CN101255191A

CN101255191A - Micro-wave promoted solid-phase synthesis of glucagons-like peptide-1(GLP-1) analogue and uses thereof

Info

Publication number: CN101255191A
Application number: CNA2008100196825A
Authority: CN
Inventors: 黄文龙; 张惠斌; 迟玉石; 周金培; 周映红; 倪帅键; 钱海
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2008-03-12
Filing date: 2008-03-12
Publication date: 2008-09-03

Abstract

The invention relates to new type GLP-1 analogues and a microwave promotive solid phase synthesis process of the same. GLP-1 analogues with longer pharmacological action time can be obtained by modifying 8, 9, 16, 22, 27 or 37 sites of natural GLP-1, the chemical synthesis is highly effectively and rapidly realized by microwave promotive solid phase synthesis process, the crude product is purified by highly effective liquid phase, and GLP-1 analogues is obtained after lyophilized.

Description

Microwave promotes solid phase synthesis glucagon-like-peptide-1 (GLP-1) analogue and application thereof

Technical field

The present invention relates to glucagon-like-peptide-1 (GLP-1) analogue and microwave thereof and promote solid phase synthesis process.

Background technology

Diabetes are Chronic Non-Communicable Diseasess of the third-largest serious threat human health after tumour, cardiovascular disorder.At present, the whole world has 200,000,000 diabetic subjects approximately, expects 2025 and will increase to 300,000,000.2007, in state-owned 3,000 ten thousand diabetic subjects, expect the year two thousand thirty, China diabetic subject number will break through 5,000 ten thousand, China become diabetes second big country that is only second to India, wherein diabetes B accounts for 90% of diabetic subject's total number of persons.Now the effective means of treatment diabetes B is an insulin injection.Adopt the method for insulin strengthening treatment to delay the diabetes process clinically, insulinize can reverse the pancreatic functional lesion to a certain extent in lowering blood glucose.But be to use Regular Insulin hypoglycemic danger can occur.Be subjected to after dosage size, injection site, injecting pathway, the injection of individual diversity XOR the influence of factor such as not feed,, serious hypoglycemia side effect will occur if use Regular Insulin is careless slightly.

Glucagon-like-peptide-1 (GLP-1) is a kind of glucose dependency intestines blood sugar lowing polypeptide hormone, GLP-1 stimulates insulin secretion and hypoglycemia do not occur, the dependent insulin secretion accelerating characteristic of this glucose, avoided the danger of the normal generation hypoglycemia that exists in the treating diabetes, these physiological functions make exploitation GLP-1 have broad prospects as a kind of diabetes B medicine.

Glucagon-like-peptide-1 (GLP-1) is main by the secreted glucose dependency intestines blood sugar lowing polypeptide hormone of the L cell of terminal jejunum, ileum and colon, and multiple existence form is arranged in vivo.It is long-armed that the Proglucagon gene is positioned at No. 2 karyomit(e)s, form by 6 exons and 5 introns, and at pancreas and enteron aisle L cell inner expression, the Proglucagon that generation is made up of 160 amino acid (proglucagon, PG).The product that Proglucagon transforms after the cracking in pancreas and enteron aisle is different.PG mainly is cracked in enteron aisle: enteroglucagon (Glicentin:PG1～69), enteroglucagon molecule continue to be cracked into GRPP (PG1～30) and oxyntomodulin (Oxyntomodulin:PG33～69); Insert peptide-2 (IP-2:PG111～123); Glucagon-like-peptide-2 (GLP-2:PG126～158); And GLP-1 (1～37)-OH (PG 72～108).GLP-1 (1～37)-OH is the peptide chain of non-activity, need enzymolysis excision N to hold 6 peptides, become GLP-1 (7～37)-OH with physiologically active, its C-terminal glycine can be used as the substrate of amidating enzyme, so promptly generates GLP-1 (7～the 36)-NH with high activity after the C-terminal amidation of GLP-1 (7～37)-OH ₂, aminoacid sequence is HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH ₂The aminoacid sequence of GLP-1 (7～37)-OH is HAEGTFTSDVSSLEGQAAKEFIAWLVKGRG-COOH.GLP-1 (7～36)-NH ₂Be the main natural form of GLP-1 in the human body, account for 80%, it promotes that acting in the GLP-1 peptide of insulin secretion is the strongest.GLP-1 (7～37)-OH accounts for 20% in addition, and both have identical physiological function.

GLP-1 promotes secretion of insulin by acting on the acceptor GLP-1 acceptor (GLP-1R) on the pancreatic film.GLP-1R highly expresses on pancreas beta cell film, is made up of 463 amino acid, belongs to the G-protein linked receptor family of striding film for seven times, combines with the GLP-1 high degree of specificity.Can increase the activity of islet cells adenylate cyclase after GLP-1 and its receptors bind, the increase of the second messenger cAMP in the irritation cell causes cytolemma K ⁺Pathway closure, cell depolarization brings out the Ca of voltage-dependent ²⁺Channel opener, extracellular Ca ²⁺Interior stream, cytoplasm Ca ²⁺Concentration raises and triggers the release of Regular Insulin.The cAMP level raises in addition, activates protein kinase A and Starch phosphorylase that cAMP relies on again, and then stimulates transcribing and translating of beta cell insulin gene, stimulates the increment and the differentiation of beta cell.

Glucagon-like-peptide-1 (GLP-1) has the various biological effect.As follows:

1, has the dependent incretin secretion of blood sugar;

2, stop the pancreas beta cell to be degenerated, stimulate the increment and the differentiation of beta cell;

3, induce the proinsulin gene transcription, promote the biosynthesizing of proinsulin;

4, increase the susceptibility of Regular Insulin;

5, increase Somatostatin secretion, the generation of glucagon suppression (this effect also is the blood sugar dependency);

6, gastric acid inhibitory secretion postpones stomach emptying;

7, by acting on the maincenter depress appetite of hypothalamus, reduce effects such as food intake.

Yet though natural GLP-1 has above plurality of advantages on the treatment diabetes, it in vivo can be by DPP IV (dipeptidyl peptidase IV, DPPIV) degraded fast ^[6]But terminal second the L-Ala Ala residue of DPP IV specific recognition GLP-1N excises dipeptides from the 2nd L-Ala of peptide chain N-terminal (Ala), makes its form that changes non-activity into, and the transformation period is only about 5 minutes in its body.GLP-1 molecule N end is and the combining site of GLP-1 acceptor that its histidine residues forfeiture causes GLP-1 to lose biological activity fully.So this needs us that natural GLP-1 is transformed, search out with expectation and can resist DPP-IV, the GLP-1 analogue that biological half-life is long.

Polypeptide can obtain by the method for engineered method or chemosynthesis.Engineered method has superiority than chemical process obtaining on long peptide (amino-acid residue length is greater than 50) or the protein, but the chemical synthesis process of polypeptide is especially after the solid phase synthesis strategy occurs, handiness, diversity and the high efficiency etc. that have gene engineering method to hardly match on less than 40 polypeptide or little peptide in preparation amino-acid residue length.Merrifield in 1963 found and have developed the method for solid-phase synthetic peptide.Solid-phase polypeptide is synthetic generally two kinds of strategies: i.e. Boc/Bzl orthogonally protect solid phase synthesis strategy and Fmoc/tBu orthogonally protect solid phase synthesis strategy, the solid-phase synthesis that Merrifield foundes is Boc/Bzl orthogonally protect solid phase synthesis strategy.But, have some shortcomings in the Boc/Bzl orthogonally protect solid phase synthesis strategy, many as side reaction, condition is harsh and in the process of prolongation peptide chain polypeptide chain can lose from solid phase etc.The Fmoc/tBu orthogonally protect solid phase synthesis strategy of Chu Xianing reaction conditions gentleness many then subsequently.After microwave technology applies in the solid phase synthesis of polypeptide in recent years, make the synthetic technology of polypeptide produce a leap.Microwave promotes chemical reaction to be because it makes polar molecule fast rotational in microwave field, makes fast 10 to 1000 times of the more conventional heating means of some speed of reaction, and productive rate improves greatly.People's reported first such as Gedye in 1986 with microwave application in organic synthesis, microwave can promote many chemical reactions, as the Diels-Alder reaction, saponification reaction etc. can be accelerated speed of reaction significantly and improve yield.In 1992, Hui-ming Yu etc. reported at first with microwave application that in polypeptide solid phase synthesis field they have finished the synthetic of acyl carrier protein segment decapeptide (acyl carrier protein, ACP 65～74).

How synthetic efficiently long peptide (greater than 30 peptides) remain a challenge in the world, because polypeptide is along with the increase of peptide chain, its synthetic difficulty is multiplied.With the synthetic long peptide of traditional solid phase method, the more purification difficult of the often very low impurity of its yield, thereby say from practical angle and to have lost the synthetic meaning.Secondly synthesis cycle is long, and 30 peptides nearly time in a week of general needs just can not finished synthesis cycle under the centre does not encounter difficulties the situation of peptide preface, if difficult peptide preface, often just can't synthesize and obtains target polypeptides.And use microwave to promote solid-phase synthetic peptide, can overcome difficulties the peptide preface, the polypeptide that synthetic traditional solid phase method can't obtain, and generated time shortens greatly, and significantly improve polypeptide crude product purity and yield, make that the purifying of product is convenient greatly, as long as just can obtain the purity high product through a preparation HPLC; Can carry out pointed decoration with D type amino acid and alpha-non-natural amino acid etc. to polypeptide very easily in addition, remove to seek active higher, polypeptide that biological half-life is longer, this especially genetic engineering technique can't accomplish.Therefore, our employing this kind method synthetic GLP-1 serial analogs that obtains rapidly and efficiently in GLP-1 analogue synthetic.

Summary of the invention

Glucagon-like-peptide-1 (GLP-1) is the dependent insulin secretion accelerating intestines of a glucose blood sugar lowing polypeptide hormone, and it has the advantage of treatment diabetes outstanding, but its biological half rate phase is short.

First purpose of the present invention is easily by DPP IV (dipeptidyl peptidase IV at it, DPP IV) shortcoming of other organized enzyme (as neutral endopeptidase neutral endopeptidase 24.11 hydrolysis) and in the body, its amino acid that easily is hydrolyzed the position is replaced with other amino acid or alpha-non-natural amino acid, reach the effect of prolong half-life with expectation, synthesized a class GLP-1 derivative.It is characterized in that its structure has following form:

His-Xaa ₁-Xaa ₂-Gly-Thr-Phe-Thr-Ser-Asp-Xaa ₃-Ser-Ser-Tyr-Leu-Glu-Xaa ₄-Gln-Ala-Ala-Lys-Xaa ₅-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa ₆(SEQ.ID?NO：1)

Xaa ₁：Asp，Asn，Arg，Lys，His，Glu，Gln，Trp，Phe，Tyr，Met，D-Gly，D-VaL，D-Leu，D-Ile，D-Ser，D-Thr

Xaa ₂：Glu，Pro

Xaa ₃：Val，Leu

Xaa ₄：Gly，Glu，Gln，Trp，Phe，Tyr，Met，D-Gly，D-VaL，D-Leu，D-Ile，D-Ser，D-Glu，D-Gln，D-Trp，D-Met

Xaa ₅：Glu，Leu

Xaa ₆：Gly，-NH ₂

Second purpose of the present invention provided the method for preparing solid phase of GLP-1 derivative, and the present invention adopts microwave to promote Fmoc/tBu orthogonally protect solid phase synthesis strategy efficiently to synthesize apace and obtains serial GLP-1 analogue.

It is characterized in that: adopt microwave to promote Fmoc/tBu orthogonally protect solid phase synthesis strategy, earlier syntheticly on solid phase carrier obtain being loaded with first Fmoc and protect amino acid whose resin, ninhydrin method detects sloughs the resin that the Fmoc protecting group obtains being loaded with first amino-acid residue after negative; Enter next coupling circulation again; repeat the step of coupling and deprotection with different protection amino acid according to corresponding peptide preface; prolong required aminoacid sequence successively, the synthetic resin that obtains being loaded with corresponding polypeptide cuts down polypeptide with cracking agent at last and obtains the polypeptide crude product from resin.Crude product is through the preparation scale high-efficient liquid phase chromatogram purification, and freeze-drying gets the pure product of polypeptide.

Be loaded with the preparation method that first Fmoc protects amino acid whose resin; it is characterized in that by obtaining with the solid phase carrier coupling again after the activated dose of activation earlier of Fmoc protection amino acid under the microwave irradiation condition; add 1-hydroxyl-benzotriazole (HOBT) or derivatives thereof in the reaction and suppress racemization, and use the acidity of organic bases neutralization reaction.Being loaded with first Fmoc, to protect the employed solid phase carrier of amino acid whose resin be Rink resin, Wang resin or 2-chlorine trityl chloride resin.Activator is dicyclohexylcarbodiimide (DCC), N, N '-DIC (DIC), N, N " carbonyl dimidazoles (CDI); 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl); 2-(7-azo benzotriazole)-N; N; N '; N '-tetramethyl-urea phosphofluoric acid ester (HATU), benzotriazole-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester (HBTU) or 1-hydroxyl-benzotriazole (HOBT) derivative that uses are N-hydroxy-succinamide (HOSU), 1-hydroxyl-7-azo benzotriazole (HOAT) or 3-hydroxyl-1,2,3-phentriazine-4 (3H)-ketone (HOOBT).Organic bases is triethylamine (TEA), N-methylmorpholine (NMM) or diisopropylethylamine (DIEA); Microwave promotion condition is: microwave frequency 2450MHz, temperature of reaction: 20～100 ℃, the reaction times is 5～15min.

Removing of Fmoc protecting group is to contain 0.1molL by use ^-1Hexahydropyridine solution reaction under microwave promotes of 1-hydroxyl-benzotriazole (HOBT), selecting dimethyl formamide (DM F), methyl-sulphoxide (DMSO) or N-Methyl pyrrolidone (NMP) for use is reaction solvent.Microwave promotion condition is: microwave frequency 2450MHz, temperature of reaction: 20～100 ℃, the reaction times is 1～10min.

The invention has the advantages that:

1. a kind of GLP-1 analogue of Ti Chuing can improve the stability of GLP-1 on the basis that keeps hypoglycemic activity, prolongs action time.Have enzymolysis such as anti-DPP IV, the biological half rate phase is long than the GLP-1 prototype.

2. microwave promotes solid phase synthesis GLP-1 analogue to improve coupled reaction speed greatly, and amino acid of the abundant coupling of conventional solid phase synthesis process goes to resin, often needs do not wait by 20 hours in 2 hours, even longer.Microwave promotes then average to need about 10 minutes; Conventional solid phase synthesis process takes off the Fmoc protecting group, often needs do not wait by 1 hour in 30 minutes, and microwave promotes then on average only to need about 5 minutes, and this has improved polypeptide synthetic efficient greatly, has shortened synthesis cycle.

3. the purity of the synthetic crude product that obtains of microwave promotion solid phase synthesis GLP-1 analogue is greater than 60%, and more conventional solid phase synthesis process improves greatly, and this has made things convenient for follow-up purifying work, only need be through once preparing the liquid phase purifying, and freeze-drying can obtain the pure product of target.

4. microwave promotes the synthetic GLP-1 analogue of solid phase method, and its cost is low because coupling efficiency is higher, required protection amino acid is average only need 2 times excessive, more conventional solid phase synthesis process needs 4 to 5 times excessively greatly to reduce.

5. microwave promotes solid phase synthesis GLP-1 analogue method easily to be automated, to change on a large scale, and this makes it be more suitable for suitability for industrialized production.

Therefore promote the GLP-1 analogue that solid phase synthesis technique prepares with microwave provided by the invention, the yield height, synthesis cycle is short, purifying crude is easy, and production cost is low, be easy to industrial automation production.The GLP-1 analogue for preparing, more stable than natural GLP-1, be suitable as the activeconstituents for the treatment of diabetes medicament.

Description of drawings

Above the present invention has been done general description, below accompanying drawing be used to illustrate specific embodiments of the present invention.Wherein:

That Fig. 1 shows is GLP-1 (7～36)-NH ₂Incubate the HPLC analysis of spectra of 0h and 4h with DPP IV temperature;

That Fig. 2 shows is GLP-1 (7～36)-NH ₂Incubate the HPLC analysis of spectra of 0h and 4h with the human plasma temperature;

That Fig. 3 shows is improved D-Gly ₈-GLP-(7～36)-NH ₂Incubate the HPLC analysis of spectra of 0h and 4h with DPP IV temperature;

That Fig. 4 shows is improved D-Gly ₈-GLP-(7～36)-NH ₂Incubate the HPLC analysis of spectra of 0h and 4h with the human plasma temperature;

Embodiment

Adopt following abbreviation in this specification:

Et ₃N: triethylamine; The NMM:N-methylmorpholine; DIEA:N, N '-diisopropylethylamine; DMF: dimethyl formamide; DM SO: methyl-sulphoxide; DCM: methylene dichloride; The Fmoc:N-9 fluorenylmethyloxycarbonyl; DIC:N, N '-DIC; CDI:N, N '-carbonyl dimidazoles; The DMAP:4-Dimethylamino pyridine; The HOSU:N-N-Hydroxysuccinimide; EDC.HCl:1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride; HATU:2-(7-azo benzotriazole)-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester; HBTU: benzotriazole-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester; HCTU:6-chlorobenzene and triazole-1,1,3,3-tetramethyl-urea phosphofluoric acid ester; HOAT:1-hydroxyl-7-azo benzotriazole; HOBT:1-hydroxyl-benzotriazole; PyBOP: phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus; HPLC: high performance liquid chromatography; ESI-MS: electrospray ionization mass spectrum; Gly: glycine; Ser: Serine; Ala: L-Ala; Thr: Threonine; Val: Xie Ansuan; Ile: Isoleucine; Leu: leucine; Tyr: tyrosine; Phe: phenylalanine; His: Histidine; Pro: proline(Pro); Asp: aspartic acid; Met: methionine(Met); Glu: L-glutamic acid; Trp: tryptophane; Lys: Methionin; Arg: arginine.Asn: l-asparagine; Gln: glutamine.

The present invention describes by following implementation column, but these embodiment do not do any restriction explanation of the present invention.

Embodiment 1

His- D-Gly-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-NH ₂The microwave of (SEQ.ID NO:2) promotes solid phase synthesis

(1) swelling of resin

Take by weighing Fmoc-rink amide-MBHA Resin 50mg (replacement amount 0.4mmol/g), through 7mL DCM swelling 30min, suction filtration removes DCM, uses 10mLNMP swelling 30min again, uses NMP at last respectively, DCM, and NMP 7mL rinses well.

(2) microwave promotes removing of Fmoc protecting group

The resin that swelling is good is put into reactor, adds 25% piperidines/NMP (V/V) solution that 7mL contains 0.1M HOBT, reacts 1min in microwave reactor, microwave power is 15W, temperature of reaction is controlled in 50 ℃, uses the cooling of air compressor pressurized air, and reaction finishes back elimination solution; Add 25% piperidines/NMP (V/V) solution that 7mL contains 0.1M HOBT again and react 4min again in microwave reactor, microwave power is 25W, and temperature of reaction is controlled at 50 ℃, uses the cooling of air compressor pressurized air.Reaction finishes back elimination solution, uses the NMP washes clean.Obtain sloughing the resin of the Fmoc protecting group of initial connection.

(3) microwave promotes synthesizing of Fmoc-Arg (Pbf)-rink amide-MBHA Resin

With Fmoc-Arg (Pbf)-OH (0.04mmol), HBTU (0.04mmol), HOBT (0.04mmol) and DIPEA (0.08mmol) are dissolved among the 10mL NMP, in the resin above again this solution being added, in microwave reactor, react 7min, microwave power is 25W, and temperature of reaction is controlled at 50 ℃, uses the cooling of air compressor pressurized air.Reaction finishes back filtering reaction solution, uses DCM and each 7mL washing resin of NMP 3 times.

(4) detection of coupling efficiency

With the coupling efficiency of ninhydrin method or bromjophenol blue method qualitative detection resin, color reaction is negative can to enter next coupling circulation.

Ninhydrin method: the resin particle washing with alcohol takes a morsel, put into transparent bottle and add respectively 2 of 5% triketohydrindene hydrate ethanol, KCN pyridine solution (2ml 0.001M KCN is diluted in the 98ml pyridine), 80% phenol ethanolic solns, in 100 ℃ of heating 5 minutes, promptly positive if resin shows blueness.

The bromjophenol blue method: the resin particle that takes a morsel washs with two formyl ethanamides, puts into the tetrabromophenol sulfonphthalein dimethylacetamide solution that transparent bottle adds 3 1%, and jolting is 3 minutes under the normal temperature, and is promptly positive if resin shows blueness.

(5) prolongation of peptide chain

According to D-Gly ₈-GLP-(7～36)-NH ₂Sequence, the steps in sequence that repeats above-mentioned deprotection and coupling is connected corresponding amino acid, the coupling microwave promotes reaction times 5～20min not wait.Obtain being connected with D-Gly ₈-GLP-(7～36)-NH ₂Resin.

(6) cracking of polypeptide on the resin

With above-mentioned obtain be connected with D-Gly ₈-GLP-(7～36)-NH ₂Resin put into reaction flask, each adds cracking agent Regeant K, and (V/V) 10mL earlier at 0 ℃ of following jolting 30min, reacts 3h more at normal temperatures for TFA/ thioanisole/water/phenol/EDT, 82.5:5:5:5:2.5.Reaction finishes the back suction filtration, adds a small amount of TFA and DCM washing three times, merging filtrate.Filtrate added in a large amount of ice ether separate out white flocks, frozen centrifugation obtains the crude product of target polypeptides.Finally obtain D-Gly ₈-GLP-(7～36)-NH ₂Crude product 63.2mg, yield are 94.3%.

(7) D-Gly ₈-GLP-(7～36)-NH ₂The purifying of crude product

With the D-Gly that obtains above ₈-GLP-(7～36)-NH ₂Crude product is dissolved in a spot of water, prepares type reversed-phase HPLC purifying crude product with Tianjin, island.Adopt the anti-phase preparative column of C18 (340mm * 28mm, 5 μ m) in the purifying; Mobile phase A: 0.1% TFA/ water (V/V), Mobile phase B: 0.1%TFA/ acetonitrile (V/V); Eluent gradient: Mobile phase B 20%～65%, 40min; Flow velocity is 6mL/min; The detection wavelength is 214nm.The solution freeze-drying of collecting gets pure product, finally obtains pure product 29.7mg.ESI-MS?m/z：found[M+3H] ³⁺1095.5，[M+4H] ⁴⁺821.9，[M+5H] ⁵⁺657.8；calu[M+3H] ³⁺1095.5，[M+4H] ⁴⁺821.9，[M+5H] ⁵⁺657.7。Theoretical relative molecular mass is 3283.6, and actual relative molecular weight is 3283.6.

Embodiment 2～28

According to embodiment 1 described method, according to the synthetic polypeptide that obtains embodiment 2～28 of corresponding sequence, by electrospray ionization mass spectrum (ESI-MS) conclusive evidence molecular weight separately.

Embodiment 2

His- Asn-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-NH ₂(SEQ.ID NO:3); Theoretical relative molecular mass is 3340.7.ESI-MS?m/z：found[M+3H] ³⁺1114.5，[M+4H] ⁴⁺836.1，[M+5H] ⁵⁺669.1；calu[M+3H] ³⁺1114.6，[M+4H] ⁴⁺836.3，[M+5H] ⁵⁺669.1。

Embodiment 3

His- Trp-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-NH ₂(SEQ.ID NO:4); Theoretical relative molecular mass is 3412.8.ESI-MSm/z：found[M+3H] ³⁺1138.6，[M+4H] ⁴⁺854.2，[M+5H] ⁵⁺683.6；calu[M+3H] ³⁺1138.6，[M+4H] ⁴⁺854.2，[M+5H] ⁵⁺683.6。

Embodiment 4

His- Tyr-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-NH ₂(SEQ.ID NO:5); Theoretical relative molecular mass is 3389.8.ESI-MS?m/z：found?calu[M+3H] ³⁺1130.8，[M+4H] ⁴⁺848.4，[M+5H] ⁵⁺679.0；calu[M+3H] ³⁺1130.9，[M+4H] ⁴⁺848.5，[M+5H] ⁵⁺679.0。

Embodiment 5

His- Met-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-NH ₂(SEQ.ID NO:6); Theoretical relative molecular mass is 3357.8.ESI-MSm/z：found?[M+3H] ³⁺1120.2，[M+4H] ⁴⁺840.4，[M+5H] ⁵⁺672.5；calu[M+3H] ³⁺1120.3；[M+4H] ⁴⁺840.5，[M+5H] ⁵⁺672.6。

Embodiment 6

His- Glu-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-NH ₂(SEQ.ID NO:7); Theoretical relative molecular mass is 3355.7.ESI-MSm/z：found[M+3H] ³⁺1119.3，[M+4H] ⁴⁺839.9，[M+5H] ⁵⁺672.2；calu[M+3H] ³⁺1119.6，[M+4H] ⁴⁺839.9，[M+5H] ⁵⁺672.1。

Embodiment 7

His- D-Val-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-NH ₂(SEQ.ID NO:8); Theoretical relative molecular mass is 3325.7.ESI-MSm/z：found[M+3H] ³⁺1109.8，[M+4H] ⁴⁺832.4，[M+5H] ⁵⁺665.9；calu[M+3H] ³⁺1109.6，[M+4H] ⁴⁺832.4，[M+5H] ⁵⁺666.1。

Embodiment 8

His- D-Leu-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-NH ₂(SEQ.ID NO:9); The opinion relative molecular mass is 3339.8.ESI-MS?m/z：found[M+3H] ³⁺1114.2，[M+4H] ⁴⁺836.0，[M+5H] ⁵⁺668.8；calu[M+3H] ³⁺1114.3，[M+4H] ⁴⁺835.9，[M+5H] ⁵⁺669.0。

Embodiment 9

His- D-Ser-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-NH ₂(SEQ.ID NO:10); Theoretical relative molecular mass is 3313.7.ESI-MSm/z：found[M+3H] ³⁺1105.5，[M+4H] ⁴⁺829.5，[M+5H] ⁵⁺663.7；calu[M+3H] ³⁺1105.6，[M+4H] ⁴⁺829.4，[M+5H] ⁵⁺663.7。

Embodiment 10

His- D-Ile-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-NH ₂(SEQ.ID NO:11); Theoretical relative molecular mass is 3339.8.ESI-MSm/z：found[M+3H] ³⁺1114.2，[M+4H] ⁴⁺836.0，[M+5H] ⁵⁺668.8；calu[M+3H] ³⁺1114.3，[M+4H] ⁴⁺835.9，[M+5H] ⁵⁺669.0。

Embodiment 11

His- D-Thr-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-NH ₂(SEQ.ID NO:12); Theoretical relative molecular mass is 3327.7.ESI-MS?m/z：found[M+3H] ³⁺1110.2，[M+4H] ⁴⁺832.8；calu[M+3H] ³⁺1110.2，[M+4H] ⁴⁺832.9，[M+5H] ⁵⁺666.5。

Embodiment 12

His- Ser-Pro-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-NH ₂(SEQ.ID NO:13); Theoretical relative molecular mass is 3281.7.ESI-MSm/z：found[M+3H] ³⁺1095.0，[M+4H] ⁴⁺821.5，[M+5H] ⁵⁺657.5；calu[M+3H] ³⁺1094.9，[M+4H] ⁴⁺821.4，[M+5H] ⁵⁺657.3。

