Lys 40 (Boc)-Rink Linker-Nle-MBHA Resin was stirred for 30 minutes, the solution was drained, and the washing and draining were repeated several times. The resin was purple when detected by ninhydrin color development, indicating that Lysine at position 24 was 24 The Fmoc protection at the (AEEA)3 site where the side chain ε-amino group is linked is removed to obtain a peptide chain where the (AEEA)3 site where the side chain ε-amino group is linked is deprotected.

[0208] 4. Lysine at position 24 24 The side chain ε-amino group (AEEA) is connected to γ-Glu at 3 positions

[0209] Add Fmoc-Glu(α-OtBu)-OH / HOBt / DIC / DMF solution to the reaction bottle containing the peptide chain with the side chain ε-amino group connected to (AEEA)3, and react at 10±5℃ for 2h. If necessary, add DIC to dilute it by half and continue condensation until the resin is colorless or light yellow when detected by ninhydrin color development, indicating that the condensation is complete. Drain the solution and repeat washing and draining several times to obtain Lys 24 24 The peptide compound Boc-Tyr with γ-Glu attached to the ε-amino group (AEEA) at the side chain 1 (tBu)-Aib 2 -Glu 3 (OtBu)-Gly 4 -Thr 5 (tBu)-αMePhe 6 -Thr 7 (tBu)-Ser 8 (tBu)-Asp 9 (OtBu)-Tyr 10 (tBu)-Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 (OtBu)-Lys 16 (Boc)-Gln 17 (Trt)-Ala 18 -Gln 19 (Trt)-Ala 20 -Glu 21 (OtBu)-Phe 22 -Val 23 -Lys 24(Fmoc-Glu(α-OtBu)-AEEA-AEEA-AEEA)-Trp 25 (Boc)-Leu 26 -Leu 27 -Lys 28 (Boc)-Gly 29 -Gly 30 -Pro 31 -Ser 32 (tBu)-Ser 33 (tBu)-Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 (tBu)-Lys 40 (Boc)-Rink Linker-Nle-MBHA Resin.

[0210] 5. Lysine at position 24 24 Deprotection of the (AEEA)3-γ-Glu side chain ε-amino group

[0211] Deprotection reaction formula:

[0212] Take 64.1g of docosanediolatoic acid mono-tert-butyl ester COOH-(CH2) 20 -COO-tBu and 23.3g of HOBt were added to a bottle containing DMF, and then the DIC / DMF solution was slowly added to the COOH-(CH2) 20 -COO-tBu / HOBt / DMF solution, stirred at 20±5℃ for more than 60 minutes to obtain COOH-(CH2) 20 -COO-tBu / HOBt / DIC / DMF for standby use.

[0213] Lysine at position 24 24 The peptide compound Boc-Tyr with γ-Glu attached to the ε-amino group (AEEA) at the side chain 1 (tBu)-Aib 2 -Glu 3 (OtBu)-Gly 4 -Thr 5 (tBu)-αMePhe 6 -Thr 7 (tBu)-Ser 8 (tBu)-Asp 9 (OtBu)-Tyr 10(tBu)-Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 (OtBu)-Lys 16 (Boc)-Gln 17 (Trt)-Ala 18 -Gln 19 (Trt)-Ala 20 -Glu 21 (OtBu)-Phe 22 -Val 23 -Lys 24 (Fmoc-Glu(α-OtBu)-AEEA-AEEA-AEEA)-Trp 25 (Boc)-Leu 26 -Leu 27 -Lys 28 (Boc)-Gly 29 -Gly 30 -Pro 31 -Ser 32 (tBu)-Ser 33 (tBu)-Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 (tBu)-Lys 40 Add the prepared PIP / DMF solution (volume ratio 1:4) (deprotecting agent) to the (Boc)-Rink Linker-Nle-MBHA Resin, stir and react for 30 minutes, drain the solution, and repeat the washing and draining several times. The resin turns purple when detected by ninhydrin color development, indicating that Lysine at position 24 is 24 The Fmoc at the (AEEA)3-γ-Glu linked to the side chain ε-amino group is deprotected to obtain a peptide chain having the (AEEA)3-γ-Glu linked to the side chain ε-amino group deprotected.

[0214] 6. Lysine 24 24 The side chain ε-amino group (AEEA)3-γ-Glu is connected to -CO-(CH2) 20 -COO-tBu

[0215] COOH-(CH2) 20 -COO-tBu / HOBt / DIC / DMF solution was added to the 24-lysine Lys24 The reaction temperature of the peptide chain (AEEA)3-γ-Glu with the side chain ε-amino group was controlled at 20±5℃ for 2 hours. If necessary, DIC was added to dilute the solution by half and the condensation was continued until the resin was colorless or light yellow when detected by ninhydrin color development, indicating that the condensation was complete. The solution was drained and the washing and draining were repeated several times to obtain Lys 24. 24 The side chain ε-amino group (AEEA)3-γ-Glu is connected to -CO-(CH2) 20 -COO-tBu peptide compound Boc-Tyr 1 (tBu)-Aib 2 -Glu 3 (OtBu)-Gly 4 -Thr 5 (tBu)-αMePhe 6 -Thr 7 (tBu)-Ser 8 (tBu)-Asp 9 (OtBu)-Tyr 10 (tBu)-Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 (OtBu)-Lys 16 (Boc)-Gln 17 (Trt)-Ala 18 -Gln 19 (Trt)-Ala 20 -Glu 21 (OtBu)-Phe 22 -Val 23 -Lys 24 (CO-(CH2) 20 -COO-tBu-Glu(α-OtBu)-AEEA-AEEA-AEEA)-Trp 25 (Boc)-Leu 26 -Leu 27 -Lys 28 (Boc)-Gly 29 -Gly 30 -Pro 31 -Ser 32 (tBu)-Ser 33 (tBu)-Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38-Ser 39 (tBu)-Lys 40 (Boc)-Rink Linker-Nle-MBHA Resin.

[0216] [Post-processing]

[0217] Under nitrogen protection, add H2O, TFA, ArOH, TIS, EDT, and ArSCH3 in sequence and stir evenly. Slowly add the prepared cleavage reagent TFA:H2O:EDT:TIS:ArOH:ArSCH3=81.5:5:2.5:1:5:5 (volume ratio) to the resin. After cleavage, stir the resin thoroughly and drain as much as possible to ensure that the product is fully dissolved in the cleavage reagent. After the reaction is completed, filter the reaction solution and transfer it to a concentration bottle. The filtrate is concentrated under reduced pressure at 32±2°C to 20-45% of the initial volume. The concentration time is controlled at 30-60 minutes. Add pre-cooled methyl tert-butyl ether (MTBE) to the precipitation reaction bottle (tank), stir with a stirring rod while adding the concentrate, and a white solid precipitates. Filter the liquid to obtain a white solid cleavage crude product. After dissolving and decarboxylating the crude cleavage product, filter it with a 0.45μm organic membrane and quantify it.

[0218] Preparation of pure peptide amount and purity working reference solution:

[0219] Preparation of the polypeptide compound working reference solution of the present invention (determination of pure peptide amount and purity):

[0220] Accurately weigh an appropriate amount of the polypeptide compound working reference substance of the present invention, dilute it with purified water to prepare a solution containing approximately 0.50 mg of the working reference substance per 1 ml, and shake well to prepare the working reference solution.

[0221] Sample determination method:

[0222] The full load method was used to precisely measure 50 μL of the polypeptide compound working reference solution of the present invention and the pure peptide amount and purity determination test solution through a 20 μL quantitative loop, and injected into the high performance liquid chromatography HLPC instrument. The method procedure was performed in accordance with the general rules 0512 of Part IV of the Chinese Pharmacopoeia 2020 edition.

[0223] ESI-MS (electrospray ionization mass spectrometry) characterization showed that the molecular weight of the synthesized polypeptide compound P016 was 5198.4.

[0224] Figure 1 is the H NMR spectrum of compound P016. Figure 2 is the C NMR spectrum of compound P016.

