CN103304660A - Synthetic method of liraglutide - Google Patents
Synthetic method of liraglutide Download PDFInfo
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- CN103304660A CN103304660A CN2013102921868A CN201310292186A CN103304660A CN 103304660 A CN103304660 A CN 103304660A CN 2013102921868 A CN2013102921868 A CN 2013102921868A CN 201310292186 A CN201310292186 A CN 201310292186A CN 103304660 A CN103304660 A CN 103304660A
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- fmoc
- glu
- glutamyl
- glp
- arg34lys26
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- 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
Abstract
The invention discloses a full chemical synthetic method for hybridization of a solid phase and a liquid phase of liraglutide. The method comprises the following steps: chemically synthesizing a liraglutide precursor protected by N terminal and a cetyl derivative; de-protecting to remove tail end protection to obtain a target polypeptide. The liraglutide precursor semi-protected is obtained by polypeptide solid-phase synthesis, and the precursor purified to the drug level enters into the next chemical synthesis.
Description
Technical field
The present invention relates to the chemosynthesis of medical polypeptide class bulk drug, relate in particular to a kind of solid phase and solution hybridization synthetic method of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
Background technology
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is the analogue of a kind of people's glicentin-1(GLP-1), is used for the treatment of diabetes.Molecular formula: C
172H
265N
43O
51, molecular weight: 3751.20, CAS number: 204656-20-2.
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is the medicine of approval in 2010.US Patent No. Patent No.6268343,6458924 and 7235627 have invented the synthetic Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor of a kind of DNA restructuring technique, and namely sequence 1-31 obtains target polypeptides by chemical synthesis link Pal-Glu (OSu)-OtBu material again.DNA restructuring arts demand uses complicated equipment, and may introduce virus in product, is unfavorable for the medicinal purpose of product.Chinese patent 200610110898.3 and 200510107588 have reported the method for direct solid phase synthesis Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], yet because the hydrophobic nature of hexadecyl on the peptide chain, the impurity that this sampling technology produces is difficult to remove.
Therefore, this area is in the urgent need to providing a kind of feasible Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] synthetic method to produce high-purity product, to satisfy medicinal use.
Summary of the invention
The present invention aims to provide a kind of new Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] solid phase synthesis process.
In a first aspect of the present invention, a kind of synthetic method of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is provided, described method comprises step:
(1) take Fmoc-Gly-solid phase synthesis resin as starting raw material, repeat Deprotection, use condensing agent to connect the step of reactive polypeptide according to solid phase synthesis process, connect successively protected amino acid or protection polypeptide with protecting group, cut peptide and obtain N-terminal with the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor of protecting group;
(2) N-terminal is carried out chemosynthesis with Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor and hexadecyl-N-hydroxy-succinamide ester, hexadecanoic acid Acibenzolar or the hexadecanoic acid acid anhydrides of protecting group in liquid phase, remove again the N-terminal blocking group, obtain the target polypeptides Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
In another preference, described target polypeptides is by Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor and hexadecyl-N-hydroxy-succinamide ester, hexadecanoic acid Acibenzolar or hexadecanoic acid acid anhydrides and condensation reagent in-situ activation and get, and described condensation reagent is selected from DCC, DIC, HBTU or TBTU.
In another preference, described N-terminal with protecting group be selected from 2-Cl-carbobenzoxy-(Cbz) (namely claiming the Cl-Z blocking group), carbobenzoxy-(Cbz) (namely claiming the Z blocking group) or fluorenylmethyloxycarbonyl (namely claiming the Fmoc blocking group);
Adopt palladium charcoal or metal catalyst hydrogenation to remove Cl-Z blocking group and Z blocking group;
Adopt the alkaline solution hydrolysis to remove the Fmoc blocking group.
In another preference, described alkaline solution is selected from piperidines or DBU.
In another preference; 20 amino acids are Fmoc-Lys (γ-Glu-OtBu); N-terminal amino acid adopts Cl-Z-His (Trt)-OH; Z-His (Trt)-OH; Alloc-His (Trt)-OH or Fmoc-His (Trt)-OH Acibenzolar method condensation; 31 peptide resins that are protected use synchronously the deprotection agent agent to take off Side chain protective group and cut peptide, obtain N-terminal with the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor of Cl-Z, Z, Alloc or Fmoc protection.
In another preference, 20 amino acids are Fmoc-Lys (γ-Glu-OtBu), N-terminal amino acid adopts Fmoc-His (Trt)-OH Acibenzolar method condensation and gets, after N-terminal Fmoc removes, adopt Cl-Z-OSu, Z-OSu or Alloc-OSu to carry out end capping, thereby 31 peptide resins that are protected take off synchronously Side chain protective group and cut peptide, obtain the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor that N-terminal is protected with Cl-Z, Z or Alloc; More preferably, 20 amino acids are Fmoc-Lys (Alloc)-OH, and after the Alloc protecting group removed with the hydrogenation of tetraphenylphosphonium palladium, with Boc-γ-Glu-OtBu reaction, 20 amino acids were Fmoc-Lys (Boc-γ-Glu-OtBu); Best, 20 amino acids are Fmoc-Lys (Dde)-OH, and after the Dde protecting group removed with 2% hydrazine hydrate/DMF, with Boc-γ-Glu-OtBu reaction, 20 amino acids were Fmoc-Lys (Boc-γ-Glu-OtBu).
In another preference; 20 amino acids are Fmoc-Lys (Boc)-OH; N-terminal amino acid adopts Cl-Z-His (Trt)-OH, Z-His (Trt)-OH, Alloc-His (Trt)-OH or Fmoc-His (Trt)-OH Acibenzolar method condensation and gets; thereby 31 peptide resins that are protected; take off synchronously Side chain protective group and cut peptide, obtain the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group) that N-terminal is protected with Cl-Z, Z, Alloc or Fmoc.
In another preference; 20 amino acids are Fmoc-Lys (Boc)-OH; N-terminal amino acid adopts Fmoc-His (Trt)-OH Acibenzolar method condensation; after N-terminal Fmoc removes; adopt Cl-Z-OSu, Z-OSu or Alloc-OSu to carry out end capping; thereby 31 peptide resins that are protected take off synchronously Side chain protective group and cut peptide, obtain the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group) that N-terminal is protected with Cl-Z, Z or Alloc.
In another preference, with Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group) and the N of N-terminal-fluorenylmethyloxycarbonyl (Fmoc blocking group) protection
α-Oxohexadecyl-Glu (OSu)-OBzl (is in Pal-Glu (OSu)-OBzl) liquid phase and carries out the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that acylation reaction is protected; The Fmoc protecting group is removed with alkali, and hydrogenation removes benzyl (Bzl);
Alkaline solution is selected from piperidines or DBU alkaline solution;
Palladium carbon catalyst catalysis is adopted in hydrogenation.
In another preference, Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group) and Pal-Glu (OSu)-OBzl that N-terminal-2-benzyloxycarbonylchloride base (Cl-Z blocking group) is protected carry out the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that acylation reaction is protected; Hydrogenation removes Cl-Z protection and benzyl;
Palladium carbon catalyst catalysis is adopted in hydrogenation.
In another preference, will carry out the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that acylation reaction is protected in the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group) of N-terminal-carbobenzoxy-(Cbz) (Z blocking group) protection and Pal-Glu (the OSu)-OBzl liquid phase; Hydrogenation removes Z protection and benzyl again;
The catalysis of palladium charcoal catalytic reagent is adopted in hydrogenation.
In another preference, will carry out the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that acylation reaction is protected in the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (20 amino acids side chains do not contain γ-Glu group) of N-terminal-carbobenzoxy-(Cbz) (Z blocking group) protection or N-terminal-2-benzyloxycarbonylchloride base (Cl-Z blocking group) protection and Pal-Glu (the OSu)-OtBu liquid phase; Hydrogenation removes Z protection or Cl-Z protection, and the tBu group is removed in hydrolysis under acidic conditions;
Palladium carbon catalyst catalysis is adopted in hydrogenation;
Acidic conditions can be HCl or TFA acidic solution.
In another preference, will carry out the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that acylation reaction is protected in the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group) of N-terminal-allyloxycarbonyl protection (Alloc blocking group) and Pal-Glu (the OSu)-OAllyl liquid phase; Hydrogenation removes Alloc protection and OAllyl group;
The catalysis of tetraphenylphosphonium palladium catalytic reagent is adopted in hydrogenation.
In another preference, will carry out the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that acylation reaction is protected in the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group) of N-terminal-allyloxycarbonyl (Alloc blocking group) protection and Pal-Glu (the OSu)-OBzl liquid phase; Hydrogenation removes Alloc protection and benzyl;
The catalysis of tetraphenylphosphonium palladium catalytic reagent is adopted in hydrogenation.
In another preference, will carry out the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that acylation reaction is protected in the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group) of N-terminal-allyloxycarbonyl (Alloc blocking group) protection and Pal-Glu (the OSu)-OtBu liquid phase; Hydrogenation removes to be hydrolyzed under Alloc protection and the acidic conditions removes the tBu group;
Palladium carbon catalyst catalysis is adopted in hydrogenation;
Acidic conditions is selected from HCl or TFA acidic solution.
In another preference, protect Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group) and Pal-Glu (the OSu)-OtBu of (Boc blocking group) to carry out the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that acylation reaction is protected N-terminal-tertbutyloxycarbonyl; Boc and tBu group are removed in hydrolysis under the acidic conditions;
Palladium carbon catalyst catalysis is adopted in hydrogenation;
Acidic conditions is selected from HCl or TFA acidic solution.
In another preference, the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor is purified by cationic exchange or anion exchange method, and ultrafiltration process or preparative chromatography method of purification are changed salt, and obtain after the freeze-drying.
In another preference, the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor can be purified to the purity greater than 70.0%HPLC; Preferably, can be purified to the purity greater than 80.0%HPLC; More preferably, can be purified to the purity greater than 90.0%HPLC; More preferably, can be purified to the purity greater than 95.0%HPLC; Best, can be purified to the purity greater than 98.0%HPLC.
In another preference, the target polypeptides Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] can be purified by preparative high performance liquid chromatography, adopt C18, C8, C4 or other polystyrene resin isochromatic spectrum fillers, the moving phase of pH2-7, such as acetonitrile and the acetonitrile of aqueous systems, triethylamine and phosphoric acid and acetonitrile and the aqueous systems of aqueous systems, sodium phosphate salt or potassium phosphate salt of trifluoroacetic acid, or the acetonitrile of Spirit of Mindererus and aqueous systems; Methyl alcohol or other alcohol can replace acetonitrile; Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] can purify to the purity greater than 95.0%HPLC; Preferably, purify to the purity greater than 96.0%HPLC; More preferably, can purify to the purity greater than 97.0%HPLC; More preferably, can purify to the purity greater than 98.0%HPLC; Best, can purify to the purity greater than 99.0%HPLC.
In another preference, the substitution value of described solid phase synthesis resin is 0.1-0.8mmol/g.
In another preference, setting-up point is room temperature, and optimum condition is 10-30 ℃.
In another preference, setting-up point is high temperature, and optimum condition is 30-80 ℃.
In another preference, described solid phase synthesis resin is selected from hydroxy resin, 2-Cl-Trt resin or chloromethyl resin; More preferably, described hydroxy resin is from king's resin or PL king's resin.
In another preference, described protected amino acid is respectively:
30#：Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Mtr)-OH、Fmoc-Arg(Pmc)-OH；
29#：Fmoc-Gly-OH；
28#：Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Mtr)-OH、Fmoc-Arg(Pmc)-OH；
27#：Fmoc-Val-OH；
26#：Fmoc-Leu-OH；
25#：Fmoc-Trp(Boc)-OH、Fmoc-Trp-OH；
24#：Fmoc-Ala-OH；
23#：Fmoc-Ile-OH；
22#：#Fmoc-Phe-OH；
21#：Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OAll)-OH、Fmoc-Glu(OBzl)-OH；
20#:Fmoc-Lys(Dde)-OH、Fmoc-Lys(Boc-Glu-OtBu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Alloc)-OH；
19#：Fmoc-Ala-OH；
18#：Fmoc-Ala-OH；
17#：Fmoc-Gln(Trt)-OH、Fmoc-Gln(Xan)-OH、Fmoc-Gln-OH；
16#：Fmoc-Gly-OH；
15#：Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OAll)-OH、Fmoc-Glu(OBzl)-OH；
14#：Fmoc-Leu-OH；
13#：Fmoc-Tyr(tBu)-OH；
12#：Fmoc-Ser(tBu)-OH、Fmoc-Ser(Trt)-OH、Fmoc-Ser-OH、Fmoc-Ser(OBzl)-OH；
11#：Fmoc-Ser(tBu)-OH、Fmoc-Ser(Trt)-OH、Fmoc-Ser-OH、Fmoc-Ser(OBzl)-OH；
10#：Fmoc-Val-OH；
9#：Fmoc-Asp(OtBu)-OH、Fmoc-Asp(oall)-OH、Fmoc-Asp(OBzl)-OH；
8#：Fmoc-Ser(tBu)-OH、Fmoc-Ser(Trt)-OH；
7#：Fmoc-Thr(tBu)-OH、Fmoc-Thr(oBzl)-OH；
6#：Fmoc-Phe-OH；
5#：Fmoc-Thr(tBu)-OH、Fmoc-Thr(oBzl)-OH；
4#：Fmoc-Gly-OH；
3#：Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OAll)-OH、Fmoc-Glu(OBzl)-OH；
2#：Fmoc-Ala-OH；
1#：Boc-His(Trt)-OH、Z-His(Trt)-OH、Boc-His(Boc)-OH,Cl-Z-His(Trt)-OH,Alloc-His(Trt)-OH,Fmoc-His(Trt)-OH,Boc-His-OH、Z-His-OH、Boc-His-OH,Cl-Z-His-OH,Alloc-His-OH,and?Fmoc-His-OH。
Accordingly, the invention provides a kind of feasible Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] synthetic method and produce high-purity product, to satisfy medicinal use.
Embodiment
The contriver based on the difference of 20 bit substituents, adopts full solid phase or solid liquid phase synthesis method through extensive and deep research, has obtained highly purified Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product (content 50-70%).
