CN106916219A

CN106916219A - A kind of synthetic method of cold labeling alpha-lactalbumin feature peptide fragment

Info

Publication number: CN106916219A
Application number: CN201710228093.7A
Authority: CN
Inventors: 徐仲杰; 罗勇; 钟佳琪; 涂亚辉; 王浩然; 孙雯; 卢伟京; 杨维成
Original assignee: Shanghai Research Institute of Chemical Industry SRICI
Current assignee: Shanghai Research Institute of Chemical Industry SRICI
Priority date: 2017-04-10
Filing date: 2017-04-10
Publication date: 2017-07-04

Abstract

The present invention relates to the synthetic method of cold labeling alpha-lactalbumin feature peptide fragment, using liquid phase synthesis polypeptide method, with cold labeling or non-marked valine as initiation material, the non-marked or marked glycine protected with Fmoc, in the presence of condensing agent, synthesis dipeptides, then protection group Fmoc is taken off, the non-marked or mark isoleucine protected with Fmoc again, in the presence of condensing agent, generation tripeptides, the non-marked or mark asparagine protected with Fmoc successively, tyrosine, tryptophan, leucine, alanine, histidine and the non-marked protected with Cbz or mark lysine are reacted, ultimately generate cold labeling alpha-lactalbumin feature peptide fragment VGINYWLAHK.Up to more than 99%, isotope abundance can fully meet the detection needs of anaphylactogen alpha-lactalbumin in food to the product chemical purity of present invention synthesis more than 99%.

Description

A kind of synthetic method of cold labeling ALA feature peptide fragment

Technical field

The present invention relates to isotope marks field, more particularly, to a kind of cold labeling ALA feature peptide The synthetic method of section.

Background technology

Food allergen refers to that human body cell can be selectively activated present in food, and inducing producing specificity antibody should Answer, cause the antigenicity substance of allergy.In general, food allergen is protein or glycoprotein, is belonging respectively to difference Protein families.Current about 160 numerous foods contain and can cause anaphylactoid anaphylactogen, about 260 kinds points Son is marked as food allergen.Epidemiological study shows that 90% food hypersenstivity is caused by 8 group foods, generally quilt Referred to as " 8 is big " sensitization food, including milk, eggs, soybean, peanut, nut, wheat, fish and shellfish.The symptom of food hypersenstivity Including nettle rash, vomiting and asthma etc., even entail dangers to life when serious.In the threat human health of WHO Report Factor in, food hypersenstivity comes the 4th.Allergen issue quantity is arranged in the relevant warning circular of domestic and international food in recent years Name the 2nd, is only second to microorganism pollution, the industrial hot spot as international common concern.

Cow's milk contains rich in protein, deep to be favored by people, is daily life especially for infant and the elderly Nutriment source main in work.But meanwhile, cow's milk is again one of main allergenic foods.Investigation shows that Milk allergy exists The incidence of disease in crowd is 0.3-7.5%, wherein the incidence of disease in one-year-old Infants Below reaches 2-3%.α in cow's milk-milky white Albumen belongs to bacteriolyze enzyme family, is to cause one of major allergen protein of Milk allergy, according to incompletely statistics, more than 51% Milk allergy is caused by ALA.ALA is about with the homology of ALA in human milk in cow's milk 74%, separately there is 6% amino acid residue chemical property similar, it is of high nutritive value but sensitization is strong.Cow's milk is used for frequently as raw material Various food, so carrying out the detection for ALA in food with important theory and realistic meaning.

Mainly there are immunoassay, PCR methods and instrument point for the method that allergy aurochs ALA is detected at present Analysis method, high performance liquid chromatography-tandem mass method (LC/MS/MS) application is most in instrumental method.But, the base in food Mass effect and unscientific pre-treating method can cause relatively large deviation to analysis result.

Isotope dilution mass spectrometry IDMS (Isotope Dilution Mass Spectrometry) is used and determinand With identical molecular structure stable isotope (²H、¹³C、¹⁵N) organic matter of mark is used as internal standard, after being mixed using mass spectroscopy The ratio between mole number of ions of internal standard and determinand, is little affected by the interference of various physics and chemical factor in sample, can be effective Eliminate the influences of the factor to analysis result such as matrix effect and pre-treatment.At present, developed country is for anaphylactogen α-breast in food Albumin is typically detected using isotope dilution mass spectrometry.

The ALA feature peptide fragment VGINYWLAHK of cold labeling has become present analysis test experience The running stores that room must be purchased.But ALA feature peptide fragment internal standard reagent is completely dependent on import, expensive, serious limitation The application in the field such as its food security and inspection and quarantine.And stable isotope ALA feature peptide fragment is succeeded in developing, Standard reagent will be provided for more accurate quantitative analysis ALA, improve China's food safety detection system.

The external isotope company with CIL Corp. of the U.S. as representative has formed more complete in terms of isotope labeling reagent Technology and product chain, but the reason such as monopolize for technical know-how and product, about all kinds of skills of Isotopic Internal Standard tube- nursery Art document and the nearly no complete report of patent.

Chinese patent CN103642895A discloses the method and reagent of a kind of quantitative determination people ALA content Box.The specific polypeptide sequence CELSQLLK obtained after being digested using people's ALA, designs the special of isotope marks The sequence of peptide and isotope marks internal standard compound, and based on internal standard method provide a kind of brand-new accurate quantitative analysis detection method to people α- Lactoalbumin carries out quantitative determination.But should be patent disclosed that a kind of detection method, protein characteristic peptide fragment not be disclosed Synthetic method.

The content of the invention

The invention aims to fill up domestic cold labeling ALA feature peptide fragment VGINYWLAHK Blank, and provide that a kind of process route is simple, be swift in response, easily prepared cold labeling ALA feature peptide The synthetic method of section VGINYWLAHK.

The purpose of the present invention can be achieved through the following technical solutions：

Using liquid phase synthesis polypeptide method, with cold labeling or non-marked valine (Val) as initiation material, and The non-marked or marked glycine (Gly) of Fmoc protections, in the presence of condensing agent, synthesize dipeptides, then take off protection group Fmoc, then non-marked or mark isoleucine (Ile) with Fmoc protections, in the presence of condensing agent, generate tripeptides, take off Protection group Fmoc, the non-marked protected with Fmoc successively in this manner or mark asparagine (Asn), tyrosine (Tyr), tryptophan (Trp), leucine (Leu), alanine (Ala), histidine (His) and with Cbz protect non-marked or Person marks lysine (Lys) to be reacted, and ultimately generates cold labeling ALA feature peptide fragment VGINYWLAHK, Specifically use following steps：

1) with cold labeling or non-marked valine (H-Val-MH₂) it is initiation material, it is non-with what Fmoc was protected Mark or marked glycine (Fmoc-Gly-OH) carry out coupling reaction in the presence of condensing agent, are then dried successively, Vacuum distillation, is recrystallized to give dipeptides Fmoc-Gly-Val-NH₂, then take off protection group Fmoc and obtain H-Gly-Val-NH₂；

2) step 1) H-Gly-Val-NH for preparing₂The non-marked or mark isoleucine protected with Fmoc (Fmoc-Ile-OH) coupling reaction is carried out in the presence of condensing agent, is then dried successively, vacuum distillation is recrystallized To tripeptides Fmoc-Ile-Gly-Val-NH₂, take off protection group Fmoc and obtain H-Ile-Gly-Val-NH₂；

3) step 2) H-Ile-Gly-Val-NH for preparing₂The non-marked or mark asparagine protected with Fmoc (Fmoc-Asn-OH) coupling reaction is carried out in the presence of condensing agent, is then dried successively, vacuum distillation is recrystallized To tetrapeptide Fmoc-Asn-Ile-Gly-Val-NH₂, take off protection group Fmoc and obtain H-Asn-Ile-Gly-Val-NH₂；

4) step 3) H-Asn-Ile-Gly-Val-NH for preparing₂The non-marked or mark junket ammonia protected with Fmoc Sour (Fmoc-Tyr-OH) carries out coupling reaction in the presence of condensing agent, is then dried successively, vacuum distillation, recrystallizes Obtain pentapeptide Fmoc-Tyr-Asn-Ile-Gly-Val-NH₂, take off protection group Fmoc and obtain H-Tyr-Asn-Ile-Gly-Val- NH₂；

5) step 4) H-Tyr-Asn-Ile-Gly-Val-NH for preparing₂The non-marked or mark protected with Fmoc Tryptophan (Fmoc-Trp-OH) carries out coupling reaction in the presence of condensing agent, is then dried successively, vacuum distillation, weight Crystallization obtains hexapeptide Fmoc-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, take off protection group Fmoc and obtain H-Trp-Tyr-Asn- Ile-Gly-Val-NH₂；

6) step 5) H-Trp-Tyr-Asn-Ile-Gly-Val-NH for preparing₂With Fmoc protect non-marked or Labelled leucine (Fmoc-Leu-OH) carries out coupling reaction in the presence of condensing agent, is then dried successively, and decompression is steamed Evaporate, be recrystallized to give heptapeptide Fmoc-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, take off protection group Fmoc and obtain H- Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂；

7) step 6) H-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH for preparing₂The non-marked protected with Fmoc Or mark alanine (Fmoc-Ala-OH) carries out coupling reaction in the presence of condensing agent, is then dried successively, depressurize Distillation, is recrystallized to give octapeptide Fmoc-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, take off protection group Fmoc and obtain To H-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂；

8) step 7) H-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH for preparing₂It is non-with what Fmoc was protected Mark or mark histidine (Fmoc-His-OH) carry out coupling reaction in the presence of condensing agent, are then dried successively, Vacuum distillation, is recrystallized to give nonapeptide Fmoc-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, take off protection Base Fmoc obtains H-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂；

9) step 8) H-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH for preparing₂Protected with Cbz Non-marked or mark lysine (Cbz-Lys-OH) coupling reaction is carried out in the presence of condensing agent, then done successively Dry, vacuum distillation is recrystallized to give decapeptide Cbz-Lys-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, take off Fall protection group Cbz and obtain H-Lys-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, as ALA is special Levy peptide fragment VGINYWLAHK.

With non-marked valine (H-Val-MH₂) for initiation material when, step 1) to step 9) in Fmoc protections 9 kinds of ammonia Base acid and the lysine of Cbz protections, at least one kind is stable isotope¹³C、¹⁵The amino acid of one or more marks of N, D.

Step 1) in, described cold labeling or non-marked valine (H-Val-MH₂) and Fmoc protection it is non- Mark or the process conditions that are coupled of marked glycine (Fmoc-Gly-OH) are：

The condensing agent for using is one or two mixtures and HBTU, HCTU, HOBt, HOCt in EDCI, DMAP, DCC In one or two kinds of mixture mixed liquor, solvent is chloroform, and reaction temperature is 0-20 DEG C,

Cold labeling or non-marked valine (H-Val-MH₂), Fmoc protect glycine (Fmoc-Gly-OH) and The mol ratio of condensing agent is 1:1:1.1-1:1:2；

Removing Fmoc-Gly-Val-NH₂The reagent that middle Fmoc protection groups are used is in triethylamine, diethylamine, piperidines Plant or several, solvent is one or more in dichloromethane, chloroform, ether, Fmoc-Gly-Val-NH₂, removing reagent and The mol ratio of solvent is 1:1:5-1:3:15,0-20 DEG C of controlling reaction temperature, reaction time 2-6 hours.

Step 2) in, described H-Gly-Val-NH₂The non-marked or mark isoleucine (Fmoc- protected with Fmoc Ile-OH the process conditions) being coupled are：Condensing agent be EDCI, DMAP, DCC in one or two mixtures and HBTU, The mixed liquor of the one or two kinds of mixture in HCTU, HOBt, HOCt, solvent is chloroform, and reaction temperature is 0-20 DEG C,

H-Gly-Val-NH₂The non-marked or mark isoleucine (Fmoc-Ile-OH) and condensing agent protected with Fmoc Mol ratio be 1:1:1.1-1:1:2；

Removing Fmoc-Ile-Gly-Val-NH₂The reagent that middle Fmoc protection groups are used is in triethylamine, diethylamine, piperidines One or more, solvent be dichloromethane, chloroform, ether in one or more, Fmoc-Ile-Gly-Val-NH₂, removing It is 1 with the mol ratio of reagent and solvent:1:5-1:3:15,0-20 DEG C of reaction temperature, reaction time 2-6 hours.

Step 3) in, described H-Ile-Gly-Val-NH₂The non-marked or mark asparagine protected with Fmoc (Fmoc-Asn-OH) process conditions being coupled are：Condensing agent be EDCI, DMAP, DCC in one or two mixtures with The mixed liquor of the one or two kinds of mixture in HBTU, HCTU, HOBt, HOCt, solvent is chloroform, and reaction temperature is 0-20 DEG C,

H-Ile-Gly-Val-NH₂The non-marked or mark asparagine (Fmoc-Asn-OH) protected with Fmoc and contracting The mol ratio of mixture is 1:1:1.1-1:1:2；

Removing Fmoc-Asn-Ile-Gly-Val-NH₂The reagent that middle Fmoc protection groups are used is triethylamine, diethylamine, piperazine One or more in pyridine, solvent is one or more in dichloromethane, chloroform, ether, Fmoc-Asn-Ile-Gly-Val- NH₂, removing reagent and solvent mol ratio be 1:1:5-1:3:15,0-20 DEG C of reaction temperature, reaction time 2-6 hours.

