CN106432468A - Solid-phase synthesis method for preparing exenatide - Google Patents

Solid-phase synthesis method for preparing exenatide Download PDF

Info

Publication number
CN106432468A
CN106432468A CN201610950754.2A CN201610950754A CN106432468A CN 106432468 A CN106432468 A CN 106432468A CN 201610950754 A CN201610950754 A CN 201610950754A CN 106432468 A CN106432468 A CN 106432468A
Authority
CN
China
Prior art keywords
fmoc
resin
pro
ser
glu
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
Application number
CN201610950754.2A
Other languages
Chinese (zh)
Inventor
竺剑峰
徐红岩
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.)
Shanghai GL peptide Ltd
Binhai Gl Peptide Co Ltd
Original Assignee
Shanghai GL peptide Ltd
Binhai Gl Peptide 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 Shanghai GL peptide Ltd, Binhai Gl Peptide Co Ltd filed Critical Shanghai GL peptide Ltd
Priority to CN201610950754.2A priority Critical patent/CN106432468A/en
Publication of CN106432468A publication Critical patent/CN106432468A/en
Pending legal-status Critical Current

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
    • C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
    • 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)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Endocrinology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Vascular Medicine (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses a method for preparing exenatide, aiming at mainly solving the technical problems in the prior art that synthesis steps are complicated, the coupling difficulty is great, post-period separation is difficult, and the like. According to the technical scheme, the method comprises the following steps: 1) mixing Fmoc-Rink Amide Linder and PEG (Polyethylene Glycol) Matrix resin, and reacting to obtain Fmoc-Rink Amide PEG Matrix resin; 2) gradually coupling amino acids with protective groups and proline pseudodipeptide segments by adopting a Fmoc solid-phase synthesis method, so as to obtain side-chain full-protection polypeptide resin; and 3) cutting the side-chain full-protection polypeptide resin, cracking polypeptides from the resin, removing side-chain protection groups so as to obtain an exenatide crude product, and carrying out high performance liquid chromatography separation and purification and freeze-drying on the crude peptide to obtain the exenatide. The process method for preparing the exenatide, provided by the invention, has the characteristics of simplicity and convenience for operation, high crude peptide purity and high comprehensive yield.

Description

A kind of solid phase synthesis process preparing Exenatide
Technical field
The present invention relates to Solid-phase synthesis peptides field, more particularly, to a kind of solid phase synthesis process of Exenatide.
Background technology
Exenatide (exenatide) one kind is from South America Monster (Gilka monster, Heloderma The glucagon of a kind of polypeptide containing 39 aminoacid separated in saliva Horridum), its structure and people (Glucagon) there is 48% homology, have 53% homology with the GLP-1 (glucagon-like-peptide-1) of people.
Research shows, as the analog of GLP-1, Exendin-4 can be with GLP-1 receptor acting, by stimulating islets of langerhans β cell regeneration, promotes insulin secretion, the release of glucagon suppression, slows down gastric emptying rate, suppresses food intake.Its Promoting insulin secretion is carried out according to blood sugar level, therefore can reduce hypoglycemic incidence rate, and to other pancreotropic hormones The insensitive type ii diabetes patient of secretogogue still has blood sugar reducing function, and GLP-1 can also mitigate type ii diabetes patient's simultaneously Body weight, is the brand-new intestinal blood sugar lowering element class medicine of a class.Intestinal blood sugar lowering element class medicine is a kind of novel therapies for type ii diabetes, It can imitate the anti-diabetic reaction of natural gut hormone in human body or the reaction reducing concentration of glucose, these reaction bags Include and stimulate internal generation insulin, the secretion of glucagon suppression after the meal in blood glucose rise, slow down blood and absorb nourishment Speed and reduce food intake dose.Exenatide (Exenatide) is the type ii diabetes medicine of brand new class.Subcutaneous Injection, daily medication 2 times, can not fill for metformin, sulfonylurea or metformin and sulfonylurea use in conjunction The type ii diabetes patient of sub-control blood glucose.
The synthetic method of Exenatide has two methods of solid phase method and solid liquid phase set at present, and CN102174082A is open A kind of method of solid phase synthesis Exenatide, using conventional Fmoc-Rink Amide AM Resin resin as carrier, The solid method of Fmoc is coupled each aminoacid one by one and obtains Exenatide, be directly synthesized due to peptide sequence longer, be coupled difficulty by One increase, easily produces residue deletions, and the peptide purification separation for the later stage causes great difficulty.CN103333237A discloses A kind of method that solid liquid phase collection fragments synthesize Exenatide, solid liquid phase synthesis step is complicated, and amplification item controlled is many, unstable Fixed, and the by-product producing is many, the removal of impurity will repeatedly be washed or other means purification, brings the feature of high cost, unfavorable In industrialization.
Content of the invention
It is an object of the invention to provide a kind of high yield, low cost, reaction condition are gentle, environmental pollution is little, be conducive to Realize the solid phase synthesis process of the Exenatide of industrialization, mainly solve synthesis step complexity, coupling hardly possible that prior art exists Degree is big, the later stage separates the technical problems such as difficulty.
