WO1991006558A1 - Procede pour la synthese de peptides et ses substrats - Google Patents

Procede pour la synthese de peptides et ses substrats Download PDF

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Publication number
WO1991006558A1
WO1991006558A1 PCT/EP1990/001900 EP9001900W WO9106558A1 WO 1991006558 A1 WO1991006558 A1 WO 1991006558A1 EP 9001900 W EP9001900 W EP 9001900W WO 9106558 A1 WO9106558 A1 WO 9106558A1
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WO
WIPO (PCT)
Prior art keywords
carrier
fmoc
amino acid
peptides
solution
Prior art date
Application number
PCT/EP1990/001900
Other languages
German (de)
English (en)
Inventor
Ronald Frank
Susanne Krause
Original Assignee
GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF)
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 GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF) filed Critical GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF)
Publication of WO1991006558A1 publication Critical patent/WO1991006558A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/042—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers characterised by the nature of the carrier
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00504—Pins
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/0059—Sequential processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • Proteins are made up of one or several linear polypeptide chains. They accomplish their diverse functions by three-dimensional folding and association of these chains. Folding and 3D structure is determined by the primary amino acid sequence of the chains. The variety of protein structures in nature is created by the correlation of twenty amino acid building blocks. Short partial sequences (oligopeptides) can to some extent imitate the local structure and function of this sequence in the overall protein structure. In turn, oligopeptides can be produced by chemical synthesis. Synthetic oligopeptides are therefore an important aid in the structural function analysis of proteins.
  • Biological test actions such as enzymatic conversion, antibody binding etc. are very sensitive, so that only small amounts (ng - ⁇ g) of peptic substrates are sufficient.
  • the peptide substrate is immobilized on a solid support material and light can be removed from the reaction solution. The success of a reaction with the peotide can then be measured quantitatively either in the oiling reaction solution or by subsequent analysis of the carrier-bound material.
  • Rods made of suitable material are used as support elements.
  • the rods are provided with a number on the upper enoe, which denotes the peptiosis sequence to be synthesized in each case.
  • the bars remain mobile and can be hung in a suitable frame.
  • the holes in the rack are preferably compatible with the arrangement of holes in a microtiter plate. According to a special embodiment, glass round rods are used as carrier elements
  • Two O-rings made from solvent-resistant Viton material are preferably installed at a distance of approx. 1 cm and approx. 5 cm from the upper flat end.
  • a suitable vessel eg beaker
  • the peptides are then built up step by step and according to the predetermined sequences according to known solid-phase peptide synthesis methods (see Merrifield, Wünsch Patent Simultane Peptide Synthesis) to the aminopropyl.
  • the Fmoc / tBu method is preferably used [C, -D. Chang, J. Meienhofer. 1978. Int. J. Peptide Protein Res. 11, 246; E. Atherton, H. Fox, D. Harkiss, C.J. Logan, R.C. Sheppard, B.J. Williams. 1978. J. Chem. Soc. Chem. Commun. 537].
  • any modified and non-natural amino acids can also be incorporated.
  • the necessary amino acid building blocks are concentrated (0.1-0.3 H) solutions in a suitable solvent, preferably with a high boiling point and lower Evaporation rate (e.g. N-methyl-pyrrolidinone, bp 203 ° C) specified in small closable containers.
  • a suitable solvent preferably with a high boiling point and lower Evaporation rate (e.g. N-methyl-pyrrolidinone, bp 203 ° C) specified in small closable containers.
  • a t- (Fmoc-amino) alkyl carboxylic acid can preferably be coupled as a spacer (spacsr) as the first amino acid: z.
  • the N-terminal amino groups are preferably blocked by acetylation with acetic anhydride.
  • the chopsticks hooked into the frame are used together for e.g. B. 3 minutes in a small pan with acetylation (eg 5% acetic anhydride, 5% diisopropylethylamine in DMF) and then washed with DMF and dichloromethane (squirt bottle).
  • the protective groups on the amino acid side chains are then cleaved off by suitable acid treatment. To do this, hang those in the frame
  • a so-called linker reagent (cf. E. Atherton et al., J. Chem. Soc. Perkin Trans. I, 1981, 538-546) can also be linked to the carrier functions before the actual peptide synthesis.
  • the peptide which is in turn built up on it, can then be split off according to the chemical nature of the peptide linker bond under suitable reaction conditions.
  • the support rods thus loaded with specific peptide sequences can now be suspended in any arrangement required for the biological test, so that their tips reach into the holes of a microtiter plate and are available for the test reaction.
  • the peptides are split off individually from support rods and used in soluble form for test purposes.
  • Figure 1 Amino acid primary sequence of the cloned, immunogenic teli-section CMV26. Overlapping decapeptide sequences shifted by one amino acid were derived from this.
  • Figure 2 List of all decapeptides to be synthesized with the assigned numbering and instructions for the simultaneous, parallel implementation of the amino acid coupling.
  • Figure 3 Schematic representation of the ELISA test, the surface-bound peptides (P) are incubated with the rabbit anti-CMV26 serum, excess serum washed off, and then with an anti-rabbit antibody from the goat, softer covalently with the enzyme ß-galactosidase linked, incubated. Only if the anti-CMY26 antibodies can bind the peptide is the second
  • Antibody bound the enzyme this can then be detected spectroscopically by its activity in the cleavage of p-nitrophenyl- ⁇ -D-galactoside in p-nitrophenolate (absorption at the wavelength 414nm)
  • Figure 4 Graphical representation of the test results. The peptides on the glass rods of numbers 14-16 and 31-33 reacted. Positive. Comparison is:
  • 55 fusion protein without CMV26 sequence.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention se rapporte à un procédé pour la synthèse rapide et éventuellement simultanée de peptides ainsi qu'à des substrats appropriés en forme de baguettes.
PCT/EP1990/001900 1989-10-25 1990-10-25 Procede pour la synthese de peptides et ses substrats WO1991006558A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19893935572 DE3935572A1 (de) 1989-10-25 1989-10-25 Verfahren zur peptidsynthese und traeger dafuer
DEP3935572.1 1989-10-25

Publications (1)

Publication Number Publication Date
WO1991006558A1 true WO1991006558A1 (fr) 1991-05-16

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ID=6392201

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1990/001900 WO1991006558A1 (fr) 1989-10-25 1990-10-25 Procede pour la synthese de peptides et ses substrats

Country Status (2)

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DE (1) DE3935572A1 (fr)
WO (1) WO1991006558A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992013881A1 (fr) * 1991-02-12 1992-08-20 Luminis Pty. Ltd. Peptides
EP0519640A1 (fr) * 1991-06-18 1992-12-23 Eli Lilly And Company Préparation rapide et triage des peptides-mimétiques
EP0586600A1 (fr) * 1991-05-24 1994-03-16 Harvard College Reacteur sequentiel et parallele.
CN100522993C (zh) * 2006-05-30 2009-08-05 中国科学院昆明动物研究所 无指盘臭蛙抗菌肽
CN102516382A (zh) * 2011-12-26 2012-06-27 大连理工大学 一种海南湍蛙抗微生物肽Hainanenin-5及其基因、分离纯化、化学合成和应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0672049A4 (fr) * 1992-11-06 1997-10-15 Chiron Mimotopes Pty Ltd Support pour la synthese de polymeres modulaires.
US6265219B1 (en) * 1996-10-30 2001-07-24 Mitokor Transponder tagging of constituents used in compound synthesis
US6518067B1 (en) * 1997-02-17 2003-02-11 Gesellschaft Fuer Biotechnologische Forschung Mbh (Gbf) Automated chemical synthesis apparatus
DE19844988A1 (de) * 1998-09-30 2000-04-13 Stefan Seeger Parallele Festphasensynthese

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1984003564A1 (fr) * 1983-03-08 1984-09-13 Commw Serum Lab Commission Procede de determination de sequences d'acides amines antigeniquement actives
WO1988007052A1 (fr) * 1987-03-11 1988-09-22 Steel Samuel L Synthese de composes analogues de peptides
WO1990009395A1 (fr) * 1989-02-17 1990-08-23 Coselco Mimotopes Pty. Ltd. Procede d'utilisation et de synthese de peptides

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1984003564A1 (fr) * 1983-03-08 1984-09-13 Commw Serum Lab Commission Procede de determination de sequences d'acides amines antigeniquement actives
WO1988007052A1 (fr) * 1987-03-11 1988-09-22 Steel Samuel L Synthese de composes analogues de peptides
WO1990009395A1 (fr) * 1989-02-17 1990-08-23 Coselco Mimotopes Pty. Ltd. Procede d'utilisation et de synthese de peptides

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992013881A1 (fr) * 1991-02-12 1992-08-20 Luminis Pty. Ltd. Peptides
EP0586600A1 (fr) * 1991-05-24 1994-03-16 Harvard College Reacteur sequentiel et parallele.
EP0586600A4 (fr) * 1991-05-24 1994-04-13 The President And Fellows Of Harvard College
EP0519640A1 (fr) * 1991-06-18 1992-12-23 Eli Lilly And Company Préparation rapide et triage des peptides-mimétiques
CN100522993C (zh) * 2006-05-30 2009-08-05 中国科学院昆明动物研究所 无指盘臭蛙抗菌肽
CN102516382A (zh) * 2011-12-26 2012-06-27 大连理工大学 一种海南湍蛙抗微生物肽Hainanenin-5及其基因、分离纯化、化学合成和应用
CN102516382B (zh) * 2011-12-26 2014-04-16 大连理工大学 一种海南湍蛙抗微生物肽Hainanenin-5及其基因、应用

Also Published As

Publication number Publication date
DE3935572A1 (de) 1991-05-02

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