EP3768665A1 - Luminescent lanthanide chelates and their use - Google Patents
Luminescent lanthanide chelates and their useInfo
- Publication number
- EP3768665A1 EP3768665A1 EP19711533.0A EP19711533A EP3768665A1 EP 3768665 A1 EP3768665 A1 EP 3768665A1 EP 19711533 A EP19711533 A EP 19711533A EP 3768665 A1 EP3768665 A1 EP 3768665A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- alkyl
- compound
- group
- substituted
- nhc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 229910052747 lanthanoid Inorganic materials 0.000 title abstract description 17
- 150000002602 lanthanoids Chemical class 0.000 title abstract description 17
- 150000001875 compounds Chemical class 0.000 claims abstract description 92
- 238000000034 method Methods 0.000 claims abstract description 41
- 230000027455 binding Effects 0.000 claims abstract description 38
- 239000000376 reactant Substances 0.000 claims abstract description 38
- 238000001514 detection method Methods 0.000 claims abstract description 37
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 34
- 238000002372 labelling Methods 0.000 claims abstract description 19
- 239000012491 analyte Substances 0.000 claims abstract description 18
- 150000003839 salts Chemical class 0.000 claims abstract description 16
- 239000007787 solid Substances 0.000 claims abstract description 12
- 150000001408 amides Chemical class 0.000 claims abstract description 9
- 239000000463 material Substances 0.000 claims abstract description 9
- -1 substituted Chemical class 0.000 claims description 60
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 25
- 125000001072 heteroaryl group Chemical group 0.000 claims description 25
- 238000004020 luminiscence type Methods 0.000 claims description 25
- 230000001268 conjugating effect Effects 0.000 claims description 21
- 239000001257 hydrogen Substances 0.000 claims description 21
- 229910052739 hydrogen Inorganic materials 0.000 claims description 21
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 18
- 125000001424 substituent group Chemical group 0.000 claims description 18
- 229910021644 lanthanide ion Inorganic materials 0.000 claims description 17
- 239000000523 sample Substances 0.000 claims description 17
- 239000003446 ligand Substances 0.000 claims description 16
- 125000003118 aryl group Chemical group 0.000 claims description 15
- 230000005284 excitation Effects 0.000 claims description 15
- 125000006850 spacer group Chemical group 0.000 claims description 15
- 238000000159 protein binding assay Methods 0.000 claims description 13
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea Chemical compound NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 claims description 12
- 108091034117 Oligonucleotide Proteins 0.000 claims description 11
- 150000002772 monosaccharides Chemical class 0.000 claims description 11
- 230000009870 specific binding Effects 0.000 claims description 10
- 150000003431 steroids Chemical class 0.000 claims description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 8
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 8
- 108020003175 receptors Proteins 0.000 claims description 8
- 102000005962 receptors Human genes 0.000 claims description 8
- PXQLVRUNWNTZOS-UHFFFAOYSA-N sulfanyl Chemical class [SH] PXQLVRUNWNTZOS-UHFFFAOYSA-N 0.000 claims description 8
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 claims description 8
- 125000004122 cyclic group Chemical group 0.000 claims description 7
- 150000002148 esters Chemical class 0.000 claims description 7
- LNBHUCHAFZUEGJ-UHFFFAOYSA-N europium(3+) Chemical compound [Eu+3] LNBHUCHAFZUEGJ-UHFFFAOYSA-N 0.000 claims description 7
- 125000000843 phenylene group Chemical group C1(=C(C=CC=C1)*)* 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 claims description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Natural products NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 6
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 6
- 108091033319 polynucleotide Proteins 0.000 claims description 6
- 102000040430 polynucleotide Human genes 0.000 claims description 6
- 239000002157 polynucleotide Substances 0.000 claims description 6
- 230000005855 radiation Effects 0.000 claims description 6
- 150000003568 thioethers Chemical class 0.000 claims description 6
- 150000003852 triazoles Chemical class 0.000 claims description 6
- 125000002344 aminooxy group Chemical group [H]N([H])O[*] 0.000 claims description 5
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 claims description 5
- 239000012954 diazonium Substances 0.000 claims description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-O diazynium Chemical compound [NH+]#N IJGRMHOSHXDMSA-UHFFFAOYSA-O 0.000 claims description 5
- 150000002016 disaccharides Chemical class 0.000 claims description 5
- 229910052736 halogen Inorganic materials 0.000 claims description 5
- 125000005842 heteroatom Chemical group 0.000 claims description 5
- 125000001261 isocyanato group Chemical group *N=C=O 0.000 claims description 5
- 125000001810 isothiocyanato group Chemical group *N=C=S 0.000 claims description 5
- 229920001282 polysaccharide Polymers 0.000 claims description 5
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 claims description 5
- KAKZBPTYRLMSJV-UHFFFAOYSA-N Butadiene Chemical group C=CC=C KAKZBPTYRLMSJV-UHFFFAOYSA-N 0.000 claims description 4
- 229910006069 SO3H Inorganic materials 0.000 claims description 4
- 239000000427 antigen Substances 0.000 claims description 4
- 102000036639 antigens Human genes 0.000 claims description 4
- 108091007433 antigens Proteins 0.000 claims description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 4
- 238000003384 imaging method Methods 0.000 claims description 4
- 238000000338 in vitro Methods 0.000 claims description 4
- 150000003904 phospholipids Chemical class 0.000 claims description 4
- HKCRVXUAKWXBLE-UHFFFAOYSA-N terbium(3+) Chemical compound [Tb+3] HKCRVXUAKWXBLE-UHFFFAOYSA-N 0.000 claims description 4
- 125000001544 thienyl group Chemical group 0.000 claims description 4
- 102000014914 Carrier Proteins Human genes 0.000 claims description 3
- 108020003215 DNA Probes Proteins 0.000 claims description 3
- 239000003298 DNA probe Substances 0.000 claims description 3
- 102000004856 Lectins Human genes 0.000 claims description 3
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- 102000015636 Oligopeptides Human genes 0.000 claims description 3
- 108010038807 Oligopeptides Proteins 0.000 claims description 3
- 108020004518 RNA Probes Proteins 0.000 claims description 3
- 239000003391 RNA probe Substances 0.000 claims description 3
- 108091008324 binding proteins Proteins 0.000 claims description 3
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 claims description 3
- 229940079593 drug Drugs 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- IOIFRTZBJMZZFO-UHFFFAOYSA-N dysprosium(3+) Chemical compound [Dy+3] IOIFRTZBJMZZFO-UHFFFAOYSA-N 0.000 claims description 3
- 125000002541 furyl group Chemical group 0.000 claims description 3
- 150000004676 glycans Chemical class 0.000 claims description 3
