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WO2009115644A1 - Chélates de lanthanide(iii) comprenant des bêta-dicétones et conjugués issus de ceux-ci - Google Patents

Chélates de lanthanide(iii) comprenant des bêta-dicétones et conjugués issus de ceux-ci Download PDF

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Publication number
WO2009115644A1
WO2009115644A1 PCT/FI2009/050208 FI2009050208W WO2009115644A1 WO 2009115644 A1 WO2009115644 A1 WO 2009115644A1 FI 2009050208 W FI2009050208 W FI 2009050208W WO 2009115644 A1 WO2009115644 A1 WO 2009115644A1
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group
aryl
iii
carbon atoms
azacycloalkane
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English (en)
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Jari Hovinen
Veli-Matti Mukkala
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Wallac Oy
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Wallac Oy
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
    • C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1003—Carbocyclic compounds
    • C09K2211/1007—Non-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1092—Heterocyclic compounds characterised by ligands containing sulfur as the only heteroatom
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/18—Metal complexes
    • C09K2211/182—Metal complexes of the rare earth metals, i.e. Sc, Y or lanthanide

Definitions

  • This invention relates to luminescent lanthanide(III) chelates based on ⁇ -diketones tethered to azacycloalkanes and biomolecule conjugates and solid supports based on these chelates.
  • the commercially available DELFIA ® assay method uses a non-luminescent europium(III) chelate as the label [US 4,565,790; US 4,808,541].
  • europium(III) ion is dissociated from the non-luminescent chelate by lowering pH to 3.2 and the luminescence is enhanced with a mixture of ⁇ -diketone (4,4,4-trifluoro-l-(2-naphtyl)-butane-l,3-dione), detergent (Triton X-IOO) and chelator (trioctylphosphine oxide, TOPO).
  • the new chelate formed has a very high luminescence, giving detection sensitivity of ca 5 • 10 "16 M.
  • the trifluorinated methyl group of the ⁇ -diketone shifts the keto-enol equilibrium efficiently towards the strongly chelating enolic form, and thus enables chelate formation even in acidic solutions. More strongly acidic ⁇ -diketones allow the use of even more acidic measurement solutions [US 7,211,440].
  • chelator TOPO is essential for high luminescence.
  • stable luminescent lanthanide(III) chelates consist of a ligand with a reactive group for covalent conjugation to bioactive molecules, an aromatic structure, which absorbs the excitation energy and transfers it to the lanthanide ion and additional chelating groups, such as carboxylic acid moieties and amines. Unlike organic chromophores, these molecules do not suffer from Raman or Rayleigh scattering or concentration quenching.
  • ⁇ -diketones have also been used to enhance luminescence in a mixed ligand system with EDTA type ligands [US 4,374,120], macrocyclic complexes [US 6,750,005], as well as with chelates tethered to various aromatic ligands as luminescence sensitizers [US 6,344,360].
  • the present technology concerns luminescent lanthanide(III) chelates comprising at least three ⁇ -diketone subunits, and an azacycloalkane backbone.
  • L 1 is a linker connecting Ar to the azacycloalkane
  • L 2 is a linker connecting G to the azacycloalkane or is not present
  • G is a reactive group or is not present
  • Ar is an aryl group, optionally mono or multisubstituted
  • R is a straight or branched alkyl chain with 1 to 9 carbon atoms substituted with three or more fluorine atoms and optionally substituted with other substituents than fluorine.
  • this disclosure concerns a bioactive complex, i.e. biomolecule conjugated with a chelate according to this technology.
  • this disclosure concerns a solid support conjugated with a chelate according to this technology.
  • this disclosure concerns a solid support conjugated with a bioactive molecule labelled with a chelate according to this technology.
  • Fig. 1 shows an emission spectrum of a stable luminescent lanthanide chelate [an europium(III) chelate based on 6,6'-(lH-pyrazole-l,3-diyl)bis(pyridine)].
  • the emission spectrum is devided into several lines. ⁇ em (rel int %) 593 (14); 615 (50) 654 (4), 692 (32).
  • Fig 2 shows an emission spectum of an europium(III) chelate according to this invention
  • Lanthanide(III) chelates based on ⁇ -diketones including an azacycloalkane backbone are new.
  • the molecules of the present technology are luminescent even in the absence of additional chelators such as phosphine oxides.
  • the chelates do not need to be saturated with an excess of metal ions.
  • the present technology concerns luminescent lanthanide(III) chelates including ⁇ -diketone subunits and a azacycloalkane backbone.
  • the azacycloalkane backbone according to this technology includes a cyclic N-containing hydrocarbon chain with three or more nitrogen atoms.
  • the azacycloalkanes are 1,4,7-triazacyclononane, 1,4,7-triazacyclodecane, 1,5,9-triazacyclododecane, cyclen and cyclam.
  • the azacycloalkane backbone includes an aromatic residue.
  • the aromatic residues is pyridine.
  • the excitation wavelength of the chelate can be tuned by modifying the structure (i.e. the chromophore or the alkyl substituent of the chromophore) of the ⁇ -diketone subunit.
  • a high excitation wavelength is useful, since the choice of materials of the measuring equipments is more flexible and the background luminescence is less significant. For example, excitation wavelengths over 350 nm allow the use of cheap LED lasers as the light source.
  • L 1 is a linker between Ar and the azacycloalkane
  • L 2 is a linker between G and the azacycloalkane or is not present
  • G is a reactive group or is not present, Ar is an aryl group, optionally mono or multisubstituted,
  • R is a straight or branched alkyl chain with 2 to 9 carbon atoms substituted with three or more fluorine atoms optionally substituted with other substituents than fluorine.
  • the linker L 1 includes one to three moieties each moiety being selected from an alkylene containing 1-3 carbon atoms, ether (-O-), thioether (-S-), amide (-CO-
  • linker L 1 is selected from methylene and ethylene.
  • An exeplary linker L 1 is methylene.
  • the aryl group Ar is selected from the group consisting of phenyl, 9H-fluoren-2-yl, 1-naphthyl, 2-naphthyl, 2-phenanthroyl, 2-furyl, 3-furyl, 2-benzofuryl, 3- benzofuryl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-thiazolyl, 4-thiazolyl, 2- benzothiazolyl, 2-benzo[a]thienyl, 3-benzo[a]thienyl, 2-pyrimidyl, 4-pyrimidyl and 5-pyrimidyl.
  • the aryl group Ar is mono- or multisubstituted.
  • each substituent is independently selected from a group consisting of straight or branched alkyl, alkoxy, aryl, aroyl, aryloxy, nitro, amino, cyano, hydroxyl, carboxy, chloro, bromo, iodo, fluoro and acyl. If these substitutions include atoms that can be substituted these can in turn be substituted.
  • the alkyl chain R is substituted with 3 to 9 fluorine atoms. In a particular embodiment R is substituted with 3 to 7 fluorine atoms.
  • R is mono- or multisubstituted with other substituents than fluorine and each substituent is independently selected from a group consisting of straight or branched alkyl, alkoxy, aryl, aroyl, aryloxy, nitro, amino, cyano, hydroxyl, carboxy, chloro, bromo, iodo, fluoro or acyl. If these substitutions include atoms that can be substituted these can in turn be substituted.
  • ⁇ -diketones are selected form a group consisting of 4,4,4- trifluoro- 1 -aryl- 1 ,3-butanedione, 4,4,5,5,5-pentafluoro- 1 -aryl- 1 ,3-pentanedione and 4,4,5,5,6,6,6-heptafluoro-l-aryl-l,3-hexanedione, wherein the aryl group is selected from a group consisting of thienyl, furyl, benzolfuryl, benzothienyl and phenyl.
  • the chelate must bear a reactive group G in order to enable covalent binding of the chelate to a bioactive molecule or to a solid support.
  • the reactive group G is selected from the group consisting of isothiocyanate, haloacetamido, maleimido, dichlorotriazinylamino, pyridyldithio, thioester, aminooxy, hydrazide, amino, a polymerizing group, and carboxylic acid, carboxylic acid halide and active ester of carboxylic acid.
  • An exemplary active ester is N-hydroxysuccinimide.
  • the reactive group G is a polymerizable group.
  • An exemplary polymerizable group is methacroyl group.
  • the reactive group G has to be either azide or terminal alkyne.
  • the reactive group G has to be a cycloalkylalkyne group.
  • Chelating compounds of this invention can also be used in applications where no reactive groups in the chelate are needed.
  • One example of this kind of technology is demonstrated in [J. Immunological Methods 1996, 193, 199].
  • Another example where no reactive group G is needed is the separation of eosinophilic and basophilic cells [WO2006/072668].
  • positively and negatively charged chelates bind negatively and positively charged cell surfaces, respectively.
  • hydrophilic moieties can be e.g. carboxylic acids [HeIv. Chim. Acta, 1996, 79, 789], carbohydrates [Anal. Chem., 2003, 75, 3193], and sulfonic acids [Bioconjugate Chem. 2008, 19, 279].
  • lanthanide(III) ions are selected from europium(III), terbium(III), samarium(III) and dysprosium(III).
  • lantanide(III) ions are europium(III) and samarium(III).
  • G is a reactive group and is selected from the group consisting of isothiocyanate, bromoacetamido, iodoacetamido, maleimido, 4,6-dichloro-l,3,5-triazinyl-2-amino, pyridyldithio, thioester, aminooxy, hydrazide, amino, azido, alkyne, a polymerizing group, carboxylic acid, carboxylic acid halide, active ester of carboxylic acid, and wherein A is cleaving ligand comprising Cl, (CH 3 ) 2 SO, H 2 O, and NO 3 " and wherein - is the position of linker L 2 ;
  • Ln is lanthanide selected from a group consisting of europium, terbium, samarium and dysprosium; and n is 1 or 2.
  • this technology concerns a bioactive complex including a bioactive molecule conjugated with a chelate according to this technology.
  • the bioactive molecule is selected from the group consisting of an oligopeptide, oligonucleotide, DNA, RNA, modified oligo- or polynucleotide, protein, oligosaccaride, polysaccaride, phospholipide, PNA, LNA, antibody, steroid, hapten, drug, receptor binding ligand and lectine.
  • this technology concerns a solid support conjugated with a chelate according to this technology.
  • the solid support is selected from the group consisting of a nanoparticle, a microparticle, a slide and a plate.
  • this technology concerns a bioactive complex according to this technology conjugated to a solid support.
  • the solid support is selected from the group consisting of nanoparticle, a microparticle, a slide and a plate.
  • Schemes 1-7 illustrate the structures and synthetic routes employed in the experimental part.
  • Scheme 1 illustrates the synthesis of the of (Z)-l-(3-(bromomethyl)phenyl)-4,4,4-trifluoro-3-hydroxybut-2-en-l-one, 2.
  • the experimental details are given in Examples 1-2.
  • Scheme 2 illustrates the synthesis the europium(II) chelate based on 1,5,9-triazacyclododecane 4.
  • Experimental details are given in Examples 3 and 4.
  • Schemes 3-5 illustrate the synthesis of the acyclic derivatives of ⁇ -diketones 7, 10 and 11. Experimental details are given in Examples 5-11.
  • Scheme 6 illustrates the synthesis a thiophene derivatives 12-17. Experimental details are given in Examples 12-16.
  • Schemes 7 and 8 illustrate the synthesis of a mercapto selective biomolecule labelling reactants (21, 27) based on a ⁇ -diketone according to this invention.
  • Photophysical properties of chelates according to this invention are shown in Table 1.
  • the photophysical properties of the chelates were determined by measuring excitation and emission spectra and fluorescence lifetime in TS buffer (50 mM tris, 150 mM NaCl, pH 7.75) with LS-55 luminescence spectrometer (PerkinElmer Instruments, Connecticut, USA).
  • Table 1 Photophysical properties of the chelates synthesized
  • the results can be summarized as follows: the acyclic analogues comprising several ⁇ -diketone subunits are practically nonluminescent in the absence of an additional chelator. the cyclic analogues comprising several ⁇ -diketone subunits (i.e. those based on azacycloalkane backbone) are luminescent even in the absense of an additional chelator. - the excitation maximum can be adjusted by changing the structure of the ⁇ -diketone subunit.

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Abstract

Cette invention porte sur des chélates de lanthanide(III) luminescents à base de β-dicétones comprenant un squelette d'azacycloalcane ainsi que sur des conjugués de biomolécules et sur des supports solides comprenant ces chélates. A la différence des chélates de lanthanide à base de β-dicétones monomères et des chélates de lanthanide(III) à base d'azaalcanes acycliques comprenant plusieurs sous-unités β-dicétones, les molécules de la présente technologie sont luminescentes même en l'absence de chélatants supplémentaires tels que des oxydes de phosphine.
PCT/FI2009/050208 2008-03-20 2009-03-18 Chélates de lanthanide(iii) comprenant des bêta-dicétones et conjugués issus de ceux-ci Ceased WO2009115644A1 (fr)

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FI20080230A FI20080230A0 (fi) 2008-03-20 2008-03-20 Lantanidi(III) kelaatteja ja niistä johdettuja konjugaatteja
FI20080230 2008-03-20

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014147288A1 (fr) 2013-01-31 2014-09-25 Kaivogen Oy Complexes de chélates de lanthanides luminescents à base de triazacyclononane à titre de réactifs de marquage
WO2015059363A1 (fr) * 2013-10-23 2015-04-30 Turun Yliopisto Procédé d'analyse d'échantillons gazeux

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007128874A1 (fr) * 2006-05-05 2007-11-15 Wallac Oy Réactifs de marquage de biomolécules à base d'azacycloalcanes et leurs conjugués dérivés

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007128874A1 (fr) * 2006-05-05 2007-11-15 Wallac Oy Réactifs de marquage de biomolécules à base d'azacycloalcanes et leurs conjugués dérivés

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
EVANGELISTA, R.A. ET AL.: "A New Europium Chelate for Protein Labelling and Time-Resolved Fluorometric Applications.", CLIN. BIOCHEM., vol. 21, 1988, pages 173 - 178 *
SAVITSKY, A.P. ET AL.: "Novel Fluorescent Chelate for Eu", PROC. SPIE, vol. 2388, 1995, pages 429 - 434 *
YUAN, J. ET AL.: "A New Tetradentate beta-Diketonate-Europium Chelate That Can Be Covalently Bound to Proteins for Time-Resolved Fluoroimmunoassay", ANAL. CHEM., vol. 70, 1998, pages 596 - 601 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014147288A1 (fr) 2013-01-31 2014-09-25 Kaivogen Oy Complexes de chélates de lanthanides luminescents à base de triazacyclononane à titre de réactifs de marquage
WO2015059363A1 (fr) * 2013-10-23 2015-04-30 Turun Yliopisto Procédé d'analyse d'échantillons gazeux

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