Chemical synthesis method of elastin-like polypeptide

Abstract

The invention belongs to the field of chemical synthesis, and particularly relates to a chemical synthesis method of elastin-like polypeptide. The invention provides a chemical synthesis method of elastin-like, which comprises the following steps of: constructing a pentapeptide repetitive sequence (valine-proline-glycine-X-glycine, X is amino acid except proline, and the amino acid except glycine is respectively and independently selected from levoamino acid or dextroamino acid) of elastin-like polypeptide through liquid phase reaction; the elastin-like polypeptide can be prepared with the target molecular weight of 1000-500000 by adding amino groups and carboxyl groups into an organic solvent, forming orthogonal protection on the amino groups and the carboxyl groups, then selectively removing amino or carboxyl end protecting groups respectively, accurately coupling the amino groups and the carboxyl groups in a solution and repeating the deprotection and coupling processes for multiple times. The preparation method can realize accurate regulation and control of the molecular weight, sequence, chirality and topological structure of the elastin-like protein, improves the synthesis efficiency of the high-molecular-weight elastin-like polypeptide, has the advantages of large synthesis scale, low cost and the like, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of chemical synthesis, specifically relating to a chemical synthesis method for an elastin-like polypeptide. Background Technology

[0002] Elastin is an important extracellular matrix protein widely found in connective tissues of humans and animals, especially in tissues that need to withstand repeated stretching and rebound, such as skin, lungs, arteries, ligaments, and elastic cartilage. The characteristic pentapeptide repeat sequence valine-proline-glycine-X-glycine (VPGXG, where X is any amino acid other than proline) has been extracted from the disordered region of elastin, and elastin-like polypeptides (ELPs) have been prepared. ELPs have wide applications in drug delivery, protein purification, and tissue engineering due to their excellent biocompatibility, biodegradability, and unique temperature-responsive properties.

[0003] Currently, elastin-like peptides are mainly obtained through recombinant expression. First, the ELP gene is edited on demand, and the ELP gene fragment is excised using enzyme digestion. Then, a portion of the plasmid is removed, and the ELP gene fragment is ligated into the excised plasmid. Subsequently, using *E. coli* as the parent organism, the edited ELP gene is incorporated into the plasmid and overexpressed in vivo. Finally, the elastin-like peptide (purity >95%) is obtained through purification using an inverse temperature phase transition (ITC) method.

[0004] During recombinant expression in *E. coli*, several issues may arise, including rapid promoter-driven production, mRNA instability, codon bias, and a lack of distinct subcellular compartments in the *E. coli* cytoplasm. These issues can lead to decreased protein solubility and affect the soluble synthesis of elastin-like peptides (ELPs). Recombinant ELPs may also contain toxic proteins and other substances, posing potential physiological toxicity. Furthermore, due to the limitations of expressing the parent ribosome, *E. coli* struggles to use non-natural amino acids as building blocks, making it difficult to introduce dextrorotatory amino acids and other functional groups into the sequence. This hinders precise control of the ELP chemical structure, and current biosynthetic methods are insufficient to produce structurally accurate elastin-like peptides suitable for large-scale production. Summary of the Invention

[0005] This invention belongs to the field of chemical synthesis, specifically relating to a chemical synthesis method for an elastin-like polypeptide.

[0006] A method for preparing an elastin-like polypeptide includes the following steps:

[0007] Step 1: Prepare a pentapeptide synthesis unit protected by a protecting group; the structure of the pentapeptide synthesis unit protected by the protecting group is R1-valine-proline-glycine-X-glycine-R2;

[0008] Step 2: Using the pentapeptide synthesis unit protected by the protecting group obtained in Step 1 as raw material, selectively remove the amino or carboxyl terminus protecting groups, and then couple it in a liquid phase system. Repeat the above deprotection and coupling process to prepare an elastin-like polypeptide; the elastin-like polypeptide is R1-(valine-proline-glycine-X-glycine). n -R2, whose structure is shown in Equation I;

[0009]

[0010] Wherein, R1 is an amino protecting group, R2 is a carboxyl protecting group, X is an amino acid other than proline, R3 is the side chain of amino acid X, R4 is the side chain protecting group of amino acid X, n is 2 to the power of y, and y is an integer greater than or equal to 1.

[0011] Preferably, the deprotection processes of R1 and R2 must be completely orthogonal, and R3 and R4 remain unchanged during the deprotection and coupling processes.

[0012] Preferably, R1 is selected from at least one of triphenylmethyl, trimethylsilylethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, and allyloxycarbonyl; R2 is selected from at least one of benzyloxy and alkoxy; and R4 is selected from at least one of triphenylmethyl, tert-butoxycarbonyl, tert-butyl, 2,2,4,6,7-pentamethylbenzofuran-5-sulfonyl, p-methoxytriphenylmethyl, trimethoxybenzyl, and N-methyltriphenylmethyl.

[0013] Preferably, when X is an inert side-chain amino acid, R1 and R2 are orthogonally protected protecting groups;

[0014] And / or, when X is a side-chain functionalized amino acid, R1, R2 and the side-chain protecting group R4 of X are all orthogonally protected protecting groups.

[0015] Preferably, R1 is selected from tert-butyloxycarbonyl, R2 is selected from benzyloxy, and X is an inert side-chain amino acid that does not contain halogen or sulfur; or R1 is selected from tert-butyloxycarbonyl, R2 is selected from methoxy or ethoxy, and X is an inert side-chain amino acid; or R1 is selected from 9-fluorenylmethoxycarbonyl, R2 is selected from benzyloxy, and X is an inert side-chain amino acid that does not contain halogen or sulfur.

[0016] Alternatively, R1 is selected from at least one of 9-fluorenylmethoxycarbonyl, trimethylsilylethoxycarbonyl, and 2,2,2-trichloroethoxycarbonyl, R2 is selected from at least one of benzyloxy, methoxy, and ethoxy, and X is a side-chain functionalized amino acid that does not contain halogen or sulfur; or, R1 is selected from at least one of allyloxycarbonyl, trimethylsilylethoxycarbonyl, and 2,2,2-trichloroethoxycarbonyl, R2 is selected from at least one of methoxy and ethoxy, and X is an amino acid other than proline.

[0017] Preferably, R1 is selected from tert-butoxycarbonyl, R2 is selected from benzyloxy, methoxy, or ethoxy, and X is selected from at least one of glycine, alanine, valine, leucine, isoleucine, and phenylalanine.

[0018] And / or, R1 is selected from 9-fluorenylmethoxycarbonyl, R2 is selected from benzyloxy, and X is selected from at least one of glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, arginine, serine, and threonine.

[0019] And / or, R1 is selected from allyloxycarbonyl, R2 is selected from ethoxy, and X is at least one of glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, arginine, serine, threonine, cysteine, and methionine.

[0020] Preferably, R1 is selected from tert-butoxycarbonyl, R2 is selected from benzyloxy, and X is at least one of glycine, aspartic acid, valine, and phenylalanine.

[0021] And / or, R1 is selected from 9-fluorenylmethoxycarbonyl, R2 is selected from benzyloxy, and X is asparagine;

[0022] And / or, R1 is selected from allyloxycarbonyl, R2 is selected from ethoxy, and X is cysteine;

[0023] All of the above-mentioned amino acids, except for glycine, can be either levorotatory or dextrorotatory.

[0024] Preferably, in step 1, the method for preparing the pentapeptide synthesis unit protected by the protecting group includes the following steps:

[0025] (1) The protected proline, activator and condensing agent are dissolved in an organic solvent to obtain an activation system. The protected glycine and alkaline reagent are dissolved in an organic solvent and added to the above activation system. After purification, the protected dipeptide product 1, namely R1-proline-glycine-R2, is obtained.

[0026] (2) Dissolve the dipeptide product 1 protected by the protecting group in a deprotecting agent to carry out the deprotection reaction, and obtain the deprotected dipeptide product 1, namely NH2-proline-glycine-R2.

[0027] (3) Dissolve the valine protected by the protecting group, the activator and the condensing agent in an organic solvent to obtain an activation system. Then dissolve the deprotected dipeptide product 1 obtained in step (2) and the alkaline reagent in an organic solvent, add the above activation system, and after purification, obtain the tripeptide product protected by the protecting group, namely R1-valine-proline-glycine-R2.

[0028] (4) The tripeptide product protected by the protecting group is dissolved in an organic solvent, a catalyst and a reducing agent are added, and after purification, the tripeptide product without the protecting group is obtained, namely R1-valine-proline-glycine-OH.

[0029] (5) Dissolve the X protected by the protecting group, the activator and the condensing agent in an organic solvent to obtain an activation system. Then dissolve the glycine protected by the protecting group and the basic reagent in an organic solvent, add them to the activation system, and after purification, obtain the dipeptide product 2 protected by the protecting group, namely R1-X-glycine-R2.

[0030] (6) Dissolve the dipeptide product 2 protected by the protecting group in a deprotecting agent to carry out the deprotection reaction, and obtain the deprotected dipeptide product 2, namely NH2-X-glycine-R2.

[0031] (7) Dissolve the deprotected tripeptide product, activator, and condensing agent obtained in step (4) in an organic solvent to obtain an activation system. Then dissolve the deprotected dipeptide product 2 obtained in step (6) and an alkaline reagent in an organic solvent, add them to the activation system, and after purification, obtain a pentapeptide synthesis unit protected by a protecting group.

[0032] Preferably, in step (1), the proline protected by the protecting group is at least one of Boc-D-proline and Boc-L-proline; the activator is selected from at least one of 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate, benzotriazole-1-oxytripyrrolylphosphine hexafluorophosphate, pentafluorophenol, N-hydroxysuccinimide, 1-hydroxy-7-azabenzotriazole-tetramethylurea hexafluorophosphate, and ethyl 2-oxime cyanophosphate; and the condensing agent is selected from 1-ethyl-(3-dimethylaminopropyl) The reagent comprises at least one of carbodiimide hydrochloride, N,N'-diisopropylcarbodiimide, and N,N'-dicyclohexylcarbodiimide; the glycine protected by the protecting group is at least one of glycine benzyl ester hydrochloride, glycine benzyl ester trifluoroacetate, and glycine benzyl ester acetate; the basic reagent is at least one of N,N-diisopropylethylamine, triethylamine, diethylamine, N-methylmorpholine, and 2,4,6-trimethylpyridine; and the organic solvent is at least one of N,N-dimethylformamide, dichloromethane, tetrahydrofuran, and dimethyl sulfoxide.

[0033] And / or, in step (2), the deprotecting agent is selected from at least one of trifluoroacetic acid, dioxane hydrochloride solution, p-toluenesulfonic acid, methanol hydrochloride solution, trimethylsilyl trifluoromethanesulfonate, hydrogen bromide acetic acid solution, dilute sulfuric acid, and boron trifluoride ether.

[0034] And / or, in step (3), the valine protected by the protecting group is at least one of Boc-D-valine and Boc-L-valine; the activator is selected from at least one of 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate, benzotriazole-1-oxytripyrrolylphosphine hexafluorophosphate, pentafluorophenol, N-hydroxysuccinimide, 1-hydroxy-7-azabenzotriazole-tetramethylurea hexafluorophosphate, and ethyl 2-oxime cyanophosphate. The condensing agent is selected from at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-diisopropylcarbodiimide, and N,N'-dicyclohexylcarbodiimide; the alkaline reagent is at least one of N,N-diisopropylethylamine, triethylamine, diethylamine, N-methylmorpholine, and 2,4,6-trimethylpyridine; the organic solvent is at least one of N,N-dimethylformamide, dichloromethane, tetrahydrofuran, and dimethyl sulfoxide.

[0035] And / or, in step (4), the organic solvent is selected from at least one of tetrahydrofuran, ethyl acetate, acetonitrile, and methanol; the catalyst is selected from at least one of carbon-supported palladium catalyst and carbon-supported palladium hydroxide; the reducing agent is selected from at least one of hydrogen, formic acid, ammonium formate, cyclohexene, and 1,4-cyclohexadiene.

[0036] And / or, in step (5), the activator is selected from at least one of 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate, benzotriazole-1-oxytripyrrolylphosphine hexafluorophosphate, pentafluorophenol, N-hydroxysuccinimide, 1-hydroxy-7-azabenzotriazole-tetramethylurea hexafluorophosphate, and ethyl 2-oxime cyanophosphate; the condensing agent is selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-diisopropylcarbodiimide... The protective group is at least one of imine and N,N'-dicyclohexylcarbodiimine; the glycine protected by the protecting group is at least one of glycine benzyl ester hydrochloride, glycine benzyl ester trifluoroacetate, and glycine benzyl ester acetate; the basic reagent is at least one of N,N-diisopropylethylamine, triethylamine, diethylamine, N-methylmorpholine, and 2,4,6-trimethylpyridine; the organic solvent is at least one of N,N-dimethylformamide, dichloromethane, tetrahydrofuran, and dimethyl sulfoxide.

[0037] And / or, in step (6), the deprotecting agent is selected from at least one of 1,4-dioxane hydrochloride, trifluoroacetic acid, p-toluenesulfonic acid, methanol hydrochloric acid solution, trimethylsilyl trifluoromethanesulfonate, acetic acid solution of hydrogen bromide, dilute sulfuric acid, boron trifluoride ether, piperidine, tetramethylpiperidine, piperazine, 1,8-diazabicycloundec-7-ene, triethylamine, diethylamine, potassium carbonate, and N,N-diisopropylethylamine;

[0038] And / or, in step (7), the activator is selected from at least one of 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate, benzotriazole-1-oxytripyrrolylphosphine hexafluorophosphate, pentafluorophenol, N-hydroxysuccinimide, 1-hydroxy-7-azabenzotriazole-tetramethylurea hexafluorophosphate, and ethyl 2-oxime cyanoacetate; the condensing agent is selected from at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-diisopropylcarbodiimide, and N,N'-dicyclohexylcarbodiimide; the alkaline reagent is at least one of N,N-diisopropylethylamine, triethylamine, diethylamine, N-methylmorpholine, and 2,4,6-trimethylpyridine; and the organic solvent is at least one of N,N-dimethylformamide, dichloromethane, tetrahydrofuran, and dimethyl sulfoxide.

[0039] Preferably, in step (1), the equivalent ratio of the protecting group-protected proline: activator: condensing agent: basic catalyst: protecting group-protected glycine is 1:(0.5-5):(0.5-5):(1-10):(0.5-3);

[0040] And / or, in step (2), the equivalent ratio of the dipeptide product 1 protected by the protecting group to the deprotecting agent is 1:1-20;

[0041] And / or, in step (3), the equivalent ratio of the valine protected by the protecting group: activator: condensing agent: basic catalyst: deprotected dipeptide product 1 is 1:(0.5-5):(0.5-5):(1-10):(0.5-3);

[0042] And / or, in step (4), the equivalent ratio of the tripeptide product protected by the protecting group to the catalyst is 1:0.02-0.2;

[0043] And / or, in step (5), the equivalent ratio of the protected group X: activator: condensing agent: basic catalyst: protected group glycine is 1:(0.5-5):(0.5-5):(1-10):(0.5-3);

[0044] And / or, in step (6), the equivalent ratio of the dipeptide product 2 protected by the protecting group to the deprotecting agent is (1:1-20);

[0045] And / or, in step (7), the equivalent ratio of the deprotected tripeptide product: activator: condensing agent: basic catalyst: deprotected dipeptide product 2 is 1:(0.5-5):(0.5-5):(1-10):(0.5-3).

[0046] Preferably, step 2 is performed according to one of the following operations:

[0047] (a) Using a pentapeptide synthesis unit protected by a protecting group as a raw material, a deprotection reaction was carried out with a metal catalyst and a reducing agent in solvent 1. After purification, intermediate product 1, namely R1-(valine-proline-glycine-X-glycine), was obtained. n -OH;

[0048] (b1) Intermediate 1 was dissolved in a deprotecting agent to carry out a deprotection reaction. After purification, intermediate 2 was obtained, namely NH2-(valine-proline-glycine-X-glycine). n -R2;

[0049] (c1) Dissolve intermediate 1 in solvent 2, add activator and condensing agent to obtain activation system, then add intermediate 2 and alkaline reagent to activation system, and after purification, obtain R1-(valine-proline-glycine-X-glycine). 2n -R2;

[0050] (d1) Repeat step (a-c1) to obtain elastin-like polypeptides;

[0051] Alternatively, (a) using a pentapeptide synthesis unit protected by a protecting group as a raw material, a metal catalyst and a reducing agent are carried out in solvent 1 for deprotection reaction, and after purification, intermediate product 1 is obtained, namely R1-(valine-proline-glycine-X-glycine).n -OH;

[0052] (b2) Dissolve intermediate product 1 in solvent 2, add activator and condensing agent to obtain activated system, add solvent 3 to precipitate, centrifuge, dissolve the obtained solid 1 in deprotecting agent, add solvent 3 to precipitate, dissolve the obtained solid 2 in solvent 2, add basic reagent, react, add solvent 3 to precipitate, purify to obtain cyclic elastin-like polypeptide, namely c(valine-proline-glycine-X-glycine). n .

[0053] Preferably, in step (a), the metal catalyst is selected from at least one of carbon-supported palladium catalyst and carbon-supported palladium hydroxide catalyst; the reducing agent is selected from at least one of hydrogen, formic acid, ammonium formate, cyclohexene, and 1,4-cyclohexadiene; and the solvent 1 is selected from at least one of methanol, ethyl acetate, acetonitrile, water, and tetrahydrofuran.

[0054] And / or, in step (b1), the deprotecting agent is selected from at least one of trifluoroacetic acid, dioxane hydrochloride solution, p-toluenesulfonic acid, methanol hydrochloride solution, trimethylsilyl trifluoromethanesulfonate, acetic acid hydrogen bromide solution, dilute sulfuric acid, boron trifluoride ether, piperidine, tetramethylpiperidine, piperazine, 1,8-diazabicycloundec-7-ene, triethylamine, diethylamine, potassium carbonate, and N,N-diisopropylethylamine;

[0055] And / or, in step (c1), the solvent 2 is selected from at least one of dimethyl sulfoxide, N,N-dimethylformamide, and dichloromethane; the activator is selected from at least one of 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate, benzotriazole-1-oxytripyrrolylphosphine hexafluorophosphate, pentafluorophenol, N-hydroxysuccinimide, 1-hydroxy-7-azabenzotriazole-tetramethylurea hexafluorophosphate, and ethyl 2-oxime cyanoacetate; the condensing agent is selected from at least one of N,N'-diisopropylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N,N'-dicyclohexylcarbodiimide; and the basic reagent is at least one of N,N-diisopropylethylamine, triethylamine, diethylamine, N-methylmorpholine, and 2,4,6-trimethylpyridine.

[0056] And / or, in step (b2), the solvent 2 is selected from at least one of dimethyl sulfoxide, N,N-dimethylformamide, and dichloromethane; the activator is selected from at least one of 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate, benzotriazole-1-oxytripyrrolylphosphine hexafluorophosphate, pentafluorophenol, N-hydroxysuccinimide, 1-hydroxy-7-azabenzotriazole-tetramethylurea hexafluorophosphate, and ethyl 2-oxime cyanophosphate; the condensing agent is selected from N,N'-diisopropylcarbodiimide, The solvent 3 is anhydrous diethyl ether; the deprotecting agent is selected from at least one of trifluoroacetic acid, dioxane hydrochloride solution, p-toluenesulfonic acid, methanol hydrochloride solution, trimethylsilyl trifluoromethanesulfonate, acetic acid hydrogen bromide solution, dilute sulfuric acid, and boron trifluoride diethyl ether; the alkaline reagent is at least one of N,N-diisopropylethylamine, triethylamine, diethylamine, N-methylmorpholine, and 2,4,6-trimethylpyridine.

[0057] Preferably, in step (a), the equivalence ratio of the pentapeptide synthesis unit protected by the protecting group to the catalyst is 1:0.01-1;

[0058] And / or, in step (b1), the equivalent ratio of intermediate product 1 to deprotecting agent is 1:2-100; preferably, the equivalent ratio of intermediate product 1 to deprotecting agent is 1:10-20.

[0059] And / or, in step (c1), the equivalent ratio of intermediate product 1 activator: condensing agent: basic catalyst: intermediate product 2 is 1:(0.5-5):(0.5-5):(1-10):(0.5-3).

[0060] definition:

[0061] Orthogonal protection: Using two or more different protecting groups to protect multiple reactive functional groups in the same molecule. A specific deprotection condition will only act on one of the protecting groups and will not affect the other protecting groups. These protecting groups will not undergo deprotection reactions at the same time. This is called orthogonal protection.

[0062] Inert side-chain amino acids: Amino acids whose side chains do not contain reactive functional groups and do not require R4 groups to protect R3. For example, amino acids whose side chains do not contain carboxyl, amino, amide, hydroxyl, guanidine, or thiol groups; specifically, such as glycine, alanine, valine, leucine, isoleucine, phenylalanine, etc.

[0063] Amino acids with side-chain functionalization: Amino acids with reactive functional groups on their side chains require an R4 group to protect R3. Examples include amino acids with side chains containing carboxyl, amino, amide, hydroxyl, guanidine, or thiol groups; specifically, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, arginine, serine, threonine, cysteine, and methionine. R4 is at least one of triphenylmethyl, tert-butoxycarbonyl, tert-butyl, 2,2,4,6,7-pentamethylbenzofuran-5-sulfonyl, p-methoxytriphenylmethyl, trimethoxybenzyl, and N-methyltriphenylmethyl.

[0064] This invention provides a chemical synthesis method for elastin-like peptides. The method involves constructing a pentapeptide repeating sequence (valine-proline-glycine-X-glycine, where X is any amino acid other than proline, and all amino acids except glycine are independently selected from either L- or D-amino acids) via a liquid-phase reaction. Orthogonal protection is then formed on the amino and carboxyl groups, and the protecting groups at the amino or carboxyl termini are selectively removed. The carboxyl termini are then activated in the liquid phase and precisely coupled to the amino termini to prepare elastin-like peptides with a molecular weight range of 1000-500000. This method allows for precise control of the molecular weight, sequence, chirality, and topological structure of elastin-like peptides, improving the synthesis efficiency of high molecular weight elastin-like peptides and offering advantages such as large-scale synthesis and low cost.

[0065] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0066] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0067] Figure 1 This is a schematic diagram of the deprotection and repeated condensation of elastin-like polypeptides.

[0068] Figure 2 HPLC and LC-MS chromatograms of the synthetic units for the Boc / Bn protection strategy series.

[0069] Figure 3 The HPLC, SEC, and MALDI-ToF MS chromatograms of Boc-(VPGVG)4-OBn are shown.

[0070] Figure 4The HPLC and LC-MS chromatograms of Boc-(VPGGG)4-OBn are shown.

[0071] Figure 5 The HPLC and LC-MS chromatograms of Boc-(VPGAG)4-OBn are shown.

[0072] Figure 6 The HPLC and LC-MS chromatograms of Boc-(VPGFG)4-OBn are shown.

[0073] Figure 7 The HPLC and LC-MS chromatograms of Fmoc-(VPGNG)4-OBn are shown.

[0074] Figure 8 For chiral ELPs, with (vpGvG) n The HPLC and LC-MS chromatograms of the synthetic unit are shown as an example.

[0075] Figure 9 For chiral ELPs with different degrees of polymerization, (vpGvG) n Separation and mass spectrometry analysis results are shown as an example.

[0076] Figure 10 HPLC chromatograms of cyclic ELP samples with different degrees of polymerization.

[0077] Figure 11 The results of separation and mass spectrometry analysis of cyclic ELP samples with different degrees of polymerization are shown. Detailed Implementation

[0078] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0079] Example 1: Preparation method of elastin polypeptide

[0080] The general formula for the pentapeptide synthesis unit of elastin-like polypeptides is: R1-VPGXG-R2.

[0081]

[0082] Where R1 represents an amino protecting group, R2 represents a carboxyl protecting group, R3 is the side chain of amino acid X, and R4 is the side chain protecting group of amino acid X. X represents an amino acid other than proline. When X is an inert side chain amino acid that does not contain halogens or sulfur (e.g., glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), phenylalanine (F)), R1 = tert-butyloxycarbonyl (Boc), and R2 = benzyloxy (OBn).

[0083] Except for achiral glycine, all amino acids at each position can be replaced with dextrorotatory amino acids.

[0084] Step 1: Synthesis of the pentapeptide unit Boc-VPGXG-OBn

[0085] (1) Preparation of dipeptide Boc-PG-OBn: Boc-L-proline (Boc-P-OH) was mixed with 1-hydroxybenzotriazole (HOBT) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and dissolved in N,N-dimethylformamide (DMF) for activation. Glycine benzyl ester hydrochloride (HCl·NH2-G-OBn) and N,N-diisopropylethylamine (DIEA) were mixed and dissolved in N,N-dimethylformamide (DMF) and then added to the activation system. The reaction was carried out for 2 hours; the equivalence ratio of Boc-P-OH:HOBT:EDC·HCl:DIEA:HCl·NH2-G-OBn was 1:1:1.1:1.01:1.01. After the reaction, an equal volume of water was added to the reaction solution, and the product was extracted by forward extraction with ethyl acetate (EA). Excess DMF in EA was removed by back-extraction three times with pure water and saturated brine. Anhydrous sodium sulfate was used to absorb the water, and the product was evaporated to dryness with silica gel. Purification was then performed by column chromatography with a gradient of PE:EA 45%-60%. Boc-PG-OBn was obtained. The yield of this step was greater than 95%.

[0086] (2) Preparation of TFA·NH2-PG-OBn: The Boc-PG-OBn obtained in (1) was dissolved in trifluoroacetic acid and stirred at room temperature for 1 hour; the equivalent ratio of Boc-PG-OBn:TFA was 1:10. After the reaction was completed, the solution was evaporated to dryness. The yield of this step was 100%.

[0087] (3) Preparation of tripeptide Boc-VPG-OBn: Boc-L-valine (Boc-V-OH) was activated by dissolving it in DMF with HOBT and EDC·HCl. The TFA·NH2-PG-OBn obtained in (2) was added to the activation system after mixing with DIEA and DMF, wherein the equivalent ratio of Boc-V-OH:HOBT:EDC·HCl:DIEA:TFA·NH2-PG-OBn = 1:1:1.1:5:1.01. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, an equal volume of water was added to the reaction solution and the product was extracted by forward extraction with ethyl acetate (EA). Excess DMF in EA was removed by back-extraction three times with pure water and saturated brine. Anhydrous sodium sulfate was used to absorb water, silica gel was added to evaporate the solution, and the product was purified by column chromatography with a gradient of PE:EA 50%-70% to obtain Boc-VPG-OBn. The yield of this step was greater than 95%.

[0088] (4) Preparation of Boc-VPG-OH: The Boc-VPG-OBn obtained in (3) was dissolved in tetrahydrofuran (THF), and a catalyst supported on carbon palladium (Pd / C) was added, wherein the equivalent ratio of Boc-VPG-OBn:Pd / C = 1:0.02. The mixture was stirred for 4-6 hours under hydrogen atmosphere. After the reaction was completed, the filtrate was filtered, and the solvent was removed by rotary evaporation to obtain Boc-VPG-OH. The yield of this step was greater than 95%.

[0089] (5) Preparation of dipeptide Boc-XG-OBn: Boc-LX (Boc-X-OH) was mixed with 1-hydroxybenzotriazole (HOBT) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and dissolved in N,N-dimethylformamide (DMF) for activation. Glycine benzyl ester hydrochloride (HCl·NH2-G-OBn) and N,N-diisopropylethylamine (DIEA) were mixed and dissolved in N,N-dimethylformamide (DMF) and then added to the activated solution. In the system, the equivalence ratio of Boc-X-OH:HOBT:EDC·HCl:DIEA:HCl·NH2-G-OBn is 1:1:1.1:1.01:1.01. The mixture is stirred at room temperature for 2 hours. After the reaction is complete, an equal volume of water is added to the reaction solution, and the product is extracted by forward extraction with ethyl acetate (EA). Excess DMF in EA is removed by back-extraction three times with pure water and saturated brine. The product is then dehydrated with anhydrous sodium sulfate, evaporated to dryness with silica gel, and purified by column chromatography with a gradient of PE:EA 20%-50% to obtain Boc-VG-OBn. The yield of this step is greater than 95%.

[0090] (6) Preparation of HCl·NH2-XG-OBn: The Boc-XG-OBn obtained in (5) was added to a 1,4-dioxane hydrochloride solution and stirred at room temperature for 2 hours to precipitate the product, wherein the ratio of Boc-XG-OBn to 1,4-dioxane hydrochloride was 1:2 (w:v). Diethyl ether was added, the mixture was stirred, filtered, and the filter cake was dried in a vacuum oven to obtain HCl·NH2-XG-OBn. The yield of this step was greater than 95%.

[0091] (7) Preparation of the pentapeptide Boc-VPGXG-OBn: The Boc-VPG-OH obtained in (4) was mixed with 2,3,4,5,6-pentafluorophenol (PFP) and N,N'-diisopropylcarbodiimide (DIC) and activated in dichloromethane (DCM). The HCl·NH2-VG-OBn and N,N-diisopropylethylamine (DIEA) obtained in (6) were mixed and dissolved in N,N-dimethylformamide (DCM) and then added to the activation system. The ratio of Boc-VPG-OH:PFP:DIC:DIEA:HCl NH2-XG-OBn was 1:1:1.1:1.01:1.01. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the insoluble matter was removed by filtration, and the mixture was evaporated to dryness using silica gel. The mixture was then purified by column chromatography with a gradient of MeOH:DCM 2.5%-5%. Boc-VPGXG-OBn was obtained. The yield of this step was greater than 80%. Purity and molecular weight were confirmed by HPLC and LC-MS.

[0092] Step 2: Elastin-like peptide Boc-(VPGXG) n -OBn synthesis

[0093] (1) Debenzylidene esterification: Boc-(VPGXG) n When -OBn is mixed with Pd / C, the equivalence ratio is Boc-(VPGXG). n -OBn:Pd / C = 1:0.02, MeOH was added, and OBn was removed under hydrogen atmosphere. After the reaction was complete, the mixture was filtered, and the filtrate was evaporated under reduced pressure to obtain Boc-(VPGXG). n -OH, the yield of this step is greater than 95%.

