CN114276222B

CN114276222B - Diaryl benzyl alcohol compound as polypeptide liquid phase synthesis carrier and preparation method and application thereof

Info

Publication number: CN114276222B
Application number: CN202111676811.XA
Authority: CN
Inventors: 李小清; 段亚亚; 张芳东; 张琦; 熊卫宝; 孔明; 李锋
Original assignee: Hangzhou Aosainuo Biotechnology Co ltd
Current assignee: Hangzhou Aosainuo Biotechnology Co ltd
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2024-04-09
Anticipated expiration: 2041-12-31
Also published as: CN114276222A

Abstract

The invention provides a diaryl benzyl alcohol compound as a polypeptide liquid phase synthesis carrier, a preparation method and application thereof, comprising a synthesis method of the compound, a method for constructing a polypeptide chain by sequentially coupling amino acids as an initial template reagent, and a method for removing the template under mild conditions without removing other protecting groups on the peptide chain; and removing the template and the protecting group from the lysate. The long-chain alkane substituted compound developed by the invention has the characteristics of simple synthesis method, convenient operation, low cost, easy storage and easy removal, is very suitable for being used as an initial template for liquid phase synthesis of polypeptide chains, and is also expected to realize large-scale industrial production.

Description

Diaryl benzyl alcohol compound as polypeptide liquid phase synthesis carrier and preparation method and application thereof

Technical Field

The invention relates to the field of polypeptide synthesis, in particular to a diaryl benzyl alcohol compound, a preparation method thereof and application thereof in polypeptide liquid phase synthesis.

Background

The somalundum is a new generation GLP-1 (glucagon-like peptide-1) analogue developed by Daneno and Norde company (NovoNordisk), is a long-acting dosage form developed based on the basic structure of liraglutide, and has better effect of treating type 2 diabetes.

Structural information of somalupeptide is as follows: chemical name: the structural formula of the somalupeptide Sermaglutide is shown as formula II:

the molecular formula: c (C) ₁₈₇ H ₂₉₁ N ₄₅ O ₅₉ The method comprises the steps of carrying out a first treatment on the surface of the Molecular weight: 4113.58; CAS accession number: 910463-68-2.

At present, fmoc strategy is mostly adopted for solid-phase synthesis of the somalunin, and the solid-phase synthesis has certain advantages in the aspect of post-treatment of reaction, but the disadvantages are obvious: 1. fmoc-protected amino acid dosing of 2-5 fold and even higher is required. 2. After coupling an amino acid, a large amount of solvent DMF is required for washing (typically 4-6 washes). 3. The reaction belongs to heterogeneous reaction. For solid phase cyclization, coupling of hydrophobic amino acid and the like, the coupling effect is poor due to large steric hindrance, and secondary and even tertiary feeding is needed. 4. Because the volume of the resin is larger, the solvent consumption is larger, the reaction kettle efficiency is lower, and the batch yield is lower.

Therefore, the liquid phase synthesis method of the somalundin, which is simple and convenient to operate and has high yield, has important practical significance.

In addition, in most solid phase synthesis strategies reported so far, the final step of removing the solid phase synthesis template material requires the use of stronger acids, such as: higher concentrations of trifluoroacetic acid. The protecting group on the polypeptide chain is correspondingly removed at the same time of removing the template. The synthesis method of the fully protected polypeptide chain is relatively less. Therefore, the synthesis method of the full-protection polypeptide chain, which is simple and convenient to operate and high in yield, is provided, and has important practical significance.

Disclosure of Invention

The first aim of the invention is to solve the problems of the prior Fmoc strategy in solid phase synthesis of polypeptides, and provide a diaryl benzyl alcohol compound as a carrier for liquid phase synthesis of polypeptides, which is used as a carrier starting end for synthesizing polypeptide chains, thereby facilitating difficult amino acid coupling and cyclization, convenient deprotection and high yield.

The second purpose of the invention is to provide the preparation method of the diaryl benzyl alcohol compound, which has the advantages of simple and easily obtained raw materials, mild preparation conditions, simple steps, easy realization and low cost.

The third object of the present invention is to provide the application of the above diaryl benzyl alcohol compound, which is used as hydrophobic carrier to synthesize fully protected polypeptide or fully deprotected polypeptide in liquid phase.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the invention provides a compound, the structure of which is shown as a formula I:

r is selected from C12-C22 fatty chain, X is H, OCH3 and halogen.

In some embodiments of the invention, the compound has the structure shown in formula III:

the molecular formula of the carrier compound shown in the formula III is as follows: c (C) ₆₇ H ₁₂₀ O ₆ Mol.wt.: 989.69, temporarily designated HO-TAGA.

In some embodiments of the invention, the polypeptide is a somalundin.

The invention also provides a preparation method of the compound, which is prepared from the compound with the structure shown in the formula 1 as a raw material through the steps of esterification, substitution, reduction and the like.

A compound of formula 1, formula C ₇ H ₆ O ₅ ，Mol.wt：170.12。

The synthetic route of the compound shown in formula I is shown in figure 5.

The synthesis method comprises the following steps:

a) Dissolving a substance shown in a formula 1 in methanol, and adding an esterification reagent to perform esterification reaction to generate a compound 2;

b) Adding the compound 2, an acid binding agent and a substitution reaction reagent into a solvent, and reacting to generate a compound 3 of the formula;

c) Dissolving the compound 3, adding a reducing agent a, and carrying out a reduction reaction to generate a compound 4;

d) Adding an oxidant a and an oxidant b into dichloromethane to be used as a solution A; dissolving the compound 4 in triethylamine and dichloromethane, dropwise adding the solution into a solution A system, and carrying out oxidation reaction to generate a compound 5;

e) And dissolving the compound 5, adding a solution containing a format reaction reagent, and reacting to obtain the compound shown in the formula III.

In a preferred embodiment of the present invention, in the step a), the esterification reagent comprises acetyl chloride, thionyl chloride or concentrated sulfuric acid, and the molar ratio of the substance represented by formula 1 to the esterification reagent is 1:1.5 to 3.0.

As a preferable scheme of the invention, in the step b), the acid-binding agent comprises one or more than two of potassium carbonate, sodium carbonate, potassium hydroxide, sodium hydroxide, diisopropylethylamine and triethylamine, the substitution reaction reagent is halogenated alkane RX, R is C12-C22 fatty chain, and X is halogen; the molar ratio of the compound 2, the substitution reaction reagent and the acid binding agent is 1.0:3.2 to 4.0:8.0 to 10.0.

As a preferred embodiment of the invention, in step c), reducing agent a comprises red aluminum or lithium aluminum hydride, and the molar ratio of compound 3 to reducing agent a is 1:2.5 to 4.0.

As a preferred embodiment of the present invention, in step d), the oxidizing agent a comprises pyridine sulfur trioxide, oxalyl chloride or NaClO, the oxidizing agent b comprises DMSO or TEMPO, and the molar ratio of compound 4, oxidizing agent a and oxidizing agent b is 1:3.0 to 5.0:10.0 to 12.0;

in step e), the reagent for the grignard reaction comprises aryl magnesium chloride, aryl magnesium bromide or aryl lithium, and one or more of tetrahydrofuran, methyltetrahydrofuran and toluene as solvents; the molar ratio of compound 5 to the formative reagent is 1:2.0 to 3.0.

The method comprises the following specific steps:

(a) Dissolving a substance shown in the formula (1) in methanol, and dropwise adding acetyl chloride to perform esterification reaction to generate a compound shown in the formula (2); wherein the temperature of dripping acetyl chloride is 15-35 ℃, then the acetyl chloride is reacted at 25-35 ℃ in a heat preservation way, and then the compound 2, namely the compound represented by the formula (2), is obtained after post-treatment.

(b) Adding a substance shown in the formula (2) into DMF, completely dissolving, adding potassium carbonate and 1-bromooctadecane, heating to 85-90 ℃, carrying out heat preservation reaction, and carrying out aftertreatment to obtain a compound 3 shown in the formula (3).

(c) Dissolving a substance shown in a formula (3) in toluene, dropwise adding 70% of red aluminum toluene solution, carrying out reduction reaction, wherein the temperature of dropwise adding red aluminum toluene is 10-30 ℃, the reaction temperature is 20-30 ℃, carrying out heat preservation reaction, and then carrying out post-treatment to obtain a compound 4, namely a compound shown in the formula (4).

(d) Adding DMSO and pyridine sulfur trioxide into dichloromethane, dissolving a substance shown in a formula (4) into triethylamine and dichloromethane, dropwise adding the mixture into the system, wherein the dropwise adding temperature is 5-15 ℃, the reaction temperature is 20-30 ℃, carrying out oxidation heat preservation reaction, and then carrying out aftertreatment to obtain a compound 5, namely the compound shown in the formula (5).

(e) Dissolving a substance shown in a formula (5) in toluene, dropwise adding a 2M phenylmagnesium chloride tetrahydrofuran solution, wherein the dropwise adding temperature is 5-15 ℃, the reaction temperature is 20-30 ℃, carrying out heat preservation reaction, and then carrying out aftertreatment to obtain a compound shown in a formula III.

The invention also provides application of the compound shown in the formula I or the compound shown in the formula I prepared by the preparation method in polypeptide synthesis.

The invention also provides a liquid phase synthesis method of the somalundum, which takes the formula II as a raw material, and the method comprises the steps of coupling amino acid in sequence, deprotecting to obtain the somalundum full-protection peptide, and then cracking to obtain the somalundum.

The synthetic route is shown in FIG. 6.

The synthesis of the somalupeptide comprises the following steps:

1) Coupling amino acids: the molar ratio of the substance represented by formula I to the amino acid is 1:1.1 to 1.5, wherein the coupling reagent is HOBt/DIC (1.1 to 1.5 equiv), HOBt/DCC (1.1 to 1.5 equiv), HOBt/EDC.HCl (1.1 to 1.5 equiv), and HOBt/DIC is preferable. The solvent used is chloroform or dichloromethane or tetrahydrofuran, dichloromethane being preferred.

2) Removing Fmoc: the Fmoc-removing reagent is DBU or piperidine, wherein DBU is preferable; the solvent used is chloroform or dichloromethane or tetrahydrofuran, dichloromethane being preferred.

Compared with the prior art, the invention has the following beneficial effects:

1) Up to now, the preparation of the compound shown in the formula I is used as a raw material, the liquid phase synthesis method is used for preparing the cable Ma Lutai, the data at home and abroad are not reported in detail, and the preparation of the compound is described in detail for the first time;

2) The raw materials used in the invention are simple and easy to obtain, and the cost is low;

3) The synthesis steps in the invention are simple to operate and easy to realize.

Drawings

FIG. 1 is a block diagram of formula III prepared according to the method of example 1 ¹ H NMR spectrum.

FIG. 2 is an HPLC plot of full-protection hexapeptide prepared according to the method of example 1.

FIG. 3 is an HPLC plot of the full protection decapeptide prepared according to the method of example 1.

Fig. 4 is an HPLC profile of somalunin prepared according to the method of example 1.

FIG. 5 shows the synthetic route of the compounds of formula I of the present invention.

Fig. 6 is a synthetic route for the inventive cord Ma Lutai.

Detailed Description

The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1:

1264.0mL of MeOH and 216.0g of trihydroxybenzoic acid are added into a 2L three-port reaction bottle under the protection of nitrogen, and the mixture is stirred for dissolving, and then cooled to below 15 ℃. 180.5mL of acetyl chloride is added dropwise, and the reaction control measurement is completed after the dropwise addition. Keeping the temperature below 35 ℃ for reaction for 6 hours; and removing the solvent after the heat preservation reaction is finished. 2.0L of EA was added to the mixture, followed by extraction once with 500.0mL of water. EA phase with 15% NaHCO ₃ Extracting with water solution for 2 times, and extracting the combined water phases with 300mL EA for 3 times; merging EA phase, washing once with 300mL saturated sodium chloride aqueous solution, adding 100.0g anhydrous sodium sulfate for drying, filtering, removing solvent to obtain 230.0g crude product, heating to dissolve clear with 240.0mL ethyl acetate, then dripping 840.0mL petroleum ether, cooling to 10-15 ℃, filtering, drying to obtain 212.8g product of formula 2, yield 91.0%, purity 99.8%.

Under the protection of nitrogen, adding 1056.0 mL of DMF and 49.0g of compound of formula (2) into a 3.0L three-necked flask, stirring to dissolve, adding K ₂ CO ₃ 293.8g of white turbidity, 310.5g of 1-bromooctadecane are added and heated to more than 90 ℃ for reaction for 16h. 1300mL of toluene is added, the temperature is raised to 70 ℃ for dissolving, the solution is filtered while the solution is hot, the temperature of the filtrate is raised to 70 ℃ and the solution is slowly cooled under mechanical stirring, and the solid is separated out. Filtering and drying to obtain 204.8g of the product of the formula 3, and the yield is 81.8%.

2500.0mL of toluene and 500.0g of compound formula 3 are added into a 3L three-port reaction bottle under the protection of nitrogen, the temperature is raised to be higher than 45 ℃ to dissolve, the temperature is reduced to be below 25 ℃, 306.8g of 70% of red aluminum toluene solution is added in batches, and the reaction is carried out for 1h under the heat preservation. After the heat preservation is finished, 1000.0mL of water is added dropwise for quenching, and then the pH=4-5 is adjusted by concentrated hydrochloric acid; the layers were separated, and the organic phase was washed with 400.0mL of saturated brine. 100.0g of anhydrous sodium sulfate is dried, filtered and desolventized, when about 1000.0mL is remained, 1500.0mL of acetonitrile is added, solid is separated out, the temperature is slowly reduced to about 5-10 ℃, stirring is carried out for 20min, filtering and drying are carried out, 422.0g of the product of the formula 4 is obtained, and the yield is 87.0%.

To a 1L three-port reaction flask, 35.0mL of DCM and 27.0g of DMSO were added under nitrogen protection, and the temperature was controlled below 15 ℃. 17.8g of pyridine sulfur trioxide are added in portions. After the addition was completed, stirring was carried out for 1 hour. 32.4g of the compound of formula 4 and 21.3g of TEA were dissolved in 320.0mL of DCM and added to the reaction flask. The temperature is controlled below 15 ℃. Stirring is carried out for 1 hour. Then the temperature is raised to between 25 and 30 ℃ and the reaction is kept for 12 hours. 450.0mL of 12% aqueous citric acid was added and the reaction quenched. Layering; the aqueous phase was extracted once with 120.0mL DCM. The organic phases were combined and washed 2 times with 120.0ml of 6% aqueous sodium bicarbonate; the mixture was washed once with 120.0mL of saturated brine. 30.0g of anhydrous sodium sulfate, and filtering. Desolvation is stopped when the residual amount is about 60.0 mL; raising the temperature to 40-45 ℃ and completely dissolving the solid. 180.0mL of ethyl acetate was added dropwise. Then slowly cooling to 5-10 ℃. Stirring for 30 minutes. Filtering and drying to obtain 24.3g of the product of the formula 5, and the yield is 74.8%.

Into a 1L three-port reaction flask, 200.0mL of THF and 20.0g of compound of formula 5 were charged under nitrogen atmosphere, and the temperature was raised to 35 ℃. Dissolving the solid, and cooling to below 25 ℃. 28.0mL of phenylmagnesium chloride was added dropwise, and the temperature was controlled below 25 ℃. Preserving heat and reacting for 1 hour; quenching the reaction by adding 20.0ml of 10% aqueous citric acid solution; THF was removed under reduced pressure, 240.0mL DCM and 80.0mL of 10% aqueous citric acid were added, the layers separated, and the organic phase was washed with 120.0mL of water and 120.0mL of saturated brine. 30.0g of anhydrous sodium sulfate, and filtering. When the solvent is removed to about 70.0mL, stopping removing the solvent, heating to 40-45 ℃, and dropwise adding 200.0mL of methanol after the solvent is dissolved. Cooling to 20-25 deg.c and stirring for 30 min; filtering and drying to obtain 19.6g of the product of the formula III, and the yield is 90.2%. ¹ H-NMR(500MHz，CDCl ₃ ) Delta (ppm): 7.35 (5H), 6.56 (2H), 5.74 (1H), 3.92 (6H), 1.75 (6H), 1.43 (6H), 1.30 (84H), 0.87 (9H), see FIG. 1.

