CN101463081B - GLP-1 derivative - Google Patents
GLP-1 derivative Download PDFInfo
- Publication number
- CN101463081B CN101463081B CN2009100451880A CN200910045188A CN101463081B CN 101463081 B CN101463081 B CN 101463081B CN 2009100451880 A CN2009100451880 A CN 2009100451880A CN 200910045188 A CN200910045188 A CN 200910045188A CN 101463081 B CN101463081 B CN 101463081B
- Authority
- CN
- China
- Prior art keywords
- fmoc
- glp
- ala
- verivate
- ser
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- 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 GLP-1 derivant, which is characterized in that the molecular structural formula of the derivant is any one of the following structural formulas: aGLP-1(7-36), namely Seq ID No.2; aGLP-1(7-37), namely Seq ID No.3; or aGLP-1(7-38), namely Seq ID No.4. The invention also discloses a preparation method for the GLP-1 derivant by solid phase chemosynthesis and the application in preparing drugs for curing diabetes.
Description
Technical field
The present invention relates to a kind ofly have human glucagon-like-peptide-1 (Glucagon-likePeptide 1, and GLP-1) active human glucagon-like-peptide-1 verivate (GLP-1 verivate) and preparation thereof and use belong to technical field of bioengineering.
Background technology
Human glucagon-like-peptide-1 (glucagon-like peptide-1; GLP-1) be mainly by a kind of 31 amino acid whose polypeptide hormones of the L emiocytosis of far-end ileum, colon and rectum; Discharge and glucagon suppression through the release of glucose dependency pancreotropic hormone, stimulating growth chalone; Be a kind of especially polypeptide drugs of type ii diabetes of mellitus of treating, have social benefit and huge economic benefit widely.
But GLP-1 is easy in vivo be degraded to by two acyltransferase polypeptide peptase IV (DipeptidylPeptidase 4, DPP IV) and sloughs N end His
7-Ala
8The GLP-1 of the non-activity of-residue (9-37) or GLP-1 (9-36)-NH
2The intravenous injection GLP-1 transformation period in vivo is merely 3～5 minutes, has limited the clinical application of GLP-1.
One of research direction of the mellitus of GLP-1 treatment at present is to change through the structure to GLP-1, obtains the GLP-1 verivate, with these derivatives for treatment mellitus, reaches the purpose of prolong drug EDD in vivo.Existing GLP-1 verivate all demonstrates stability to be increased, and the character that biological activity still can be kept in the body.For example on the 8th, carry out amino acid change as L-Ala Ala being changed into the GLP-1 verivate of glycocoll Gly.The derivative of fatty acid that and for example prepares GLP-1, representative drugs have the NN2211 (trade(brand)name Liraglutide) of Nuo De company of Novartis development, on the Lys26 of GLP-1, carry out acidylate.Moreover, the GLP-1 molecule is carried out polyoxyethylene glycol (PEG) modify, as modifying the GLP-1 molecule with PEG20000.Also have direct CJC-1131, increase the body internal stability that strengthens GLP-1 through molecular weight GLP-1 molecule and albumin macromolecule fusion.
Though there is prolongation the GLP-1 verivate transformation period for preparing through the amino acid that changes on the 8th of the GLP-1, the 20th Lys, 28 Lys and 30 degradation site that Arg is some proteolytic enzyme in vivo still have the possibility that is degraded in vivo.Though through transformation period prolongation in the GLP-1 verivate body of methods such as lipid acid or polyethyleneglycol modified GLP-1 preparation; But the site of lipid acid or polyethyleneglycol modified GLP-1 and degree of modification often are difficult to control; Cause subsequent purification and quality control all to compare difficulty, preparation process is loaded down with trivial details, yield is low, cost is high.Therefore, existing GLP-1 verivate still exists preparation and purification difficult or transformation period still to fall short of or is difficult to defective such as oral administration, presses for novel glp-1-1 product that development is grown biological half-life.
Background technology has proposed a kind of GLP-1 verivate and preparation method thereof; See that application number and denomination of invention that the applicant applies for are respectively " 200610024355.X " and " a kind of human glucagon-like-peptide-1 verivate and preparation and application ", and application number and denomination of invention are respectively the patent of invention of " 200610029646.8 " and " a kind of human glucagon-like-peptide-1 verivate and solid state chemistry thereof are synthetic ".Background technology prepares the GLP-1 verivate of gained, and output is high, and purifying process is simplified, and production cost is lower, and this verivate long half time is in the transformation period of GLP-1.But the verivate of background technology preparation also has following shortcoming: for example, the 2nd amino acids is still lost activity by two acyltransferase polypeptide peptase IV degraded in vivo easily.The 20th Lys, 28 Lys and 30 degradation site that Arg is some proteolytic enzyme in vivo still have the possibility that is degraded in vivo.
