Search Images Maps Play YouTube News Gmail Drive More »
Sign in
Screen reader users: click this link for accessible mode. Accessible mode has the same essential features but works better with your reader.

Patents

  1. Advanced Patent Search
Publication numberWO2014147288 A1
Publication typeApplication
Application numberPCT/FI2014/050075
Publication date25 Sep 2014
Filing date31 Jan 2014
Priority date31 Jan 2013
Publication numberPCT/2014/50075, PCT/FI/14/050075, PCT/FI/14/50075, PCT/FI/2014/050075, PCT/FI/2014/50075, PCT/FI14/050075, PCT/FI14/50075, PCT/FI14050075, PCT/FI1450075, PCT/FI2014/050075, PCT/FI2014/50075, PCT/FI2014050075, PCT/FI201450075, WO 2014/147288 A1, WO 2014147288 A1, WO 2014147288A1, WO-A1-2014147288, WO2014/147288A1, WO2014147288 A1, WO2014147288A1
InventorsHarri Takalo, Jaana Rosenberg
ApplicantKaivogen Oy
Export CitationBiBTeX, EndNote, RefMan
External Links: Patentscope, Espacenet
Luminescent triazacyclononane-based lanthanide chelate complexes as labelling reagents
WO 2014147288 A1
Abstract
The invention relates to a detectable molecule comprising a biospecific binding reactant attached to a macrocyclic luminescent lanthanide chelate comprising a lanthanide ion and a chelating ligand of the formula (I) wherein, R1 consists of a linker coupling the chelate to the biospecific binding reactant and optionally a spacer; either R3 or R4 is H; and the lanthanide ion Ln3+ is europium(lll) or samarium(lll). The invention also relates to corresponding lanthanide chelates. The invention further relates to the use of the detectable molecule of the invention.
Claims  (OCR text may contain errors)
1 . A detectable molecule comprising a biospecific binding reactant attached to a luminescent lanthanide chelate comprising a lanthanide ion and a chelating ligand of the formula (I)
wherein, a) Ri consists of a linker Zi coupling said chelate to said biospecific binding reactant selected from the group consisting of thiourea (-NH-CS-NH-), aminoacetamide (-NH-CO-CH2-NH-), amide (-NH-CO- and -CO-NH-, -NCH3-CO- and -CO-NCH3-), aliphatic thioether (-S-), disulfide (-S-S-), 6-substituted-1 ,3,5- triazine-2,4-diamine, 1 ,2,3-triazole, wherein n = 1 -6;
and optionally a spacer, i.e. a distance-making biradical between the phenyl and linker Zi , and said spacer is a group consisting of one to four moieties, each moiety being selected from the group consisting of phenylene, alkylene containing 1 -16 carbon atoms, ethenylene (-CH=CH-), ethynediyl (-C≡C-), ether (-O-), thioether (-S-), amide (-CO-NH- and -NH-CO-, -NCH3-CO- and -CO- NCH3-), carbonyl (-CO-); b) either R3 or R is H; i) when R3 = H, R2 = R4 or R2≠ R4, and R2 and R4 are substituents independently selected from the group consisting of -COOH, -COO", -SO3H, -SO3", -CI, -Br, -F, -I, -CF3, -CN, -OCH3, -SCH3, -O(CH2)nCOOH, -O(CH2)nCOO", -O(CH2)nCONH(CH2)mSO3H, -O(CH2)nCONH(CH2)mSO3", -O(CH2)nPO3H, -O(CH2)nPO3 "-O(CH2CH2O)nOH, -O(CH2CH2O)nOCH3, -S(CH2)nCOOH, -S(CH2)nCOO", -S(CH2)nCONH(CH2)mSO3H, -S(CH2)nCONH(CH2)mSO3 ", -S(CH2)nPO3H, -S(CH2)nPO3 ", -S(CH2CH2O)nOH, -S(CH2CH2O)nOCH3, wherein n = 1-6 and m =1-6; -NHCOR5, -COR5, -CONH2, -CONHR5, -CON(R5)2, wherein R5 is an alkyl with 1 to 6 carbon atoms and optionally contains an additional -COOH, -COO", -SO3H, -SO3 ", -PO3H or -PO3 " group1 ii) when R = H;
R2 = R3 or R2≠ R3, and R2 and R3 are substituents independently selected from the group consisting of -COOH, -COO", -SO3H, -SO3 ", -CI, -Br, -F, -I, - CF3, -CN, -OCH3, -SCH3, -O(CH2)nCOOH, -O(CH2)nCOO", -O(CH2)nCONH(CH2)mSO3H, -O(CH2)nCONH(CH2)mSO3 ", -O(CH2)nPO3H, -O(CH2)nPO3 ", -O(CH2CH2O)nOH, -O(CH2CH2O)nOCH3, -S(CH2)nCOOH, -S(CH2)nCOO", -S(CH2)nCONH(CH2)mSO3H, -S(CH2)nCONH(CH2)mSO3 ", -S(CH2)nPO3H, -S(CH2)nPO3 ", -S(CH2CH2O)nOH, -S(CH2CH2O)nOCH3, wherein n = 1-6 and m = 1-6, -NHCOR5, -COR5, -CONH2, -CONHR5, -CON(R5)2, wherein R5 is an alkyl with 1 to 6 carbon atoms and optionally contains an additional -COOH, -COO", -SO3H, -SO3 ", -PO3H or -PO3 " group; or
R2 = H and R3 is a substituent selected from the group consisting of -O(CH2)nCONH(CH2)mSO3H, -O(CH2)nCONH(CH2)mSO3-, -O(CH2)nPO3H, -O(CH2)nPO3-, -S(CH2)nCONH(CH2)mSO3H, -S(CH2)nCONH(CH2)mSO3-, -S(CH2)nPO3H, -S(CH2)nPO3 ", wherein n = 1-6 and m =1-6; and NHCOR5, -COR5, -CONH2, -CONHR5, -CON(R5)2, wherein R5 is an alkyl with 1 to 6 carbon atoms and contains an additional -COOH, -COO", -SO3H, -SO3 ", -PO3H or -PO3 " group; and c) the lanthanide ion Ln3+ is europium(lll) or samarium(lll).
2. The detectable molecule according to claim 1 characterized in that the biospecific binding reactant is selected from the group consisting of antibodies, antigens, receptor ligands, specific binding proteins or peptides, nucleic acid molecules, deoxyribonucleic acid (DNA) probes, ribonucleic acid (RNA) probes, nucleic acid derivatives and chimeric molecules comprising nucleic acids (DNA and/or RNA) and/or nucleic acid derivatives.
3. The detectable molecule according to claim 1 or 2, characterized in that it is
OCH2COONa
4. A luminescent lanthanide chelate comprising a lanthanide ion and a chelating ligand of formula (I)
wherein, a) Ri consists of a linker Z2selected from the group consisting of azido (-N3), alkylene (-C≡CH), amino (-NH2), aminooxy (-ONH2), carboxyl (-COOH), aldehyde
(-CHO), mercapto (-SH), groups or activated derivatives
thereof, and optionally a spacer, i.e. a distance-making biradical, between the phenyl and linker Z2, and said spacer is a group consisting of one to four moieties, each moiety being selected from the group consisting of phenylene, alkylene containing 1-16 carbon atoms, ethenylene (-CH=CH-), ethynediyl (-C≡C-), ether (-O-), thioether (-S-), amide (-CO-NH- and -NH-CO-, -NCH3-CO- and -CO- NCH3-), carbonyl (-CO-); b) either R3 or R is H; i) when R3 = H, R2 = R4 or R2≠ R4, and R2 and R4 are substituents independently selected from the group consisting of -COOH, -COO", -SO3H, -SO3", -CI, -Br, -F, -I, -CF3, -CN, -OCH3, -SCH3, -O(CH2)nCOOH, -O(CH2)nCOO", -O(CH2)nCONH(CH2)mSO3H, -O(CH2)nCONH(CH2)mSO3", -O(CH2)nPO3H, -O(CH2)nPO3 ", -O(CH2CH2O)nOH, -O(CH2CH2O)nOCH3, -S(CH2)nCOOH, -S(CH2)nCOO", -S(CH2)nCONH(CH2)mSO3H, -S(CH2)nCONH(CH2)mSO3 ", -S(CH2)nPO3H, -S(CH2)nPO3 " -S(CH2CH2O)nOH, -S(CH2CH2O)nOCH3, wherein n = 1-6 and m = 1-6, -NHCOR5, -COR5, -CONH2, -CONHR5, -CON(R5)2, wherein R5 is an alkyl with 1 to 6 carbon atoms and optionally contains an additional -COOH, -COO", -SO3H, -SO3 ", -PO3H or -PO3 " group; ii) when R = H,
R2 = R3 or R2≠ R3, and R2 and R3 are substituent independently selected from the group consisting of -COOH, -COO", -SO3H, -SO3 ", -CI, -Br, -F, -I, -CF3, -CN, -OCH3, -SCH3, -O(CH2)nCOOH, -O(CH2)nCOO", -O(CH2)nCONH(CH2)mSO3H, -O(CH2)nCONH(CH2)mSO3 ", -O(CH2)nPO3H, -O(CH2)nPO3 ", -O(CH2CH2O)nOH, -O(CH2CH2O)nOCH3, -S(CH2)nCOOH, -S(CH2)nCOO", -S(CH2)nCONH(CH2)mSO3H, -S(CH2)nCONH(CH2)mSO3 ", -S(CH2)nPO3H, -S(CH2)nPO3 ", -S(CH2CH2O)nOH, -S(CH2CH2O)nOCH3, wherein n = 1-6 and m = 1-6, -NHCOR5, -COR5, -CONH2, -CONHR5, -CON(R5)2, wherein R5 is an alkyl with 1 to 6 carbon atoms and optionally contains an additional -COOH, -COO", -SO3H, -SO3 ", -PO3H or -PO3 " group; or
R2 = H and R3 is a substituent selected from the group consisting of -O(CH2)nCONH(CH2)mSO3H, -O(CH2)nCONH(CH2)mSO3 ", -O(CH2)nPO3H, -O(CH2)nPO3 ", -S(CH2)nCONH(CH2)mSO3H, -S(CH2)nCONH(CH2)mSO3 ", -S(CH2)nPO3H, -S(CH2)nPO3 ", wherein n = 1-6 and m = 1-6, NHCOR5, -COR5, -CONH2, -CONHR5, -CON(R5)2, wherein R5 is an alkyl with 1 to 6 carbon atoms and contains an additional -COOH, -COO., -SO3H, -SO3", -PO3H or -PO3" group; and c) the lanthanide ion Ln3+ is europium(lll) or samarium(lll).
5. The chelate according to claim 4 characterized in that the linker Z2 is an activated derivative of an amino, aminooxy, carbonyl, aldehyde or mercapto group, preferably selected from the group consisting of an isocyanato, isothiocyanato, diazonium, bromoacetamido, iodoacetamido, reactive esters, pyridyl-2-dithio, and 6-substituted 4-chloro-1 ,3,5-triazin-2-ylamino.
6. The chelate according to claim 5, characterized in that it is selected from the group consisting of
and
7. The chelate according to claim 5 characterized in that the linker Z2 is an activated derivative of 6-substituted 4-chloro-1 ,3,5-triazin-2-ylamino, wherein the substituents are selected from the group consisting of hydrogen, halogen, alkoxy, aryloxy, amino, alkyl with 1 to 6 carbon atoms, substituted amino or thioethers, and preferable selected from the group consisting of chloro, fluoro, ethoxy, 2-methoxyethoxy, 2-cyanoethoxy, 2,2,2-trifluoroethoxy, thiophenoxy or ethoxycarbonylthiomethoxy.
8. The chelate according to claim 7 characterized in that the linker Z2 is a maleimido group, an azido group or an alkylene group.
9. Use of the detectable molecule according to claims 1 , 2 or 3 in a biospecific binding assay.
10. An assay method comprising the steps of a) contacting a sample with a detectable molecule comprising a biospecific binding reactant attached to a luminescent chelate according to any of claims 1 , 2 or 3, b) exciting said detectable molecule, and c) measuring the luminescence of said detectable molecule.
Description  (OCR text may contain errors)

LUMINESCENT LANTHANIDE LABELLING REAGENTS AND THEIR USE

FIELD OF THE INVENTION

The present invention relates to detectable lanthanide chelates to be attached to a biospecific binding reactant to form detectable molecules and use of the detectable molecules in bioaffinity based binding assays. The present invention particularly relates to detectable molecules with macrocyclic lanthanide chelates.

BACKGROUND OF THE INVENTION

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.

Time-resolved fluorometry (TRF) employing long-lifetime emitting luminescence lanthanide chelates has been applied in many specific binding assays, such as e.g. immunoassays, DNA hybridization assays, receptor-binding assays, enzymatic assays, immunocytochemical, immunohistochemical assays or cell based assays to measure wanted analyte at very low concentration (see e.g. Hemmila, I., Mukkala, V.-M., 2001 , Crit. Rev. Clin. Lab. Sci., 38, 441 , and current trends see e.g. Eliseeva, S.V., Bunzli, J.-C. G., 2010, Chem. Soc. Rev., 39, 189; Thibon, A., Pierre, V.C., 2009, Anal. Bioanal. Chem., 394, 107). Besides time- resolved fluorometry, lanthanide chelates have been used in magnetic resonance imaging (MRI) or positron emission tomography (PET).

A luminescent lanthanide chelate has to fulfil several requirements to be applicable in TRF methods 1 ) it has to be photochemical stable both in ground and excited state, 2) it has to be kinetically and chemically stable, 3) the excitation wavelength has to be as high as possible, preferably over 300 nm, 4) it has to have efficient cation emission i.e. high luminescence yield (excitation coefficient x quantum yield, εΦ), 5) the luminescence decay time has to be long, 6) the chelate has to have good water solubility, 6) for labelling it should have a reactive group to allow covalent attachment to a biospecific binding reactant, and 7) the affinity and nonspecific binding properties of the labelled biomolecules have to be retained.

The commonly observed sensitization mechanism for luminescent lanthanide chelates involves a triplet pathway, in which the excited energy transfers from a chelate ligand to a specific lanthanide ion through the ligand's triplet state. However, if a ligand presents a low-energy charge-transfer (CT) state, the sensitization can occur directly from the relaxed CT state without any participation of the triplet (Andraud, C, Maury, O., Eur. J. Inorg. Chem. 2009, 4357). Moreover, a singlet pathway for the sensitization of lanthanide luminescence has been demonstrated (see e.g. Yang, C, et al., 2004, Angew. Chem. Int. Ed., 5010), in which the excited-state energy of a ligand is directly transferred from its singlet state to the luminescence states of the lanthanide ion.

The challenge is to prepare a chelate label to fulfil all requirements in one molecule, and therefore, certain compromises are generally maid in the development of suitable labels. As consequence, a number of attempts have been made to tune the photo-physical properties of the chelate labels suitable for time- resolved fluorometric applications. These include e.g. chelate derivatives of pyridine, bipyridine, terpyridine analogous structures (see the review Hovinen., J., et al., 2009, Bioconjugate Chem., 20, 404; Eliseeva, S.V., Bunzli, J.-C. G., 2010, Chem. Soc. Rev., 39, 189; Parker, D., et al., 2010, Eur. J. Inorg. Chem., 3961 ; D'Aleo, A., et al., 2010, C.R. Chemie, 13, 681 ; Bourdolle, A., et al., 201 1 , Inorg. Chem., 4987; WO 2006/120444), phenantroline (WO2009/0213917; Quici, S., et al., 2005, Inorg. Chim. Acta, 44, 529) and various phenolic compounds (US 4,670,572; US 4,794,191 ; IT 1235668; US 2003/0027189) as the energy mediating groups (i.e. chromophore) and polycarboxylic acids as chelating parts. Moreover, various dicarboxylic derivatives (Shavaleev, N.M., et al ., 2010, Inorg. Chem., 49, 3927; Eliseeva, S.V., Bunzli, J.-C.G., 2010, Chem. Soc. Rev, 39, 189; US 5,032,677; US 5,055,578; US 4,772,563), macrocyclic Schiff bases and cryptates (see also Hovinen, J., et al., 2009, Bioconjugate Chem.; US 2008/0213917), calixarenes (Sato, N., et al., 1993, J. Chem. Soc. Perkin Trans. 2, 621 ; Steemers, F.J., et al., 1995, J. Am. Chem., 1 17, 9408), DTPA carbostyril 124 conjugate (Selvin, P.R., et al., 1994, J. Am. Chem. Soc, 1 16, 6029), DOTA based β-diketones (WO 2009/1 15644) and DOTA phenols (D'Aleo, A., et al., 2009, Helv, Chim. Acta, 92, 2439) have been disclosed. Earlier it has been shown that complexes with three individual chromophores such as 4-phenylethynylpyridines coupled in the 1 ,4,7-triazacyclononane have notable good luminescence properties with Eu(lll) ions (Takalo, H., et al. 1996, Helv. Chim. Acta, 79, 789; Latva, M, et al., 1997, J. Luminescence, 75, 149). Since the publications, the designed chelating structures have been applied in several labels containing the substituted 4-(phenylethynyl)pyridine, substituted fur-2-ylpyridine, thien-2-ylpyridine, N-alkylpyrrolylpyridine, 1 ,2,3-triazolylpyridine and trimethoxyphenylpyridine moieties (FI 8670U1 ; WO 2005/021538; US 2005/084451 ; WO 2009/030819; WO 2010/055207; WO 2008/025886; WO 2005/058877; DE 202008013312; DE 202008013314; US 2008/0167443; WO 2008/0201 13; WO 2007/128874; US 7,018,851 ; Jaakkola, L, et al., 2005, Bioconjugate Chem., 16, 700; von Lode, P., et al., 2003, Anal. Chem. 75, 3193). However, the patent applications do not disclose any practical information if improved luminescence has been obtained with the three chromophore containing 1 ,4,7-triazacyclononane derivatives compared to labels having two chromophores labels designs disclosed in the patent applications as has been obtained with the basic structures (Takalo, H., et al., 1996 Helv. Chim. Acta, 79, 789, Latva, M, et al., 1997, J. Luminescence, 75, 149).

Andraud, C, et al. (Eur. J. Inorg. Chem 2009, 4357; Inorg. Chem. 201 1 , 4987) has lately shown that ligands, which contain conjugated π-electron structure such as 4-(phenylethynyl)pyridine moiety substituted by an electron-donating substituent (such as alkoxy, alkylthio, amino groups), can be excited through ligand's CT-state to be applied in two-photon imaging. The electron-releasing substituents in the para-position of the 4-(phenylethynyl)pyridine together with electron with-drawing groups (such as carboxylic acids) in the other end of the chromophore moiety will cause a charge separation inside the ligand chromophore based on the mesomeric effect of the electron-releasing substituents. When such a CT-state exists, the excited energy can transfer through the CT state giving high quantum yield but only in apolar solvents, and thus, the luminescence is decreased in highly polar aqueous media. Moreover, when the CT-state is situated low enough compared to the excited levels of lanthanide ion, the energy back-transfer from the excited lanthanide leads to lowered quantum yield. Lately prepared two different Eu(lll) labels based on para-substituted 4-(phenylethynyl)pyridine moieties in the 1 ,4,7-triazacyclononane gave surprisingly low luminescence when used in a model immunoassay (Takalo, H., et al., 2010, a poster presentation in the 1 st International Conference on luminescence of Lanthanides Odessa, Ukraine) and the observed luminescence intensities were clearly below the other labels, which contain only two similar individual chromophores. The studied 4- (phenylethynyl)pyridine based triazacyclononanes contained electron-releasing substituents and the substituted ligand chromophores have close structural similarities with the molecules studied by Andraud et al.. Thus, the reason for the low luminescence intensities can be explained by the low-lying CT-state of the ligand, as has been published with similar type of pyridine dicarboxylic acids. Moreover, it can be assumed that any such chelate structures, both cyclic and open chain designs will suffer from the same phenomenon, and therefore, this limits chromophore structure possibilities especially if the excitation wavelength of chelate should be high (i.e. > 330 nm).

