CN104004083A - Method for synthesizing liraglutide - Google Patents

Method for synthesizing liraglutide Download PDF

Info

Publication number
CN104004083A
CN104004083A CN201410265582.6A CN201410265582A CN104004083A CN 104004083 A CN104004083 A CN 104004083A CN 201410265582 A CN201410265582 A CN 201410265582A CN 104004083 A CN104004083 A CN 104004083A
Authority
CN
China
Prior art keywords
coupling
fmoc
protecting group
resin
side chain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201410265582.6A
Other languages
Chinese (zh)
Other versions
CN104004083B (en
Inventor
李向群
董华建
郭德文
文永均
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CHENGDU SHENGNUO BIOTEC Co Ltd
Original Assignee
CHENGDU SHENGNUO BIOTEC Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by CHENGDU SHENGNUO BIOTEC Co Ltd filed Critical CHENGDU SHENGNUO BIOTEC Co Ltd
Priority to CN201410265582.6A priority Critical patent/CN104004083B/en
Publication of CN104004083A publication Critical patent/CN104004083A/en
Application granted granted Critical
Publication of CN104004083B publication Critical patent/CN104004083B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Endocrinology (AREA)

Abstract

The invention relates to the field of medicine synthesis, and discloses a method for synthesizing liraglutide. The method includes the steps that three polypeptide fragments of the first to the fourth amino acid, the fifteenth to the sixteenth amino acid and the seventeenth to thirty-first amino acid are synthesized at first according to the amino acid sequence from N end to C end of a main chain of liraglutide, and then the three polypeptide fragments and the other amino acid are connected according to the sequence from the C end to the N end in a coupling mode to synthesize the liraglutide. According to the method, the three fragments can be synthesized simultaneously, coupling and acidolysis with much carrier resin are avoided, the use quantity of resin carriers is greatly reduced, the synthesis cycle is shortened, and the complexity level of synthetic process is simplified on the premise that high total recovery and purity are guaranteed.

