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WO2003031984A2 - Procede d'analyse sequentielle de polypeptides - Google Patents

Procede d'analyse sequentielle de polypeptides Download PDF

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Publication number
WO2003031984A2
WO2003031984A2 PCT/EP2002/011257 EP0211257W WO03031984A2 WO 2003031984 A2 WO2003031984 A2 WO 2003031984A2 EP 0211257 W EP0211257 W EP 0211257W WO 03031984 A2 WO03031984 A2 WO 03031984A2
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
halogen
detection
phenyl isothiocyanate
reaction
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.)
Ceased
Application number
PCT/EP2002/011257
Other languages
German (de)
English (en)
Other versions
WO2003031984A3 (fr
Inventor
Christian Wurzel
Ralf KRÜGER
Barbara Zu Lynar
Barbara Popke
Brigitte Wittmann Liebold
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.)
CONSEQUENCE GmbH
Original Assignee
CONSEQUENCE GmbH
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 CONSEQUENCE GmbH filed Critical CONSEQUENCE GmbH
Priority to AU2002346975A priority Critical patent/AU2002346975A1/en
Publication of WO2003031984A2 publication Critical patent/WO2003031984A2/fr
Publication of WO2003031984A3 publication Critical patent/WO2003031984A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • G01N33/6824—Sequencing of polypeptides involving N-terminal degradation, e.g. Edman degradation