- 239000002523 lectin Substances 0.000 claims description 3
- 239000005017 polysaccharide Substances 0.000 claims description 3
- DOSGOCSVHPUUIA-UHFFFAOYSA-N samarium(3+) Chemical compound [Sm+3] DOSGOCSVHPUUIA-UHFFFAOYSA-N 0.000 claims description 3
- 125000002529 biphenylenyl group Chemical group C1(=CC=CC=2C3=CC=CC=C3C12)* 0.000 claims description 2
- 125000005678 ethenylene group Chemical group [H]C([*:1])=C([H])[*:2] 0.000 claims description 2
- 125000004957 naphthylene group Chemical group 0.000 claims description 2
- 125000005565 oxadiazolylene group Chemical group 0.000 claims description 2
- 125000005564 oxazolylene group Chemical group 0.000 claims description 2
- 125000005550 pyrazinylene group Chemical group 0.000 claims description 2
- 125000005551 pyridylene group Chemical group 0.000 claims description 2
- 125000005576 pyrimidinylene group Chemical group 0.000 claims description 2
- 125000005557 thiazolylene group Chemical group 0.000 claims description 2
- 125000005556 thienylene group Chemical group 0.000 claims description 2
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 claims 1
- 239000013522 chelant Substances 0.000 abstract description 34
- 230000015572 biosynthetic process Effects 0.000 description 73
- 238000003786 synthesis reaction Methods 0.000 description 70
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 44
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 32
- 239000000203 mixture Substances 0.000 description 30
- 239000000047 product Substances 0.000 description 28
- 229910001868 water Inorganic materials 0.000 description 17
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N DMSO Substances CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 16
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 15
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 14
- 238000004128 high performance liquid chromatography Methods 0.000 description 14
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 14
- 239000000741 silica gel Substances 0.000 description 14
- 229910002027 silica gel Inorganic materials 0.000 description 14
- 238000003556 assay Methods 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 10
- 238000007792 addition Methods 0.000 description 10
- 125000004432 carbon atom Chemical group C* 0.000 description 10
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 9
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 9
- 238000001704 evaporation Methods 0.000 description 9
- 230000008020 evaporation Effects 0.000 description 9
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 9
- 150000007523 nucleic acids Chemical class 0.000 description 9
- 238000003756 stirring Methods 0.000 description 9
- 238000005160 1H NMR spectroscopy Methods 0.000 description 8
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 description 8
- 238000003018 immunoassay Methods 0.000 description 7
- 150000002500 ions Chemical class 0.000 description 7
- 108020004707 nucleic acids Proteins 0.000 description 7
- 102000039446 nucleic acids Human genes 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- AOSZTAHDEDLTLQ-AZKQZHLXSA-N (1S,2S,4R,8S,9S,11S,12R,13S,19S)-6-[(3-chlorophenyl)methyl]-12,19-difluoro-11-hydroxy-8-(2-hydroxyacetyl)-9,13-dimethyl-6-azapentacyclo[10.8.0.02,9.04,8.013,18]icosa-14,17-dien-16-one Chemical compound C([C@@H]1C[C@H]2[C@H]3[C@]([C@]4(C=CC(=O)C=C4[C@@H](F)C3)C)(F)[C@@H](O)C[C@@]2([C@@]1(C1)C(=O)CO)C)N1CC1=CC=CC(Cl)=C1 AOSZTAHDEDLTLQ-AZKQZHLXSA-N 0.000 description 6
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 6
- IWZSHWBGHQBIML-ZGGLMWTQSA-N (3S,8S,10R,13S,14S,17S)-17-isoquinolin-7-yl-N,N,10,13-tetramethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-amine Chemical compound CN(C)[C@H]1CC[C@]2(C)C3CC[C@@]4(C)[C@@H](CC[C@@H]4c4ccc5ccncc5c4)[C@@H]3CC=C2C1 IWZSHWBGHQBIML-ZGGLMWTQSA-N 0.000 description 5
- 229940126657 Compound 17 Drugs 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 239000008346 aqueous phase Substances 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 238000006862 quantum yield reaction Methods 0.000 description 5
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 4
- JHUUPUMBZGWODW-UHFFFAOYSA-N 3,6-dihydro-1,2-dioxine Chemical compound C1OOCC=C1 JHUUPUMBZGWODW-UHFFFAOYSA-N 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 4
- VFOJFRHATPMNFQ-UHFFFAOYSA-N N-(2-aminoethyl)-4-bromo-6-(hydroxymethyl)pyridine-2-carboxamide Chemical compound NCCNC(C1=NC(=CC(=C1)Br)CO)=O VFOJFRHATPMNFQ-UHFFFAOYSA-N 0.000 description 4
- 229910018828 PO3H2 Inorganic materials 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 4
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- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 4
- 239000011343 solid material Substances 0.000 description 4
- ASGMFNBUXDJWJJ-JLCFBVMHSA-N (1R,3R)-3-[[3-bromo-1-[4-(5-methyl-1,3,4-thiadiazol-2-yl)phenyl]pyrazolo[3,4-d]pyrimidin-6-yl]amino]-N,1-dimethylcyclopentane-1-carboxamide Chemical compound BrC1=NN(C2=NC(=NC=C21)N[C@H]1C[C@@](CC1)(C(=O)NC)C)C1=CC=C(C=C1)C=1SC(=NN=1)C ASGMFNBUXDJWJJ-JLCFBVMHSA-N 0.000 description 3
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- YJLIKUSWRSEPSM-WGQQHEPDSA-N (2r,3r,4s,5r)-2-[6-amino-8-[(4-phenylphenyl)methylamino]purin-9-yl]-5-(hydroxymethyl)oxolane-3,4-diol Chemical compound C=1C=C(C=2C=CC=CC=2)C=CC=1CNC1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O YJLIKUSWRSEPSM-WGQQHEPDSA-N 0.000 description 3
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
Definitions
- the present invention relates to the field of luminescent lanthanide chelates suitable as label for detection in bioassay applications.
- Time-resolved fluorometry employing long-lifetime emitting luminescent lanthanide chelates has been applied in many specific binding assays, such as immunoassays, DNA hybridization assays, receptor-binding assays, enzymatic assays, bio-imaging such as immunocytochemical, immunohistochemical assays or cell based assays to measure analytes even at very low concentration.
- specific binding assays such as immunoassays, DNA hybridization assays, receptor-binding assays, enzymatic assays, bio-imaging such as immunocytochemical, immunohistochemical assays or cell based assays to measure analytes even at very low concentration.
- lanthanide chelates have been used in magnetic resonance imaging (MRI) and position emission tomography (PET).
- an optimal label has to fulfill several requirements.
- the excitation wavelength has to be as high as possible, preferably above 300 nm. It needs an efficient cation emission i.e. a high luminescence yield (excitation coefficient x quantum yield, eF).
- the observed luminescence decay time has to be rather long, and the chelate has to have good water solubility.
- the challenge is to prepare a chelate label to fulfill all requirements in one molecule, and therefore, certain compromises are generally made in the development of suitable labels.
- a number of attempts have been made to tune the photo -physical properties of the chelate labels suitable for time-resolved fluorometric applications.