[0225] [Example 2] One by one synthesis method

[0226] The polypeptide compound P016 was prepared in a similar manner to Example 1, except that a single amino acid was used instead of the dipeptide fragment in Example 1.

[0227] [Example 3] Dipeptide fragment synthesis method (4+32 fragment coupling)

[0228] The polypeptide compound P016 was prepared in a similar manner to Example 1, except that a single amino acid was used instead of Glu in Example 1. 3 -Gly 4 and Thr 5 -αMePhe 6 dipeptide fragment.

[0229] [Example 4] Large fragment synthesis method (1+26 fragment coupling)

[0230] The polypeptide compound P016 was prepared by a method similar to that of Example 1, except that 15←40 was synthesized one by one, and 1-14 was synthesized using a large fragment Boc-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 Instead of the single amino acid and dipeptide fragments in Example 1.

[0231] [Example 5] Dipeptide Fragment Synthesis (6+28 Fragment Coupling)

[0232] The polypeptide compound P016 was prepared in a similar manner to that of Example 1, except that Alloc was used as the ε-amino protecting group of lysine at position 24 instead of Mtt in Example 1.

[0233] [Example 6] Dipeptide Fragment Synthesis (6+28 Fragment Coupling)

[0234] The polypeptide compound P016 was prepared in a similar manner to that of Example 1, except that Dde was used as the ε-amino protecting group of lysine at position 24 instead of Mtt in Example 1.

[0235] [Example 7] Dipeptide Fragment Synthesis (6+28 Fragment Coupling)

[0236] The polypeptide compound P016 was prepared by a method similar to that of Example 1, except that after the fragments were coupled to form a peptide chain, the side chain modification portion COOH-(CH2) was first formed. 20 -COO-tBu-Glu(αOtBu)-AEEA-AEEA-AEEA, and then modify the side chain part COOH-(CH2) 20 -COO-tBu-Glu(αOtBu)-AEEA-AEEA-AEEA as a whole was directly coupled to the ε-amino group of lysine at position 24 of the peptide chain.

[0237] The preparation methods and product test results of the polypeptide compound P016 of Examples 1 to 6 are recorded in Table 4 below.

[0238] In Table 4 above, the synthesis yield is calculated by dividing the molar weight of crude P016 by the molar weight of resin. The molar weight of crude P016 is calculated by dividing the mass of crude P016 by the molecular weight of P016, 5198.4. Because crude P016 contains impurities, the synthesis yield may exceed 100%.

[0239] Crude purity refers to the purity of the crude cleavage product.

[0240] The final yield was calculated by dividing the molar weight of the pure peptide P016 after HPLC purification by the molar weight of the resin. The molar weight of the pure peptide P016 was the mass of the pure peptide P016 divided by the molecular weight of P016, 5198.4.

[0241] In Table 4 above, 15←28 refers to coupling from amino acid 28 to amino acid 15 one by one; 31←40 refers to coupling from amino acid 40 to amino acid 31 one by one; 15←40 refers to coupling from amino acid 40 to amino acid 15 one by one; 1←40 refers to coupling from amino acid 40 to amino acid 1 one by one; 1-14 refers to amino acid 1 to amino acid 14 as a whole as a fragment; 1-2, 3-4, 5-6, 10-11, 12-13, and 29-30 are dipeptide fragments.

[0242] In the dipeptide fragment synthesis method (6+28 fragment coupling) of Example 1, the peptide chain is first synthesized, and then the side chain ε-amino group of lysine at position 24 is modified. In the peptide chain synthesis, Tyr 1 -Aib 2 Glu 3 -Gly 4 、Thr 5 -αMePhe 6 、Tyr 10 -Aib 11 、Ile 12 -Aib 13、Gly 29 -Gly 30 There are a total of six dipeptide fragments, and Mtt is used to protect the side chain ε-amino group of lysine at position 24, while achieving excellent final yield and crude purity of the polypeptide compound. As can be seen from Table 4 above, Example 2 adopts a one-by-one synthesis method, Example 4 adopts a large fragment condensation method, the fragment synthesis method of Example 3 (4+32 fragment coupling) only uses part of the dipeptide fragment, Example 5 and Example 6 respectively use Alloc and Dde as the side chain ε-amino protecting group of lysine at position 24, and Example 7 first synthesizes the side chain modification part, and then directly couples the side chain modification part to the peptide chain as a whole. The final yields of Examples 2-7 are all significantly lower than the dipeptide fragment synthesis method of Example 1, the crude purity of the polypeptide compound of Example 2 is very low, the crude purity of the polypeptide compound of Examples 3 and 4 is significantly lower than that of Example 1, and the crude purity of the polypeptide compound of Examples 5, 6 and 7 is equivalent to that of Example 1.

[0243] [Example 8] Scale-up experiment of dipeptide fragment synthesis method

[0244] Polypeptide compound P016 was prepared using a method similar to that of Example 1, except that, based on the molar weight of the resin injected, the scale of Example 1 was scaled up from 10 mmol to 50 mmol (pilot batch), 300 mmol (pilot scale batch), and 300 mmol (GMP batch). The test results are reported in Table 5 below. The operating conditions for the pilot scale and GMP batches were essentially the same, with the difference being that the GMP batches were produced in a GMP workshop, requiring more meticulous temperature control and other procedures.

[0245] Table 5 Amplification experiment results

[0246] As can be seen from Table 5 above, the method of the present invention can be stably scaled up to large-scale production of 50 mmol to 300 mmol without sacrificing the final yield and the purity of the pure product.

[0247] [Biological activity of the polypeptide compound P016 of the present invention]

[0248] 1. In vitro binding activity to human GLP-1 and GIP receptors

[0249] hGLP-1 and the polypeptide compound of the present invention were dissolved in DMSO and stored at -80°C. 89 μL of membranes (5 μg / well) dissolved in binding buffer (50 mM Hepes, pH 7.4, 5 mM MgCl2, 5 mM EDTA, 0.005% TWEEN, 0.005% HSA) were transferred to a 96-well assay plate. Compounds were diluted in DMSO, and then 1 μL of the diluted compound or 100% DMSO was added to the assay plate containing the membrane solution. 10 μL of [125I]GLP-1 (final reaction concentration 0.15 nM) was then added. The assay plate was incubated at room temperature for 90 minutes. The membrane complexes were harvested using a Cell Harvester onto GF / B plates pre-coated with 0.5% PEI and rinsed three times with 500 μL of 4°C pre-chilled elution buffer (50 mM Hepes, pH 7.4, 500 mM NaCl). After drying at 37°C for 2 hours, 50 μL of scintillation fluid was added to each well, sealed, and left for at least 1 hour. The wells were then read using Microbet2 to determine the level of membrane-bound radioligand.

[0250] The absolute IC was calculated by nonlinear regression of the percentage of [125I]GLP-1 bound and the concentration of the added compound. 50 Concentration. Use the Cheng-Prusoff formula to calculate IC 50 The concentration was converted to Ki (Ki is the inhibition constant).

[0251] hGIP and the polypeptide compounds of the present invention were dissolved in DMSO and stored at -80°C. 98 μL of membranes (15 μg / well) dissolved in binding buffer (50 mM HEPES pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.1% BSA, 0.005% Tween-20) were transferred to a 96-well assay plate. Compounds were diluted in DMSO, and then 2 μL of the diluted compound or 100% DMSO was added to the assay plate containing the membrane solution. 100 μL of [125I]GIP (final reaction concentration 0.0315 nM) was then added. The assay plate was incubated at room temperature for 90 minutes. The membrane complexes were harvested using a Cell Harvester onto GF / B plates pre-coated with 0.5% PEI and rinsed three times with 500 μL of 4°C pre-chilled elution buffer (50 mM Tris-HCl pH 7.4, 125 mM NaCl). After drying at 37°C for 2 hours, 50 μL of scintillation fluid was added to each well, sealed, and left for at least 1 hour. The wells were then read using a Microbeta2 to determine the level of membrane-bound radioligand.

[0252] The absolute IC50 concentration was calculated by nonlinear regression of the percentage of [125I]GIP bound and the concentration of the added compound. The IC50 was calculated using the Cheng-Prusoff formula. 50 The concentration was converted to Ki (Ki is the inhibition constant).

[0253] Table 6 Receptor binding affinity of the polypeptide compound P016 of the present invention, Ki ratio

[0254] Ki ratio: the ratio of the Ki value of the endogenous ligand to the Ki value of the test compound

[0255] 2. Agonist activity on hGLP-1R and hGIPR

[0256] The cAMP reporter gene method was used to determine the agonist activity of the polypeptide compound of the present invention on human GLP-1R.

[0257] HEK293 / CRE / GLP-1R cells were seeded at 50,000 cells / well (80 μL / well) in a 96-well plate and cultured overnight in a 37°C, 5% CO2 incubator. Compound-containing assay medium (DMEM with 0.1% casein) was added to the 96-well plate at 20 μL / well and incubated in a 37°C, CO2 incubator for another 6 hours. After equilibration to room temperature, the supernatant was removed and 50 μL / well of Bright-Glo reagent was added. The cells were shaken and lysed at room temperature for 10 minutes. Luminescence was read using an Envision microplate reader to measure luciferase activity.

[0258] The response value of 100 nM GLP-1 was set as 100% response value, and nonlinear regression was performed using GraphPad based on the response percentage and the added compound concentration to obtain the EC of the compound. 50 value.

[0259] The LANCE Ultra cAMP Kit was used to determine the agonist activity of the compounds of the present invention on human GIPR.

[0260] The in vitro agonist activity of the compounds of the present invention against GIPR was determined in HEK293 cells stably expressing human GIPR (HEK293 / GIPR cells). HEK293 / GIPR cells were prepared with HBSS buffer (0.1% Casein, 500 μM IBMX, 5 mM HEPES) and seeded at 1000 cells / well (5 μL / well) in 384-well cell culture plates. 5 μL of HBSS buffer containing the compound was added to the 384-well cell culture plates. The plates were sealed and incubated in a 37°C, 5% CO2 incubator for approximately 30 minutes. After incubation, 5 μL of cAMP-Eu working solution and 5 μL of cAMP-Ulight working solution were added, followed by shaking to mix. The plates were incubated at 25°C for 1 hour. The signals at 665 nm and 615 nm were read on an Envision microplate reader. The 665 nm / 615 nm ratio was calculated and converted to cAMP concentration using a cAMP standard curve. The response value of 1 μM GIP was set as 100% response value, and nonlinear regression was performed using GraphPad based on the response percentage and the added compound concentration to obtain the EC value of the polypeptide compound of the present invention. 50 value.

[0261] Table 7 Agonist activity on human GLP-1 and GIP receptors, EC50 values

[0262] EC 50 Value: Endogenous ligand EC 50 EC values ​​of the tested compounds 50 Value ratio

[0263] 3. Pharmacodynamics in db / db mice: To study the effect of the polypeptide compound of the present invention on blood glucose in diabetic model mice (db / db mice)

[0264] This study used a single subcutaneous administration of the compound to db / db mice and measured changes in blood glucose, food intake, and body weight to elucidate the hypoglycemic effect and duration of efficacy of the compound of the present invention. In this study, male db / db mice aged 8-9 weeks were used. The db / db mice were housed in an independent ventilated cage IVC facility with controlled temperature (20-26°C) and humidity (40-70%), a 12h:12h light / dark cycle, and free access to food and water. Blood was collected from the tip of the tail and basal blood glucose was measured with a Roche blood glucose meter. The mice were randomly divided into groups (n=6 / group) based on initial blood glucose and initial body weight, and each group had similar body weight and blood glucose.

[0265] The compound of the present invention (10 nmol / kg) was dissolved in a solvent (PBS containing 0.1% Tween 20, pH 7.2-7.4). After a single subcutaneous administration, the set time points (0-120 h) were recorded. The duration of the hypoglycemic effect of the polypeptide compound was significantly different from that of the vehicle group.

