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  • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
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  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
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  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
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  • C09K2211/18—Metal complexes
  • C09K2211/182—Metal complexes of the rare earth metals, i.e. Sc, Y or lanthanide

Definitions

• This invention relates to a group of chelating agents and chelates including 4-(N- alkylpyrrolo)pyridine subunit, biomolecules labeled with these chelates and chelating agents as well as solid supports conjugated with these chelates, chelating agents and labeled biomolecules.
• time-resolved fluorescence based on lanthanide(III) chelates has become a successful detection technology, and it has been used in in vitro diagnostics for over two decades.
• Time-resolved fluorescence quenching assays based on energy transfer from a lanthanide(III) chelate to a nonfluorescent quencher have been applied in various assays of hydrolyzing enzymes as well as for nucleic acid detection.
• the different photochemical properties of europium, terbium, dysprosium and samarium chelates even enable the development multiparametric homogenous assays.
• 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 or phosphonic acid moieties and amines. Unlike organic chromophores, luminescent lanthanide(III) chelates allow multilabeling. In addition, development of chelates bearing several light absorbing moieties is possible.
• a luminescent lanthanide(III) chelate has to fulfill several requirements a) the molecule has to be photochemically stable both in the ground and excited states, b) the molecule has to be kinetically stable, c) the molecule has to be chemically stable, d) the excitation wavelength has to be as high as possible e) the molecule must have a high excitation coefficient in the excitation wavelength, f) the energy transfer from the ligand to the central ion has to be efficient, g) the luminescence decay time has to be long, h) the chelate should be readily soluble in water, i) the bioactive molecules have to retain their affinities after the coupling to the lanthanide chelate.
• Pyridine moiety is by far the most common chromophoric subunit in luminescent lanthanide chelates. Since a single unsubstituted pyridine moiety is not efficient enough to serve as light-absorbing and triplet-sensitizing aromatic group in stable fluorescent chelates, pyridine has often been substituted with various energy absorbing groups. Pyridine analogues conjugated to five membered heteroaromatic rings have also been prepared. The chromophores of stable chelates are often composed from one to three conjugated pyridines or the pyridine moieties are connected to each other via N, O, or S-containing hydrocarbon chains. Pyridine may also be a part of a polyaromatic structure such as phenantronine.
• lanthanide chelates disclosed in art including 4-substituted pyridine subunits have the excitation maxima only somewhat over 300 nm; a higher excitation wavelength would be desirable while developing simpler and less expensive detection instruments. The higher excitation wavelength would also reduce the significance of the background luminescence signal. Furthermore, shorter wavelengths are absorbed by biological materials such as nucleic acids and aromatic amino acids.
• covalent conjugation of the chelate to bioactive molecules is required. Most commonly, this is performed in solution by allowing an amino or mercapto group of a bioactive molecule to react with isothiocyanato, haloacetamido, maleimido or iV-hydroxysuccinimido derivatives of the label [Fichna, J., Janecka, A., Bioconjugate Chem., 2003, 14, 3]. Since in almost all biomolecule labelings the reaction is performed with an excess of an activated label, laborious purification procedures cannot be avoided. Especially, when the attachment of several label molecules, or site-specific labeling in the presence of several functional groups of similar reactivity is required, the isolation and characterization of the desired biomolecule conjugate is extremely difficult, and often practically impossible.
• biomolecule conjugates used in many applications have to be extremely pure, since even small amounts of fluorescent impurities considerably increase the luminescence background and reduce the detection sensitivity.
• it is highly desirable to perform the conjugation of biomolecules on solid phase since most of the impurities can be removed by washings while the biomolecule is still anchored to the solid support, and once released into the solution, only one chromatographic purification is required.
• the technology disclosed here provides chelating agents and lanthanide chelates useful for labeling biomolecules for use as probes in time-resolved fluorescence spectroscopy, wherein the chromophore includes at least one (TV-alkylpyrrole)pyridyl group.
• the pyrrole group may also include other substituents such as a carboxylic or sulfonic acid group or an ester, an amide or a salt of these acids, and aldehydes.
• the pyrrole group may also been conjugated with another pyrrole group.
• the disclosure provides chelates which give fluorescence with different chelated lanthanide ions.
• the disclosure also provides chelates and chelating agents suitable for labeling of biomolecules in solution.
• the disclosure provides chelating agents suitable for labeling oligopeptides, oligonucleotides and other molecules simultaneously with their synthesis on a solid phase.
• the disclosure provides biomolecules and solid supports labeled with the chelates and chelating agents according to this technology.
• the disclosure concerns lanthanide chelates including (TV-alkylpyrrolo)pyridyl subunit.
• the aqueous solubility of the chelates can be enhanced by carboxylic or sulfonic acid functions in the pyrrole ring.
• the carbon atoms of the pyrrole ring can have other substituents also, such as aldehydes and nitro groups.
• the pyrrole group can be conjugated to another pyrrole group that can also be substituted.
• the invention concerns chelates including - a lanthanide ion, Ln 3+
• a chromophoric moiety including one or more aromatic units, wherein at least one of the aromatic units is (TV-alkylpyrrolo)pyridiyl group, wherein the carbon atoms of the iV-alkylpyrrole group are optionally substituted, and wherein the aromatic moieties are tethered directly to each other to form a bipyridine or terpyridyl group or are tethered to each other via a cyclic or acyclic iV-containing hydrocarbon chain, - a chelating part including at least two carboxylic acid or phosphonic acid groups, or esters, amides or salts of the acids, attached to an aromatic unit of the chromophoric moiety, either directly or via a cyclic or acyclic N-containing hydrocarbon chain, and
• a reactive group A tethered to the chromophoric moiety or to the chelating part either directly or via a linker L, the reactive group A enabling binding to a biomolecule or to a functional group on a solid phase.
• this invention concerns a chelating agent including a (N- alkylpyrrole)pyridine subunit.
• the chelating agent includes
• chromophoric moiety including one or more aromatic units, wherein at least one of the aromatic units is (TV-pyrrolo)pyridiyl group, wherein the carbon atoms of the N- alkylpyrrole group are optionally substituted, wherein the aromatic moieties are tethered directly to each other to form a terpyridyl group or tethered to each other via a cyclic or acyclic N-containing hydrocarbon chain,
• a chelating part including at least two carboxylic acid or phosphonic acid groups, or esters or amides of the acids, attached to an aromatic unit of the chromophoric moiety, either directly or via a cyclic or acyclic N-containing hydrocarbon chain, and
• a reactive group A tethered to the chromophoric moiety or to the chelating part either directly or via a linker L, the reactive group A enabling binding to a biomolecule or to a functional group on a solid phase.
• the invention concerns a biomolecule conjugated with a chelate or a chelating agent according to this invention.
• the invention concerns a solid support conjugated with a chelate, a chelating agent or a biomolecule labeled according to this invention.
• this invention concerns a labeled oligopeptide, or an organic molecule obtained by synthesis on a solid phase, by introduction of an appropriate chelating agent according to this invention into the oligopeptide structure on an oligopeptide synthesizer, followed by deprotection and optionally also the introduction of a metal ion.
• this invention concerns a labeled oligonucleotide, obtained by synthesis on a solid phase, by introduction of an appropriate chelating agent according to this invention into the oligonucleotide structure on an oligonucleotide synthesizer, followed by deprotection and optionally also the introduction of a metal ion.
• lanthanide chelates including 4-(N- alkylpyrrole)pyridine subunit have higher excitation wavelenght than several prior art chelates including 4-substituted pyridine subunit (Table 1).
• the 4-(N- alkylpyrrole)pyridyl group is capable of absorbing light or energy and transferring the excitation energy to the chelated lanthanide ion, giving rise to fluorescence.
• alkyl group can be linear or branched, like methyl, ethyl, n- propyl, i-propyl, w-butyl, £-butyl and sec-butyl group.
• the alkyl group can be tethered also to other groups like hydroxyl, carboxylic acid and sulfonic acid groups if these groups are separated from the pyrrole ring by one or more methylene groups.
• the pyrrole group can also include other substituents.
• a "chelating agent” is a precursor of a lanthanide(III) chelate that can be converted to the corresponding lanthanide(III) chelate after protecting groups of the chelating agent are removed and the ligand is treated with lanthanide(III) ion.
• active ester is an aryl ester, vinyl ester, or hydroxyamine ester.
• exemplary active esters are nitrophenyl ester, pentafluorophenyl ester and N- hydroxysuccinimidyl ester.
• transient protecting group is a group that is selectively removed between the coupling steps of oligonucleotide and oligopeptide syntheses. In oligopeptide synthesis the ⁇ -amino group is protected with a transient protecting group, such as Fmoc that can be removed by ⁇ -elimination. In oligonucleotide synthesis, in turn, the 5 '-hydroxy group is masked with a transient protecting group such as DMTr-group removable by mild acidolysis.
• polymerizable group is a monomomer capable of forming a polymer.
• exemplary polymerizable groups are methacroyl, vinyl, styrene and acrylonitrile.
• the chromophoric moiety includes one, two or three pyridyl groups, wherein at least one of them is iV-alkylpyrrole substituted.
• the alkyl groups are linear or branched alkyl groups, such as methyl, ethyl, w-propyl, i-propyl, w-butyl, £-butyl and sec-butyl.
• the chromophoric unit may include unsubstituted pyridyl groups, pyridyl groups bearing other substituents and/or other aromatic groups.
• the pyridyl groups can be tethered directly to each other to form a bipyridine or terpyridyl group. Alternatively, the pyridyl groups are tethered to each other via N- containing hydrocarbon chains.
• the N-containing hydrocarbon chain can be either cyclic or acyclic. In a particular embodiment the N-containing hydrocarbon chain is cyclic.
• the chromophore may also consists of a single 4-(7V-alkylpyrrole)pyridine unit.
• the chromophoric moiety includes one, two or three carboxylic or sulfonic acid groups or esters, amides or salts of these acids.
• the carboxylic acid and sulfonic acid group enhances the aqueous solubility of the chelate.
• These groups can also be used for covalent or noncovalent coupling of the chelate to bioactive molecules and solid supports.
• the chelating agent or chelate must bear a reactive group A in order to enable covalent binding of the chelating agent or chelate to a biomolecule or to a solid support. However, there exist applications where no such covalent binding is necessary. Chelating compounds of this invention can also be used in applications where no reactive groups in the chelate are needed.
• a reactive group A could, in principle, be attached directly to the chromophoric group or to the chelating part, it is desirable, for steric reasons, to have a linker L between the reactive group A and the chromophoric group or chelating part.
• the linker is especially important in case the chelate should be used in solid phase syntheses of oligopeptides and oligonucleotides, but it is desirable also when labeling biomolecules in solution.
• the reactive group A is selected from the group consisting of isothiocyanate, bromoacetamido, iodoacetamido, maleimido, 4,6- dichloro-l,3,5-triazinyl-2-amino, pyridyldithio, thioester, aminooxy, azide, hydrazide, amino, alkyne, a polymerizing group, and a carboxylic acid or acid halide or an active ester.
• the reactive group A is a polymerizable group, such as methacroyl group.
• the reactive group A has to be either azide or terminal alkyne.
• A' is cleaving group like Cl, (CHs) 2 SO, H 2 O, and NOs .
• Bioactive molecules can be labeled statistically also using label molecules tethered to titanocenes [WO2007/ 12291]. These molecules react predominantly with phosphate residues. In this case the reactive group A is
• linker L is the position of linker L.
• the group A-L- can be tethered to the molecule in different ways. It can be tethered to the chelating part, to the N-containing chain joining the aromatic units together, or to an aromatic unit.
• the chelated metal ion Ln 3+ is europium(III), samarium(III), terbium(III) or dysprosium(III). In a particular embodiment the chelated metal ion Ln 3+ is europium(III) and samarium(III).
• Exemplary specific chelates according to this invention are the following structures:
• R is an alkyl group
• n is 1 or 2
• m is 1, 2 or 3
• k is 1 or 2
• L is a linker as defined above
• A is a reactive group as defined above
• the linker L replaces one, two or three hydrogen atoms of formula I, one or two of the hydrogen atoms of formula IV and one of the hydrogen atoms of formulas II, III, and V to VIII.
• the chelating agent according to this technology is suitable for use in the synthesis of an oligopeptide on solid phase.
• the reactive group A is connected to the chelating agent via a linker L, and A is a carboxylic acid or its salt, acid halide, an active ester or an amino acid residue - CH(NHR 3 )R 4 where R 3 is a transient protecting group and R 4 is a carboxylic acid or its salt, acid halide or an active ester.
• exemplary chelating agents are the following structures:
• R , 1 , r R > 2 , L, and Z are defined as above, n is 1 or 2, and A is a carboxylic acid or its salt, acid halide, an active ester or an amino acid residue -CH(NHR 3 )R 4 where R 3 is a transient protecting group and R 4 is a carboxylic acid or its salt, acid halide or an active ester, R" is an alkyl ester or an allyl ester and R is an alkyl group, and wherein L replaces one of the hydrogen atoms of any of the formulas IX to XVI.
• the transient protecting group is selected from a group consisting of Fmoc (fluorenylmethoxycarbonyl), Boc (tert-butyloxycarbonyl), or Bsmoc (l,l-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl), and R 4 is a carboxylic acid or its salt, acid halide or an active ester.
• the chelating agent can be introduced into biomolecules with the aid of a peptide synthesizer or manually.
• the chelating agent can be coupled to an amino tethered solid support or immobilized amino acid in the presence of an activator.
• the condensation step is completed the transient amino protecting group of the labeling reagent is selectively removed while the material is still attached to the solid support
• a chelating agent or other reagent e.g. appropriately protected amino acid, steroid, hapten or organic molecule
• the material is detached from the solid support and deprotected. Purification can be performed by HPLC techniques. Finally, the purified ligand is converted into the corresponding lanthanide(III) chelate by the addition of a known amount of lanthanide(III) ion.
• the chelating agent according to this invention is suitable for use in the synthesis of an oligonucleotide.
• the reactive group A is connected to the chelating agent via a linker L, and A is
• Z 3 is chloro or NR 6 R 7
• R 5 is a protecting group
• R 6 and R 7 are alkyl groups including 1-8 carbons
• Z 2 is absent or is a radical of a purine base or a pyrimidine base or any other modified base suitable for use in the synthesis of modified oligonucleotides, and the base is connected to the oxygen atom either via i) a hydrocarbon chain, which is substituted with a protected hydroxymethyl group, or via ii) a furan ring or pyrane ring or any modified furan or pyrane ring, suitable for use in the synthesis of modified oligonucleotides .
• the chelating agent can be introduced into oligonucleotides with the aid of an oligonucleotide synthesizer.
• a useful method is disclosed in US 6,949,639 and EP 1 308 452. These patent publications disclose a method for direct attachment of a desired number of conjugate groups to the oligonucleotide structure during chain assembly.
• the key reaction in the synthesis strategy towards nucleosidic and acyclonucleosidic oligonucleotide building blocks is the Mitsunobu alkylation which allows introduction of various chelating agents to the acyclonucleoside or nucleoside, and finally to the oligonucleotide structure.
• the chelating agents are introduced during the chain assembly. Conversion to the lanthanide chelate takes place after the synthesis during the deprotection steps.
• Z 2 is a radical of any of the bases thymine, uracil, adenine, guanine or cytosine, and the base is connected to the oxygen atom via i) a hydrocarbon chain, which is substituted with a protected hydroxymethyl group, or via ii) a furan ring having a protected hydroxymethyl group in its 4-position and optionally a hydroxyl, protected hydroxyl or modified hydroxyl group in its 2- position.
• the reactive group - Z 2 -O-P(NR 6 R 7 )-O-R 5 is selected from the group consisting of:
• R" is an alkyl ester or an allyl ester of a carboxylic acid
• R is an alkyl group
• n is 1 or 2
• L is as defined above
• A is - Z 2 -O-P(NR 6 R 7 )-O-R 5 as defined above, and wherein L replaces one hydrogen atom of the formulas IX to XVI.
• Z 2 can be omitted from the structure.
• the chromophore of the chelates or chelating agents according to this invention can be synthesized using methods disclosed in the art.
• An exemplary synthetic route comprises a palladium catalyzed Heck reaction between iV-alkylpyrrole and a molecule including 4-halogenopyridine subunit, wherein the halogen is preferably bromine or iodine.
• reaction intermediates are obtainable also via Stille and Suzuki reaction using iV-alkylpyrrole boronates and stannates, respectively.
• the biomolecule conjugated with a chelating agent or a chelate according to this invention is an oligopeptide, oligonucleotide, DNA, RNA, modified oligo- or polynucleotide, such as phosphoromonothioate, phosphorodithioate, phosphoroamidate and/or sugar- or base modified oligo- or polynucleotide, protein, oligosaccaride, polysaccaride, phospholipide, PNA, LNA, antibody, steroid, hapten, drug, receptor binding ligand and lectine.
• modified oligo- or polynucleotide such as phosphoromonothioate, phosphorodithioate, phosphoroamidate and/or sugar- or base modified oligo- or polynucleotide, protein, oligosaccaride, polysaccaride, phospholipide, PNA, LNA, antibody, steroid, hapten, drug, receptor
• the chelates, chelating agents and biomolecules according to this invention may be conjugated on a solid support.
• the solid support may be a particle such as a nanoparticle or microparticle, a slide, a plate or a resin suitable for solid phase oligonucleotide or oligopeptide synthesis.
• the chelate or chelating agent may be introduced in the solid support, for example a particle, simultaneously with the preparation of the particles [Org. Biomol. Chem. 2006, 4, 1383].
• the chelate is tethered to an azide group and the solid support is derivatized with terminal alkynes or vice versa.
• the biomolecule conjugated with the solid support is a labeled oligopeptide obtained by synthesis on a solid phase by introduction of a chelating agent according to this invention into the oligopeptide structure on an oligopeptide synthesizer, followed by deprotection and optionally introduction of a metal ion.
• the biomolecule conjugated with the solid support is a labeled oligonucleotide obtained by synthesis on a solid phase by introduction of a chelating agent according to this invention into the oligonucleotide structure on an oligonucleotide synthesizer, followed by deprotection and optionally also introduction of a metal ion.
• the biomolecule conjugated with solid support is DNA, RNA, oligopeptide, oligonucleotide, polypeptide, polynucleotide or protein labeled with a chelate according to this invention.
• Schemes 1-6 illustrate the structures and synthetic routes employed in the experimental part.
• Schemes 1 and 2 illustrate the synthesis of the chelates 3a-c, 5 and 10a,b.
• the experimental details are given in Examples 1-12.
• Schemes 3 illustrates the synthesis of the 10-dentate europium(III) chelate 14
• Scheme 4 illustrates the synthesis of the macrocyclic europium(III) chelate tethered to a carboxylic acid group 18.
• Experimental details are given Examples 13 and 14.
• Synthesis of oligopeptide (22) and oligonucleotide (25, 27) labeling reactants according to this invention are illustrated in Schemes 5 and 6, respectively. Experimental details are given in Examples 15-17.
• Adsorption column chromatography was performed on columns packed with silica gel 60 (Merck). NMR spectra were recorded either on a Brucker 250 or on a Jeol LA-600 spectrometer operating at 250 and 600 MHz for 1 H, respectively. Me 4 Si was used as an internal reference. Coupling constants are given in Hz. Electrospray mass spectra were recorded on an Applied Biosystems Mariner ESI-TOF instrument. HPLC purifications were performed using a Shimadzu LC 10 AT instrument equipped with a diode array detector, a fraction collector and a reversed phase column (LiChrocart 125-3 Purospher RP-18e 5 ⁇ m).
• the title compound was synthesized using the method of Example 11 but using samarium(III) citrate.
• Compound 19 (synthesis disclosed in J. Peptide Sci. 2006, 12, 199) is converted to compound 20 using the method disclosed in Example 2.
• Compound 20 is further converted to the oligopeptide labeling reactant 22 using the methods disclosed in J. Peptide ScL, 2006, 12, 199.

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