- One generally used method to improve luminescence intensity is to prepare chelate ligands with several independent chromophoric moieties combined in structure designs, which offer high stabilities and luminescence quantum yields.
- Chelates which contain two and three separate 4-(phenylethynyl)pyridines are published by Takalo, H., et al, Helv. Chim. Acta., 79 (1996) 789. More recent examples of lanthanide chelates and chelating ligands are those disclosed in e.g. EP 1 447 666, WO 2010/055207, WO 2010/006605 and WO 2008/020113.
- lanthanide chelates have only moderate total molar absorptivity i.e. below 27,000 with four chromophores, whereas e.g. chelates with only one phenylethynylpyridine subunit normally have absorptivities of 25, GOO- 35, 000 cm- 1 (Latva, M., et al, J. Luminescence, 75 (1997)149) depending on the substituents in the chromophore.
- WO 2013/026790 Al describes luminescent lanthanide chelates having three chromophores and the use thereof Picot et al. (Inorg. Chem. 46 (2007)2659) describe pyridine-dicarboxamide ligands and related D3 symmetric europium (III) complexes.
- 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 labeling process and non-specific binding properties of labeled biomolecules. Aggregates will produce purification problems and reduced yield of labeled material. Moreover, increased non-specific binding of labeled bio molecule will enhance background luminescence of biospecific assays and thus reduces assay sensitivity.
- each of Gi, G 2 and G 3 is independently selected from i) a conjugating group and ii) a single bond; provided that at least one of Gi, G 2 and G 3 is independently a conjugating group;
- each of Ri, R 2 , and R 3 is independently selected from i) a reactive group Z, ii) a hydrophilic group, and iii) hydrogen;
- each of Ai and A 2 is independently selected from i) a reactive group Z, ii) a hydrophilic group, and iii) hydrogen or Ci- 6 -alkyl;
- each R A and R B is independently selected from
- aryl or heteroaryl is optionally substituted by one, two or three substituents selected from the group consisting of -NH 2 , -Ci- 12 -alkyl, -C 2-12 - alkenyl, -C 2-i2 -alkinyl, halogen, -CF 3 , -Ci-i 2 -alkyl-NH 2 , -OH, -SH, -CN, -NCS, heteroaryl, and -NH-(optionally substituted 5 to l2-membered heteroaryl);
- Ri, R2, R3, R A , R B , A I or A2 is a reactive group Z
- Ln 3+ is a lanthanide ion.
- the objects are solved by a compound of formula (II)
- each of Gi, G 2 , G 3 , Ri, R 2 , R 3 , Ai and A 2 represents the groups Gi, G2, G 3 , Ri, R2, R 3 , Chi, Ch 2 , Ai and A2 as defined above.
- the objects are solved by a detection agent comprising a biospecific binding reactant conjugated to a compound of formula (I) or a salt thereof or a compound of formula (II) or a salt thereof.
- the objects are solved by a method of detecting an analyte in a biospecific binding assay, said method comprising the steps of:
- the objects are solved by a method of labelling a biospecific binding reactant with a compound of the invention, comprising the steps of
- the objects are solved by using a detection agent of the invention in a specific bioaffinity based binding assay utilizing time-resolved fluorometric determination of a specific luminescence.
- the objects are solved by using a compound of the invention or the detection agent of the invention for the in vitro detection of an analyte in a sample or for bio-imaging applications.
- the objects are solved by providing solid support material conjugated with a compound of the invention or the detection agent of the invention.
- the present invention relates to compounds of formula (I)
- each of Gi, G 2 and G 3 is independently selected from i) a conjugating group and ii) a single bond; provided that at least one of Gi, G 2 and G 3 is independently a conjugating group; each of Ri, R 2 , and R 3 is independently selected from i) a reactive group Z, ii) a hydrophilic group, and iii) hydrogen;
- each of Ai and A 2 is independently selected from i) a reactive group Z, ii) a hydrophilic group, and iii) hydrogen or Ci- 6 -alkyl;
- each R A and R B is independently selected from
- aryl or heteroaryl is optionally substituted by one, two or three substituents selected from the group consisting of -NH 2 , -Ci-i 2 -alkyl, -C 2-i2 - alkenyl, -C 2-i2 -alkinyl, halogen, -CF 3 , -Ci-i 2 -alkyl-NH 2 , -OH, -SH, -CN, -NCS, heteroaryl, and -NH-(optionally substituted 5- to l2-membered heteroaryl);
- Ri, R 2 , R 3 , R A , R B , A I or A 2 is a reactive group Z, and wherein Ln 3+ is a lanthanide ion.
- the compounds may be considered as lanthanide chelate labels suitable for use in specific bioaffinity based binding assays, such as immunoassays (both homogeneous and heterogeneous), nucleic acid hydridization assays, receptor binding assays, enzymatic assays, immunocytochemical, immunohistochemical assays and cell based assays utilizing fluorometric or time-resolved fluorometric determination of specific luminescence.
- One advantage of the lanthanide chelates of the present invention is that they provide a surprisingly high absorbance and luminescence intensity, i.e. brightness, a rather long lifetime (decay time) t as well as a high excitation wavelength above 340 nm.
- the chelates of the present inventions are thus suitable for use in light emitting diode (LED)-based excitation and instrumentation setup. Overall, the compounds are broadly applicable for use in the abovementioned bioassays.
- the chelates of the present invention comprise three individual chromophore moieties around an emitting lanthanide ion. It is to be understood that the lanthanide ion is bound by nine coordinate bonds, most likely as follows:
- At least two of Gi, G 2 and G3 are independently a conjugating group.
- each of Gi, G 2 and G3 is selected as a conjugating group.
- (-Het/Ar) e.g. phenylene, biphenylene, naphthylene, pyridylene, pyrazinylene, pyrimidinylene, pyridazinylene, furylene, thienylene, pyrrolylene, imidazolylene, pyrazolylene, thiazolylene, isothiazolylene, oxazolylene, isoxazolylene,
- the groups are arranged so as to be conjugated with each other and are attached to the respective pyridine in such a way that the conjugating group is bound to the pyridine.
- Each of the biradicals of (hetero)aromatic ring or ring systems may be unsubstituted or mono-R 4 -substituted, di-R 4 ,R5-substituted, tri-R 4 ,R 5 , Re-substituted, tctra-R 4 ,R5,R6 .
- Rv-substitutcd or pcnta-R 4 ,R 5 ,R 6 ,R 7 ,Rs-substitutcd, wherein each of such possible substituents R 4 , Rs, Re, R 7 , and Rs independently are selected from Ci_ 12-alkyl, -COOH, -Ci- 6 -alkyl-COOH, -COO , -Ci-e-alkyl-COO , -SO3H, -Ci-e-alkyl- SO3H, -S0 3 , -Ci- 6 -alkylS0 3- ,-Ci- 6 -alkyl-0-P0 3 2 ⁇ , -Ci- 6 -alkyl-P0 3 2 -, -Ci-e-alklyl-O- PO3H2, -Ci- 6 -alkyl-P0 3 H 2 , -Ci-e-alkyl-OH, -
- the excitation wavelength of the chelates may be shifted to a desired longer wavelength.