Definitions

  • the conversion rates of the individual sub-steps are of crucial importance for the analysis and identification of the polypeptide.
  • the highest possible turnover rate preferably of over 90% and preferably of over 95%, should be achieved in order to be able to clearly identify a large number of degradation stages.
  • the incomplete degradation means that in the subsequent degradation steps, the amino acid that is now split off is overlaid by the background of the amino acids from the previous steps, the so-called overlap, so that the sequence can no longer be clearly identified.
  • protein sequencers are currently being used, such as, for example, the “Procise cLc” device from Applied Biosystems, Foster City, USA, or the “Knauer 910” device from Knauermaschinemaschinemaschine, Berlin, used. (Technical descriptions of the devices can be found in the respective operating instructions [12, 13]).
  • the present invention has for its object to provide a compound which can be used instead of the phenyl isothiocyanate used in the prior art in the context of Edman degradation and overcomes the disadvantages described in the prior art.
  • Another object on which the present invention is based is that a reagent is provided which replaces the phenyl isothiocyanate used in the prior art in the context of Edman degradation and is intended to further reduce the detection limit for the overall process compared to the prior art.
  • At least one of the radicals R] to R 5 is a halogen and / or contains a halogen
  • the object is achieved in a second aspect by the use of 3,5-bis (trifluoromethyl) phenyl isothiocyanate for the derivatization and / or for the detection of primary and / or secondary amines.
  • the object is achieved according to the invention by using a halogen-substituted phenyl isothiocyanate of the formula
  • the derivatized amine is detected and / or identified as a further step, as is provided in the context of the method according to the invention.
  • Derivatized amine as used herein is the primary or secondary amine which is reacted with the halogen substituted phenyl isothiocyanate as described herein, or the reaction product thereof.
  • anilinothiazolinone amino acid is extracted from the reaction mixture and converted into a phenylhydantoin amino acid.
  • the amino acid derivative is detected by means of an ECD detector, UV detection, retention time, mass information and / or retention time and mass information, or by means of negative chemical ionization.
  • the 3,5-bis (trifluoromethyl) phenyl isothiocyanate according to the invention has a high proportion of fluorine atoms in the molecule and this by suitable coupling to detectors such as ECD (electron capture detection) or by negative chemical ionization (NCI) or chemical ionization (CI) can be specifically detected.
  • detectors such as ECD (electron capture detection) or by negative chemical ionization (NCI) or chemical ionization (CI)
  • HPLC a separation and characterization of the amino acid derivatives obtained by means of gas chromatography with reference to standardized could Retention times take place.
  • the same also applies in principle to those of the phenysothiocyanates disclosed or described herein which have fewer fluorine atoms but still have the above physico-chemical properties.
  • the halogen-substituted phenyl isothiocyanate disclosed herein can react with a primary amine of a peptide, in particular from the terminal amino acid of the peptide and, after cleavage of the said terminal amino acid, can be detected selectively. It is within the scope of the present invention that the reaction with the halogen-substituted phenyl isothiocyanate described herein is not limited to the reaction with peptides. Rather, a reaction generally takes place with primary or secondary amines. In the broadest sense, this reaction is therefore a derivatization reaction of primary and / or secondary amines.
  • the ratio of signal to noise is increased many times over, so that a much more sensitive analysis of the target substances, ie of primary and secondary amines, is made possible.
  • the detection of primary and secondary amines is to be carried out down to the attomole range or in even smaller amounts, and thus ultimately possible to identify the primary or secondary amine containing compound.
  • the halogen-substituted phenyhsothiocyanates described herein primary and secondary amines are now accessible for direct gas chromatographic measurement.
  • the sample to be analyzed can be any sample. Accordingly, the sample can come from the technical or analytical area.
  • the analytical area includes in particular the areas of biology, biochemistry and medicine as well as forensics as well as that of process technology.
  • the samples are preferably of biological origin and in particular blood, urine, stool, lymph fluid or cerebrospinal fluid.
  • the sample can also be an air sample, a soil sample, or a water sample.
  • the sample can also be a solid substance, such as hair or a solid sample in general.
  • the amine contained in the sample in particular primary or secondary amine, or presumably contained therein primary and / or secondary amine, can preferably be such an amine as is used in starting materials for the production of industrial products, such as solvents, for example.
  • amines as they can be detected or detected according to the invention also represent constituents or reactive groups of pharmaceuticals, including drugs.
  • Amines as they can be detected or detected according to the invention also represent, for example, various metabolic products. Of particular interest in In connection with the present invention are primary and secondary amines which are biogenic amines.
  • biogenic amines can be detected particularly preferably or the samples containing them can be analyzed using the method according to the invention.
  • Tyramine, dopamine, noradrenaline and adrenaline which are neurotransmitters, hormones or tissue hormones and which are the decarboxylation product of the amino acid tyrosine.
  • Tryptamine, serotonin and melatonin which are neurotransmitters, hormones and tissue hormones and which are the decarboxylation product of tryptophan.
  • Aminoacteon which arises from L-2-aminoacetoacetic acid and is a precursor for vitamin B12.
  • 5- Amino-4-oxovaleric acid which is formed from succinylglycine and is a precursor for porphyrins.
  • Cadaverine which is made from L-lysine and is a precursor for alkaloids.
  • Dopamine which is derived from L-dopa and is a neurotransmitter, precursor for the catecholamines, L-noradrenaline and L-adrenaline, and is also a proto-alkaloid.
  • Serotonin which arises from 5-hydroxy-L-tryptophan and is a neurotransmitter and precursor of the hormone melatonin. Tryptamine, which arises from L-tryptophan and causes contraction of the smooth muscles and has a growth-promoting effect on plants. Tyramine, which arises from L-tyrosine and causes the contraction of the smooth muscles.
  • Example 1 Determining the clutch speed of FM-PITC:
  • HPLC system was used for detection purposes:

Landscapes

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

Abstract

L'invention concerne l'utilisation d'un phénylisothiocyanate substitué halogène de formule (I): dans laquelle R1 à R5 sont sélectionnés indépendamment l'un de l'autre dans le groupe comprenant hydrogène, halogène, alkyle et halogénure d'alcoyle, halogène étant sélectionné dans le groupe constitué par fluor, chlore, brome et iode, et alkyle étant sélectionné dans le groupe constitué par méthyle, éthyle, n-propyle et isopropyle, dans la mesure où au moins un des restes R1 à R5 est et/ou contient un halogène. Cette utilisation sert à créer des dérivés et/ou à déceler la présence d'amines primaires et/ou secondaires.
PCT/EP2002/011257 2001-10-08 2002-10-08 Procede d'analyse sequentielle de polypeptides Ceased WO2003031984A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002346975A AU2002346975A1 (en) 2001-10-08 2002-10-08 Method for analysing the sequence of polypeptides

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2001149568 DE10149568A1 (de) 2001-10-08 2001-10-08 Verfahren zur Sequenzanalyse von Polypeptiden
DE10149568.4 2001-10-08

Publications (2)

Publication Number Publication Date
WO2003031984A2 true WO2003031984A2 (fr) 2003-04-17
WO2003031984A3 WO2003031984A3 (fr) 2003-07-31

Family

ID=7701781

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2002/011257 Ceased WO2003031984A2 (fr) 2001-10-08 2002-10-08 Procede d'analyse sequentielle de polypeptides

Country Status (3)

Country Link
AU (1) AU2002346975A1 (fr)
DE (1) DE10149568A1 (fr)
WO (1) WO2003031984A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009145286A1 (fr) 2008-05-30 2009-12-03 武田薬品工業株式会社 Composé hétérocyclique
KR20170067791A (ko) 2014-10-24 2017-06-16 다케다 야쿠힌 고교 가부시키가이샤 헤테로시클릭 화합물

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR1528249A (fr) * 1966-06-22 1968-06-07 Ciba Geigy Agents de destruction des acarides et des mollusques
DE2608488A1 (de) * 1975-03-05 1976-09-16 Ciba Geigy Ag Iso(thio)harnstoffe
IT1237964B (it) * 1990-02-01 1993-06-19 Minnesota Mining & Mfg Elementi fotografici agli alogenuri d'argento sensibili all'infrarosso
US5534440A (en) * 1991-02-22 1996-07-09 Biomedical Research Centre Limited Compounds and methods for sequencing amino acids
DE69328080T2 (de) * 1992-07-27 2000-12-14 Kurabo Industries Ltd., Kurashiki Verfahren zum hochempfindlichen nachweis von aminosäurederivaten

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009145286A1 (fr) 2008-05-30 2009-12-03 武田薬品工業株式会社 Composé hétérocyclique
US8338622B2 (en) 2008-05-30 2012-12-25 Takeda Pharmaceutical Company Limited Heterocyclic compound
KR20170067791A (ko) 2014-10-24 2017-06-16 다케다 야쿠힌 고교 가부시키가이샤 헤테로시클릭 화합물
EP3210973A4 (fr) * 2014-10-24 2018-04-25 Takeda Pharmaceutical Company Limited Composé hétérocyclique
US10214498B2 (en) 2014-10-24 2019-02-26 Takeda Pharmaceutical Company Limited Heterocyclic compound
JP2020007347A (ja) * 2014-10-24 2020-01-16 武田薬品工業株式会社 複素環化合物
US10544111B2 (en) 2014-10-24 2020-01-28 Takeda Pharmaceutical Company Limited Heterocyclic compound
US10975043B2 (en) 2014-10-24 2021-04-13 Takeda Pharmaceutical Company Limited Heterocyclic compound
EP3848353A1 (fr) 2014-10-24 2021-07-14 Takeda Pharmaceutical Company Limited Médicaments pour le traitement de maladies ophtalmiques
JP6995094B2 (ja) 2014-10-24 2022-02-21 武田薬品工業株式会社 複素環化合物
KR102490156B1 (ko) 2014-10-24 2023-01-18 다케다 야쿠힌 고교 가부시키가이샤 헤테로시클릭 화합물

Also Published As

Publication number Publication date
WO2003031984A3 (fr) 2003-07-31
AU2002346975A1 (en) 2003-04-22
DE10149568A1 (de) 2003-04-24

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