GB2169901A - 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. Download PDF

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Publication number
GB2169901A
GB2169901A GB08431178A GB8431178A GB2169901A GB 2169901 A GB2169901 A GB 2169901A GB 08431178 A GB08431178 A GB 08431178A GB 8431178 A GB8431178 A GB 8431178A GB 2169901 A GB2169901 A GB 2169901A
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United Kingdom
Prior art keywords
amino acid
amino
alpha
protected
group
Prior art date
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GB08431178A
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GB8431178D0 (en
Inventor
Robert Sheppard
Eric Atherton
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Pharmacia and Upjohn Cambridge Ltd
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LKB Biochrom Ltd
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Publication date
Application filed by LKB Biochrom Ltd filed Critical LKB Biochrom Ltd
Priority to GB08431178A priority Critical patent/GB2169901A/en
Publication of GB8431178D0 publication Critical patent/GB8431178D0/en
Priority to PCT/GB1985/000573 priority patent/WO1986003494A1/en
Priority to AU52054/86A priority patent/AU5205486A/en
Priority to EP86900197A priority patent/EP0204801A1/en
Publication of GB2169901A publication Critical patent/GB2169901A/en
Withdrawn legal-status Critical Current

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    • 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

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  • 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

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 liner combination has been obtained. According to the invention the said reactive protected amino acid derivative has the acyl group used to form the peptide bond activated as a pentafluorophenyl ester.

Description

SPECIFICATION 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 (s) 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 acitvated 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 pnitrophenyl 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 catalyst. 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 DRA WING 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.lle.Asp.Tyr.lle.Asn.Gly.OH 1 10 was selected. Earlier attempts to assemble this sequence using tnitrophenyl 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 functionalised with an internal reference norleucine residue and with the acid-labile palkoxybenzyl 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 trinitrobenzenesulphonic 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-isoluecine pentafluornrphenyl ester reacted with the resulting asparaginyl-glycyl resin much mote 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; lle, 1.80; Tyr, 0.93; Ala, 1.90; Gln, 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 t-Bondapack C,8, 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 (3)

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-aipha-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) untii the said linear combinatiion 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 there is used as catalyst in step (b) 1 -hydroxybenzotriazole.
3. A method as claimed in Claim 1 and substantially as hereinbefore described.
GB08431178A 1984-12-11 1984-12-11 method for a solid phase synthesis of a linear combination of amino acid residues. Withdrawn GB2169901A (en)

Priority Applications (4)

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.
PCT/GB1985/000573 WO1986003494A1 (en) 1984-12-11 1985-12-11 Method for a solid phase synthesis of a linear combination of amino acid residues
AU52054/86A AU5205486A (en) 1984-12-11 1985-12-11 Method for a solid phase synthesis of a linear combination of amino acid residues
EP86900197A EP0204801A1 (en) 1984-12-11 1985-12-11 Method for a solid phase synthesis of a linear combination of amino acid residues

Applications Claiming Priority (1)

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.

Publications (2)

Publication Number Publication Date
GB8431178D0 GB8431178D0 (en) 1985-01-23
GB2169901A true GB2169901A (en) 1986-07-23

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GB08431178A Withdrawn GB2169901A (en) 1984-12-11 1984-12-11 method for a solid phase synthesis of a linear combination of amino acid residues.

Country Status (4)

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

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0287882A1 (en) * 1987-04-08 1988-10-26 Hoechst Aktiengesellschaft Synthesis of peptidamides by the solid phase method using anchor groups unstable as to acids

Families Citing this family (5)

* 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
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
CH551948A (en) * 1969-04-05 1974-07-31 Hoechst Ag PROCESS FOR THE MANUFACTURING OF PEPTIDES.
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

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0287882A1 (en) * 1987-04-08 1988-10-26 Hoechst Aktiengesellschaft Synthesis of peptidamides by the solid phase method using anchor groups unstable as to acids

Also Published As

Publication number Publication date
EP0204801A1 (en) 1986-12-17
GB8431178D0 (en) 1985-01-23
AU5205486A (en) 1986-07-01
WO1986003494A1 (en) 1986-06-19

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