- two of Gi, G 2 and G 3 are independently selected as the conjugating groups phenylethynyl, phenyl, thienyl and furyl, which each are optionally substituted.
- two of Gi, G 2 and G 3 are independently selected phenylethynyl (-CoC-C 6 H 5 -).
- Gi, G 2 and G 3 is each an optionally substituted phenylethynyl, which each may be substituted with the substituents described above.
- all three of Gi, G 2 and G 3 are each independently selected from mono-R 4 -substituted ethynediyl-phenylene (-CoC-C 6 H5-), di-R 4 ,R 5 - substituted ethynediyl-phenylene, or tri-R 4 ,R 5 , Re-substituted ethynediyl-phenylene.
- R 4 ,R 5 and/or Re are each independently -OR9, wherein preferably R9 is -Ci- 6 -alkyl-COO and more preferably - CH 2 -COO .
- two of Gi, G 2 and G 3 are each independently selected from mono-R 4 -substituted ethynediyl-phenylene, di-R 4 ,Rs-substituted ethynediyl-phenylene, or tri-R 4 ,R 5 , Re-substituted ethynediyl-phenylene.
- R 4 , R5 and/or Re are each independently -OR9, wherein preferably R9 is -Ci- 6 -alkyl-COO and more preferably -CH 2 -COO ; and the third of Gi, G 2 and G 3 is an unsubstituted ethynediyl-phenylene, wherein at least one of Ri, R 2 and R 3 is a reactive group Z.
- Ri, R 2 and/or R 3 are hydrogen.
- at least one reactive group Z is required in the chelate molecule of the invention.
- the number of Z groups in the chelate molecule is 1, 2, or 3, in particular 1 or 2, more preferably 1.
- all three of Ri, R 2 and R 3 are hydrogens, it follows that the at least one of Ai, A 2 , Chi or Ch 2 comprises a reactive group Z, wherein it is preferred that at least one of Chi or Ch 2 comprises a reactive group Z.
- the reactive group Z is facilitating the labelling of a biospecific binding reactant, or is facilitating the formation of a covalent bond to a solid support material.
- the chelate may be introduced in the solid support, e.g. a particle, simultaneously with the preparation of the particles.
- the substituents in 6-substituted 4-chloro-l,3,5-triazin-2-ylamino can be selected from the group consisting of hydrogen, halogen, alkoxy, aryloxy, amino, alkyl with one to six carbon atoms, substituted amino or thioethers, and are preferably selected from the group consisting of chloro, fluoro, ethoxy, 2-methoxyethoxy, 2- cyanoethoxy, 2,2,2-trifluoroethoxy, thiophenoxy or ethoxycarbonyl-thiomethoxy.
- the substituted amino or thioether is preferably mono- or disubstituted each substituent being preferably independently selected from the group consisting of an alkyl or alkoxy with one to six carbon atoms, phenyl, carbonyl or carboxyl.
- n 1-6; and a triazole (e.g. formed by the so-called“click” chemistry).
- the reactive group Z comprises an amino group, -NCS, or a 6-substituted 4-chloro-l,3,5-triazin-2-ylamino group. It is particularly preferred that the reactive group Z comprises a 4,6-dichloro-l,3,5-triazin-2-ylamino group. In another preferred embodiment, the reactive group Z consists of azido (-
- the spacer consists of one to five moieties, each moiety is selected from the group consisting of phenylene, -(CH 2 ) I-IO -, an ethynediyl (-CoC-),
- the spacer - if necessary or desirable - may position the reactive group Z in a position accessible for reaction with the biospecific binding reactant, thereby facilitating the labelling reaction.
- the compound of the invention comprises a single reactive group Z, in particular as the substituent R 2, R A or R B .
- the hydrophilic group may include a spacer as described above.
- the spacer may be readily introduced in the course of the synthesis of the ligand or the chelate.
- the hydrophilic group is selected from monosaccharides, disaccharides, -(CH 2 ) i _ 3 -0-(CH 2 CH 2 0)o- 5 -H, -(CH 2 ) i _ 3 -0-(CH 2 CH 2 0)o- 5 -C i -4 - alkyl, -0-(CH 2 CH 2 0)i_ 6 -H, and -0-(CH 2 CH 2 0)i- 6 -Ci- 4 -alkyl.
- hydrophilic group comprises monosaccharides.
- the hydrophilic group comprises monosaccharides or disaccharides.
- Ai or A 2 is hydrogen. It is particularly preferred that both Ai and A 2 are hydrogen.
- the lanthanide ion, Ln 3+ is selected from europium(III), terbium(III), dysprosium(III) and samarium(III).
- the lanthanide ion is europium(III).
- RA and/or RB is selected as a reactive group Z.
- the reactive group Z comprises a spacer.
- the spacer comprises three to five of the above moieties.
- the reactive group Z when RA and/or RB is selected as a reactive group Z, the reactive group Z also comprises an amino group, -NCS, or a 6-substituted 4-chloro-l,3,5-triazin-2-ylamino group.
- R A and/ or R B is selected as optionally
- R A and / or R B is optionally substituted -
- the aryl or heteroaryl is an optionally substituted 6- to l2-membered aryl and preferably an optionally substituted phenyl. It is particularly preferred that the phenyl is substituted with at least one substituent, preferably a substituent in the para-position.
- the substituent is selected from -NH2, -NCS, or -NH-(optionally substituted 5- to l2-membered heteroaryl), wherein preferably the optionally substituted heteroaryl is a 6-substituted 4-chloro-l,3,5-triazin-2-ylamino group, preferably a 4,6-dichloro-l,3,5-triazin-2-ylamino group as described above.
- R AI is -Ci - 6 -alkyl.
- R A 2 is a bond
- R A is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoe
- R A is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoe
- Xi may be hydrogen or a reactive group suitable for the reaction with a bio specific binding reactant in order to establish a link to the said biospecific binding reactant as described for reactive group Z.
- Xi is selected from -NH 2 , -NCS, -NHCOCF 3 , and -NH- (optionally substituted 5- to l2-membered heteroaryl). It is preferred that Li is attached in the para-position of the phenyl-ring.
- Ai, A 2 , Ri, R 2 and R 3 are hydrogen.
- the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
- the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
- the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
- the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
- the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
- the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
- the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
- the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
- the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
- the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
- the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
- the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
- the present invention relates to compounds of formula (II)
- each of Gi, G 2 , G 3 , Ri, R 2 , R 3 , Chi, Ch 2 , Ai and A 2 represents the groups Gi, G 2 , G 3 , Ri, R 2 , R 3 , Chi, Ch 2 , Ai and A 2 as defined above for the compounds of formula (I).
- the compounds of formula (II) may be precursor molecules for producing compounds of formula (I). If the ligand is to be used in peptide or oligonucleotide synthesis as a means to prepare a labeled peptide or a labeled oligonucleotide as described in e.g. US 2005/0181393, it is preferable to use one functional group for attaching the ligand to the peptide’s or oligonucleotide’s back-bone during the synthesis in Ri, R 2 , FU, Ai,
- the present invention relates to a detection agent comprising a biospecific binding reactant conjugated to a compound of formula (I) or a salt thereof or a compound of formula (II) or a salt thereof
- the detection agent is a detectable molecule comprising a biospecific binding reactant conjugated to a luminescent lanthanide chelate of formula (I) or a precursor of formula (II) of the present invention. Conjugation, i.e. the formation of a covalent bond, is typically obtained by means of a reactive group of said chelate.
- the biospecific binding reactant should be capable of specifically binding an analyte of interest for the purpose of quantitative or qualitative analysis of said analyte in a sample.
- biospecific binding reactants are those selected from an antibody, an antigen, a receptor ligand, a specific binding protein, a DNA probe, an RNA probe, an oligopeptide, an oligonucleotide, a modified oligonucleotide (e.g. a locked nucleic acid (LNA) modified oligonucleotide), a modified polynucleotide (e.g. an LNA modified polynucleotide), a protein, an oligosaccaride, a polysaccharide, a phospholipid, a PNA, a steroid, a hapten, a drug, a receptor binding ligand, and lectin.
- LNA locked nucleic acid
- the biospecific binding reactant is selected from antibodies, e.g. Troponin I antibodies (anti-Tnl).
- the present invention relates to a method of detecting an analyte in a biospecific binding assay, said method comprising the steps of:
- the present invention relates to a method of labelling a biospecific binding reactant with a compound of the invention, comprising the steps of
- the resulting compound may be a detection agent of the invention.
- the conjugation may occur via the reaction group Z of the compound of formula (I) or formula (II).
- the present invention relates to the use of a compound of formula (I) or formula (II) of the invention for the in vitro detection of an analyte in a sample.
- the present invention thus also relates to the use of a detection agent of the invention in a specific bioaffinity based binding assay, e.g. utilizing time-resolved fluorometric determination of a specific luminescence.
- the specific bioaffinity based binding assay is a heterogeneous immunoassay, a homogenous immunoassay, a DNA hybridization assay, a receptor binding assay, an immunocytochemical or an immunohistochemical assay.
- the present invention relates to the use of a compound of formula (I) or formula (II) of the invention or the detection agent of the invention in bio- imaging applications.
- a use is particularly advantageous if the compound of formula (I) or formula (II) of the invention is a molecule with a neutral net charge or almost neutral net charge (i.e. the molecule comprises an overall net charge of from -3 to +5).
- the compounds of the invention or the detection agent of the invention may for example be used as a contrasting agent.
- the contrasting agent may e.g. be used in MRI or PET applications.
- the compound of the invention or the detection agent of the invention may further be used for microscopy applications, e.g. in cell culture experiments, such as in confocal laser scanning microscopy and or hybridization experiments.
- Still another aspect of the invention relates to a solid support material conjugated with a compound of formula (I) or formula (II) of the invention or the detection agent of the invention.
- the compound or the detection agent of the invention is typically immobilized to the solid support material either covalently or non-covalently.
- the solid support material is selected from a nanoparticle, a microparticle, a slide, a plate, and a solid phase synthesis resin.
- conjugating group refers to a moiety connecting two other moieties by at least two covalent bonds. Therefore, a conjugating group is a biradical group.
- the term“reactive group” as in“reactive group Z” refers to a functional group that may react in a labelling reaction of a compound of the invention with a bio specific binding reactant, or is facilitating the formation of a covalent bond to a solid support material.
- the chelate may be introduced in the solid support, e.g. a particle, simultaneously with the preparation of the particles.
- the reactive group Z establishes a link to said biospecific binding reactant.
- n 1-6; and a triazole (e.g. formed by the so-called
- hydrophilic group refers to a moiety that is present in order to improve the water solubility of the chelate.
- a compound comprising a hydrophilic group as a substituent has a higher solubility in water than the corresponding compound not comprising said hydrophilic group.
- hydrophilic groups are mono- and oligosaccharides, such as
- oligoalkylene glycols e.g. those having 1-20 repeating units
- oligoethylene glycol and oligopropylene glycol and the like.
- the term“chelating group” refers to a moiety of a chelate that inter alia coordinates the metal ion of the chelate.
- the term“-Ci -12-alkyl” refers to straight-chain and branched non- cyclic saturated hydrocarbons having from 1 to 12 carbon atoms.
- Representative straight chain -C1 -12 alkyl groups include -methyl, -ethyl, -n-propyl, -n-butyl, -n- pentyl, -n-hexyl, n-heptyl, n-octyl, n-nonyl and n-decyl.
- branched -(Ci-Ci 2 )alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, neopentyl, l-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1- dimethylpropyl, 1 ,2-dimethylpropyl, l-methylpentyl, 2-methylpentyl, 3- methylpentyl, 4-methylpentyl, l-ethylbutyl, 2-ethylbutyl, 3-ethylbutyl, 1,1- dimethylbutyl, 1 ,2-dimethylbutyl, l,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3- dimethylbutyl, 3,3-dimethylbutyl, 5-methylhexyl, 6-methylheptyl, and the like.
- -Ci- 6 -alkyl refers to straight-chain and branched non- cyclic saturated hydrocarbons having from 1 to 6 carbon atoms.
- Representative straight chain -Ci - 6 -alkyl groups include -methyl, -ethyl, -n-propyl, -n-butyl, -n- pentyl, and -n-hexyl.
- Representative branched-chain -Ci- 6 -alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, neopentyl, l-methylbutyl, 2- methylbutyl, 3-methylbutyl, l,l-dimethylpropyl, and 1 ,2-dimethylpropyl, methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-mehtylpentyl, l-ethylbutyl, 2- ethylbutyl, 3-ethylbutyl, l,l-dimethylbutyl, 1 ,2-dimethylbutyl, l,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, and the like.
- the term“-C2 12-alkenyl” refers to straight chain and branched non- cyclic hydrocarbons having from 2 to 12 carbon atoms and including at least one carbon-carbon double bond.
- Representative straight chain and branched -C2-12- alkenyl groups include -vinyl, -allyl, -l-butenyl, -2-butenyl, -isobutylenyl, -1- pentenyl, -2-pentenyl, -3 -methyl- l-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2- butenyl, -l-hexenyl, -2-hexenyl, 3-hexenyl, and the like.
- -C2-i2-alkinyl refers to straight chain and branched non- cyclic hydrocarbons having from 2 to 12 carbon atoms and including at least one carbon-carbon triple bond.
