Solid-phase synthesis method for preparing exenatide

A technology for exenatide and solid-phase synthesis, which is applied in the preparation methods of peptides, chemical instruments and methods, peptides, etc., can solve the problems of complex synthesis steps, difficult separation in the later stage, and difficult coupling, etc., and achieves reduction of residual peptides. The formation of impurities, the elimination of β-sheet secondary space structure, and the effect of improving yield

Inactive Publication Date: 2017-02-22
滨海吉尔多肽有限公司 +1
6 Cites 2 Cited by

AI-Extracted Technical Summary

Problems solved by technology

[0005] The object of the present invention is to provide a high yield, low cost, mild reaction conditions, less environmental pollution, and a solid-phase synthesis method for exenatide th...
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

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.

Application Domain

Peptide preparation methodsVasoactive intestinal peptide +1

Technology Topic

Side chainExenatide +11

Image

  • Solid-phase synthesis method for preparing exenatide
  • Solid-phase synthesis method for preparing exenatide

Examples

  • Experimental program(3)

Example Embodiment

[0061] Example 1
[0062] (1) Preparation of Fmoc-Rink Amide AM PEGMatrix resin
[0063] Weigh 25 grams (0.8mmol/g, 20mmol) of AM PEGMatrix resin, soak it in 500ml DCM for 30 minutes to fully swell the resin, drain it, and 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, DMF/NMP 500ml with a volume ratio of 1:1, react for 4 hours, drain it, and use DMF Wash 3 times, drain dry, then add 500ml of acetic anhydride:pyridine:DMF mixed solution with a volume ratio of 2:2:1, react for 1 hour, drain dry, wash 6 times with DMF, drain dry to obtain Fmoc-RinkAmide AM PEGMatrix Resin.
[0064] (2) Preparation of 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
[0065] In the Fmoc-Rink Amide AM PEGMatrix resin obtained in step (1), add 500ml of deprotection reagent, react for 30 minutes, drain, wash 5 times with DMF, and 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, reaction for 1 hour, ninhydrin detection The resin is colorless and transparent, drained, washed 3 times with DMF, drained to obtain Fmoc-Ser(tbu)-Rink Amide AM PEGMatrix resin; then add deprotection reagent, deprotection reaction, and then add the Fmoc protecting group Amino acid and pseudo-dipeptide fragments are repeated until the last threonine is connected, DMF is washed 3 times, then methanol and dichloromethane are alternately washed 3 times, finally methanol is added to shrink the resin, drained, and vacuum dried to 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)-RinkAmide AM PEGMatrix resin total 152g;
[0066] The amounts of amino acids and pseudo-dipeptide fragments X, Y, and Z added in each step of the condensation reaction are:
[0067] Fmoc-Pro-OH (MW: 337.4, 40mmol) 13.5g,
[0068] Fmoc-Pro-OH (MW: 337.4, 40mmol) 13.5g,
[0069] Fmoc-Pro-OH (MW: 337.4, 40mmol) 13.5g,
[0070] Fmoc-Ala-OH (MW: 311.3, 40mmol) 12.5g,
[0071] Fmoc-Gly-OH (MW: 297.3, 40mmol) 11.9g,
[0072] X=Fmoc-Ser(tbu)-Ser{psi(me,me)pro}-OH(MW:510.58, 40mmol) 20.4g,
[0073] Fmoc-Pro-OH (MW: 337.4, 40mmol) 13.5g,
[0074] Fmoc-Gly-OH (MW: 297.3, 40mmol) 11.9g,
[0075] Fmoc-Gly-OH (MW: 297.3, 40mmol) 11.9g,
[0076] Fmoc-Asn(trt)-OH (MW: 596.7, 40mmol) 23.9g,
[0077] Fmoc-Lys(boc)-OH (MW: 468.5, 40mmol) 18.7g,
[0078] Fmoc-Leu-OH (MW: 353.4, 40mmol) 14.2g,
[0079] Fmoc-Trp(boc)-OH (MW: 526.6, 40mmol) 21.1g,
[0080] Fmoc-Glu(otbu)-OH (MW: 425.5, 40mmol) 17g,
[0081] Fmoc-Ile-OH (MW: 353.4, 40mmol) 14.2g,
[0082] Fmoc-Phe-OH (MW: 387.4, 40mmol) 15.5g,
[0083] Fmoc-Leu-OH (MW: 353.4, 40mmol) 14.2g,
[0084] Fmoc-Arg(pbf)-OH (MW: 648.8, 40mmol) 26.0g,
[0085] Fmoc-Val-OH (MW: 339.4, 40mmol) 13.6g,
[0086] Fmoc-Ala-OH (MW: 311.3, 40mmol) 12.5g,
[0087] Fmoc-Glu(otbu)-OH (MW: 425.5, 40mmol) 17g,
[0088] Fmoc-Glu(otbu)-OH (MW: 425.5, 40mmol) 17g,
[0089] Fmoc-Glu(otbu)-OH (MW: 425.5, 40mmol) 17g,
[0090] Fmoc-Met-OH (MW: 371.5, 40mmol) 14.9g,
[0091] Fmoc-Gln(trt)-OH (MW: 610.7, 40mmol) 24.5g,
[0092] Fmoc-Lys(boc)-OH (MW: 468.5, 40mmol) 18.7g,
[0093] Y=Fmoc-Leu-Ser{psi(me,me)pro}-OH(MW:480.58,40mmol) 19.2g,
[0094] Fmoc-Asp(otbu)-OH (MW: 411.5, 40mmol) 16.5g,
[0095] Fmoc-Ser(tbu)-OH (MW: 383.4, 40mmol) 15.4g,
[0096] Fmoc-Thr(tbu)-OH (MW: 397.5, 40mmol) 15.9g
[0097] Fmoc-Phe-OH (MW: 387.4, 40mmol) 15.5g,
[0098] Z=Fmoc-Gly-Thr{psi(me,me)pro}-OH(MW:438.48,40mmol)17.5g,
[0099] Fmoc-Glu(otbu)-OH (MW: 425.5, 40mmol) 17g,
[0100] Fmoc-Gly-OH (MW: 297.3, 40mmol) 11.9g,
[0101] Fmoc-His(trt)-OH (MW: 619.7, 40mmol) 24.8g,
[0102] Fmoc-Ser(tbu)-OH (MW: 383.4, 40mmol) 15.4g;
[0103] The amount of condensing agent used in each step of the condensation reaction is: HBTU (MW: 379.2, 40mmol) 15.2g, HOBT (MW: 135.1, 40mmol) 5.4g, the amount of organic base added in each step of the condensation reaction: NMM( MW: 102, 80mmol) 9.0ml.
[0104] (3) Preparation of Exenatide
[0105] Take a total of 152g of the fully protected peptide resin in step (2) and place it in a 2L round-bottomed flask, add 1.5L of cutting reagent, and the ratio is TFA: thioanisole: phenol: ethanedithiol: H 2 O=87.5:5:2.5:2.5:2.5(v/v), place in a constant temperature shaker at 25℃ and shake for 2.5 hours, filter out the resin particles with suction, collect the filtrate, then add 7.5L ether to crystallize precipitation, and centrifuge to collect Precipitate, wash with ether for 3-6 times, dry in vacuum to obtain 85g of exenatide crude product, and then separate and purify by high performance liquid chromatography and freeze-dry to obtain pure exenatide product 38g (such as figure 1 , Shown in 2), the total yield reached 45%.

Example Embodiment

[0106] Example 2
[0107] Step (1) AM PEGMatrix resin reacts with Fmoc-Rink Amide linker and DIEA for 8 hours, then add acetic anhydride: pyridine: DMF mixed solution to react for 0.5 hours, PEGMatrix resin substitution degree is 1.0mmol/g; Step (2) Fmoc -Rink Amide AM PEGMatrix resin, add deprotection reagent, react for 45 minutes; add Fmoc-Ser(tbu)-OH, NMM, reaction time is 0.5 hours; step (3) add to the peptide chain resin with full side chain protection Peptide cleavage reagent, react for 2.5 hours. The rest is the same as in Example 1.

Example Embodiment

[0108] Example 3
[0109] Step (1) AM PEGMatrix resin reacts with Fmoc-Rink Amide linker and TBTU for 2 hours, then add acetic anhydride: pyridine: DMF mixed solution to react for 2 hours, PEGMatrix resin substitution degree is 0.5mmol/g; step (2) Fmoc -Rink Amide AM PEGMatrix resin, add deprotection reagent, react for 60 minutes; add Fmoc-Ser(tbu)-OH, NMM, reaction time is 2 hours; step (3) add to the peptide chain resin with full side chain protection Cleavage reagent, react for 3 hours. The rest is the same as in Example 1.
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

no PUM

Description & Claims & Application Information

We can also present the details of the Description, Claims and Application information to help users get a comprehensive understanding of the technical details of the patent, such as background art, summary of invention, brief description of drawings, description of embodiments, and other original content. On the other hand, users can also determine the specific scope of protection of the technology through the list of claims; as well as understand the changes in the life cycle of the technology with the presentation of the patent timeline. Login to view more.
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Similar technology patents

Vascular hemophilia factor detection reagent, preparation method and application thereof

InactiveCN107677832AHigh coupling efficiencyLow costDisease diagnosisBiological testingChemistryMicrosphere
Owner:北京众驰伟业科技发展有限公司

Preparation method of teduglutide

PendingCN113480633AHigh coupling efficiencyEfficient couplingPeptide preparation methodsBulk chemical productionTeduglutideCombinatorial chemistry
Owner:CHENGDU SINTANOVO BIOTECHNOLOGV CO LTD

High-hardness modified PC (polycarbonate) and carbon fiber composite material and preparation method thereof

InactiveCN109438956AHigh coupling efficiencyLink structure is stablePolycarbonatePolypropylene
Owner:ANHUI XUSHENG NEW CONCEPT MATERIAL CO LTD

Nano carbon black with metallic luster and preparation method thereof

ActiveCN112480719AHigh coupling efficiencyImprove appearanceOrganic-compounds/hydrides/coordination-complexes catalystsInksMaterials scienceOxidative coupling of methane
Owner:唐山黑猫炭黑有限责任公司

Classification and recommendation of technical efficacy words

  • High coupling efficiency

Riemerella anatipestifer surface antigen D15 truncated recombinant protein sensitization latex reagent as well as preparation method and application thereof

ActiveCN103926414AGood repeatability and sensitivityHigh coupling efficiencyBacteria peptidesBiological testingCarboxylationChemistry
Owner:SICHUAN AGRI UNIV

Method for labeling antibody with DNA

ActiveCN105372415AThe reaction steps are simpleHigh coupling efficiencyMaterial analysisImmuno pcrCovalent bond
Owner:WUHAN GENECREATE BIOLOGICAL ENG CO LTD

High-hardness modified PC (polycarbonate) and carbon fiber composite material and preparation method thereof

InactiveCN109438956AHigh coupling efficiencyLink structure is stablePolycarbonatePolypropylene
Owner:ANHUI XUSHENG NEW CONCEPT MATERIAL CO LTD

Nano carbon black with metallic luster and preparation method thereof

ActiveCN112480719AHigh coupling efficiencyImprove appearanceOrganic-compounds/hydrides/coordination-complexes catalystsInksMaterials scienceOxidative coupling of methane
Owner:唐山黑猫炭黑有限责任公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products