Therefore, still need develop GLP-1 verivate, be used for treating diabetes better with longer action time.
Summary of the invention
GLP-1 has two kinds of forms in vivo, and a kind of is GLP-1 (7-36)-NH2, is made up of 30 amino-acid residues, and another kind is GLP-1 (7-37), is made up of 31 amino-acid residues, and the two has identical BA.The GLP-1 that the present invention relates to refers to GLP-1 (7-37), and its sequence (Seq ID No.1) is enumerated as follows: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly.
Because structure and the function of GLP-1 are fully aware of at present: the N end is to keep active institute essential, and the C end responsible with the combining of acceptor.GLP-1 (7-34), GLP-1 (7-35), GLP-1 (7-36), GLP-1 (7-37) have proved have hypoglycemic activity, this shows that the C end of GLP-1 has plasticity-to BA.
Relation according to the 26S Proteasome Structure and Function of GLP-1 peptide C end function " plasticity-"; With biologically stable and prolong half-life in the body that increases GLP-1 is design philosophy; We have carried out number of research projects; Adopt new way that the aminoacid sequence of GLP-1 is designed and transforms, prepare long lasting novel glp-1-1 verivate.
First purpose of the present invention provides a kind of novel glp-1-1 verivate aGLP-1; It is characterized in that; The molecular structural formula of this verivate is following three kinds of structural formula: aGLP-1 (7-36), i.e. Seq ID No.2, aGLP-1 (7-37); Be Seq ID No.3 and aGLP-1 (7-38), i.e. one of Seq ID No.4.
Wherein, aGLP-1 (7-36), promptly Seq ID No.2 sequence is following:
His-Xaa2-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Xaa20-Glu-Phe-Ile-Ala-Trp-Leu-Val-Xaa28-Gly-Xaa30。
AGLP-1 (7-37), promptly Seq ID No.3 sequence is following:
His-Xaa2-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Xaa20-Glu-Phe-Ile-Ala-Trp-Leu-Val-Xaa28-Gly-Xaa30-Xaa31。
AGLP-1 (7-38), promptly Seq ID No.4 sequence is following:
His-Xaa2-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Xaa20-Glu-Phe-Ile-Ala-Trp-Leu-Val-Xaa28-Gly-Xaa30-Xaa31-Xaa32。
Wherein Xaa2 is Ser, any one amino acid among His and the D-Ala, and Xaa20 is Ser; His, any one amino acid among Gln and the Ala, Xaa28 is Ser; His, any one amino acid among Asp and the Ala, Xaa30 are any one amino acid among Gly and the Cys; And when Xaa30 was C-terminal amino acid, the C-terminal of Gly and Cys can be carboxyl-OH or acid amides-NH2, and Xaa31 is any one amino acid among Gly and the Cys; And when Xaa31 was C-terminal amino acid, the C-terminal of Gly and Cys can be carboxyl-OH or acid amides-NH2, and Xaa32 is any one amino acid among Gly and the Cys; And when Xaa32 was C-terminal amino acid, the C-terminal of Gly and Cys can be carboxyl-OH or acid amides-NH2.This verivate has not only kept natural GLP-1 activity in vivo; Also obviously prolonged EDD in the body, EDD is sustainable at least 7 hours in the body, as gives this verivate of higher concentration; EDD will be longer in the body, have good clinical use value.
In addition, this verivate can be through the method or the preparation of solid state chemistry compound method of recombinant DNA technology, technology maturation, and cost is low, and suitable popularizing used.
Second purpose of the present invention provides the method for the synthetic said derivative of solid state chemistry.
It is very ripe that the solid state chemistry synthesis method is less than 40 amino acid whose little peptide technologies in preparation, has quick, easy, the low cost and other advantages of purifying.For realizing above-mentioned purpose, technical scheme of the present invention selects for use the solid state chemistry synthesis method to prepare GLP-1 verivate aGLP-1.
Specify technical scheme of the present invention at present.