- Representative straight chain and branched -C2-i2-alkinyl groups include -acetylenyl, -propynyl, -l-butynyl, -2-butynyl, -l-pentynyl, -2- pentynyl, -3 -methyl- l-butynyl, -4-pentynyl, -l-hexynyl, -2-hexynyl, -5-hexynyl, and the like.
- -(5- to l2-membered)heteroaryl or“-Het” means an aromatic heterocycle ring of 5 to 12 members, including both mono- and bicyclic ring systems, where at least one carbon atom (of one or both of the rings) is replaced with a heteroatom independently selected from nitrogen, oxygen, and sulfur, or at least two carbon atoms of one or both of the rings are replaced with a heteroatom independently selected from nitrogen, oxygen, and sulfur.
- one of the bicyclic -(5- to l2-membered)heteroaryl rings contains at least one carbon atom.
- both of the bicyclic -(5- to l2-membered)heteroaryl rings contain at least one carbon atom.
- Representative -(5- to l2-membered)heteroaryls include pyridyl, furyl, benzofuranyl, thiophenyl, benzothiophenyl, quinolinyl, isoquinolinyl, pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isoxazolyl, oxadiazolinyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidyl, pyrimidinyl, pyrazinyl, thiadiazolyl, triazinyl, thienyl, thiadiazolyl, cinnolinyl, phthalazinyl
- a "-(6- to l4-membered)aryl” or“-Ar“ means an aromatic carbocyclic ring containing 6 to 14 carbon atoms, including both mono- and bicyclic ring systems.
- Representative -(6- to l4-membered)aryl groups include -indenyl, - phenyl, -naphthyl, -anthracenyl and the like.
- the term“optionally substituted” refers to a group that is either unsubstituted or substituted.
- Optional substituents on optionally substituted groups include 1, 2, 3, 4 or 5 groups each independently selected from the group consisting of Ci-12-alkyl, -COOH, -Ci-e-alkyl-COOH, -COO -Ci -e-alky l-COO , -S0 3 H, -Ci-e- alkyl-SOsH, -S0 3 , -Ci -e-alkylS 0 3 , -Ci -e-alky l-0-P0 3 2 , -Ci- 6 -alkyl-P0 3 2 , -Ci-e- alklyl-0-P0 3 H 2 , -Ci- 6 -alkyl-P0 3 H 2 , -Ci-e-alkyl-OH, -
- the term“lanthanide ion” or“Ln 3+ ” is intended to mean a trivalent ion of the lanthanide series of the Periodic Table of Elements, e.g. europium(III), terbium(III), samarium(III) and dysprosium(III), i.e. Eu 3+ , Tb 3+ , Sm 3+ or Dy 3 .
- europium(III) (Eu 3+ ) and terbium(III) (Tb 3+ ) are preferred.
- the basic structure of the lanthanide chelate of the formula (I) (as well as the lanthanide chelating ligand of the formula (II)) comprises at least two negative charges, and even more negative charges depending on the substituent in formula (I) or (II).
- the compounds, respectively, in addition to what is illustrated in formula (I) and formula (II), may be further associated with one or more cations as counter ions as“salts”. Examples of such counter ions are Na + , Ca 2+ , K + . Particularly preferred are Na + and K + .
- the counter ions are those from Groups IA and IIA of the periodic table of elements.
- the metal ion Ln 3+ which is bound by coordinate bonds in the chelate, is not considered as a counter ion in a salt.
- the term“reactive ester” means activated carboxylic acids to improve their reactivity generally known e.g. in peptide synthesis and biomolecule labeling, e.g. esters with N-hydroxysuccinimide etc. (see e.g. Montalbetti, C., et. al.,
- spacer is intended to mean a distance-making group between, e.g., a conjugating group or a pyridine moiety of the core structure within the reactive group Z or a hydrophilic group.
- the spacer typically has a length of 1-20 bonds between the attachment point and reactive group (or hydrophilic group), such as 3-15 bonds, or 5-12 bonds.
- the term“distance-making biradical” refers to a group that forms bonds to two other groups with the purpose to separate the two other groups from each other, e.g. as a linker between the two other groups, e.g. to facilitate positioning a reactive group in a position accessible for reaction with a biospecific binding reactant.
- the term“monosaccharide” is intended to mean C5-C7 carbohydrates being either in the acyclic or in cyclic form.
- monosaccharides are C 6 carbohydrates, e.g. those selected from
- oligosaccharide refers to a saccharide polymer containing a small number, typically from 3 to 10 units of monosaccharides mentioned above, which are preferably linked together by glycosidic bonds.
- polysaccharide refers to a saccharide polymer containing more than 10 units of monosaccharides, preferably linked together by glycosidic bonds.
- biospecific binding reactant is a compound capable of specifically binding an analyte of interest for the purpose of quantitative or qualitative analysis of said analyte in a sample (e.g. a sample of a bodily fluid).
- the term“antibody” refers to the commonly known Y-shaped protein produced mainly by plasma cells that is used by the immune system to neutralize pathogens such as pathogenic bacteria and viruses.
- the term“antibody” as used in the context of the present invention also comprises molecules derived from such antibodies, such as a Fab-fragment, Fab2-ffagment, Fc-fragment, diabodies and the like.
- an“antigen” is a molecule capable of inducing an immune response to produce an antibody.
- an antigen may be a molecule binding to an antibody.
- a“receptor ligand” is a molecule that is known to bind to a cell receptor.
- receptor ligands are neurotransmitters, hormones, growth factors or the like.
- corresponding receptors are G-protein coupled receptors, protein kinase receptors and the like.
- the term“DNA probe” or“RNA probe” refers to a deoxynucleic acid or a ribonucleic acid that may hybridize as a“probe” with a target nucleic acid sequence.
- the probe may comprise a complementary sequence to a target nucleic acid sequence.
- protein covers any type of protein, including enzymes or specific binding proteins that interact specifically with one or more target molecules.
- protein refers to any polymer of amino acids of any length, and therefore also covers peptides e.g. oligopeptides which only comprise 2 to 100 amino acids.
- phospholipid refers to a class of lipids that are a major component of all cell membranes.
- the structure of the phospholipid molecule generally consists of two hydrophobic fatty acid "tails" and a hydrophilic "head” consisting of a phosphate group. The two components are joined together by a glycerol molecule.
- the phosphate groups can be modified with simple organic molecules such as choline.
- PNA or“peptide nucleic acid” refers to an artificially synthesized polymer similar to RNA or DNA.
- DNA and RNA have a deoxyribose and ribose sugar backbone, respectively, whereas PNA's backbone is composed of repeating -N-(2-aminoethyl)-glycine units linked by peptide bonds.
- the term“steroid” refers to an organic compound with four rings arranged in a specific molecular configuration. Examples include the dietary lipid cholesterol, the sex hormones estradiol and testosterone and the anti-inflammatory drug dexamethasone. Steroids have two principal biological functions: certain steroids (such as cholesterol) are important components of cell membranes which alter membrane fluidity, and many steroids are signaling molecules which activate steroid hormone receptors.
- the steroid core structure is composed of seventeen carbon atoms, bonded in four "fused" rings: three six-membered cyclohexane rings and one five-membered cyclopentane ring. Steroids vary by the functional groups attached to this four-ring core and by the oxidation state of the rings.
- Sterols are forms of steroids with a hydroxyl group at position three and a skeleton derived from cholestane. They can also vary more markedly by changes to the ring structure as for example in ring scissions which produce secosteroids such as vitamin D3.
- hapten refers to a molecule that elicits an immune response only when attached to a large carrier molecule such as a protein; wherein the carrier may be one that preferably does not elicit an immune response by itself.
- drug refers to a compound with pharmacological properties that may be used in the preventive or therapeutic treatment of a disease.
- lectin refers to carbohydrate-binding proteins, i.e.
- oligonucleotide refers to short DNA or RNA molecules (comprising less than 30 monomers), oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids can be manufactured as single-stranded molecules with any user-specified sequence, and so are vital for artificial gene synthesis, polymerase chain reaction (PCR), DNA sequencing, library construction and as molecular probes. In nature, oligonucleotides are usually found as small RNA molecules that function in the regulation of gene expression (e.g. microRNA), or are degradation intermediates derived from the breakdown of larger nucleic acid molecules.
- Oligonucleotides are characterized by the sequence of nucleotide residues that make up the entire molecule.
- Modified oligonucleotides refer to oligonucleotides that comprise one or more non-natural nucleic acid, i.e. a nucleic acid that does not comprise cytosine, guanine, adenine, thymine or uracil as base.
- A“polynucleotide” refers to a biopolymer composed of 30 or more nucleotide monomers covalently bonded in a chain.
- A“modified polynucleotide” refers to polynucleotide that comprises one or more non-natural nucleic acid monomers.
- the term“analyte” refers to a target parameter of interest to be determined qualitatively and/or quantitatively in a sample.
- the term“biospecific binding assay” or“specific bioaffinity based binding assay” means an in vitro assay wherein a specific complex is formed between a biomolecule and a target molecule and the presence of the complex, i.e. the binding, may be detectable by standard biochemical methods.
- the term“biospecific binding reactant” means a biomolecule that may specifically bind to an analyte of interest under conditions present in a biospecific binding assay.
- the term“detection agent” means a compound that is detectable e.g. in an in vitro assay format, e.g. by luminescence or UV-VIS absorbance.
- the term“specific luminescence” refers to the luminescence of a detection agent, wherein the luminescence is measured in a way that ensures that the luminescence is specifically related to the detection agent, e.g. by selecting a wavelength for light measurement where the detection agent shows a high emission of light, e.g. close to the emission maximum in the spectra of the detection agent.
- luminescence refers to the emission of light by a substance not resulting from heat. Examples of luminescence are
- the activated derivatives include, but are not limited to, isocyanato (-NCO),
- sample refers to a sample collected from a patient for use in in vitro bioassays to determine an analyte parameter of interest, wherein the sample is typically a bodily fluid, such as blood, saliva, urine, cerebrospinal fluid, or a tissue sample such as a biopsy sample.
- a bodily fluid such as blood, saliva, urine, cerebrospinal fluid, or a tissue sample such as a biopsy sample.
- Fluorescence efficiencies were determined with Perkin-Elmer Wallac Victor plate fluorometer. Eu-content of Eu-chelates and labelled antibodies were measured by using ICP-MS instrument, PerkinElmer 6100 DRC Plus, in quantitative mode. The excitation, emission spectra and decay times were recorded on a Varian Cary Eclipse fluorescence spectrometer.
- Trifluoroacetic anhydride (11.1 ml, 80 mmol) was added to a cold solution of 4- aminophenylacetic acid (3.02 g, 20 mmol) in trifluoroacetic acid (30 ml). After stirring for 15 min at 0 °C and 2 h at RT, H 2 0 (50 ml) was added, the cooled mixture was filtrated and the product washed with H 2 0 (3 x 50 ml). Yield: 3.94 g (80 %).
- N-hydroxysuccinimide (0.69 g, 6.0 mmol) and N,N-dicyclohexylcarbodiimide (1.23 g, 6.0 mmol) was added to a solution of compound 2 (1.48 g, 6.0 mol) in dry 1,4- dioxane (20 ml). After stirring for 4.5 h at RT, the mixture was filtrated, the solid material washed with l,4-dioxane (2 x 5 ml and 10 ml) and the filtrate was evaporated to dryness.
- N-hydroxysuccinimide (0.51 g, 4.42 mmol) and N,N-dicyclohexylcarbodiimide (0.91 g, 4.42 mmol) was added to a solution of compound 2 (1.09 g, 4.42 mol) in dry 1,4- dioxane (10 ml). After stirring for 5 h at RT, the mixture was filtrated, the solid material washed with 1 ,4-dioxane (4 x 5 ml) and the filtrate was evaporated to dryness. The residue was dissolved in dry DMF (15 ml) and after an addition of compound 5 (1.21 g, 4.42 mmol) in dry DMF (15 ml), the mixture was stirred overnight at RT.
- MALDI TOF-MS mass calculated (M+l + ) 566.99, 568.99 and 570.98; found 565.67, 567.67 and 569.68.
- This compound 12 was synthesized from the compound 9 using a method analogous to the synthesis described in the Example 8. The product was purified by FC (silica gel, 10% MeOH/DCM). Yield: 65%.
- MALDI TOF-MS mass calculated (M+H + ) 680.06, 682.07 and 684.07; found 680.27, 682.18 and 684.36.
- This compound 15 was synthesized from the compound 11 and 13 using a method analogous to the synthesis described in the Example 11.
- the product (70%) was purified by FC (silica gel, 7.5% EtOH/DCM).
- MALDI TOF-MS mass calculated (M+H + ) 1171.16 and 1173.16; found 1171.40 and 1173.22.
- Example 13 Synthesis of compound 16 This compound 16 was synthesized from the compound 12 and 13 in dry DMF at 70 °C using a method analogous to the synthesis described in the Example 11. The product (26%) was purified by FC (silica gel, 5% EtOH/DCM/l% TEA). TOF-MS mass: calculated (M+EE) 1230.20 and 1232.19; found 1230.27 and 1232.26
- This compound 19 was synthesized from the compound 15 and 17 using a method analogous to the synthesis described in the Example 14. The product (61%) was purified by FC (silica gel, from 8% to 10% EtOH/DCM). MAFDI TOF-MS mass: calculated (M + ) 2153.80, (M+K + ) 2192.80; found 2152.01 and 2191.97.
- This compound 20 was synthesized from the compound 16 and 17 using a method analogous to the synthesis described in the Example 14. The product (64%) was purified by FC (silica gel, 10% EtOH/DCM). MAFDI TOF-MS mass: calculated (M+H + ) 2213.85; found 2212.64.
- Example 21 Synthesis of compound 25 This compound 25 was synthesized from the compound 22 using a method analogous to the synthesis described in the Example 20.
- Example 22 Synthesis of compound 26 This compound 26 was synthesized from the compound 23 using a method analogous to the synthesis described in the Example 20. R f (HPLC): 15.10 min.
- Example 23 Synthesis of compound 27 2,4,6-Tricloro-l,3,5-triazine (6 mg, 31 umol) was added to a mixture of compound 21 (57 mg, 21 umol) in EEO (0.5 ml) and acetone (0.25 ml) and the pH was adjusted to 7.0 with suitable additions of solid NaHCCE. After stirring for 30 min, the product was precipitated with acetone, centrifuged and washed with acetone. R f (HPLC): 13.32 min. UV: 360 nm.
- Example 24 Synthesis of compound 28 This compound 28 was synthesized from the compound 22 using a method analogous to the synthesis described in the Example 23.
- MALDI TOF-MS mass calculated (M+H + ) 2019.39; found 2018.47.
- Example 27 Synthesis of compound 32 A mixture of compound 30 (0.76 g, 1.21 mmol) and PBn (135 m ⁇ , 1.45 mmol) in dry l,4-dioxane (125 ml) was stirred for 2 h at RT, concentrated, dissolved in 10% EtOH/DCM (60 ml) and neutralized with 5% NaHC0 3 (20 ml). The aqueous phase was extracted with 10% EtOH/DCM (2 x 15 ml), the combined organic phases were washed with H 2 0 (30 ml) and dried with Na 2 S0 4 . After evaporation the product (0.74 g, 89%) was used in the next step without any further purifications.
- MALDI MALDI
- TOF-MS mass calculated (M+Na + ) 711.12 and 713.12; found 711.39 and 713.57.
- Example 28 Synthesis of compound 33 This compound 33 was synthesized from the compound 31 using a method analogous to the synthesis described in the Example 27. Yield: 63%.
- Example 29 Synthesis of compound 34 A mixture of compound 32 (0.22 g, 0.32 mmol), 13 (0.20 g, 0.32 mmol; Sund, H., et al, Molecules 22(2017)1807), dry K2CO3 (0.18 g, 1.29 mmol) and dry DMF (7.5 ml) was stirred at 60 °C for 2 days. After filtration and washes with DMF, the product (0.19 g, 43%) was purified by FC (silica gel, from 7.5% EtOH/DCM). MALDI TOF- MS mass: calculated (M+H + ) 1239.25; found 1238.80.
- This compound 35 was synthesized from the compound 33 and 13 using a method analogous to the synthesis described in the Example 29 and using dry MeCN as the reaction solvent. Yield: 79%.
- MALDI TOF-MS mass calculated (M+Na + ) 1317.31; found 1318.52.
- This compound 37 was synthesized from the compound 35 and 17 using a method analogous to the synthesis described in the Example 31. Yield: 23%.
- MALDI TOF- MS mass calculated (M+H + ) 1950.75; found 1949.21.
- This compound 38 was synthesized from the compound 36 using a method analogous to the synthesis described in the Example 17. Yield: 100%. R f (HPLC): 13.35 min.
- This compound 41 was synthesized from the compound 39 using a method analogous to the synthesis described in the Example 20.
- This compound 47 was synthesized from the compound 46 and 17 using a method analogous to the synthesis described in the Example 14.
- the product (85%) was purified by FC (silica gel, from 5% to 10% MeOH/DCM).
- This compound 49 was synthesized from the compound 48 using a method analogous to the synthesis described in the Example 20.
- Example 42 Conjugate reaction of the activated reagents 24-28, 40, 41 or 49 with taurine and photochemical measurements of the corresponding products
- ICP-MS calibration standard 1 A commercial multi- standard from Ultra Scientific, IMS-101, ICP-MS calibration standard 1 was used for the calibration.
- the sample preparation for the ICP-MS was performed by using a digestion procedure i.e. a microwave digestion system from Anton Paar, Microwave Sample preparation System, Multiwave 3000.
- the Eu chelate in the 50 mM TRIS buffer was digested with microwave in mixture of Suprapur acids, HNO 3 (5 ml) and H2O2 (1 ml). Afterwards the sample was diluted with deionized water (100 ml).
- the R f values reflect improved water solubility as all of them have lower R f values compared to the corresponding value of the reference chelate.
- the synthesis of taurine conjugate from the reference chelate is described e.g. in Sund, H., et ah, Molecules 22(2017)1807.
- the reference Eu(III) chelate was prepared according to von Lode P. et al., Anal. Chem. 75(2003)3193. All in all, the novel Eu(III) chelates are preferable labels compared to the commercial used reference label.
- the UV absorption and excitation wavelengths are exceptionally high being the highest ones reported for Eu(III) labelling reagents, and thus, they perfectly fit to be used in instrumentation designs based on cheap UV light emitting diode excitation at 370 nm.
- the absorptivities are also high (e.g. 90 000 M 'cm 1 for 25 and rest are almost at same level) which enable the moderate luminescence brightness regardless of the low quantum yields.
- the main reason behind the low quantum yield is the excitation energy back- flow from the excited Eu(III) ion due to the low lying intra ligand charge transfer (ILCT) state which has been observed with Eu(III) chelate with similar 4-phenylethynyl pyridine chromophores (see e.g. Andraud, C, et al.,
- Tnl antibody Labeling of a Tnl antibody was performed similarly as described in von Lode P. et al, Anal. Chem. 75(2003)3193 by using 350 mM Na 2 CC> 3 buffer (pH 9.8) as reaction buffer and 300 fold excess of the labelling reagents 25, 28, 41 or 49. The reactions were carried out overnight at RT.
- the labeled antibody was separated from the excess of chelates on Superdex 200 GL 10/30 gel filtration column (GE healthcare) by using TRIS-saline-azide buffer (50 mM TRIS, 0.9% NaCl, pH 7.75) as an eluent. The fractions containing the antibody were pooled and the Eu concentration was measured by UV and secured by ICP-MS described in the Example 42.
- the Tnl antibody labeled with the chelate 25, 28, 41 and 49 was tested in sandwich immunoassay for cardiac troponin I.
- a Tnl antibody labelled with a-gal-9-D Eu von Lode P. et al., Anal. Chem. 75(2003)3193
- 10 m ⁇ of diluted tracer antibody f 5 ng/m ⁇ 10 m ⁇ of diluted tracer antibody f 5 ng/m ⁇
- 20 m ⁇ of Tnl standard solution were pipetted to a pre-coated assay well (single wells in 96 well plate format, wells coated with streptavidin and a biotinylated capture antibody against Tnl, Radiometer Turku Oy).
- the reaction mixtures were incubated for 20 min at 36°C with shaking.
- the wells were washed 6 times and dried prior to measurement with VictorTM Plate fluorometer.
- the results are summarized in Table 2.
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