Patent Application
20210395606
The invention relates to an azamacrocyclic lanthanide chelate design having substituted 4-(phenylethynyl)pyridine chromophores around an emitting lanthanide core. The chromophores have high molar absorptivity and luminescence with lanthanide ions. The invention also relates to the ligand from which the chelate is prepared, and to chelates attached to a biospecific reactant, and their use in various assays.
WO2013/011236 discloses luminescent lanthanide chelates having three 4-(phenylethynyl)pyridine chromophoric groups tethered to a triazamacrocyclic core. The 4-(phenylethynyl)pyridine chromophoric groups are substituted at the para-position of the phenyl ring with an electron donating group.
The scientific literature (Tetrahedron Letters, 55, 2014, 1357-1361) acknowledges that the triazamacrocyclic ligands of the type disclosed in WO2013/011236 have relatively poor aqueous solubility. Attempts to improve aqueous solubility by appending a PEG group to the electron donating para-substituent were of limited success.
WO2013/092992 discloses luminescent lanthanide chelates having three 4-(phenylethynyl)pyridine chromophoric groups tethered to an acyclic core. In some embodiments, one chromophoric group comprises a reactive group and the other two chromophoric groups comprise two or three —OCH2CO2H groups in the ortho and/or para positions.
WO2014/147288 discloses triazacyclononane-based lanthanide chelate complexes useful as labelling reagents. The disclosed chelates have three 4-(phenylethynyl)pyridine chromophoric groups, one of which chromophoric groups comprises a reactive group; the other two chromophoric groups have either (i) two carboxyl (—CO2H) substituents on the phenyl ring in the meta and para positions, or (ii) two —OCH2CO2H groups on the phenyl ring in the meta positions.
A first aspect of the invention relates to a luminescent lanthanide chelate of formula (I) or a salt or solvate thereof:
wherein a, b, and c are independently selected from 0 and 1; and
Ln3+ is selected from Eu3+ , Tb3+, Dy3+, and Sm3+; and
Chrom1, Chrom2, and Chrom3 are of formula (II):
wherein Che is a chelating group independently selected from —CO2H, —PO3H2, —PO(OH)R2, —CH2PO3H2, and —CONR3R4,
R2 is selected from phenyl, benzyl, methyl, ethyl, propyl, n-butyl, iso-butyl, sec-butyl or tert-butyl,
R3 and R4 are independently selected from hydrogen and —L1—Z1, wherein L1 is a direct bond or a spacer group, and Z1 is a reactive group enabling the chelate to be linked to biospecific reactant; and
d is 1, 2, 3, 4, or 5;
R1 is one or more substituents independently selected from any one of the group consisting of:
(i) hydrogen,
(ii) an electron donating solubilising group selected from —X—R5 wherein X is an oxygen atom, a sulphur atom, or —N(R6)CO—, R6 is hydrogen or —C1-6alkyl, and R5 is selected from hydrogen, —C1-6alkyl, —(CH2)1-6OH, —(CH2)1-6OC1-6alkyl, —(CH2)1-6CO2H, —(CH2)1-6CONR7R8, —(CH2)1-6SO3H, —(CH2)1-6NH2, —(CH2)1-6N(CH3)2, —(CH2)1-6N(CH3)2+—(CH2)1-6SO3− and polyethylene glycol, wherein R7 and R8 are each independently selected from hydrogen, C1-6alkyl, —(CH2)1-6OH, —CH(CH2OH)2, and —CH(CH2OH)3, (iii) a group selected from C1-6alkyl, —(CH2)1-6OH, —(CH2)1-6OCH3, —(CH2)1-6SCH3 (iv) —L2—Z2, wherein L2 is a direct bond or a spacer group, and Z2 is a reactive group enabling the chelating agent to be linked to a molecule to be labelled,
wherein at least one of the groups Chrom1, Chrom2, and Chrom3 has two or more R1 substituents selected from group (ii) in the para and ortho positions in relation to the acetylene group; and
provided that the chelate of formula (I) has no more than one reactive group selected from Z1 and Z2.
