CN110938115A - Whole liquid phase synthesis method of sinapultide - Google Patents

Whole liquid phase synthesis method of sinapultide Download PDF

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CN110938115A
CN110938115A CN201811113515.7A CN201811113515A CN110938115A CN 110938115 A CN110938115 A CN 110938115A CN 201811113515 A CN201811113515 A CN 201811113515A CN 110938115 A CN110938115 A CN 110938115A
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leu
boc
lys
fmoc
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陈永汉
汪伟
宓鹏程
陶安进
袁建成
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Hybio Pharmaceutical Co Ltd
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    • C07K—PEPTIDES
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Abstract

The invention relates to a whole liquid-phase synthesis method of sinapultide, which comprises the steps of firstly synthesizing Fmoc-Leu-Leu-Leu-Lys (Boc) -OH, synthesizing a whole protection fragment Fmoc-Leu-Leu-Leu-Lys (Boc) -Leu-Leu-Leu-Leu-Lys (Boc) -OH by two times on the basis of the fragment, and then carrying out liquid-phase coupling on the Boc-Lys (Boc) -OH to completely protect the sinapultide; and cracking under liquid phase condition to remove protecting group Boc. The preparation method is simple, the yield and the purity are high, the solvent is saved, the purpose of environmental protection is achieved, and the preparation method is suitable for large-scale production.

Description

Whole liquid phase synthesis method of sinapultide
Technical Field
The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a method for synthesizing sinapultide in a full liquid phase.
Background
Neonatal respiratory distress syndrome (neonatal NRDS) refers to the occurrence of transient (minutes to hours) spontaneous respiration after birth of a newborn, followed by progressive dyspnea, cyanosis, moans or other acute respiratory distress symptoms and respiratory failure. The formation of hyaline membrane in the lung of an infant patient is also known as hyaline membrane disease of newborn.
On day 6/3 2012, lucinactant (surfaxin) was approved by the FDA in the united states for use in premature Respiratory Distress Syndrome (RDS). Surfaxin (Lucinactant, ruxintan) is a product designed on the basis of 21-amino acid peptide KL4(sinapultide ) according to the characteristics of natural human lung surfactant, and is used for simulating human lung surfactant protein B (SP-B). Therefore, unlike other animal-derived products, the product of lurasiracetam can be unlimited, not only to pharmaceutical levels, but also without the risk of spreading potential animal-related diseases.
The cinacalpeptide, having the english name Sinapultide and a molecular weight of 2469.40, is an artificially designed polypeptide containing 21 amino acids, and has the following structure:
Figure BDA0001809878390000011
regarding the preparation method of the sinapultide, the U.S. Pat. No. 5,97,73 adopts the gene recombination technology to synthesize the sinapultide, but the method has the disadvantages of complicated operation, extremely high technical requirements and production cost, and is not beneficial to the industrialized mass production.
The patent CN 102850440A is the first domestic patent related to the synthesis method of Sinapu, and adopts a fragment synthesis method. Firstly, Fmoc-Lys (Boc) -Leu-Leu-Leu-Leu-OH is synthesized in a solid phase, and then peptide resin is synthesized in the solid phase and the peptide is cracked to obtain the sinapultide. The method needs a large amount of expensive resin carriers, has a long production period, is easy to shrink in the peptide resin synthesis process, and has low crude peptide purity and high cost.
Patent CN 104098656A adopts Fmoc solid phase synthesis method, coupling each protected amino acid in sequence to obtain protected 21 peptide resin, and obtaining the sinapultide through cracking reaction. The method has the advantages of long production period, easy shrinkage in the peptide resin synthesis process, low purity of crude peptide, low total yield and high production cost.
Patent CN 104817631A describes a method of synthesizing Fmoc-Lys- (Boc) -Wang resin by Fmoc solid phase synthesis, followed by alternate coupling of protected Fmoc- (Leu)4-OH and protected amino acid Fmoc-Lys (Boc) -OH to prepare the sinapultide resin, and finally preparing the sinapultide through a cracking reaction. The method has the advantages of easy shrinkage in the process of synthesizing the peptide resin, low purity of crude peptide and low total yield.
The patent CN 105384799A firstly prepares a polypeptide segment I in a liquid phase, namely Fmoc-Leu-Leu-Leu-Lys (Boc) -OH, then sequentially couples four polypeptide segments on a carrier resin by adopting an Fmoc solid phase synthesis method, finally couples protective amino acid Lys, and prepares the sinapultide after cracking reaction. The method has the advantages of easy shrinkage in the process of synthesizing the peptide resin, low purity of crude peptide and low total yield.
Disadvantages of solid phase synthesis: a solid carrier is needed and the reaction is ensured to be complete through excessive feeding, so that the cost is high; the peptide resin can shrink obviously in the synthesis process, so that the coupling effect is poor; the scale of the solid-phase synthesis method is greatly affected by equipment and is difficult to scale up.
Disclosure of Invention
The invention aims to shorten the production period and simultaneously avoid the problems of low solid-phase synthesis yield and difficult scale amplification by adopting the synthesis of a fully protected peptide fragment and then synthesizing the sinapultide by a liquid phase method.
The same refined peptide product is produced, the dosage of Fmoc amino acid raw materials can be effectively reduced by adopting a liquid phase fragment synthesis method, and simultaneously, the dosage of organic solvents (DMF and DCM) in solid phase synthesis can be reduced, so that the effect of waste reduction is achieved;
the preparation method of the sinapultide comprises the following steps:
A. synthesis of the full protection peptide:
synthesis of fully protected peptide fragment 1:
Figure BDA0001809878390000031
synthesis of fully protected peptide fragment 2:
Figure BDA0001809878390000032
synthesis of fully protected peptide fragment 3:
Figure BDA0001809878390000033
synthesis of fully protected peptide fragment 4:
Figure BDA0001809878390000034
synthesis of fully protected peptide fragment 5:
Figure BDA0001809878390000041
synthesis of fully protected peptide fragment 6:
Figure BDA0001809878390000042
synthesis of fully protected peptide fragment 7:
Figure BDA0001809878390000043
B. cracking to obtain crude peptide product
Figure BDA0001809878390000051
The invention provides a liquid phase preparation method of sinapultide, which comprises the following steps:
1) performing carboxyl activation by Fmoc-Leu-OH, and coupling with H-Leu-OH to obtain Fmoc-Leu-Leu-OH;
2) removing the Fmoc group of the Fmoc-Leu-Leu-OH to obtain H-Leu-Leu-OH;
3) performing carboxyl activation by Fmoc-Leu-Leu-OH, and coupling with H-Leu-Leu-OH to obtain Fmoc-Leu-Leu-Leu-Leu-OH;
4) performing carboxyl activation by Fmoc-Leu-Leu-Leu-Leu-OH, and coupling with H-Lys (Boc) -OH to obtain Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH;
5) removing Fmoc group of Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH to obtain H-Leu-Leu-Leu-Leu-Lys (Boc) -OH;
6) performing carboxyl activation by Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH, and coupling with H-Leu-Leu-Leu-Lys (Boc) -OH to obtain Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH;
7) removing Fmoc group of Fmoc-Leu-Leu-Leu-Lys (Boc) -OH to obtain H-Leu-Leu-Leu-Leu-Lys (Boc) -OH;
8) performing carboxyl activation by Fmoc-Leu-Leu-Leu-Lys (Boc) -Leu-Leu-Leu-Leu-Lys (Boc) -OH, and coupling with H-Leu-Leu-Leu-Leu-Lys (Boc) -OH to obtain Fmoc-Leu-Leu-Leu-Leu-Leu-Lys (Boc) -Leu-Leu-Leu-Leu-Lys (Boc) -OH;
9) removing Fmoc group of Fmoc-Leu-Leu-Leu-Lys (Boc) -Leu-Leu-Leu-Leu-Lys (Boc) -OH to obtain H-Leu-Leu-Leu-Leu-Leu-Lys (Boc);
10) performing carboxyl activation with Boc-Lys (Boc) -OH, and coupling with H-Leu-Leu-Leu-Leu-Lys (Boc) -Leu-Leu-Leu-Leu-Lys (Boc) -OH to obtain Boc-Lys (Boc) -Leu-Leu-Leu-Leu-Leu-Lys (Boc) -OH.
11) Cleaving Boc-Lys (Boc) -Leu-Leu-Leu-Leu-Lys (Boc) -Boc protecting group of OH to obtain H-Lys-Leu-Leu-Leu-Lys-Leu-Leu-Leu-Leu-Leu-Lys-OH.
In the technical scheme of the invention, the steps 1) to 11) are all completed in a liquid phase.
In the technical scheme of the invention, the Fmoc group removing reagent used for removing the Fmoc group is Lewis acid, and the Lewis acid is selected from AlCl3、BF3、AlCl3And N-methylmorpholine, AlCl3Composition of toluene and BF3And toluene.
In the technical scheme of the invention, the molar ratio of the Fmoc group removing reagent used for removing the Fmoc group to the Fmoc group compound to be removed is the Fmoc group compound to be removed, namely AlCl31:1-3 (preferably 1:2), the compound to be Fmoc group removed AlCl3N-methylmorpholine-1: 1-3:1-2 (preferably 1:2: 1.5).
In the technical scheme of the invention, the method for removing the Fmoc group comprises the step of adding AlCl into an organic solution of a substrate to be subjected to Fmoc group removal3Adding an acidic aqueous solution for acidification after the reaction is completed, adjusting the pH value to 1-3, adding diethyl ether for extraction to remove impurities, taking a water phase, and adjusting the water phase to be neutral to obtain a compound with the Fmoc group removed; or
Adding AlCl into the organic solution of the substrate to be removed with Fmoc group3And N-methylmorpholine until the reaction is completed, precipitating in methyl tertiary ether, and collecting the precipitate to obtain the compound with the Fmoc group removed.
In the technical scheme of the invention, carboxyl activation is to perform activation treatment on a compound to be subjected to carboxyl activation by a carboxyl activating agent, wherein the carboxyl activating agent is selected from one or more of HOSu, HONb, DCC and EDC.HCl, and is preferably a composition of HOSu and DCC or a composition of HONb and DCC.
In the technical scheme of the invention, the ratio of the compound to be activated by carboxyl to the carboxyl activating agent is 1: 1-1.5.
In the technical scheme of the invention, the carboxyl activated intermediate is coupled after being separated and purified, the carboxyl activated intermediate is separated and purified into a carboxyl activating agent and a compound to be activated by carboxyl, the compound is reacted completely, the filtrate is concentrated after being filtered, and the carboxyl activated intermediate is obtained by crystallizing with ethyl acetate and ether or ethyl acetate and methyl tertiary ether.
The carboxyl activated intermediate is a compound after carboxyl is activated.
In the technical scheme of the invention, the cleavage method is to treat a cleavage reagent to obtain a product for removing the Boc protecting group, wherein the cleavage reagent is an aqueous solution containing trifluoroacetic acid, and the concentration of the trifluoroacetic acid in the aqueous solution of the trifluoroacetic acid is more than 80-99%, preferably more than 90-95%.
In the technical scheme of the invention, the cracking method comprises the steps of treating with trifluoroacetic acid aqueous solution, reacting to be complete, adding into glacial ethyl ether or methyl tert-ether, separating out precipitate, and filtering the precipitate to obtain a cracking product.
The English and abbreviation meanings in the invention are as follows:
abbreviations and English Means of
Fmoc 9-fluorenylmethoxycarbonyl group
HONb N-hydroxy-5-norbornene-2, 3-dicarboximides
DCC Dicyclohexylcarbodiimide
HOSu N-hydroxysuccinimide
AlCl3 Aluminium trichloride
TFA Trifluoroacetic acid
Boc Tert-butyloxycarbonyl radical
DMF N, N-dimethylformamide
DCM Methylene dichloride
HOBt 1-hydroxybenzotriazoles
DIC N, N' -diisopropylcarbodiimide
NMM N-methylmorpholine
Advantageous effects
1) The sinapultide contains a large amount of hydrophobic amino acid Leu, so that resin shrinkage can be caused in solid phase synthesis, the sinapultide is synthesized by adopting a gradual coupling method, and when the sinapultide is coupled to the 6 th amino acid Leu, the peptide resin begins to shrink, so that the coupling difficulty is obviously increased; the invention adopts liquid phase synthesis to effectively solve the problem of difficult coupling caused by shrinkage of solid phase synthetic resin, and simultaneously adopts liquid phase fragment synthesis to improve the purity of crude peptide, simplify the steps and greatly reduce the production period.
2) The same refined peptide product is produced, the dosage of Fmoc amino acid raw materials can be effectively reduced by adopting a liquid phase fragment synthesis method, and simultaneously, the dosage of organic solvents (DMF and DCM) in solid phase synthesis can be reduced, so that the effect of waste reduction is achieved.
3) The method is simple and suitable for large-scale production.
Drawings
FIG. 1 is an HPLC chromatogram of the product of example 16.
FIG. 2 is an HPLC chromatogram of the product of example 17.
FIG. 3 is an HPLC chromatogram of the product of example 18.
FIG. 4 is an HPLC chromatogram of the product of example 19.
Detailed Description
Example 1 Synthesis of fully protected peptide 1
Preparation of Fmoc-Leu-ONb: Fmoc-Leu-OH 3534g (10mol) and HONb 1969g (11mol) were weighed out and dissolved in 15L tetrahydrofuran, and stirred in an ice-water bath. 2476g (12.0mol) of DCC was weighed, dissolved in 10L of tetrahydrofuran, added dropwise to the solution slowly, and the reaction was monitored by TLC with stirring. After the reaction is finished, suction filtration is carried out, the reaction solution is concentrated to 8L, then 50L of methyl tert-ether is added into the concentrated solution, a large amount of white solid is separated out, and the solution is kept stand for 4h at the temperature of minus 20 ℃. And (3) carrying out suction filtration, dissolving the solid with 5L ethyl acetate, adding 50L methyl tert-ether for crystallization, carrying out suction filtration, and drying the solid in vacuum to obtain 4837g of Fmoc-Leu-ONb with the purity of more than 99% and the yield of 94.0%.
Preparation of Fmoc-Leu-Leu-OH: H-Leu-OH1233g (9.4mol) and sodium carbonate 1091g (10.3mol) were weighed out and dissolved in 10L water, 10L of Fmoc-Leu-ONb 4837g (9.4mol) tetrahydrofuran solution was slowly added under ice-water bath, the reaction was stirred and TLC was used to monitor the end point of the reaction. And after the reaction is completed, filtering, adding 1N hydrochloric acid aqueous solution into the filtrate in ice-water bath, adjusting the pH value of the solution to 2-3, adding 5L of ethyl acetate for extraction for 2 times, combining organic phases, carrying out rotary evaporation and concentration to 5L, adding 30L of methyl tert-ether into the concentrated solution, carrying out crystallization at 4 ℃ to obtain Fmoc-Leu-Leu-OH, filtering, drying, washing with methyl tert-ether, and carrying out vacuum drying to obtain Fmoc-Leu-Leu-OH 4083g, wherein the purity is more than 99%, and the yield is 93.1%.
Preparation of Fmoc-Leu-Leu-ONb: Fmoc-Leu-Leu-OH 2041g (4.4mol) and HONb868g (4.8mol) were weighed out and dissolved in 8L tetrahydrofuran, and stirred in an ice-water bath. DCC 1094g (5.3mol) was weighed, dissolved in 5L tetrahydrofuran, added dropwise slowly to the above solution, stirred for reaction, and the end of the reaction was monitored by TLC. After the reaction is finished, carrying out suction filtration, concentrating the reaction solution to 4L, adding 25L of methyl tert-ether into the concentrated solution, precipitating a large amount of white solid, and standing the solution for 1h at-20 ℃. Standing, performing suction filtration, dissolving the solid with 4L ethyl acetate, adding 20L methyl tert-ether, crystallizing, performing suction filtration, and vacuum drying the solid to obtain 2569g of Fmoc-Leu-Leu-ONb with the purity of more than 99% and the yield of 93.0%.
Preparation of H-Leu-Leu-OH 2041g (4.4mol) of Fmoc-Leu-Leu-OH was weighed, dissolved with 20L of dichloromethane with stirring, and 1173g of AlCl was weighed3(8.8mol) and 667g N-methylmorpholine (6.6mol) were added to the reaction solution and stirred for 3 hours, and the end of the reaction was monitored by TLC. And (3) carrying out rotary evaporation on the reaction liquid to 4L, adding the reaction liquid into 40L methyl tert-ether for precipitation, centrifuging to collect solid precipitate, and carrying out vacuum drying to obtain H-Leu-Leu-OH 987.7g with the purity of more than 99% and the yield of 92%.
Fmoc-Leu-Leu-Leu-Leu-OH preparation: H-Leu-Leu-OH 977g (4.0mol) and sodium carbonate 466g (4.4mol) are accurately weighed and dissolved in 5L water, 5L tetrahydrofuran solution of Fmoc-Leu-Leu-ONb 2569g (4.0mol) is slowly added in ice water bath, the reaction is stirred and the end point of the reaction is monitored by TLC. And after the reaction is completed, filtering, adding 1N hydrochloric acid aqueous solution into the filtrate in an ice-water bath, adjusting the pH value of the solution to 2-3, adding 5L of ethyl acetate for extraction for 2 times, merging organic phases, carrying out rotary evaporation and concentration to 5L, adding the concentrated solution into 30L of methyl tert-ether, carrying out crystallization at 4 ℃ to obtain a solid product, filtering, washing with methyl tert-ether, and carrying out vacuum drying to obtain Fmoc-Leu-Leu-Leu-Leu-OH 2606g, wherein the purity is more than 99%, and the yield is 94.0%.
Preparation of Fmoc-Leu-Leu-Leu-Leu-ONb: Fmoc-Leu-Leu-Leu-Leu-OH 2606g (3.7mol) and HONb 735g (4.1mol) were weighed out and dissolved in 8L tetrahydrofuran and stirred in an ice-water bath. DCC 915g (4.4mol) was weighed, dissolved in 5L tetrahydrofuran, added dropwise to the solution slowly, stirred and monitored by TLC. After the reaction is finished, carrying out suction filtration, concentrating the reaction solution to 4L, adding 30L of methyl tert-ether into the concentrated solution, separating out a large amount of white solid, and standing the solution for 4h at-20 ℃. Standing, performing suction filtration, dissolving the solid with 4L ethyl acetate, adding 25L methyl tert-ether for crystallization, performing suction filtration, and vacuum drying the solid to obtain 2942g of Fmoc-Leu-Leu-Leu-Leu-ONb, wherein the purity is more than 99%, and the yield is 93.1%.
Preparation of Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH: H-Lys (Boc) -OH 862g (3.5mol) and sodium carbonate 413g (3.9 mol) were weighed out and dissolved in 5L water, 5L tetrahydrofuran solution of Fmoc-Leu-Leu-Leu-Leu-ONb2942g (3.5mol) was added slowly in ice-water bath, the reaction was stirred and monitored by TLC. And (3) filtering after complete reaction, adding 1N hydrochloric acid aqueous solution into the filtrate in an ice-water bath, adjusting the pH value of the solution to 2-3, adding 5L ethyl acetate for extraction for 2 times, merging organic phases, carrying out rotary evaporation and concentration to 4L, adding 25L methyl tert-ether into the concentrated solution, carrying out crystallization at 4 ℃ to obtain a solid product, filtering, drying, washing with the methyl tert-ether, and carrying out vacuum drying to obtain Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH 2940g, wherein the purity is more than 99% and the yield is 92.8%.
Example 2 Synthesis of fully protected peptide 2
Preparation of H-Leu-Leu-Leu-Leu-Lys (Boc) -OH: Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH1470g (1.6mmol) was weighed, dissolved with stirring in 8L of dichloromethane, and 427g of AlCl was weighed3(3.2mmol) and 242g of N-methylmorpholine (2.4mol) were added to the reaction solution and the reaction was stirred for 3 hours and monitored by TLC. The reaction solution is evaporated to 1.6L, added into 16L methyl tert-ether for precipitation, centrifuged to collect solid precipitate, and dried in vacuum to obtain H-Leu-Leu-Leu-Leu-Lys (Boc) -OH 1029g with purity of more than 98.5% and yield of 92%.
Example 3 Synthesis of fully protected peptide 3
Preparation of Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -ONb: Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH1470g (1.6mmol) and HONb 322g (1.8mol) were weighed out and dissolved in 4L tetrahydrofuran and stirred in an ice-water bath. DCC396g (1.9mol) was weighed, dissolved in 4L tetrahydrofuran, added dropwise slowly to the above solution, stirred and monitored by TLC. After the reaction is finished, carrying out suction filtration, concentrating the reaction solution to 3L, adding 20L of methyl tert-ether into the concentrated solution, separating out a large amount of white solid, and standing the solution for 4h at-20 ℃. Standing, performing suction filtration, dissolving the solid with 3L ethyl acetate, adding 25L methyl tert-ether, crystallizing, performing suction filtration, and vacuum drying the filter cake to obtain 1538g of Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -ONb, wherein the purity is more than 98.5%, and the yield is 93.7%.
Preparation of Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH: weighing H-Leu-Leu-Leu-Leu-Lys (Boc) -OH 1029g (1.5mol) and sodium carbonate 180g (1.7mol) and dissolving in 5L acetonitrile/water (1:1), slowly adding in ice water bath to obtain 5L tetrahydrofuran solution of Fmoc-Leu-Leu-Leu-Lys (Boc) -ONb 1515g (1.4mol), stirring for reaction, and monitoring the reaction by TLC. And filtering after complete reaction, adding 1N hydrochloric acid aqueous solution into the filtrate in an ice-water bath, adjusting the pH value of the solution to 2-3, adding 5L ethyl acetate for extraction for 2 times, merging organic phases, carrying out rotary evaporation and concentration to 4L, adding 30L methyl tert-ether into the concentrated solution for precipitation to obtain a solid product, filtering, drying, washing the methyl tert-ether, and carrying out vacuum drying to obtain 2059g of Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH, wherein the purity is higher than 95% and the yield is 91.8%.
Example 4, synthesis of fully protected peptide fragment 4:
Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH 1025g (640mmol) was weighed, dissolved with 8L dichloromethane with stirring, and 173g AlCl was weighed3(1.3mmol) and 97g N-methylmorpholine (960mmol) were added to the reaction solution and the reaction was stirred for 3 hours and monitored by TLC. The reaction solution is evaporated to 1.6L, added into 16L methyl tert-ether for precipitation, centrifuged to collect solid precipitate, and dried in vacuum to obtain H-Leu-Leu-Leu-Leu-Lys (Boc) -OH 820g, the purity is higher than 95%, and the yield is 92.8%.
Example 5, synthesis of fully protected peptide fragment 5:
preparation of Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -ONb: Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH 1025g (640mmol) and HONb126g (704mmol) were weighed and dissolved in 5L tetrahydrofuran, and stirred in an ice-water bath. DCC 158g (768mmol) was weighed, dissolved in 5L tetrahydrofuran, added dropwise slowly to the above solution, stirred and monitored by TLC. After the reaction is finished, suction filtration is carried out, the reaction solution is concentrated to 3L, 25L of methyl tert-ether is added into the concentrated solution, a large amount of white solid is separated out, and the solution is kept stand for 4h at the temperature of minus 20 ℃. Standing, vacuum-filtering, dissolving the solid with 3L ethyl acetate, adding 25L methyl tert-ether for precipitation, vacuum-filtering, and vacuum-drying to obtain Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -ONb 1033g with purity higher than 95% and yield 91.5%.
Preparation of Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH:
H-Leu-Leu-Leu-Leu-Lys (Boc) -Leu-Leu 820g (594mmol) and 75g (704mmol) of sodium carbonate were dissolved in 5L of water, and 5L of a tetrahydrofuran solution containing 5L of Fmoc-Leu-Leu-Leu-Lys (Boc) -Leu-Leu-Leu-Leu-Lys (Boc) -ONb 1033g (587mol) was added slowly in an ice-water bath, followed by stirring and TLC to monitor the reaction. And filtering after the reaction is completed, adding 1N hydrochloric acid aqueous solution into the filtrate in an ice-water bath, adjusting the pH value of the solution to 2-3, adding 5L ethyl acetate for extraction for 2 times, merging organic phases, carrying out rotary evaporation and concentration to 4L, adding 30L methyl tert-ether into the concentrated solution for precipitation to obtain a solid product, filtering, draining, washing the methyl tert-ether for 3 times, and carrying out vacuum drying to obtain Fmoc-Leu-Leu-Leu-Lys (Boc) -Leu-Leu-Leu-Leu-Leu-Lys (Boc) -OH 1613g, wherein the purity is more than 92 percent and the yield is 92.7 percent.
Example 6, synthesis of fully protected peptide fragment 6:
Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH 1613g (544mmol) was weighed, dissolved with stirring in 8L dichloromethane, and 146g AlCl was weighed3(1.1mmol) and 824g N-methylmorpholine (846mmol) were added to the reaction solution and the reaction was stirred for 3 hours and monitored by TLC. The reaction liquid is spirally steamed to 1.6L, added into 16L methyl tert-ether for precipitation, centrifuged to collect solid precipitate, and vacuum dried to obtain H-Leu-Leu-Leu-Leu-Lys (Boc) -OH 1373g, the purity is more than 90%, and the yield is 92%.
Example 7, synthesis of fully protected peptide fragment 7:
preparation of Boc-Lys (Boc) -ONb: Boc-Lys (Boc) -OH 208g (600mmol) and HONb118g (660mmol) were weighed accurately and dissolved in 3L tetrahydrofuran and stirred in an ice-water bath. DCC 148g (720mol) was weighed out accurately, dissolved in 2L tetrahydrofuran, added dropwise slowly to the above solution, stirred for reaction, and monitored by TLC. After the reaction is finished, suction filtration is carried out, the reaction solution is concentrated to 2L, 16L of methyl tert-ether is added into the concentrated solution, a large amount of white solid is separated out, and the solution is kept stand for 4h at the temperature of minus 20 ℃. Standing, performing suction filtration, dissolving the solid with 2L ethyl acetate, adding 15L methyl tertiary ether, crystallizing, performing suction filtration, and drying the solid in vacuum to obtain Boc-Lys (Boc) -ONb 287g with the purity of more than 99% and the yield of 94.6%.
Preparation of Boc-Lys (Boc) -Leu-Leu-Leu-Leu-Leu-Lys (Boc) -OH:
H-Leu-Leu-Leu-Leu-Lys (Boc) -OH 1373g (500mmol) and sodium carbonate 64g (600mmol) were dissolved in 5L acetonitrile/water (1:1), Boc-Lys (Boc) -ONb 253g (500mmol) in tetrahydrofuran solution 3L was added slowly in an ice-water bath, the reaction was stirred and TLC monitored. After the reaction is completed, filtering, adding 1N hydrochloric acid aqueous solution into the filtrate in ice-water bath, adjusting the pH value of the solution to 2-3, adding 5L ethyl acetate for extraction for 2 times, merging organic phases, carrying out rotary evaporation and concentration to 4L, adding 30L methyl tert-ether into the concentrated solution for precipitation to obtain a solid product, filtering and pumping, washing the methyl tert-ether for 3 times, and carrying out vacuum drying to obtain a solid product Boc-Lys (Boc) -Leu-Leu-Leu-Leu-Lys (Boc) -OH 1324g, wherein the purity is more than 90% and the yield is 91.7%.
Example 8 crude peptide cleavage
1324g of the crude fully protected sinapultide peptide from example 7 (430mmol) was charged to a 20L cleavage kettle and 13.3L of the formulated cleavage reagent (TFA: H)2O95: 5) was added to the above cleavage kettle, and the reaction was stirred at room temperature for 2 hours. After the reaction is finished, the lysate is filtered, poured into 133L of glacial methyl-tert-ether, a large amount of white precipitate is separated out, centrifuged, washed, dried and weighed, and the crude peptide 1041g of the sinapultide, the yield is 98.1%, the HPLC purity is 85.7%, and the MS 2470.1 is obtained.
Example 9 Synthesis of fully protected peptide 8
Preparation of Fmoc-Leu-OSu: Fmoc-Leu-OH 3534g (10mol) and HOSu 1266g (11mol) were weighed out and dissolved in 15L1, 4-dioxane, and stirred in an ice-water bath. DCC 2476g (12.0mol) was weighed, dissolved in 10L1, 4-dioxane, added dropwise slowly to the above solution, stirred and monitored by TLC. After the reaction is finished, suction filtration is carried out, the reaction solution is concentrated to 8L, then 50L of ether is added into the concentrated solution, a large amount of white solid is separated out, and the solution is kept stand for 4h at the temperature of minus 20 ℃. And (3) carrying out suction filtration, dissolving the solid with 5L ethyl acetate, adding 50L diethyl ether for crystallization, carrying out suction filtration, and drying the solid in vacuum to obtain Fmoc-Leu-OSu 4140g with the purity of more than 99% and the yield of 92.0%.
Preparation of Fmoc-Leu-Leu-OH: H-Leu-OH 1179g (9.0mol) and sodium carbonate 1091g (9.9mol) were weighed and dissolved in 10L of water, 10L of Fmoc-Leu-OSu 4050g (9.0mol) 1, 4-dioxane solution was slowly added under ice-water bath, and the reaction was stirred and TLC was used to monitor the end point of the reaction. And after the reaction is completed, filtering, adding 1N hydrochloric acid aqueous solution into the filtrate in an ice-water bath, adjusting the pH value of the solution to 2-3, adding 5L of ethyl acetate for extraction for 2 times, merging organic phases, carrying out rotary evaporation and concentration to 5L, adding 30L of diethyl ether into the concentrated solution, carrying out crystallization at 4 ℃ to obtain Fmoc-Leu-Leu-OH, filtering, drying, washing with diethyl ether, and carrying out vacuum drying to obtain 3837g of Fmoc-Leu-OH with the purity of more than 99% and the yield of 91.5%.
Preparation of Fmoc-Leu-Leu-OSu: Fmoc-Leu-Leu-OH 1864g (4.0mol) and HOSu 506g (4.4mol) were weighed out and dissolved in 8L1, 4-dioxane, and stirred in an ice-water bath. DCC 1094g (4.8mol) was weighed, dissolved in 5L1, 4-dioxane, added dropwise slowly to the above solution, stirred for reaction, and the end of the reaction was monitored by TLC. After the reaction is finished, carrying out suction filtration, concentrating the reaction solution to 4L, adding 25L of diethyl ether into the concentrated solution, separating out a large amount of white solid, and standing the solution for 1h at-20 ℃. Standing, performing suction filtration, dissolving the solid with 4L ethyl acetate, adding 20L diethyl ether, crystallizing, performing suction filtration, and vacuum drying the solid to obtain Fmoc-Leu-Leu-OSu2073g with the purity of more than 99% and the yield of 92.0%.
Preparation of H-Leu-Leu-OH 1864g (4.0mmol) of Fmoc-Leu-Leu-OH was weighed, dissolved in 20L of toluene with stirring, and 1066g of AlCl was weighed3(8.0mmol) was added to the reaction solution and stirred for 3 hours, and the end of the reaction was monitored by TLC. The mixture was acidified by addition of 1N aqueous HCl, adjusted to pH 2.0, and extracted 3 times with 4L portions of ether. Collecting the aqueous phase, adding saturated NaHCO3Neutralizing the water solution, and freeze-drying to obtain H-Leu-Leu-OH 899g with purity higher than 99% and yield 92%.
Fmoc-Leu-Leu-Leu-Leu-OH preparation: 855g (3.5mol) of H-Leu-Leu-OH and 408g (3.9 mol) of sodium carbonate are accurately weighed and dissolved in 5L of water, 5L of 1, 4-dioxane solution of 1971g (3.5mol) of Fmoc-Leu-Leu-OSu is slowly added in an ice water bath, the reaction is stirred, and the end point of the reaction is monitored by TLC. And after the reaction is completed, filtering, adding 1N hydrochloric acid aqueous solution into the filtrate in an ice water bath, adjusting the pH value of the solution to 2-3, adding 5L of ethyl acetate for extraction for 2 times, merging organic phases, carrying out rotary evaporation and concentration to 5L, adding the concentrated solution into 30L of diethyl ether, carrying out crystallization at 4 ℃ to obtain a solid product, filtering, washing with cold diethyl ether, and carrying out vacuum drying to obtain Fmoc-Leu-Leu-Leu-OH 2249g, wherein the purity is more than 99% and the yield is 92.8%.
Preparation of Fmoc-Leu-Leu-Leu-Leu-OSu: Fmoc-Leu-Leu-Leu-Leu-OH 2216g (3.2mol) and HOSu 405g (3.5mol) were weighed out and dissolved in 8L1, 4-dioxane, stirred in ice water bath. 783g (3.8mol) of DCC was weighed, dissolved in 5L1, 4-dioxane, added dropwise slowly to the above solution, stirred for reaction, and monitored by TLC. After the reaction is finished, carrying out suction filtration, concentrating the reaction solution to 4L, adding 30L of diethyl ether into the concentrated solution, separating out a large amount of white solid, and standing the solution for 4h at the temperature of minus 20 ℃. Standing, performing suction filtration, dissolving the solid with 4L ethyl acetate, adding 25L diethyl ether for crystallization, performing suction filtration, and vacuum drying the solid to obtain Fmoc-Leu-Leu-Leu-Leu-OSu2337g with the purity of more than 99% and the yield of 92.5%.
Preparation of Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH: H-Lys (Boc) -OH 739g (3.0mol) and sodium carbonate 350g (3.3 mol) were weighed and dissolved in 5L of water, and then 5L of 1, 4-dioxane solution of Fmoc-Leu-Leu-Leu-Leu-OSu2337g (3.0mol) was added slowly in ice-water bath, followed by stirring and TLC monitoring. And (3) filtering after complete reaction, adding 1N hydrochloric acid aqueous solution into the filtrate in an ice-water bath, adjusting the pH value of the solution to 2-3, adding 5L ethyl acetate for extraction for 2 times, merging organic phases, carrying out rotary evaporation and concentration to 4L, adding 25L diethyl ether into the concentrated solution, carrying out crystallization at 4 ℃ to obtain a solid product, filtering, drying, washing with diethyl ether, and carrying out vacuum drying to obtain Fmoc-Leu-Leu-Leu-Lys (Boc) -OH 2554g, wherein the purity is more than 99% and the yield is 92.5%.
Example 10 Synthesis of fully protected peptide 9
Preparation of H-Leu-Leu-Leu-Leu-Lys (Boc) -OH: Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH1243g (1.35mmol) was weighed, dissolved with 8L toluene with stirring, and 360g AlCl was weighed3(2.7mmol) was added to the reaction solution and stirred for 3 hours, and the reaction was monitored by TLC. Acidifying with 1N HCl aqueous solution, adjusting pH to 2.0, and extracting with diethyl etherTake 3 times, 2L each time. Collecting the aqueous phase, adding saturated NaHCO3Neutralizing the water solution, and freeze-drying to obtain H-Leu-Leu-Leu-Leu-Lys (Boc) -OH 868g with purity higher than 98.5% and yield 92%.
Example 11 Synthesis of fully protected peptide 10
Preparation of Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OSu: Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH1243g (1.35mmol) and HOSu 173g (1.5mol) were weighed out and dissolved in 4L1, 4-dioxane, stirred in an ice-water bath. DCC 334g (1.6mol) was weighed, dissolved in 4L1, 4-dioxane, added dropwise slowly to the above solution, stirred for reaction, and monitored by TLC. After the reaction is finished, carrying out suction filtration, concentrating the reaction solution to 3L, adding 20L of diethyl ether into the concentrated solution, separating out a large amount of white solid, and standing the solution for 4h at the temperature of minus 20 ℃. Standing, performing suction filtration, dissolving the solid with 3L ethyl acetate, adding 25L diethyl ether, crystallizing, performing suction filtration, and vacuum drying the filter cake to obtain Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OSu 1277g with the purity of more than 98.5% and the yield of 93.0%.
Preparation of Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH: H-Leu-Leu-Leu-Leu-Lys (Boc) -OH 838g (1.2mol) and sodium carbonate 138g (1.3mol) were dissolved in 5L acetonitrile/water (1:1), and 5L of 1, 4-dioxane solution of Fmoc-Leu-Leu-Leu-Lys (Boc) -OSu 1221g (1.2mol) was slowly added in an ice-water bath, followed by stirring and TLC monitoring. Filtering after complete reaction, adding 1N hydrochloric acid water solution into the filtrate in ice-water bath, adjusting the pH value of the solution to 2-3, adding 5L ethyl acetate for extraction for 2 times, merging organic phases, carrying out rotary evaporation and concentration to 4L, adding 30L diethyl ether into the concentrated solution, carrying out crystallization at 4 ℃ to obtain a solid product, filtering, drying, washing with diethyl ether, and carrying out vacuum drying to obtain Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH 1777g, wherein the purity is higher than 95% and the yield is 92.5%.
Example 12, synthesis of the fully protected peptide fragment 11:
Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH 880g (550mmol) was weighed, dissolved in 6L toluene with stirring, and 146g AlCl was weighed3(1.3mmol) was added to the reaction solution and stirred for 3 hours, and the reaction was monitored by TLC. Acidifying with 1N HCl aqueous solution, adjusting pH to 2.0, extracting with diethyl ether for 3 times (1 each time)And L. Collecting the aqueous phase, adding saturated NaHCO3Neutralizing the water solution, and freeze-drying to obtain H-Leu-Leu-Leu-Leu-Lys (Boc) -OH 701g with purity higher than 95% and yield 92.5%.
Example 13, synthesis of the fully protected peptide fragment 12:
preparation of Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OSu: Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH 880g (550mmol) and HOSu70g (605mmol) were weighed and dissolved in 5L1, 4-dioxane and stirred in an ice-water bath. DCC 136g (660mmol) was weighed, dissolved in 5L1, 4-dioxane, added dropwise slowly to the above solution, stirred for reaction, and monitored by TLC. After the reaction is finished, suction filtration is carried out, the reaction solution is concentrated to 3L, 25L of diethyl ether is added into the concentrated solution, a large amount of white solid is separated out, and the solution is kept stand for 4h at the temperature of minus 20 ℃. Standing, performing suction filtration, dissolving the solid with 3L ethyl acetate, adding 25L diethyl ether for precipitation, performing suction filtration, and performing vacuum drying on the solid to obtain 857g of Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OSu, wherein the purity is more than 95 percent, and the yield is 91.8 percent.
Preparation of Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH:
H-Leu-Leu-Leu-Leu-Lys (Boc) -OH 690g (506mmol) and sodium carbonate 65g (610mmol) were dissolved in 5L water, and 5L of 1, 4-dioxane solution of Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OSu 857g (506mol) was slowly added in an ice-water bath, followed by stirring and TLC monitoring. Filtering after the reaction is completed, adding 1N hydrochloric acid water solution into the filtrate in ice-water bath, adjusting the pH value of the solution to 2-3, adding 5L ethyl acetate for extraction for 2 times, merging organic phases, carrying out rotary evaporation and concentration to 4L, adding 30L diethyl ether into the concentrated solution for precipitation to obtain a solid product, filtering, pumping, washing with diethyl ether for 3 times, and carrying out vacuum drying to obtain Fmoc-Leu-Leu-Leu-Leu-Leu-Lys (Boc) -Leu-Leu-Leu-Leu-Leu-Lys (Boc) -OH 1376g, wherein the purity is more than 92 percent and the yield is 91..
Example 14, synthesis of fully protected peptide fragment 13:
weighing Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -Leu-Leu-Leu-Leu-Lys (Boc) -OH 1376g (464mmol) was dissolved in 8L toluene with stirring, and 123g AlCl was weighed3(928mmol) was added to the reaction solution and stirred for 3 hours, and the reaction was monitored by TLC. The mixture was acidified by addition of 1N aqueous HCl, adjusted to pH 2.0, and extracted 3 times with 2L portions of ether. Collecting the aqueous phase, adding saturated NaHCO3Neutralizing the water solution, and freeze-drying to obtain H-Leu-Leu-Leu-Leu-Lys (Boc) -OH 1170g with purity higher than 90% and yield 92.1%.
Example 15, synthesis of the fully protected peptide fragment 14:
preparation of Boc-Lys (Boc) -OSu: Boc-Lys (Boc) -OH 173g (500mmol) and HOSu63.3g (550mmol) were weighed out and dissolved in 3L1, 4-dioxane, and stirred in an ice-water bath. DCC 124g (600mol) was weighed out accurately, dissolved in 2L of 1, 4-dioxane, added dropwise slowly to the above solution, stirred for reaction, and monitored by TLC. After the reaction is finished, suction filtration is carried out, the reaction solution is concentrated to 2L, 16L of ether is added into the concentrated solution, a large amount of white solid is separated out, and the solution is kept stand for 4h at the temperature of minus 20 ℃. Standing, performing suction filtration, dissolving the solid with 2L ethyl acetate, adding 15L diethyl ether, crystallizing, performing suction filtration, and vacuum drying the solid to obtain Boc-Lys (Boc) -OSu 209g with the purity of more than 99% and the yield of 94.5%.
Preparation of Boc-Lys (Boc) -Leu-Leu-Leu-Leu-Leu-Lys (Boc) -OH:
H-Leu-Leu-Leu-Leu-Lys (Boc) -OH 1170g (426mmol) and 54g (511 mmol) of sodium carbonate were dissolved in 5L of acetonitrile/water (1:1), and 3L of a 1, 4-dioxane solution of Boc-Lys (Boc) -OSu 189g (426mmol) was slowly added in an ice-water bath, followed by stirring and TLC to monitor the reaction. After the reaction is completed, filtering, adding 1N hydrochloric acid water solution into the filtrate in ice-water bath, adjusting the pH value of the solution to 2-3, adding 5L ethyl acetate for extraction for 2 times, merging organic phases, carrying out rotary evaporation and concentration to 4L, adding 30L diethyl ether into the concentrated solution for precipitation to obtain a solid product, filtering, pumping, washing with diethyl ether for 3 times, carrying out vacuum drying to obtain a solid product, filtering, pumping, washing with diethyl ether for 3 times, and carrying out vacuum drying to obtain Boc-Lys (Boc) -Leu-Leu-Leu-Leu-Lys (Boc) -Leu-Leu-Leu-Lys (Boc-Leu-Leu-Lys) (Boc-Leu-Leu-Lys (Boc-Leu-Leu-.
Example 16 crude peptide cleavage
1324g of the crude fully protected sinapultide peptide from example 7 (430mmol) was charged to a 20L cleavage kettle and 13.3L of the formulated cleavage reagent (TFA: H)2O95: 5) was added to the above cleavage kettle, and the reaction was stirred at room temperature for 2 hours. After the reaction is finished, the lysate is filtered, poured into 133L of ethyl glacial ice ether, a large amount of white precipitate is separated out, centrifuged, washed, dried and weighed, and the crude sinapultide peptide 1041g, the yield is 98.1%, the HPLC purity is 85.7%, and MS 2470.1 are obtained.
Example 17 crude peptide cleavage
1199g of crude fully protected sinapultide peptide from example 15 (391mmol) was charged to a 20L cleavage kettle and 12.0L of the formulated cleavage reagent (TFA: H)2O-90: 10) was added to the above cleavage kettle, and the reaction was stirred at room temperature for 2 hours. After the reaction is finished, the lysate is filtered, poured into 120L of ethyl glacial ice ether, a large amount of white precipitate is separated out, centrifuged, washed, dried and weighed, 927g of crude sinapultide is obtained, the yield is 96.0%, the HPLC purity is 83.6%, and MS 2470.3 is obtained.
Example 18 preparation of purified sinapultide
1041g of crude sinapultide peptide from example 16 was purified using high performance liquid preparative chromatography, the crude peptide was dissolved with 10% glacial acetic acid + 10% acetonitrile + 80% water.
First-step purification: mobile phase A: 2% phosphoric acid, pH adjusted to 2.3 with sodium hydroxide, mobile phase B: pure acetonitrile, detection wavelength: 230nm, chromatographic packing: c8-10 μm, B phase elution gradient: 26-36% (60min), collecting qualified fraction, and performing second-step purification;
and a second step of purification: mobile phase A: 50mmpl/L ammonium bicarbonate, pH adjusted to 8.3 with glacial acetic acid, mobile phase B: 95% acetonitrile + 5% isopropanol, detection wavelength: 230nm, chromatographic packing: c8-10 μm, B phase elution gradient: 28 to 40 percent (60min), collecting qualified fractions,
the third step is salt conversion: the mobile phase A is: 0.1% acetic acid, mobile phase B: acetonitrile, detection wavelength: 230nm, chromatographic packing: isocratic elution with C8-10 μm 95% A + 5% B for 20min, gradient elution with B phase for 5% -50% (20min), concentration, and freeze drying to obtain sinapultide acetate 670g with HPLC purity of 99.4%, yield of 64.3%, and MS 2469.3.
Example 19 preparation of purified sinapultide
927g of crude sinapultide peptide from example 17 was purified using high performance liquid preparative chromatography, the crude peptide was dissolved with 10% glacial acetic acid + 10% acetonitrile + 80% water.
First-step purification: mobile phase A: 2% phosphoric acid, pH adjusted to 2.3 with sodium hydroxide, mobile phase B: pure acetonitrile, detection wavelength: 230nm, chromatographic packing: c8-10 μm, B phase elution gradient: 26-36% (60min), collecting qualified fraction, and performing second-step purification;
and a second step of purification: mobile phase A: 50mmpl/L ammonium bicarbonate, pH adjusted to 8.3 with glacial acetic acid, mobile phase B: 95% acetonitrile + 5% isopropanol, detection wavelength: 230nm, chromatographic packing: c8-10 μm, B phase elution gradient: 28 to 40 percent (60min), collecting qualified fractions,
the third step is salt conversion: the mobile phase A is: 0.1% acetic acid, mobile phase B: acetonitrile, detection wavelength: 230nm, chromatographic packing: isocratic elution with C8-10 μm 95% A + 5% B for 20min, gradient elution with B phase for 5% -50% (20min), concentration, and freeze drying to obtain refined peptide 582g of sinapultide with HPLC purity of 99.0%, yield of 60.3%, and MS 2469.2.
Example 20 (comparative experiment 1) solid phase Synthesis of Sinapsin
1000g of Wang resin (500mmol) with a substitution of 0.5mmol/g was used as a starting resin, washed twice with 7.0L of DMF and swollen with 7.0L of DMF for 30 minutes, 937g of Fmoc-Lys (Boc) -OH (2mol), 64.8g of HOBt (2.4mmol) and 9.0g of DMAP (200mmol) were weighed out, dissolved in 5L of DMF, charged into a reaction column, 60.4g of DIC (2.4mol) were added, and the reaction was stirred at room temperature for 3 hours. Washed 6 times with 5L of DMF and blocked for 6 hours by adding 5L of acetic anhydride/pyridine (1:1) solution. The blocking solution was pipetted and washed 3 times with 5L DCM and 3 times with 5L DMF.
Fmoc was removed twice 10 minutes each time by adding 7L of 20% piperidine/DMF (V/V) solution and the resin was washed 6 times with 5L DMF after removal. 532.5g of Fmoc-Leu-OH (1.5mmol) and 243g of HOBt (1.8mol) were weighed, dissolved in 2.5L of DMF, activated for 5 minutes by adding 226g of DIC (1.8mol) in an ice-water bath, and the mixture was charged into a reaction column and reacted at room temperature for 2 hours. The resin was washed 3 times with 5L DMF, Fmoc was removed twice for 10 minutes each time by adding 5L of 20% piperidine/DMF (V/V) solution, and the resin was washed 6 times with 5L DMF after removal.
Subsequent Fmoc-Leu-OH (532.5g), Fmoc-Lys (Boc) -OH (702.8g), Fmoc-Leu-OH (532.5g), Fmoc-Lys (Boc) -OH (702.8g), Fmoc-Leu-OH (532.5g), Fmoc-Lys (Boc) -OH (702.8g), Fmoc-Leu-OH (532.5g), Fmoc-Leu-OH (532.5g), Fmoc-Lys (Boc) -OH (702.8g), all amino acids at 1.5mmol, reaction time 2 h, 7L DMF washes 3 times. Fmoc was removed twice for 10 min by addition of 7L of 20% piperidine/DMF (V/V) solution and the resin was washed 6 times with 7L DMF after removal. After the peptide resin was coupled, it was shrunk 3 times with 7L of methanol for 10 minutes each. Then, vacuum drying was carried out to obtain 2180g of a peptide resin.
2180g of the above-mentioned sinapultide peptide resin (500mmol) was put into a 30L cleavage vessel, and 21.8L of a prepared cleavage reagent (TFA: H)2O95: 5) was added to the above cleavage kettle, and reacted at room temperature for 2 hours. After the reaction is finished, the lysate is filtered, poured into 218L of ethyl glacial ether, a large amount of white precipitate is separated out, centrifuged, washed, dried and weighed, and the crude peptide 1112g of the sinapultide is obtained with the yield of 90.1 percent, the HPLC purity of 70.7 percent and MS 2649.8.
And purifying 1112g of the crude sinapultide by using a high performance liquid chromatography, concentrating and freeze-drying to obtain 565g of fine sinapultide, wherein the HPLC purity is 99.1%, the yield is 50.8% and the MS2469.7 is obtained.
Example 21 (comparative experiment 2) Synthesis of Sinapsin by solid-liquid combination
Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH 2300g (2.5mol) was synthesized by the method of example 1, using 400g of 2CTC resin (200mmol) with substitution degree of 0.5mmol/g as starting resin, washed twice with 3.0L DMF, swollen with 3.0L DMF for 30 min, weighing Fmoc-Leu-Leu-Leu-Lys (Boc) -OH 552g (600mmol), dissolved in 1.5L DMF, charged to the reaction column, 116g of DIPEA (900mmol) was added, reacted at room temperature with stirring for 3 h, and blocked with 128g of methanol for 30 min. The reaction solution was removed by suction and washed 6 times with 2.0L of DMF. Fmoc was removed twice 10 minutes each time by adding 2.0L of 20% piperidine/DMF (V/V) solution and the resin was washed 6 times with 2.0L DMF after removal.
Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH 552g (600mmol) and 97g HOBt (720mmol) were weighed, dissolved in 1.5L DMF, activated for 5 minutes by adding 91g DIC (720mmol) in an ice-water bath, and the mixture was put into a reaction column and reacted at room temperature for 2 hours. Washing with 2.0L DMF 3 times, adding 2.0L 20% piperidine/DMF (V/V) solution to remove Fmoc twice for 10 min each time, and washing the resin 6 times with 2.0L DMF after removal.
The same procedure was used to recouple Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH 552g (600mmol), Fmoc-Leu-Leu-Leu-Lys (Boc) -OH 552g (600mmol) and Fmoc-Lys (Boc) -OH 281g (600 mmol). The reaction time was 2 hours and 2.0L DMF was washed 3 times. Fmoc was removed twice 10 minutes each time by adding 2.0L of 20% piperidine/DMF (V/V) solution and the resin was washed 6 times with 2.0L DMF after removal. After the peptide resin was coupled, it was shrunk 3 times with 2.0L of methanol for 10 minutes each. Then, vacuum drying was carried out to obtain 992g of a peptide resin. In the coupling process, 5 peptide fragments are used for coupling,
992g of the above-mentioned sinapultide peptide resin (200mmol) was charged into a 20L cleavage vessel, and 9.92L of the prepared cleavage reagent (TFA: H)2O95: 5) was added to the above cleavage kettle, and reacted at room temperature for 2 hours. After the reaction is finished, the lysate is filtered, poured into 10L of ethyl glacial ether, a large amount of white precipitate is separated out, centrifuged, washed, dried and weighed to obtain 464g of crude sinapultide, the yield is 94.1%, the HPLC purity is 77.6%, and MS 2469.3 is obtained.
And (3) purifying 1112g of the crude sinapultide peptide by using a high performance liquid chromatography, concentrating and freeze-drying to obtain 280g of fine sinapultide peptide, wherein the HPLC purity is 99.0%, the yield is 56.8%, and the MS2649.7 is obtained.

Claims (8)

1. A liquid phase preparation method of sinapultide, which comprises the following steps:
1) performing carboxyl activation by Fmoc-Leu-OH, and coupling with H-Leu-OH to obtain Fmoc-Leu-Leu-OH;
2) removing the Fmoc group of the Fmoc-Leu-Leu-OH to obtain H-Leu-Leu-OH;
3) performing carboxyl activation by Fmoc-Leu-Leu-OH, and coupling with H-Leu-Leu-OH to obtain Fmoc-Leu-Leu-Leu-Leu-OH;
4) performing carboxyl activation by Fmoc-Leu-Leu-Leu-Leu-OH, and coupling with H-Lys (Boc) -OH to obtain Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH;
5) removing Fmoc group of Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH to obtain H-Leu-Leu-Leu-Leu-Lys (Boc) -OH;
6) performing carboxyl activation by Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH, and coupling with H-Leu-Leu-Leu-Lys (Boc) -OH to obtain Fmoc-Leu-Leu-Leu-Leu-Lys (Boc) -OH;
7) removing Fmoc group of Fmoc-Leu-Leu-Leu-Lys (Boc) -OH to obtain H-Leu-Leu-Leu-Leu-Lys (Boc) -OH;
8) performing carboxyl activation by Fmoc-Leu-Leu-Leu-Lys (Boc) -Leu-Leu-Leu-Leu-Lys (Boc) -OH, and coupling with H-Leu-Leu-Leu-Leu-Lys (Boc) -OH to obtain Fmoc-Leu-Leu-Leu-Leu-Leu-Lys (Boc) -Leu-Leu-Leu-Leu-Lys (Boc) -OH;
9) removing Fmoc group of Fmoc-Leu-Leu-Leu-Lys (Boc) -Leu-Leu-Leu-Leu-Lys (Boc) -OH to obtain H-Leu-Leu-Leu-Leu-Leu-Lys (Boc);
10) performing carboxyl activation with Boc-Lys (Boc) and coupling with H-Leu-Leu-Leu-Leu-Lys (Boc) -OH to give Boc-Lys (Boc) -Leu-Leu-Leu-Leu-Leu-Lys (Boc) -OH;
11) cleaving Boc-Lys (Boc) -Leu-Leu-Leu-Leu-Lys (Boc) -Boc protecting group of OH to obtain H-Lys-Leu-Leu-Leu-Lys-Leu-Leu-Leu-Leu-Leu-Lys-OH.
2. The liquid phase preparation method of claim 1, wherein the Fmoc group removing reagent used for removing Fmoc groups is a Lewis acid selected from AlCl3、BF3、AlCl3And N-methylmorpholine, AlCl3Composition of toluene and BF3And toluene.
3. The liquid phase preparation method according to any one of claims 1 to 2, wherein the molar ratio of the Fmoc group removing reagent used for removing the Fmoc group to the Fmoc group compound to be removed is 1:1-3 (preferably 1:2) to the Fmoc group compound to be removed, AlCl3, and 1:1-3:1-2 (preferably 1:2:1.5) to the Fmoc group compound to be removed, AlCl3, N-methylmorpholine.
4. The liquid-phase preparation method of any one of claims 1 to 3, wherein the Fmoc group is removed by adding AlCl to an organic solution of the substrate from which the Fmoc group is to be removed3Adding an acidic aqueous solution for acidification after the reaction is completed, adjusting the pH value to 1-3, adding diethyl ether for extraction to remove impurities, taking a water phase, and adjusting the water phase to be neutral to obtain a compound with the Fmoc group removed; or
Adding AlCl into the organic solution of the substrate to be removed with Fmoc group3And N-methylmorpholine until the reaction is completed, precipitating in methyl tertiary ether, and collecting the precipitate to obtain the compound with the Fmoc group removed.
5. The liquid phase preparation process according to any one of claims 1 to 4, wherein the carboxyl group activation is a treatment of activating the compound to be activated by a carboxyl group activating agent selected from one or more of HOSu, DCC, HONb, EDC. HCl, preferably a combination of HOSu and DCC, or a combination of HONb and DCC.
6. The liquid-phase preparation process according to any one of claims 1 to 5, wherein the ratio of the compound to be activated by carboxyl groups to the carboxyl group activating agent is from 1:1 to 1.5.
7. The liquid-phase preparation process according to any one of claims 1 to 6, wherein the carboxyl-activated intermediate is isolated and purified and then coupled, the carboxyl-activated intermediate is isolated and purified to complete the reaction between the carboxyl-activating agent and the compound to be activated, the filtrate is concentrated after filtration, and the carboxyl-activated intermediate is obtained by crystallization from ethyl acetate and diethyl ether, or from ethyl acetate and methyl tert-ether.
8. The liquid phase preparation process according to any one of claims 1 to 7, wherein the cleavage is carried out by treating with a cleavage reagent to obtain a product for removing the Boc protecting group, wherein the cleavage reagent is an aqueous solution containing trifluoroacetic acid, and the concentration of trifluoroacetic acid in the aqueous trifluoroacetic acid solution is 80% to 99% or more, preferably 90% to 95% or more.
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