DE202008013314U1 - lanthanide chelates - Google Patents

lanthanide chelates Download PDF

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DE202008013314U1
DE202008013314U1 DE202008013314U DE202008013314U DE202008013314U1 DE 202008013314 U1 DE202008013314 U1 DE 202008013314U1 DE 202008013314 U DE202008013314 U DE 202008013314U DE 202008013314 U DE202008013314 U DE 202008013314U DE 202008013314 U1 DE202008013314 U1 DE 202008013314U1
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chelate
bioactive substance
amino
lanthanide
terbium
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/36—Radicals substituted by singly-bound nitrogen atoms

Abstract

Chelat der Formel (I), das für Markierung von bioaktiver Substanz in Lösung geeignet ist,

Figure 00000001
dadurch gekennzeichnet, dass
– A eine reaktive Gruppe für Bindung an die bioaktive Substanz darstellt, gewählt aus einer Gruppe bestehend aus Isothiocyanat, Bromoacetamido, Jodoacetamido, Maleimid, 4,6-Dichlor-1,3,5-triazinyl-2-amino, Pyridyldithiol, Thioester, Aminooxy, Azid, Alkyn, Hydrazid, Amino, Carboxylsäure, Ester oder Säurehalid.Chelate of formula (I) suitable for labeling bioactive substance in solution,
Figure 00000001
characterized in that
A represents a reactive group for binding to the bioactive substance selected from a group consisting of isothiocyanate, bromoacetamido, iodoacetamido, maleimide, 4,6-dichloro-1,3,5-triazinyl-2-amino, pyridyldithiol, thioester, aminooxy , Azide, alkyne, hydrazide, amino, carboxylic acid, ester or acid halide.

Description

GEBIET DER ERFINDUNGFIELD OF THE INVENTION

Vorliegende Erfindung betrifft neue Lanthanidchelate, die für die Markierung bioaktiver Substanzen in Lösungsphase geeignet sind.This The invention relates to novel lanthanide chelates useful for labeling bioactive substances in solution phase are suitable.

HINTERGRUND DER ERFINDUNGBACKGROUND OF THE INVENTION

Die Publikationen und das sonstige Material, worauf hier verwiesen wird, werden zur Veranschaulichung des Hintergrundes der Erfindung verwendet, und insbesondere dienen die Fälle, auf die hier Bezug genommen wird, zur Bereitstellung weiterer Details bezüglich der Technik.The Publications and other material referred to here are used to illustrate the background of the invention, and in particular, the cases referred to herein serve will provide further details regarding the Technology.

Von einem lumineszierenden Lanthanidchelat werden häufig verschiedene physikalische und chemische Sondereigenschaften erwartet. Das Molekül sollte sowohl im Grund- als auch im angeregten Zustand thermodynamisch, kinetisch und photochemisch stabil sein. Die Energieübertragung vom Liganden auf das Zentralion sollte schnell und effektiv sein. Das Chelat sollte oft auch wasserlöslich sein. Das Chelat sollte seine positiven Eigenschaften auch noch nach seiner Kopplung an ein bioaktives Molekül beibehalten. Ein gutes Markierungsreagens verändert nicht die Eigenschaften der von ihm gebundenen bioaktiven Substanz.From A luminescent lanthanide chelate often becomes different expected physical and chemical special properties. The molecule should be thermodynamically, both in the ground and in the excited state, be kinetically and photochemically stable. The energy transfer from the ligand to the central ion should be fast and effective. The chelate should often be water-soluble. The chelate should its positive properties even after its coupling retained on a bioactive molecule. A good labeling reagent does not change the properties of his bound bioactive substance.

Bei zahlreichen Anwendungen ist eine kovalente Bindung des Markierungsmoleküls an das bioaktive Molekül notwendig. Bei der Markierung der Zielmoleküle, wie Proteine, Oligonukleotide und Oligopeptide, werden oft Isothiocyanat-, N-Hydroxysuccinimid-, Haloacetamid-, Dichlortriazin- oder Maleimidderivate eines Chelats verwendet. Da dann die Markierungsreaktion in Anwesenheit eines Überschusses der aktivierten Markierung durchgeführt wird, können arbeitsintensive Reinigungsverfahren nicht vermieden werden. Obwohl synthetische bioaktive Moleküle auf der Oberfläche des festen Trägers markiert werden können, ist die Markierung in Lösung bei großen bioaktiven Substanzen, wie Proteinen, unvermeidbar. Dann sollte die Markierungsreaktion möglichst effektiv und ortsspezifisch sein.at numerous applications is a covalent binding of the tag molecule necessary to the bioactive molecule. At the mark the target molecules, such as proteins, oligonucleotides and oligopeptides, often isothiocyanate, N-hydroxysuccinimide, haloacetamide, Dichlortriazin or maleimide derivatives of a chelate used. There then the labeling reaction in the presence of an excess the activated mark can be performed Labor-intensive cleaning procedures can not be avoided. Even though synthetic bioactive molecules on the surface of the solid support is the label in solution for large bioactive Substances, such as proteins, unavoidable. Then the labeling reaction should be be as effective and site specific as possible.

Es ist beobachtet worden, dass die Elektronen abgebenden Gruppen eines aromatischen Ringes die lichtphysikalischen Eigenschaften bestimmter, von 4-Phenyl-2,6-bis[N,N-di(carboxyalkyl)aminoalkyl]pyridinen abgeleiteter Lanthanid(III)chelaten verbessern ( US 4 761 481 ). Später ist beobachtet worden, dass oben genannte Eigenschaften durch Chelatisieren mehrerer aromatischer Gruppen an ein Lanthanidion verbessert werden können (US Patentanmeldung 11/004 061). Dies steigert ebenfalls die Stabilität der Chelatstruktur.It has been observed that the electron-donating groups of an aromatic ring improve the light-physical properties of certain lanthanide (III) chelates derived from 4-phenyl-2,6-bis [N, N-di (carboxyalkyl) aminoalkyl] pyridines ( US 4,761,481 ). Later, it has been observed that the above properties can be improved by chelating a plurality of aromatic groups to a lanthanide ion (US Patent Application 11 / 004,061). This also increases the stability of the chelate structure.

DARSTELLUNG DER ERFINDUNGPRESENTATION OF THE INVENTION

Hauptsächliches Ziel vorliegender Erfindung ist es, aus 4-Phenylpyridin abgeleitete Chelate zur Verwendung bereit zu stellen, welche die Markierung bioaktiver Substanzen in Lösungsphase ermöglichen. Die Wasserlöslichkeit hat durch Kopplung von Carboxylsäure-, Phosphorsäure- oder Sulfonsäuregruppen an die Chromophoren verbessert werden können. Die so erhaltenen Biokonjugate eignen sich besonders gut zur Bestimmung basierend auf zeitlich getrennter Fluoreszenz.Cardinal The aim of the present invention is derived from 4-phenylpyridine To provide chelates for use, which is the label allow bioactive substances in solution phase. The water solubility has by coupling of carboxylic acid, Phosphoric or sulfonic acid groups to the chromophores can be improved. The bioconjugates thus obtained are particularly good for determining based on time separate fluorescence.

Die charakteristischen Merkmale der Erfindung gehen aus Schutzanspruch 1 hervor.The Characteristic features of the invention are based on protection claim 1 forth.

Vorliegende Erfindung betrifft also ein Chelat, das für die Markierung einer bioaktiven Substanz in Lösungsphase geeignet ist, und das die Strukturformel (I) aufweist

Figure 00020001
und worin

  • – Ln ein Lanthanid ist,
  • – A eine reaktive Gruppe für Bindung an eine bioaktive Substanz darstellt, gewählt aus einer Gruppe bestehend aus Isothiocyanat, Bromoacetamido, Jodoacetamido, Maleimid, 4,6-Dichlor-1,3,5-triazinyl-2-amino, Aminooxy, Thioester, Pyridyldithiol, Azid, Alkyn, Hydrazid, Amino, Carboxylsäure, Ester oder Säurehalid.
The present invention thus relates to a chelate which is suitable for the labeling of a bioactive substance in solution phase, and which has the structural formula (I)
Figure 00020001
and in which
  • - Ln is a lanthanide,
  • A represents a reactive group for binding to a bioactive substance selected from a group consisting of isothiocyanate, bromoacetamido, iodoacetamido, maleimide, 4,6-dichloro-1,3,5-triazinyl-2-amino, aminooxy, thioester, pyridyldithiol , Azide, alkyne, hydrazide, amino, carboxylic acid, ester or acid halide.

DETAILLIERTE BESCHREIBUNG DER ERFINDUNGDETAILED DESCRIPTION OF THE INVENTION

Das Lanthanid Ln ist vorzugsweise Terbium, Dysprosium, Europium oder Samarium. Besonders vorteilhafte Lanthanide sind Terbium und Dysprosium.The Lanthanide Ln is preferably terbium, dysprosium, europium or Samarium. Particularly advantageous lanthanides are terbium and dysprosium.

Die reaktive Gruppe A ist ausgehend von der Etylgruppe an die orto-, para- oder meta-Stellung des Phenylringes angeschlossen.The reactive group A is derived from the etyl group to the ortho, para or meta position of the phenyl ring connected.

