Design and synthesis method of tumor dual-targeting polypeptide

Abstract

The invention discloses a design and synthesis method of tumor dual-targeting polypeptide, the synthesis of the polypeptide adopts an Fmoc manual solid-phase synthesis technology, and the method specifically comprises the following steps: S1, amino acid protection; s2, swelling the resin; s3, removing an amino protecting group of the resin; s4, grafting a first amino acid; s5, removing an Fmoc protecting group of an amino group; s6, grafting other amino acids according to the designed polypeptide sequence; s7, performing disulfide bond cyclization; s8, shrinking; s9, cutting is conducted; and S10, product purification. The Fmoc manual solid-phase synthesis technology is adopted, operation is easy and convenient, synthesis efficiency is high, the method is suitable for large-scale production, and a new effective tool is provided for tumor targeted therapy and diagnosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for designing and synthesizing a tumor dual-targeting polypeptide. Background Art

[0002] With the development of cell biology, scientists have discovered that antigens, receptors or mRNAs related to tumor growth and proliferation have become tumor biomarkers, providing a material basis for targeted diagnosis and treatment of tumors. Antibodies, ligands and antisense nucleic acids corresponding to these biomarkers have become key tools for targeted therapy. Designing targeted drug delivery systems using the specific mutual recognition characteristics between receptors and ligands has become one of the effective means to improve drug targeting and reduce toxic side effects.

[0003] Traditional tumor treatment and diagnosis drugs are usually evenly distributed throughout the body due to their lack of targeting, resulting in low drug concentrations reaching the tumor site. This low concentration not only reduces the therapeutic effect and diagnostic resolution, but may also have toxic side effects on healthy cells. To solve this problem, researchers have developed peptide-based targeting carriers that increase the binding force between drugs and targets, allowing therapeutic or diagnostic agents to accumulate in the tumor, thereby effectively improving the effect of tumor treatment or diagnosis.

[0004] However, there are still some problems with the existing peptide-based ligand design. The expression of receptors on the surface of tumor cells is usually limited. When all receptors bind to ligands, the cell's adsorption of drugs reaches saturation. At this point, even if the drug dose is increased, the drug concentration at the target site cannot be further increased, which seriously limits the therapeutic or diagnostic effect of the drug. Therefore, how to break through the limitation of receptor expression and increase the concentration of drugs at the tumor site has become a key issue that needs to be solved urgently. Summary of the invention

[0005] 1. Technical Problems Solved

[0006] The technical problems to be solved by the present invention are the various problems mentioned in the above background technology, and a method for designing and synthesizing a tumor dual-targeting polypeptide is provided.

[0007] 2. Technical Solution

[0008] In order to solve the above technical problems, the technical solution provided by the present invention is: a method for designing and synthesizing a tumor dual-targeting peptide, wherein the peptide is synthesized using Fmoc manual solid phase synthesis technology, and the specific steps include:

[0009] S1. Amino acid protection: reacting a compound containing an amino group with Fmoc-anhydrous hydrofluoric anhydride and a base to generate an Fmoc-amino protected compound.

[0010] S2, resin swelling: add a proper amount of purified N,N-dimethylformamide (DMF) into a solid phase reactor, add the weighed resin into the solvent, stir and swell with nitrogen for 0.5 to 2 hours, preferably 1 hour, and then drain the solvent for later use.

[0011] S3. Removal of the amino protecting group of the resin. In order to remove the amino protecting group Fmoc, add an appropriate amount of piperidine / DMF solution to the swollen resin reaction mixture in the first step under nitrogen conditions, and stir for about 5 minutes to remove the Fmoc protecting group on the resin; wash alternately with N,N-dimethylformamide and dichloromethane, and detect with Kaiser reagent. If there are resin particles that have not changed color, it indicates that the removal is not complete, and the above deprotection reaction needs to be repeated. When all the resins appear blue or black, it indicates that the removal is complete. Drain the solvent for later use.