A kind of solid state chemistry compound method of GLP-1 verivate is characterized in that, the concrete operations step:
(1) synthetic polypeptide resin
Earlier resin is inserted conventional Peptide synthesizer; To press the aminoacid sequence of said GLP-1 verivate again with the amino acid monomer of protection base; Be arranged in the said conventional synthesizer to the N-end from the C-end, under 25 ℃, take off Fmoc protection, activation, connection; Circulation repeatedly then synthetic obtains the polypeptide resin with Side chain protective group;
(2) deprotection base and cut-out resin
After the polypeptide resin of above-mentioned band Side chain protective group carried out scission reaction, through filtering, washing of precipitate, drying obtains GLP-1 verivate bullion;
(3) HPLC separation and purification, lyophilize
Above-mentioned bullion is carried out separation and purification with preparation HPLC,, obtain the GLP-1 verivate again through lyophilize;
Wherein, the amino acid monomer of said band protection base comprises: Fmoc-L-Ala-OH, Fmoc-D-Ala-OH, Fmoc-L-His (Trt)-OH, Fmoc-L-Val-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Tyr (tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Leu-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Gln (Trt)-OH, Fmoc-L-Phe-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Ile-OH, Fmoc-L-Asp (OtBu)-OH, Fmoc-L-Trp-OH, Fmoc-L-Cys (Trt)-OH;
Wherein, when the peptide C end is carboxyl, select Wang resin as said solid phase carrier resin; When the peptide C end is acid amides, select conventional aminoresin as said solid phase carrier resin.
When described GLP-1 verivate when only comprising the polypeptide by genetic code amino acids coding residue, also can prepare verivate through the DNA recombinant technology.For example, press the aminoacid sequence synthetic gene fragment of GLP-1 verivate; Mode to be suitable for expressing independent protein or fusion rotein is put into expression vector with encoding sequence; Expression vector with the GLP-1 verivate gene order that contains GLP-1 verivate or fusion transforms suitable procaryotic host cell, obtains engineering strain; Engineering strain through liquid fermenting, is contained GLP-1 verivate fusion rotein or contains the proteic wet thallus of GLP-1 verivate separately through centrifugal acquisition; With the wet thallus broken wall, obtain to contain GLP-1 verivate fusion rotein or contain the proteic bullion of GLP-1 verivate separately through separating; Purified, lyophilize makes product G LP-1 verivate.
The 3rd purpose of the present invention is the application that proposes the GLP-1 verivate, and this verivate is made the activeconstituents of this medicine in the medicine of preparation treatment mellitus.
The invention has the advantages that: the GLP-1 verivate that the present invention proposes has the transformation period of being longer than GLP-1; Working method is easy, and cost is lower; Be suitable for treating the activeconstituents of diabetes medicament.
Embodiment
Below in conjunction with embodiment, technical scheme of the present invention is detailed further.The amino acid monomer of used band protection base and other chemical reagent etc. in specification sheets and following examples; All can buy and obtain from associated companies; The experimental technique of unreceipted actual conditions can be undertaken by normal condition, or is undertaken by the condition that goods supplier is advised.All embodiment all operate according to the step of the compound method in summary of the invention regulation, and all embodiment are only enumerated and the relevant committed step of product separately.
Embodiment 1
The solid state chemistry synthesis method is synthesized GLP-1 verivate of the present invention, and the molecular structural formula of this verivate is: aGLP-1 (7-36), i.e. Seq ID No.2; Xaa2=D-Ala wherein, Xaa20=Ser, Xaa28=Ala; Xaa30=Cys, and the C-terminal of Cys is acid amides-NH2, operation steps:
1, the amino acid monomer of the band of usefulness protection base has 16; They are: Fmoc-L-Ala-OH, Fmoc-D-Ala-OH, Fmoc-L-His (Trt)-OH, Fmoc-L-Val-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Tyr (tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Leu-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Gln (Trt)-OH, Fmoc-L-Phe-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Ile-OH, Fmoc-L-Asp (OtBu)-OH, Fmoc-L-Trp-OH, Fmoc-L-Cys (Trt)-OH, and wherein abbreviation expression:
The Fmoc:9-fluorenylmethyloxycarbonyl
Trt: trityl, i.e. trityl
OtBu: tertiary butyl ester
TBU: the tertiary butyl, i.e. tert-butyl;
2, need the plant and instrument and the reagent of usefulness
Instrument: SYMPHONY type 12 passage Peptide synthesizers, model: SYMPHONY, U.S.'s product;
Reagent: N-Methyl pyrrolidone, methylene dichloride, hexahydropyridine, methyl alcohol; Dimethylamino pyridine, i.e. Dimethylaminopyridine/DMF N, N-diisopropylethylamine, i.e. N; N-diisopropylethylamine/NMP, HBTU 100mmol/0.5M HOBT inDMF N, N-NSC 57182; Be N, N-Dicyclohexylcarbodiimide/NMP
Wherein: DMF is N, dinethylformamide
NMP is a N-Methyl pyrrolidone
HOBT is an I-hydroxybenzotriazole
HBTU is 2-(1 hydrogen benzotriazole base)-1,1,3,3-tetramethyl-urea hexafluorophosphate, i.e. 2-(1H-benzotriazole-y1-1,3,3-tetramethyl-Uroniumhexafluorophosphate;
3, operation
Synthesizing of the first step polypeptide resin
When the peptide C end is carboxyl, select Wang resin, when the peptide C end is acid amides, select conventional aminoresin, select conventional aminoresin in the present embodiment.
With the 0.25mmol scale is example, takes by weighing conventional aminoresin 0.25g, inserts in the reactor drum on the SYMPHONY type 12 passage Peptide synthesizers; The amino acid monomer of band protection base is taken by weighing the 1mmol bottling, press the aminoacid sequence of GLP-1 verivate, i.e. Seq IDNo.2; Be arranged in the described synthesizer to the N-end from C-end, under 25 ℃, take off Fmoc protection, activation, connection automatically by computer program control; And then carry out next round circulation; So accomplish and synthesize, obtain polypeptide resin, on described synthesizer, dry up, weigh with Side chain protective group;
Wherein, amino acid monomer putting in order as follows to what N-held from the C-end:
Fmoc-L-Cys (Trt)-OH; Fmoc-L-Gly-OH; Fmoc-L-Ala-OH; Fmoc-L-Val-OH; Fmoc-L-Leu-OH; Fmoc-L-Trp-OH; Fmoc-L-Ala-OH; Fmoc-L-Ile-OH; Fmoc-L-Phe-OH; Fmoc-L-Glu (OtBu)-OH; Fmoc-L-Ser (tBu)-OH; Fmoc-L-Ala-OH; Fmoc-L-Ala-OH; Fmoc-L-Gln (Trt)-OH; Fmoc-L-Gly-OH; Fmoc-L-Glu (OtBu)-OH; Fmoc-L-Leu-OH; Fmoc-L-Tyr (tBu)-OH; Fmoc-L-Ser (tBu)-OH; Fmoc-L-Ser (tBu)-OH; Fmoc-L-Val-OH; Fmoc-L-Asp (OtBu)-OH; Fmoc-L-Ser (tBu)-OH; Fmoc-L-Thr (tBu)-OH; Fmoc-L-Phe-OH; Fmoc-L-Thr (tBu)-OH; Fmoc-L-Gly-OH; Fmoc-L-Glu (OtBu)-OH; Fmoc-D-Ala-OH and Fmoc-L-His (Trt)-OH
The second step deprotection base and cut-out resin
The polypeptide resin of the band Side chain protective group that the first step is obtained places tool plug Erlenmeyer flask, adds following lytic reagent:
, under 30 ℃, induction stirring reaction 2 hours is filtered then, collection filtrating, and resin washs with trifluoroacetic acid, merges to collect liquid and washings, adds ether generation deposition, filters, and deposition is with the ether washing, and drying gets bullion;
The HPLC separation and purification of the 3rd step, lyophilize
The bullion that second step was obtained carries out separation and purification with preparation HPLC, again through lyophilize, gets product G LP-1 verivate.
The product G LP-1 verivate that makes has the aminoacid sequence of the GLP-1 verivate of above-mentioned proposition.
Embodiment 2 solid state chemistry synthesis methods are synthesized GLP-1 verivate of the present invention, and the molecular structural formula of this verivate is aGLP-1 (7-37), i.e. Seq ID No.3; Xaa2=D-Ala wherein, Xaa20=Ser, Xaa28=Ala; Xaa30=Gly; Xaa31=Cys, and the C-terminal of Cys is carboxyl-OH, selects Wang resin in the present embodiment.
In the first step of operation steps, the amino acid monomer of band protection base is taken by weighing the 1mmol bottling, press the aminoacid sequence of GLP-1 verivate, i.e. Seq ID No.3, hold from C-to be arranged in the SYMPHONY type 12 passage Peptide synthesizers to N one end:
Fmoc-L-Cys (Trt)-OH; Fmoc-L-Gly-OH; Fmoc-L-Gly-OH; Fmoc-L-Ala-OH; Fmoc-L-Val-OH; Fmoc-L-Leu-OH; Fmoc-L-Trp-OH; Fmoc-L-Ala-OH; Fmoc-L-Ile-OH; Fmoc-L-Phe-OH; Fmoc-L-Glu (OtBu)-OH; Fmoc-L-Ser (tBu)-OH; Fmoc-L-Ala-OH; Fmoc-L-Ala-OH; Fmoc-L-Gln (Trt)-OH; Fmoc-L-Gly-OH; Fmoc-L-Glu (OtBu)-OH; Fmoc-L-Leu-OH; Fmoc-L-Tyr (tBu)-OH; Fmoc-L-Ser (tBu)-OH; Fmoc-L-Ser (tBu)-OH; Fmoc-L-Val-OH; Fmoc-L-Asp (OtBu)-OH; Fmoc-L-Ser (tBu)-OH; Fmoc-L-Thr (tBu)-OH; Fmoc-L-Phe-OH; Fmoc-L-Thr (tBu)-OH; Fmoc-L-Gly-OH; Fmoc-L-Glu (OtBu)-OH; Fmoc-D-Ala-OH and Fmoc-L-His (Trt)-OH
Other operation stepss and experiment condition are identical with embodiment 1.
Embodiment 3 solid state chemistry synthesis methods prepare GLP-1 verivate of the present invention, and the molecular structural formula of this verivate is aGLP-1 (7-38), i.e. Seq ID No.4; Xaa2=Ser wherein, Xaa20=Gln, Xaa28=Asp; Xaa30=Gly, Xaa31=Gly, Xaa32=Cys; And the C-terminal of Cys is carboxyl-OH, selects Wang resin in the present embodiment.
In the first step of operation steps, the amino acid monomer of band protection base is taken by weighing the 1mmol bottling, press the aminoacid sequence of GLP-1 verivate, i.e. Seq ID No.4, hold to N-from the C-end to be arranged in the SYMPHONY type 12 passage Peptide synthesizers:
Fmoc-L-Cys (Trt)-OH; Fmoc-L-Gly-OH; Fmoc-L-Gly-OH; Fmoc-L-Gly-OH; Fmoc-L-Asp (OtBu)-OH; Fmoc-L-Val-OH; Fmoc-L-Leu-OH; Fmoc-L-Trp-OH; Fmoc-L-Ala-OH; Fmoc-L-Ile-OH; Fmoc-L-Phe-OH; Fmoc-L-Glu (OtBu)-OH; Fmoc-L-Gln (Trt)-OH; Fmoc-L-Ala-OH; Fmoc-L-Ala-OH; Fmoc-L-Gln (Trt)-OH; Fmoc-L-Gly-OH; Fmoc-L-Glu (OtBu)-OH; Fmoc-L-Leu-OH; Fmoc-L-Tyr (tBu)-OH; Fmoc-L-Ser (tBu)-OH; Fmoc-L-Ser (tBu)-OH; Fmoc-L-Val-OH; Fmoc-L-Asp (OtBu)-OH; Fmoc-L-Ser (tBu)-OH; Fmoc-L-Thr (tBu)-OH; Fmoc-L-Phe-OH; Fmoc-L-Thr (tBu)-OH; Fmoc-L-Gly-OH; Fmoc-L-Glu (OtBu)-OH; Fmoc-L-Ser (tBu)-OH and Fmoc-L-His (Trt)-OH
Other operation stepss and experiment condition are identical with embodiment 1.
The hypoglycemic activity of embodiment 4GLP-1 verivate.
Experiment material and method:
Male and healthy kunming mice (cleaning level, Fudan University in Shanghai medical college animal center provides);
50% glucose solution;
0.9%NaCl solution;
GLP-1；
AGLP-1 has the structure of the said GLP-1 verivate of embodiment 1-3;
Blood glucose monitoring system (the newly upright medicine equipment ltd in Shanghai produces).
Male and healthy kunming mice overnight fasting is divided into 5 groups (n=8).1, the glucose control group; 2, GLP-1 administration control group; 3～5, aGLP-1 administration group, concrete sequential structure is the structure described in the embodiment 1-3.The glucose solution of GLP-1 administration control group 2 abdominal injections 100 μ L18mmol/kg and the GLP-1 of 6nmol/kg; AGLP-1 administration group 3～5; The glucose solution of every group of difference abdominal injection 100 μ L 18mmol/kg and the aGLP-1 of 6nmol/kg, note was at this moment zero moment.30min replenishes 200 μ L, 50% glucose solution before surveying blood sugar, and mouse tail vein measuring blood sugar of blood extracting behind the per injection 30min is with check aGLP-1 hypoglycemic activity.The glucose control group is only injected 50% glucose solution, does not give GLP-1 and aGLP-1, by identical time interval determination blood sugar.
The result is as shown in table 1, shown in numerical value be the average of n=8.Compare with the glucose group mouse, GLP-1 and aGLP-1 group can both reduce mouse blood sugar after administration, but the lasting hypoglycemic time of GLP-1 can only keep about 100 minutes.And aGLP-1 group lasting hypoglycemic time after administration was 440 minutes, and the transformation period is than GLP-1 significant prolongation in the display body.As giving the aGLP-1 of greater concn, EDD will be longer in the body, show that aGLP-1 has good clinical use value.
The hypoglycemic activity of table 1aGLP-1
The aminoacid sequence of the GLP-1 verivate that the present invention relates to is in respect of following 4.Be shown in following sequence table at present in detail.
Sequence table
< 110>East China Normal University
< 120>a kind of GLP-1 verivate
<160>4
<210>1
<211>31
<212>PRT
< 213>people
<400>1
His?Ala?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu
5 10 15
Gly?Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg
20 25 30
Gly
<210>2
<211>30
<212>PRT
< 213>artificial sequence
<220>
< 221>synthetic construct
<222>(2)
< 223>Xaa=Ser, His or D-Ala
<220>
< 221>synthetic construct
<222>(20)
< 223>Xaa=Ser, His, Gln or Ala
<220>
< 221>synthetic construct
<222>(28)
< 223>Xaa=Ser, His, Asp or Ala
<220>
< 221>synthetic construct
<222>(30)
< 223>Xaa=Gly or Cys
<400>2
His?Xaa?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu
5 10 15
Gly?Gln?Ala?Ala?Xaa?Glu?Phe?Ile?Ala?Trp?Leu?Val?Xaa?Gly?Xaa
20 25 30
<210>3
<211>31
<212>PRT
< 213>artificial sequence
<220>
< 221>synthetic construct
<222>(2)
< 223>Xaa=Ser, His or D-Ala
<220>
< 221>synthetic construct
<222>(20)
< 223>Xaa=Ser, His, Gln or Ala
<220>
< 221>synthetic construct
<222>(28)
< 223>Xaa=Ser, His, Asp or Ala
<220>
< 221>synthetic construct
<222>(30)
< 223>Xaa=Gly or Cys
<220>
< 221>synthetic construct
<222>(31)
< 223>Xaa=Gly or Cys
<400>3
His?Xaa?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu
5 10 15
Gly?Gln?Ala?Ala?Xaa?Glu?Phe?Ile?Ala?Trp?Leu?Val?Xaa?Gly?Xaa
20 25 30
Xaa
<210>4
<211>32
<212>PRT
< 213>artificial sequence
<220>
< 221>synthetic construct
<222>(2)
< 223>Xaa=Ser, His or D-Ala
<220>
< 221>synthetic construct
<222>(20)
< 223>Xaa=Ser, His, Gln or Ala
<220>
< 221>synthetic construct
<222>(28)
< 223>Xaa=Ser, His, Asp or Ala
<220>
< 221>synthetic construct
<222>(30)
< 223>Xaa=Gly or Cys
<220>
< 221>synthetic construct
<222>(31)
< 223>Xaa=Gly or Cys
<220>
< 221>synthetic construct
<222>(32)
< 223>Xaa=Gly or Cys
<400>4
His?Xaa?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu
5 10 15
Gly?Gln?Ala?Ala?Xaa?Glu?Phe?Ile?Ala?Trp?Leu?Val?Xaa?Gly?Xaa
20 25 30
Xaa?Xaa
Claims (3)
1. a GLP-1 verivate is characterized in that, the molecular structural formula of this verivate is following:
AGLP-1 (7-36), i.e. Seq ID No.2,
Wherein, said aGLP-1 (7-36) is that Xaa2 is D-Ala among the Seq ID No.2, and Xaa20 is Ser, and Xaa28 is Ala, and Xaa30 is Cys, and the C-terminal of Cys is acid amides-NH
2
2. the solid state chemistry synthesis preparation method of the described GLP-1 verivate of claim 1; Earlier resin is inserted Peptide synthesizer; To be arranged in the said Peptide synthesizer to the N-end from the C-end with the aminoacid sequence of the amino acid monomer of protecting base again, obtain polypeptide resin through synthesizing according to the said GLP-1 verivate of claim 1; Through deprotection base, cut-out resin, HPLC purifying, lyophilize, obtain said GLP-1 verivate again;
Wherein, When the amino acid monomer of said band protection base is followed successively by Fmoc-L-Cys (Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Ala-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH, Fmoc-L-Phe-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L-Gln (Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr (tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp (OtBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-D-Ala-OH and Fmoc-L-His (Trt)-OH; Gained GLP-1 verivate aminoacid sequence is Seq ID No.2; In this sequence; Xaa2=D-Ala; Xaa20=Ser; Xaa28=Ala; Xaa30=Cys, and the C-terminal of Cys is acid amides-NH
2
Wherein, when GLP-1 verivate C-terminal is acid amides, select conventional aminoresin as said solid phase carrier resin.
3. the described GLP-1 verivate of claim 1 is in the purposes of preparation in the hypoglycemic drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100451880A CN101463081B (en) | 2009-01-12 | 2009-01-12 | GLP-1 derivative |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100451880A CN101463081B (en) | 2009-01-12 | 2009-01-12 | GLP-1 derivative |
Related Child Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012100131244A Division CN102558340B (en) | 2009-01-12 | 2009-01-12 | Glucagon-like peptide (GLP)-1 derivative |
| CN201210012881XA Division CN102558339B (en) | 2009-01-12 | 2009-01-12 | GLP-1(Glucagon-like Peptide 1) derivative |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101463081A CN101463081A (en) | 2009-06-24 |
| CN101463081B true CN101463081B (en) | 2012-07-04 |
Family
ID=40803899
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009100451880A Expired - Fee Related CN101463081B (en) | 2009-01-12 | 2009-01-12 | GLP-1 derivative |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101463081B (en) |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK2513140T3 (en) * | 2009-12-16 | 2016-01-18 | Novo Nordisk As | Double-acylated GLP-1 derivatives |
| KR20130093470A (en) * | 2010-04-30 | 2013-08-22 | 가부시키가이샤산와카가쿠켄큐쇼 | Peptide for improving in vivo stability of physiologically active substance or the like and physiologically active substance with improved in vivo stability |
| DK3326620T3 (en) | 2010-12-16 | 2020-05-25 | Novo Nordisk As | SOLID COMPOSITIONS CONTAINING A GLP-1 AGONIST AND SALT OF N- (8- (2- HYDROXYBENZOYL) AMINO) CAPRYLIC ACID |
| MX355361B (en) | 2011-04-12 | 2018-04-17 | Novo Nordisk As | Double-acylated glp-1 derivatives. |
| CN102180963B (en) * | 2011-04-22 | 2014-06-25 | 中国药科大学 | Glucagons like peptide-1 (GLP-1) analog and application thereof |
| AU2013234496B2 (en) | 2012-03-22 | 2017-07-27 | Novo Nordisk A/S | Compositions of GLP-1 peptides and preparation thereof |
| HUE042757T2 (en) | 2012-03-22 | 2019-07-29 | Novo Nordisk As | Compositions comprising a delivery agent and preparation thereof |
| ES2871328T3 (en) | 2012-06-20 | 2021-10-28 | Novo Nordisk As | Tablet formulation comprising a peptide and a delivery agent |
| DK2976325T3 (en) | 2013-03-21 | 2017-06-06 | Sanofi Aventis Deutschland | SYNTHESIS OF PEPTIDE PRODUCTS CONTAINING CYCLIC IMID |
| HUE034308T2 (en) | 2013-03-21 | 2018-02-28 | Sanofi Aventis Deutschland | Synthesis of hydantoin containing peptide products |
| CN104098689A (en) * | 2013-04-12 | 2014-10-15 | 华东师范大学 | GLP-1 derivative DLG3312 and solid-phase chemical synthesis method thereof |
| CN103333238A (en) * | 2013-06-18 | 2013-10-02 | 华东师范大学 | mPEG-MAL-aGLP-1 composite |
| CN104587453B (en) * | 2014-12-24 | 2017-01-18 | 华东师范大学 | GLP-1 derivative particle compound as well as preparation method and application thereof |
| IL275778B2 (en) | 2018-02-02 | 2023-12-01 | Novo Nordisk As | Solid compositions comprising a glp-1 agonist, a salt of n-(8-(2-hydroxybenzoyl)amino)caprylic acid and a lubricant |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0955314B1 (en) * | 1998-04-10 | 2003-12-03 | Les Laboratoires Servier | Peptide analogues of glucagon-like-peptide-1 (7-37), their preparation and pharmaceutical compositions |
| CN1703424A (en) * | 2002-10-11 | 2005-11-30 | 株式会社三和化学研究所 | GLP-1 derivatives and transmicosal absorption preparations thereof |
| CN1816622A (en) * | 2003-03-28 | 2006-08-09 | 独立行政法人农业生物资源研究所 | Process for producing plant storage organ with high production of recombinant protein and novel recombinant protein |
| CN1982336A (en) * | 2006-03-03 | 2007-06-20 | 华东师范大学 | Human pancreas glucagon sample peptide-1-derivative, its production and use |
-
2009
- 2009-01-12 CN CN2009100451880A patent/CN101463081B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0955314B1 (en) * | 1998-04-10 | 2003-12-03 | Les Laboratoires Servier | Peptide analogues of glucagon-like-peptide-1 (7-37), their preparation and pharmaceutical compositions |
| CN1703424A (en) * | 2002-10-11 | 2005-11-30 | 株式会社三和化学研究所 | GLP-1 derivatives and transmicosal absorption preparations thereof |
| CN1816622A (en) * | 2003-03-28 | 2006-08-09 | 独立行政法人农业生物资源研究所 | Process for producing plant storage organ with high production of recombinant protein and novel recombinant protein |
| CN1982336A (en) * | 2006-03-03 | 2007-06-20 | 华东师范大学 | Human pancreas glucagon sample peptide-1-derivative, its production and use |
Non-Patent Citations (1)
| Title |
|---|
| 李宗杰.促胰岛素释放肽和GLP-1类似物的研究进展.《生命科学研究》.2006, * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101463081A (en) | 2009-06-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101463081B (en) | GLP-1 derivative | |
| KR101087859B1 (en) | Insulinotropic peptide synthesis | |
| CN101993485B (en) | Peptide analog homologous dimer capable of accelerating insulin secretion and application thereof | |
| CN102532301A (en) | Novel Exendin-4 analogues and preparation method thereof | |
| KR101297942B1 (en) | Synthesis of glucagon-like peptide | |
| CN111410686B (en) | Molecular modification of GLP-1R activator and application of dimer thereof in treating metabolic diseases | |
| AU2012237899B2 (en) | Site-directed mono-substituted PEGylated Exendin analog and preparation method therefor | |
| CN101463078B (en) | Exendin-4 derivative and solid phase chemical synthesis thereof | |
| CN103224558B (en) | A kind of preparation method of Exenatide | |
| CN106029088A (en) | Stable glucagon analogues and use for treatment of hypoglycaemia | |
| CN101747426B (en) | Method for synthesizing pramlintide | |
| CN110317258A (en) | A kind of novel polypeptide segment of Suo Malu peptide and preparation method thereof | |
| CN113135991B (en) | Method for preparing somaglutide | |
| CN111944039A (en) | Synthetic method of somaglutide | |
| CN106866788B (en) | Preparation of octreotide acetate and octreotide acetate injection pharmaceutical composition | |
| CN109432394B (en) | Octreotide acetate injection pharmaceutical composition and application thereof | |
| CN1904150B (en) | Human glucagon peptide/derivative and its solid phase chemical synthesis | |
| CN112679602A (en) | Solid-phase synthesis method of Somaloutide | |
| CN106860854B (en) | Octreotide acetate injection pharmaceutical composition and octreotide acetate | |
| CN108070030B (en) | Preparation method of loxenapeptide and analogue thereof | |
| CN102558340B (en) | Glucagon-like peptide (GLP)-1 derivative | |
| CN109248323A (en) | Acylated GLP-1 derivative | |
| CN106432468A (en) | Solid-phase synthesis method for preparing exenatide | |
| CN102558339B (en) | GLP-1(Glucagon-like Peptide 1) derivative | |
| CN104428314A (en) | Solid phase peptide synthesis of insulin using side chain anchored lysine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120704 Termination date: 20220112 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |