CN105732798B

CN105732798B - A kind of synthetic method of Liraglutide

Info

Publication number: CN105732798B
Application number: CN201510735332.9A
Authority: CN
Inventors: 谷海涛; 徐峰; 刘标; 王良友; 伏帅; 孙美禄
Original assignee: Jiangsu Sinopep Macao Zaino Biological Pharmaceutical Ltd By Share Ltd
Current assignee: Jiangsu Sinopep Macao Zaino Biological Pharmaceutical Ltd By Share Ltd
Priority date: 2015-11-03
Filing date: 2015-11-03
Publication date: 2018-10-02
Anticipated expiration: 2035-11-03
Also published as: CN105732798A

Abstract

The present invention relates to field of medicaments products, are a kind of synthetic methods of Liraglutide, include the following steps：Fragment condensation is at all kinds of Liraglutide intermediates；The deprotection of full guard Liraglutide, cracking；The purifying and freeze-drying of Liraglutide.This method uses the synthesis mode of 4+5+7+6+9,Carry out the synthesis of 5 segments simultaneously,Substantially reduce the generated time of product,Simultaneously by His Ala Glu Gly,Thr‑Phe‑Thr‑Ser‑Asp,Val‑Ser‑Ser‑Tyr‑Leu‑Glu‑Gly,Gln‑Ala‑Ala‑N6‑[N‑(1‑oxohexadecyl)‑Glu]‑Lys‑Glu‑Phe,The substep of the composition-factors such as Ile Ala Trp Leu Val Arg Gly Arg Gly OH parses,Reduce the synthesis of difficult peptide sequence in synthesis in solid state,Solves the difficulty amplified in batches in synthesis in solid state,Improve combined coefficient,Since segment synthesis is that purifying difficulty effectively reduces,Greatly reduce production cost simultaneously.

Description

A kind of synthetic method of Liraglutide

Technical field

The present invention is Peptide systhesis preparation field, the more particularly to synthetic method of Liraglutide.

Background technology

Liraglutide is the medicine of approval in 2010：Its structural formula is as follows：

Prepared by the biological methods such as current useful genetic engineering, but put into high, equipment complexity, and difficulty is big.Simultaneously United States Patent (USP) US6268343B1 and US6458924B2 report the solid-liquid synthetic method of Liraglutide, intermediate GLP-1 (7-37)- OH be required to reversed-phase HPLC purifying, then under liquid-phase condition with N^α- alkanoyl-Glu (ONSu)-OtBu reacts, and due to GLP-1 (7-37)-OH N-terminals are unprotected and Side chain protective group is totally removed, and can cause to generate many impurity, it is difficult to purify, grasp Make cumbersome.A kind of DNA recombinations technique synthesis profit of US Patent 7235627 inventions of No.6268343,6458924 and is drawn Shandong peptide precursor, i.e. sequence 1-31, then Pal-Glu (OSu)-OtBu substances are linked by chemical synthesis and obtain target polypeptides.DNA It recombinates technique to need using complicated equipment, and virus may be introduced in the product, be unfavorable for the pharmaceutical purpose of product.It is Chinese special Sharp CN201110271342.3, CN200610110898.3, CN201210369966.3 and CN200510107588 are reported The method of direct synthesis in solid state Liraglutide, however, due to the hydrophobicity of cetyl on peptide chain, the impurity of this sampling technology generation It is difficult to remove to be.The existing synthetic method of Liraglutide needs two-step purifying there are cumbersome, and synthesis cycle is long, and waste liquid is more, It is unfavorable for environmental protection, need to spends a large amount of acetonitriles, it is with high costs and the shortcomings that be unfavorable for large-scale production.

Invention content

The technical problem to be solved by the present invention is in view of the deficiencies of the prior art, provide a kind of method it is more reasonable, at Originally it substantially reduces, and the synthetic method of the Liraglutide suitable for industrialized production.

The technical problem to be solved by the present invention is to what is realized by technical solution below.The present invention is a kind of profit drawing The synthetic method of Shandong peptide, its main feature is that：Include the following steps：

A, fragment condensation is at all kinds of Liraglutide intermediates：

（1）The synthesis of R-His (R1)-Ala-Glu (R2)-Gly-R3；

（2）The synthesis of R-Thr (R4)-Phe-Thr (R4)-Ser (R4)-Asp (R5)-R3；

（3）The synthesis of R-Val-Ser (R4)-Ser (R4)-Tyr (R4)-Leu-Glu (R2)-Gly-R3；

（4）R-Gln-Ala-Ala-N6- [N- (1-oxohexadecyl)-Glu (R2)]-Lys-Glu (R2)-Phe-R3's Synthesis；

（5）The synthesis of R-Ile-Ala-Trp (R6)-Leu-Val-Arg (R7)-Gly-Arg (R7)-Gly-R3；

（6）R-His (R1)-Ala-Glu (R2)-Gly-Thr (R4)-Phe-Thr (R4)-Ser (R4)-Asp (R5)-R3's Synthesis；

（7）R-His(R1)-Ala-Glu(R2)-Gly-Thr(R4)-Phe-Thr(R4)-Ser(R4)-Asp(R5)- The synthesis of Val-Ser (R4)-Ser (R4)-Tyr (R4)-Leu-Glu (R2)-Gly-R3；

（8）R-His(R1)-Ala-Glu(R2)-Gly-Thr(R4)-Phe-Thr(R4)-Ser(R4)-Asp(R5)- Val-Ser(R4)-Ser(R4)-Tyr(R4)-Leu-Glu(R2)-Gly-Gln-Ala-Ala-N6-[N-(1- Oxohexadecyl)-Glu (R2)]-Lys-Glu (R2)-Phe-R3 synthesis；

（9）R-Thr(R4)-Phe-Thr(R4)-Ser(R4)-Asp(R5)-Val-Ser(R4)-Ser(R4)-Tyr(R4)- Leu

The synthesis of-Glu (R2)-Gly-R3；

（10）R-Thr(R4)-Phe-Thr(R4)-Ser(R4)-Asp(R5)-Val-Ser(R4)-Ser(R4)-Tyr (R4)-Leu-

Glu(R2)-Gly- Gln-Ala-Ala-N6-[N-(1-oxohexadecyl)-Glu(R2)]-Lys-Glu(R2)- The synthesis of Phe-R3；

（11）R-Thr(R4)-Phe-Thr(R4)-Ser(R4)-Asp(R5)-Val-Ser(R4)-Ser(R4)-Tyr (R4)-Leu

-Glu(R2)-Gly- Gln-Ala-Ala-N6-[N-(1-oxohexadecyl)-Glu(R2)]-Lys-Glu (R2)-Phe-Ile-Ala

The synthesis of-Trp (R6)-Leu-Val-Arg (R7)-Gly-Arg (R7)-Gly-R3；

（12）Val-Ser(R4)-Ser(R4)-Tyr(R4)-Leu-Glu(R2)-Gly- Gln-Ala-Ala-N6-

The synthesis of [N- (1-oxohexadecyl)-Glu (R2)]-Lys-Glu (R2)-Phe-R3；

（13）Val-Ser(R4)-Ser(R4)-Tyr(R4)-Leu-Glu(R2)-Gly- Gln-Ala-Ala-N6

-[N-(1-oxohexadecyl)-Glu(R2)]-Lys-Glu(R2)-Phe-Ile-Ala-Trp(R6)-Leu- The synthesis of Val-Arg (R7)-Gly-Arg (R7)-Gly-R3；

（14）R-His(R1)-Ala-Glu(R2)-Gly-Thr(R4)-Phe-Thr(R4)-Ser(R4)-Asp(R5)- Val-Ser(R4)

-Ser(R4)-Tyr(R4)-Leu-Glu(R2)-Gly-Gln-Ala-Ala-N6-[N-(1-oxohexadecyl)- Glu (R2)]-Lys-Glu (R2)-Phe-Ile-Ala-Trp (R6)-Leu-Val-Arg (R7)-Gly-Arg (R7)-Gly-R3 Synthesis；

After above-described various protected amino acid fragment condensations, the various intermediate fragments of Liraglutide of formation；Wherein： R be selected from Z, Boc, Cl-Z, Fmoc, oNBS, dNBS, Troc, Dts, pNZ, oNZ, NVOC, NPPOC, HFA, Ddz, Bpoc, Nps, Nsc, Bsmoc, α-Nsmoc, ivDde, Fmoc*, MTT, Alloc wherein any one；

R1 be selected from TOS, TRT, Mtt, Mmt, Boc, Bom, Bum, Fmoc, Dmbz, Dnp wherein any one；

It is wherein arbitrary that R2 is selected from H, Otbu, Bn, cHX, Mpe, 2-ph1pr, TEGbn, Damb, Al, pNB, pTMSE, Dmnb One；

It is wherein arbitrary that R3 is selected from H, Otbu, Bn, cHX, Mpe, 2-ph1pr, TEGbn, Damb, Al, pNB, pTMSE, Dmnb One；

R4 be selected from Bn, cHx, tBu, Trt, TBDMS, Dmnb, Poc wherein any one；

It is wherein arbitrary that R5 is selected from H, Otbu, Bn, cHX, Mpe, 2-ph1pr, TEGbn, Damb, Al, pNB, pTMSE, Dmnb One；

R6 be selected from Z, Boc, Cl-Z, Fmoc, For, Hoc, Mts, Alloc wherein any one；

R7 be selected from TOS, Pbf, Pmc, Mts, Mtr, Mis wherein any one；

R8 is selected from MTT, Alloc, Z, Cl-Z, oNBS, dNBS, Troc, Dts, pNZ, oNZ, NVOC, NPPOC, HFA wherein Any one；

B, the deprotection of full guard Liraglutide, cracking,

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln- Ala-Ala-N6-[N-(1-oxohexadecyl)-Glu]-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly- Arg-Gly-OH；

C, the purifying and freeze-drying of Liraglutide.

A kind of synthetic method of Liraglutide of the present invention, further preferred technical solution are：Described 14 The coupling system used in the building-up process of polypeptide fragment is selected from：Single condensing agent：DIC, DCC, EDC, chloracetyl chloride, Azide Object, TBTU, PyBOP, HBTU；Or two system condensing agent：DIC/HOBT、DCC/HOBT、EDC/HOBT.

A kind of synthetic method of Liraglutide of the present invention, further preferred technical solution are：

（1）The synthesis of R-Gln-Ala-Ala-N6- [N- (1-oxohexadecyl)-Glu]-Lys-Glu (R2)-Phe-R3 Using pure liquid phase synthesis, including a variety of segment synthetic methods, specific segment are as follows：

R-Gln-Ala-OH、R-Gln-Ala-Ala-OH、R-Gln-Ala-Ala-N6-[N-(1-oxohexadecyl)- Glu]-Lys(R8)-OH、R-Gln-Ala-Ala-N6-[N-(1-oxohexadecyl)-Glu]-Lys(R8)-Glu(R2)-R3、 R-Phe-R3、R-Glu(R2)-Phe-R3、 R- N6-[N-(1-oxohexadecyl)-Glu]-Lys(R8)-Glu(R2)- Phe-R3、R-Ala- N6-[N-(1-oxohexadecyl)-Glu]-Lys(R8)-Glu(R2)-Phe-R3、R-Ala-Ala- N6-[N-(1-oxohexadecyl)-Glu]-Lys(R8)-Glu(R2)-R3；

（2）N- (1-oxohexadecyl)-Glu (R2)]-R3 synthesis：

Using NH2-Glu (R2)-OH and stearic active ester carry out that purpose product is obtained by the reaction；In active ester method synthesis The single condensing agent and condensing agent compound used be：DIC、DCC、EDC、TBTU、PyBOP、HBTU、DIC/HOBT、DCC/HOBT、 EDC/HOBT；Or it with stearic acid anhydrides carries out that product is obtained by the reaction using NH2-Glu (R2)-OH；Acid anhydrides method uses chloroethene Acyl chlorides, ethyl chloroformate, isobutylchloroformate, isobutyl chlorocarbonate；Or using sulphur benzyl ester first with stearic acid at thioesters, later Ester exchange reaction is carried out with NH2-Glu (R2)-OH, obtains product；Or azide is formed using stearic acid, later and NH2- Glu (R2)-OH forms product；

（3）, R-N6- [N- (1-oxohexadecyl)-Glu]-Lys-R3 synthesis

Using N- (1-oxohexadecyl)-Glu (R2)] the active ester of-OH with Z-Lys-OH carries out that purpose is obtained by the reaction Product；The single condensing agent used and condensing agent compound are in active ester method synthesis：DIC、DCC、EDC、TBTU、PyBOP、 HATU、HBTU、DIC/HOBT、DCC/HOBT、EDC/HOBT；Or use N- (1-oxohexadecyl)-Glu (R2)]-OH Acid anhydrides with Fmoc-Lys-OH carries out that product is obtained by the reaction；Acid anhydrides method using chloracetyl chloride, ethyl chloroformate, isobutylchloroformate, Isobutyl chlorocarbonate；Or using sulphur benzyl ester first with N- (1-oxohexadecyl)-Glu (R2)]-OH is at thioesters, later and Z- Lys-OH carries out ester exchange reaction, obtains product；Or use N- (1-oxohexadecyl)-Glu (R2)]-OH formation nitrine Compound forms product with Z-Lys-OH later；

(4), the synthesis of NH2-N6- [N- (1-oxohexadecyl)-Glu]-Lys-OH：

Using the method for catalytic hydrogenation, deprotection base；Or use alkali deprotection base；Or using sour deprotection Base.

A kind of synthetic method of Liraglutide of the present invention, further preferred technical solution are：Each fragment condensation Method uses active ester method, mixed anhydride method, azido compound method or sulphur benzyl ester exchange process.

In the method for the present invention, each explanation of nouns see the table below：

Fmoc	9-fluorenylmethyloxycarbonyl
		HBTU	O- benzotriazole-N- tetramethylurea hexafluorophosphates
HATU	O- (7- azo benzotriazole -1- oxygen)-N- tetramethylurea hexafluorophosphates
		PyBOP	(benzotriazole -1- oxygen) tripyrrole alkane subbase phosphorus hexafluorophosphate
DIC	Diisopropylcarbodiimide
		HOBt	I-hydroxybenzotriazole
HOAt	1- hydroxyl -7- azo benzotriazole
		DCC	Dicyclohexylcarbodiimide
TMP	2.4.6- trimethylpyridine
		Pbf	2,2,4,6,7- pentamethyl Dihydrobenzofuranes -5- sulfonyls
Trt	Trityl
		tBu	Tertiary butyl
OtBu	Tertiary butyl oxygen
		DMF	N,N-dimethylformamide
DCM	Dichloromethane
		Z-Cl	Benzyloxycarbonylchloride base
TFA	Trifluoroacetic acid
		Alloc	Allyloxycarbonyl
Z	Benzyloxycarbonyl group
		Boc	Tertbutyloxycarbonyl
oxohexadecyl	Palmityl
		AM	Aminomethyl
EDT	Dithioglycol
		HOSU	Succinimide oxygroup
Ac	Acetate

Compared with prior art, this method synthesizes Liraglutide product using pure liquid phase process.The method of the present invention uses 4+5 The synthesis mode of+7+6+9, while carrying out the synthesis of 5 segments, the generated time of product is substantially reduced, while by right His-Ala-Glu-Gly、Thr-Phe-Thr-Ser-Asp、Val-Ser-Ser-Tyr-Leu-Glu-Gly、Gln-Ala-Ala- N6-[N-(1-oxohexadecyl)-Glu]

The substep solution of the composition-factors such as-Lys-Glu-Phe, Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH Analysis reduces the synthesis of difficult peptide sequence in synthesis in solid state, solves the difficulty amplified in batches in synthesis in solid state, improves synthesis effect Rate is that purifying difficulty effectively reduces while greatly reducing production cost due to segment synthesis.

Description of the drawings

Fig. 1 is the thick peptide HPLC spectrograms of Liraglutide；

Fig. 2 is Liraglutide fine peptide HPLC spectrograms；

Fig. 3 is Liraglutide fine peptide first mass spectrometric figure；

Fig. 4 is Liraglutide fine peptide second order ms figure；

Fig. 5 is that Liraglutide fine peptide second order ms retrieve data comparison shot chart：

Fig. 6 is Liraglutide fine peptide second order ms retrieval matching figure.

Specific implementation mode

Referring to the drawings, further describe the specific technical solution of the present invention, in order to those skilled in the art into One step the present invention is understood, without constituting the limitation to its right.

A kind of embodiment 1, synthetic method of Liraglutide, includes the following steps：

A, fragment condensation is at all kinds of Liraglutide intermediates：

（1）The synthesis of R-His (R1)-Ala-Glu (R2)-Gly-R3；

（2）The synthesis of R-Thr (R4)-Phe-Thr (R4)-Ser (R4)-Asp (R5)-R3；

（3）The synthesis of R-Val-Ser (R4)-Ser (R4)-Tyr (R4)-Leu-Glu (R2)-Gly-R3；

（9）R-Thr(R4)-Phe-Thr(R4)-Ser(R4)-Asp(R5)-Val-Ser(R4)-Ser(R4)-Tyr(R4)- Leu

The synthesis of-Glu (R2)-Gly-R3；

（10）R-Thr(R4)-Phe-Thr(R4)-Ser(R4)-Asp(R5)-Val-Ser(R4)-Ser(R4)-Tyr (R4)-Leu-

（11）R-Thr(R4)-Phe-Thr(R4)-Ser(R4)-Asp(R5)-Val-Ser(R4)-Ser(R4)-Tyr (R4)-Leu

The synthesis of-Trp (R6)-Leu-Val-Arg (R7)-Gly-Arg (R7)-Gly-R3；

（12）Val-Ser(R4)-Ser(R4)-Tyr(R4)-Leu-Glu(R2)-Gly- Gln-Ala-Ala-N6-

The synthesis of [N- (1-oxohexadecyl)-Glu (R2)]-Lys-Glu (R2)-Phe-R3；

（13）Val-Ser(R4)-Ser(R4)-Tyr(R4)-Leu-Glu(R2)-Gly- Gln-Ala-Ala-N6

R4 be selected from Bn, cHx, tBu, Trt, TBDMS, Dmnb, Poc wherein any one；

R6 be selected from Z, Boc, Cl-Z, Fmoc, For, Hoc, Mts, Alloc wherein any one；

R7 be selected from TOS, Pbf, Pmc, Mts, Mtr, Mis wherein any one；

B, the deprotection of full guard Liraglutide, cracking,

C, the purifying and freeze-drying of Liraglutide.

Embodiment 2, in a kind of synthetic method of Liraglutide described in embodiment 1：The conjunction of 14 polypeptide fragments It is selected from the coupling system used in the process：Single condensing agent：DIC, DCC, EDC, chloracetyl chloride, azide, TBTU, PyBOP、HBTU；Or two system condensing agent：DIC/HOBT、DCC/HOBT、EDC/HOBT.

Embodiment 3, in a kind of synthetic method of Liraglutide described in embodiment 1：（1）R-Gln-Ala-Ala-N6-[N- (1-oxohexadecyl)-Glu]-Lys-Glu (R2)-Phe-R3 synthesis use pure liquid phase synthesis, comprising a variety of segments synthesis Method, specific segment are as follows：

（2）N- (1-oxohexadecyl)-Glu (R2)]-R3 synthesis：

（3）, R-N6- [N- (1-oxohexadecyl)-Glu]-Lys-R3 synthesis

(4), the synthesis of NH2-N6- [N- (1-oxohexadecyl)-Glu]-Lys-OH：

Embodiment 4, in a kind of synthetic method of Liraglutide described in embodiment 1 or 2：Each fragment condensation is using activation Ester process, mixed anhydride method, azido compound method or sulphur benzyl ester exchange process.

Embodiment 5, the synthetic method experiment of Liraglutide

One, the synthesis of Fmoc-His (trt)-Ala-Glu (Otbu)-Gly-OH

(1), the synthesis of Fmoc-His (trt)-Ala-OH

Solution 1：Fmoc-His (trt)-OH of 100 mmoles, the HOSu of 120 mmoles are weighed, the round-bottomed flask of 500ml is placed in In, 200mlTHF dissolvings are added, be cooled under magnetic agitation 0-5 DEG C it is spare.

Solution 2：The DCC for weighing 120 mmoles, is placed in small beaker, is dissolved with the THF of 100ml, be cooled to 0-5 DEG C it is spare

Solution 3：The NH2-Ala-OH for weighing 130 mmoles is dissolved in 10% sodium carbonate liquor of 100ml, spare at room temperature

At 0-5 DEG C, by DCC solution（Solution 2）It is slowly added drop-wise in the solution 1 of stirring, it is anti-at reaction 15 minutes, 25 DEG C It answers 2 hours, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.It is filtered to remove white precipitate, is used 30mlTHF washings are precipitated and are filtered, merging filtrate, and revolving removes THF, obtains a large amount of white solids, uses saturated sodium bicarbonate 500ml mashing washings, filter, wash repeatedly 3 times, obtain white solid.White solid is dissolved in 200mlTHF, is used at 25 DEG C Constant pressure funnel being added drop-wise in solution 3 slowly, the reaction was continued 1 hour for reaction after being added dropwise, and reaction terminates（It is subject to contact plate）. With dilute citric acid tune pH value to 7, revolving removes THF, heavy to a large amount of whites between 3-4, are obtained with dilute citric acid tune pH value again Shallow lake, filtering are beaten with dilute citric acid 300ml and are washed, and filtering obtains white solid after washing repeatedly 3 times.Product is that white is solid Body, purity：98%

(2)、Fmoc-His(trt)-Ala-Glu(Otbu)-OH

Solution 1：Fmoc-His (trt)-Ala-OH of 100 mmoles, the HOSu of 120 mmoles are weighed, the round bottom of 500ml is placed in In flask, 200mlTHF dissolvings are added, be cooled under magnetic agitation 0-5 DEG C it is spare.

Solution 3：NH2- Glu (the Otbu)-OH for weighing 130 mmoles, is dissolved in 10% sodium carbonate liquor of 100ml, room It is spare under temperature

At 0-5 DEG C, by DCC solution（Solution 2）It is slowly added drop-wise in the solution 1 of stirring, it is anti-at reaction 15 minutes, 25 DEG C It answers 2 hours, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.It is filtered to remove white precipitate, is used 30mlTHF washings are precipitated and are filtered, merging filtrate, and revolving removes THF, obtains a large amount of white solids, uses saturated sodium bicarbonate 500ml mashing washings, filter, wash repeatedly 3 times, obtain white solid.White solid is dissolved in 200mlTHF, is used at 25 DEG C Constant pressure funnel being added drop-wise in solution 3 slowly, the reaction was continued 1 hour for reaction after being added dropwise, and reaction terminates（It is subject to contact plate）. With dilute citric acid tune pH value to 7, revolving removes THF, heavy to a large amount of whites between 3-4, are obtained with dilute citric acid tune pH value again Shallow lake, filtering are beaten with dilute citric acid 300ml and are washed, and filtering obtains white solid after washing repeatedly 3 times.Product is that white is solid Body, purity：97.8%

(3), the synthesis of Fmoc-His (trt)-Ala-Glu (Otbu)-Gly-OH

Solution 1：Fmoc-His (trt)-Ala-Glu (Otbu)-OH of 100 mmoles, the HOSu of 120 mmoles are weighed, is placed in In the round-bottomed flask of 500ml, 200mlTHF dissolvings are added, be cooled under magnetic agitation 0-5 DEG C it is spare.

Solution 3：The NH2-Gly-OH for weighing 130 mmoles is dissolved in 10% sodium carbonate liquor of 100ml, spare at room temperature

At 0-5 DEG C, by DCC solution（Solution 2）It is slowly added drop-wise in the solution 1 of stirring, it is anti-at reaction 15 minutes, 25 DEG C It answers 2 hours, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.It is filtered to remove white precipitate, is used 30mlTHF washings are precipitated and are filtered, merging filtrate, and revolving removes THF, obtains a large amount of white solids, uses saturated sodium bicarbonate 500ml mashing washings, filter, wash repeatedly 3 times, obtain white solid.White solid is dissolved in 200mlTHF, is used at 25 DEG C Constant pressure funnel being added drop-wise in solution 3 slowly, the reaction was continued 1 hour for reaction after being added dropwise, and reaction terminates（It is subject to contact plate）. With dilute citric acid tune pH value to 7, revolving removes THF, heavy to a large amount of whites between 3-4, are obtained with dilute citric acid tune pH value again Shallow lake, filtering are beaten with dilute citric acid 300ml and are washed, and filtering obtains white solid after washing repeatedly 3 times.Product is that white is solid Body, purity：97.6%

Two, the synthesis of Fmoc-Thr (tbu)-Phe-Thr (tbu)-Ser (tbu)-Asp (Otbu)-OH

(1)、Fmoc-Thr(tbu)-Phe-OH

Solution 1：Fmoc-Thr (tbu)-OH of 100 mmoles, the HOSu of 120 mmoles are weighed, the round-bottomed flask of 500ml is placed in In, 200mlTHF dissolvings are added, be cooled under magnetic agitation 0-5 DEG C it is spare.

Solution 3：The NH2-Phe-OH for weighing 130 mmoles is dissolved in 10% sodium carbonate liquor of 100ml, spare at room temperature

At 0-5 DEG C, by DCC solution（Solution 2）It is slowly added drop-wise in the solution 1 of stirring, it is anti-at reaction 15 minutes, 25 DEG C It answers 2 hours, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.It is filtered to remove white precipitate, is used 30mlTHF washings are precipitated and are filtered, merging filtrate, and revolving removes THF, obtains a large amount of white solids, uses saturated sodium bicarbonate 500ml mashing washings, filter, wash repeatedly 3 times, obtain white solid.White solid is dissolved in 200mlTHF, is used at 25 DEG C Constant pressure funnel being added drop-wise in solution 3 slowly, the reaction was continued 1 hour for reaction after being added dropwise, and reaction terminates（It is subject to contact plate）. With dilute citric acid tune pH value to 7, revolving removes THF, heavy to a large amount of whites between 3-4, are obtained with dilute citric acid tune pH value again Shallow lake, filtering are beaten with dilute citric acid 300ml and are washed, and filtering obtains white solid after washing repeatedly 3 times.Product is that white is solid Body, purity：97.5%

(2), the synthesis of Fmoc-Thr (tbu)-Phe-Thr (tbu)-OH

Solution 1：Fmoc-Thr (tbu)-Phe-OH of 100 mmoles, the HOSu of 120 mmoles are weighed, the round bottom of 500ml is placed in In flask, 200mlTHF dissolvings are added, be cooled under magnetic agitation 0-5 DEG C it is spare.

Solution 3：NH2-Thr (the tbu)-OH for weighing 130 mmoles, is dissolved in 10% sodium carbonate liquor of 100ml, room temperature Under it is spare

(3), the synthesis of Fmoc-Thr (tbu)-Phe-Thr (tbu)-Ser (tbu)-OH

Solution 1：Fmoc-Thr (tbu)-Phe-Thr (tbu)-OH of 100 mmoles, the HOSu of 120 mmoles are weighed, is placed in In the round-bottomed flask of 500ml, 200mlTHF dissolvings are added, be cooled under magnetic agitation 0-5 DEG C it is spare.

Solution 3：NH2-Ser (the tbu)-H for weighing 130 mmoles, is dissolved in 10% sodium carbonate liquor of 100ml, at room temperature It is spare

At 0-5 DEG C, by DCC solution（Solution 2）It is slowly added drop-wise in the solution 1 of stirring, it is anti-at reaction 15 minutes, 25 DEG C It answers 2 hours, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.It is filtered to remove white precipitate, is used 30mlTHF washings are precipitated and are filtered, merging filtrate, and revolving removes THF, obtains a large amount of white solids, uses saturated sodium bicarbonate 500ml mashing washings, filter, wash repeatedly 3 times, obtain white solid.White solid is dissolved in 200mlTHF, is used at 25 DEG C Constant pressure funnel being added drop-wise in solution 3 slowly, the reaction was continued 1 hour for reaction after being added dropwise, and reaction terminates（It is subject to contact plate）. With dilute citric acid tune pH value to 7, revolving removes THF, heavy to a large amount of whites between 3-4, are obtained with dilute citric acid tune pH value again Shallow lake, filtering are beaten with dilute citric acid 300ml and are washed, and filtering obtains white solid after washing repeatedly 3 times.Product is that white is solid Body, purity：97.1%

(4)、Fmoc-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(Otbu)-OH

Solution 1：Weigh Fmoc-Thr (tbu)-Phe-Thr (tbu)-Ser (tbu)-OH of 100 mmoles, 120 mmoles HOSu is placed in the round-bottomed flask of 500ml, and 200mlTHF dissolvings are added, be cooled under magnetic agitation 0-5 DEG C it is spare.

Solution 3：NH2-Asp (the Otbu)-OH for weighing 130 mmoles, is dissolved in 10% sodium carbonate liquor of 100ml, room temperature Under it is spare

At 0-5 DEG C, by DCC solution（Solution 2）It is slowly added drop-wise in the solution 1 of stirring, it is anti-at reaction 15 minutes, 25 DEG C It answers 2 hours, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.It is filtered to remove white precipitate, is used 30mlTHF washings are precipitated and are filtered, merging filtrate, and revolving removes THF, obtains a large amount of white solids, uses saturated sodium bicarbonate 500ml mashing washings, filter, wash repeatedly 3 times, obtain white solid.White solid is dissolved in 200mlTHF, is used at 25 DEG C Constant pressure funnel being added drop-wise in solution 3 slowly, the reaction was continued 1 hour for reaction after being added dropwise, and reaction terminates（It is subject to contact plate）. With dilute citric acid tune pH value to 7, revolving removes THF, heavy to a large amount of whites between 3-4, are obtained with dilute citric acid tune pH value again Shallow lake, filtering are beaten with dilute citric acid 300ml and are washed, and filtering obtains white solid after washing repeatedly 3 times.Product is that white is solid Body, purity：97.2%

Three, the synthesis of Fmoc-Val-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-Glu (Otbu)-Gly-OH

(1), the synthesis of Fmoc-Val-Ser (tbu)-OH

Solution 1：The Fmoc-Val-OH of 100 mmoles, the HOSu of 120 mmoles are weighed, is placed in the round-bottomed flask of 500ml, adds Enter 200mlTHF dissolvings, be cooled under magnetic agitation 0-5 DEG C it is spare.

Solution 3：NH2-Ser (the tbu)-OH for weighing 130 mmoles, is dissolved in 10% sodium carbonate liquor of 100ml, room temperature Under it is spare

At 0-5 DEG C, by DCC solution（Solution 2）It is slowly added drop-wise in the solution 1 of stirring, it is anti-at reaction 15 minutes, 25 DEG C It answers 2 hours, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.It is filtered to remove white precipitate, is used 30mlTHF washings are precipitated and are filtered, merging filtrate, and revolving removes THF, obtains a large amount of white solids, uses saturated sodium bicarbonate 500ml mashing washings, filter, wash repeatedly 3 times, obtain white solid.White solid is dissolved in 200mlTHF, is used at 25 DEG C Constant pressure funnel being added drop-wise in solution 3 slowly, the reaction was continued 1 hour for reaction after being added dropwise, and reaction terminates（It is subject to contact plate）. With dilute citric acid tune pH value to 7, revolving removes THF, heavy to a large amount of whites between 3-4, are obtained with dilute citric acid tune pH value again Shallow lake, filtering are beaten with dilute citric acid 300ml and are washed, and filtering obtains white solid after washing repeatedly 3 times.Product is that white is solid Body, purity：98.5%

(2), the synthesis of Fmoc-Val-Ser (tbu)-Ser (tbu)-OH

Solution 1：Fmoc-Val-Ser (tbu)-OH of 100 mmoles, the HOSu of 120 mmoles are weighed, the round bottom of 500ml is placed in In flask, 200mlTHF dissolvings are added, be cooled under magnetic agitation 0-5 DEG C it is spare.

At 0-5 DEG C, by DCC solution（Solution 2）It is slowly added drop-wise in the solution 1 of stirring, it is anti-at reaction 15 minutes, 25 DEG C It answers 2 hours, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.It is filtered to remove white precipitate, is used 30mlTHF washings are precipitated and are filtered, merging filtrate, and revolving removes THF, obtains a large amount of white solids, uses saturated sodium bicarbonate 500ml mashing washings, filter, wash repeatedly 3 times, obtain white solid.White solid is dissolved in 200mlTHF, is used at 25 DEG C Constant pressure funnel being added drop-wise in solution 3 slowly, the reaction was continued 1 hour for reaction after being added dropwise, and reaction terminates（It is subject to contact plate）. With dilute citric acid tune pH value to 7, revolving removes THF, heavy to a large amount of whites between 3-4, are obtained with dilute citric acid tune pH value again Shallow lake, filtering are beaten with dilute citric acid 300ml and are washed, and filtering obtains white solid after washing repeatedly 3 times.Product is that white is solid Body, purity：98.2%

(3), the synthesis of Fmoc-Val-Ser (tbu)-Ser (tbu)-Tyr (tbu)-OH

Solution 1：Fmoc-Val-Ser (tbu)-Ser (tbu)-OH of 100 mmoles, the HOSu of 120 mmoles are weighed, is placed in In the round-bottomed flask of 500ml, 200mlTHF dissolvings are added, be cooled under magnetic agitation 0-5 DEG C it is spare.

Solution 3：NH2-Tyr (the tbu)-OH for weighing 130 mmoles, is dissolved in 10% sodium carbonate liquor of 100ml, room temperature Under it is spare

At 0-5 DEG C, by DCC solution（Solution 2）It is slowly added drop-wise in the solution 1 of stirring, it is anti-at reaction 15 minutes, 25 DEG C It answers 2 hours, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.It is filtered to remove white precipitate, is used 30mlTHF washings are precipitated and are filtered, merging filtrate, and revolving removes THF, obtains a large amount of white solids, uses saturated sodium bicarbonate 500ml mashing washings, filter, wash repeatedly 3 times, obtain white solid.White solid is dissolved in 200mlTHF, is used at 25 DEG C Constant pressure funnel being added drop-wise in solution 3 slowly, the reaction was continued 1 hour for reaction after being added dropwise, and reaction terminates（It is subject to contact plate）. With dilute citric acid tune pH value to 7, revolving removes THF, heavy to a large amount of whites between 3-4, are obtained with dilute citric acid tune pH value again Shallow lake, filtering are beaten with dilute citric acid 300ml and are washed, and filtering obtains white solid after washing repeatedly 3 times.Product is that white is solid Body, purity：98%.

(4), the synthesis of Fmoc-Val-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-OH

Solution 1：Weigh Fmoc-Val-Ser (tbu)-Ser (tbu)-Tyr (tbu)-OH of 100 mmoles, 120 mmoles HOSu is placed in the round-bottomed flask of 500ml, and 200mlTHF dissolvings are added, be cooled under magnetic agitation 0-5 DEG C it is spare.

Solution 3：The NH2-Leu-OH for weighing 130 mmoles is dissolved in 10% sodium carbonate liquor of 100ml, spare at room temperature

(5), the synthesis of Fmoc-Val-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-Glu (Otbu)-OH

Solution 1：Weigh Fmoc-Val-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-OH, 120 mmoles of 100 mmoles HOSu, be placed in the round-bottomed flask of 500ml, 200mlTHF dissolvings be added, be cooled under magnetic agitation 0-5 DEG C it is spare.

Solution 3：NH2-Glu (the Otbu)-OH for weighing 130 mmoles, is dissolved in 10% sodium carbonate liquor of 100ml, room temperature Under it is spare

(6), the synthesis of Fmoc-Val-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-Glu (Otbu)-Gly-OH

Solution 1：Weigh Fmoc-Val-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-Glu (Otbu)-of 100 mmoles The HOSu of OH, 120 mmoles, are placed in the round-bottomed flask of 500ml, and 200mlTHF dissolvings are added, 0-5 DEG C is cooled under magnetic agitation It is spare.

Four, Fmoc-Gln-Ala-Ala-N6- [N- (1-oxohexadecyl)-Glu (otbu)]-Lys-Glu (Otbu)

The synthesis of-Phe-OH

(1)、Fmoc-Gln-Ala-OH

Solution 1：The Fmoc-Gln-OH of 100 mmoles, the HOSu of 120 mmoles are weighed, is placed in the round-bottomed flask of 500ml, adds Enter 200mlTHF dissolvings, be cooled under magnetic agitation 0-5 DEG C it is spare.

Solution 2：The DCC for weighing 120 mmoles, is placed in small beaker, is dissolved with the THF of 100ml, be cooled to 0-5 DEG C it is spare.

Solution 3：The NH2-Ala-OH for weighing 130 mmoles is dissolved in 10% sodium carbonate liquor of 100ml, standby at room temperature With.

At 0-5 DEG C, by DCC solution（Solution 2）It is slowly added drop-wise in the solution 1 of stirring, it is anti-at reaction 15 minutes, 25 DEG C It answers 2 hours, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.It is filtered to remove white precipitate, is used 30mlTHF washings are precipitated and are filtered, merging filtrate, and revolving removes THF, obtains a large amount of white solids, uses saturated sodium bicarbonate 500ml mashing washings, filter, wash repeatedly 3 times, obtain white solid.White solid is dissolved in 200mlTHF, is used at 25 DEG C Constant pressure funnel being added drop-wise in solution 3 slowly, the reaction was continued 1 hour for reaction after being added dropwise, and reaction terminates（It is subject to contact plate）. With dilute citric acid tune pH value to 7, revolving removes THF, heavy to a large amount of whites between 3-4, are obtained with dilute citric acid tune pH value again Shallow lake, filtering are beaten with dilute citric acid 300ml and are washed, and filtering obtains white solid after washing repeatedly 3 times.Product is that white is solid Body, purity：98.5%.

(2)、Fmoc-Gln-Ala-Ala-OH

Solution 1：The Fmoc-Gln-Ala-OH of 100 mmoles, the HOSu of 120 mmoles are weighed, the round-bottomed flask of 500ml is placed in In, 200mlTHF dissolvings are added, be cooled under magnetic agitation 0-5 DEG C it is spare.

At 0-5 DEG C, by DCC solution（Solution 2）It is slowly added drop-wise in the solution 1 of stirring, it is anti-at reaction 15 minutes, 25 DEG C It answers 2 hours, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.It is filtered to remove white precipitate, is used 30mlTHF washings are precipitated and are filtered, merging filtrate, and revolving removes THF, obtains a large amount of white solids, uses saturated sodium bicarbonate 500ml mashing washings, filter, wash repeatedly 3 times, obtain white solid.White solid is dissolved in 200mlTHF, is used at 25 DEG C Constant pressure funnel being added drop-wise in solution 3 slowly, the reaction was continued 1 hour for reaction after being added dropwise, and reaction terminates（It is subject to contact plate）. With dilute citric acid tune pH value to 7, revolving removes THF, heavy to a large amount of whites between 3-4, are obtained with dilute citric acid tune pH value again Shallow lake, filtering are beaten with dilute citric acid 300ml and are washed, and filtering obtains white solid after washing repeatedly 3 times.Product is that white is solid Body, purity：98.3%.

(3), the synthesis of N6- [N- (1-oxohexadecyl)-Glu (otbu)]-OH

Solution 1：Weigh the cetyl acid of 100 mmoles（Palmitic acid）, 120 mmoles HOSu, be placed in 500ml round bottom burn In bottle, 200mlTHF dissolvings are added, be cooled under magnetic agitation 0-5 DEG C it is spare.

Solution 3：NH2-Glu (the otbu)-OH for weighing 130 mmoles, is dissolved in 10% sodium carbonate liquor of 100ml, room temperature Under it is spare.

At 0-5 DEG C, by DCC solution（Solution 2）It is slowly added drop-wise in the solution 1 of stirring, it is anti-at reaction 15 minutes, 25 DEG C It answers 2 hours, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.It is filtered to remove white precipitate, is used 30mlTHF washings are precipitated and are filtered, merging filtrate, and revolving removes THF, obtains a large amount of white solids, uses saturated sodium bicarbonate 500ml mashing washings, filter, wash repeatedly 3 times, obtain white solid.White solid is dissolved in 200mlTHF, is used at 25 DEG C Constant pressure funnel being added drop-wise in solution 3 slowly, the reaction was continued 1 hour for reaction after being added dropwise, and reaction terminates（It is subject to contact plate）. With dilute citric acid tune pH value to 7, revolving removes THF, heavy to a large amount of whites between 3-4, are obtained with dilute citric acid tune pH value again Shallow lake, filtering are beaten with dilute citric acid 300ml and are washed, and filtering obtains white solid after washing repeatedly 3 times.Product is that white is solid Body, purity：96.5%.

(4), the synthesis of Z-N6- [N- (1-oxohexadecyl)-Glu (otbu)]-Lys-OH

Solution 1：Weigh N6- [N- (1-oxohexadecyl)-Glu (otbu)]-OH of 100 mmoles, 120 mmoles HOSu is placed in the round-bottomed flask of 500ml, and 200mlDMF dissolvings are added, be cooled under magnetic agitation 0-5 DEG C it is spare.

Solution 2：The DCC for weighing 120 mmoles, is placed in small beaker, is dissolved with the DMF of 100ml, be cooled to 0-5 DEG C it is spare.

Solution 3：The Z-Lys-OH for weighing 130 mmoles is dissolved in 10% sodium carbonate liquor of 100ml, spare at room temperature.

At 0-5 DEG C, by DCC solution（Solution 2）It is slowly added drop-wise in the solution 1 of stirring, it is anti-at reaction 15 minutes, 25 DEG C It answers 2 hours, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.It is filtered to remove white precipitate, is used 30mlDMF washings are precipitated and are filtered, merging filtrate, and a large amount of pure water are added, a large amount of white solids occur, and it is solid that white is obtained by filtration Body is filtered, is washed repeatedly 3 times, obtained white solid with saturated sodium bicarbonate 500ml mashing washings.White solid is dissolved in In 200mlTHF, at 25 DEG C with constant pressure funnel being added drop-wise in solution 3 slowly, after being added dropwise reaction the reaction was continued 1 hour, instead It should terminate（It is subject to contact plate）.With dilute citric acid tune pH value to 7, revolving removes THF, again with dilute citric acid tune pH value to 3-4 Between, a large amount of white precipitates are obtained, are filtered, is beaten and is washed with dilute citric acid 300ml, filtering obtains white after washing repeatedly 3 times Solid.Product is white solid, purity：96.5%

(6), the synthesis of N6- [N- (1-oxohexadecyl)-Glu (otbu)]-Lys-OH

Z-N6- [N- (1-oxohexadecyl)-Glu (otbu)]-Lys-OH for weighing 100 mmoles, is placed in the hydrogenation of 5L In kettle, it is added the dissolving of 200ml methanol, the formic acid of 2ml, 5% palladium carbon 10g maintains forcing press when pressurized with hydrogen is to 3M at 30 DEG C Tool is stirred to react 8 hours.Detection determines that the reaction was complete, terminates reaction.It is filtered to remove palladium carbon, takes filtrate, it is molten that revolving removes methanol Liquid is beaten washing precipitation with 200ml anhydrous ethers, is repeated 3 times, and acquisition product is white solid, purity 98%.

(7), the synthesis of Fmoc-Gln-Ala-Ala-N6- [N- (1-oxohexadecyl)-Glu (otbu)]-Lys-OH

Solution 1：The Fmoc-Gln-Ala-Ala-OH of 100 mmoles, the HOSu of 120 mmoles are weighed, the round bottom of 500ml is placed in In flask, 200mlDMF dissolvings are added, be cooled under magnetic agitation 0-5 DEG C it is spare.

Solution 2：The DCC for weighing 120 mmoles, is placed in small beaker, is dissolved with the DMF of 100ml, be cooled to 0-5 DEG C it is spare

Solution 3：N6- [N- (1-oxohexadecyl)-Glu (otbu)]-Lys-OH for weighing 130 mmoles, is dissolved in 100ml 10% sodium carbonate liquor in, it is spare at room temperature

At 0-5 DEG C, by DCC solution（Solution 2）It is slowly added drop-wise in the solution 1 of stirring, it is anti-at reaction 15 minutes, 25 DEG C It answers 2 hours, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.It is filtered to remove white precipitate, is used 30mlDMF washings are precipitated and are filtered, merging filtrate, and a large amount of pure water are added, a large amount of white solids occur, and it is solid that white is obtained by filtration Body is filtered, is washed repeatedly 3 times, obtained white solid with saturated sodium bicarbonate 500ml mashing washings.White solid is dissolved in In 200mlTHF, at 25 DEG C with constant pressure funnel being added drop-wise in solution 3 slowly, after being added dropwise reaction the reaction was continued 1 hour, instead It should terminate（It is subject to contact plate）.With dilute citric acid tune pH value to 7, revolving removes THF, again with dilute citric acid tune pH value to 3-4 Between, a large amount of white precipitates are obtained, are filtered, is beaten and is washed with dilute citric acid 300ml, filtering obtains white after washing repeatedly 3 times Solid.Product is white solid, purity：96.7%

(8)、Fmoc-Gln-Ala-Ala-N6-[N-(1-oxohexadecyl)-Glu(otbu)]-Lys-Glu(Otbu)- OH

Solution 1：Weigh Fmoc-Gln-Ala-Ala-N6- [N- (1-oxohexadecyl)-Glu of 100 mmoles (otbu)]-Lys-OH, 120 mmoles HOSu, be placed in the round-bottomed flask of 500ml, be added 200mlDMF dissolving, magnetic agitation Under be cooled to 0-5 DEG C it is spare.

At 0-5 DEG C, by DCC solution（Solution 2）It is slowly added drop-wise in the solution 1 of stirring, it is anti-at reaction 15 minutes, 25 DEG C It answers 2 hours, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.It is filtered to remove white precipitate, is used 30mlDMF washings are precipitated and are filtered, merging filtrate, and a large amount of pure water are added, a large amount of white solids occur, and it is solid that white is obtained by filtration Body is filtered, is washed repeatedly 3 times, obtained white solid with saturated sodium bicarbonate 500ml mashing washings.White solid is dissolved in In 200mlTHF, at 25 DEG C with constant pressure funnel being added drop-wise in solution 3 slowly, after being added dropwise reaction the reaction was continued 1 hour, instead It should terminate（It is subject to contact plate）.With dilute citric acid tune pH value to 7, revolving removes THF, again with dilute citric acid tune pH value to 3-4 Between, a large amount of white precipitates are obtained, are filtered, is beaten and is washed with dilute citric acid 300ml, filtering obtains white after washing repeatedly 3 times Solid.Product is white solid, purity：96.3%

(9)、Fmoc-Gln-Ala-Ala-N6-[N-(1-oxohexadecyl)-Glu(otbu)]-Lys-Glu(Otbu)- Phe-OH

Solution 1：Weigh Fmoc-Gln-Ala-Ala-N6- [N- (1-oxohexadecyl)-Glu of 100 mmoles (otbu)]-Lys-Glu (Otbu)-OH, 120 mmoles HOSu, be placed in the round-bottomed flask of 500ml, be added 200mlDMF it is molten Solution, be cooled under magnetic agitation 0-5 DEG C it is spare.

At 0-5 DEG C, by DCC solution（Solution 2）It is slowly added drop-wise in the solution 1 of stirring, it is anti-at reaction 15 minutes, 25 DEG C It answers 2 hours, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.It is filtered to remove white precipitate, is used 30mlDMF washings are precipitated and are filtered, merging filtrate, and a large amount of pure water are added, a large amount of white solids occur, and it is solid that white is obtained by filtration Body is filtered, is washed repeatedly 3 times, obtained white solid with saturated sodium bicarbonate 500ml mashing washings.White solid is dissolved in In 200mlTHF, at 25 DEG C with constant pressure funnel being added drop-wise in solution 3 slowly, after being added dropwise reaction the reaction was continued 1 hour, instead It should terminate（It is subject to contact plate）.With dilute citric acid tune pH value to 7, revolving removes THF, again with dilute citric acid tune pH value to 3-4 Between, a large amount of white precipitates are obtained, are filtered, is beaten and is washed with dilute citric acid 300ml, filtering obtains white after washing repeatedly 3 times Solid.Product is white solid, purity：97.1%

Five, the synthesis of Fmoc-Ile-Ala-Trp (Boc)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-otbu

(1)、Fmoc-Arg(pbf)-Gly-otbu

Solution 1, the NH2-Gly-otbu for weighing 120 mmoles, Fmoc-Arg (pbf)-OH of 100 mmoles, 120 mmoles HOBT is placed in the round-bottomed flask of 500ml, is dissolved with the DMF of 200ml, be cooled to 0 DEG C it is spare.

At 0-5 DEG C, by DCC solution（Solution 2）It is slowly added drop-wise in the solution 1 of stirring, it is anti-at reaction 15 minutes, 25 DEG C It answers 2 hours, contact plate determines that reaction terminates（The specific reaction time is subject to the contact plate time）.It is filtered to remove white precipitate, is used 30mlDMF washings are precipitated and are filtered, merging filtrate, and a large amount of pure water are added, a large amount of white solids occur, and it is solid that white is obtained by filtration Body.White solid 1L ethyl acetate dissolves, and is fitted into separatory funnel, is washed 3 times with dilute citric acid 150ml respectively, with saturation Sodium bicarbonate 150ml is washed 3 times, is washed 2 times with saturated salt solution 200ml, 2 hours dry, and revolving removes solvent and obtains product. Purity is 98.5%.

(2), the synthesis of Fmoc-Gly-Arg (pbf)-Gly-otbu

Solution 1：Fmoc-Arg (the pbf)-Gly-otbu for weighing 100 mmoles is placed in the round-bottomed flask of 500ml, and magnetic force stirs Mix lower addition piperidines：Dichloromethane=1：4 solution dissolving, is deprotected 30 minutes, bulk petroleum ether is added at room temperature, is precipitated big Amount precipitation, filters, and for several times with petroleum ether, obtains white solid product（NH2-Arg(pbf)-Gly-otbu）, purity is 99%.The solid DMF of 100ml dissolves, be cooled to 0-5 DEG C it is spare

Solution 2：The Fmoc-Gly-OH of 100 mmoles, the HOBT of 120 mmoles are weighed, is placed in the round-bottomed flask of 500ml, is used The DMF of 200ml dissolves, be cooled to 0 DEG C it is spare.

Solution 3：The DCC for weighing 120 mmoles, is placed in small beaker, is dissolved with the DMF of 100ml, be cooled to 0-5 DEG C it is spare

At 0-5 DEG C, by DCC solution（Solution 3）It is slowly added drop-wise in the solution 2 of stirring, reacts 15 minutes, by solution 1 It is slowly added drop-wise in reaction solution, is reacted 2 hours at being reacted 15 minutes, 25 DEG C at 0-5 DEG C, contact plate determines that reaction terminates（Specifically Reaction time is subject to the contact plate time）.It is filtered to remove white precipitate, is washed with 30mlDMF and precipitates and filter, merging filtrate adds Enter a large amount of pure water, a large amount of white solids occurs, white solid is obtained by filtration.White solid 1L ethyl acetate dissolves, and is packed into and divides It in liquid funnel, is washed 3 times with dilute citric acid 150ml respectively, is washed 3 times with saturated sodium bicarbonate 150ml, use saturated salt solution 200ml is washed 2 times, 2 hours dry, and revolving removes solvent and obtains product.Purity is 98.3%.

(3)、Fmoc-Arg(pbf)-Gly-Arg(pbf)-Gly-otbu

Solution 1：Fmoc-Gly-Arg (the pbf)-Gly-otbu for weighing 100 mmoles is placed in the round-bottomed flask of 500ml, magnetic Power is added with stirring piperidines：Dichloromethane=1：4 solution dissolving, is deprotected 30 minutes at room temperature, and bulk petroleum ether, analysis is added Go out a large amount of precipitations, filter, for several times with petroleum ether, obtains white solid product（NH2-Gly-Arg(pbf)-Gly-otbu）, Purity is 98.5%.The solid DMF of 100ml dissolves, be cooled to 0-5 DEG C it is spare

Solution 2：Fmoc-Arg (pbf)-OH of 100 mmoles, the HOBT of 120 mmoles are weighed, the round-bottomed flask of 500ml is placed in In, dissolved with the DMF of 200ml, be cooled to 0 DEG C it is spare.

At 0-5 DEG C, by DCC solution（Solution 3）It is slowly added drop-wise in the solution 2 of stirring, reacts 15 minutes, by solution 1 It is slowly added drop-wise in reaction solution, is reacted 2 hours at being reacted 15 minutes, 25 DEG C at 0-5 DEG C, contact plate determines that reaction terminates（Specifically Reaction time is subject to the contact plate time）.It is filtered to remove white precipitate, is washed with 30mlDMF and precipitates and filter, merging filtrate adds Enter a large amount of pure water, a large amount of white solids occurs, white solid is obtained by filtration.White solid 1L ethyl acetate dissolves, and is packed into and divides It in liquid funnel, is washed 3 times with dilute citric acid 150ml respectively, is washed 3 times with saturated sodium bicarbonate 150ml, use saturated salt solution 200ml is washed 2 times, 2 hours dry, and revolving removes solvent and obtains product.Purity is 98%.

(4)、Fmoc-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-otbu

Solution 1：Fmoc-Arg (pbf)-Gly-Arg (the pbf)-Gly-otbu for weighing 100 mmoles is placed in the round bottom of 500ml In flask, piperidines is added under magnetic agitation：Dichloromethane=1：4 solution dissolving, is deprotected 30 minutes at room temperature, is added a large amount of Petroleum ether is precipitated a large amount of precipitations, filters, and for several times with petroleum ether, obtains white solid product（NH2- Arg(pbf )- Gly-Arg(pbf)-Gly-otbu）, purity 98.2%.The solid DMF of 100ml dissolves, be cooled to 0-5 DEG C it is spare

Solution 2：The Fmoc-Val-OH of 100 mmoles, the HOBT of 120 mmoles are weighed, is placed in the round-bottomed flask of 500ml, is used The DMF of 200ml dissolves, be cooled to 0 DEG C it is spare.

(5)、Fmoc-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-otbu

Solution 1：Fmoc-Val-Arg (pbf)-Gly-Arg (the pbf)-Gly-otbu for weighing 100 mmoles is placed in 500ml's In round-bottomed flask, piperidines is added under magnetic agitation：Dichloromethane=1：4 solution dissolving, is deprotected 30 minutes, is added at room temperature Bulk petroleum ether is precipitated a large amount of precipitations, filters, and for several times with petroleum ether, obtains white solid product（NH2- Val-Arg (pbf)-Gly-Arg(pbf)-Gly-otbu）, purity 97.8%.The solid DMF of 100ml dissolves, be cooled to 0-5 DEG C it is spare

Solution 2：The Fmoc-Leu-OH of 100 mmoles, the HOBT of 120 mmoles are weighed, is placed in the round-bottomed flask of 500ml, is used The DMF of 200ml dissolves, be cooled to 0 DEG C it is spare.

(6), the synthesis of Fmoc-Trp (Boc)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-otbu

Solution 1：Fmoc-Leu-Val-Arg (pbf)-Gly-Arg (the pbf)-Gly-otbu for weighing 100 mmoles is placed in In the round-bottomed flask of 500ml, piperidines is added under magnetic agitation：Dichloromethane=1：4 solution dissolving, is deprotected 30 points at room temperature Bulk petroleum ether is added in clock, and a large amount of precipitations are precipitated, filter, and for several times with petroleum ether, obtains white solid product（NH2- Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-otbu）, purity 98.8%.The solid DMF of 100ml dissolves, cooling It is spare to 0-5 DEG C

Solution 2：Fmoc-Trp (Boc)-OH of 100 mmoles, the HOBT of 120 mmoles are weighed, the round-bottomed flask of 500ml is placed in In, dissolved with the DMF of 200ml, be cooled to 0 DEG C it is spare.

(7), the synthesis of Fmoc-Ala-Trp (boc)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-otbu

Solution 1：Weigh 100 mmoles Fmoc-Trp (Boc)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-otbu

In the round-bottomed flask of 500ml, piperidines is added under magnetic agitation：Dichloromethane=1：4 solution dissolving, at room temperature Bulk petroleum ether is added in deprotection 30 minutes, and a large amount of precipitations are precipitated, filter, and for several times with petroleum ether, obtains white solid production Product（NH2-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-otbu）, purity 98.6%.Solid is used The DMF of 100ml dissolves, be cooled to 0-5 DEG C it is spare

(8), the conjunction of Fmoc-Ile-Ala-Trp (boc)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-otbu At

Solution 1：Weigh Fmoc-Ala-Trp (boc)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-of 100 mmoles Gly-otbu is placed in the round-bottomed flask of 500ml, and piperidines is added under magnetic agitation：Dichloromethane=1：4 solution dissolving, room temperature Lower deprotection 30 minutes, is added bulk petroleum ether, and a large amount of precipitations are precipitated, filter, and for several times with petroleum ether, obtains white solid Product（NH2-Ala-Trp(boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-otbu）, purity 99%.Solid Dissolved with the DMF of 100ml, be cooled to 0-5 DEG C it is spare

Solution 2：The Fmoc-Ile-OH of 100 mmoles, the HOBT of 120 mmoles are weighed, is placed in the round-bottomed flask of 500ml, is used The DMF of 200ml dissolves, be cooled to 0 DEG C it is spare.

At 0-5 DEG C, by DCC solution（Solution 3）It is slowly added drop-wise in the solution 2 of stirring, reacts 15 minutes, by solution 1 It is slowly added drop-wise in reaction solution, is reacted 2 hours at being reacted 15 minutes, 25 DEG C at 0-5 DEG C, contact plate determines that reaction terminates（Specifically Reaction time is subject to the contact plate time）.It is filtered to remove white precipitate, is washed with 30mlDMF and precipitates and filter, merging filtrate adds Enter a large amount of pure water, a large amount of white solids occurs, white solid is obtained by filtration.White solid 1L ethyl acetate dissolves, and is packed into and divides It in liquid funnel, is washed 3 times with dilute citric acid 150ml respectively, is washed 3 times with saturated sodium bicarbonate 150ml, use saturated salt solution 200ml is washed 2 times, 2 hours dry, and revolving removes solvent and obtains product.Purity is 96%.

Six, segment Fmoc-Gln-Ala-Ala-N6- [N- (1-oxohexadecyl)-Glu (otbu)]-Lys-Glu (Otbu)-Phe

The synthesis of-Ile-Ala-Trp (boc)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-otbu

Weigh Fmoc-Ile-Ala-Trp (boc)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly- of 100 mmoles Otbu is placed in the round-bottomed flask of 500ml, and piperidines is added under magnetic agitation：Dichloromethane=1：4 solution dissolving, takes off at room temperature Bulk petroleum ether is added in protection 30 minutes, and a large amount of precipitations are precipitated, filter, and for several times with petroleum ether, obtains white solid production Product, purity 94%.

Weigh Fmoc-Gln-Ala-Ala-N6- [N- (1-oxohexadecyl)-Glu (otbu)]-Lys-Glu (Otbu)

110 mmoles of-Phe-OH segments, NH2-Ile-Ala-Trp (boc)-Leu-Val-Arg (pbf)-Gly-Arg (pbf) 100 mmoles of-Gly-otbu segments and 130 mmoles of triethylamine are dissolved in 2700mlDMF, and diphenyl phosphinylidyne is added at 0 DEG C 120 mmoles of trinitride.It is stirred 6 hours at 0 DEG C, then 25 DEG C are stirred 20 hours.300ml dilute hydrochloric acid to be slowly added at 0 DEG C molten later Under stirring, there are a large amount of white precipitates, solid is obtained by filtration in liquid.Solid 400ml ethyl acetate dissolves, and separatory funnel carries out Washing.Respectively dilute sodium bicarbonate 50mlX3, dilute hydrochloric acid 30mlX3 finally use saturated common salt water washing 50mlX3, solution transfer To triangular flask, anhydrous sodium sulfate is added or anhydrous magnesium sulfate is dried.It is about 10 hours dry.Revolving removes solvent（Recycling It utilizes）, obtain white solid.Purity is 92%.

Seven, Fmoc-Val-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-Glu (Otbu)-Gly-Gln-Ala-Ala- N6-

[N-(1-oxohexadecyl)-Glu(otbu)]-Lys-Glu(Otbu)-Phe-Ile-Ala-Trp(boc)- The synthesis of Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-otbu

Weigh Fmoc-Gln-Ala-Ala-N6- [N- (1-oxohexadecyl)-Glu (otbu)]-Lys of 100 mmoles

-Glu(Otbu)-Phe-Ile-Ala-Trp(boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly- Otbu is placed in the round-bottomed flask of 500ml, and piperidines is added under magnetic agitation：Dichloromethane=1：4 solution dissolving, takes off at room temperature Bulk petroleum ether is added in protection 30 minutes, and a large amount of precipitations are precipitated, filter, and for several times with petroleum ether, obtains white solid production Product, purity 92.5%.

Weigh 110 milli of Fmoc-Val-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-Glu (Otbu)-Gly-OH segments It rubs, NH2-Gln-Ala-Ala-N6- [N- (1-oxohexadecyl)-Glu (otbu)]-Lys-Glu (Otbu)

100 milli of-Phe-Ile-Ala-Trp (boc)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-otbu segments It rubs and 130 mmoles of triethylamine is dissolved in 2700mlDMF, 120 mmoles of diphenylphosphoryl azide object are added at 0 DEG C.6 are stirred at 0 DEG C Hour, then 25 DEG C stir 20 hours.300ml dilute hydrochloric acid solutions are slowly added at 0 DEG C later, under stirring, it is heavy a large amount of whites occur It forms sediment, solid is obtained by filtration.Solid 400ml ethyl acetate dissolves, and separatory funnel is washed.Respectively dilute sodium bicarbonate 50mlX3, dilute hydrochloric acid 30mlX3 finally use saturated common salt water washing 50mlX3, solution to be transferred to triangular flask, anhydrous slufuric acid are added Sodium or anhydrous magnesium sulfate are dried.It is about 10 hours dry.Revolving removes solvent（It recycles）, obtain white solid.It is pure Degree is 89%.

Eight, Fmoc-Thr (tbu)-Phe-Thr (tbu)-Ser (tbu)-Asp (Otbu)-Val-Ser (tbu)-Ser (tbu)

-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln-Ala-Ala-N6-[N-(1-oxohexadecyl)-Glu (otbu)]-Lys-Glu(Otbu)-Phe-Ile-Ala-Trp(boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly- The synthesis of otbu

Weigh 100 mmoles Fmoc-Val-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-Glu (Otbu)-Gly-Gln

-Ala-Ala-N6-[N-(1-oxohexadecyl)-Glu(otbu)]-Lys-Glu(Otbu)-Phe-Ile-Ala- Trp (boc)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-otbu is placed in the round-bottomed flask of 500ml, magnetic agitation Lower addition piperidines：Dichloromethane=1：4 solution dissolving, is deprotected 30 minutes, bulk petroleum ether is added at room temperature, is precipitated a large amount of Precipitation filters, and for several times with petroleum ether, obtains white solid product, purity 90.5%.

Weigh 110 milli of Fmoc-Val-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-Glu (Otbu)-Gly-OH segments It rubs, NH2-Gln-Ala-Ala-N6- [N- (1-oxohexadecyl)-Glu (otbu)]-Lys-Glu (Otbu)-Phe

100 mmoles of-Ile-Ala-Trp (boc)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-otbu segments, It is dissolved in 2700mlDMF with 130 mmoles of triethylamine, 120 mmoles of diphenylphosphoryl azide object is added at 0 DEG C.Stirring 6 is small at 0 DEG C When, then 25 DEG C stir 20 hours.300ml dilute hydrochloric acid solutions are slowly added at 0 DEG C later, under stirring, a large amount of white precipitates occur, Solid is obtained by filtration.Solid 400ml ethyl acetate dissolves, and separatory funnel is washed.Respectively dilute sodium bicarbonate 50mlX3, Dilute hydrochloric acid 30mlX3 finally uses saturated common salt water washing 50mlX3, solution to be transferred to triangular flask, anhydrous sodium sulfate or nothing is added Water magnesium sulfate is dried.It is about 10 hours dry.Revolving removes solvent（It recycles）, obtain white solid.Purity is 85%.

Nine, Fmoc-His (trt)-Ala-Glu (Otbu)-Gly-Thr (tbu)-Phe-Thr (tbu)-Ser (tbu)-Asp (Otbu)- Val-Ser(tbu)-Ser(tbu)-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln-Ala-Ala-N6-[N- (1-oxohexadecyl)-Glu(otbu)]-Lys-Glu(Otbu)-Phe-Ile-Ala-Trp(boc)-Leu-Val-Arg (pbf)-Gly-Arg(pbf)

The synthesis of-Gly-otbu

Weigh Fmoc-Thr (tbu)-Phe-Thr (tbu)-Ser (tbu)-Asp (Otbu)-Val-Ser of 100 mmoles (tbu)-Ser(tbu)-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln-Ala-Ala-N6-[N-(1-oxohexadecyl)- Glu(otbu)]-Lys-Glu(Otbu)-Phe-Ile-Ala-Trp(boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)- Gly-otbu is placed in the round-bottomed flask of 500ml, and piperidines is added under magnetic agitation：Dichloromethane=1：4 solution dissolving, room temperature Lower deprotection 30 minutes, is added bulk petroleum ether, and a large amount of precipitations are precipitated, filter, and for several times with petroleum ether, obtains white solid Product, purity 88.5%.

Weigh 110 mmoles of Fmoc-His (trt)-Ala-Glu (Otbu)-Gly-OH segments, NH2-Thr (tbu)-Phe- Thr(tbu)-Ser(tbu)-Asp(Otbu)- Val-Ser(tbu)-Ser(tbu)-Tyr(tbu)

-Leu-Glu(Otbu)-Gly-Gln-Ala-Ala-N6-[N-(1-oxohexadecyl)-Glu(otbu)]-Lys- Glu (Otbu)-Phe-Ile-Ala-Trp (boc)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-otbu segments 100 Mmoles and 130 mmoles of triethylamine are dissolved in 2700mlDMF, and 120 mmoles of diphenylphosphoryl azide object are added at 0 DEG C.It is stirred at 0 DEG C It mixes 6 hours, then 25 DEG C are stirred 20 hours.300ml dilute hydrochloric acid solutions are slowly added at 0 DEG C later, under stirring, a large amount of whites occur Precipitation, is obtained by filtration solid.Solid 400ml ethyl acetate dissolves, and separatory funnel is washed.Respectively dilute sodium bicarbonate 50mlX3, dilute hydrochloric acid 30mlX3 finally use saturated common salt water washing 50mlX3, solution to be transferred to triangular flask, anhydrous slufuric acid are added Sodium or anhydrous magnesium sulfate are dried.It is about 10 hours dry.Revolving removes solvent（It recycles）, obtain white solid.It is pure Degree is 80%.

Ten, NH2-His (trt)-Ala-Glu (Otbu)-Gly-Thr (tbu)-Phe-Thr (tbu)-Ser (tbu)-Asp (Otbu)- Val-Ser(tbu)-Ser(tbu)-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln-Ala-Ala-N6-[N- (1-oxohexadecyl)-Glu(otbu)]-Lys-Glu(Otbu)-Phe-Ile-Ala-Trp(boc)-Leu-Val-Arg (pbf) synthesis of-Gly-Arg (pbf)-Gly-otbu

Weigh Fmoc-His (trt)-Ala-Glu (Otbu)-Gly-Thr (tbu)-Phe-Thr (tbu)-of 100 mmoles Ser(tbu)-Asp(Otbu)

-Val-Ser(tbu)-Ser(tbu)-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln-Ala-Ala-N6-[N- (1-oxohexadecyl)-Glu(otbu)]-Lys-Glu(Otbu)-Phe-Ile-Ala-Trp(boc)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-otbu is placed in the round-bottomed flask of 500ml, and piperidines is added under magnetic agitation：Dichloromethane= 1：4 solution dissolving, is deprotected 30 minutes, bulk petroleum ether is added at room temperature, and a large amount of precipitations are precipitated, filters, is washed with petroleum ether It washs for several times, obtains white solid product, purity 88.5%.

11, the synthesis of the thick peptide of Liraglutide

Weigh NH2-His (trt)-Ala-Glu (Otbu)-Gly-Thr (tbu)-Phe-Thr (tbu)-Ser of 100 mmoles (tbu)-Asp(Otbu)- Val-Ser(tbu)-Ser(tbu)-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln-Ala- Ala-N6-[N-(1-oxohexadecyl)-Glu(otbu)]-Lys-Glu(Otbu)-Phe-Ile-Ala-Trp(boc)-Leu- Val-Arg (pbf)-Gly-Arg (pbf)-Gly-otbu is placed in the reaction kettle of 50L, and deprotecting regent is added at room temperature （TFA：Thioanisole：Water：EDT=92:4:2:2）30L is cracked 1.5 hours at room temperature, and lysate poured into and has been pre-cooled （0℃）In anhydrous ether, stirring sedimentation 3 hours, centrifugation is washed 5 times with anhydrous ether, is dried in vacuo, is obtained white solid.It is pure Degree is 75.3%, yield 89%.

12, prepared by the purifying of Liraglutide product

Weigh 100 mmoles the thick peptide acetonitrile of Liraglutide and water mixed liquor dissolving, using C8 or C18 prepare column into 3 purifying of row simultaneously obtain the product that purity is more than 99.9%, molecular weight 3751, peptide sequence measurement knot by turning salt and freeze-drying By for：Detect that the overall amino acid sequence of test sample Liraglutide is：HAEGTFTSDVSSYLE GQAAK(γ-Pal) EFIAWLVRGRG。

Specific mobile phase, equipment, system are as follows：

One pure equipment：LC6000 3# U3000 2# UV220nm

System：Mobile phase A is that 0.1% acetic acid Mobile phase B is acetonitrile

Flow velocity：60ml/ minutes

Gradient：B mobile phases 5% maintain 5 minutes, 2 minutes mobile phase 5%-35% used times of B, the B mobile phase 35%-55% used times 40 Minute

Two pure equipment：LC6000 3# U3000 2# UV220nm

System：Mobile phase A is that 0.1%TFA Mobile phase Bs are acetonitrile

Flow velocity：50ml/ minutes

Gradient：B mobile phases 5% maintain 5 minutes, 1 minute mobile phase 5%-45% used time of B, the B mobile phase 45%-55% used times 40 Minute.

As a result referring to Fig.1-6.The method of the present invention obtains product from purifying is synthesized to, and the total recovery of Liraglutide is rubbed for 35% That yield, yield maintains an equal level with existing literature report or slightly above reports substantially, but overall time cost, and Material Cost etc. is significantly It reduces, and is suitable for industrialized production.

Claims

1. a kind of synthetic method of Liraglutide, it is characterised in that：The synthetic method is completed in pure liquid-phase system, including such as Lower step：

A, fragment condensation is at all kinds of Liraglutide intermediates：

（1）The synthesis of R-His (R1)-Ala-Glu (R2)-Gly-R3；

（2）The synthesis of R-Thr (R4)-Phe-Thr (R4)-Ser (R4)-Asp (R5)-R3；

（3）The synthesis of R-Val-Ser (R4)-Ser (R4)-Tyr (R4)-Leu-Glu (R2)-Gly-R3；

（4）The conjunction of R-Gln-Ala-Ala-N6- [N- (1-oxohexadecyl)-Glu (R2)]-Lys-Glu (R2)-Phe-R3 At；

（6）The conjunction of R-His (R1)-Ala-Glu (R2)-Gly-Thr (R4)-Phe-Thr (R4)-Ser (R4)-Asp (R5)-R3 At；

（7）R-His(R1)-Ala-Glu(R2)-Gly-Thr(R4)-Phe-Thr(R4)-Ser(R4)-Asp(R5)- Val-Ser (R4) synthesis of-Ser (R4)-Tyr (R4)-Leu-Glu (R2)-Gly-R3；

（8）R-His(R1)-Ala-Glu(R2)-Gly-Thr(R4)-Phe-Thr(R4)-Ser(R4)-Asp(R5)- Val-Ser (R4)-Ser(R4)-Tyr(R4)-Leu-Glu(R2)-Gly-Gln-Ala-Ala-N6-[N-(1-oxohexadecyl)-Glu (R2)] synthesis of-Lys-Glu (R2)-Phe-R3；

（9）R-Thr(R4)-Phe-Thr(R4)-Ser(R4)-Asp(R5)-Val-Ser(R4)-Ser(R4)-Tyr(R4)-Leu

The synthesis of-Glu (R2)-Gly-R3；

（10）R-Thr(R4)-Phe-Thr(R4)-Ser(R4)-Asp(R5)-Val-Ser(R4)-Ser(R4)-Tyr(R4)- Leu-

Glu(R2)-Gly- Gln-Ala-Ala-N6-[N-(1-oxohexadecyl)-Glu(R2)]-Lys-Glu(R2)-Phe- The synthesis of R3；

（11）R-Thr(R4)-Phe-Thr(R4)-Ser(R4)-Asp(R5)-Val-Ser(R4)-Ser(R4)-Tyr(R4)-Leu

-Glu(R2)-Gly- Gln-Ala-Ala-N6-[N-(1-oxohexadecyl)-Glu(R2)]-Lys-Glu(R2)- Phe-Ile-Ala

The synthesis of-Trp (R6)-Leu-Val-Arg (R7)-Gly-Arg (R7)-Gly-R3；

（12）Val-Ser(R4)-Ser(R4)-Tyr(R4)-Leu-Glu(R2)-Gly- Gln-Ala-Ala-N6-

The synthesis of [N- (1-oxohexadecyl)-Glu (R2)]-Lys-Glu (R2)-Phe-R3；

（13）Val-Ser(R4)-Ser(R4)-Tyr(R4)-Leu-Glu(R2)-Gly- Gln-Ala-Ala-N6

-[N-(1-oxohexadecyl)-Glu(R2)]-Lys-Glu(R2)-Phe-Ile-Ala-Trp(R6)-Leu-Val-Arg (R7) synthesis of-Gly-Arg (R7)-Gly-R3；

（14）R-His(R1)-Ala-Glu(R2)-Gly-Thr(R4)-Phe-Thr(R4)-Ser(R4)-Asp(R5)-Val-Ser (R4)

-Ser(R4)-Tyr(R4)-Leu-Glu(R2)-Gly-Gln-Ala-Ala-N6-[N-(1-oxohexadecyl)-Glu (R2)] conjunction of-Lys-Glu (R2)-Phe-Ile-Ala-Trp (R6)-Leu-Val-Arg (R7)-Gly-Arg (R7)-Gly-R3 At；

After above-described various protected amino acid fragment condensations, the various intermediate fragments of Liraglutide of formation；Wherein：R is selected From Z, Boc, Cl-Z, Fmoc, oNBS, dNBS, Troc, Dts, pNZ, oNZ, NVOC, NPPOC, HFA, Ddz, Bpoc, Nps, Nsc, Bsmoc, α-Nsmoc, ivDde, Fmoc*, MTT, Alloc wherein any one；

R2 be selected from H, Otbu, Bn, cHX, Mpe, 2-ph1pr, TEGbn, Damb, Al, pNB, pTMSE, Dmnb wherein any one；

R3 be selected from H, Otbu, Bn, cHX, Mpe, 2-ph1pr, TEGbn, Damb, Al, pNB, pTMSE, Dmnb wherein any one；

R4 be selected from Bn, cHx, tBu, Trt, TBDMS, Dmnb, Poc wherein any one；

R5 be selected from H, Otbu, Bn, cHX, Mpe, 2-ph1pr, TEGbn, Damb, Al, pNB, pTMSE, Dmnb wherein any one；

R6 be selected from Z, Boc, Cl-Z, Fmoc, For, Hoc, Mts, Alloc wherein any one；

R7 be selected from TOS, Pbf, Pmc, Mts, Mtr, Mis wherein any one；

It is wherein arbitrary that R8 is selected from MTT, Alloc, Z, Cl-Z, oNBS, dNBS, Troc, Dts, pNZ, oNZ, NVOC, NPPOC, HFA One；

B, the deprotection of full guard Liraglutide, cracking,

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala- Ala-N6-[N-(1-oxohexadecyl)-Glu]-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg- Gly-OH；

C, the purifying and freeze-drying of Liraglutide；

The concrete operations of above-mentioned steps A-C are as follows：

（1）The synthesis of R-Gln-Ala-Ala-N6- [N- (1-oxohexadecyl)-Glu]-Lys-Glu (R2)-Phe-R3 uses Pure liquid phase synthesis, including a variety of segment synthetic methods, specific segment are as follows：

R-Gln-Ala-OH、R-Gln-Ala-Ala-OH、R-Gln-Ala-Ala-N6-[N-(1-oxohexadecyl)-Glu]- Lys(R8)-OH、R-Gln-Ala-Ala-N6-[N-(1-oxohexadecyl)-Glu]-Lys(R8)-Glu(R2)-R3、R- Phe-R3、R-Glu(R2)-Phe-R3、 R- N6-[N-(1-oxohexadecyl)-Glu]-Lys(R8)-Glu(R2)-Phe- R3、R-Ala- N6-[N-(1-oxohexadecyl)-Glu]-Lys(R8)-Glu(R2)-Phe-R3、R-Ala-Ala- N6- [N-(1-oxohexadecyl)-Glu]-Lys(R8)-Glu(R2)-R3；

（2）N- (1-oxohexadecyl)-Glu (R2)]-R3 synthesis：

Using NH2-Glu (R2)-OH and stearic active ester carry out that purpose product is obtained by the reaction；It is used in active ester method synthesis Single condensing agent and condensing agent compound be：DIC、DCC、EDC、TBTU、PyBOP、HBTU、DIC/HOBT、DCC/HOBT、EDC/ HOBT；Or it with stearic acid anhydrides carries out that product is obtained by the reaction using NH2-Glu (R2)-OH；Acid anhydrides method using chloracetyl chloride, Ethyl chloroformate, isobutylchloroformate, isobutyl chlorocarbonate；Or using sulphur benzyl ester first with stearic acid at thioesters, Zhi Houyu NH2-Glu (R2)-OH carries out ester exchange reaction, obtains product；Or azide is formed using stearic acid, later and NH2- Glu (R2)-OH forms product；

（3）, R-N6- [N- (1-oxohexadecyl)-Glu]-Lys-R3 synthesis

Using N- (1-oxohexadecyl)-Glu (R2)] the active ester of-OH with Z-Lys-OH carries out that purpose product is obtained by the reaction； The single condensing agent used and condensing agent compound are in active ester method synthesis：DIC、DCC、EDC、TBTU、PyBOP、HATU、 HBTU、DIC/HOBT、DCC/HOBT、EDC/HOBT；Or use N- (1-oxohexadecyl)-Glu (R2)]-OH acid anhydrides It carries out that product is obtained by the reaction with Fmoc-Lys-OH；Acid anhydrides method is using chloracetyl chloride, ethyl chloroformate, isobutylchloroformate, chloromethane Sour isobutyl ester；Or using sulphur benzyl ester first with N- (1-oxohexadecyl)-Glu (R2)]-OH is at thioesters, later and Z-Lys- OH carries out ester exchange reaction, obtains product；Or use N- (1-oxohexadecyl)-Glu (R2)]-OH formation azide, Later product is formed with Z-Lys-OH；

(4), the synthesis of NH2-N6- [N- (1-oxohexadecyl)-Glu]-Lys-OH：

2. a kind of synthetic method of Liraglutide according to claim 1, it is characterised in that：14 polypeptide fragments Building-up process in the coupling system used be selected from：

（1）Single condensing agent：DIC, DCC, EDC, chloracetyl chloride, azide, TBTU, PyBOP, HBTU；Or

（2）Two system condensing agents：DIC/HOBT、DCC/HOBT、EDC/HOBT.

3. the synthetic method of Liraglutide according to claim 1, it is characterised in that：Each fragment condensation uses Acibenzolar Method, mixed anhydride method, azido compound method or sulphur benzyl ester exchange process.