Step 4) in, described H-Asn-Ile-Gly-Val-NH₂The non-marked or mark tyrosine protected with Fmoc (Fmoc-Tyr-OH) process conditions being coupled are：Condensing agent be EDCI, DMAP, DCC in one or two mixtures with The mixed liquor of the one or two kinds of mixture in HBTU, HCTU, HOBt, HOCt, solvent is chloroform, and reaction temperature is 0-20 DEG C,

H-Asn-Ile-Gly-Val-NH₂With Fmoc protect non-marked or mark tyrosine (Fmoc-Tyr-OH) and The mol ratio of condensing agent is 1:1:1.1-1:1:2；

Removing Fmoc-Tyr-Asn-Ile-Gly-Val-NH₂The reagent that middle Fmoc protection groups are used is triethylamine, diethyl One or more in amine, piperidines, solvent is one or more in dichloromethane, chloroform, ether, Fmoc-Tyr-Asn- Ile-Gly-Val-NH₂, removing reagent and solvent mol ratio be 1:1:5-1:3:15,0-20 DEG C of reaction temperature, during reaction Between 2-6 hours.

Step 5) in, described H-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or mark color protected with Fmoc The process conditions that propylhomoserin (Fmoc-Trp-OH) is coupled are：Condensing agent is one or two mixing in EDCI, DMAP, DCC The mixed liquor of the one or two kinds of mixture in thing and HBTU, HCTU, HOBt, HOCt, solvent is chloroform, and reaction temperature is 0- 20 DEG C,

H-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or mark tryptophan (Fmoc-Trp- protected with Fmoc OH) and condensing agent mol ratio be 1:1:1.1-1:1:2；

Removing Fmoc-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The reagent that middle Fmoc protection groups are used is triethylamine, two One or more in ethamine, piperidines, solvent is one or more in dichloromethane, chloroform, ether, Fmoc-Trp-Tyr- Asn-Ile-Gly-Val-NH₂, removing reagent and solvent mol ratio be 1:1:5-1:3:15,0-20 DEG C of reaction temperature, instead 2-6 hours between seasonable.

Step 6) in, described H-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or mark protected with Fmoc Remember that the process conditions that leucine (Fmoc-Leu-OH) is coupled are：Condensing agent be EDCI, DMAP, DCC in one or two The mixed liquor of the one or two kinds of mixture in mixture and HBTU, HCTU, HOBt, HOCt, solvent is chloroform, reaction temperature It is 0-20 DEG C,

H-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or labelled leucine (Fmoc- protected with Fmoc Leu-OH) and condensing agent mol ratio be 1:1:1.1-1:1:2；

Removing Fmoc-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The reagent that middle Fmoc protection groups are used is three second One or more in amine, diethylamine, piperidines, solvent is one or more in dichloromethane, chloroform, ether, Fmoc-Leu- Trp-Tyr-Asn-Ile-Gly-Val-NH₂, removing reagent and solvent mol ratio be 1:1:5-1:3:15, reaction temperature 0- 20 DEG C, reaction time 2-6 hours.

Step 7) in, described H-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂With Fmoc protect non-marked or Person marks alanine (Fmoc-Ala-OH) process conditions for being coupled to be：Condensing agent be EDCI, DMAP, DCC in one kind or The mixed liquor of the one or two kinds of mixture in two kinds of mixtures and HBTU, HCTU, HOBt, HOCt, solvent is chloroform, reaction Temperature is 0-20 DEG C,

H-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or mark alanine protected with Fmoc (Fmoc-Ala-OH) and condensing agent mol ratio be 1:1:1.1-1:1:2；

Removing Fmoc-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The reagent that middle Fmoc protection groups are used for One or more in triethylamine, diethylamine, piperidines, solvent is one or more in dichloromethane, chloroform, ether, Fmoc- Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, removing reagent and solvent mol ratio be 1:1:5-1:3:15, 0-20 DEG C of reaction temperature, reaction time 2-6 hours.

Step 8) in, described H-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂It is nonstandard with what Fmoc was protected Remember or mark that the process conditions that histidine (Fmoc-His-OH) is coupled are：Condensing agent is in EDCI, DMAP, DCC The mixed liquor of the one or two kinds of mixture in kind or two kinds of mixtures and HBTU, HCTU, HOBt, HOCt, solvent is chloroform, Reaction temperature is 0-20 DEG C,

H-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or mark histidine protected with Fmoc (Fmoc-His-OH) and condensing agent mol ratio be 1:1:1.1-1:1:2；

Removing Fmoc-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The examination that middle Fmoc protection groups are used Agent is one or more in triethylamine, diethylamine, piperidines, and solvent is one or more in dichloromethane, chloroform, ether, Fmoc-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, removing reagent and solvent mol ratio be 1:1:5- 1:3:15,0-20 DEG C of reaction temperature, reaction time 2-6 hours.

Step 9) in, described H-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂It is non-with what Cbz was protected Mark or mark lysine (Cbz-Lys-OH) process conditions for being coupled are：Condensing agent is in EDCI, DMAP, DCC The mixed liquor of the one or two kinds of mixture in one or two mixtures and HBTU, HCTU, HOBt, HOCt, solvent is chlorine Imitative, reaction temperature is 0-20 DEG C,

H-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or mark protected with Cbz rely The mol ratio of propylhomoserin (Cbz-Lys-OH) and condensing agent is 1:1:1.1-1:1:2；

Removing Cbz-Lys-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂Middle Cbz protection groups are in gold Hydrogen or hydrogen bromide/acetic acid mixed solvent are passed through under category palladium chtalyst；Cbz-Lys-His-Ala-Leu-Trp-Tyr-Asn-Ile- Gly-Val-NH₂, Metal Palladium and hydrogen mol ratio be 1:0.01:1-1:0.05:15, reaction temperature is 0-50 DEG C, reaction time 1-4 hours；Cbz-Lys-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, hydrogen bromide/acetic acid mol ratio be 1:1-1:5, reaction temperature is 0-50 DEG C, reaction time 1-4 hours.

The ALA feature peptide fragment VGINYWLAHK for preparing is cold labeling¹³C、¹⁵Tri- kinds of N, D is same One or more marks in the element of position, its molecular structural formula is as follows：H-Lys-His-Ala-Leu-Trp-Tyr-Asn- Ile-Gly-Val-NH₂, wherein,

H-Lys-NH₂For¹³C、¹⁵One or more marks in tri- kinds of isotopes of N, D；

H-His-NH₂For¹³C、¹⁵One or more marks in tri- kinds of isotopes of N, D；

H-Ala-NH₂For¹³C、¹⁵One or more marks in tri- kinds of isotopes of N, D；

H-Leu-NH₂For¹³C、¹⁵One or more marks in tri- kinds of isotopes of N, D；

H-Trp-NH₂For¹³C、¹⁵One or more marks in tri- kinds of isotopes of N, D；

H-Tyr-NH₂For¹³C、¹⁵One or more marks in tri- kinds of isotopes of N, D；

H-Asn-NH₂For¹³C、¹⁵One or more marks in tri- kinds of isotopes of N, D；

H-Ile-NH₂For¹³C、¹⁵One or more marks in tri- kinds of isotopes of N, D；

H-Gly-NH₂For¹³C、¹⁵One or more marks in tri- kinds of isotopes of N, D；

H-Val-NH₂For¹³C、¹⁵One or more marks in tri- kinds of isotopes of N, D.

Compared with prior art, the present invention has advantages below：

1st, synthesize present invention firstly discloses a kind of cold labeling ALA feature peptide fragment VGINYWLAHK Method, the country there are no the isotope marks synthesis report of this peptide fragment, and reason is mainly and is limited by price and application, now with The internal standard polypeptide of state's object detection method, needs the peptide fragment for making isotope marks just to carry out anaphylactogen this peptide fragment Detection, therefore isotope marks are needed, without report, the application is first time report for the country；

2nd, the present invention is simple using process route, and the reaction time is short, it is easy to synthesize, and stable isotope atom utilization is high；

3rd, the easily separated purification of product of the present invention, liquid phase method synthesis polypeptide be from solvent separate out crystal, therefore purity compared with Good, more than 99%, isotope abundance can fully meet anaphylactogen α-milky white egg in food to product chemical purity more than 99% The demand of white trace detection；

4th, economy of the present invention and use value are good, with preferable market promotion prospect.

Specific embodiment

The synthetic method of cold labeling ALA feature peptide fragment, using liquid phase synthesis polypeptide method, with steady It is initiation material to determine isotope marks or non-marked valine (Val), and Fmoc protections non-marked or marked glycine (Gly), in the presence of condensing agent, synthesize dipeptides, then take off protection group Fmoc, then non-marked or mark with Fmoc protections Note isoleucine (Ile), in the presence of condensing agent, generate tripeptides, take off protection group Fmoc, in this manner successively and The non-marked of Fmoc protections or mark asparagine (Asn), tyrosine (Tyr), tryptophan (Trp), leucine (Leu), third Propylhomoserin (Ala), histidine (His) and the non-marked protected with Cbz or mark lysine (Lys) are reacted, most throughout one's life Into cold labeling ALA feature peptide fragment VGINYWLAHK, specifically using following steps：

6) step 5) H-Trp-Tyr-Asn-Ile-Gly-Val-NH for preparing₂With Fmoc protect non-marked or Labelled leucine (Fmoc-Leu-OH) carries out coupling reaction in the presence of condensing agent, is then dried successively, and decompression is steamed Evaporate, be recrystallized to give heptapeptide Fmoc-Leu-Trp-Tyr-Asn-Ile-Gly-Val-

NH₂, take off protection group Fmoc and obtain H-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂；

It should be noted that step 1) in if with non-marked valine (H-Val-MH₂) for initiation material when, step 1) To step 9) in Fmoc protection 9 kinds of amino acid and Cbz protection lysine, at least one kind is stable isotope¹³C、¹⁵N、D One or more mark amino acid.

Step 1) in, cold labeling or non-marked valine (H-Val-MH₂) and Fmoc protection non-marked or The process conditions that person's marked glycine (Fmoc-Gly-OH) is coupled are：The condensing agent for using is in EDCI, DMAP, DCC The mixed liquor of the one or two kinds of mixture in one or two mixtures and HBTU, HCTU, HOBt, HOCt, solvent is chlorine Imitative, reaction temperature is 0-20 DEG C, cold labeling or non-marked valine (H-Val-MH₂), Fmoc protection glycine (Fmoc-Gly-OH) and condensing agent mol ratio be 1:1:1.1-1:1:2；Removing Fmoc-Gly-Val-NH₂Middle Fmoc protection groups The reagent for using is one or more in triethylamine, diethylamine, piperidines, and solvent is the one kind in dichloromethane, chloroform, ether Or several, Fmoc-Gly-Val-NH₂, removing reagent and solvent mol ratio be 1:1:5-1:3:15, controlling reaction temperature 0- 20 DEG C, reaction time 2-6 hours.

Step 2) in, H-Gly-Val-NH₂The non-marked or mark isoleucine (Fmoc-Ile-OH) protected with Fmoc The process conditions being coupled are：Condensing agent be EDCI, DMAP, DCC in one or two mixtures and HBTU, HCTU, The mixed liquor of the one or two kinds of mixture in HOBt, HOCt, solvent is chloroform, and reaction temperature is 0-20 DEG C, H-Gly- Val-NH₂The non-marked or the mol ratio of mark isoleucine (Fmoc-Ile-OH) and condensing agent protected with Fmoc are 1:1: 1.1-1:1:2；Removing Fmoc-Ile-Gly-Val-NH₂The reagent that middle Fmoc protection groups are used is triethylamine, diethylamine, piperidines In one or more, solvent be dichloromethane, chloroform, ether in one or more, Fmoc-Ile-Gly-Val-NH₂, it is de- Except being 1 with the mol ratio of reagent and solvent:1:5-1:3:15,0-20 DEG C of reaction temperature, reaction time 2-6 hours.

Step 3) in, H-Ile-Gly-Val-NH₂The non-marked or mark asparagine (Fmoc- protected with Fmoc Asn-OH the process conditions) being coupled are：Condensing agent be EDCI, DMAP, DCC in one or two mixtures and HBTU, The mixed liquor of the one or two kinds of mixture in HCTU, HOBt, HOCt, solvent is chloroform, and reaction temperature is 0-20 DEG C, H- Ile-Gly-Val-NH₂With Fmoc protect non-marked or mark asparagine (Fmoc-Asn-OH) and condensing agent mole Than being 1:1:1.1-1:1:2；Removing Fmoc-Asn-Ile-Gly-Val-NH₂The reagent that middle Fmoc protection groups are used for triethylamine, One or more in diethylamine, piperidines, solvent is one or more in dichloromethane, chloroform, ether, Fmoc-Asn- Ile-Gly-Val-NH₂, removing reagent and solvent mol ratio be 1:1:5-1:3:15,0-20 DEG C of reaction temperature, during reaction Between 2-6 hours.

Step 4) in, H-Asn-Ile-Gly-Val-NH₂The non-marked or mark tyrosine (Fmoc- protected with Fmoc Tyr-OH the process conditions) being coupled are：Condensing agent be EDCI, DMAP, DCC in one or two mixtures and HBTU, The mixed liquor of the one or two kinds of mixture in HCTU, HOBt, HOCt, solvent is chloroform, and reaction temperature is 0-20 DEG C, H- Asn-Ile-Gly-Val-NH₂With the non-marked or mark tyrosine (Fmoc-Tyr-OH) of Fmoc protections and rubbing for condensing agent You are than being 1:1:1.1-1:1:2；Removing Fmoc-Tyr-Asn-Ile-Gly-Val-NH₂The reagent that middle Fmoc protection groups are used for One or more in triethylamine, diethylamine, piperidines, solvent is one or more in dichloromethane, chloroform, ether, Fmoc- Tyr-Asn-Ile-Gly-Val-NH₂, removing reagent and solvent mol ratio be 1:1:5-1:3:15, reaction temperature 0-20 DEG C, reaction time 2-6 hours.

Step 5) in, H-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or mark tryptophan protected with Fmoc (Fmoc-Trp-OH) process conditions being coupled are：Condensing agent be EDCI, DMAP, DCC in one or two mixtures with The mixed liquor of the one or two kinds of mixture in HBTU, HCTU, HOBt, HOCt, solvent is chloroform, and reaction temperature is 0-20 DEG C, H-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or mark tryptophan (Fmoc-Trp-OH) protected with Fmoc and contracting The mol ratio of mixture is 1:1:1.1-1:1:2；Removing Fmoc-Trp-Tyr-Asn-Ile-Gly-Val-NH₂Middle Fmoc protection groups The reagent for using is one or more in triethylamine, diethylamine, piperidines, and solvent is the one kind in dichloromethane, chloroform, ether Or several, Fmoc-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, removing reagent and solvent mol ratio be 1:1:5-1:3: 15,0-20 DEG C of reaction temperature, reaction time 2-6 hours.

Step 6) in, H-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or the bright ammonia of mark protected with Fmoc The process conditions that sour (Fmoc-Leu-OH) is coupled are：Condensing agent is one or two mixtures in EDCI, DMAP, DCC With the mixed liquor of the one or two kinds of mixture in HBTU, HCTU, HOBt, HOCt, solvent is chloroform, and reaction temperature is 0-20 DEG C, H-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or labelled leucine (Fmoc-Leu-OH) protected with Fmoc And the mol ratio of condensing agent is 1:1:1.1-1:1:2；Removing Fmoc-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂In The reagent that Fmoc protection groups are used is one or more in triethylamine, diethylamine, piperidines, and solvent is dichloromethane, chloroform, second One or more in ether, Fmoc-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, removing reagent and solvent mole Than being 1:1:5-1:3:15,0-20 DEG C of reaction temperature, reaction time 2-6 hours.

Step 7) in, H-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or mark protected with Fmoc The process conditions that alanine (Fmoc-Ala-OH) is coupled are：Condensing agent is mixed for one or two in EDCI, DMAP, DCC The mixed liquor of the one or two kinds of mixture in compound and HBTU, HCTU, HOBt, HOCt, solvent is chloroform, and reaction temperature is 0-20 DEG C, H-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or mark alanine protected with Fmoc (Fmoc-Ala-OH) and condensing agent mol ratio be 1:1:1.1-1:1:2；Removing Fmoc-Ala-Leu-Trp-Tyr-Asn- Ile-Gly-Val-NH₂The reagent that middle Fmoc protection groups are used is one or more in triethylamine, diethylamine, piperidines, solvent It is one or more in dichloromethane, chloroform, ether, Fmoc-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂、 The mol ratio of removing reagent and solvent is 1:1:5-1:3:15,0-20 DEG C of reaction temperature, reaction time 2-6 hours.

Step 8) in, H-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂With Fmoc protect non-marked or Mark histidine (Fmoc-His-OH) process conditions for being coupled are：Condensing agent is the one kind or two in EDCI, DMAP, DCC The mixed liquor of the one or two kinds of mixture in mixture and HBTU, HCTU, HOBt, HOCt is planted, solvent is chloroform, reaction temperature It is 0-20 DEG C to spend, H-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or mark group protected with Fmoc The mol ratio of propylhomoserin (Fmoc-His-OH) and condensing agent is 1:1:1.1-1:1:2；Removing Fmoc-His-Ala-Leu-Trp- Tyr-Asn-Ile-Gly-Val-NH₂The reagent that middle Fmoc protection groups are used is one kind in triethylamine, diethylamine, piperidines or several Kind, solvent is one or more in dichloromethane, chloroform, ether, Fmoc-His-Ala-Leu-Trp-Tyr-Asn-Ile- Gly-Val-NH₂, removing reagent and solvent mol ratio be 1:1:5-1:3:15,0-20 DEG C of reaction temperature, reaction time 2-6 Hour.

Step 9) in, H-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂With Cbz protect non-marked or Person marks lysine (Cbz-Lys-OH) process conditions for being coupled to be：Condensing agent be EDCI, DMAP, DCC in one kind or The mixed liquor of the one or two kinds of mixture in two kinds of mixtures and HBTU, HCTU, HOBt, HOCt, solvent is chloroform, reaction Temperature is 0-20 DEG C, H-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or mark protected with Cbz The mol ratio of note lysine (Cbz-Lys-OH) and condensing agent is 1:1:1.1-1:1:2；Removing Cbz-Lys-His-Ala-Leu- Trp-Tyr-Asn-Ile-Gly-Val-NH₂Middle Cbz protection groups are that hydrogen or hydrogen bromide/acetic acid are passed through under catalyzing by metal palladium is mixed Bonding solvent；Cbz-Lys-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, Metal Palladium and hydrogen mol ratio be 1: 0.01:1-1:0.05:15, reaction temperature is 0-50 DEG C, reaction time 1-4 hours；Cbz-Lys-His-Ala-Leu-Trp- Tyr-Asn-Ile-Gly-Val-NH₂, hydrogen bromide/acetic acid mol ratio be 1:1-1:5, reaction temperature is 0-50 DEG C, during reaction Between 1-4 hours.

H-Lys-NH₂For¹³C、¹⁵One or more marks in tri- kinds of isotopes of N, D；H-His-NH₂For¹³C、¹⁵N、D One or more marks in three kinds of isotopes；H-Ala-NH₂For¹³C、¹⁵One or more marks in tri- kinds of isotopes of N, D Note；H-Leu-NH₂For¹³C、¹⁵One or more marks in tri- kinds of isotopes of N, D；H-Trp-NH₂For¹³C、¹⁵Tri- kinds of N, D is same One or more marks in the element of position；H-Tyr-NH₂For¹³C、¹⁵One or more marks in tri- kinds of isotopes of N, D；H- Asn-NH₂For¹³C、¹⁵One or more marks in tri- kinds of isotopes of N, D；H-Ile-NH₂For¹³C、¹⁵Tri- kinds of isotopes of N, D In one or more mark；H-Gly-NH₂For¹³C、¹⁵One or more marks in tri- kinds of isotopes of N, D；H-Val- NH₂For¹³C、¹⁵One or more marks in tri- kinds of isotopes of N, D.

With reference to specific embodiment, the present invention is described in detail.Following examples will be helpful to the technology of this area Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that to the ordinary skill of this area For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention Protection domain.

Embodiment 1

Cold labeling H-Lys-¹³C₆-His-¹³C₆-Ala-¹³C₃-Leu-¹³C₆-Trp-¹³C₁₁-Tyr-¹³C₉-Asn -¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂Synthesis

1、Fmoc-Gly-¹³C₂-Val-¹³C₅-NH₂Synthesis

By H-Val-¹³C₅-NH₂1.22g is dissolved in 10ml chloroforms, and stirring is lower to add DIEA1.29g, and dissolving is stand-by； Weigh Fmoc-Gly-¹³C₂- OH 3.0g and HOCt 3.0g are dissolved in 15ml chloroforms, and stirring is lower to be added dropwise DIC1.5g, reaction 10 minutes, then this solution is added in the mixed liquor for starting, room temperature reaction 3 hours, thin-layer chromatography detection reaction is complete, reaction Washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after liquid filtering, then dried with anhydrous magnesium chloride, be concentrated under reduced pressure, Ether crystallization is added to obtain white solid as target product 3.45g, yield 85.5%, purity 99.0%, abundance 99.1atom%¹³C。

2、H-Gly-¹³C₂-Val-¹³C₅-NH₂Synthesis

Take Fmoc-Gly-¹³C₂-Val-¹³C₅-NH₂4.0g is dissolved in 50ml chloroforms, adds triethylamine 5ml, and reaction 4 is small When, after thin-layer chromatography monitoring reaction completely, decompression removal solvent and triethylamine are dissolved, successively with watery hydrochloric acid, carbon with dichloromethane Acid sodium aqueous solution, saturated common salt water washing, anhydrous magnesium sulfate are dried, and are concentrated under reduced pressure, and the isolated white solid of preparative chromatography is It is target product 1.56g, yield 86.2%, purity 98.5%, abundance 99.1atom%¹³C。

3、Fmoc-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂Synthesis

By H-Gly-¹³C₂-Val-¹³C₅-NH₂1.81g is dissolved in 10ml chloroforms, and DIEA1.29g is added under stirring, molten Solution, it is stand-by；Weigh Fmoc-Ile-¹³C₆- OH 3.53g and HOCt 3.0g are dissolved in 15ml chloroforms, and stirring is lower to be added dropwise DIC1.5g, is reacted 10 minutes, then adds this solution in the mixed liquor for starting, and room temperature reaction 3 hours, thin-layer chromatography is detected Reaction is complete, is washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering, then uses anhydrous magnesium chloride Dry, be concentrated under reduced pressure, add ether crystallization to obtain white solid as target product 4.37g, yield 84.8%, purity 98.8%, abundance 99.1atom%¹³C。

4、H-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂Synthesis

Take Fmoc-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂5.15g is dissolved in 50ml chloroforms, adds diethylamine 5ml, Reaction 4 hours, after thin-layer chromatography monitoring reaction completely, decompression removal solvent and diethylamine are dissolved, successively with dilute with dichloromethane Hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate are dried, and are concentrated under reduced pressure, the isolated white of preparative chromatography Solid is target product 2.56g, yield 85.2%, purity 98.5%, abundance 99.1atom%¹³C。

5、Fmoc-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂Synthesis

By H-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂3.0g is dissolved in 10ml chloroforms, and stirring is lower to be added DIEA1.29g, dissolving is stand-by；Weigh Fmoc-Asn-¹³C₄- OH 3.54g and HOCt 3.0g are dissolved in 15ml chloroforms, are stirred Lower dropwise addition DIC1.5g is mixed, is reacted 10 minutes, in the mixed liquor for then starting the addition of this solution, room temperature reaction 3 hours, thin layer Chromatogram detection reaction is complete, is washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering, then with nothing Aqueous magnesium chloride is dried, and is concentrated under reduced pressure, and adds ether crystallization to obtain white solid as target product 5.80g, and yield 88.1% is pure Degree 98.6%, abundance 99.1atom%¹³C。

6、H-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂Synthesis

Take Fmoc-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂6.58g is dissolved in 50ml chloroforms, adds two Ethamine 5ml, is reacted 4 hours, and after thin-layer chromatography monitoring reaction completely, decompression removal solvent and diethylamine are dissolved with dichloromethane, Watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing are used successively, and anhydrous magnesium sulfate is dried, and is concentrated under reduced pressure, and preparative chromatography is separated Obtain white solid as target product 3.54g, yield 84.7%, purity 98.5%, abundance 99.1atom%¹³C。

7、Fmoc-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂Synthesis

By H-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂4.18g is dissolved in 10ml chloroforms, stirring Lower addition DIEA1.29g, dissolving is stand-by；Weigh Fmoc-Tyr-¹³C₉- OH 4.12g and HOCt 3.0g are dissolved in the chloromethanes of 15ml tri- In alkane, stirring is lower to be added dropwise DIC1.5g, reacts 10 minutes, then adds this solution in the mixed liquor for starting, and room temperature reaction 3 is small When, thin-layer chromatography detection reaction is complete, is washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering, so Dried with anhydrous magnesium chloride afterwards, be concentrated under reduced pressure, add ether crystallization to obtain white solid as target product 6.81g, yield 83.9%, purity 98.8%, abundance 99.1atom%¹³C。

8、H-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂Synthesis

Take Fmoc-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂8.11g is dissolved in 50ml chloroforms In, diethylamine 5ml is added, to react 4 hours, after thin-layer chromatography monitoring reaction completely, decompression removal solvent and diethylamine use dichloro Methane is dissolved, and successively with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate is dried, and is concentrated under reduced pressure, system Standby chromatographic isolation obtains white solid as target product 5.05g, yield 85.5%, purity 98.5%, abundance 99.1atom%¹³C。

9、Fmoc-Trp-¹³C₁₁-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂Synthesis

By H-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂5.90g is dissolved in 10ml chloroforms In, stirring is lower to add DIEA1.29g, and dissolving is stand-by；Weigh Fmoc-Trp-¹³C₁₁- OH 4.37g and HOCt 3.0g are dissolved in In 15ml chloroforms, stirring is lower to be added dropwise DIC1.5g, reacts 10 minutes, in the mixed liquor for then starting the addition of this solution, room Temperature reaction 3 hours, thin-layer chromatography detection reaction is complete, after reacting liquid filtering with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt according to Secondary washing, is then dried with anhydrous magnesium chloride, is concentrated under reduced pressure, and adds ether crystallization to obtain white solid as target product 8.84g, yield 87.5%, purity 98.6%, abundance 99.1atom%¹³C。

10、H-Trp-¹³C₁₁-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂Synthesis

Take Fmoc-Trp-¹³C₁₁-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂10.1g is dissolved in In 50ml chloroforms, diethylamine 5ml is added, reacted 4 hours, after thin-layer chromatography monitoring reaction completely, decompression removal solvent and diethyl Amine, is dissolved with dichloromethane, and successively with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate is dried, and is subtracted Pressure concentration, the isolated white solid of preparative chromatography is target product 6.67g, yield 84.6%, purity 98.7%, abundance 99.1atom%¹³C。

11、Fmoc-Leu-¹³C₆-Trp-¹³C₁₁-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂'s Synthesis