[0266] Table 8: Duration of hypoglycemic effect of the polypeptide compound of the present invention

[0267] The above is a detailed introduction to the polypeptide compound and its application provided by the present invention.

[0268] Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The above examples are only intended to help understand the methods and core concepts of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and such improvements and modifications are also protected by the claims of the present invention. Amino acid sequence:

Claims

1. Use of a dipeptide fragment in solid phase synthesis of a polypeptide compound, characterized in that: The dipeptide fragment is selected from the following structures: Gly-Gly, Ile-Aib, Tyr-Aib, Thr-αMePhe and Glu-Gly, and the polypeptide compound is a compound of formula AI and has the following structure: H-Tyr 17 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 [[ID=​​​​​​​​​​​​​​​​​​​ Optionally, at any lysine selected from positions 16, 24, 28 and 40, the side chain ε-amino group is chemically modified via ([2-(2-amino-ethoxy)-ethoxy]-acetyl)a-(γ-Glu)b-CO-(CH2)c-COOH, wherein each a is independently an integer from 0 to 5, each b is independently an integer from 0 to 5, and each c is independently an integer from 10 to 24.

2. The use according to claim 1, characterized in that The polypeptide compound is selected from the following compounds: Compound P001: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2(SEQ ID NO:1) Compound P007: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 (AEEA-AEEA-γGlu-CO-(CH2) 16 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:2) Compound P008:H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 (AEEA-AEEA-γGlu-CO-(CH2) 16 -COOH)-Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:3) Compound P013: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 (AEEA-AEEA-γGlu-CO-(CH2) 18 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:4) Compound P014: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 (AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:5) Compound P015: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 (AEEA-AEEA-γGlu-γGlu-γGlu-CO-(CH2) 16 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:6) Compound P016: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 (AEEA-AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:7) Compound P017:H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 (AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:8) Compound P018:H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 (AEEA-AEEA-γGlu-γGlu-γGlu-CO-(CH2) 16 -COOH)-Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:9) Compound P019: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 (AEEA-AEEA-AEEA-γGlu-CO-(CH2) 16 -COOH)-Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:10) Compound P020: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 (AEEA-AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:11) wherein AEEA represents [2-(2-amino-ethoxy)-ethoxy]-acetyl.

3. The use according to claim 1 or 2, characterized in that The polypeptide compound is: Compound P016: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 (AEEA-AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:7) 4. The use according to any one of claims 1 to 3, characterized in that The free α-amino group of the dipeptide fragment has an Fmoc protecting group, or a Boc protecting group, or both an Fmoc protecting group and a Boc protecting group.

5. The use according to claim 4, characterized in that The dipeptide fragment having an Fmoc protecting group is selected from the group consisting of: Fmoc-Gly-Gly-OH, Fmoc-Ile-Aib-OH, Fmoc-Tyr(tBu)-Aib-OH, Fmoc-Thr(tBu)-αMePhe-OH and Fmoc-Glu(OtBu)-Gly-OH.

6. The use according to claim 4, characterized in that The dipeptide fragment having a Boc protecting group is selected from the group consisting of: Boc-Gly-Gly-OH, Boc-Ile-Aib-OH, Boc-Tyr(tBu)-Aib-OH, Boc-Thr(tBu)-αMePhe-OH and Boc-Glu(OtBu)-Gly-OH, preferably Boc-Tyr(tBu)-Aib-OH.

7. The use according to claim 4, characterized in that The dipeptide fragment having both an Fmoc protecting group and a Boc protecting group is selected from the group consisting of: Fmoc-Gly-Gly-OH, Fmoc-Ile-Aib-OH, Fmoc-Tyr(tBu)-Aib-OH, Fmoc-Thr(tBu)-αMePhe-OH, Fmoc-Glu(OtBu)-Gly-OH and Boc-Tyr(tBu)-Aib-OH; Preferably, the dipeptide fragment having both an Fmoc protecting group and a Boc protecting group is a combination of Fmoc-Gly-Gly-OH, Fmoc-Ile-Aib-OH, Fmoc-Tyr(tBu)-Aib-OH, Fmoc-Thr(tBu)-αMePhe-OH, Fmoc-Glu(OtBu)-Gly-OH and Boc-Tyr(tBu)-Aib-OH.

