CN113372429A

CN113372429A - Preparation method of human serum amyloid A beta 1-42

Info

Publication number: CN113372429A
Application number: CN202110252951.8A
Authority: CN
Inventors: 姚爱红; 杨浩; 万靖; 刘承; 张鑫
Original assignee: Jiangsu Genscript Biotech Co Ltd
Current assignee: Jiangsu Genscript Biotech Co Ltd
Priority date: 2020-03-09
Filing date: 2021-03-09
Publication date: 2021-09-10

Abstract

The invention relates to the technical field of polypeptide synthesis, in particular to a preparation method of human serum amyloid A beta 1-42. The preparation method of the human serum amyloid A beta 1-42 provided by the invention takes Boc-Ser (Fmoc-Gly) -OH as one of raw materials, 26-oxoacyl iso A beta 1-42 is prepared according to the amino acid sequence of the human serum amyloid A beta 1-42, and the human serum amyloid A beta 1-42 is obtained through acyl migration; the method takes amino acid with fully-protected side chain active groups as raw materials, thereby reducing the probability of side reaction in the synthesis process; in each step, synthesis parameters are controlled, so that the yield of the product is improved, and the occurrence of side reactions is reduced.

Description

Preparation method of human serum amyloid A beta 1-42

Technical Field

The invention relates to the technical field of polypeptide synthesis, in particular to a preparation method of human serum amyloid A beta 1-42.

Background

Alzheimer Disease (AD), is a disease associated with aging characterized by extensive death of brain nerve cells, with the result that brain nerve cells are gradually lost, ultimately resulting in impairment of memory, judgment and decision-making ability, orientation, attention, and language ability. In recent years, the incidence rate of the Alzheimer disease is in a remarkable rising trend, the incidence rate of China is 0.71%, and about 1000 million patients with the Alzheimer disease exist. By the year 2050, the number of Alzheimer's disease patients in China is estimated to reach 2700 ten thousand. Since alzheimer's disease is an age-closely related disease, about 10% of people are affected in the 65-year-old population, and about 50% in the 85-year-old population.

The pathological features of alzheimer's disease mainly include the extracellular appearance of senile plaques and intracellular neurofibrillary tangles with beta-amyloid (a β) as a core in cerebral cortex and hippocampal region. The A beta is polypeptide containing 39-43 amino acids generated by the proteolysis of amyloid precursor protein through beta-and gamma-secretases, and the large deposition of the A beta is a main reason for the degeneration and death of senile plaque peripheral neurons in the brain of an AD patient. A β exists in many forms in the brain, such as: low molecular weight a β monomers, a β oligomers, insoluble a β fibrils, and the like. The A beta monomer is a soluble low molecular substance, but when the intracellular environment is changed, the A beta monomer can be induced to rapidly aggregate to form a dimer, a trimer or a soluble A beta oligomer, and the A beta oligomer has poor stability and is easily converted into an A beta fiber body. The types of the A beta are divided into A beta 1-39, A beta 1-40, A beta 1-41, A beta 1-42 and A beta 1-43, wherein the A beta 1-42 has the highest toxicity, has strong water-based property, easy aggregation and strong neurotoxicity, and is a core pathogenic substance of AD. Therefore, the study of A β 1-42 is the current focus.

The amino acid sequence of A beta 1-42 is from N end to C end: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA are provided.

Oligomers synthesized at home and abroad from Abeta 1-42 are unstable, amyloid peptide is easy to randomly form various mixed products with high molecular weight or low molecular weight in vitro, and the accuracy and reliability of experimental results are directly influenced by the heterogeneous mixture of components. In recent years, many researchers have used a variety of methods to prepare a β 1-42 oligomers, but all have low yields, unstable intermediates, and complex compositions. Because of the high hydrophobicity and easy aggregation formation of the A beta 1-42, the A beta 1-42 is difficult to prepare by the conventional solid phase synthesis mode. Further developing a method for solid-phase synthesis of the polypeptide Abeta 1-42, can lay a good foundation for AD scientific research experiments and also provide an effective basis for the pathogenesis and pathological process of AD and the search and screening of effective drugs.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a method for preparing human serum amyloid A beta 1-42, wherein the method uses amino acid with fully protected side chain active groups as raw materials, thereby reducing the probability of side reactions in the synthesis process; in each step, synthesis parameters are controlled, and reasonable reaction reagents are selected, so that the yield of the product is improved, and side reactions are reduced.

The preparation method of the human serum amyloid A beta 1-42 provided by the invention comprises the following steps:

Boc-Ser (Fmoc-Gly) -OH is used as one of raw materials, 26-oxoacyl iso-Abeta 1-42 is prepared according to the amino acid sequence of human serum amyloid Abeta 1-42, and human serum amyloid Abeta 1-42 is obtained through acyl migration;

after coupling any amino acid or polypeptide in 1-25 positions, 8-12 vol% piperidine/DMF solution is adopted in the step of removing Fmoc.

In the invention, 26-oxoacyl-iso-Abeta 1-42 is synthesized by adopting a one-by-one coupling mode, wherein Boc-Ser (Fmoc-Gly) -OH is used as a raw material for coupling amino acids at the 25 th to the 26 th positions. The Fmoc protected amino acid raw materials are adopted for coupling other amino acids. Boc-Ser (Fmoc-Gly) -OH is used as a raw material, so that the coupling difficulty in the step is reduced, the reaction efficiency can be improved, and the purity of the product is improved. However, since the peptide chain has an oxyacyl foreign group after synthesis using Boc-Ser (Fmoc-Gly) -OH as a starting material, the conditions for removing the Fmoc protecting group must be optimized in the subsequent coupling step in order to maintain the stability of the group.

In the embodiment of the invention, the preparation method is a solid-phase synthesis method. In the invention, the solid-phase synthesized carrier is CTC resin or wang resin. 2-chlorotrityl chloride resin is adopted as a solid phase carrier, so that the risk of racemization caused by esterification of C-terminal amino acid and the resin is avoided; in some embodiments, the carrier is a CTC resin.

In some embodiments, the synthesis of the polypeptide of the invention specifically comprises the following steps:

step 1: preparing Fmoc-Ala-CTC resin;

step 2: sequentially coupling amino acids on the basis of Fmoc-Ala-CTC resin, wherein Boc-Ser (Fmoc-Gly) -OH is used as a raw material for coupling 25-26-bit amino acid to prepare 26-oxoacyl-iso-Abeta 1-42-peptide resin;

and step 3: after cutting peptide resin, purifying the crude 26-oxoacyl iso Abeta 1-42, and transferring acyl by amino migration to obtain human serum amyloid Abeta 1-42.

In the present invention, in the step of coupling the 41 th amino acid, the coupling agent is HATU/DIEA, or Pybop/DIEA.

In some embodiments, the molar ratio of HATU to DIEA is 1: 3; the molar ratio of Pybop to DIEA was 1: 3.

In the coupling step of Boc-Ser (Fmoc-Gly) -OH, DIC/HOBt and HATU/HOBT/DIEA are used as coupling reagents in sequence, and two coupling reactions are carried out.

In the step of coupling the 32-position and the 31-position, DIC/HOBt and HATU/HOBT/DIEA are used as coupling reagents in sequence, and two coupling reactions are carried out.

In the invention, the coupling agent of other coupling steps is selected from DIC/HOBt, HATU/HOBT/DIEA, HBTU/HOBT/DIEA or PyBOP/HOBT/DIEA besides amino acids at positions 41, 32, 31 and 25-26.

In some embodiments, the coupling agent for the other coupling step is DIC/HOBt.

In the present invention, in the step of coupling the amino acids at positions 32 and 31, the charged amount of the amino acids is 5 eq.

In the present invention, the amount of amino acids charged in the coupling step is 3eq, except for the coupling of the amino acids at positions 32 and 31.

In the present invention, the coupling solvent is selected from DMF, CH2Cl2, NMP.

In some embodiments, the coupling solvent is DMF.

In the invention, after coupling any amino acid or polypeptide in 1-25 positions, the Fmoc removal reagent is 10 vol% piperidine/DMF solution, and the removal is carried out for 2 times at 20-25 ℃ for 5 min;

after coupling any amino acid or polypeptide in 27-42 positions, the Fmoc removal reagent is 20 vol% piperidine/DMF solution under the conditions of 20-25 ℃ and 5-30 min.

In some embodiments, the cleavage agent for cleaving the resin after the solid phase synthesis is a mixture of TFA and at least one of thioanisole, phenol, EDT, ammonium iodide, and water.

The concentration of TFA in the cleavage agent is not less than 80%. The generation of oxidation impurity Met (O) can be avoided by adding ammonium iodide into the cutting reagent. In some embodiments, the cleavage agent consists of TFA, thioanisole, phenol, EDT, water, and an iodinated amine. Wherein TFA: thioanisole: phenol: EDT (electro-thermal transfer coating): water 87.5: 2.5: 2.5: 5: 2.5; the concentration of the amine iodide was 1 mg/mL.

The cracking steps are as follows: adding a cracking agent into the 26-oxoacyl-iso Abeta 1-42-resin compound for reaction at the temperature of 0-40 ℃ for 1-4 hours.

In the invention, the acyl migration is to dissolve 26-oxoacyl iso A beta 1-42 in acetonitrile aqueous solution, adjust the pH value to 7.4 by ammonia water, and react for 4 hours at 25 ℃ to prepare the A beta 1-42.

The preparation method of the human serum amyloid A beta 1-42 provided by the invention takes Boc-Ser (Fmoc-Gly) -OH as one of raw materials, 26-oxoacyl iso A beta 1-42 is prepared according to the amino acid sequence of the human serum amyloid A beta 1-42, and the human serum amyloid A beta 1-42 is obtained through acyl migration; the method takes amino acid with fully-protected side chain active groups as raw materials, thereby reducing the probability of side reaction in the synthesis process; in each step, synthesis parameters are controlled, so that the yield of the product is improved, and the occurrence of side reactions is reduced.

Drawings

FIG. 1 shows a high performance liquid chromatogram of a crude product of 26-oxoacyliso A beta 1-42;

FIG. 2 shows a high performance liquid chromatogram of pure A beta 1-42;

FIG. 3 shows the pure A β 1-42 mass spectrum;

FIG. 4 shows a high performance liquid chromatogram of pure A β 1-42;

FIG. 5 shows the pure A β 1-42 mass spectrum;

FIG. 6 shows a high performance liquid chromatogram of pure A β 1-42;

FIG. 7 shows the pure A β 1-42 mass spectrum;

FIG. 8 shows an A β 27-42 mass spectrum;

FIG. 9 shows the mass spectrum of crude 26-oxoacylisoAbeta 1-42;

FIG. 10 shows a high performance liquid chromatogram of crude 26-oxoacyliso A beta 1-42.

Detailed Description

The invention provides a preparation method of human serum amyloid A beta 1-42, and a person skilled in the art can realize the preparation method by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

In the present invention, the "polypeptide" or "peptide" has its ordinary meaning in the art and may refer to an amide from two or more aminocarboxylic acid molecules (the same or different) that forms a covalent bond by formally losing water from the carbonyl carbon of one aminocarboxylic acid molecule and the nitrogen atom of another aminocarboxylic acid molecule. "amino acid residue" also has its ordinary meaning in the art and refers to the composition of an amino acid (as a single amino acid or as part of a peptide) after it is combined with a peptide, another amino acid, or an amino acid residue. Generally, when an amino acid is combined with another amino acid or amino acid residue, water is removed, and the remaining amino acid is referred to as an amino acid residue.

The "amino acid" also has its ordinary meaning in the art and can include both proteinogenic amino acids and non-proteinogenic amino acids. The abbreviations for amino acid residues in the present invention are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

The "protecting group" or "protecting group" as referred to herein has its ordinary meaning in the art. A protecting group includes a chemical moiety that is attached to or configured to attach to a reactive group (i.e., a protected group) within a molecule (e.g., a peptide) such that the protecting group prevents or otherwise inhibits the protected group from participating in a reaction. Protection may be performed by attaching a protecting group to the molecule. Deprotection can occur when a protecting group is removed from a molecule, for example, by chemical transformation to remove the protecting group.

In the amino acid raw materials adopted by the invention for synthesizing the polypeptide, alpha amino groups of all amino acids are Fmoc protecting groups, and serine is except for 26 th amino acid (Ser)²⁶) In addition, the remaining side chain hydroxyl protecting groups are t-butyl, the aspartic acid and glutamic acid side chain carboxyl protecting groups are t-butyl, the glutamine and asparagine side chain amide protecting groups are trityl, the histidine side chain imidazole protecting groups are trityl or t-butoxycarbonyl, the lysine and tryptophan side chain protecting groups are t-butoxycarbonyl, and the arginine side chain guanidino protecting group is 2,2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl. Specifically, the amino acid raw materials adopted by the invention comprise: glycine (Gly, G): Fmoc-Gly-OH; arginine (Arg, R): Fmoc-Arg (Pbf) -OH; valine (Val, V): Fmoc-Val-OH; leucine (Leu, L): Fmoc-Leu-OH; tryptophan (Trp, W): Fmoc-Trp (Boc) -OH(ii) a Alanine (Ala, a): Fmoc-Ala-OH; isoleucine (Ile, I): Fmoc-Ile-OH; phenylalanine (Phe, F): Fmoc-Phe-OH; glutamic acid (Glu, E): Fmoc-Glu (OtBu) -OH; alanine (Ala, a): Fmoc-Ala-OH; glutamine (Gln, Q): Fmoc-Gln (Trt) -OH; methionine (Met, M): Fmoc-Met-OH; serine (Ser, S): Fmoc-Ser (tBu) -OH; leucine (Leu, L): Fmoc-Leu-OH; threonine (Thr, T): Fmoc-Thr (tBu) -OH; lysine (Lys, K): Fmoc-Lys (Boc) -OH; histidine (His, H): Boc-His (Trt) -OH; gly²⁵-Ser²⁶Boc-Ser (Fmoc-Gly) -OH; tyrosine (Tyr, Y): Fmoc-Tyr (tBu) -OH; aspartic acid (Asp, D): Fmoc-Asp (OtBu) -OH.

The term "coupling" as used herein refers to the process of adding a new amino acid to a bound amino acid or peptide. The synthesis of polypeptides includes solid phase synthesis and liquid phase synthesis, and solid phase and liquid phase combination methods have been reported. In the field, the coupling can be performed by adopting a mode of coupling one by one amino acid sequence, or a mode of coupling a plurality of polypeptide fragments by adopting a segmented coupling mode according to a peptide sequence.

Boc-Ser (Fmoc-Gly) -OH is used as one of raw materials, 26-oxoacyl iso-Abeta 1-42 is prepared according to the amino acid sequence of human serum amyloid Abeta 1-42, and human serum amyloid Abeta 1-42 is obtained through acyl migration; after coupling any amino acid or polypeptide in 1-25 positions, adopting 10 vol% piperidine/DMF solution in the step of removing Fmoc.

step 1: preparing Fmoc-Ala-CTC resin;

Specifically, in the present embodiment, the sequential coupling means that Fmoc-Ala-CTC is first synthesized according to the peptide sequence of human serum amyloid a β 1-42, and then Ile, Val, Gly, Val, Met, Leu, Gly, Ile, Ala, Gly, Lys, Asn, Gly-Ser, Val, Leu, Lys, gin, His, Val, gin, Tyr, Gly, Ser, Asp, His, Arg, Phe, Glu, Ala, and Asp are prepared into 26-oxoacyl iso a β 1-42-CTC resin.

In the present invention, each of the coupling reactions comprises: removing Fmoc protection and carrying out condensation reaction. For the step of removing Fmoc protection, before the 25 th-26 th amino acid is coupled, namely before Boc-Ser (Fmoc-Gly) -OH is coupled, the Fmoc removal reagent in each step adopts 20 vol% piperidine/DMF solution, and the conditions for removing Fmoc are 20-25 ℃ and 5min for 2 times; in the Fmoc removal step after coupling Boc-Ser (Fmoc-Gly) -OH, a removal reagent is 10 vol% piperidine/DMF solution, and the removal conditions are 20-25 ℃ and 5-30 min.

The research of the invention finds that the coupling reaction of the 42 th amino acid and the 41 th amino acid is very unstable, the reaction efficiency is low and byproducts are easy to generate. At present, the specific structure of the byproduct is not clarified, and the molecular weight is larger than the target molecular weight through mass spectrum detection. After modification of the coupling conditions of this step, this instability problem is solved and no by-products are obtained in the product. In the step of coupling the 41 th amino acid, the coupling agent adopted is HATU/DIEA or Pybop/DIEA. Experiments show that the normal running of the reaction can be maintained by adopting the two coupling agents. In some embodiments, the molar ratio of HATU to DIEA is 1: 3; the molar ratio of Pybop to DIEA was 1: 3. In the reaction for coupling the amino acid at position 41, the molar ratio of amino acid to DIEA was 1: 3.

In the step of coupling Boc-Ser (Fmoc-Gly) -OH, DIC/HOBt and HATU/HOBT/DIEA are sequentially used as coupling reagents, and two coupling reactions are carried out; the solvent for the first coupling is DMF, and the solvent for the second coupling is DMF and CH₂Cl₂Wherein the ratio of DMF: CH (CH)₂Cl₂Is 4: 1. The time for the first coupling was 40min, followed by washing with DMF; the time for the second coupling was 40 min. Wherein, the molar ratio of DIC to HOBt is 1: 1.25; the molar ratio of HATU, HOBT and DIEA is 1:1: 2.

in the present invention, eq is a molar equivalent, and is a unit used in chemical or biological science, and is used to represent the amount of a substance. In the general coupling step, the amount of amino acid added was 3eq, but in the step of coupling the amino acids at positions 32 and 31, the condensation reaction did not easily occur, and if the amount of amino acid charged was insufficient, the synthesis efficiency was seriously decreased. The research of the invention shows that the condensation reaction efficiency is obviously improved after the feeding amount of the amino acid is increased. Therefore, in the present invention, the charged amount of amino acid in the step of coupling the amino acids at positions 32 and 31 is 5 eq. In addition to the coupling of the amino acids at positions 32 and 31, the amount of amino acids charged in the other coupling steps was 3 eq.

In addition, the coupling effect of the amino acids at the 32 th position and the 31 th position is closely related to the selection of the coupling agent, in the coupling of the two amino acids, DIC/HOBt and HATU/HOBT/DIEA are used as the coupling agents of each amino acid in sequence, and two coupling reactions are carried out; the solvent for the first coupling is DMF, and the solvent for the second coupling is DMF and CH₂Cl₂Wherein the ratio of DMF: CH (CH)₂Cl₂Is 4: 1. The time for the first coupling was 40min, followed by washing with DMF; the time for the second coupling was 40 min. Wherein, the molar ratio of DIC to HOBt is 1: 1.25; the molar ratio of HATU, HOBT and DIEA is 1:1: 2.

in the invention, the coupling agent in other coupling steps is any one or more of DIC/HOBt, HATU/HOBT/DIEA, HBTU/HOBT/DIEA or PyBOP/HOBT/DIEA besides the amino acids at the 41 th, 32 th, 31 th and 25-26 th positions. In some embodiments, the coupling agent for the other coupling steps is DIC/HOBt, and the ratio of the amounts of species of each reactant in these coupling steps is n amino acids: HOBT: DIC 1: 1.25: 1. the solvent for coupling is selected from DMF and CH₂Cl₂NMP. In some embodiments, the coupling solvent is DMF.

Specifically, in the present invention, the preparation of the 26-oxoacyliso a β 1-42-CTC resin comprises:

preparing Fmoc-Ala-CTC resin, removing Fmoc protective groups by using 20% of Pip/DMF for 25min, coupling with Fmoc-Ile-OH, wherein a coupling agent is DIEA/HATU (molar ratio is 3:1) or Pybop/DIEA, a solvent is DMF, and the feeding amount of amino acid is 3 eq;

coupling Ile, Val, Gly, Val, Met, Leu and Gly in sequence; performing coupling in each step by using 20% of Pip/DMF to react for 25min to remove Fmoc protection, wherein a coupling agent is DIC/HOBt (molar ratio is 8.6:6.9) or a coupling agent is Pybop/HOBT (molar ratio is 1: 1), a solvent is DMF, and the feeding amount of amino acid is 3 eq;

thirdly, coupling the 31 st amino acid and the 32 nd amino acid, reacting with 20 percent of Pip/DMF for 25min to remove Fmoc protection and Fmoc-Ile in each step of coupling³¹-OH and Fmoc-Ile³²The charging amount of-OH is 5eq, DIC/HOBt (molar ratio 1:1.25) is used as a coupling agent and DMF is used as a solvent in the coupling process, after coupling is carried out for 40min, washing is carried out by DMF, HATU/HOBT/DIEA (molar ratio 1:1: 2) is used as a coupling agent and DMF is used as a solvent, and coupling is carried out for 40 min;

sequentially coupling Ala, Gly, Lys and Asn, reacting 20% Pip/DMF for 25min to remove Fmoc protection in each coupling step, wherein a coupling agent is DIC/HOBt (molar ratio is 8.6:6.9) or Pybop/HOBT (molar ratio is 1: 1), a solvent is DMF, and the feeding amount of each amino acid is 3 eq;

coupling Gly-Ser, reacting 20% Pip/DMF for 25min to remove Fmoc protection, feeding Boc-Ser (Fmoc-Gly) -OH with 3eq, taking DIC/HOBt (molar ratio 1:1.25) as a coupling agent and DMF as a solvent in the coupling process, washing with DMF after coupling for 40min, taking HATU/HOBT/DIEA (molar ratio 1:1: 2) as a coupling agent and DMF/DCM as a solvent (volume ratio 4:1), and coupling for 40 min;

sixthly, sequentially coupling Val, Asp, Glu, Ala, Phe, Val, Leu, Lys, Gln, His, Val, Glu, Tyr, Gly, Ser, Asp, His, Arg, Phe, Glu, Ala and Asp, reacting for 5min by 10 percent Pip/DMF in each coupling step, and repeatedly removing Fmoc protection for 2 times, wherein a coupling agent is DIC/HOBt (molar ratio of 8.6:6.9) or a coupling agent is Pybop/HOBT (molar ratio of 1: 1), a solvent is DMF, and the feeding amount of each amino acid is 3 eq.

In the invention, after the solid phase synthesis, the cleavage agent for cutting the resin is a mixed solution of TFA and at least one of methyl sulfide, phenol, EDT, ammonium iodide and water. The concentration of TFA in the cleavage agent is not less than 80%. The generation of oxidation impurity Met (O) can be avoided by adding ammonium iodide into the cutting reagent. In some embodiments, the cleavage agent consists of TFA, thioanisole, phenol, EDT, water, and an iodinated amine. Wherein TFA: thioanisole: phenol: EDT (electro-thermal transfer coating): water 87.5: 2.5: 2.5: 5: 2.5; the concentration of the amine iodide was 1 mg/mL. The cracking steps are as follows: adding a cracking agent into the 26-oxoacyl-iso Abeta 1-42-resin compound for reaction at the temperature of 0-40 ℃ for 1-4 hours.

After resin cutting, crude 26-oxoacyl iso Abeta 1-42 is obtained and purified by high performance liquid chromatography, and pure 26-oxoacyl iso Abeta 1-42 is obtained. The chromatographic conditions for purification were, column: 30mm of polymer (polystyrene, particle size 10um 300A, column length 25cm, diameter 30mm) or C18 column, mobile phase a: 0.25% aqueous formic acid, mobile phase B: 0.1% TFA/acetonitrile solution; phase B is changed from 5% to 22% in 0-5 min; changing the phase B from 22% to 42% within 5-50min, operating for 45min, and controlling the temperature of the mobile phase at 60 ℃.

In a specific embodiment, the preparation method of the present invention further preferably comprises the steps of:

(1) Fmoc-Ala-resin complexes were prepared.

The resin was added to a cup-shaped reaction vessel with a filter unit. The solvent DCM was added to the vessel to swell the resin for 30min, after dissolving Fmoc-Ala-OH and the esterification reagents in DCM, the resin was added and reacted at 25 ℃ for 70 min. After the reaction is finished, a methanol/DIEA mixed solution (volume ratio is 1: 1) which is one-third of the volume of the resin is added, the reaction is carried out for 30min at room temperature, and then the mixture is filtered. The solvent DMF was then added, the resin was washed five times and filtered with suction. Wherein the resin is 2-chlorotrityl chloride resin, the esterification reagent is DIEA, and the proportion of substances is n (Fmoc-Ala-OH): n (DIEA): n (2-chlorotrityl chloride resin) ═ 0.3: 1:1.

(2) preparing a protected 26-oxoacyl-iso-Abeta 1-42 resin compound.

The Fmoc-Ala-resin complex was added with a 20% Pip/DMF solution by volume to carry out deprotection reaction at 25 ℃ for 25 min. After the reaction, suction filtration is carried out. The resin was then washed 5 times with DMF.

Adding a second amino acid Fmoc-Ile-OH at the C terminal and a condensation reagent into the reactor, and reacting for 70min at 25 ℃. After the reaction, DMF was added to wash the resin, and the mixture was filtered. The condensation reagent is preferably HATU/DIEA, Pybop/DIEA. Wherein the ratio of the amounts of the reactant materials is: nFmoc-Ile-OH: HATU: DIEA 1:1: 3.

sequentially coupling amino acids from the C end to the N end of the 26-oxoacyl iso A beta 1-42, and carrying out cyclic deprotection reaction and condensation reaction, wherein the condensation reagent can be one or more of DIC/HOBt, HATU/HOBT/DIEA, HBTU/HOBT/DIEA and PyBOP/HOBT/DIEA, and the solvent is one or a mixture of two of DMF, CH2Cl2 and NMP, and the 26-oxoacyl iso A beta 1-42-resin compound is synthesized, wherein the preferable amount ratio of reactant substances is as follows: n amino acids: HOBT: DIC 1: 1.25: 1.

the preferred coupling method of using Boc-Ser (Fmoc-Gly) -OH as a raw material for Gly25-Ser26 comprises coupling DIC/HOBt with DMF for 40min, suction-filtering, washing resin with DMF for 1 time, and coupling with HATU/HOBT/DIEA for 40min again, wherein the solvent is DMF: CH (CH)₂Cl₂Is 4: 1.

The Fmoc removal of the amino acids 1-25 is preferably performed in a 10% piperidine/DMF solution for 2 times 5 min.

(3) Preparing a crude product of 26-oxoacyl iso Abeta 1-42.

Adding a cleavage reagent to the protected 26-oxoacyl-iso-A beta 1-42 resin complex, wherein the cleavage reagent is a mixed solution of TFA and at least one of TFA, thioanisole, phenol, EDT, ammonium iodide and water, and the concentration of TFA in the mixed solution is not less than 80%; adding a cutting reagent into the protected 26-oxoacyl-iso-Abeta 1-42-resin compound for reaction at the temperature of 0-40 ℃ for 1-4 hours. The preferred ratios are TFA: thioanisole: phenol: EDT (electro-thermal transfer coating): water 87.5: 2.5: 2.5: 5: 2.5, adding ammonium iodide (1mg/mL), and pre-cooling the cracking reagent at 0-10 ℃; adding a cracking reagent into the 26-oxoacyl-iso-Abeta 1-42 resin peptide in an ice water bath, reacting for 30min, gradually raising the temperature to 25 ℃, and continuing cracking for 2 h. Adding precooled ether into the lysate for centrifugal precipitation to obtain a crude 26-oxoacyl-iso-Abeta 1-42 product;

(4) preparing the A beta 1-42 pure product.

The crude 26-oxoacyl iso Abeta 1-42 is freeze-dried. Dissolving the freeze-dried solid with 5% methanol or acetonitrile aqueous solution by volume ratio, and purifying by preparative high performance liquid chromatography to obtain a refined 26-oxoacyl iso Abeta 1-42 product. The chromatographic conditions for purification were 30mm polymer column (polystyrene, particle size 10um 300A, column length 25cm, diameter 30mm), mobile phase A as 0.25% aqueous formic acid, and phase B as 0.1% TFA/acetonitrile; phase B is changed from 5% to 22% in 0-5 min; changing the phase B from 22% to 42% within 5-50min, operating for 45min, and controlling the temperature of the mobile phase at 60 ℃.

In addition, some abbreviations commonly used in the present invention have the following meanings:

the English abbreviations of the condensation reagents related to the invention have the following meanings:

2-CTC resin: 2-chloro-triphenyl chloride resin

DIEA: n, N-diisopropylethylamine

Wang resin: p-alkoxy benzyl alcohol resin

HBTU: benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate

HATU: 2- (7-azabenzotriazole) -tetramethylurea hexafluorophosphate

Pybop: benzotriazol-1-yl-oxytripyrrolidine hexafluorophosphate

DIC: n, N-diisopropylcarbodiimide

TFA: trifluoroacetic acid

HOBT: 1-hydroxybenzotriazoles

DCM: methylene dichloride

NMP: n-methyl pyrrolidone

And Pip: piperidine derivatives

Fmoc: fmoc acyl

DBU: 1, 8-diazabicycloundec-7-enes

EDT (electro-thermal transfer coating): 1, 2-ethanedithiol

DMF: n, N-dimethylformamide

Obzl: Boc-L-serine benzyl ester

EDC. HCl: 1-Ethyl- (3-dimethylaminopropyl) carbodiimides hydrochloride

DMAP: 4-dimethylaminopyridine

Ar: argon gas

PE: EA Petroleum Ether: ethyl acetate

The invention is further illustrated by the following examples:

example 1: synthesis of Boc-Ser (Fmoc-Gly) -OH

The compound Fmoc-Gly-OH (2.97g, 10mmol), the compound Boc-Ser-OBzl (2.95g, 88.4mmol) and EDC.HCl (2.3g, 12mmol) are added into a 1L single-mouth bottle, 500ml DCM is added to dissolve completely, DMAP (0.122g, 0.2mmol) and Ar protection are added to react for about 5h, and a sample is taken and HPLC detects that the reaction is complete; washing the reaction solution with 1mol/L HCl aqueous solution for 2 times, washing with saturated salt for 2 times, drying with anhydrous magnesium sulfate, and evaporating to dryness to obtain white solid 5.1 g; dissolving the white solid in 100mL of MeOH, adding 10% Pd/C, reacting for 2.5H after H2 is replaced, and detecting by a sampling point plate until the compound completely reacts; filtering to remove Pd/C, evaporating to dryness, and passing through a silica gel column; developing agent: PE, EA is 3:1, and 1 drop of acetic acid is added; eluent: PE, EA is 5: 1; thus, 2.84g of a pure product was obtained.

Example 2: preparation of Abeta 1-42

(1) And (3) synthesizing Fmoc-Ala-CTC resin.

Weighing 10g of 2-CTC resin with the substitution degree of 0.98mmol/g, adding the resin into a cup-shaped reaction column, swelling the resin with DCM for 30 minutes, and filtering to remove DCM; weighing 3mmol of Fmoc-Ala-OH, adding DCM and DIEA into a reaction cup, and carrying out nitrogen bubbling reaction for 90 min; adding 3mL of methanol and DIEA to perform end-capping reaction for 30min, washing with DMF for 4 times, washing with DCM for 1 time, and then draining with methanol to obtain Fmoc-Gly-CTC resin; a small amount of the resin was used and the degree of substitution was measured by UV spectrophotometer to be 0.23mmol/g (three times average).

(2)2-CTC resin Ile⁴¹Coupling of (3).

Fmoc-Ala-CTC resin with a degree of substitution of 0.23mmol/g, prepared in example 1, was placed in a cup-shaped reaction column, the resin was swollen with DCM for 30min, after which DCM was removed by suction filtration; adding 20% of Pip/DMF to remove the Fmoc protecting group, wherein the removal time is 25min, adding DMF to wash for 5 times, DCM to wash for 1 time, and DMF to wash for 1 time, and ninhydrin detecting the color development of the resin, and simultaneously ensuring that piperidine is washed cleanly without residue. 2.4g of Fmoc-Ile-OH (6.9mmol), 2.6g of HATU (6.9mmol) were weighed out, dissolved in 50mL of DMF, and 3.5mL of DIEA (21mmol) were added thereto, reacted for 2min, poured into a cup-shaped reaction column, and subjected to nitrogen bubbling for 70 min.

(3) Preparing a protected 26-oxoacyl-iso-Abeta 1-42 resin compound.

100mL of 20% Pip/DMF solution was added to the reactor and reacted at room temperature for 25min, then DMF was added and washed 6 times, DCM was washed 1 time, and ninhydrin detection was performed to detect the color development of the resin. 6.9mmol Fmoc-Val-OH, 8.6mmol HOBt and 6.9mmol DIC were dissolved in 50mL DMF, and the resulting solution was added to the reactor and reacted at 25 ℃ for 70 min. After the reaction was completed, the mixture was filtered. Then 80mL of DMF was added and the resin was washed six times.

According to the sequence from the C end to the N end of 26-oxygen acyl iso-Abeta 1-42, amino acid is coupled in sequence, and cyclic deprotection reaction and condensation reaction are carried out. Wherein Fmoc-Ile³¹-OH，Fmoc-Ile³²The coupling method of-OH is that amino acid is fed by 5eq, the reaction concentration is 0.3mmol/mL, DIC/HOBt is a coupling reagent (amino acid: DIC: HOBT ═ 1:1:1.25), a solvent is DMF, coupling is carried out for 40min, then suction filtration is carried out, DMF is used for washing resin for 1 time, and then HATU/HOBT/DIEA is selected for secondary coupling for 40min, and the solvent is DMF.

The coupling method of Boc-Ser (Fmoc-Gly) -OH comprises the steps of feeding 3eq amino acid, coupling the amino acid with the reaction concentration of 0.3mmol/mL, coupling DIC/HOBt with a coupling reagent and DMF as a solvent for 40min, then carrying out suction filtration, washing the resin with DMF for 1 time, then selecting HATU/HOBT/DIEA for secondary coupling for 40min, and carrying out secondary coupling on the resin with DMF as a solvent: DCM 4: 1.

The Fmoc removal of the amino acids at positions 1-25 was performed in a 10% piperidine/DMF solution for 2 times 5 min.

And removing other amino acids Fmoc once by using 20% piperidine/DMF solution for 25 min.

(4) Preparing a crude product of the protected 26-oxoacyl iso Abeta 1-42.

Placing 1g of the 26-oxoacyl-iso-Abeta 1-42 resin compound in an ice-water bath, pre-cooling a cleavage reagent (TFA: benzylthioether: phenol: EDT: water: 87.5: 2.5: 2.5: 5: 2.5, ammonium iodide (1mg/mL)) at 0-10 ℃, and adding the cleavage reagent into 26-oxoacyl-iso-Abeta 1-42 resin peptide; after the reaction is carried out in an ice-water bath for 30min, the temperature is gradually increased to 25 ℃, and the cracking is continued for 2 h. Adding precooled diethyl ether with the volume 15 times of the lysate for centrifugal precipitation to obtain 270mg of a 26-oxoacyl-iso Abeta 1-42 crude product, wherein an analysis chart of the crude product is shown in figure 1;

(5) preparing the A beta 1-42 pure product.

Dissolving 270mg of crude 26-oxoacyl iso Abeta 1-42 product in 5% methanol aqueous solution by volume ratio, and purifying by high performance liquid chromatography to obtain refined 26-oxoacyl iso Abeta 1-42 product, wherein the purification conditions comprise: high performance liquid chromatography with 30mm polymer (polystyrene, particle size 10um 300A, column length 25cm, diameter 30mm) as column, mobile phase A of 0.25% formic acid water solution, and phase B of 0.1% TFA/acetonitrile solution; phase B is changed from 5% to 22% in 0-5 min; changing the phase B from 22% to 42% within 5-50min, operating for 45min, and controlling the temperature of the mobile phase at 60 ℃. After purification, 64.8mg of pure product with the purity of 98.798 percent and the total yield of 12.49 percent is obtained after freeze drying.

Dissolving the pure product in acetonitrile/water (volume ratio of 1: 1) until the concentration of the pure product is 1mg/mL, adding one thousandth of ammonia water to adjust the pH value to 7.4, reacting at 25 ℃ for 4 hours to obtain Abeta 1-42, and freeze-drying to obtain 58.3mg of solid with the purity of 98.298%. The high performance liquid chromatogram is shown in FIG. 2, and the mass spectrum is shown in FIG. 3.

Example 3: preparation of Abeta 1-42

(1) And (3) synthesizing Fmoc-Ala-CTC resin.

Weighing 10g of 2-CTC resin with the substitution degree of 0.98mmol/g, adding the resin into a cup-shaped reaction column, swelling the resin with DCM for 30 minutes, and filtering to remove DCM; weighing 1mmol of Fmoc-Ala-OH, adding DCM and DIEA into a reaction cup, and carrying out nitrogen bubbling reaction for 90 min; adding 3mL of methanol and DIEA to perform end-capping reaction for 30min, washing with DMF for 4 times, washing with DCM for 1 time, and then draining with methanol to obtain Fmoc-Gly-CTC resin; a small amount of the resin was taken and the degree of substitution was measured by a UV spectrophotometer to be 0.07mmol/g (three-time average).

(2)2-CTC resin Ile⁴¹Coupling of (3).

Fmoc-Ala-CTC resin with a degree of substitution of 0.07mmol/g, prepared in example 1, was placed in a cup-shaped reaction column, the resin was swollen with DCM for 30min, after which DCM was removed by suction filtration; adding 20% of Pip/DMF to remove the Fmoc protecting group, wherein the removal time is 25min, adding DMF to wash for 5 times, DCM to wash for 1 time, the Chinese medicine DMF to wash for 1 time, and ninhydrin detection to detect the color development of the resin, and simultaneously ensuring that piperidine is washed cleanly and has no residue. 0.74g of Fmoc-Ile-OH (2.1mmol), 0.78g of HATU (2.1mmol) were weighed out, dissolved in 15mL of DMF, and 1.05mL of DIEA (6.3mmol) was added thereto, reacted for 2min, poured into a cup-shaped reaction column, and subjected to nitrogen bubbling for 70 min.

(3) Preparing a protected 26-oxoacyl-iso-Abeta 1-42 resin compound.

100mL of 20% Pip/DMF solution was added to the reactor and reacted at room temperature for 25min, then DMF was added and washed 6 times, DCM was washed 1 time, and ninhydrin detection was performed to detect the color development of the resin. 2.1mmol of Fmoc-Val-OH, 2.1mmol of HATU, 2.1mmol of HOBT and 6.3mmol of DIEA were dissolved in 15mL of DMF, and the resulting solution was added to a reactor and reacted at 25 ℃ for 70 min. After the reaction was completed, the mixture was filtered. Then 25mL of DMF was added and the resin was washed six times.

According to the sequence from the C end to the N end of 26-oxygen acyl iso-Abeta 1-42, amino acid is coupled in sequence, and cyclic deprotection reaction and condensation reaction are carried out. When the length of the peptide chain is 16 amino acids, a small amount of resin is taken for mass spectrum detection, the mass spectrum is shown in figure 4, the target molecular weight of A beta 27-42 is 1511.9, the detected molecular weight is 1511.6 (bivalent is 756.8), and 1397.8 (bivalent is 699.9) is the molecular weight of deletion Ile. The coupling method of Boc-Ser (Fmoc-Gly) -OH comprises the steps of feeding 3eq amino acid, coupling the amino acid with the reaction concentration of 0.3mmol/mL, coupling DIC/HOBt with a coupling reagent and DMF as a solvent for 40min, then carrying out suction filtration, washing the resin with DMF for 1 time, then selecting HATU/HOBT/DIEA for secondary coupling for 40min, and carrying out secondary coupling on the resin with DMF as a solvent: DCM 4: 1.

The Fmoc removal of the amino acids at positions 1-25 was performed in a 10% piperidine/DMF solution for 2 times 5 min. And removing other amino acids Fmoc once by using 20% piperidine/DMF solution for 25 min.

(4) Preparing a crude product of the protected 26-oxoacyl iso Abeta 1-42.

Placing 1g of the 26-oxoacyl-iso-A beta 1-42 resin compound in an ice water bath, adding a cracking reagent into 26-oxoacyl-iso-A beta 1-42 resin peptide, wherein the mass ratio is TFA: thioanisole: phenol: EDT (electro-thermal transfer coating): water 87.5: 2.5: 2.5: 5: 2.5, adding ammonium iodide (1mg/mL), and pre-cooling the cracking reagent at 0-10 ℃; after the reaction is carried out in an ice-water bath for 30min, the temperature is gradually increased to 25 ℃, and the cracking is continued for 2 h. Adding precooled diethyl ether with the volume 15 times of the lysate for centrifugal precipitation to obtain 112mg of crude 26-oxoacyl iso Abeta 1-42 product;

(5) preparing the A beta 1-42 pure product.

Dissolving the crude 26-oxoacyl iso Abeta 1-42 product in 5% methanol water solution by volume ratio, and purifying by high performance liquid chromatography to obtain refined 26-oxoacyl iso Abeta 1-42 product, wherein the purification conditions are as follows: high performance liquid chromatography with 30mm polymer (polystyrene, particle size 10um 300A, column length 25cm, diameter 30mm) as column, mobile phase A of 0.25% formic acid water solution, and phase B of 0.1% TFA/acetonitrile solution; phase B is changed from 5% to 22% in 0-5 min; changing the phase B from 22% to 42% within 5-50min, operating for 45min, and controlling the temperature of the mobile phase at 60 ℃. After purification, 11.8mg of pure product with the purity of 98.703 percent and the total yield of 6.72 percent is obtained after freeze drying. The pure product was dissolved in acetonitrile/water (volume ratio 1: 1) at a concentration of 1mg/mL, and one thousandth of ammonia water was added to adjust pH to 7.4, and reacted at 25 ℃ for 4 hours to obtain a β 1-42, which was lyophilized to obtain 10.3mg of a solid with a purity of 97.651%. The high performance liquid chromatogram is shown in FIG. 4, and the mass spectrum is shown in FIG. 5.

Example 4: preparation of Abeta 1-42

(1) And (3) synthesizing Fmoc-Ala-CTC resin.

Weighing 10g of 2-CTC resin with the substitution degree of 0.98mmol/g, adding the resin into a cup-shaped reaction column, swelling the resin with DCM for 30 minutes, and filtering to remove DCM; weighing 5mmol of Fmoc-Ala-OH, adding DCM and DIEA into a reaction cup, and carrying out nitrogen bubbling reaction for 90 min; adding 3mL of methanol and DIEA to perform end-capping reaction for 30min, washing with DMF for 4 times, washing with DCM for 1 time, and then draining with methanol to obtain Fmoc-Gly-CTC resin; a small amount of the resin was used and the degree of substitution was measured by UV spectrophotometer to be 0.33mmol/g (three times average).

(2)2-CTC resin Ile⁴¹Coupling of (3).

Fmoc-Ala-CTC resin with a degree of substitution of 0.33mmol/g, prepared in example 1, was placed in a cup-shaped reaction column, the resin was swollen with DCM for 30min, after which DCM was removed by suction filtration; adding 20% of Pip/DMF to remove the Fmoc protecting group, wherein the removal time is 25min, adding DMF to wash for 5 times, DCM to wash for 1 time, the Chinese medicine DMF to wash for 1 time, and ninhydrin detection to detect the color development of the resin, and simultaneously ensuring that piperidine is washed cleanly and has no residue. 3.5g of Fmoc-Ile-OH (10mmol) and 5.2g of Pybop (10mmol) were weighed out and dissolved in 70mL of DMF, 5mL of DIEA (30mmol) was added and reacted for 2min, and then poured into a cup-shaped reaction column and reacted for 70min with nitrogen bubbling.

(3) Preparing a protected 26-oxoacyl-iso-Abeta 1-42 resin compound.

100mL of 20% Pip/DMF solution was added to the reactor and reacted at room temperature for 25min, then DMF was added and washed 6 times, DCM was washed 1 time, and ninhydrin detection was performed to detect the color development of the resin. 10mmol Fmoc-Val-OH, 10mmol Pybop, 10mmol HOBT and 30mmol DIEA were dissolved in 70mL DMF, and the resulting solution was added to the reactor and reacted at 25 ℃ for 70 min. After the reaction was completed, the mixture was filtered. Then 100mL of DMF was added and the resin was washed six times.

(4) Preparing a crude product of the protected 26-oxoacyl iso Abeta 1-42.

Placing the 26-oxoacyl-iso-A beta 1-42 resin compound in an ice water bath, adding a cracking reagent into 26-oxoacyl-iso-A beta 1-42 resin peptide, wherein the mass is TFA: thioanisole: phenol: EDT (electro-thermal transfer coating): water 87.5: 2.5: 2.5: 5: 2.5, adding ammonium iodide (1mg/mL), and pre-cooling the cracking reagent at 0-10 ℃; after the reaction is carried out in an ice-water bath for 30min, the temperature is gradually increased to 25 ℃, and the cracking is continued for 2 h. Adding precooled diethyl ether with the volume 15 times of the volume of the lysate for centrifugal precipitation to obtain 320mg of a 26-oxoacyl iso Abeta 1-42 crude product;

(5) preparing the A beta 1-42 pure product.

Dissolving the crude 26-oxoacyl iso Abeta 1-42 product in 5% methanol water solution, purifying by high performance liquid chromatography to obtain refined 26-oxoacyl iso Abeta 1-42 product, and freeze drying to obtain 72.6mg product with purity of 97.361% and total yield of 12.20%. The purification conditions were: 30mm of polymer (polystyrene, particle size 10um 300A, column length 25cm, diameter 30mm), mobile phase A of 0.25% aqueous formic acid solution, and phase B of 0.1% TFA/acetonitrile solution; phase B is changed from 5% to 22% in 0-5 min; changing the phase B from 22% to 42% within 5-50min, operating for 45min, and controlling the temperature of the mobile phase at 60 ℃. The polypeptide was dissolved in acetonitrile/water at a concentration of 1mg/mL, and a thousandth of ammonia water was added thereto to adjust the pH to 7.4, followed by reaction at 25 ℃ for 4 hours to obtain 64.3mg of a β 1-42 as a solid after lyophilization, the purity of which was 97.153%. The high performance liquid chromatogram is shown in FIG. 6, and the mass spectrum is shown in FIG. 7.

Example 5: preparation of Abeta 1-42

(1) And (3) synthesizing Fmoc-Ala-CTC resin.

Weighing 3g of 2-CTC resin with the substitution degree of 0.98mmol/g, adding the resin into a cup-shaped reaction column, swelling the resin with DCM for 30 minutes, and filtering to remove DCM; weighing 1mmol of Fmoc-Ala-OH, adding DCM and DIEA into a reaction cup, and carrying out nitrogen bubbling reaction for 90 min; adding 3mL of methanol and DIEA to perform end-capping reaction for 30min, washing with DMF for 4 times, washing with DCM for 1 time, and then draining with methanol to obtain Fmoc-Gly-CTC resin; a small amount of the resin was used and the degree of substitution was measured by UV spectrophotometer to be 0.25mmol/g (three times average).

(2)2-CTC resin Ile⁴¹Coupling of (3).

Fmoc-Ala-CTC resin with a degree of substitution of 0.25mmol/g, prepared in example 1, was placed in a cup-shaped reaction column, the resin was swollen with DCM for 30min, after which DCM was removed by suction filtration; adding 20% of Pip/DMF to remove the Fmoc protecting group, wherein the removal time is 25min, adding DMF to wash for 5 times, DCM to wash for 1 time, and DMF to wash for 1 time, and ninhydrin detecting the color development of the resin, and simultaneously ensuring that piperidine is washed cleanly without residue. 2.4g of Fmoc-Ile-OH (2.25mmol) and 2.6g of HATU (2.25mmol) were weighed out, dissolved in 15mL of DMF, and 1.1mL of DIEA (6.75mmol) was added thereto, reacted for 2min, poured into a cup-shaped reaction column, and subjected to nitrogen bubbling for 70 min.

(3) Preparing a protected 26-oxoacyl-iso-Abeta 1-42 resin compound.

40mL of 20% Pip/DMF solution was added to the reactor and reacted at room temperature for 25min, then DMF was added and washed 6 times, DCM was washed 1 time, and ninhydrin detection was performed to detect the color development of the resin. 2.25mmol of Fmoc-Val-OH, 2.8mmol of HOBt and 2.25mmol of DIC were dissolved in 15mL of DMF, and the resulting solution was added to a reactor and reacted at 25 ℃ for 70 min. After the reaction was completed, the mixture was filtered. Then 50mL of DMF was added and the resin was washed six times.

Amino acids are coupled in sequence from C end to N end of 26-oxoacyl iso Abeta 1-42, and cyclic deprotection reaction (Fmoc protective group is removed by 20% Pip/DMF for 25min and 1 time) and condensation reaction are carried out until the synthesis of peptide chain is finished. Wherein Fmoc-Ile³¹-OH，Fmoc-Ile³²The coupling method of-OH is that amino acid is fed by 5eq, the reaction concentration is 0.3mmol/mL, DIC/HOBt is used as a coupling reagent, DMF is used as a solvent, the coupling is carried out for 40min, then suction filtration is carried out, HATU/HOBT/DIEA is used for coupling again for 40min after DMF is used for washing resin for 1 time, and the solvent is DMF.

All amino acids Fmoc were removed once in 20% piperidine/DMF for 25 min.

(4) Preparing a crude product of the protected 26-oxoacyl iso Abeta 1-42.

Placing 1g of the 26-oxoacyl-iso-Abeta 1-42 resin compound in an ice-water bath, pre-cooling a cleavage reagent (ammonium iodide (1mg/mL) is added in a ratio of TFA to benzylsulfide to phenol to EDT to water of 87.5: 2.5: 2.5: 5: 2.5) at 0-10 ℃, and adding the cleavage reagent into 26-oxoacyl-iso-Abeta 1-42 resin peptide; after the reaction is carried out in an ice-water bath for 30min, the temperature is gradually increased to 25 ℃, and the cracking is continued for 2 h. Adding precooled diethyl ether with the volume 15 times of the lysate, and obtaining 130mg of crude 26-oxoacyl-iso-Abeta 1-42 after centrifugal precipitation, wherein the mass chromatogram of the crude product is shown in figure 9, and the high performance liquid chromatogram is shown in figure 10; it is difficult to prepare a β 1-42 pure product because the mass-to-charge ratio obtained is the residual peptide after cleavage of the ester bond (molecular weight: 1598, bivalent 799.70) without the correct target molecular weight.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims

1. A method for preparing human serum amyloid A beta 1-42, comprising:

2. The method according to claim 1, wherein the coupling agent is HATU/DIEA or Pybop/DIEA in the step of coupling the 41 st amino acid.

3. The method of claim 1, wherein the coupling step of coupling the amino acids at positions 32 and 31 or Boc-Ser (Fmoc-Gly) -OH comprises performing two coupling reactions using DIC/HOBt and HATU/HOBT/DIEA as coupling reagents.

4. The process according to claim 2 or 3, wherein the coupling agent for the coupling step is selected from DIC/HOBt, HATU/HOBT/DIEA, HBTU/HOBT/DIEA or PyBOP/HOBT/DIEA, in addition to the amino acids 41, 32, 31, 25-26.

5. The process according to any one of claims 1 to 4, wherein the amino acid is charged in an amount of 5eq in the step of coupling the amino acids at positions 32 and 31.

6. The method according to any one of claims 1 to 5, wherein the coupling solvent is selected from DMF and CH₂Cl₂、NMP。

7. The method according to any one of claims 1 to 5, wherein after coupling any amino acid or polypeptide in 1 to 25 positions, the Fmoc removal is performed at 20 to 25 ℃ for 5min for 2 times;

8. The method according to any one of claims 1 to 6, wherein the synthesis is solid phase synthesis, and the carrier is CTC resin or Wang resin.

9. The method according to claim 8, wherein the cleavage agent for cleaving the resin after the solid phase synthesis is a mixture of TFA and at least one of thioanisole, phenol, EDT, ammonium iodide and water.

10. The production method according to any one of claims 1 to 9, wherein the acyl group transfer is carried out by dissolving 26-oxoacyliso a β 1-42 in an aqueous acetonitrile solution, adjusting pH to 7.4 with ammonia water, and reacting at 25 ℃ for 4 hours to obtain a β 1-42.