A second aspect of the invention relates to a detectable molecule comprising a bio-specific binding reagent conjugated to a luminescent lanthanide chelate according to the first aspect of the invention.
A third aspect of the invention relates to the lanthanide chelating ligand from which the chelate of the first aspect of the invention is prepared.
A fourth aspect of the invention relates to a method of carrying out a biospecific binding assay, said method comprising the steps of:
a) forming a biocomplex between an analyte and a biospecific binding reactant labelled with a luminescent lanthanide chelate according to the first aspect of the invention;
b) exciting said biocomplex with radiation having an excitation wavelength, thereby forming an excited biocomplex; and
c) detecting emission radiation emitted from said excited biocomplex.
A fifth aspect of the invention relates to a use of a detectable molecule according to the second aspect of the invention in a specific bioaffinity based binding assay utilizing time-resolved fluorometric determination of a specific luminescence-resolved fluorometric determination of a specific luminescence.
A sixth aspect of the invention relates to a solid support material conjugated with a luminescent lanthanide chelate according to the first aspect of the invention or a lanthanide chelating ligand according to the third aspect of the invention.
The lanthanide chelates and the detectable molecules of the present invention have advantageously high aqueous solubility. Detectable molecules having high aqueous solubility are useful in, for example, bioassays which benefit from a high concentration of detectable molecules. A higher concentration of detectable molecules enables a more sensitive assay, and necessitates a reduced volume of assay media. It is advantageous also because the detectable molecules have high solubility in aqueous samples requiring analysis such as blood plasma, saliva, other body fluids, and preparations thereof.
The lanthanide chelates and the detectable molecules of the present invention have advantageously high luminescence yields i.e. brightness (ccD), especially when dry. Examples of antibodies labelled with the claimed chelate (see Examples 18 and 19) demonstrate an exceptionally high luminescence yield of up to 69500 M−1 cm−1 when dry. This high luminescence enables a very sensitive assay because the bright biomolecule-detectable molecule conjugate is easily detected. The surprising 80-100 fold improvement in the luminescence of the dry detectable molecule compared to an aqueous solution of the same enables the skilled person to significantly increase the sensitivity of an assay by simply adding a drying step.
The ligands of the claimed invention form surprisingly stable complexes with lanthanide ions. Therefore the claimed luminescent lanthanide chelates and detectable molecules have an advantageously high stability. By ‘high stability’ it is meant that the complexed lanthanide ion has a reduced tendency to escape from the ligand or to be exchanged by an alternative ion. High stability is advantageous because the loss of the lanthanide ion from the ligand results in a loss of detectable luminescence, and therefore a reduced utility in the assays of the present invention. This high stability is especially useful when the chelates or detectable molecules are used in conditions having a high concentration of alternative metal ions and/or other chelates. The high stability enables the chelates of the present invention to be used together with other labelled chelates for example when two or more different probes are used in immunoassays or DNA hybridisation assays. The high stability is advantageous because the claimed chelates and detectable molecules can be used in conditions requiring an elevated temperature such as Polymerase Chain Reaction (PCR) assays, especially during the multiplication cycles.
Furthermore, the chelates and detectable molecules can tolerate long incubation times in the presence of additional metal ions and/or at high temperatures.
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 immuno-assays (both homogeneous and heterogeneous), nucleic acid hybridization assays, receptor-binding assays, enzymatic assays, immunocytochemical, immunohistochemical assays and cell based assays utilizing fluorometric or time-resolved fluorometric determination of specific luminescence based on one or two photon-excitation. Chelates of the present invention provide means to obtain improved bioaffinity based binding assays even at wavelengths above 340 nm. The present invention makes available new ligands, chelates and detectable molecules having, for example, improved solubility, improved assay sensitivity, improved luminescence, improved high temperature stability, and improved stability in the presence of other ions and chelates.
One aspect of the present invention relates to a luminescent lanthanide chelate of formula (I) or a salt thereof:
In the triazamacrocyclic ring of the present invention the units a, b, and c are independently selected from 0 and 1. In an embodiment a=b=c=0.
Ln3+ is a trivalent lanthanide ion selected from europium (III) (Eu3+), terbium (III) (Tb3+), dysprosium (III) (Dy3+), and samarium (III) (Sm3+). In a preferred embodiment Ln3+ is Eu3+.
The chelates of the present invention have three chromophoric groups of formula (II), namely Chrom1, Chrom2, and Chrom3.
The group Che is a chelating group independently selected from —CO2H, —PO3H2, —PO(OH)R2, —CH2PO3H2, and —CONR3R4 such as —CONH2. In a preferred embodiment the group Che is —CO2H. In embodiments where Che is ionisable, such as where Che=CO2H, the Che group can exist in ionised (e.g. —CO2−) or non-ionised (CO2H) forms.
The group R2 is selected from phenyl, benzyl, methyl, ethyl, propyl, n-butyl, iso-butyl, sec-butyl or tert-butyl.
R3 and R4 are independently selected from hydrogen and —L1—Z1, wherein L1 is a direct bond or a spacer group, and Z1 is a reactive group enabling the chelate to be linked to a molecule biospecific reactant. In an embodiment, the groups R3 and R4 are both hydrogen.
The phenyl rings of the chromophoric groups Chrom1, Chrom2, and Chrom3 are each substituted with 1, 2, 3, 4, or 5 R1 groups.
The 1, 2, 3, 4 or 5 R1 groups are each individually selected from any one of the groups consisting of:
(i) hydrogen,
(ii) an electron donating solubilising group selected from —X—R5 wherein X is an oxygen atom, a sulphur atom, or —N(R6)CO—, R6 is hydrogen or C1-6alkyl such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl or t-butyl, and R5 is selected from hydrogen, —C1-6alkyl such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl or t-butyl, —(CH2)1-6OH such as —CH2OH or —(CH2)2OH, —(CH2)1-6OC1-6alkyl such as —(CH2)OCH3 or —(CH2)2OCH3, —(CH2)1-6CO2H such as —CH2CO2H or —(CH2)2CO2H, —(CH2)1-6CONR7R8 such as —CH2CONH2, —(CH2)1-6SO3H, —(CH2)1-6NH2, —(CH2)1-6N(CH3)2, —(CH2)1-6N(CH3)2+—(CH2)1-6SO3− and polyethylene glycol such as —(CH2CH2O)1-4OCH2CH2OH, or —(CH2CH2O)1-4OCH2CH2OCH3;
wherein R7 and R8 are each independently selected from hydrogen, C1-6alkyl, such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl or t-butyl, —(CH2)1-6OH such as —CH2OH or —(CH2)2OH, —CH(CH2OH)2, and —CH(CH2OH)3,
in one embodiment an electron donating solubilising group selected from —X—R5 wherein X is an oxygen atom or —N(R6)CO—, R6 is hydrogen or C1-6alkyl, and R5 is selected from hydrogen, —(CH2)1-6OH, —(CH2)1-6CO2H, —(CH2)1-6CONR7R8, and —(CH2)1-6SO3H, wherein R7 and R8 are each independently selected from hydrogen, C1-6alkyl-OH, —CH(CH2OH)2, and —CH(CH2OH)3,
(iii) a group selected from C1-6alkyl such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl or t-butyl, —(CH2)1-6OH such as —CH2OH or —(CH2)2OH, —(CH2)1-6OCH3 such as —(CH2)OCH3 or —(CH2)2OCH3, or —(CH2)1-6SCH3,
in one embodiment a group selected from C1-6alkyl such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl or t-butyl, —(CH2)1-6OH such as —CH2OH or —(CH2)2OH, (iv) —L2—Z2, wherein L2 is a direct bond or a spacer group, and Z2 is a reactive group enabling the chelating agent to be linked to a biospecific reactant.