8. The use according to any one of claims 1 to 7, characterized in that The polypeptide compound of formula AI is divided into the following 6 fragments: Tyr 1 -Aib 2 Glu 3 -Gly 4 、Thr 5 -αMePhe 6 、Tyr 10 -Aib 11 、Ile 12 -Aib 13 、Gly 29 -Gly 30 , and single amino acids at the remaining 28 sites were coupled.

9. A method for solid phase synthesis of dipeptide fragments of a polypeptide compound, characterized in that: The polypeptide compound is a compound of formula AI and has the following structure: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38- Ser 39 -Lys 40 -NH2 formula AI (SEQ ID NO:12), wherein optionally, at any one lysine selected from positions 16, 24, 28 and 40, the side chain ε-amino group is chemically modified via ([2-(2-amino-ethoxy)-ethoxy]-acetyl)a-(γ-Glu)b-CO-(CH2)c-COOH, wherein each a is independently an integer from 0 to 5, each b is independently an integer from 0 to 5, and each c is independently an integer from 10 to 24, The dipeptide fragment is selected from the following structures: Gly-Gly, Ile-Aib, Tyr-Aib, Thr-αMePhe and Glu-Gly.

10. The method according to claim 9, wherein The polypeptide compound is a compound selected from the following: Compound P001: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 -Ala 18 -Gln 19 -Ala 20 -Glu 21 -Phe 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 [[ID=5​​​​​​​​​​​​​​​​​​​​​​​ Compound P007: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 (AEEA-AEEA-γGlu-CO-(CH2) 16 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:2) Compound P008:H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 (AEEA-AEEA-γGlu-CO-(CH2) 16 -COOH)-Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:3) Compound P013: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 (AEEA-AEEA-γGlu-CO-(CH2) 18 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:4) Compound P014: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 (AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:5) Compound P015: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 (AEEA-AEEA-γGlu-γGlu-γGlu-CO-(CH2) 16 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:6) Compound P016: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 (AEEA-AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:7) Compound P017:H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 (AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:8) Compound P018:H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 (AEEA-AEEA-γGlu-γGlu-γGlu-CO-(CH2) 16 -COOH)-Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:9) Compound P019: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 (AEEA-AEEA-AEEA-γGlu-CO-(CH2) 16 -COOH)-Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:10) Compound P020: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 -Trp 25 -Leu 26 -Leu 27 -Lys 28 (AEEA-AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:11) wherein AEEA represents [2-(2-amino-ethoxy)-ethoxy]-acetyl.

11. The method according to claim 9 or 10, wherein: The polypeptide compound is: Compound P016: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 - Ser 8 -Asp 9 -Tyr 10 -Aib 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Gln 17 - Wake up 18 -Gln 19 - Wake up 20 -Glu 21 - Phew 22 -Val 23 -Lys 24 (AEEA-AEEA-AEEA-γGlu-CO-(CH2) 20 -COOH)-Trp 25 -Leu 26 -Leu 27 -Lys 28 -Gly 29 -Gly 30 - Pro 31 - Ser 32 - Ser 33 -Gly 34 - Wake up 35 - Pro 36 - Pro 37 - Pro 38- Ser 39 -Lys 40 -NH2 (SEQ ID NO:7) 12. The method according to any one of claims 9 to 11, wherein The free α-amino group of the dipeptide fragment has an Fmoc protecting group, or a Boc protecting group, or both an Fmoc protecting group and a Boc protecting group.

13. The method according to claim 12, wherein: The dipeptide fragment having an Fmoc protecting group is selected from the group consisting of: Fmoc-Gly-Gly-OH, Fmoc-Ile-Aib-OH, Fmoc-Tyr(tBu)-Aib-OH, Fmoc-Thr(tBu)-αMePhe-OH and Fmoc-Glu(OtBu)-Gly-OH.

