FIELD

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.

BACKGROUND

The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.

The use of long life-time emitting lanthanide(III) chelate labels or probes together with time-resolved fluorometry in detection provides a method to generate sensitive bioaffinity assays. Indeed, 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.

Although organic chelators and their substituents have a significant effect on the photophysical properties of lanthanide(III) chelates, no general rules for the estimation of these effects are available. Finding a highly fluorescent chelate structure to fulfill all the requirements set for a label in respect to signal, conjugation, stability and biocompactibility as remains a challenge. A great number of stable fluorescent chelates have been developed at numerous laboratories. The chelate structures disclosed in prior art can be categorized as a) pyridine analogues and their multimeric forms [US 4,920,195, US 4,801,722, US 4,761,481, US 4,459,186, EP 0770610, US 5,216,134, US 4,859,777, US 5,202,423, US 5,324,825, US Pat. Appl. 11/004,061; US Pat. Appl. 10/928,143]; b) DTPA conjugates with light-absorbing groups c) polymacrocyclic cage-type complexes such as cryptates [US 4,927,923, EP 493745A], d) and others like calizarenes, podants, helicates and cyclic Schiff s bases [EP 0369000A].

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. While bipyridine, terpyridine and phenantronine moieties are commercially available, preparation of chromophores based on 4-substituted pyridines and pyridines conjugated to fϊve- membered hetereocyclic rings require numerous synthetic manipulations.

Numerous 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.

In several applications, 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.

The biomolecule conjugates used in many applications, such as homogenous quenching assays, have to be extremely pure, since even small amounts of fluorescent impurities considerably increase the luminescence background and reduce the detection sensitivity. Thus, 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.

Solution phase labeling of large biomolecules, such as proteins, cannot be avoided. In these cases, the labeling reaction has to be as selective, and the purification of the biomolecule conjugates as effective as possible. SUMMARY

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.

In one aspect, 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.

In another aspect the disclosure provides chelating agents suitable for labeling oligopeptides, oligonucleotides and other molecules simultaneously with their synthesis on a solid phase.

In another aspect the disclosure provides biomolecules and solid supports labeled with the chelates and chelating agents according to this technology.

Thus, according to one aspect, 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.

According to one aspect, 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

- optionally 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.

According to one aspect this invention concerns a chelating agent including a (N- alkylpyrrole)pyridine subunit.

According to another aspect the the chelating agent includes

- a 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.

According to another aspect, the invention concerns a biomolecule conjugated with a chelate or a chelating agent according to this invention.

According to another aspect, the invention concerns a solid support conjugated with a chelate, a chelating agent or a biomolecule labeled according to this invention.

According to another aspect, 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.

According to another aspect, 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.

DETAILED DESCRIPTION OF THE INVENTION

In this invention, it was observed that 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.

As defined herein, "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.

As defined herein, 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.

As defined herein "active ester" is an aryl ester, vinyl ester, or hydroxyamine ester. Exemplary active esters are nitrophenyl ester, pentafluorophenyl ester and N- hydroxysuccinimidyl ester. As defined herein "transient protecting group" is a group that is selectively removed between the coupling steps of oligonucleotide and oligopeptide syntheses. In oligopeptide synthesis the α-amino group is protected with a transient protecting group, such as Fmoc that can be removed by β-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.

As defined herein "polymerizable group" is a monomomer capable of forming a polymer. Exemplary polymerizable groups are methacroyl, vinyl, styrene and acrylonitrile.

According to one embodiment, the chromophoric moiety includes one, two or three pyridyl groups, wherein at least one of them is iV-alkylpyrrole substituted. In a particular embodiment, the alkyl groups are linear or branched alkyl groups, such as methyl, ethyl, w-propyl, i-propyl, w-butyl, £-butyl and sec-butyl. In addition to (N- alkylpyrrole)pyridyl group or groups, 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.

According to one embodiment 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. One example of this kind of technology is demonstrated e.g. in Blomberg, et al., in J. Immunological Methods 1996, 193, 199. Another example where no reactive group A is needed is the separation of eosinophilic and basophilic cells [WO2006/072668]. In this application positively and negatively charged chelates bind with negatively and positively charged cell surfaces, respectively.

Yet another example where no linker is needed is the preparation of highly luminescent beads simply by swelling chelates into the polymer [e.g. Soukka et al., Anal. Chem. 2001, 73, 2254].

Although in many applications 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.

According to one embodiment the linker L is formed from one to ten moieties, each moiety being selected from the group consisting of phenylene, alkylene containing 1- 12 carbon atoms, ethynydiyl (-C≡C-), ethylenediyl (-C=C-), ether (-O-), thioether (-S- ), amide (-CO-NH-, -CO-NR -, -NH-CO- and -NR -CO-), carbonyl (-CO-), ester (- COO- and -OOC-), disulfide (-SS-), sulfonamide (-SO ₂ -NH-, -SO ₂ -NR'-), sulfone (- SO ₂ -), phosphate (-0-PO ₂ -O-), diaza (-N=N-), and tertiary amine, wherein R' represents an alkyl group containing less than 5 carbon atoms.

According to one embodiment, 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.

In case the chelate or chelating agent should be attached to a microparticle or nanoparticle during the manufacturing process of the particles, the reactive group A is a polymerizable group, such as methacroyl group.

In case the chelate or chelating agent is to be attached to solid supports including nanomaterials, biomolecules, and various organic molecules using copper(I) catalyzed Huisgen-Sharpless dipolar [2+3] cycloaddition reaction, the reactive group A has to be either azide or terminal alkyne.

It has been proposed [US 5,985,566] that oligonucleotides, DNA, RNA, oligopeptides, proteins and lipids can be transformed statistically by using label molecules tethered to platinum derivatives. In this case the reactive group A is

wherein 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

wherein - 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.

According to one embodiment, 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:

wherein 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, and wherein 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. According to one embodiment, the chelating agent according to this technology is suitable for use in the synthesis of an oligopeptide on solid phase. In this application, 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:

wherein 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.

In a preferable embodiment 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. When 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

(e.g. with piperidine in the case of Fmoc-protecting group). Then, if desired, a second coupling of a chelating agent or other reagent (e.g. appropriately protected amino acid, steroid, hapten or organic molecule) is performed as above. When the synthesis of the desired molecule is completed, 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.

According to another embodiment, the chelating agent according to this invention is suitable for use in the synthesis of an oligonucleotide. In this case the reactive group A is connected to the chelating agent via a linker L, and A is

-Z ² -O-PZ ³ -O-R ⁵

wherein one of the oxygen atoms optionally is replaced by sulfur, 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, and 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.

According to one embodiment 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.

According to one embodiment the reactive group - Z ² -O-P(NR ⁶ R ⁷ )-O-R ⁵ is selected from the group consisting of:

where - is the position of the linker L and DMTr is dimethoxytrityl.

According to one embodiment the chelating agent for this use is selected from one of the specific structures disclosed below

where 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. For the preparation of oligonucleotide conjugates tethered to a single label molecule 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. These 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.

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.

In case the chelate or chelating agent has a polymerizing group as a reactive group, then 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]. When copper(I) catalyzed Huisgen-Sharpless reaction is used for derivatization, the chelate is tethered to an azide group and the solid support is derivatized with terminal alkynes or vice versa.

According to one embodiment, the biomolecule conjugated with the solid support, either covalently or noncovalently, 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.

According to another embodiment, the biomolecule conjugated with the solid support, either covalently or noncovalently, 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.

According to another embodiment, the biomolecule conjugated with solid support, either covalently or noncovalently, is DNA, RNA, oligopeptide, oligonucleotide, polypeptide, polynucleotide or protein labeled with a chelate according to this invention.

The invention is further elucidated by the following examples. The structures and synthetic routes employed in the experimental part are depicted in Schemes 1-6. 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 μm). Mobile phase: (Buffer A): 0.02 M triethylammonium acetate (pH 7.0); (Buffer B): A in 50 % (v/v) acetonitrile. Gradient: from 0 to 1 min 95% A, from 1 to 31 min from 95% A to 100% B. Flow rate was 0.6 mL min. ^"1 AU dry solvents were from Merck and they were used as received. Fluorescence spectra were recorded on a PerkinElmer LS-55 instrument.

Example 1

Synthesis of tetra(te/t-butyl) {[4-(l '-methylpyrrol-2'-yl)pyridin-2,6- diyl]bis(methylenenitrilo)}tetrakis(acetate) (2a)

A mixture of tetra(teτt-butyl) [(4-bromopyridin-2,6- diyl)bis(methylenenitrilo)]tetrakis(acetate) (1; 278 mg, 0.41 mmol), l-methyl-2- (tributylstannyl)pyrrole (153 mg, 0.41 mmol), bis(triphenylphosphine)palladium(II) dichloride (15 mg, 21 μmol) and DMF (5 ml) was dearated with argon and stirred overnight in the dark at 100 ⁰ C. The reaction mixture was filtered, evaporated to dryness and the product purified by silica column chromatography (15% MeOH/CH ₂ Cl ₂ ). Yield 155 mg (56 %). ESI-TOF-MS [M+H ⁺ ]: calc. for C ₃₆ H ₅₇ N ₄ O ₈ ⁺ 673.42, found 673.47. ¹ H NMR (600 MHz, CDCl ₃ ): δ 7.58 (s, 2H); 6.74 (m, IH); 6.46 (m, IH); 6.18 (m, IH); 4.04 (s, 4H); 3.78 (s, 3H); 3.51 (s, 8H); 1.45 (s, 36H)

Example 2

Synthesis of tetra(teτt-butyl) { [4-(2 ' -methoxycarbonyl- 1 ' -methylpyrrol-5 ' -yl)pyridin- 2,6-diyl]bis(methylenenitrilo)}tetrakis(acetate) (2b)

A mixture of tetra(teτt-butyl) [(4-bromopyridin-2,6- diyl)bis(methylenenitrilo)]tetrakis(acetate) (1; 128 mg, 0.19 mmol), methyl 1- methylpyrrole-2-carboxylate (265 mg, 1.9 mmol), potassium acetate (37 mg, 0.38 mmol), tetrabutyl ammonium iodide (70 mg, 0.19 mmol), bis(triphenylphosphine)palladium(II) chloride (6.7 mg, 9.5 μmol), triphenylphosphine (5.0 mg, 19 μmol) and DMF (3.8 ml) was deaerated with argon and stirred overnight in the dark at 100 ⁰ C. The reaction mixture was filtered, evaporated to dryness and the product purified by silica column chromatography (10 % MeOH/CH ₂ Cl ₂ ). Yield 66 mg (48 %). ESI-TOF-MS [M+H ⁺ ]: calc. for C ₃₈ H ₅₉ N ₄ Oi ₀ ⁺ 731.42, found 731.47. ¹ H NMR (600 MHz, CDCl ₃ ): δ 8.22 (s, 2H); 7.06 (d, J = AA Hz, IH); 6.70 (d, J = 4.2 Hz, IH); 6.18 (m, IH); 4.60 (s, 4H); 4.11 (s, 3H); 3.89 (s, 3H); 3.60 (s, 8H); 1.45 (s, 36H)

Example 3

Synthesis of tetramethyl {[4-(2'-formyl-l '-methylpyrrol-5'-yl)pyridin-2,6- diyl]bis(methylenenitrilo)}tetrakis(acetate) (Ic)

A mixture of tetramethyl [(4-bromopyridin-2,6- diyl)bis(methylenenitrilo)]tetrakis(acetate) (160 mg, 0.33 mmol), l-methylpyrrole-2- carboxaldehyde (356 mg, 3.3 mmol), potassium acetate (65 mg, 0.66 mmol), tetrabutyl ammonium iodide (121 mg, 0.33 mmol), palladium(II) acetate (3.7 mg, 17 μmol), trifurylphosphine (8.7 mg, 33 μmol) and DMF (4 ml) was deaerated with argon and stirred overnight in the dark at 120 ⁰ C. The reaction mixture was filtered, evaporated to dryness and the product purified by silica column chromatography (10 % MeOH/CH ₂ Cl ₂ + 1% TEA). Yield 60 mg (34 %). ESI-TOF-MS [M-HH ⁺ ]: calc. for C ₂₅ H ₃₃ N ₄ O ₉ ⁺ 533.22, found 533.21.

Example 4

Synthesis of tetramethyl {[(4-pyrrol-5'-yl)pyridin-2,6- diyl]bis(methylenenitrilo)}tetrakis(acetate) (2d)

A mixture of tetramethyl [(4-bromopyridin-2,6- diyl)bis(methylenenitrilo)]tetrakis(acetate) (1; 76 mg, 0.15 mmol), pyrrole (202 mg, 3.0 mmol), potassium acetate (30 mg, 0.30 mmol), tetrabutyl ammonium iodide (56 mg, 0.15 mmol), palladium(II) acetate (1.7 mg, 7.5 μmol), trifurylphosphine (3.9 mg, 15 μmol) and DMF (1.5 ml) was deaerated with argon and stirred overnight in the dark at 120 ⁰ C. The reaction mixture was filtered, evaporated to dryness and the product purified by silica column chromatography (PE:EA:TEA 3:5:1). Yield 7 mg (10 %). ESI-TOF-MS [M+H ⁺ ]: calc. for C ₂₃ H ₃ IN ₄ O ₈ ⁺ 491.21, found 491.23. Example 5.

Synthesis of the europium(III) chelates 3a-c

Compounds 2a-c were deprotected and converted to the corresponding europium(III) chelates 3a-c using the methods disclosed in US2005/084451.

Example 6.

Synthesis of the methine ligand 4.

A mixture of compound 2c (24 mg, 45.1 μmol), methyl 2-pyrrole carboxylate (5.64 mg, 45.1 μmol), and phosphorus oxychloride (4.3 μL, 47.3 μmol) and DIPEA (32.5 μlin dry dichloromethane (400 μl) was heated for 30 min at reflux. The mixture was cooled to rt, diluted with dichloromethane, washed with Na ₂ SO ₄ and dried. Purification was performed on preparative TLC.

Example 7.

Synthesis of the europium chelate 5.

Compound 4 was deprotected and converted to the corresponding europium(III) chelate 5 using the methods disclosed in US2005/084451.

Example 8

Synthesis of ethyl 4-(2'-methoxycarbonyl-l '-methylpyrrol-5'-yl)-6-

(hydroxymethyl)pyridine-2-carboxylate (7)

A mixture of compound 6 (172 mg, 0.61 mmol), methyl l-methylpyrrole-2- carboxylate (842 mg, 6.1 mmol), potassium acetate (119 mg, 1.2 mmol), tetrabutyl ammonium iodide (224 mg, 0.61 mmol), palladium(II) acetate (6.8 mg, 30 μmol), trifurylphosphine (15.9 mg, 60 μmol) and DMF (3.5 ml) was deaerated with argon and stirred overnight in the dark at 110 ⁰ C. The reaction mixture was filtered, evaporated to dryness and the product purified by aluminum oxide column chromatography (EtOH/CH ₂ Cl ₂ 1% → 5%). Yield 75 mg (39 %). ESI-TOF-MS [M+H ⁺ ]: calc. for Ci ₆ Hi ₉ N ₂ O ₅ ⁺ 319.13, found 319.11.

Example 9

Synthesis of Ethyl 4-(2'-methoxycarbonyl-l '-methylpyrrol-5'-yl)-6-

(chloromethyl)pyridine-2-carboxylate (8)

A mixture of compound 7 (75 mg, 0.24 mmol), phosphorus trichloride (33 mg, 0.24 mmol) and DMF (5 ml) was stirred for 1 h at room temperature. The reaction mixture was then neutralized with saturated aqueous NaHCθ3, evaporated to dryness and the product purified by preparative TLC (PE:EA 1 :1). Yield 35 mg (43 %). ESI-TOF-MS [M+H ⁺ ]: calc. for Ci ₆ Hi ₈ ClN ₂ O ₄ ⁺ 337.10, found 337.08.

Example 10

Synthesis of l,4,7-tris{[6'-ethoxycarbonyl-4'-(4"-methoxycarbonyl-l '-methylpyrrol- 5 ' ' -yl)pyridine-2 ' -yljmethyl} - 1 ,4,7-triazacyclononane (9)

A mixture of compound 9 (35 mg, 0.104 mmol), 1,4,7-triazacyclononane (4.4 mg, 0.034), potassium carbonate (29 mg, 0.208 mmol) and DMF (2 ml) was stirred overnight at room temperature. The reaction mixture was filtered, evaporated to dryness and the product purified by preparative TLC (15 % EtOH/CH ₂ Cl ₂ + 1% TEA). Yield was 20 mg (57 %).

Example 11 Synthesis of 6,6',6"-[(octahydro-lH-l,4,7-triazonine-l,4,7- triyl)tris(methylene)]tris [4-(4-carboxy- 1 -methylpyrrol-5 -yl)pyridine-2-carboxylic acid] europium(III) (10a)

Compound 9 (8 mg, 8 μmol) was dissolved in 2 ml of 2 M KOH/MeOH solution. 0.1 ml of water was added and the mixture was stirred for 3 days at 40 ⁰ C. The methanol was removed in vacuo, the pH of the reaction mixture was adjusted to 8.5 with 2 M HCl and 40 μl of 0.2 M europium(III) citrate was added. The mixture was stirred overnight at room temperature and purified by HPLC. ESI-TOF-MS [M+H ⁺ ]: calc. for C ₄₅ H ₄₃ EuN ₉ Oi ₂ ⁺ 1054.22, found 1054.35.

Example 12

Synthesis of 6,6',6"-[(octahydro-lH-l,4,7-triazonine-l,4,7- triyl)tris(methylene)]tris [4-(4-carboxy- 1 -methylpyrrol-5 -yl)pyridine-2-carboxylic acid] samarium(III) (10b)

The title compound was synthesized using the method of Example 11 but using samarium(III) citrate.

Example 13.

Preparation of the 10-dentate europium chelate 14.

Compound 11 (synthesis disclosed in Helvetica Chimica Acta 1996, 79, 789) is allowed to react with methyl l-methylpyrrole-2-carboxylate as disclosed in Example 2 to give the ligand 12. Deprotection followed by treatment with europium(III) chloride gives rise to chelate 13, treatment of which with N-hydroxysuccimimide in the presence of EDAC as disclosed in J. Am. Chem.Soc. 126, 4888-4896 gives the chelate 14.

Example 14. Preparation of the azamacrocycle 18.

Compound 15 (synthesis disclosed in Tetrahedron Lett. 2005, 46, 4387) is allowed to react with methyl l-methylpyrrole-2-carboxylate as disclosed in Example 2 to give the ligand 12. Removal of the nosyl groups and carboxymethylation using the method disclosed in WO2007128874 gives compound 17. Saponification followed by treatment with europium chloride gives rise to the chelate 18.

Example 15

Preparation of the oligopeptide labeling reactant 22.

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.

Example 16.

Preparation of the non-nucleosidic oligonucleotide labeling reactant 25.

Compound 23 (synthesis disclosed in Bioconjugate Chem., 2005, 17, 700) is converted to compound 24 using the method disclosed in Example 2. Phosphitylation gives rise to compound 25.

Example 17.

Schematic preparation of the non-nucleosidic oligonucleotide labeling reactant 27.

Reaction of 2'-deoxy-5'-O-(4,4'-dimethoxytrityl)uridine and compound 24 under Mitsunobu conditions followed by phosphitylation as disclosed in Bioconjugate Chem., 2005, 17, 700 gives rise to compound 27. Table 1. Emission wavelenghts of europium(III) chelates including 4-substituted pyridine subunits.

a) data from /. Luminescence 1997, 75, 149-169. b) data from US2005/084451.

Sn(Bu) ₃ X = H = H

SCHEME 1

SCHEME 2

13 14

R = COO-f-Bu

SCHEME 3

MeOO

SCHEME 4 SCHEME 5

phosphitylation

Scheme 6 It will be appreciated that the methods of the present invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. Although only the synthesis of europium(III) chelates is presented here, it is clear that an artisan can prepare the corresponding samarium(III), terbium(III) and dysprosium(III) chelates using the methods disclosed here by substituting the europium(III) salt with the desired lanthanide(III) salt. It will be apparent to an expert skilled in the field that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive.