CN102532302A - Method for preparing exenatide with natural coupling method - Google Patents
Method for preparing exenatide with natural coupling method Download PDFInfo
- Publication number
- CN102532302A CN102532302A CN2011103960502A CN201110396050A CN102532302A CN 102532302 A CN102532302 A CN 102532302A CN 2011103960502 A CN2011103960502 A CN 2011103960502A CN 201110396050 A CN201110396050 A CN 201110396050A CN 102532302 A CN102532302 A CN 102532302A
- Authority
- CN
- China
- Prior art keywords
- ser
- glu
- gly
- leu
- pro
- 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.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/605—Glucagons
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a method for preparing exenatide. The method comprises the following steps of: (1) splitting exenatide into a plurality of peptide fragments, wherein peptide fragments of which the ends N are Thr or Ser and other peptide fragments needed by exenatide are obtained with a solid phase synthesis method; (2) forming an acetal structure by using the ends C of peptide fragments coupled to the ends N of the peptide fragments of which the ends N are Thr or Ser, and removing side chain protecting groups; (3) removing side chain protecting groups and section N protecting groups of the peptide fragments of which the ends N are Thr or Ser; (4) coupling the peptide fragments in the step (2) with the peptide fragments in the step (3) in a liquid phase to obtain exenatide of which the end N is protected; and (5) removing end N amine protecting groups and purifying to obtain exenatide. A process disclosed by the invention has the characteristics of simple reaction operation, intermediate controllability, short reaction period, high yield, low cost and the like, and has a considerable economic and practical value and a wide application prospect.
Description
Technical field
The present invention relates to a kind of preparation method of polypeptide drugs, relate in particular to the method that the nature coupling method prepares Exenatide.
Background technology
Exenatide; English exenatide by name, its molecular structure is His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH
2
Exenatide is first incretin analogue by the common research and development of Lilly Co., Eli. and Amylin company; Be the polypeptide of forming by 39 amino acid of synthetic, similar with endogenous incretin such as glucagon-like-peptide-1 (GLP-1) effect, have the insulin secretion that promotes that glucose relies on; Recover first o'clock phase insulin secretion; The secretion of glucagon suppression, the emptying of the gastric content that slows down improves the effects such as function of pancreatic beta cell.
Among prior art US6924264, US7157555, the US6902744, the method for solid phase synthesis is all adopted in the preparation of Exenatide.This kind method is because Exenatide is to contain 39 amino acid whose long peptides, has that technical difficulty is big, synthesis cycle is long, production cost is high, the process midbody is not because the product purity of can not purifying bringing is not high, is unfavorable for problems such as scale operation.
Reported in the WO2011006644A2 patent that the method that adopts solid-liquid combination carries out the synthetic of Exenatide.Exenatide is divided into 1-10,11-21,22-29, four fragments of 30-39, and solid phase method synthesizes each full guard peptide fragment respectively, then the coupling in liquid phase of several fragments is obtained Exenatide.Therefore this method is because each fragment all is the full guard peptide, has that hydrophobicity is strong, peptide chain is poorly soluble, fragment purification is difficult, the low inferior problem of the intersegmental coupling efficiency of sheet.
There is complex operation in the existing compound method of Exenatide, and synthesis cycle is long, and waste liquid is many, is unfavorable for environmental protection, the with high costs and shortcoming that is unfavorable for scale operation.
Summary of the invention
1) Exenatide is divided into M bar peptide fragment, M=2 or 3; Wherein there is L bar N end to be Thr or Ser peptide fragment, L=1 or 2, the T bar constitutes other required peptide fragment of Exenatide, L+T=M; Obtain the peptide fragment of described M bar side chain full guard with solid phase synthesis process;
2) will be coupled to the peptide fragment C end formation ethylidene ether structure of step 1) N end for Thr or Ser peptide fragment N end; Remove the side chain protected group, form peptide fragment C end ethylidene ether structure;
3) remove side chain protected group and the N segment protect group of N end, get the N end and be Thr or Ser peptide fragment for Thr or Ser peptide fragment;
4) utilizing step 2) described peptide fragment C end ethylidene ether structure and the described N end of step 3) obtain the Exenatide of N end protection for Thr or the coupling in liquid phase of Ser peptide fragment;
5) remove N end amino protecting group, purification process gets Exenatide.
As further improvement of the present invention; The said solid phase synthesis process of said step 1); Employed resin is aminoresin or 2-CTC resin; Wherein aminoresin is applicable to the solid phase synthesis that contains Exenatide C end amide structure, and aminoresin comprises Sieber resin, Rink Amide resin or Rink Amide-MBHA resin.
The 2-CTC resin is applicable to that the C end is the peptide fragment solid phase synthesis of carboxyl structure.
