CN107090015A - A kind of target molecule polypeptide for specifically binding signal transduction factor and preparation method thereof - Google Patents
A kind of target molecule polypeptide for specifically binding signal transduction factor and preparation method thereof Download PDFInfo
- Publication number
- CN107090015A CN107090015A CN201610090128.0A CN201610090128A CN107090015A CN 107090015 A CN107090015 A CN 107090015A CN 201610090128 A CN201610090128 A CN 201610090128A CN 107090015 A CN107090015 A CN 107090015A
- Authority
- CN
- China
- Prior art keywords
- gly
- resin
- tbu
- fmoc
- dmf
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to a kind of target molecule polypeptide for specifically binding signal transduction factor and preparation method thereof.The amino acid sequence of the target molecule polypeptide is SNFYMPLGGGSK.In the preparation method technique of the present invention, using gentle Fmoc routes, the PIP that de- Fmoc only uses 20% is often walked, often step condensation reaction time is short, substantially reduces the production cycle；Using reasonable sealing technique, the purity of thick peptide product is improved；Cutting uses TFA, it is to avoid conventional hydrogen fluoride, reduces the three wastes produced in production, is conducive to industrialized production.
Description
Technical field
The present invention relates to a kind of target molecule polypeptide of specific binding signal transduction factor (EPCAM) and its preparation side
Method.Specifically, the target molecule polypeptide of the biological activity combined the invention provides the targeting with higher EPCAM.
Background technology
Barrett esophagus
The Esophageal Mucosa that gastroesophageal reflux (Gastroesophageal Reflux Disease, GERD) is triggered with
The intestinal metaplasia of goblet cell, i.e. Barrett oesophaguses (Barrett ' s Esophagus, BE), research show that adenocarcinoma of esophagus is several
BE is derived from, more than 60% gastroesophageal junction gland cancer and cardia cancer derive from BE.The main cause that BE is produced is long
Phase chronic gastroesophageal reflux (Gastroesophageal Reflux Disease, GERD).Length of the epithelium of esophagus in bile acid
Along intestinal metaplasia (BE)-minuent dysplasia (Low Grade Dysplasia, LGD)-high grade dysplasia under phase erosion
Canceration occurs for the trend of (High Grade Dysplasia, HGD)-gland cancer.At present, recommend to carry out for BE patient to peep in periodically
Mirror is monitored, and HGD and early stage EA requires row surgery excision.However, HGD and early stage EA flat structure, without obvious textural anomaly
And tubercle, in Microendoscopic it is difficult to distinguish.The four-quadrant grab sample biopsy or the orientation work in suspicious region generally used
Inspection is easily failed to pinpoint a disease in diagnosis.Therefore, with the intensification recognized BE molecular biology and the defect of traditional remedies, BE detection and treatment
Increasingly to targetingization and individuation.
Progress of the micromolecule polypeptide in targeted therapy
Using small-molecular peptides as guiding, building treatment or diagnostic targeted drug has cost low, and immunogenicity is small, group
The advantage of good penetrability is knitted, and the affinity of many micromolecule polypeptides and its part is very high, no less than the parent of antibody and antigen
With.Two major classes can be included as the small-molecular peptides being oriented to, a class is naturally occurring, such as growth hormone-release inhibiting factor
(Somatostatin), vasoactive intestinal peptide (Vasoactive Intestinal Peptide, VIP) etc., they are respectively in god
Specific diagnosis and treatment through endocrine tumors, tumor in digestive tract etc. have played good guidance quality effect.It is another kind of, be
Non-naturally occurring peptide, compared with native peptides, the species of such peptide is more, and identification is more convenient, and be possible to than native peptides with
The affinity of its corresponding acceptor is higher, therefore such peptide turns into focus of concern in recent years.Peptide storehouse sieve is presented in phage random
Choosing is considered as the rapidly and effectively method for such peptide screening.
Application of the micromolecule polypeptide in targeted therapy
The micromolecule polypeptide of targeting can be coupled radionuclide as diagnostic reagent, and isotope, fluorescein etc. is combined
PET, ECT or fluorescence endoscope carry out medical diagnosis on disease.Used as treatment, they can be coupled chemotherapeutics, engineered protein
Medicine, gene therapy medicament builds tumor-targeting new drug, and increase antineoplastic improves anti-in the drug concentration of tumor by local
Tumor effect, reduces system toxicity.The RGD- adriamycins of target tumor neovascular endothelium cell surface integrin molecule up to
While to identical treatment effect, consumption is only the 1/40 of simple adriamycin group；Target tumor neovascular endothelium cell surface
The NGR- human tumour necrosis factors combination with cisplatin treatment Refractory solid tumor of CD13 molecules has system toxicity low, patient's compliance
The characteristics of good；NGR- adriamycins are 40% more than common adriamycin suppression tumour growth；Target the SIGYPLP- of human umbilical vein cell
The efficiency gene transfection of adenovirus is 15.5 times of simple adenovirus vector；RGD-TNF can improve tumor by local medicine
Thing concentration, increases the therapeutic index of TNF.Substantial amounts of basis and clinical and experimental study confirm micromolecule polypeptide
It is directed at the potential in terms of neoplasm targeted therapy.
The Research Prospects of the target molecule of the micromolecule polypeptide of adenocarcinoma of esophagus specific binding effect
Current research data display BE is developed into during EA, and very polymolecular expression changes.For example：In squamous
Skin transcription factor P63, Sox2, Pax9 are expressed in HGD tissues and lowered；Gut epithelium transcription factor Cdx2 is through bile acid treatment
Squamous cell up-regulated expression；BMP BMP4 up-regulated expressions in the esophageal epithelial cell of GERD patient；BMP4
Upstream signaling molecule SHH EA tissue expressions raise；A kind of newfound Farnesoid X receptor TGR5 is raised in EA tissue expressions
Deng.In addition, a variety of skin factor acceptors such as c-erbB2, EGFR, SRC, K-ras, cyclinD1, p16, p27, APC, p53, egg
White kinases, proto-oncogene are found related to BE cancerations.Achievement in research is although numerous, but EA is so far without clearly specificity
Tumor markers, is that neoplasm targeted therapy and polypeptide are oriented to antineoplastic research and bring difficulty.Adenocarcinoma of esophagus is specifically bound
The target molecule of the micromolecule polypeptide of effect is exactly probably the specific tumour mark of adenocarcinoma of esophagus, is controlled for cancer target
Treat significant, also occur development mechanism accumulation clue for adenocarcinoma of esophagus.
The content of the invention
It is an object of the present invention to provide a kind of specific binding signal transduction factor (being abbreviated as EPCAM)
Target molecule polypeptide, the target molecule polypeptide includes amino acid sequence SNFYMPLGGGSK, or the target molecule polypeptide includes ammonia
Replace, lack or add the amino acid sequence of one or several amino acid in base acid sequence SNFYMPLGGGSK and with specificity
The polypeptide of junctional epithelium cell adhesion molecule activity.
Preferably, the amino acid sequence of the target molecule polypeptide is SNFYMPLGGGSK.
Another object of the present invention is to provide the preparation method of above-mentioned target molecule polypeptide, methods described is included with Fmoc-
Solid phase polypeptide synthesis synthesizes crude product polypeptide.
Preferably, methods described is also isolated and purified including the use of C18 or C8 posts to the crude product polypeptide.
Preferably, in the Fmoc- solid phase polypeptide synthesis, with triphen chloromethyl resin, 4- methyl-triphen chloromethyl
Any one of resin, 4- methoxyl groups-triphen chloromethyl resin, the chloro- triphen chloromethyl resins of 2- or Wang resin carry for solid phase
Body.
Preferably, it is an object of the present invention to by with N, N- Diisopropylcarbodiimides/1- hydroxyls-nitrogen of benzo-three
Azoles (DIC/HOBt), N, N- Diisopropylcarbodiimides/N- hydroxyl -7- azos BTAs (DIC/HOAt), 7- azepines
Benzothiazole -1- bases-oxygen-(three-(dimethylamino) phosphines) hexafluorophosphate/1- hydroxyls-benzo-triazole (BOP/HOBt), 7- nitrogen
Miscellaneous benzothiazole -1- bases-oxygen-(three-(dimethylamino) phosphines) hexafluorophosphate/1- hydroxyls-benzo-triazole/N- hydroxyl -7- azos
BTA (BOP/HOAt), BTA-N, N, N ', N '-tetramethylurea hexafluorophosphoric acid ester/1- hydroxyls-nitrogen of benzo-three
Azoles (HBTU/HOBt), BTA-N, N, N ', N '-tetramethylurea hexafluorophosphoric acid ester/N- hydroxyl -7- azo BTAs
(HBTU/HOAt), 2- (7- azos BTA)-N, N, N ', N '-tetramethylurea hexafluorophosphoric acid ester/1- hydroxyls-benzo-three
Nitrogen azoles (HATU/HOBt), 2- (7- azos BTA)-N, N, N ', N '-tetramethylurea hexafluorophosphoric acid ester/N- hydroxyls -7- are even
Nitrogen BTA (HATU/HOAt) or 6- Chloro-Benzotriazole -1,1,3,3- tetramethylureas hexafluorophosphoric acids ester/1- hydroxyls-benzene
And a kind of in-triazole (HCTU/HOBt) is that one kind that condensing agent carries out in peptide reaction is that condensing agent carries out peptide reaction,
Obtain the SNFYMPLGGGSK dodecapeptide resins of protection.
Preferably, it is synchronous to carry out de- side chain protecting group and cut peptide after the target molecule polypeptide resin protected, obtain
Target molecule polypeptide crude product.Again through C18 (or C8) column separating purification, after freeze-drying, the one kind of purity up to more than 98% is made to obtain
Specifically bind the target molecule polypeptide (hereinafter abbreviated as dodecapeptide or SNF dodecapeptides) of signal transduction factor.
Preferably, wherein crude product through C18 or C8 chromatogram column separating purifications the step of be：
Target molecule polypeptide crude product is dissolved in the aqueous solution, filtered, filtrate has two through C18 or C8 chromatographies, mobile phase
Kind：A：0.1% trifluoroacetic acid adds 99.9% water；B：0.1% trifluoroacetic acid adds 99.9% acetonitrile；Gradient elution；Flow velocity is 1-
150ml/min；Detection wavelength is：215~285nm；The efflux required for collecting is tracked with liquid chromatograph, is freezed, is obtained
Finished product.
The adenocarcinoma of esophagus specific binding polypeptide obtained has biological activity, with the targeting knot higher with EPCAM
Conjunction ability.Tested by RNA interference, fluorescence polarization experiment, immunofluorescence experiment determines the protein active and targeting combination energy
Power, as a result shows, adenocarcinoma of esophagus specific binding polypeptide SNFYMPL has higher EPCAM targeting binding ability.And the party
Method just completes isolating and purifying for destination protein using only a chromatographic column, and step is simple, quick, cost is low, is adenocarcinoma of esophagus
Effect in diagnosing tumor treatment of diagnosis and treatment and signal transduction factor established further research and extensively
The basis of application.
In addition, the targeting in order to carry out adenocarcinoma of esophagus specific binding dodecapeptide SNF and signal transduction factor EPCAM
Property checking, further verify expression of the target molecule in esophageal adenocarcinoma cells using transcript profile sequencing result, using fluorescence polarization,
The affinity of RNA interference and immunofluorescence assay dodecapeptide and target molecule, identifies the target molecule of dodecapeptide.
In addition, the unique advantage of chemical synthesis that the present invention is provided.Its advantage is：SNF dodecapeptides solid phase of the present invention
Synthesis preparation method has the characteristics that：Raw and auxiliary material convenient sources, process stabilizing, high income is quality controllable, product purity
Height, with short production cycle, cost is low, is produced on a large scale.The great market competition of SNF dodecapeptides prepared using the inventive method
Power, its production scale is fully able to meet the application demand of SNF dodecapeptides, and its quality is fully able to reach answering for SNF dodecapeptides
With requiring.
In addition, in the technique of the present invention, using gentle Fmoc routes, often walk de- Fmoc only with 20% PIP, often walk
Condensation reaction time is short, substantially reduces the production cycle；Using reasonable sealing technique, the purity of thick peptide product is improved；Cutting is adopted
With TFA, it is to avoid conventional hydrogen fluoride, the three wastes produced in production are reduced, are conducive to industrialized production.
Brief description of the drawings
Fig. 1 fluorescence polarization results
Expression of the EPCAM in mRNA level in-site in esophageal adenocarcinoma cells after Fig. 2 RNA interferences
Expression of the EPCAM in protein level in esophageal adenocarcinoma cells after Fig. 3 RNA interferences
The immunofluorescence results of esophageal adenocarcinoma cells after Fig. 4 RNA interferences
Embodiment
Illustrate the present invention in more detail by the following examples, but this does not imply that protection scope of the present invention is by this
A little embodiment limitations.
It is according to the technical solution adopted in the present invention：By with triphen chloromethyl resin, 4- methyl-triphen chloromethyl tree
Any one of fat, 4- methoxyl groups-triphen chloromethyl resin, the chloro- triphen chloromethyl resins of 2- or Wang resin are initiation material,
With DIC/HOBt or DIC/HOAt or BOP/HOBt or BOP/HOAt or HBTU/HOBt or HBTU/HOAt or HATU/HOBt or
One kind in HATU/HOAt or HCTU/HOBt carries out peptide reaction for condensing agent, obtains protection SNFYMPLGGGSK dodecapeptide trees
Fat, it is thereafter, synchronous to carry out de- side chain protecting group and cut peptide, SNFYMPLGGGSK crude products are obtained, then through C18 (or C8) post separation
Purifying, after freeze-drying, is made to obtain SNF dodecapeptide of the purity up to more than 98%.The adenocarcinoma of esophagus specific binding obtained is more
Peptide has biological activity, with the targeting binding ability higher with EPCAM.Tested by RNA interference, fluorescence polarization experiment,
Immunofluorescence experiment, determines the protein active and targeting binding ability, as a result shows, adenocarcinoma of esophagus specific binding polypeptide
SNFYMPLGGGSK has higher EPCAM targeting binding ability.And this method just completes mesh using only a chromatographic column
Albumen isolate and purify, step is simple, quick, cost is low, is the diagnosis and treatment of adenocarcinoma of esophagus and epithelial cell stick
Further research and wide variety of basis have been established in effect of the molecule in diagnosing tumor treatment.
Above-mentioned technical proposal is realized, overall plan is followed the steps below：
1) Peptide systhesis.
(1) Fmoc-Lys (Boc)-resin is prepared：
It is chloro- with triphen chloromethyl resin, 4- methyl-triphen chloromethyl resin, 4- methoxyl groups-triphen chloromethyl resin, 2-
Triphen chloromethyl resin or Wang resin one kind therein make resin, with DMF or dichloromethane immersion 10~
60 minutes, in mixture, the bulking value concentration of resin was 5-20ml/g；
Then in above-mentioned resin, N, N'- diisopropylethylamine (DIEA), N, N- Diisopropylcarbodiimides are added
(DIC) or DMAP (DMAP), Fmoc-Val-OH, 10~50 DEG C are reacted 0.5~5 hour, add closed reagent
10~50 DEG C of methanol, chlorobenzoyl chloride or pyridine are reacted 0.2-3 hours, and resin is washed with isopropanol, DMF, is obtained
Obtain Fmoc-Lys (Boc)-resin；
DIEA or DMAP molal quantity is 2~20 times of resin；
Fmoc-Lys (Boc)-OH molal quantity is 1~10 times of resin：
In reaction solution, the concentration of resin is 0.5~5ml/g；
(2) Fmoc-Ser (tBu)-Lys (Boc)-resin is prepared：
In Fmoc-Lys (Boc)-resin of step (1), add 10~50 DEG C of reagent of raising one's hat and react 5~30 minutes, use
Vavuum pump is drained, and is rejoined 10~50 DEG C of reagent of raising one's hat and is reacted 10~60 minutes, drains, steamed with isopropanol, DMF, weight
DMF is washed, and is added in Fmoc-Ser (tBu)-OH, DIEA and TBTU/HOBt, HBTU/HOBt or the BOP/HOBt dissolved with DMF
At least one mixture, 10~50 DEG C are reacted 10~60 minutes, are drained with vavuum pump, with isopropanol, DMF, the steamed DMF of weight
Washing, is drained, and obtains Fmoc-Ser (tBu)-Lys (Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Ser (tBu)-OH molal quantity is 1-5 times of resin；
The ratio of the weight of Fmoc-Lys (Boc)-resin and the addition for reagent of raising one's hat is 5~20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(3) Fmoc-Gly-Ser (tBu)-Lys (Boc)-resin is prepared：
In Fmoc-Ser (tBu)-Lys (Boc)-resin of step (2), addition raise one's hat 10~50 DEG C of reagent reaction 5~
30 minutes, drained with vavuum pump, rejoin 10~50 DEG C of the reagent of raising one's hat and react 10~60 minutes, drain, with isopropanol, DMF,
The steamed DMF washings of weight, add Fmoc-Ser (the tBu)-OH dissolved with DMF, DIEA and TBTU/HOBt, HBTU/HOBt or
At least one of BOP/HOBt mixtures, 10~50 DEG C react 10~60 minutes, drained with vavuum pump, with isopropanol, DMF,
The steamed DMF washings of weight, drain, obtain Fmoc-Gly-Ser (tBu)-Lys (Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Gly-OH molal quantitys are 2~5 times of resin；
The ratio of the weight of Fmoc-Ser (tBu)-Lys (Boc)-resin and the addition for reagent of raising one's hat is 5~20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(4) Fmoc-Gly-Gly-Ser (tBu)-Lys (Boc)-resin is prepared：
In Fmoc-Gly-Ser (tBu)-Lys (Boc)-resin of step (3), 10~50 DEG C of reactions of reagent of raising one's hat are added
5~30 minutes, drained with vavuum pump, rejoin 10~50 DEG C of the reagent of raising one's hat and react 10~60 minutes, drain, with isopropanol,
The steamed DMF washings of DMF, weight, add Fmoc-Ser (tBu)-OH, DIEA and TBTU/HOBt, the HBTU/HOBt dissolved with DMF
Or at least one of BOP/HOBt mixture, 10~50 DEG C are reacted 10~60 minutes, are drained with vavuum pump, with isopropanol,
The steamed DMF washings of DMF, weight, drain, obtain Fmoc-Gly-Gly-Ser (tBu)-Lys (Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Gly-OH molal quantitys are 2~5 times of resin；
The ratio of the weight of Fmoc-Gly-Ser (tBu)-Lys (Boc)-resin and the addition for reagent of raising one's hat for 5~
20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(5) Fmoc-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin is prepared：
In Fmoc-Gly-Gly-Ser (tBu)-Lys (Boc)-resin of step (4), 10~50 DEG C of reagent of raising one's hat is added
Reaction 5~30 minutes, is drained with vavuum pump, is rejoined 10~50 DEG C of reagent of raising one's hat and is reacted 10~60 minutes, drains, use isopropyl
The steamed DMF washings of alcohol, DMF, weight, add Fmoc-Ser (tBu)-OH, DIEA and TBTU/HOBt, the HBTU/ dissolved with DMF
At least one of HOBt or BOP/HOBt mixture, 10~50 DEG C are reacted 10~60 minutes, are drained with vavuum pump, are used isopropyl
The steamed DMF washings of alcohol, DMF, weight, drain, obtain Fmoc-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Gly-OH molal quantitys are 2~5 times of resin；
The ratio of the weight of Fmoc-Gly-Gly-Ser (tBu)-Lys (Boc)-resin and the addition for reagent of raising one's hat is 5
~20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(6) Fmoc-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin is prepared：
In Fmoc-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin of step (5), addition raise one's hat reagent 10~
50 DEG C are reacted 5~30 minutes, are drained with vavuum pump, are rejoined 10~50 DEG C of reagent of raising one's hat and are reacted 10~60 minutes, drain, use
The steamed DMF washings of isopropanol, DMF, weight, add dissolved with DMF Fmoc-Ser (tBu)-OH, DIEA and TBTU/HOBt,
At least one of HBTU/HOBt or BOP/HOBt mixture, 10~50 DEG C are reacted 10~60 minutes, are drained with vavuum pump, are used
The steamed DMF washings of isopropanol, DMF, weight, drain, obtain Fmoc-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-tree
Fat；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Leu-OH molal quantitys are 2~5 times of resin：
The ratio of the weight of Fmoc-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin and the addition for reagent of raising one's hat
For 5~20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(7) Fmoc-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin is prepared：
In Fmoc-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin of step (6), reagent of raising one's hat is added
10~50 DEG C are reacted 5~30 minutes, are drained with vavuum pump, are rejoined 10~50 DEG C of reagent of raising one's hat and are reacted 10~60 minutes, take out
It is dry, washed with the steamed DMF of isopropanol, DMF, weight, add Fmoc-Ser (tBu)-OH, DIEA and the TBTU/ dissolved with DMF
At least one of HOBt, HBTU/HOBt or BOP/HOBt mixture, 10~50 DEG C are reacted 10~60 minutes, use vacuum pumping
It is dry, washed, drained with the steamed DMF of isopropanol, DMF, weight, acquisition Fmoc-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-
Lys (Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Pro-OH molal quantitys are 2~5 times of resin：
The weight of Fmoc-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin and the addition for reagent of raising one's hat
Ratio is 5~20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(8) Fmoc-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin is prepared：
In Fmoc-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin of step (7), addition is raised one's hat
10~50 DEG C of reagent reacts 5~30 minutes, is drained with vavuum pump, rejoins 10~50 DEG C of reagent of raising one's hat and reacts 10~60 points
Clock, is drained, and wash with isopropanol, DMF, weight steamed DMF, Fmoc-Ser (tBu)-OH, DIEA that addition is dissolved with DMF and
At least one of TBTU/HOBt, HBTU/HOBt or BOP/HOBt mixture, 10~50 DEG C are reacted 10~60 minutes, use vacuum
Pumping is done, and is washed with the steamed DMF of isopropanol, DMF, weight, is drained, obtain Fmoc-Met-Pro-Leu-Gly-Gly-Gly-Ser
(tBu)-Lys (Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Met-OH molal quantitys are 2~5 times of resin：
The addition of the weight of Fmoc-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin and reagent of raising one's hat
The ratio of amount is 5~20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(9) Fmoc-Tyr (tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin is prepared：
In Fmoc-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin of step (8), add
10~50 DEG C of reagent of raising one's hat reacts 5~30 minutes, is drained with vavuum pump, rejoins 10~50 DEG C of reactions 10~60 of reagent of raising one's hat
Minute, drain, wash with isopropanol, DMF, weight steamed DMF, Fmoc-Ser (tBu)-OH, DIEA that addition is dissolved with DMF and
At least one of TBTU/HOBt, HBTU/HOBt or BOP/HOBt mixture, 10~50 DEG C are reacted 10~60 minutes, use vacuum
Pumping is done, and is washed with the steamed DMF of isopropanol, DMF, weight, is drained, obtain Fmoc-Tyr (tBu)-Met-Pro-Leu-Gly-
Gly-Gly-Ser (tBu)-Lys (Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Tyr (tBu)-OH molal quantitys are 2~5 times of resin：
The weight of Fmoc-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin and reagent of raising one's hat
The ratio of addition is 5~20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(10) prepare
Fmoc-Phe-Tyr (tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin：
In step (9)
In Fmoc-Tyr (tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin, add de-
10~50 DEG C of cap reagent reacts 5~30 minutes, is drained with vavuum pump, rejoins 10~50 DEG C of reagent of raising one's hat and reacts 10~60 points
Clock, is drained, and wash with isopropanol, DMF, weight steamed DMF, Fmoc-Ser (tBu)-OH, DIEA that addition is dissolved with DMF and
At least one of TBTU/HOBt, HBTU/HOBt or BOP/HOBt mixture, 10~50 DEG C are reacted 10~60 minutes, use vacuum
Pumping is done, and is washed with the steamed DMF of isopropanol, DMF, weight, is drained, obtain Fmoc-Phe-Tyr (tBu)-Met-Pro-Leu-
Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Phe-OH molal quantitys are 2~5 times of resin：
The weight of Fmoc-Tyr (tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin with
Raise one's hat reagent addition ratio be 5~20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(11) prepare
Fmoc-Asn(Trt)-Phe-Tyr(tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBu)-Lys(Boc)-
Resin：
In step (10)
In Fmoc-Phe-Tyr (tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin, plus
10~50 DEG C of the reagent of entering to raise one's hat reacts 5~30 minutes, is drained with vavuum pump, rejoin 10~50 DEG C of reactions 10 of reagent of raising one's hat~
60 minutes, drain, washed with the steamed DMF of isopropanol, DMF, weight, add Fmoc-Ser (tBu)-OH, the DIEA dissolved with DMF
With at least one of TBTU/HOBt, HBTU/HOBt or BOP/HOBt mixture, 10~50 DEG C are reacted 10~60 minutes, with true
Empty pumping is done, and is washed with the steamed DMF of isopropanol, DMF, weight, is drained, obtain Fmoc-sn (Trt)-Phe-Tyr (tBu)-Met-
Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Asn (Trt)-OH molal quantitys are 2~5 times of resin：
The weight of Fmoc-Phe-Tyr (tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin
Measure and raise one's hat reagent addition ratio be 5~20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(12) prepare
Fmoc-Ser(tBu)-Asn(Trt)-Phe-Tyr(tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBu)-
Lys (Boc)-resin：
In step (11)
Fmoc-Asn(Trt)-Phe-Tyr(tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBu)-Lys(Boc)-
In resin, add 10~50 DEG C of reagent of raising one's hat and react 5~30 minutes, drained with vavuum pump, rejoin reagent 10~50 of raising one's hat
DEG C reaction 10~60 minutes, drains, wash with isopropanol, DMF, weight steamed DMF, the Fmoc-Ser that addition is dissolved with DMF
(tBu)-OH, DIEA, TBTU/HOBt or HBTU/HOBt or BOP/HOBt mixture, 10~50 DEG C are reacted 10~60 minutes,
Drained with vavuum pump, washed with the steamed DMF of isopropanol, DMF, weight, drain, obtain
Fmoc-Ser(tBu)-Asn(Trt)-Phe-Tyr(tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBu)-
Lys (Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Ser (tBu)-OH molal quantitys are 2~5 times of resin：
Fmoc-Asn(Trt)-Phe-Tyr(tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBu)-Lys(Boc)-
The ratio of the weight of resin and the addition for reagent of raising one's hat is 5~20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(13) prepare
Ser(tBu)-Asn(Trt)-Phe-Tyr(tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBu)-Lys
(Boc)-resin：
In step (12)
Fmoc-Ser(tBu)-Asn(Trt)-Phe-Tyr(tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBu)-
In Lys (Boc)-resin, add 10~50 DEG C of reagent of raising one's hat and react 5~30 minutes, drained with vavuum pump, rejoin examination of raising one's hat
10~50 DEG C of agent is reacted 10~60 minutes, is drained, and is washed with the steamed DMF of isopropanol, DMF, weight, methanol is washed 2 times, uses methanol
Immersion 0.5~3 hour, is drained, and dries up resin into dry particle with nitrogen, resulting resin is Ser (tBu)-Asn
(Trt)-Phe-Tyr (tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin；
(14) acquisition of crude product
Drying
Ser(tBu)-Asn(Trt)-Phe-Tyr(tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBu)-Lys
(Boc)-resin poured into the cutting bottle of glass, is added and pre- be cooled to -20~10 DEG C and cut peptide reagent (TFA/EDT/H2O/Thio=
90-95/2-5/2-5/1-5, volume ratio), 10 DEG C -50 DEG C are reacted 1-5 hours, are filtered to remove resin, plus -20~10 DEG C of ice second
Ether is precipitated, and sediment is collected by centrifugation, is washed with ether 1~6 time, frozen drying 10~24 hours, and acquisition is used as dodecapeptide
Target molecule polypeptide crude product；
Cut in peptide reagent, the concentration of resin is：5~50ml/g.
Preferably it also includes the step of crude product is through C18 or C8 chromatogram column separating purifications.
It is further preferred that wherein crude product through C18 or C8 chromatogram column separating purifications the step of be：
SNF dodecapeptide crude products are dissolved in the aqueous solution, filtered, filtrate has two through C18 or C8 chromatographies, mobile phase
Kind：A：0.1% trifluoroacetic acid adds 99.9% water；B：0.1% trifluoroacetic acid adds 99.9% acetonitrile；Gradient elution；Flow velocity is 1-
150ml/min；Detection wavelength is：215~285nm；The efflux required for collecting is tracked with liquid chromatograph, is freezed, is obtained
Finished product.
Herein, such as unspecified, " normal temperature " expression room temperature, i.e., 25 DEG C.
The beneficial effects of the invention are as follows：SNF dodecapeptides solid phase synthesis preparation method thereof of the present invention has the characteristics that：Former auxiliary material
Expect convenient sources, process stabilizing, high income is quality controllable, product purity is high, and with short production cycle, cost is low, can scale metaplasia
Production.The great market competitiveness of SNF dodecapeptides prepared using the inventive method, its production scale is fully able to meet SNF 12
The application demand of peptide, its quality is fully able to reach the application requirement of SNF dodecapeptides.
In addition, in the technique of the present invention, using gentle Fmoc routes, often walk de- Fmoc only with 20% PIP, often walk
Condensation reaction time is short, substantially reduces the production cycle；Using reasonable sealing technique, the purity of thick peptide product is improved；Cutting is adopted
With TFA, it is to avoid conventional hydrogen fluoride, the three wastes produced in production are reduced, are conducive to industrialized production.
The Chinese and English list 1 of raw material employed in aforementioned process is as follows：
Table 1
The SNF dodecapeptide building-up processes of the 1g rank dichloro resins (2ClResin) of embodiment 1
(1) Fmoe-Lys (Boc) -2ClResin is prepared
Weigh dichloro resin 1g (gill biochemistry (Shanghai) Co., Ltd.；Loadding：1.0mmol/g) pour into reaction column
In, about 10mlDCM is added, immersion is drained for 30 minutes after (normal temperature) with vavuum pump, adds Fmoc-Lys (Boc)-OH 0.25g,
DIEA0.9ml, DCM20ml, in normal-temperature reaction 2 hours, add closed reagent methanol, normal-temperature reaction 3 hours, resin isopropyl
Alcohol is washed 3 times, and DMF is washed 3 times, and the steamed DMF of weight is washed 2 times, is drained, and obtains Fmoc-Lys (Boc) -2ClResin；
(2) Fmoc-Ser (tBn)-Lys (Boc) -2ClResin is prepared
In Fmoe-Ser (tBn)-Lys (Boc) -2ClResin of step (1), addition raise one's hat reagent (20% piperidines
DMF solution), normal-temperature reaction 5 minutes is drained, and rejoins the agent normal-temperature reaction 20 minutes of raising one's hat, 3 times, DMF are washed with isopropanol
Washing 3 times, the steamed DMF of weight is washed 2 times, is drained, and adds Fmoc-Ser (tBn)-OH 0.7g, DIEA0.4ml,
TBTU0.34g, HOBT1ml, DMF10ml normal-temperature reaction 1.5 hours, is drained with vavuum pump, is washed with isopropanol 3 times, DMF washings
3 times, the steamed DMF of weight is washed 2 times, is drained, and obtains Fmoc-Ser (tBn)-Lys (Boc) -2ClResin；
(3) Fmoc-Gly-Ser (tBn)-Lys (Boc) -2ClResin is prepared
In Fmoc-Ser (tBn)-Lys (Boc) -2ClResin of step (2), addition raise one's hat reagent (20% piperidines
DMF solution), normal-temperature reaction 5 minutes is drained, and rejoins the agent normal-temperature reaction 20 minutes of raising one's hat, 3 times, DMF are washed with isopropanol
Washing 3 times, the steamed DMF of weight is washed 2 times, is drained, and adds Fmoc-Gly-OH0.37g, DIEA0.4ml, TBTU0.38g,
DMF10ml normal-temperature reaction 37min, are drained with vavuum pump, are washed with isopropanol 3 times, and DMF is washed 3 times, the steamed DMF washings 2 of weight
It is secondary, drain, obtain Fmoc-Gly-Ser (tBn)-Lys (Boc) -2ClResin；
(4) Fmoc-Gly-Gly-Ser (tBn)-Lys (Boc) -2ClResin is prepared
In Fmoc-Gly-Ser (tBn)-Lys (Boc) -2ClResin of step (3), reagent (20% piperazine of raising one's hat is added
The DMF solution of pyridine), normal-temperature reaction 5 minutes is drained, and is rejoined the agent normal-temperature reaction 20 minutes of raising one's hat, is washed with isopropanol 3 times,
DMF is washed 3 times, and the steamed DMF of weight is washed 2 times, is drained, and adds Fmoc-Gly-OH0.36g, DIEA0.4ml, TBTU0.38g,
DMF10ml normal-temperature reaction 71min, are drained with vavuum pump, are washed with isopropanol 3 times, and DMF is washed 3 times, the steamed DMF washings 2 of weight
It is secondary, drain, obtain Fmoc-Gly-Gly-Ser (tBn)-Lys (Boc) -2ClResin；
(5) Fmoc-Gly-Gly-Gly-Ser (tBn)-Lys (Boc) -2ClResin is prepared
In Fmoc-Gly-Gly-Ser (tBn)-Lys (Boc) -2ClResin of step (4), reagent of raising one's hat is added
(DMF solution of 20% piperidines), normal-temperature reaction 5 minutes, is drained, and is rejoined the agent normal-temperature reaction 20 minutes of raising one's hat, is used isopropanol
Washing 3 times, DMF is washed 3 times, and the steamed DMF of weight is washed 2 times, is drained, and adds Fmoc-Gly-OH0.38g, DIEA0.4ml,
TBTU0.42g, DMF10ml normal-temperature reaction 94min, are drained with vavuum pump, are washed with isopropanol 3 times, and DMF is washed 3 times, and weight is steamed
DMF wash 2 times, drain, obtain Fmoc-Gly-Gly-Gly-Ser (tBn)-Lys (Boc) -2ClResin；
(6) Fmoc-Leu-Gly-Gly-Gly-Ser (tBn)-Lys (Boc) -2ClResin is prepared
In Fmoc-Gly-Gly-Gly-Ser (tBn)-Lys (Boc) -2ClResin of step (5), reagent of raising one's hat is added
(DMF solution of 20% piperidines), normal-temperature reaction 5 minutes, is drained, and is rejoined the agent normal-temperature reaction 20 minutes of raising one's hat, is used isopropanol
Washing 3 times, DMF is washed 3 times, and the steamed DMF of weight is washed 2 times, is drained, and adds Fmoc-Leu-OH0.43g, DIEA0.4ml,
TBTU0.37g, DMF10ml normal-temperature reaction 74min, are drained with vavuum pump, are washed with isopropanol 3 times, and DMF is washed 3 times, and weight is steamed
DMF wash 2 times, drain, obtain Fmoc-Leu-Gly-Gly-Gly-Ser (tBn)-Lys (Boc) -2ClResin；
(7) Fmoc-Pro-Leu-Gly-Gly-Gly-Ser (tBn)-Lys (Boc) -2ClResin is prepared
In Fmoc-Leu-Gly-Gly-Gly-Ser (tBn)-Lys (Boc) -2ClResin of step (6), addition is raised one's hat
Reagent (DMF solution of 20% piperidines), normal-temperature reaction 5 minutes is drained, and rejoins the agent normal-temperature reaction 20 minutes of raising one's hat, with different
Propanol rinse 3 times, DMF is washed 3 times, and the steamed DMF of weight is washed 2 times, is drained, and adds Fmoc-Pro-OH0.38g,
DIEA0.4ml, TBTU0.37g, DMF10ml normal-temperature reaction 71min, are drained with vavuum pump, are washed with isopropanol 3 times, DMF washings
3 times, the steamed DMF of weight is washed 2 times, is drained, and acquisition Fmoc--Pro-Leu-Gly-Gly-Gly-Ser (tBn)-Lys (Boc)-
2ClResin；
(8) Fmoc-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBn)-Lys (Boc) -2ClResin is prepared
In Fmoc-Pro-Leu-Gly-Gly-Gly-Ser (tBn)-Lys (Boc) -2ClResin of step (7), add
Raise one's hat reagent (DMF solution of 20% piperidines), normal-temperature reaction 5 minutes is drained, rejoin the agent normal-temperature reaction 20 minutes of raising one's hat,
Washed with isopropanol 3 times, DMF is washed 3 times, the steamed DMF of weight is washed 2 times, is drained, and adds Fmoc-Met-OH0.43g,
DIEA0.4ml, TBTU0.37g, DMF10ml normal-temperature reaction 81min, are drained with vavuum pump, are washed with isopropanol 3 times, DMF washings
3 times, the steamed DMF of weight is washed 2 times, is drained, and obtains Fmoc-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBn)-Lys
(Boc)-2ClResin；
(9) prepare
Fmoc-Tyr(tBn)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBn)-Lys(Boc)-2ClResin
In step (8)
In Fmoc-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBn)-Lys (Boc) -2ClResin, examination of raising one's hat is added
Agent (DMF solution of 20% piperidines), normal-temperature reaction 5 minutes is drained, and is rejoined the agent normal-temperature reaction 20 minutes of raising one's hat, is used isopropyl
Alcohol is washed 3 times, and DMF is washed 3 times, and the steamed DMF of weight is washed 2 times, is drained, and adds Fmoc-Tyr (tBn)-OH0.52g,
DIEA0.4ml, TBTU0.36g, DMF10ml normal-temperature reaction 81min, are drained with vavuum pump, are washed with isopropanol 3 times, DMF washings
3 times, the steamed DMF of weight is washed 2 times, is drained, and obtains Fmoc-Tyr (tBn)-Met-Pro-Leu-Gly-Gly-Gly-Ser
(tBn)-Lys(Boc)-2ClResin；
(10) prepare
Fmoc-Phe-Tyr(tBn)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBn)-Lys(Boc)-2ClResin
Fmoc- in step (9)
Tyr(tBn)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBn)-Lys(Boc)-
In 2ClResin, reagent (DMF solution of 20% piperidines) of raising one's hat is added, normal-temperature reaction 5 minutes is drained, added again
Enter to raise one's hat agent normal-temperature reaction 20 minutes, is washed with isopropanol 3 times, and DMF is washed 3 times, and the steamed DMF of weight is washed 2 times, is drained, plus
Enter Fmoc-Phe-OH0.42g, DIEA0.4ml, TBTU0.36g, DMF10ml normal-temperature reaction 71min, drained with vavuum pump, with different
Propanol rinse 3 times, DMF is washed 3 times, and the steamed DMF of weight is washed 2 times, is drained, and obtains Fmoc-Phe-Tyr (tBn)-Met-Pro-
Leu-Gly-Gly-Gly-Ser(tBn)-Lys(Boc)-2ClResin；
(11) prepare
Fmoc-Asn(Trt)-Phe-Tyr(tBn)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBn)-Lys(Boc)-
2ClResin
In step (10)
Fmoc-Phe-Tyr(tBn)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBn)-Lys(Boc)-2ClResin
In, reagent (DMF solution of 20% piperidines) of raising one's hat is added, normal-temperature reaction 5 minutes is drained, rejoins agent normal-temperature reaction of raising one's hat
20 minutes, washed with isopropanol 3 times, DMF is washed 3 times, the steamed DMF of weight is washed 2 times, is drained, addition Fmoc-Asn (Trt)-
OH0.65g, DIEA0.4ml, TBTU0.39g, DMF10ml normal-temperature reaction 96min, are drained with vavuum pump, 3 are washed with isopropanol
Secondary, DMF is washed 3 times, and the steamed DMF of weight is washed 2 times, is drained, and obtains Fmoc-Asn (Trt)-Phe-Tyr (tBn)-Met-Pro-
Leu-Gly-Gly-Gly-Ser(tBn)-Lys(Boc)-2ClResin
(12) prepare
Fmoc-Ser(tBn)-Asn(Trt)-Phe-Tyr(tBn)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBn)-
Lys(Boc)-2ClResin
Fmoc- in step (11)
Asn(Trt)-Phe-Tyr(tBn)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBn)-Lys(Boc)-
In 2ClResin, reagent (DMF solution of 20% piperidines) of raising one's hat is added, normal-temperature reaction 5 minutes is drained, rejoins agent of raising one's hat
Normal-temperature reaction 20 minutes, is washed 3 times with isopropanol, and DMF is washed 3 times, and the steamed DMF of weight is washed 2 times, is drained, and adds Fmoc-
Ser (tBn)-OH0.42g, DIEA0.4ml, TBTU0.38g, DMF10ml normal-temperature reaction 67min, is drained with vavuum pump, uses isopropyl
Alcohol is washed 3 times, and DMF is washed 3 times, and the steamed DMF of weight is washed 2 times, is drained, and obtains Fmoc-Ser (tBn)-Asn (Trt)-Phe-
Tyr(tBn)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBn)-Lys(Boc)-2ClResin；
(13) prepare
Ser(tBn)-Asn(Trt)-Phe-Tyr(tBn)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBn)-Lys
(Boc)-2ClResin
In step (12)
Fmoc-Ser(tBn)-Asn(Trt)-Phe-Tyr(tBn)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBn)-
In Lys (Boc) -2ClResin, reagent (DMF solution of 20% piperidines) of raising one's hat is added, normal-temperature reaction 5 minutes is drained, added again
Enter to raise one's hat agent normal-temperature reaction 20 minutes, is washed with DMF 3 times, and isopropanol is washed 3 times, and the steamed DMF of weight is washed 2 times, methanol washing
2 times, soaked 1.5 hours, drained with methanol, dry up resin into dry particle with nitrogen, resulting resin is：
Ser(tBn)-Asn(Trt)-Phe-Tyr(tBn)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBn)-Lys
(Boc)-2ClResin；
(14) crude product is prepared
Drying
Ser(tBn)-Asn(Trt)-Phe-Tyr(tBn)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBn)-Lys
(Boc) -2ClResin is poured into the cutting bottle of glass, add it is pre- be cooled to -20 DEG C cut peptide reagent (TFA9.5ml,
H2O0.5ml), normal-temperature reaction 1.5 hours, are filtered to remove resin, and ether 500ml on the rocks is precipitated, and 3500 turns are collected by centrifugation for 3 minutes
Sediment, is washed 3 times, after being dissolved with pure water with ether, frozen drying 24 hours, obtains SNFYMPLGGGSK peptide crude products
1.26g, it is 78% (note to determine purity through HPLC：0.69/1.23 should be more than)；
(15) purify
The SNF dodecapeptide crude products 1.23g of step (17) is dissolved in 13ml water, filtered, filtrate is splined on C18 chromatographic columns,
Mobile phase is the mixture of following A, B liquid：A：0.1% trifluoroacetic acid adds 99.9% water；B：0.1% trifluoroacetic acid adds 99.9%
Acetonitrile.The gradient elution since A liquid accounts for mixture ratio 100%, period B liquid accounts for mixture ratio and gradually risen, until
100% is accounted for, flow velocity is 10ml/min, Detection wavelength is：215nm.Target peak is collected (with being given birth under the same terms with laboratory level
A small amount of sterling of production does control and indicates target peak) efflux, it is concentrated freeze-dried, obtain finished product, finished product 0.69g (MW obtained altogether：
1419.6), determine purity through HPLC and be more than 98%.
The specific binding checking of the adenocarcinoma of esophagus of embodiment 2
Esophageal adenocarcinoma cells OE33 is chosen, is compared with people's esophageal epithelial cell Q-htert cells.
Fluorescence polarization determination adhesion
By EPCAM active fragments (ABCAM companies) PBS (27mM KCl, 137mM NaCl, 10.1mM, 1.8mM, pH
7.4) the gradually half-and-half dilution 10 times to 0.001 μM from 10 μM of concentration, is separately added into the orifice plate of black lucifuge 96 (Corning Life
Science, Nonbinding Surface or NBS) in, per the μ l of hole 60.40 μ l 100nM SNF-FITC room temperatures are added per hole
It is incubated 60 minutes, fluorescence microplate reader (Tecan Infinite M1000fluorescence plate reader) read plate, polarization
Value is set to λ ex=490nm and λ em=530nm.The polarization value of gained various concentrations is calculated by below equation：
Kd polarization curve can be obtained, and calculates Kd values for Kd=172nmol/L (R^2=0.997).As shown in figure 1, fluorescence
Polarization surveys SNF dodecapeptides and EPCAM adhesion result is shown, adhesion Kd values are Kd=172nmol/L (R^2=0.997),
SNF dodecapeptides and EPCAM have combination, and adhesion is higher..
RNA interference
In Shanghai, bioengineering Co., Ltd builds synthesis siRNA.By plating cells (6 orifice plate 4x105Individual cell, 8 hole EZ
Slide 3x104Individual cell), overnight, culture medium is changed for OPTI-MEM.By LipofectamineTM2000 (6 orifice plate 5ul/ holes；
8 holes EZ Slide 1ul/ holes) and OPTI-MEM (6 orifice plate 250ul/ holes；8 holes EZ Slide 30ul/ holes) mix, stand
5min.Simultaneously by siRNA (6 orifice plate 5ul/ holes；8 holes EZ Slide 1ul/ holes) and OPTI-MEM (6 orifice plate 250ul/ holes；8 holes
EZ Slide 30ul/ holes) mix.The Lipofectamine of later half volume will be mixedTM2000 mix with the siRNA after mixing
Close, stand 20min.Control group only adds LipofectamineTM2000 (6 orifice plate 255ul/ holes；8 hole EZ Slide 31ul/
Hole), experimental group adds LipofectamineTM2000 and siRNA mixture (6 orifice plate 510ul/ holes；8 hole EZ Slide
62ul/ holes).Result of interference, rna level expression in 2 days, 3 days protein expressions.
Qrt-PCR
1st, cell total rna is extracted
Plating cells (6 orifice plate), after cell is covered with, precooling PBS (pH7.4) washings cell three times adds Trazol
(1ml/ holes), stands 5min, pipette tips piping and druming on ice.Each hole Trazol is drawn onto in 1.5ml EP pipes, precooling chloroform is added
0.2ml/ is managed, and firmly shakes 15s.5min is stood on ice, is centrifuged (4 DEG C, 12000rpm, 20min).Liquid is divided into three layers after centrifugation
(upper strata-colourless water sample layer is RNA, and middle level white is DNA and bottom red is protein), it is careful to draw upper strata colourless liquid
Move into a new EP pipes.Isometric different pre- cold isopropanol is added, 0.4-0.5ml is mixed, and 10min, centrifugation (4 are stood on ice
DEG C, 12000rpm, 15min).Supernatant is removed, precipitation adds pre-cooled ethanol 1ml, and concussion shakes up, centrifugation (4 DEG C, 12000rpm,
10min).It is careful to remove precipitation standing and drying 20min in supernatant, pipe.The dissolving of 20ul DEPC water is added, the ultraviolet survey RNA of ELIASA is dense
Degree and purity.
2nd, reverse transcription
RNA dilutes, and regulation concentration is 200ng/ul.Sample-adding：10mM dNTP mix 2μl；The μ l of sample (RNA) 2；DEPC water
6μl.Reverse transcription：37 DEG C of 15min, 85 DEG C of 5s, 4 DEG C of 30min.Gained sample adds 90 μ l DEPC water to dilute.
3、qRT-PCR
Sample-adding：Each 1 μ l/ holes of upstream and downstream primer；The μ l/ holes of cDNA 2；The μ l/ holes of Green 10.
Program：Originate (50 DEG C of 2min；95℃2min)；40 circulation (95 DEG C of 3s；60 DEG C of 30) data analyses：Obtain 2(-ΔΔCt)Value, is compared, as shown in Fig. 2 EPCAM is shown in the expression of results of mRNA level in-site, in mRNA level in-site, OE33 cells
Middle EPCAM expression is far above the expression in Q-htert cells..
Western blot test (Western Blot)
1st, cultivate cell and carry out RNA interference.
2nd, total protein of cell is extracted, 5X Loading Buffer buffer solutions is added, boils sample 10minutes.
3rd, it is separated by electrophoresis：The μ g to 10% of loading 20 SDS-PAGE glue (10cm x 10cm) electrophoresis.
4th, transferring film：
(1) the size clip film and filter paper 6 according to glue, glue and pvdf membrane are dipped in transfering buffering liquid and balance 15min.
Pvdf membrane need to first soak saturation with pure methanol and move into again in transfering buffering liquid for 5 seconds.
(2) assembling transfer sandwich：The metafiltration of sponge+3 paper+glue+metafiltration of film+3 paper+sponge, after every layer is put well, gas of rushing
Bubble, glue is put in negative pole face (black side).
(3) transfer groove is placed in ice bath, is put into sandwich (black side is to black side), plus transfering buffering liquid, plugs electricity
Pole, constant current, 300mA 1h.
5th, immuning hybridization and colour developing：
(1) film is put in 25ml Block buffers, and 4 DEG C overnight, slow to shake.15ml TBST are washed 3 times (10min/ times).
(2) (1 added after dilution：1000) primary antibody, 4 DEG C overnight, slow to shake.
(3) 15ml TBST are washed 3 times (10min/ times).
(4) (1 added after dilution：5000) secondary antibody of HRPO (HRP) mark, is incubated at room temperature 1h, slowly shakes
It is dynamic.
(5) 15ml TBST are washed 3 times (10min/ times).
(6) Protein Detection, machine sweeps film.EPCAM is shown in the expression of results of protein level as shown in Figure 3, in albumen water
Flat, EPCAM expression is far above the expression in Q-htert cells in OE33 cells.
Immunofluorescence
1st, cultivate cell and carry out RNA interference.
2nd, precooling PBS is washed twice, lowlenthal serum closing 20min
3rd, immunofluorescence dyeing, antibodyome (control group) is indirect antibody staining：Each hole adds 200 μ l primary antibodies (1：
400), 4 DEG C of rocked overnights；500 μ l PBS/ holes, 5min/ times, are cleaned three times；Each hole adds 200 μ l fluorescence secondary antibodies (1：
800), it is incubated at room temperature 30min；500 μ l PBS/ holes, 5min/ times, are cleaned three times；The paraformaldehydes of 200 μ l 4% fix 20min；
500 μ l PBS/ holes, 5min/ times, are cleaned one time；100μl DAPI(1：100) 7min is dyed；500 μ l PBS/ holes, 5min/ times,
Cleaning three times；The anti-fluorescence quenching mountings of 10 μ l.SNF groups (experimental group)：Each hole adds 200 μ l SNF (800 μ g/ml), room
Temperature is incubated 30s；500 μ l PBS/ holes, 5min/ times, are cleaned three times；200 μ l4% paraformaldehydes fix 20min；500μl PBS/
Hole, 5min/ times, is cleaned one time；100μl DAPI(1：100) 7min is dyed；500 μ l PBS/ holes, 5min/ times, are cleaned three times；
The anti-fluorescence quenching mountings of 10 μ l.
4th, inverted fluorescence microscope sees piece.Immunofluorescence experiment detects that dodecapeptide is shown to EPCAM targeting result,
The SNF dodecapeptides combined on the high OE33 cell surfaces of EPCAM expression quantity are far above the binding capacity in Q-htert cell surfaces.
This confirms that SNF dodecapeptides increase with EPCAM increase the targeting of cell.
It is described above, only it is several embodiments of the application, any type of limitation is not done to the application, although this Shen
Please disclosed as above with preferred embodiment, but and be not used to limit the application, any those skilled in the art are not taking off
In the range of technical scheme, make a little variation using the technology contents of the disclosure above or modification is equal to
Case study on implementation is imitated, is belonged in the range of technical scheme.
Claims (10)
1. a kind of target molecule polypeptide for specifically binding signal transduction factor, it is characterised in that the target molecule polypeptide bag
SNFYMPLGGGSK containing amino acid sequence, or the target molecule polypeptide include amino acid sequence SNFYMPLGGGSK in substitution,
Lack or add the amino acid sequence of one or several amino acid and with specific binding signal transduction factor activity
Polypeptide.
2. the target molecule polypeptide described in claim 1, it is characterised in that the amino acid sequence of the target molecule polypeptide is
SNFYMPLGGGSK。
3. the preparation method of the target molecule polypeptide described in claim 1 or 2, it is characterised in that methods described includes solid with Fmoc-
Phase polypeptide synthesis synthesizes crude product polypeptide.
4. method according to claim 3, it is characterised in that methods described is also including the use of C18 or C8 posts to described thick
Product polypeptide is isolated and purified.
5. method according to claim 3, it is characterised in that in the Fmoc- solid phase polypeptide synthesis, with triphen chlorine
Methyl resin, 4- methyl-triphen chloromethyl resin, 4- methoxyl groups-triphen chloromethyl resin, the chloro- triphen chloromethyl resins of 2- or
Any one of Wang resin is solid phase carrier.
6. method according to claim 3, it is characterised in that in the Fmoc- solid phase polypeptide synthesis, with N, N- bis-
Isopropyl carbodiimides/1- hydroxyls-benzo-triazole (DIC/HOBt), N, N- Diisopropylcarbodiimides/N- hydroxyls-
7- azos BTA (DIC/HOAt), 7- azepine benzothiazole -1- bases-oxygen-(three-(dimethylamino) phosphines) hexafluorophosphate/
1- hydroxyls-benzo-triazole (BOP/HOBt), 7- azepine benzothiazole -1- bases-oxygen-(three-(dimethylamino) phosphines) hexafluorophosphoric acid
Salt/1- hydroxyls-benzo-triazole/N- hydroxyl -7- azos BTAs (BOP/HOAt), BTA-N, N, N ', N ' -
Tetramethylurea hexafluorophosphoric acid ester/1- hydroxyls-benzo-triazole (HBTU/HOBt), BTA-N, N, N ', N '-tetramethyl
Urea hexafluorophosphoric acid ester/N- hydroxyl -7- azos BTAs (HBTU/HOAt), 2- (7- azos BTA)-N, N, N ',
N '-tetramethylurea hexafluorophosphoric acid ester/1- hydroxyls-benzo-triazole (HATU/HOBt), 2- (7- azos BTA)-N, N,
N ', N '-tetramethylurea hexafluorophosphoric acid ester/N- hydroxyl -7- azos BTAs (HATU/HOAt) or 6- Chloro-Benzotriazoles -
One kind in 1,1,3,3- tetramethylureas hexafluorophosphoric acid ester/1- hydroxyls-benzo-triazole (HCTU/HOBt) is carried out for condensing agent
One kind in peptide reaction carries out peptide reaction, the target molecule polypeptide resin protected for condensing agent.
7. method according to claim 6, it is characterised in that after the target molecule polypeptide resin protected, same to stepping
Row takes off side chain protecting group and cuts peptide, obtains target molecule polypeptide crude product.
8. method according to claim 3, it is characterised in that methods described comprises the following steps：
(1) Fmoc-Lys (Boc)-resin is prepared：
With triphen chloromethyl resin, 4- methyl-triphen chloromethyl resin, 4- methoxyl groups-triphen chloromethyl resin, the chloro- triphens of 2-
Chloromethyl resin or Wang resin one kind therein make resin, and 10~60 points are soaked with DMF or dichloromethane
In clock, mixture, the bulking value concentration of resin is 5-20ml/g；
Then in above-mentioned resin, N, N'- diisopropylethylamine (DIEA), N, N- Diisopropylcarbodiimides are added
(DIC) or DMAP (DMAP), Fmoc-Val-OH, 10~50 DEG C are reacted 0.5~5 hour, add closed reagent
10~50 DEG C of methanol, chlorobenzoyl chloride or pyridine are reacted 0.2-3 hours, and resin is washed with isopropanol, DMF, is obtained
Obtain Fmoc-Lys (Boc)-resin；
DIEA or DMAP molal quantity is 2~20 times of resin；
Fmoc-Lys (Boc)-OH molal quantity is 1~10 times of resin：
In reaction solution, the concentration of resin is 0.5~5ml/g；
(2) Fmoc-Ser (tBu)-Lys (Boc)-resin is prepared：
In Fmoc-Lys (Boc)-resin of step (1), add 10~50 DEG C of reagent of raising one's hat and react 5~30 minutes, use vacuum
Pumping is done, and is rejoined 10~50 DEG C of reagent of raising one's hat and is reacted 10~60 minutes, drain, is washed with the steamed DMF of isopropanol, DMF, weight
Wash, add in Fmoc-Ser (tBu)-OH, DIEA and TBTU/HOBt, HBTU/HOBt or the BOP/HOBt dissolved with DMF extremely
A kind of few mixture, 10~50 DEG C are reacted 10~60 minutes, are drained with vavuum pump, are washed with the steamed DMF of isopropanol, DMF, weight
Wash, drain, obtain Fmoc-Ser (tBu)-Lys (Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Ser (tBu)-OH molal quantity is 1-5 times of resin；
The ratio of the weight of Fmoc-Lys (Boc)-resin and the addition for reagent of raising one's hat is 5~20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(3) Fmoc-Gly-Ser (tBu)-Lys (Boc)-resin is prepared：
In Fmoc-Ser (tBu)-Lys (Boc)-resin of step (2), add 10~50 DEG C of reagent of raising one's hat and react 5~30 points
Clock, is drained with vavuum pump, is rejoined 10~50 DEG C of reagent of raising one's hat and is reacted 10~60 minutes, drains, with isopropanol, DMF, steam again
The DMF washings crossed, add Fmoc-Ser (tBu)-OH, DIEA and TBTU/HOBt, HBTU/HOBt or the BOP/ dissolved with DMF
At least one of HOBt mixtures, 10~50 DEG C are reacted 10~60 minutes, are drained, with isopropanol, DMF, steamed again with vavuum pump
The DMF washings crossed, drain, obtain Fmoc-Gly-Ser (tBu)-Lys (Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Gly-OH molal quantitys are 2~5 times of resin；
The ratio of the weight of Fmoc-Ser (tBu)-Lys (Boc)-resin and the addition for reagent of raising one's hat is 5~20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(4) Fmoc-Gly-Gly-Ser (tBu)-Lys (Boc)-resin is prepared：
In Fmoc-Gly-Ser (tBu)-Lys (Boc)-resin of step (3), addition raise one's hat 10~50 DEG C of reagent reaction 5~
30 minutes, drained with vavuum pump, rejoin 10~50 DEG C of the reagent of raising one's hat and react 10~60 minutes, drain, with isopropanol, DMF,
The steamed DMF washings of weight, add Fmoc-Ser (the tBu)-OH dissolved with DMF, DIEA and TBTU/HOBt, HBTU/HOBt or
At least one of BOP/HOBt mixtures, 10~50 DEG C react 10~60 minutes, drained with vavuum pump, with isopropanol, DMF,
The steamed DMF washings of weight, drain, obtain Fmoc-Gly-Gly-Ser (tBu)-Lys (Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Gly-OH molal quantitys are 2~5 times of resin；
The ratio of the weight of Fmoc-Gly-Ser (tBu)-Lys (Boc)-resin and the addition for reagent of raising one's hat is 5~20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(5) Fmoc-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin is prepared：
In Fmoc-Gly-Gly-Ser (tBu)-Lys (Boc)-resin of step (4), 10~50 DEG C of reactions of reagent of raising one's hat are added
5~30 minutes, drained with vavuum pump, rejoin 10~50 DEG C of the reagent of raising one's hat and react 10~60 minutes, drain, with isopropanol,
The steamed DMF washings of DMF, weight, add Fmoc-Ser (tBu)-OH, DIEA and TBTU/HOBt, the HBTU/HOBt dissolved with DMF
Or at least one of BOP/HOBt mixture, 10~50 DEG C are reacted 10~60 minutes, are drained with vavuum pump, with isopropanol,
The steamed DMF washings of DMF, weight, drain, obtain Fmoc-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Gly-OH molal quantitys are 2~5 times of resin；
The ratio of the weight of Fmoc-Gly-Gly-Ser (tBu)-Lys (Boc)-resin and the addition for reagent of raising one's hat for 5~
20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(6) Fmoc-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin is prepared：
In Fmoc-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin of step (5), 10~50 DEG C of reagent of raising one's hat is added
Reaction 5~30 minutes, is drained with vavuum pump, is rejoined 10~50 DEG C of reagent of raising one's hat and is reacted 10~60 minutes, drains, use isopropyl
The steamed DMF washings of alcohol, DMF, weight, add Fmoc-Ser (tBu)-OH, DIEA and TBTU/HOBt, the HBTU/ dissolved with DMF
At least one of HOBt or BOP/HOBt mixture, 10~50 DEG C are reacted 10~60 minutes, are drained with vavuum pump, are used isopropyl
The steamed DMF washings of alcohol, DMF, weight, drain, obtain Fmoc-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Leu-OH molal quantitys are 2~5 times of resin：
The ratio of the weight of Fmoc-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin and the addition for reagent of raising one's hat is 5
~20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(7) Fmoc-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin is prepared：
In Fmoc-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin of step (6), addition raise one's hat reagent 10~
50 DEG C are reacted 5~30 minutes, are drained with vavuum pump, are rejoined 10~50 DEG C of reagent of raising one's hat and are reacted 10~60 minutes, drain, use
The steamed DMF washings of isopropanol, DMF, weight, add dissolved with DMF Fmoc-Ser (tBu)-OH, DIEA and TBTU/HOBt,
At least one of HBTU/HOBt or BOP/HOBt mixture, 10~50 DEG C are reacted 10~60 minutes, are drained with vavuum pump, are used
The steamed DMF washings of isopropanol, DMF, weight, drain, obtain Fmoc-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys
(Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Pro-OH molal quantitys are 2~5 times of resin：
The ratio of the weight of Fmoc-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin and the addition for reagent of raising one's hat
For 5~20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(8) Fmoc-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin is prepared：
In Fmoc-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin of step (7), reagent of raising one's hat is added
10~50 DEG C are reacted 5~30 minutes, are drained with vavuum pump, are rejoined 10~50 DEG C of reagent of raising one's hat and are reacted 10~60 minutes, take out
It is dry, washed with the steamed DMF of isopropanol, DMF, weight, add Fmoc-Ser (tBu)-OH, DIEA and the TBTU/ dissolved with DMF
At least one of HOBt, HBTU/HOBt or BOP/HOBt mixture, 10~50 DEG C are reacted 10~60 minutes, use vacuum pumping
It is dry, washed with the steamed DMF of isopropanol, DMF, weight, drain, obtain Fmoc-Met-Pro-Leu-Gly-Gly-Gly-Ser
(tBu)-Lys (Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Met-OH molal quantitys are 2~5 times of resin：
The weight of Fmoc-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin and the addition for reagent of raising one's hat
Ratio is 5~20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(9) Fmoc-Tyr (tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin is prepared：
In Fmoc-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin of step (8), addition is raised one's hat
10~50 DEG C of reagent reacts 5~30 minutes, is drained with vavuum pump, rejoins 10~50 DEG C of reagent of raising one's hat and reacts 10~60 points
Clock, is drained, and wash with isopropanol, DMF, weight steamed DMF, Fmoc-Ser (tBu)-OH, DIEA that addition is dissolved with DMF and
At least one of TBTU/HOBt, HBTU/HOBt or BOP/HOBt mixture, 10~50 DEG C are reacted 10~60 minutes, use vacuum
Pumping is done, and is washed with the steamed DMF of isopropanol, DMF, weight, is drained, obtain Fmoc-Tyr (tBu)-Met-Pro-Leu-Gly-
Gly-Gly-Ser (tBu)-Lys (Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Tyr (tBu)-OH molal quantitys are 2~5 times of resin：
The addition of the weight of Fmoc-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin and reagent of raising one's hat
The ratio of amount is 5~20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(10) prepare
Fmoc-Phe-Tyr (tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin：
In step (9)
In Fmoc-Tyr (tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin, examination of raising one's hat is added
10~50 DEG C of agent is reacted 5~30 minutes, is drained with vavuum pump, is rejoined 10~50 DEG C of reagent of raising one's hat and is reacted 10~60 minutes,
Drain, washed with the steamed DMF of isopropanol, DMF, weight, add Fmoc-Ser (tBu)-OH, DIEA and the TBTU/ dissolved with DMF
At least one of HOBt, HBTU/HOBt or BOP/HOBt mixture, 10~50 DEG C are reacted 10~60 minutes, use vacuum pumping
It is dry, washed with the steamed DMF of isopropanol, DMF, weight, drain, obtain
Fmoc-Phe-Tyr (tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Phe-OH molal quantitys are 2~5 times of resin：
The weight of Fmoc-Tyr (tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin is with raising one's hat
The ratio of the addition of reagent is 5~20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(11) prepare
Fmoc-Asn (Trt)-Phe-Tyr (tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-tree
Fat：
In step (10)
In Fmoc-Phe-Tyr (tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin, add de-
10~50 DEG C of cap reagent reacts 5~30 minutes, is drained with vavuum pump, rejoins 10~50 DEG C of reagent of raising one's hat and reacts 10~60 points
Clock, is drained, and wash with isopropanol, DMF, weight steamed DMF, Fmoc-Ser (tBu)-OH, DIEA that addition is dissolved with DMF and
At least one of TBTU/HOBt, HBTU/HOBt or BOP/HOBt mixture, 10~50 DEG C are reacted 10~60 minutes, use vacuum
Pumping is done, and is washed with the steamed DMF of isopropanol, DMF, weight, is drained, obtain
Fmoc-sn (Trt)-Phe-Tyr (tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Asn (Trt)-OH molal quantitys are 2~5 times of resin：
The weight of Fmoc-Phe-Tyr (tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin with
Raise one's hat reagent addition ratio be 5~20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(12) prepare
Fmoc-Ser(tBu)-Asn(Trt)-Phe-Tyr(tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBu)-Lys
(Boc)-resin：
In step (11)
Fmoc-Asn (Trt)-Phe-Tyr (tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin
In, add 10~50 DEG C of reagent of raising one's hat and react 5~30 minutes, drained with vavuum pump, rejoin 10~50 DEG C of reagent of raising one's hat anti-
Answer 10~60 minutes, drain, washed with the steamed DMF of isopropanol, DMF, weight, the Fmoc-Ser (tBu) that addition is dissolved with DMF-
OH, DIEA, TBTU/HOBt or HBTU/HOBt or BOP/HOBt mixture, 10~50 DEG C are reacted 10~60 minutes, use vacuum
Pumping is done, and is washed with the steamed DMF of isopropanol, DMF, weight, is drained, obtain
Fmoc-Ser(tBu)-Asn(Trt)-Phe-Tyr(tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBu)-Lys
(Boc)-resin；
The component and volume ratio of described reagent of raising one's hat be：PIP：DMF=1：2-5；
Fmoc-Ser (tBu)-OH molal quantitys are 2~5 times of resin：
Fmoc-Asn (Trt)-Phe-Tyr (tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser (tBu)-Lys (Boc)-resin
Weight and raise one's hat reagent addition ratio be 5~20ml/g；
TBTU/HBTU/BOP molal quantity is 2-5 times of resin；
HOBT/HOAT molal quantitys are 2-5 times of resin；
(13) prepare
Ser(tBu)-Asn(Trt)-Phe-Tyr(tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBu)-Lys(Boc)-
Resin：
In step (12)
Fmoc-Ser(tBu)-Asn(Trt)-Phe-Tyr(tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBu)-Lys
(Boc) in-resin, add 10~50 DEG C of reagent of raising one's hat and react 5~30 minutes, drained with vavuum pump, rejoin reagent of raising one's hat
10~50 DEG C are reacted 10~60 minutes, are drained, and are washed with the steamed DMF of isopropanol, DMF, weight, methanol is washed 2 times, is soaked with methanol
Bubble 0.5~3 hour, is drained, and dries up resin into dry particle with nitrogen, resulting resin is
Ser(tBu)-Asn(Trt)-Phe-Tyr(tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBu)-Lys(Boc)-
Resin；
(14) acquisition of crude product
Drying
Ser(tBu)-Asn(Trt)-Phe-Tyr(tBu)-Met-Pro-Leu-Gly-Gly-Gly-Ser(tBu)-Lys(Boc)-
Resin poured into the cutting bottle of glass, is added and pre- be cooled to -20~10 DEG C and cut peptide reagent (TFA/EDT/H2O/Thio=90-95/
2-5/2-5/1-5, volume ratio), 10 DEG C -50 DEG C are reacted 1-5 hours, are filtered to remove resin, plus -20~10 DEG C of ice ether sinks
Form sediment, sediment is collected by centrifugation, is washed with ether 1~6 time, frozen drying 10~24 hours obtains the target as dodecapeptide
Molecular polypeptide crude product；
Cut in peptide reagent, the concentration of resin is：5~50ml/g.
9. method according to claim 7, it is characterised in that also including by crude product through C18 or C8 chromatogram column separating purifications
The step of.
10. the step of method according to claim 4 or 9, wherein crude product are through C18 or C8 chromatogram column separating purifications is：
Target molecule polypeptide crude product is dissolved in the aqueous solution, filtered, filtrate is through C18 or C8 chromatographies, and mobile phase there are two kinds：A：
0.1% trifluoroacetic acid adds 99.9% water；B：0.1% trifluoroacetic acid adds 99.9% acetonitrile；Gradient elution；Flow velocity is 1-150ml/
min；Detection wavelength is：215~285nm；The efflux required for collecting is tracked with liquid chromatograph, is freezed, finished product is obtained.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610090128.0A CN107090015A (en) | 2016-02-18 | 2016-02-18 | A kind of target molecule polypeptide for specifically binding signal transduction factor and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610090128.0A CN107090015A (en) | 2016-02-18 | 2016-02-18 | A kind of target molecule polypeptide for specifically binding signal transduction factor and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107090015A true CN107090015A (en) | 2017-08-25 |
Family
ID=59648698
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610090128.0A Pending CN107090015A (en) | 2016-02-18 | 2016-02-18 | A kind of target molecule polypeptide for specifically binding signal transduction factor and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107090015A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113583093A (en) * | 2021-08-03 | 2021-11-02 | 陕西师范大学 | Polypeptide specifically bound with tumor stem cell marker EpCAM and application thereof |
| CN114106111A (en) * | 2021-11-28 | 2022-03-01 | 中国人民解放军军事科学院军事医学研究院 | High-adhesion polypeptide and application thereof |
| CN114774395A (en) * | 2022-06-02 | 2022-07-22 | 亿彤科技发展(福建)有限公司 | High-purity chitinase 3-like protein 1 epitope peptide for liver disease detection and preparation method thereof |
| CN114957438A (en) * | 2022-06-28 | 2022-08-30 | 亿彤科技发展(福建)有限公司 | Human Abeta 1-42 antigenic determinant polypeptide for detecting Alzheimer disease and preparation method thereof |
| CN115181187A (en) * | 2022-07-18 | 2022-10-14 | 吾奇生物医疗科技(江苏)有限公司 | Polypeptide for promoting epithelial cell adhesion, functional collagen material, and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010121266A1 (en) * | 2009-04-18 | 2010-10-21 | The Regents Of The University Of Michigan | Targeted detection of dysplasia in barrett's esophagus with a novel fluorescence-labeled polypeptide |
| CN102229649A (en) * | 2011-05-05 | 2011-11-02 | 中国人民解放军第四军医大学 | Preparation method of body protection polypeptide (BPC 157 peptide) |
| CN102229645A (en) * | 2011-06-03 | 2011-11-02 | 程云 | Method for preparing immune seven peptides |
-
2016
- 2016-02-18 CN CN201610090128.0A patent/CN107090015A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010121266A1 (en) * | 2009-04-18 | 2010-10-21 | The Regents Of The University Of Michigan | Targeted detection of dysplasia in barrett's esophagus with a novel fluorescence-labeled polypeptide |
| CN102229649A (en) * | 2011-05-05 | 2011-11-02 | 中国人民解放军第四军医大学 | Preparation method of body protection polypeptide (BPC 157 peptide) |
| CN102229645A (en) * | 2011-06-03 | 2011-11-02 | 程云 | Method for preparing immune seven peptides |
Non-Patent Citations (2)
| Title |
|---|
| MENG LI ET AL.: "Affinity Peptide for Targeted Detection of Dysplasia in Barrett"s Esophagus", 《GASTROENTEROLOGY》 * |
| N A C S WONG ET AL.: "EpCAM and gpA33 are markers of Barrett’s metaplasia", 《J CLIN PATHOL》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113583093A (en) * | 2021-08-03 | 2021-11-02 | 陕西师范大学 | Polypeptide specifically bound with tumor stem cell marker EpCAM and application thereof |
| CN114106111A (en) * | 2021-11-28 | 2022-03-01 | 中国人民解放军军事科学院军事医学研究院 | High-adhesion polypeptide and application thereof |
| CN114774395A (en) * | 2022-06-02 | 2022-07-22 | 亿彤科技发展(福建)有限公司 | High-purity chitinase 3-like protein 1 epitope peptide for liver disease detection and preparation method thereof |
| CN114774395B (en) * | 2022-06-02 | 2023-08-22 | 亿彤科技发展(福建)有限公司 | High-purity chitinase 3-like protein 1 epitope peptide for liver disease detection and preparation method thereof |
| CN114957438A (en) * | 2022-06-28 | 2022-08-30 | 亿彤科技发展(福建)有限公司 | Human Abeta 1-42 antigenic determinant polypeptide for detecting Alzheimer disease and preparation method thereof |
| CN114957438B (en) * | 2022-06-28 | 2024-04-02 | 福建亿彤生物科技有限公司 | Human Abeta 1-42 epitope polypeptide for detecting Alzheimer disease and preparation method thereof |
| CN115181187A (en) * | 2022-07-18 | 2022-10-14 | 吾奇生物医疗科技(江苏)有限公司 | Polypeptide for promoting epithelial cell adhesion, functional collagen material, and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107090015A (en) | A kind of target molecule polypeptide for specifically binding signal transduction factor and preparation method thereof | |
| CN104130315B (en) | A kind of polypeptide of special target HER2 albumen | |
| CN105085631B (en) | A kind of polypeptide of special target HER2 albumen and its application | |
| CN112500457B (en) | Dual-targeting FOXM1/CDK1 anti-tumor polypeptide | |
| CN105524159B (en) | Polypeptide molecule with cancer cell selective killing and migration inhibition effects and application | |
| CN105085632A (en) | Specific EGFR (epidermal growth factor receptor)-protein-targeted polypeptide and application thereof | |
| US20220340623A1 (en) | Novel micropeptide hmmw and application thereof | |
| RU2001127668A (en) | ADHESIVE VESSEL MOLECULES AND MODULATION OF THEIR FUNCTIONS | |
| CN105330725B (en) | A kind of pH response and polypeptide and its application for targeting human tumour vascular markers VEGFR2 | |
| CN102671185A (en) | Application of scolopendra mutilans neurotoxin peptide omega-SLPTX-Ssmla | |
| CN109467588A (en) | It is a kind of target people N-cadherin albumen polypeptide and its application | |
| CN106699850B (en) | RBBP4 targeting polypeptide and anti-tumor polypeptide and application thereof | |
| CN104177476B (en) | The polypeptide of a kind of targeted human cancerous cell and application thereof | |
| CN113072622B (en) | Polypeptide for selectively degrading PD-L1 protein on tumor cell membrane and application | |
| CN109652555A (en) | A kind of molecular marker of diagnosis and treatment malignant tumour | |
| CN108530521A (en) | Recombinate the preparation and application of hepatitis C antigen | |
| WO2013162760A1 (en) | Novel cyclotides with anticancer activity | |
| CN103204921A (en) | Secretory protein with chemotactic activity, and coding sequence and application thereof | |
| CN106566813B (en) | Hybridoma cell strain XA272-918, antibody and its application | |
| CN107098963B (en) | Short peptide for inhibiting tumor and coding gene and application thereof | |
| CN109942715A (en) | A kind of recombination fusion protein of targeting therapy on tumor and its preparation method and application | |
| RU2550223C1 (en) | THROMBOCYTE AGGREGATION-INHIBITING HETEROMERIC PEPTIDES BASED ON IMIDAZO[4,5-e]BENZO[1,2-c;3,4-c']DIFUROXANE | |
| CN106831953A (en) | A kind of polypeptide and its application | |
| CN118638189A (en) | Bicyclic polypeptide and preparation method and application thereof | |
| WO2014046732A1 (en) | Cyclotide-based cxcr4 antagonists with anti-hiv activity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170825 |
|
| RJ01 | Rejection of invention patent application after publication |