WO1990004183A1 - Procede de preparation de thiohydantoines et d'analyse de sequences de proteines - Google Patents

Procede de preparation de thiohydantoines et d'analyse de sequences de proteines Download PDF

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Publication number
WO1990004183A1
WO1990004183A1 PCT/AU1989/000433 AU8900433W WO9004183A1 WO 1990004183 A1 WO1990004183 A1 WO 1990004183A1 AU 8900433 W AU8900433 W AU 8900433W WO 9004183 A1 WO9004183 A1 WO 9004183A1
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WO
WIPO (PCT)
Prior art keywords
peptide
amino acid
protein
thiohydantoin
terminal
Prior art date
Application number
PCT/AU1989/000433
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English (en)
Inventor
Adam Sinclair Inglis
Franca Casagranda
John Francis Kelly Wilshire
Original Assignee
Commonwealth Scientific And Industrial Research Organisation
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.)
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Publication date
Application filed by Commonwealth Scientific And Industrial Research Organisation filed Critical Commonwealth Scientific And Industrial Research Organisation
Publication of WO1990004183A1 publication Critical patent/WO1990004183A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D233/86—Oxygen and sulfur atoms, e.g. thiohydantoin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6818—Sequencing of polypeptides
    • G01N33/6821—Sequencing of polypeptides involving C-terminal degradation

Definitions

  • This invention relates to a method for the preparation of amino acid thiohydantoins and for the determination of the amino acid sequence of a protein or peptide from the C-terminal end thereof.
  • Such decomposition not only lowers the yield of amino acid thiohydantoin (IV) produced by the cleavage reaction, but also causes the formation of by-products which are likely to interfere with the unequivocal identification of the thiohydantoin (IV) released in each cycle, a step which is crucial for the success of the sequencing method.
  • this method also, there is provided a process for the preparation of amino acid thiohydantoins utilising this Schlack-Kumpf procedure.
  • a method for the C-terminal degradation of a protein or peptide which comprises the steps of:
  • the shortened protein or peptide produced in the cleavage reaction is suitable for a further degradation cycle.
  • the cleavage reaction has been attempted using either acids of varying strengths or mild organic bases such as acetohydroxamic acid or triethylamine. These approaches were apparently based on the belief that mild conditions were mandatory, particularly when basic reagents were being used. However, it has now been found that cleavage with a strong inorganic base at a suitable concentration (such as aqueous 0.5M potassium or other alkali metal hydroxide) in the presence of a water-miscible organic solvent (such as methanol), and optionally an antioxidant (such as dithioerythritol or dithiothreitol), gives excellent results provided that the reaction time is short.
  • a strong inorganic base at a suitable concentration (such as aqueous 0.5M potassium or other alkali metal hydroxide) in the presence of a water-miscible organic solvent (such as methanol), and optionally an antioxidant (such as dithioerythritol or dithiothreitol), gives
  • the C-terminal amino acid carboxyl group is activated prior to said coupling reaction.
  • Activation with acetic anhydride and acetic acid is preferred, however other known activation procedures such as that described by Kubo et.al. (1971) and variations thereof, may also be used for the activation step.
  • an aqueous solution of the strong inorganic base is used, such as a solution having a concentration of >0.2M, and preferred solutions comprise alkali metal hydroxides such as sodium or potassium hydroxide of >0.2M in aqueous methanol.
  • Preferred cleavage conditions in accordance with the present invention include the use of 0.5 potassium hydroxide in 33% aqueous methanol in the presence of dithioerythritol as antioxidant, however it will be appreciated that other strong inorganic base/organic solvent/antioxidant combinations may be used without departing from the broad ambit of the invention.
  • a method for the determination of the C- terminal amino acid of a protein or peptide which comprises the steps of C-terminal degradation of the protein or peptide by the method broadly described above, followed by identification of the C-terminal amino acid thiohydantoin formed by the cleavage reaction.
  • the method of this aspect of the invention enables the successful sequencing of the original protein or peptide from the C-terminal end by subjecting the shortened protein or peptide to one or more further degradation cycles in accordance with this invention, followed by identification of the (or each) C-terminal amino acid thiohydantoin as it is formed by the cleavage reaction. While this aspect of the invention is exemplified in the present specification with a procedure suitable for manual sequencing, it will be apparent to those skilled in the art that the simple operations involved can readily be automated using currently available technology, for example that used for automatic N-terminal amino acid sequencing, with appropriate programming of the controlling systems.
  • the determination of the amino acid sequence of the protein or peptide is carried out with the protein or peptide covalently attached by its N-terminal end to a solid support.
  • the solid support may, for example, consist of activated glass, glassfibre or other appropriate polymeric material.
  • the proteins or peptides may also be sequenced after they have been strongly adsorbed (i.e. non-covalently bound) to the solid support.
  • appropriate reaction conditions will need to be employed, for example reagents that could cause dissolution of the sample should just wet out the support, then be dried off before introducing further solvents or other reagents.
  • R x represents the residue of an amino acid, which comprises the step of coupling the carboxyl group of a compound of the general formula (V):
  • R x is as defined above and R 2 represents H or R 3 -CO- in which R 3 represents a lower alkyl (for example, methyl) group, an amino acid residue or a peptide residue, with thiocyanic acid or a thiocyanate to form a substituted thiohydantoin derivative of the general formula (Ila):
  • R x may represent the residue of any of the 20 common amino acids, namely alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine ( ), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) or valine (V).
  • the method of this aspect of the invention is of particular utility in that it enables the preparation of several amino acid thiohydantoins which previously could not be prepared readily, for example the thiohydantoins of serine, arginine, threonine and proline.
  • the amino acid thiohydantoins can be readily prepared in accordance with the present invention by a microscale in situ procedure, for example from the corresponding acyl amino acid such as the N-acetyl amino acid or the corresponding propionyl or benzoyl amino acid.
  • Acetylserine (100 nmoles) was added to a mixture of acetic anhydride/acetic acid (4:1, 120 ⁇ l) in a screw cap Eppendorf vial and the resultant mixture allowed to react for 5 minutes at 80°C.
  • Thiocyanic acid (HSCN) ( ⁇ 1.8M, 30 ⁇ l) was then added and the mixture heated at 80°C for 30 minutes and then dried under vacuum.
  • the residue was treated at room temperature with base (0.2M KOH in 33% methanol/water) containing dithioerythrit ⁇ l, the mixture stirred, and the cleavage reagent removed immediately and analysed for the corresponding thiohydantoin by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • HPLC provides a rapid and sensitive method for the identification and quantitation of amino acid thiohydantoins, and the following Table sets out retention times for a number of the products prepared in accordance with this Example (elution from a HP 1090 HPLC using a Water Pico Tag column of a flow rate of 1.0 ml/min and a column temperature of 40°C).
  • Figure 1 shows a typical HPLC profile for the thiohydantoins of the amino acids: aspartic acid (AT-asp), asparagine (AT-asn), glutamine (AT-gln), glutamic acid (AT-glu), glycine (AT-gly), alanine (AT-ala), lysine (AT-lys), serine (AT-ser), threonine (AT-thr), tyrosine (AT-tyr), valine (AT-val), proline (AT-pro), methionine (AT-met), isoleucine (AT-ile), leucine (AT-leu), phenylalanine (AT-phe) and tryptophan (AT-trp).
  • aspartic acid AT-asp
  • asparagine AT-asn
  • glutamine AT-gln
  • glutamic acid AT-glu
  • glycine AT-gly
  • alanine AT-ala
  • Peptides were covalently bound to activated glass beads (usually in 60-80% yield) by the method described by euth et.al. (1982).
  • Beads (1.5-4.0mg) containing approximately 20 nmol peptide were subjected to a modified sequencing procedure which is based on that of Meuth et.al., (1982), i.e. using activation with acetic acid and acetic anhydride and with thiocyanic acid as reagent.
  • Cleavage was achieved with 0.5M KOH in 33% aqueous methanol in the presence of dithioerythritol, and the cleavage solution analysed by HPLC for thiohydantoin content (see Experimental Protocol).

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Physics & Mathematics (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Un procédé de préparation de thiohydantoïnes d'acides aminés et de séquençage d'une protéine ou d'un peptide consiste en la dégradation de la terminaison c d'une protéine ou d'un peptide par i) accouplement du groupe carboxyle à terminaison c avec de l'acide thiocyanique ou un thiocyanate afin de former un dérivé de thiohydrantoïne à substitution et ii) clivage du dérivé de thiohydantoïne à substitution avec une base inorganique forte en présence d'un solvant organique miscible à l'eau, afin d'obtenir une protéine ou un peptide raccourci ainsi qu'une thiohydantoïne d'acide aminé à terminaison c que l'on peut identifier à des fins de séquençage.
PCT/AU1989/000433 1988-10-07 1989-10-06 Procede de preparation de thiohydantoines et d'analyse de sequences de proteines WO1990004183A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
AUPJ084088 1988-10-07
AUPJ0840 1988-10-07
AUPJ093988 1988-10-12
AUPJ0939 1988-10-12

Publications (1)

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WO1990004183A1 true WO1990004183A1 (fr) 1990-04-19

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996007916A1 (fr) * 1994-09-08 1996-03-14 The Perkin-Elmer Corporation Activation par anhydride acetique destinee au sequençage c-terminal d'une proteine
JP2006521377A (ja) * 2003-03-27 2006-09-21 ランケナー インスティテュート フォー メディカル リサーチ 新型ｉｄｏ阻害剤とその使用方法
AU2004315596B2 (en) * 2003-08-29 2011-11-24 President And Fellows Of Harvard College Inhibitors of cellular necrosis
US9499521B2 (en) 2014-12-11 2016-11-22 President And Fellows Of Harvard College Inhibitors of cellular necrosis and related methods
US9586880B2 (en) 2008-12-23 2017-03-07 President And Fellows Of Harvard College Small molecule inhibitors of necroptosis
US9725452B2 (en) 2013-03-15 2017-08-08 Presidents And Fellows Of Harvard College Substituted indoles and pyrroles as RIP kinase inhibitors

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3959307A (en) * 1974-02-15 1976-05-25 Wittmann Brigitte Method to determine automatically the sequence of amino acids
EP0012381A1 (fr) * 1978-12-16 1980-06-25 Bayer Ag Procédé de préparation de composés contenant des groupes hydantoine

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3959307A (en) * 1974-02-15 1976-05-25 Wittmann Brigitte Method to determine automatically the sequence of amino acids
EP0012381A1 (fr) * 1978-12-16 1980-06-25 Bayer Ag Procédé de préparation de composés contenant des groupes hydantoine

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HOPPE-SEYLER'S ZEITSCHRIFT FUR PHYSIOLOGISCHE CHEMIE, Volume 154, 5 February 1926 (05.02.26), P. SCHLACK and W. KUMPF: "Uber eine neue Methode zur Ermittlung der Konstitution von Peptiden", pp 125-170; whole document, especially p 133, p 139 and p 142-157. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996007916A1 (fr) * 1994-09-08 1996-03-14 The Perkin-Elmer Corporation Activation par anhydride acetique destinee au sequençage c-terminal d'une proteine
JP2006521377A (ja) * 2003-03-27 2006-09-21 ランケナー インスティテュート フォー メディカル リサーチ 新型ｉｄｏ阻害剤とその使用方法
AU2004315596B2 (en) * 2003-08-29 2011-11-24 President And Fellows Of Harvard College Inhibitors of cellular necrosis
US8143300B2 (en) 2003-08-29 2012-03-27 President And Fellows Of Harvard College Inhibitors of cellular necrosis
US8741942B2 (en) 2003-08-29 2014-06-03 President And Fellows Of Harvard College Inhibitors of cellular necrosis
US9586880B2 (en) 2008-12-23 2017-03-07 President And Fellows Of Harvard College Small molecule inhibitors of necroptosis
US9725452B2 (en) 2013-03-15 2017-08-08 Presidents And Fellows Of Harvard College Substituted indoles and pyrroles as RIP kinase inhibitors
US9499521B2 (en) 2014-12-11 2016-11-22 President And Fellows Of Harvard College Inhibitors of cellular necrosis and related methods
US9944628B2 (en) 2014-12-11 2018-04-17 President And Fellows Of Harvard College Inhibitors of cellular necrosis and related methods
US10508102B2 (en) 2014-12-11 2019-12-17 President And Fellows Of Harvard College Inhibitors of cellular necrosis and related methods

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