Description

A kind of method of synthetic Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]
Technical field
The present invention relates to the synthetic field of medicine, be specifically related to a kind of method of synthetic 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], English Liraglutide by name, is a kind of medicine for the treatment of type ii diabetes that Denmark Novo Nordisk Co.,Ltd develops.Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is a kind of people glicentin-1 (GLP-1) analogue, and it can play good reduction blood sugar effect as GLP-1 receptor stimulant, and peptide order is as follows:
NH 2-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N α-PAL-γ-Glu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-COOH
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] has been compared an amino acid difference with natural GLP-1 molecular structure, and has increased by 16 carbon palmityl fatty acid side chains, has 97% homology with natural human GLP-1.Different from natural GLP-1, pharmacokinetics and the pharmacodynamic characteristics of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] in human body is all applicable to dosage regimen once a day.After subcutaneous injection administration, the mechanism that its extends action time comprises: make to absorb the self association that slows down, with albumin bound, DPP IV (DPP-IV) and neutral endopeptidase (NEP) are had to higher enzyme stability, thereby have longer plasma half-life.
In the existing synthetic method of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], 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 the domestic barms that cannot obtain is produced.Patent US6268343B1, US6458924B2 and document " Journal of Medicinal Chemistry43,1664-1669,2000 " all disclose and have utilized intermediate GLP-1 (7-37)-OH and N α-alkanoyl-Glu (ONSu)-OtBu prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], but in these three kinds of prior aries, intermediate GLP-1 (7-37)-OH all needs reversed-phase HPLC purifying, then under liquid-phase condition with N α-alkanoyl-Glu (ONSu)-OtBu reaction; and because GLP-1 (7-37)-OH N holds not protection and Side chain protective group, all remove, can cause producing much impurity, be difficult to purifying; complex operation; cycle is long, and waste liquid is many, is unfavorable for environmental protection; and two-step purifying; need a large amount of acetonitriles of cost, with high costs, be unfavorable for scale operation etc.
Chinese patent CN102286092A discloses a kind of total synthesis method, adopt 2-CTC resin or king's resin, according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] peptide order coupling amino acid one by one, finally by crossing reverse purifying, obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], its do not need two-step purifying and also impurity less, be better than above-mentioned several existing preparation method.But this method needs coupling amino acid one by one, synthesis cycle is long, and total recovery is lower, is only 15% left and right (referring to CN102286092A embodiment 12-14), still needs further raising.
In order to address the above problem, application number is a kind of method that 201210369966.3 Chinese patent discloses synthetic Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it is held to the amino-acid sequence of C end according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain N, first synthetic the 1st to the 4th amino acid, the 5th is to ten amino acid, the 11st to the 16th amino acid, the 17th to the 24th amino acid and the 25th to the 31st amino acid whose 5 polypeptide fragments, then 5 polypeptide fragments of coupling synthesize Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], shorten synthesis cycle, after the purifying of raising Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], total recovery surpasses 30%, and purity surpasses 99%.But the method needs to synthesize 5 fragments with resin carrier respectively, and then carries out coupling, wherein relate to connection and cracking resin carrier repeatedly, not easy on operational degree, and coupling and cracking resin carrier all need to spend many time, and expend too much resin carrier.In addition, this patent is at foreign matter content context of detection the unexposed content on the larger maximum single contaminant of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] quality impact.
At Peptides Synthesis, different synthesis strategies has a significant impact the purity of the finished product, maximum single contaminant content and yield, how to guarantee under the prerequisite of purity and yield, reducing as much as possible the complexity of synthesis technique, maximum single contaminant and shorten synthesis cycle, is technician's research emphasis instantly.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of method of synthetic Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], make the method for the invention guaranteeing, under the prerequisite of its total recovery and purity, to reduce complexity and the maximum single contaminant of synthetic method, shorten synthesis cycle.
For achieving the above object, the invention provides following technical scheme:
A method for synthetic Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], comprises the following steps:
Step 1, synthesize the polypeptide fragment 1 that on aminoacid sequence N shown in SEQ ID NO:1 end, His side chain and on Glu side chain coupling has protecting group;
Synthesize the polypeptide fragment 2 that on aminoacid sequence N shown in SEQ ID NO:2 end, Glu side chain coupling has protecting group;
Synthesize in the coupling of aminoacid sequence C shown in SEQ ID NO:3 end have resin carrier, on Gln side chain, on Glu side chain, on Trp side chain, on Arg side chain coupling have protecting group and on Lys side chain coupling have N αthe polypeptide fragment 3 of-PAL-γ-Glu (α-OtBu);
Step 2, by the C end coupling of the N end of polypeptide fragment 3 and polypeptide fragment 2, after coupling, remove the N end protecting group of polypeptide fragment 2, obtain polypeptide resin I;
Step 3, the order of holding N end according to aminoacid sequence C shown in SEQ ID NO:4, first N is held coupling to have the leucic C section of protecting group and the coupling of the N of polypeptide resin I end, then one by one other corresponding protected amino acids are extended to coupling successively, after coupling, remove N end protecting group, obtain polypeptide resin II;
Step 4, by the N end coupling of the C end of polypeptide fragment 1 and polypeptide resin II, after coupling, remove the N end protecting group of polypeptide fragment 1, obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resin;
Step 5, Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resinous acid solution remove C end resin and all protecting groups obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product, and purifying crude obtains Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain amino acid has 31, adopts sheet phase method to synthesize and has a variety of forms, but only have the total recovery purity of the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that suitable sheet phase method guarantee is higher, can reduce the complexity of synthesis technique again simultaneously.For this reason, applicant, according to long-term experimental study and amino acid racemization situation, has proposed the method for the invention and has prepared Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], is guaranteeing, under the prerequisite of its total recovery and purity, to reduce the complexity of synthetic method.。
In the method for the invention, first according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain peptide order, be divided into 4 parts, be divided into synthetic and other amino acid whose couplings one by one of 3 fragments, with Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain N, hold the amino-acid sequence numbering of C end, as shown in the formula:
NH 2-His 1-Ala 2-Glu 3-Gly 4-Thr 5-Phe 6-Thr 7-Ser 8-Asp 9-Val 10-Ser 11-Ser 12-Tyr 13-L?eu 14-Glu 15-Gly 16-Gln 17-Ala 18-Ala 19-Lys 20(N α-PAL-γ-Glu)-Glu 21-Phe 22-Ile 23-Ala 24-Trp 25-Leu 26-Val 27-Arg 28-Gly 29-Arg 30-Gly 31-COOH
Shown in SEQ ID NO:1, aminoacid sequence is the peptide sequence of numbering 1-4 in above formula, shown in SEQ ID NO:2, aminoacid sequence is the peptide sequence of numbering 15-16 in above formula, shown in SEQ ID NO:3, aminoacid sequence is the peptide sequence of numbering 17-31 in above formula, and shown in SEQ ID NO:4, aminoacid sequence is the peptide sequence of numbering 5-14 in above formula.
The present invention's synthetic polypeptide fragment 1 in step 1 is on aminoacid sequence basis shown in SEQ ID NO:1, difference coupling protecting group on its N end, His side chain and on Glu side chain; Synthetic polypeptide fragment 2 is on aminoacid sequence basis shown in SEQ ID NO:2, difference coupling protecting group on its N end, Glu side chain; Synthetic polypeptide fragment 3 is on aminoacid sequence basis shown in SEQ ID NO:3, in the coupling of its C end, have resin carrier, on Gln side chain, on Glu side chain, on Trp side chain, on Arg side chain coupling have protecting group and on Lys side chain coupling have N α-PAL-γ-Glu (α-OtBu).And remaining other amino acid; to form after polypeptide resin I in polypeptide fragment 3 and polypeptide fragment 2 couplings; according to aminoacid sequence C shown in SEQ ID NO:4, hold the order of N end; first N is held coupling to have the leucic C section of protecting group and the coupling of the N of polypeptide resin I end; then one by one other corresponding protected amino acids are extended to coupling successively; after coupling, remove N end protecting group, obtain polypeptide resin II.Then polypeptide resin II and polypeptide fragment 1 carry out last coupling again to complete the peptide chain of whole Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] synthetic.
Protecting group of the present invention is the blocking group that amino, carboxyl etc. disturb synthetic group on the conventional protected amino acid main chain in the synthetic field of amino acid and side chain, prevent that amino, carboxyl etc. from reacting in preparing target product process, generate impurity, amino acid for the side chain that needs protection in the present invention, its side-chain structure as well known to those skilled in the art and knowing adopts conventional protecting group to carry out the groups such as amino on protected amino acid side chain, carboxyl, as preferably, the present invention is by the side chain of Trt protecting group protection group propylhomoserin, glutamine; By the side chain of OtBu protecting group protection L-glutamic acid, aspartic acid; By the side chain of tBu protecting group protection Threonine, Serine, tyrosine; By the side chain of Boc protecting group sematic color propylhomoserin; By Pdf protecting group, protect arginic side chain.In addition, in the amino acid relating in the method for the invention, amino acid N end is all preferably protected by Fmoc protecting group, and Histidine also can be protected by Boc protecting group.And the amino acid of protected base protection is called protected amino acid.
As preferred version, synthetic polypeptide fragment 1 is specially described in step 1:
Under coupling reagent exists, after N is held to glycine (Fmoc-Gly-OH) that coupling has a Fmoc protecting group and resin carrier coupling, de-Fmoc protecting group obtains H-Gly-resin carrier, then adopt activating reagent and condensation reagent, according to aminoacid sequence C shown in SEQ ID NO:1, hold the order of N end, one by one N is held successively coupling to have Fmoc protecting group and side chain coupling to have the L-glutamic acid (Fmoc-Glu (OtBu)-OH) of OtBu protecting group, the coupling of N end has the L-Ala (Fmoc-Ala-OH) of Fmoc protecting group, the coupling of N end has Boc protecting group or Fmoc protecting group and side chain coupling to have the Histidine (Fmoc/Boc-His (Trt)-OH) of Trt protecting group to extend coupling, lysate cracking after coupling, deresinate carrier obtains polypeptide fragment 1 (Fmoc/Boc-His (Trt)-Ala-Glu (OtBu)-Gly-OH).
As preferred version, synthetic polypeptide fragment 2 is specially described in step 1:
Under coupling reagent exists; after N is held to glycine (Fmoc-Gly-OH) that coupling has a Fmoc protecting group and resin carrier coupling, de-Fmoc protecting group obtains H-Gly-resin carrier; then adopt activating reagent and condensation reagent; according to aminoacid sequence C shown in SEQ ID NO:2, hold the order of N end; hold coupling to have Fmoc protecting group and side chain coupling to have the L-glutamic acid (Fmoc-Glu (OtBu)-OH) of OtBu protecting group to extend coupling N; lysate cracking after coupling, deresinate carrier obtains polypeptide fragment 2 (Fmoc-Glu (OtBu)-Gly-OH).
As preferred version, synthetic polypeptide fragment 3 is specially described in step 1:
Under coupling reagent exists, after N is held to glycine (Fmoc-Gly-OH) that coupling has a Fmoc protecting group and resin carrier coupling, de-Fmoc protecting group obtains H-Gly-resin carrier, then adopt activating reagent and condensation reagent, according to aminoacid sequence C shown in SEQ ID NO:3, hold the order of N end, one by one N is held successively coupling to have Fmoc protecting group and side chain coupling to have the arginine (Fmoc-Arg (Pdf)-OH) of Pdf protecting group, the coupling of N end has the glycine (Fmoc-Gly-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the arginine (Fmoc-Arg (Pdf)-OH) of Pdf protecting group, the coupling of N end has the α-amino-isovaleric acid (Fmoc-Val-OH) of Fmoc protecting group, the coupling of N end has the leucine (Fmoc-Leu-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the tryptophane (Fmoc-Trp (Boc)-OH) of Boc protecting group, the coupling of N end has L-Ala (Fmoc-Ala-OH) the N end coupling of Fmoc protecting group to have the Isoleucine (Fmoc-Ile-OH) of Fmoc protecting group, the coupling of N end has the phenylalanine (Fmoc-Phe-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the L-glutamic acid (Fmoc-Glu (OtBu)-OH) of OtBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have N αmethionin (Fmoc-Lys (the N of-PAL-γ-Glu (α-OtBu) α-PAL-γ-Glu (α-OtBu))-OH), the coupling of two N end has the L-Ala (Fmoc-Ala-OH) of Fmoc protecting group, the coupling of N end to have Fmoc protecting group and side chain coupling to have the glutamine (Fmoc-Gln (Trt)-OH) of Trt protecting group to extend coupling, remove N section with Fmoc protecting group obtain polypeptide fragment 3 (Gln (Trt)-Ala-Ala-Lys (N-ε-(N α-PAL-γ-Glu (α-OtBu))-Glu (OtBu)-Phe-Ile-Ala-Tr p (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-resin carrier).
Wherein, described N αin-PAL-γ-Glu (α-OtBu); OtBu is the protecting group of α carboxyl on L-glutamic acid (being the carboxyl on main chain); in order to make the amino condensation coupling on carboxyl on L-glutamic acid side chain and lysine side-chain form paddy acyl group, finally to complete the synthetic of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] lysine side-chain.
As preferred version, step 3 is specially:
Adopt activating reagent and condensation reagent, according to aminoacid sequence C shown in SEQ ID NO:4, hold the order of N end, after first N being held coupling to have the C end of leucine (Fmoc-Leu-OH) of Fmoc protecting group and the coupling of the N of polypeptide resin I end, de-Fmoc protecting group obtains H-Leu-polypeptide resin I, then one by one N is held successively coupling to have Fmoc protecting group and side chain coupling to have the tyrosine (Fmoc-Tyr (tBu)-OH) of tBu protecting group, the coupling of two N ends has Fmoc protecting group and side chain coupling to have the Serine (Fmoc-Ser (tBu)-OH) of tBu protecting group, the coupling of N end has the glycine (Fmoc-Val-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the aspartic acid (Fmoc-Asp (OtBu)-OH) of OtBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Serine (Fmoc-Ser (tBu)-OH) of tBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Threonine (Fmoc-Thr (tBu)-OH) of tBu protecting group, the coupling of N end has the phenylalanine (Fmoc-Phe-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Threonine (Fmoc-Thr (tBu)-OH) of tBu protecting group to extend coupling, after coupling, remove N end with Fmoc protecting group, obtain polypeptide resin II (Thr (tBu)-Phe-Thr (tBu)-Ser (tBu)-Asp (OtBu)-Val-Ser (tBu)-Ser (tBu)-Tyr (tBu)-Leu-polypeptide resin I).
In the preferred version of above-mentioned synthetic polypeptide fragment 1-3, the formed polypeptide resin substitution value of first protected amino acid and resin carrier coupling is preferably 0.2～1.0mmol/g polypeptide resin, and preferred substitution value is 0.3～0.5mmol/g polypeptide resin.
Extension coupling of the present invention refers to after first amino acid and resin carrier coupling, and remaining amino acid carries out coupling with the amino acid generation condensation reaction of previous coupling (condensation reaction of the amino and carboxyl of main chain) one by one according to the order of sequence separately.During coupling of the present invention, each protected amino acid or polypeptide fragment consumption are preferably 1-6 times of polypeptide resin mole number, and more preferably 2.5-3.5 doubly; The described linked reaction time is preferably 60～300 minutes, more preferably 100～140 minutes.
In extending coupling, because each amino acid N end has protecting group, therefore need to first remove the coupling again of N end protecting group, this is common practise for a person skilled in the art.The present invention preferably uses PIP/DMF (piperidines/DMF) mixing solutions to remove N end protecting group, in mixing solutions, containing piperidines, is 10～30% (V), and all the other are DMF.Removing the consumption of N end protecting group reagent is every gram of polypeptide resin 5～15mL, is more preferably every gram of polypeptide resin 8～12mL; Going the N end protecting group time is 10～60 minutes, preferably 15～25 minutes.
It should be noted that; polypeptide resin of the present invention refers to the polypeptide resin that any number protected amino acid is connected with resin carrier according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] amino-acid sequence and forms, and this wherein also comprises polypeptide resin I, polypeptide resin II, Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resin, polypeptide fragment 3 in independent claim.
In the preferred version of above-mentioned synthetic polypeptide fragment 1-3, the resin carrier adopting during described polypeptide fragment is synthetic is Trityl-Cl (trityl chloride) resinoid or hydroxy kind resin.More preferably, described Trityl-Cl resinoid is Trityl-Cl resin, 4-Methyltrityl-Cl (4-methyl trityl chloride) resin, 4-Methoxytrityl-Cl (4-methoxyl group trityl chloride) resin or 2-Cl Trity-Cl (2-chlorine trityl chloride) resin; Described carboxyl resinoid is Wang (king) resin or HMP (to hydroxymethyl phenoxy methylated polystyrene) resin.
Wherein, further preferably, when resin carrier is Trityl-Cl resinoid, the coupling method of protected amino acid and vector resin is: the carboxyl of protected amino acid and the Cl-substituted alkyl in resin under the effect of alkali, esterification occur and access protected amino acid;
In the preferred version of above-mentioned synthetic polypeptide fragment 1-3, the dichloromethane solution of the trifluoroethanol of the further preferred volume per-cent 25% of described lysate.
As preferably, described condensation reagent is preferably N, N-DIC (DIC), N, N-dicyclohexylcarbodiimide (DCC), phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus/organic bases (PyBOP/ organic bases), 2-(7-azepine-1H-benzotriazole-1-yl)-1, 1, 3, 3-tetramethyl-urea phosphofluoric acid ester/organic bases (HATU/ organic bases), benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate/organic bases (HBTU/ organic bases), O-benzotriazole-N, N, N', a kind of in N'-tetramethyl-urea Tetrafluoroboric acid ester/organic bases (TBTU/ organic bases).The mole dosage of described condensation reagent is preferably 1～6 times of amino total mole number in polypeptide resin, more preferably 2.5～3.5 times.
It should be noted that, described PyBOP/ organic bases, HATU/ organic bases, HBTU/ organic bases, TBTU/ organic bases, the condensation reagent that belongs in the present invention four kinds of binary systems, be that PyBOP, HATU, HBTU need to combine and become a kind of condensation reagent use with organic bases respectively in use, the mol ratio of wherein said organic bases and PyBOP, HATU, HBTU, TBTU is preferably as 1.3-3.0:1, more preferably 1.3-1.5:1.
As preferably, the organic bases in described condensation reagent is preferably DIPEA (DIPEA), triethylamine (TEA) or N-methylmorpholine (NMM), more preferably DIPEA.
As preferably, described activating reagent is I-hydroxybenzotriazole (HOBt) or N-hydroxyl-7-azepine benzotriazole (HOAt).The consumption of described activating reagent is preferably 1.2～6 times of amino total mole number in polypeptide resin, more preferably 2.5～3.5 times.
As preferably, coupling reagent of the present invention is DIPEA (DIPEA), N, one of two kinds of N-DIC/DMAPs (DIC/DMAP).
Meanwhile, the step 2 of synthetic method of the present invention and step 4 preferably adopt above-mentioned condensation reagent and activating reagent to carry out coupling.
In building-up process of the present invention, preferably adopt DMF solvent to dissolve.
Except the above-mentioned synthetic method of enumerating, the present invention also can adopt liquid phase synthesizing method to synthesize according to segment condense strategy of the present invention.
In the method for the invention step 5, as preferred version, to adopt the TFA that is 80-95% by volume percent, EDT, the surplus that volume percent is 1-10% be the mixing acid hydrolysis solution acidolysis that water forms in acidolysis described in step 5.More preferably, use by volume percent the TFA that is 90%, EDT, the surplus that volume percent is 5% be the mixing acid hydrolysis solution acidolysis that water forms.Described mixing acid hydrolysis solution consumption is preferably every gram of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resin needs 4～15mL, more preferably 9～11mL.The time of described acidolysis is preferably under room temperature condition 1～6 hour, more preferably 3～4 hours.
As preferably, described purifying is specially:
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product, adds water and stirs, and with ammoniacal liquor, adjusts pH8.5 to dissolving completely, 0.45 μ m filtering with microporous membrane for solution, and purifying is standby;
Adopt high performance liquid chromatography to carry out purifying, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, flow phase system is the 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, the chromatographic column flow velocity of 77mm*250mm is 90mL/min, adopts gradient system wash-out, circulation sample introduction purifying, getting crude product solution is splined in chromatographic column, start moving phase wash-out, collect main peak and boil off after acetonitrile, get profit and draw Shandong peptide purification intermediate concentrated solution;
Get Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] purify intermediates concentrated solution, with 0.45 μ m filter membrane, filter standby;
Adopt high performance liquid chromatography to change salt, flow phase system is 1% acetic acid/aqueous solution-acetonitrile, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, the chromatographic column flow velocity of 77mm*250mm is 90mL/min, adopt gradient elution, quadrat method in circulation, is splined in chromatographic column, starts moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collects and changes salt main peak and with analyzing Liquid Detection purity, merge and change salt main peak solution, concentrating under reduced pressure, obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] aqueous acetic acid, lyophilize, obtains Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] sterling.
The Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] being synthesized by the method for the invention detects through HPLC, and purity is more than 99%, and total recovery is more than 30%, and in the method for the invention, 3 fragments can be synthesized simultaneously, compare with the technical scheme of existing synthesizing amino acid one by one, shortened synthesis cycle, raise the efficiency.
Compare with the Chinese patent that application number is 201210369966.3, by 5 fragments, reduce to 3 fragments, resin carrier consumption greatly reduces, and has reduced complexity and the cost of technique, avoids too much and coupling vector resin and acidolysis, shortens synthesis cycle.
From above technical scheme, the method of the invention can be carried out the synthetic of 3 fragments simultaneously, and avoid too much and coupling vector resin and acidolysis, resin carrier consumption greatly reduces, shortened synthesis cycle, guaranteeing, under the prerequisite of higher total recovery and purity, to simplify the complexity of synthesis technique.
Embodiment
The invention discloses a kind of method of synthetic Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], those skilled in the art can use for reference content herein, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Method of the present invention is described by preferred embodiment, related personnel obviously can be within not departing from content of the present invention, spirit and scope to compound as herein described with preparation method changes or suitably change and combination, realize and apply the technology of the present invention.
In the specific embodiment of the invention; all couplings all can be by commercially available acquisition by the amino acid of protecting group; protected amino acid in the present invention is purchased from Hui Rong bio tech ltd, Chengdu; resin used is purchased from Shangyu pul resin company limited; wherein polypeptide fragment 1 of the present invention, polypeptide fragment 2 also can be by commercially available acquisitions, and the Chinese implication that in application documents, english abbreviation used is corresponding is in Table 1.
The lexical or textual analysis of table 1 english abbreviation
Below in conjunction with embodiment, further set forth the present invention.
Embodiment 1: polypeptide fragment 1 synthetic
Taking substitution degree is the 2-CTC resin 1Kg of 0.5mol/g, join in solid phase synthetic instrument reactor, with DMF washing 2 times, with DMF swelling resin, after 30 minutes, drain, getting 0.75mol Fmoc-Gly-OH dissolves with DMF, add in the above-mentioned reaction column that resin is housed, then add 1.5mol DIPEA, react and drain after 2 hours, add the DMF solution that contains 1.5mol anhydrous methanol, stirring reaction 1 hour, with DMF washing 6 times, obtains Fmoc-Gly-2-CTC resin.
With every gram of resin 10mL20% piperidines/DMF solution, remove Fmoc protecting group in Fmoc-Gly-2-CTC resin 20 minutes, then, with DMF washing 6 times, obtain H-Gly-2-CTC resin.
Get 1.5mol Fmoc-Glu (OtBu)-OH and 1.5mol HOBt, with DMF, dissolve, under stirring, add 1.5mol DIC, continue stirring reaction 1 hour, add in solid phase synthetic instrument reactor, (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 2h, if resin water white transparency reacts completely, resin colour developing, represent that reaction not exclusively, need extend the linked reaction time again), obtain Fmoc-Glu (OtBu)-Gly-2-CTC resin.
According to aminoacid sequence C shown in SEQ ID NO:1, hold the amino-acid sequence of N end, repeat above-mentioned deprotection base and the step that adds corresponding amino acid coupling, complete successively the extension coupling of Fmoc-Ala-OH, Fmoc-His (Trt)-OH.
Reaction finishes rear methanol wash 6 times of using, and resin vacuum-drying is spent the night, and weighs and obtains the polypeptide fragment 1 that 1.45Kg does not remove 2-CTC resin, is added in 25L glass reactor.
Configuration lysate 25%TFE/ dichloromethane solution 15L, pours lytic reagent in flask into room temperature reaction 2h.Reaction finishes, and filters resin, collects filtrate.Filtrate volume is revolved after evaporate to dryness, add 1.5L DCM, mixed solution is dropped in 15L ether, separate out white solid, filter collecting precipitation, anhydrous diethyl ether washing, and vacuum-drying, obtains 0.43Kg polypeptide fragment 1, Fmoc-His (Trt)-Ala-Glu (OtBu)-Gly-OH, purity 96.3%, yield 92.1%.
Embodiment 2: polypeptide fragment 1 synthetic
Taking substitution degree is the 2-CTC resin 1Kg of 0.5mol/g, join in solid phase synthetic instrument reactor, with DMF washing 2 times, with DMF swelling resin, after 30 minutes, drain, getting 0.75mol Fmoc-Gly-OH dissolves with DMF, add in the above-mentioned reaction column that resin is housed, then add 1.5mol DIPEA, react and drain after 2 hours, add the DMF solution that contains 1.5mol anhydrous methanol, stirring reaction 1 hour, with DMF washing 6 times, obtains Fmoc-Gly-2-CTC resin.
With every gram of resin 10mL20% piperidines/DMF solution, remove Fmoc protecting group in Fmoc-Gly-2-CTC 20 minutes, then, with DMF washing 6 times, obtain H-Gly-2-CTC resin.
Get 1.5mol Fmoc-Glu (OtBu)-OH and 1.5mol HOBt, with DMF, dissolve, under stirring, add 1.43mol HBTU, continue stirring reaction and after 1 hour, add again 2molDIPEA, after mixing, join in solid phase synthetic instrument reactor, (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 2h, if resin water white transparency, react completely, resin colour developing, represent that reaction not exclusively, need extend the linked reaction time again), obtain Fmoc-Glu (OtBu)-Gly-2-CTC resin.
According to aminoacid sequence C shown in SEQ ID NO:1, hold the amino-acid sequence of N end, repeat above-mentioned deprotection base and the step that adds corresponding amino acid coupling, complete successively the extension coupling of Fmoc-Ala-OH, Fmoc-His (Trt)-OH.
Reaction finishes rear methanol wash 6 times of using, and resin vacuum-drying is spent the night, and weighs and obtains the polypeptide fragment 1 that 1.43Kg does not remove 2-CTC resin, is added in 25L glass reactor.
Configuration lytic reagent 25%TFE/ dichloromethane solution 15L, pours lytic reagent in flask into room temperature reaction 2h.Reaction finishes, and filters resin, collects filtrate.Filtrate volume is revolved after evaporate to dryness, add 1.5L DCM, mixed solution is dropped in 15L ether, separate out white solid, filter collecting precipitation, anhydrous diethyl ether washing, and vacuum-drying, obtains 0.42Kg polypeptide fragment 1, Fmoc-His (Trt)-Ala-Glu (OtBu)-Gly-OH, purity 94.7%, yield 89.9%.
Embodiment 3: polypeptide fragment 2 synthetic
Taking substitution degree is the 2-CTC resin 1Kg of 0.5mol/g, join in solid phase synthetic instrument reactor, with DMF washing 2 times, with DMF swelling resin, after 30 minutes, drain, getting 0.75mol Fmoc-Gly-OH dissolves with DMF, add in the above-mentioned reaction column that resin is housed, then add 1.5mol DIPEA, react and drain after 2 hours, add the DMF solution that contains 1.5mol anhydrous methanol, stirring reaction 1 hour, with DMF washing 6 times, obtains Fmoc-Gly-2-CTC resin.
With every gram of resin 10mL20% piperidines/DMF solution, remove Fmoc protecting group in Fmoc-Gly-2-CTC resin 20 minutes, then, with DMF washing 6 times, obtain H-Gly-2-CTC resin.
Get 1.5mol Fmoc-Glu (OtBu)-OH and 1.5mol HOBt, with DMF, dissolve, under stirring, add 1.5mol DIC, continue stirring reaction 1 hour, add in solid phase synthetic instrument reactor, (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 2h, if resin water white transparency reacts completely, resin colour developing, represent that reaction not exclusively, need extend the linked reaction time again), obtain Fmoc-Glu (OtBu)-Gly-2-CTC resin.
Reaction finishes rear methanol wash 6 times of using, and resin vacuum-drying is spent the night, and weighs and obtains the polypeptide fragment 2 that 1.24Kg does not remove 2-CTC resin, is added in 25L glass reactor.
Configuration lytic reagent 25%TFE/ dichloromethane solution 15L, pours lytic reagent in flask into room temperature reaction 2h.Reaction finishes, and filters resin, collects filtrate.Filtrate volume is revolved after evaporate to dryness, add 1.2L DCM, mixed solution is dropped in 12L ether, separate out white solid, filter collecting precipitation, anhydrous diethyl ether washing, and vacuum-drying, obtain 0.22Kg polypeptide fragment 2, Fmoc-Glu (OtBu)-Gly-OH, purity 97.6%, yield 91.3%.
Embodiment 4: polypeptide fragment 2 synthetic
Taking substitution degree is the 2-CTC resin 1Kg of 0.5mol/g, join in solid phase synthetic instrument reactor, with DMF washing 2 times, with DMF swelling resin, after 30 minutes, drain, getting 0.75mol Fmoc-Gly-OH dissolves with DMF, add in the above-mentioned reaction column that resin is housed, then add 1.5mol DIPEA, react and drain after 2 hours, add the DMF solution that contains 1.5mol anhydrous methanol, stirring reaction 1 hour, with DMF washing 6 times, obtains Fmoc-Gly-2-CTC resin.
With every gram of resin 10mL20% piperidines/DMF solution, remove Fmoc protecting group in Fmoc-Gly-2-CTC resin 20 minutes, then, with DMF washing 6 times, obtain H-Gly-2-CTC resin.
Get 1.5mol Fmoc-Glu (OtBu)-OH and 1.5mol HOBt, with DMF, dissolve, under stirring, add 1.43mol HBTU, continue stirring reaction and after 1 hour, add again 2molDIPEA, after mixing, join in solid phase synthetic instrument reactor, (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 2h, if resin water white transparency, react completely, resin colour developing, represent that reaction not exclusively, need extend the linked reaction time again), obtain Fmoc-Glu (OtBu)-Gly-2-CTC resin.
Reaction finishes rear methanol wash 6 times of using, and resin vacuum-drying is spent the night, and weighs and obtains the polypeptide fragment 2 that 1.25Kg does not remove 2-CTC resin, is added in 25L glass reactor.
Configuration lytic reagent 25%TFE/ dichloromethane solution 15L, pours lytic reagent in flask into room temperature reaction 2h.Reaction finishes, and filters resin, collects filtrate.Filtrate volume is revolved after evaporate to dryness, add 1.2L DCM, mixed solution is dropped in 12L ether, separate out white solid, filter collecting precipitation, anhydrous diethyl ether washing, and vacuum-drying, obtain 0.23Kg polypeptide fragment 2, Fmoc-Glu (OtBu)-Gly-OH, purity 97.1%, yield 95.4%.
Embodiment 5: polypeptide fragment 3 synthetic
Taking substitution degree is the Wang resin 0.5Kg of 0.4mol/g, join in solid phase synthetic instrument reactor, with DMF washing 2 times, with DMF swelling resin, after 30 minutes, drain, getting 0.6mol Fmoc-Gly-OH dissolves with DMF, add in the above-mentioned reaction column that resin is housed, add again 0.6mol DIC and 0.06mol4-Dimethylamino pyridine (DMAP), react and drain after 6 hours, add the DMF solution that contains 1.5mol anhydrous methanol, stirring reaction 1 hour, with DMF washing 6 times, obtains Fmoc-Gly-Wang resin.
With every gram of resin 10mL20% piperidines/DMF solution, remove Fmoc protecting group in Fmoc-Gly-Wang resin 20 minutes, then, with DMF washing 6 times, obtain H-Gly-Wang resin.
Get 0.6mol Fmoc-Arg (Pdf)-OH and 0.6mol HOBt, with DMF, dissolve, under stirring, add 0.6mol DIC, continue stirring reaction 1 hour, add in solid phase synthetic instrument reactor, (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 3h, if resin water white transparency reacts completely, resin colour developing, represent that reaction not exclusively, need extend the linked reaction time again), obtain Fmoc-Arg (Pdf)-Gly-Wang resin.
According to aminoacid sequence C shown in SEQ ID NO:3, hold the amino-acid sequence of N end, repeat above-mentioned deprotection base and the step that adds corresponding amino acid coupling, one by one N is held successively again coupling to have the glycine (Fmoc-Gly-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the arginine (Fmoc-Arg (Pdf)-OH) of Pdf protecting group, the coupling of N end has the α-amino-isovaleric acid (Fmoc-Val-OH) of Fmoc protecting group, the coupling of N end has the leucine (Fmoc-Leu-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the tryptophane (Fmoc-Trp (Boc)-OH) of Boc protecting group, the coupling of N end has L-Ala (Fmoc-Ala-OH) the N end coupling of Fmoc protecting group to have the Isoleucine (Fmoc-Ile-OH) of Fmoc protecting group, the coupling of N end has the phenylalanine (Fmoc-Phe-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the L-glutamic acid (Fmoc-Glu (OtBu)-OH) of OtBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have N αmethionin (Fmoc-Lys (the N of-PAL-γ-Glu (α-OtBu) α-PAL-γ-Glu (α-OtBu))-OH), the coupling of two N ends has the L-Ala (Fmoc-Ala-OH) of Fmoc protecting group, the coupling of N end to have Fmoc protecting group and side chain coupling to have the glutamine (Fmoc-Gln (Trt)-OH) of Trt protecting group to extend coupling, finally with every gram of polypeptide resin 15mL PIP (20%)/DMF (80%) mixing solutions remove N section with Fmoc protecting group 20min, then with DMF washing 6 times, obtain polypeptide fragment 3 (Gln (Trt)-Ala-Ala-Lys (N-ε-(N α-PAL-γ-Glu (α-OtBu))-Glu (OtBu)-Phe-Ile-Ala-Tr p (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-Wang resin),
Embodiment 6: polypeptide fragment 3 synthetic
Taking substitution degree is the Wang resin 0.5Kg of 0.4mol/g, join in solid phase synthetic instrument reactor, with DMF washing 2 times, with DMF swelling resin, after 30 minutes, drain, getting 0.6mol Fmoc-Gly-OH dissolves with DMF, add in the above-mentioned reaction column that resin is housed, add again 0.6mol DIC and 0.06mol4-Dimethylamino pyridine (DMAP), react and drain after 6 hours, add the DMF solution that contains 1.5mol anhydrous methanol, stirring reaction 1 hour, with DMF washing 6 times, obtains Fmoc-Gly-Wang resin.
With every gram of resin 10mL20% piperidines/DMF solution, remove Fmoc protecting group in Fmoc-Gly-Wang resin 20 minutes, then, with DMF washing 6 times, obtain H-Gly-Wang resin.
Get 0.6mol Fmoc-Arg (Pdf)-OH and 0.6mol HOBt, with DMF, dissolve, under stirring, add 0.57mol HBTU, continue stirring reaction and after 1 hour, add again 0.9molDIPEA, after mixing, join in solid phase synthetic instrument reactor, (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 3h, if resin water white transparency, react completely, resin colour developing, represent that reaction not exclusively, need extend the linked reaction time again), obtain Fmoc-Arg (Pdf)-Gly-Wang resin.
According to aminoacid sequence C shown in SEQ ID NO:3, hold the amino-acid sequence of N end, repeat above-mentioned deprotection base and the step that adds corresponding amino acid coupling, one by one N is held successively again coupling to have the glycine (Fmoc-Gly-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the arginine (Fmoc-Arg (Pdf)-OH) of Pdf protecting group, the coupling of N end has the α-amino-isovaleric acid (Fmoc-Val-OH) of Fmoc protecting group, the coupling of N end has the leucine (Fmoc-Leu-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the tryptophane (Fmoc-Trp (Boc)-OH) of Boc protecting group, the coupling of N end has L-Ala (Fmoc-Ala-OH) the N end coupling of Fmoc protecting group to have the Isoleucine (Fmoc-Ile-OH) of Fmoc protecting group, the coupling of N end has the phenylalanine (Fmoc-Phe-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the L-glutamic acid (Fmoc-Glu (OtBu)-OH) of OtBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have N αmethionin (Fmoc-Lys (the N of-PAL-γ-Glu (α-OtBu) α-PAL-γ-Glu (α-OtBu))-OH), the coupling of two N ends has the L-Ala (Fmoc-Ala-OH) of Fmoc protecting group, the coupling of N end to have Fmoc protecting group and side chain coupling to have the glutamine (Fmoc-Gln (Trt)-OH) of Trt protecting group to extend coupling, finally with every gram of polypeptide resin 15mL PIP (20%)/DMF (80%) mixing solutions remove N section with Fmoc protecting group 20min, then with DMF washing 6 times, obtain polypeptide fragment 3 (Gln (Trt)-Ala-Ala-Lys (N-ε-(N α-PAL-γ-Glu (α-OtBu))-Glu (OtBu)-Phe-Ile-Ala-Tr p (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-Wang resin),
Embodiment 7: polypeptide resin I synthetic
Get 0.44mol polypeptide fragment 2, 0.44mol polypeptide fragment 3 and 0.44mol HOAt, with DMF, dissolve, under stirring, add 0.44mol DIC, continue stirring reaction 1 hour, join in solid phase synthetic instrument reactor, (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 6h, if resin water white transparency, react completely, resin colour developing, represent that reaction not exclusively, the linked reaction time need be extended again), use again every gram of resin 10mL PIP (20%)/DMF (80%) mixing solutions to take off Fmoc protecting group, time is 20min, with DMF washing 6 times, obtain polypeptide resin I, be NH 2-Glu (OtBu)-Gly-Gln (Trt)-Ala-Ala-Lys (N-ε-(N α-PAL-γ-Glu (α-OtBu))-Glu (Ot Bu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-Wang resin,
Embodiment 8: polypeptide resin I synthetic
Get 0.44mol polypeptide fragment 2, 0.44mol polypeptide fragment 3 and 0.44mol HOAt, with DMF, dissolve, under stirring, add 0.42mol HATU, continue stirring reaction and after 1 hour, add again 0.65molDIPEA, after mixing, join in solid phase synthetic instrument reactor, (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 6h, if resin water white transparency, react completely, resin colour developing, represent that reaction not exclusively, the linked reaction time need be extended again), use again every gram of resin 10mL PIP (20%)/DMF (80%) mixing solutions to take off Fmoc protecting group, time is 20min, with DMF washing 6 times, obtain polypeptide resin I, be NH 2-Glu (OtBu)-Gly-Gln (Trt)-Ala-Ala-Lys (N-ε-(N α-PAL-γ-Glu (α-OtBu))-Glu (Ot Bu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-Wang resin,
Embodiment 9: polypeptide resin II synthetic
Get 0.6mol protected amino acid, 0.6mol polypeptide resin I and 0.6mol HOBt; with DMF, dissolve; under stirring, add 0.6mol DIC, continue stirring reaction 1 hour, add in solid phase synthetic instrument reactor; (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 3h; if resin water white transparency, reacts completely, resin colour developing; represent that reaction not exclusively, extends the linked reaction time).
Protected amino acid is held the order of N end according to aminoacid sequence C shown in SEQ ID NO:4, after first N being held coupling to have the C end of leucine (Fmoc-Leu-OH) of Fmoc protecting group and the coupling of the N of polypeptide resin I end, de-Fmoc protecting group obtains H-Leu-polypeptide resin I, then one by one N is held successively coupling to have Fmoc protecting group and side chain coupling to have the tyrosine (Fmoc-Tyr (tBu)-OH) of tBu protecting group, the coupling of two N ends has Fmoc protecting group and side chain coupling to have the Serine (Fmoc-Ser (tBu)-OH) of tBu protecting group, the coupling of N end has the glycine (Fmoc-Val-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the aspartic acid (Fmoc-Asp (OtBu)-OH) of OtBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Serine (Fmoc-Ser (tBu)-OH) of tBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Threonine (Fmoc-Thr (tBu)-OH) of tBu protecting group, the coupling of N end has the phenylalanine (Fmoc-Phe-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Threonine (Fmoc-Thr (tBu)-OH) of tBu protecting group to extend coupling, after coupling, with every gram of resin 10mL PIP (20%)/DMF (80%) mixing solutions, take off Fmoc protecting group, time is 20min, with DMF, wash, obtain polypeptide resin II, that is: NH 2-Thr (tBu)-Phe-Thr (tBu)-Ser (tBu)-Asp (OtBu)-Val-Ser (tBu)-Ser (tBu)-Tyr (t Bu)-Leu-Glu (OtBu)-Gly-Gln (Trt)-Ala-Ala-Lys (N-ε-(N α-PAL-γ-Glu (α-OtBu))-Glu (OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-Wang resin.
Embodiment 10: polypeptide resin II synthetic
Get 0.6mol protected amino acid, 0.6mol polypeptide resin I and 0.6mol HOBt; with DMF, dissolve; under stirring, add 0.57mol HBTU, continue stirring reaction and after 1 hour, add again 0.9molDIPEA, after mixing, join in solid phase synthetic instrument reactor; (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 3h; if resin water white transparency, reacts completely, resin colour developing; represent that reaction not exclusively, extends the linked reaction time).
Protected amino acid is held the order of N end according to aminoacid sequence C shown in SEQ ID NO:4, after first N being held coupling to have the C end of leucine (Fmoc-Leu-OH) of Fmoc protecting group and the coupling of the N of polypeptide resin I end, de-Fmoc protecting group obtains H-Leu-polypeptide resin I, then one by one N is held successively coupling to have Fmoc protecting group and side chain coupling to have the tyrosine (Fmoc-Tyr (tBu)-OH) of tBu protecting group, the coupling of two N ends has Fmoc protecting group and side chain coupling to have the Serine (Fmoc-Ser (tBu)-OH) of tBu protecting group, the coupling of N end has the glycine (Fmoc-Val-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the aspartic acid (Fmoc-Asp (OtBu)-OH) of OtBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Serine (Fmoc-Ser (tBu)-OH) of tBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Threonine (Fmoc-Thr (tBu)-OH) of tBu protecting group, the coupling of N end has the phenylalanine (Fmoc-Phe-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Threonine (Fmoc-Thr (tBu)-OH) of tBu protecting group to extend coupling, after coupling, with every gram of resin 10mL PIP (20%)/DMF (80%) mixing solutions, take off Fmoc protecting group, time is 20min, with DMF, wash, obtain polypeptide resin II, that is: NH 2-Thr (tBu)-Phe-Thr (tBu)-Ser (tBu)-Asp (OtBu)-Val-Ser (tBu)-Ser (tBu)-Tyr (t Bu)-Leu-Glu (OtBu)-Gly-Gln (Trt)-Ala-Ala-Lys (N-ε-(N α-PAL-γ-Glu (α-OtBu))-Glu (OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-Wang resin.
Embodiment 11: Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resin synthetic
Get 0.44mol polypeptide fragment 1, 0.44mol polypeptide resin II and 0.44mol HOAt, with DMF, dissolve, under stirring, add 0.44mol DIC, continue stirring reaction 1 hour, join in solid phase synthetic instrument reactor, (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 6h, if resin water white transparency, react completely, resin colour developing, represent that reaction not exclusively, the linked reaction time need be extended again), use again every gram of resin 10mL PIP (20%)/DMF (80%) mixing solutions to take off Fmoc protecting group, time is 20min, with DCM washing 6 times, vacuum decompression is dry, get profit and draw Shandong peptide resin 1445g, be NH 2-His (Trt)-Ala-Glu (OtBu)-Gly-Thr (tBu)-Phe-Thr (tBu)-Ser (tBu)-Asp (OtBu)-Va l-Ser (tBu)-Ser (tBu)-Tyr (tBu)-Leu-Glu (OtBu)-Gly-Gln (Trt)-Ala-Ala-Lys (N-ε-(N α-PAL-γ-Glu (α-OtBu))-Glu (OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Ar g (Pbf)-Gly-Wang resin.
Embodiment 12: Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resin synthetic
Get 0.44mol polypeptide fragment 1, 0.44mol polypeptide resin II and 0.44mol HOAt, with DMF, dissolve, under stirring, add 0.42mol HATU, continue stirring reaction and after 1 hour, add again 0.65molDIPEA, after mixing, join in solid phase synthetic instrument reactor, (reaction end detects and is as the criterion with ninhydrin method room temperature reaction 6h, if resin water white transparency, react completely, resin colour developing, represent that reaction not exclusively, the linked reaction time need be extended again), use again every gram of resin 10mL PIP (20%)/DMF (80%) mixing solutions to take off Fmoc protecting group, time is 20min, with DCM washing 6 times, vacuum decompression is dry, get profit and draw Shandong peptide resin 1381g, be NH 2-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 (N-ε-(N α-PAL-γ-Glu (α-OtBu))-Glu (OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-Wang resin.
Embodiment 13: the preparation of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product
Get embodiment 11 gained Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resins, adding volume ratio is the mixing acid hydrolysis solution (consumption 10mL/ gram of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resin) of TFA ︰ water ︰ EDT=90 ︰ 5 ︰ 5, stir, stirring at room reaction 3 hours, reaction mixture is used sand core funnel to filter, collect filtrate, resin is again with a small amount of TFA washing 3 times, concentrating under reduced pressure after merging filtrate, add anhydrous diethyl ether precipitation, then wash precipitation 3 times with anhydrous diethyl ether, drain to obtain off-white powder, vacuum decompression is dried to constant weight, obtains Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product 863g.
Embodiment 14: the preparation of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product
Get embodiment 12 gained Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resins, adding volume ratio is the mixing acid hydrolysis solution (consumption 10mL/ gram of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resin) of TFA ︰ water ︰ EDT=90 ︰ 5 ︰ 5, stir, stirring at room reaction 3 hours, reaction mixture is used sand core funnel to filter, collect filtrate, resin is again with a small amount of TFA washing 3 times, concentrating under reduced pressure after merging filtrate, add anhydrous diethyl ether precipitation, then wash precipitation 3 times with anhydrous diethyl ether, drain to obtain off-white powder, vacuum decompression is dried to constant weight, obtains Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product 797g.
Embodiment 15: Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] purifying crude
Get embodiment 13 gained Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude products, add water and stir, with ammoniacal liquor, adjust pH8.5 to dissolving completely, 0.45 μ m filtering with microporous membrane for solution, purifying is standby;
Adopt high performance liquid chromatography to carry out purifying, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, flow phase system is the 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, the chromatographic column flow velocity of 77mm*250mm is 90mL/min, adopts gradient system wash-out, circulation sample introduction purifying, getting crude product solution is splined in chromatographic column, start moving phase wash-out, collect main peak and boil off after acetonitrile, get profit and draw Shandong peptide purification intermediate concentrated solution;
Get Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] purify intermediates concentrated solution, with 0.45 μ m filter membrane, filter standby;
Adopt high performance liquid chromatography to change salt, flow phase system is 1% acetic acid/aqueous solution-acetonitrile, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, the chromatographic column flow velocity of 77mm*250mm is 90mL/min, adopt gradient elution, quadrat method in circulation, be splined in chromatographic column, start moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collection is changed salt main peak and is used and analyze Liquid Detection purity, merge and change salt main peak solution, concentrating under reduced pressure, obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] aqueous acetic acid, lyophilize, obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] sterling 270.9g, total recovery is 36.1%, molecular weight: 3751.2 (100%M+H), purity: 99.3%, maximum single contaminant 0.12%.
Embodiment 16: Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] purifying crude
Get embodiment 14 gained Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude products, add water and stir, with ammoniacal liquor, adjust pH8.5 to dissolving completely, 0.45 μ m filtering with microporous membrane for solution, purifying is standby;
Adopt high performance liquid chromatography to carry out purifying, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, flow phase system is the 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, the chromatographic column flow velocity of 77mm*250mm is 90mL/min, adopts gradient system wash-out, circulation sample introduction purifying, getting crude product solution is splined in chromatographic column, start moving phase wash-out, collect main peak and boil off after acetonitrile, get profit and draw Shandong peptide purification intermediate concentrated solution;
Get Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] purify intermediates concentrated solution, with 0.45 μ m filter membrane, filter standby;
Adopt high performance liquid chromatography to change salt, flow phase system is 1% acetic acid/aqueous solution-acetonitrile, purifying is the anti-phase C18 of 10 μ m with chromatograph packing material, the chromatographic column flow velocity of 77mm*250mm is 90mL/min, adopt gradient elution, quadrat method in circulation, be splined in chromatographic column, start moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collection is changed salt main peak and is used and analyze Liquid Detection purity, merge and change salt main peak solution, concentrating under reduced pressure, obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] aqueous acetic acid, lyophilize, obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] sterling 237.8g, total recovery is 31.7%, molecular weight: 3751.6 (100%M+H), purity: 99.0%, maximum single contaminant 0.10%.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (10)

