CN103864918B - A kind of solid phase synthesis process of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] - Google Patents

A kind of solid phase synthesis process of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] Download PDF

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CN103864918B
CN103864918B CN201410125860.8A CN201410125860A CN103864918B CN 103864918 B CN103864918 B CN 103864918B CN 201410125860 A CN201410125860 A CN 201410125860A CN 103864918 B CN103864918 B CN 103864918B
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fmoc
otbu
tbu
glu
gly
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CN103864918A (en
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冷国庆
余荣熹
王艳
王浩
迟帅
刘宇帅
田辉
张琪
李迎新
肖明辉
李喜全
赵民喜
苏宏健
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HARBIN JIXIANGLONG BIOLOGICAL TECHNOLOGY Co Ltd
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HARBIN JIXIANGLONG BIOLOGICAL TECHNOLOGY Co Ltd
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Abstract

The invention discloses the solid phase synthesis process of a kind of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], comprise the following steps that 1) first synthesize the 1st to ten amino acid fragment the 3, the 11st to the 19th amino acid fragment the 2, the 20th to the 31st amino acid fragment 1, wherein the 20th upper lysine uses Fmoc Lys (Mtt) OH, and the 1st upper histidine uses Boc His (Trt) OH；2) pal Glu (OH) Otbu is synthesized with liquid phase synthesizing method；3) by the Mtt protection group on the 20th lysine of 5%TFA selectively removing, the side chain of pal Glu (OH) Otbu and the 20th lysine is connected and obtains fragment 4；4) successively fragment 2, fragment 3 are connected with fragment 4, obtain the peptide resin of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] full guard；5) by cracking, purification, lyophilizing, Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] fine work is obtained.

Description

A kind of solid phase synthesis process of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]
Technical field
The present invention relates to the synthetic method of a peptide species, be specifically related to the solid phase synthesis process of a kind of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
Background technology
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (Liraglutide) is human glucagon-like-peptide-1 (GLP-1) analog researched and developed by Novo Nordisk Co., Ltd, is made up of 31 amino acid residues, and the structure of its molecule is as follows:
H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-ε-(N-α-Palmitoyl-L-γ-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (Liraglutide) is the GLP-1 analog researched and developed by Novo Nordisk Co., Ltd.This medicine lists in U.S.'s approval on January 25th, 2010, and dosage form is subcutaneous injection, only needs every day subcutaneous injection can play good blood sugar reducing function 1 time.
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is the medicine according to natural GLP-1 (7-37) sequent synthesis.With the homology that GLP-1 has 95%, remain the biological activity of GLP-1 (7-37), its molecular structure the 34th lysine unlike natural GLP-1 molecule is replaced by arginine, the 16 carbon palmityl fatty acid side chains connected by g glutamic acid are increased on 26th lysine, it is made after being combined in vivo, to form reversible medicine-albumin complex with plasma albumin, complex is difficult to be degraded, can also delay Slow release makes distribution time extend, overcoming the degradable shortcoming of natural GLP-1 (7-37), the half-life reaches 13 hours.
Report about Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] preparation report is a lot of both at home and abroad, and Novo Nordisk Co., Ltd is mainly by gene recombination technology, utilizes yeast production Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]；But owing to needs use gene recombination technology; technical difficulty is big, and cost is of a relatively high, simultaneously because Arg34-GLP-1(7-37) side chain of-OH is in unprotected state; more impurity can be produced when reacting with N α-hexadecanoyl-Glu (ONSu)-OtBu, lose bigger.Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] solid phase synthesis patented method (CN103087181A) of domestic report, use king's resin, according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] peptide sequence coupling amino acid one by one, side chain is also coupling one by one, eventually pass anti-phase purification and obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], this method needs coupling amino acid one by one, and synthesis cycle is long, and total recovery is relatively low.Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] solid phase synthesis patented method (CN102875665A) of domestic report; use and be divided into 5 fragment synthesis Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]s; although the method can shorten synthesis cycle; improve efficiency; but the fragment divided is more; resin consumptions is big; relatively costly; and the 20th lysine uses Fmoc-Lys (Alloc)-OH; and selectively removing Alloc needs heavy metal catalyst Pd (Ph3P) 4; it is generally required to the lower reaction of nitrogen protection, industrially production can be restricted.
The present invention is directed to the problems referred to above, find a solution, i.e. it is respectively synthesized the method being connected to together by the synthesis of main chain being divide into 3 fragments, wherein the 20th lysine uses Fmoc-Lys (Mtt)-OH, the removing of Mtt has only to 5%TFA/5%Tis and just can carry out in DCM, simple, industrialized production is the most unrestricted.Main chain synthesis be divide into 3 fragments by the present invention, can shorten synthesis cycle, improves efficiency, also can relative reduction cost, side chain is connected with lysine with pal-Glu (OH)-Otbu for overall, it is possible to increase synthesis yield.
Summary of the invention
It is an object of the invention to provide the synthetic method of a kind of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], improve the combined coefficient of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], reduce synthesis cost, improve total recovery, in order to industrialized production.
The present invention provides the synthetic method of a kind of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], comprises the following steps:
1) according to the amino acid sequence of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain N end to C end, first synthesis the 1st to ten amino acid be fragment the 3, the 11st to the 19th aminoacid be fragment the 2, the 20th to the 31st aminoacid be fragment 1, totally 3 fragments, wherein the 20th upper lysine uses Fmoc-Lys (Mtt)-OH, and the 1st upper histidine uses Boc-His (Trt)-OH.
2) pal-Glu (OH)-Otbu is synthesized with liquid phase synthesizing method.
3) by the Mtt protection group on the 20th lysine of 5%TFA selectively removing, the lysine of pal-Glu (OH)-Otbu with the in fragment 1 the 20th is connected and obtains fragment 4.
4) successively fragment 2, fragment 3 are connected with fragment 4, obtain the peptide resin of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] full guard.
5) by cracking, purification, lyophilizing, Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] fine work is obtained.
Wherein, described step 1) is the preparation of 3 fragments of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], and step is as follows:
Wherein the preferred solid phase carrier of fragment 1 is king's resin.With king's resin as solid phase carrier, carry out successively condensation reaction connect following aminoacid:
Fmoc-Gly-OH, Fmoc-Arg (pbf)-OH, 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 (mtt)-OH, obtain fragment 1 full guard resin:
Fmoc-Lys (mtt)-Glu (Otbu)-Phe-Ile-Ala-Trp (BOC)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-king's resin
The condensing agent that wherein first aminoacid Fmoc-Gly-OH is used is HOBT/DIC/DMAP, and the condensation solvent used is DMF.The preferred HOBT/DIC of condensing agent that remaining aminoacid is used, de-FMOC reagent preferably 25% piperidines/DMF solution.
Wherein the preferred solid phase carrier of fragment 2 is dichloro trityl chloride resin.With dichloro trityl chloride resin as solid phase carrier; carry out condensation reaction successively and connect following aminoacid: 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, obtain fragment 2 full guard resin:
Fmoc-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-Glu (Otbu)-Gly-Gln (trt)-Ala-Ala-CTC resin.With 1%TFA/DCM crack fragment 2 full guard resin 1 hour, obtain fragment 2 full guard peptide Fmoc-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-Glu (Otbu)-Gly-Gln (trt)-Ala-Ala-OH.
The condensing agent that wherein first aminoacid Fmoc-Ala-OH is used is DIEA, and the condensation solvent used is DMF.The preferred HOBT/DIC of condensing agent that remaining aminoacid is used, de-FMOC reagent preferably 25% piperidines/DMF solution.
Wherein the preferred solid phase carrier of fragment 3 is dichloro trityl chloride resin.With dichloro trityl chloride resin as solid phase carrier, carry out successively condensation reaction connect following aminoacid:
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, Fmoc-Ala-OH, BOC-His (trt)-OH, obtain fragment 3 full guard resin:
BOC-His (trt)-Ala-Glu (Otbu)-Gly-Thr (tbu)-Phe-Thr (tbu)-Ser (tbu)-Asp (otbu)-Val-CTC resin.With 1%TFA/DCM crack fragment 3 full guard resin 1 hour, obtain fragment 3 full guard peptide:
BOC-His(trt)-Ala-Glu(Otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Val-OH
The condensing agent that wherein first aminoacid Fmoc-Val-OH is used is DIEA, and the condensation solvent used is DMF.The preferred HOBT/DIC of condensing agent that remaining aminoacid is used, de-FMOC reagent preferably 25% piperidines/DMF solution.
Wherein, described step 2) synthesize pal-Glu (OH)-Otbu with liquid phase synthesizing method, step is as follows:
Under ice bath, Glu (OH)-Otbu is dissolved in THF solution, adds the TEA of 2 times amount, then Hexadecanoyl chloride joins above-mentioned solution reaction 3 hours, is subsequently adding the water of 10 times amount, and regulation PH is 4-5, extracting with ethyl acetate, saturated NaHCO3 solution washs, washing, saline washs, it is dried, is spin-dried for, with ethyl acetate petroleum ether recrystallization, sucking filtration, dry, stand-by.
Wherein, described step 3) step is as follows:
By fragment 1 full guard resin:
Fmoc-Lys (mtt)-Glu (Otbu)-Phe-Ile-Ala-Trp (BOC)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-king's resin, use cutting reagent A:(TFA:Tis:DCM=5:5:90), shake 2 times under room temperature, each 30 minutes, 2 times are respectively washed successively with DCM, methanol, DMF, DCM, drain, obtain
Fmoc-Lys-Glu (Otbu)-Phe-Ile-Ala-Trp (BOC)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-king's resin; then by pal-Glu (OH)-Otbu with DIC; join in the DMF solution of above-mentioned resin after HOBT activation; react 120 minutes; sucking filtration; respectively wash 2 times with DCM, methanol, DMF, DCM successively, obtain fragment 4 full guard resin:
Fmoc-Lys(pal-Glu (otbu))-Glu (Otbu)-Phe-Ile-Ala-Trp (BOC)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-king's resin.
Wherein, described step 4) step is as follows:
Fragment 4 full guard resin; add 25% piperidines/DMF solution to react 10 minutes each 2 times; obtain H2N-Lys(pal-Glu (otbu))-Glu (Otbu)-Phe-Ile-Ala-Trp (BOC)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-king's resin; by full guard fragments of peptides 2 with DIC; join in the DMF solution of above-mentioned resin after HOBT activation; react 120 minutes; sucking filtration; respectively wash 2 times with DCM, methanol, DMF, DCM successively, obtain fragment 5 full guard resin
Fmoc-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-Glu (Otbu)-Gly-Gln (trt)-Ala-Ala-Lys(pal-Glu (otbu))-Glu (Otbu)-Phe-Ile-Ala-Trp (BOC) Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-king's resin; fragment 5 full guard resin; add 25% piperidines/DMF solution to react 10 minutes each 2 times, obtain
H2N-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-Glu (Otbu)-Gly-Gln (trt)-Ala-Ala-Lys(pal-Glu (otbu))-Glu (Otbu)-Phe-Ile-Ala-Trp (BOC)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-king's resin; by full guard fragments of peptides 3 with DIC; join in the DMF solution of above-mentioned resin after HOBT activation; react 120 minutes; sucking filtration; respectively wash 2 times with DCM, methanol, DMF, DCM successively, obtain the peptide resin of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] full guard:
BOC-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 (trt)-Ala-Ala-Lys(pal-Glu (otbu))-Glu (Otbu)-Phe-Ile-Ala-Trp (BOC)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-king's resin.
Wherein, described step 5) step is as follows:
Cleavage step, every gram of resin adds 10ml lysate, lysate volume ratio TFA:EDT:H2O=90-95:2.5-5:2.5-5, cracks 2 hours, and add diethyl ether precipitation, is filtrated to get thick peptide.Thick peptide, through reversed-phase high-performance liquid chromatography purification, lyophilization, obtains fine peptide.
Compared with prior art, advantages of the present invention is mainly manifested in:
This synthetic method can shorten synthesis cycle, improves efficiency, also can reduce cost, and side chain is connected with lysine with pal-Glu (OH)-Otbu for entirety, it is possible to increase synthesis yield, it is simple to industrialized production.
Below by way of experimental data beneficial effects of the present invention is described:
Wherein the computational methods of yield are as follows: amount * molecular mass yield=(the fine peptide quality/theory peptide content after lyophilizing) * 100% of theoretical peptide content=theory material
The detection method of purity is as follows: with acetonitrile: TFA(1000:1) as mobile phase A, with water: TFA(1000:1) as Mobile phase B, chromatographic column is C18 post, detects wavelength 210nm, column temperature 40 DEG C, flow velocity is 1.0m l/min, carries out gradient elution.Original state is 40% mobile phase A, mobile phase A is risen to 80% in 40 minutes, and A phase rose in 5 minutes 100%, keeps 5 minutes, then returned to original state in 2 minutes, keeps 10 minutes.
The method of the present invention; which use following reagent; as the 20th upper lysine uses Fmoc-Lys (Mtt)-OH, the 1st upper histidine uses Boc-His (Trt)-OH, by the Mtt protection group etc. on the 20th lysine of 5%TFA selectively removing.The use of these reagent obtains through screening, and screening process is as follows:
20th upper lysine uses the screening of Fmoc-Lys (Mtt)-OH
The Side chain protective groups such as general lysine have following several: BOC, Alloc, Dde, and BOC protection group needs higher acid concentration to remove, and easily other protection group on peptide chain is removed, thus affects synthesis yield.And selectively removing Alloc needs heavy metal catalyst Pd (Ph3P) 4, it is generally required to the lower reaction of nitrogen protection, industrially production can be restricted.Selectively removing Dde, needs with alkaline reagent, the easy racemization of peptide chain under alkaline environment.And selectively removing Mtt, it is only necessary to the acid of low concentration just can remove, and neither affects other protection group on peptide chain, also will not make peptide chain that other side reaction occurs.
1st upper histidine uses the screening of Boc-His (Trt)-OH
Histidine on 1st can also use Fmoc-His (Trt)-OH, but it is the use of Fmoc-His (Trt)-OH, after peptide chain has synthesized, final step also needs to remove Fmoc with 25% piperidines/DMF, and use Boc-His (Trt)-OH final step just without deprotection, directly BOC can be removed by cracking, the most both save reagent, and also simplify operating procedure.
Screening by the Mtt protection group on the 20th lysine of 5%TFA selectively removing
Removing to Mtt protection group is screened, and has groped 1%TFA and 5%TFA, TLC and has detected discovery, and under the conditions of 5%TFA, within 30 minutes, Mtt can remove completely, and under the conditions of 1%TFA, within 30 minutes, Mtt can not remove completely.
Knowable to result above, the embodiment of the present invention ten effect is optimum.
Detailed description of the invention
Embodiment 1: the synthesis of fragment 1
Weigh the king resin 10g that substitution degree is 1.08mmol/g, join in solid state reaction post, after DMF swellable resins 30 minutes, weigh 3.21g Fmoc-Gly-OH, 1.46gHOBt and 0.13gDMAP DMF to dissolve, add 1.84mL DIC to activate 5 minutes, add above-mentioned equipped with in the reaction column of resin, after reacting 3 hours, add 20mL pyridine and 20ml acetic anhydride is closed 8 hours.Washing 6 times with DMF, methanol washs 2 times, and detection substitution degree is 0.35mmol/g.
With the FMOC in 25% piperidines/DMF removing FMOC-Gly-king's resin, each 10 minutes, twice totally, deprotection terminated to wash 6 times with DMF., weigh
Fmoc-Arg (Pbf)-OH6.81g, HOBt1.42g, dissolve with 40mlDMF, adds DIC1.63ml and activates 5 minutes, joins in reaction column and react 2 hours, obtain
Fmoc-Arg (Pbf)-Gly-king's resin.Repeat above-mentioned de-FMOC and the step of amino acid couplings; carry out successively, 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, the coupling of Fmoc-Lys (mtt)-OH, obtain fragment 1 full guard resin:
Fmoc-Lys (mtt)-Glu (Otbu)-Phe-Ile-Ala-Trp (BOC)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-king's resin
Embodiment 2: the synthesis of fragment 2
Weigh the CTC resin 20g that substitution degree is 1.0mmol/g, join in solid state reaction post, after DMF swellable resins 30 minutes, take 12.44g Fmoc-Ala-OH DMF to dissolve, after adding 26.4mLDIEA activation, add above-mentioned equipped with in the reaction column of resin, after reacting 2 hours, add 20mL absolute methanol to close 1 hour, wash 6 times with DMF, obtain Fmoc-Ala-CTC.
With the FMOC in 25% piperidines/DMF removing Fmoc-Ala-CTC, each 10 minutes, twice totally, deprotection terminated to wash 6 times with DMF., weigh Fmoc-Ala-OH18.68g, HOBt8.1g, dissolve with 80mlDMF, add DIC9.3ml and activate 5 minutes, join in reaction column and react 2 hours, obtain Fmoc-Ala-Ala-CTC.Repeat above-mentioned de-FMOC and the step of amino acid couplings; carry out 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 successively, obtain fragment 2 full guard resin:
Fmoc-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-Glu (Otbu)-Gly-Gln (trt)-Ala-Ala-CTC resin 48 grams.With 1%TFA/DCM480ml crack fragment 2 full guard resin 1 hour, filtering, filtrate carries out rotation and steams, and when filtrate volume remains 1/5, the ether adding 10 times amount precipitates, and filters, and filter cake ether washs 5 times, carries out vacuum drying and obtains fragment 2 full guard peptide
Fmoc-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-Glu (Otbu)-Gly-Gln (trt)-Ala-Ala-OH, weight: 23.5 grams of purity: 95.4%
Embodiment 3: the synthesis of fragment 2
Weigh the CTC resin 20g that substitution degree is 1.0mmol/g, join in solid state reaction post, after DMF swellable resins 30 minutes, take 12.44g Fmoc-Ala-OH DMF to dissolve, after adding 26.4mLDIEA activation, add above-mentioned equipped with in the reaction column of resin, after reacting 2 hours, add 20mL absolute methanol to close 1 hour, wash 6 times with DMF, obtain Fmoc-Ala-CTC.
With the FMOC in 25% piperidines/DMF removing Fmoc-Ala-CTC, each 10 minutes, twice totally, deprotection terminated to wash 6 times with DMF., weigh Fmoc-Ala-OH18.68g, TBTU19.26g, dissolve with 80mlDMF, add DIEA19.8ml and activate 5 minutes, join in reaction column and react 2 hours, obtain Fmoc-Ala-Ala-CTC.Repeat above-mentioned de-FMOC and the step of amino acid couplings; carry out 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 successively, obtain fragment 2 full guard resin:
Fmoc-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-Glu (Otbu)-Gly-Gln (trt)-Ala-Ala-CTC resin 46.3 grams.With 1%TFA/DCM463ml crack fragment 2 full guard resin 1 hour, filtering, filtrate carries out rotation and steams, and when filtrate volume remains 1/5, the ether adding 10 times amount precipitates, and filters, and filter cake ether washs 5 times, carries out vacuum drying and obtains fragment 2 full guard peptide
Fmoc-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-Glu (Otbu)-Gly-Gln (trt)-Ala-Ala-OH, weight: 22.3 grams of purity: 89.1%
Embodiment 4: the synthesis of fragment 2
Weigh the CTC resin 20g that substitution degree is 1.0mmol/g, join in solid state reaction post, after DMF swellable resins 30 minutes, take 12.44g Fmoc-Ala-OH DMF to dissolve, after adding 26.4mLDIEA activation, add above-mentioned equipped with in the reaction column of resin, after reacting 2 hours, add 20mL absolute methanol to close 1 hour, wash 6 times with DMF, obtain Fmoc-Ala-CTC.
With the FMOC in 25% piperidines/DMF removing Fmoc-Ala-CTC, each 10 minutes, twice totally, deprotection terminated to wash 6 times with DMF., weigh Fmoc-Ala-OH18.68g, HBTU22.76g, dissolve with 80mlDMF, add DIEA19.8ml and activate 5 minutes, join in reaction column and react 2 hours, obtain Fmoc-Ala-Ala-CTC.Repeat above-mentioned de-FMOC and the step of amino acid couplings; carry out 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 successively, obtain fragment 2 full guard resin:
Fmoc-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-Glu (Otbu)-Gly-Gln (trt)-Ala-Ala-CTC resin 45.3 grams.With 1%TFA/DCM453ml crack fragment 2 full guard resin 1 hour, filtering, filtrate carries out rotation and steams, and when filtrate volume remains 1/5, the ether adding 10 times amount precipitates, and filters, and filter cake ether washs 5 times, carries out vacuum drying and obtains fragment 2 full guard peptide
Fmoc-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-Glu (Otbu)-Gly-Gln (trt)-Ala-Ala-OH, weight: 21.8 grams of purity: 91.6%
Embodiment 5: the synthesis of fragment 3
Weigh the CTC resin 20g that substitution degree is 1.0mmol/g, join in solid state reaction post, after DMF swellable resins 30 minutes, take 13.58g Fmoc-Val-OH DMF to dissolve, after adding 26.4mLDIEA activation, add above-mentioned equipped with in the reaction column of resin, after reacting 2 hours, add 20mL absolute methanol to close 1 hour, wash 6 times with DMF, obtain Fmoc-Val-CTC.
With the FMOC in 25% piperidines/DMF removing Fmoc-Val-CTC, each 10 minutes, twice totally, deprotection terminated to wash 6 times with DMF., weigh Fmoc-Asp (otbu)-OH24.7g, HOBt8.1g, dissolve with 80mlDMF, add DIC9.3ml and activate 5 minutes, join in reaction column and react 2 hours, obtain Fmoc-Asp (otbu)-Val-CTC.Repeat above-mentioned de-FMOC and the step of amino acid couplings; carry out 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 successively, obtain fragment 3 full guard resin:
BOC-His (trt)-Ala-Glu (Otbu)-Gly-Thr (tbu)-Phe-Thr (tbu)-Ser (tbu)-Asp (otbu)-Val-CTC resin 50.6 grams.With 1%TFA/DCM506ml crack fragment 3 full guard resin 1 hour, filtering, filtrate carries out rotation and steams, and when filtrate volume remains 1/5, the ether adding 10 times amount precipitates, and filters, and filter cake ether washs 5 times, carries out vacuum drying and obtains fragment 3 full guard peptide
BOC-His(trt)-Ala-Glu(Otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-A Sp (otbu)-Val-OH, weight: 24.6 grams of purity: 94.8%
Embodiment 6: the synthesis of fragment 3
Weigh the CTC resin 20g that substitution degree is 1.0mmol/g, join in solid state reaction post, after DMF swellable resins 30 minutes, take 13.58g Fmoc-Val-OH DMF to dissolve, after adding 26.4mLDIEA activation, add above-mentioned equipped with in the reaction column of resin, after reacting 2 hours, add 20mL absolute methanol to close 1 hour, wash 6 times with DMF, obtain Fmoc-Val-CTC.