Need be under the weak acid situation in fragment peptide building-up process the peptide chain fragment be separated down and do not influenced the amino acid side chain blocking group from pitch shake.And these side chain protected groups to be converted into the ethylidene ether structure in the carboxyl structure of C end be to exist in the past, be used for guaranteeing that the acetate aldehyde structure is created in C end carboxyl rather than on side chain carboxyl group.The applicant finds that unexpectedly the 2-CTC resin can satisfy the requirement of this building-up reactions.
As further improvement of the present invention, in the said step 1), to participate in fragment link coupled Thr or Ser and use the amido protecting group, the amido protecting group is Boc.
As further improvement of the present invention, said step is with 1) in, be TFE with peptide fragment cracking from the resin used weak acid that gets off.
As further improvement of the present invention, said step 2) in, peptide fragment C end forms the ethylidene ether structure, and employed aldehyde is salicylic aldehyde.
As further improvement of the present invention, said step 2) comprise the steps:
2a) will be coupled to the peptide fragment C end formation ethylidene ether structure of step 1) N end for Thr or Ser peptide fragment N end; Wherein, peptide fragment C end forms the ethylidene ether structure, and employed aldehyde is salicylic aldehyde.
2b) adopt acid cleavage that all segmental Side chain protective groups are removed, form side chain unprotected peptide fragment.Wherein, the acid that the protection base is removed is TFA.
As further improvement of the present invention, in the said step 4), coupling obtains the Exenatide of N end protection in liquid phase, and solvent is pyridine/acetic acid.
M bar peptide fragment is selected from following peptide fragment structure among the present invention:
SEQ?ID?No.1:His-Gly-Glu-Gly-OH
SEQ?ID?No.2:Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-OH
SEQ?ID?No.3:Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH
2
SEQ?ID?No.4:His-Gly-Glu-Gly-Thr-Phe-OH
SEQ?ID?No.5:Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-OH
SEQ?ID?No6:Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-OH
SEQ?ID?No.7:Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH
2
SEQ?ID?No.8:His-Gly-Glu-Gly-Thr-Phe-Thr-OH
SEQ?ID?No.9:Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-OH
SEQ?ID?No.10:Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-OH
SEQ?ID?No.11:His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-OH
SEQ?ID?No.12:Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-OH
SEQ?ID?No.13:Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-OH
SEQ?ID?No.14:Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH
2
SEQ?ID?No.15:Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH
2
SEQ?ID?No.16:Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH
2。
As further improvement of the present invention, in the said step 5), remove N end amino protecting group, purification process gets Exenatide; N end amino protecting group is Fmoc, adopts DBLK to remove, and said DBLK is that 1: 4 piperidines and DMF forms by volume ratio.Purification process is this area technique means commonly used, can comprise freeze-drying in case of necessity.
During the coupling of prior art polypeptide liquid-phase fragment, because problem optionally when synthetic, the segmental amino acid side chain of peptide chain must be protected to prevent that the coupling site from going wrong.Because segmental amino acid side chain needs protection, peptide fragment solubleness is very low; If side chain no protective is synthesized, must be that peptide chain contains this coupling site of Cys, because it is less relatively to contain the peptide chain of Cys, limited technological use.Contain a plurality of Ser/Thr in the Exenatide peptide chain; The present invention finds unexpectedly; Adopt the peptide chain that the N end contains Ser or Thr can under the situation of not protecting side chain, optionally carry out fragment coupling, the selectivity when ethylidene ether structure significant feature is to guarantee the fragment coupling.Carry out side chain no protective synthetic advantage and be, solved problems such as segmental dissolving, purifying, improved yield.And the efficient during to coupling is greatly improved.
Compared with prior art, the invention has the beneficial effects as follows: simple to operate, synthesis cycle is short, and cost is low, and midbody is prone to purifying, and aftertreatment is easy, and by product is few, and product yield is high, and the finished product purity is high, is beneficial to the scale operation of Exenatide.
Description of drawings
Fig. 1: the process flow sheet that the present invention is total
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is explained further details.
Aminoresin and 2-CTC resin available from Tianjin Nankai with become ltd, various protection amino acid are available from the biochemical ltd of gill.During the implication of the english abbreviation that uses is listed in the table below in specification sheets and claims:
During the implication of employed abbreviation is listed in the table below in specification sheets and claims:
Fmoc | 9-fluorenylmethyloxycarbonyl |
The Sieber resin | 3-hydroxyl-9H-xanthenes-9-ketone resin |
The 2-CTC resin | 2-trityl chloride resin |
Boc | Tertbutyloxycarbonyl |
tBu | The tertiary butyl |
DMF | N, dinethylformamide |
DCM | Methylene dichloride |
DBLK | 20% hexahydropyridine/DMF solution |
Ser | Serine |
Thr | Threonine |
DIEA | N, the N-diisopropylethylamine |
TFE | Trifluoroethanol |
Salicyladehyde | Salicylic aldehyde |
TIS | Tri isopropyl silane |
Being divided into SEQ ID No.11, SEQ ID No.12, SEQ ID No.3 with Exenatide below is that example specifies.
Embodiment 1: full guard peptide fragment 11 preparations of Fmoc protection are arranged at the N end
SEQ ID No.11 is also referred to as peptide fragment 11 among the present invention, its molecular structure is His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-OH; The peptide fragment 11 of full guard is meant and contains blocking group tBu, and the peptide fragment 11 of OtBu protection is identical with " Exenatide (1-10) " implication in the present invention.
The preparation of A.Fmoc-Leu-CTC resin
Taking by weighing substitution degree is the 2-CTC resin 20g of 0.5mmol/g, joins in the solid state reaction post, with DMF washing 2 times, with DMF swelling resin 30 minutes; Get 7.1g Fmoc-Leu-OH and dissolve with DMF, ice-water bath adds 5.22mL DIEA activation down; Add then in the above-mentioned reaction column that resin is housed, reacted 2 hours; Add 8mL anhydrous methanol sealing 1 hour.With DMF washing 3 times, DCM washes 3 times, and with anhydrous methanol sealing 30 minutes, contraction was drained, and obtains the Fmoc-Leu-CTC resin, and the detection substitution degree is 0.42mmol/g.
The B.N end has the preparation of the full guard peptide fragment 11 of Fmoc protection
1. amino acid whose coupling
Taking by weighing substitution degree is the Fmoc-Leu-CTC resin 19g of 0.42mmol/g, adds in the solid state reaction post, with DMF washing 2 times, with DMF swelling Fmoc-Leu-CTC resin 30 minutes, removes the Fmoc protection with DBLK, washs 4 times with DMF then, and DCM washes 2 times.With 10.21g Fmoc-Asp (OtBu)-OH (24mmol), it is 1: 1 DCM and DMF mixing solutions that 3.89g HOBt (28.8mmol), 3.63g DIC (28.8mmol) are dissolved in volume ratio; Add in the solid state reaction post, (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if the resin water white transparency; Then react completely; The resin colour developing, the expression reaction not exclusively needs linked reaction 1h again).Repeat the above-mentioned Fmoc of removing protection and add corresponding amino acid link coupled step; Order according to peptide fragment 11; Accomplish Fmoc-Ser (tBu)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr (tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Gly-OH, Fmoc-His (Trt)-OH coupling successively, wherein last amino acid His coupling need not remove Fmoc after finishing.Reaction finishes the back and shrinks resin drying under vacuum overnight, the peptide fragment 11-CTC resin 30.91g that weighs and obtain the Fmoc-full guard with methyl alcohol.
2. the peptide fragment 11-CTC resin of cracking Fmoc-full guard, the peptide fragment 11 that obtains full guard takes by weighing Fmoc-Exenatide (1-10)-CTC resin 30g, is added in the 500ml flask.Configuration lytic reagent 300ml (volume ratio, TFE: DCM=1: 4), lytic reagent is poured in the flask into room temperature reaction 2h.Reaction finishes, and filters resin, collects filtrating.Filtrate volume revolved steam to volume ratio<25%, drop to that (normal hexane: ether volume ratio=1: 4) centrifugal, anhydrous diethyl ether washing, and vacuum-drying obtain the thick peptide 14.28g of Fmoc-Exenatide (1-10)-OH in the 500ml precipitation reagent.
The preparation of embodiment 2:Fmoc-Exenatide (1-10)-O-bigcatkin willow aldehydo-ester
With Fmoc-Exenatide (1-10)-thick peptide of OH (10.9g, 6mmol), salicylic aldehyde (3.1ml, 30mmol) and DCC (2.5g 12mmol) is dissolved among the 400ml DCM room temperature reaction 2h.Reaction finishes, and the reaction solution volume is revolved steam to volume ratio<25%, adds in 100 times of amount anhydrous diethyl ethers, and centrifugal, anhydrous diethyl ether washing, and vacuum-drying obtain Fmoc-Exenatide (1-10)-O-bigcatkin willow aldehydo-ester 10.73g.
Embodiment 3: remove Fmoc-Exenatide (1-10)-O-bigcatkin willow aldehydo-ester protection base
(9.6g 5mmol) places reaction vessel, adds (TFA: H in the 200ml lysate with Fmoc-Exenatide (1-10)-O-bigcatkin willow aldehydo-ester
2O: TIS volume ratio=95: 2.5: 2.5), stirring at room reaction 2h.After reaction finishes; Reaction solution is poured in the 2L anhydrous diethyl ether, centrifugal, the anhydrous diethyl ether washing; And vacuum-drying; Obtain Fmoc-peptide fragment 11-O-bigcatkin willow aldehydo-ester, i.e. Fmoc-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-O-salicylal dehyde ester, product weight 6.85g.
Embodiment 4: the preparation of Boc protection Exenatide (11-31) is arranged at the N end
SEQ ID No.12, the present invention is also referred to as peptide fragment 12, is meant the 11-31 peptide fragment of Exenatide C end, and molecular structure does; Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-OH; The peptide fragment 12 of full guard is meant and contains blocking group Trt, and the peptide fragment 12 of OtBu, Pbf, tBt, Boc protection is identical with " Exenatide (11-31) " implication in the present invention.
The preparation of A.Fmoc-Pro-CTC resin
Taking by weighing substitution degree is the 2-CTC resin 25g of 0.4mmol/g, joins in the solid state reaction post, with DMF washing 2 times, with DMF swelling resin 30 minutes; Get 6.75g Fmoc-Pro-OH and dissolve with DMF, ice-water bath adds down 5.22mL DIEA activation, adds in the above-mentioned reaction column that resin is housed, and reacts after 2 hours; Add 8mL anhydrous methanol sealing 1 hour.With DMF washing 3 times, DCM washes 3 times, and with anhydrous methanol sealing 30 minutes, contraction was drained, and obtains the Fmoc-Pro-CTC resin, and the detection substitution degree is 0.35mmol/g.
The B.N end has the preparation of the full guard fragment 12 of Boc protection
1. amino acid whose coupling
Taking by weighing substitution degree is the Fmoc-Pro-CTC resin 25.7g of 0.35mmol/g, adds in the solid state reaction post, with DMF washing 2 times, with DMF swelling Fmoc-Pro-CTC resin 30 minutes, removes the Fmoc protection with DBLK, washs 4 times with DMF then, and DCM washes 2 times.With 8.03g Fmoc-Gly-OH (27mmol), it is 1: 1 DCM and DMF mixing solutions that 4.38g HOBt (32.4mmol), 4.08g DIC (32.4mmol) are dissolved in volume ratio; Add in the solid state reaction post, (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if the resin water white transparency; Then react completely; The resin colour developing, the expression reaction not exclusively needs linked reaction 1h again).Repeat the above-mentioned Fmoc of removing protection and add corresponding amino acid link coupled step; Order according to peptide fragment 12; Accomplish Fmoc-Gly-OH, Fmoc-Asn (Trt)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc)-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Met-OH, Fmoc-Gln (Trt)-OH, Fmoc-Lys (Boc)-OH, Boc-Ser (tBu)-OH successively, wherein the N of last amino acid Ser end protection base is Boc.Reaction finishes the back shrinks with methyl alcohol, the resin drying under vacuum overnight, and weighing obtains Boc-Exenatide (11-31)-CTC resin 50.77g.
2. cracking Boc-Exenatide (11-31)-CTC resin obtains full guard peptide fragment 12
Take by weighing Boc-Exenatide (11-31)-CTC resin 50.0g, be added in the 1000ml flask.Configuration lytic reagent 500ml (TFE: DCM volume ratio=1: 4), lytic reagent is poured in the flask into room temperature reaction 2h.Reaction finishes, and filters resin, collects filtrating.Filtrate volume revolved steam to volume ratio<25%, drop to that (normal hexane: ether volume ratio=1: 4) centrifugal, anhydrous diethyl ether washing, and vacuum-drying obtain the thick peptide 29.22g of Boc-Exenatide (11-31)-OH in the 2000ml precipitation reagent.
Embodiment 5: the bigcatkin willow aldehydo-ester of preparation Boc-Exenatide (11-31)-OH
With Boc-Exenatide (11-31)-thick peptide of OH (23.31g, 6mmol), salicylic aldehyde (3.1ml, 30mmol) and DCC (2.5g 12mmol) is dissolved among the 400ml DCM room temperature reaction 2h.Reaction finishes, and the reaction solution volume is revolved steam to volume ratio<25v%, adds in 50 times of amount anhydrous diethyl ethers, and centrifugal, anhydrous diethyl ether washing, and vacuum-drying obtain Boc-Exenatide (11-31)-O-bigcatkin willow aldehydo-ester 22.13g.
Embodiment 6: Exenatide (11-31)-basic the removing of O-bigcatkin willow aldehydo-ester protection
(19.94g 5mmol) places reaction vessel, adds (TFA: H in the 300ml lysate with Boc-Exenatide (11-31)-thick peptide of O-bigcatkin willow aldehydo-ester
2O: TIS volume ratio=95: 2.5: 2.5), stirring at room reaction 2h.After reaction finishes; Reaction solution is poured in the 3L anhydrous diethyl ether, centrifugal, the anhydrous diethyl ether washing; And vacuum-drying; Obtain peptide fragment 12-O-bigcatkin willow aldehydo-ester, i.e. Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-O-salicylaldehyde ester, product weight 11.65g.
Embodiment 7: peptide fragment 3 preparations
SEQ ID No.3, the present invention is also referred to as peptide fragment 3, is meant the peptide fragment of Exenatide C end 32-39, and its molecular structure is Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH
2The peptide fragment 3 of full guard is meant the peptide fragment that contains blocking group tBu protection, and is identical with " Exenatide (32-39) " implication in the present invention.
1.Boc-the preparation of Exenatide (32-39)-Sieber resin
Taking by weighing substitution degree is the sieber resin 33.3g of 0.3mmol/g, joins in the solid state reaction post, with DMF washing 2 times, with DMF swelling resin 30 minutes, removes the Fmoc protection with DBLK, washs 4 times with DMF then, and DCM washes 2 times.With Fmoc-Pro-OH (10.12g, 30mmol), HOBt (4.86g, 36mmol); DIC (4.54g, 36mmol) being dissolved in volume ratio is 1: 1 DCM and DMF mixing solutions, adds in the solid state reaction post; (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if the resin water white transparency then reacts completely; The resin colour developing, the expression reaction not exclusively needs linked reaction 1h again).Repeat the above-mentioned Fmoc of removing protection and add corresponding amino acid link coupled step; Order according to peptide fragment 3; Accomplish Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH Fmoc-Ser (tBu)-OH, Boc-Ser (tBu)-OH successively, wherein the N of last amino acid Ser end protection base is Boc.Reaction finishes the back shrinks with methyl alcohol, the resin drying under vacuum overnight, and weighing obtains Boc-Exenatide (32-39)-Sieber resin 42.04g.
2.Boc-the cracking of Exenatide (32-39)-Sieber resin
Boc-Exenatide (32-39)-Sieber resin 42g is placed reaction vessel, add (TFA: H in the 420ml lysate
2O: TIS volume ratio=95: 2.5: 2.5), stirring at room reaction 2h.Reaction is poured reaction solution in the 4.2L anhydrous diethyl ether into after finishing, and is centrifugal, and anhydrous diethyl ether washing, and vacuum-drying obtain product S er-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH
26.26g.
Embodiment 8: peptide fragment 3 preparations
1.Boc-the preparation of Exenatide (32-39)-Rink Amide resin
Taking by weighing substitution degree is the Rink Amide resin 26.3g of 0.38mmol/g, joins in the solid state reaction post, with DMF washing 2 times, with DMF swelling resin 30 minutes, removes the Fmoc protection with DBLK, washs 4 times with DMF then, and DCM washes 2 times.With Fmoc-Pro-OH (10.12g, 30mmol), HOBt (4.86g, 36mmol); DIC (4.54g, 36mmol) being dissolved in volume ratio is 1: 1 DCM and DMF mixing solutions, adds in the solid state reaction post; (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if the resin water white transparency then reacts completely; The resin colour developing, the expression reaction not exclusively needs linked reaction 1h again).Repeat the above-mentioned Fmoc of removing protection and add corresponding amino acid link coupled step; According to the order of peptide fragment 3, accomplish Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH Fmoc-Ser (tBu)-OH, Boc-Ser (tBu)-OH successively.Wherein the N of last amino acid Ser end protection base is Boc.Reaction finishes the back shrinks with methyl alcohol, the resin drying under vacuum overnight, and weighing obtains Boc-Exenatide (32-39)-Rink Amide resin 35.03g.
2.Boc-the cracking of Exenatide (32-39)-Rink Amide resin
Boc-Exenatide (32-39)-Rink Amide resin 35g is placed reaction vessel, add (TFA: H in the 350ml lysate
2O: TIS volume ratio=95: 2.5: 2.5), stirring at room reaction 2h.Reaction is poured reaction solution in the 3.5L anhydrous diethyl ether into after finishing, and is centrifugal, and anhydrous diethyl ether washing, and vacuum-drying obtain product S er-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH
26.32g.
Embodiment 9: peptide fragment 3 preparations
1.Boc-the preparation of Exenatide (32-39)-Rink Amide-MBHA resin
Taking by weighing substitution degree is the Rink Amide-MBHA resin 23.2g of 0.43mmol/g, joins in the solid state reaction post, with DMF washing 2 times, with DMF swelling resin 30 minutes, removes the Fmoc protection with DBLK, washs 4 times with DMF then, and DCM washes 2 times.With Fmoc-Pro-OH (10.12g, 30mmol), HOBt (4.86g, 36mmol); DIC (4.54g, 36mmol) being dissolved in volume ratio is 1: 1 DCM and DMF mixing solutions, adds in the solid state reaction post; (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if the resin water white transparency then reacts completely; The resin colour developing, the expression reaction not exclusively needs linked reaction 1h again).Repeat the above-mentioned Fmoc of removing protection and add corresponding amino acid link coupled step; According to the order of peptide fragment 3, accomplish Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH Fmoc-Ser (tBu)-OH, Boc-Ser (tBu)-OH successively.Wherein the N of last amino acid Ser end protection base is Boc.Reaction finishes the back shrinks with methyl alcohol, the resin drying under vacuum overnight, and weighing obtains Boc-Exenatide (32-39)-Rink Amide-MBHA resin 31.2g.
2.Boc-the cracking of Exenatide (32-39)-Rink Amide-MBHA resin
Boc-Exenatide (32-39)-Rink Amide-MBHA resin 31g is placed reaction vessel, add (TFA: H in the 310ml lysate
2O: TIS volume ratio=95: 2.5: 2.5), stirring at room reaction 2h.Reaction is poured reaction solution in the 3.1L anhydrous diethyl ether into after finishing, and is centrifugal, and anhydrous diethyl ether washing, and vacuum-drying obtain product S er-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH
26.23g.
Embodiment 10: the segmental preparation of Exenatide (1-31) of Fmoc protection is arranged at the N end
Exenatide (1-31) fragment is meant according to the fragment of Exenatide composition order from 1-31 peptide of C end beginning.
Fmoc-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-O-salicylal dehyde ester (5.56g with preparation among the embodiment 3; 4mmol) with embodiment 6 in the Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-O-salicylaldehyde est er (10.26g for preparing; 4mmol) mixed dissolution is in 40ml pyridine/acetic acid (mol ratio; 1: 1), stirring at room reaction 4h.Acetic acid in the reaction solution is revolved dried, be added in the 200ml ether, centrifugal, the anhydrous diethyl ether washing, and vacuum-drying obtains white solid.Then; Add 100ml reaction solution (TFA: H2O: TIS volume ratio=94: 5: 1) behind the room temperature reaction 5min; Reaction solution is added in the 1000ml ether; Centrifugal; The anhydrous diethyl ether washing, and vacuum-drying obtains product Fmoc-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-O-salicylaldehyde ester 12.36g, yield 80.6%.MALDI-TOF:(M+H)
+=3833.2。
Embodiment 11: the preparation of the Exenatide of Fmoc protection is arranged at the N end
With the Fmoc-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-O-salicylaldehyde ester of preparation among the embodiment 10 (11.50g, 3mmol) with the foregoing description in the H-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH for preparing
2(2.09g, 3mmol) mixed dissolution is in 40ml pyridine/acetic acid (mol ratio, 1: 1), and stirring at room is reacted 4h.Acetic acid in the reaction solution is revolved dried, be added in the 200ml ether, centrifugal, the anhydrous diethyl ether washing, and vacuum-drying obtains white solid.Then; Add 100ml reaction solution (TFA: H2O: TIS volume ratio=94: 5: 1) behind the room temperature reaction 5min; Reaction solution is added in the 1000ml ether; Centrifugal, the anhydrous diethyl ether washing, and vacuum-drying obtains product Fmoc-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH
210.31g, yield 77.9%.MALDI-TOF:(M+H)
+=4409.0。
Embodiment 12: the preparation of Exenatide
With the Fmoc-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln that obtains among the embodiment 11-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH
28.82g be dissolved among the 50ml DBLK; Behind the room temperature reaction 30min; Add in the 1000ml anhydrous diethyl ether; It is centrifugal to separate out white precipitate, the anhydrous diethyl ether washing, and vacuum-drying obtains product Exenatide His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH
2, product weight 8.23g, yield 98.3%.MALDI-TOF:(M+H)
+=4186.9。
The preparation of the smart peptide acetate of embodiment 13 Exenatides
Take by weighing the thick peptide of 8.0g Exenatide with the 400ml water dissolution after, adopt Waters 2545RP-HPLC system, wavelength 230nm; Chromatographic column is 50 * 250mm anti-phase C18 post; Conventional 0.2%TFA/ acetonitrile moving phase purifying is collected purpose peak cut, obtains purity greater than 98.5% smart peptide.Smart peptide solution is adopted Waters 2545RP-HPLC system, and chromatographic column is 50 * 250mm anti-phase C18 post, and 0.2% acetum/acetonitrile moving phase is changeed salt; Collect purpose peak cut; Rotary evaporation concentrates, and freeze-drying obtains the smart peptide 2.33g of Exenatide acetate, HPLC purity 98.9%.
Above content is to combine concrete preferred implementation to the further explain that the present invention did, and can not assert that practical implementation of the present invention is confined to these explanations.For the those of ordinary skill of technical field under the present invention, under the prerequisite that does not break away from the present invention's design, can also make some simple deduction or replace, all should be regarded as belonging to protection scope of the present invention.
Claims (8)
1. the preparation method of an Exenatide is characterized in that: may further comprise the steps:
1) Exenatide is divided into M bar peptide fragment, M=2 or 3; Wherein there is L bar N end to be Thr or Ser peptide fragment, L=1 or 2, the T bar constitutes other required peptide fragment of Exenatide, L+T=M; Obtain the peptide fragment of described M bar side chain full guard with solid phase synthesis process;
2) will be coupled to the peptide fragment C end formation ethylidene ether structure of step 1) N end for Thr or Ser peptide fragment N end; Remove the side chain protected group, form peptide fragment C end ethylidene ether structure;
3) remove side chain protected group and the N segment protect group of N end, get the N end and be Thr or Ser peptide fragment for Thr or Ser peptide fragment;
4) utilizing step 2) described peptide fragment C end ethylidene ether structure and the described N end of step 3) obtain the Exenatide of N end amido protecting for Thr or the coupling in liquid phase of Ser peptide fragment;
5) remove N end amino protecting group, purification process gets Exenatide.
2. preparation method according to claim 1; It is characterized in that: the said solid phase synthesis process of step 1); Employed resin is aminoresin or 2-CTC resin; Wherein aminoresin is applicable to the solid phase synthesis that contains Exenatide C end amide structure, and the 2-CTC resin is applicable to that the C end is the peptide fragment solid phase synthesis of carboxyl structure.
3. preparation method according to claim 2 is characterized in that: aminoresin comprises Sieber resin, Rink Amide resin or Rink Amide-MBHA resin.
4. preparation method according to claim 1 is characterized in that: participate in fragment link coupled Thr or Ser and use the amido protecting group, the amido protecting group is Boc.
5. preparation method according to claim 1 is characterized in that: peptide fragment C end forms the ethylidene ether structure, and employed aldehyde is salicylic aldehyde.
6. preparation method according to claim 1 is characterized in that: the said N end of step 5) amino protecting group is Fmoc, and removing reagent is DBLK.
7. preparation method according to claim 1 is characterized in that: M bar peptide fragment is selected from following peptide fragment structure:
SEQ?ID?No.1:His-Gly-Glu-Gly-OH
SEQ?ID?No.2:Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-OH
SEQ?ID?No.3:Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH
2
SEQ?ID?No.4:His-Gly-Glu-Gly-Thr-Phe-OH
SEQ?ID?No.5:Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-OH
SEQ?ID?No6:Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-OH
SEQ?ID?No.7:Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH
2
SEQ?ID?No.8:His-Gly-Glu-Gly-Thr-Phe-Thr-OH
SEQ?ID?No.9:Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-OH
SEQ?ID?No.10:Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-OH
SEQ?ID?No.11:His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-OH
SEQ?ID?No.12:Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-OH
SEQ?ID?No.13:Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-OH
SEQ?ID?No.14:Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH
2
SEQ?ID?No.15:Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH
2
SEQ?ID?No.16:Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH
2?。
8. according to claim 1 or 6 described preparing methods, it is characterized in that: the N end is that Thr or Ser peptide fragment are SEQ ID No.3 or SEQ ID No.12, constitutes required other peptide fragment SEQ ID No.11 of Exenatide.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2011103960502A CN102532302A (en) | 2011-12-02 | 2011-12-02 | Method for preparing exenatide with natural coupling method |
PCT/CN2012/080760 WO2013078889A1 (en) | 2011-12-02 | 2012-08-30 | The method for preparing exenatide by natural coupling |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2011103960502A CN102532302A (en) | 2011-12-02 | 2011-12-02 | Method for preparing exenatide with natural coupling method |
Publications (1)
Publication Number | Publication Date |
---|---|
CN102532302A true CN102532302A (en) | 2012-07-04 |
Family
ID=46340453
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN2011103960502A Pending CN102532302A (en) | 2011-12-02 | 2011-12-02 | Method for preparing exenatide with natural coupling method |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN102532302A (en) |
WO (1) | WO2013078889A1 (en) |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013078889A1 (en) * | 2011-12-02 | 2013-06-06 | 深圳翰宇药业股份有限公司 | The method for preparing exenatide by natural coupling |
CN103304659A (en) * | 2013-06-19 | 2013-09-18 | 深圳翰宇药业股份有限公司 | Solid-phase preparation method of liraglutide |
CN103333237A (en) * | 2013-05-07 | 2013-10-02 | 海南双成药业股份有限公司 | Synthesis of exenatide through solid phase fragment method |
CN106167521A (en) * | 2016-08-29 | 2016-11-30 | 杭州湃肽生化科技有限公司 | A kind of solid phase synthesis process of Exenatide |
CN106749610A (en) * | 2016-12-29 | 2017-05-31 | 陕西慧康生物科技有限责任公司 | A kind of preparation method of Exenatide and products thereof |
CN110372784A (en) * | 2019-07-29 | 2019-10-25 | 深圳佳肽生物科技有限公司 | The synthetic method and application of Exenatide |
CN111918872A (en) * | 2018-03-29 | 2020-11-10 | 株式会社钟化 | Method for producing long-chain peptide |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101538324A (en) * | 2009-01-13 | 2009-09-23 | 深圳市翰宇药业有限公司 | Method for preparing Exenatide |
WO2011006644A2 (en) * | 2009-07-15 | 2011-01-20 | Lonza Ltd | Process for the production of exenatide and of an exenatide analogue |
WO2011017837A1 (en) * | 2009-08-12 | 2011-02-17 | Xuechen Li | Native chemical ligation at serine and threonine sites |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2205624B1 (en) * | 2007-10-27 | 2016-09-07 | Corden Pharma Colorado, Inc. | Insulinotropic peptide synthesis using solid and solution phase combination techniques |
CN102532302A (en) * | 2011-12-02 | 2012-07-04 | 深圳翰宇药业股份有限公司 | Method for preparing exenatide with natural coupling method |
-
2011
- 2011-12-02 CN CN2011103960502A patent/CN102532302A/en active Pending
-
2012
- 2012-08-30 WO PCT/CN2012/080760 patent/WO2013078889A1/en active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101538324A (en) * | 2009-01-13 | 2009-09-23 | 深圳市翰宇药业有限公司 | Method for preparing Exenatide |
WO2011006644A2 (en) * | 2009-07-15 | 2011-01-20 | Lonza Ltd | Process for the production of exenatide and of an exenatide analogue |
WO2011017837A1 (en) * | 2009-08-12 | 2011-02-17 | Xuechen Li | Native chemical ligation at serine and threonine sites |
Non-Patent Citations (2)
Title |
---|
于佳一: "艾塞那肽合成方法学研究", 《中国优秀博硕学位论文全文数据库(硕士)工程科技I辑》 * |
翁晓燕: "艾塞那肽在治疗2型糖尿病临床分析", 《中外健康文摘》 * |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013078889A1 (en) * | 2011-12-02 | 2013-06-06 | 深圳翰宇药业股份有限公司 | The method for preparing exenatide by natural coupling |
CN103333237A (en) * | 2013-05-07 | 2013-10-02 | 海南双成药业股份有限公司 | Synthesis of exenatide through solid phase fragment method |
CN103304659A (en) * | 2013-06-19 | 2013-09-18 | 深圳翰宇药业股份有限公司 | Solid-phase preparation method of liraglutide |
CN103304659B (en) * | 2013-06-19 | 2015-12-02 | 深圳翰宇药业股份有限公司 | The method for preparing solid phase of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] |
CN106167521A (en) * | 2016-08-29 | 2016-11-30 | 杭州湃肽生化科技有限公司 | A kind of solid phase synthesis process of Exenatide |
CN106749610A (en) * | 2016-12-29 | 2017-05-31 | 陕西慧康生物科技有限责任公司 | A kind of preparation method of Exenatide and products thereof |
CN111918872A (en) * | 2018-03-29 | 2020-11-10 | 株式会社钟化 | Method for producing long-chain peptide |
CN110372784A (en) * | 2019-07-29 | 2019-10-25 | 深圳佳肽生物科技有限公司 | The synthetic method and application of Exenatide |
Also Published As
Publication number | Publication date |
---|---|
WO2013078889A1 (en) | 2013-06-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN102286092B (en) | Solid-phase synthesis method of liraglutide | |
CN103497245B (en) | Method for synthesizing thymalfasin | |
CN106699871B (en) | Preparation method of liraglutide | |
CN102532302A (en) | Method for preparing exenatide with natural coupling method | |
CN104004083B (en) | A kind of method synthesizing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] | |
CN103848910B (en) | The solid phase synthesis process of a kind of Sa Molutai | |
CN102875665B (en) | Method for synthesizing liraglutide | |
CN108034004A (en) | A kind of synthetic method of Suo Malu peptides | |
CN104650219B (en) | The method that fragment condensation prepares Liraglutide | |
CN109627317A (en) | The method that fragment condensation prepares Suo Malu peptide | |
CN103224558B (en) | A kind of preparation method of Exenatide | |
CN101747426B (en) | Method for synthesizing pramlintide | |
CN108203462A (en) | A kind of method for preparing Suo Malu peptides | |
CN105384809A (en) | Method for preparing teriparatide by fragment method and solid-liquid combination | |
CN102731643A (en) | Method for preparing polypeptide used for treating osteoporosis | |
CN107903317A (en) | A kind of synthetic method of Liraglutide | |
CN110922470A (en) | Preparation method of somaglutide | |
CN101357938B (en) | Method for synthesizing Exenatide from solid phase polypeptide | |
CN104072603B (en) | It is a kind of to synthesize for the method for degree Shandong peptide | |
CN107022021A (en) | A kind of solid-phase synthesis of Liraglutide | |
CN106478805A (en) | A kind of preparation method of GLP-1 derivant | |
CN104098688A (en) | Method for synthesizing thymalfasin | |
CN113754753A (en) | Synthetic method of somaglutide | |
CN117106055A (en) | Synthesis method of telipopeptide | |
CN112028986A (en) | Synthesis method of semaglutide |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C12 | Rejection of a patent application after its publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20120704 |