Embodiment 13

His- Val-Pro-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-NH ₂(SEQ.ID NO:14); Theoretical relative molecular mass is 3293.7.ESI-MSm/z：found[M+3H] ³⁺1098.9，[M+4H] ⁴⁺824.5；calu[M+3H] ³⁺1098.9，[M+4H] ⁴⁺824.4。

Embodiment 14

His- Leu-Pro-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-NH ₂(SEQ.ID NO:15); Theoretical relative molecular mass is 3307.8.ESI-MSm/z：found[M+3H] ³⁺1103.8，[M+4H] ⁴⁺828.0，[M+4H+Na] ⁵⁺667.0；calu[M+3H] ³⁺1103.6，[M+4H] ⁴⁺827.9，[M+4H+Na] ⁵⁺667.0。

Embodiment 15

His- Gly-Pro-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-NH ₂(SEQ.ID NO:16); Theoretical relative molecular mass is 3251.7.ESI-MSm/z：found[M+3H] ³⁺1084.8，[M+4H] ⁴⁺813.8，[M+5H] ⁵⁺651.2；calu[M+3H] ³⁺1084.9，[M+4H] ⁴⁺813.9，[M+5H] ⁵⁺651.3。

Embodiment 16

His- Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Leu-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID NO:17); Theoretical relative molecular mass is 3297.7.ESI-MSm/z：found[M+3H] ³⁺1100.3，[M+4H] ⁴⁺825.3，[M+5H] ⁵⁺660.7；calu[M+3H] ³⁺1100.2，[M+4H] ⁴⁺825.4，[M+5H] ⁵⁺660.5。

Embodiment 17

His- Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Leu-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID NO:18); Theoretical relative molecular mass is 3327.7.ESI-MSm/z：found[M+3H] ³⁺1110.2，[M+4H] ⁴⁺832.8，[M+5H] ⁵⁺666.6；calu[M+3H] ³⁺1110.2，[M+4H] ⁴⁺832.9，[M+5H] ⁵⁺666.5。

Embodiment 18

His- Thr-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Leu-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID NO:19); Theoretical relative molecular mass is 3341.7.ESI-MSm/z：calu[M+3H] ³⁺1115.1，[M+4H] ⁴⁺836.5，[M+5H] ⁵⁺669.4；calu[M+3H] ³⁺1114.9，[M+4H] ⁴⁺836.4，[M+5H] ⁵⁺669.3。

Embodiment 19

His- Leu-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Leu-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID NO:20); Theoretical relative molecular mass is 3353.8.ESI-MSm/z：found[M+3H] ³⁺1118.9，[M+4H] ⁴⁺839.5，[M+5H] ⁵⁺672.0；calu[M+3H] ³⁺1118.9，[M+4H] ⁴⁺839.5，[M+5H] ⁵⁺671.8。

Embodiment 20

His- Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys- Leu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID NO:21); Theoretical relative molecular mass is 3297.7.ESI-MS?m/z：found[M+3H] ³⁺1100.2，[M+4H] ⁴⁺825.4，[M+4H+Na] ⁵⁺665.0；calu[M+3H] ³⁺1100.3，[M+4H] ⁴⁺825.5，[M+4H+Na] ⁵⁺665.0。

Embodiment 21

His- Ile-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Leu-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID?NO：22)；。ESI-MS?m/z：found[M+3H] ³⁺1118.9，[M+4H] ⁴⁺839.5，[M+5H] ⁵⁺672.0；calu[M+3H] ³⁺1118.9，[M+4H] ⁴⁺839.5，[M+5H] ⁵⁺671.8。

Embodiment 22

His- Leu-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys- Leu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID NO:23); Theoretical relative molecular mass is 3323.8.ESI-MSm/z：found[M+3H] ³⁺1108.9，[M+4H] ⁴⁺831.9，[M+5H] ⁵⁺666.0；calu[M+3H] ³⁺1108.9，[M+4H] ⁴⁺832.0，[M+5H] ⁵⁺665.8。

Embodiment 23

His- Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys- Leu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID NO:24); Theoretical relative molecular mass is 3267.7.ESI-MSm/z：found[M+3H] ³⁺1090.2，[M+4H] ⁴⁺817.9，[M+5H] ⁵⁺654.5；calu[M+3H] ³⁺1090.2，[M+4H] ⁴⁺817.9，[M+5H] ⁵⁺654.5。

Embodiment 24

His- Phe-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu- Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID NO:25); Theoretical relative molecular mass is 3345.8.ESI-MSm/z：found[M+3H] ³⁺1149.4，[M+4H] ⁴⁺862.4；calu[M+3H] ³⁺1149.6，[M+4H] ⁴⁺862.5。

Embodiment 25

His- Ile-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu- Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID NO:26); Theoretical relative molecular mass is 3411.8.ESI-MSm/z：found[M+3H] ³⁺1138.2，[M+4H] ⁴⁺854.0，[M+5H] ⁵⁺683.4；calu[M+3H] ³⁺1138.3，[M+4H] ⁴⁺854.0，[M+5H] ⁵⁺683.4。

Embodiment 26

His- Val-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu- Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID NO:27); Theoretical relative molecular mass is 3397.8.ESI-MSm/z：found[M+3H] ³⁺1133.5，[M+4H] ⁴⁺850.5，[M+5H] ⁵⁺680.5；calu[M+3H] ³⁺1133.6，[M+4H] ⁴⁺850.4，[M+5H] ⁵⁺680.6。

Embodiment 27

His- Lys-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu- Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID NO:28); Theoretical relative molecular mass is 3426.8.ESI-MSm/z：found[M+3H] ³⁺1143.2，[M+4H] ⁴⁺857.7，[M+5H] ⁵⁺686.6；calu[M+3H] ³⁺1143.3，[M+4H] ⁴⁺857.7，[M+5H] ⁵⁺686.4。

Embodiment 28

His- D-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu- Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID NO:29); Theoretical relative molecular mass is 3355.7.ESI-MS?m/z：found[M+3H] ³⁺1119.4，[M+4H] ⁴⁺839.8，[M+4H+K] ⁵⁺679.8；calu[M+3H] ³⁺1119.6，[M+4H] ⁴⁺839.9，[M+4H+K] ⁵⁺672.7。

Embodiment 29

His- D-Leu-Glu-Gly-Thr-Phe-Thr-Ser-Asp- LeuThe microwave of-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-OH (SEQ.ID NO:30) promotes solid phase synthesis

(1) swelling of resin

Take by weighing Wang Resin 50mg (replacement amount 0.4mmol/g), through 7mL DCM swelling 30min, suction filtration removes DCM, uses 10mLNMP swelling 30min again, uses NMP at last respectively, DCM, and NMP 7mL rinses well.

(2) Fmoc-Gly-Wang Resin's is synthetic

With Fmoc-Gly-OH (0.08mmol), HBTU (0.08mmol), HOBT (0.08mmol) and DIPEA (0.08mmol) are dissolved among the 10mLNMP, in the resin above again this solution being added, in microwave reactor, react 7min, microwave power is 25W, and temperature of reaction is controlled at 50 ℃, uses the cooling of air compressor pressurized air.Reaction finishes back filtering reaction solution, uses DCM and each 7mL washing resin of NMP 3 times.

The resin that obtains is added in aceticanhydride/DCM solution of 10ml 15%, add 0.01g DMAP again, jolting 3h under the room temperature, reaction finishes back filtering reaction solution, uses DCM and each 7mL washing resin of NMP 3 times.

(3) microwave promotes removing of Fmoc protecting group

(4) microwave promotes synthesizing of Fmoc-Arg (Pbf)-Gly-Wang Resin

(4) detection of coupling efficiency

(5) prolongation of peptide chain

According to D-Leu ₈-Leu ₁₆The sequence of-GLP-(7～37)-OH, the steps in sequence that repeats above-mentioned deprotection and coupling is connected corresponding amino acid, and the coupling microwave promotes that reaction times 5～20min does not wait.Obtain being connected with D-Leu ₈-Leu ₁₆The resin of-GLP-(7～37)-OH.

(6) cracking of polypeptide on the resin

With above-mentioned obtain be connected with D-Leu ₈-Leu ₁₆The resin of-GLP-(7～37)-OH is put into reaction flask, and each adds cracking agent Regeant K, and (TFA/ thioanisole/water/phenol/EDT, 82.5: 5: 5: 5: 2.5, V/V) 10mL earlier at 0 ℃ of following jolting 30min, reacted 3h more at normal temperatures.Reaction finishes the back suction filtration, adds a small amount of TFA and DCM washing three times, merging filtrate.Filtrate added in a large amount of ice ether separate out white flocks, frozen centrifugation obtains the crude product of target polypeptides.Finally obtain D-Leu ₈-Leu ₁₆-GLP-(7～37)-OH crude product 59.8mg, yield is 92.4%.

(7) D-Leu ₈-Leu ₁₆The purifying of-GLP-(7～37)-OH crude product

With the D-Leu that obtains above ₈-Leu ₁₆-GLP-(7～37)-OH crude product is dissolved in a spot of water, prepares type reversed-phase HPLC purifying crude product with Tianjin, island.Adopt the anti-phase preparative column of C18 (340mm * 28mm, 5 μ m) in the purifying; Mobile phase A: 0.1%TFA/ water (V/V), Mobile phase B: 0.1%TFA/ acetonitrile (V/V); Eluent gradient: Mobile phase B 20%～65%, 40min; Flow velocity is 6mL/min; The detection wavelength is 214nm.The solution freeze-drying of collecting gets pure product, finally obtains pure product 29.7mg.ESI-MSm/z:found[M+3H] ³⁺1138.2, [M+4H] ⁴⁺854.0, [M+5H] ⁵⁺683.5; Calu[M+3H] ³⁺1138.3, [M+4H] ⁴⁺854.0, [M+5H] ⁵⁺683.4. actual relative molecular weight is 3411.8.

Embodiment 30～33

According to embodiment 29 described methods, according to the synthetic polypeptide that obtains embodiment 30～31 of corresponding sequence, by electrospray ionization mass spectrum (ESI-MS) conclusive evidence molecular weight separately.

Embodiment 30

His- Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Leu-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-OH (SEQ.ID NO:31); Theoretical relative molecular mass is 3385.8.ESI-MS?m/z：found[M+3H] ³⁺1129.4，[M+4H] ⁴⁺847.5，[M+5H] ⁵⁺678.2；calu[M+3H] ³⁺1129.6，[M+4H] ⁴⁺847.4，[M+5H] ⁵⁺678.2。

Embodiment 31

His- D-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Leu-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-OH (SEQ.ID NO:32); Theoretical relative molecular mass is 3341.7.ESI-MS?m/z：found[M+3H] ³⁺1119.7，[M+4H] ⁴⁺839.7，[M+5H] ⁵⁺672.1；calu[M+3H] ³⁺1119.7，[M+4H] ⁴⁺839.9，[M+5H] ⁵⁺672.2。

Embodiment 32

GLP-1 and analogue thereof are to the stability experiment of DPP IV

Through GLP-1 or its analogue D-Gly behind the purifying ₈-GLP-(7～36)-NH ₂The DPP IV of 5nmol and 5mU is in the Tris-HCL buffered soln of 50mM in 200 μ L concentration, and 37 ℃ of temperature are incubated 4h, and pH 7.4.The acetonitrile/water solution termination reaction that adds 10 μ L 20% at last.Get 0h respectively, the temperature that 4h is ordered is incubated solution, and is centrifugal, gets supernatant liquor, advances HPLC and analyzes; The post tail is collected degraded product GLP-1 (9～36)-NH2.Analyze and adopt C18 reversed-phase column (150mm * 4.6mm, 5 μ m); Mobile phase A: 0.1%TFA/ water (V/V), Mobile phase B: 0.1%TFA/ acetonitrile (V/V); Eluent gradient: Mobile phase B 10%～45%, 22min; Flow velocity is 1mL/min; Column temperature is 40 ℃; The detection wavelength is 214nm.

As Fig. 1, shown in Figure 2, the result shows without the natural GLP-1 that transforms after incubating 4h with the DPPIV temperature, all is hydrolyzed to GLP-1 (9～the 36)-NH of non-activity basically ₂, complete peptide chain is less than 10%.And improved GLP-1 analogue and DPP IV temperature all still keep prototype after incubating 4h basically, and not seeing has degraded, and complete peptide chain is greater than 90%.The result show by to GLP-1 easily by the position transformation of DPP IV hydrolysis, can make the enzymolysis of GLP-1 analogue opposing DPP IV, thereby can keep the integrity of peptide chain.

Embodiment 33

GLP-1 and analogue thereof are to the stability experiment of human plasma

Through GLP-1 or its analogue D-Gly behind the purifying ₈-GLP-(7～36)-NH ₂5nmol and 10 μ L human plasmas are in the Tris-HCL buffered soln of 50mM in 200 μ L concentration, and 37 ℃ of temperature are incubated 4h, and pH 7.4.The acetonitrile solution termination reaction that adds 10 μ L 20% at last.Get 0h respectively, the temperature that 4h is ordered is incubated solution, and is centrifugal, gets supernatant liquor, advances HPLC and analyzes.Analyze and adopt C18 reversed-phase column (150mm * 4.6mm, 5 μ m); Mobile phase A: 0.1%TFA/ water (V/V), Mobile phase B: 0.1%TFA/ acetonitrile (V/V); Eluent gradient: Mobile phase B 0%, 3min; 0%～24%, 17min; 24%～48%, 30min; 48%～80%, 1min.Flow velocity is 1mL/min; Column temperature is 40 ℃; The detection wavelength is 214nm.

As Fig. 3, shown in Figure 4, the result shows without the natural GLP-1 that transforms after incubating 4h with the human plasma temperature, all is hydrolyzed to GLP-1 (9～the 36)-NH of non-activity basically ₂, complete peptide chain is less than 15%.And improved GLP-1 analogue and human plasma temperature all still keep prototype after incubating 4h basically, and not seeing has degraded, and complete peptide chain is greater than 90%.The result show by to GLP-1 easily by the position transformation of enzymic hydrolysis, can make the enzymolysis in the GLP-1 analogue opposing blood plasma, thereby can keep the integrity of peptide chain.