By H-Trp-¹³C₁₁-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂7.89g is dissolved in 10ml In chloroform, stirring is lower to add DIEA1.29g, and dissolving is stand-by；Weigh Fmoc-Leu-¹³C₆- OH3.59g and HOCt 3.0g It is dissolved in 15ml chloroforms, stirring is lower to be added dropwise DIC1.5g, reacts 10 minutes, and then this solution added the mixed liquor for starting In, room temperature reaction 3 hours, thin-layer chromatography detection reaction is complete, is eaten with sodium bicarbonate solution, hydrochloric acid, saturation after reacting liquid filtering Salt solution is washed successively, is then dried with anhydrous magnesium chloride, is concentrated under reduced pressure, and is added ether crystallization to obtain white solid as target and is produced Thing 9.74g, yield 86.2%, purity 98.9%, abundance 99.1atom%¹³C。

12、H-Leu-¹³C₆-Trp-¹³C₁₁-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂Conjunction Into

Take Fmoc-Leu-¹³C₆-Trp-¹³C₁₁-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂ 11.3g is dissolved in 50ml chloroforms, adds diethylamine 5ml, is reacted 4 hours, and after thin-layer chromatography monitoring reaction completely, decompression removal is molten Agent and diethylamine, are dissolved, successively with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate with dichloromethane Dry, be concentrated under reduced pressure, the isolated white solid of preparative chromatography is target product 7.94g, yield 87.4%, purity 98.6%, abundance 99.1atom%¹³C。

13、Fmoc-Ala-¹³C₃-Leu-¹³C₆-Trp-¹³C₁₁-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val -¹³C₅-NH₂Synthesis

By H-Trp-¹³C₁₁-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂9.09g is dissolved in 10ml In chloroform, stirring is lower to add DIEA1.29g, and dissolving is stand-by；Weigh Fmoc-Ala-¹³C₃- OH3.59g and HOCt 3.0g It is dissolved in 15ml chloroforms, stirring is lower to be added dropwise DIC1.5g, reacts 10 minutes, and then this solution added the mixed liquor for starting In, room temperature reaction 3 hours, thin-layer chromatography detection reaction is complete, is eaten with sodium bicarbonate solution, hydrochloric acid, saturation after reacting liquid filtering Salt solution is washed successively, is then dried with anhydrous magnesium chloride, is concentrated under reduced pressure, and is added ether crystallization to obtain white solid as target and is produced Thing 10.6g, yield 88.0%, purity 98.4%, abundance 99.1atom%¹³C。

14、H-Ala-¹³C₃-Leu-¹³C₆-Trp-¹³C₁₁-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂Synthesis

Take Fmoc-Ala-¹³C₃-Leu-¹³C₆-Trp-¹³C₁₁-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂12.0g is dissolved in 50ml chloroforms, adds diethylamine 5ml, is reacted 4 hours, after thin-layer chromatography monitoring reaction completely, is subtracted Pressure removal solvent and diethylamine, are dissolved, successively with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, nothing with dichloromethane Water magnesium sulfate is dried, and is concentrated under reduced pressure, and the isolated white solid of preparative chromatography is target product 8.51g, and yield 86.5% is pure Degree 98.6%, abundance 99.1atom%¹³C。

15、Fmoc-His-¹³C₆-Ala-¹³C₃-Leu-¹³C₆-Trp-¹³C₁₁-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly -¹³C₂-Val-¹³C₅-NH₂Synthesis

By H-Ala-¹³C₃-Leu-¹³C₆-Trp-¹³C₁₁-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅- NH₂9.84g is dissolved in 10ml chloroforms, and stirring is lower to add DIEA1.29g, and dissolving is stand-by；Weigh Fmoc-His-¹³C₆-OH 3.83g and HOCt 3.0g are dissolved in 15ml chloroforms, and stirring is lower to be added dropwise DIC1.5g, reacts 10 minutes, then by this solution Add in the mixed liquor for starting, room temperature reaction 3 hours, thin-layer chromatography detection reaction is complete, and sodium acid carbonate is used after reacting liquid filtering Solution, hydrochloric acid, saturated aqueous common salt are washed successively, are then dried with anhydrous magnesium chloride, are concentrated under reduced pressure, and add ether crystallization to obtain white Color solid is target product 12.07g, yield 89.5%, purity 98.9%, abundance 99.1atom%¹³C。

16、H-His-¹³C₆-Ala-¹³C₃-Leu-¹³C₆-Trp-¹³C₁₁-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂Synthesis

Take Fmoc-His-¹³C₆-Ala-¹³C₃-Leu-¹³C₆-Trp-¹³C₁₁-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂12.0g is dissolved in 50ml chloroforms, adds diethylamine 5ml, is reacted 4 hours, and thin-layer chromatography monitoring has been reacted Quan Hou, decompression removal solvent and diethylamine, is dissolved, successively with watery hydrochloric acid, aqueous sodium carbonate, saturated aqueous common salt with dichloromethane Washing, anhydrous magnesium sulfate is dried, and is concentrated under reduced pressure, and the isolated white solid of preparative chromatography is target product 9.78g, yield 86.7%, purity 98.6%, abundance 99.1atom%¹³C。

17、Fmoc-Lys-¹³C₆-His-¹³C₆-Ala-¹³C₃-Leu-¹³C₆-Trp-¹³C₁₁-Tyr-¹³C₉-Asn-¹³C₄-Ile -¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂Synthesis

By H-His-¹³C₆-Ala-¹³C₃-Leu-¹³C₆-Trp-¹³C₁₁-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂- Val-¹³C₅-NH₂11.28g is dissolved in 10ml chloroforms, and stirring is lower to add DIEA1.29g, and dissolving is stand-by；Weigh Fmoc- Lys-¹³C₆- OH 3.74g and HOCt 3.0g are dissolved in 15ml chloroforms, the lower dropwise addition DIC1.5g of stirring, reaction 10 minutes, so This solution is added afterwards in the mixed liquor for starting, room temperature reaction 3 hours, thin-layer chromatography detection reaction is complete, after reacting liquid filtering Washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt, then dried with anhydrous magnesium chloride, be concentrated under reduced pressure, add ether Crystallization obtains white solid as target product 12.98g, yield 87.5%, purity 98.9%, abundance 99.1atom%¹³C。

18、H-Lys-¹³C₆-His-¹³C₆-Ala-¹³C₃-Leu-¹³C₆-Trp-¹³C₁₁-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂Synthesis

Take Fmoc-Lys-¹³C₆-His-¹³C₆-Ala-¹³C₃-Leu-¹³C₆-Trp-¹³C₁₁-Tyr-¹³C₉-Asn-¹³C₄-Ile-¹³C₆-Gly-¹³C₂-Val-¹³C₅-NH₂14.84g is dissolved in 50ml chloroforms, adds diethylamine 5ml, is reacted 4 hours, thin-layer chromatography Monitoring reaction completely after, decompression removal solvent and diethylamine, with dichloromethane dissolve, successively with watery hydrochloric acid, aqueous sodium carbonate, Saturated common salt water washing, anhydrous magnesium sulfate is dried, and is concentrated under reduced pressure, and the isolated white solid of preparative chromatography is target product 10.8g, yield 85.5%, purity 98.7%, abundance 99.1atom%¹³C。

Embodiment 2

Cold labeling H-Lys-¹⁵N₂-His-¹⁵N₃-Ala-¹⁵N-Leu-¹⁵N-Trp-¹⁵N₂-Tyr-¹⁵N-Asn-¹⁵N- Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂Synthesis

1、Fmoc-Gly-¹⁵N-Val-¹⁵N-NH₂Synthesis

By H-Val-¹⁵N-NH₂1.17g is dissolved in 10ml chloroforms, and stirring is lower to add DIEA1.29g, and dissolving is stand-by； Weigh Fmoc-Gly-¹⁵N-OH 3.0g and HOCt 3.0g are dissolved in 15ml chloroforms, and stirring is lower to be added dropwise DIC 1.5g, reaction 10 minutes, then this solution is added in the mixed liquor for starting, room temperature reaction 3 hours, thin-layer chromatography detection reaction is complete, reaction Washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after liquid filtering, then dried with anhydrous magnesium chloride, be concentrated under reduced pressure, Ether crystallization is added to obtain white solid as target product 3.48g, yield 87.7%, purity 98.9%, abundance 99.5atom%¹⁵N。

2、H-Gly-¹⁵N-Val-¹⁵N-NH₂Synthesis

Take Fmoc-Gly-¹⁵N-Val-¹⁵N-NH₂4.00g is dissolved in 50ml chloroforms, adds triethylamine 5ml, is reacted 4 hours, After thin-layer chromatography monitoring reaction completely, decompression removal solvent and triethylamine are dissolved, successively with watery hydrochloric acid, carbonic acid with dichloromethane Sodium water solution, saturated common salt water washing, anhydrous magnesium sulfate are dried, and are concentrated under reduced pressure, and the isolated white solid of preparative chromatography is Target product 1.53g, yield 88.2%, purity 98.5%, abundance 99.5atom%¹⁵N。

3、Fmoc-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂Synthesis

By H-Gly-¹⁵N-Val-¹⁵N-NH₂1.74g is dissolved in 10ml chloroforms, and DIEA1.29g is added under stirring, molten Solution, it is stand-by；Weigh Fmoc-Ile-¹⁵N-OH 3.53g and HOCt 3.0g are dissolved in 15ml chloroforms, and stirring is lower to be added dropwise DIC 1.5g, is reacted 10 minutes, then adds this solution in the mixed liquor for starting, and room temperature reaction 3 hours, thin-layer chromatography detection is reacted Completely, washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering, then dried with anhydrous magnesium chloride, It is concentrated under reduced pressure, add ether crystallization to obtain white solid as target product 4.30g, yield 84.3%, purity 99.0%, abundance 99.5atom%¹⁵N。

4、H-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂Synthesis

Take Fmoc-Gly-¹⁵N-Val-¹⁵N-NH₂5.10g is dissolved in 50ml chloroforms, adds diethylamine 5ml, is reacted 4 hours, After thin-layer chromatography monitoring reaction completely, decompression removal solvent and diethylamine are dissolved, successively with watery hydrochloric acid, carbonic acid with dichloromethane Sodium water solution, saturated common salt water washing, anhydrous magnesium sulfate are dried, and are concentrated under reduced pressure, and the isolated white solid of preparative chromatography is Target product 2.57g, yield 89.0%, purity 98.7%, abundance 99.5atom%¹⁵N。

5、Fmoc-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂Synthesis

By H-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂2.87g is dissolved in 10ml chloroforms, and stirring is lower to add DIEA 1.29g, dissolving is stand-by；Weigh Fmoc-Asn-¹⁵N-OH 3.54g and HOCt 3.0g are dissolved in 15ml chloroforms, under stirring DIC 1.5g are added dropwise, react 10 minutes, in the mixed liquor for then starting the addition of this solution, room temperature reaction 3 hours, thin-layer chromatography Detection reaction is complete, is washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering, then uses anhydrous chlorine Change magnesium to dry, be concentrated under reduced pressure, add ether crystallization to obtain white solid as target product 5.35g, yield 85.9%, purity 98.3%, abundance 99.5atom%¹⁵N。

6、H-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂Synthesis

Take Fmoc-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂6.23g is dissolved in 50ml chloroforms, adds diethyl Amine 5ml, is reacted 4 hours, and after thin-layer chromatography monitoring reaction completely, decompression removal solvent and diethylamine are dissolved with dichloromethane, according to Secondary use watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate are dried, and are concentrated under reduced pressure, and preparative chromatography is separated Target product 3.42g, yield 85.3%, purity 98.6%, abundance 99.5atom% are to white solid¹⁵N。

7、Fmoc-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂Synthesis

By H-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂4.01g is dissolved in 10ml chloroforms, and stirring is lower to be added Enter DIEA 1.29g, dissolve, it is stand-by；Weigh Fmoc-Tyr-¹⁵N-OH 4.12g and HOCt3.0g are dissolved in 15ml chloroforms, Stirring is lower to be added dropwise DIC 1.5g, reacts 10 minutes, then adds this solution in the mixed liquor for starting, and room temperature reaction 3 hours is thin Layer chromatography detection reaction is complete, is washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering, Ran Houyong Anhydrous magnesium chloride is dried, and is concentrated under reduced pressure, and adds ether crystallization to obtain white solid as target product 6.68g, yield 84.9%, Purity 98.4%, abundance 99.5atom%¹⁵N。

8、H-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂Synthesis

Take Fmoc-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂7.87g is dissolved in 50ml chloroforms, plus Enter diethylamine 5ml, react 4 hours, after thin-layer chromatography monitoring reaction completely, decompression removal solvent and diethylamine use dichloromethane Dissolving, successively with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate is dried, and is concentrated under reduced pressure, and prepares color Compose isolated white solid as target product 4.92g, yield 87.2%, purity 98.8%, abundance 99.5atom%¹⁵N。

9、Fmoc-Trp-¹⁵N₂-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂Synthesis

By H-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂5.65g is dissolved in 10ml chloroforms, Stirring is lower to add DIEA 1.29g, and dissolving is stand-by；Weigh Fmoc-Trp-¹⁵N₂- OH 4.37g and HOCt 3.0g are dissolved in 15ml tri- In chloromethanes, stirring is lower to be added dropwise DIC 1.5g, reacts 10 minutes, then adds this solution in the mixed liquor for starting, and room temperature is anti- Answer 3 hours, thin-layer chromatography detection reaction is complete, is washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering Wash, then dried with anhydrous magnesium chloride, be concentrated under reduced pressure, add ether crystallization to obtain white solid as target product 8.39g, receive Rate 86.1%, purity 98.2%, abundance 99.5atom%¹⁵N。

10、H-Trp-¹⁵N₂-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂Synthesis

Take Fmoc-Trp-¹⁵N₂-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂9.74g is dissolved in 50ml In chloroform, diethylamine 5ml is added, reacted 4 hours, after thin-layer chromatography monitoring reaction completely, decompression removal solvent and diethylamine are used Dichloromethane is dissolved, and successively with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate is dried, and is depressurized dense Contracting, the isolated white solid of preparative chromatography is target product 6.46g, yield 85.9%, purity 97.7%, abundance 99.5atom%¹⁵N。

11、Fmoc-Leu-¹⁵N-Trp-¹⁵N₂-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂Synthesis

By H-Trp-¹⁵N₂-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂7.51g is dissolved in 10ml trichlorines In methane, stirring is lower to add DIEA 1.29g, and dissolving is stand-by；Weigh Fmoc-Leu-¹⁵N-OH 3.59g and HOCt 3.0g are dissolved in In 15ml chloroforms, stirring is lower to be added dropwise DIC 1.5g, reacts 10 minutes, in the mixed liquor for then starting the addition of this solution, Room temperature reaction 3 hours, thin-layer chromatography detection reaction is complete, and sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt are used after reacting liquid filtering Wash successively, then dried with anhydrous magnesium chloride, be concentrated under reduced pressure, add ether crystallization to obtain white solid as target product 9.27g, yield 85.3%, purity 98.9%, abundance 99.5atom%¹⁵N。

12、H-Leu-¹⁵N-Trp-¹⁵N₂-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂Synthesis

Take Fmoc-Leu-¹⁵N-Trp-¹⁵N₂-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂ 10.87g Be dissolved in 50ml chloroforms, add diethylamine 5ml, react 4 hours, thin-layer chromatography monitoring reaction completely after, decompression removal solvent and Diethylamine, is dissolved with dichloromethane, and successively with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate is done It is dry, it is concentrated under reduced pressure, the isolated white solid of preparative chromatography is target product 7.42g, yield 85.8%, purity 98.4%, Abundance 99.5atom%¹⁵N。

13、Fmoc-Ala-¹⁵N-Leu-¹⁵N-Trp-¹⁵N₂-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N- NH₂Synthesis

By H-Leu-¹⁵N-Trp-¹⁵N₂-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂8.65g is dissolved in In 10ml chloroforms, stirring is lower to add DIEA1.29g, and dissolving is stand-by；Weigh Fmoc-Ala-¹⁵N-OH 3.59g and HOCt 3.0g is dissolved in 15ml chloroforms, and DIC1.5g is added dropwise under stirring, is reacted 10 minutes, then adds this solution what is started to mix In conjunction liquid, room temperature reaction 3 hours, thin-layer chromatography detection reaction is complete, sodium bicarbonate solution, hydrochloric acid is used after reacting liquid filtering, is satisfied Washed successively with saline solution, then dried with anhydrous magnesium chloride, be concentrated under reduced pressure, add ether crystallization to obtain white solid as mesh Mark product 10.07g, yield 87.0%, purity 97.7%, abundance 99.5atom%¹⁵N。

14、H-Ala-¹⁵N-Leu-¹⁵N-Trp-¹⁵N₂-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂'s Synthesis

Take Fmoc-Ala-¹⁵N-Leu-¹⁵N-Trp-¹⁵N₂-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N- NH₂-NH₂11.58g is dissolved in 50ml chloroforms, adds diethylamine 5ml, is reacted 4 hours, after thin-layer chromatography monitoring reaction completely, is subtracted Pressure removal solvent and diethylamine, are dissolved, successively with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, nothing with dichloromethane Water magnesium sulfate is dried, and is concentrated under reduced pressure, and the isolated white solid of preparative chromatography is target product 9.17g, and yield 86.9% is pure Degree 98.3%, abundance 99.5atom%¹⁵N。

15、Fmoc-His-¹⁵N₃-Ala-¹⁵N-Leu-¹⁵N-Trp-¹⁵N₂-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N- Val-¹⁵N-NH₂Synthesis

By H-Ala-¹⁵N-Leu-¹⁵N-Trp-¹⁵N₂-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂ 10.56g is dissolved in 10ml chloroforms, and stirring is lower to add DIEA1.29g, and dissolving is stand-by；Weigh Fmoc-His-¹⁵N₃-OH 3.83g and HOCt 3.0g are dissolved in 15ml chloroforms, and stirring is lower to be added dropwise DIC 1.5g, reacts 10 minutes, then by this solution Add in the mixed liquor for starting, room temperature reaction 3 hours, thin-layer chromatography detection reaction is complete, and sodium acid carbonate is used after reacting liquid filtering Solution, hydrochloric acid, saturated aqueous common salt are washed successively, are then dried with anhydrous magnesium chloride, are concentrated under reduced pressure, and add ether crystallization to obtain white Color solid is target product 12.55g, yield 88.5%, purity 96.9%, abundance 99.5atom%¹⁵N。

16、H-His-¹⁵N₃-Ala-¹⁵N-Leu-¹⁵N-Trp-¹⁵N₂-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val -¹⁵N-NH₂Synthesis

Take Fmoc-His-¹⁵N₃-Ala-¹⁵N-Leu-¹⁵N-Trp-¹⁵N₂-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N- Val-¹⁵N-NH₂14.18g is dissolved in 50ml chloroforms, adds diethylamine 5ml, is reacted 4 hours, and thin-layer chromatography monitoring reaction is complete Afterwards, decompression removal solvent and diethylamine, are dissolved with dichloromethane, are washed with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt successively Wash, anhydrous magnesium sulfate is dried, be concentrated under reduced pressure, the isolated white solid of preparative chromatography is target product 10.21g, yield 85.4%, purity 97.6%, abundance 99.5atom%¹⁵N。

17、Fmoc-Lys-¹⁵N₂-His-¹⁵N₃-Ala-¹⁵N-Leu-¹⁵N-Trp-¹⁵N₂-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N- Gly-¹⁵N-Val-¹⁵N-NH₂Synthesis

By H-His-¹⁵N₃-Ala-¹⁵N-Leu-¹⁵N-Trp-¹⁵N₂-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N-Gly-¹⁵N-Val-¹⁵N-NH₂11.96g is dissolved in 10ml chloroforms, and stirring is lower to add DIEA1.29g, and dissolving is stand-by；Weigh Fmoc-Lys-¹⁵N-OH 3.74g and HOCt 3.0g are dissolved in 15ml chloroforms, and stirring is lower to be added dropwise DIC 1.5g, reacts 10 minutes, then will This solution is added in the mixed liquor for starting, room temperature reaction 3 hours, and thin-layer chromatography detection reaction is complete, and carbon is used after reacting liquid filtering Sour hydrogen sodium solution, hydrochloric acid, saturated aqueous common salt are washed successively, are then dried with anhydrous magnesium chloride, are concentrated under reduced pressure, and add ether crystallization Obtain white solid as target product 13.53g, yield 87.5%, purity 98.0%, abundance 99.5atom%¹⁵N。

18、H-Lys-¹⁵N₂-His-¹⁵N₃-Ala-¹⁵N-Leu-¹⁵N-Trp-¹⁵N₂-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N- Gly-¹⁵N-Val-¹⁵N-NH₂Synthesis

Take Fmoc-Lys-¹⁵N₂-His-¹⁵N₃-Ala-¹⁵N-Leu-¹⁵N-Trp-¹⁵N₂-Tyr-¹⁵N-Asn-¹⁵N-Ile-¹⁵N- Gly-¹⁵N-Val-¹⁵N-NH₂15.46g is dissolved in 50ml chloroforms, adds diethylamine 5ml, is reacted 4 hours, and thin-layer chromatography monitoring is anti- After answering completely, decompression removal solvent and diethylamine are dissolved with dichloromethane, use watery hydrochloric acid, aqueous sodium carbonate, saturation to eat successively Salt water washing, anhydrous magnesium sulfate is dried, and is concentrated under reduced pressure, and the isolated white solid of preparative chromatography is target product 11.40g, Yield 86.1%, purity 97.9%, abundance 99.5atom%¹⁵N。

Embodiment 3

Cold labeling H-Lys-D₂-His-D₂-Ala-D₃-Leu-D₆-Trp-D₂-Tyr-D₂-Asn-D₂-Ile-D₆- Gly-D₂-Val-D₆-NH₂Synthesis

1、Fmoc-Gly-D₂-Val-D₆-NH₂Synthesis

By H-Val-D₆-NH₂1.23g (0.01mol) is dissolved in 10ml chloroforms, and DIEA1.29g is added under stirring, molten Solution, it is stand-by；Weigh Fmoc-Gly-D₂- OH 3.0g and HOCt 3.0g are dissolved in 15ml chloroforms, and stirring is lower to be added dropwise DIC1.5g, is reacted 10 minutes, then adds this solution in the mixed liquor for starting, and room temperature reaction 3 hours, thin-layer chromatography is detected Reaction is complete, is washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering, then uses anhydrous magnesium chloride Dry, be concentrated under reduced pressure, add ether crystallization to obtain white solid as target product 3.46g, yield 86.0%, purity 99.0%, abundance 98.5atom%D.

2、H-Gly-D₂-Val-D₆-NH₂Synthesis

Take Fmoc-Gly-D₂-Val-D₆-NH₂4.0g is dissolved in 50ml chloroforms, adds triethylamine 5ml, is reacted 4 hours, thin After layer chromatography monitoring reaction completely, decompression removal solvent and triethylamine are dissolved, successively with watery hydrochloric acid, sodium carbonate with dichloromethane The aqueous solution, saturated common salt water washing, anhydrous magnesium sulfate are dried, and are concentrated under reduced pressure, and the isolated white solid of preparative chromatography is mesh Mark product 1.55g, yield 85.5%, purity 98.5%, abundance 98.5atom%D.

3、Fmoc-Ile-D₆-Gly-D₂-Val-¹³D₆-NH₂Synthesis

By H-Gly-D₂-Val-¹³D₆-NH₂1.82g is dissolved in 10ml chloroforms, and DIEA1.29g is added under stirring, molten Solution, it is stand-by；Weigh Fmoc-Ile-D₆- OH 3.53g and HOCt 3.0g are dissolved in 15ml chloroforms, and stirring is lower to be added dropwise DIC1.5g, is reacted 10 minutes, then adds this solution in the mixed liquor for starting, and room temperature reaction 3 hours, thin-layer chromatography is detected Reaction is complete, is washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering, then uses anhydrous magnesium chloride Dry, be concentrated under reduced pressure, add ether crystallization to obtain white solid as target product 4.45g, yield 86.2%, purity 98.8%, abundance 98.5atom%D.

4、H-Ile-D₆-Gly-D₂-Val-¹³D₆-NH₂Synthesis

Take Fmoc-Ile-D₆-Gly-D₂-Val-¹³D₆-NH₂5.17g is dissolved in 50ml chloroforms, adds diethylamine 5ml, instead Answer 4 hours, after thin-layer chromatography monitoring reaction completely, decompression removal solvent and diethylamine are dissolved with dichloromethane, and dilute salt is used successively Acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate are dried, and are concentrated under reduced pressure, and the isolated white of preparative chromatography is solid Body is target product 2.50g, yield 85.0%, purity 98.5%, abundance 98.5atom%D.

5、Fmoc-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂Synthesis

By H-Ile-D₆-Gly-D₂-Val-¹³D₆-NH₂2.95g is dissolved in 10ml chloroforms, and stirring is lower to be added DIEA1.29g, dissolving is stand-by；Weigh Fmoc-Asn-D₂- OH 3.56g and HOCt 3.0g are dissolved in 15ml chloroforms, are stirred Lower dropwise addition DIC1.5g is mixed, is reacted 10 minutes, in the mixed liquor for then starting the addition of this solution, room temperature reaction 3 hours, thin layer Chromatogram detection reaction is complete, is washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering, then with nothing Aqueous magnesium chloride is dried, and is concentrated under reduced pressure, and adds ether crystallization to obtain white solid as target product 5.72g, and yield 88.1% is pure Degree 98.6%, abundance 98.5atom%D.

6、H-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂Synthesis

Take Fmoc-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂6.49g is dissolved in 50ml chloroforms, adds diethylamine 5ml, is reacted 4 hours, and after thin-layer chromatography monitoring reaction completely, decompression removal solvent and diethylamine are dissolved, successively with dichloromethane With watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate drying, concentrated under reduced pressure, preparative chromatography is isolated White solid is target product 3.62g, yield 85.0%, purity 98.5%, abundance 98.5atom%D.

7、Fmoc-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂Synthesis

By H-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂4.26g is dissolved in 10ml chloroforms, and stirring is lower to be added DIEA1.29g, dissolving is stand-by；Weigh Fmoc-Tyr-D₂- OH 4.05g and HOCt 3.0g are dissolved in 15ml chloroforms, are stirred Lower dropwise addition DIC1.5g is mixed, is reacted 10 minutes, in the mixed liquor for then starting the addition of this solution, room temperature reaction 3 hours, thin layer Chromatogram detection reaction is complete, is washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering, then with nothing Aqueous magnesium chloride is dried, and is concentrated under reduced pressure, and adds ether crystallization to obtain white solid as target product 7.0g, and yield 86.0% is pure Degree 98.8%, abundance 98.5atom%D.

8、H-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂Synthesis

Take Fmoc-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂8.14g is dissolved in 50ml chloroforms, is added Diethylamine 5ml, reacts 4 hours, and after thin-layer chromatography monitoring reaction completely, decompression removal solvent and diethylamine are molten with dichloromethane Solution, successively with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate is dried, and is concentrated under reduced pressure, preparative chromatography Isolated white solid is target product 5.03g, yield 85.0%, purity 98.5%, abundance 98.5atom%D.

9、Fmoc-Trp-D₂-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂Synthesis

By H-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂5.92g is dissolved in 10ml chloroforms, stirring Lower addition DIEA1.29g, dissolving is stand-by；Weigh Fmoc-Trp-D₂- OH 4.28g and HOCt3.0g are dissolved in 15ml chloroforms In, stirring is lower to be added dropwise DIC1.5g, reacts 10 minutes, in the mixed liquor for then starting the addition of this solution, room temperature reaction 3 hours, Thin-layer chromatography detection reaction is complete, is washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering, then Dried with anhydrous magnesium chloride, be concentrated under reduced pressure, add ether crystallization to obtain white solid as target product 8.62g, yield 86.0%, purity 98.6%, abundance 98.5atom%D.

10、H-Trp-D₂-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂Synthesis

Take Fmoc-Trp-D₂-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂10.0g is dissolved in 50ml chloroforms In, diethylamine 5ml is added, to react 4 hours, after thin-layer chromatography monitoring reaction completely, decompression removal solvent and diethylamine use dichloro Methane is dissolved, and successively with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate is dried, and is concentrated under reduced pressure, system Standby chromatographic isolation obtains white solid as target product 6.68g, yield 85.6%, purity 98.7%, abundance 98.5atom% D。

11、Fmoc-Leu-D₆-Trp-D₂-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂Synthesis

By H-Trp-D₂-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂7.80g is dissolved in 10ml chloroforms In, stirring is lower to add DIEA1.29g, and dissolving is stand-by；Weigh Fmoc-Leu-D₆- OH 3.59g and HOCt 3.0g are dissolved in 15ml In chloroform, stirring is lower to be added dropwise DIC1.5g, reacts 10 minutes, then adds this solution in the mixed liquor for starting, and room temperature is anti- Answer 3 hours, thin-layer chromatography detection reaction is complete, is washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering Wash, then dried with anhydrous magnesium chloride, be concentrated under reduced pressure, add ether crystallization to obtain white solid as target product 9.69g, receive Rate 86.5%, purity 98.9%, abundance 98.5atom%D.

12、H-Leu-D₆-Trp-D₂-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂Synthesis

Take Fmoc-Leu-D₆-Trp-D₂-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂11.2g is dissolved in In 50ml chloroforms, diethylamine 5ml is added, reacted 4 hours, after thin-layer chromatography monitoring reaction completely, decompression removal solvent and diethyl Amine, is dissolved with dichloromethane, and successively with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate is dried, and is subtracted Pressure concentration, the isolated white solid of preparative chromatography is target product 7.82g, yield 87.0%, purity 98.6%, abundance 98.5atom%D.

13、Fmoc-Ala-D₃-Leu-D₆-Trp-D₂-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂Conjunction Into

By H-Leu-D₆-Trp-D₂-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂8.99g is dissolved in 10ml tri- In chloromethanes, stirring is lower to add DIEA1.29g, and dissolving is stand-by；Weigh Fmoc-Ala-^D3- OH3.59g and HOCt 3.0g are dissolved in In 15ml chloroforms, stirring is lower to be added dropwise DIC1.5g, reacts 10 minutes, in the mixed liquor for then starting the addition of this solution, room Temperature reaction 3 hours, thin-layer chromatography detection reaction is complete, after reacting liquid filtering with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt according to Secondary washing, is then dried with anhydrous magnesium chloride, is concentrated under reduced pressure, and adds ether crystallization to obtain white solid as target product 11.0g, yield 89.0%, purity 98.4%, abundance 98.5atom%D.

14、H-Ala-D₃-Leu-D₆-Trp-D₂-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂Synthesis

Take Fmoc-Ala-D₃-Leu-D₆-Trp-D₂-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂12.4g Be dissolved in 50ml chloroforms, add diethylamine 5ml, react 4 hours, thin-layer chromatography monitoring reaction completely after, decompression removal solvent and Diethylamine, is dissolved with dichloromethane, and successively with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate is done It is dry, it is concentrated under reduced pressure, the isolated white solid of preparative chromatography is target product 8.75g, yield 86.0%, purity 98.6%, Abundance 98.5atom%D.

15、Fmoc-His-D₂-Ala-D₃-Leu-D₆-Trp-D₂-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆- NH₂Synthesis

By H-Ala-D₃-Leu-D₆-Trp-D₂-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂10.2g is molten In 10ml chloroforms, stirring is lower to add DIEA1.29g, and dissolving is stand-by；Weigh Fmoc-His-D₂- OH 3.79g and HOCt 3.0g is dissolved in 15ml chloroforms, and DIC1.5g is added dropwise under stirring, is reacted 10 minutes, then adds this solution what is started to mix In conjunction liquid, room temperature reaction 3 hours, thin-layer chromatography detection reaction is complete, sodium bicarbonate solution, hydrochloric acid is used after reacting liquid filtering, is satisfied Washed successively with saline solution, then dried with anhydrous magnesium chloride, be concentrated under reduced pressure, add ether crystallization to obtain white solid as mesh Mark product 11.95g, yield 86.6%, purity 98.9%, abundance 98.5atom%D.

16、H-His-D₂-Ala-D₃-Leu-D₆-Trp-D₂-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂ Synthesis

Take Fmoc-His-D₂-Ala-D₃-Leu-D₆-Trp-D₂-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆- NH₂13.8g is dissolved in 50ml chloroforms, adds diethylamine 5ml, is reacted 4 hours, and after thin-layer chromatography monitoring reaction completely, decompression is gone Except solvent and diethylamine, dissolved with dichloromethane, successively with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous sulphur Sour magnesium is dried, and is concentrated under reduced pressure, and the isolated white solid of preparative chromatography is target product 9.90g, yield 85.5%, purity 98.6%, abundance 98.5atom%D.

17、Fmoc-Lys-D₂-His-D₂-Ala-D₃-Leu-D₆-Trp-D₂-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂- Val-D₆-NH₂Synthesis

By H-His-D₂-Ala-D₃-Leu-D₆-Trp-D₂-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val-D₆-NH₂ 11.57g is dissolved in 10ml chloroforms, and stirring is lower to add DIEA1.29g, and dissolving is stand-by；Weigh Fmoc-Lys-D₂-OH 3.70g and HOCt 3.0g are dissolved in 15ml chloroforms, and stirring is lower to be added dropwise DIC1.5g, reacts 10 minutes, then by this solution Add in the mixed liquor for starting, room temperature reaction 3 hours, thin-layer chromatography detection reaction is complete, and sodium acid carbonate is used after reacting liquid filtering Solution, hydrochloric acid, saturated aqueous common salt are washed successively, are then dried with anhydrous magnesium chloride, are concentrated under reduced pressure, and add ether crystallization to obtain white Color solid is target product 11.69g, yield 88.0%, purity 98.9%, abundance 98.5atom%D.

18、H-Lys-D₂-His-D₂-Ala-D₃-Leu-D₆-Trp-D₂-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂-Val- D₆-NH₂Synthesis

Take Fmoc-Lys-D₂-His-D₂-Ala-D₃-Leu-D₆-Trp-D₂-Tyr-D₂-Asn-D₂-Ile-D₆-Gly-D₂- Val-D₆-NH₂15.09g is dissolved in 50ml chloroforms, adds diethylamine 5ml, is reacted 4 hours, and thin-layer chromatography monitoring reaction is complete Afterwards, decompression removal solvent and diethylamine, are dissolved with dichloromethane, are washed with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt successively Wash, anhydrous magnesium sulfate is dried, be concentrated under reduced pressure, the isolated white solid of preparative chromatography is target product 11.2g, yield 86.6%, purity 98.7%, abundance 98.5atom%D.

Embodiment 4

Cold labeling H-Lys-His-Ala-Leu-¹³C₆-Trp-Tyr-Asn-Ile-Gly-Val-NH₂Synthesis

1、Fmoc-Leu-¹³C₆-Trp-Tyr-Asn-Ile-Gly-Val-NH₂Synthesis

By H-Trp-Tyr-Asn-Ile-Gly-Val-NH₂7.51g is dissolved in 10ml chloroforms, and stirring is lower to be added DIEA1.29g, dissolving is stand-by；Weigh Fmoc-Leu-¹³C₆- OH 3.59g and HOCt 3.0g are dissolved in 15ml chloroforms, are stirred Lower dropwise addition DIC1.5g is mixed, is reacted 10 minutes, in the mixed liquor for then starting the addition of this solution, room temperature reaction 3 hours, thin layer Chromatogram detection reaction is complete, is washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering, then with nothing Aqueous magnesium chloride is dried, and is concentrated under reduced pressure, and adds ether crystallization to obtain white solid as target product 9.45g, and yield 86.5% is pure Degree 98.6%, abundance 99.1atom%¹³C。

2、H-Leu-¹³C₆-Trp-Tyr-Asn-Ile-Gly-Val-NH₂Synthesis

Take Fmoc-Leu-¹³C₆-Trp-Tyr-Asn-Ile-Gly-Val-NH₂10.92g is dissolved in 50ml chloroforms, is added Diethylamine 5ml, reacts 4 hours, and after thin-layer chromatography monitoring reaction completely, decompression removal solvent and diethylamine are molten with dichloromethane Solution, successively with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate is dried, and is concentrated under reduced pressure, preparative chromatography Isolated white solid is target product 7.5g, yield 86.2%, purity 98.5%, abundance 99.1atom%¹³C。

3、Fmoc-Lys-His-Ala-Leu-¹³C₆-Trp-Tyr-Asn-Ile-Gly-Val-NH₂Synthesis

By H-Leu-¹³C₆-Trp-Tyr-Asn-Ile-Gly-Val-NH₂8.71g is dissolved in 10ml chloroforms, stirring Lower addition DIEA1.29g, dissolving is stand-by；Weigh Fmoc Lys-His-Ala-OH 3.53g and HOCt3.0g and be dissolved in 15ml trichlorines In methane, stirring is lower to be added dropwise DIC1.5g, reacts 10 minutes, in the mixed liquor for then starting the addition of this solution, room temperature reaction 3 Hour, thin-layer chromatography detection reaction is complete, is washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering, Then dried with anhydrous magnesium chloride, be concentrated under reduced pressure, add ether crystallization to obtain white solid as target product 12.08g, yield 84.5%, purity 98.8%, abundance 99.1atom%¹³C。

4、H-Lys-His-Ala-Leu-¹³C₆-Trp-Tyr-Asn-Ile-Gly-Val-NH₂Synthesis

Take Fmoc-Lys-His-Ala-Leu-¹³C₆-Trp-Tyr-Asn-Ile-Gly-Val-NH₂14.29g is dissolved in 50ml In chloroform, diethylamine 5ml is added, reacted 4 hours, after thin-layer chromatography monitoring reaction completely, decompression removal solvent and diethylamine are used Dichloromethane is dissolved, and successively with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate is dried, and is depressurized dense Contracting, the isolated white solid of preparative chromatography is target product 10.44g, yield 86.4%, purity 98.5%, abundance 99.1atom%¹³C。

Embodiment 5

Cold labeling H-Lys-His-Ala-Leu-D₆-Trp-Tyr-Asn-Ile-D₈-Gly-Val-NH₂Conjunction Into

1、Fmoc-Ile-D₈-Gly-Val-NH₂Synthesis

By H-Gly-Val-NH₂1.74g is dissolved in 10ml chloroforms, and stirring is lower to add DIEA1.29g, and dissolving is treated With；Weigh Fmoc-Ile-D₈- OH 3.61g and HOCt 3.0g are dissolved in 15ml chloroforms, and stirring is lower to be added dropwise DIC1.5g, instead Answer 10 minutes, then add this solution in the mixed liquor for starting, room temperature reaction 3 hours, thin-layer chromatography detection reaction is complete, instead Answer liquid to be washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after filtering, then dried with anhydrous magnesium chloride, depressurize dense Contracting, adds ether crystallization to obtain white solid as target product 4.56g, yield 88.2%, purity 98.8%, abundance 99.1atom%D.

2、H-Ile-D₈-Gly-Val-NH₂Synthesis

Take Fmoc-Ile-D₈-Gly-Val-NH₂5.18g is dissolved in 50ml chloroforms, adds triethylamine 5ml, is reacted 4 hours, After thin-layer chromatography monitoring reaction completely, decompression removal solvent and triethylamine are dissolved, successively with watery hydrochloric acid, carbonic acid with dichloromethane Sodium water solution, saturated common salt water washing, anhydrous magnesium sulfate are dried, and are concentrated under reduced pressure, and the isolated white solid of preparative chromatography is Target product 2.59g, yield 87.5%, purity 98.5%, abundance 99.1atom%D.

3、Fmoc-Trp-Tyr-Asn-Ile-D₈-Gly-Val-NH₂Synthesis

By H-Ile-D₈-Gly-Val-NH₂2.96g is dissolved in 10ml chloroforms, and DIEA1.29g is added under stirring, molten Solution, it is stand-by；Weigh Fmoc-Trp-Tyr-Asn-OH 7.39g and HOCt 3.0g to be dissolved in 15ml chloroforms, stirring is lower to be added dropwise DIC1.5g, is reacted 10 minutes, then adds this solution in the mixed liquor for starting, and room temperature reaction 3 hours, thin-layer chromatography is detected Reaction is complete, is washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering, then uses anhydrous magnesium chloride Dry, be concentrated under reduced pressure, add ether crystallization to obtain white solid as target product 9.08g, yield 89.2%, purity 98.5%, abundance 99.1atom%D.

4、H-Trp-Tyr-Asn-Ile-D₈-Gly-Val-NH₂Synthesis

Take Fmoc-Trp-Tyr-Asn-Ile-D₈-Gly-Val-NH₂10.18g is dissolved in 50ml chloroforms, adds triethylamine 5ml, is reacted 4 hours, and after thin-layer chromatography monitoring reaction completely, decompression removal solvent and triethylamine are dissolved, successively with dichloromethane With watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate drying, concentrated under reduced pressure, preparative chromatography is isolated White solid is target product 6.74g, yield 84.7%, purity 98.5%, abundance 99.1atom%D.

5、Fmoc-Leu-D₆-Trp-Tyr-Asn-Ile-D₈-Gly-Val-NH₂Synthesis

By H-Trp-Tyr-Asn-Ile-D₈-Gly-Val-NH₂7.96g is dissolved in 10ml chloroforms, and stirring is lower to be added DIEA1.29g, dissolving is stand-by；Weigh Fmoc-Leu-D₆- OH 3.59g and HOCt 3.0g are dissolved in 15ml chloroforms, are stirred Lower dropwise addition DIC1.5g is mixed, is reacted 10 minutes, in the mixed liquor for then starting the addition of this solution, room temperature reaction 3 hours, thin layer Chromatogram detection reaction is complete, is washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering, then with nothing Aqueous magnesium chloride is dried, and is concentrated under reduced pressure, and adds ether crystallization to obtain white solid as target product 9.86g, and yield 86.7% is pure Degree 98.5%, abundance 99.1atom%D.

6、H-Leu-D₆-Trp-Tyr-Asn-Ile-D₈-Gly-Val-NH₂Synthesis

Take Fmoc-Leu-D₆-Trp-Tyr-Asn-Ile-D₈-Gly-Val-NH₂11.37g is dissolved in 50ml chloroforms, is added Triethylamine 5ml, reacts 4 hours, and after thin-layer chromatography monitoring reaction completely, decompression removal solvent and triethylamine are molten with dichloromethane Solution, successively with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate is dried, and is concentrated under reduced pressure, preparative chromatography Isolated white solid is target product 7.80g, yield 85.3%, purity 98.7%, abundance 99.1atom%D.

7、Fmoc-Lys-His-Ala-Leu-D₆-Trp-Tyr-Asn-Ile-D₈-Gly-Val-NH₂Synthesis

By H-Leu-D₆-Trp-Tyr-Asn-Ile-D₈-Gly-Val-NH₂9.15g is dissolved in 10ml chloroforms, stirring Lower addition DIEA1.29g, dissolving is stand-by；Weigh Fmoc-Lys-His-Ala-OH 6.12g and HOCt3.0g and be dissolved in 15ml trichlorines In methane, stirring is lower to be added dropwise DIC1.5g, reacts 10 minutes, in the mixed liquor for then starting the addition of this solution, room temperature reaction 3 Hour, thin-layer chromatography detection reaction is complete, is washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering, Then dried with anhydrous magnesium chloride, be concentrated under reduced pressure, add ether crystallization to obtain white solid as target product 13.15g, yield 87.1%, purity 98.5%, abundance 99.1atom%D.

8、H-Lys-His-Ala-Leu-D₆-Trp-Tyr-Asn-Ile-D₈-Gly-Val-NH₂Synthesis

Take Fmoc-Lys-His-Ala-Leu-D₆-Trp-Tyr-Asn-Ile-D₈-Gly-Val-NH₂15.09g is dissolved in In 50ml chloroforms, triethylamine 5ml is added, reacted 4 hours, after thin-layer chromatography monitoring reaction completely, decompression removal solvent and three second Amine, is dissolved with dichloromethane, and successively with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate is dried, and is subtracted Pressure concentration, the isolated white solid of preparative chromatography is target product 11.47g, yield 89.0%, purity 98.5%, abundance 99.1atom%D.

Embodiment 6

Cold labeling H-Lys-¹³C₆-His-Ala-Leu-D₆-Trp-Tyr-Asn-Ile-¹⁵N₁-Gly-Val-NH₂ Synthesis

1、Fmoc-Ile-¹⁵N₁-Gly-Val-NH₂Synthesis

By H-Gly-Val-NH₂1.74g is dissolved in 10ml chloroforms, and stirring is lower to add DIEA1.29g, and dissolving is treated With；Weigh Fmoc-Ile-¹⁵N₁- OH 3.54g and HOCt 3.0g are dissolved in 15ml chloroforms, and stirring is lower to be added dropwise DIC1.5g, Reaction 10 minutes, then adds this solution in the mixed liquor for starting, and room temperature reaction 3 hours, thin-layer chromatography detection reaction is complete, Washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering, then dried with anhydrous magnesium chloride, decompression Concentration, adds ether crystallization to obtain white solid as target product 4.41g, yield 86.4%, purity 98.6%, abundance 99.1atom%¹⁵N。

2、H-Ile-¹⁵N₁-Gly-Val-NH₂Synthesis

Take Fmoc-Ile-¹⁵N₁-Gly-Val-NH₂5.10g is dissolved in 50ml chloroforms, adds diethylamine 5ml, and reaction 4 is small When, after thin-layer chromatography monitoring reaction completely, decompression removal solvent and diethylamine are dissolved, successively with watery hydrochloric acid, carbon with dichloromethane Acid sodium aqueous solution, saturated common salt water washing, anhydrous magnesium sulfate are dried, and are concentrated under reduced pressure, and the isolated white solid of preparative chromatography is It is target product 4.37g, yield 85.6%, purity 98.5%, abundance 99.1atom%¹⁵N。

3、Fmoc-Trp-Tyr-Asn-Ile-¹⁵N₁-Gly-Val-NH₂Synthesis

By H-Ile-¹⁵N₁-Gly-Val-NH₂2.89g is dissolved in 10ml chloroforms, and stirring is lower to add DIEA1.29g, Dissolving, it is stand-by；Weigh Fmoc-Trp-Tyr-Asn-OH 7.39g and HOCt 3.0g to be dissolved in 15ml chloroforms, the lower drop of stirring Plus DIC1.5g, react 10 minutes, then this solution is added in the mixed liquor for starting, room temperature reaction 3 hours, thin-layer chromatography is examined Survey reaction complete, washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering, then use anhydrous chlorination Magnesium is dried, and is concentrated under reduced pressure, and adds ether crystallization to obtain white solid as target product 8.84g, yield 87.5%, purity 98.5%, abundance 99.1atom%¹⁵N。

4、H-Trp-Tyr-Asn-Ile-¹⁵N₁-Gly-Val-NH₂Synthesis

Take Fmoc-Trp-Tyr-Asn-Ile-¹⁵N₁-Gly-Val-NH₂10.10g is dissolved in 50ml chloroforms, adds three second Amine 5ml, is reacted 4 hours, and after thin-layer chromatography monitoring reaction completely, decompression removal solvent and triethylamine are dissolved with dichloromethane, according to Secondary use watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate are dried, and are concentrated under reduced pressure, and preparative chromatography is separated Target product 6.82g, yield 86.4%, purity 98.5%, abundance 99.1atom% are to white solid¹⁵N。

5、Fmoc-Leu-D₆-Trp-Tyr-Asn-Ile-¹⁵N₁-Gly-Val-NH₂Synthesis

By H-Trp-Tyr-Asn-Ile-¹⁵N₁-Gly-Val-NH₂7.89g is dissolved in 10ml chloroforms, and stirring is lower to be added Enter DIEA1.29g, dissolve, it is stand-by；Weigh Fmoc-Leu-D₆- OH 3.59g and HOCt 3.0g are dissolved in 15ml chloroforms, Stirring is lower to be added dropwise DIC1.5g, reacts 10 minutes, then adds this solution in the mixed liquor for starting, and room temperature reaction 3 hours is thin Layer chromatography detection reaction is complete, is washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering, Ran Houyong Anhydrous magnesium chloride is dried, and is concentrated under reduced pressure, and adds ether crystallization to obtain white solid as target product 10.04g, yield 88.8%, purity 98.5%, abundance 99.1atom%¹⁵N,D。

6、H-Leu-D₆-Trp-Tyr-Asn-Ile-¹⁵N₁-Gly-Val-NH₂Synthesis

Take Fmoc-Leu-D₆-Trp-Tyr-Asn-Ile-¹⁵N₁-Gly-Val-NH₂11.31g is dissolved in 50ml chloroforms, plus Enter diethylamine 5ml, react 4 hours, after thin-layer chromatography monitoring reaction completely, decompression removal solvent and triethylamine use dichloromethane Dissolving, successively with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate is dried, and is concentrated under reduced pressure, and prepares color Compose isolated white solid as target product 7.98g, yield 87.7%, purity 98.7%, abundance 99.1atom%¹⁵N,D。

7、Fmoc-His-Ala-Leu-D₆-Trp-Tyr-Asn-Ile-¹⁵N₁-Gly-Val-NH₂Synthesis

By H-Leu-D₆-Trp-Tyr-Asn-Ile-¹⁵N₁-Gly-Val-NH₂9.10g is dissolved in 10ml chloroforms, is stirred Lower addition DIEA1.29g is mixed, is dissolved, it is stand-by；Weigh Fmoc-His-Ala-OH 4.66g and HOCt3.0g and be dissolved in the chloromethanes of 15ml tri- In alkane, stirring is lower to be added dropwise DIC1.5g, reacts 10 minutes, then adds this solution in the mixed liquor for starting, and room temperature reaction 3 is small When, thin-layer chromatography detection reaction is complete, is washed successively with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt after reacting liquid filtering, so Dried with anhydrous magnesium chloride afterwards, be concentrated under reduced pressure, add ether crystallization to obtain white solid as target product 11.48g, yield 84.5%, purity 98.5%, abundance 99.1atom%¹⁵N,D。

8、H-His-Ala-Leu-D₆-Trp-Tyr-Asn-Ile-¹⁵N₁-Gly-Val-NH₂Synthesis

Take Fmoc-His-Ala-Leu-D₆-Trp-Tyr-Asn-Ile-¹⁵N₁-Gly-Val-NH₂13.58g is dissolved in 50ml chlorine In imitative, triethylamine 5ml is added, reacted 4 hours, after thin-layer chromatography monitoring reaction completely, decompression removal solvent and triethylamine, with two Chloromethanes is dissolved, and successively with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate is dried, and is concentrated under reduced pressure, The isolated white solid of preparative chromatography is target product 9.86g, yield 86.7%, purity 98.5%, abundance 99.1atom%¹⁵N,D。

9、Fmoc-Lys-¹³C₆-His-Ala-Leu-D₆-Trp-Tyr-Asn-Ile-¹⁵N₁-Gly-Val-NH₂Synthesis

By H-His-Ala-Leu-D₆-Trp-Tyr-Asn-Ile-¹⁵N₁-Gly-Val-NH₂11.37g is dissolved in 10ml trichlorines In methane, stirring is lower to add DIEA1.29g, and dissolving is stand-by；Weigh Fmoc-Lys-¹³C₆- OH 3.74g and HOCt 3.0g are dissolved in In 15ml chloroforms, stirring is lower to be added dropwise DIC1.5g, reacts 10 minutes, in the mixed liquor for then starting the addition of this solution, room Temperature reaction 3 hours, thin-layer chromatography detection reaction is complete, after reacting liquid filtering with sodium bicarbonate solution, hydrochloric acid, saturated aqueous common salt according to Secondary washing, is then dried with anhydrous magnesium chloride, is concentrated under reduced pressure, and adds ether crystallization to obtain white solid as target product 13.02g, yield 87.2%, purity 98.5%, abundance 99.1atom%¹³C,¹⁵N,D。

10、H-Lys-¹³C₆-His-Ala-Leu-D₆-Trp-Tyr-Asn-Ile-¹⁵N₁-Gly-Val-NH₂Synthesis

Take Fmoc-Lys-¹³C₆-His-Ala-Leu-D₆-Trp-Tyr-Asn-Ile-¹⁵N₁-Gly-Val-NH₂14.93g is molten In 50ml chloroforms, triethylamine 5ml is added, reacted 4 hours, after thin-layer chromatography monitoring reaction completely, decompression removal solvent and three Ethamine, is dissolved with dichloromethane, and successively with watery hydrochloric acid, aqueous sodium carbonate, saturated common salt water washing, anhydrous magnesium sulfate is dried, Concentrated under reduced pressure, the isolated white solid of preparative chromatography is target product 10.98g, and yield 86.3%, purity 98.5% is rich Degree 99.1atom%¹³C,¹⁵N,D。

Specific embodiment of the invention is described above.It is to be appreciated that the invention is not limited in above-mentioned Particular implementation, those skilled in the art can within the scope of the claims make various deformations or amendments, this not shadow Sound substance of the invention.

Claims

1. a kind of synthetic method of cold labeling ALA feature peptide fragment, it is characterised in that utilize liquid phase synthesis Polypeptide method, the amino acid with cold labeling or non-marked as initiation material, and Fmoc protection non-marked or mark Note amino acid, in the presence of condensing agent, synthesizes dipeptides, then takes off protection group Fmoc, then the non-marked protected with Fmoc or The different aminoacids of person's mark repeat above-mentioned reaction, final synthesizing stable isotope marks ALA feature peptide fragment VGINYWLAHK。

2. the synthetic method of a kind of cold labeling ALA feature peptide fragment according to claim 1, it is special Levy and be, the method specifically uses following steps：

1) with cold labeling or non-marked valine (H-Val-MH₂) be initiation material, with Fmoc protect non-marked or Person's marked glycine (Fmoc-Gly-OH) carries out coupling reaction in the presence of condensing agent, is then dried successively, and decompression is steamed Evaporate, be recrystallized to give dipeptides Fmoc-Gly-Val-NH₂, then take off protection group Fmoc and obtain H-Gly-Val-NH₂；

2) step 1) H-Gly-Val-NH for preparing₂The non-marked or mark isoleucine (Fmoc- protected with Fmoc Ile-OH coupling reaction) is carried out in the presence of condensing agent, is then dried successively, vacuum distillation is recrystallized to give tripeptides Fmoc-Ile-Gly-Val-NH₂, take off protection group Fmoc and obtain H-Ile-Gly-Val-NH₂；

4) step 3) H-Asn-Ile-Gly-Val-NH for preparing₂The non-marked or mark tyrosine protected with Fmoc (Fmoc-Tyr-OH) coupling reaction is carried out in the presence of condensing agent, is then dried successively, vacuum distillation is recrystallized To pentapeptide Fmoc-Tyr-Asn-Ile-Gly-Val-NH₂, take off protection group Fmoc and obtain H-Tyr-Asn-Ile-Gly-Val- NH₂；

5) step 4) H-Tyr-Asn-Ile-Gly-Val-NH for preparing₂The non-marked or mark color ammonia protected with Fmoc Sour (Fmoc-Trp-OH) carries out coupling reaction in the presence of condensing agent, is then dried successively, vacuum distillation, recrystallizes Obtain hexapeptide Fmoc-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, take off protection group Fmoc and obtain H-Trp-Tyr-Asn-Ile- Gly-Val-NH₂；

6) step 5) H-Trp-Tyr-Asn-Ile-Gly-Val-NH for preparing₂The non-marked or mark protected with Fmoc Leucine (Fmoc-Leu-OH) carries out coupling reaction in the presence of condensing agent, is then dried successively, vacuum distillation, weight Crystallization obtains heptapeptide Fmoc-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, take off protection group Fmoc and obtain H-Leu-Trp- Tyr-Asn-Ile-Gly-Val-NH₂；

7) step 6) H-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH for preparing₂With Fmoc protect non-marked or Mark alanine (Fmoc-Ala-OH) carries out coupling reaction in the presence of condensing agent, is then dried successively, and decompression is steamed Evaporate, be recrystallized to give octapeptide Fmoc-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, take off protection group Fmoc and obtain H-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂；

8) step 7) H-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH for preparing₂The non-marked protected with Fmoc Or mark histidine (Fmoc-His-OH) carries out coupling reaction in the presence of condensing agent, is then dried successively, depressurize Distillation, is recrystallized to give nonapeptide Fmoc-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, take off protection group Fmoc obtains H-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂；

9) step 8) H-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH for preparing₂It is nonstandard with what Cbz was protected Note or mark lysine (Cbz-Lys-OH) carry out coupling reaction in the presence of condensing agent, are then dried successively, subtract Pressure distillation, is recrystallized to give decapeptide Cbz-Lys-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, take off protection Base Cbz obtains H-Lys-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, as ALA feature peptide fragment VGINYWLAHK。

3. the synthetic method of a kind of cold labeling ALA feature peptide fragment according to claim 1 and 2, its It is characterised by, with non-marked valine (H-Val-MH₂) for initiation material when, step 1) to step 9) in 9 kinds of Fmoc protections At least one kind is stable isotope in amino acid and the lysine of Cbz protections¹³C、¹⁵The amino of one or more marks of N, D Acid.

4. the synthetic method of a kind of cold labeling ALA feature peptide fragment according to claim 2, it is special Levy and be, step 1) in,

Described cold labeling or non-marked valine (H-Val-MH₂) and Fmoc protection non-marked or mark it is sweet The process conditions that propylhomoserin (Fmoc-Gly-OH) is coupled are：

The condensing agent for using is in one or two mixtures and HBTU, HCTU, HOBt, HOCt in EDCI, DMAP, DCC The mixed liquor of one or two kinds of mixture, solvent is chloroform, and reaction temperature is 0-20 DEG C,

Cold labeling or non-marked valine (H-Val-MH₂), Fmoc protect glycine (Fmoc-Gly-OH) and condensation The mol ratio of agent is 1:1:1.1-1:1:2；

Removing Fmoc-Gly-Val-NH₂The reagent that middle Fmoc protection groups are used is one kind in triethylamine, diethylamine, piperidines or several Kind, solvent is one or more in dichloromethane, chloroform, ether, Fmoc-Gly-Val-NH₂, removing reagent and solvent Mol ratio is 1:1:5-1:3:15,0-20 DEG C of controlling reaction temperature, reaction time 2-6 hours.

5. the synthetic method of a kind of cold labeling ALA feature peptide fragment according to claim 2, it is special Levy and be, step 2) in,

Described H-Gly-Val-NH₂The non-marked or mark isoleucine (Fmoc-Ile-OH) protected with Fmoc are coupled Process conditions be：Condensing agent is in one or two mixtures and HBTU, HCTU, HOBt, HOCt in EDCI, DMAP, DCC One or two kinds of mixture mixed liquor, solvent is chloroform, and reaction temperature is 0-20 DEG C,

H-Gly-Val-NH₂With Fmoc protect non-marked or mark isoleucine (Fmoc-Ile-OH) and condensing agent mole Than being 1:1:1.1-1:1:2；

Removing Fmoc-Ile-Gly-Val-NH₂The reagent that middle Fmoc protection groups are used is the one kind in triethylamine, diethylamine, piperidines Or it is several, solvent is one or more in dichloromethane, chloroform, ether, Fmoc-Ile-Gly-Val-NH₂, removing reagent It is 1 with the mol ratio of solvent:1:5-1:3:15,0-20 DEG C of reaction temperature, reaction time 2-6 hours.

6. the synthetic method of a kind of cold labeling ALA feature peptide fragment according to claim 2, it is special Levy and be, step 3) in,

Described H-Ile-Gly-Val-NH₂The non-marked or mark asparagine (Fmoc-Asn-OH) protected with Fmoc are carried out The process conditions of coupling are：Condensing agent be EDCI, DMAP, DCC in one or two mixtures and HBTU, HCTU, HOBt, The mixed liquor of the one or two kinds of mixture in HOCt, solvent is chloroform, and reaction temperature is 0-20 DEG C,

H-Ile-Gly-Val-NH₂The non-marked or mark asparagine (Fmoc-Asn-OH) and condensing agent protected with Fmoc Mol ratio is 1:1:1.1-1:1:2；

Removing Fmoc-Asn-Ile-Gly-Val-NH₂The reagent that middle Fmoc protection groups are used is in triethylamine, diethylamine, piperidines One or more, solvent is one or more in dichloromethane, chloroform, ether, Fmoc-Asn-Ile-Gly-Val-NH₂, it is de- Except being 1 with the mol ratio of reagent and solvent:1:5-1:3:15,0-20 DEG C of reaction temperature, reaction time 2-6 hours.

7. the synthetic method of a kind of cold labeling ALA feature peptide fragment according to claim 2, it is special Levy and be, step 4) in,

Described H-Asn-Ile-Gly-Val-NH₂The non-marked or mark tyrosine (Fmoc-Tyr-OH) protected with Fmoc are entered Row coupling process conditions be：Condensing agent be EDCI, DMAP, DCC in one or two mixtures and HBTU, HCTU, HOBt, The mixed liquor of the one or two kinds of mixture in HOCt, solvent is chloroform, and reaction temperature is 0-20 DEG C,

H-Asn-Ile-Gly-Val-NH₂The non-marked or mark tyrosine (Fmoc-Tyr-OH) and condensing agent protected with Fmoc Mol ratio be 1:1:1.1-1:1:2；

Removing Fmoc-Tyr-Asn-Ile-Gly-Val-NH₂The reagent that middle Fmoc protection groups are used is triethylamine, diethylamine, piperidines In one or more, solvent be dichloromethane, chloroform, ether in one or more, Fmoc-Tyr-Asn-Ile-Gly- Val-NH₂, removing reagent and solvent mol ratio be 1:1:5-1:3:15,0-20 DEG C of reaction temperature, the reaction time, 2-6 was small When.

8. the synthetic method of a kind of cold labeling ALA feature peptide fragment according to claim 2, it is special Levy and be, step 5) in,

Described H-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or mark tryptophan (Fmoc-Trp- protected with Fmoc OH the process conditions) being coupled are：Condensing agent be EDCI, DMAP, DCC in one or two mixtures and HBTU, HCTU, The mixed liquor of the one or two kinds of mixture in HOBt, HOCt, solvent is chloroform, and reaction temperature is 0-20 DEG C,

H-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or mark tryptophan (Fmoc-Trp-OH) protected with Fmoc and contracting The mol ratio of mixture is 1:1:1.1-1:1:2；

Removing Fmoc-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The reagent that middle Fmoc protection groups are used for triethylamine, diethylamine, One or more in piperidines, solvent is one or more in dichloromethane, chloroform, ether, Fmoc-Trp-Tyr-Asn- Ile-Gly-Val-NH₂, removing reagent and solvent mol ratio be 1:1:5-1:3:15,0-20 DEG C of reaction temperature, during reaction Between 2-6 hours.

9. the synthetic method of a kind of cold labeling ALA feature peptide fragment according to claim 2, it is special Levy and be, step 6) in,

Described H-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or labelled leucine (Fmoc- protected with Fmoc Leu-OH the process conditions) being coupled are：Condensing agent be EDCI, DMAP, DCC in one or two mixtures and HBTU, The mixed liquor of the one or two kinds of mixture in HCTU, HOBt, HOCt, solvent is chloroform, and reaction temperature is 0-20 DEG C,

H-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or labelled leucine (Fmoc-Leu-OH) protected with Fmoc And the mol ratio of condensing agent is 1:1:1.1-1:1:2；

Removing Fmoc-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The reagent that middle Fmoc protection groups are used is triethylamine, two One or more in ethamine, piperidines, solvent is one or more in dichloromethane, chloroform, ether, Fmoc-Leu-Trp- Tyr-Asn-Ile-Gly-Val-NH₂, removing reagent and solvent mol ratio be 1:1:5-1:3:15, reaction temperature 0-20 DEG C, reaction time 2-6 hours.

10. the synthetic method of a kind of cold labeling ALA feature peptide fragment according to claim 2, it is special Levy and be, step 7) in,

Described H-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or mark alanine protected with Fmoc (Fmoc-Ala-OH) process conditions being coupled are：Condensing agent be EDCI, DMAP, DCC in one or two mixtures with The mixed liquor of the one or two kinds of mixture in HBTU, HCTU, HOBt, HOCt, solvent is chloroform, and reaction temperature is 0-20 DEG C,

H-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or mark alanine (Fmoc- protected with Fmoc Ala-OH) and condensing agent mol ratio be 1:1:1.1-1:1:2；

Removing Fmoc-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The reagent that middle Fmoc protection groups are used is three second One or more in amine, diethylamine, piperidines, solvent is one or more in dichloromethane, chloroform, ether, Fmoc-Ala- Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, removing reagent and solvent mol ratio be 1:1:5-1:3:15, reaction temperature 0-20 DEG C, reaction time 2-6 hours of degree.

A kind of 11. synthetic methods of cold labeling ALA feature peptide fragment according to claim 2, it is special Levy and be, step 8) in,

Described H-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or mark group ammonia protected with Fmoc The process conditions that sour (Fmoc-His-OH) is coupled are：Condensing agent is one or two mixtures in EDCI, DMAP, DCC With the mixed liquor of the one or two kinds of mixture in HBTU, HCTU, HOBt, HOCt, solvent is chloroform, and reaction temperature is 0-20 DEG C,

Removing Fmoc-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The reagent that middle Fmoc protection groups are used is three One or more in ethamine, diethylamine, piperidines, solvent is one or more in dichloromethane, chloroform, ether, Fmoc- His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, removing reagent and solvent mol ratio be 1:1:5-1:3: 15,0-20 DEG C of reaction temperature, reaction time 2-6 hours.

A kind of 12. synthetic methods of cold labeling ALA feature peptide fragment according to claim 2, it is special Levy and be, step 9) in,

Described H-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or mark protected with Cbz rely The process conditions that propylhomoserin (Cbz-Lys-OH) is coupled are：Condensing agent is one or two mixing in EDCI, DMAP, DCC The mixed liquor of the one or two kinds of mixture in thing and HBTU, HCTU, HOBt, HOCt, solvent is chloroform, and reaction temperature is 0- 20 DEG C,

H-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂The non-marked or mark lysine protected with Cbz (Cbz-Lys-OH) and condensing agent mol ratio be 1:1:1.1-1:1:2；

Removing Cbz-Lys-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂Middle Cbz protection groups are urged in Metal Palladium Hydrogen or hydrogen bromide/acetic acid mixed solvent are passed through under change；

Cbz-Lys-His-Ala-Leu-Trp-Tyr-Asn-Ile-Gly-Val-NH₂, Metal Palladium and hydrogen mol ratio be 1: 0.01:1-1:0.05:15, reaction temperature is 0-50 DEG C, reaction time 1-4 hours；Cbz-Lys-His-Ala-Leu-Trp- Tyr-Asn-Ile-Gly-Val-NH₂, hydrogen bromide/acetic acid mol ratio be 1:1-1:5, reaction temperature is 0-50 DEG C, during reaction Between 1-4 hours.

A kind of 13. synthetic methods of cold labeling ALA feature peptide fragment according to claim 1 and 2, Characterized in that, the ALA feature peptide fragment VGINYWLAHK for preparing is cold labeling¹³C、¹⁵Tri- kinds of N, D One or more marks in isotope, its molecular structural formula is as follows：H-Lys-His-Ala-Leu-Trp-Tyr- Asn-Ile-Gly-Val-NH₂, wherein,

H-Val-NH₂For¹³C、¹⁵One or more marks in tri- kinds of isotopes of N, D.