With the FMOC in 25% piperidines/DMF removing Fmoc-Val-CTC, each 10 minutes, twice totally, deprotection terminated to wash 6 times with DMF., weigh Fmoc-Asp (otbu)-OH24.7g, TBTU19.26g, dissolve with 80mlDMF, add DIEA19.8ml and activate 5 minutes, join in reaction column and react 2 hours, obtain Fmoc-Asp (otbu)-Val-CTC.Repeat above-mentioned de-FMOC and the step of amino acid couplings; carry out 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 successively, obtain fragment 3 full guard resin:
BOC-His (trt)-Ala-Glu (Otbu)-Gly-Thr (tbu)-Phe-Thr (tbu)-Ser (tbu)-Asp (otbu)-Val-CTC resin 47.6 grams.With 1%TFA/DCM476ml crack fragment 3 full guard resin 1 hour, filtering, filtrate carries out rotation and steams, and when filtrate volume remains 1/5, the ether adding 10 times amount precipitates, and filters, and filter cake ether washs 5 times, carries out vacuum drying and obtains fragment 3 full guard peptide
BOC-His (trt)-Ala-Glu (Otbu)-Gly-Thr (tbu)-Phe-Thr (tbu)-Ser (tbu)-Asp (otbu)-Val-OH, weight: 23.2 grams of purity: 87.6%
Embodiment 7: the synthesis of fragment 3
Weigh the CTC resin 20g that substitution degree is 1.0mmol/g, join in solid state reaction post, after DMF swellable resins 30 minutes, take 13.58g Fmoc-Val-OH DMF to dissolve, after adding 26.4mLDIEA activation, add above-mentioned equipped with in the reaction column of resin, after reacting 2 hours, add 20mL absolute methanol to close 1 hour, wash 6 times with DMF, obtain Fmoc-Val-CTC.
With the FMOC in 25% piperidines/DMF removing Fmoc-Val-CTC, each 10 minutes, twice totally, deprotection terminated to wash 6 times with DMF., weigh Fmoc-Asp (otbu)-OH24.7g, HBTU22.76g, dissolve with 80mlDMF, add DIEA19.8ml and activate 5 minutes, join in reaction column and react 2 hours, obtain Fmoc-Asp (otbu)-Val-CTC.Repeat above-mentioned de-FMOC and the step of amino acid couplings; carry out 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 successively, obtain fragment 3 full guard resin:
BOC-His (trt)-Ala-Glu (Otbu)-Gly-Thr (tbu)-Phe-Thr (tbu)-Ser (tbu)-Asp (otbu)-Val-CTC resin 46.2 grams.With 1%TFA/DCM462ml crack fragment 3 full guard resin 1 hour, filtering, filtrate carries out rotation and steams, and when filtrate volume remains 1/5, the ether adding 10 times amount precipitates, and filters, and filter cake ether washs 5 times, carries out vacuum drying and obtains fragment 3 full guard peptide
BOC-His (trt)-Ala-Glu (Otbu)-Gly-Thr (tbu)-Phe-Thr (tbu)-Ser (tbu)-Asp (otbu)-Val-OH, weight: 22.7 grams of purity: 90.4%
The synthesis of embodiment 8:pal-Glu (OH)-Otbu
Weigh 4.06 grams of Glu (OH)-Otbu to be dissolved in THF solution, under ice bath, add the TEA of 4.56ml, then 6.03 grams of Hexadecanoyl chlorides being joined above-mentioned solution reaction 3 hours, be subsequently adding the water of 10 times amount, regulation PH is 4-5, extracting with ethyl acetate, saturated NaHCO3 solution washs, washing, saline washs, it is dried, is spin-dried for, with ethyl acetate petroleum ether recrystallization, sucking filtration, dry, obtain solid weight: 7.96 grams of purity: 98.6%
Embodiment 9: the synthesis of fragment 4
Weigh fragment 1
Fmoc-Lys (mtt)-Glu (Otbu)-Phe-Ile-Ala-Trp (BOC)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-king's resin 20 grams, use cutting reagent A:(TFA:Tis:DCM=5:5:90), shake 2 times under room temperature, 200ml every time, each 30 minutes, respectively wash 2 times with DCM, methanol, DMF, DCM successively, drain, obtain
nullFmoc-Lys-Glu (Otbu)-Phe-Ile-Ala-Trp (BOC)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-king's resin，Then pal-Glu (OH)-Otbu4.8 gram is weighed，HOBT1.42 gram，Dissolve with DMF，Add 1.63mlDIC,Activate and join in above-mentioned resin for 5 minutes，React 120 minutes，Sucking filtration，Use DCM successively、Methanol、DMF、DCM respectively washs 2 times，Obtain fragment 4 full guard resin: Fmoc-Lys(pal-Glu (otbu))-Glu (Otbu)-Phe-Ile-Ala-Trp (BOC)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-king's resin.
Embodiment 10: the synthesis of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] peptide resin
By the fragment 4 full guard resin in embodiment 9, add 25% piperidines/DMF solution to react 10 minutes each 2 times, obtain H2N-Lys(pal-Glu (otbu))-Glu (Otbu)-Phe-Ile-Ala-Trp (BOC)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-king's resin, weigh the full guard fragments of peptides 2 in embodiment 2, 16.95 grams, HOBT1.42 gram, dissolve with DMF, add 1.63mlDIC, activate and within 5 minutes, join in above-mentioned resin, react 120 minutes, sucking filtration, successively with DCM, methanol, DMF, DCM respectively washs 2 times, obtain fragment 5 full guard resin
Fmoc-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-Glu (Otbu)-Gly-Gln (trt)-Ala-Ala-Lys(pal-Glu (otbu))-Glu (Otbu)-Phe-Ile-Ala-Trp (BOC) Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-king's resin; fragment 5 full guard resin; add 25% piperidines/DMF solution to react 10 minutes each 2 times, obtain
nullH2N-Ser (tbu)-Ser (tbu)-Tyr (tbu)-Leu-Glu (Otbu)-Gly-Gln (trt)-Ala-Ala-Lys(pal-Glu (otbu))-Glu (Otbu)-Phe-Ile-Ala-Trp (BOC) Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-king's resin，Weigh the full guard fragments of peptides 3 in embodiment 5，17.7 grams,HOBT1.42 gram，Dissolve with DMF，Add 1.63mlDIC,Activate and join in above-mentioned resin for 5 minutes，React 120 minutes，Sucking filtration，Use DCM successively、DMF、DCM、Methanol respectively washs 2 times，Vacuum drying，Obtain the peptide resin of 32.4 grams of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] full guard
BOC-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 (trt)-Ala-Ala-Lys(pal-Glu (otbu))-Glu (Otbu)-Phe-Ile-Ala-Trp (BOC)-Leu-Val-Arg (pbf)-Gly-Arg (pbf)-Gly-king's resin.
Embodiment 11: the cracking of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] peptide resin
32.4 grams of peptide resins embodiment 10 obtained, join in 324ml lysate and react 2 hours, lysate volume ratio TFA:EDT:H2O=90:5:5, reaction terminates, and filters resin, and filtrate adds in 2L ice ether to be precipitated, and filters, and filter cake ether washs 5 times, and filter cake is vacuum dried, and obtains thick peptide: 13.2 grams, purity: 63%
Embodiment 12: the preparation of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] fine peptide
Thick peptide is dissolved in acetic acid: in water (90:10), after thick peptide is completely dissolved, dilute, make the ratio of acetic acid be down to 10%, with the membrane filtration of 0.45 micron, prepare sample introduction.Chromatographic column is 50*250mm, and filler is C8, wavelength 220nm, flow velocity 60ml/min, and flowing be 0.1%TFA/ acetonitrile mutually, and collection main peak carries out concentrating, lyophilizing, obtains fine peptide: 5.12 grams, purity: 99.2% total recovery: 38.9%.

Claims (9)

1. the solid phase synthesis process of an Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it is characterised in that comprise the steps:
Step 1, amino acid sequence according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain N end to C end, first synthesis the 1st To ten amino acid fragment 3, the 11st to the 19th amino acid fragment 2, the 20th to the 31st amino Acid fragment 1, totally 3 fragments, wherein the 20th upper lysine uses Fmoc-Lys (Mtt)-OH, the 1st upper histidine uses Boc-His (Trt)-OH；
Step 2, synthesize pal-Glu (OH)-Otbu with liquid phase synthesizing method；
Mtt protection group on step 3, use the 20th lysine of 5%TFA selectively removing, will Pal-Glu (OH)-Otbu is connected with the in fragment 1 the 20th lysine and obtains fragment 4；
Step 4, first fragment 2 is connected with fragment 4, then is connected with fragment 3, obtain Li Lalu The peptide resin of peptide full guard；
Step 5, by cracking, purification, lyophilizing, obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] fine work.
Method the most according to claim 1, it is characterised in that described in step 1, solid phase synthesis is many Fragments of peptides 1, step is as follows:
With king's resin as solid phase carrier, carry out successively condensation reaction connect following aminoacid:
Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、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(Mtt)-OH,
Obtain fragment 1 full guard resin:
Fmoc-Lys(Mtt)-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)- Gly-Arg (Pbf)-Gly-king's resin, described king's resin and first aminoacid
The coupling agent that Fmoc-Gly-OH coupling is used is HOBT/DIC/DMAP.
Method the most according to claim 1, it is characterised in that solid phase synthesis described in step 1 Polypeptide fragment 2, step is as follows:
With 2-chlorine trityl chloride resin as solid phase carrier, carry out condensation reaction successively and connect as follows Aminoacid:
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, obtain fragment 2 full guard resin:
Fmoc-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(Otbu)-Gly-Gln(Trt)-Ala- Ala-CTC resin, with 1%TFA/DCM crack fragment 2 full guard resin 1 hour, obtains Fragment 2 full guard peptide Fmoc-Ser (tBu)-Ser (tBu)-Tyr (tBu)-Leu-Glu (Otbu)- Gly-Gln (Trt)-Ala-Ala-OH, described 2-chlorine trityl chloride resin and first The coupling agent that amino acid couplings is used is DIEA.
Method the most according to claim 1, it is characterised in that solid phase synthesis described in step 1 Polypeptide fragment 3, step is as follows:
With 2-chlorine trityl chloride resin as solid phase carrier, carry out condensation reaction successively and connect as follows Aminoacid:
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、Fmoc-Ala-OH、
Boc-His (Trt)-OH, obtains fragment 3 full guard resin:
Boc-His(Trt)-Ala-Glu(Otbu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)- Asp (Otbu)-Val-CTC resin, with 1%TFA/DCM crack fragment 3 full guard resin 1 Hour, obtain fragment 3 full guard peptide:
Boc-His(Trt)-Ala-Glu(Otbu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)- Asp (Otbu)-Val-OH, described 2-chlorine trityl chloride resin and first aminoacid The coupling agent that coupling is used is DIEA.
Method the most according to claim 1, it is characterised in that use liquid phase synthesis described in step 2 Method synthesis pal-Glu (OH)-Otbu, step is as follows:
Under ice bath, Glu (OH)-Otbu is dissolved in THF solution, adds the TEA of 2 times amount, so Rear Hexadecanoyl chloride joins above-mentioned solution reaction 3 hours, is subsequently adding the water of 10 times amount, Regulation pH is 4-5, extracts with ethyl acetate, saturated NaHCO3Solution washs, washing, Saline washs, and is dried, is spin-dried for, and with ethyl acetate petroleum ether recrystallization, sucking filtration, dries, Stand-by.
Method the most according to claim 1, it is characterised in that described in step 3, solid phase synthesis is many Fragments of peptides 4, step is as follows:
By fragment 1 full guard resin:
Fmoc-Lys(Mtt)-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)- Gly-Arg (Pbf)-Gly-king's resin, uses cutting reagent A:(TFA:TIS:DCM=5:5:90), Shake under room temperature 2 times, each 30 minutes, use DCM, methanol, DMF, DCM successively Each washing 2 times, drains, obtains Fmoc-Lys-Glu (Otbu)-Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-king's resin, then will
Pal-Glu (OH)-Otbu joins the DMF of above-mentioned resin with DIC, HOBT after activating In solution, react 120 minutes, sucking filtration, successively with DCM, methanol, DMF, DCM Each washing 2 times, obtains fragment 4 full guard resin: Fmoc-Lys (pal-Glu (Otbu)) -Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)- Gly-king's resin.
Method the most according to claim 1, it is characterised in that obtain Li Lalu described in step 4 The peptide resin of peptide full guard, step is as follows:
Fragment 4 full guard resin, adds 25% piperidines/DMF solution and reacts 10 minutes each 2 times, To H2N-Lys (pal-Glu (Otbu))-Glu (Otbu)-Phe-Ile-Ala-Trp (Boc)- Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-king's resin, by full guard fragments of peptides 2 with Join after DIC, HOBT activation in the DMF solution of above-mentioned resin, react 120 minutes, Sucking filtration, respectively washs 2 times with DCM, methanol, DMF, DCM successively, obtains fragment 5 Full guard resin
Fmoc-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(Otbu)-Gly-Gln(Trt)-Ala- Ala-Lys(pal-Glu(Otbu))-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu- Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-king's resin, fragment 5 full guard resin, add 25% Piperidines/DMF solution reacts 10 minutes each 2 times, obtains
H2N-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(Otbu)-Gly-Gln(Trt)-Ala- Ala-Lys(pal-Glu(Otbu))-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)- Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-king's resin, by full guard fragments of peptides 3 with Join after DIC, HOBT activation in the DMF solution of above-mentioned resin, react 120 minutes, Sucking filtration, respectively washs 2 times with DCM, methanol, DMF, DCM successively, obtains Li La The peptide resin of Shandong peptide full guard
Boc-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 (Trt)-Ala-Ala-Lys(pal-Glu(Otbu))-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc) -Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-king's resin.
8. according to method described in Claims 2 or 3 or 4, it is characterised in that described polypeptide fragment In solid phase synthesis, in addition to first amino acid whose coupling, remaining amino acid couplings is made Condensing agent be HOBT/DIC, TBTU/DIEA or HBTU/DIEA.
Method the most according to claim 1, it is characterised in that cracking described in step 5 uses body Amass and compare TFA:EDT:H2The decomposition agent of O=90-95:2.5-5:2.5-5.
CN201410125860.8A 2014-03-31 2014-03-31 A kind of solid phase synthesis process of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] Active CN103864918B (en)

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Denomination of invention: A Solid-phase synthesis Method of Liraglutide

Effective date of registration: 20230707

Granted publication date: 20160817

Pledgee: Harbin Kechuang Financing Guarantee Co.,Ltd.

Pledgor: HARBIN JIXIANGLONG BIOLOGICAL TECHNOLOGY Co.,Ltd.

Registration number: Y2023230000063