Die Alkoxygruppen sind ausgehend von der Pyridingruppe an die orto-, para- oder meta-Stellung des Phenylringes angeschlossen.The Alkoxy groups are derived from the pyridine group to the ortho, para or meta position of the phenyl ring connected.

Die zu markierende bioaktive Substanz ist beispielsweise ein Oligopeptid, Oligonukleotid, Polypeptid, Polynukleotid, Protein, Oligosaccharid, Polysaccharid, Phospholipid, PNA, LNA, Antikörper, Hapten, Arzneimittel, Molekül, das an einen Rezeptor bindet, oder Lektin.The bioactive substance to be labeled is, for example, an oligopeptide, Oligonucleotide, polypeptide, polynucleotide, protein, oligosaccharide, Polysaccharide, phospholipid, PNA, LNA, antibodies, hapten, Drug, molecule that binds to a receptor, or Lectin.

Die Erfindung wird mittels des folgenden, nicht einschränkenden experimentellen Teils veranschaulicht.The Invention is by means of the following, non-limiting experimental part illustrated.

EXPERIMENTELLER TEILEXPERIMENTAL PART

Die Erfindung wird mittels folgender Beispiele näher erläutert.The Invention will be explained in more detail by means of the following examples.

BeispieleExamples

Beispiel 1. Herstellung von Terbium(III)chelatExample 1. Preparation of terbium (III) chelate

Verbindung 1 wurde mit dem in der Publikation [ US 5 055 578 ] beschriebenen Verfahren hergestellt, jedoch mit 2,6-Dimethoxy-4-hydroxybenzaldehyd als Ausgangssubstanz. Die Hydroxygruppe der Verbindung 1 wurde mit tert-Butylester der Bromessigsäure in Acetonitril in Anwesenheit von Caliumcarbonat alkylisiert.Compound 1 was compared with that in publication [ US 5 055 578 ], but with 2,6-dimethoxy-4-hydroxybenzaldehyde as the starting material. The hydroxy group of compound 1 was alkylated with tert-butyl ester of bromoacetic acid in acetonitrile in the presence of potassium carbonate.

Das Verfahren ist in der Publikation [ US 6 080 838 ] beschrieben. Diester 2 wurde mit Natriumborohydrid zu Dimethanol 3 reduziert, wie in der Publikation [ Acta Chem. Scand. B, 1988, 373 ] beschrieben. Bromisierung mit Phosphortribromid in trockenem DMF ergab Dibromid 4. Das Verfahren ist in der Publikation [ Bioconjugate Chem. 2005, 16, 700 ] beschrieben.The method is described in the publication [ US Pat. No. 6,080,838 ]. Diester 2 was reduced with sodium borohydride to dimethanol 3 as described in the publication [ Acta Chem. Scand. B, 1988, 373 ]. Bromination with phosphorus tribromide in dry DMF gave dibromide 4. The method is described in the publication [ Bioconjugate Chem. 2005, 16, 700 ].

Terbium(III)chelat 8 wurde unter Nutzung des in der Publikation [ Helv. Chim. Acta, 79, 1996, 789 ] beschriebenen Verfahrens hergestellt. Kurz gesagt wurde Verbindung 4 zuerst mit Di-tert-Butylester der Iminodiessigsäure und danach mit Aminoethylanilin in Acetonitril in Anwesenheit von Caliumcarbonat umgesetzt. Die Entfernung der Schutzgruppen mit Trifluoressigsäure und die Behandlung mit Terbiumchlorid ergab Chelat 7, das mit Thiophosgen zu Verbindung 8 aktiviert wurde. Das Aktivierungsverfahren ist in der Publikation [ Bioconjugate Chem. 1994, 5, 278 ] beschrieben.Terbium (III) chelate 8 was synthesized using the method described in the publication [ Helv. Chim. Acta, 79, 1996, 789 ] described method. Briefly, compound 4 was reacted first with di-tert-butyl ester of iminodiacetic acid and then with aminoethylaniline in acetonitrile in the presence of potassium carbonate. The Removal of the protecting groups with trifluoroacetic acid and treatment with terbium chloride gave chelate 7, which was activated with thiophosgene to give compound 8. The activation procedure is described in the publication [ Bioconjugate Chem. 1994, 5, 278 ].

Figure 00040001
Figure 00040001

ZITATE ENTHALTEN IN DER BESCHREIBUNGQUOTES INCLUDE IN THE DESCRIPTION

Diese Liste der vom Anmelder aufgeführten Dokumente wurde automatisiert erzeugt und ist ausschließlich zur besseren Information des Lesers aufgenommen. Die Liste ist nicht Bestandteil der deutschen Patent- bzw. Gebrauchsmusteranmeldung. Das DPMA übernimmt keinerlei Haftung für etwaige Fehler oder Auslassungen.This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Zitierte PatentliteraturCited patent literature

  • - US 4761481 [0005] - US 4761481 [0005]
  • - US 5055578 [0015] US 5055578 [0015]
  • - US 6080838 [0016] - US 6080838 [0016]

Zitierte Nicht-PatentliteraturCited non-patent literature

  • - Acta Chem. Scand. B, 1988, 373 [0016] - Acta Chem. Scand. B, 1988, 373 [0016]
  • - Bioconjugate Chem. 2005, 16, 700 [0016] - Bioconjugate Chem. 2005, 16, 700 [0016]
  • - Helv. Chim. Acta, 79, 1996, 789 [0017] - Helv. Chim. Acta, 79, 1996, 789. [0017]
  • - Bioconjugate Chem. 1994, 5, 278 [0017] - Bioconjugate Chem. 1994, 5, 278 [0017]

Claims (4)

Chelat der Formel (I), das für Markierung von bioaktiver Substanz in Lösung geeignet ist,
Figure 00050001
dadurch gekennzeichnet, dass – A eine reaktive Gruppe für Bindung an die bioaktive Substanz darstellt, gewählt aus einer Gruppe bestehend aus Isothiocyanat, Bromoacetamido, Jodoacetamido, Maleimid, 4,6-Dichlor-1,3,5-triazinyl-2-amino, Pyridyldithiol, Thioester, Aminooxy, Azid, Alkyn, Hydrazid, Amino, Carboxylsäure, Ester oder Säurehalid.Chelate of formula (I) suitable for labeling bioactive substance in solution,
Figure 00050001
characterized in that - A represents a reactive group for binding to the bioactive substance selected from a group consisting of isothiocyanate, bromoacetamido, iodoacetamido, maleimide, 4,6-dichloro-1,3,5-triazinyl-2-amino, pyridyldithiol , Thioester, aminooxy, azide, alkyn, hydrazide, amino, carboxylic acid, ester or acid halide. Chelat nach Schutzanspruch 1, dadurch gekennzeichnet, dass das Lanthanid Ln Terbium, Dysprosium, Europium oder Samarium ist.Chelate according to protection claim 1, characterized that the lanthanide Ln terbium, dysprosium, europium or samarium is. Chelat nach Schutzanspruch 1 und 2, dadurch gekennzeichnet, dass das Lanthanid Ln Terbium oder Dysprosium ist.Chelate according to protection claims 1 and 2, characterized that the lanthanide Ln is terbium or dysprosium. Chelat nach Schutzanspruch 1–3, dadurch gekennzeichnet, dass die bioaktive Substanz ein Oligopeptid, Oligonukleotid, Polypeptid, Polynukleotid, Protein, Oligosaccharid, Polysaccharid, Phospholipid, PNA, LNA, Antikörper, Hapten, Arzneimittel, Molekül, das an einen Rezeptor bindet, oder Lektin ist.Chelate for protection claim 1-3, by in that the bioactive substance is an oligopeptide, oligonucleotide, Polypeptide, polynucleotide, protein, oligosaccharide, polysaccharide, Phospholipid, PNA, LNA, antibodies, hapten, drugs, Molecule that binds to a receptor or is lectin.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014147288A1 (en) 2013-01-31 2014-09-25 Kaivogen Oy Luminescent triazacyclononane-based lanthanide chelate complexes as labelling reagents

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4761481A (en) 1985-03-18 1988-08-02 Baxter Travenol Laboratories, Inc. Substituted pyridine derivatives
US5055578A (en) 1987-11-06 1991-10-08 Baxter Diagnostics Inc. Fluorescent poly(arylpyridine) rare earth chelates
US6080838A (en) 1997-11-25 2000-06-27 University Of Virginia Patent Foundation Peptidomimetic of helix-turn-helix or gamma-turn

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4761481A (en) 1985-03-18 1988-08-02 Baxter Travenol Laboratories, Inc. Substituted pyridine derivatives
US5055578A (en) 1987-11-06 1991-10-08 Baxter Diagnostics Inc. Fluorescent poly(arylpyridine) rare earth chelates
US6080838A (en) 1997-11-25 2000-06-27 University Of Virginia Patent Foundation Peptidomimetic of helix-turn-helix or gamma-turn

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Acta Chem. Scand. B, 1988, 373
Bioconjugate Chem. 1994, 5, 278
Bioconjugate Chem. 2005, 16, 700
Helv. Chim. Acta, 79, 1996, 789

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014147288A1 (en) 2013-01-31 2014-09-25 Kaivogen Oy Luminescent triazacyclononane-based lanthanide chelate complexes as labelling reagents

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