At least one (i.e. one, two or all three) of the groups Chrom1, Chrom2, and Chrom3 has two or more R1 substituents selected from group (ii) in the para and ortho positions in relation to the acetylene group. In a preferred embodiment, two of the groups Chrom1, Chrom2, and Chrom3 have two or three R1 substituents selected from group (ii) in the para and ortho positions in relation to the acetylene group, and the third chromophoric group is substituted with —L2—Z2.
In an embodiment X is an oxygen atom. In a preferred embodiment X is an oxygen atom and R5 is —(CH2)1-6CO2H such as —CH2CO2H, or —(CH2)1-6SO3H, or —(CH2)1-6N(CH3)2+—(CH2)1-6—SO3.
In an embodiment one or two of the chromophoric groups Chrom1, Chrom2, and Chrom3 are independently selected from the chromophoric groups of formula (IIa), (IIb) or (IIc) in which the groups R1A, R1AA, R1AAA, R1B, R1BB, R1C, and R1CC are each independently selected from R1 group (ii) as defined hereinbefore.
In a preferred embodiment the groups R1A, R1AA, R1AAA, R1B, R1BB, R1C, and R1CCC are —OCH2CO2H.
In a preferred embodiment the chelating agents of formula (I) have only one reactive group. In a preferred embodiment the Che group does not comprise a reactive group. Rather, the reactive group Z2 is connected via L2 to the phenyl ring of a chromophoric group selected from Chrom1, Chrom2, and Chrom3
In a preferred embodiment, two of the chromophoric groups Chrom1, Chrom2, and Chrom3 are selected from formula (IIa), (IIb) or (IIc) as defined hereinbefore, and the third chromophoric group is selected from (IId), (IIe), or (IIf):
wherein R1A, R1AA, R1AAA, R1B, R1BB, R1C, and R1CCC are each independently selected from R1 group (ii) as defined hereinbefore. In a preferred embodiment L2 is a direct bond and Z2 is an isothiocyanato (—NCS) group. In a preferred embodiment the chromophoric group comprising the reactive group has formula (IIg).
As used herein, the term C1-6alkyl includes, but is not limited to, the following alkyl groups: methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl and t-butyl.
As used herein, the terms ‘ortho’ and ‘para’ when used to describe the substitution pattern of a 6-membered ring (e.g. phenyl) mean substituted at the 2- and 4-positions respectively. For example, a phenyl ring substituted with three substituents at the ortho and para positions is a 2, 4, 6 substituted ring.
It should be understood that when the ligands and chelates of the present invention comprise ionisable groups such as carboxylates, sulfonates and the like, the chelates may be present in ionised (e.g. —CO2−) or non-ionised (e.g. —CO2H) forms, and if ionised may include cations as counter ions, e.g. Na+, K+, Ca2+ and the like.
The chelates and ligands of the present invention comprise a reactive group (Z1 or Z2), optionally linked to the ligand or chelate by a spacer (L1 or L2). In such instances, the reactive group is facilitating the labelling of a biospecific binding reactant, or is facilitating the formation of a covalent bond to a solid support material. In case the chelate has a polymerizing group as reactive group, then the chelate may be introduced in the solid support, e.g. a particle, simultaneously with the preparation of the particles.
If present, the reactive group is typically selected from azido (—N3), alkynyl (—C═CH), alkylene (—CH═CH2), amino (—NH2), aminooxy (—O—NH2), carboxyl (—CO2H), aldehyde (—CHO), mercapto (—SH), maleimido, activated derivatives of maleimido, isocyanato (—NCO), isothiocyanato (—NCS), diazonium (—N+N), bromoacetamido, iodoacetamido, reactive esters, pyridyl-2-dithio, and 6-substituted 4-chloro-1,3,5-triazin-2-ylamino, in particular, the reactive group comprises a isothiocyanato (—NCS) group.