Technical solution of the present invention：A kind of solid-phase synthesis of Exenatide, comprise the steps：
（1）Fmoc-Rink Amide Linker and AM PEGMatrix reaction resin mixture are obtained Fmoc-Rink Amide AM PEGMatrix resin；
（2）It is coupled 33 fluorenylmethyloxycarbonyl protection ammonia using Fmoc solid-phase synthesis according to the order from C-terminal to N-terminal one by one Base acid and three class proline vacation dipeptide fragment X, Y, Z, obtain the polypeptide resin of side chain full guard, shown in formula I；
His(trt)-Gly-Glu(otbu)-X-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Y-Lys(boc)-Gln (trt)-Met-Glu(otbu)-Glu(otbu)-Glu(otbu)-Ala-Val-Arg(pbf)-Leu-Phe-Ile-Glu (otbu)-Trp(boc)-Leu-Lys(boc)-Asn(trt)-Gly-Gly-Pro-Z-Gly-Ala-Pro-Pro-Pro-Ser (tbu)-Rink Amide PEGMatrix resin
Formulas I
In formula：X= Gly-Thr(psi(me,me)pro)；Y= Leu-Ser(psi(me,me)pro)；Z= Ser(tbu)-Ser (psi(me,me)pro)；
（3）Addition is cut peptide reagent and the peptide chain resin of side chain full guard is cracked, and 30 nonapeptides are cleaved from resin And remove side chain protecting group, open the five-membered ring of three false dipeptide fragment in sequence simultaneously, obtain exenatide crude product, as formula Shown in II；Crude product is refining to obtain Exenatide through high performance liquid chromatography separation purification, lyophilizing again；
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-NH2
Formula II.
Comprise the following steps that：
（1）Take AM PEGMatrix resin DCM to soak, make resin fully swelling, drain, add Fmoc- Rink Amide Linker, DIEA, select TBTU/HOBT as catalyst, select volume ratio to be 1:1 DMF/NMP mixed solution as solvent, Reaction 2-8 hour, drains, is washed with DMF 3 times, add volume ratio 2:2:1 acetic anhydride：Pyridine：The mixed solution of DMF, instead Answer 0.5-2 hour, drain, washed with DMF 6 times, drain, obtain Fmoc-Rink Amide AM PEGMatrix resin；
The substitution value of PEGMatrix resin between 0.5mmol/g-1.0mmol/g, preferably 0.8mmol/g, Fmoc-Rink The amount of Amide Linker is 2 times of AM PEGMatrix resin mole；The amount of TBTU/HOBT is AM PEGMatrix resin 2 times of mole, the amount of DIEA is 4 times of AM PEGMatrix resin mole；
（2）In step（1）In the Fmoc-Rink Amide AM PEGMatrix resin obtaining, add deprotecting regent, reaction 30-60 minute, drains, and is washed with DMF 5 times, drains；Add Fmoc-Ser (tbu)-OH, NMM, select HBTU/HOBT as contracting Mixture, using DMF as solvent, reacts 0.5-2 hour, ninhydrin method detects reaction end, drains, is washed with DMF 3 times, drain, Obtain Fmoc-Ser (tbu)-Rink Amide PEGMatrix resin；Then add deprotecting regent, deprotection reaction, then Add and there is fluorenylmethyloxycarbonyl protected amino acid and class proline vacation dipeptide fragment, so repeatedly, until having connected last Soviet Union Propylhomoserin.Washed with DMF 3 times, again with methanol and dichloromethane replace washing 3 times, finally plus methanol shrinkage resin, drain, vacuum It is dried, obtain the peptide chain resin of side chain full guard, i.e. His (trt)-Gly-Glu (otbu)-{ Gly-Thr (psi (me, me) pro)}-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-{Leu-Ser(psi(me,me)pro)}-Lys(boc)-Gln (trt)-Met-Glu(otbu)-Glu(otbu)-Glu(otbu)-Ala-Val-Arg(pbf)-Leu-Phe-Ile-Glu (otbu)-Trp(boc)-Leu-Lys(boc)-Asn(trt)-Gly-Gly-Pro-{Ser(tbu)-Ser(psi(me,me) Pro) }-Gly-Ala-Pro-Pro-Pro-Ser (tbu)-Rink Amide AM PEGMatrix resin；
