CN106699871A - Preparation method of liraglutide - Google Patents
Preparation method of liraglutide Download PDFInfo
- Publication number
- CN106699871A CN106699871A CN201611223233.3A CN201611223233A CN106699871A CN 106699871 A CN106699871 A CN 106699871A CN 201611223233 A CN201611223233 A CN 201611223233A CN 106699871 A CN106699871 A CN 106699871A
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- fmoc
- fragments
- peptides
- resin
- liraglutide
- Prior art date
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- YSDQQAXHVYUZIW-QCIJIYAXSA-N Liraglutide Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCNC(=O)CC[C@H](NC(=O)CCCCCCCCCCCCCCC)C(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=C(O)C=C1 YSDQQAXHVYUZIW-QCIJIYAXSA-N 0.000 title claims abstract description 90
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- 230000035800 maturation Effects 0.000 description 1
- 125000001160 methoxycarbonyl group Chemical group [H]C([H])([H])OC(*)=O 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
- WOOWBQQQJXZGIE-UHFFFAOYSA-N n-ethyl-n-propan-2-ylpropan-2-amine Chemical compound CCN(C(C)C)C(C)C.CCN(C(C)C)C(C)C WOOWBQQQJXZGIE-UHFFFAOYSA-N 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- TYPIQBXHJBDEBC-HNNXBMFYSA-N tert-butyl (3s)-3-(9h-fluoren-9-ylmethoxycarbonylamino)-4-oxobutanoate Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OC(C)(C)C)C=O)C3=CC=CC=C3C2=C1 TYPIQBXHJBDEBC-HNNXBMFYSA-N 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 1
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/605—Glucagons
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a preparation method of liraglutide, and belongs to the technical field of medicines. The method comprises the following steps of (1) carrying out solid-phase synthesis to form four peptide segments of the liraglutide, wherein the peptide segment 1 is the (1-9)th amino acids in a liraglutide main chain sequence, the peptide segment 2 is the (10-14)th amino acids in the liraglutide main chain sequence, the peptide segment 3 is the (15-23)th amino acids in the liraglutide main chain sequence and the peptide segment 4 is the (24-31)th amino acids in the liraglutide main chain sequence; (2) stepwise coupling various peptide segments to obtain an all-protected liraglutide main chain peptide; (3) removing a protecting group of the 20th lysine and completing connection of side chains to obtain all-protected liraglutide peptide resin; and (4) carrying out cracking, purification and freeze-drying to obtain the liraglutide. The preparation method is high in production efficiency and stable in process, and a product is high in purity, high in yield and suitable for industrial production.
Description
Technical field
The present invention relates to a kind of preparation method of Liraglutide, belong to pharmaceutical technology field.
Background technology
Liraglutide is developed by Novo Nordisk Co., Ltd, is a kind of analog of human pancreatic glucagons like polypeptide -1 (GLP-1), with
Naive GLP-1 homologys up to 97%, with GLP-1 receptor agonisms.With hypoglycemic, mitigation weight, promote pancreas islet
The multiple action such as cytothesis and protection cardiovascular system, can be with other antidiabetic medicines combination treatment type ii diabetes.Profit
Draw Shandong peptide to be researched and developed after 10 years, listing is approved in European Union and the U.S. respectively in July, 2009 and in January, 2010, and in 2011 3
The moon is in Discussion on Chinese Listed.Novo Nordisk is based on unique fatty acid chain modification technique platform of its exploitation, by by natural GLP-1 points
34 lysines of son are substituted by arginine and increase by a 16 carbon fatty acid side chains on 26 lysines and obtain Li Lalu
Peptide.Due to the presence of the fatty acid side chain, the medicine is caused to be combined increase with the invertibity non-covalent bond of plasma albumin, this is not only
Enable that the medicine temporarily hides the degradation of dipeptidyl peptidase-4 (DPP-4), also cause the medicine because forming reversible with albumin
Complex and slowly discharge, cause its body absorption to slow down with distributed process, half-life period more natural GLP-1 is obviously prolonged, and gives
Medicine frequency reaches once a day.Newest LEADER research display, Liraglutide causes mortality risk reduction by 22%, is the
It is a kind of to have the cardiovascular GLP-1 medicines for benefiting.
The molecular formula of Liraglutide is C172H265N43O51, molecular weight is 3751.20, and No. CAS is 204656-20-2, its knot
Structure is as follows：
NH2-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-
Gln-Ala-Ala-Lys(N-ε-(Nα-Palmitoyl-γ-Glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-
Arg-Gly-Arg-Gly-COOH
The production technology of Novo Nordisk development is, by gene recombination technology, Liraglutide to be obtained using saccharomycetes to make fermentation.
Patent CN104592381 discloses one kind by gene recombination technology, and with Escherichia coli as carrier, fermentation obtains Liraglutide
Technique.On the whole, the technical difficulty of genetic recombination is larger, and complex process, production cost is higher.And obtained due to fermentation
The side chain of GLP-1 (7-37)-OH is in unprotected state, impurity is also easy to produce in the course of reaction of connection side chain, so as to cause
Purifying is more difficult, and technique is more complicated.
The maturation of polypeptide techniques is prepared with solid-phase synthesis, increasing Liraglutide synthesis technique is developed
Come.The patent reports such as CN102286092, CN103145828 and CN103980358 utilize Fmoc Preservation tactics, are progressively coupled
Solid phase synthesis process prepare Liraglutide.Due to there is 15 hydrophobic amino acids in Liraglutide sequence, in coupling process
The contraction of resin is more serious, is also easy to produce disappearance peptide, causes yield to reduce.Simultaneously impurity close with product property in thick peptide compared with
It is many, purify extremely difficult.Therefore, the generation of impurity is controlled in building-up process, the number of times of purifying products is reduced, there is provided Yi Zhongshou
Rate is high, cycle is short Liraglutide preparation method has important practical significance.
The content of the invention
To solve the deficiencies in the prior art, the present invention provides a kind of preparation method of Liraglutide.The method contracts for fragment
It is legal, have the advantages that yield is higher, synthesis cycle is short, good stability, it is suitable to large-scale industrial production.
It is an object of the invention to provide a kind of preparation method of Liraglutide, the method comprises the following steps：
1) 4 fragments of peptides of synthesis in solid state Liraglutide.Wherein fragments of peptides 1 is the 1-9 in Liraglutide backbone sequence
Amino acids；Fragments of peptides 2 is the 10-14 amino acids in Liraglutide backbone sequence；Fragments of peptides 3 is Liraglutide main chain sequence
15-23 amino acids in row；Fragments of peptides 4 is the 24-31 amino acids in Liraglutide backbone sequence.
2) each fragments of peptides is progressively coupled, obtains the Liraglutide main chain peptide of full guard.
3) the 20th protection group of lysine of removing, and the connection of side chain is completed, obtain the Liraglutide peptide tree of full guard
Fat.
4) through cracking, purifying, freeze, Liraglutide is obtained.
The above method specifically includes following steps：
1) solid-phase synthesis are used, with resin as carrier, in the presence of condensing agent and activator, Li Lalu is respectively synthesized
The resin of the fragments of peptides 1,2,3,4 of peptide；Wherein fragments of peptides 1 is the 1-9 amino acids in Liraglutide backbone sequence；Fragments of peptides
2 is the 10-14 amino acids in Liraglutide backbone sequence；Fragments of peptides 3 is 15-23 in Liraglutide backbone sequence
Amino acid；Fragments of peptides 4 is the 24-31 amino acids in Liraglutide backbone sequence；
2) in the presence of lysate, fragments of peptides 1-3 is cleaved from resin respectively, obtains full guard fragments of peptides 1-
3；The amino protecting group on the resin of fragments of peptides 4 is sloughed, in the presence of condensing agent and activator, by fragments of peptides 3 and the tree of fragments of peptides 4
Fat is coupled, and obtains the resin of fragments of peptides 5；The amino protecting group on the resin of fragments of peptides 5 is sloughed, in the effect of condensing agent and activator
Under, fragments of peptides 2 and the resin of fragments of peptides 5 are coupled, obtain the resin of fragments of peptides 6；The amino protecting group on the resin of fragments of peptides 6 is sloughed,
In the presence of condensing agent and activator, fragments of peptides 1 and the resin of fragments of peptides 6 are coupled, obtain Liraglutide main chain peptide resin.
3) the lysine side-chain protection of Liraglutide main chain peptide resin the 20th is sloughed, and connects side chain and obtain Liraglutide
Peptide resin.
4) in the presence of lysate, cracking Liraglutide peptide resin obtains the thick peptide of Liraglutide；The thick peptide warp of Liraglutide
Purify, freeze, obtain Liraglutide.
Preferably, step 1) resin that uses of synthesis in solid state fragments of peptides is Wang resin or 2- chlorine trityl chloride resins, more
Preferably 2- chlorine trityl chloride resin.Wherein using the substitution degree preferably 0.3~0.8mmol/g of resin, more preferably 0.4~
0.6mmol/g。
Preferably, step 1) synthesis in solid state fragments of peptides condensing agent and the activator selected be combined as the mixed of DIC and HOBt
The mixture or DIEA and the mixture of HATU of compound, DIEA and TBTU, the mixture of more preferably DIEA and TBTU.Condensing agent with
The mol ratio of activator is 1.5~2.0:1, the solvent for using is DMF, and it is that percent by volume is 20% piperidines to take off Fmoc protection liquid
DMF solution.
Preferably, step 2) described in lysate for percent by volume for 1%TFA DCM solution.
Preferably, step 2) mixture for being combined as DIC and HOBt for being coupled the condensing agent and activator selected,
The mixture of DIEA and TBTU, the mixture of more preferably DIC and HOBt, condensing agent is 1.0~1.5 with the mol ratio of activator:
1。
Preferably, step 3) described in the reagent for sloughing lysine side-chain Dde protections be the mixture of hydrazine hydrate and DMF,
The volume ratio of the two is 1:(10~13).
Preferably, step 4) described in lytic reagent be TFA, EDT, PhSMe, TIS and H2The mixture of O；Above-mentioned TFA,
EDT, PhSMe, TIS and H2The volume ratio of O is (80~85):(3~6):(3~5):(1~2):(2~4).
Preferably, step 4) described in purification step include RPLC purify and regulation isoelectric point separate out,
Sedimentation.
The above method, some preferred schemes specifically include following steps：
1) in the presence of coupling reagent, by the 2- that Fmoc-Asp (Otbu)-OH and substitution degree are 0.4~0.6mmol/g
After the coupling of chlorine trityl chloride resin, slough Fmoc protection groups and obtain Gly- resin carriers.DIEA/TBTU is subsequently adding, successively
It is coupled following amino acid：Fmoc-Ser(tbu)-OH、Fmoc-Thr(tbu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tbu)-
OH, Fmoc-Gly-OH, Fmoc-Glu (Otbu)-OH, Fmoc-Ala-OH, BOC-His (trt)-OH, obtain the resin of fragments of peptides 1
BOC-His (trt)-Ala-Glu (Otbu)-Gly-Thr (tbu)-Phe-Thr (tbu)-Ser (tbu)-Asp (Otbu)-resin；
In the presence of coupling reagent, by the 2- chlorine trityl chloride resins that Fmoc-Leu-OH and substitution degree are 0.4~0.6mmol/g
After coupling, slough Fmoc protection groups and obtain Leu- resin carriers.DIEA/TBTU is subsequently adding, following amino acid is coupled successively：
Fmoc-Tyr (tbu)-OH, Fmoc-Ser (tbu)-OH, Fmoc-Ser (tbu)-OH, Fmoc-Val-OH, obtain the tree of fragments of peptides 2
Fat Fmoc-Val-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu- resins；In the presence of coupling reagent, by Fmoc-Ile-
After OH is coupled with the 2- chlorine trityl chloride resin that substitution degree is 0.4~0.6mmol/g, sloughs Fmoc protection groups and obtain Ile- trees
Fat carrier.DIEA/TBTU is subsequently adding, following amino acid is coupled successively：Fmoc-Phe-OH、Fmoc-Glu(Otbu)-OH、
Fmoc-Lys(Dde)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(trt)-OH、Fmoc-Gly-OH、Fmoc-
Glu (Otbu)-OH, obtains fragments of peptides 3 resin Fmoc-Glu (Otbu)-Gly-Gln (trt)-Ala-Ala-Lys (Dde)-Glu
(Otbu)-Phe-Ile- resins；It is 0.4~0.6mmol/g by Fmoc-Gly-OH and substitution degree in the presence of coupling reagent
2- chlorine trityl chloride resin coupling after, slough Fmoc protection groups and obtain Gly- resin carriers.It is subsequently adding DIEA/TBTU,
Following amino acid is coupled successively：Fmoc-Arg(Pbf)-OH、Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、
Fmoc-Leu-OH, Fmoc-Trp (BOC)-OH, Fmoc-Ala-OH, obtain the resin Fmoc-Ala-Trp (BOC) of fragments of peptides 4-
Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly- resins；
2) use the DCM solution that percent by volume is 1%TFA as lytic reagent, the resin of fragments of peptides 1,2,3 is entered respectively
Row cracking, obtains fragments of peptides 1, fragments of peptides 2, the fragments of peptides 3 of full guard；Use the DMF solution that percent by volume is 20% piperidines
The amino protecting group on the resin of fragments of peptides 4 is sloughed, after fragments of peptides 3 is activated with the mixture of DIC and HOBt, above-mentioned tree is added to
In the DMF solution of fat, reaction obtains the resin of fragments of peptides 5；Fragments of peptides is sloughed using the DMF solution that percent by volume is 20% piperidines
Amino protecting group on 5 resins, after fragments of peptides 2 is activated with the mixture of DIC and HOBt, the DMF for being added to above-mentioned resin is molten
In liquid, reaction obtains the resin of fragments of peptides 6；Sloughed on the resin of fragments of peptides 6 using the DMF solution that percent by volume is 20% piperidines
Amino protecting group, after fragments of peptides 1 is activated with the mixture of DIC and HOBt, is added in the DMF solution of above-mentioned resin, reacts
Obtain Liraglutide main chain peptide resin；
3) the use of volume ratio is 1:13 hydrazine hydrate and DMF mixed solutions, slough Liraglutide main chain peptide resin the 20th
Lysine Dde protection.Then the lysine of Fmoc-Glu-OtBu and 20 is coupled, after sloughing Fmoc protections, is added
Palmitoyl chloride, completes side chain connection.
4) the use of TFA, EDT, PhSMe, TIS and H2O volume ratio is 85:5:4:2:4 mixed solution is lysate, cracking
Liraglutide peptide resin, obtains the thick peptide of Liraglutide；After the inverted high-efficient liquid phase chromatogram purification of the thick peptide of Liraglutide, solution is adjusted
To isoelectric point, after separating out product, centrifugal sedimentation is freezed pH value, obtains Liraglutide.
The english abbreviation lexical or textual analysis that this patent is used：
| Abbreviation and English | Lexical or textual analysis |
| Fmoc | 9- Cong methoxycarbonyl groups |
| Boc | Tertbutyloxycarbonyl |
| tBu | The tert-butyl group |
| trt | Trityl |
| Pbf | 2,2,4,6,7- pentamethyl benzofuran -5- sulfonyls |
| Dde | 1- (4,4- dimethyl -2,6- dioxo hexamethylenes methylene) -3- methyl butyls |
| DMF | N,N-dimethylformamide |
| DIEA | N, N- diisopropylethylamine |
| DIC | N, N- DIC |
| DCM | Dichloromethane |
| TBTU | O- BTAs-N, N, N', N'- tetramethylurea tetrafluoro boric acid |
| HOBt | I-hydroxybenzotriazole |
| HATU | 2- (7- aoxidizes BTA)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester |
| TFA | Trifluoroacetic acid |
| PhSMe | Thioanisole |
| EDT | 1,2- dithioglycols |
| PhOH | Phenol |
| TIS | Tri isopropyl silane |
Beneficial effect of the present invention：
The present invention passes through technical optimization, there is provided a kind of efficiency high, low cost, yield are higher, be suitable for industrialized production
Liraglutide preparation method, its advantage is mainly reflected in：1) process stabilizing of the present invention, obtained Liraglutide is pure
Degree can reach more than 99.5%, and total recovery can reach more than 40%；2) technique of the present invention can be with 4 polypeptide fragments simultaneously
Synthesized, can effectively be shortened synthesis cycle, improve production efficiency.3) because Liraglutide peptide chain is long, hydrophobic amino
Acid is more, is also easy to produce disappearance peptide.Multiple clips synthetic method of the present invention, can reduce the generation of disappearance peptide, its major impurity
It is not missing from peptide, but the fragment not being coupled, impurity differs more with the amino acid sequence of principal component so that product is easier to pure
Change.4) buffer solution after thick peptide purification, the method desalination separated out using regulation pH value to isoelectric point, are taken off relative to using chromatographic column
The method of salt, present method avoids silica gel absorption problem, reduces product loss, and yield is higher.
Specific embodiment
With reference to specific embodiment, the present invention will be further described, but the present invention should not be limited by the examples.
Embodiment 1：
A kind of preparation method of Liraglutide is present embodiments provided, is prepared in accordance with the following steps：
1st, prepared by fragments of peptides
The preparation of 1.1 fragments of peptides 1
Substitution degree is weighed for 1.2mmol/g resins 300g (0.36mol), is added into Peptide systhesis reactor, add 2L
DCM swellable resins 30 minutes, washed once with DCM.Weigh 119.3g Fmoc-Asp (Otbu)-OH (0.29mol) and 69.8g
DIEA (0.54mol) DCM are dissolved, and add in above-mentioned reactor, and reaction is drained after 1.5 hours, is washed with DCM 1 time.Add body
Product is than being 10:1 DCM and methanol solution, stirring closing 30 minutes.Solvent is drained, DCM is washed 3 times, methyl alcohol shrinkage resin 3 times,
Fmoc-Asp (Otbu) chloro- trityl resins of -2- are obtained after vacuum drying, after testing, resin substitution degree is 0.45mmol/g.
The chloro- trityl resin 360g of Fmoc-Asp (Otbu) -2- are weighed, with the DMF that percent by volume is 20% piperidines
Solution removal Fmoc protection groups, 10 minutes every time, altogether twice, are then washed 6 times with DMF, obtain H-Asp (Otbu) -2- chloro- three
Benzyl resin.
Fmoc-Ser (tbu)-OH (2eq), TBTU (2eq) and DIEA (2eq) are weighed, is dissolved in appropriate DMF, stir-activating
5 minutes, add into reactor, then again to DIEA (1eq) is added in reactor, (reaction end was with indenes in 2 hours for room temperature reaction
The detection of triketone method is defined).Solvent is drained, is washed twice with DMF, obtain Fmoc-Fmoc-Ser (tbu)-Asp (Otbu) -2- chloro-
Trityl resin.
The step of repeating above-mentioned de- Fmoc and amino acid couplings, is sequentially completed Fmoc-Thr (tbu)-OH, Fmoc-Phe-
OH、Fmoc-Thr(tbu)-OH、Fmoc-Gly-OH、Fmoc-Glu(Otbu)-OH、Fmoc-Ala-OH、BOC-His(trt)-OH
Coupling, washed with DMF 3 times, DCM wash 3 times, methyl alcohol shrink 3 times, vacuum drying, obtain the resin of fragments of peptides 1 of full guard.
1 resin of fragments of peptides is cracked with the DCM solution that percent by volume is 1%TFA 1 hour, resin is filtered to remove, by filtrate
Revolving is subsequently added into 10L ether to the 1/4 of original volume, separates out white solid, and precipitation, absolute ether washing 5 is collected by filtration
Secondary, vacuum drying obtains 256.3g fragments of peptides 1：BOC-His(trt)-Ala-Glu(Otbu)-Gly-Thr(tbu)-Phe-Thr
(tbu)-Ser (tbu)-Asp (Otbu), purity 95.2%, yield 94.8%.
The preparation of 1.2 fragments of peptides 2
Substitution degree is weighed for 1.2mmol/g resins 300g (0.36mol), is added into Peptide systhesis reactor, add 2L
DCM swellable resins 30 minutes, washed once with DCM.Weigh 102.5g Fmoc-Leu-OH (0.29mol) and 69.8g DIEA
(0.54mol) is dissolved with DCM, is added in above-mentioned reactor, and reaction is drained after 1.5 hours, is washed with DCM 1 time.Add volume ratio
It is 10:1 DCM and methanol solution, stirring closing 30 minutes.Solvent is drained, DCM is washed 3 times, methyl alcohol shrinkage resin 3 times, vacuum
The chloro- trityl resins of Fmoc-Leu-2- are obtained after drying, after testing, resin substitution degree is 0.50mmol/g.
The chloro- trityl resin 360g of Fmoc-Leu-2- are weighed, it is de- with the DMF solution that percent by volume is 20% piperidines
Except Fmoc protection groups, 10 minutes every time, altogether twice, then washed with DMF 6 times, obtain the chloro- trityl resins of Leu-2-.
Fmoc-Tyr (tbu)-OH (2eq), TBTU (2eq) and DIEA (2eq) are weighed, is dissolved in appropriate DMF, stir-activating
5 minutes, add into reactor, then again to DIEA (1eq) is added in reactor, (reaction end was with indenes in 2 hours for room temperature reaction
The detection of triketone method is defined).Solvent is drained, is washed twice with DMF, obtain the chloro- trityl trees of Fmoc-Tyr (tbu)-Leu-2-
Fat.
The step of repeating above-mentioned de- Fmoc and amino acid couplings, is sequentially completed Fmoc-Ser (tbu)-OH, Fmoc-Ser
(tbu) coupling of-OH, Fmoc-Val-OH, is washed 3 times with DMF, and DCM is washed 3 times, and methyl alcohol shrinks 3 times, and vacuum drying is obtained
The resin of fragments of peptides 2 of full guard.
2 resin of fragments of peptides is cracked with the DCM solution that percent by volume is 1%TFA 1 hour, resin is filtered to remove, by filtrate
Revolving is subsequently added into 5L ether to the 1/4 of original volume, separates out white solid, and precipitation, absolute ether washing 5 is collected by filtration
Secondary, vacuum drying obtains 131.8g fragments of peptides 2：Fmoc-Val-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu, purity
96.8%, yield 95.9%.
The preparation of 1.3 fragments of peptides 3
Substitution degree is weighed for 1.2mmol/g resins 300g (0.36mol), is added into Peptide systhesis reactor, add 2L
DCM swellable resins 30 minutes, washed once with DCM.Weigh 102.5g Fmoc-Ile-OH (0.29mol) and 69.8g DIEA
(0.54mol) is dissolved with DCM, is added in above-mentioned reactor, and reaction is drained after 1.5 hours, is washed with DCM 1 time.Add volume ratio
It is 10:1 DCM and methanol solution, stirring closing 30 minutes.Solvent is drained, DCM is washed 3 times, methyl alcohol shrinkage resin 3 times, vacuum
The chloro- trityl resins of Fmoc-Ile-2- are obtained after drying, after testing, resin substitution degree is 0.49mmol/g.
The chloro- trityl resin 360g of Fmoc-Ile-2- are weighed, it is de- with the DMF solution that percent by volume is 20% piperidines
Except Fmoc protection groups, 10 minutes every time, altogether twice, then washed with DMF 6 times, obtain the chloro- trityl resins of Ile-2-.
Fmoc-Phe-OH (2eq), TBTU (2eq) and DIEA (2eq) are weighed, is dissolved in appropriate DMF, 5 points of stir-activating
Clock, adds into reactor, and then again to DIEA (1eq) is added in reactor, (reaction end was with indenes three in 2 hours for room temperature reaction
The detection of ketone method is defined).Solvent is drained, is washed twice with DMF, obtain the chloro- trityl resins of Fmoc-Phe-Ile-2-.
The step of repeating above-mentioned de- Fmoc and amino acid couplings, is sequentially completed Fmoc-Glu (Otbu)-OH, Fmoc-Lys
(Dde)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(Otbu)-
The coupling of OH, is washed 3 times with DMF, and DCM is washed 3 times, and methyl alcohol shrinks 3 times, and vacuum drying obtains the tree of fragments of peptides 3 of full guard
Fat.
3 resin of fragments of peptides is cracked with the DCM solution that percent by volume is 1%TFA 1 hour, resin is filtered to remove, by filtrate
Revolving is subsequently added into 10L ether to the 1/4 of original volume, separates out white solid, and precipitation, absolute ether washing 5 is collected by filtration
Secondary, vacuum drying obtains 304.6g fragments of peptides 3：Fmoc-Glu(Otbu)-Gly-Gln(trt)-Ala-Ala-Lys(Dde)-Glu
(Otbu)-Phe-Ile, purity 95.7%, yield 95.3%.
The preparation of 1.4 fragments of peptides 4
Substitution degree is weighed for 1.2mmol/g resins 300g (0.36mol), is added into Peptide systhesis reactor, add 2L
DCM swellable resins 30 minutes, washed once with DCM.Weigh 86.2g Fmoc-Gly-OH (0.29mol) and 69.8g DIEA
(0.54mol) is dissolved with DCM, is added in above-mentioned reactor, and reaction is drained after 1.5 hours, is washed with DCM 1 time.Add volume ratio
It is 10:1 DCM and methanol solution, stirring closing 30 minutes.Solvent is drained, DCM is washed 3 times, methyl alcohol shrinkage resin 3 times, vacuum
The chloro- trityl resins of Fmoc-Gly-2- are obtained after drying, after testing, resin substitution degree is 0.54mmol/g.
The chloro- trityl resin 350g of Fmoc-Gly-2- are weighed, it is de- with the DMF solution that percent by volume is 20% piperidines
Except Fmoc protection groups, 10 minutes every time, altogether twice, then washed with DMF 6 times, obtain the chloro- trityl resins of Gly-2-.
Fmoc-Arg (Pbf)-OH (2eq), TBTU (2eq) and DIEA (2eq) are weighed, is dissolved in appropriate DMF, stir-activating
5 minutes, add into reactor, then again to DIEA (1eq) is added in reactor, (reaction end was with indenes in 2 hours for room temperature reaction
The detection of triketone method is defined).Solvent is drained, is washed twice with DMF, obtain the chloro- trityl trees of Fmoc-Arg (Pbf)-Gly-2-
Fat.
The step of repeating above-mentioned de- Fmoc and amino acid couplings, be sequentially completed Fmoc-Gly-OH, Fmoc-Arg (Pbf)-
OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (BOC)-OH, the coupling of Fmoc-Ala-OH, are washed 3 times with DMF,
DCM is washed 3 times, and methyl alcohol shrinks 3 times, and vacuum drying obtains the resin of fragments of peptides 4 of full guard.
2nd, the coupling of fragments of peptides
The resin of fragments of peptides 4 of full guard is taken, the DMF solution removing Fmoc protections that percent by volume is 20% piperidines are added
Base, 10 minutes every time, altogether twice, is then washed 6 times with DMF, obtains NH2-Ala-Trp(BOC)-Leu-Val-Arg(Pbf)-
Gly-Arg (Pbf)-Gly- resins.98.8g fragments of peptides 3 (57mmol) is weighed, 10.4g HOBt (68mmol) are dissolved in appropriate DMF
In, add 8.6g DIC (68mmol) stir-activating 5 minutes, add into reactor, room temperature reaction 4 hours (reaction end with
Ninhydrin method detection is defined).Solvent is drained, is washed twice with DMF, obtain the resin of fragments of peptides 5 of full guard.
Add percent by volume be 20% piperidines DMF solution removing Fmoc protection groups, 10 minutes every time, altogether twice, with
Washed with DMF 6 times afterwards, obtain NH2-Glu(Otbu)-Gly-Gln(trt)-Ala-Ala-Lys(Dde)-Glu(Otbu)-Phe-
Ile-Ala-Trp (BOC)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly- resins.Weigh 42.1g fragments of peptides 2
(57mmol), 10.4g HOBt (68mmol), is dissolved in appropriate DMF, adds 8.6g DIC (68mmol) stir-activating 5 minutes,
Add into reactor, room temperature reaction 3.5 hours (reaction end is defined by ninhydrin method detection).Solvent is drained, is washed with DMF
Twice, the resin of fragments of peptides 6 of full guard is obtained.
Add percent by volume be 20% piperidines DMF solution removing Fmoc protection groups, 10 minutes every time, altogether twice, with
Washed with DMF 6 times afterwards, obtain NH2-Val-Ser(tbu)-Ser(tbu)-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln
(trt)-Ala-Ala-Lys(Dde)-Glu(Otbu)-Phe-Ile-Ala-Trp(BOC)-Leu-Val-Arg(Pbf)-Gly-
Arg (Pbf)-Gly- resins.90.6g fragments of peptides 1 (57mmol) is weighed, 10.4g HOBt (68mmol) are dissolved in appropriate DMF,
Add 8.6g DIC (68mmol) stir-activating 5 minutes, add into reactor, (reaction end was with indenes in 3.5 hours for room temperature reaction
The detection of triketone method is defined).Solvent is drained, is washed twice with DMF, obtain the Liraglutide main chain peptide resin of full guard.
3rd, the connection of deprotection and side chain
To addition 1L hydrazine hydrates in the Liraglutide main chain peptide resin of above-mentioned full guard and DMF mixed solutions (volume ratio 1:
13), stir 15 minutes at room temperature, slough the Dde protections of the lysine of Liraglutide main chain peptide resin the 20th, then use DMF
Washing 6 times.
Weigh 48.5g Fmoc-Glu-OtBu (114mmol), 36.6g TBTU (114mmol) and 14.7g DIEA
(114mmol), is dissolved in appropriate DMF, stir-activating 5 minutes, adds in reactor, then again to adding 7.4g in reactor
DIEA (57mmol), room temperature reaction 2 hours (reaction end is defined by ninhydrin method detection).Solvent is drained, two are washed with DMF
It is secondary, the DMF solution removing Fmoc protection groups that percent by volume is 20% piperidines are added, 10 minutes every time, altogether twice, then use
DMF is washed 6 times.
29.2g palmitic acids (114mmol), 36.6g TBTU (114mmol) and 22.1g DIEA (171mmol) are weighed, it is molten
In appropriate DMF, stir-activating 5 minutes is added into reactor, and (reaction end is detected room temperature reaction with ninhydrin method within 2 hours
It is defined).Solvent is drained, is washed with DMF 4 times, DCM is washed 4 times, methyl alcohol shrinks 4 times, vacuum drying obtains Liraglutide peptide tree
Fat 241g.
4th, crack and purify
Above-mentioned Liraglutide peptide resin 241g is taken, 2.5L lysates (TFA is added：EDT：PhSMe：TIS：H2O volume ratios are
85:5:4:2:4), stirring reaction 2.5 hours, filtering at room temperature, collects filtrate, and resin is washed 3 times with a small amount of TFA, and filtrate merges
Add afterwards into the absolute ether of 20L precoolings, standing sedimentation 2 hours.Filtering, filter cake washs 3 times with appropriate absolute ether, very
Sky is dried, and obtains the thick peptide 101.6g of Liraglutide, purity 73.4%, thick peptide yield 82.2%.
Liraglutide crude product is taken, the stirring of 20L purified waters is added, between ammoniacal liquor regulation solution ph to 8~8.5, stirred
Until product is completely dissolved.It is to be purified then with 0.45 μm of membrane filtration.Purification condition：50*250mm chromatographic columns, with eight alkyl
Silane group silica gel is fixing phase, and Detection wavelength 220nm, mobile phase A is the 0.1%TFA/ aqueous solution, and Mobile phase B is 0.1%
TFA/ acetonitrile solutions, flow velocity is 50~60ml/min, and gradient is 35%B~75%B, and main peak is collected in circulation sample introduction purifying.
Qualified product will be purified to merge, add the pH value of appropriate weak aqua ammonia regulation solution to 4.9, separate out consolidating for white
Body, after stirring 30 minutes, is centrifuged off supernatant, and the purifying water washing of solid precooling 3 times is then dispersed to appropriate purifying
In water, freeze, obtain Liraglutide sterling 37.9g, purity 99.6%, purifying yield is 50.6%, total recovery 41.6%.
Embodiment 2
Difference with embodiment 1 is：The substitution degree of Fmoc-Asp (Otbu) chloro- trityl resins of -2- is in step 1
0.32mmol/g；The substitution degree of the chloro- trityl resins of Fmoc-Leu-2- is 0.31mmol/g；The chloro- triphens of Fmoc-Ile-2-
The substitution degree of methyl resin is 0.35mmol/g；The substitution degree of the chloro- trityl resins of Fmoc-Gly-2- is 0.31mmol/g；
In step 1 condensing agent and activator be combined as condensing agent and activator in DIEA/HATU, step 2 be combined as DIEA/
TBTU。
Step 4 vacuum drying obtains the thick peptide 92.8g of Liraglutide, purity 72.8%, thick peptide yield 74.5%.
Step 4 after purification, finally gives Liraglutide sterling 33.8g, and purity 99.5%, purifying yield is 49.8%, always
Yield 37.1%.
Embodiment 3
Difference with embodiment 2 is：The substitution degree of Fmoc-Asp (Otbu) chloro- trityl resins of -2- is in step 1
0.78mmol/g；The substitution degree of the chloro- trityl resins of Fmoc-Leu-2- is 0.72mmol/g；The chloro- triphens of Fmoc-Ile-2-
The substitution degree of methyl resin is 0.75mmol/g；The substitution degree of the chloro- trityl resins of Fmoc-Gly-2- is 0.78mmol/g；
Condensing agent and activator is combined as DIC/HOBt in step 1.
Step 4 vacuum drying obtains the thick peptide 118.5g of Liraglutide, purity 57.6%, thick peptide yield 75.2%.
Step 4 after purification, finally gives Liraglutide sterling 32.5g, and purity 99.3%, purifying yield is 47.3%, always
Yield 35.6%.
Embodiment 4
Difference with embodiment 2 is：The carrier used in step 1 is the Wang resin of 1.02mmol/g for substitution degree.System
The substitution degree for obtaining Fmoc-Asp (Otbu)-Wang resin is 0.48mmol/g；The substitution degree of Fmoc-Leu- Wang resins is
0.52mmol/g；The substitution degree of Fmoc-Ile- Wang resins is 0.50mmol/g；The substitution degree of Fmoc-Gly- Wang resins is
0.55mmol/g；Condensing agent and activator is combined as DIEA/TBTU in step 2.
Step 4 vacuum drying obtains the thick peptide 95.8g of Liraglutide, purity 56.7%, thick peptide yield 59.9%.
Step 4 after purification, finally gives Liraglutide sterling 25.5g, and purity 99.5%, purifying yield is 46.8%, always
Yield 28.0%.
Comparative example 1
Difference with embodiment 1 is：The qualified products obtained in step 4, desalting and purifying is carried out using chromatographic column.Specifically
Purification condition is as follows：50*250mm chromatographic columns, are fixing phase, Detection wavelength 220nm, mobile phase with eight alkyl silane bonded silica gels
A is 0.05% ammonia/water solution, and Mobile phase B is acetonitrile, and flow velocity is 50~60ml/min, and gradient is 30%B~60%B.Revolving
Organic solvent is removed, is freezed, obtain Liraglutide sterling 30.7g, purity 99.6%, purifying yield is 41.0%.
Comparative example 2
1st, the synthesis of main chain peptide resin
Substitution degree is weighed for 1.2mmol/g resins 100g (0.12mol), is added into Peptide systhesis reactor, added
700mL DCM swellable resins 30 minutes, washed once with DCM.Weigh 29.7g Fmoc-Gly-OH (0.10mol) and 23.3g
DIEA (0.27mol) DCM are dissolved, and add in above-mentioned reactor, and reaction is drained after 1.5 hours, is washed with DCM 1 time.Add body
Product is than being 10:1 DCM and methanol solution, stirring closing 30 minutes.Solvent is drained, DCM is washed 3 times, methyl alcohol shrinkage resin 3 times,
The chloro- trityl resins of Fmoc-Gly-2- are obtained after vacuum drying, after testing, resin substitution degree is 0.56mmol/g.
The chloro- trityl resin 102g of Fmoc-Gly-2- are weighed, it is de- with the DMF solution that percent by volume is 20% piperidines
Except Fmoc protection groups, 10 minutes every time, altogether twice, then washed with DMF 6 times, obtain the chloro- trityl resins of Gly-2-.
Fmoc-Arg (Pbf)-OH (2eq), TBTU (2eq) and DIEA (2eq) are weighed, is dissolved in appropriate DMF, stir-activating
5 minutes, add into reactor, then again to DIEA (1eq) is added in reactor, (reaction end was with indenes in 2 hours for room temperature reaction
The detection of triketone method is defined).Solvent is drained, is washed twice with DMF, obtain the chloro- trityl trees of Fmoc-Arg (Pbf)-Gly-2-
Fat.
The step of repeating above-mentioned de- Fmoc and amino acid couplings, according to Liraglutide main chain be sequentially completed Fmoc-Gly-OH,
Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Trp(BOC)-OH、Fmoc-Ala-OH、Fmoc-
Ile-OH、Fmoc-Phe-OH、Fmoc-Glu(Otbu)-OH、Fmoc-Lys(Dde)-OH、Fmoc-Ala-OH、Fmoc-Ala-
OH、Fmoc-Gln(trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(Otbu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tbu)-
OH、Fmoc-Ser(tbu)-OH、Fmoc-Ser(tbu)-OH、Fmoc-Val-OH、Fmoc-Asp(Otbu)-OH、Fmoc-Ser
(tbu)-OH、Fmoc-Thr(tbu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tbu)-OH、Fmoc-Gly-OH、Fmoc-Glu
(Otbu)-OH, the coupling of Fmoc-Ala-OH, BOC-His (trt)-OH, are washed 3 times with DMF, and DCM is washed 3 times, and methyl alcohol shrinks 3
Secondary, vacuum drying obtains the Liraglutide main chain peptide resin of full guard.
2nd, the connection of deprotection and side chain
To addition 1L hydrazine hydrates in the Liraglutide main chain peptide resin of above-mentioned full guard and DMF mixed solutions (volume ratio 1:
13), stir 15 minutes at room temperature, slough the Dde protections of the lysine of Liraglutide main chain peptide resin the 20th, then use DMF
Washing 6 times.
Weigh 48.5g Fmoc-Glu-OtBu (114mmol), 36.6g TBTU (114mmol) and 14.7g DIEA
(114mmol), is dissolved in appropriate DMF, stir-activating 5 minutes, adds in reactor, then again to adding 7.4g in reactor
DIEA (57mmol), room temperature reaction 2 hours (reaction end is defined by ninhydrin method detection).Solvent is drained, two are washed with DMF
It is secondary, the DMF solution removing Fmoc protection groups that percent by volume is 20% piperidines are added, 10 minutes every time, altogether twice, then use
DMF is washed 6 times.
29.2g palmitic acids (114mmol), 36.6g TBTU (114mmol) and 22.1g DIEA (171mmol) are weighed, it is molten
In appropriate DMF, stir-activating 5 minutes is added into reactor, and (reaction end is detected room temperature reaction with ninhydrin method within 2 hours
It is defined).Solvent is drained, is washed with DMF 4 times, DCM is washed 4 times, methyl alcohol shrinks 4 times, vacuum drying obtains Liraglutide peptide tree
Fat 232g.
4th, crack and purify
Above-mentioned Liraglutide peptide resin 232g is taken, 2.4L lysates (TFA is added：EDT：PhSMe：TIS：H2O volume ratios are
85:5:4:2:4), stirring reaction 2.5 hours, filtering at room temperature, collects filtrate, and resin is washed 3 times with a small amount of TFA, and filtrate merges
Add afterwards into the absolute ether of 20L precoolings, standing sedimentation 2 hours.Filtering, filter cake washs 3 times with appropriate absolute ether, very
Sky is dried, and obtains the thick peptide 96.9g of Liraglutide, purity 47.8%, thick peptide yield 51.1%.
Liraglutide crude product is taken, the stirring of 20L purified waters is added, between ammoniacal liquor regulation solution ph to 8~8.5, stirred
Until product is completely dissolved.It is to be purified then with 0.45 μm of membrane filtration.Purification condition：50*250mm chromatographic columns, with eight alkyl
Silane group silica gel is fixing phase, and Detection wavelength 220nm, mobile phase A is the 0.1%TFA/ aqueous solution, and Mobile phase B is 0.1%
TFA/ acetonitrile solutions, flow velocity is 50~60ml/min, and gradient is 35%B~75%B, and main peak is collected in circulation sample introduction purifying.
Qualified product will be purified to merge, add the pH value of appropriate weak aqua ammonia regulation solution to 4.9, separate out consolidating for white
Body, after stirring 30 minutes, is centrifuged off supernatant, and the purifying water washing of solid precooling 3 times is then dispersed to appropriate purifying
In water, freeze, obtain Liraglutide sterling 22.5g, purity 99.4%, purifying yield is 48.3%, total recovery 24.7%.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this
The people of technology, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore protection of the invention
What scope should be defined by claims is defined.
Claims (10)
1. a kind of preparation method of Liraglutide, it is characterised in that comprise the following steps：
1) 4 fragments of peptides of synthesis in solid state Liraglutide, wherein fragments of peptides 1 are 1-9 ammonia in Liraglutide backbone sequence
Base acid；Fragments of peptides 2 is the 10-14 amino acids in Liraglutide backbone sequence；Fragments of peptides 3 is in Liraglutide backbone sequence
15-23 amino acids；Fragments of peptides 4 is the 24-31 amino acids in Liraglutide backbone sequence；
2) each fragments of peptides is progressively coupled, obtains the Liraglutide main chain peptide of full guard；
3) the 20th protection group of lysine of removing, and the connection of side chain is completed, obtain the Liraglutide peptide resin of full guard；
4) through cracking, purifying, freeze, Liraglutide is obtained.
2. method according to claim 1, it is characterised in that comprise the following steps：
1) solid-phase synthesis are used, with resin as carrier, in the presence of condensing agent and activator, Liraglutide is respectively synthesized
The resin of fragments of peptides 1,2,3,4；Wherein fragments of peptides 1 is the 1-9 amino acids in Liraglutide backbone sequence；Fragments of peptides 2 is
10-14 amino acids in Liraglutide backbone sequence；Fragments of peptides 3 is 15-23 ammonia in Liraglutide backbone sequence
Base acid；Fragments of peptides 4 is the 24-31 amino acids in Liraglutide backbone sequence；
2) in the presence of lysate, fragments of peptides 1-3 is cleaved from resin respectively, obtains full guard fragments of peptides 1-3；It is de-
The amino protecting group gone on the resin of fragments of peptides 4, in the presence of condensing agent and activator, by fragments of peptides 3 and the resin idol of fragments of peptides 4
Connection, obtains the resin of fragments of peptides 5；The amino protecting group on the resin of fragments of peptides 5 is sloughed, in the presence of condensing agent and activator, will
Fragments of peptides 2 is coupled with the resin of fragments of peptides 5, obtains the resin of fragments of peptides 6；The amino protecting group on the resin of fragments of peptides 6 is sloughed, in condensation
In the presence of agent and activator, fragments of peptides 1 and the resin of fragments of peptides 6 are coupled, obtain Liraglutide main chain peptide resin；
3) the lysine side-chain protection of Liraglutide main chain peptide resin the 20th is sloughed, and connects side chain and obtain Liraglutide peptide tree
Fat；
4) in the presence of lysate, cracking Liraglutide peptide resin obtains the thick peptide of Liraglutide；The thick peptide of Liraglutide is purified,
It is lyophilized, obtain Liraglutide.
3. method according to claim 2, it is characterised in that step 1) resin that uses of synthesis in solid state fragments of peptides is Wang Shu
Fat or 2- chlorine trityl chloride resins, wherein the use of the substitution degree of resin being 0.3~0.8mmol/g.
4. method according to claim 2, it is characterised in that step 1) condensing agent selected of synthesis in solid state fragments of peptides and work
The mixture of the mixture, the mixture of DIEA and TBTU or DIEA and HATU that are combined as DIC and HOBt of agent, condensing agent and
The mol ratio of activator is 1.5~2.0:1, the solvent for using is DMF, and it is that percent by volume is 20% piperidines to take off Fmoc protection liquid
DMF solution.
5. method according to claim 2, it is characterised in that step 2) described in lysate for percent by volume be 1%
The DCM solution of TFA.
6. method according to claim 2, it is characterised in that step 2) coupling condensing agent and activator selected
Mixture, the mixture of DIEA and TBTU of DIC and HOBt are combined as, condensing agent is 1.0~1.5 with the mol ratio of activator:
1。
7. method according to claim 2, it is characterised in that step 3) described in slough lysine side-chain Dde protections
Reagent is the mixture of hydrazine hydrate and DMF, and the volume ratio of the two is 1:(10~13).
8. method according to claim 2, it is characterised in that step 4) described in lytic reagent be TFA, EDT, PhSMe,
TIS and H2The mixture of O；Above-mentioned TFA, EDT, PhSMe, TIS and H2The volume ratio of O is (80~85):(3~6):(3~5):(1
~2):(2~4).
9. method according to claim 2, it is characterised in that step 4) described in purification step include RP-HPLC
Chromatogram purification and regulation isoelectric point are separated out, sedimentation.
10. method according to claim 2, it is characterised in that specifically include following steps：
1) in the presence of coupling reagent, by the 2- chlorine three that Fmoc-Asp (Otbu)-OH and substitution degree are 0.4~0.6mmol/g
After the coupling of benzyl chlorine resin, slough Fmoc protection groups and obtain Gly- resin carriers, be subsequently adding DIEA/TBTU, be coupled successively
Following amino acid：Fmoc-Ser(tbu)-OH、Fmoc-Thr(tbu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tbu)-OH、
Fmoc-Gly-OH, Fmoc-Glu (Otbu)-OH, Fmoc-Ala-OH, BOC-His (trt)-OH, obtain the resin B OC- of fragments of peptides 1
His (trt)-Ala-Glu (Otbu)-Gly-Thr (tbu)-Phe-Thr (tbu)-Ser (tbu)-Asp (Otbu)-resin；In idol
In the presence of joint-trial agent, by the 2- chlorine trityl chloride resin coupling that Fmoc-Leu-OH and substitution degree are 0.4~0.6mmol/g
Afterwards, Fmoc protection groups are sloughed and obtains Leu- resin carriers, be subsequently adding DIEA/TBTU, following amino acid is coupled successively：Fmoc-
Tyr (tbu)-OH, Fmoc-Ser (tbu)-OH, Fmoc-Ser (tbu)-OH, Fmoc-Val-OH, obtain the resin of fragments of peptides 2
Fmoc-Val-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu- resins；In the presence of coupling reagent, by Fmoc-Ile-OH
After the 2- chlorine trityl chloride resin coupling that substitution degree is 0.4~0.6mmol/g, slough Fmoc protection groups and obtain Ile- resins
Carrier, is subsequently adding DIEA/TBTU, and following amino acid is coupled successively：Fmoc-Phe-OH、Fmoc-Glu(Otbu)-OH、Fmoc-
Lys(Dde)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(trt)-OH、Fmoc-Gly-OH、Fmoc-Glu
(Otbu)-OH, obtains fragments of peptides 3 resin Fmoc-Glu (Otbu)-Gly-Gln (trt)-Ala-Ala-Lys (Dde)-Glu
(Otbu)-Phe-Ile- resins；It is 0.4~0.6mmol/g by Fmoc-Gly-OH and substitution degree in the presence of coupling reagent
2- chlorine trityl chloride resin coupling after, slough Fmoc protection groups and obtain Gly- resin carriers, be subsequently adding DIEA/TBTU,
Following amino acid is coupled successively：Fmoc-Arg(Pbf)-OH、Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、
Fmoc-Leu-OH, Fmoc-Trp (BOC)-OH, Fmoc-Ala-OH, obtain the resin Fmoc-Ala-Trp (BOC) of fragments of peptides 4-
Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly- resins；
2) use the DCM solution that percent by volume is 1%TFA as lytic reagent, the resin of fragments of peptides 1,2,3 is split respectively
Solution, obtains fragments of peptides 1, fragments of peptides 2, the fragments of peptides 3 of full guard；Sloughed using the DMF solution that percent by volume is 20% piperidines
Amino protecting group on the resin of fragments of peptides 4, after fragments of peptides 3 is activated with the mixture of DIC and HOBt, is added to above-mentioned resin
In DMF solution, reaction obtains the resin of fragments of peptides 5；The tree of fragments of peptides 5 is sloughed using the DMF solution that percent by volume is 20% piperidines
Amino protecting group on fat, after fragments of peptides 2 is activated with the mixture of DIC and HOBt, is added to the DMF solution of above-mentioned resin
In, reaction obtains the resin of fragments of peptides 6；The ammonia on the resin of fragments of peptides 6 is sloughed using the DMF solution that percent by volume is 20% piperidines
Base protection group, after fragments of peptides 1 is activated with the mixture of DIC and HOBt, is added in the DMF solution of above-mentioned resin, reacts
To Liraglutide main chain peptide resin；
3) the use of volume ratio is 1:13 hydrazine hydrate and DMF mixed solutions, slough relying for Liraglutide main chain peptide resin the 20th
, then be coupled for the lysine of Fmoc-Glu-OtBu and 20 by the Dde protections of propylhomoserin, after sloughing Fmoc protections, adds palm
Acyl chlorides, completes side chain connection；
4) TFA, EDT, PhSMe, TIS and H are used2O volume ratios are 85:5:4:2:4 mixed solution is lysate, and cracking profit is drawn
Shandong peptide peptide resin, obtains the thick peptide of Liraglutide；After the inverted high-efficient liquid phase chromatogram purification of the thick peptide of Liraglutide, solution ph is adjusted
To isoelectric point, after separating out product, centrifugal sedimentation is freezed, and obtains Liraglutide.
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