[0094] (2) Remove Boc: Remove Boc-(VPGXG) n -OBn dissolves in TFA, where the equivalent ratio of Boc-(VPGXG) is... n -OBn:TFA = 1:10, stir and react for 2 hours; after the reaction is complete, add diethyl ether and stir to precipitate. After the product is completely precipitated, filter and dry the residue. The obtained product can be used directly without further purification.

[0095] (3) Coupling: The Boc-(VPGXG) obtained in step two (1) is coupled together. n -OH was dissolved in DMSO and stirred. PFP and DIC were added successively and stirred to activate the product, resulting in an activated intermediate. DIEA was then added to the TFA·NH2-(VPGXG) obtained in step 2). n -OBn, stir well and then add to the activation system, wherein the equivalent ratio of Boc-(VPGXG) is... n -OH:PFP:DIC:DIEA:TFA·NH2-(VPGXG) n-OBn = 1:1.01:1.1:1.5:1.01, stir the reaction for 2-3 hours, and then separate and purify to prepare Boc-(VPGXG). 2n -OBn.

[0096] (4) Repeat step two (4-6) to prepare Boc-(VPGXG). n -OBn (n is 2, 4, 8, 16, 32, 64, 128) achieves an exponential increase in the molecular weight of ELP.

[0097] (5) If n = 1 or 2, the separation and purification in step (3) is a rapid preparative liquid chromatography with dichloromethane / methanol as the mobile phase.

[0098] (6) If n is greater than 2, the separation and purification in step (3) is as follows: first, precipitate the reaction solution in anhydrous diethyl ether, centrifuge at 7000 rpm for 5 min, repeat twice to obtain crude product, and then use high performance liquid chromatography for preparation and purification.

[0099] Example 2: Preparation method of elastin polypeptide

[0100] The general formula for the pentapeptide synthesis unit of elastin-like polypeptides is: R1-VPGXG-R2.

[0101]

[0102] Where R1 represents an amino protecting group, R2 represents a carboxyl protecting group, R3 is the side chain of amino acid X, R4 is the side chain protecting group of amino acid X, and X represents an amino acid other than proline. When X is tryptophan (W), tyrosine (Y), aspartate, histidine (H), asparagine (N), glutamic acid (E), lysine (K), glutamine (Q), arginine (R), serine (S), threonine (T), R1 = 9-fluorenylmethoxycarbonyl (Fmoc), and R2 = benzyloxy (OBn).

[0103] Except for achiral glycine, all amino acids at each position can be replaced with dextrorotatory amino acids.

[0104] Step 1: Synthesis of the pentapeptide unit Fmoc-VPGXG-OBn

[0105] (1) Preparation of tripeptide Fmoc-VPG-OBn: Fmoc-L-valine (Fmoc-V-OH) was activated by dissolving it in DMF with HOBT and EDC·HCl. The TFA NH2-PG-OBn obtained in step (2) of Example 1 was added to the activation system after mixing with DIEA and DMF, wherein the equivalent ratio of Fmoc-V-OH:HOBT:EDC·HCl:DIEA:TFA·NH2-PG-OBn = 1:1:1.1:5:1.01. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, an equal volume of water was added to the reaction solution and the product was extracted by forward extraction with ethyl acetate (EA). Excess DMF in EA was removed by back-extraction three times with pure water and saturated brine. Anhydrous sodium sulfate was used to absorb water, silica gel was added to evaporate the solution, and the product was purified by column chromatography with a gradient of PE:EA 50%-70% to obtain Fmoc-VPG-OBn. The yield of this step was greater than 95%.

[0106] (2) Preparation of Fmoc-VPG-OH: The Fmoc-VPG-OBn obtained in (1) was dissolved in THF, and a catalyst supported on carbon palladium (Pd / C) was added. The mixture was stirred in a hydrogen atmosphere for 4-6 hours. After the reaction was completed, the filtrate was filtered, and the solvent was removed by rotary evaporation to obtain Fmoc-VPG-OH. The yield of this step was greater than 95%.

[0107] (3) Preparation of dipeptide Fmoc-XG-OBn: Fmoc-LX (Fmoc-X-OH) was mixed with 1-hydroxybenzotriazole (HOBT) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and activated in N,N-dimethylformamide (DMF). Glycine benzyl ester hydrochloride (HCl·NH2-G-OBn) and N,N-diisopropylethylamine (DIEA) were mixed and dissolved in N,N-dimethylformamide (DMF) and then... The product was added to the activation system with an equivalent ratio of Fmoc-X-OH:HOBT:EDC·HCl:DIEA:HCl·NH2-G-OBn = 1:1:1.1:1.01:1.01. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, an equal volume of water was added to the reaction solution and the product was extracted by forward extraction with dichloromethane (DCM). Excess DMF in the DCM was removed by back-extraction three times with pure water and saturated brine. Anhydrous sodium sulfate was used to absorb the water. The obtained product could be used directly without further purification.

[0108] (4) Preparation of NH2-XG-OBn: Dissolve the Fmoc-XG-OBn obtained in (3) in DMF, add potassium fluoride (KF) and triethylamine (NEt3), wherein the equivalent ratio of NH2-XG-OBn:KF:NEt3 = 1:5:10, and stir for 2 hours. After the reaction is completed, add an equal volume of water to the reaction solution and extract the product by forward extraction with dichloromethane (DCM). Remove excess DMF and triethylamine in DCM by back-extraction three times with dilute hydrochloric acid water and saturated brine. Use anhydrous sodium sulfate to absorb water. The obtained product can be used directly without further purification.

[0109] (5) Preparation of the pentapeptide Fmoc-VPGXG-OBn: The Fmoc-VPG-OH obtained in (2) was mixed with 2,3,4,5,6-pentafluorophenol (PFP) and N,N'-diisopropylcarbodiimide (DIC) and activated in dichloromethane (DCM). The NH2-XG-OBn obtained in (11) was dissolved in N,N-dimethylformamide (DMF) and added to the activation system. The ratio of Fmoc-VPG-OH:PFP:DIC:NH2-XG-OBn was 1:1:1.1:1:1.01. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the insoluble matter was removed by filtration, and the mixture was evaporated to dryness using silica gel. The mixture was then purified by column chromatography with a gradient of DCM:EA 30%-60%. Fmoc-VPGXG-OBn was obtained. The yield of this step was greater than 80%. The purity and molecular weight were confirmed by HPLC and LC-MS.

[0110] Step 2: Develop the elastin-like peptide Fmoc-(VPGXG) n -OBn synthesis

[0111] (1) Debenzylidene esterification: Fmoc-(VPGXG) n When -OBn is mixed with Pd / C, the equivalent ratio of Fmoc-(VPGXG) is... n -OBn:Pd / C = 1:0.02, MeOH was added, and OBn was removed under hydrogen atmosphere. After the reaction was complete, the mixture was filtered through a membrane, and the filtrate was evaporated under reduced pressure to obtain Fmoc-(VPGXG). n -OH, the yield of this step is greater than 95%.

[0112] (2) De-Boc: Remove Fmoc-(VPGXG) n -OBn is dissolved in DMF, and DBU is added, wherein the equivalent ratio of Fmoc-(VPGXG) is... n -OBn:DBU = 1:2, stir and react for 2 hours; after the reaction is complete, add an equal volume of water to the reaction solution and extract the product by forward extraction with dichloromethane (DCM). Remove excess DMF and triethylamine from the DCM by back-extraction three times with dilute hydrochloric acid and saturated brine. Use anhydrous sodium sulfate to absorb water. The obtained product can be used directly without further purification.

[0113] (3) Coupling: The Fmoc-(VPGXG) obtained in step two (1) is coupled together. n -OH was dissolved in DCM and stirred. PFP and DIC were added successively and stirred to activate the intermediate, and then NH2-(VPGXG) obtained in step 2 (2) was added. n -OBn, stir well and then add to the activation system, wherein the equivalent ratio of Fmoc-(VPGXG) is... n -OH:PFP:DIC:TFA·NH2-(VPGXG) n -OBn = 1:1.01:1.1:1.01, stir and react for 2-3 hours. After the reaction is complete, add an equal volume of water to the reaction solution and extract the product by forward extraction with dichloromethane (DCM). Use anhydrous sodium sulfate to absorb the water, remove the solvent by rotary evaporation, and then purify by high performance liquid chromatography (HPLC) to prepare Fmoc-(VPGXG). 2n -OBn.

[0114] (4) Repeat step two (1-3) to prepare Fmoc-(VPGXG). n -OBn (n is 2, 4, 8, 16, 32, 64, 128).

[0115] Example 3: Preparation method of elastin polypeptide

[0116] The general formula for the pentapeptide synthesis unit of elastin-like polypeptides is: R1-VPGXG-R2.

[0117]

[0118] Where R1 represents an amino protecting group, R2 represents a carboxyl protecting group, R3 is the side chain of amino acid X, R4 is the side chain protecting group of amino acid X, and X represents an amino acid other than proline. When X is a side chain functionalized amino acid or an amino acid containing halogen or sulfur (e.g., cysteine), R1 = allyloxycarbonyl (Alloc), R2 = methoxy (OMe) or ethoxy (OEt).

[0119] Except for achiral glycine, all amino acids at each position can be replaced with dextrorotatory amino acids.

[0120] Step 1: Synthesis of the pentapeptide unit Alloc-VPGXG-Et

[0121] (1) Preparation of dipeptide Boc-PG-Et: Boc-L-proline (Boc-P-OH) was mixed with 1-hydroxybenzotriazole (HOBT) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and dissolved in N,N-dimethylformamide (DMF) for activation. Glycine ethyl ester hydrochloride (HCl·NH2-G-Et) and N,N-diisopropylethylamine (DIEA) were mixed and dissolved in N,N-dimethylformamide (DMF) and then added to the activation system. The reaction should last for 2 hours; the equivalence ratio of Boc-P-OH:HOBT:EDC·HCl:DIEA:HCl·NH2-G-Et is 1:1:1.1:1.01:1.01. After the reaction, an equal volume of water is added to the reaction solution, and the product is extracted by forward extraction with ethyl acetate (EA). Excess DMF in EA is removed by back-extraction three times with pure water and saturated brine. Water is absorbed using anhydrous sodium sulfate, and the solution is evaporated to dryness using silica gel. Purification is then performed by column chromatography with a gradient of PE:EA 45%-60%. Boc-PG-Et is obtained. The yield of this step is greater than 95%.

[0122] (2) Preparation of TFA·NH2-PG-Et: The Boc-PG-Et obtained in (1) was dissolved in trifluoroacetic acid and stirred at room temperature for 1 hour. The equivalent ratio of Boc-PG-Et:TFA was 1:10. After the reaction was completed, the solution was evaporated to dryness. The yield of this step was 100%.

[0123] (3) Preparation of Alloc-V-OH: L-valine (NH2-V-OH) was mixed with potassium carbonate (K2CO3), and H2O / THF (v:v = 1:1) was added. The mixture was stirred until dissolved, and then allyl chloroformate (Alloc-Cl) was added, wherein NH2-V-OH:K2CO3:Alloc-C = 1:1.5:1.2. The mixture was stirred for 16 h. After the reaction was completed, tetrahydrofuran (THF) was removed by rotary evaporation, and the mixture was extracted with ethyl acetate (EA). The aqueous phase was taken and the pH was adjusted to 2. Dichloromethane (DCM) was then added for extraction. The dichloromethane (DCM) phase was collected and washed with saturated brine. Anhydrous sodium sulfate was used to absorb the water. The final product was obtained by rotary evaporation to remove DCM. The final product can be used directly without further purification.

[0124] (4) Preparation of the tripeptide Alloc-VPG-Et: Alloc-L-valine (Alloc-V-OH) obtained in (3) was dissolved in DMF with HOBT and EDC·HCl for activation. TFA NH2-PG-Et obtained in step (2) was mixed with DIEA and DMF and added to the activation system. The equivalent ratio of Alloc-V-OH:HOBT:EDC·HCl:DIEA:TFA·NH2-PG-Et was 1:1:1.1:5:1.01. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, an equal volume of water was added to the reaction solution and the product was extracted by forward extraction with ethyl acetate (EA). Excess DMF in EA was removed by back-extraction three times with pure water and saturated brine. Anhydrous sodium sulfate was used to absorb water, silica gel was added and evaporated to dryness, and the product was purified by column chromatography with a gradient of PE:EA 50%-70% to obtain Alloc-VPG-Et. The yield of this step was greater than 95%.

[0125] (5) Preparation of Alloc-VPG-OH: The Alloc-VPG-Et obtained in (4) was dissolved in THF, and NaOH aqueous solution was added; after the reaction was completed, THF was removed by rotary evaporation, ethyl acetate was added to extract the product by forward extraction, NaOH was neutralized with dilute hydrochloric acid solution, and then washed with pure water and saturated brine, dried with anhydrous sodium sulfate, and ethyl acetate was removed by rotary evaporation to obtain Alloc-VPG-OH. The yield of this step was greater than 95%.

[0126] (6) Preparation of dipeptide Fmoc-XG-Et: Fmoc-L-cysteine ​​(Fmoc-X-OH) was mixed with 1-hydroxybenzotriazole (HOBT) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and activated in N,N-dimethylformamide (DMF). Glycine benzyl ester hydrochloride (HCl·NH2-G-Et) and N,N-diisopropylethylamine (DIEA) were mixed and dissolved in N,N-dimethylformamide (DMF). Then add it to the activation system, where the equivalent ratio of Fmoc-X-OH:HOBT:EDC·HCl:DIEA:HCl·NH2-G-Et is 1:1:1.1:1.01:1.01, and stir at room temperature for 2 hours. After the reaction is completed, add an equal volume of water to the reaction solution and extract the product by forward extraction with dichloromethane (DCM). Remove excess DMF from the DCM by back-extraction three times with pure water and saturated brine. Use anhydrous sodium sulfate to absorb water. The obtained product can be used directly without further purification.

[0127] (7) Preparation of NH2-XG-Et: Dissolve the Fmoc-XG-Et obtained in (6) in DMF, add potassium fluoride (KF) and triethylamine (NEt3), wherein the equivalent ratio of Fmoc-XG-Et:KF:NEt3 is 1:5:10, and stir the reaction for 2 hours; after the reaction is completed, add an equal volume of water to the reaction solution and extract the product by forward extraction with dichloromethane (DCM), and back-extract the excess DMF and triethylamine in DCM three times by dilute hydrochloric acid water and saturated brine, and use anhydrous sodium sulfate to absorb water. The obtained product can be used directly without further purification.

[0128] (8) Preparation of the pentapeptide Alloc-VPGXG-Et: Alloc-VPG-OH obtained in (5) was mixed with 2,3,4,5,6-pentafluorophenol (PFP) and N,N'-diisopropylcarbodiimide (DIC) and activated in dichloromethane (DCM). NH2-XG-Et was dissolved in N,N-dimethylformamide (DMF) and added to the activation system. The ratio of Alloc-VPG-OH:PFP:DIC:NH2-NG-Et was 1:1:1.1:1:1.01. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the insoluble matter was removed by filtration, and the mixture was evaporated to dryness using silica gel. The mixture was then purified by column chromatography with a gradient of DCM:EA 30%-60%. Alloc-VPGXG-Et was obtained. The yield of this step was greater than 80%. The purity and molecular weight were confirmed by HPLC and LC-MS.

[0129] Step 2: Alloc-(VPGXG), a polypeptide similar to elastin. n Synthesis of -Et

[0130] (1) Preparation of Alloc-(VPGXG) n -OH: Alloc-(VPGXG) n -Et is dissolved in THF, and NaOH aqueous solution is added; the reaction is stirred for 2 hours. After the reaction is complete, THF is removed by rotary evaporation, and the product is extracted by forward extraction with dichloromethane. The NaOH is neutralized with dilute hydrochloric acid solution, and then washed with pure water and saturated brine. The product is dried over anhydrous sodium sulfate, and ethyl acetate is removed by rotary evaporation. The yield of this step is greater than 95%.

[0131] (2) Remove Alloc: Remove Alloc-(VPGXG) n -Et is mixed with triphenylphosphine palladium, wherein the equivalent ratio of Alloc-(VPGXG) is... n -Et:triphenylphosphinepalladium = 1:0.02, add MeOH, stir until alloc is completely removed, after the reaction is complete, filter using a filter membrane, and evaporate the filtrate under reduced pressure to obtain NH2-(VPGXG). n -Et, the yield in this step is greater than 95%.

[0132] (3) Coupling: Alloc-(VPGXG) obtained in step 2(1) n -OH was dissolved in dimethyl sulfoxide and stirred. PFP and DIC were added successively and stirred to activate the mixture, thus obtaining an activated intermediate. Then, NH2-(VPGXG) obtained in step two (2) was added. n -Et, stir well and then add to the activation system, wherein the equivalent ratio of Alloc-(VPGXG) is... n -OH:PFP:DIC:TFA·NH2-(VPGXG) n -Et = 1:1.01:1.1:1.01 The mixture was stirred for 2-3 hours. After the reaction was complete, an equal volume of water was added to the reaction solution, and the product was extracted by dichloromethane (DCM) in the forward direction. Anhydrous sodium sulfate was used to absorb the water, and the solvent was removed by rotary evaporation. The product was then purified by high-performance liquid chromatography (HPLC) to obtain Alloc-(VPGXG). 2n -Et.

[0133] (4) Repeat step two (1-3) to prepare Alloc-(VPGXG). n -Et (n is 2, 4, 8, 16, 32, 64, 128).

[0134] Example 4: Class 4 Elastin Polypeptide - (VPGVG) n Preparation method of -

[0135]

[0136] in, n = 2, 4, 8, 16, 32, 64, 128.

[0137] Following the preparation method of Example 1, where X is valine (V), Boc-(VPGVG) was prepared. n -OBn, (n=2,4,8,16,32,64,128), the short peptides prepared above were tested by high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), gel permeation chromatography (SEC), and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MA LDI-TOP MS). Figure 2 For intermediates, HPLC and LC-MS were performed. Figure 3The images show the HPLC, SEC, and MALDI-ToF MS chromatograms of Boc-(VPGVG)4-OBn. The purity of Boc-VG-OBn is greater than 98%, with a molecular weight of 364.2; the purity of HCl-NH2-VG-OBn is greater than 98%, with a molecular weight of 299.6; the purity of Boc-VPG-OBn is greater than 98%, with a molecular weight of 461.2; the purity of Boc-VPG-OH is greater than 90%, with a molecular weight of 371.1; the purity of Boc-(VPGVG)4-OBn is greater than 98%, with a molecular weight of 1845.0; the purity of Boc-(VPGVG)8-OBn is greater than 98%, with a molecular weight of 3481.9; and the purity of Boc-(VPGVG)4-OBn is greater than 98%, with a molecular weight of 3481.9. 16 -OBn purity greater than 98%, molecular weight 6755.8; Boc-(VPGVG) 32 -OBn purity greater than 98%, molecular weight 13303.5; Boc-(VPGVG) 64 -OBn purity greater than 98%, molecular weight 26398.9; Boc-(VPGVG) 128 -OBn has a purity greater than 98% and a molecular weight of 52589.8.

[0138] Example 5: Class 5 Elastin Peptide - (VPGGG) n Preparation method of -

[0139]

[0140] Where R3 = H, n = 2, 4, 8, 16.

[0141] Following the preparation method of Example 1, where X is glycine (G), Boc-(VPGGG) was prepared. n -OBn (n=2,4,8,16), the above-prepared elastin-like peptides were tested by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Figure 4 The images show the HPLC and LC-MS chromatograms of Boc-(VPGGG)4-OBn, where the purity of Boc-(VPGGG)4-OBn is greater than 98% and the molecular weight is 1676.8.

[0142] Example 6: Class 6 Elastin Polypeptide (VPGAG) n Preparation method of -

[0143]

[0144] in, n = 2, 4, 8.

[0145] Following the preparation method of Example 1, where X is aspartate, Boc-(VPGAG) was prepared. n -OBn (n=2,4,8) were tested using high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Figure 5 The HPLC and LC-MS chromatograms of Boc-(VPGAG)4-OBn are shown. The purity of Boc-(VPGAG)4-OBn is greater than 98%, and its molecular weight is 1732.9.

[0146] Example 7: Elastin Polypeptide (VPGFG) n Preparation method of -

[0147]

[0148] in, n = 2, 4, 8.

[0149] Following the preparation method of Example 1, where X is phenylalanine (F), Boc-(VPGFG) was prepared. n -OBn (n=2,4,8) were tested using high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Figure 6 The images show the HPLC and LC-MS chromatograms of Boc-(VPGFG)4-OBn, where the purity of Boc-(VPGFG)4-OBn is greater than 98% and the molecular weight is 2037.0.

[0150] Example 8: Class 8 Elastin Polypeptide-(VPGNG) n Preparation method of -

[0151]

[0152] in, n = 2, 4, 8.

[0153] Following the preparation method of Example 2, where X is asparagine (N), Fmoc-(VPGNG) was prepared. n -OBn (n=2,4,8) were tested using high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Figure 7 The figures show the HPLC and LC-MS chromatograms of Fmoc-(VPGNG)4-OBn, where the purity of Fmoc-(VPGNG)4-OBn is greater than 98% and the molecular weight is 2995.3.

[0154] Example 9: Class 9 Elastin Polypeptide-(VPGSG)n Preparation method of -

[0155]

[0156] in, n = 2, 4, 8.

[0157] Following the preparation method of Example 3, where X is cysteine ​​(C), Alloc-(VPGCG) was prepared. n -Et(n=2,4,8).

[0158] Example 10: Preparation method of chiral elastin-like polypeptides

[0159] (vpGvG) n As a representative:

[0160] in, n = 2, 4, 8, 16, 32.

[0161] 1) Preparation of the dipeptide Boc-pG-OBn: Boc-D-proline (Boc-p-OH) was mixed with 1-hydroxybenzotriazole (HOBT) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and dissolved in N,N-dimethylformamide (DMF) for activation. Glycine benzyl ester hydrochloride (HCl·NH2-G-OBn) and N,N-diisopropylethylamine (DIEA) were mixed and dissolved in N,N-dimethylformamide (DMF) and then added to the activation system. The reaction should last for 2 hours; the equivalence ratio of Boc-p-OH:HOBT:EDC·HCl:DIEA:HCl·NH2-G-OBn is 1:1:1.1:1.01:1.01. After the reaction, an equal volume of water is added to the reaction solution, and the product is extracted by forward extraction with ethyl acetate (EA). Excess DMF in EA is removed by back-extraction three times with pure water and saturated brine. Water is absorbed using anhydrous sodium sulfate, and the solution is evaporated to dryness using silica gel. Purification is then performed by column chromatography with a gradient of PE:EA 45%-60%. Boc-pG-OBn is obtained. The yield of this step is greater than 95%.

[0162] 2) Preparation of TFA·NH2-pG-OBn: The Boc-pG-OBn obtained in step 1) was dissolved in trifluoroacetic acid and stirred at room temperature for 1 hour; after the reaction was completed, the solution was evaporated to dryness. The yield of this step was 100%.

[0163] 3) Preparation of the tripeptide Boc-vpG-OBn: Boc-D-valine (Boc-v-OH) was activated by dissolving it in DMF with HOBT and EDC·HCl. The TFA·NH2-PG-OBn obtained in step 2) was mixed with DIEA and DMF and added to the activation system, where the equivalent ratio of Boc-v-OH:HOBT:EDC·HCl:DIEA:TFA·NH2-pG-OBn was 1:1:1.1:5:1.01. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, an equal volume of water was added to the reaction solution, and the product was extracted by forward extraction with ethyl acetate (EA). Excess DMF in EA was removed by back-extraction three times with pure water and saturated brine. Anhydrous sodium sulfate was used to absorb the water, and the mixture was evaporated to dryness using silica gel. The product was then purified by column chromatography with a gradient of PE:EA 50%-70% to obtain Boc-vpG-OBn. The yield of this step was greater than 95%.

[0164] 4) Preparation of Boc-vpG-OH: The Boc-vpG-OBn obtained in step 3) was dissolved in THF, and a carbon-supported palladium (Pd / C) catalyst was added, wherein the equivalent ratio of Boc-vpG-OBn:Pd / C = 1:0.02. The mixture was stirred under hydrogen atmosphere for 4-6 hours. After the reaction was completed, the filtrate was filtered, and the solvent was removed by rotary evaporation to obtain Boc-vpG-OH. The yield of this step was greater than 95%.

[0165] 5) Preparation of dipeptide Boc-vG-OBn: Boc-D-valine (Boc-v-OH) was mixed with 1-hydroxybenzotriazole (HOBT) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and dissolved in N,N-dimethylformamide (DMF) for activation. Glycine benzyl ester hydrochloride (HCl·NH2-G-OBn) and N,N-diisopropylethylamine (DIEA) were mixed and dissolved in N,N-dimethylformamide (DMF) and then added to the activation system. The equivalence ratio was... The reaction mixture of Boc-v-OH:HOBT:EDC·HCl:DIEA:HCl·NH2-G-OBn was stirred at room temperature for 2 hours. After the reaction was complete, an equal volume of water was added to the reaction solution, and the product was extracted by forward extraction with ethyl acetate (EA). Excess DMF in EA was removed by back-extraction three times with pure water and saturated brine. The solution was then dehydrated with anhydrous sodium sulfate, evaporated to dryness with silica gel, and purified by column chromatography with a gradient of PE:EA 20%-50% to obtain Boc-vG-OBn. The yield of this step was greater than 95%.

[0166] 6) Preparation of HCl·NH2-vG-OBn: The Boc-vG-OBn obtained in step 5) was added to a 1,4-dioxane hydrochloride solution and stirred at room temperature for 2 hours to precipitate the product, wherein the ratio of Boc-vG-OBn to 1,4-dioxane hydrochloride was 1:2 (w:v). Diethyl ether was added, the mixture was stirred, filtered, and the filter cake was dried in a vacuum oven to obtain HCl·NH2-vG-OBn. The yield of this step was greater than 95%.

[0167] 7) Preparation of the pentapeptide Boc-vpGvG-OBn: The Boc-vpG-OH obtained in step 4) was mixed with 2,3,4,5,6-pentafluorophenol (PFP) and N,N'-diisopropylcarbodiimide (DIC) and activated in dichloromethane (DCM). HCl·NH2-vG-OBn and N,N-diisopropylethylamine (DIEA) were mixed and dissolved in N,N-dimethylformamide (DCM) and added to the activation system. The ratio of Boc-vpG-OH:PFP:DIC:DIEA:HCl NH2-vG-OBn was 1:1:1.1:1.01:1.01. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, insoluble matter was removed by filtration, and the mixture was evaporated to dryness using silica gel. Purification was then performed by column chromatography with a gradient of MeOH:DCM 2.5%-5%. Boc-vpGvG-OBn was obtained. The yield of this step was greater than 80%. Purity and molecular weight were confirmed by HPLC and LC-MS.

[0168] 8) Preparation of Boc-(vpGvG) n -OH: This refers to the Boc-(vpGvG) group. n When -OBn is mixed with Pd / C, the equivalence ratio is Boc-(vpGvG). n -OBn:Pd / C = 1:0.02, MeOH is added, and OBn is removed under hydrogen atmosphere. After the reaction is complete, the mixture is filtered through a filter membrane, and the filtrate is evaporated under reduced pressure to obtain Boc-(vpGvG)n-OH. The yield of this step is greater than 95%.

[0169] 9) Preparation of TFA·NH2-(vpGvG) n -OBn: This will change the Boc to (vpGvG). n -OBn dissolves in TFA, where the equivalence ratio is Boc-(vpGvG). n -OBn:TFA = 1:10, stir and react for 2 hours; after the reaction is complete, add diethyl ether and stir to precipitate. After the product is completely precipitated, filter and dry the residue. The obtained product can be used directly without further purification.

[0170] 10) Preparation of Boc-(vpGvG) n -OBn: Subtract (vpGvG) from the Boc obtained in step 8). n-OH is dissolved in DMSO and stirred. PFP and DIC are added successively and stirred to activate the mixture, resulting in an activated intermediate. DIEA is then added to the TFA·NH2-(vpGvG) obtained in step 9). n -OBn, stir well and then add to the activation system, wherein the equivalent ratio of Boc-(vpGvG) is... n -OH:PFP:DIC:DIEA:TFA·NH2-(vpGvG) n -OBn = 1:1.01:1.1:1.5:1.01, stir and react for 2-3 hours. After the reaction is complete, evaporate the reaction liquid under reduced pressure, transfer it to a centrifuge tube, add anhydrous diethyl ether to precipitate, centrifuge at 7000 rpm for 5 minutes, repeat twice, purify using high performance liquid chromatography (HPLC) and collect the pure fraction, then freeze-dry to obtain Boc-(vpGvG). 2n -OBn(n=2,4,8).

[0171] 11) Repeat steps (8-10) to prepare Boc-(vpGvG) n -OBn (n=2,4,8) achieves an exponential increase in the molecular weight of ELP.

[0172] 12) The prepared elastin-like peptides and intermediates were tested using high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), gel permeation chromatography (SEC), and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-ToF MS). Figure 8 and Figure 9 Among them, Boc-vG-OBn has a purity greater than 98% and a molecular weight of 364.2; HCl NH2-vG-OBn has a purity greater than 98% and a molecular weight of 299.6; Boc-vpG-OBn has a purity greater than 98% and a molecular weight of 461.2; Boc-vpG-OH has a purity greater than 90% and a molecular weight of 371.1; Boc-(vpGvG)4-OBn has a purity greater than 98% and a molecular weight of 1845.0; Boc-(vpGvG)8-OBn has a purity greater than 98% and a molecular weight of 3481.9; Boc-(vpGvG) 16 -OBn purity greater than 98%, molecular weight 6755.8; Boc-(vpGvG) 32 -OBn has a purity greater than 98% and a molecular weight of 13303.5.

[0173] Example 11: Preparation method of cyclic elastin-like polypeptides

[0174] Represented by c(VPGVG)n:

[0175]

[0176] in, n = 4, 8, 16, 32.

[0177] Step 1: Synthesis of the pentapeptide unit Boc-VPGXG-OBn

[0178] Following the preparation method in step one of Example 1, where X is valine (V), Boc-VPGVG-OBn was prepared.

[0179] Step 2: Synthesis of cyclic elastin-like polypeptides

[0180] 1) Preparation of Boc-(VPGVG) n -OH: This refers to Boc-(VPGVG) n After mixing -OBn with Pd / C, MeOH is added to remove OBn under hydrogen atmosphere. After the reaction is complete, the mixture is filtered through a membrane, and the filtrate is evaporated under reduced pressure to obtain Boc-(VPGNG)n-OH. The yield of this step is greater than 95%.

[0181] 2) Preparation of c(VPGVG) n The Boc-(VPGVG) obtained in step 1) n -OH and PFP were mixed and dissolved in DMSO. DIC was added and stirred for 2 hours. Anhydrous diethyl ether was added to precipitate the mixture. The mixture was centrifuged at 7000 rpm for 5 minutes, and this process was repeated twice. The obtained solid was then added to trifluoroacetic acid and stirred for 10 minutes. Anhydrous diethyl ether was added again to precipitate the mixture, and the mixture was centrifuged at 7000 rpm for 5 minutes, and this process was repeated twice. The obtained solid was dissolved in DMSO. DIEA was added and the mixture was stirred and reacted for 10 minutes. After the reaction was completed, anhydrous diethyl ether was added to precipitate the mixture, and the mixture was centrifuged for 5 minutes, and this process was repeated twice. The mixture was purified by high-performance liquid chromatography (HPLC), and the purified fraction was collected and lyophilized to obtain c(VPGVG). n (n = 2, 4, 8, 16).

[0182] 3) The prepared elastin-like peptides and intermediates were tested using high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), gel permeation chromatography (SEC), and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-ToF MS). Figure 10 and Figure 11 The purity of Boc-(VPGVG)4-OH is greater than 98%; the purity of Boc-(VPGVG)8-OH is greater than 98%; and the purity of Boc-(VPGVG)4-OH is greater than 98%. 16 -OH purity greater than 98%; Boc-(VPGVG) 32The purity of -OH is greater than 98%; the purity of c(VPGVG)4 is greater than 98%, and the molecular weight is 1636.9; the purity of c(VPGVG)8 is greater than 98%, and the molecular weight is 3273.8; c(VPGVG) 16 The purity is greater than 98%, and the molecular weight is 6547.7; c(VPGVG) 32 The purity is greater than 98%, and the molecular weight is 13095.3.

[0183] In summary, this invention provides a chemical synthesis method for elastin-like peptides, which constructs a pentapeptide repeat sequence (VPGXG) of an elastin-like peptide through a liquid-phase reaction. n The method involves orthogonally protecting the amino and carboxyl groups, selectively removing the protecting groups at the amino or carboxyl terminus, activating the carboxyl terminus in a liquid phase, and then precisely coupling it with the amino terminus to prepare high molecular weight elastin-like peptides. This method allows for precise control of the molecular weight, sequence, and chirality of elastin-like peptides, improving the synthesis efficiency of long-chain elastin-like peptides and offering advantages such as large-scale synthesis and low cost.

Claims

1. A method for preparing an elastin-like polypeptide, characterized in that, Includes the following steps: Step 1: Prepare a pentapeptide synthesis unit protected by a protecting group; the structure of the pentapeptide synthesis unit protected by the protecting group is R1-valine-proline-glycine-X-glycine-R2; Step 2: Using the pentapeptide synthesis unit protected by the protecting group obtained in Step 1 as raw material, selectively remove the amino or carboxyl terminus protecting groups, and then couple it in a liquid phase system. Repeat the above deprotection and coupling process to prepare an elastin-like polypeptide; the elastin-like polypeptide is R1-(valine-proline-glycine-X-glycine). n -R2, whose structure is shown in Equation I; Wherein, R1 is an amino protecting group, R2 is a carboxyl protecting group, X is an amino acid other than proline, R3 is the side chain of amino acid X, R4 is the side chain protecting group of amino acid X, n is 2 to the power of y, and y is an integer greater than or equal to 1.

2. The method for preparing the elastin-like polypeptide according to claim 1, characterized in that: The deprotection processes of R1 and R2 must be completely orthogonal, and R3 and R4 remain unchanged during the deprotection and coupling processes.

3. The method for preparing the elastin-like polypeptide according to claim 1 or 2, characterized in that: R1 is selected from at least one of triphenylmethyl, trimethylsilylethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, and allyloxycarbonyl; R2 is selected from at least one of benzyloxy and alkoxy; R4 is selected from at least one of triphenylmethyl, tert-butoxycarbonyl, tert-butyl, 2,2,4,6,7-pentamethylbenzofuran-5-sulfonyl, p-methoxytriphenylmethyl, trimethoxybenzyl, and N-methyltriphenylmethyl.

4. The method for preparing the elastin-like polypeptide according to claim 1, characterized in that: When X is an inert side-chain amino acid, R1 and R2 are orthogonally protected protecting groups; And / or, when X is a side-chain functionalized amino acid, R1, R2 and the side-chain protecting group R4 of X are all orthogonally protected protecting groups.

5. The method for preparing the elastin-like polypeptide according to claim 4, characterized in that: R1 is selected from tert-butyloxycarbonyl, R2 is selected from benzyloxy, and X is an inert side-chain amino acid that does not contain halogen or sulfur; R1 is selected from tert-butyloxycarbonyl, R2 is selected from methoxy or ethoxy, and X is an inert side-chain amino acid; or R1 is selected from 9-fluorenylmethoxycarbonyl, R2 is selected from benzyloxy, and X is an inert side-chain amino acid that does not contain halogen or sulfur. Alternatively, R1 is selected from at least one of 9-fluorenylmethoxycarbonyl, trimethylsilylethoxycarbonyl, and 2,2,2-trichloroethoxycarbonyl, R2 is selected from at least one of benzyloxy, methoxy, and ethoxy, and X is a side-chain functionalized amino acid that does not contain halogen or sulfur; or, R1 is selected from at least one of allyloxycarbonyl, trimethylsilylethoxycarbonyl, and 2,2,2-trichloroethoxycarbonyl, R2 is selected from at least one of methoxy and ethoxy, and X is an amino acid other than proline.

6. The method for preparing the elastin-like polypeptide according to claim 5, characterized in that: R1 is selected from tert-butoxycarbonyl, R2 is selected from benzyloxy, methoxy, or ethoxy, and X is selected from at least one of glycine, alanine, valine, leucine, isoleucine, and phenylalanine. And / or, R1 is selected from 9-fluorenylmethoxycarbonyl, R2 is selected from benzyloxy, and X is selected from at least one of glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, arginine, serine, and threonine. And / or, R1 is selected from allyloxycarbonyl, R2 is selected from ethoxy, and X is at least one of glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, arginine, serine, threonine, cysteine, and methionine. Preferably, R1 is selected from tert-butoxycarbonyl, R2 is selected from benzyloxy, and X is at least one of glycine, aspartic acid, valine, and phenylalanine. And / or, R1 is selected from 9-fluorenylmethoxycarbonyl, R2 is selected from benzyloxy, and X is asparagine; And / or, R1 is selected from allyloxycarbonyl, R2 is selected from ethoxy, and X is cysteine; All of the above-mentioned amino acids, except for glycine, can be either levorotatory or dextrorotatory.

7. The method for preparing the elastin-like polypeptide according to any one of claims 1-6, characterized in that: In step 1, the preparation method of the pentapeptide synthesis unit protected by the protecting group includes the following steps: (1) The protected proline, activator and condensing agent are dissolved in an organic solvent to obtain an activation system. The protected glycine and alkaline reagent are dissolved in an organic solvent and added to the above activation system. After purification, the protected dipeptide product 1, namely R1-proline-glycine-R2, is obtained. (2) Dissolve the dipeptide product 1 protected by the protecting group in a deprotecting agent to carry out the deprotection reaction, and obtain the deprotected dipeptide product 1, namely NH2-proline-glycine-R2. (3) Dissolve the valine protected by the protecting group, the activator and the condensing agent in an organic solvent to obtain an activation system. Then dissolve the deprotected dipeptide product 1 obtained in step (2) and the alkaline reagent in an organic solvent, add the above activation system, and after purification, obtain the tripeptide product protected by the protecting group, namely R1-valine-proline-glycine-R2. (4) The tripeptide product protected by the protecting group is dissolved in an organic solvent, a catalyst and a reducing agent are added, and after purification, the tripeptide product without the protecting group is obtained, namely R1-valine-proline-glycine-OH. (5) Dissolve the X protected by the protecting group, the activator and the condensing agent in an organic solvent to obtain an activation system. Then dissolve the glycine protected by the protecting group and the basic reagent in an organic solvent, add them to the activation system, and after purification, obtain the dipeptide product 2 protected by the protecting group, namely R1-X-glycine-R2. (6) Dissolve the dipeptide product 2 protected by the protecting group in a deprotecting agent to carry out the deprotection reaction, and obtain the deprotected dipeptide product 2, namely NH2-X-glycine-R2. (7) Dissolve the deprotected tripeptide product, activator, and condensing agent obtained in step (4) in an organic solvent to obtain an activation system. Then dissolve the deprotected dipeptide product 2 obtained in step (6) and an alkaline reagent in an organic solvent, add them to the activation system, and after purification, obtain a pentapeptide synthesis unit protected by a protecting group.

8. The method for preparing the elastin-like polypeptide according to claim 7, characterized in that: In step (1), the proline protected by the protecting group is at least one of Boc-D-proline and Boc-L-proline; the activator is selected from at least one of 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate, benzotriazole-1-oxytripyrrolylphosphine hexafluorophosphate, pentafluorophenol, N-hydroxysuccinimide, 1-hydroxy-7-azabenzotriazole-tetramethylurea hexafluorophosphate, and ethyl 2-oxime cyanophosphate; the condensing agent is selected from 1-ethyl-(3-dimethylaminopropyl)carbamate. The reagent comprises at least one of imine hydrochloride, N,N'-diisopropylcarbodiimide, and N,N'-dicyclohexylcarbodiimide; the glycine protected by the protecting group is at least one of glycine benzyl ester hydrochloride, glycine benzyl ester trifluoroacetate, and glycine benzyl ester acetate; the basic reagent is at least one of N,N-diisopropylethylamine, triethylamine, diethylamine, N-methylmorpholine, and 2,4,6-trimethylpyridine; and the organic solvent is at least one of N,N-dimethylformamide, dichloromethane, tetrahydrofuran, and dimethyl sulfoxide. And / or, in step (1), the equivalent ratio of the protecting group-protected proline: activator: condensing agent: basic catalyst: protecting group-protected glycine is 1:(0.5-5):(0.5-5):(1-10):(0.5-3); And / or, in step (2), the deprotecting agent is selected from at least one of trifluoroacetic acid, dioxane hydrochloride solution, p-toluenesulfonic acid, methanol hydrochloride solution, trimethylsilyl trifluoromethanesulfonate, hydrogen bromide acetic acid solution, dilute sulfuric acid, and boron trifluoride ether. And / or, in step (2), the equivalent ratio of the dipeptide product 1 protected by the protecting group to the deprotecting agent is 1:1-20; And / or, in step (3), the valine protected by the protecting group is at least one of Boc-D-valine and Boc-L-valine; the activator is selected from at least one of 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate, benzotriazole-1-oxytripyrrolylphosphine hexafluorophosphate, pentafluorophenol, N-hydroxysuccinimide, 1-hydroxy-7-azabenzotriazole-tetramethylurea hexafluorophosphate, and ethyl 2-oxime cyanophosphate. The condensing agent is selected from at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-diisopropylcarbodiimide, and N,N'-dicyclohexylcarbodiimide; the alkaline reagent is at least one of N,N-diisopropylethylamine, triethylamine, diethylamine, N-methylmorpholine, and 2,4,6-trimethylpyridine; the organic solvent is at least one of N,N-dimethylformamide, dichloromethane, tetrahydrofuran, and dimethyl sulfoxide. And / or, in step (3), the equivalent ratio of the valine protected by the protecting group: activator: condensing agent: basic catalyst: deprotected dipeptide product 1 is 1:(0.5-5):(0.5-5):(1-10):(0.5-3); And / or, in step (4), the organic solvent is selected from at least one of tetrahydrofuran, ethyl acetate, acetonitrile, and methanol; the catalyst is selected from at least one of carbon-supported palladium catalyst and carbon-supported palladium hydroxide; the reducing agent is selected from at least one of hydrogen, formic acid, ammonium formate, cyclohexene, and 1,4-cyclohexadiene. And / or, in step (4), the equivalent ratio of the tripeptide product protected by the protecting group to the catalyst is 1:0.02-0.2; And / or, in step (5), the activator is selected from at least one of 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate, benzotriazole-1-oxytripyrrolylphosphine hexafluorophosphate, pentafluorophenol, N-hydroxysuccinimide, 1-hydroxy-7-azabenzotriazole-tetramethylurea hexafluorophosphate, and ethyl 2-oxime cyanophosphate; the condensing agent is selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-diisopropylcarbodiimide... The protective group is at least one of imine and N,N'-dicyclohexylcarbodiimine; the glycine protected by the protecting group is at least one of glycine benzyl ester hydrochloride, glycine benzyl ester trifluoroacetate, and glycine benzyl ester acetate; the basic reagent is at least one of N,N-diisopropylethylamine, triethylamine, diethylamine, N-methylmorpholine, and 2,4,6-trimethylpyridine; the organic solvent is at least one of N,N-dimethylformamide, dichloromethane, tetrahydrofuran, and dimethyl sulfoxide. And / or, in step (5), the equivalent ratio of the protected group X: activator: condensing agent: basic catalyst: protected group glycine is 1:(0.5-5):(0.5-5):(1-10):(0.5-3); And / or, in step (6), the deprotecting agent is selected from at least one of 1,4-dioxane hydrochloride, trifluoroacetic acid, p-toluenesulfonic acid, methanol hydrochloric acid solution, trimethylsilyl trifluoromethanesulfonate, acetic acid solution of hydrogen bromide, dilute sulfuric acid, boron trifluoride ether, piperidine, tetramethylpiperidine, piperazine, 1,8-diazabicycloundec-7-ene, triethylamine, diethylamine, potassium carbonate, and N,N-diisopropylethylamine; And / or, in step (6), the equivalent ratio of the dipeptide product 2 protected by the protecting group to the deprotecting agent is (1:1-20); And / or, in step (7), the activator is selected from at least one of 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate, benzotriazole-1-oxytripyrrolylphosphine hexafluorophosphate, pentafluorophenol, N-hydroxysuccinimide, 1-hydroxy-7-azabenzotriazole-tetramethylurea hexafluorophosphate, and ethyl 2-oxime cyanoacetate; the condensing agent is selected from at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-diisopropylcarbodiimide, and N,N'-dicyclohexylcarbodiimide; the basic reagent is at least one of N,N-diisopropylethylamine, triethylamine, diethylamine, N-methylmorpholine, and 2,4,6-trimethylpyridine; and the organic solvent is at least one of N,N-dimethylformamide, dichloromethane, tetrahydrofuran, and dimethyl sulfoxide. And / or, in step (7), the equivalent ratio of the deprotected tripeptide product: activator: condensing agent: basic catalyst: deprotected dipeptide product 2 is 1:(0.5-5):(0.5-5):(1-10):(0.5-3).

9. The method for preparing the elastin-like polypeptide according to claim 1, characterized in that: Step 2 is performed according to one of the following operations: (a) Using a pentapeptide synthesis unit protected by a protecting group as a raw material, a deprotection reaction was carried out with a metal catalyst and a reducing agent in solvent 1. After purification, intermediate product 1, namely R1-(valine-proline-glycine-X-glycine), was obtained. n -OH; (b1) Intermediate 1 was dissolved in a deprotecting agent to carry out a deprotection reaction. After purification, intermediate 2 was obtained, namely NH2-(valine-proline-glycine-X-glycine). n -R2; (c1) Dissolve intermediate 1 in solvent 2, add activator and condensing agent to obtain activation system, then add intermediate 2 and alkaline reagent to activation system, and after purification, obtain R1-(valine-proline-glycine-X-glycine). 2n -R2; (d1) Repeat step (a-c1) to obtain elastin-like polypeptides; Alternatively, (a) using a pentapeptide synthesis unit protected by a protecting group as a raw material, a metal catalyst and a reducing agent are carried out in solvent 1 for deprotection reaction, and after purification, intermediate product 1 is obtained, namely R1-(valine-proline-glycine-X-glycine). n -OH; (b2) Dissolve intermediate product 1 in solvent 2, add activator and condensing agent to obtain activated system, add solvent 3 to precipitate, centrifuge, dissolve the obtained solid 1 in deprotecting agent, add solvent 3 to precipitate, dissolve the obtained solid 2 in solvent 2, add basic reagent, react, add solvent 3 to precipitate, purify to obtain cyclic elastin-like polypeptide, namely c(valine-proline-glycine-X-glycine). n .

10. The method for preparing the elastin-like polypeptide according to claim 9, characterized in that: In step (a), the metal catalyst is selected from at least one of carbon-supported palladium catalyst and carbon-supported palladium hydroxide catalyst; the reducing agent is selected from at least one of hydrogen, formic acid, ammonium formate, cyclohexene, and 1,4-cyclohexadiene; and the solvent 1 is selected from at least one of methanol, ethyl acetate, acetonitrile, water, and tetrahydrofuran. And / or, in step (a), the equivalence ratio of the pentapeptide synthesis unit protected by the protecting group to the catalyst is 1:0.01-1; And / or, in step (b1), the deprotecting agent is selected from at least one of trifluoroacetic acid, dioxane hydrochloride solution, p-toluenesulfonic acid, methanol hydrochloride solution, trimethylsilyl trifluoromethanesulfonate, acetic acid hydrogen bromide solution, dilute sulfuric acid, boron trifluoride ether, piperidine, tetramethylpiperidine, piperazine, 1,8-diazabicycloundec-7-ene, triethylamine, diethylamine, potassium carbonate, and N,N-diisopropylethylamine; And / or, in step (b1), the equivalent ratio of the intermediate product 1 to the deprotecting agent is 1:2-100; And / or, in step (c1), the solvent 2 is selected from at least one of dimethyl sulfoxide, N,N-dimethylformamide, and dichloromethane; the activator is selected from at least one of 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate, benzotriazole-1-oxytripyrrolylphosphine hexafluorophosphate, pentafluorophenol, N-hydroxysuccinimide, 1-hydroxy-7-azabenzotriazole-tetramethylurea hexafluorophosphate, and ethyl 2-oxime cyanoacetate; the condensing agent is selected from at least one of N,N'-diisopropylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N,N'-dicyclohexylcarbodiimide; and the basic reagent is at least one of N,N-diisopropylethylamine, triethylamine, diethylamine, N-methylmorpholine, and 2,4,6-trimethylpyridine. And / or, in step (c1), the equivalent ratio of intermediate product 1 activator: condensing agent: basic catalyst: intermediate product 2 is 1:(0.5-5):(0.5-5):(1-10):(0.5-3); And / or, in step (b2), the solvent 2 is selected from at least one of dimethyl sulfoxide, N,N-dimethylformamide, and dichloromethane; the activator is selected from at least one of 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate, benzotriazole-1-oxytripyrrolylphosphine hexafluorophosphate, pentafluorophenol, N-hydroxysuccinimide, 1-hydroxy-7-azabenzotriazole-tetramethylurea hexafluorophosphate, and ethyl 2-oxime cyanophosphate; the condensing agent is selected from N,N'-diisopropylcarbodiimide, The solvent 3 is anhydrous diethyl ether; the deprotecting agent is selected from at least one of trifluoroacetic acid, dioxane hydrochloride solution, p-toluenesulfonic acid, methanol hydrochloride solution, trimethylsilyl trifluoromethanesulfonate, acetic acid hydrogen bromide solution, dilute sulfuric acid, and boron trifluoride diethyl ether; the alkaline reagent is at least one of N,N-diisopropylethylamine, triethylamine, diethylamine, N-methylmorpholine, and 2,4,6-trimethylpyridine.

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