14. The method according to claim 12, wherein: The dipeptide fragment having a Boc protecting group is selected from the group consisting of: Boc-Gly-Gly-OH, Boc-Ile-Aib-OH, Boc-Tyr(tBu)-Aib-OH, Boc-Thr(tBu)-αMePhe-OH and Boc-Glu(OtBu)-Gly-OH, preferably Boc-Tyr(tBu)-Aib-OH.

15. The method according to claim 12, wherein The dipeptide fragment having both an Fmoc protecting group and a Boc protecting group is selected from the group consisting of: Fmoc-Gly-Gly-OH, Fmoc-Ile-Aib-OH, Fmoc-Tyr(tBu)-Aib-OH, Fmoc-Thr(tBu)-αMePhe-OH, Fmoc-Glu(OtBu)-Gly-OH and Boc-Tyr(tBu)-Aib-OH; Preferably, the dipeptide fragment having both an Fmoc protecting group and a Boc protecting group is a combination of Fmoc-Gly-Gly-OH, Fmoc-Ile-Aib-OH, Fmoc-Tyr(tBu)-Aib-OH, Fmoc-Thr(tBu)-αMePhe-OH, Fmoc-Glu(OtBu)-Gly-OH and Boc-Tyr(tBu)-Aib-OH.

16. The method according to any one of claims 9 to 15, wherein: The polypeptide compound of formula AI is divided into the following 6 fragments: Tyr 1 -Aib 2 Glu 3 -Gly 4 、Thr 5 -αMePhe 6 、Tyr 10 -Aib 11 、Ile 12 -Aib 13 、Gly 29 -Gly 30 , and single amino acids at the remaining 28 sites were coupled.

17. The method according to any one of claims 9 to 16, comprising the steps of: The peptide chain of the polypeptide compound of formula AI is prepared by a dipeptide fragment solid phase synthesis method, and Optionally, the side chain ε-amino group is chemically modified at any one lysine selected from positions 16, 24, 28 and 40 via ([2-(2-amino-ethoxy)-ethoxy]-acetyl)a-(γ-Glu)b-CO-(CH2)c-COOH, wherein each a is independently an integer from 0 to 5, each b is independently an integer from 0 to 5, and each c is independently an integer from 10 to 24.

18. The method according to claim 17, comprising the steps of: The peptide chain of the polypeptide compound of formula AI is prepared by a dipeptide fragment solid phase synthesis method, wherein at any one lysine selected from positions 16, 24, 28 and 40, there is Mtt, Alloc or Dde as a side chain ε-amino protecting group, and the remaining three lysines have Boc as a side chain ε-amino protecting group; For lysine using Mtt, Alloc or Dde as the side chain ε-amino protecting group, deprotection is performed; and The side chain ε-amino group of the deprotected lysine was chemically modified via ([2-(2-amino-ethoxy)-ethoxy]-acetyl)a-(γ-Glu)b-CO-(CH2)c-COOH to obtain a polypeptide compound of formula AI.

19. The method according to claim 18, comprising the steps of: The peptide chain of the polypeptide compound of formula AI is prepared by a dipeptide fragment solid phase synthesis method, wherein the 24th lysine has Mtt as a side chain ε-amino protecting group, and the 16th, 28th and 40th lysines have Boc as side chain ε-amino protecting groups; Deprotecting the side chain ε-amino group of lysine at position 24; and At the side chain ε-amino group of the deprotected lysine at position 24, 2-(2-amino-ethoxy)-ethoxy]-acetyl was linked by using Fmoc-AEEA-OH, γ-Glu was linked by using Fmoc-Glu(α-OtBu)-OH, and -OC-(CH2)c-COO-tBu was linked by using HOOC-(CH2)c-COO-tBu, thereby forming a 3-hydroxy-1-([2-(2-amino-ethoxy)-ethoxy]-acetyl)3-(γ-Glu)1-CO-(CH2) 20 -COO-tBu to chemically modify the side chain ε-amino group of lysine 24; and After post-treatment, polypeptide compound P016 was obtained.

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