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] solid phase synthesis process provided by the invention passes through following step take Fmoc-Gly-solid phase synthesis resin as starting raw material:
Deprotection steps: with protected amino acid or polypeptide-solid phase synthesis resin with DCM mixes, swelling, add piperidines/DMF solution deprotection in reactor;
Condensation step feeds intake: will pour in the above-mentioned reactor after the dissolving in the mixing solutions of DMF/DCM with protected amino acid, HOBt and the DIC of protecting group, detect to judge with triketohydrindene hydrate whether condensation is complete; DMF/DCM (30%-70%)
Repeat deprotection, condensation step, peptide chain is extended to the N end from the C end, until 1# amino acid is synthetic complete, obtain the direct-connected peptide with protecting group.
In above-mentioned deprotection steps, need abundant swelling;
In the above-mentioned condensation step that feeds intake, the mixing solutions of described DMF/DCM is DMF/DCM (30%-70%).
In one embodiment of the invention, in the above-mentioned condensation step that feeds intake, 20 protected amino acid can be selected Fmoc-Lys (Dde)-OH, terminal Fmoc-His (the Trt)-OH that selects; 20 protecting group Dde is removed in alternative cutting, and Boc-Glu (Osu)-OBzl reaction obtains the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor again, and final and Pal-OSu reacts and obtains crude product.
In another embodiment of the present invention; in the above-mentioned condensation step that feeds intake; 20 protected amino acid can be selected Fmoc-Lys (Dde)-OH, Fmoc-Lys (Boc)-OH; Fmoc-Lys (Alloc)-OH complete resection protecting group is not contained the peptide of protecting group; terminal Z-His (the Trt)-OH that selects is directly synthetic; behind the preliminary purification again and Pal-Glu (OSu)-OBzl synthetic in solid phase or liquid phase, hydrogenation reaction deprotection base obtains crude product.
In another embodiment of the present invention; in the above-mentioned condensation step that feeds intake; 20 protected amino acid can be selected Fmoc-Lys (Boc-Glu-OtBu)-OH; the complete resection protecting group is not contained the peptide of protecting group; react with Pal-OSu behind the preliminary purification, hydrogenation reaction deprotection base obtains crude product again.
The implication of employed abbreviation or English full name is listed in the table below among the present invention:
As used herein, " solid phase synthesis " or " Solid-phase Polypeptide synthesizes (solid phase peptide synthesis) " is a kind of peptide synthesis technology well known in the art, includes but not limited to following method: with the protected amino acid of amino covalently bound (bonding) on solid phase carrier; Going in the presence of the protective material, the protecting group of desamidizate is received on the solid phase carrier first amino acid; Then amino be closed (protection) second amino acid whose carboxyl by activation, second amino acid that carboxyl is activated forms peptide bond with first the amino acid whose amino reaction (condensation) that is connected on solid phase carrier again, has just generated like this a dipeptides with protecting group on solid phase carrier; Repeat above-mentioned peptide bond and form reaction, peptide chain is grown, until reach needed peptide chain length from the C end to the N end; The protecting group of last deaminize, the ester bond (cutting) between hydrolysis peptide chain and the solid phase carrier obtains synthetic peptide.
As used herein, " removing protective material " or " deprotection agent " can Alternate, all refers to be connected to the chemical reagent that the amino protecting agent on the amino acid is removed, described amino protecting agent can make well known in the art, such as but not limited to, Fmoc, Boc; It is described that to go protective material to make well known in the art, such as but not limited to, in its cumulative volume, be the DMF solution that contains the 15-25v/v% piperidines.
As used herein, " condensing agent ", " activator ", " activating reagent " or " condensation activator " can Alternates, all be to instigate an amino acid whose amino and another amino acid whose carboxyl condensation to form the chemical reagent of peptide bond, preferably use in the present invention DIC/HOBt.
As used herein, " cutting agent " refer to the polypeptide of resin-bonded and the chemical reagent of resin isolation, can make well known in the art, such as but not limited to, the weakly acidic solution, the HCl solution that contain TFA preferably contain the phenol solution of TFA, TIS and EDT.
As used herein, " HPLC purity " refers to the polypeptide products that will prepare, detect through HPLC, according to resulting chromatogram collection of illustrative plates, carry out area normalization method and percentage ratio that the peak area of the target polypeptides compound that obtains occupies in all peak area summations.
As used herein, " Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product " refers to that HPLC purity is at the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] product of 40%-70%.
The structural formula of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is suc as formula shown in the I:
H-His
1-Ala
2-Glu
3-Gly
4-Thr
5-Phe
6-Thr
7-Ser
8-Asp
9-Val
10-Ser
11-Ser
12-Tyr
13-Leu
14-Glu
15-Gly
16-Gln
17-Ala
18-Ala
19-Lys
20[N-(1-oxohexadecyl)-L-γⅠ-glutamyl]-Glu
21-Phe
22-Ile
23-Ala
24-Trp
25–Leu
26-Val
27-Arg
28–Gly
29–A?rg
30-Gly
31-OH
Relevant ninhydrin (Kaiser), ninhydrin test (Ninhydrin test), and monitoring method can be referring to document VIRENDER K.SARIN, et al. " Quantitative Monitoring of Solid-Phase Peptide Synthesis by the Ninhydrin Reaction " ANALYTICAL BIOCHEMISTRY117,147-157(1981), E.KAISER, et al. " Color Test for Detection of Free Terminal Amino Groups in the Solid-Phase Synthesis of Peptides " SHORT COMMUNICATIONS595-598 (Received October28,1969), with THORKILD CHRISTENSEN " A Qualitative Test for Monitoring Coupling Completeness in Solid Phase Peptide Synthesis Using Chloranil " Acta Chemica Scandinavica B33 (1979) 763-766.
The above-mentioned feature that the present invention mentions, or the feature that embodiment mentions can arbitrary combination.All features that this case specification sheets discloses can with any composition forms and usefulness, each feature that discloses in the specification sheets can anyly provide the alternative characteristics of identical, impartial or similar purpose to replace.Therefore except special instruction is arranged, the feature that discloses only is the general example of equalization or similar features.
Major advantage of the present invention is:
1, synthetic polypeptide method provided by the invention is taked N end temporary protection, improves the selectivity of Pal condensing site, and the crude product purity that obtains is higher.
2, the present invention can change the high purification difficult that causes of Pal hydrophobicity on peptide chain, finally improves sterling purity.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to normal condition or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise all percentage ratio, ratio, ratio or umber by weight.
Unit in the percent weight in volume among the present invention is well-known to those skilled in the art, for example refers to the weight of solute in 100 milliliters solution.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The usefulness that better implementation method described in the literary composition and material only present a demonstration.
The Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] HPLC analytical procedure that relates among the following embodiment is as follows:
Stationary phase: Kromasil C18,5um, 30 ℃ of 4.6*150mm column temperatures
Moving phase: A phosphoric acid triethylamine solution (pH=6.5), B acetonitrile
Flow velocity: 1.0ml/min
Gradient: 32-52%B, 30 minutes
Detect wavelength: 215nm
The Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product HPLC purification process that relates among the following embodiment is as follows:
Stationary phase: Kromasil C18,10um, 30 ℃ of column temperatures
Moving phase: A phosphoric acid triethylamine solution (pH=7.0), B acetonitrile
Flow velocity: 20ml/min
Gradient: 26-48%B, 100 minutes
Detect wavelength: 215nm
Embodiment 1
Take by weighing 19.23g (5mmol) Fmoc-Gly-Wang Resin and drop in the reaction column, add the solution deprotection of 20% piperidines/DMF(V/V) twice.Washing, the resin indenes that takes a morsel after taking out inspection is positive.Take by weighing 9.73gFmoc-Arg (Pbf)-OH and 2.43gHOBt, add approximately 50mLDMF dissolving, add the 2.8mLDIC activation, be poured into room temperature reaction 2h in the reaction column, triketohydrindene hydrate detects.Washing.Repeat above-mentioned deprotection, washing and condensation step, until N-terminal Fmoc-His (Trt)-OH condensation is complete, 20 protected amino acids are Fmoc-Lys (Boc)-OH, remove N-terminal Fmoc protecting group and add the DMF washing.Take by weighing 4.25gZ (2-Cl)-OSu and also react with joining in the above-mentioned resin after an amount of DMF dissolving, triketohydrindene hydrate detects, and drying obtains Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] intermediate peptide resin 41.2g, and the peptide resin yield is about 91%.Above-mentioned resin joins cracking in the cracking prescription reagent, and filtrate joins separates out thick peptide in the anhydrous diethyl ether, get 15.7g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] intermediate crude product, and yield is 88.4%.With crude product freeze-drying behind high-pressure liquid phase (HPLC) purifying, obtain 6.47g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group).It is 98.8%, ESI-MS (+) that HPLC detects purity: [M+3H]
+=1185.2, purification yield 41.2%.
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group) is dissolved in approximately among the 100mLDMF and ice-water bath, adds 1.1g Pal-Glu (OSu)-OBzl reaction and spends the night, and monitors reaction process with HPLC.Add vinegar acid for adjusting pH to 5 after reaction finishes, add 0.8gPd/C and make catalyzer and pass into hydrogen, remove Z (2-Cl) and Obzl protecting group, equally by HPLC monitoring reaction process.After hydrogenation finished, filtering Pd/C catalyzer joined filtrate in the freezing anhydrous diethyl ether, has solid to separate out, centrifugal, washing, drying obtains the final crude product 6.42g(of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] yield 95.6%).With the thick peptide HPLC of 6.42g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] method purifying, obtain 4.3g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] finished product.It is 99.0%, ESI-MS (+) that HPLC detects purity: [M+3H]
+=1251.6, total recovery 23.0%.
Embodiment 2
Take by weighing 27.0g (10mmol) Fmoc-Gly-WangResin and drop in the reaction column, add an amount of DCM swelling, suction filtration.Add the solution deprotection of 20% piperidines/DMF(V/V) twice.Add again DMF, MeOH, DCM washs successively, the resin indenes that takes a morsel after taking out inspection is positive.Take by weighing 12.98gFmoc-Arg (Pbf)-OH, 7.21gHBTU and 2.70gHOBt add approximately the 80mLDMF dissolving, behind the ice-water bath 2min, add after 3.5mLDIPEA activates approximately 10min, are poured into room temperature reaction 2h in the reaction column, and triketohydrindene hydrate detects.Reaction is taken out reaction solution after finishing, and adds DMF washing 2 times, repeats above-mentioned deprotection, washing and condensation step, until N-terminal His condensation is complete.20# and 1# protected amino acid are respectively Fmoc-Lys (Dde)-OH and Z-His (Trt)-OH.
Configure 2% hydrazine hydrate/DMF solution 100mL and join in the intermediate peptide resin and react, remove Lys20 side chain Dde protecting group, triketohydrindene hydrate detects.After removing end, washing resin.HPLC method purifying after the resin cutting obtains Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group)
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group) is dissolved in approximately among the 100mLDMF and ice-water bath, adds 2.5g Pal-Glu (OSu)-OBzl reaction and spends the night, and monitors reaction process with HPLC.Add vinegar acid for adjusting pH to 5 after reaction finishes, add 1.7gPd/C and make catalyzer and pass into hydrogen, remove Z and OBzl protecting group, equally by HPLC monitoring reaction process.After hydrogenation finished, filtering Pd/C catalyzer joined filtrate in the freezing anhydrous diethyl ether, has solid to separate out, centrifugal, washing, drying obtains the final crude product 14.0g(of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] yield 96%).With the thick peptide HPLC of 14g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] method purifying, obtain 9.0g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] finished product.It is 98.8%, ESI-MS (+) that HPLC detects purity: [M+3H]
+=1251.6, total recovery 24.10%.
Embodiment 3
Take by weighing 47.6g (20mmol) Fmoc-Gly-WangResin and drop in the reaction column, add the solution deprotection of 20% piperidines/DMF(V/V) twice.Add again DMF, MeOH, DCM washs successively, the resin indenes that takes a morsel after taking out inspection is positive.Take by weighing 32.4gFmoc-Arg (Pbf)-OH, 26.0gPyBOP and 6.75gHOBt add approximately the 160mLDMF dissolving, behind the ice-water bath 2min, add after 8.7mLDIPEA activates approximately 10min, and reaction 2h, triketohydrindene hydrate detects.After reaction finishes, the DMF washing.Repeat above-mentioned deprotection, washing and condensation step, until N-terminal Fmoc-His (Trt)-OH condensation is complete, dry rear gained intermediate peptide resin heavily is 145.0g (peptide resin yield 93%).Lys wherein
20(Boc-γ-Glu-OtBu)-OH is as the condensation monomer to adopt Fmoc-Lys.Intermediate peptide resin cutting purifying obtains 31.4g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor, and it is 92.8%, ESI-MS (+) that HPLC detects purity: [M+3H]
+=1246.0, purification yield 47.6%.
The 31.4g precursor of gained is dissolved in approximately among the 200mLDMF and ice-water bath, adds 3.25g(9.2mmol) the Pal-OSu reaction spends the night, and monitors reaction process with HPLC.Reaction finishes to add in the backward reaction solution approximately 40mL piperidines continuation stirred 1～2 hour, to remove N-terminal Fmoc group, monitored reaction process with HPLC.Again filtrate is joined in the freezing anhydrous diethyl ether, have solid to separate out, centrifugal, washing, drying obtains the final crude product 28.4g(of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] yield 90%).The thick peptide HPLC of 28.4 Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]s purifying is obtained 15.3g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] finished product.It is 99.1%, ESI-MS (+) that HPLC detects purity: [M+3H]
+=1251.6, total recovery 20.4%.
The above only is preferred embodiment of the present invention, be not to limit essence technology contents scope of the present invention, essence technology contents of the present invention is broadly to be defined in the claim scope of application, any technology entity or method that other people finish, if defined identical with the claim scope of application, also or a kind of change of equivalence, all will be regarded as being covered by among this claim scope.
Claims (20)
1. the synthetic method of an Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], described method comprises step:
(1) take Fmoc-Gly-solid phase synthesis resin as starting raw material, repeat Deprotection, use condensing agent to connect the step of reactive polypeptide according to solid phase synthesis process, connect successively protected amino acid or protection polypeptide with protecting group, cut peptide and obtain N-terminal with the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor of protecting group;
(2) N-terminal is carried out chemosynthesis with Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor and hexadecyl-N-hydroxy-succinamide ester, hexadecanoic acid Acibenzolar or the hexadecanoic acid acid anhydrides of protecting group in liquid phase, remove again the N-terminal blocking group, obtain the target polypeptides Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
2. synthetic method as claimed in claim 1, it is characterized in that, described target polypeptides is by Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor and hexadecyl-N-hydroxy-succinamide ester, hexadecanoic acid Acibenzolar or hexadecanoic acid acid anhydrides and condensation reagent in-situ activation and get, described condensation reagent is selected from DCC, DIC, HBTU or TBTU.
3. synthetic method as claimed in claim 1, it is characterized in that, described N-terminal with protecting group be selected from 2-Cl-carbobenzoxy-(Cbz) (namely claiming the Cl-Z blocking group), carbobenzoxy-(Cbz) (namely claiming the Z blocking group) or fluorenylmethyloxycarbonyl (namely claiming the Fmoc blocking group);
Adopt palladium charcoal or metal catalyst hydrogenation to remove Cl-Z blocking group and Z blocking group;
Adopt the alkaline solution hydrolysis to remove the Fmoc blocking group.
4. synthetic method as claimed in claim 1; it is characterized in that; 20 amino acids are Fmoc-Lys (γ-Glu-OtBu); N-terminal amino acid adopts Cl-Z-His (Trt)-OH; Z-His (Trt)-OH; Alloc-His (Trt)-OH or Fmoc-His (Trt)-OH Acibenzolar method condensation; 31 peptide resins that are protected; use synchronously the deprotection agent agent to take off Side chain protective group and cut peptide, obtain the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor that N-terminal is protected with Cl-Z, Z, Alloc or Fmoc.
5. synthetic method as claimed in claim 4; it is characterized in that, 20 amino acids are Fmoc-Lys (Alloc)-OH, after the Alloc protecting group removes with the hydrogenation of tetraphenylphosphonium palladium; with Boc-γ-Glu-OtBu reaction, 20 amino acids are Fmoc-Lys (Boc-γ-Glu-OtBu).
6. synthetic method as claimed in claim 4; it is characterized in that, 20 amino acids are Fmoc-Lys (Dde)-OH, after the Dde protecting group removes with 2% hydrazine hydrate/DMF; with Boc-γ-Glu-OtBu reaction, 20 amino acids are Fmoc-Lys (Boc-γ-Glu-OtBu).
7. synthetic method as claimed in claim 1; it is characterized in that; 20 amino acids are Fmoc-Lys (Boc)-OH; N-terminal amino acid adopts Cl-Z-His (Trt)-OH, Z-His (Trt)-OH, Alloc-His (Trt)-OH or Fmoc-His (Trt)-OH Acibenzolar method condensation and gets; thereby 31 peptide resins that are protected; take off synchronously Side chain protective group and cut peptide, obtain the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group) that N-terminal is protected with Cl-Z, Z, Alloc or Fmoc.
8. synthetic method as claimed in claim 1 is characterized in that, with Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group) and the N of N-terminal-fluorenylmethyloxycarbonyl (Fmoc blocking group) protection
α-Oxohexadecyl-Glu (OSu)-OBzl (is in Pal-Glu (OSu)-OBzl) liquid phase and carries out the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that acylation reaction is protected; The Fmoc protecting group is removed with alkali, and hydrogenation removes benzyl (Bzl);
Alkaline solution is selected from piperidines or DBU alkaline solution;
Palladium carbon catalyst catalysis is adopted in hydrogenation.
9. synthetic method as claimed in claim 1, it is characterized in that, Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group) and Pal-Glu (OSu)-OBzl that N-terminal-2-benzyloxycarbonylchloride base (Cl-Z blocking group) is protected carry out the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that acylation reaction is protected; Hydrogenation removes Cl-Z protection and benzyl;
Palladium carbon catalyst catalysis is adopted in hydrogenation.
10. synthetic method as claimed in claim 1, it is characterized in that, will carry out the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that acylation reaction is protected in the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group) of N-terminal-carbobenzoxy-(Cbz) (Z blocking group) protection and Pal-Glu (the OSu)-OBzl liquid phase; Hydrogenation removes Z protection and benzyl again;
The catalysis of palladium charcoal catalytic reagent is adopted in hydrogenation.
11. synthetic method as claimed in claim 1, it is characterized in that, will carry out the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that acylation reaction is protected in the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (20 amino acids side chains do not contain γ-Glu group) of N-terminal-carbobenzoxy-(Cbz) (Z blocking group) protection or N-terminal-2-benzyloxycarbonylchloride base (Cl-Z blocking group) protection and Pal-Glu (the OSu)-OtBu liquid phase; Hydrogenation removes Z protection or Cl-Z protection, and the tBu group is removed in hydrolysis under acidic conditions;
Palladium carbon catalyst catalysis is adopted in hydrogenation;
Acidic conditions can be HCl or TFA acidic solution.
12. synthetic method as claimed in claim 1, it is characterized in that, will carry out the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that acylation reaction is protected in the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group) of N-terminal-allyloxycarbonyl protection (Alloc blocking group) and Pal-Glu (the OSu)-OAllyl liquid phase; Hydrogenation removes Alloc protection and OAllyl group;
The catalysis of tetraphenylphosphonium palladium catalytic reagent is adopted in hydrogenation.
13. synthetic method as claimed in claim 1, it is characterized in that, will carry out the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that acylation reaction is protected in the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group) of N-terminal-allyloxycarbonyl (Alloc blocking group) protection and Pal-Glu (the OSu)-OBzl liquid phase; Hydrogenation removes Alloc protection and benzyl;
The catalysis of tetraphenylphosphonium palladium catalytic reagent is adopted in hydrogenation.
14. synthetic method as claimed in claim 1, it is characterized in that, will carry out the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that acylation reaction is protected in the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group) of N-terminal-allyloxycarbonyl (Alloc blocking group) protection and Pal-Glu (the OSu)-OtBu liquid phase; Hydrogenation removes to be hydrolyzed under Alloc protection and the acidic conditions removes the tBu group;
Palladium carbon catalyst catalysis is adopted in hydrogenation;
Acidic conditions is selected from HCl or TFA acidic solution.
15. synthetic method as claimed in claim 1, it is characterized in that, protect Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain does not contain γ-Glu group) and Pal-Glu (the OSu)-OtBu of (Boc blocking group) to carry out the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that acylation reaction is protected N-terminal-tertbutyloxycarbonyl; Boc and tBu group are removed in hydrolysis under the acidic conditions;
Palladium carbon catalyst catalysis is adopted in hydrogenation;
Acidic conditions is selected from HCl or TFA acidic solution.
16. such as 1 described synthetic method, it is characterized in that, the substitution value of described solid phase synthesis resin is 0.1-0.8mmol/g.
17. synthetic method as claimed in claim 1 is characterized in that, setting-up point is room temperature, and optimum condition is 10-30 ℃.
18. synthetic method as claimed in claim 1 is characterized in that, setting-up point is high temperature, and optimum condition is 30-80 ℃.
19. synthetic method as claimed in claim 1 is characterized in that, described solid phase synthesis resin is selected from hydroxy resin, 2-Cl-Trt resin or chloromethyl resin; Preferably, described hydroxy resin is from king's resin or PL king's resin.
20. synthetic method as claimed in claim 1 is characterized in that, described protected amino acid is respectively:
30#：Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Mtr)-OH、Fmoc-Arg(Pmc)-OH；
29#：Fmoc-Gly-OH；
28#：Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Mtr)-OH、Fmoc-Arg(Pmc)-OH；
27#：Fmoc-Val-OH；
26#：Fmoc-Leu-OH；
25#：Fmoc-Trp(Boc)-OH、Fmoc-Trp-OH；
24#：Fmoc-Ala-OH；
23#：Fmoc-Ile-OH；
22#：#Fmoc-Phe-OH；
21#：Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OAll)-OH、Fmoc-Glu(OBzl)-OH；
20#:Fmoc-Lys(Dde)-OH、Fmoc-Lys(Boc-Glu-OtBu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Alloc)-OH；
19#：Fmoc-Ala-OH；
18#：Fmoc-Ala-OH；
17#：Fmoc-Gln(Trt)-OH、Fmoc-Gln(Xan)-OH、Fmoc-Gln-OH；
16#：Fmoc-Gly-OH；
15#：Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OAll)-OH、Fmoc-Glu(OBzl)-OH；
14#：Fmoc-Leu-OH；
13#：Fmoc-Tyr(tBu)-OH；
12#：Fmoc-Ser(tBu)-OH、Fmoc-Ser(Trt)-OH、Fmoc-Ser-OH、Fmoc-Ser(OBzl)-OH；
11#：Fmoc-Ser(tBu)-OH、Fmoc-Ser(Trt)-OH、Fmoc-Ser-OH、Fmoc-Ser(OBzl)-OH；
10#：Fmoc-Val-OH；
9#：Fmoc-Asp(OtBu)-OH、Fmoc-Asp(oall)-OH、Fmoc-Asp(OBzl)-OH；
8#：Fmoc-Ser(tBu)-OH、Fmoc-Ser(Trt)-OH；
7#：Fmoc-Thr(tBu)-OH、Fmoc-Thr(oBzl)-OH；
6#：Fmoc-Phe-OH；
5#：Fmoc-Thr(tBu)-OH、Fmoc-Thr(oBzl)-OH；
4#：Fmoc-Gly-OH；
3#：Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OAll)-OH、Fmoc-Glu(OBzl)-OH；
2#：Fmoc-Ala-OH；
1#：Boc-His(Trt)-OH、Z-His(Trt)-OH、Boc-His(Boc)-OH,Cl-Z-His(Trt)-OH,Alloc-His(Trt)-OH,Fmoc-His(Trt)-OH,Boc-His-OH、Z-His-OH、Boc-His-OH,Cl-Z-His-OH,Alloc-His-OH,and?Fmoc-His-OH。
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