Synthesis of cord Ma Lutai:

1) Fmoc-Gly-O-TAGA (coupling of the first amino acid):

to a 250mL three-port reaction flask, 150.0mL of methylene chloride, 0.43g of DMAP and 15.0g of compound of formula III were added under nitrogen atmosphere, and the solid solution was stirred. 5.4g Fmoc-Gly-OH was added and 4.56g DCC was added in portions. The temperature is controlled below 30 ℃. The reaction was incubated for 5 hours and monitored by TLC (EA: PE=1:4); and (5) filtering. When the solvent is removed until about 80.0g is remained, stopping removing the solvent, raising the temperature to 40-45 ℃, and dripping 180.0mL of methanol after the solid is dissolved. Then slowly cooling to 10-15 deg.C. Filtering and drying to obtain 19.1g of Fmoc-Gly-O-TAGA. The yield thereof was found to be 99.3%.

2) H-Gly-O-TAGA (Fmoc removal):

into a 250mL three-port reaction flask, 148.0mL of methylene chloride and 19.1g of Fmoc-Gly-TAGA were added under nitrogen atmosphere, and the solid solution was stirred. 44.4mL of DMF was added, the system was cooled to below 15℃and 2.56g of DBU was added. The reaction was monitored by TLC (EA: PE=1:4) at 10-15℃for 30 min. 60.0mL of 5% aqueous citric acid was added, and the mixture was washed and separated. Further, the mixture was washed with 60.0mL of water and 60.0mL of a 5% aqueous sodium hydrogencarbonate solution. 60.0mL of water was used for washing, and 60.0mL of saturated brine was used for washing.

3) Fmoc-Arg (Pbf) -Gly-O-TAGA (coupling of second amino acid):

under the protection of nitrogen, 11.7g Fmoc-Arg (Pbf) -OH and 2.46g HOBt were added to the three-port flask, and the solid was dissolved by stirring, followed by 2.7g DIC. The temperature is controlled below 30 ℃. The reaction was incubated for 3 hours. Desolventizing to a viscous state, stopping desolventizing, and adding 180.0mL of methanol. Stirring for 30 minutes. Filtering and drying to obtain the Fmoc-Arg (Pbf) -Gly-O-TAGA 24.7g product. The yield thereof was found to be 97.8%.

4) Coupling and deprotection of amino acids 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 to amino acid Fmoc-Gln (Trt) -OH were repeated 2-3 to give Fmoc-Gln (Trt) -Ala-Ala-Lys [ Oct (OtBu) -Glu (AEEa-eeA) -OtBu-Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-O-TAGA.

5) Synthesis of a fully protected hexapeptide fragment (Fmoc-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-OH):

to a 250mL three-port reaction flask, 150.0mL of methylene chloride, 0.43g of DMAP and 15.0g of compound of formula III were added under nitrogen atmosphere, and the solid solution was stirred. 5.4g Fmoc-Gly-OH was added and 4.56g DCC was added in portions. The temperature is controlled below 30 ℃. The reaction was kept at the temperature for 5 hours and filtered. When the solvent is removed until about 80.0g is remained, stopping removing the solvent, raising the temperature to 40-45 ℃, and dripping 180.0mL of methanol after the solid is dissolved. Then slowly cooling to 10-15 deg.C. Filtering and drying to obtain 19.1g of product. The yield thereof was found to be 99.3%. Repeating the coupling and deprotection of the 2 nd, 3 rd, 4 th and 5 th amino acids to the coupling of the 6 th amino acid Fmoc-Ser (tBu) -OH to obtain Fmoc-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-O-TAGA; 150.0mL of tetrahydrofuran and 15.0g of the above fully protected hexapeptide containing a carrier were charged into a 500mL hydrogenation reactor, the solid was dissolved and purged with nitrogen, and then 1.5g of 10% wet palladium on carbon (50% aqueous solution) was added thereto, and after the nitrogen substitution, the hydrogen substitution was performed. The hydrogenation reaction is carried out under the condition of below 35 ℃ and below 0.04 MPa. The reaction was incubated for 5 hours and monitored by TLC (EA: PE=1:4); filtration and concentration of the mother liquor to a solid under reduced pressure. 200.0mL of methanol was added, stirred for 30 minutes, and filtered. The mother liquor was concentrated to a solid under reduced pressure, 60.0mL of n-heptane was added and stirred for 30 minutes. Filtering and drying to obtain fully-protected hexapeptide Fmoc-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-OH 7.65g, yield 98.8%, and HPLC liquid phase purity 96.0% (peak time: 3.9 min), as shown in figure 2.

6) Synthesis of the fully protected decapeptide fragment (Boc-His (Trt) -Aib-Glu (OtBu) -Gly-Thr (OtBu) -Phe-Thr (OtBu) -Ser (OtBu) -Gln (OtBu) -Val-OH):

to a 250mL three-port reaction flask, 150.0mL of methylene chloride, 0.43g of DMAP and 15.0g of compound of formula III were added under nitrogen atmosphere, and the solid solution was stirred. 6.2g Fmoc-Val-OH was added and 4.56g DCC was added in portions. The temperature is controlled below 30 ℃. The reaction was kept at the temperature for 5 hours and filtered. When the solvent is removed until about 80.0g is remained, stopping removing the solvent, raising the temperature to 40-45 ℃, and dripping 180.0mL of methanol after the solid is dissolved. Then slowly cooling to 10-15 deg.C. Filtering and drying to obtain 19.6g of product. The yield thereof was found to be 98.6%. Repeating the coupling and deprotection of the 2 nd, 3 th, 4 th, 5 th, 6 th, 7 th, 8 th and 9 th amino acids to the coupling of the 10 th amino acid Boc-His (Trt) -OH to obtain Boc-His (Trt) -Aib-Glu (OtBu) -Gly-Thr (OtBu) -Phe-Thr (OtBu) -Ser (OtBu) -Gln (OtBu) -Val-O-TAGA; 150.0mL of tetrahydrofuran and 15.0g of the above fully protected decapeptide containing carrier were charged into a 500mL hydrogenation reactor, the solid was dissolved and purged with nitrogen, and then 1.5g of 10% wet palladium on carbon (50% aqueous solution) was added thereto, and after nitrogen substitution, hydrogen substitution was performed. The hydrogenation reaction is carried out under the condition of below 35 ℃ and below 0.04 MPa. The reaction was incubated for 5 hours and monitored by TLC (EA: PE=1:4); filtration and concentration of the mother liquor to a solid under reduced pressure. 200.0mL of methanol was added, stirred for 30 minutes, and filtered. The mother liquor was concentrated to a solid under reduced pressure, 60.0mL of n-heptane was added and stirred for 30 minutes. Filtering and drying to obtain the fully-protected decapeptide product Boc-His (Trt) -Aib-Glu (OtBu) -Gly-Thr (OtBu) -Phe-Thr (OtBu) -Ser (OtBu) -Gln (OtBu) -Val-OH 9.14g, with a yield of 98.0%, and an HPLC liquid phase purity (peak time: 3.4 min) of 84.8%, as shown in figure 3.

7) Synthesis of Diundecapeptide Fmoc-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys [ Oct (OtBu) -Glu (AEEA-AEEA) -OtBu ] -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-O-TAGA:

to a 250mL three-port reaction flask, 148.0mL of methylene chloride and 18.5g of the above fully protected pentadecapeptide containing carrier were added under nitrogen protection, and the solid solution was stirred. 44.4mL of DMF was added, the system was cooled to below 15℃and 0.72g of DBU was added. The reaction was monitored by TLC (EA: PE=1:4) for 30 min below 15 ℃.60.0mL of 5% aqueous citric acid was added, and the mixture was washed and separated. Further, the mixture was washed with 60.0mL of water and 60.0mL of a 5% aqueous sodium hydrogencarbonate solution. 60.0mL of water was used for washing, and 60.0mL of saturated brine was used for washing.

Into the three-port reaction flask, 5.8g of the above fully protected hexapeptide and 0.74g of HOBt were added, and the solid solution was stirred and then 0.84g of DIC was added. The temperature is controlled below 30 ℃. The reaction was incubated for 3 hours. Desolventizing to a viscous state, stopping desolventizing, and adding 180.0mL of methanol. Stirring for 30 minutes. Filtering and drying to obtain the product Fmoc-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys [ Oct (OtBu) -Glu (AEEA-AEEA) -OtBu ] -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-O-TAGA 20.8g. The yield thereof was found to be 95.0%.

8) Synthesis of Triundecapeptide, boc-His (Trt) -Aib-Glu (OtBu) -Gly-Thr (OtBu) -Phe-Thr (OtBu) -Ser (OtBu) -Gln (OtBu) -Val-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys [ Oct (OtBu) -Glu (AEEa-eeA) -OtBu ] -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-OH:

to a 250mL three-port reaction flask, 166.0mL of methylene chloride and 20.8g of the above fully protected di-undecapeptide containing carrier were added under nitrogen protection, and the solid solution was stirred. 44.4mL of DMF was added, the system was cooled to below 15℃and 0.85g of DBU was added. The reaction was monitored by TLC (EA: PE=1:4) for 30 min below 15 ℃.60.0mL of 5% aqueous citric acid was added, and the mixture was washed and separated. Further, the mixture was washed with 60.0mL of water and 60.0mL of a 5% aqueous sodium hydrogencarbonate solution. 60.0mL of water was used for washing, and 60.0mL of saturated brine was used for washing.

7.6g of the fully protected decapeptide and 0.61g of HOBt were added to the three-port flask, and the solid solution was stirred and then added with 0.97g of DIC. The temperature is controlled below 30 ℃. The reaction was incubated for 3 hours. Desolventizing to a viscous state, stopping desolventizing, and adding 180.0mL of methanol. Stirring for 30 minutes. Filtering and drying to obtain the product Boc-His (Trt) -Aib-Glu (OtBu) -Gly-Thr (OtBu) -Phe-Thr (OtBu) -Ser (OtBu) -Gln (OtBu) -Val-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys [ Oct (OtBu) -Glu (AEEa-eea) -OtBu ] -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-O-TAGA 24.4g. The yield thereof was found to be 92.9%. 150.0mL of tetrahydrofuran, 15.0g of the above fully protected triundecapeptide containing a carrier were charged into a 500mL hydrogenation reactor, the solid was stirred to dissolve, the solution was replaced with nitrogen, and 1.5g of 10% wet palladium on carbon (50% aqueous solution) was added thereto, and after the replacement with nitrogen, the hydrogen was replaced. The hydrogenation reaction is carried out under the condition of below 35 ℃ and below 0.04 MPa. The reaction was incubated for 5 hours and monitored by TLC (EA: PE=1:4); filtration and concentration of the mother liquor to a solid under reduced pressure. 200.0mL of methanol was added, stirred for 30 minutes, and filtered. The mother liquor was concentrated to a solid under reduced pressure, 60.0mL of n-heptane was added and stirred for 30 minutes. The product was filtered and dried to give the fully protected thirty-first peptide product Boc-His (Trt) -Aib-Glu (OtBu) -Gly-Thr (OtBu) -Phe-Thr (OtBu) -Ser (OtBu) -Gln (OtBu) -Val-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys [ Oct (OtBu) -Glu (AEEa-eeA) -OtBu ] -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-OH 12.39g in 98.0%.

9) Cracking: boc-His (Trt) -Aib-Glu (OtBu) -Gly-Thr (OtBu) -Phe-Thr (OtBu) -Ser (OtBu) -Gln (OtBu) -Val-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys [ Oct (OtBu) -Glu (AEEA-AEEA) -OtBu ] -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-OH 12.3g was added to a 250.0mL three-port reaction flask, followed by 74.0g of TFA: EDT: tis=90:5:5 (volume ratio) lysate was cleaved below 30℃for 2 hours, added dropwise to 370.0g isopropyl ether, precipitated and filtered. The filter cake was again quenched with 120.0g acetonitrile: water = 2:1 (volume ratio), dissolving, filtering, purifying the crude peptide in mother liquor by column. 5.9g of the Somamunon is obtained, the yield is 69.7%, and the purity of HPLC liquid phase (peak time: 21.1 min) is 99.8%, see figure 4.

Example 2:

under the protection of nitrogen, 1264.0mL of MeOH and 216.0g of trihydroxybenzoic acid are added into a 2L three-port reaction bottle, the solution is stirred and cleaned, and the temperature is reduced to 5-10 ℃. 180.5mL of acetyl chloride is added dropwise, and the reaction control measurement is completed after the dropwise addition. The reaction is carried out for 6 hours at the temperature of 25-35 ℃; and removing the solvent after the heat preservation reaction is finished. 2.0L of EA was added to the mixture, followed by extraction once with 500.0mL of water. The EA phase was extracted 2 times with 15% aqueous NaHCO3 and the combined aqueous phases were extracted 3 times with 300mL EA; merging EA phase, washing once with 300mL saturated sodium chloride aqueous solution, adding 100.0g anhydrous sodium sulfate for drying, filtering, removing solvent to obtain 230.0g crude product, heating to dissolve clear with 240.0mL ethyl acetate, then dripping 840.0mL petroleum ether, cooling to 10-15 ℃, filtering, and drying to obtain 215.1g product of formula 2 with 92.0% yield.

Under the protection of nitrogen, adding 1056.0 mL of DMF and 49.0g of compound of formula (2) into a 3.0L three-necked flask, stirring to dissolve, adding K ₂ CO ₃ 293.8g of white turbidity, 310.5g of 1-bromooctadecane were added and reacted at a temperature of 90 ℃ for 16h. 1300mL of toluene is added, the temperature is raised to 70 ℃ for dissolving, the solution is filtered while the solution is hot, the temperature of the filtrate is raised to 70 ℃ and the solution is slowly cooled under mechanical stirring, and the solid is separated out. Filtering and drying to obtain 200.3g of the product of the formula 3 with the yield of 80.0%.

2500.0mL of toluene and 500.0g of compound formula 3 are added into a 3L three-port reaction bottle under the protection of nitrogen, the temperature is raised to 45 ℃ for dissolving, the temperature is reduced to 25 ℃, 306.8g of 70% red aluminum toluene solution is added in batches, and the reaction is carried out for 1h under the heat preservation. After the heat preservation is finished, 1000.0mL of water is added dropwise for quenching, and then the pH=4-5 is adjusted by concentrated hydrochloric acid; the layers were separated, and the organic phase was washed with 400.0mL of saturated brine. 100.0g of anhydrous sodium sulfate is dried, filtered and desolventized, when about 1000.0mL is remained, 1500.0mL of acetonitrile is added, solid is separated out, the temperature is slowly reduced to about 5-10 ℃, stirring is carried out for 20min, filtering and drying are carried out, 417.1g of the product of the formula 4 is obtained, and the yield is 86.0%.

Under the protection of nitrogen, 35.0mL of DCM and 27.0g of DMSO are added into a 1L three-port reaction flask, and the temperature is controlled between 5 ℃ and 10 ℃. 17.8g of pyridine sulfur trioxide are added in portions. After the addition was completed, stirring was carried out for 1 hour. 32.4g of the compound of formula 4 and 21.3g of TEA were dissolved in 320.0mLDCM, which was added to the above-mentioned reaction flask. The temperature is controlled to be 5-15 ℃. Stirring is carried out for 1 hour. Then the temperature is raised to between 25 and 30 ℃ and the reaction is kept for 12 hours. 450.0mL of 12% aqueous citric acid was added and the reaction quenched. Layering; the aqueous phase was extracted once with 120.0ml dcm. The organic phases were combined and washed 2 times with 120.0ml of 6% aqueous sodium bicarbonate; the mixture was washed once with 120.0mL of saturated brine. 30.0g of anhydrous sodium sulfate, and filtering. Desolvation is stopped when the residual amount is about 60.0 mL; raising the temperature to 40-45 ℃ and completely dissolving the solid. 180.0mL of ethyl acetate was added dropwise. Then slowly cooling to 5-10 ℃. Stirring for 30 minutes. Filtering and drying to obtain 24.4g of the product of the formula 5 with the yield of 75.0%.

Into a 1L three-port reaction flask, 200.0mL of THF and 20.0g of compound of formula 5 were added under nitrogen protection, and the temperature was raised to 30-35 ℃. Dissolving the solid, and cooling to 20-25 ℃. 28.0mL of phenylmagnesium chloride is added dropwise, and the temperature is controlled between 20 and 25 ℃. Preserving heat and reacting for 1 hour; quenching the reaction by adding 20.0ml of 10% aqueous citric acid solution; THF was removed under reduced pressure, 240.0mL of ldcm and 80.0mL of 10% aqueous citric acid were added, the layers were separated, and the organic phase was washed with 120.0mL of water and 120.0mL of saturated brine. 30.0g of anhydrous sodium sulfate, and filtering. When the solvent is removed to about 70.0mL, stopping removing the solvent, heating to 40-45 ℃, and dropwise adding 200.0mL of methanol after the solvent is dissolved. Cooling to 20-25 deg.c and stirring for 30 min; filtering and drying to obtain 19.3g of the product of the formula III, and the yield is 89.0%.

Synthesis of cord Ma Lutai:

1) Fmoc-Gly-O-TAGA (coupling of the first amino acid):

to a 250mL three-port reaction flask, 150.0mL of methylene chloride, 0.43g of DMAP and 15.0g of compound of formula III were added under nitrogen atmosphere, and the solid solution was stirred. 5.4g Fmoc-Gly-OH was added and 2.76g DIC was added in portions. The temperature is controlled between 25 and 30 ℃. The reaction was incubated for 5 hours and monitored by TLC (EA: PE=1:4); and (5) filtering. When the solvent is removed until about 80.0g is remained, stopping removing the solvent, raising the temperature to 40-45 ℃, and dripping 180.0mL of methanol after the solid is dissolved. Then slowly cooling to 10-15 ℃. Filtering and drying to obtain 19.0g of Fmoc-Gly-O-TAGA. The yield thereof was found to be 98.8%.

2) H-Gly-O-TAGA (Fmoc removal):

into a 250mL three-port reaction flask, 148.0mL of methylene chloride and 19.0g of Fmoc-Gly-TAGA were added under nitrogen atmosphere, and the solid solution was stirred. 44.4mL of DMF was added, the temperature of the system was reduced to 10-15℃and 14.8g of piperidine was added. The reaction was monitored by TLC (EA: PE=1:4) at 10-15℃for 30 min. 60.0mL of 5% aqueous citric acid was added, and the mixture was washed and separated. Further, the mixture was washed with 60.0mL of water and 60.0mL of a 5% aqueous sodium hydrogencarbonate solution. 60.0mL of water was used for washing, and 60.0mL of saturated brine was used for washing.

3) Fmoc-Arg (Pbf) -Gly-O-TAGA (coupling of second amino acid):

under the protection of nitrogen, 11.5g Fmoc-Arg (Pbf) -OH and 2.45g HOBt were added to the three-port flask, and the solid was dissolved by stirring, followed by adding 4.4g DCC. The temperature is controlled below 30 ℃. The reaction was incubated for 3 hours. Desolventizing to a viscous state, stopping desolventizing, and adding 180.0mL of methanol. Stirring for 30 minutes. Filtering and drying to obtain 24.5g of Fmoc-Arg (Pbf) -Gly-O-TAGA. The yield thereof was found to be 97.0%.

to a 250mL three-port reaction flask, 150.0mL of methylene chloride, 0.43g of DMAP and 15.0g of compound of formula III were added under nitrogen atmosphere, and the solid solution was stirred. 5.4g Fmoc-Gly-OH was added and 2.76g DIC was added in portions. The temperature is controlled between 25 and 30 ℃. The reaction was kept at the temperature for 5 hours and filtered. When the solvent is removed until about 80.0g is remained, stopping removing the solvent, raising the temperature to 40-45 ℃, and dripping 180.0mL of methanol after the solid is dissolved. Then slowly cooling to 10-15 ℃. Filtering and drying to obtain 19.1g of product. The yield thereof was found to be 99.3%. The coupling and deprotection of amino acids 2, 3, 4, 5 to amino acid Fmoc-Ser (tBu) -OH of 6 were repeated 2-3. Obtaining Fmoc-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-O-TAGA; 150.0mL of tetrahydrofuran and 15.0g of the above fully protected hexapeptide containing a carrier were charged into a 500mL hydrogenation reactor, the solid was dissolved and purged with nitrogen, and then 1.5g of 10% wet palladium on carbon (50% aqueous solution) was added thereto, and after the nitrogen substitution, the hydrogen substitution was performed. Hydrogenation reaction is carried out under the conditions of 30-35 ℃ and 0.02-0.04 MPa. The reaction was incubated for 5 hours and monitored by TLC (EA: PE=1:4); filtration and concentration of the mother liquor to a solid under reduced pressure. 200.0mL of methanol was added, stirred for 30 minutes, and filtered. The mother liquor was concentrated to a solid under reduced pressure, 60.0mL of n-heptane was added and stirred for 30 minutes. Filtering and drying to obtain the fully protected hexapeptide Fmoc-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-OH 7.66g with the yield of 99.0%.

to a 250mL three-port reaction flask, 150.0mL of methylene chloride, 0.43g of DMAP and 15.0g of compound of formula III were added under nitrogen atmosphere, and the solid solution was stirred. 5.8g Fmoc-Val-OH was added and 2.6g DIC was added in portions. The temperature is controlled between 25 and 30 ℃. The reaction was kept at the temperature for 5 hours and filtered. When the solvent is removed until about 80.0g is remained, stopping removing the solvent, raising the temperature to 40-45 ℃, and dripping 180.0mL of methanol after the solid is dissolved. Then slowly cooling to 10-15 ℃. Filtering and drying to obtain 19.3g of product. The yield thereof was found to be 98.5%. The coupling and deprotection of amino acids 2, 3, 4, 5, 6, 7, 8, 9 to amino acid 10 Boc-His (Trt) -OH were repeated 2-3. Obtaining Boc-His (Trt) -Aib-Glu (OtBu) -Gly-Thr (OtBu) -Phe-Thr (OtBu) -Ser (OtBu) -Gln (OtBu) -Val-O-TAGA; 150.0mL of tetrahydrofuran and 15.0g of the above fully protected decapeptide containing carrier were charged into a 500mL hydrogenation reactor, the solid was dissolved and purged with nitrogen, and then 1.5g of 10% wet palladium on carbon (50% aqueous solution) was added thereto, and after nitrogen substitution, hydrogen substitution was performed. Hydrogenation reaction is carried out under the conditions of 30-35 ℃ and 0.02-0.04 MPa. The reaction was incubated for 5 hours and monitored by TLC (EA: PE=1:4); filtration and concentration of the mother liquor to a solid under reduced pressure. 200.0mL of methanol was added, stirred for 30 minutes, and filtered. The mother liquor was concentrated to a solid under reduced pressure, 60.0mL of n-heptane was added and stirred for 30 minutes. Filtering and drying to obtain the fully-protected decapeptide product Boc-His (Trt) -Aib-Glu (OtBu) -Gly-Thr (OtBu) -Phe-Thr (OtBu) -Ser (OtBu) -Gln (OtBu) -Val-OH 9.2g, with the yield of 98.6%.

to a 250mL three-port reaction flask, 148.0mL of methylene chloride and 18.5g of the above fully protected pentadecapeptide containing carrier were added under nitrogen protection, and the solid solution was stirred. 44.4mL of DMF was added, the temperature of the system was reduced to 10-15℃and 14.8g of piperidine was added. The reaction was monitored by TLC (EA: PE=1:4) at 10-15℃for 30 min. 60.0mL of 5% aqueous citric acid was added, and the mixture was washed and separated. Further, the mixture was washed with 60.0mL of water and 60.0mL of a 5% aqueous sodium hydrogencarbonate solution. 60.0mL of water was used for washing, and 60.0mL of saturated brine was used for washing.

To the three-port reaction flask, 5.5g of the fully protected hexapeptide and 0.68g of HOBt were added, and the solid was dissolved by stirring, followed by adding 1.26g of DCC. The temperature is controlled between 25 and 30 ℃. The reaction was incubated for 3 hours. Desolventizing to a viscous state, stopping desolventizing, and adding 180.0mL of methanol. Stirring for 30 minutes. Filtering and drying to obtain the product Fmoc-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys [ Oct (OtBu) -Glu (AEEA-AEEA) -OtBu ] -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-O-TAGA 21.0g. The yield thereof was found to be 95.9%.

8) Synthesis of Triundecapeptide, boc-His (Trt) -Aib-Glu (OtBu) -Gly-Thr (OtBu) -Phe-Thr (OtBu) -Ser (OtBu) -Gln (OtBu) -Val-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys [ Oct (OtBu) -Glu (AEEa-eeA) -OtBu ] -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-OH: to a 250mL three-port reaction flask, 166.0mL of methylene chloride and 20.8g of the above fully protected di-undecapeptide containing carrier were added under nitrogen protection, and the solid solution was stirred. 44.4mL of DMF was added, the system was cooled to 10-15℃and 0.85g of DBU was added. The reaction was monitored by TLC (EA: PE=1:4) at 10-15℃for 30 min. 60.0mL of 5% aqueous citric acid was added, and the mixture was washed and separated. Further, the mixture was washed with 60.0mL of water and 60.0mL of a 5% aqueous sodium hydrogencarbonate solution. 60.0mL of water was used for washing, and 60.0mL of saturated brine was used for washing.

7.6g of the fully protected decapeptide product and 0.61g of HOBt were added to the three-port flask, the solid was dissolved with stirring, and 1.59g of DCC was added. The temperature is controlled between 25 and 30 ℃. The reaction was incubated for 3 hours. Desolventizing to a viscous state, stopping desolventizing, and adding 180.0mL of methanol. Stirring for 30 minutes. The product Boc-His (Trt) -Aib-Glu (OtBu) -Gly-Thr (OtBu) -Phe-Thr (OtBu) -Ser (OtBu) -Gln (OtBu) -Val-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys [ Oct (OtBu) -Glu (AEEa-eeA) -OtBu ] -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-OTAGa 24.6g is obtained after filtration and drying. The yield thereof was found to be 93.7%. 150.0mL of tetrahydrofuran, 15.0g of the above fully protected triundecapeptide containing a carrier were charged into a 500mL hydrogenation reactor, the solid was stirred to dissolve, the solution was replaced with nitrogen, and 1.5g of 10% wet palladium on carbon (50% aqueous solution) was added thereto, and after the replacement with nitrogen, the hydrogen was replaced. Hydrogenation reaction is carried out under the conditions of 30-35 ℃ and 0.02-0.04 MPa. The reaction was incubated for 5 hours and monitored by TLC (EA: PE=1:4); filtration and concentration of the mother liquor to a solid under reduced pressure. 200.0mL of methanol was added, stirred for 30 minutes, and filtered. The mother liquor was concentrated to a solid under reduced pressure, 60.0mL of n-heptane was added and stirred for 30 minutes. The product was filtered and dried to give the fully protected thirty-first peptide product Boc-His (Trt) -Aib-Glu (OtBu) -Gly-Thr (OtBu) -Phe-Thr (OtBu) -Ser (OtBu) -Gln (OtBu) -Val-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys [ Oct (OtBu) -Glu (AEEa-eeA) -OtBu ] -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-OH 12.4g in 96.9%.

10 Cracking: boc-His (Trt) -Aib-Glu (OtBu) -Gly-Thr (OtBu) -Phe-Thr (OtBu) -Ser (OtBu) -Gln (OtBu) -Val-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys [ Oct (OtBu) -Glu (AEEA-AEEA) -OtBu ] -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-OH 12.4g was added to a 250.0mL three-port reaction flask, followed by 146.4g of TFA: EDT: tis=90:5:5 (volume ratio) lysate was cleaved at 25-30℃for 2 hours, added dropwise to 500.0g isopropyl ether, precipitated and filtered. The filter cake was again quenched with 250.0g acetonitrile: water = 2:1 (volume ratio), washing, filtering, purifying the crude peptide in mother liquor by column. 5.64g of the obtained somalundin is obtained, and the yield is 66.0%.

Example 3:

under the protection of nitrogen, 1264.0mL of MeOH and 216.0g of trihydroxybenzoic acid are added into a 2L three-port reaction bottle, the solution is stirred and cleared, and the temperature is reduced to 0-5 ℃. 180.5mL of acetyl chloride is added dropwise, and the reaction control measurement is completed after the dropwise addition. The reaction is carried out for 6 hours at the temperature of 20-30 ℃; and removing the solvent after the heat preservation reaction is finished. 2.0L of EA was added to the mixture, followed by extraction once with 500.0mL of water. EA phase with 15% NaHCO ₃ Extracting with water solution for 2 times, and extracting the combined water phases with 300mL EA for 3 times; merging EA phase, washing once with 300mL saturated sodium chloride aqueous solution, adding 100.0g anhydrous sodium sulfate for drying, filtering, removing solvent to obtain 230.0g crude product, heating to dissolve clear with 240.0mL ethyl acetate, then dripping 840.0mL petroleum ether, cooling to 10-15 ℃, filtering, and drying to obtain 213.2g product of formula 2 with the yield of 91.2%.

Under the protection of nitrogen, adding 1056.0 mL of DMF and 49.0g of compound of formula (2) into a 3.0L three-necked flask, stirring to dissolve, adding K ₂ CO ₃ 293.8g of white turbidity, 310.5g of 1-bromooctadecane were added and reacted at 100℃for 16h. 1300mL of toluene is added, the temperature is raised to 70 ℃ for dissolving, the solution is filtered while the solution is hot, the temperature of the filtrate is raised to 70 ℃ and the solution is slowly cooled under mechanical stirring, and the solid is separated out. Filtering and drying to obtain 203.5g of the product of the formula 3 with the yield of 81.3%.

2500.0mL of toluene and 500.0g of compound formula 3 are added into a 3L three-port reaction bottle under the protection of nitrogen, the temperature is raised to 50 ℃ for dissolving, the temperature is reduced to 20 ℃, 306.8g of 70% red aluminum toluene solution is added in batches, and the reaction is carried out for 1h under the heat preservation. After the heat preservation is finished, 1000.0mL of water is added dropwise for quenching, and then the pH=4-5 is adjusted by concentrated hydrochloric acid; the layers were separated, and the organic phase was washed with 400.0mL of saturated brine. 100.0g of anhydrous sodium sulfate is dried, filtered and desolventized, when about 1000.0mL is remained, 1500.0mL of acetonitrile is added, solid is separated out, the temperature is slowly reduced to 5-10 ℃, stirring is carried out for 20min, 421.0g of the product of the formula 4 is obtained by filtering and drying, and the yield is 86.8%.

Under the protection of nitrogen, 35.0mL of DCM and 27.0g of DMSO are added into a 1L three-port reaction flask, and the temperature is controlled between 0 and 5 ℃. 17.8g of pyridine sulfur trioxide are added in portions. After the addition was completed, stirring was carried out for 1 hour. 32.4g of the compound of formula 4 and 21.3g of TEA were dissolved in 320.0mL of DCM and added to the reaction flask. The temperature is controlled to be 5-10 ℃. Stirring is carried out for 1 hour. Then the temperature is raised to 30 to 35 ℃ at room temperature, and the reaction is kept for 15 hours. 450.0mL of 12% aqueous citric acid was added and the reaction quenched. Layering; the aqueous phase was extracted once with 120.0mL DCM. The organic phases were combined and washed 2 times with 120.0ml of 6% aqueous sodium bicarbonate; the mixture was washed once with 120.0mL of saturated brine. 30.0g of anhydrous sodium sulfate, and filtering. Desolvation is stopped when the residual amount is about 60.0 mL; raising the temperature to 40-45 ℃ and completely dissolving the solid. 180.0mL of ethyl acetate was added dropwise. Then slowly cooling to 5-10 ℃. Stirring for 30 minutes. Filtering and drying to obtain 24.5g of the product of the formula 5 with the yield of 75.4%.

200.0mL of 2-MeTHF and 20.0g of the compound of formula 7 were added to a 1L three-port reaction flask under nitrogen atmosphere, and the temperature was raised to 35 to 40 ℃. Dissolving the solid, and cooling to 25-30 ℃. 28.0mL of phenylmagnesium chloride is added dropwise, and the temperature is controlled between 20 and 25 ℃. Preserving heat and reacting for 1 hour; quenching the reaction by adding 20.0ml of 10% aqueous citric acid solution; the 2-MeTHF was removed under reduced pressure, 240.0mL of DCM and 80.0mL of 10% aqueous citric acid were added, the layers were separated, and the organic phase was washed with 120.0mL of water and 120.0mL of saturated brine. 30.0g of anhydrous sodium sulfate, and filtering. When the solvent is removed to about 70.0mL, stopping removing the solvent, heating to 40-45 ℃, and dropwise adding 200.0mL of methanol after the solvent is dissolved. Cooling to 20-25 deg.c and stirring for 30 min; filtering and drying to obtain 20.0g of the product of the formula III with the yield of 92.0%.

Synthesis of cord Ma Lutai:

1) Fmoc-Gly-O-TAGA (coupling of the first amino acid):

to a 250mL three-port reaction flask, 150.0mL of methylene chloride, 0.43g of DMAP and 15.0g of compound of formula III were added under nitrogen atmosphere, and the solid solution was stirred. 5.4g Fmoc-Gly-OH was added and 2.76g DIC was added in portions. The temperature is controlled between 15 and 20 ℃. The reaction was incubated for 7 hours and monitored by TLC (EA: PE=1:4); and (5) filtering. When the solvent is removed until about 80.0g is remained, stopping removing the solvent, raising the temperature to 40-45 ℃, and dripping 180.0mL of methanol after the solid is dissolved. Then slowly cooling to 10-15 ℃. Filtering and drying to obtain 19.2g of Fmoc-Gly-O-TAGA. The yield thereof was found to be 99.8%.

2) H-Gly-O-TAGA (Fmoc removal):

into a 250mL three-port reaction flask, 148.0mL of methylene chloride and 19.0g of Fmoc-Gly-TAGA were added under nitrogen atmosphere, and the solid solution was stirred. 44.4mL of DMF was added, the temperature of the system was reduced to 20-25℃and 14.8g of piperidine was added. The reaction was monitored by TLC (EA: PE=1:4) at 20-25℃for 30 min. 60.0mL of 5% aqueous citric acid was added, and the mixture was washed and separated. Further, the mixture was washed with 60.0mL of water and 60.0mL of a 5% aqueous sodium hydrogencarbonate solution. 60.0mL of water was used for washing, and 60.0mL of saturated brine was used for washing.

3) Fmoc-Arg (Pbf) -Gly-O-TAGA (coupling of second amino acid): under the protection of nitrogen, 11.5g Fmoc-Arg (Pbf) -OH and 2.45g HOBt were added to the three-port flask, and the solid was dissolved by stirring, followed by adding 4.4g DCC. The temperature is controlled to be 20-25 ℃. The reaction was incubated for 3 hours. Desolventizing to a viscous state, stopping desolventizing, and adding 180.0mL of methanol. Stirring for 30 minutes. Filtering and drying to obtain the Fmoc-Arg (Pbf) -Gly-O-TAGA 24.3g product. The yield thereof was found to be 96.2%.

5) Synthesis of a fully protected hexapeptide fragment (Fmoc-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-OH): to a 250mL three-port reaction flask, 150.0mL of methylene chloride, 0.43g of DMAP and 15.0g of compound of formula III were added under nitrogen atmosphere, and the solid solution was stirred. 5.4g Fmoc-Gly-OH was added and 2.76g DIC was added in portions. The temperature is controlled to be 20-25 ℃. The reaction was incubated for 7 hours and filtered. When the solvent is removed until about 80.0g is remained, stopping removing the solvent, raising the temperature to 40-45 ℃, and dripping 180.0mL of methanol after the solid is dissolved. Then slowly cooling to 10-15 ℃. Filtering and drying to obtain 18.9g of product. The yield thereof was found to be 98.3%. Repeating the coupling and deprotection of the 2 nd, 3 rd, 4 th and 5 th amino acids to the coupling of the 6 th amino acid Fmoc-Ser (tBu) -OH to obtain Fmoc-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-O-TAGA; 150.0mL of tetrahydrofuran and 15.0g of the above fully protected hexapeptide containing a carrier were charged into a 500mL hydrogenation reactor, the solid was dissolved and purged with nitrogen, and then 1.5g of 10% wet palladium on carbon (50% aqueous solution) was added thereto, and after the nitrogen substitution, the hydrogen substitution was performed. Hydrogenation reaction is carried out under the conditions of 35-40 ℃ and 0.01-0.02 MPa. The reaction was incubated for 5 hours and monitored by TLC (EA: PE=1:4); filtration and concentration of the mother liquor to a solid under reduced pressure. 200.0mL of methanol was added, stirred for 30 minutes, and filtered. The mother liquor was concentrated to a solid under reduced pressure, 60.0mL of n-heptane was added and stirred for 30 minutes. Filtering and drying to obtain the fully protected hexapeptide Fmoc-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-OH 7.7g with the yield of 99.5%.

6) Synthesis of the fully protected decapeptide fragment (Boc-His (Trt) -Aib-Glu (OtBu) -Gly-Thr (OtBu) -Phe-Thr (OtBu) -Ser (OtBu) -Gln (OtBu) -Val-OH): to a 250mL three-port reaction flask, 150.0mL of methylene chloride, 0.43g of DMAP and 15.0g of compound of formula III were added under nitrogen atmosphere, and the solid solution was stirred. 5.6g Fmoc-Val-OH was added and 2.7g DIC was added in portions. The temperature is controlled to be 20-25 ℃. The reaction was kept at the temperature for 5 hours and filtered. When the solvent is removed until about 80.0g is remained, stopping removing the solvent, raising the temperature to 40-45 ℃, and dripping 180.0mL of methanol after the solid is dissolved. Then slowly cooling to 10-15 ℃. Filtering and drying to obtain 19.5g of product. The yield thereof was found to be 99.4%. The coupling and deprotection of amino acids 2, 3, 4, 5, 6, 7, 8, 9 to amino acid 10 Boc-His (Trt) -OH were repeated 2-3. Obtaining Boc-His (Trt) -Aib-Glu (OtBu) -Gly-Thr (OtBu) -Phe-Thr (OtBu) -Ser (OtBu) -Gln (OtBu) -Val-O-TAGA; 150.0mL of tetrahydrofuran and 15.0g of the above fully protected decapeptide containing carrier were charged into a 500mL hydrogenation reactor, the solid was dissolved and purged with nitrogen, and then 1.5g of 10% wet palladium on carbon (50% aqueous solution) was added thereto, and after nitrogen substitution, hydrogen substitution was performed. Hydrogenation reaction is carried out under the conditions of 35-40 ℃ and 0.01-0.02 MPa. The reaction was incubated for 5 hours and monitored by TLC (EA: PE=1:4); filtration and concentration of the mother liquor to a solid under reduced pressure. 200.0mL of methanol was added, stirred for 30 minutes, and filtered. The mother liquor was concentrated to a solid under reduced pressure, 60.0mL of n-heptane was added and stirred for 30 minutes. Filtering and drying to obtain the fully-protected decapeptide product Boc-His (Trt) -Aib-Glu (OtBu) -Gly-Thr (OtBu) -Phe-Thr (OtBu) -Ser (OtBu) -Gln (OtBu) -Val-OH 9.1g, with the yield of 97.5%.

7) Synthesis of Diundecapeptide Fmoc-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys [ Oct (OtBu) -Glu (AEEA-AEEA) -OtBu ] -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-O-TAGA: to a 250mL three-port reaction flask, 148.0mL of methylene chloride and 18.5g of the above fully protected pentadecapeptide containing carrier were added under nitrogen protection, and the solid solution was stirred. 44.4mL of DMF was added, the temperature of the system was reduced to 10-15℃and 14.8g of piperidine was added. The reaction was monitored by TLC (EA: PE=1:4) at 20-25℃for 30 min. 60.0mL of 5% aqueous citric acid was added, and the mixture was washed and separated. Further, the mixture was washed with 60.0mL of water and 60.0mL of a 5% aqueous sodium hydrogencarbonate solution. 60.0mL of water was used for washing, and 60.0mL of saturated brine was used for washing.

To the three-port reaction flask, 5.6g of the fully protected hexapeptide and 0.68g of HOBt were added, and the solid was dissolved by stirring, followed by adding 1.26g of DCC. The temperature is controlled to be 20-25 ℃. The reaction was incubated for 3 hours. Desolventizing to a viscous state, stopping desolventizing, and adding 180.0mL of methanol. Stirring for 30 minutes. Filtering and drying to obtain the product Fmoc-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys [ Oct (OtBu) -Glu (AEEA-AEEA) -OtBu ] -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-O-TAGA 21.2g. The yield thereof was found to be 96.8%.

8) Synthesis of Triundecapeptide, boc-His (Trt) -Aib-Glu (OtBu) -Gly-Thr (OtBu) -Phe-Thr (OtBu) -Ser (OtBu) -Gln (OtBu) -Val-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys [ Oct (OtBu) -Glu (AEEa-eeA) -OtBu ] -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-OH: to a 250mL three-port reaction flask, 166.0mL of methylene chloride and 20.8g of the above fully protected di-undecapeptide containing carrier were added under nitrogen protection, and the solid solution was stirred. 44.4mL of DMF was added, the system was cooled to 20-25℃and 0.9g of DBU was added. The reaction was monitored by TLC (EA: PE=1:4) at 15-20℃for 30 min. 60.0mL of 5% aqueous citric acid was added, and the mixture was washed and separated. Further, the mixture was washed with 60.0mL of water and 60.0mL of a 5% aqueous sodium hydrogencarbonate solution. 60.0mL of water was used for washing, and 60.0mL of saturated brine was used for washing. 7.7g of the fully protected decapeptide and 0.61g of HOBt were added to the three-port flask, the solid was dissolved with stirring, and 1.59g of DCC was added. The temperature is controlled between 25 and 30 ℃. The reaction was incubated for 3 hours. Desolventizing to a viscous state, stopping desolventizing, and adding 180.0mL of methanol. Stirring for 30 minutes. The product Boc-His (Trt) -Aib-Glu (OtBu) -Gly-Thr (OtBu) -Phe-Thr (OtBu) -Ser (OtBu) -Gln (OtBu) -Val-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys [ Oct (OtBu) -Glu (AEEa-eeA) -OtBu ] -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-O-TAGA 24.8g was obtained after filtration and drying. The yield thereof was found to be 94.4%.

150.0mL of tetrahydrofuran, 15.0g of the above fully protected triundecapeptide containing a carrier were charged into a 500mL hydrogenation reactor, the solid was stirred to dissolve, the solution was replaced with nitrogen, and 1.5g of 10% wet palladium on carbon (50% aqueous solution) was added thereto, and after the replacement with nitrogen, the hydrogen was replaced. Hydrogenation reaction is carried out under the conditions of 35-40 ℃ and 0.01-0.02 MPa. The reaction was incubated for 5 hours and monitored by TLC (EA: PE=1:4); filtration and concentration of the mother liquor to a solid under reduced pressure. 200.0mL of methanol was added, stirred for 30 minutes, and filtered. The mother liquor was concentrated to a solid under reduced pressure, 60.0mL of n-heptane was added and stirred for 30 minutes. The product was filtered and dried to give the fully protected thirty-first peptide product Boc-His (Trt) -Aib-Glu (OtBu) -Gly-Thr (OtBu) -Phe-Thr (OtBu) -Ser (OtBu) -Gln (OtBu) -Val-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys [ Oct (OtBu) -Glu (AEEa-eeA) -OtBu ] -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-OH 12.5g in 97.6%.

9) Cracking: boc-His (Trt) -Aib-Glu (OtBu) -Gly-Thr (OtBu) -Phe-Thr (OtBu) -Ser (OtBu) -Gln (OtBu) -Val-Ser (OtBu) -Ser (OtBu) -Tyr (OtBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys [ Oct (OtBu) -Glu (AEEA-AEEA) -OtBu ] -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-OH 12.4g was added to a 250.0mL three-port reaction flask, followed by 146.4g of TFA: EDT: tis=90:5:5 (volume ratio) lysate was cleaved at 30-35℃for 2 hours, added dropwise to 500.0g isopropyl ether, precipitated and filtered. The filter cake was again quenched with 250.0g acetonitrile: water = 2:1 (volume ratio), washing, filtering, purifying the crude peptide in mother liquor by column. 5.7g of the obtained somalundin is obtained, and the yield is 66.7%.

In the present invention, the amino acid sequences of the polypeptides involved are shown in Table 1.

Table 1: amino acid sequence listing

While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and additions may be made without departing from the scope of the invention. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. The diaryl benzyl alcohol compound as polypeptide liquid phase synthesis carrier is characterized in that the structural formula of the diaryl benzyl alcohol compound is shown as the formula I:r is selected from C12-C22 alkyl, X is H, -OCH ₃ Or halogen;

the preparation method of the diaryl benzyl alcohol compound comprises the steps of taking a compound with a structure shown in a formula 1 as a raw material, carrying out esterification reaction, substitution reaction, reduction reaction, oxidation reaction and addition, and then carrying out hydrolysis reaction and precipitation to obtain the compound shown in the formula I; The method comprises the steps of carrying out a first treatment on the surface of the The method comprises the following specific steps:

a) Dissolving a substance shown in a formula 1 in methanol, and adding an esterification reagent to perform esterification reaction to generate a compound 2;；

b) Adding the compound 2, an acid binding agent and a substitution reaction reagent into a solvent, and reacting to generate a compound 3 of the formula;；

c) Dissolving the compound 3, adding a reducing agent a, and carrying out a reduction reaction to generate a compound 4; ；

d) Adding an oxidant a and an oxidant b into dichloromethane to be used as a solution A; dissolving the compound 4 in triethylamine and dichloromethane, dropwise adding the solution into a solution A system, and carrying out oxidation reaction to generate a compound 5; ；

e) And dissolving the compound 5, adding a solution containing a Grignard reagent, and reacting to obtain the compound shown in the formula I.

2. The diaryl benzyl alcohol compound as polypeptide liquid phase synthesis carrier as claimed in claim 1, wherein the diaryl benzyl alcohol is characterized in thatThe structural formula of the class of compounds is shown as formula III:。

3. the diarylbenzenes of claim 1, wherein in step a), the esterification reagent comprises acetyl chloride, thionyl chloride or concentrated sulfuric acid, and the molar ratio of the substance represented by formula 1 to the esterification reagent is 1:1.5-3.0.

4. The diaryl benzyl alcohol compound according to claim 1, wherein in the step b), the acid-binding agent comprises one or more of potassium carbonate, sodium carbonate, potassium hydroxide, sodium hydroxide, diisopropylethylamine and triethylamine, the substitution reaction agent is halogenated alkane RX, R is C12-C22 alkyl, and X is halogen; the molar ratio of the compound 2, the substitution reaction reagent and the acid binding agent is 1.0:3.2-4.0:8.0-10.0.

5. The diaryl benzyl alcohol-based compound according to claim 1, wherein in step c), the reducing agent a comprises red aluminum or lithium aluminum hydride, and the molar ratio of the compound 3 to the reducing agent a is 1:2.5-4.0.

6. The diaryl benzyl alcohol-based compound according to claim 1, wherein in step d), the oxidizing agent a comprises pyridine sulfur trioxide, oxalyl chloride or NaClO, the oxidizing agent b comprises DMSO or TEMPO, and the molar ratio of compound 4, oxidizing agent a and oxidizing agent b is 1:3.0-5.0:10.0 to 12.0;

in the step e), the reagent of the Grignard reaction comprises aryl magnesium chloride, aryl magnesium bromide or aryl lithium, and one or more of tetrahydrofuran, methyltetrahydrofuran and toluene as a solvent; the molar ratio of compound 5 to grignard reagent is 1:2.0-3.0.

7. The use of the diaryl benzyl alcohol compound as defined in claim 1, characterized in that the use of the compound as defined in claim 1 as a hydrophobic carrier for the synthesis of fully protected polypeptides or deprotected polypeptides in liquid phase.

8. The use of diaryl benzyl alcohol compound as defined in claim 7, wherein the compound shown in the formula I is used as hydrophobic carrier, amino acid is coupled in turn through condensation reaction to prepare fully protected polypeptide chain containing initial end carrier, and then the fully protected polypeptide chain without initial end carrier is prepared through deprotection.