A synthetic method of tilportide

Abstract

The present invention provides a method for synthesizing tilpoxetine, wherein the target peptide chain is divided into 6 fragments, and bis(4-C18-C28 alkoxyphenyl)methylamine, 2,4-bis(18-C28 alkoxy)-benzyl alcohol and NH2-Asp(OTAG)-OR 1 As a hydrophobic tag carrier to assist in synthesis. The side chain carboxyl group of aspartic acid was innovatively used as the anchoring position of the hydrophobic tag, a new fragment connection route was designed, and the fully protected telport peptide conjugate was synthesized through the synergistic effect of multiple tags. Finally, the protecting group and tag were removed with TFA cleavage solution. Compared with the solid-phase synthesis method, this method is carried out in a homogeneous reaction, and the amount of amino acids and condensing agents is reduced from 2 to 3 times to 1 to 1.2 times, which significantly reduces the cost. Compared with the existing hydrophobic tag method, its advantage is that multiple tags can be introduced as protecting groups to improve the yield and purity of medium and long-chain polypeptide synthesis, while solving the problem of hydrophobicity attenuation caused by peptide chain extension, which meets the requirements of green chemical process.

Description

Technical Field

[0001] The present invention relates to the field of polypeptide drug preparation, and in particular to a method for fully synthesizing tilpotide based on a soluble hydrophobic tag as a carrier. Background Art

[0002] Tirzepatide (trade name Mounjaro™) is a new dual GIP / GLP-1 receptor agonist developed by Eli Lilly and Company with a molecular weight of 4813.45. Its molecular structure contains 39 amino acids and a C20 fatty acid side chain. It is used to improve blood sugar control in adult patients with type 2 diabetes (T2DM). The drug works synergistically by activating GIP and GLP-1 receptors at the same time, promoting the secretion of GIP and GLP-1 hormones. GIP hormones play a key role in nutrient metabolism and energy balance, while GLP-1 hormones regulate blood sugar by delaying gastric emptying, suppressing appetite and improving satiety. Studies have shown that dual activation of these two receptors can enhance the hypoglycemic effect and promote weight loss. At the same time, Tirzepatide's molecular structure has an alkyl side chain composed of C20 fatty acids and polyethylene glycol, which can bind to albumin in the body to prolong its half-life, thereby achieving subcutaneous injection once a week. The structural formula of tirzepatide is as follows: Figure 7 shown.

[0003] Patent CN 113330024 A proposes a fragment-based synthesis process for tilpoxetine, dividing the peptide chain into four fragments: AA1-14, 15-21, 22-29, and 30-39. Each fragment is obtained through peptide solid-phase synthesis. The key step is the use of an -ALLOC protection strategy on the lysine side chain of the AA15-21 fragment, followed by precise installation of the drug alkyl chain after deprotection. Fragment condensation utilizes a continuous flow liquid-phase reaction, and the innovative introduction of a ceramic-based nanofiltration membrane with a pore size of 200-450 Da replaces the traditional precipitation method, effectively solving the problem of residual low-molecular-weight byproducts such as dibenzofulvene. The process optimizes compatibility through a DMSO / ACN / DMF mixed solvent, combines a continuous flow reactor (PFR) with a real-time HPLC monitoring system at 36-minute intervals, and dynamically controls reaction parameters, ultimately achieving kilogram-scale production. However, the industrialization of this technology faces three core bottlenecks: although ceramic membranes have outstanding purification efficiency, their high procurement and maintenance costs significantly increase production costs; at the same time, the integrated system of continuous flow reactors and HPLC online monitoring has strict requirements on equipment sealing and operational expertise, which greatly increases the technical threshold for large-scale production.

[0004] Patent CN 116731154A proposes a strategy for synthesizing tilpotide using a hydrophobic tag. The strategy involves dividing tilpotide into six fragments, namely AA1-4, 5-14, 15-21, 22-29, 30-39, and an alkyl chain. The hydrophobic tag OTAG is then used to anchor the C-terminus of the first amino acid in the fragment, and the polypeptide fragments OTAG-AA1(22-29)-Fmoc, OTAG-AA2(15-21)-Fmoc, OTAG-AA3(5-14)-Fmoc, and OTAG-AA4(1-4)-Boc are synthesized, respectively. The hydrophobic tag is then removed by soft cleavage to expose the C-terminus of the polypeptide fragment. Finally, the fragments are condensed one by one to obtain a fully protected tilpotide-tag conjugate. However, our experiments show that the hydrophobicity of peptide chains in hydrophobic tag-assisted synthesis decreases as the length of the peptide chain increases. When the length of the peptide chain exceeds 10 AA, the hydrophobicity decreases significantly and even becomes soluble in highly polar solvents (acetonitrile). The low yield of the product in the examples of this patent also shows that this method has certain problems in the synthesis of medium-length peptide drugs.

[0005] Based on the above existing technical problems, the purpose of the present invention is to provide a method for synthesizing tilpotide. This method innovatively utilizes the side chain carboxyl group of aspartic acid as the anchoring position of the hydrophobic tag, and designs a new fragment connection route. Compared with the solid-phase synthesis method, the method of the present invention can monitor the reaction process, is simple to post-process, has high purity, and uses less solvent. Compared with the hydrophobic tag polypeptide fragment connection method, this method can realize the introduction of multiple hydrophobic tags as protective groups during the synthesis of medium and long-chain polypeptides, while improving the product yield and purity, it effectively overcomes the hydrophobic attenuation problem caused by peptide chain extension in the traditional method, and is in line with the green chemical production process method. Summary of the Invention

[0006] Based on the above existing technical problems, the object of the present invention is to provide a method for synthesizing tilpotide.

[0007] In order to achieve the above technical objectives, the technical solutions adopted by the present invention are as follows:

[0008] The present invention provides a method for synthesizing tilpotide, which comprises first dividing the main chain of tilpotide into five polypeptide fragments: positions 30-39, positions 22-29, positions 16-21, positions 10-15, and positions 1-9, wherein the side chain polypeptide is recorded as fragment 6; and comprising the following steps:

[0009] In the first step, amino acid 39 was coupled to the amide hydrophobic tag carrier NTAG-NH2. Then, amino acids 39 to 30 were coupled one by one to obtain fragment 1, which is denoted as Fmoc-AA (30 - 39)-NTAG, where AA represents amino acids 30 to 39. The nomenclature has the same meaning as follows;

[0010] In the second step, the 29th amino acid was first coupled to the ester-based hydrophobic tag carrier OTAG-OH, and then the 29th amino acid was coupled one by one to the 22nd amino acid to obtain fragment 2 Fmoc-AA (22 - 29)-OTAG, where AA (22 - 29) represents the amino acids at positions 22-29.

[0011] In the third step, fragment 2 Fmoc-AA (22-29)-OTAG was used for soft cleavage to remove the tag carrier connected to amino acid 29 to obtain Fmoc-AA (22-29)-OH;

[0012] In the fourth step, the amino acid at position 21 was first coupled to the ester-based hydrophobic tag carrier OTAG-OH, and then the amino acids at position 21 were coupled one by one to the amino acids at position 16 to obtain fragment 3 Fmoc-AA(16-21)-OTAG;

[0013] In the fifth step, fragment 3 Fmoc-AA(16-21)-OTAG was used for soft cleavage to remove the tag carrier connected to amino acid 21 to obtain Fmoc-AA(16-21)-OH;

[0014] In the sixth step, after removing Fmoc from the first step fragment 1 Fmoc-AA(30-39)-NTAG, it was sequentially connected with Fmoc-AA(22-29)-OH and Fmoc-AA(16-21)-OH to obtain Fmoc-AA(16-39)-NTAG;

[0015] In the seventh step, the side chain carboxyl group of aspartic acid was innovatively used as the anchoring position of the hydrophobic tag to prepare a new hydrophobic tag compound NH2-Asp(OTAG)-OR 1 , its main body is Asp amino acid, and its C-terminus contains substituent R 1 The side chain substituent is OTAG. The hydrophobic tag compound NH2-Asp(OTAG)-OR 1 As a starting point, amino acids were coupled sequentially to obtain fragment 4 Fmoc-AA(10 - 14)-Asp(OTAG)-OR 1 ;

[0016] Step 8: Similarly, NH2-Asp(OTAG)-OR 1 As a starting point, amino acids were coupled sequentially to obtain fragment 5 Boc-AA(1-8)-Asp(OTAG)-OR 1 ;

[0017] Step 9: Fragment 5 Boc-AA(1-8)-Asp(OTAG)-OR 1 Remove R 1 The seventh step of the fragment 4 Fmoc - AA (10 - 14) -Asp (OTAG) - OR 1 After removing Fmoc, 1 Fmoc-AA(1-8)-Asp(OTAG)-OH was coupled to obtain Boc-AA(1-8)-Asp(OTAG)-AA(10-14)-Asp(OTAG)-OR 1 , and then remove R 1 treatment;

[0018] Step 10: Remove Fmoc from the Fmoc-AA(16-39)-NTAG obtained in step 6 and 1 The Boc-AA(1-8)-Asp(OTAG)-AA(10-14)-Asp(OTAG)-OH was connected to obtain a fully protected tilpotide backbone;

[0019] In the eleventh step, with the assistance of the ester-based hydrophobic tag carrier OTAG-OH, amino acids were sequentially coupled to the carrier to obtain fragment 6, whose structure is Ara(Ot-Bu) - Glu (α- Ot-Bu) - AEEA - AEEA - OTAG, which was then softly cleaved to obtain Ara(Ot-Bu) - Glu(α- Ot-Bu) - AEEA - AEEA - OH;

[0020] In the twelfth step, after removing the Lys side chain protecting group from the fully protected tilpotide main chain obtained in the tenth step, the main chain is coupled with Ara(Ot-Bu)-Glu(α-Ot-Bu)-AEEA-AEEA-OH obtained by soft cleavage to obtain fully protected tilpotide;

[0021] Step 13: acid cleavage of the fully protected tilpotide in Step 12 to obtain crude tilpotide;

[0022] Step 14: purify the crude tilpotide and dry it to obtain pure tilpotide.

[0023] Furthermore, the structure of the amide hydrophobic tag NTAG-NH2 is as follows:

[0024] ; R 2 is a straight-chain saturated alkyl group of 18 to 28 carbon atoms.

[0025] The structure of the ester-based hydrophobic tag OTAG-OH is as follows:

[0026] ; R 2 is a straight-chain saturated alkyl group of 18 to 28 carbon atoms.

[0027] The hydrophobic tag compound NH2-Asp(OTAG)-OR 1 The structure is as follows:

[0028] ;

[0029] R 2 is a straight chain saturated alkyl group of 18 to 28 carbon atoms; R 1 is a C1-C6 alkyl group or a silyl group, wherein R 3 、R 4 、R 5 are each independently selected from C1-C10 alkyl, aryl or hydrogen, and R 3 、R 4 、R 5 Not entirely hydrogen.

[0030] Furthermore, the hydrophobic tag compound NH2-Asp(OTAG)-OR 1 Substituent R in the structure 1 Preferred are methyl, ethyl, propyl, butyl, TMSE, TBDMSE, TPSE or TBSE substituents.

[0031] Furthermore, the hydrophobic tag compound NH2-Asp(OTAG)-OR 1 The synthesis method is:

[0032] S1: N-terminal contains Fmoc, C-terminal contains substituent R 1 Asp is denoted as Fmoc-Asp-OR 1 , Fmoc-Asp-OR 1Mix with 2,4-di(C18-C28alkoxy)benzyl alcohol and DMAP in tetrahydrofuran, add EDCI dropwise in an ice bath to react, and TLC detects the reaction to be complete; remove most of the solvent under reduced pressure, add a large amount of acetonitrile and stir, then filter and dry to obtain Fmoc-Asp(OTAG)-OR 1 ;

[0033] Fmoc-Asp-OR 1 The molar ratio of DMAP to 2,4-di(C18-C28alkoxy)benzyl alcohol is 1-1.5:1, and the molar amount of DMAP is Fmoc- Asp- OR 1 10-20% of the molar amount, EDCI and Fmoc-Asp-OR 1 The molar ratio is 0.8-1.2:1;

[0034] S2: Fmoc-Asp(OTAG)-OR from step S1 1 Further Fmoc removal was performed to obtain the hydrophobic tag NH2-Asp(OTAG)-OR 1 .

[0035] In the present invention, the coupling of amino acids AA on the hydrophobic tag carrier and the coupling connection between polypeptide fragments are both conventional operations.

[0036] Taking the process of coupling amino acids to the hydrophobic tag carrier OTAG-OH or NTAG-NH2 to prepare amino acids NH2-AA-OTAG or NH2-AA-NTAG containing a hydrophobic tag carrier at the C-terminus as an example, the preparation steps are as follows:

[0037] 1) Dissolve the hydrophobic tag carrier OTAG-OH or NTAG-NH2 in the reaction solvent, add Fmoc-AA-OH, additives, condensing agents, and base in sequence, and react in an ice-water bath for 0.2-1 h. Move to room temperature and continue the reaction. Monitor the reaction endpoint by TLC. Drain the solvent and add a poor solvent to precipitate the product to obtain Fmoc-AA-OTAG or Fmoc-AA-NTAG.

[0038] 2) Dissolve Fmoc-AA-OTAG or Fmoc-AA-NTAG in a reaction solvent, add a de-Fmoc reagent, and stir the reaction. Monitor the reaction endpoint by TLC. Drain the solvent and add a poor solvent to precipitate the product to obtain NH2-AA-OTAG or NH2-AA-NTAG.

[0039] Taking the process of connecting a long polypeptide fragment sequence Fmoc-AA(1-8)-Asp(OTAG)-AA(10-14)-Asp(OTAG)-OH with NH2-AA(16-39)-NTAG to generate a fully protected tilpotide backbone as an example, the preparation steps are as follows: NH2-AA(16-39)-NTAG is dissolved in a reaction solvent, and Fmoc-AA(1-8)-Asp(OTAG)-AA(10-14)-Asp(OTAG)-OH, additives, condensing agents and base are added in sequence. The reaction is carried out in an ice-water bath for 0.2-5 hours, and the reaction is continued at room temperature. The reaction endpoint is monitored by TLC. The solvent is then dried, and a poor solvent is added to precipitate the product to obtain the fully protected tilpotide backbone.

[0040] Furthermore, the additive is one or more of HOBt, 6-NO2-HOBt, 6-CF3-HOBt, HOAt, 6-Cl-HOBt, 6-HOAt, 4-HOAt, HODhat, HODhad, HOSu, HONB, HOCt, HOPy, HOBI, HOI, 6-Cl-HOBI, and Oxyma; preferably one of HOBt, HOAt, HOSu, and HONB.

[0041] Furthermore, the condensing agent is at least one of carbodiimides, tetramethylammonium salts, and phosphonium salts; the carbodiimide condensing agent is one or more of DCC, DIC, EDCI, CIC, BMC, BEC, and CPC; the tetramethylammonium salt condensing agent is one or more of HBTU, TBTU, TDTU, HDTU, TDATU, HDATU, TPTU, HPTU, TSTU, HSTU, TPFTU, HPFTU, CF3-HBTU, HATU, TATU, HATTU, HOTT, TOTU, and HOTU; and the phosphonium salt condensing agent is one or more of BOP, BrOP, PyClOP, PyBrOP, ClOP, PyBOP, AOP, PyAOP, PyOxm, PyNOP, PyFOP, PyFNBOP, and PyCloK.

[0042] Furthermore, the condensing agent is preferably one or more of DIC, EDCI, CIC, TBTU, HBTU, and PyBOP.

[0043] Furthermore, the base is one or more of NMM, TEA, DIPEA, DMAP, and K2CO3, and more preferably the base is one of NMM and DIPEA.

[0044] Furthermore, in step 1), the molar ratio of the hydrophobic tag carrier OTAG-OH or NTAG-NH2, Fmoc-AA-OH, condensing agent, additive and base is 1:1.2~1.5:1.1~1.5:1.1~1.5:3~10.

[0045] Furthermore, the reaction solvent in step 2) is one or more of dichloromethane, tetrahydrofuran, and chloroform; and the poor solvent is one or more of methanol, acetonitrile, and water.

[0046] Furthermore, the Fmoc removal reagent in step 2) is one or more of piperidine, DBU, diethylamine, and sodium hydroxide.

[0047] Furthermore, the molar ratio of NH2-AA(16-39)-NTAG, Fmoc-AA(1-8)-Asp(OTAG)-AA(10-14)-Asp(OTAG)-OH, condensing agent, additive and base is 1:1.2~1.5:1.1~1.5:1.1~1.5:3~10.

[0048] Furthermore, the multiple polypeptide fragments of the present invention are synthesized by coupling one by one by condensation in a liquid phase, and the amino termini, carboxyl termini and side chains of the amino acids or peptides in the synthesis process of telportide are all protected by protecting groups, as follows:

[0049] (1) The protecting group used at the amino terminus is one of Cbz, Boc, Fmoc, and Ac;

[0050] (2) The protecting groups used for the carboxyl end are methyl ester, benzyl ester, tert-butyl ester, allyl ester, nitrobenzyl ester, propargyl ester, 9-fluorenylmethyl ester, and ferrocenyl ester;

[0051] (3) The side chain protecting group of Lys can be one or more of Dde, Boc, Ac, and Cbz.

[0052] Furthermore, the hydrophobic tag NH2-Asp(OTAG)-OR 1 Medium R 1 The deprotection reagent can be one of a 0.01M-0.1M aqueous lithium hydroxide solution, a tetrahydrofuran solution containing 0.05M-0.1M lithium bromide and 0.02M-0.2M triethylamine, a 0.05-1M TBAF tetrahydrofuran solution, a 0.05-1M HF tetrahydrofuran solution, a tetrahydrofuran solution containing 5%-20% HF and 5%-20% PIP, preferably a tetrahydrofuran solution containing 0.05M-0.1M lithium bromide, 0.02M-0.2M triethylamine and a 0.05-0.5M TBAF tetrahydrofuran solution.

[0053] The substituent R at the C-terminus of the amino acid in the polypeptide fragment of the present invention 1 Removal of hydrophobic tag NH2-Asp(OTAG)-OR 1 Medium R 1 The removal method is the same.

[0054] Furthermore, the soft cleavage method only removes the hydrophobic tag without affecting other side chain protecting groups. The method is as follows: the soft cleavage reaction is carried out in a TFA cleavage liquid system, and the reaction is carried out at room temperature for 2 to 5 hours. After the reaction is completed, a poor solvent is added to the system for crystallization, and the peptide fragment is filtered and washed to obtain a peptide fragment. The TFA cleavage liquid system of the soft cleavage method is a solution containing a volume fraction of 0.8-1.5% TFA and 4-8% TEA.

[0055] Furthermore, the acid cleavage method is to remove multiple hydrophobic tags and other side chain protecting groups to obtain crude tilpoitide peptide, and the method is as follows: the acid cleavage reaction is carried out in a TFA cleavage solution system, and the reaction is carried out at room temperature for 2 to 5 hours. After the reaction is completed, a poor solvent is added to the system for crystallization, and the crude tilpoitide peptide is filtered and washed to obtain the crude tilpoitide peptide; the TFA cleavage solution system for the acid cleavage reaction is a solution containing 80-85% TFA and 3-6% EDT by volume.

[0056] Compared with the prior art, the present invention has the following advantages:

[0057] 1. This invention utilizes multiple hydrophobic tags to collaboratively assist the total synthesis of tilpoxetine. This innovative development of multiple large hydrophobic groups to jointly support the solution to the problem of decreased hydrophobicity allows for the targeted quantitative total synthesis of the mid-peptide chain without cleaving the hydrophobic tags to separate the naked peptide fragments.

[0058] 2. The present invention innovatively utilizes the side chain carboxyl group of aspartic acid as the anchoring position of the hydrophobic tag, and designs a new fragment connection route. Compared with the solid-phase synthesis method, the method of the present invention has simple post-processing, high purity, and low solvent consumption. Compared with the hydrophobic tag polypeptide fragment connection method, this method can introduce multiple hydrophobic tags as protective groups during the synthesis of medium and long-chain polypeptides. While improving the product yield and purity, it effectively overcomes the hydrophobicity attenuation problem caused by peptide chain extension in traditional methods, and complies with the green chemical production process method. 3. The present invention adopts the innovative combination of hydrophobic tags and hydrophobic groups to synthesize the full synthesis of telportopeptide, which greatly enhances the hydrophobicity of the polypeptide fragments and greatly improves the synthesis yield and purity of the polypeptide fragments.

[0059] 4. The present invention utilizes a soluble hydrophobic tag as a carrier for the total synthesis of tipol peptide. The reaction is carried out in solution, making it a homogeneous reaction. Compared to traditional solid-phase peptide synthesis methods, the reaction progress can be monitored by TLC, enabling quantitative completion of the reaction and avoiding fragment loss during the reaction.

[0060] 5. In the reaction process of each step of condensation and deprotection, the post-treatment can be carried out by adding a poor solvent to precipitate the product, and the product is washed with the poor solvent; the post-treatment is relatively simple and the solvent used is relatively environmentally friendly and the amount used is small, which conforms to the green chemical production process.

[0061] 6. The present invention uses a hydrophobic tag as a carrier, which can remove impurities generated by the raw materials during post-processing. Compared with traditional solid-phase peptide synthesis methods, impurities generated by some difficult sequences can be easily removed. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is a high-resolution mass spectrum of the polypeptide fragment 1 obtained by removing the label carrier from the polypeptide fragment according to the acid cleavage experimental steps of Example 13;

[0063] Figure 2 This is the high-resolution mass spectrum of polypeptide fragment 2 after the label carrier is removed;

[0064] Figure 3 This is the high-resolution mass spectrum of peptide fragment 3 after the tag carrier is removed;

[0065] Figure 4 This is the high-resolution mass spectrum of peptide fragment 4 after the tag carrier is removed;

[0066] Figure 5 This is the high-resolution mass spectrum of the peptide fragment of Boc - AA(1 - 8) - Asp(OTAG) - AA(10 - 14) - Asp(OTAG) - OTMSE after the tag carrier is removed;

[0067] Figure 6 This is the high-resolution correlation data of tilportide;

[0068] Figure 7 is the structural formula of tilpotide. DETAILED DESCRIPTION

[0069] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely illustrative of the present invention. The experimental methods used in the following examples are generally performed under conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0070] The abbreviations used in this invention and their corresponding meanings in Chinese are as follows:

[0071] Table 1 Abbreviations and Chinese equivalents of amino acid reagents used

[0072] English abbreviation Chinese name AA amino acids Aib -2-aminoisobutyric acid Ala -Alanine Asp -Aspartic acid Gln -Glutamine Glu -Glutamate Gly -Glycine Trp -Tryptophan Phe -Phenylalanine Ala -Alanine Ile -Isoleucine Leu -Leucine Lys -Lysine Ser -serine Pro -Proline Thr -Threonine Val -Valine AEEA 2-(2-(2-aminoethoxy)ethoxy)acetic acid C20 acid eicosanedioic acid .

[0073] Table 2 Abbreviations of condensation reagents used and their Chinese equivalents

[0074] DIC 1,3-Diisopropylcarbodiimide DCC ,-Dicyclohexylcarboximide EDCI 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride CIC -Cyclohexyl, -isopropylcarbodiimide BMC -tert-Butyl,-methylcarbodiimide BEC -tert-Butyl,-ethylcarbodiimide CPC ,-Dicyclopentylcarbodiimide BOP Benzotriazole-1-oxytris(dimethylamino)phosphine hexafluorophosphate PyBOP Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate PyClOP Chlorotripyrrolidinyl hexafluorophosphate PyBrOP Tripyrrolidinylphosphonium bromide hexafluorophosphate DMT-MM 4-(4,6-Dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride ClOP Chlorotris(dimethylamino)phosphonium hexafluorophosphate AOP 7-Azabenzotriazol-1-yloxytris(dimethylamino)phosphine hexafluorophosphate PyOxm (Z)-(((1-Cyano-2-ethoxy-2-oxoethylidene)amino)oxy)tris(pyrrolidin-1-yl)phosphonium hexafluorophosphate PyAOP 7-Azabenzotriazole-1-oxy)tripyrrophosphonium hexafluorophosphate COMU (2-Hydromino-ethyl cyanoacetate)-N,N-dimethyl-morpholinourea hexafluorophosphate Oxyma Pure Ethyl 2-oximecyanoacetate HBTU Benzotriazole-,,,-tetramethyluronium hexafluorophosphate TBTU 2-(1-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate TPTU 2-(2-Pyridon-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate HPTU 1,1,3,3-Tetramethyl-2-(2-oxopyridin-1(2H)-yl)isourea hexafluorophosphate TSTU 2-Succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate HSTU ,,,-Tetramethylurea-(-succinimidyl)hexafluorophosphate HATU 2-(7-Azobenzotriazole)-,,,-tetramethyluronium hexafluorophosphate TATU 2-(7-Azabenzotriazole)-,,,-tetramethyluronium tetrafluoroborate HOBT 1-Hydroxybenzotriazole <![CDATA[6-NO2-HOBt]]> 6-Nitro-1-hydroxybenzotriazole <![CDATA[6-CF3-HOBt]]> 6-Trifluoromethyl-1-hydroxybenzotriazole HOAt -Hydroxy-7-azabenzotriazole 6-Cl-HOBt 6-Chloro-1-hydroxybenzotriazole HOSu -Hydroxysuccinimide HOCt 1-Hydroxy-1-1,2,3-triazole-4-carboxylic acid ethyl ester HONB -Hydroxy-5-norbornene-2,3-dicarboximide .

[0075] Table 3 Abbreviations of silicon-containing reagents used and their Chinese equivalents

[0076] TBDMS tert-Butyldimethylsilyl TMSE trimethylsilylethyl TES triethylsilyl DIPS Diisopropylmethoxysilyl TIPDS triisopropylsilyl .

[0077] Table 4 Abbreviations of acid and base reagents used and their Chinese equivalents

[0078] DBU 1,8-Diazabicycloundec-7-ene DIPEA N,N-Diisopropylethylamine TEA Triethylamine NMM N-methylmorpholine DMAP 4-Dimethylaminopyridine TBAF Tetrabutylammonium fluoride HF hydrogen fluoride .

[0079] Table 5 Abbreviations of protective groups used and their Chinese equivalents

[0080] Boc tert-Butyloxycarbonyl Trt Trityl Fmoc 9-Fluorenylmethoxycarbonyl t-Bu tert-butyl Me methyl Mmt Monomethoxytriphenylphosphine Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl .

[0081] Example 1 Fmoc-Gly-Pro-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-NTAG

[0082] 1.1. Fmoc-Ser(t-Bu)-NTAG was synthesized by coupling Ser amino acid to the amide hydrophobic tag carrier NTAG-NH2

[0083] 921 mg (2.4 mmol) of Fmoc-Ser(t-Bu)-OH, 325 mg (2.4 mmol) of HOBT, and 1664 mg (2 mmol) of bis(4-docosyloxyphenyl)methanamine were dissolved in 40 mL of dichloromethane. 344 mg (4.0 mmol) of NMM was added, followed by 494 mg (2.4 mmol) of DCC in an ice bath. The reaction was allowed to react for 2 hours, and TLC confirmed the reaction was complete. Most of the solvent was removed under reduced pressure, and a large amount of acetonitrile was added and stirred to form a solid. The solid was stirred with acetonitrile for a period of time, then filtered and dried to obtain 2.548 g of pure Fmoc-Ser(t-Bu)-NTAG in a 98.6% yield.

[0084] 1.2. Removal of Fmoc to obtain NH2-Ser(t-Bu)-NTAG

[0085] The resulting Fmoc-Ser(t-Bu)-NTAG was dissolved in 20% piperidine in tetrahydrofuran and stirred at room temperature for 40 minutes. The reaction progress was monitored by TLC. After completion of the reaction, the pH was adjusted to 4-5 with 4M hydrochloric acid. A large amount of acetonitrile was added to precipitate a solid. Filtration then yielded 1.97 g of solid NH2-Ser(t-Bu)-NTAG in a 99.2% yield.

[0086] 1.3. Two amino acids coupled to form Fmoc-Pro-Ser(t-Bu)-NTAG

[0087] Dissolve NH2-Ser(t-Bu)-NTAG, 810 mg (2.4 mmol) of Fmoc-Pro-OH, and 325 mg (2.4 mmol) of HOBT (obtained in step 1.2) in 40 mL of dichloromethane. Add 344 mg (4.0 mmol) of NMM and 494 mg (2.4 mmol) of DCC in an ice bath. Allow to react for 2 hours, and the reaction is complete by TLC. Remove the majority of the solvent under reduced pressure, then add a large amount of acetonitrile and stir to form a solid. After stirring the solid with acetonitrile for a period of time, filter, and dry the mixture to obtain 2.42 g of pure Fmoc-Pro-Ser(t-Bu)-NTAG (97.0% yield).

[0088] 1.4. Removal of Fmoc to obtain NH2-Pro-Ser(t-Bu)-NTAG

[0089] The resulting Fmoc-Pro-Ser(t-Bu)-NTAG was dissolved in 20% piperidine in tetrahydrofuran and stirred at room temperature for 40 minutes. The reaction progress was monitored by TLC. After completion of the reaction, the pH was adjusted to 4-5 with 4M hydrochloric acid. A large amount of acetonitrile was added to precipitate a solid. Filtration then yielded 1.98 g of solid NH2-Pro-Ser(t-Bu)-NTAG, a yield of 98.7%.

[0090] The amino acids were coupled sequentially according to the method of steps 1.3-1.4 to finally obtain fragment 1 containing amino acids 30-39 and an amide hydrophobic tag carrier, denoted as Fmoc-AA (30-39)-NTAG, with a structure of Fmoc-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-NTAG.

[0091] The peptide fragment 1 was subjected to the acid cleavage experimental procedure of Example 13 to remove the label carrier on the peptide fragment. The high-resolution mass spectrum of the peptide fragment 1 after acid cleavage was as follows: Figure 1 shown.

[0092] Fmoc was removed from Fragment 1 Fmoc-AA (30 - 39)-NTAG of Example 1 according to the method of Step 1.2 of Example 1 to obtain NH2-AA (30 - 39)-NTAG for use.

[0093] Example 2 Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OTAG

[0094] 2.1. Synthesis of Fmoc-Gly-OTAG

[0095] To a solution of 558 mg (2.4 mmol) of Fmoc-Gly-OH, 49 mg (0.4 mmol) of DMAP, and 1515 mg (2 mmol) of 2,4-di(docosyloxy)benzyl alcohol in 40 mL of dichloromethane, 504 mg (2.4 mmol) of DIC was added dropwise under an ice bath. The reaction was allowed to react for 2 hours, and the reaction was complete by TLC. Most of the solvent was removed under reduced pressure, and a large amount of acetonitrile was added and stirred to form a solid. The solid was stirred with acetonitrile for a period of time, then filtered and air-dried to obtain 2.037 g of pure Fmoc-Gly-OTAG (99.6% yield).

[0096] 2.2 Synthesis of NH2-Gly-OTAG

[0097] The resulting Fmoc-Gly-OTAG was dissolved in a 20% piperidine-tetrahydrofuran solution as a deprotecting agent and stirred at room temperature for 40 minutes. The reaction progress was monitored by TLC. After completion of the reaction, the pH was adjusted to 4-5 with 4M hydrochloric acid. A large amount of acetonitrile was added to precipitate a solid. Filtration then yielded 1.572 g of solid NH2-Gly-OTAG, a yield of 98.7%.

[0098] 2.3 Synthesis of Fmoc-Ala-Gly-OTAG

[0099] 1.886 g of NH2-Gly-OTAG, 517 mg (4 mmol) of DIPEA, and 603 mg (2.4 mmol) of Fmoc-Ala-OH were mixed in 40 mL of dichloromethane. 504 mg (2.4 mmol) of DIC was added dropwise under an ice bath. The reaction was allowed to react for 2 hours, and TLC confirmed the reaction was complete. Most of the solvent was removed under reduced pressure, and a large amount of acetonitrile was added and stirred to form a solid. The solid was stirred with acetonitrile for a period of time, filtered, and air-dried to obtain 2.093 g of pure Fmoc-Ala-Gly-OTAG, with a yield of 97.6%.

[0100] 2.4. Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OTAG

[0101] According to the above method, amino acids were coupled sequentially, and NH2-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OTAG was obtained after multiple coupling and deprotection.

[0102] 2.861 g of NH2-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OTAG, 922 mg (2.4 mmol) of Fmoc-Phe-OH, and 517 mg (4 mmol) of DIPEA were dissolved in 40 mL of dichloromethane. PyBOP (1249 mg (2.4 mmol) was added under ice-cooling and allowed to react for 2 hours. TLC confirmed the reaction was complete. The solvent was mostly removed under reduced pressure, and a large amount of acetonitrile was added and stirred to form a solid. The solid was stirred with acetonitrile for a period of time, filtered, and air-dried. This fragment 2, containing amino acids 22-29 and an ester-based hydrophobic tag carrier, was obtained, designated Fmoc-AA (22-29)-OTAG. Its structure was Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OTAG, with a yield of 75.3%.

[0103] The polypeptide fragment 2 was soft-cleaved according to the method of 7.2 of Example 7 to remove the label carrier on the polypeptide fragment. The high-resolution mass spectrum of the polypeptide fragment 2 after the carrier was removed was as follows: Figure 2 shown.

[0104] Example 3 Synthesis of Fmoc-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Dde)-Ala-OTAG

[0105] 3.1 Synthesis of Fmoc-Ala-OTAG

[0106] To a solution of 603 mg (2.4 mmol) of Fmoc-Ala-OH, 49 mg (0.4 mmol) of DMAP, and 1515 mg (2 mmol) of 2,4-di(docosyloxy)benzyl alcohol in 40 mL of dichloromethane, 504 mg (2.4 mmol) of DIC was added dropwise under an ice bath. The reaction was allowed to react for 2 hours, and TLC confirmed the reaction to be complete. Most of the solvent was removed under reduced pressure, and a large amount of acetonitrile was added and stirred to form a solid. The solid was stirred with acetonitrile for a period of time, filtered, and air-dried to obtain 2.003 g of pure Fmoc-Ala-OTAG (99.3% yield).

[0107] 3.2 Synthesis of NH2-Ala-OTAG

[0108] The resulting Fmoc-Ala-OTAG was dissolved in 20% piperidine in tetrahydrofuran and stirred at room temperature for 40 minutes. The reaction progress was monitored by TLC. After completion of the reaction, the pH was adjusted to 4-5 with 4M hydrochloric acid. A large amount of acetonitrile was added to precipitate a solid. Filtration then yielded 1.546 g of solid NH2-Gly-OTAG, a yield of 99.2%.

[0109] 3.3. Synthesis of Fmoc- Lys(Dde)-Ala-OTAG

[0110] 1.995 g of NH2-Ala-OTAG, 517 mg (4 mmol) of DIPEA, and 812 mg (2.4 mmol) of Fmoc-Lys(Dde)-OH were mixed in 40 mL of dichloromethane. 504 mg (2.4 mmol) of DIC was added dropwise under an ice bath. The reaction was allowed to react for 2 hours, and TLC confirmed the reaction was complete. Most of the solvent was removed under reduced pressure, and a large amount of acetonitrile was added and stirred to form a solid. The solid was stirred with acetonitrile for a period of time, filtered, and air-dried to obtain 2.143 g of pure Fmoc-Lys(Dde)-Ala-OTAG, with a yield of 98.0%.

[0111] 3.4. Fmoc-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Dde)-Ala-OTAG

[0112] According to the above method, amino acids were coupled sequentially, and NH2-Ile-Ala-Gln(Trt)-Lys(Dde)-Ala-OTAG was obtained after multiple coupling and deprotection.

[0113] 2.355 g of NH2-Ile-Ala-Gln(Trt)-Lys(Dde)-Ala-OTAG, 922 mg (2.4 mmol) of Fmoc-Phe-OH, and 517 mg (4 mmol) of DIPEA were dissolved in 40 mL of dichloromethane. PyBOP (1249 mg (2.4 mmol) was added under ice-cooling and allowed to react for 2 hours. TLC confirmed the reaction was complete. The solvent was mostly removed under reduced pressure, and a large amount of acetonitrile was added and stirred to form a solid. The solid was stirred with acetonitrile for a period of time, filtered, and air-dried to yield fragment 3, designated Fmoc-AA(16-21)-OTAG. Its structure is Fmoc-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(Dde)-Ala-OTAG, and the yield is 72.5%.

[0114] The polypeptide fragment 3 was soft-cleaved according to the method of 7.2 of Example 7 to remove the label carrier on the polypeptide fragment. The high-resolution mass spectrum of the polypeptide fragment 3 after the carrier was removed was as follows: Figure 3 shown.

[0115] Example 4 Fmoc-Tyr(t-Bu)-Ser(t-Bu)-Ile-Aib-Leu-Asp(OTAG)-OTMSE

[0116] 4.1 Synthesis of Fmoc-Asp(OTAG)-OTMSE

[0117] Fmoc-Asp-OTMSE (1093 mg (2.4 mmol), DMAP (49 mg (0.4 mmol), and 2,4-di(docosyloxy)benzyl alcohol) (1515 mg (2 mmol)) were mixed in 40 mL of tetrahydrofuran. EDCI (460 mg (2.4 mmol)) was added dropwise under an ice bath. The reaction was allowed to react for 2 hours, and TLC confirmed the reaction was complete. Most of the solvent was removed under reduced pressure, and a large amount of acetonitrile was added and stirred to form a solid. The solid was stirred with acetonitrile for a period of time and then filtered to obtain 2.360 g of pure Fmoc-Asp(OTAG)-OTMSE in a 98.7% yield.

[0118] 4.2 Synthesis of NH2-Asp(OTAG)-OTMSE

[0119] The resulting Fmoc-Asp(OTAG)-OTMSE was dissolved in 20% piperidine in tetrahydrofuran and stirred at room temperature for 40 minutes. The reaction progress was monitored by TLC. After completion of the reaction, the pH was adjusted to 4-5 with 4M hydrochloric acid, and a large amount of acetonitrile was added for precipitation. Filtering then yielded 1.960 g of solid NH2-Asp(OTAG)-OTMSE in a 98.5% yield.

[0120] 4.3 Synthesis of Fmoc-Leu-Asp(OTAG)-OTMSE

[0121] 848 mg (2.4 mmol) of Fmoc-Leu-OH, 517 mg (4 mmol) of DIPEA, and NH2-Asp(OTAG)-OTMSE were dissolved in 40 mL of tetrahydrofuran. PyBOP (1249 mg (2.4 mmol) was added under ice-cooling and allowed to react for 2 hours. TLC confirmed the reaction was complete. Most of the solvent was removed under reduced pressure, and a large amount of acetonitrile was added and stirred to form a solid. The solid was stirred with acetonitrile for a period of time, filtered, and air-dried to yield 2.538 g of Fmoc-Leu-Asp(OTAG)-OTMSE, a yield of 97.2%.

[0122] 4.4. Synthesis of Fmoc-Tyr(t-Bu)-Ser(t-Bu)-Ile-Aib-Leu-Asp(OTAG)-OTMSE

[0123] According to the above method, amino acids were coupled sequentially, and NH2-Ser(t-Bu)-Ile-Aib-Leu-Asp(OTAG)-OTMSE was obtained after multiple coupling and deprotection.

[0124] 2.311 g of NH2-Ser(t-Bu)-Ile-Aib-Leu-Asp(OTAG)-OTMSE, 1243 mg (2.4 mmol) of Fmoc-Tyr(t-Bu)-OH, and 517 mg (4 mmol) of DIPEA were dissolved in 40 mL of dichloromethane. 1249 mg (2.4 mmol) of PyBOP was added under ice-cooling and allowed to react for 2 hours. TLC confirmed the reaction was complete. Most of the solvent was removed under reduced pressure, and a large amount of acetonitrile was added and stirred to form a solid. The solid was stirred with acetonitrile for a period of time, filtered, and air-dried to obtain fragment 4 (Fmoc-Tyr(t-Bu)-Ser(t-Bu)-Ile-Aib-Leu-Asp(OTAG)-OTMSE) in an 80.5% yield.

[0125] The polypeptide fragment 4 was soft-cleaved according to the method of 7.2 of Example 7 to remove the label carrier on the polypeptide fragment. The high-resolution mass spectrum of the polypeptide fragment 4 after the carrier was removed was as follows: Figure 4 shown.

[0126] Fragment 4 of Example 4 was subjected to Fmoc removal according to the method of Step 4.2 of Example 4 to obtain NH2-Tyr(t-Bu)-Ser(t-Bu)-Ile-Aib-Leu-Asp(OTAG)-OTMSE for use.

[0127] Example 5 Synthesis of Boc-Tyr(t-Bu)-Aib-Glu(t-Bu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Asp(OTAG)-OTMSE

[0128] 5.1 Synthesis of Fmoc-Asp(OTAG)-OTMSE

[0129] Fmoc-Asp-OTMSE (1093 mg (2.4 mmol), DMAP (49 mg (0.4 mmol), and 2,4-di(docosyloxy)benzyl alcohol) (1515 mg (2 mmol)) were mixed in 40 mL of tetrahydrofuran. EDCI (460 mg (2.4 mmol)) was added dropwise under an ice bath. The reaction was allowed to react for 2 hours, and TLC confirmed the reaction was complete. Most of the solvent was removed under reduced pressure, and a large amount of acetonitrile was added and stirred to form a solid. The solid was stirred with acetonitrile for a period of time, filtered, and air-dried to obtain 2.382 g of pure Fmoc-Asp(OTAG)-OTMSE (99.7% yield).

[0130] The NMR characterization data of Fmoc-Asp(OTAG)-OTMSE are as follows:

[0131] .

[0132] 1H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 7.6 Hz, 2H), 7.59 (d, J = 7.6Hz, 2H), 7.41 (t, J = 7.2 Hz, 2H), 7.32 (t, J = 7.2 Hz, 2H), 7.19 (d, J = 8.0Hz, 1H), 6.44 - 6.40 (m, 2H), 5.87 (d, J = 8.8 Hz, 1H), 5.13 (s, 2H), 4.64 -4.60 (m, 1H), 4.43 (q, J = 3.2 Hz, 1H), 4.25 - 4.18 (m, 3H), 3.92 (q, J= 10.0Hz, 4H), 3.09 (dd, J = 4.4, 4.4Hz, 1H), 2.85 (dd, J = 4.4, 4.8 Hz, 1H), 1.80- 1.72 (m, 4H), 1.45 - 1.39 (m, 4H), 1.26 (br, 72H), 0.97 (t, 2H), 0.89 (t, J= 6.8 Hz, 6H), 0.03 (s, 9H).

[0133] 13 C NMR (101 MHz, CDCl3) δ 170.9, 161.1, 159.0, 155.5, 144.0, 143.9,141.4, 131.3, 127.8, 127.2, 125.0, 120.1, 115.7, 104.7, 100.0, 68.4, 68.2,67.4, 64.4, 62.7, 50.7, 47.3, 36.9, 31.5, 29.9 29.8, 29.7, 29.6, 29.5, 29.5,29.4, 29.2, 26.2, 26.1, 23.5, 22.8, 17.4, 14.3, -1.4.

[0134] 5.2 Synthesis of NH2-Asp(OTAG)-OTMSE

[0135] The resulting product was dissolved in 20% piperidine in tetrahydrofuran and stirred at room temperature for 40 minutes. The reaction progress was monitored by TLC. After completion of the reaction, the pH was adjusted to 4-5 with 4M hydrochloric acid. A large amount of acetonitrile was added for precipitation. Filtration then yielded 1.913 g of solid NH2-Asp(OTAG)-OTMSE in a 98.3% yield.

[0136] 5.3 Synthesis of Fmoc-Thr(t-Bu)-Asp(OTAG)-OTMSE

[0137] NH2-Asp(OTAG)-OTMSE, 954 mg (2.4 mmol) of Fmoc-Thr(t-Bu)-OH, 517 mg (4 mmol) of DIPEA, and NH2-Asp(OTAG)-OTMSE were dissolved in 40 mL of dichloromethane. PyBOP (1249 mg (2.4 mmol) was added under ice-cooling and allowed to react for 2 hours. TLC confirmed the reaction was complete. The solvent was mostly removed under reduced pressure, and a large amount of acetonitrile was added and stirred to form a solid. The solid was stirred with acetonitrile for a period of time, then filtered and dried to yield 2.716 g of Fmoc-Aib-Asp(OTAG)-OTMSE (97.0% yield).

[0138] 5.4. Boc-Tyr(t-Bu)-Aib-Glu(t-Bu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Asp(OTAG)-OTMSE synthesis

[0139] According to the above method, amino acids were coupled sequentially, and after multiple coupling and deprotection, NH2-Aib-Glu(t-Bu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Asp(OTAG)-OTMSE was obtained.

[0140] 2.830 g of NH2-Aib-Glu(t-Bu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Asp(OTAG)-OTMSE, 810 mg (2.4 mmol) of Boc-Tyr(t-Bu)-OH, and 517 mg (4 mmol) of DIPEA were dissolved in 40 mL of dichloromethane. PyBOP (1249 mg (2.4 mmol) was added under ice-cooling and allowed to react for 2 hours. TLC confirmed the reaction was complete. Most of the solvent was removed under reduced pressure, and a large amount of acetonitrile was added and stirred to form a solid. The solid was stirred with acetonitrile for a period of time, then filtered and dried to obtain fragment 5Boc-Tyr(t-Bu)-Aib-Glu(t-Bu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Asp(OTAG)-OTMSE in a yield of 73.4%.

[0141] Example 6 Synthesis of Fragment 6 Ara(Ot-Bu) - Glu(α-Ot-Bu) - AEEA - AEEA - OTAG

[0142] Referring to the procedures of Examples 2 and 3, the hydrophobic tag OTAG-OH (2,4-di(docosyloxy)benzyl alcohol) was anchored to the C-terminus of Fmoc-AEEA-OH. After multiple coupling and deprotection steps, Ara(Ot-Bu)-Glu(α-Ot-Bu)-AEEA-AEEA-OTAG was obtained with an overall yield of 81.46% in seven steps.

[0143] Example 7 Deprotection of each fragment

[0144] 7.1. Removal of Silicon-Containing Protective Groups from Fragment 5

[0145] Fragment 5 was reacted in 1M TBAF in tetrahydrofuran for 0.5 h, and the reaction progress was monitored by TLC. After the reaction, a large amount of acetonitrile was added with stirring, and a white solid precipitated, yielding the peptide sequence Boc-Tyr(t-Bu)-Aib-Glu(t-Bu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Asp(OTAG)-OH in a yield of 94.6%.

[0146] 7.2 Soft Cracking of Fragment 2

[0147] Fragment 2 was cleaved in a dichloromethane solution containing 1% TFA and 5% TEA for 1 hour, with reaction progress monitored by TLC. After completion of the reaction, insoluble byproducts were removed by filtration through diatomaceous earth, and the filtrate was collected to obtain the peptide sequence 2, Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OH, which is Fmoc-AA(22-29)-OH, in a yield of 98.7%.

[0148] 7.3 Soft Cracking of Fragment 3

[0149] Fragment 3 was cleaved in dichloromethane containing 1% TFA and 5% TEA for 1 hour, with reaction progress monitored by TLC. After completion of the reaction, insoluble byproducts were removed by filtration through diatomaceous earth, and the filtrate was collected to obtain the peptide sequence 3: Fmoc- -Lys(Boc)-Ile-Ala-Gln-Lys(Dde)-Ala-OH, which is Fmoc-AA(16-21)-OH, in a yield of 97.9%.

[0150] Example 8 Synthesis of Fmoc-AA (16-39) – NTAG

[0151] 8.1. Synthesis of Fmoc-AA (22-39) – NTAG

[0152] 183 mg of NH2-AA(30-39)-NTAG, 235 mg (0.15 mmol) of Fmoc-AA(22-29)-OH, and 20 mg (0.15 mmol) of HOBT, obtained by deprotection in Example 1, were dissolved in 40 mL of dichloromethane. 26 mg (0.3 mmol) of NMM was added, followed by 62 mg (0.3 mmol) of DCC in an ice bath. The reaction was allowed to react for 2 hours, and TLC confirmed the reaction was complete. Most of the solvent was removed under reduced pressure, and a large amount of acetonitrile was added and stirred to form a solid. The solid was stirred with acetonitrile for a period of time, then filtered and dried to obtain 329 mg of pure Fmoc-AA(22-39)-NTAG in a 97.0% yield.

[0153] 8.2. Removal of Fmoc to obtain NH2-AA (22-39) - NTAG

[0154] The resulting Fmoc-AA (22-39) - NTAG was dissolved in 20% piperidine in tetrahydrofuran and stirred at room temperature for 40 minutes. The reaction progress was monitored by TLC. After completion of the reaction, the pH was adjusted to 4-5 with 4M hydrochloric acid. A large amount of acetonitrile was added to precipitate a solid. Filtration then yielded 302 mg of solid NH2-AA (22-39) - NTAG, a 99.7% yield.

[0155] 8.3. According to Example 8.1, Fmoc-AA(16-21)-OH was introduced using the same method to obtain Fmoc-AA(16-39)-NTAG with a yield of 96.5%.

[0156] Fmoc-AA (16-39) - NTAG was removed from Fmoc according to the method in step 8.2 of Example 8 to obtain NH2-AA (16-39) - NTAG for use.

[0157] Example 9 Synthesis of Fmoc-AA(1-8)-Asp(OTAG)-AA(10-14)-Asp(OTAG)-OTMSE

[0158] 1.646 g of NH2-Tyr(t-Bu)-Ser(t-Bu)-Ile-Aib-Leu-Asp(OTAG)-OTMSE obtained in Example 4 and 2.123 g of Boc-Tyr(t-Bu)-Aib-Glu(t-Bu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Asp(OTAG)-OH obtained in Example 7 were dissolved in 40 mL of dichloromethane. 173 mg (2 mmol) of NMM was added, and 826 mg (4 mmol) of DCC was added under ice-cooling. The reaction was allowed to react for 2 hours. TLC confirmed the reaction was complete. Most of the solvent was removed under reduced pressure, and a large amount of acetonitrile was added with stirring to form a solid. The solid was stirred with acetonitrile for a period of time, filtered, and then pulled dry to obtain 3702 mg of pure Boc-AA(1-8)-Asp(OTAG)-AA(10-14)-Asp(OTAG)-OTMSE (yield 98.7%).

[0159] Boc - AA (1 - 8) - Asp (OTAG) - AA (10 - 14) - Asp (OTAG) - OTMSE was softly cleaved according to the method of 7.2 of Example 7 to remove the label carrier on the polypeptide fragment. The high-resolution mass spectrum of the polypeptide fragment after the carrier was removed is shown in FIG. Figure 5 shown.

[0160] Boc-AA(1-8)-Asp(OTAG)-AA(10-14)-Asp(OTAG)-OTMSE was subjected to removal of the silicon-containing protecting group according to the method of step 7.1 of Example 7 to obtain Boc-AA(1-8)-Asp(OTAG)-AA(10-14)-Asp(OTAG)-OH.

[0161] Example 10 Synthesis of fully protected tilpotide backbone

[0162] 418 mg (0.1 mmol) of the hydrophobic tag peptide sequence NH2-AA(16-39)-NTAG and 368 mg (0.1 mmol) of Boc-AA(1-8)-Asp(OTAG)-AA(10-14)-Asp(OTAG)-OH were dissolved in 40 ml of a mixture of tetrahydrofuran and N,N'-dimethylformamide. 33 mg (0.25 mmol) of HOBt and 95 mg (0.25 mmol) of HBTU were added under ice-cooling. After stirring for half an hour, the mixture was heated to 40°C and allowed to react for 5 hours. TLC confirmed the reaction was complete. The majority of the solvent was removed under reduced pressure, and a large amount of acetonitrile was added and stirred to form a solid. The solid was stirred with acetonitrile for a period of time and then filtered to obtain 745 mg of the fully protected telpotide backbone in a yield of 94.8%.

[0163] Example 11 Removal of the Dde protecting group from the Lys side chain of the fully protected telpotide main chain

[0164] 392.1 mg of the fully protected tilpotide backbone was dissolved in 0.5% hydrazine hydrate in tetrahydrofuran and stirred for half an hour. TLC confirmed the reaction was complete. After completion of the reaction, the pH was adjusted to 4-5 with an appropriate amount of hydrochloric acid. A large amount of acetonitrile was added to precipitate the solution, which was then filtered to obtain the tilpotide backbone with exposed lysine side chains in a yield of 97.5%.

[0165] Example 12 Synthesis of fully protected tilpoxetine

[0166] Referring to the coupling operation methods in Examples 2 and 3, the fully protected tilpotide backbone after removal of Dde was coupled with fragment 6 by the condensation method of Examples 2 and 3 to obtain fully protected tilpotide with a yield of 93.7%.

[0167] Example 13 Acid cleavage of fully protected tilpotide to obtain crude drug

[0168] Fully protected tilpotide was dissolved in an acid lysis buffer (DCM:EDT:TFA:HO = 5:5:85:5, volume ratio) and stirred at room temperature for 5 h. The reaction endpoint was monitored by TLC. After completion of the reaction, the product was filtered through celite, and the filtrate was precipitated by adding methyl tert-butyl ether. The resulting white solid was filtered to obtain crude tilpotide with a yield of 91.3% and a purity of 62.5%. Pure tilpotide was obtained through preparative separation.

[0169] HR-MS (ESI): m / z calcd. for C 225 H 348 N 48 O 68[M-4] / 4: 1603.5090, found:1603.1682.

[0170] The high-resolution correlation data of tilpotide after preparation are Figure 6 shown.

[0171] Example 14 Synthesis of a novel hydrophobic tag polypeptide conjugate Fmoc-Asp(OTAG)-OH

[0172] Synthesis of Fmoc-Asp(OTAG)-OMe

[0173] 14.1. Combine 886 mg (2.4 mmol) of Fmoc-Asp-OMe, 49 mg (0.4 mmol) of DMAP, and 1515 mg (2 mmol) of 2,4-di(docosyloxy)benzyl alcohol in 40 mL of tetrahydrofuran. Add 460 mg (2.4 mmol) of EDCI dropwise in an ice bath. Allow to react for 2 hours. TLC confirms the reaction is complete. Remove most of the solvent under reduced pressure. Add a large amount of acetonitrile and stir to form a solid. Stir the solid with acetonitrile for a period of time, filter, and air-dry to obtain 2.21 g of pure Fmoc-Asp(OTAG)-OMe (98.1% yield).

[0174] The NMR characterization data of Fmoc-Asp(OTAG)-OMe are as follows:

[0175] .

[0176] 1 H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 7.6 Hz, 2H), 7.58 (d, J = 7.6Hz, 2H), 7.40 (t, J = 7.2 Hz, 2H), 7.30 (t, J = 7.2 Hz, 2H), 7.17 (d, J = 8.0Hz, 1H), 6.42 - 6.39 (m, 2H), 5.64 (d, J = 8.8 Hz, 1H), 5.12 (s, 2H), 4.63 -4.60 (m, 1H), 4.42 (m, 2H),4.36 (q, J = 3.2 Hz, 1H), 3.90 (q, J= 10.0 Hz,4H), 1.81 - 1.72 (m, 4H), 3.60 (s, 3H), 1.45 - 1.37 (m, 4H), 1.26 (br, 72H), 0.89 (t, J = 6.8 Hz, 6H).

[0177] 13 C NMR (101 MHz, CDCl3) δ 171.2, 162.0, 158.7, 154.3, 145.2, 144.7,142.3, 131.3, 128.7, 127.3, 125.5, 121.4, 116.3, 104.7, 69.3, 68.9, 67.5,65.8, 63.2, 51.5, 48.2, 37.3, 32.0, 29.9, 29.8, 29.7, 29.6, 29.5, 29.5, 29.4,29.2, 26.3, 26.2, 22.7, 17.7, 14.3.

[0178] 14.2 Synthesis of Fmoc-Asp(OTAG)-OH

[0179] 2.21 g of Fmoc-Asp(OTAG)-OMe was dissolved in 20 mL of 0.05 M lithium bromide and 0.075 M triethylamine in tetrahydrofuran. The mixture was allowed to react on ice for 10 hours, and TLC confirmed the reaction was complete. After completion, the pH was adjusted to 4-5 with 4 M hydrochloric acid, and a large amount of acetonitrile was added for precipitation. Filtration then afforded 2.143 g of solid Fmoc-Asp(OTAG)-OH in a 98.2% yield.

Claims

1. A method for synthesizing tilpoxetine, characterized in that: The following steps are involved: (1) The main chain of telportin was divided into five polypeptide fragments: positions 30-39, 22-29, 16-21, 10-15, and 1-9, and the side chain polypeptides were designated as fragment 6; (2) With the assistance of the amide hydrophobic tag carrier NTAG-NH2, amino acids are sequentially coupled on the carrier to obtain the 30-39 fragment 1 containing the tag carrier, which is recorded as Fmoc-AA(30-39)-NTAG; (3) With the assistance of the ester-based hydrophobic tag carrier OTAG-OH, amino acids were sequentially coupled to the carrier to synthesize fragments 22-29 and 16-21 of the tag carrier; (4) Fragments 2 and 3 were soft-cleaved to remove the carrier, and Fmoc was removed from fragment 1. The Fmoc chemical synthesis method was then used to sequentially couple the fragments 2 and 3 after the carrier was removed to obtain Fmoc-AA(16-39)-NTAG; (5) Using the hydrophobic tag compound NH2-Asp(OTAG)-OR 1 , which uses the Asp side chain carboxyl group as the anchor position of the hydrophobic tag OTAG, R 1 is a C1-C6 alkyl group or a silyl group, and the amino acids are coupled sequentially to synthesize the 10-15 fragment 4 and the 1-9 fragment 5 respectively; (6) After the coupling of fragments 4 and 5, they were coupled with Fmoc-AA(16-39)-NTAG to obtain the fully protected tilpoxetine backbone; (7) removing the Lys side chain protecting group from the fully protected tilpotide main chain and coupling it with the side chain polypeptide to obtain fully protected tilpotide; then acid cleavage and purification are performed in sequence, and drying is performed to obtain pure tilpotide; The structure of the amide hydrophobic tag NTAG-NH2 is as follows: R 2 is a straight-chain saturated alkyl group of 18 to 28 carbon atoms; The structure of the ester-based hydrophobic tag OTAG-OH is as follows: R 2 is a straight-chain saturated alkyl group of 18 to 28 carbon atoms; In step (5), the hydrophobic tag compound NH2-Asp(OTAG)-OR 1 The structure is as follows: R 2 is a straight-chain saturated alkyl group of 18 to 28 carbon atoms; R 1 is a C1-C6 alkyl group or a silyl group, wherein R 3 、R 4 、R 5 are each independently selected from C1-C10 alkyl or hydrogen, and R 3 、R 4 、R 5 Not entirely hydrogen.

2. The method for synthesizing telpotide according to claim 1, wherein: The operation process of step (2) is as follows: first, the 39th amino acid is coupled to the amide hydrophobic tag carrier NTAG-NH2, and then the 39th amino acid is coupled one by one to the 30th amino acid to obtain fragment 1, which is recorded as Fmoc-AA(30-39)-NTAG.

3. The method for synthesizing telpotide according to claim 1, wherein: The synthesis process of fragment 2 in step (3) is as follows: first, the 29th amino acid is coupled to the ester-based hydrophobic tag carrier OTAG-OH, and then the 29th amino acid is coupled one by one to the 22nd amino acid to obtain fragment 2Fmoc-AA(22-29)-OTAG.

4. The method for synthesizing telpotide according to claim 1, wherein: The synthesis process of fragment 3 in step (3) is as follows: first, the 21st amino acid is coupled to the ester-based hydrophobic tag carrier OTAG-OH, and then the 21st amino acid is coupled one by one to the 16th amino acid to obtain fragment 3Fmoc-AA(16-21)-OTAG.

5. The method for synthesizing tilpoxetine according to claim 1, wherein: In step (5), the structure of fragment 4 is Fmoc-AA(10-14)-Asp(OTAG)-OR 1 ; Structure of fragment 5 Fmoc-AA(1-8)-Asp(OTAG)-OR 1 .

6. The method for synthesizing telpotide according to claim 1, wherein: Fragment 6 was synthesized using the ester-based hydrophobic tag carrier OTAG-OH, whose structure is Ara(OtBu)-Glu(α-OtBu)-AEEA-AEEA-OTAG; fragment 6 was then soft-cleaved to remove the carrier tag to obtain Ara(OtBu)-Glu(α-OtBu)-AEEA-AEEA-OH, which was used for coupling with the fully protected telpotide backbone after removal of the Lys side chain protecting group.

7. The method for synthesizing telpotide according to claim 1, wherein: The structure of fragment 1 is: Fmoc-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NTAG; The structure of fragment 2 is: Fmoc-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-OTAG; The structure of fragment 3 is: Fmoc-Lys(R 3 )-Ile-Ala-Gln-Lys(R 3 )-Ala-OTAG, Lys side chain protecting group R 3 is one or more of Dde, Boc, Ac, and Cbz; The structure of fragment 4 is: Fmoc-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OTAG)-OR 1 ; The structure of the fragment 5 is: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OTAG)-OR 1 ; The structure of the fragment 6 is: Ara(OtBu)-Glu(α-OtBu)-AEEA-AEEA-OTAG.

8. The method for synthesizing telpotide according to claim 1, wherein: The amino termini, carboxyl termini and side chains of amino acids or peptide fragments in the synthesis of telportide are protected by protecting groups, as follows: (1) The protecting group used at the amino terminus is one or more of Cbz, Boc, Fmoc, Ac, and Trt; (2) The protecting group used at the carboxyl end is one of methyl ester, benzyl ester, tert-butyl ester, trimethylsilyl alcohol ester, allyl ester, nitrobenzyl ester, propargyl ester, 9-fluorenylmethyl ester, and ferrocenyl ester; (3) The protecting group used in the side chain is one or more of Trt, Boc, tBu, Cbz, Dde, Bn, and Ac; The Lys side chain protecting group is one or more of Dde, Boc, Ac, and Cbz.

9. The method for synthesizing telpotide according to claim 1, wherein: The condensation reagents in the coupling reaction include one or more of DIC / Oxyma pure, DIC / HOBt, DMT-MM, HBTU / HOBt, COMU, Pyoxime or HATU / HOAt, PyBOP.

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