CN110684077B - Large-scale synthesis method of achirelin - Google Patents

Large-scale synthesis method of achirelin Download PDF

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CN110684077B
CN110684077B CN201910991436.4A CN201910991436A CN110684077B CN 110684077 B CN110684077 B CN 110684077B CN 201910991436 A CN201910991436 A CN 201910991436A CN 110684077 B CN110684077 B CN 110684077B
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arg
glu
pbf
gln
met
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CN110684077A (en
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李晨
梁强
王昕�
张忠旗
陈科
杨小琳
赵金礼
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Shaanxi HuiKang Bio Tech Co Ltd
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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Abstract

The invention discloses a large-scale synthesis method of Archimedes, which adopts 2-Chlorotrityl Chloride Resin as a carrier, synthesizes a full-protection fragment peptide acid in a solid phase, takes the full-protection peptide acid fragment as a raw material, carries out liquid-phase amidation reaction at a carboxyl terminal to obtain full-protection amide, and obtains a target peptide by deprotection. Compared with the commonly used amino resin in the prior synthesis method, the resin carrier adopted by the invention has low price, and the loading amount is increased by nearly one time; the selected ammonia reagent for the amidation reaction is cheap and easy to obtain, the amidation reaction time is short, and the hydrolysis of the ester is inhibited. The anhydrous ether is discarded in the post-cutting treatment process, so that the safety is high, and the environment is protected. The method overcomes the problems of high cost and low loading capacity of the traditional classical solid-phase synthetic resin, overcomes the defects of complex liquid-phase synthetic process, long synthetic period and low efficiency, has the advantages of low synthetic cost, high synthetic efficiency and simple synthetic method, and can be used for large-scale synthesis of the achirelin.

Description

Large-scale synthesis method of achirelin
Technical Field
The invention belongs to the technical field of polypeptide synthesis, and particularly relates to a solid-liquid combined archipellin synthesis method.
Background
The archirelin-Argireline is an active polypeptide with the wrinkle-removing function, and the sequence is Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2The botulinum toxin is a safe substitute of botulinum toxin, and can block information conduction between nerve muscles, avoid muscle over-contraction, rearrange subcutaneous newly synthesized collagen, and relieve or even eliminate wrinkles. Therefore, it is often used as an anti-wrinkle component in cosmetics.
At present, the synthetic methods of the ayorelin mainly comprise a solid-phase synthetic method and a liquid-phase synthetic method. The several methods for synthesizing the archipelene disclosed at present have different defects. For example, in the preparation method described in patent document CN 103694316a, Rink Amide-MBHA Resin is used as a Resin carrier for solid phase synthesis, the substitution degree is only 0.5mmol/g, and the method is expensive, inevitably causes high cost, and is not suitable for large-scale production.
The preparation method of patent document with publication number CN 106632609A is characterized by that it uses the preferential liquid phase synthesis of Fmoc-Arg (Pbf) -OH fragment, then connects it with Rink Amide-AM Resin, and makes them be successively assembled and synthesized, and said combination mode is connected with solid phase in turn, and has no difference, and the solid phase carrier used is expensive, and the arginine dipeptide fragment is used as difficult sequence, and the liquid phase synthesis is difficult and can not ensure purity, and the solid phase assembly can bring trouble to purification, and is not suitable for large-scale synthesis.
The synthesis method disclosed in publication No. CN 103613642a adopts liquid phase stepwise synthesis, which reduces the cost of raw materials, but has long synthesis period, low efficiency, high purification cost, and is not suitable for large-scale synthesis.
The synthesis method described in publication No. CN 102603869a requires the preparation of an amino resin, but ammonia gas as a polar molecule has low solubility in a nonpolar solvent, dichloromethane, and thus it is difficult to prepare an amino resin, and the loading amount is low, which is not suitable for large-scale synthesis.
The synthesis method disclosed in publication No. CN 196749529A is characterized in that benzotriazole-1-yl-oxypyrrolidinophosphonium hexafluorophosphate and 1-hydroxyphenyltriazole are selected to synthesize the activated ester, ammonium chloride is selected as an ammonia reagent, but the ammonium chloride is selected as an inorganic salt, and the solubility of the ammonium chloride in an organic solvent N, N-dimethylformamide is very low, so that the conversion rate of amidation reaction is not high, raw materials are remained, the synthesis yield is low, the purity cannot be guaranteed, and the large-scale synthesis is not facilitated.
Disclosure of Invention
The invention aims to solve the technical problems of high carrier cost and low loading capacity of the traditional classical solid-phase synthesis method and the defects of long synthesis period and low efficiency of the liquid-phase synthesis method in the prior art, and provides the synthetic method of the archipellin, which has the advantages of simple process method, low production cost, easy purification and high efficiency.
Specifically, the invention provides a large-scale synthesis method of the argireline, which adopts 2-Chlorotrityl Chloride Resin as a carrier, synthesizes a full-protection fragment peptide acid in a solid phase manner, takes the full-protection peptide acid fragment as a raw material, carries out liquid-phase amidation reaction at a carboxyl terminal to obtain full-protection amide, and obtains the target argireline by purifying and deprotecting the full-protection amide.
In order to solve the technical problem, the invention adopts the synthesis of Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2Dissolving Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -OH in N, N-dimethylformamide, adding N-hydroxysuccinimide and N, N' -diisopropylcarbodiimide at low temperature, keeping low temperature for reaction, adding an ammonia reagent, naturally heating for reaction, precipitating precipitates after the reaction is finished, filtering, washing the precipitates, and drying at low temperature to obtain Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Pbf- (Pbf) -NH2。
More preferably, in the scaled synthesis of ayrelin, Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH is synthesized2Dissolving Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -OH in N, N-dimethylformamide, adding N-hydroxysuccinimide and N, N' -diisopropylcarbodiimide at 0-5 ℃, keeping the temperature at 0-5 ℃, and reacting 6Adding an ammonia reagent for 8 hours, naturally heating for reaction for 2-4 hours, adding saturated saline water with 2 times volume of the reaction solution, separating out precipitate, filtering, washing the precipitate for 3 times by pure water, and drying at 40-45 ℃ to obtain Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2。
More specifically, the technical solution adopted to solve the above technical problems consists of the following steps;
(1) synthesis of Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin
Synthesizing Fmoc-Arg (Pbf) -2-Chlorotrityl Chloride Resin
Swelling 2-Chlorotrityl Chloride Resin with dichloromethane for 10 minutes, carrying out suction filtration on the solvent, adding dichloromethane, Fmoc-Arg (Pbf) -OH and N, N' -diisopropylethylamine, carrying out stirring reaction at normal temperature for 2 hours under the protection of nitrogen, carrying out blocking for 0.5 hour with anhydrous methanol, carrying out suction filtration, and washing with isopropanol and N, N-dimethylformamide sequentially to obtain Fmoc-Arg (Pbf) -2-Chlorotrityl Chloride Resin.
② Fmoc-Arg (Pbf) -2-Chlorotrityl Chloride Resin
Fmoc-Arg (Pbf) -2-Chlorotrityl Chloride Resin is subjected to Fmoc protection group removal by using an N, N-dimethylformamide solution of piperidine with the volume concentration of 20% for 40 minutes, suction filtration is carried out, a filter cake is washed by isopropanol and N, N-dimethylformamide in sequence, ninhydrin detection is positive, N-dimethylformamide, Fmoc-Arg (Pbf) -OH, 1-hydroxy phenylpropyl triazole and N, N' -diisopropyl carbodiimide are added, stirring reaction is carried out for 2 hours at normal temperature under the protection of nitrogen, ninhydrin detection is negative, suction filtration is carried out, and the filter cake is washed by isopropanol and N, N-dimethylformamide in sequence to obtain Fmoc-Arg (Pbf) -2-Chlorotrityl Chloride Resin.
③ Synthesis of Fmoc-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin
Fmoc-Gln (Trt) -OH, Fmoc-Met-OH, Fmoc-Glu (OtBu) -OH and Fmoc-Glu (OtBu) -OH are sequentially connected to Fmoc-Arg (Pbf) -2-Chlorotrityl Chloride Resin according to the method of the step (II) to obtain Fmoc-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin.
Synthesizing Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin
Removing Fmoc protecting groups from Fmoc-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl-Chloride Resin by using an N, N-dimethylformamide solution of piperidine with the volume concentration of 20% for 40 minutes, performing suction filtration, washing filter cakes by using isopropanol and N, N-dimethylformamide sequentially, detecting the ninhydrin to be positive, adding a mixed solution of dichloromethane, acetic anhydride and N, N-diisopropylethylamine in a volume ratio of 9:1:0.5, stirring for 30 minutes at normal temperature under the protection of nitrogen, and detecting the ninhydrin to be negative. Filtering, washing the filter cake with isopropanol, N-dimethylformamide and anhydrous methanol in sequence, and drying to obtain Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin.
(2) Synthesis of Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -OH
Adding a cutting fluid into Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin, cracking Resin, wherein the cutting fluid of the cracking Resin is a mixed solution of trifluoroethanol, acetic acid and dichloromethane in a volume ratio of 2:0.75:7.25, stirring and reacting for 2 hours, filtering, washing a filter cake, washing the combined filtrate to pH =6 by using a saturated sodium bicarbonate solution, adjusting the pH =7 by using a saturated saline solution, combining all water phases and the filtrate, removing an organic solvent under reduced pressure, precipitating, filtering, washing the filter cake by using pure water, and drying at 40 ℃ to obtain Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -OH.
(3) Synthesis of Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2
Dissolving Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -OH in N, N-dimethylformamide, adding N-hydroxysuccinimide and N, N' -diisopropylcarbodiimide at 0-5 ℃, reacting for 6-8 hours under the condition of keeping at 0-5 ℃, adding an ammonia reagent, naturally heating for reacting for 2-4 hours, adding 2 times of volume of saturated solution into the reaction solutionAnd adding saline water, precipitating, filtering, washing the precipitate with pure water for 3 times, and drying at 40-45 ℃ to obtain Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2. The reaction temperature is required to be 0-5 ℃, at the temperature, although a small part of raw materials are not completely reacted, racemization can be greatly inhibited, and a racemization product is inevitably generated by the normal-temperature reaction, which is the key point of the invention. The added ammonia reagent can be one of ammonia gas, ammonia solution and ammonia water, and the ammonia reagent must form a mutual soluble system with the selected reaction solvent, otherwise, the reaction efficiency is low. The cost is comprehensively considered, the ammonia water is cheap and easy to obtain, and the ammonia water is preferably used as the ammonia reagent.
The obtained Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2Dissolving in dichloromethane, adding a little methanol, carrying out column chromatography by using 100-200 mesh silica gel, eluting with a dichloromethane/methanol system at an elution solvent ratio of 20:1, wherein the raw material cannot be washed out under the polarity, and the most optimal elution ratio is obtained, wherein a small amount of large-polarity raw material points can be eluted by using methanol and repeatedly used, and the eluent is concentrated to obtain Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2And (5) purifying.
(4) Synthesis of Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2
To Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2And adding cutting fluid into the pure product to remove side chain groups, wherein the side chain group removing cutting fluid is mixed solution of trifluoroacetic acid, triisopropyl silane, benzylsulfide and water in a volume ratio of 88:5:4: 3. Stirring for reaction for 2 hours, removing the solvent under reduced pressure, adding cold ethyl acetate into the concentrated solution, separating out a precipitate, and washing with petroleum ether to obtain a crude product of the target peptide. Purifying the crude target peptide product by reverse phase chromatography, and freeze-drying to obtain a pure product of ayorelin, namely Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2。
The invention adopts 2-Chlorotrityl Chloride Resin as a carrier, synthesizes a full-protection fragment peptide acid in a solid phase, takes the full-protection peptide acid fragment as a raw material, carries out liquid-phase amidation reaction at a carboxyl terminal to obtain full-protection amide, and obtains target achirelin by deprotection, and the invention has the following beneficial effects:
(1) the substitution degree of the 2-Chlorotrityl Chloride Resin selected by the invention is 1.6mmol/g, the price is 7000 yuan/kg, the Rink Amide-AM Resin used in the classical synthesis has the substitution degree of 0.9mmol, the price is 15000 yuan/kg, and the selected 2-Chlorotrityl Chloride Resin has the advantages of relatively low price, nearly doubled uploading amount, increased Resin peptide yield and increased total target peptide yield under the same feeding amount.
(2) In the present invention, Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -OH is used to synthesize Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH)2When the method is used, the reaction temperature is 0-5 ℃, the temperature condition is the only necessary condition for inhibiting racemization in the liquid-phase amidation reaction, and the difficulty in preparation and purification is reduced; the selected ammonia reagent is ammonia water which is cheap and easy to obtain, and is mutually soluble with N, N-dimethylformamide, so that the reaction time is shortened, the hydrolysis of ester is inhibited, and the method is suitable for industrial production.
(3) In the present invention, Ac-Glu-Glu-Met-Gln-Arg-Arg-NH is synthesized2The side chain is removed to protect the cutting fluid, the classical components of phenol and dithioglycol are abandoned, and the original dithioglycol has foul smell and is not friendly to the environment; after cutting, the cutting fluid is concentrated, the classical precipitation agent, namely the easily prepared toxic solvent, namely the anhydrous ether is abandoned, and the ethyl acetate is used instead, so that the physical discomfort caused by mistaken ether inhalation in the operation process is avoided. The precipitant can be concentrated and recycled after being added with sodium bicarbonate to neutralize acid, thus being beneficial to industrial production.
(4) After column chromatography, the purity of the crude product of the archipellin obtained by deprotection is more than 80 percent and is higher than that of the crude product synthesized by a classical solid phase.
Drawings
FIG. 1 is a mass spectrum of the synthetic Archimedes according to example 6.
FIG. 2 is a HPLC analysis chart of the synthetic ayorelin of example 6.
Detailed Description
The invention will be described in more detail below with reference to exemplary embodiments and the drawings, but the scope of the invention is not limited to these exemplary embodiments.
The following description is made for brevity in the specification
2-Chlorotrityl Chloride Resin, which is named as 2-Chlorotrityl Chloride Resin and has a degree of substitution of 1.6 mmol/g;
OtBu: a tert-butoxy group; trt: a trityl group;
pbf 2,2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl
The sources of the respective substances used in the examples are described below, and if not otherwise specified, the raw materials and instruments used are commercially available, and the instruments and raw materials conventionally used in the art are used as long as they meet the experimental requirements
The substitution degree of the 2-chlorotrityl chloride resin is 1.6mmol/g, and the resin is commercially available from Tianjin Nankai and science and technology Limited.
The analytical high performance liquid chromatograph is Hitachi full-automatic L2000.
The preparative high performance liquid chromatograph is Innovative Hengtong LC3000, the C18 analytical chromatographic column is Dalian physical chemistry research institute 4.6mm multiplied by 250mm, and the C18 preparative chromatographic column is Chengdu science popularization biology limited company 14cm multiplied by 75 cm.
The amino acids, 1-hydroxy phenylpropyl triazole, N '-diisopropyl carbodiimide, N' -diisopropyl ethylamine and N-hydroxy succinimide were purchased from Shanghai Jier Biochemical Co., Ltd.
The mass spectrometer was a Waters UPLC-Mass spectrometer.
All the organic solvents and organic reagents used in the experiment are domestic AR or CP.
Example 1 Large-scale synthesis method of Archerelin
The large-scale synthesis method of the achillea comprises the following steps:
(1) synthesizing Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin;
(2) synthesizing Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -OH;
(3) synthesis of Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2；
(4) Synthesis of Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2。
Example 2 Synthesis of Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin
On the basis of the technical solution provided in the first embodiment, the present embodiment further relates to the synthesis of Ac-glu (otbu) -Met-gln (trt) -arg (pbf) -2-Chlorotrityl Chloride Resin, and the specific synthesis steps are as follows:
(1) synthesis of Fmoc-Arg (Pbf) -2-Chlorotrityl Chloride Resin
Adding 100.0g of 2-Chlorotrityl Chloride Resin into a synthesizer, adding 1000mL of dichloromethane for soaking, swelling for 15 minutes, performing suction filtration, adding 1000mL of dichloromethane, 114.2g (0.176 mol) of Fmoc-Arg (Pbf) -OH and 105mL (0.64 mol) of N, N' -diisopropylethylamine into the synthesizer, stirring and reacting for 2 hours at normal temperature under the protection of nitrogen, blocking with anhydrous methanol for 0.5 hour, performing suction filtration, washing twice with isopropanol (800 mL each time) and twice with N, N-dimethylformamide (800 mL each time) in sequence, and obtaining Fmoc-Arg (Pbf) -2-Chlorotrityl Chloride Resin.
(2) Synthesis of Fmoc-Arg (Pbf) -2-Chlorotrityl Chloride Resin
Fmoc-Arg (Pbf) -2-Chlorotrityl Chloride Resin in a synthesizer is used for removing Fmoc protecting groups by 1L of N, N-dimethylformamide solution of piperidine with the volume concentration of 20 percent for 40 minutes, mixed solution is removed by suction filtration, filter cakes are washed twice by isopropanol in sequence, 800ml and N, N-dimethylformamide are used for washing twice (800 ml for each time), ninhydrin detects positive, and Resin shows black. 1L N, N-dimethylformamide, 207.6g (0.32 mol) of Fmoc-Arg (Pbf) -OH, 64.9 g (0.48 mol) of 1-hydroxyphenyltriazole and 74.4ml (0.48 mol) of N, N' -diisopropylcarbodiimide are added into a synthesizer, the mixture is stirred and reacted for 2 hours at normal temperature under the protection of nitrogen, ninhydrin detection is negative, the Resin is colorless and transparent, suction filtration is carried out, and a filter cake is washed by isopropanol and N, N-dimethylformamide sequentially and 800ml each time, so that Fmoc-Arg (Pbf) -2-Chlorotrityl Chloride Resin is obtained.
(3) Synthesis of Fmoc-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin
According to the method of step (2), 195.4 g (0.32 mol) of Fmoc-Gln (Trt) -OH, 118.8 g (0.32 mol) of Fmoc-Met-OH, 136.2g (0.32 mol) of Fmoc-Glu (OtBu) -OH, and the other reagents were used in the amounts of: 64.9 g (0.48 mol) of 1-hydroxyphenyltriazole, 74.4mL (0.48 mol) of N, N' -diisopropylcarbodiimide, and washing the Resin with isopropanol and N, N-dimethylformamide each 2 times, 800mL each time, to give Fmoc-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin.
(4) Synthesis of Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin
Fmoc-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl-Chloride Resin in a synthesizer is subjected to Fmoc protection group removal for 40 minutes by using 1L of N, N-dimethylformamide solution of piperidine with the volume concentration of 20 percent, mixed liquid is removed by suction filtration, filter cakes are washed twice by isopropanol and N, N-dimethylformamide sequentially, 800ml of the mixed liquid is obtained each time, ninhydrin is used for positive detection, and the Resin is black. Adding a mixed solution of 900ml of dichloromethane, 100ml of acetic anhydride and 50ml of N, N' -diisopropylethylamine into a synthesizer, stirring for 30 minutes at normal temperature under the protection of nitrogen, detecting negative ninhydrin, performing colorless and transparent Resin, performing suction filtration, washing a filter cake twice with isopropanol and N, N-dimethylformamide sequentially, 800ml of anhydrous methanol and three times of 700ml each time, performing suction filtration, and drying to obtain Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin.
Example 3: synthesis of Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -OH
On the basis of the technical solution provided in the first embodiment, the present embodiment further relates to the synthesis of Ac-glu (otbu) -Met-gln (trt) -arg (pbf) -OH, and the specific synthesis steps are as follows:
adding dried Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin into a 2L beaker, adding 200ml of trifluoroethanol, 75ml of acetic acid and 725ml of dichloromethane cutting fluid, cracking the resin, stirring and reacting for 2 hours, filtering, washing the filter cake with dichloromethane three times, each time 100ml, combining filtrates, washing the filtrate twice with saturated sodium bicarbonate solution, each time 700ml with saturated sodium bicarbonate solution to pH =6, adjusting pH =7 with saturated saline solution 800ml, combining aqueous phase and filtrate, transferring into eggplant-shaped bottle, adding saturated saline solution 500ml, and decompressing to remove the organic solvent, precipitating, filtering, washing filter cake twice with pure water, drying at 40 ℃ to obtain 221.2g of Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -OH.
Example 4: synthesis of Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2
On the basis of the technical solution provided in the first embodiment, the first embodiment further encompasses Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2The specific synthesis steps are as follows:
(1) 221.2g (0.127 mol) of Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -OH was transferred to a 5L reaction flask, dissolved in 1800ml of N, N-dimethylformamide, adding 29.1g (0.253 mol) of N-hydroxysuccinimide and 39.2ml (0.253 mol) of N, N' -diisopropylcarbodiimide at 0-5 deg.C, reacting for 6-8 hours while maintaining the temperature at 0-5 deg.C, TLC monitoring reaction is basically completed, 120ml of ammonia water is added, the reaction is naturally heated for 2 to 4 hours, adding 2 times volume of saturated saline solution into the reaction solution, precipitating, centrifuging, filtering, washing the precipitate with pure water for 3 times, and drying at 40-45 ℃ to obtain 210.4g of Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH.2The yield thereof was found to be 95.5%.
(2) 210.2g of Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH obtained2Dissolving in 150ml dichloromethane, adding a little methanol, carrying out column chromatography by using 100-200 mesh silica gel, eluting with a dichloromethane/methanol system with an elution solvent ratio of 20:1, and concentrating the eluent to obtain 190.5g Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH)2And (5) purifying.
Example 5: synthesis of Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2
Based on the technical scheme provided in the first embodiment, the first embodiment further encompasses Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2The specific synthesis steps are as follows:
(1) to 190.5gAc-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2The side chain protecting group of the pure product is removed by adding a mixed solution of 440ml of trifluoroacetic acid, 25ml of triisopropylsilane, 20ml of benzylsulfide and 15ml of water into the pure product. Stirring for reaction for 2 hours, removing the solvent under reduced pressure, adding 2000ml of cold ethyl acetate into the concentrated solution, separating out a precipitate, filtering, and washing a filter cake with petroleum ether to obtain the trifluoroacetate of the crude product of the archipellin.
(2) Dissolving the trifluoroacetate of the crude product of the archipelene with 0.01M acetic acid/sodium acetate solution with pH =4, filtering, fixing volume, purifying by using a cation exchange column (filler: sp strong cation exchange resin), collecting eluent of target peptide, purifying by using reverse phase chromatography on the eluent after ion exchange (the filler is Gel reverse phase polymer), performing chromatographic procedures of A phase 0.1% acetic acid/water, B phase 0.1% acetic acid/methanol, balancing A phase, and sampling, and collecting the eluent. Freeze drying to obtain pure archipellin acetate 88.6g, namely Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2. The purity is more than 98 percent, and the yield is 59 percent.
Example 6:
based on the experiments of the above examples 1 to 5, the structure of the synthesized product is characterized by adopting a mass spectrometer, and as can be seen from the attached drawing 1, the molecular weight and the molecular ion peak of the synthesized product are consistent with those of the archipelene, which indicates that the synthesized product is the archipelene; referring to the attached figure 2, the purity of the coarse product of the archipellin synthesized by HPLC analysis is more than 80 percent and higher than the purity of the classical solid phase synthesis.
Example 7:
in step (1), on the basis of the example I, Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -OH-2-Chlorotrityl Chloride Resin is synthesized, and the experimental charge is 50g, so that Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin is obtained. The experimental procedure used was the same as in example 1.
Example 8:
on the basis of the first embodiment, in the technical step (3) provided in the first embodiment, the molar ratio of Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -OH to N-hydroxysuccinimide and N, N' -diisopropylcarbodiimide used is 1:1.5: 2.0; separating column chromatography is carried out to obtain 128.6g of full-protection peptide amide fragment with the yield of 93 percent. Deprotection to obtain trifluoroacetate, ion exchange, and reverse phase preparation to obtain 56g of achillene acetate with the yield of 73%.
As can be seen from the series of examples, the 2-Chlorotrityl Chloride Resin selected by the invention has the substitution degree of 1.6mmol/g and the price of 7000 yuan/kg, the Rink Amide-AM Resin used in the classical synthesis has the substitution degree of 0.9mmol and the price of 15000 yuan/kg, and the 2-Chlorotrityl Chloride Resin selected by the invention has the relatively low price, the uploading amount is increased by nearly one time, and under the same feeding amount, the yield of the Resin peptide is increased and the total yield of the target peptide is increased; synthesizing Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -OH into Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2When the method is used, the reaction temperature is 0-5 ℃, the temperature condition is the only necessary condition for inhibiting racemization in the liquid-phase amidation reaction, and the difficulty in preparation and purification is reduced; the selected ammonia reagent is ammonia water which is cheap and easy to obtain, and is mutually soluble with N, N-dimethylformamide, so that the reaction time is shortened, the hydrolysis of ester is inhibited, and the method is suitable for industrial production; in the synthesis of Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2In the method, the side chain is removed to protect the cutting fluid, the classical components of phenol and ethanedithiol are abandoned, and the ethanedithiol has a foul taste and is environment-friendly; after cutting, the cutting fluid is concentrated, the classical precipitation agent, namely the easily prepared toxic solvent, namely the anhydrous ether is abandoned, and the ethyl acetate is used instead, so that the physical discomfort caused by mistaken ether inhalation in the operation process is avoided. The precipitant can be concentrated and recycled after being added with sodium bicarbonate to neutralize acid, thus being beneficial to industrial production. Therefore, the synthetic method of the achillene provided by the application is suitable for industrial large-scale production and has a very large industrial application prospect.
As described above, the present invention can be preferably implemented, and the above-mentioned embodiments only describe the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the design spirit of the present invention should fall within the protection scope determined by the present invention.

Claims (8)

1. A large-scale synthesis method of Archimedes Henryi is characterized by comprising the following specific steps: (1) synthesizing Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin by using 2-Chlorotrityl Chloride Resin as a carrier; (2) synthesizing Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -OH; (3) synthesis of Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2Dissolving Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -OH in N, N-dimethylformamide, adding N-hydroxysuccinimide and N, N' -diisopropylcarbodiimide at 0-5 ℃, keeping at 0-5 ℃, reacting for 6-8 hours, adding an ammonia reagent, naturally heating for reacting for 2-4 hours, adding 2 times volume of saturated saline solution into the reaction solution, precipitating, filtering, washing the precipitate for 3 times with pure water, and drying at 40-45 ℃ to obtain Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2Wherein the ammonia reagent is ammonia water; the obtained Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2Dissolving in dichloromethane, adding a little methanol, carrying out silica gel sample stirring column chromatography, eluting with a dichloromethane/methanol system, and concentrating the eluent to obtain Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2Pure products; (4) synthesis of Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2To Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2Adding cutting fluid into the pure product to remove side chain groups, stirring for reaction for 2 hours, removing the solvent under reduced pressure, adding ethyl acetate into the concentrated solution, separating out a precipitate, and washing with petroleum ether to obtain a crude product of the target peptide; purifying the crude target peptide product by reverse phase chromatography, and freeze-drying to obtain a pure product of ayorelin, namely Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2。
2. The large-scale synthesis method of ayorelin according to claim 1, wherein in the step (3), the amount of ammonia added is Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -OH: ammonia =1 mmol: 1 ml.
3. The method for the large-scale synthesis of ayorelin according to claim 1, characterized in that it consists of the following steps:
(1) synthesis of Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin:
synthesizing Fmoc-Arg (Pbf) -2-Chlorotrityl Chloride Resin: swelling carrier 2-Chlorotrityl Chloride Resin with dichloromethane for 10 minutes, adding dichloromethane, Fmoc-Arg (Pbf) -OH, N' -diisopropylethylamine, stirring and reacting at normal temperature for 2 hours under the protection of nitrogen, blocking with anhydrous methanol for 0.5 hour, performing suction filtration, and washing with isopropanol and N, N-dimethylformamide sequentially to obtain Fmoc-Arg (Pbf) -2-Chlorotrityl Chloride Resin;
(ii) Fmoc-Arg (Pbf) -2-Chlorotrityl Chloride Resin: will be provided with
Fmoc-Arg (Pbf) -2-Chlorotrityl Chloride Resin removes Fmoc protecting groups by using an N, N-dimethylformamide solution of piperidine with the volume concentration of 20 percent for 40 minutes, suction filtration is carried out, filter cakes are washed by isopropanol and N, N-dimethylformamide in sequence, then N, N-dimethylformamide, Fmoc-Arg (Pbf) -OH, 1-hydroxy phenylpropyl triazole and N, N' -diisopropyl carbodiimide are added, the mixture is stirred and reacted for 2 hours at normal temperature under the protection of nitrogen, the suction filtration is carried out, the filter cakes are washed by isopropanol and N, N-dimethylformamide in sequence, and Fmoc-Arg (Pbf) -2-Chlorotrityl Chloride Resin is obtained
Chloride Resin；
③ Synthesis of Fmoc-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin: Fmoc-Gln (Trt) -OH, Fmoc-Met-OH, Fmoc-Glu (OtBu) -OH and Fmoc-Glu (OtBu) -OH are connected to Fmoc-Arg (Pbf) -2-Chlorotrityl Chloride Resin in sequence to obtain Fmoc-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin;
synthesizing Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin: Fmoc-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin is subjected to Fmoc protective group removal by using a solution of piperidine with the volume concentration of 20% in N, N-dimethylformamide for 40 minutes, suction filtration is carried out, a filter cake is washed by isopropanol and N, N-dimethylformamide in sequence, adding mixed solution of dichloromethane, acetic anhydride and N, N' -diisopropylethylamine with the volume ratio of 9:1:0.5, stirring for 30 minutes at normal temperature under the protection of nitrogen, performing suction filtration, washing a filter cake by isopropanol, N-dimethylformamide and anhydrous methanol in sequence, and then drying to obtain Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin;
(2) synthesis of Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -OH:
adding a cutting fluid into Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -2-Chlorotrityl Chloride Resin, cracking the Resin, stirring for reaction for 2 hours, filtering, washing the filtrate by using a saturated sodium bicarbonate solution until the pH is =6, washing the filtrate by using a saturated saline solution until the pH is =7, adding a saturated saline solution and removing an organic solvent under reduced pressure, precipitating, filtering, washing filter cakes by using pure water, and drying at 40 ℃ to obtain Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Arg Pbf-OH;
(3) synthesis of Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2：
Dissolving Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -OH in N, N-dimethylformamide, adding N-hydroxysuccinimide and N, N' -diisopropylcarbodiimide at 0-5 ℃, keeping at 0-5 ℃, reacting for 6-8 hours, adding an ammonia reagent, naturally heating for reacting for 2-4 hours, adding 2 times volume of saturated saline solution into the reaction solution, precipitating, filtering, washing the precipitate for 3 times with pure water, and drying at 40-45 ℃ to obtain Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2；
The obtained Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2Dissolving in dichloromethane, silica gel chromatography, andeluting with chloromethane/methanol system, concentrating the eluate to obtain Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2Pure products;
(4) synthesis of Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2：
To Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH2Adding cutting fluid into the pure product to remove side chain groups, stirring for reaction for 2 hours, removing the solvent under reduced pressure, adding ethyl acetate into the concentrated solution, precipitating, washing with petroleum ether to obtain a target peptide crude product, purifying the target peptide crude product by reverse phase chromatography, and freeze-drying to obtain the pure product of ayurrelin, namely Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2。
4. The large-scale synthesis method of ayorelin according to claim 3, characterized in that: in the (1) of the (1), the molar ratio of the 2-Chlorotrityl Chloride Resin to Fmoc-Arg (Pbf) -OH, N' -diisopropylethylamine is 1- (1.1-1.2): 4.
5. The large-scale synthesis method of ayorelin according to claim 3, characterized in that: in the step (1), the molar ratio of the 2-Chlorotrityl Chloride Resin to Fmoc-Arg (Pbf) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Met-OH, Fmoc-Glu (OtBu) -OH and Fmoc-Glu (OtBu) -OH is 1: 2; the molar ratio of the 2-Chlorotrityl Chloride Resin to the 1-hydroxyphenyltriazole and the N, N' -diisopropylcarbodiimide is 1:3: 3.
6. The large-scale synthesis method of ayorelin according to claim 3, wherein in the step (2), the cleavage resin cutting fluid is a mixed solution of trifluoroethanol, acetic acid and dichloromethane in a volume ratio of 2:0.75: 7.25.
7. The large-scale synthesis method of ayorelin as claimed in claim 3, wherein in step (3), said Ac-Glu (OtBu) -Met-Gln (Trt) -Gln
The molar ratio of Arg (Pbf) -OH to N-hydroxysuccinimide to N, N' -diisopropylcarbodiimide is 1 (1.5-2.0) to 2.0 (3.0); the reaction temperature is 0-5 ℃; the column chromatography eluent is dichloromethane and methanol with the volume ratio of 20: 1.
8. The large-scale synthesis method of the ayorelin according to claim 3, characterized in that in the step (4), the side chain group removal cutting fluid is a mixed solution of trifluoroacetic acid, triisopropylsilane, phenylmethylsulfide and water in a volume ratio of 88:5:4: 3.
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