1. a method for synthetic Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], is characterized in that, comprises the following steps:
Step 1, synthesize the polypeptide fragment 1 that on aminoacid sequence N shown in SEQ ID NO:1 end, His side chain and on Glu side chain coupling has protecting group;
Synthesize the polypeptide fragment 2 that on aminoacid sequence N shown in SEQ ID NO:2 end, Glu side chain coupling has protecting group;
Synthesize in the coupling of aminoacid sequence C shown in SEQ ID NO:3 end have resin carrier, on Gln side chain, on Glu side chain, on Trp side chain, on Arg side chain coupling have protecting group and on Lys side chain coupling have N αthe polypeptide fragment 3 of-PAL-γ-Glu (α-OtBu);
Step 2, by the C end coupling of the N end of polypeptide fragment 3 and polypeptide fragment 2, after coupling, remove the N end protecting group of polypeptide fragment 2, obtain polypeptide resin I;
Step 3, the order of holding N end according to aminoacid sequence C shown in SEQ ID NO:4, first N is held coupling to have the leucic C section of protecting group and the coupling of the N of polypeptide resin I end, then one by one other corresponding protected amino acids are extended to coupling successively, after coupling, remove N end protecting group, obtain polypeptide resin II;
Step 4, by the N end coupling of the C end of polypeptide fragment 1 and polypeptide resin II, after coupling, remove the N end protecting group of polypeptide fragment 1, obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resin;
Step 5, Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resinous acid solution remove C end resin and all protecting groups obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product, and purifying crude obtains Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
2. method according to claim 1, is characterized in that, synthetic polypeptide fragment 1 is specially described in step 1:
Under coupling reagent exists, after N is held to glycine (Fmoc-Gly-OH) that coupling has a Fmoc protecting group and resin carrier coupling, de-Fmoc protecting group obtains H-Gly-resin carrier, then adopt activating reagent and condensation reagent, according to aminoacid sequence C shown in SEQ ID NO:1, hold the order of N end, one by one N is held successively coupling to have Fmoc protecting group and side chain coupling to have the L-glutamic acid (Fmoc-Glu (OtBu)-OH) of OtBu protecting group, the coupling of N end has the L-Ala (Fmoc-Ala-OH) of Fmoc protecting group, the coupling of N end has Boc protecting group or Fmoc protecting group and side chain coupling to have the Histidine (Fmoc/Boc-His (Trt)-OH) of Trt protecting group to extend coupling, lysate cracking after coupling, deresinate carrier obtains polypeptide fragment 1 (Fmoc/Boc-His (Trt)-Ala-Glu (OtBu)-Gly-OH).
3. method according to claim 1, is characterized in that, synthetic polypeptide fragment 2 is specially described in step 1:
Under coupling reagent exists; after N is held to glycine (Fmoc-Gly-OH) that coupling has a Fmoc protecting group and resin carrier coupling, de-Fmoc protecting group obtains H-Gly-resin carrier; then adopt activating reagent and condensation reagent; according to aminoacid sequence C shown in SEQ ID NO:2, hold the order of N end; hold coupling to have Fmoc protecting group and side chain coupling to have the L-glutamic acid (Fmoc-Glu (OtBu)-OH) of OtBu protecting group to extend coupling N; lysate cracking after coupling, deresinate carrier obtains polypeptide fragment 2 (Fmoc-Glu (OtBu)-Gly-OH).
4. method according to claim 1, is characterized in that, synthetic polypeptide fragment 3 is specially described in step 1:
Under coupling reagent exists, after N is held to glycine (Fmoc-Gly-OH) that coupling has a Fmoc protecting group and resin carrier coupling, de-Fmoc protecting group obtains H-Gly-resin carrier, then adopt activating reagent and condensation reagent, according to aminoacid sequence C shown in SEQ ID NO:3, hold the order of N end, one by one N is held successively coupling to have Fmoc protecting group and side chain coupling to have the arginine (Fmoc-Arg (Pdf)-OH) of Pdf protecting group, the coupling of N end has the glycine (Fmoc-Gly-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the arginine (Fmoc-Arg (Pdf)-OH) of Pdf protecting group, the coupling of N end has the α-amino-isovaleric acid (Fmoc-Val-OH) of Fmoc protecting group, the coupling of N end has the leucine (Fmoc-Leu-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the tryptophane (Fmoc-Trp (Boc)-OH) of Boc protecting group, the coupling of N end has L-Ala (Fmoc-Ala-OH) the N end coupling of Fmoc protecting group to have the Isoleucine (Fmoc-Ile-OH) of Fmoc protecting group, the coupling of N end has the phenylalanine (Fmoc-Phe-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the L-glutamic acid (Fmoc-Glu (OtBu)-OH) of OtBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have N αmethionin (Fmoc-Lys (the N of-PAL-γ-Glu (α-OtBu) α-PAL-γ-Glu (α-OtBu))-OH), the coupling of two N end has the L-Ala (Fmoc-Ala-OH) of Fmoc protecting group, the coupling of N end to have Fmoc protecting group and side chain coupling to have the glutamine (Fmoc-Gln (Trt)-OH) of Trt protecting group to extend coupling, remove N section with Fmoc protecting group obtain polypeptide fragment 3 (Gln (Trt)-Ala-Ala-Lys (N-ε-(N α-PAL-γ-Glu (α-OtBu))-Glu (OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-resin carrier).
5. method according to claim 1, is characterized in that, step 3 is specially:
Adopt activating reagent and condensation reagent, according to aminoacid sequence C shown in SEQ ID NO:4, hold the order of N end, after first N being held coupling to have the C end of leucine (Fmoc-Leu-OH) of Fmoc protecting group and the coupling of the N of polypeptide resin I end, de-Fmoc protecting group obtains H-Leu-polypeptide resin I, then one by one N is held successively coupling to have Fmoc protecting group and side chain coupling to have the tyrosine (Fmoc-Tyr (tBu)-OH) of tBu protecting group, the coupling of two N ends has Fmoc protecting group and side chain coupling to have the Serine (Fmoc-Ser (tBu)-OH) of tBu protecting group, the coupling of N end has the glycine (Fmoc-Val-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the aspartic acid (Fmoc-Asp (OtBu)-OH) of OtBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Serine (Fmoc-Ser (tBu)-OH) of tBu protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Threonine (Fmoc-Thr (tBu)-OH) of tBu protecting group, the coupling of N end has the phenylalanine (Fmoc-Phe-OH) of Fmoc protecting group, the coupling of N end has Fmoc protecting group and side chain coupling to have the Threonine (Fmoc-Thr (tBu)-OH) of tBu protecting group to extend coupling, after coupling, remove N end with Fmoc protecting group, obtain polypeptide resin II (Thr (tBu)-Phe-Thr (tBu)-Ser (tBu)-Asp (OtBu)-Val-Ser (tBu)-Ser (tBu)-Tyr (tBu)-Leu-polypeptide resin I).
6. according to method described in claim 1-4 any one, it is characterized in that, described resin carrier is Trityl-Cl resinoid or hydroxy kind resin.
7. according to method described in claim 2-4 any one, it is characterized in that, described lysate is the dichloromethane solution of the trifluoroethanol of volume percent 25%.
8. according to method described in claim 1-5 any one, it is characterized in that, described condensation reagent is preferably N, N-DIC, N, N-dicyclohexylcarbodiimide, phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus/N, N-diisopropylethylamine, 2-(7-azepine-1H-benzotriazole-1-yl)-1, 1, 3, 3-tetramethyl-urea phosphofluoric acid ester/N, N-diisopropylethylamine, benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate/N, N-diisopropylethylamine, O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid ester/N, a kind of in N-diisopropylethylamine.
9. according to method described in claim 1-5 any one, it is characterized in that, described activating reagent is I-hydroxybenzotriazole or N-hydroxyl-7-azepine benzotriazole.
10. method according to claim 1, is characterized in that, to adopt the TFA that is 80-95% by volume percent, EDT, the surplus that volume percent is 1-10% be the mixing acid hydrolysis solution acidolysis that water forms in acidolysis described in step 5.
CN201410265582.6A 2014-06-13 2014-06-13 A kind of method synthesizing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] Active CN104004083B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201410265582.6A CN104004083B (en) 2014-06-13 2014-06-13 A kind of method synthesizing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201410265582.6A CN104004083B (en) 2014-06-13 2014-06-13 A kind of method synthesizing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]

Publications (2)

Publication Number Publication Date
CN104004083A true CN104004083A (en) 2014-08-27
CN104004083B CN104004083B (en) 2016-10-05

Family

ID=51365011

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201410265582.6A Active CN104004083B (en) 2014-06-13 2014-06-13 A kind of method synthesizing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]

Country Status (1)

Country Link
CN (1) CN104004083B (en)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104650219A (en) * 2015-02-15 2015-05-27 兰州大学 Method for preparing liraglutide by convergent synthesis
WO2016046753A1 (en) 2014-09-23 2016-03-31 Novetide, Ltd. Synthesis of glp-1 peptides
WO2016067271A1 (en) * 2014-10-31 2016-05-06 Auro Peptides Ltd A process for the preparation of liraglutide
CN106478805A (en) * 2015-08-28 2017-03-08 甘李药业股份有限公司 A kind of preparation method of GLP-1 derivant
CN107286234A (en) * 2016-03-31 2017-10-24 深圳翰宇药业股份有限公司 A kind of method for reducing and/or removing default peptide in Solid-phase synthesis peptides
WO2018033127A1 (en) * 2016-08-19 2018-02-22 深圳市健元医药科技有限公司 Synthesis method for low-racemization impurity liraglutide
CN107903317A (en) * 2017-12-29 2018-04-13 江苏诺泰澳赛诺生物制药股份有限公司 A kind of synthetic method of Liraglutide
CN108107103A (en) * 2017-12-14 2018-06-01 华中师范大学 The MS probe of glutamate receptor and its space distribution rule detection method in brain tissue
WO2019069274A1 (en) 2017-10-04 2019-04-11 Chemical & Biopharmaceutical Laboratories Of Patras S.A. A process for preparing a glucagon-like peptide
CN110894227A (en) * 2018-09-13 2020-03-20 南京华威医药科技集团有限公司 Solid-phase synthesis method of liraglutide
WO2020074583A1 (en) 2018-10-09 2020-04-16 Fresenius Kabi Ipsum S.R.L. Process for the manufacture of glp-1 analogues
WO2020127476A1 (en) 2018-12-19 2020-06-25 Krka, D.D., Novo Mesto Pharmaceutical composition comprising glp-1 analogue
WO2021123228A1 (en) 2019-12-18 2021-06-24 Krka, D.D., Novo Mesto Pharmaceutical composition comprising glp-1 analogue

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102875665A (en) * 2012-09-28 2013-01-16 深圳翰宇药业股份有限公司 Method for synthesizing liraglutide
CN103275208A (en) * 2013-05-27 2013-09-04 成都圣诺生物制药有限公司 Preparation method for liraglutide
CN103288951A (en) * 2013-06-19 2013-09-11 深圳翰宇药业股份有限公司 Preparation method of liraglutide

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102875665A (en) * 2012-09-28 2013-01-16 深圳翰宇药业股份有限公司 Method for synthesizing liraglutide
CN103275208A (en) * 2013-05-27 2013-09-04 成都圣诺生物制药有限公司 Preparation method for liraglutide
CN103288951A (en) * 2013-06-19 2013-09-11 深圳翰宇药业股份有限公司 Preparation method of liraglutide

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016046753A1 (en) 2014-09-23 2016-03-31 Novetide, Ltd. Synthesis of glp-1 peptides
WO2016067271A1 (en) * 2014-10-31 2016-05-06 Auro Peptides Ltd A process for the preparation of liraglutide
CN104650219B (en) * 2015-02-15 2017-11-14 兰州大学 The method that fragment condensation prepares Liraglutide
CN104650219A (en) * 2015-02-15 2015-05-27 兰州大学 Method for preparing liraglutide by convergent synthesis
CN106478805B (en) * 2015-08-28 2021-05-04 甘李药业股份有限公司 Preparation method of GLP-1 derivative
CN106478805A (en) * 2015-08-28 2017-03-08 甘李药业股份有限公司 A kind of preparation method of GLP-1 derivant
CN107286234A (en) * 2016-03-31 2017-10-24 深圳翰宇药业股份有限公司 A kind of method for reducing and/or removing default peptide in Solid-phase synthesis peptides
CN107286234B (en) * 2016-03-31 2021-06-08 深圳翰宇药业股份有限公司 Method for reducing and/or removing default peptide in polypeptide solid phase synthesis
WO2018033127A1 (en) * 2016-08-19 2018-02-22 深圳市健元医药科技有限公司 Synthesis method for low-racemization impurity liraglutide
WO2018032521A1 (en) * 2016-08-19 2018-02-22 深圳市健元医药科技有限公司 Method for synthesizing liraglutide
US11518794B2 (en) 2016-08-19 2022-12-06 Shenzhen JYMed Technology Co., Ltd. Synthesis method for liraglutide with low racemate impurity
WO2019069274A1 (en) 2017-10-04 2019-04-11 Chemical & Biopharmaceutical Laboratories Of Patras S.A. A process for preparing a glucagon-like peptide
CN108107103B (en) * 2017-12-14 2020-10-16 华中师范大学 Mass spectrum probe of glutamate receptor and method for detecting spatial distribution rule of glutamate receptor in brain tissue
CN108107103A (en) * 2017-12-14 2018-06-01 华中师范大学 The MS probe of glutamate receptor and its space distribution rule detection method in brain tissue
CN107903317A (en) * 2017-12-29 2018-04-13 江苏诺泰澳赛诺生物制药股份有限公司 A kind of synthetic method of Liraglutide
CN110894227A (en) * 2018-09-13 2020-03-20 南京华威医药科技集团有限公司 Solid-phase synthesis method of liraglutide
WO2020074583A1 (en) 2018-10-09 2020-04-16 Fresenius Kabi Ipsum S.R.L. Process for the manufacture of glp-1 analogues
US11396533B2 (en) 2018-10-09 2022-07-26 Fresenius Kabi Ipsum S.R.L. Process for the manufacture of GLP-1 analogues
WO2020127476A1 (en) 2018-12-19 2020-06-25 Krka, D.D., Novo Mesto Pharmaceutical composition comprising glp-1 analogue
WO2021123228A1 (en) 2019-12-18 2021-06-24 Krka, D.D., Novo Mesto Pharmaceutical composition comprising glp-1 analogue

Also Published As

Publication number Publication date
CN104004083B (en) 2016-10-05

Similar Documents

Publication Publication Date Title
CN104004083A (en) Method for synthesizing liraglutide
DK2757107T3 (en) A process for the solid phase synthesis of liraglutide
CN109311961B (en) Synthesis method of Somalutide
CN102875665B (en) Method for synthesizing liraglutide
CN103497245B (en) Method for synthesizing thymalfasin
CN101563364B (en) Insulinotropic peptide synthesis
CN105384809B (en) A kind of method that segment method solid-liquid combination prepares Teriparatide
CN110294800B (en) Preparation method of somaglutide
CN106146648B (en) Synthetic method of parathyroid hormone analogue
CN103224558B (en) A kind of preparation method of Exenatide
CN106699871A (en) Preparation method of liraglutide
CN109180801A (en) A method of synthesis Suo Malu peptide
CN111732651B (en) Method for preparing Somalutide through continuous flow solid phase reaction
CN103848910A (en) Solid synthetic method of semaglutide
CN101747426A (en) Method for synthesizing pramlintide
CN104098688A (en) Method for synthesizing thymalfasin
CN102532302A (en) Method for preparing exenatide with natural coupling method
CN104788546A (en) Preparation method of linear peptides containing 24 amino acid residues
CN104844706B (en) A method of synthesis lixisenatide
CN106478805B (en) Preparation method of GLP-1 derivative
CN106749614A (en) A kind of fragment method solid-liquid combination is prepared for the method for degree Shandong peptide
CN104177491B (en) A kind of preparation method of Tesamorelin
CN104072603A (en) Teduglutide synthesis method
CN112679602A (en) Solid-phase synthesis method of Somaloutide
CN113754753A (en) Synthetic method of somaglutide

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant