EP0204801A1 - Method for a solid phase synthesis of a linear combination of amino acid residues - Google Patents

Method for a solid phase synthesis of a linear combination of amino acid residues

Info

Publication number
EP0204801A1
EP0204801A1 EP86900197A EP86900197A EP0204801A1 EP 0204801 A1 EP0204801 A1 EP 0204801A1 EP 86900197 A EP86900197 A EP 86900197A EP 86900197 A EP86900197 A EP 86900197A EP 0204801 A1 EP0204801 A1 EP 0204801A1
Authority
EP
European Patent Office
Prior art keywords
amino acid
amino
alpha
protected
group
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.)
Withdrawn
Application number
EP86900197A
Other languages
German (de)
French (fr)
Inventor
Robert Sheppard
Eric Atherton
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.)
Pharmacia and Upjohn Cambridge Ltd
Original Assignee
LKB Biochrom 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 LKB Biochrom Ltd filed Critical LKB Biochrom Ltd
Publication of EP0204801A1 publication Critical patent/EP0204801A1/en
Withdrawn legal-status Critical Current

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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
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/08—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents
    • C07K1/088—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents containing other elements, e.g. B, Si, As
    • 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

Definitions

  • the present invention relates to a method for a solid phase synthesis of a linear combination of amino acid residues, linked via peptide bonds, starting with an amino acid residue covalently linked to a support and protected by an N-alpha-amino protecting group, comprising the following steps: (a) removing the N-alpha-amino protecting group to obtain an N-alpha-amino group,
  • step (b) adding an amino acid residue protected by an N-alpha-amino protecting group, via a peptide bond, to the N-alpha-amino group obtained in step (a) by use of a reactive protected amino acid derivative and, where necessary, a catalyst,

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)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Procédé de synthèse en phase solide d'une combinaison linéaire de résidus d'acides aminés, liés par des liaisons peptides, commençant par un résidu d'acide aminé lié de manière covalente à un support et protégé par un groupe protecteur N-alpha-amino, consistant à a) éliminer le groupe protecteur N-alpha-amino pour obtenir un groupe N-alpha-amino; b) ajouter un résidu d'acide aminé protégé par un groupe protecteur N-alpha-amino, via une liaison peptide, au groupe N-alpha-amino obtenu dans l'étage a), en utilisant un dérivé d'acide aminé réactif protégé et, si nécessaire, un catalyseur; c) répéter les étapes a) et b) jusqu'à obtention de ladite combinaison linéaire. Selon l'invention, dans le dérivé d'acide aminé réactif protégé, le groupe acyle utilisé pour former la liaison peptide est activé comme un ester de pentafluorophényle.Method for the solid phase synthesis of a linear combination of amino acid residues linked by peptide bonds, starting with an amino acid residue covalently linked to a support and protected by an N-alpha-amino protective group , consisting in a) removing the N-alpha-amino protecting group to obtain an N-alpha-amino group; b) add an amino acid residue protected by an N-alpha-amino protective group, via a peptide bond, to the N-alpha-amino group obtained in stage a), using a protected reactive amino acid derivative and, if necessary, a catalyst; c) repeating steps a) and b) until said linear combination is obtained. According to the invention, in the protected reactive amino acid derivative, the acyl group used to form the peptide bond is activated like a pentafluorophenyl ester.

Description

"METHOD FOR A SOLID PHASE SYNTHESIS OF A LINEAR COMBINATION OF AMINO ACID RESIDUES" TECHNICAL FIELD
The present invention relates to a method for a solid phase synthesis of a linear combination of amino acid residues, linked via peptide bonds, starting with an amino acid residue covalently linked to a support and protected by an N-alpha-amino protecting group, comprising the following steps: (a) removing the N-alpha-amino protecting group to obtain an N-alpha-amino group,
(b) adding an amino acid residue protected by an N-alpha-amino protecting group, via a peptide bond, to the N-alpha-amino group obtained in step (a) by use of a reactive protected amino acid derivative and, where necessary, a catalyst,
(c) repeating steps (a) and (b) until the said linear combination has been obtained.
BACKGROUND ART Traditionally, the above solid phase peptide synthesis has been carried out using t-butoxycarbonyl (Boc) amino-acids activated in situ with an equivalent amount of dicyclohexyl-carbodiimide. A significant advance was the introduction of preformed Boc and fluorenylmethoxycarbonyl (Fmoc) amino-acid anhydrides in both polystyrene and polyamide-based solid phase synthesis, avoiding contact of the reactive resin-bound amino group with the activating reagent. Acylation reactions are rapid, especially in polar media such as dimethylformamide.
Activated esters (particularly p-nitrophenyl or trichlorophenyl derivatives) have also been used from time to time in solid phase synthesis but reaction rates may be low even in the presence of catalysts. Again polar reaction media are to be preferred. This last consideration is particularly relevant in polyamide-based synthesis since this resin support is totally compatible with a wide range of aprotic polar organic solvents. SUMMARY OF THE INVENTION
The object of the present invention is to seek alternative activated Fmoc-amino-acid derivatives which would combine the high reaction rates and freedom from side reactions achievable with symmetric anhydrides with the crystallinity, stability, and ease of handling of activated esters in order to avoid manual pre-activation at each step in the synthesis and substantially simplify mechanisation.
This is attained by the method according to the invention in that the said reactive protected amino acid derivative has the acyl group used to form the peptide bond activated as a pentafluorophenyl ester. BRIEF DESCRIPTION OF DRAWING
The single figure is an HPLC diagram of a peptide assembled in accordance with the method of this invention.
DETAILED DESCRIPTION For a test case the very difficult acyl carrier protein 65-74 sequence
H. Val.Gln.Ala.Ala.Ile.Asp.Tyr.Ile.Asn.Gly.OH 1 10
was selected. Earlier attempts to assemble this sequence using p-nitrophenyl esters in the presence of catalyst 1-hydroxybenzotriazole were quite unsuccessful, although excellent syntheses were achieved using symmetrical anhydrides.
Fmoc-amino-acid pentafluorophenyl esters were prepared following the procedures of Kisfaludy and Schoen in Synthesis, 1983, 325.
The polydimethylacrylamide resin
was functicnalised with an internal reference norleucine residue and with the acid-labile p-alkoxybenzyl alcohol linkage agent as known per se.
Esterification of the C-terminal Fmoc-glycine residue utilised the preformed symmetric anhydride with dimethylaminopyridine catalysis.
The progress of all subsequent acylation reactions was monitored by sensitive ninhydrin and trinitrobenzene-sulphonic acid colour tests for residual amine and by later amino-acid analysis.
Fmoc-asparagine pentafluorophenyl ester (five-fold escess in dimethylformamide) had reacted completely with deprotected glycyl resin at the time of the first colour test (25 min). The reaction was allowed to continue for a total of 50 min. Sterically hindered Fmoc-isolueucine pentafluororphenyl ester reacted with the resulting asparaginyl-glycyl resin much more sluggishly. Positive colour tests were obtained after 49 min. After 60 min 1-hydroxybenzotriazole catalyst was added. Only very faint colour tests were obtained after an additional 45 min and the reaction was terminated after a total reaction period of three hours. O-t-butyl-Fmoc-tyrosine pentafluorophenyl ester reacted completely within 25 min. This reactivity pattern was confirmed with the succeeding residues. Complete acylation was indicated at the first colour test for t-butyl aspartate-6 (10 min), alanine-4 (13 min), and alanine-3 (5 min). Isoleucine-5 again required catalysis by 1-hydroxybenzotriazole which was added after 43 min and gave complete reaction after an additional 50 min. An anomalous result was obtained after addition of Fmoc-glutamine-2 pentafluorophenyl ester with very occasional resin beads giving positive colour tests for residual amine in a generally colourless bulk background. Some shrinkage of the resin also occurred at this stage. Reaction was complete 35 min after the addition of catalyst. The terminal valine residue was nearly complete after 25 min and was left overnight without catalysis.
The completed decapeptide was cleaved from the resin with 95% trifluoroacetic acid. Residual resin analysis showed cleavage to be 96% complete. No significant peptide was lost from the resin during the course of the synthesis. The isolated yield of crude decapeptide determined by quantitative amino-acid analysis was 91%
(Found: Gly, 1.00; Asp, 1.89; lie, 1.80; Tyr , 0.93; Ala, 1.90; Gin, 0.98; Val, 0.97).
Analytical HPLC of total crude decapeptide on
Aquapore RP-300 is shown in the figure. Reservoir A contained 0.1% aq. trifluoroacetic acid; B contained 90% acetonitrile, 10% A. After 2 min isocratic elution with 5% B, a linear gradient of 5-60% B was developed over 40 min at a flow rate of 1.5 ml/min. On μ-Bondapack C18, reservoir A contained 0.01 M ammonium acetate, pH 4.5; B, 90% acetonitrile, 10% A. After 2 min at 15% B, a linear gradient of 15-35% over 40 min eluted the decapeptide at 11.5 min.
The retention time of the principal peak is identical with that of previous preparations.
From this it can be concluded that Fmoc-amino-acid pentafluorophenyl esters are valuable alternatives to symmetrical anhydrides for solid phase peptide synthesis. Their crystallinity and apparent stability greatly simplify and speed the conduct of solid phase synthesis. Reaction rates are substantially accelerated by catalyst 1-hydroxybenzotriazole and appear likely to be adequate for most sequences. The high purity (> 90%) of the crude reaction product obtained above shows that serious side reactions are not induced by the reagent.

Claims

CLAIMS :
1. A method for a solid phase synthesis of a linear combination of amino acid residues, linked via peptide bonds, starting with an amino acid residue covalently linked to a support and protected by an N-alpha-amino protecting group, comprising the following steps:
(a) removing the N-alpha-amino protecting group to obtain an N-alpha-amino group,
(b) adding an amino acid residue protected by an N-alpha- amino protecting group, via a peptide bond, to the
N-alpha-amino group obtained in step (a) by use of a reactive protected amino acid derivative and, when necessary, a catalyst,
(c) repeating steps (a) and (b) until the said linear combination has been obtained, characterised in that the said reactive protected amino acid derivative has the acyl group used to form the peptide bond activated as a pentafluorophenyl ester.
2. A method according to Claim 1, characterised in that thene is used as catalyst in step (b)
1-hydroxybenzotriazole.
3. A method as claimed in Claim 1 and substantially as hereinbefore described.
EP86900197A 1984-12-11 1985-12-11 Method for a solid phase synthesis of a linear combination of amino acid residues Withdrawn EP0204801A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB08431178A GB2169901A (en) 1984-12-11 1984-12-11 method for a solid phase synthesis of a linear combination of amino acid residues.
GB8431178 1984-12-11

Publications (1)

Publication Number Publication Date
EP0204801A1 true EP0204801A1 (en) 1986-12-17

Family

ID=10570981

Family Applications (1)

Application Number Title Priority Date Filing Date
EP86900197A Withdrawn EP0204801A1 (en) 1984-12-11 1985-12-11 Method for a solid phase synthesis of a linear combination of amino acid residues

Country Status (4)

Country Link
EP (1) EP0204801A1 (en)
AU (1) AU5205486A (en)
GB (1) GB2169901A (en)
WO (1) WO1986003494A1 (en)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4701304A (en) * 1985-04-19 1987-10-20 Applied Protein Technologies, Inc. Apparatus for automated synthesis of peptides
GB2187461A (en) * 1986-02-03 1987-09-09 Medical Res Council Monitoring method for the synthesis of a linear of amino acid residues
US4859736A (en) * 1987-03-30 1989-08-22 Ciba-Geigy Corporation Synthetic polystyrene resin and its use in solid phase peptide synthesis
DE3711866A1 (en) * 1987-04-08 1988-10-27 Hoechst Ag SYNTHESIS OF PEPTIDAMIDES BY SOLID PHASE METHOD USING SAEURELABILE ANCHOR GROUPS
HU202130B (en) * 1988-07-29 1991-02-28 Sp K Byuro Biolog Priborostr Reactor for solid-phase synthesis of biopolymers
AU670108B2 (en) 1992-09-11 1996-07-04 Becton Dickinson & Company Improved antibodies to plasmodium falciparum

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
HU164903B (en) * 1969-04-05 1974-05-28
US4105602A (en) * 1975-02-10 1978-08-08 Armour Pharmaceutical Company Synthesis of peptides with parathyroid hormone activity
US4108846A (en) * 1977-02-01 1978-08-22 Hoffmann-La Roche Inc. Solid phase synthesis with base N alpha-protecting group cleavage
US4427827A (en) * 1982-10-20 1984-01-24 Usv Pharmaceutical Corporation Synthesis of hormone fragments
EP0161468A3 (en) * 1984-05-07 1988-10-26 Pennwalt Corporation Process for the solid phase synthesis of peptides which contain sulfated tyrosine

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8603494A1 *

Also Published As

Publication number Publication date
GB2169901A (en) 1986-07-23
WO1986003494A1 (en) 1986-06-19
AU5205486A (en) 1986-07-01
GB8431178D0 (en) 1985-01-23

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