CN104231059B - One peptide species and its production and use - Google Patents

One peptide species and its production and use Download PDF

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Publication number
CN104231059B
CN104231059B CN201310243117.8A CN201310243117A CN104231059B CN 104231059 B CN104231059 B CN 104231059B CN 201310243117 A CN201310243117 A CN 201310243117A CN 104231059 B CN104231059 B CN 104231059B
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fmoc
glu
polypeptide
leu
nle
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CN104231059A (en
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戴政清
刘剑
马亚平
袁建成
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Hybio Pharmaceutical Co Ltd
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Hybio Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
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  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The present invention relates to Peptides Synthesis, particularly to peptide species and its production and use.The aminoacid sequence of this polypeptide is as shown in SEQ ID NO:1, and the N end of polypeptide is through acetylation modification；From N end, the X of the 22nd in polypeptide8Main chain and the X of the 25th9Side chain carries out amido link cyclisation.The polypeptide that the present invention provides remains the biologic activity of Astressin B, and its metabolic stability is higher, can effectively prevent and hair growth；The preparation method of this polypeptide has the advantage that production cost is low, yield is high.

Description

One peptide species and its production and use
Technical field
The present invention relates to Peptides Synthesis, particularly to peptide species and its production and use.
Background technology
Alopecia refers to the phenomenon of alopecia, including physiologic alopecia and pathologic alopecia.Physiologic alopecia refers to be in The phenomenon of the alopecia of catagen and resting stage, is constantly in dynamically owing to entering the hair of catagen and newly entering trophophase Balance, therefore the hair of normal quantity can be maintained.Pathologic alopecia refers to hair exception or excessive coming off, and main cause is human body Pyretic toxicity in blood is arranged the most out, so that some disease occurs in human body, shows on hair to be exactly hair follicle atrophy, and hair takes off Falling, easily broken, how nonelastic oil is, the most serious situation be general de-, entirely take off.Different by pathogenesis, pathologic alopecia mainly divides For nervous alopecia, endocrine alopecia, trophism alopecia, physical property alopecia, alopecia of chemical origin, infectious baldness, symptomatic de- Send out, congenital alopecia, immunity alopecia, seasonal alopecia etc..Alopecia can affect the image of patient, and then has influence on inter personal contact And mental health, bring great misery to patient.
At present, the drug main minoxidil to be had of hair growth, finasteride etc., but both medicines all can cause seriously Untoward reaction.Wherein, minoxidil is for taking the effect that can play blood pressure lowering orally, if outside heavy dose of use, this medicine percutaneous Skin absorption penetrates into internal, is easily caused headache, hypotension；The secondary work that long-term finasteride tablet hair growth for oral administration may result in With including: male chest increases, mammiplasia, impotence, penis is red and swollen, and scrotum pain, more serious is possible to cause forever The internal reproductive organ sudden change of property, affects sperm quality for a long time, ultimately results in fetal development deformity, can produce after groups of people's long-term taking The side effect such as depression.Therefore, the medicine developing novel, safe hair growth is still that the focus of current study of pharmacy.
Astressin B is newly discovered to prevent the polypeptide drug with hair growth, and molecular formula is C199H298N52O49, molecular weight is 3962.65, its structure shown in formula I, wherein, the Glu of the 22nd and the 25th from N end Lys carries out amido link cyclisation by side chain.
Ac-Asp1-Leu2-Thr3-D-Phe4-His5-Leu6-Leu7-Arg8-Glu9-Val10-Leu11-Glu12-Nle13- Ala14-Arg15-Ala16-Glu17-Gln18-Gml19-Ala20-Gln21-cyclo(Glu22-Ala23-His24-Lys25)-Asn26- Arg27-Lys28-Leu29-Nle30-Glu31-Cml32-Ile33-NH2
Formulas I
In the building-up process of Astressin B, general employing Fmoc solid phase synthesis strategy synthesizes.Astressin There is Cml residue in B, the amino acid residue raw material used in building-up process is Fmoc-Cml-OH, and its market price is more Costliness, and in coupling process, there is the situation of coupling difficulty, the yield causing Astressin B is on the low side；From N end 22nd amino acids of Astressin B is Glu, and the Lys of this Glu and 25 carries out amido link cyclisation by side chain, in solid phase The amino acid residue raw material used in building-up process is Fmoc-Glu (OAll)-OH, and its market price is the most costly.Based on this 2 reasons so that the production cost of Astressin B is high, and yield is relatively low.Therefore, low for production cost, yield is high The polypeptide drug with hair growth can be prevented still to have demand.
Summary of the invention
In view of this, the invention provides peptide species and its production and use.This polypeptide is remaining While the biologic activity of Astressin B, its metabolic stability is higher, can effectively prevent and hair growth；This polypeptide Preparation method has the advantage that production cost is low, yield is high.
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
The invention provides a peptide species, its aminoacid sequence as shown in SEQ ID NO:1, particularly as follows:
Asp-Leu-Thr-X1-His-X2-Leu-Arg-X3-Val-Leu-X4-X5-Ala-Arg-Ala-X6-Gln-X7- Ala-Gln-X8-Ala-His-X9-X10-Arg-Lys-Leu-X11-Glu-X7-Ile；
Wherein, X7For N-Me-Leu；
X8For γ-Glu or β-Asp；
X1For D-Phe or Phe；
X2For Ile or Leu；
X3For Asp or Glu；
X4For Asp or Glu；
X5For Ile or Nle；
X6For Asp or Glu；
X9For Orn or Lys；
X10For Gln or Asn；
X11For Ile or Nle；
The N end of polypeptide is through acetylation modification；
From N end, the X of the 22nd in polypeptide8Main chain and the X of the 25th9Side chain carries out amido link cyclisation.
In some embodiments that the present invention provides, X in the aminoacid sequence of polypeptide8For γ-Glu, X1For D-Phe, X2For Leu, X3For Glu, X4For Glu, X5For Nle, X6For Glu, X9For Lys, X10For Asn, X11For Nle, its aminoacid sequence such as SEQ Shown in ID NO:2.
In other embodiments that the present invention provides, X in the aminoacid sequence of polypeptide8For γ-Glu, X1For Phe, X2For Leu, X3For Glu, X4For Glu, X5For Nle, X6For Glu, X9For Lys, X10For Asn, X11For Nle, its aminoacid sequence such as SEQ Shown in ID NO:3.
In other embodiments that the present invention provides, X in the aminoacid sequence of polypeptide8For γ-Glu, X1For D-Phe, X2 For Ile, X3For Glu, X4For Glu, X5For Nle, X6For Glu, X9For Lys, X10For Asn, X11For Nle, its aminoacid sequence is such as Shown in SEQ ID NO:4.
In other embodiments that the present invention provides, X in the aminoacid sequence of polypeptide8For γ-Glu, X1For D-Phe, X2 For Leu, X3For Asp, X4For Glu, X5For Nle, X6For Glu, X9For Lys, X10For Asn, X11For Nle, its aminoacid sequence is such as Shown in SEQ ID NO:5.
In other embodiments that the present invention provides, X in the aminoacid sequence of polypeptide8For γ-Glu, X1For D-Phe, X2 For Leu, X3For Glu, X4For Asp, X5For Nle, X6For Glu, X9For Lys, X10For Asn, X11For Nle, its aminoacid sequence is such as Shown in SEQ ID NO:6.
In other embodiments that the present invention provides, X in the aminoacid sequence of polypeptide8For γ-Glu, X1For D-Phe, X2 For Leu, X3For Glu, X4For Glu, X5For Ile, X6For Glu, X9For Lys, X10For Asn, X11For Nle, its aminoacid sequence is such as Shown in SEQ ID NO:7.
In other embodiments that the present invention provides, X in the aminoacid sequence of polypeptide8For γ-Glu, X1For D-Phe, X2 For Leu, X3For Glu, X4For Glu, X5For Nle, X6For Asp, X9For Lys, X10For Asn, X11For Nle, its aminoacid sequence is such as Shown in SEQ ID NO:8.
In other embodiments that the present invention provides, X in the aminoacid sequence of polypeptide8For β-Asp, X1For D-Phe, X2 For Leu, X3For Glu, X4For Glu, X5For Nle, X6For Glu, X9For Lys, X10For Asn, X11For Nle, its aminoacid sequence is such as Shown in SEQ ID NO:9.
In other embodiments that the present invention provides, X in the aminoacid sequence of polypeptide8For γ-Glu, X1For D-Phe, X2 For Leu, X3For Glu, X4For Glu, X5For Nle, X6For Glu, X9For Orn, X10For Asn, X11For Nle, its aminoacid sequence is such as Shown in SEQ ID NO:10.
In other embodiments that the present invention provides, X in the aminoacid sequence of polypeptide8For γ-Glu, X1For D-Phe, X2 For Leu, X3For Glu, X4For Glu, X5For Nle, X6For Glu, X9For Lys, X10For Gln, X11For Nle, its aminoacid sequence is such as Shown in SEQ ID NO:11.
In other embodiments that the present invention provides, X in the aminoacid sequence of polypeptide8For γ-Glu, X1For D-Phe, X2 For Leu, X3For Glu, X4For Glu, X5For Nle, X6For Glu, X9For Lys, X10For Asn, X11For Ile, its aminoacid sequence is such as Shown in SEQ ID NO:12.
Present invention also offers the preparation method of the polypeptide that a kind of present invention provides, comprise the steps:
Step A: take Ile and resin coupling, obtain Ile-resin；
Step B: take Ile-resin through progressively coupling, obtain polypeptide fragment-resin；
Step C: the N end of polypeptide fragment-resin is through acetylation modification；
Step D: removing the 22nd and protection group of the 25th amino acids residue from N end, through cyclisation, cracking, purification, freezes Dry, to obtain final product；
From N end, polypeptide the 22nd amino acids raw material uses Fmoc-Asp-OAll or Fmoc-Glu-OAll；
The sequence of the polypeptide in polypeptide fragment-resin is as shown in SEQ ID NO:1.
As preferably, the mixture that reagent is acetic anhydride and DIEA that in step C, acetylation modification uses.
As preferably, the reagent acetic anhydride that in step C, acetylation modification uses is 1:2 with the mol ratio of DIEA.
As preferably, the reagent removing employing in step D is phenyl silane and Pd (PPh3)4Mixture.
As preferably, the mixture that reagent is PyBOP, HOBt and DIEA of the cyclized by treatment employing of step D.
As preferably, in the reagent of the cyclized by treatment employing of step D, PyBOP, HOBt are 5:6:10 with the mol ratio of DIEA.
As preferably, the reagent cracking employing in step D is TFA.
The polypeptide that present invention also offers the present invention provides is preparing the application prevented and treated in alopecia agent.
Present invention also offers a kind of pharmaceutical preparation, polypeptide provided by the present invention and pharmaceutically acceptable adjuvant composition.
As preferably, the dosage form of pharmaceutical preparation is injectable powder, injection, microsphere, microcapsule, liposome, microemulsion, tablet, ball Agent, capsule, granule or powder.
The invention provides peptide species and its production and use.The aminoacid sequence of this polypeptide such as SEQ ID NO: Shown in 1, particularly as follows: Asp-Leu-Thr-X1-His-X2-Leu-Arg-X3-Val-Leu-X4-X5-Ala-Arg-Ala-X6-Gln- X7-Ala-Gln-X8-Ala-His-X9-X10-Arg-Lys-Leu-X11-Glu-X7-Ile；Wherein, X7For N-Me-Leu；X8For γ-Glu or β-Asp；X1For D-Phe or Phe；X2For Ile or Leu；X3For Asp or Glu；X4For Asp or Glu；X5For Ile or Nle；X6For Asp or Glu；X9For Orn or Lys；X10For Gln or Asn；X11For Ile or Nle；The N end of polypeptide is repaiied through acetylation Decorations；From N end, the X of the 22nd in polypeptide8Main chain and the X of the 25th9Side chain carries out amido link cyclisation.By in vitro to cell The influence research of interior cAMP activity understands, and the polypeptide that Astressin B and the present invention provide all can dose-dependently increase PC- 12 intracellular cAMP amount, show that the polypeptide that the present invention provides remains the biologic activity of Astressin B；By to polypeptide Metabolic stability Journal of Sex Research understand, half-life of polypeptide that the present invention provides is more longer than the Astressin B time, shows its generation Thank to stability higher；By drug efficacy study, it is in the mice under higher pressure state and injects what the present invention provided respectively Polypeptide and after Astressin B12 week, test mice alopecia area the most only accounts for about the 20% of whole back area, and only injects The control mice group the most whole back hair of normal saline all takes off light, shows that the polypeptide that the present invention provides can effective pre-anticreep Send out, and essentially the same with the pre-Anti-hair loss effect of Astressin B；By the drug efficacy study to alopecia mice, hair takes off The mice of light is after the polypeptide 12 weeks that the injection present invention provides, and the rate of growth of hair is about 75%, simple injecting normal saline The rate of growth of mouse hair still maintains about 5%, and the rate of growth of the mouse hair of injection Astressin B is about 70%, Result show polypeptide that the present invention provides can effective hair growth, and the effect of hair growth and Astressin B hair growth Effect basically identical or the most better；In the aminoacid sequence of the polypeptide that the present invention provides, N-Me-Leu is used to replace Cml in Astressin B peptide sequence, uses γ-Glu or β-Asp to replace looped Glu in Astressin B peptide sequence, it is possible to decrease Production cost, improves the yield of product.As can be seen here, the polypeptide that the present invention provides remains lives the biology of Astressin B Property, its metabolic stability is higher, can effectively prevent and hair growth；The preparation method of this polypeptide has that production cost is low, yield High advantage.
Accompanying drawing explanation
Fig. 1 shows the HPLC collection of illustrative plates of the prepared polypeptide crude product of embodiment 1；
Fig. 2 shows the HPLC collection of illustrative plates of the prepared polypeptide fine work of embodiment 1；
Fig. 3 shows the mass spectrum of the prepared polypeptide fine work of embodiment 1；
Fig. 4 shows Astressin B that embodiment 12 provides and the polypeptide that embodiment 1 the prepares shadow to intracellular cAMP activity Ring；Wherein, line 1 shows the Astressin B impact on intracellular cAMP activity；Line 2 shows that the prepared polypeptide of embodiment 1 is to intracellular The impact of cAMP activity.
Detailed description of the invention
The invention discloses peptide species and its production and use, in those skilled in the art can use for reference herein Hold, be suitably modified technological parameter and realize.Special needs to be pointed out is, all similar replacements and change are to those skilled in the art For be apparent from, they are considered as being included in the present invention.Preferably enforcement has been passed through in the method for the present invention and application Example is described, related personnel substantially can in without departing from present invention, spirit and scope to method described herein and Application is modified or suitably changes and combine, and realizes and applies the technology of the present invention.
Abbreviation concrete meaning used in specification and claims is as follows:
Abbreviation and English implication
Alloc allyloxycarbonyl
All pi-allyl
Cml C-Alpha-Methyl-leucine
N-Me-Leu N-methylleucine
Biotin biotin
DIC DIC
DIEA diisopropylethylamine
DMF N,N-dimethylformamide
DCM dichloromethane
EDTA ethylenediaminetetraacetic acid
Fmoc 9-fluorenylmethyloxycarbonyl
HBTU O-benzotriazole-N, N, N ', N '-tetramethylurea hexafluorophosphate
HOBt I-hydroxybenzotriazole
MPEG poly glycol monomethyl ether
MPEG-NH2 amino-polyethyleneglycols monomethyl ether
Pd (PPh3) 4 tetra-triphenylphosphine palladium
PhSiH3 phenylsilane
PyBOP (benzotriazole-1-oxygen) tripyrrole alkane subbase phosphorus hexafluorophosphate
TFA trifluoroacetic acid
In polypeptide that the present invention provides and its production and use, raw materials used, adjuvant and reagent all can be buied by market.
Below in conjunction with embodiment, the present invention it is expanded on further:
The preparation of embodiment 1 polypeptide
With the solid phase carrier of Rink Amide resinous type, selecting Fmoc protection amino strategy synthesis, its operating procedure is:
Weigh Rink Amide resin 25g(10mmol), join solid state reaction post, wash 2～3 times with DMF, use DMF Swelling 30 minutes.By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100 mL, removing Fmoc protection group 20 minutes, use 1,2,3-indantrione monohydrate Whether method detection Fmoc removes completely, and resin has color, shows that Fmoc removes.
Weighing Fmoc-Ile-OH 10.6g, DIEA is activator, activates 3～5 minutes, adds reaction column reaction 1～3 little Time, obtain Fmoc-Ile-Rink Amide resin.
By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, use ninhydrin method Whether detection Fmoc removes completely, and resin has color, shows that Fmoc removes.
According to the method for above-mentioned coupling Fmoc-Ile-OH, coupling successively on Fmoc-Ile-Rink Amide resin Fmoc-N-Me-Leu-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Nle-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、 Fmoc-Arg(Pbf)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Alloc)-OH、Fmoc-His(Trt)-OH、Fmoc- Ala-OH、Fmoc-Glu-OAll、Fmoc-Gln(Trt)-OH、Fmoc-Ala-OH、Fmoc-N-Me-Leu-OH、Fmoc-Gln (Trt)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Ala-OH、Fmoc-Nle- OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Val-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Arg(Pbf)- OH、Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-His(Trt)-OH、Fmoc-D-Phe-OH、Fmoc-Thr(tBu)-OH、 Fmoc-Leu-OH、Fmoc-Asp(OtBu)-OH。
Add acetic anhydride and the DIEA of 20m mol of 15m mol, complete the acetylation of N-end.
OAll protection group on removing lysine Alloc Side chain protective group and glutamic acid main chain, concrete operations are: DCM washes 3 Secondary, add 400mmol phenyl silane, react 5 minutes, add 2mmolPd (PPh3) 4, react 120 minutes, use ninhydrin method Detection, resin has color, shows that Alloc removes.
Intramolecular amide bond is cyclized, addition 50mmol PyBOP, 60mmol HOBt, 100mmol DIEA, coupling 6 hours, Shrink, drain, obtain peptide resin.
Being cracked by peptide resin TFA, react 1～5 hour, be cleaved by polypeptide from resin, removing side chain is protected simultaneously Protect base.
To the polypeptide solution ether sedimentation being cleaved, obtain polypeptide crude product, the sequence of polypeptide such as SEQ ID NO:2 institute Showing, detect its purity and content by HPLC method, its HPLC collection of illustrative plates is as shown in Figure 1；Testing result is: the purity of polypeptide crude product is 78%, yield is 98.6%, and content is 69.5%.
Using C18 chromatographic column, with conventional flowing phase eluting, collect component, lyophilizing obtains polypeptide fine work, examines by HPLC method Surveying its purity and content, by mass spectrum and its sequence of Edman degradation analysis, its HPLC collection of illustrative plates is as in figure 2 it is shown, its mass spectrum such as Fig. 3 Shown in.Testing result is: the purity of polypeptide fine work is 96.8%, and total recovery is 64.5%, and content is 93.7%, mass spectrographic detection knot Fruit is 3962.65 for 3962.058(target molecular weight).
The preparation of embodiment 2 polypeptide
With the solid phase carrier of Rink Amide resinous type, selecting Fmoc protection amino strategy synthesis, its operating procedure is:
Weigh Rink Amide resin 25g, join solid state reaction post, with DMF wash 2～3 times, swelling 30 points with DMF Clock.By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, detect by ninhydrin method Whether Fmoc removes completely, and resin has color, shows that Fmoc removes.
Weighing Fmoc-Ile-OH10.6g, DIEA is activator, activates 3～5 minutes, adds reaction column reaction 1～3 little Time, obtain Fmoc-Ile-Rink Amide resin.
By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, use ninhydrin method Whether detection Fmoc removes completely, and resin has color, shows that Fmoc removes.
According to the method for above-mentioned coupling Fmoc-Ile-OH, coupling successively on Fmoc-Ile-Rink Amide resin Fmoc-N-Me-Leu-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Nle-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、 Fmoc-Arg(Pbf)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Alloc)-OH、Fmoc-His(Trt)-OH、Fmoc- Ala-OH、Fmoc-Glu-OAll、Fmoc-Gln(Trt)-OH、Fmoc-Ala-OH、Fmoc-N-Me-Leu-OH、Fmoc-Gln (Trt)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Ala-OH、Fmoc-Nle- OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Val-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Arg(Pbf)- OH、Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-His(Trt)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、 Fmoc-Leu-OH、Fmoc-Asp(OtBu)-OH。
Add acetic anhydride and the DIEA of 20mmol of 15mmol, complete the acetylation of N-end.
OAll protection group on removing lysine Alloc Side chain protective group and glutamic acid main chain, concrete operations are: DCM washes 3 Secondary, add 400mmol phenyl silane, react 5 minutes, add 2mmol Pd (PPh3) 4, react 120 minutes, use ninhydrin method Detection, resin has color, shows that Alloc removes.
Intramolecular amide bond is cyclized, addition 50mmol PyBOP, 60mmol HOBt, 100mmol DIEA, coupling 6 hours, Shrink, drain, obtain peptide resin.
Being cracked by peptide resin TFA, react 1～5 hour, be cleaved by polypeptide from resin, removing side chain is protected simultaneously Protect base.
To the polypeptide solution ether sedimentation being cleaved, obtain polypeptide crude product, the sequence of polypeptide such as SEQ ID NO:3 institute Show, detect its purity and content by HPLC method.Testing result is: the purity of polypeptide crude product is 76.5%；Yield is 99.7%；Contain Amount is 65.5%.
Using C18 chromatographic column, with conventional flowing phase eluting, collect component, lyophilizing obtains polypeptide fine work, examines by HPLC method Survey its purity and content, by mass spectrum and its sequence of Edman degradation analysis.Testing result is: the purity of polypeptide fine work is 98.8%, Total recovery is 62.5%, and content is 90.6%, mass spectrographic testing result be 3963.051(target molecular weight be 3962.65).
The preparation of embodiment 3 polypeptide
With the solid phase carrier of Rink Amide resinous type, selecting Fmoc protection amino strategy synthesis, its operating procedure is:
Weigh Rink Amide resin 25g, join solid state reaction post, with DMF wash 2～3 times, swelling 30 points with DMF Clock.By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, detect by ninhydrin method Whether Fmoc removes completely, and resin has color, shows that Fmoc removes.
Weighing Fmoc-Ile-OH10.6g, DIEA is activator, activates 3～5 minutes, adds reaction column reaction 1～3 little Time, obtain Fmoc-Ile-Rink Amide resin.
By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, use ninhydrin method Whether detection Fmoc removes completely, and resin has color, shows that Fmoc removes.
According to the method for above-mentioned coupling Fmoc-Ile-OH, coupling successively on Fmoc-Ile-Rink Amide resin Fmoc-N-Me-Leu-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Nle-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、 Fmoc-Arg(Pbf)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Alloc)-OH、Fmoc-His(Trt)-OH、Fmoc- Ala-OH、Fmoc-Glu-OAll、Fmoc-Gln(Trt)-OH、Fmoc-Ala-OH、Fmoc-N-Me-Leu-OH、Fmoc-Gln (Trt)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Ala-OH、Fmoc-Nle- OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Val-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Arg(Pbf)- OH、Fmoc-Leu-OH、Fmoc-Ile-OH、Fmoc-His(Trt)-OH、Fmoc-D-Phe-OH、Fmoc-Thr(tBu)-OH、 Fmoc-Leu-OH、Fmoc-Asp(OtBu)-OH。
Add acetic anhydride and the DIEA of 20mmol of 15mmol, complete the acetylation of N-end.
OAll protection group on removing lysine Alloc Side chain protective group and glutamic acid main chain, concrete operations are: DCM washes 3 Secondary, add 400mmol phenyl silane, react 5 minutes, add 2mmol Pd (PPh3) 4, react 120 minutes, use ninhydrin method Detection, resin has color, shows that Alloc removes.
Intramolecular amide bond is cyclized, addition 50mmol PyBOP, 60mmol HOBt, 100mmol DIEA, coupling 6 hours, Shrink, drain, obtain peptide resin.
Being cracked by peptide resin TFA, react 1～5 hour, be cleaved by polypeptide from resin, removing side chain is protected simultaneously Protect base.
To the polypeptide solution ether sedimentation being cleaved, obtain polypeptide crude product, the sequence of polypeptide such as SEQ ID NO:4 institute Show, detect its purity and content by HPLC method.Testing result is: the purity of polypeptide crude product is 72.5%；Yield is 96.7%；Contain Amount is 60.3%.
Using C18 chromatographic column, with conventional flowing phase eluting, collect component, lyophilizing obtains polypeptide fine work, examines by HPLC method Survey its purity and content, by mass spectrum and its sequence of Edman degradation analysis.Testing result is: the purity of polypeptide fine work is 96.9%, Total recovery is 61.5%, and content is 88.4%, mass spectrographic testing result be 3962.854(target molecular weight be 3962.65).
The preparation of embodiment 4 polypeptide
With the solid phase carrier of Rink Amide resinous type, selecting Fmoc protection amino strategy synthesis, its operating procedure is:
Weigh Rink Amide resin 25g, join solid state reaction post, with DMF wash 2～3 times, swelling 30 points with DMF Clock.By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, detect by ninhydrin method Whether Fmoc removes completely, and resin has color, shows that Fmoc removes.
Weighing Fmoc-Ile-OH10.6g, DIEA is activator, activates 3～5 minutes, adds reaction column reaction 1～3 little Time, obtain Fmoc-Ile-Rink Amide resin.
By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, use ninhydrin method Whether detection Fmoc removes completely, and resin has color, shows that Fmoc removes.
According to the method for above-mentioned coupling Fmoc-Ile-OH, coupling successively on Fmoc-Ile-Rink Amide resin Fmoc-N-Me-Leu-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Nle-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、 Fmoc-Arg(Pbf)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Alloc)-OH、Fmoc-His(Trt)-OH、Fmoc- Ala-OH、Fmoc-Glu-OAll、Fmoc-Gln(Trt)-OH、Fmoc-Ala-OH、Fmoc-N-Me-Leu-OH、Fmoc-Gln (Trt)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Ala-OH、Fmoc-Nle- OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Arg(Pbf)- OH、Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-His(Trt)-OH、Fmoc-D-Phe-OH、Fmoc-Thr(tBu)-OH、 Fmoc-Leu-OH、Fmoc-Asp(OtBu)-OH。
Add acetic anhydride and the DIEA of 20mmol of 15mmol, complete the acetylation of N-end.
OAll protection group on removing lysine Alloc Side chain protective group and glutamic acid main chain, concrete operations are: DCM washes 3 Secondary, add 400mmol phenyl silane, react 5 minutes, add 2mmol Pd (PPh3) 4, react 120 minutes, use ninhydrin method Detection, resin has color, shows that Alloc removes.
Intramolecular amide bond is cyclized, addition 50mmol PyBOP, 60mmol HOBt, 100mmol DIEA, coupling 6 hours, Shrink, drain, obtain peptide resin.
Being cracked by peptide resin TFA, react 1～5 hour, be cleaved by polypeptide from resin, removing side chain is protected simultaneously Protect base.
To the polypeptide solution ether sedimentation being cleaved, obtain polypeptide crude product, the sequence of polypeptide such as SEQ ID NO:5 institute Show, detect its purity and content by HPLC method.Testing result is: the purity of polypeptide crude product is 77.2%；Yield is 96.7%；Contain Amount is 59.6%.
Using C18 chromatographic column, with conventional flowing phase eluting, collect component, lyophilizing obtains polypeptide fine work, examines by HPLC method Survey its purity and content, by mass spectrum and its sequence of Edman degradation analysis.Testing result is: the purity of polypeptide fine work is 96.8%, Total recovery is 59.4%, and content is 88.9%, mass spectrographic testing result be 3949.135(target molecular weight be 3948.62).
The preparation of embodiment 5 polypeptide
With the solid phase carrier of Rink Amide resinous type, selecting Fmoc protection amino strategy synthesis polypeptide, it operates step Suddenly it is:
Weigh Rink Amide resin 25g, join solid state reaction post, with DMF wash 2～3 times, swelling 30 points with DMF Clock.By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, detect by ninhydrin method Whether Fmoc removes completely, and resin has color, shows that Fmoc removes.
Weighing Fmoc-Ile-OH10.6g, DIEA is activator, activates 3～5 minutes, adds reaction column reaction 1～3 little Time, obtain Fmoc-Ile-Rink Amide resin.
By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, use ninhydrin method Whether detection Fmoc removes completely, and resin has color, shows that Fmoc removes.
According to the method for above-mentioned coupling Fmoc-Ile-OH, coupling successively on Fmoc-Ile-Rink Amide resin Fmoc-N-Me-Leu-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Nle-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、 Fmoc-Arg(Pbf)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Alloc)-OH、Fmoc-His(Trt)-OH、Fmoc- Ala-OH、Fmoc-Glu-OAll、Fmoc-Gln(Trt)-OH、Fmoc-Ala-OH、Fmoc-N-Me-Leu-OH、Fmoc-Gln (Trt)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Ala-OH、Fmoc-Nle- OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Arg(Pbf)- OH、Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-His(Trt)-OH、Fmoc-D-Phe-OH、Fmoc-Thr(tBu)-OH、 Fmoc-Leu-OH、Fmoc-Asp(OtBu)-OH。
Add acetic anhydride and the DIEA of 20mmol of 15mmol, complete the acetylation of N-end.
OAll protection group on removing lysine Alloc Side chain protective group and glutamic acid main chain, concrete operations are: DCM washes 3 Secondary, add 400mmol phenyl silane, react 5 minutes, add 2mmol Pd (PPh3) 4, react 120 minutes, use ninhydrin method Detection, resin has color, shows that Alloc removes.
Intramolecular amide bond is cyclized, addition 50mmol PyBOP, 60mmol HOBt, 100mmol DIEA, coupling 6 hours, Shrink, drain, obtain peptide resin.
Being cracked by peptide resin TFA, react 1～5 hour, be cleaved by polypeptide from resin, removing side chain is protected simultaneously Protect base.
To the polypeptide solution ether sedimentation being cleaved, obtain polypeptide crude product, the sequence of polypeptide such as SEQ ID NO:6 institute Show, detect its purity and content by HPLC method.Testing result is: the purity of polypeptide crude product is 80.3%, and yield is 94.7%, contains Amount is 68.5%.
Using C18 chromatographic column, with conventional flowing phase eluting, collect component, lyophilizing obtains polypeptide fine work, examines by HPLC method Survey its purity and content, by mass spectrum and its sequence of Edman degradation analysis.Testing result is: the purity of polypeptide fine work is 97.8%, Total recovery is 66.5%, and content is 93.7%, mass spectrographic testing result be 3949.209(target molecular weight be 3948.62).
The preparation of embodiment 6 polypeptide
With the solid phase carrier of Rink Amide resinous type, selecting Fmoc protection amino strategy synthesis, its operating procedure is:
Weigh Rink Amide resin 25g, join solid state reaction post, with DMF wash 2～3 times, swelling 30 points with DMF Clock.By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, detect by ninhydrin method Whether Fmoc removes completely, and resin has color, shows that Fmoc removes.
Weighing Fmoc-Ile-OH10.6g, DIEA is activator, activates 3～5 minutes, adds reaction column reaction 1～3 little Time, obtain Fmoc-Ile-Rink Amide resin.
By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, use ninhydrin method Whether detection Fmoc removes completely, and resin has color, shows that Fmoc removes.
According to the method for above-mentioned coupling Fmoc-Ile-OH, coupling successively on Fmoc-Ile-Rink Amide resin Fmoc-N-Me-Leu-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Nle-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、 Fmoc-Arg(Pbf)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Alloc)-OH、Fmoc-His(Trt)-OH、Fmoc- Ala-OH、Fmoc-Glu-OAll、Fmoc-Gln(Trt)-OH、Fmoc-Ala-OH、Fmoc-N-Me-Leu-OH、Fmoc-Gln (Trt)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Ala-OH、Fmoc-Ile- OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Val-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Arg(Pbf)- OH、Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-His(Trt)-OH、Fmoc-D-Phe-OH、Fmoc-Thr(tBu)-OH、 Fmoc-Leu-OH、Fmoc-Asp(OtBu)-OH。
Add acetic anhydride and the DIEA of 20mmol of 15mmol, complete the acetylation of N-end.
OAll protection group on removing lysine Alloc Side chain protective group and glutamic acid main chain, concrete operations are: DCM washes 3 Secondary, add 400mmol phenyl silane, react 5 minutes, add 2mmol Pd (PPh3) 4, react 120 minutes, use ninhydrin method Detection, resin has color, shows that Alloc removes.
Intramolecular amide bond is cyclized, addition 50mmol PyBOP, 60mmol HOBt, 100mmol DIEA, coupling 6 hours, Shrink, drain, obtain peptide resin.
Being cracked by peptide resin TFA, react 1～5 hour, be cleaved by polypeptide from resin, removing side chain is protected simultaneously Protect base.
To the polypeptide solution ether sedimentation being cleaved, obtain polypeptide crude product, the sequence of polypeptide such as SEQ ID NO:7 institute Show, detect its purity and content by HPLC method.Testing result is: the purity of polypeptide crude product is 77.5%, and yield is 96.7%, contains Amount is 64.8%.
Using C18 chromatographic column, with conventional flowing phase eluting, collect component, lyophilizing obtains polypeptide fine work, examines by HPLC method Survey its purity and content, by mass spectrum and its sequence of Edman degradation analysis.Testing result is: the purity of polypeptide fine work is 96.9%, Total recovery is 59.8%, and content is 86.4%, mass spectrographic testing result be 3962.817(target molecular weight be 3962.65).
The preparation of embodiment 7 polypeptide
With the solid phase carrier of Rink Amide resinous type, selecting Fmoc protection amino strategy synthesis polypeptide, it operates step Suddenly it is:
Weigh Rink Amide resin 25g, join solid state reaction post, with DMF wash 2～3 times, swelling 30 points with DMF Clock.By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, detect by ninhydrin method Whether Fmoc removes completely, and resin has color, shows that Fmoc removes.
Weighing Fmoc-Ile-OH10.6g, DIEA is activator, activates 3～5 minutes, adds reaction column reaction 1～3 little Time, obtain Fmoc-Ile-Rink Amide resin.
By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, use ninhydrin method Whether detection Fmoc removes completely, and resin has color, shows that Fmoc removes.
According to the method for above-mentioned coupling Fmoc-Ile-OH, coupling successively on Fmoc-Ile-Rink Amide resin Fmoc-N-Me-Leu-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Nle-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、 Fmoc-Arg(Pbf)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Alloc)-OH、Fmoc-His(Trt)-OH、Fmoc- Ala-OH、Fmoc-Glu-OAll、Fmoc-Gln(Trt)-OH、Fmoc-Ala-OH、Fmoc-N-Me-Leu-OH、Fmoc-Gln (Trt)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Ala-OH、Fmoc-Nle- OH, Fmoc-Glu (OtBu)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Arg (Pbf)- OH、Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-His(Trt)-OH、Fmoc-D-Phe-OH、Fmoc-Thr(tBu)-OH、 Fmoc-Leu-OH、Fmoc-Asp(OtBu)-OH。
Add acetic anhydride and the DIEA of 20mmol of 15mmol, complete the acetylation of N-end.
OAll protection group on removing lysine Alloc Side chain protective group and glutamic acid main chain, concrete operations are: DCM washes 3 Secondary, add 400mmol phenyl silane, react 5 minutes, add 2mmol Pd (PPh3) 4, react 120 minutes, use ninhydrin method Detection, resin has color, shows that Alloc removes.
Intramolecular amide bond is cyclized, addition 50mmol PyBOP, 60mmol HOBt, 100mmol DIEA, coupling 6 hours, Shrink, drain, obtain peptide resin.
Being cracked by peptide resin TFA, react 1～5 hour, be cleaved by polypeptide from resin, removing side chain is protected simultaneously Protect base.
To the polypeptide solution ether sedimentation being cleaved, obtain polypeptide crude product, the sequence of polypeptide such as SEQ ID NO:8 institute Show, detect its purity and content by HPLC method.Testing result is: the purity of polypeptide crude product is 68.8%, and yield is 99.2%, contains Amount is 56.5%.
Using C18 chromatographic column, with conventional flowing phase eluting, collect component, lyophilizing obtains polypeptide fine work, examines by HPLC method Survey its purity and content, by mass spectrum and its sequence of Edman degradation analysis.Testing result is: the purity of polypeptide fine work is 97.2%, Total recovery is 58.1%, and content is 91.8%, mass spectrographic testing result be 3948.906(target molecular weight be 3948.62).
The preparation of embodiment 8 polypeptide
With the solid phase carrier of Rink Amide resinous type, selecting Fmoc protection amino strategy synthesis, its operating procedure is:
Weigh Rink Amide resin 25g, join solid state reaction post, with DMF wash 2～3 times, swelling 30 points with DMF Clock.By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, detect by ninhydrin method Whether Fmoc removes completely, and resin has color, shows that Fmoc removes.
Weighing Fmoc-Ile-OH10.6g, DIEA is activator, activates 3～5 minutes, adds reaction column reaction 1～3 little Time, obtain Fmoc-Ile-Rink Amide resin.
By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, use ninhydrin method Whether detection Fmoc removes completely, and resin has color, shows that Fmoc removes.
According to the method for above-mentioned coupling Fmoc-Ile-OH, coupling successively on Fmoc-Ile-Rink Amide resin Fmoc-N-Me-Leu-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Nle-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、 Fmoc-Arg(Pbf)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Alloc)-OH、Fmoc-His(Trt)-OH、Fmoc- Ala-OH、Fmoc-Asp-OAll、Fmoc-Gln(Trt)-OH、Fmoc-Ala-OH、Fmoc-N-Me-Leu-OH、Fmoc-Gln (Trt)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Ala-OH、Fmoc-Nle- OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Val-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Arg(Pbf)- OH、Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-His(Trt)-OH、Fmoc-D-Phe-OH、Fmoc-Thr(tBu)-OH、 Fmoc-Leu-OH、Fmoc-Asp(OtBu)-OH。
Add acetic anhydride and the DIEA of 20mmol of 15mmol, complete the acetylation of N-end.
OAll protection group on removing lysine Alloc Side chain protective group and glutamic acid main chain, concrete operations are: DCM washes 3 Secondary, add 400mmol phenyl silane, react 5 minutes, add 2mmol Pd (PPh3) 4, react 120 minutes, use ninhydrin method Detection, resin has color, shows that Alloc removes.
Intramolecular amide bond is cyclized, addition 50mmol PyBOP, 60mmol HOBt, 100mmol DIEA, coupling 6 hours, Shrink, drain, obtain peptide resin.
Being cracked by peptide resin TFA, react 1～5 hour, be cleaved by polypeptide from resin, removing side chain is protected simultaneously Protect base.
To the polypeptide solution ether sedimentation being cleaved, obtain polypeptide crude product, the sequence of polypeptide such as SEQ ID NO:9 institute Show, detect its purity and content by HPLC method.Testing result is: the purity of polypeptide crude product is 71.8%, and yield is 96.3%, contains Amount is 64.3%.
Using C18 chromatographic column, with conventional flowing phase eluting, collect component, lyophilizing obtains polypeptide fine work, examines by HPLC method Survey its purity and content, by mass spectrum and its sequence of Edman degradation analysis.Testing result is: the purity of polypeptide fine work is 96.9%, Total recovery is 64.1%, and content is 89.7%, mass spectrographic testing result be 3949.005(target molecular weight be 3948.62).
The preparation of embodiment 9 polypeptide
With the solid phase carrier of Rink Amide resinous type, selecting Fmoc protection amino strategy synthesis, its operating procedure is:
Weigh Rink Amide resin 25g, join solid state reaction post, with DMF wash 2～3 times, swelling 30 points with DMF Clock.By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, detect by ninhydrin method Whether Fmoc removes completely, and resin has color, shows that Fmoc removes.
Weighing Fmoc-Ile-OH10.6g, DIEA is activator, activates 3～5 minutes, adds reaction column reaction 1～3 little Time, obtain Fmoc-Ile-Rink Amide resin.
By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, use ninhydrin method Whether detection Fmoc removes completely, and resin has color, shows that Fmoc removes.
According to the method for above-mentioned coupling Fmoc-Ile-OH, coupling successively on Fmoc-Ile-Rink Amide resin Fmoc-N-Me-Leu-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Nle-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、 Fmoc-Arg(Pbf)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Orn(Alloc)-OH、Fmoc-His(Trt)-OH、Fmoc- Ala-OH、Fmoc-Glu-OAll、Fmoc-Gln(Trt)-OH、Fmoc-Ala-OH、Fmoc-N-Me-Leu-OH、Fmoc-Gln (Trt)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Ala-OH、Fmoc-Nle- OH, Fmoc-Glu (OtBu)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Arg (Pbf)- OH、Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-His(Trt)-OH、Fmoc-D-Phe-OH、Fmoc-Thr(tBu)-OH、 Fmoc-Leu-OH、Fmoc-Asp(OtBu)-OH。
Add acetic anhydride and the DIEA of 20mmol of 15mmol, complete the acetylation of N-end.
OAll protection group on removing lysine Alloc Side chain protective group and glutamic acid main chain, concrete operations are: DCM washes 3 Secondary, add 400mmol phenyl silane, react 5 minutes, add 2mmol Pd (PPh3) 4, react 120 minutes, use ninhydrin method Detection, resin has color, shows that Alloc removes.
Intramolecular amide bond is cyclized, addition 50mmol PyBOP, 60mmol HOBt, 100mmol DIEA, coupling 6 hours, Shrink, drain, obtain peptide resin.
Being cracked by peptide resin TFA, react 1～5 hour, be cleaved by polypeptide from resin, removing side chain is protected simultaneously Protect base.
To the polypeptide solution ether sedimentation being cleaved, obtain polypeptide crude product, the sequence of polypeptide such as SEQ ID NO:10 Shown in, detect its purity and content by HPLC method.Testing result is: the purity of polypeptide crude product is 72.3%, and yield is 97.3%, Content is 65.7%.
Using C18 chromatographic column, with conventional flowing phase eluting, collect component, lyophilizing obtains polypeptide fine work, examines by HPLC method Survey its purity and content, by mass spectrum and its sequence of Edman degradation analysis.Testing result is: the purity of polypeptide fine work is 98.9%, Total recovery is 67.4%, and content is 92.1%, mass spectrographic testing result be 3949.364(target molecular weight be 3948.62).
The preparation of embodiment 10 polypeptide
With the solid phase carrier of Rink Amide resinous type, selecting Fmoc protection amino strategy synthesis, its operating procedure is:
Weigh Rink Amide resin 25g, join solid state reaction post, with DMF wash 2～3 times, swelling 30 points with DMF Clock.By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, detect by ninhydrin method Whether Fmoc removes completely, and resin has color, shows that Fmoc removes.
Weighing Fmoc-Ile-OH10.6g, DIEA is activator, activates 3～5 minutes, adds reaction column reaction 1～3 little Time, obtain Fmoc-Ile-Rink Amide resin.
By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, use ninhydrin method Whether detection Fmoc removes completely, and resin has color, shows that Fmoc removes.
According to the method for above-mentioned coupling Fmoc-Ile-OH, coupling successively on Fmoc-Ile-Rink Amide resin Fmoc-N-Me-Leu-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Nle-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、 Fmoc-Arg(Pbf)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Alloc)-OH、Fmoc-His(Trt)-OH、Fmoc- Ala-OH、Fmoc-Glu-OAll、Fmoc-Gln(Trt)-OH、Fmoc-Ala-OH、Fmoc-N-Me-Leu-OH、Fmoc-Gln (Trt)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Ala-OH、Fmoc-Nle- OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Val-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Arg(Pbf)- OH、Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-His(Trt)-OH、Fmoc-D-Phe-OH、Fmoc-Thr(tBu)-OH、 Fmoc-Leu-OH、Fmoc-Asp(OtBu)-OH。
Add acetic anhydride and the DIEA of 20mmol of 15mmol, complete the acetylation of N-end.
OAll protection group on removing lysine Alloc Side chain protective group and glutamic acid main chain, concrete operations are: DCM washes 3 Secondary, add 400mmol phenyl silane, react 5 minutes, add 2mmol Pd (PPh3) 4, react 120 minutes, use ninhydrin method Detection, resin has color, shows that Alloc removes.
Intramolecular amide bond is cyclized, addition 50mmol PyBOP, 60mmol HOBt, 100mmol DIEA, coupling 6 hours, Shrink, drain, obtain peptide resin.
Being cracked by peptide resin TFA, react 1～5 hour, be cleaved by polypeptide from resin, removing side chain is protected simultaneously Protect base.
To the polypeptide solution ether sedimentation being cleaved, obtain polypeptide crude product, the sequence of polypeptide such as SEQ ID NO:11 Shown in, detect its purity and content by HPLC method.Testing result is: the purity of polypeptide crude product is 79.1%, and yield is 96.7%, Content is 59.5%.
Using C18 chromatographic column, with conventional flowing phase eluting, collect component, lyophilizing obtains polypeptide fine work, examines by HPLC method Survey its purity and content, by mass spectrum and its sequence of Edman degradation analysis.Testing result is: the purity of polypeptide fine work is 97.8%, Total recovery is 59.5%, and content is 89.2%, mass spectrographic testing result be 3976.950(target molecular weight be 3976.68).
The preparation of embodiment 11 polypeptide
With the solid phase carrier of Rink Amide resinous type, selecting Fmoc protection amino strategy synthesis, its operating procedure is:
Weigh Rink Amide resin 25g, join solid state reaction post, with DMF wash 2～3 times, swelling 30 points with DMF Clock.By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, detect by ninhydrin method Whether Fmoc removes completely, and resin has color, shows that Fmoc removes.
Weighing Fmoc-Ile-OH10.6g, DIEA is activator, activates 3～5 minutes, adds reaction column reaction 1～3 little Time, obtain Fmoc-Ile-Rink Amide resin.
By the piperidines that volume ratio is 1:4 and DMF mixed liquor 100mL, removing Fmoc protection group 20 minutes, use ninhydrin method Whether detection Fmoc removes completely, and resin has color, shows that Fmoc removes.
According to the method for above-mentioned coupling Fmoc-Ile-OH, coupling successively on Fmoc-Ile-Rink Amide resin Fmoc-N-Me-Leu-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、 Fmoc-Arg(Pbf)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Alloc)-OH、Fmoc-His(Trt)-OH、Fmoc- Ala-OH、Fmoc-Glu-OAll、Fmoc-Gln(Trt)-OH、Fmoc-Ala-OH、Fmoc-N-Me-Leu-OH、Fmoc-Gln (Trt)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Ala-OH、Fmoc-Nle- OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Val-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Arg(Pbf)- OH、Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-His(Trt)-OH、Fmoc-D-Phe-OH、Fmoc-Thr(tBu)-OH、 Fmoc-Leu-OH、Fmoc-Asp(OtBu)-OH。
Add acetic anhydride and the DIEA of 20mmol of 15mmol, complete the acetylation of N-end.
OAll protection group on removing lysine Alloc Side chain protective group and glutamic acid main chain, concrete operations are: DCM washes 3 Secondary, add 400mmol phenyl silane, react 5 minutes, add 2mmol Pd (PPh3) 4, react 120 minutes, use ninhydrin method Detection, resin has color, shows that Alloc removes.
Intramolecular amide bond is cyclized, addition 50mmol PyBOP, 60mmol HOBt, 100mmol DIEA, coupling 6 hours, Shrink, drain, obtain peptide resin.
Being cracked by peptide resin TFA, react 1～5 hour, be cleaved by polypeptide from resin, removing side chain is protected simultaneously Protect base.
To the polypeptide solution ether sedimentation being cleaved, obtain polypeptide crude product, the sequence of polypeptide such as SEQ ID NO:12 Shown in, detect its purity and content by HPLC method.Testing result is: the purity of polypeptide crude product is 73.7%, and yield is 98.2%, Content is 64.1%.
Using C18 chromatographic column, with conventional flowing phase eluting, collect component, lyophilizing obtains polypeptide fine work, examines by HPLC method Survey its purity and content, by mass spectrum and its sequence of Edman degradation analysis.Testing result is: the purity of polypeptide fine work is 96.9%, Total recovery is 63.8%, and content is 88.2%, mass spectrographic testing result be 3963.333(target molecular weight be 3962.65).
Embodiment 12 polypeptide is in vitro on the impact that intracellular cAMP is active
PC-12 cell (Adrenal Pheochromocytoma cell) is cultivated 48 hours, discards culture fluid, and use buffer solution for cleaning 2～3 times, add 1mL and contain the buffer of 1% bovine serum albumin, the polypeptide that Astressin B and embodiment 1 are provided respectively with Concentration is that the IBMX of 100 μMs is hatched half an hour jointly, discards culture medium, adds hydrochloric acid and terminates the enzyme degraded to cAMP.Collect thin Born of the same parents, ultrasonic degradation cell, measure cellular protein content by BCA detection method.Operate by the explanation of cAMP enzyme linked immunological kit, Set up the standard substance group of variable concentrations with Criterion curve, under 450nm wavelength, measure light in microplate reader after having reacted and inhale Receipts value, reads the cAMP concentration of correspondence on standard curve, finally calculates cAMP concentration in sample with this absorbance value, and result is such as Shown in Fig. 4.
From above-mentioned result of the test, Astressin B in dose-dependently increasing PC-12 intracellular cAMP amount, Increase cAMP maximum (Emax) be 161.2 ± 7.2pmol/100 μ g(albumen), EC50Value is 1.7 × 10-9M；Embodiment 1 provides The impact of polypeptide cAMP intracellular on PC-12 in vitro closely similar with Astressin B, it increases cAMP maximum (Emax) be 152.4 ± 7.8pmol/100 μ g(albumen), EC50Value is 2.1 × 10-9M, result shows the polypeptide that embodiment 1 provides Remain the biologic activity of Astressin B.
The polypeptide that Example 2～11 prepares carries out intracellular cAMP activity research, and result shows, embodiment 2～11 is made The polypeptide obtained can increase PC-12 intracellular cAMP amount equally, and in dose dependent, shows that embodiment 2～11 prepares many Peptide also remains the biologic activity of Astressin B.
The metabolic stability Journal of Sex Research of embodiment 13 polypeptide
Take Astressin B and the prepared polypeptide of embodiment 1, be dissolved in the mixed solvent of octanol/water (1:1, v/v) respectively In (0.5mg/ml), be then added in 25% rat blood serum solution, under the conditions of 37 DEG C cultivate, every 30min sampling carry out HPLC analyzes, and measures Astressin B and time that polypeptide that embodiment 1 prepares is degradable.
Result shows, Astressin B is the most degradable after 2.5h, and the polypeptide that embodiment 1 prepares is after 3.5h The most degradable.This result shows, the half-life of the polypeptide that embodiment 1 prepares is more longer than the Astressin B time, its metabolism Stability is higher.
The polypeptide that Example 2～11 prepares carries out metabolic stability Journal of Sex Research, and result shows, embodiment 2～11 prepares The half-life of polypeptide is all long than the Astressin B time, shows that its metabolic stability is high.
The effect disquisition of the pre-Anti-hair loss of embodiment 14 polypeptide
Experimental animal is female KM mice (body weight 30～38g), and before experiment, mice non-fasting prohibits water, is randomly divided into 3 groups, Often group 6.Then divide at the 0th, 1,2,4,6,8,10,12 weeks, once-through lumbar injection isopyknic normal saline (10mL/ Kg), Astressin B(5mg/kg) and the prepared polypeptide (5mg/kg) of embodiment 1.After injection, every 30min carries out one to Mus group Secondary strong illumination makes Mus group live often under scaring state.Observe the most weekly the change of mouse back hair.
The result of experiment shows, under higher pressure state (scaring state), and simple injecting normal saline in matched group Mice group after 2 weeks begin to occur depilation phenomenon, after 4 weeks, the average alopecia area of every mice accounts for whole back area About 20%, and inject Astressin B and the mice of polypeptide that embodiment 1 prepares not have obvious alopecia within 4 weeks existing As occurring.After 12 weeks, the only control mice group the most whole back hair of injecting normal saline all takes off light, and injects The test group alopecia area of the polypeptide that Astressin B and embodiment 1 prepare the most only accounts for about the 20% of whole back area.On Stating result of the test to show, the polypeptide that Astressin B and embodiment 1 prepare all has certain pre-Anti-hair loss effect, and embodiment 1 is made The polypeptide obtained and the pre-Anti-hair loss effect of Astressin B are essentially the same.
The polypeptide that Example 2～11 prepares carries out pre-Anti-hair loss effect disquisition, and result shows, embodiment 2～11 prepares Polypeptide all have certain pre-Anti-hair loss effect, and essentially the same with the pre-Anti-hair loss effect of Astressin B.
The effect disquisition of embodiment 15 polypeptide therapeutic alopecia
Take the female KM mice (body weight 25～32g) of 4～8 weeks sizes, by the ambient pressure such as illumination, noise before experiment The hair obtaining mice is stimulated substantially to take off light.Then before experiment, mice non-fasting prohibits water, is randomly divided into 3 groups, often group 6.Then Divide at the 0th, 1,2,4,6,8,10,12 weeks, the isopyknic normal saline of once-through lumbar injection (10mL/kg), Astressin And the polypeptide (5mg/kg) for preparing of embodiment 1 B(5mg/kg).After injection, keeping mice in the little life of home, every day is regular Supply water for food.Observe the most weekly the change of mouse back hair.
The result of experiment shows, after 1 week, injection has the mice of the prepared polypeptide of embodiment 1 all to have a small amount of hair to grow, its Two matched groups of Yuing have no significant change；After 2 weeks, it is left that the mouse back of the polypeptide that injection embodiment 1 prepares the most about grows 2% Right hair, and inject and have the mice of Astressin B also to have a small amount of hair to grow, the mice of simple injecting normal saline is behind Grow without any hair；After 8 weeks, the mice starting merely injecting normal saline has the hair of about 5% to grow, and injects enforcement The rate of growth of the mouse hair of the polypeptide that example 1 prepares accounts for the 60% of whole back area, the mice of injection Astressin B Hair growth rate accounts for the 50% of whole back area；After 12 weeks, the rate of growth of the mouse hair of simple injecting normal saline is still Maintaining about 5%, and the rate of growth injecting the mouse hair of Astressin B is about 70%, injection embodiment 1 prepares many The rate of growth of the mouse hair of peptide is about 75%.Result shows, the polypeptide that Astressin B and embodiment 1 prepare all has necessarily Hair growth effect, the effect of the effect of the polypeptide therapeutic alopecia that embodiment 1 the prepares hair growth than Astressin B simultaneously Fruit is the most better.
The polypeptide that Example 2～11 prepares carries out hair growth effect disquisition, and result shows, embodiment 2～11 prepares Polypeptide all have certain hair growth effect, compared with the pre-Anti-hair loss effect of Astressin B, effect is basically identical or slightly Better.
The preparation of embodiment 16 injectable powder
The polypeptide of Example 1 preparation mixes with customary adjuvant, conventionally prepares injectable powder.
The preparation of embodiment 17 injection
The polypeptide of Example 2 preparation mixes with customary adjuvant, conventionally prepares injection.
The preparation of embodiment 18 microsphere
The polypeptide of Example 3 preparation mixes with customary adjuvant, conventionally prepares microsphere.
The preparation of embodiment 19 microcapsule
The polypeptide of Example 4 preparation mixes with customary adjuvant, conventionally prepares microcapsule.
The preparation of embodiment 20 liposome
The polypeptide of Example 5 preparation mixes with customary adjuvant, conventionally prepares liposome.
The preparation of embodiment 21 microemulsion
The polypeptide of Example 6 preparation mixes with customary adjuvant, conventionally prepares microemulsion.
The preparation of embodiment 22 tablet
The polypeptide of Example 7 preparation mixes with customary adjuvant, conventionally prepares tablet.
The preparation of embodiment 23 pill
The polypeptide of Example 8 preparation mixes with customary adjuvant, conventionally prepares pill.
The preparation of embodiment 24 capsule
The polypeptide of Example 9 preparation mixes with customary adjuvant, conventionally prepares capsule.
The preparation of embodiment 25 granule
The polypeptide of Example 10 preparation mixes with customary adjuvant, conventionally prepares granule.
The preparation of embodiment 26 powder
The polypeptide of Example 11 preparation mixes with customary adjuvant, conventionally prepares powder.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For Yuan, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should It is considered as protection scope of the present invention.

Claims (9)

1. a peptide species, it is characterised in that its aminoacid sequence as shown in SEQ ID NO:1, particularly as follows:
Asp-Leu-Thr-X1-His-X2-Leu-Arg-X3-Val-Leu-X4-X5-Ala-Arg-Ala-X6-Gln-X7-Ala- Gln-X8-Ala-His-X9-X10-Arg-Lys-Leu-X11-Glu-X7-Ile；
Wherein, X7For N-Me-Leu, and, the aminoacid sequence of the polypeptide shown in SEQ ID NO:1 is particularly limited as:
X in the aminoacid sequence of described polypeptide8For γ-Glu, X1For D-Phe, X2For Leu, X3For Glu, X4For Glu, X5For Nle, X6For Glu, X9For Lys, X10For Asn, X11For Nle, its aminoacid sequence is as shown in SEQ ID NO:2；
X in the aminoacid sequence of described polypeptide8For γ-Glu, X1For Phe, X2For Leu, X3For Glu, X4For Glu, X5For Nle, X6 For Glu, X9For Lys, X10For Asn, X11For Nle, its aminoacid sequence is as shown in SEQ ID NO:3；
X in the aminoacid sequence of described polypeptide8For γ-Glu, X1For D-Phe, X2For Ile, X3For Glu, X4For Glu, X5For Nle, X6For Glu, X9For Lys, X10For Asn, X11For Nle, its aminoacid sequence is as shown in SEQ ID NO:4；
X in the aminoacid sequence of described polypeptide8For γ-Glu, X1For D-Phe, X2For Leu, X3For Asp, X4For Glu, X5For Nle, X6For Glu, X9For Lys, X10For Asn, X11For Nle, its aminoacid sequence is as shown in SEQ ID NO:5；
X in the aminoacid sequence of described polypeptide8For γ-Glu, X1For D-Phe, X2For Leu, X3For Glu, X4For Asp, X5For Nle, X6For Glu, X9For Lys, X10For Asn, X11For Nle, its aminoacid sequence is as shown in SEQ ID NO:6；
X in the aminoacid sequence of described polypeptide8For γ-Glu, X1For D-Phe, X2For Leu, X3For Glu, X4For Glu, X5For Ile, X6For Glu, X9For Lys, X10For Asn, X11For Nle, its aminoacid sequence is as shown in SEQ ID NO:7；
X in the aminoacid sequence of described polypeptide8For γ-Glu, X1For D-Phe, X2For Leu, X3For Glu, X4For Glu, X5For Nle, X6For Asp, X9For Lys, X10For Asn, X11For Nle, its aminoacid sequence is as shown in SEQ ID NO:8；
X in the aminoacid sequence of described polypeptide8For β-Asp, X1For D-Phe, X2For Leu, X3For Glu, X4For Glu, X5For Nle, X6 For Glu, X9For Lys, X10For Asn, X11For Nle, its aminoacid sequence is as shown in SEQ ID NO:9；
X in the aminoacid sequence of described polypeptide8For γ-Glu, X1For D-Phe, X2For Leu, X3For Glu, X4For Glu, X5For Nle, X6For Glu, X9For Orn, X10For Asn, X11For Nle, its aminoacid sequence is as shown in SEQ ID NO:10；
X in the aminoacid sequence of described polypeptide8For γ-Glu, X1For D-Phe, X2For Leu, X3For Glu, X4For Glu, X5For Nle, X6For Glu, X9For Lys, X10For Gln, X11For Nle, its aminoacid sequence is as shown in SEQ ID NO:11；
X in the aminoacid sequence of described polypeptide8For γ-Glu, X1For D-Phe, X2For Leu, X3For Glu, X4For Glu, X5For Nle, X6For Glu, X9For Lys, X10For Asn, X11For Ile, its aminoacid sequence is as shown in SEQ ID NO:12；
The N end of described polypeptide is through acetylation modification；
From N end, the X of the 22nd in described polypeptide8Main chain and the X of the 25th9Side chain carries out amido link cyclisation.
2. the preparation method of a polypeptide as claimed in claim 1, it is characterised in that comprise the steps:
Step A: take Ile and resin coupling, obtain Ile-resin；
Step B: take described Ile-resin through progressively coupling, obtain polypeptide fragment-resin；
Step C: the N end of described polypeptide fragment-resin is through acetylation modification；
Step D: removing the 22nd and protection group of the 25th amino acids residue from N end, through cyclisation, cracking, purification, lyophilizing, Obtain；
From N end, described polypeptide the 22nd amino acids raw material uses Fmoc-Asp-OAll or Fmoc-Glu-OAll；
The sequence of the polypeptide in described polypeptide fragment-resin is as shown in SEQ ID NO:1.
Preparation method the most according to claim 2, it is characterised in that the reagent that acetylation modification described in step C uses For acetic anhydride and the mixture of DIEA.
Preparation method the most according to claim 2, it is characterised in that the reagent removing employing described in step D is phenyl Silane and Pd (PPh3)4Mixture.
Preparation method the most according to claim 2, it is characterised in that the reagent being cyclized employing described in step D is The mixture of PyBOP, HOBt and DIEA.
Preparation method the most according to claim 2, it is characterised in that the reagent cracking employing described in step D is TFA.
7. the polypeptide as claimed in claim 1 application in preparation preventing and treating alopecia agent.
8. a pharmaceutical preparation, it is characterised in that be made up of the polypeptide described in claim 1 and pharmaceutically acceptable adjuvant.
Pharmaceutical preparation the most according to claim 8, it is characterised in that the dosage form of described pharmaceutical preparation is injectable powder, injection Liquid, microsphere, microcapsule, liposome, microemulsion, tablet, pill, capsule, granule or powder.
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WO2002005749A2 (en) * 2000-07-19 2002-01-24 Bristol-Myers Squibb Pharma Company Crf2 ligands in combination therapy
WO2012088397A3 (en) * 2010-12-22 2012-09-13 The Salk Institute For Biological Studies Cyclic crf antagonist peptides
CN102702320A (en) * 2012-06-01 2012-10-03 深圳翰宇药业股份有限公司 Method for preparing eptifibatide
CN103230346A (en) * 2013-04-15 2013-08-07 深圳市新百肽生物科技有限公司 Cosmetic containing astressin-B and preparation method thereof

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Publication number Priority date Publication date Assignee Title
WO2002005749A2 (en) * 2000-07-19 2002-01-24 Bristol-Myers Squibb Pharma Company Crf2 ligands in combination therapy
WO2012088397A3 (en) * 2010-12-22 2012-09-13 The Salk Institute For Biological Studies Cyclic crf antagonist peptides
CN102702320A (en) * 2012-06-01 2012-10-03 深圳翰宇药业股份有限公司 Method for preparing eptifibatide
CN103230346A (en) * 2013-04-15 2013-08-07 深圳市新百肽生物科技有限公司 Cosmetic containing astressin-B and preparation method thereof

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