Described has fluorenylmethyloxycarbonyl protected amino acid and false dipeptide fragment, includes Fmoc-Pro-OH, Fmoc-Pro- successively OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser (tbu)-Ser { psi (me, me) pro }-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, 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 (o
Tbu)-OH, Fmoc-Glu (otbu)-OH, Fmoc-Glu (otbu)-OH, Fmoc-Met-OH, Fmoc-Gln (trt)-OH, Fmoc-Lys (boc)-OH, Fmoc-Leu-Ser { psi (me, me) pro }-OH, Fmoc-Asp (otbu)-OH, Fmoc-Ser (tbu)-OH, Fmoc-Thr (tbu)-OH, Fmoc-Phe-OH, Fmoc-Gly-Thr { psi (me, me) pro }-OH, Fmoc- Glu (otbu)-OH, Fmoc-Gly-OH, Fmoc-His (trt)-OH；
The amount of Fmoc-Ser (tbu)-OH is 2 times of Fmoc-Rink Amide AM PEGMatrix resin mole,；Raise one's hat examination Agent is the PIP/DMF mixed solution of volume ratio 20%, and the amount of each step fluorenylmethyloxycarbonyl protected amino acid is Fmoc-Rink 2 times of Amide AM PEGMatrix resin mole, the amount of condensation reagent is Fmoc-Rink Amide AM PEGMatrix 2 times of resin mole, the amount of NMM is 4 times of Fmoc-Rink Amide AM PEGMatrix resin mole, and each step is anti- Answer detection method the used 1,2,3-indantrione monohydrate detection method of terminal；
（3）The peptide chain resin of side chain full guard adds and cuts peptide reagent, react 2-3 hour, sucking filtration filters resin particle, and receives Collection filtrate, is subsequently adding the ice crystallizing from ether precipitation of 6 times of volumes, precipitation is collected by centrifugation, then is washed with ether 5 times, vacuum drying, Obtain exenatide crude product.
Cut peptide reagent and select volume ratio TFA:Thioanisole:Phenol:Dithioglycol:H2O=87.5:5:2.5:2.5:2.5 Mix reagent.
In the present invention, some conventional abbreviations have following meanings
HBTU：O- benzotriazole-N, N, N, N- tetramethylurea hexafluorophosphate
TBTU：O- BTA-N, N, N', N'- tetramethylurea Tetrafluoroboric acid
HOBt：I-hydroxybenzotriazole
DIEA：N, N- diisopropylethylamine
NMM：N-methylmorpholine
Fmoc：Fluorenylmethyloxycarbonyl
Pbf：2,2,4,6,7- pentamethyl -2H- benzofuran -5 sulfonyl
Trt：Trityl
Tbu：The tert-butyl group
Otbu：Tert-butoxy
Pro：Proline
Thr：Threonine
Gly：Glycine
Ala：Alanine
Ser：Serine
Asn：Agedoite
Asp：Aspartic acid
Arg：Arginine
His：Histidine
Gln：L-Glutamine
Leu：Leucine
Ile：Isoleucine
Met：Methionine
Phe：Phenylalanine
Lys：Lysine
Glu：Glutamic acid
Val：L-Valine
Pip：Piperidines
DMF：N,N-dimethylformamide
DCM：Dichloromethane
TFA：Trifluoroacetic acid
NMP：N-Methyl pyrrolidone.
Beneficial effects of the present invention：Adopt AM PEGMatrix resin as synthesis material in the present invention, have stable Formation of chemical bond gel network structure, all more stable in whole synthesis and cracking process, and also the PEG chain in structure is many Reactive polypeptide provides the condition of an abundant solvation, effectively increases the coupling efficiency of polypeptide；Introduce three classes in the sequence False dipeptide fragment X, Y, the Z of proline, effectively eliminates β-pleated sheet secondorder spatial structure, at utmost decreases residual peptide impurity Formed, greatly improve the yield of later-period purification.
Brief description
Fig. 1 is product mass spectra figure of the present invention.
Fig. 2 is product chromatogram of the present invention.
Specific embodiment
Hereinafter with reference to example, the present invention is described in further detail, but the invention is not restricted to this instantiation.
Embodiment 1
（1）Preparation Fmoc-Rink Amide AM PEGMatrix resin
Weigh 25 grams of AM PEGMatrix resin (0.8mmol/g, 20mmol), soaked 30 minutes with 500ml DCM, make resin Fully swelling, drain, add Fmoc-Rink Amide linker (MW:539.58,40mmol) 21.6g, DIEA (MW: 129.24,80mmol) 13.5ml, TBTU (MW:321.1,40mmol) 12.8g, HOBT (MW:135.1,40mmol) 5.4g, body Amass and compare 1:1 DMF/NMP 500ml, reacts 4 hours, drains, washed with DMF 3 times, drain, add volume ratio 2:2:1 second Anhydride：Pyridine：The mixed solution 500ml of DMF, reacts 1 hour, drains, washed with DMF 6 times, drain, and obtains Fmoc-Rink Amide AM PEGMatrix resin.
（2）Preparation His (trt)-Gly-Glu (otbu)-{ Gly-Thr (psi (me, me) pro) }-Phe-Thr (tbu)- Ser(tbu)-Asp(otbu)-{Leu-Ser(psi(me,me)pro)}-Lys(boc)-Gln(trt)-Met-Glu(otbu)- Glu(otbu)-Glu(otbu)-Ala-Val-Arg(pbf)-Leu-Phe-Ile-Glu(otbu)-Trp(boc)-Leu-Lys (boc)-Asn(trt)-Gly-Gly-Pro-{Ser(tbu)-Ser(psi(me,me)pro)}-Gly-Ala-Pro-Pro-Pro- Ser (tbu)-Rink Amide AM PEGMatrix resin
In step（1）In the Fmoc-Rink Amide AM PEGMatrix resin obtaining, add 500ml deprotecting regent, instead Answer 30 minutes, drain, washed with DMF 5 times, drain；Add Fmoc-Ser (tbu)-OH (MW:383.4,40mmol) 15.4g, HBTU(MW:379.2,40mmol) 15.2g, HOBT (MW:135.1,40mmol) 5.4g, NMM (MW:102.1,80mmol) 9.0ml, DMF500ml, react 1 hour, and 1,2,3-indantrione monohydrate detects resin water white transparency, drains, is washed with DMF 3 times, drain, and obtain Fmoc-Ser (tbu)-Rink Amide AM PEGMatrix resin；Then add deprotecting regent, deprotection reaction, then Add the aminoacid with Fmoc blocking group and false dipeptide fragment, so repeatedly, until after having connected last threonine, DMF washs 3 times, and again with methanol and dichloromethane replace washing 3 times, finally plus methanol shrinkage resin, drains, vacuum drying, obtains Obtain His (trt)-Gly-Glu (otbu)-{ Gly-Thr (psi (me, me) pro) }-Phe-Thr (tbu)-Ser (tbu)-Asp (otbu)-{Leu-Ser(psi(me,me)pro)}-Lys(boc)-Gln(trt)-Met-Glu(otbu)-Glu(otbu)-Glu (otbu)-Ala-Val-Arg(pbf)-Leu-Phe-Ile-Glu(otbu)-Trp(boc)-Leu-Lys(boc)-Asn(trt)- Gly-Gly-Pro-{Ser(tbu)-Ser(psi(me,me)pro)}-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-Rink The common 152g of Amide AM PEGMatrix resin；
Aminoacid and false dipeptide fragment X that each step condensation reaction is added, the amount of Y, Z is respectively：
Fmoc-Pro-OH(MW:337.4,40mmol) 13.5g,
Fmoc-Pro-OH(MW:337.4,40mmol) 13.5g,
Fmoc-Pro-OH(MW:337.4,40mmol) 13.5g,
Fmoc-Ala-OH(MW:311.3,40mmol) 12.5g,
Fmoc-Gly-OH(MW:297.3,40mmol) 11.9g,
X=Fmoc-Ser(tbu)-Ser{psi(me,me)pro}-OH(MW:510.58,40mmol) 20.4g,
Fmoc-Pro-OH(MW:337.4,40mmol) 13.5g,
Fmoc-Gly-OH(MW:297.3,40mmol) 11.9g,
Fmoc-Gly-OH(MW:297.3,40mmol) 11.9g,
Fmoc-Asn(trt)-OH(MW:596.7,40mmol) 23.9g,
Fmoc-Lys(boc)-OH(MW:468.5,40mmol) 18.7g,
Fmoc-Leu-OH(MW:353.4,40mmol) 14.2g,
Fmoc-Trp(boc)-OH(MW:526.6,40mmol) 21.1g,
Fmoc-Glu(otbu)-OH(MW:425.5,40mmol) 17g,
Fmoc-Ile-OH(MW:353.4,40mmol) 14.2g,
Fmoc-Phe-OH(MW:387.4,40mmol) 15.5g,
Fmoc-Leu-OH(MW:353.4,40mmol) 14.2g,
Fmoc-Arg(pbf)-OH(MW:648.8,40mmol) 26.0g,
Fmoc-Val-OH(MW:339.4,40mmol) 13.6g,
Fmoc-Ala-OH(MW:311.3,40mmol) 12.5g,
Fmoc-Glu(otbu)-OH(MW:425.5,40mmol) 17g,
Fmoc-Glu(otbu)-OH(MW:425.5,40mmol) 17g,
Fmoc-Glu(otbu)-OH(MW:425.5,40mmol) 17g,
Fmoc-Met-OH(MW:371.5,40mmol) 14.9g,
Fmoc-Gln(trt)-OH(MW:610.7,40mmol) 24.5g,
Fmoc-Lys(boc)-OH(MW:468.5,40mmol) 18.7g,
Y=Fmoc-Leu-Ser{psi(me,me)pro}-OH(MW:480.58,40mmol) 19.2g,
Fmoc-Asp(otbu)-OH(MW:411.5,40mmol) 16.5g,
Fmoc-Ser(tbu)-OH(MW:383.4,40mmol) 15.4g,
Fmoc-Thr(tbu)-OH(MW:397.5,40mmol) 15.9g
Fmoc-Phe-OH(MW:387.4,40mmol) 15.5g,
Z=Fmoc-Gly-Thr{psi(me,me)pro}-OH(MW:438.48,40mmol) 17.5g,
Fmoc-Glu(otbu)-OH(MW:425.5,40mmol) 17g,
Fmoc-Gly-OH(MW:297.3,40mmol) 11.9g,
Fmoc-His(trt)-OH(MW:619.7,40mmol) 24.8g,
Fmoc-Ser(tbu)-OH(MW:383.4,40mmol) 15.4g；
The amount of the condensing agent that each step condensation reaction is adopted is：HBTU(MW:379.2,40mmol) 15.2g, HOBT (MW: 135.1,40mmol) 5.4g, the amount of the organic base that each step condensation reaction is added is：NMM(MW:102,80mmol) 9.0ml.
（3）Prepare Exenatide
Take step（2）In the common 152g of full guard peptide resin be placed in the round-bottomed flask of 2L, add 1.5L cutting reagent, proportioning is TFA:Thioanisole:Phenol:Dithioglycol:H2O=87.5:5:2.5:2.5:2.5 (v/v), are placed in 25 DEG C of vibrations in constant-temperature table Reaction 2.5 hours, sucking filtration filters resin particle, collects filtrate, is subsequently adding 7.5L crystallizing from ether precipitation, precipitation is collected by centrifugation, Washed 3-6 time with ether again, vacuum drying, obtain exenatide crude product 85g, then through high performance liquid chromatography separation purification, lyophilizing Obtain the Exenatide fine work 38g of purity afterwards（As Fig. 1, shown in 2）, total recovery reaches 45%.
Embodiment 2
Step（1）AM PEGMatrix resin and 8 hours Amide linker, DIEA response time of Fmoc-Rink, add Acetic anhydride：Pyridine：The mixed solution of DMF reacts 0.5 hour, and PEGMatrix resin substitution value is 1.0mmol/g；Step（2） In Fmoc-Rink Amide AM PEGMatrix resin, add deprotecting regent, react 45 minutes；Add Fmoc-Ser (tbu)-OH, NMM, the response time is 0.5 hour；Step（3）The peptide chain resin of side chain full guard adds and cuts peptide reagent, instead Answer 2.5 hours.Remaining is with embodiment 1.
Embodiment 3
Step（1）AM PEGMatrix resin and 2 hours Amide linker, TBTU response time of Fmoc-Rink, add Acetic anhydride：Pyridine：The mixed solution of DMF reacts 2 hours, and PEGMatrix resin substitution value is 0.5mmol/g；Step（2） In Fmoc-Rink Amide AM PEGMatrix resin, add deprotecting regent, react 60 minutes；Add Fmoc-Ser (tbu)-OH, NMM, the response time is 2 hours；Step（3）The peptide chain resin of side chain full guard adds and cuts peptide reagent, reaction 3 hours.Remaining is with embodiment 1.

Claims (7)

1. a kind of solid phase synthesis process preparing Exenatide is it is characterised in that comprise the steps：
（1）Fmoc-Rink Amide Linker and AM PEGMatrix reaction resin mixture are obtained Rink Amide AM PEGMatrix resin；
（2）It is coupled 33 fluorenylmethyloxycarbonyl protection ammonia using Fmoc solid-phase synthesis according to the order from C-terminal to N-terminal one by one Base acid and three class proline vacation dipeptide fragment X, Y, Z, obtain the polypeptide resin of side chain full guard, shown in formula I；
His(trt)-Gly-Glu(otbu)-X-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Y-Lys(boc)-Gln (trt)-Met-Glu(otbu)-Glu(otbu)-Glu(otbu)-Ala-Val-Arg(pbf)-Leu-Phe-Ile-Glu (otbu)-Trp(boc)-Leu-Lys(boc)-Asn(trt)-Gly-Gly-Pro-Z-Gly-Ala-Pro-Pro-Pro-Ser (tbu)-Rink Amide PEGMatrix resin
Formulas I
In formula：X= Gly-Thr(psi(me,me)pro)；Y= Leu-Ser(psi(me,me)pro)；Z= Ser(tbu)-Ser (psi(me,me)pro)；
（3）Addition is cut peptide reagent and the peptide chain resin of side chain full guard is cracked, and 30 nonapeptides are cleaved from resin And remove side chain protecting group, open the five-membered ring of three false dipeptide fragment in sequence simultaneously, obtain exenatide crude product, as formula Shown in II；Crude product is refining to obtain Exenatide through high performance liquid chromatography separation purification, lyophilizing again；
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-NH2
Formula II.
2. method according to claim 1 is it is characterised in that step（1）Particular content comprises the steps
Take AM PEGMatrix resin dichloromethane to soak, make resin fully swelling, drain, add Fmoc- Rink Amide Linker, DIPEA, select O- BTA-N, N, N', N'- tetramethylurea Tetrafluoroboric acid/1- hydroxy benzeness And triazole is as catalyst, volume ratio is selected to be 1:1 N,N-dimethylformamide/N-Methyl pyrrolidone mixed solution is made For solvent, react 2-8 hour, drain, washed with DMF 3 times, add volume ratio 2:2:1 acetic anhydride： Pyridine：The mixed solution of DMF, reacts 0.5-2 hour, drains, washed with DMF 6 times, Drain, obtain Fmoc-Rink Amide AM PEGMatrix resin.
3. method according to claim 2 is it is characterised in that the substitution value of PEGMatrix resin is in 0.5mmol/g- Between 1.0mmol/g, the amount of Fmoc-Rink Amide Linker is 2 times of AM PEGMatrix resin mole；O- benzo Triazole-N, the amount of N, N', N'- tetramethylurea Tetrafluoroboric acid/I-hydroxybenzotriazole is AM PEGMatrix resin mole 2 times, the amount of dichloromethane is 4 times of AM PEGMatrix resin mole.
4. method according to claim 1 is it is characterised in that step（2）Particular content comprises the steps：
In step（1）In the Fmoc-Rink Amide AM PEGMatrix resin obtaining, add deprotecting regent, react 30- 60 minutes, drain, washed with DMF 5 times, drain；Addition Fmoc-Ser (tbu)-OH, N-methylmorpholine, Select O- benzotriazole-N, N, N, N- tetramethylurea hexafluorophosphate/I-hydroxybenzotriazole as condensing agent, with N, N- bis- Methylformamide, as solvent, reacts 0.5-2 hour, and ninhydrin method detects reaction end, drains, uses DMF Washing 3 times, drains, and obtains Fmoc-Ser (tbu)-Rink Amide PEGMatrix resin；Then add deprotecting regent, Deprotection reaction, adds with fluorenylmethyloxycarbonyl protected amino acid and class proline vacation dipeptide fragment, so repeatedly, until connecting Last threonine complete；Washed with DMF 3 times, again with methanol and dichloromethane replace washing 3 times, finally Plus methanol shrinkage resin, drain, vacuum drying, obtain the peptide chain resin of side chain full guard, i.e. His (trt)-Gly-Glu (otbu)-{Gly-Thr(psi(me,me)pro)}-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-{Leu-Ser(psi (me,me)pro)}-Lys(boc)-Gln(trt)-Met-Glu(otbu)-Glu(otbu)-Glu(otbu)-Ala-Val-Arg (pbf)-Leu-Phe-Ile-Glu(otbu)-Trp(boc)-Leu-Lys(boc)-Asn(trt)-Gly-Gly-Pro-{Ser (tbu)-Ser(psi(me,me)pro)}-Gly-Ala-Pro-Pro-Pro-Ser(tbu)-Rink Amide AM PEGMatrix resin；
Described has fluorenylmethyloxycarbonyl protected amino acid and false dipeptide fragment, includes Fmoc-Pro-OH, Fmoc-Pro- successively OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser (tbu)-Ser { psi (me, me) pro }-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, 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 (o
Tbu)-OH, Fmoc-Glu (otbu)-OH, Fmoc-Glu (otbu)-OH, Fmoc-Met-OH, Fmoc-Gln (trt)-OH, Fmoc-Lys (boc)-OH, Fmoc-Leu-Ser { psi (me, me) pro }-OH, Fmoc-Asp (otbu)-OH, Fmoc-Ser (tbu)-OH, Fmoc-Thr (tbu)-OH, Fmoc-Phe-OH, Fmoc-Gly-Thr { psi (me, me) pro }-OH, Fmoc- Glu (otbu)-OH, Fmoc-Gly-OH, Fmoc-His (trt)-OH.
5. method according to claim 4 is it is characterised in that the amount of Fmoc-Ser (tbu)-OH is Fmoc-Rink 2 times of Amide AM PEGMatrix resin mole,；Reagent of raising one's hat is the piperidines/N of volume ratio 20%, N- dimethyl formyl Amine mixed solution, the amount of each step fluorenylmethyloxycarbonyl protected amino acid is Fmoc-Rink Amide AM PEGMatrix resin 2 times of mole, the amount of condensation reagent is 2 times of Fmoc-Rink Amide AM PEGMatrix resin mole, N- methyl The amount of morpholine is 4 times of Fmoc-Rink Amide AM PEGMatrix resin mole, the detection side of each step reaction end Method used 1,2,3-indantrione monohydrate detection method.
6. method according to claim 1 is it is characterised in that step（3）The peptide chain resin of side chain full guard adds Cut peptide reagent reaction 2-3 hour, sucking filtration filters resin particle, and collects filtrate, the ice crystallizing from ether being subsequently adding 6 times of volumes sinks Form sediment, precipitation is collected by centrifugation, then is washed with ether 5 times, vacuum drying, obtain exenatide crude product.
7. method according to claim 6 selects volume ratio trifluoroacetic acid it is characterised in that cutting peptide reagent:Thioanisole: Phenol:Dithioglycol:H2O=87.5:5:2.5:2.5:2.5 mix reagent.
CN201610950754.2A 2016-11-03 2016-11-03 Solid-phase synthesis method for preparing exenatide Pending CN106432468A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610950754.2A CN106432468A (en) 2016-11-03 2016-11-03 Solid-phase synthesis method for preparing exenatide

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610950754.2A CN106432468A (en) 2016-11-03 2016-11-03 Solid-phase synthesis method for preparing exenatide

Publications (1)

Publication Number Publication Date
CN106432468A true CN106432468A (en) 2017-02-22

Family

ID=58179162

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610950754.2A Pending CN106432468A (en) 2016-11-03 2016-11-03 Solid-phase synthesis method for preparing exenatide

Country Status (1)

Country Link
CN (1) CN106432468A (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108676070A (en) * 2018-05-11 2018-10-19 滨海吉尔多肽有限公司 A kind of solid-phase synthesis according to Nagqu 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
CN116789768A (en) * 2023-07-31 2023-09-22 中奥生物医药技术(广东)有限公司 Preparation method of F1 polypeptide

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102159587A (en) * 2008-10-07 2011-08-17 弗·哈夫曼-拉罗切有限公司 Pseudoproline dipeptides
CN102174082A (en) * 2011-02-23 2011-09-07 上海昂博生物技术有限公司 Method for preparing exenatide
CN102942625A (en) * 2012-11-30 2013-02-27 南京工业大学 Solid-phase synthesis method of exenatide
CN103224558A (en) * 2013-04-03 2013-07-31 齐鲁制药有限公司 Preparation method of exenatide
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
CN103613656A (en) * 2013-11-20 2014-03-05 陕西东大生化科技有限责任公司 Solid-phase fragment synthetic method of exenatide

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102159587A (en) * 2008-10-07 2011-08-17 弗·哈夫曼-拉罗切有限公司 Pseudoproline dipeptides
CN102174082A (en) * 2011-02-23 2011-09-07 上海昂博生物技术有限公司 Method for preparing exenatide
CN102942625A (en) * 2012-11-30 2013-02-27 南京工业大学 Solid-phase synthesis method of exenatide
CN103224558A (en) * 2013-04-03 2013-07-31 齐鲁制药有限公司 Preparation method of exenatide
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
CN103613656A (en) * 2013-11-20 2014-03-05 陕西东大生化科技有限责任公司 Solid-phase fragment synthetic method of exenatide

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
邱芊等: "固相多肽合成中氨基酸保护的研究进展", 《化工时刊》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108676070A (en) * 2018-05-11 2018-10-19 滨海吉尔多肽有限公司 A kind of solid-phase synthesis according to Nagqu 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
CN116789768A (en) * 2023-07-31 2023-09-22 中奥生物医药技术(广东)有限公司 Preparation method of F1 polypeptide
CN116789768B (en) * 2023-07-31 2024-03-15 中奥生物医药技术(广东)有限公司 Preparation method of F1 polypeptide

Similar Documents

Publication Publication Date Title
CN109311961B (en) Synthesis method of Somalutide
CN108059666B (en) A kind of method that solid-liquid combination prepares Suo Malu peptides
CN103497245B (en) Method for synthesizing thymalfasin
KR101087859B1 (en) Insulinotropic peptide synthesis
CN110372785B (en) Synthesis method of Somalutide
CN103333237B (en) Synthesis of exenatide through solid phase fragment method
CN108047329A (en) A kind of preparation method of A Bapa peptides
CN113135991B (en) Method for preparing somaglutide
CN107022021A (en) A kind of solid-phase synthesis of Liraglutide
CN103304659A (en) Solid-phase preparation method of liraglutide
CN101747426A (en) Method for synthesizing pramlintide
CN107880111A (en) A kind of method for preparing Liraglutide
CN106432468A (en) Solid-phase synthesis method for preparing exenatide
CN109734794A (en) A kind of preparation method of A Bapa peptide
CN108059667B (en) A kind of solid phase synthesis process of Lanreotide
CN112679602A (en) Solid-phase synthesis method of Somaloutide
CN112125970B (en) Synthesis method of semaglutide
CN109021087A (en) A kind of method that solid liquid phase combination prepares ziconotide
CN108070030B (en) Preparation method of loxenapeptide and analogue thereof
CN104428314A (en) Solid phase peptide synthesis of insulin using side chain anchored lysine
CN103122026A (en) Solid-phase preparation method for exenatide crude product
WO2023279323A1 (en) Method for synthesizing glp-1 analog
CN103992401B (en) Method for preparing exenatide
CN114249810A (en) Synthetic method of somaglutide
CN107417786B (en) Preparation method of thymosin alpha 1

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20170222

RJ01 Rejection of invention patent application after publication