A well-known challenge with chelates and ligands having many chromophores is to find out a suitable structure design, which offers high water solubility and at the same time being inert towards any possible bioprocesses. It is known that the addition of chromophores decreases the solubility of ligands and chelates in water, increases the formation of biospecific binding reactant aggregates during the labelling process and non-specific binding properties of labelled biomolecules. Aggregates will produce purification problems and reduced yield of labelled material. Moreover, increased non-specific binding of labelled biomolecule will enhance background luminescence of biospecific assays and thus reduces assay sensitivity. OBJECTS AND SUMMARY OF THE PRESENT INVENTION

One object of the present invention is to provide a detectable molecule comprising a biospecific binding reactant attached to a luminescent lanthanide chelate.

Another object of the present invention is to provide a luminescent lanthanide chelate.

A further object of the present invention is to provide use of the detectable molecule of the invention in a biospecific binding assay.

A still further object of the present invention is to provide an assay method employing the detectable molecule. Thus, this invention provides a detectable molecule comprising a biospecific binding reactant attached to a macrocyclic luminescent lanthanide chelate comprising a lanthanide ion and a chelating ligand of the formula (I)

wherein, a) Ri consists of a linker Zi coupling said chelate to said biospecific binding reactant selected from the group consisting of thiourea (-NH-CS-NH-), aminoacetamide (-NH-CO-CH2-NH-), amide (-NH-CO- and -CO-NH-, -NCH3-CO- and -CO-NCH3-), aliphatic thioether (-S-), disulfide (-S-S-), 6-substituted-1 ,3,5-

triazine-2,4-diamine, 1 ,2,3-triazole, or , wherein n = 1-6;

and optionally a spacer, i.e. a distance-making biradical between the phenyl and linker Zi , and said spacer is a group consisting of one to four moieties, each moiety being selected from the group consisting of phenylene, alkylene containing 1 -16 carbon atoms, ethenylene (-CH=CH-), ethynediyl (-C≡C-), ether (-O-), thioether (-S-), amide (-CO-NH- and -NH-CO-, -NCH3-CO- and -CO- NCH3-), carbonyl (-CO-); b) either R3 or R is H; i) when R3 = H, R2 = R4 or R2≠ R4, and R2 and R4 are substituents independently selected from the group consisting of -COOH, -COO", -SO3H,

-SO3", -CI, -Br, -F, -I, -CF3, -CN, -OCH3, -SCH3, -O(CH2)nCOOH, -O(CH2)nCOO", -O(CH2)nCONH(CH2)mSO3H, -O(CH2)nCONH(CH2)mSO3", -O(CH2)nPO3H, -O(CH2)nPO3 "-O(CH2CH2O)nOH, -O(CH2CH2O)nOCH3, -S(CH2)nCOOH, -S(CH2)nCOO", -S(CH2)nCONH(CH2)mSO3H, -S(CH2)nCONH(CH2)mSO3 ", -S(CH2)nPO3H, -S(CH2)nPO3 ", -S(CH2CH2O)nOH,

-S(CH2CH2O)nOCH3, wherein n = 1-6 and m =1-6; -NHCOR5, -COR5, -CONH2, -CONHR5, -CON(R5)2, wherein R5 is an alkyl with 1 to 6 carbon atoms and optionally contains an additional -COOH, -COO", -SO3H, -SO3", -PO3H or -PO3" group1 ii) when R4 = H;

R2 = R3 or R2≠ R3, and R2 and R3 are substituents independently selected from the group consisting of -COOH, -COO", -SO3H, -SO3 ", -CI, -Br, -F, -I, -CF3, -CN, -OCH3, -SCH3, -O(CH2)nCOOH, -O(CH2)nCOO", -O(CH2)nCONH(CH2)mSO3H, -O(CH2)nCONH(CH2)mSO3-, -O(CH2)nPO3H, -Ο(ΟΗ2)ηΡΟ3 ", -O(CH2CH2O)nOH, -O(CH2CH2O)nOCH3, -S(CH2)nCOOH, -S(CH2)nCOO", -S(CH2)nCONH(CH2)mSO3H, -S(CH2)nCONH(CH2)mSO3 ", -S(CH2)nPO3H, -S(CH2)nPO3 ", -S(CH2CH2O)nOH, -S(CH2CH2O)nOCH3j wherein n = 1-6 and m = 1-6, -NHCOR5, -COR5, -CONH2, -CONHR5, -CON(R5)2, wherein R5 is an alkyl with 1 to 6 carbon atoms and optionally contains an additional -COOH, -COO", -SO3H, -SO3 ", -PO3H or -PO3 " group; or

R2 = H and R3 is a substituent selected from the group consisting of -O(CH2)nCONH(CH2)mSO3H, -O(CH2)nCONH(CH2)mSO3-, -O(CH2)nPO3H, -O(CH2)nPO3-, -S(CH2)nCONH(CH2)mSO3H, -S(CH2)nCONH(CH2)mSO3-, -S(CH2)nPO3H, -S(CH2)nPO3 ", wherein n = 1-6 and m =1-6; and NHCOR5, -COR5, -CONH2, -CONHR5, -CON(R5)2, wherein R5 is an alkyl with 1 to 6 carbon atoms and contains an additional -COOH, -COO", -SO3H, -SO3 ", -PO3H or -PO3 " group; and the lanthanide ion Ln3+ is europium(lll) or samarium(lll).

The invention also provides a macrocyclic luminescent lanthanide comprising a lanthanide ion and a chelating ligand of formula (I)

wherein, a) Ri consists of a linker Z2selected from the group consisting of azido (-N3), alkylene (-C≡CH), amino (-NH2), aminooxy (-ONH2), carboxyl (-COOH), aldehyde

(-CHO), mercapto (-SH), groups or activated derivatives

thereof, and optionally a spacer, i.e. a distance-making biradical between the phenyl and linker Z2, and said spacer is a group consisting of one to four moieties, each moiety being selected from the group consisting of phenylene, alkylene containing 1 -16 carbon atoms, ethenylene (-CH=CH-), ethynediyl (-C≡C-), ether (-O-), thioether (-S-), amide (-CO-NH- and -NH-CO-, -NCH3-CO- and -CO- NCH3-), carbonyl (-CO-); b) either R3 or R is H; i) when R3 = H, R2 = R4 or R2≠ R4, and R2 and R4 are substituents independently selected from the group consisting of -COOH, -COO", -SO3H,

-SO3", -CI, -Br, -F, -I, -CF3, -CN, -OCH3, -SCH3, -O(CH2)nCOOH, -O(CH2)nCOO", -O(CH2)nCONH(CH2)mSO3H, -O(CH2)nCONH(CH2)mSO3", -O(CH2)nPO3H, -O(CH2)nPO3 ", -O(CH2CH2O)nOH, -O(CH2CH2O)nOCH3, -S(CH2)nCOOH, -S(CH2)nCOO", -S(CH2)nCONH(CH2)mSO3H, -S(CH2)nCONH(CH2)mSO3 ", -S(CH2)nPO3H, -S(CH2)nPO3 " -S(CH2CH2O)nOH,

-S(CH2CH2O)nOCH3, wherein n = 1-6 and m = 1-6, -NHCOR5, -COR5, -CONH2, -CONHR5, -CON(R5)2, wherein R5 is an alkyl with 1 to 6 carbon atoms and optionally contains an additional -COOH, -COO", -SO3H, -SO3", -PO3H or -PO3" group; ii) when R4 = H,

R2 = R3 or R2≠ R3, and R2 and R3 are substituent independently selected from the group consisting of -COOH, -COO", -SO3H, -SO3 ", -CI, -Br, -F, -I, -CF3, -CN, -OCH3, -SCH3, -O(CH2)nCOOH, -O(CH2)nCOO", -O(CH2)nCONH(CH2)mSO3H, -O(CH2)nCONH(CH2)mSO3 ", -O(CH2)nPO3H, -O(CH2)nPO3 ", -O(CH2CH2O)nOH, -O(CH2CH2O)nOCH3, -S(CH2)nCOOH,

-S(CH2)nCOO", -S(CH2)nCONH(CH2)mSO3H, -S(CH2)nCONH(CH2)mSO3 ", -S(CH2)nPO3H, -S(CH2)nPO3 ", -S(CH2CH2O)nOH, -S(CH2CH2O)nOCH3, wherein n = 1-6 and m = 1-6, -NHCOR5, -COR5, -CONH2, -CONHR5, -CON(R5)2, wherein R5 is an alkyl with 1 to 6 carbon atoms and optionally contains an additional -COOH, -COO", -SO3H, -SO3 ", -PO3H or -PO3 " group; or

R2 = H and R3 is a substituent selected from the group consisting of -O(CH2)nCONH(CH2)mSO3H, -O(CH2)nCON H(CH2)mSO3 ", -O(CH2)nPO3H, -O(CH2)nPO3 ", -S(CH2)nCONH(CH2)mSO3H, -S(CH2)nCONH(CH2)mSO3 ", -S(CH2)nPO3H, -S(CH2)nPO3 ", wherein n = 1-6 and m = 1-6, NHCOR5, -COR5, -CONH2, -CONHR5, -CON(R5)2, wherein R5 is an alkyl with 1 to 6 carbon atoms and contains an additional -COOH, -COO., -SO3H, -SO3", -PO3H or -PO3" group; and c) the lanthanide ion Ln3+ is europium(lll) or samarium(lll). The invention further provides use of the detectable molecule according to the invention in a biospecific binding assays.

The invention still further provides an assay method comprising the steps of a) contacting a sample with a detectable molecule comprising a biospecific binding reactant attached to a luminescent chelate according to any of claims 1 , 2 or 3, b) exciting said detectable molecule, and c) measuring the luminescence of said detectable molecule.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention providing a nonadentate lanthanide chelate and a labelled biomolecule based on 1 ,4,7-triazacyclononane decreases the possibility of the charge separation inside the ligand chromophores compared to prior art chelates. The existence of the low-lying CT-state is prevented, and a more luminescent lanthanide chelate and labelled biomolecule is made possible, and moreover at the same time, all important features of chelates and labeled biomolecules can be retained without any additional forming of aggregates and purification problems. The invention provides chelates which have triplet states high enough to be used for efficient lanthanide excitation. The chelates and labeled biomolecules according to the present invention have exceptional and surprisingly high luminescence and complex stability without any significant background increase and hardly any reduction of the antibody's affinity although the labeled model antibody contained even 20-30 Eu chelates/lgG.

The aim of the present invention is to provide means to obtain improved lanthanide chelate labels to be used in specific bioaffinity based binding assays, such as immunoassays (both heterogeneous and homogenous assays), nucleic acid hybridization assays, receptor binding assays, enzymatic assays, immunocytochemical, immunohistochemical or cell based assays utilizing fluorometric or time-resolved fluorometric determination of the specific luminescence.

The chelates of the present invention aim to combine several important features in a single label, such as

1 . high absorptive, preferable over 50 000 cm"1, at suitable wavelength, preferable over 300 nm, 2. three separate UV absorbing parts (chromophores) in the same ligand structure to allow the high absorptivity of the reactant,

3. effective energy transfer from the chromophores to the lanthanide ion,

4. functional groups at suitable positions in the chromophores to allow the UV excitation over 300 nm and to prevent the possibility of the charge separation through the ligand chromophores during the UV excitation,

5. functional groups to allow high water solubility and to prevent formation of aggregates during labelling of biomolecules or storing the labelled reactants and unspecific binding of labelled biomolecules,

6. a strongly chelating part to create a) the thermodynamic stability required for storing the labelled reactants for extended periods of time, and b) high kinetic stability to allow the use of labelled reactants in conditions where competing metal ions or chelating agents may be present enabling the use of the reactants e.g. in in-vivo assays without any toxic effects, or improving performance of present PCR- based assays, 7. a chelating part for as complete protection of the chelated ion against well- known luminescence quenching of water molecules as possible,

8. a lanthanide chelate can be electrically neutral, which improves the chelate's capability to penetrate through cell membranes, and thus, allows broad usability for in-vivo labelling and imaging, 9. symmetry, i.e. a ligand field with the chelate of the invention emphasizes the emission line intensity at ca. 615 nm over the other emissions lines, allowing high luminescence intensity maximizing the signal to noise ratio (sg/n) in time-resolved fluorescence resonance energy transfer (TR-FRET) assay applications over chelates presently used,

10. a functional group allowing effective coupling of the chelate to be used as a binding reactant (e.g. antibody) without destroying its binding properties and decreasing the luminescence properties of the label.

The term biospecific binding reactant as used herein refers to any substance or molecule capable of binding another molecule. The biospecific binding reactant is typically selected from a group consisting of an antibody, an antigen, a receptor ligand, a specific binding protein or peptide, a nucleic acid molecule, and a deoxyribonucleic acid (DNA) probe, a ribonucleic acid (RNA) probe, nucleic acid derivatives [such as peptide nucleic acid (PNA) and locked nucleic acid (LNA)] and chimeric molecules comprising nucleic acids (DNA and/or RNA) and/or nucleic acid derivatives, including but limited to chimeric probe molecules comprising DNA, RNA, PNA and/or LNA.

The term biospecific binding assay as used herein refers to methods and techniques that are used to determine the presence or the presence and quantity of an analyte molecule in a sample. Biospecific binding assays include but are not limited to immunoassays, nucleic acid hybridization assays, nucleic acid amplification assays, based on e.g. polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence based amplification (NASBA) or rolling circle amplification reaction, receptor-binding assays, enzymatic assays and cellular binding assays. Biospecific binding assays can be homogeneous or heterogeneous. In homogeneous assays all assay components are present in one solution phase and it is not necessary to physically separate bound label molecules from unbound label molecules to detect biospecific binding events, whereas, in heterogeneous assays, biospecific binding events only become detectable after separation of bound label from unbound label. The present invention concerns detectable molecules comprising a biospecific binding reactant attached to a luminescent lanthanide chelate as defined above.

Methods applicable for attaching the biospecific binding reactant to the luminescent lanthanide are disclosed in prior art, e.g. in Bioconjugate Techniques, G.T. Hermanson, Academic Press (1996).

The present invention also concerns the use of the detectable molecules of the invention in biospecific binding assays. In these assays the improvement in comparison to prior art comprises using the detectable molecule of the invention instead of detectable molecules of prior art. The present invention further concerns a luminescent lanthanide chelate as defined above.

If the linker Z2 of the invention is an activated derivative of an amino, aminooxy, carbonyl, aldehyde or mercapto group it can preferably be selected from the group consisting of an isocyanato, isothiocyanato, diazonium, bromoacetamido, iodoacetamido, reactive esters, pyridyl-2-dithio, and 6-substituted 4-chloro-1 ,3,5- triazin-2-ylamino. The linker Z2 can also be a maleimido group for thiol group labelling or an azido (-N3) or an alkylene (-C≡CH) group. Azido and acetylene groups are used for so-called click type labelling using copper(l) catalyzed Huisgen-Sharpless dipolar [2+3] cycloaddition reaction between azido and terminal alkyne and vice verse.

The substituents in 6-substituted 4-chloro-1 ,3,5-triazin-2-ylamino can be selected from the group consisting of hydrogen, halogen, alkoxy, aryloxy, amino, alkyl with 1 to 6 carbon atoms, substituted amino or thioethers, and preferable selected from the group consisting of chloro, fluoro, ethoxy, 2-methoxyethoxy, 2-cyanoethoxy, 2,2,2-trifluoroethoxy, thiophenoxy or ethoxycarbonylthiomethoxy. The substituted amino or thioether is preferably mono- or disubstituted each substitution being preferably independently selected from the group consisting of an alkyl or alkoxy with 1 to 6 carbon atoms, phenyl, carbonyl and carboxyl. It should be understood Ri may include a spacer, i.e. a distance-making biradical, so as - if necessary or desirable - to position the reactive group Zi or Z2 in a position accessible for reaction with the biospecific binding reactant. The spacer may be readily introduced in the course of the synthesis of the ligand or the chelate. The spacer is a group consisting of one to four moieties, each moiety being selected from the group consisting of phenylene, alkylene containing 1-16 carbon atoms, ethenylene (-CH=CH-), ethynediyl (-C≡C-), ether (-O-), thioether (-S-), amide (-CO-NH- and -NH-CO-, -NCH3-CO- and -CO-NCH3-) and carbonyl (-CO-). If the Ri group is to be attached to a solid support it preferably has a reactive group, which is a polymerizing group.

With different R2, R3 and R4 groups the excitation wavelength can be shifted to a longer wavelength without promotion of a low-lying CT-state. Preferable are R2-4 groups, which contain additional hydrophilic moieties, such as e.g. -COOH, -SO3H, -PO3H and any salt forms thereof.

If the ligand is to be used in peptide or oligonucleotide synthesis as means to prepare a label peptide or a labelled oligonucleotide as described in e.g. US 2005/0181393 it is preferable to use a functional group for attaching the ligand to the peptide's or oligonucleotide's back-bone during the synthesis in Ri as described e.g. US 2005/0181393.

OCH2COONa

Preferable detectable molecules are

and

wherein

OCH2COONa

a specific binding reactant is attached to the luminescent lanthanide chelates 21 and 22 of the examples described hereafter.

A detectable molecule according to the invention can e.g. be used in a heterogeneous immunoassay wherein an analyte to be measured is added into a well of a microtiter plate coated with a molecule that can recognize the analyte. After incubation in the well for a sufficient time the well is washed according to a suitable protocol and the detectable molecule labelled with the lanthanide chelate is added into the well. After a further incubation step the well is washed again and the signal coming from the lanthanide chelate is measured according to a suitable protocol and using suitable instrumentation.

A lanthanide chelate according to the invention can e.g. be used in a homogeneous protease assay wherein a protease substrate is labelled at one end with a chelate according to the invention and at the other end with a suitable quencher molecule. When the molecule is intact its signal is minimal. When added into a measurement chamber with protease the substrate molecule is cut and the lanthanide chelate and the quencher are separated. The activity of the protease can be measured as an increase in the signal coming from the lanthanide chelate.

A detectable molecule according to the present invention can e.g. be used in a homogeneous kinase assay. Kinases are enzymes which add phosphor groups into certain amino acids, like tyrosine, serine and threonine, in proteins. Kinase activity can be measured e.g. by using artificial substrates having biotin at one of the ends of the substrate and the amino acid to be phosphorylated at the other end. When the substrate gets phosphorylated it can be detected by using a molecule, e.g. an antibody, recognizing the phosphorylated form labelled with the lanthanide. To make the assay homogeneous a suitable acceptor molecule needs to be in the proximity of the lanthanide chelate. In the kinase assay this is possible e.g. by utilizing an acceptor labelled streptavidin. When the acceptor and the chelate are in proximity, like is the case in phosphorylated kinase substrate, the energy transfer from the lanthanide to the acceptor can be measured using suitable instrumentation.

It will be appreciated that the methods of the present invention can be incorporated in a form of a variety of embodiments, only few of which are disclosed herein. It will apparent for the specialist in the field that other embodiments exist and do not depart from the split of the invention. Thus, the described embodiments are illustrative and should not be constructed as restrictive.

Following examples further elucidate the invention. The structures and the synthetic routes employed in the experimental part are described in Schemes 1-4. The synthetic experimental details are given in Examples 1- 23. Introduction of the labelling reagents 21 and 22 into an IgG is described in Example 24. The performed immunoassay is described in Example 25. The results indicate surprising high signal compared to a reference compound. Examples

FC = Flash chromatography, RT = room temperature. Microwave synthesizer was Initiator system (Biotage). 1H NMR and 13C NMR spectra were recorded on a Bruker AVANCE 500 DRX (Bruker, Karlsruhe, Germany) by using SiMe4 as internal standard. Mass spectra were recorded on a Voyager DE Pro (Applied Biosystems, Foster City, CA) mass spectrometer. HPLC runs were performed by Dionex's Ultimate 3000 system including Dionex's Ultimate 3000 Diode Array Detector. The used column was ThermoHypersil 150x4 mm 5 μ Hypersil ODS. Runs were performed by using an eluent gradient starting from 75 % H20, 20% 100 mM TEAA and 5% CH3CN and within 30 min to 30% H20, 20% triethylammonium acetate (TEAA) and 50% CH3CN. Fluorescence measurements were performed with 1420 Victor Multilabel Counter (Perkin Elmer).

Example 1

Synthesis of diethyl 4-bromophthalate (1) A solution of 4-bromophtalic acid (2.55 g, 10.4 mmol) and sulphuric acid (0.4 ml) in dry EtOH (70ml) was refluxed 53 hours. The reaction mixture was diluted with CH2CI2 (100 ml), neutralized with 5 % aq. NaHCO3 (30 ml), washed with H2O (40 ml) and dried with Na2SO4. The product was purified by FC (silica gel, CH2CI2). Yield: 2.1 1 g (67 %). 1H NMR (CDCI3): δ (ppm) 7.82 (d, J=1 .9 Hz, 1 H), 7.66 (dd, J=1 .9 Hz, J=8.3 Hz, 1 H), 7.62 (d, J=8.3 Hz, 1 H), 4.37 (q, J=7.1 Hz, 4H), 1 .37 (t, J=7.1 Hz, 6H) 13C NMR (CDCI3): δ (ppm) 167.00, 166.77, 134.71 , 134.25, 132.16, 131 .02, 130.95, 125.97, 62.42, 62.25, 14.47. Maldi-TOF MS for Ci2Hi3BrO4: calculated 300.00 and 302.00, found 300.94 and 302.94 (M + 1 ).

Example 2 Synthesis of dimethyl 2,2'-[(5-bromo-1,3-phenylene)bis(oxy)]diacetate (2)

After addition of NaH (0.76 g, 31 .7 mmol) into a solution of 5-bromobenzene-1 ,3- diol (2.72 g, 14.4 mmol) (Hille, U.E., et al., 2009, Eur. J. Med. Chem, 44, 2765) in dry DMF (34 ml), the mixture was stirred at RT for 20 minutes. Methyl bromoacetate (3.04 ml, 31 .6 mmol) was added and the stirring was continued for 2,5 hours at RT. The reaction mixture was diluted with CH2CI2 (85 ml), washed with 5 % aq. NaHCO3 (70 ml), H2O (2x70 ml) and dried with Na2SO4. The product was purified by FC (silica gel, from CH2CI2 to 1 % MeOH in CH2CI2. Yield: 4.2 g (88 %). 1H NMR (CDCI3): δ (ppm)) 6.70 (d, J=2.3 Hz, 2H), 6.43 (t, J=2.3 Hz, 1 H), 4.59 (s, 4H), 3.81 (s, 6H). 13C NMR (CDCI3): δ (ppm) 168.68, 159.31 , 123.12, 1 1 1 .53, 101 .23, 65.31 , 52.42. Maldi-TOF MS for Ci2Hi3BrO6: calculated 331 .99 and 333.99, found 333.90 and 335.92 (M+1 ).

Example 3 Synthesis of diethyl 4-[(trimethylsilyl)ethynyl]phthalate (3)

A mixture of compound 1 (2.0 g, 6.64 mmol), bis(triphenylphosphine)palladium (II) chloride (93 mg, 0.13 mmol), Cul (50 mg, 0.26 mmol) in dry Et3N (15,0 ml) and THF (20 ml) was dearated with argon. (Trimethylsilyl)acetylene (1 .1 ml, 7.7 mmol) was added and the mixture was stirred at 55C for 2,5 hours. The mixture was filtered, the filtrate evaporated, the residue was dissolved in CHCI3 (120 ml), washed with H2O (2 x 30ml) and dried with Na2SO4. The product was purified by FC (silica gel, 10 % AcOEt in petroleum ether (b.p. 40-65 C)). Yield: 1 .55 g (73 %). 1H NMR (CDCI3): δ (ppm)) 7.77 (d, J=1 .5 Hz, 1 H), 7.68 (d, J=8.1 Hz,1 H), 7.57 (dd, J=1 .5 Hz, J=8.1 Hz, 1 H), 4.3645 (q, J=7.2 Hz, 2H), 4.358 (q, J=7.1 Hz, 2H), 1 .37 (t, J=7.2 Hz, 3H), 1 .37 (t, J=7.1 Hz 3H) 13C NMR (CDCI3): δ (ppm) 167.42, 167.28, 134.23, 133.13, 132.53, 131 .65, 130.94, 129.38, 103.37, 98.45, 62.20, 62.15, 14.49, 14.48, 0.169. Maldi-TOF MS for Ci7H22O4Si: calculated 318.44, found 319.97 (M+1 ).

Example 4 Synthesis of dimethyl 2,2'-{{5-[(trimethylsilyl)ethynyl]-1,3-phenylene}bis- (oxy)}-diacetate (4)

A mixture of compound 2 (0.53 g, 1 .59 mmol), bis(triphenylphosphine)palladium (II) chloride (56 mg, 79 μηηοΙ), Cul (15 mg, 79 μηηοΙ) in dry DMF (2.6 ml) and DEA (8.0 ml) was dearated with argon. (Trimethylsilyl)acetylene (0.30 ml, 1 .90 mmol) was added and the reaction was performed in microwave synthesizer at 120 C for 25 minutes. After evaporation to dryness, the product was purified by FC (silica gel, from CH2CI2 to 1 % MeOH in CH2CI2. Yield: 0.37 g (66 %). 1H NMR (CDCI3): δ (ppm)) 6.63 (d, J=2.3 Hz, 2H), 6.50 (t, J=2.3 Hz, 1 H), 4.60 (s, 4H), 3.81 (s, 6H), 0.24 (s, 9H). 13C NMR (CDCI3): δ (ppm) 169.03, 158.69, 124.99, 1 1 1 .36, 104.37, 103.69, 94.80, 65.40, 52.46, 0.00. Maldi-TOF MS for Ci7H22O6Si: calculated 350.12, found 352.18 (M+2).

Example 5

Synthesis of diethyl 4-ethynylphthalate (5) A solution of compound 3 (1 .55 g, 4.90 mmol) in dry CH2CI2 (50 ml) was dearated with argon. After addition of tetrabutylammonium fluoride (1 .53 g, 5.85 mmol), the mixture was stirred at RT for 1 .5 hours, washed with 10 % aq. citric acid (40 ml), H2O (4 x 60 ml) and dried with Na2SO4. The product was purified by FC (silica gel, 10 % AcOEt in petroleum ether (b.p. 40-65 C)). Yield: 0.60 g (50 %). 1H NMR (CDCI3): δ (ppm)) 7.81 (d, J=1 .5 Hz, 1 H), 7.70 (d, J=8.0 Hz,1 H), 7.62 (dd, J=1 .5 Hz, J=8.0 Hz, 1 H), 4.372 (q, J=7.1 Hz, 2H), 4.367 (q, J=7.1 Hz, 2H), 3.23 (s,1 H), 1 .373 (t, J=7.1 Hz, 3H), 1 .367 (t, J=7.1 Hz, 3H) 13C NMR (CDCI3): δ (ppm) 167.27, 167.25, 134.58, 133.09, 132.77, 132.29, 129.43, 125.66, 82.02, 80.67, 62.28, 62.23, 14.48. Maldi-TOF MS for Ci4Hi4O4: calculated 246.09, found 247.98 (M+1 ).

Example 6

Synthesis of dimethyl 2,2'-[(5-ethynyl-1,3-phenylene)bis(oxy)]diacetate (6)

This compound 6 was synthesized from the compound 4 using a method analogous to the synthesis described in the Example 5. The product was purified by FC (silica gel, 40 % AcOEt in petroleum ether (b.p. 40-65 C)). Yield: 49 %. 1H NMR (CDCI3): δ (ppm) 6.67 (d, J=2.3 Hz, 2H), 6.53 (t, J=2.3 Hz, 1 H), 4.61 (s, 4H), 3.81 (s, 6H). 13C NMR (CDCI3): δ (ppm) 168.31 , 158.08, 123.30, 1 10.93, 103.07, 82.40, 76.97, 64.71 , 51 .84. Maldi-TOF MS for Ci4Hi4O6: calculated 278.08, found 280.10 (M+2). Example 7

Synthesis of N-(4-ethynylphenyl)-2,2,2-trifluoroacetamide (7)

4-Ethynylaniline (4.0 g, 34.14 mmol) was added in small portions to a ice-cold trifluoroacetic acid anhydride (19.2 ml, 0.135 mmol). After stirring for 2.5 hours at RT, cold H2O (100 ml) was added, the mixture filtrated and the product washed with cold H2O (2 x 40 ml). Yield: 7.1 g (98 %). 1H NMR (CDCI3): δ (ppm)) 8.08 (s,br, 1 H), 7.55 (d, J=8.8 Hz, 2H), 7.51 (d, J=8.8 Hz, 2H), 3.1 (s, 1 H). 13C NMR (CDCIs): δ (ppm) 154.72, 135.40, 133.23, 129.92, 120.25, 120.51 , 82.67, 77.99. Maldi-TOF MS for Ci0H6F3NO: calculated 213.04, found 213.05. Example 8

Synthesis of ethyl 3-bromo-5-(hydroxymethyl)benzoate (8)

Diethyl 4-bromo-2,6-pyridinedicarboxylate (2.0 g, 6.62 mmol) (Takalo, H., et al., 1988, Acta Chem Scand B 42, 373) was suspended in EtOH (34 ml) and sodium borohydride (0.25 g, 6.61 mmol) was added in small portions. After stirring for 1 hour at RT, the pH was adjusted to 3-3.5 with 6 M HCI and the mixture was evaporated to dryness. After addition of H2O (20 ml), the mixture was neutralized with solid NaHCO3, extracted with 10 % EtOH in CH2CI2 (3 x 35 ml) and dried with Na2SO4. The product was purified by FC (silica gel, 2 % EtOH in CH2CI2. Yield: 0.94 g (55 %). 1H NMR (CDCI3): δ (ppm)) 8.16 (d, J=1 .7 Hz, 1 H), 7.75 (d, J=1 .7 Hz, 1 H), 4.85 (s, 2H), 4.47 (q, J=7.1 Hz, 2H), 3.45 (s, br, 1 H), 1 .43 (t, J=7.1 Hz, 3H). 13C NMR (CDCI3): δ (ppm) 163.93, 161 .79, 148.31 , 134.45, 127.13, 127.00, 62.32, 62.38, 14.27. Maldi-TOF MS for Ci0Hn BrNO3: calculated 258.98 and 260.98, found 260.97 and 262.97 (M+2). Example 9

Synthesis of diethyl 4-[(2-ethoxycarbonyl)-6-(hydroxymethyl)pyridin-4-yl]- ethynyl)phthalate (9)

A mixture of compound 5 (0.59 g, 2.39 mmol) and 8 (0.57 g, 2.17 mmol) in dry Et3N (4.3 ml) and THF (6.0 ml) was dearated with argon. After addition of bis(triphenylphosphine)palladium (II) chloride (31 mg, 44 μηηοΙ) and Cul (17 mg, 89 μΐηοΙ), the mixture was stirred overnight at 55C, filtered and the filtrate evaporated to dryness. The product was purified by FC (silica gel, 50 % AcOEt in petroleum ether (b.p. 40-65 C)). Yield was 0.65 g (70 %). 1H NMR (CDCI3): δ (ppm)) 8.10 (s, 1 H), 7.90 (d, J=1 .5 Hz, 1 H), 7.76 (d, J=8.0 Hz, 1 H), 7.70 (dd, J=1 .5 Hz, J=8.0 Hz, 1 H), 7.64(s, 1 H), 4.88 (d, J=4.2 Hz, 2H), 4.49 (q, J=7.1 Hz, 2H), 4.40 (q, J=7.2 Hz, 2H), 4.39 (q, J=7.1 Hz, 2H), 1 .45 (t, J=7.1 Hz, 3H), 1 .40 (t, J=7.2 Hz, 3H), 1 .38 (t, J=7.1 Hz, 3H). 13C NMR (CDCI3): δ (ppm) 167.19, 167.07, 164.87, 161 .16, 148.10, 134.35, 133.25, 132.90, 132.88, 132.65, 129.64, 126.10, 125.79, 125.18, 93.28, 89.06, 64.87, 62.59, 62.40, 62.36, 14.70, 14.50, 14.48. Maldi-TOF MS for C23H23NO7: calculated 425.15, found 427.1 1 (M+2).

Example 10

Synthesis of dimethyl 2,2'-{{5-{[2-(ethoxycarbonyl)-6-(hydroxymethyl)- pyridin-4-yl)ethynyl]-1,3-phenylene}bis(oxy}}diacetate (10) This compound 10 was synthesized from the compound 6 using a method analogous to the synthesis described in the Example 9. The product was purified by FC (silica gel, from 20 % to 40 % AcOEt in petroleum ether (b.p. 40-65 C)). Yield was 74 %. 1H NMR (CDCI3): δ (ppm)) 8.08 (s, 1 H), 7.61 (s, 1 H), 6.73 (d, J=2.3 Hz, 2H), 6.58 (t, J=2.3 Hz, 1 H), 4.86 (s, 2H), 4.65 (s, 4H), 4.48 (q, J=7.1 Hz, 2H), 3.83 (s, 6H), 1 .45 (t, J=7.1 Hz, 3H). 13C NMR (CDCI3): δ (ppm) 168.83, 164.59, 160.55, 158.84, 147.54, 133.00, 125.75, 125.45, 123.47, 1 1 1 .28, 104.30, 94.41 , 86.18, 65.32, 62.17, 52.44, 14.31 . Maldi-TOF MS for C23H23NO9: calculated 457.14, found 459.20 (M+2). Example 1 1

Synthesis of ethyl 6-(hydroxymethyl)-4-{[4-(2,2,2-trifluoroacetamido)phenyl]- ethynyl}picolinate (11)

This compound 1 1 was synthesized from the compound 7 using a method analogous to the synthesis described in the Example 9. The product was purified by FC (silica gel, from 2 % to 5 % MeOH in CH2CI2. Yield: 90 %. 1H NMR (CDCI3): δ (ppm)) 8.16 (s, br, 1 H), 8.09 (s, 1 H), 7.66 (d, J=8.7 Hz, 2H), 7.60 (s, 1 H), 7.59 (d, J=8.7 Hz, 2H), 4.86 (s, 2H), 4.48 (q, J=7.1 Hz, 2H), 1 .45 (t, J=7.1 Hz, 3H). 13C NMR (CDCIs): δ (ppm) 166.14, 165.00, 160.79, 155.30, 148.02, 136.53, 133.54, 126.10, 125.68, 124.23, 120.71 , 1 19.99, 94.60, 87.14, 64.80, 62.55, 14.69. Maldi- TOF MS for Ci9Hi5F3N2O4 : calculated 392.10, found 393.95 (M + 1 ).

Example 12

Synthesis of diethyl 4-[(2-bromomethyl)-6-(ethoxycarbonyl)pyridin-4-yl)- ethynyljphthalate (12) After addition of PBr3 (0.167 ml, 1 .78 mmol) in a solution of compound 9 (0.63 g, 1 .48 mmol) and dry CHCI3 (28 ml), the mixture was stirred at RT for 1 .5 hours. The mixture was neutralized with 5 % aq. NaHCO3 (-10 ml), the aqueous phase extracted with chloroform (3 x 25 ml) and the combined organic fractions dried with Na2SO4. The product was purified by FC (silica gel, from CH2CI2 to 1 % EtOH in CH2CI2. Yield: 0.49 g (68 %). 1H NMR (CDCI3): δ (ppm)) 8.12 (s, 1 H), 7.91 (d, J=1 .3 Hz, 1 H), 7.89 (s, 1 H), 7.77 (d, J=8.1 Hz, 1 H), 7.70 (dd, J=1 .3 Hz, J=8.1 Hz, 1 H), 4.65 (s, 2H), 4.50 (q, J=7.1 Hz, 2H), 4.40 (q, J=7.1 Hz, 2H), 4.39 (q, J=7.1 Hz, 2H), 1 .45 (t, J=7.1 Hz, 3H), 1 .39 (q, 6H). 13C NMR (CDCI3): δ (ppm) 166.79, 166.63, 164.27, 157.94, 148.33, 133.99, 133.06, 132.89, 132.65, 132.32, 129.28, 128.53, 126.24, 124.66, 93.33, 89.29, 62.37, 62.03, 61 .98, 32.73, 14.33, 14.13, 14.1 1 . Maldi-TOF MS for C23H22BrO6: calculated 487.06 and 489.06, found 488.67 and 490.69 (M+1 ). Example 13

Synthesis of dimethyl 2,2'-{{5-{[2-(bromomethyl)-6-(ethoxycarbonyl)pyridin- 4-yl]ethynyl}-1,3-phenylene}bis(oxy)}diacetate ( 13)

This compound 13 was synthesized from the compound 10 using a method analogous to the synthesis described in the Example 12. The product was purified by FC (silica gel, 5 % MeOH in CH2CI2.Yield: 88 %. 1H NMR (CDCI3): δ (ppm)) 8.09 (d, J=1 .3 Hz 1 H), 7.75 (d, J=1 .3 Hz 1 H), 6.74 (d, J=2.3 Hz, 2H), 6.59 (t, J=2.3 Hz, 1 H), 4.65 (s, 4H), 4.64 (s, 2H), 4.50 (q, J=7.1 Hz, 2H), 3.83 (s, 6H), 1 .45 (t, J=7.1 Hz, 3H). 13C NMR (CDCI3): δ (ppm) 168.77, 164.35, 158.90, 157.76, 148.22, 133.51 , 128.50, 126.23, 123.36, 1 1 1 .34, 104.47, 94.84, 85.85, 65.37, 62.29, 52.40, 32.80, 14.31 . Maldi-TOF MS for C23H22BrNO8: calculated 519.05 and 521 .05, found 520.95 and 522.98 (M+1 ).

Example 14

Synthesis of ethyl 6-(bromomethyl)-4-{[4-(2,2,2-trifluoroacetamido)phenyl]- ethynyl}picolinate ( 14)

This compound 14 was synthesized from the compound 11 using a method analogous to the synthesis described in the Example 12. The product was purified by FC (silica gel, from CH2CI2 to 2 % MeOH in CH2CI2.Yield: 76 %. 1H NMR (CDCI3): δ (ppm)) 8.30 (s, br, 1 H), 8.1 (d, J=1 .3 Hz, 1 H), 7.75 (d, J=1 .3 Hz, 1 H), 7.69 (d, J=8.7 Hz, 2H), 7.60 (d, J=8.7 Hz, 2H), 4.62 (s, 2H), 4.50 (q, J=7.1 Hz, 2H), 1 .45 (t, J=7.1 Hz, 3H). 13C NMR (CDCI3): δ (ppm) 164.42, 157.77, 154.81 , 148.17, 138.10, 136.34, 133.68, 133.15, 128.44, 126.17, 120.37, 1 19.40, 94.71 , 86.38, 62.33, 32.72, 14.29. Maldi-TOF MS for Ci9Hi4BrF3N2O3: calculated 454.01 , found 455.87 (M + 1 ). Example 15

Synthesis of di-tert-butyl 7-{{6-(ethoxycarbonyl)-4-{[4-(2,2,2-trifluoroacet- amido)phenyl]ethynyl}pyridin-2-yl}methyl}-1,4,7-triazacyclonane-1,4- dicarboxylate (15) A mixture of compound 14 (0.63 g, 1 .39 mmol), di-terf-butyl 1 ,4,7-triazacyclonane- 1 ,4-dicarboxylate (0.42 g, 1 .28 mmol), dry K2CO3 (0.84 g, 6.06 mmol) and dry acetonitrile (1 1 ml) was stirred at RT for 22 hours under argon. The mixture was and the filtrate evaporated to dryness. The product was purified by FC (silica gel, 2 % EtOH in CH2CI2. Yield: 0.87 g (97 %). 1H NMR (CDCI3): δ (ppm)) 8.67 (s, br,1 H), 8.05 (d, J=1 .1 Hz, 1 H), 7.91 (d, J=1 .1 Hz, 1 H), 7.70 (d, J=8.7 Hz, 2H), 7.57 (d, J=8.7 Hz, 2H), 4.47 (q, J=7.1 Hz, 2H), 3.91 (s, 2H), 3.56-3.12 (m, 8H), 2.75-2.64 (m, 4H), 1 .49 (s, 9H), 1 .45 (s, 9H), 1 .43 (t, J=7.1 Hz, 3H). 13C NMR (CDCI3): δ (ppm) 162.58, 155.88, 155,72, 155.55, 147.50, 136.31 , 133.07, 132.96, 132.90, 125.78, 120.63, 1 19.72, 1 14.49, 93.76, 87.14, 79.49, 62.38, 62.04, 51 .28, 50.66, 49.18, 28.57, 14.33. Maldi-TOF MS for C35H44F3N5O7: calculated 703.32, found 705.01 (M+2).

Example 16

Synthesis of ethyl 6-[(1,4 -triazacyclonan-1-yl)methyl]-4-{[4-(2,2,2- trifluoroacet-amido)phenyl]ethynyl}picolinate (16) A solution of compound 15 (0.44 g, 0.63 mmol) in TFA (4.4 ml) was stirred at RT for 2 hours. After evaporation the mixture was triturated with diethylether (60 ml) and centrifuged, washed with diethylether (2 x 10 ml) and dried. The compound 16 was used for the next step without purification. Yield: 0.47 g (89 %). 1H NMR (D2O): δ (ppm) 8.05 (s, 1 H), 7.59 (m, 5H), 4.50 (q, J=7.1 Hz, 2H), 4.21 (s, 2H), 3.92 (s, 4H), 3.43 (s, 4H), 3.19 (s, 4H), 1 .46 (t, J=7.1 Hz, 3H). 13C NMR (D2O): δ (ppm): 165.29, 159.59, 146.49, 136.67, 134.12, 132.94, 128.04, 125.97, 121 .13, 1 18.72, 1 16.81 , 1 14.52, 95.52, 86.08, 63.25, 57.30, 49.1 1 , 45.08, 43.83, 13.38. Maldi-TOF MS for C25H26F3N5O3: calculated 503.21 , found 505.17 (M+2). Example 17

Synthesis of Tetraethyl 4)4'-{{6,6'-{{7-{[6-(ethoxycarbonyl)-4-{[4-(2)2)2- trifluoro-acetamido)phenyl]ethynyl}pyridin-2-yl]methyl}-1,4,7- triazacyclononane-1,4-diyl}bis-(methylene)bis(2-(ethoxycarbonyl)pyridine- 6,4-diyl}}bis(ethyne-2,1-diyl)}-diphthalate (17)

The mixture of compounds 16 (100 mg, 0.12 mmol), 12 (140 mg, 0.29 mmol), diisopropylethyamine (162 μΙ, 0.95 mmol) and dry acetonitrile (2.0 ml) was stirred at RT for 40 hours. The reaction mixture was evaporated to dryness, dissolved in CH2CI2 (20 ml), washed with H2O (2 x 10 ml), and dried with Na2SO4. The product was purified by FC (silica gel, from 2 % to 5 % EtOH in 0,5 % TEA in CH2CI2. Yield: 123 mg (79 %). 1H NMR (CDCI3): δ (ppm) 8.03 (s, 2H), 7.97 (s, 1 H), 7.86 (d, J=1 .4 Hz, 2H), 7.82 (d, J=8.4 Hz, 2H), 7.79 (s, 2H), 7.74 (d, J= 8.0 Hz, 2H), 7.67 (dd, J=1 .4 Hz, J=8.0 Hz, 3H), 7.49 (d, J=8.4 Hz, 2H), 4.38 (m, 20H), 3.30 (s, br, 12H), 1 .38 (m, 21 H). 13C NMR (CDCI3): δ (ppm): 166.79, 166.76, 162.29, 162.02, 155.50, 155.20, 148.04, 147.89, 137.46, 134.06, 132.82, 132.66, 132.54, 132.23, 129.31 , 128.27, 128.06, 126.03, 124.65, 120.87, 1 16.91 , 1 14.62, 93.35, 88.39, 62.17, 62.08, 62.04, 61 .98, 45.83, 14.28, 14.09, 14.08. Maldi-TOF MS for C71 H70F3N7O15: calculated 1317.49, found 1318.50 (M+1 ).

Example 18 Synthesis of tetramethyl 2,2',2",2"'-{{{6,6'-{7-{[6-(ethoxycarbonyl)-4-[{4- (2,2,2-trifluoroacetamido)phenyl]ethynyl}pyridin-2-yl]methyl-1,4,7-tri- azacyclonane-1,4-diyl}-bis(methylene)bis[2-(ethoxycarbonyl)pyridine-6,4- diyl]}bis(ethyne-2,1-diyl)}bis-(benzene-5,3,1-triyl)}tetrakis(oxy)}te^

(18) This compound 18 was synthesized from the compound 13 using a method analogous to the synthesis described in the Example 17. The product was purified by FC (silica gel, from 5 to 10 % EtOH in 0.5 % TEA in CH2CI2. Yield: 33 %. 1H NMR (CDCI3): δ (ppm) 8.00 (s, 3H), 7.79 (s, 3H), 7.65 (s, br, 2H), 7.50 (d, J= 8.6 Hz, 2H), 6.68 (d, J=2.1 Hz, 4H), 6.54 (t, J=2.1 Hz, 2H), 4.63 (s, 8H), 4.43 (q, J=6.7 Hz, 6H), 4.10 (s, br, 6H), 3.82 (s, 12H), 3.03 (s, br, 12H), 1 .38 (t, J=6.7 Hz, 9H). 13C NMR (CDCIs): δ (ppm): 168.94, 164.63, 158.87, 158.82, 155.14, 147.93, 136.42, 132.93, 132.89, 127.75, 125.57, 123.56, 120.51 , 1 16.83, 1 14.54, 1 1 1 .34, 104.12, 93.99, 86.39, 65.31 , 62.07, 62.05, 52.44, 46.00, 14.31 . Maldi-TOF MS for C71 H70F3N7O19: calculated 1381 .47, found 1382.26 (M+1 ).

Example 19

Synthesis of Europium (III) chelate 19

A mixture of compound 17 (52 mg, 39 μηηοΙ), 0.5 M KOH in EtOH (3.0 ml) and H2O (1 .5 ml) was stirred at RT for 2 hours. After evaporation, the residue was dissolved in H2O (1 .2 ml) and the pH adjusted to 6.5 with 6 M HCI. EuCI3 hexahydrate (17.3 mg, 47 μηηοΙ) in H2O (225 μΙ) was added within 15 min and the pH maintained at 5- 7 with solid NaHCO3. After stirring for 3,5 hours at RT, the pH was raised to 8.5 with 1 M NaOH and the precipitate was centrifuged off, and the supernatant was extracted with phenol (ca. 0.75 g). The phenol phase was treated with H2O (1 .7 ml) and Et2O (7 ml) and the aqueous phase was washed with Et2O (3 x 7 ml), triturated with acetone (42 ml), the precipitate was centrifuged and washed with acetone (2 x 10 ml). The product was used for the next step without further purification. Yield: 38 mg (83 %). Maldi-TOF MS for C55H40EuN7Oi4: calculated 1 175.18, found 1 176.69 (M+1 ). Rf(HPLC): 15.9 min, UV(HPLC): 328 nm. Example 20

Synthesis of Europium (III) chelate 20

This compound 20 was synthesized from the compound 18 using a method analogous to the synthesis described in the Example 19. Yield: 74 %. Maldi-TOF MS for C63H56EuN7Oi8 (negative mode): calculated 1295.23, found 1295.49. Rf(HPLC): 16.9 min, UV(HPLC): 320 nm. Example 21

Synthesis of Europium (III) chelate 21

An aq. solution (1 .4 ml) of compound 19 (38 mg, 32 μηηοΙ) was added slowly to a mixture of thiophosgene (17 μΙ, 224 μηηοΙ), NaHCO3 (28 mg, 333 μηηοΙ) and CHCI3 (1 .4 ml). After stirring for 1 .5 hours at RT, the water phase was washed with CHCI3, the pH adjusted to 7 with 1 M HCI and the product was precipitated with acetone, centrifuged and washed acetone. Maldi-TOF MS for C56H38EuN7Oi4S (negative mode): calculated 1217.14, found 1217.27. Rf(HPLC): 23.5 min, UV(HPLC): 328 nm. Example 22

Synthesis of Europium (III) chelate 22

This compound 22 was synthesized from the compound 20 using a method analogous to the synthesis described in the Example 21 . Maldi-TOF MS for C64H5 EuN7Oi8S (negative mode): calculated 1360.17, found 1360.30. Rf(HPLC): 23.4 min, UV(HPLC): 324 nm.

Example 23

Synthesis of Europium (III) chelate 23

An aq. solution (1 ml) of compound 22 (51 mg, 42 μηηοΙ) was added to a mixture of 2-propenoyl chloride ( μΙ, 150 μηηοΙ), NaHCO3 (18 mg, 215 μηηοΙ) and CHCI3 (1 ml). After stirring for ca. 2 hours at RT, the water phase was washed with CHCI3 (3 x 2 ml), the product precipitated with acetone (40 ml) and washed with acetone (2 x 20 ml). Rf(HPLC): 18.9 min, UV(HPLC): 329 nm. Example 24

Coupling of chelate 21 and 22 to protein (cTnl antibody 817)

Labelling was performed in 10 mM borate buffer, pH 8.6-9.0 using 30-fold molar excess of chelates. Reactions were normally carried out overnight at +4 C or at room temperature. Labelled antibodies were purified on Superfex 200 HR 10/30 or Superdex 200 HiLoad 26/60 gel filtration columns using Tris-saline-azide (86.1 g/L Tris, 9.0 g/L NaCI, and 0.5 g/L NaN3), pH 7.75 as an elution buffer. The fractions containing the antibody were pooled.

Labelling ratio for the new chelates (Eu/lgG) were quantified by measuring the absorbance of the labelled antibody caused by the chelate over 300 nm and using measured absorbances of the labels (ca. 79 000).

An earlier published nonadentate label {2,2',2",2"'-{[2-(4-isothiocyanato- phenyl)ethylimino]bis(methylene)bis{4-{[4-(a-galactopyranoxy)phenyl]ethynyl}- pyridine-2,6-diyl}bis(methylenenitrilo)}tetrakis(acetato)}europium(lll) (von Lode, P., et al., 2003, Anal. Chem. 75, 3193) was used as a reference label. The europium concentrations measured against europium calibrator.

Example 25

Performed cTnl immunoassays with the chelate-labelled antibodies

The cTnl immunoassays were performed by using biotinylated capture cTnl antibodies together with the labelled cTnl detection antibodies described above and according to the main principles described in published method (von Lode, P., et al., 2003, Anal. Chem. 75, 3193). The combined assay/wash buffer contained 5 mmol HEPES, 2.1 g/L NaCI, 0.1 mmol EDTA, 0.055 g/L Tween 20 and 1g/L Gernall II, pH 7.75. The capture biotinylated antibodies were pre-incubated in assay wells. The standards followed by the detection label antibody diluted in 20 μΙ and 10 μΙ assay buffer, respectively, were applied to the wells. After one hour incubation at 36 C, the wells were washed, dried and measured. The results are in the Table 1 below, and show that the new chelates give over 200 % higher luminescence compared to the assumed 50 %.

Table 1

First test Second test

Label Reference Chelate 21 Reference Chelate 22

chelate chelate

Counts for 0 ng/ml 1 466 682 358 2 479 (cTnl standard)

Counts for 5 ng/ml 94 094 104 331 46 291 200 337 cTnl standard

Eu/lgG 12.7 6, 1 8.2 17.1

Signal/Eu 7 294 16914 5 602 1 1538

Signal 2,3 2, 1 improvement/Eu

Scheme 1

3: Rj = 3-COOEt

R2 = 4-COOEt

4: Rj = 3-OCH2COOMe

R2 = 5-OCH2COOMe

1: Rj = 3-COOEt TEA/THF

R2 = 4-COOEt

2: Rj = 3-OCH2COOMe

R2 = 5-OCH2COOMe K2C03, MeOH

or TBAF

5: Ri = 3-COOEt

R2 = 4-COOEt

6: Rj = 3-OCH2COOMe

R2 = 5-OCH2COOMe

Scheme 2

F3CCOHN

12: Rj = 3-COOEt

R2 = 4-COOEt

13: Rj = 3-OCH2COOMe

R2 = 5-OCH2COOMe

R2 = 4-CF?CONH Scheme 4A

Scheme 4C

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. It will be apparent for the specialist 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.
Patent Citations
Cited PatentFiling datePublication dateApplicantTitle
WO2005021538A127 Aug 200410 Mar 2005Wallac OyNovel chelating agents and chelates and their use
WO2005058877A115 Nov 200430 Jun 2005Wallac OyNovel chelating agents and highly luminescent and stable chelates and their use
WO2006120444A111 May 200616 Nov 2006University Of DurhamResponsive luminescent lanthanide complexes
WO2007128874A14 May 200715 Nov 2007Wallac OyBiomolecule labeling reactants based on azacycloalkanes and conjugates derived thereof
WO2008020113A117 Aug 200721 Feb 2008Abacus Diagnostica OyLuminescent lanthanide labelling reagents and their use
WO2008025886A131 Aug 20076 Mar 2008Wallac OyMetal chelates and chelating agents containing triazolyl subunits
WO2009021391A116 Jun 200819 Feb 2009Wuxi Kipor Power Co., LtdManually transportable carrier
WO2009030819A15 Sep 200812 Mar 2009Wallac OyLuminescent lanthanide (iii) chelates, chelating agents and conjugates derived thereof
WO2009115644A118 Mar 200924 Sep 2009Wallac OyLanthanide(iii) chelates comprising beta-diketones and conjugates derived thereof
WO2010055207A112 Nov 200920 May 2010Wallac OyChelating, chelating agents and conjugates deriver thereof
WO2010106222A2 *10 Mar 201023 Sep 2010Wallac OyBiotinidase assay
WO2013011236A1 *17 Jul 201224 Jan 2013Cisbio BioassaysNovel complexing agents and corresponding lanthanide complexes
DE202008013312U17 Oct 200812 Feb 2009Wallac OyMarkierungsreagenzien
DE202008013314U17 Oct 200819 Feb 2009Wallac OyLanthanidchelate
FI8670U1 Title not available
IT1235668B Title not available
US46705723 Feb 19862 Jun 1987Eastman Kodak CompanyPhenolic fluorescent labels
US47725638 Aug 198520 Sep 1988Hsc Research Development Corporation1,10-phenanthroline derivatives and use in fluorescence immunoassays
US47941913 Feb 198827 Dec 1988Eastman Kodak CompanyFluorescent chelates
US50326776 Nov 198716 Jul 1991Baxter International Inc.Fluorescent poly(arylpyridine) rare earth chelates
US50555785 Nov 19908 Oct 1991Baxter Diagnostics Inc.Fluorescent poly(arylpyridine) rare earth chelates
US701885113 Feb 200328 Mar 2006Innotrac Diagnostics OyBiospecific binding reactants labeled with new luminescent lanthanide chelates and their use
US200300271897 Jun 20026 Feb 2003The Regents Of The University Of CaliforniaSalicylamide-lanthanide complexes for use as luminescent markers
US2005008445130 Aug 200421 Apr 2005Wallac OyNovel chelating agents and chelates and their use
US200501813936 Dec 200418 Aug 2005Wallac OyNovel chelating agents and highly luminescent and stable chelates and their use
US2008016744317 Dec 200710 Jul 2008Wallac OyNovel chelating agents and highly luminescent and stable chelates and their use
US2008021391715 Aug 20074 Sep 2008The Regents Of The University Of CaliforniaLuminescent macrocyclic lanthanide complexes
Non-Patent Citations
Reference
1ANDRAUD, C. ET AL., EUR. J. INORG. CHEM, 2009, pages 4357
2ANDRAUD, C.; MAURY, 0., EUR. J. INORG. CHEM., 2009, pages 4357
3BOURDOLLE, A. ET AL., INORG. CHEM., 2011, pages 4987
4D'ALEO, A. ET AL., C.R. CHEMIE, vol. 13, 2010, pages 681
5D'ALEO, A. ET AL., HELV, CHIM. ACTA, vol. 92, 2009, pages 2439
6ELISEEVA, S.V.; BUNZLI, J.-C. G., CHEM. SOC. REV., vol. 39, 2010, pages 189
7ELISEEVA, S.V.; BUNZLI, J.-C.G., CHEM. SOC. REV, vol. 39, 2010, pages 189
8G.T. HERMANSON: "Bioconjugate Techniques", 1996, ACADEMIC PRESS
9HEMMI A, I.; MUKKALA, V.-M., CRIT. REV. CLIN. LAB. SCI., vol. 38, 2001, pages 441
10HILLE, U.E. ET AL., EUR. J. MED. CHEM, vol. 44, 2009, pages 2765
11HOVINEN, J. ET AL., BIOCONJUGATE CHEM., 2009
12HOVINEN., J. ET AL., BIOCONJUGATE CHEM., vol. 20, 2009, pages 404
13INORG. CHEM., 2011, pages 4987
14JAAKKOLA, L. ET AL., BIOCONJUGATE CHEM., vol. 16, 2005, pages 700
15 *JAMES W. WALTON ET AL: "Very bright europium complexes that stain cellular mitochondria", CHEMICAL COMMUNICATIONS, vol. 49, no. 16, 21 January 2013 (2013-01-21), pages 1600, XP055060987, ISSN: 1359-7345, DOI: 10.1039/c2cc35247h
16LATVA, M ET AL., J. LUMINESCENCE, vol. 75, 1997, pages 149
17LODE, P. ET AL., ANAL. CHEM., vol. 75, 2003, pages 3193
18PARKER, D. ET AL., EUR. J. INORG. CHEM., 2010, pages 3961
19QUICI, S. ET AL., INORG. CHIM. ACTA, vol. 44, 2005, pages 529
20SATO, N. ET AL., J. CHEM. SOC. PERKIN TRANS., vol. 2, 1993, pages 621
21SELVIN, P.R. ET AL., J. AM. CHEM. SOC., vol. 116, 1994, pages 6029
22SHAVALEEV, N.M. ET AL., INORG. CHEM., vol. 49, 2010, pages 3927
23STEEMERS, F.J. ET AL., J. AM. CHEM., vol. 117, 1995, pages 9408
24 *TAKALO H ET AL: "SYNTHESIS AND LUMINESCENCE OF NOVEL EUIII COMPLEXING AGENTS AND LABELS WITH 4-(PHENYLETHYNYL)PYRIDINE SUBUNITS", HELVETICA CHIMICA ACTA, VERLAG HELVETICA CHIMICA ACTA, vol. 79, no. 3, 1996, pages 789 - 802, XP001002467, ISSN: 0018-019X, DOI: 10.1002/HLCA.19960790321
25TAKALO, H. ET AL., 1 ST INTERNATIONAL CONFERENCE ON LUMINESCENCE OF LANTHANIDES, 2010
26TAKALO, H. ET AL., ACTA CHEM SCAND, vol. B 42, 1988, pages 373
27TAKALO, H. ET AL., HELV. CHIM. ACTA, vol. 79, 1996, pages 789
28THIBON, A.; PIERRE, V.C., ANAL. BIOANAL. CHEM., vol. 394, 2009, pages 107
29 *VIRGINIE PLACIDE ET AL: "Design and synthesis of europium luminescent bio-probes featuring sulfobetaine moieties", TETRAHEDRON LETTERS, vol. 55, no. 7, 13 January 2014 (2014-01-13), pages 1357 - 1361, XP055107089, ISSN: 0040-4039, DOI: 10.1016/j.tetlet.2014.01.025
30VON LODE, P. ET AL., ANAL. CHEM., vol. 75, 2003, pages 3193
31 *WERMUTH C G: "PREPARATION OF WATER-SOLUBLE COMPOUNDS BY COVALENT ATTACHMENT OF SOLUBILIZING MOIETIES", PRACTICE OF MEDICINAL CHEMISTRY, XX, XX, 1996, pages 755 - 776, XP001203531
32YANG, C. ET AL., ANGEW. CHEM. INT. ED., 2004, pages 5010
Referenced by
Citing PatentFiling datePublication dateApplicantTitle
WO2016066641A1 *27 Oct 20156 May 2016Dhr FinlandNew chromophoric structures for macrocyclic lanthanide chelates
Classifications
International ClassificationC07F5/00, G01N33/52, C09K11/77
Cooperative ClassificationG01N33/532, G01N33/582, C09K2211/182, C09K2211/1029, C09K11/06, C09K2211/1007, C07F5/003
Legal Events
DateCodeEventDescription
12 Nov 2014121Ep: the epo has been informed by wipo that ep was designated in this application
Ref document number: 14704172
Country of ref document: EP
Kind code of ref document: A1
31 Jul 2015NENPNon-entry into the national phase in:
Ref country code: DE
24 Feb 2016122Ep: pct application non-entry in european phase
Ref document number: 14704172
Country of ref document: EP
Kind code of ref document: A1