[0012] S4, connect the first amino acid, connect the carboxyl group of the first amino acid of the target peptide to the amino group of the resin in step 3 in the form of a covalent bond; weigh the calculated Fmoc-protected amino acid and condensing agent HBTU and HOBt and dissolve them in DMF, mix the solution evenly and pour it into the resin in the solid phase reactor, stir and react for about 2 hours with nitrogen, monitor with Kaiser reagent, if there are still blue or black resin particles, it means that the reaction is not complete, and the above reaction steps need to be repeated until all the resins return to the original color of the resin (white or very light yellow), indicating that the reaction is complete. Then drain the solvent, wash alternately with N,N-dimethylformamide and dichloromethane, and drain to prepare for the connection of the following amino acids.

[0013] S5. Removal of the Fmoc protecting group from the amino group according to the method of step 3.

[0014] S6. Connect other amino acids in the order of the designed polypeptide sequence, take the amino group of the first amino acid to be deprotected as the starting point of synthesis, make it undergo acylation reaction with the carboxyl group of the adjacent amino acid to form a peptide bond, repeat the method of removing the protecting group in step 3 and the method of connecting the amino group in step 4 until the target peptide is completed.

[0015] S7, disulfide bond ring formation, dissolve I2 in acetic acid DMF solution to prepare I2 solution, add the prepared I2 solution to the reaction mixture and react for 4 hours, then drain the solvent and the resin will appear purple. Add an excess of ascorbic acid DMF solution to remove excess I2, repeat the addition several times until the purple color of the resin fades and the original color is shown, and wash it clean. A disulfide bond ring-forming product is obtained. After removing the Fmoc protecting group according to step 3, the Kaiser reagent detects that the resin is colorless, indicating that it has been removed cleanly.

[0016] S8. Shrinkage. After cyclization, wash the resin with DCM 2-3 times, remove the solvent, add methanol and stir to shrink twice (5min+5min), remove the methanol, and continue to dry for 30-60min to shrink the peptide resin.

[0017] S9, cutting, the shrunken peptide resin and the cutting solution are subjected to a cutting reaction under stirring for 2-3 hours. After the reaction is completed, the resin residue is filtered out and washed. The filtrates are combined to precipitate a white solid, which is centrifuged to obtain a white crude product.

[0018] S10, product purification, first use analytical high performance liquid chromatography to analyze the product, then use preparative reverse phase high performance liquid chromatography, gradient elution method to liquid phase elution separation of the obtained crude peptide, and finally perform structural characterization and activity identification.

[0019] As an improvement, the base in step 1 is NaOH, Na2CO3, NaHCO3, preferably Na2CO3.

[0020] As an improvement, the resin in step 2 can be 2-chlorotributyl chloride resin, MBHA amino resin, Wang resin, etc., preferably MBHA amino resin.

[0021] As an improvement, the volume percentage of piperidine and DMF in step 3 is 10% to 30%, preferably 20%.

[0022] As an improvement, in step 4, the molar ratio of amino acid: HBTU: HOBt is 1: (0.5-1.5): (0.5-1.5), preferably 1:1:1; and DIEA is added to the above solution, wherein the molar ratio of amino acid to DIEA is 1:1-3, preferably 1:2.

[0023] As an improvement, the polypeptide sequence in step 6 has two tumor targeting units, CNRGC and RGD.

[0024] As an improvement, the solvent used for the I2 solution in step 7 is a 30% acetic acid DMF solution, which is prepared into an I2 solution with a concentration of 1 to 5 g / ml, preferably 2 g / ml.

[0025] As an improvement, the cutting fluid in step 9 is prepared in the following proportions: 70% to 90% trifluoroacetic acid, preferably 90%; 1% to 3% anisole, preferably 2%; 2% to 7% thioanisole, preferably 5%; and 2% to 5% 1,2-diethylenedithiol, preferably 3%.

[0026] As an improvement, the CNGRC fragment contained in the polypeptide forms a disulfide bond ring through the thiol groups of the cysteine ​​at both ends of the fragment, while the RGD fragment is connected by a straight-chain peptide bond.