Embodiment 34

Hypoglycemic activity experiment in GLP-1 and the similar object thereof

Get 10 the week age male mouse of kunming (body weight 18～22g), random packet, 6 every group.Only give drinking-water, overnight fasting.One group of glucose solution (concentration 20%) and physiological saline according to every kilogram of abdominal injection 18mmol of mouse body weight; Other groups are according to the glucose solution of every kilogram of abdominal injection 18mmol of mouse body weight and the GLP-1 analogue solution of 25nmol (10 μ mol/L).0,15,30,45,60 usefulness blood glucose meter are measured blood glucose value.

As shown in table 1, because the GLP-1 after modifying has the resistance to enzymolysis effect, prolong its biological half-life greatly, so hypoglycemic experiment shows that its promoting insulin secretion is not only weakened in the body, also than stronger without the natural GLP-1 that transforms.

The hypoglycemic effect of table 1 GLP-1 and analogue thereof

n＝6，x±s. ^*P＜0.05， ^**P＜0.01， ^***P＜0.001vs?saline

Sequence table

＜110〉China Medicine University

＜120〉microwave promotes solid phase synthesis glucagon-like-peptide-1 (GLP-1) analogue and application thereof

<160>32

<170>PatentIn?version?3.5

<210>1

<211>31

<212>PRT

＜213〉artificial sequence

<220>

＜221〉synthetic construct

<222>(2)..(2)

＜223〉the 2nd Xaa is Asp, Asn, Arg, Lys, His, Glu, Gln, Trp, Phe, Tyr, Met, D-Gly, D-VaL, D-Leu, D-Ile, D-Ser, or D-Thr

<220>

＜221〉synthetic construct

<222>(3)..(3)

＜223〉the 3rd Xaa is Glu, or Pro

<220>

＜221〉synthetic construct

<222>(10)..(10)

＜223〉the 10th Xaa is Val, or Leu

<220>

＜221〉synthetic construct

<222>(16)..(16)

＜223〉the 10th Xaa is Gly, Glu, Gln, Trp, Phe, Tyr, Met, D-Gly, D-VaL, D-Leu, D-Ile, D-Ser, D-Glu, D-Gln, D-Trp, or D-Met

<220>

＜221〉synthetic construct

<222>(21)..(21)

＜223〉the 21st Xaa is Glu, or Leu

<220>

＜221〉synthetic construct

<222>(31)..(31)

＜223〉the 31st Xaa is Gly, or-NH2

<400>1

His?Xaa?Xaa?Gly?Thr?Phe?Thr?Ser?Asp?Xaa?Ser?Ser?Tyr?Leu?Glu?Xaa

1 5 10 15

Gln?Ala?Ala?Lys?Xaa?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg?Xaa

20 25 30

<210>2

<211>30

<212>PRT

＜213〉artificial sequence

<220>

＜221〉synthetic construct

<222>(2)..(2)

＜223〉the 2nd Xaa is D-Gly

<400>2

His?Xaa?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>3

<211>30

<212>PRT

＜213〉artificial sequence

<400>3

His?Asn?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>4

<211>30

<212>PRT

＜213〉artificial sequence

<400>4

His?Trp?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>5

<211>30

<212>PRT

＜213〉artificial sequence

<400>5

His?Tyr?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>6

<211>30

<212>PRT

＜213〉artificial sequence

<400>6

His?Met?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>7

<211>30

<212>PRT

＜213〉artificial sequence

<400>7

His?Glu?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>8

<211>30

<212>PRT

＜213〉artificial sequence

<220>

＜221〉synthetic construct

<222>(2)..(2)

＜223〉the 2nd Xaa is D-Val

<400>8

His?Xaa?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>9

<211>30

<212>PRT

＜213〉artificial sequence

<220>

＜221〉synthetic construct

<222>(2)..(2)

＜223〉the 2nd Xaa D-Leu

<400>9

His?Xaa?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>10

<211>30

<212>PRT

＜213〉artificial sequence

<220>

＜221〉synthetic construct

<222>(2)..(2)

＜223〉the 2nd Xaa is D-Ser

<400>10

His?Xaa?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>11

<211>30

<212>PRT

＜213〉artificial sequence

<220>

＜221〉synthetic construct

<222>(2)..(2)

＜223〉the 2nd Xaa is D-Ile

<400>11

His?Xaa?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>12

<211>30

<212>PRT

＜213〉artificial sequence

<220>

＜221〉synthetic construct

<222>(2)..(2)

＜223〉the 2nd Xaa is D-Thr

<400>12

His?Xaa?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>13

<211>30

<212>PRT

＜213〉artificial sequence

<400>13

His?Ser?Pro?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>14

<211>30

<212>PRT

＜213〉artificial sequence

<400>14

His?Val?Pro?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>15

<211>30

<212>PRT

＜213〉artificial sequence

<400>15

His?Leu?Pro?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>16

<211>30

<212>PRT

＜213〉artificial sequence

<400>16

His?Gly?Pro?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>17

<211>30

<212>PRT

＜213〉artificial sequence

<400>17

His?Gly?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Leu?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>18

<21l>30

<212>PRT

＜213〉artificial sequence

<400>18

His?Ser?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Leu?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>19

<211>30

<212>PRT

＜213〉artificial sequence

<400>19

His?Thr?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Leu?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>20

<211>30

<212>PRT

＜213〉artificial sequence

<400>20

His?Leu?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Leu?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>21

<211>30

<212>PRT

＜213〉artificial sequence

<400>21

His?Ser?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Leu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>22

<211>30

<212>PRT

＜213〉artificial sequence

<400>22

His?Ile?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Leu?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>23

<211>30

<212>PRT

＜213〉artificial sequence

<400>23

His?Leu?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Leu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>24

<211>30

<212>PRT

＜213〉artificial sequence

<400>24

His?Gly?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Leu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>25

<211>30

<212>PRT

＜213〉artificial sequence

<400>25

His?Phe?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Glu

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>26

<211>30

<212>PRT

＜213〉artificial sequence

<400>26

His?Ile?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Glu

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>27

<211>30

<212>PRT

＜213〉artificial sequence

<400>27

His?Val?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Glu

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>28

<211>30

<212>PRT

＜213〉artificial sequence

<400>28

His?Lys?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Glu

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>29

<211>30

<212>PRT

＜213〉artificial sequence

<220>

＜221〉synthetic construct

<222>(2)..(2)

＜223〉the 2nd Xaa is D-Gly

<400>29

His?Xaa?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Glu

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg

20 25 30

<210>30

<211>31

<212>PRT

＜213〉artificial sequence

<220>

＜221〉synthetic construct

<222>(2)..(2)

＜223〉the 2nd Xaa is D-Leu

<400>30

His?Xaa?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Leu?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg?Gly

20 25 30

<210>31

<211>31

<212>PRT

＜213〉artificial sequence

<400>31

His?Ser?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Leu?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg?Gly

20 25 30

<210>32

<211>31

<212>PRT

＜213〉artificial sequence

<220>

＜221〉synthetic construct

<222>(2)..(2)

＜223〉the 2nd Xaa is D-Gly

<400>32

His?Xaa?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Leu?Ser?Ser?Tyr?Leu?Glu?Gly

1 5 10 15

Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg?Gly

20 25 30

Claims

1. glucagon-like-peptide-1 (GLP-1) analogue that contains formula I (SEQ.ID NO:1) structure, the glucagon-like-peptide-1 that obtains (GLP-1) analogue; It is characterized in that its structure has following form:

His-Xaa ₁-Xaa ₂-Gly-Thr-Phe-Thr-Ser-Asp-Xaa ₃-Ser-Ser-Tyr-Leu-Glu-Xaa ₄-Gln-Ala-Ala-Lys-Xaa ₅-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa ₆(SEQ.IDNO：1)

Xaa ₁: Asp, Asn, Arg, Lys, His, Glu, Gln, Trp, Phe, Tyr, Met, D-Gly, D-VaL, D-Leu, D-Ile, D-Ser, or D-Thr;

Xaa ₂: Glu, or Pro;

Xaa ₃: Val, or Leu;

Xaa ₄: Gly, Glu, Gln, Trp, Phe, Tyr, Met, D-Gly, D-VaL, D-Leu, D-Ile, D-Ser, D-Glu, D-Gln, D-Trp, or D-Met;

Xaa ₅: Glu, or Leu;

Xaa ₆: Gly, or-NH ₂

2. polypeptide according to claim 1 has following sequence:

His- D-Gly-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-NH ₂(SEQ.ID?NO：2)；

His- Asn-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-NH ₂(SEQ.ID?NO：3)；

His- Trp-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-NH ₂(SEQ.ID?NO：4)；

His- Tyr-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-NH ₂(SEQ.ID?NO：5)；

His- Met-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-NH ₂(SEQ.ID?NO：6)；

His- Glu-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-NH ₂(SEQ.ID?NO：7)；

His- D-Val-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-NH ₂(SEQ.ID?NO：8)；

His- D-Leu-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-NH ₂(SEQ.ID?NO：9)；

His- D-Ser-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-NH ₂(SEQ.ID?NO：10)；

His- D-Ile-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-NH ₂(SEQ.ID?NO：11)；

His- D-Thr-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-NH ₂(SEQ.ID?NO：12)；

His- Ser-Pro-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-NH ₂(SEQ.ID?NO：13)；

His- Val-Pro-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-NH ₂(SEQ.ID?NO：14)；

His- Leu-Pro-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-NH ₂(SEQ.ID?NO：15)；

His- Gly-Pro-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-NH ₂(SEQ.ID?NO：16)；

His- Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Leu-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID?NO：17)；

His- Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Leu-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID?NO：18)；

His- Thr-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Leu-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID?NO：19)；

His- Leu-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Leu-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID?NO：20)；

His- Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys- Leu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID?NO：21)；

His- Ile-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Leu-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID?NO：22)；

His- Leu-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys- Leu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID?NO：23)；

His- Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys- Leu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID?NO：24)；

His- Phe-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu- Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID?NO：25)；

His- Ile-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu- Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID?NO：26)；

His- Val-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu- Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID?NO：27)；

His- Lys-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu- Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Aia-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID?NO：28)；

His- D-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu- Glu-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂(SEQ.ID?NO：29)

His- D-Leu-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Leu-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-OH(SEQ.ID?NO：30)；

His- Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Leu-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-OH(SEQ.ID?NO：31)

His- D-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Leu-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-OH(SEQ.ID?NO：32)。

3. a pharmaceutical composition comprises the GLP-1 analogue described in the claim 1 for the treatment of significant quantity or its pharmacy acceptable salt and pharmaceutically acceptable carrier or thinner.

4. the GLP-1 analogue described in the claim 1 or its pharmacy acceptable salt and pharmaceutically acceptable carrier or thinner are used for the treatment of utilization in the medicine of diabetes in preparation.

5. the preparation method of the GLP-1 analogue described in the claim 1, it is characterized in that: adopt microwave to promote Fmoc/tBu orthogonally protect solid phase synthesis strategy, earlier syntheticly on solid phase carrier obtain being loaded with first Fmoc and protect amino acid whose resin, ninhydrin method detects sloughs the resin that the Fmoc protecting group obtains being loaded with first amino-acid residue after negative; Enter next coupling circulation again; repeat the step of coupling and deprotection with different protection amino acid according to corresponding peptide preface; prolong required aminoacid sequence successively, the synthetic resin that obtains being loaded with corresponding polypeptide cuts down polypeptide with cracking agent at last and obtains the polypeptide crude product from resin.Crude product is through the preparation scale high-efficient liquid phase chromatogram purification, and freeze-drying gets the pure product of polypeptide.

6. promote solid phase synthesis GLP-1 analogue method according to microwave described in the claim 5; it is characterized in that being loaded with the preparation method that first Fmoc protects amino acid whose resin; be by obtaining with the solid phase carrier coupling again after the activated dose of activation earlier of Fmoc protection amino acid under the microwave irradiation condition; add 1-hydroxyl-benzotriazole (HOBT) or derivatives thereof in the reaction and suppress racemization, and use the acidity of organic bases neutralization reaction.

7. according to claim 6ly be loaded with the preparation method that first Fmoc protects amino acid whose resin, it is characterized in that the solid phase carrier that uses is Rink resin, Wang resin or 2-chlorine trityl chloride resin; The activator that uses is dicyclohexylcarbodiimide (DCC), N, N '-DIC (DIC), N, N " carbonyl dimidazoles (CDI), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl), 2-(7-azo benzotriazole)-N; N; N '; N '-tetramethyl-urea phosphofluoric acid ester (HATU), benzotriazole-N; N; N ', N '-tetramethyl-urea phosphofluoric acid ester (HBTU) or 6-chlorobenzene and triazole-1,1,3,3-tetramethyl-urea phosphofluoric acid ester (HCTU); 1-hydroxyl-benzotriazole (HOBT) derivative that uses is N-hydroxy-succinamide (HOSU), 1-hydroxyl-7-azo benzotriazole (HOAT) or 3-hydroxyl-1,2,3-phentriazine-4 (3H)-ketone (HOOBT); The organic bases that uses is triethylamine (TEA), N-methylmorpholine (NMM) or diisopropylethylamine (DIEA); Microwave promotion condition is: microwave frequency 2450MHz, temperature of reaction: 20～100 ℃, the reaction times is 5～15min.

8. promote solid phase synthesis GLP-1 analogue synthetic method according to microwave described in the claim 5, it is characterized in that removing of Fmoc protecting group is to contain 0.1molL by use ^-1Hexahydropyridine solution reaction under microwave promotes of 1-hydroxyl-benzotriazole (HOBT), selecting dimethyl formamide (DM F), methyl-sulphoxide (DM SO) or N-Methyl pyrrolidone (NMP) for use is reaction solvent; Microwave promotion condition is: microwave frequency 2450MHz, temperature of reaction: 20～100 ℃, the reaction times is 1～10min.