[0027] As an improvement, the polypeptide design and synthesis method is to organically combine the RGD peptide ligand that binds to the integrin αvβ3 receptor and the CNGRC cyclic peptide ligand that binds to the aminopeptidase CD13 in one polypeptide molecule. The two ligands bind to the two receptors of the tumor respectively, effectively increasing the concentration of the drug at the target site, thereby ensuring the effect of treatment or diagnosis.

[0028] 3. Beneficial Effects

[0029] The present invention provides a method for designing and synthesizing a tumor dual-targeting peptide, which organically combines an RGD cyclic peptide ligand that binds to the integrin αvβ3 receptor and an NGR linear peptide ligand that binds to the aminopeptidase CD13 in one polypeptide molecule, thereby achieving simultaneous binding of the polypeptide to two different receptors on tumor cells. This dual-targeting design effectively solves the problem of drug adsorption saturation caused by limited receptor expression in traditional single-target peptides, significantly increases the concentration of the drug at the tumor site, and thus improves the effect of treatment or diagnosis. In addition, the present invention adopts Fmoc manual solid phase synthesis technology, which is easy to operate, has high synthesis efficiency, is suitable for large-scale production, and provides a new and effective tool for tumor targeted treatment and diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The present invention is a flowchart of the steps of a method for designing and synthesizing a tumor dual-targeting polypeptide.

[0031] Figure 2 This is the structure of a tumor dual-targeting polypeptide P1 in an embodiment of a method for designing and synthesizing a tumor dual-targeting polypeptide of the present invention.

[0032] Figure 3 This is a high-resolution mass spectrum of Example P1 of a design and synthesis method for a tumor dual-targeting polypeptide of the present invention.

[0033] Figure 4 This is the structure of a tumor dual-targeting polypeptide P2 in an embodiment of a design and synthesis method of a tumor dual-targeting polypeptide of the present invention. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0035] like Figure 1 As shown, a method for designing and synthesizing a tumor dual-targeting peptide, wherein the peptide is synthesized using Fmoc manual solid phase synthesis technology, and the specific steps include:

[0036] S1. Amino acid protection: reacting a compound containing an amino group with Fmoc-anhydrous hydrofluoric anhydride and a base to generate an Fmoc-amino protected compound.

[0037] S2, resin swelling: add a proper amount of purified N,N-dimethylformamide (DMF) into a solid phase reactor, add the weighed resin into the solvent, stir and swell with nitrogen for 0.5 to 2 hours, preferably 1 hour, and then drain the solvent for later use.

[0038] S3. Removal of the amino protecting group of the resin. In order to remove the amino protecting group Fmoc, add an appropriate amount of piperidine / DMF solution to the swollen resin reaction mixture in the first step under nitrogen conditions, and stir for about 5 minutes to remove the Fmoc protecting group on the resin; wash alternately with N,N-dimethylformamide and dichloromethane, and detect with Kaiser reagent. If there are resin particles that have not changed color, it indicates that the removal is not complete, and the above deprotection reaction needs to be repeated. When all the resins appear blue or black, it indicates that the removal is complete. Drain the solvent for later use.

[0039] S4, connect the first amino acid, connect the carboxyl group of the first amino acid of the target peptide to the amino group of the resin in step 3 in the form of a covalent bond; weigh the calculated Fmoc-protected amino acid and condensing agent HBTU and HOBt and dissolve them in DMF, mix the solution evenly and pour it into the resin in the solid phase reactor, stir and react for about 2 hours with nitrogen, monitor with Kaiser reagent, if there are still blue or black resin particles, it means that the reaction is not complete, and the above reaction steps need to be repeated until all the resins return to the original color of the resin (white or very light yellow), indicating that the reaction is complete. Then drain the solvent, wash alternately with N,N-dimethylformamide and dichloromethane, and drain to prepare for the connection of the following amino acids.

[0040] S5. Removal of the Fmoc protecting group from the amino group according to the method of step 3.

[0041] S6. Connect other amino acids in the order of the designed polypeptide sequence, take the amino group of the first amino acid to be deprotected as the starting point of synthesis, make it undergo acylation reaction with the carboxyl group of the adjacent amino acid to form a peptide bond, repeat the method of removing the protecting group in step 3 and the method of connecting the amino group in step 4 until the target peptide is completed.

[0042] S7, disulfide bond ring formation, dissolve I2 in acetic acid DMF solution to prepare I2 solution, add the prepared I2 solution to the reaction mixture and react for 4 hours, then drain the solvent and the resin will appear purple. Add an excess of ascorbic acid DMF solution to remove excess I2, repeat the addition several times until the purple color of the resin fades and the original color is shown, and wash it clean. A disulfide bond ring-forming product is obtained. After removing the Fmoc protecting group according to step 3, the Kaiser reagent detects that the resin is colorless, indicating that it has been removed cleanly.

[0043] S8. Shrinkage. After cyclization, wash the resin with DCM 2-3 times, remove the solvent, add methanol and stir to shrink twice (5min+5min), remove the methanol, and continue to dry for 30-60min to shrink the peptide resin.

[0044] S9, cutting, the shrunken peptide resin and the cutting solution are subjected to a cutting reaction under stirring for 2-3 hours. After the reaction is completed, the resin residue is filtered out and washed. The filtrates are combined to precipitate a white solid, which is centrifuged to obtain a white crude product.

[0045] S10, product purification, first use analytical high performance liquid chromatography to analyze the product, then use preparative reverse phase high performance liquid chromatography, gradient elution method to liquid phase elution separation of the obtained crude peptide, and finally perform structural characterization and activity identification.

[0046] The following are embodiments of the present invention in a specific process:

[0047] Example 1: Synthesis of tumor dual-targeting peptide H2N-c[CRGNC]GGKG(RGD)FK-CONH2(P1)

[0048] 1. Resin swelling and removal of amino protecting groups

[0049] 1.0 g of MBHA amino resin (amino loading rate of 0.58 mmol / g) was added to a solid phase reactor, and 10 ml of purified N,N-dimethylformamide (DMF) was added to swell the resin. The mixture was stirred for about 2 h under nitrogen gas and then the solvent was dried.

[0050] In order to remove the amino protecting group Fmoc, add 10 ml of piperidine / DMF solution (volume percentage is 20%) to the reaction mixture, stir with nitrogen for 5 minutes, then drain the solvent, wash with N,N-dimethylformamide and dichloromethane. Detect with Kaiser reagent, if there are resin particles that do not change color, it means that the removal is not complete, and the deprotection reaction needs to be repeated. When all the resins are blue or black, it means that the removal is complete.

[0051] 2. Connect the first amino acid

[0052] Weigh 0.815g (1.74mmol, 468.54g / mol) of protected amino acid Fmoc-Lys(Boc)-OH, 0.660g (1.74mmol, 379.24g / mol) of condensing agent HBTU and 0.235g (1.74mmol, 135.13g / mol) of HOBt, dissolve in 15ml DMF, add 0.449g (3.48mmol, 129g / mol) of DIEA, mix well and pour into the resin in the solid phase reactor, stir and react for about 2h with nitrogen. Monitor with Kaiser reagent. If there are still blue or black resin particles, it means that the reaction is not complete. Repeat the above reaction steps until all the resin returns to the original color of the resin (white or very light yellow). Then drain the solvent, wash with N,N-dimethylformamide and dichloromethane, and drain to prepare for the subsequent amino acid.

[0053] 3. Connect the second amino acid

[0054] The Fmoc protecting group of the first amino acid was removed by the method of removing the amino protecting group Fmoc in step 1.

[0055] Weigh 0.521 g (1.74 mmol, 299.33 g / mol) of protected amino acid Fmoc-D-Phe-OH, 0.660 g (1.74 mmol, 379.24 g / mol) of condensing agent HBTU, 0.235 g (1.74 mmol, 135.13 g / mol) of HOBt and 0.449 g (3.48 mmol, 129 g / mol) of DIEA, dissolve them in 15 ml of DMF, add the solution to the resin in the solid phase reactor, stir the reaction, test until the reaction is complete, wash and drain in preparation for the next amino acid.

[0056] 4. Connect with subsequent amino acids

[0057] Take the following protected amino acids in order: Fmoc-Asp(otBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Cys(Acm)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Cys(Acm)-OH,

[0058] Repeat step 3 until all amino acids are connected to the resin to obtain the side chain protected peptide resin Fmoc-Cys(Acm)-Arg(Pbf)-Gly-Asn(Trt)-Cys(Acm)-Gly-Gly-Lys(Boc)-Gly-Arg(Pbf)-Gly-Asp(tBu)-D-Phe-Lys(Boc)-Resin.

[0059] 5. Disulfide bond ring

[0060] Add 2g / ml I2 solution (solvent is 30% acetic acid DMF solution), stop the reaction after 4 hours, drain the solvent, and the resin will appear purple. Add excess ascorbic acid DMF solution to remove excess I2, repeat the addition several times until the purple color of the resin fades and the original color is shown, and wash it clean. The product with disulfide bonds forming a ring is obtained. After removing the Fmoc protecting group according to step 3, the Kaiser reagent detection resin is colorless, indicating that it is completely removed.

[0061] 6. Contraction

[0062] After the cyclization is completed, the resin is washed with DCM for 2-3 times, the solvent is pumped off, methanol is added and stirred to shrink twice (5min+5min), the methanol is pumped off, and the pumping is continued for 30-60min to shrink the peptide resin.

[0063] 7. Cutting

[0064] Prepare cutting solution with the following ratio: 90% trifluoroacetic acid, 2% anisole, 5% anisole thioether, 3% 1,2-diethylenedithiol. Put the prepared cutting solution into the refrigerator for later use. Take out the peptide resin from the reactor and transfer it to a round-bottom flask. Slowly add the cutting solution to the round-bottom flask under stirring. The reaction is carried out for 3 hours and the solution is light yellow. After the reaction is completed, filter to remove the resin residue, wash the resin with cutting reagent and dichloromethane for several times, and combine the filtrate. Ether precipitation, centrifugation, obtain a white crude product, and freeze for later use.

[0065] 8. Product purification

[0066] The product was first analyzed using an Agilent analytical high performance liquid chromatography (Agilent 1100) equipped with a C-18 reverse phase column (4.6×250 mm), and the elution conditions were: phase A was an aqueous solution containing 0.1% TFA, and phase B was acetonitrile containing 0.1% TFA. The gradient was a linear increase of 0 to 60% of phase B from 0 to 30 minutes to determine the peak time and approximate purity of the target product.

[0067] Then, Shimadzu LC-6AD (Japan) preparative reverse-phase high performance liquid chromatography (HPLC) equipped with Agilent C-18 (250×21.4 mm, The crude peptide (P1) was separated by liquid elution using a reverse phase column (μm) and gradient elution. The elution conditions were as follows: mobile phase A was an aqueous solution containing 0.1% TFA, and phase B was acetonitrile containing 0.1% TFA. The gradient was a linear increase from 0 to 30% in phase B from 0 to 30 minutes.

[0068] Through the above steps, the structure of P1 is as follows Figure 2 As shown, the final product is a white solid. Please note that it must be dried at low temperature during storage, otherwise it will easily lose its activity. The purity of the synthetic peptide was analyzed by high performance liquid chromatography. The retention time of HPLC analysis was 3.93min and the main peak content was 95.9%. Figure 3 This is the high-resolution mass spectrometry (HRESI-MS+) analysis of the synthesized tumor dual-targeting peptide P1. There are three peaks in the figure: 1451.6740 ([M+H]+), 726.3436 ([M+2H]2+), and 484.5666 ([M+3H]3+). These three peaks correspond one by one to the theoretical peaks of the P1 peptide, indicating that the synthesis of P1 was successful.

[0069] Example 2: Synthesis of tumor dual-targeting peptide c[CRGNC]GSSKSSDGRGK-CONH2 (P2)

[0070] 1. Resin swelling and removal of amino protecting groups

[0071] Same as Example 1.

[0072] 2. Connect the first amino acid

[0073] Same as Example 1.

[0074] 3. Connect the second amino acid

[0075] The Fmoc protecting group of the first amino acid was removed by the method of removing the amino protecting group Fmoc in step 1.

[0076] Weigh 0.091 g (1.74 mmol, 174.20 g / mol) of protected amino acid Fmoc-Gly-OH, 0.660 g (1.74 mmol, 379.24 g / mol) of condensing agent HBTU, 0.235 g (1.74 mmol, 135.13 g / mol) of HOBt and 0.449 g (3.48 mmol, 129 g / mol) of DIEA, dissolve them in 15 ml of DMF, add the solution to the resin in the solid phase reactor, stir the reaction, test until the reaction is complete, wash and drain in preparation for the next amino acid.

[0077] 4. Connect with subsequent amino acids

[0078] Take the following protected amino acids in order: Fmoc-Arg(pbf)-OH, Fmoc-Gly-OH, Fmoc-Asp(otBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Cys(Acm)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Cys(Acm)-OH,

[0079] Repeat step 3 until all amino acids are connected to the resin to obtain the side chain protected peptide resin Fmoc-Cys(Acm)-Arg(Pbf)-Gly-Asn(Trt)-Cys(Acm)-Gly-Ser(tBu)-Ser(tBu)-Lys(Dde)-Ser(tBu)-Ser(tBu)-Asp(otBu)-Gly-Arg(Pbf)-Gly-Lys(Boc)-Resin.

[0080] 5. Disulfide bond ring

[0081] Same as Example 1.

[0082] 6. Contraction

[0083] Same as Example 1.

[0084] 7. Cutting

[0085] Same as Example 1.

[0086] 8. Product purification

[0087] Same as Example 1.

[0088] The structure of P2 finally obtained through the above steps is as follows Figure 4 As shown, the synthesized products were characterized by chromatography-mass spectrometry.

[0089] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0090] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0091] The present invention and its embodiments are described above, and such description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if ordinary technicians in the field are inspired by it, without departing from the purpose of the invention, they can design a structure and embodiment similar to the technical solution without creativity, which should belong to the protection scope of the present invention.

Claims

1. A method for designing and synthesizing a tumor dual-targeting peptide, characterized in that: The polypeptide is synthesized using Fmoc manual solid phase synthesis technology, and the specific steps include: S1. Amino acid protection: reacting a compound containing an amino group with Fmoc-anhydrous hydrofluoric anhydride and a base to generate an Fmoc-amino protected compound. S2, resin swelling: add a proper amount of purified N,N-dimethylformamide (DMF) into a solid phase reactor, add the weighed resin into the solvent, stir and swell with nitrogen for 0.5 to 2 hours, preferably 1 hour, and then drain the solvent for later use. S3. Removal of the amino protecting group of the resin. In order to remove the amino protecting group Fmoc, add an appropriate amount of piperidine / DMF solution to the swollen resin reaction mixture in the first step under nitrogen conditions, and stir for about 5 minutes to remove the Fmoc protecting group on the resin; wash alternately with N,N-dimethylformamide and dichloromethane, and detect with Kaiser reagent. If there are resin particles that have not changed color, it indicates that the removal is not complete, and the above deprotection reaction needs to be repeated. When all the resins appear blue or black, it indicates that the removal is complete. Drain the solvent for later use. S4, connect the first amino acid, connect the carboxyl group of the first amino acid of the target peptide to the amino group of the resin in step 3 in the form of a covalent bond; weigh the calculated Fmoc-protected amino acid and condensing agent HBTU and HOBt and dissolve them in DMF, mix the solution evenly and pour it into the resin in the solid phase reactor, stir and react for about 2 hours with nitrogen, monitor with Kaiser reagent, if there are still blue or black resin particles, it means that the reaction is not complete, and the above reaction steps need to be repeated until all the resins return to the original color of the resin (white or very light yellow), indicating that the reaction is complete. Then drain the solvent, wash alternately with N,N-dimethylformamide and dichloromethane, and drain to prepare for the connection of the following amino acids. S5. Removal of the Fmoc protecting group from the amino group according to the method of step 3. S6. Connect other amino acids in the order of the designed polypeptide sequence, take the amino group of the first amino acid to be deprotected as the starting point of synthesis, make it undergo acylation reaction with the carboxyl group of the adjacent amino acid to form a peptide bond, repeat the method of removing the protecting group in step 3 and the method of connecting the amino group in step 4 until the target peptide is completed. S7, disulfide bond ring formation, dissolve I2 in acetic acid DMF solution to prepare I2 solution, add the prepared I2 solution to the reaction mixture and react for 4 hours, then drain the solvent and the resin will appear purple. Add an excess of ascorbic acid DMF solution to remove excess I2, repeat the addition several times until the purple color of the resin fades and the original color is shown, and wash it clean. A disulfide bond ring-forming product is obtained. After removing the Fmoc protecting group according to step 3, the Kaiser reagent detects that the resin is colorless, indicating that it has been removed cleanly. S8. Shrinkage. After cyclization, wash the resin with DCM 2-3 times, remove the solvent, add methanol and stir to shrink twice (5min+5min), remove the methanol, and continue to dry for 30-60min to shrink the peptide resin. S9, cutting, the shrunken peptide resin and the cutting solution are subjected to a cutting reaction under stirring for 2-3 hours. After the reaction is completed, the resin residue is filtered out and washed. The filtrates are combined to precipitate a white solid, which is centrifuged to obtain a white crude product. S10, product purification, first use analytical high performance liquid chromatography to analyze the product, then use preparative reverse phase high performance liquid chromatography, gradient elution method to liquid phase elution separation of the obtained crude peptide, and finally perform structural characterization and activity identification.

2. The method for designing and synthesizing a tumor dual-targeting polypeptide according to claim 1, characterized in that: In step 1, the base is NaOH, Na2CO3, NaHCO3, preferably Na2CO3.

3. The method for designing and synthesizing a tumor dual-targeting polypeptide according to claim 3, characterized in that: The resin in step 2 can be 2-chlorotributyl chloride resin, MBHA amino resin, Wang resin, etc., preferably MBHA amino resin.

4. The method for designing and synthesizing a tumor dual-targeting polypeptide according to claim 1, characterized in that: In step 3, the volume percentage of piperidine and DMF is 10% to 30%, preferably 20%.

5. The method for designing and synthesizing a tumor dual-targeting polypeptide according to claim 1, characterized in that: In step 4, the molar ratio of amino acid: HBTU: HOBt is 1: (0.5-1.5): (0.5-1.5), preferably 1: 1: 1; and then DIEA is added to the above solution, wherein the molar ratio of amino acid to DIEA is 1: 1-3, preferably 1:

2.

6. The method for designing and synthesizing a tumor dual-targeting polypeptide according to claim 1, characterized in that: In step 6, the polypeptide sequence has two tumor targeting units, CNRGC and RGD.

7. The method for designing and synthesizing a tumor dual-targeting polypeptide according to claim 1, characterized in that: The solvent used for the I2 solution in step 7 is a 30% acetic acid DMF solution, which is prepared into an I2 solution with a concentration of 1 to 5 g / ml, preferably 2 g / ml.

8. The method for designing and synthesizing a tumor dual-targeting polypeptide according to claim 1, characterized in that: The cutting solution in step 9 is prepared in the following proportions: 70% to 90% trifluoroacetic acid, preferably 90%; 1% to 3% anisole, preferably 2%; 2% to 7% thioanisole, preferably 5%; and 2% to 5% 1,2-diethylenedithiol, preferably 3%.

9. The method for designing and synthesizing a tumor dual-targeting polypeptide according to claim 1, characterized in that: The CNGRC fragment contained in the polypeptide forms a disulfide bond ring through the sulfhydryl groups of the cysteine ​​at both ends of the fragment, while the RGD fragment is connected by a straight-chain peptide bond.

10. The method for designing and synthesizing a tumor dual-targeting polypeptide according to claim 1, characterized in that: The polypeptide design and synthesis method is to organically combine the RGD peptide ligand that binds to the integrin αvβ3 receptor and the CNGRC cyclic peptide ligand that binds to the aminopeptidase CD13 in one polypeptide molecule, and the two ligands are respectively bound to the two receptors of the tumor, thereby effectively increasing the concentration of the drug at the target site, thereby ensuring the effect of treatment or diagnosis.

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