The substituents in 6-substituted 4- chloro-1,3,5-triazin-2-ylamino can be selected from the group consisting hydrogen, halogen, alkoxy, aryloxy, amino, alkyl with one to six 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 preferable mono- or disubstituted each substituent being preferable independently selected from C1-6-alkyl, C1-6-alkyl-O—, phenyl, carbonyl or carboxyl.
It follows that upon reaction with a biospecific binding reactant, the reactive group establishes a link to said biospecific binding reactant, e.g. of one of the following types: a thiourea (—NH—C(═S)—NH—), an aminoacetamide (—NH—CO—CH2—NH—), an amide (—NH—CO—, —CO—NH—, —NCH3—CO— and —CO—NCH3—), oxime (—O—N═CH—), hydrazone (—CO—NH—NH═CH—) (and aliphatic thioether (—S—), a disulfide (—S—S—), a 6-substituted-1,3,5-triazine-2,4-diamine,
a wherein n=1-6; and a triazole (e.g. formed by the so-called “click” chemistry).
It should be understood that when a reactive group (e.g. Z1 or Z2) is present, the group may include a spacer (e.g. L1 or L2), i.e. a distance-making biradical, so as—if necessary or desirable—to position the reactive group 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 term “spacer” is intended to mean a distance-making group between, e.g., a conjugating group or a pyridine moiety of the core structure and, e.g. the reactive group. The spacer typically has a length of 1-20 bonds between the attachment point and reactive group, such as 3-15 bonds, or 5-12 bonds. The said spacer is formed of one to five moieties, each moiety selected from the group consisting of phenylene, alkylene containing 1-10 carbon atoms, an ethynediyl (—C═C—), an ether (—O—), a thioether (—S—), a disulfide (—S—S—), an amide (—C(═O)—NH—, —NH—C(═O)—, —C(═O)—NCH3— and —NCH3—C(═O)—), a thiourea (—NH—C(═S)—NH—) and a triazole.
In an preferred embodiment, the chelate of the present invention has formula (111a) wherein R1AA is hydrogen or —OCH2CO2−:
In another preferred embodiment, the chelate of the present invention has the formula (IIIb)
Lanthanide Chelating Ligand
Another aspect of the invention relates to a lanthanide chelating ligand of formula (IV) wherein a, b, c, Chrom1, Chrom2, and Chrom3 are as defined for formula (I).
Still another aspect of the present invention relates to a detectable molecule comprising a biospecific binding reactant conjugated to a luminescent lanthanide chelate as defined hereinabove. Conjugation 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.
Examples of biospecific binding reactants are those selected from an antibody, an antigen, a receptor ligand, a specific binding protein, a DNA probe, a RNA probe, an oligopeptide, an oligonucleotide, a modified oligonucleotide (e.g. an 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 lectine. In a preferred embodiment, the biospecific binding reactant is selected from antibodies, e.g. Troponin I antibodies (anti-Tni).
A still further aspect of the invention relates to a method of carrying out a biospecific binding assay, wherein the method comprises the steps of:
a) forming a biocomplex between an analyte and a biospecific binding reactant labelled with a lanthanide chelate as defined herein;
b) exciting said biocomplex with radiation having an excitation wavelength, thereby forming an excited biocomplex; and
c) detecting emission radiation emitted from said excited biocomplex.
In step b), the excitation wavelength is preferably 300 nm or longer, e.g. around 320-360 nm.
The method follows the conventional assay steps as will be evident for the skilled person.
This being said, a further aspect of the invention relates to the use of a detectable molecule as defined above in a specific bioaffinity based binding assay utilizing time-resolved fluorometric determination of a specific luminescence based on one or two photon-excitation. In one embodiment, 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.
In an alternative embodiment, one or more of steps a), b), and c) is performed at an elevated temperature such as above 40° C., above 50° C., above 60° C., above 70° C., above 80° C., above 90° C. or above 100° C. In an embodiment step, step a) (i.e the formation of the biocomplex) is performed at an elevated temperature as defined above.
In an alternative embodiment, the method for carrying out a biospecific binding assay comprises an additional step of drying the biocomplex. In a preferred embodiment, the drying step occurs after step a) and before step b).
Still another aspect of the invention relates to a solid support material conjugated with a luminescent lanthanide chelate as defined hereinabove. The luminescent lanthanide chelate is typically immobilized to the solid support material either covalently or non-covalently.
In some interesting embodiments, the solid support material is selected from a nano-particle, a microparticle, a slide, a plate, and a solid phase synthesis resin.
The novel lanthanide chelates ligands and the corresponding luminescent lanthanide chelates and labelled biospecific binding reactant are based on a cyclic ligand structure which provides surprisingly efficiently excitation of the chelated lanthanide ion. At the same time, all important features of the luminescent lanthanide chelate and labelled biospecific binding reactant can be retained without any additional formation of aggregates and purification problems.
The chelates of the present invention aim to combine several important features in a single label such as:
(a) Broad excitation wavelengths at around 350 nm (see the Examples) enables the use of UV LEDs as an excitation source which will provide a cost reduction in instrument manufacturing, and the possibility of instrument miniaturization.
(b) The chelates are applicable to different lanthanides.
(c) The high luminescence of the ligands means that it is possible to decrease the labeling degree without loss of signal.
(d) The lower degree of labeling can improve the affinity of the biomolecule and decrease unspecific binding during the assay. Thus faster kinetic is possible and lower background is seen which can also improve the assay sensitivity.
(e) Reduction of unwanted adsorption properties of the chromophore moiety with improved aqueous solubility, especially concerning chelates with several aromatic chromophore moieties. This should reduce the unspecific binding of the labeled antibody and give improved assay sensitivity.
(f) Improved stability of the chelate means that more demanding assay conditions can be used such as high temperature, long incubation times and high concentrations of additional metal ions.
The following non-limiting examples are aimed to further demonstrate the invention. The structures and synthetic routes employed are presented in Schemes 1-5.
1H-NMR spectra were recorded with Bruker AVANCE DRX 500 MHz. Tetramethyl silane was used as internal reference. Mass spectra were recorded PerSeptive Biosystems Voyager DE-PRO MALDI-TOF instrument using α-cyano-4-cinnamic acid matrix. UV-Vis spectra were recorded on Pharmacia Ultrospec 3300 pro. Fluorescence efficiencies were determined with Perkin-Elmer Wallac Victor platefluorometer. 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.
Conditions for HPLC purification runs: Reversed phase HPLC (RP-18 column). The solvents were A: triethyl ammonium acetate buffer (20 mM, pH7) and B: 50% acetonitrile in triethyl ammonium acetate buffer (20 mM, pH7). The gradient was started from 5% of solvent B and the amount of solvent B was linearly raised to 100% in 30 minutes. Column chromatography was performed with columns packed with silica gel 60 (Merck). FC=Flash chromatography, RT=room temperature.
A mixture of the compound 1 (0.34 g, 1.60 mmol; WO2011026790) and 2 (0.47 g, 2.15 mmol; Takalo, H., et al., Helv. Chim. Acta, 79(1996)789) in dry TEA (5 ml) and THF (10 ml) was de-aerated with argon. After addition of bis(triphenylphosphine)palladium(II) chloride (19 mg, 27 μmol) and Cul (10 mg, 53 μmol), the mixture was stirred for 24 hours at 55° C. After evaporation to dryness, the product (0.49 g, 94%) was purified by FC (silica gel, 10% EtOH/DCM/1% TEA). 1H-NMR (CDCl3): 8.49; (1H, s), 8.08; (1H, s), 7.66; (2H, d, J=8.7 Hz), 7.60; (1 H, s,), 7.59; (2H, d, J=8.7 Hz), 4.85; (2H, s), 4.49; (2 H, q, J=7.1 Hz), 1.43; (3H, t, J=7.1 Hz). 13C-NMR (CDCl3): 164.55, 160.42, 155.24, 154.94, 154.64, 154.34, 147.42, 136.24, 133.10, 132.99, 125.58, 125.27, 120.30, 119.36, 118.94, 116.65, 114.35, 112.06, 94.24, 87.57, 64.30, 62.10, 14.18. MALDI TOF-MS mass: calculated (M+H+) 393.18; found 394.16
A mixture of compound 3 (0.47 g, 1.20 mmol) and PBr3 (0.17 ml, 1.80 mmol) in dry CHCl3 (40 ml) was stirred for 18 h at +55° C., neutralized with 5% NaHCO3 solution (20 ml), the aqueous phase was extracted with CHCl3 (2×10 ml) and the combined organic phases were dried with Na2SO4. The product (0.43 g, 78%) was purified by FC (silica gel, 10% EtOH/DCM). 1H-NMR (CDCl3): 8.25; (1H, s), 8.01; (1H, d, J=1.1 Hz), 7.75; (1H, d, J=1.1 Hz); 7.68; (2H, d, J=8.7 Hz), 7.65; (2H, d, J=8.7 Hz); 4.62; (2H, s), 4.50; (2H, q, J=7.1 Hz), 1.45; (3H, t, J=7.1 Hz). 13C-NMR (CDCl3): 164.32, 157.68, 155.16, 154.85, 154.55, 154.25, 148.06, 136.21, 133.59, 133.06, 128.36, 126.09, 120.26, 119.32, 118.92, 116.62, 114.32, 112.03, 94.61, 86.29, 62.25, 32.62, 14.20. MALDI TOF-MS mass: calculated (M+H+) 455.02 and 457.02; found 455.78 and 457.73.
A mixture of compound 4 (0.41 g, 0.90 mmol), 5 (0.14 g, 0.82 mmol), dry K2CO3 (0.23 g, 1.62 mmol) and dry MeCN (8 ml) was stirred for 24 h at RT. After filtration and washing the solid material with DCM, the filtrate was evaporated to dryness. The product (0.31 g, 53%) was purified by FC (silica gel, from 1% to 3% EtOH/DCM). 1H-NMR (D6-DMSO): 11.48; (1 H,s), 7.97; (1 H, s), 7.78-7.85; (3H, m), 7.66; (2H, d, J=8.3 Hz), 4.38; (2H, q, J=7.1 Hz); 3.80-3.85; (2H, m), 3.10-3.45; (8H, m), 2.65-2.75; (2H, m), 2.65-2.55; (2H, m), 1.43; (3H, s), 1.42; (3H, s), 1.40; (6H, s), 1.39 (6H, s), 1.34; (3 H, t, J=7.1 Hz). 13C-NMR (D6-DMSO): 164.09, 155.78, 154.96, 154.80, 154.70, 154.56, 154.37, 154.08, 147.22, 137.51, 132.74, 132. 54, 129.33, 127.03, 124.69, 120.85, 118.97, 116.69, 114.39, 112.62, 93.89, 86.35, 78.71, 61.44, 61.29, 51.42, 50.18, 49,69, 28.03, 14.02. Both spectra indicate the existence of rigid compound having different structural isomers. MALDI TOF-MS mass: calculated (M+H+) 704.33; found 705.09.
A mixture of compound 6 (0.29 g, 0.41 mmol) and TFA (2 ml) was stirred for 2 h at RT, evaporated to dryness and triturated with Et2O (40 ml). The product (0.34 g, 89%) was centrifuged, washed with Et2O (2×15 ml) and dried. 1H NMR (D6-DMSO): 11.54; (1H, s), 7.82; (2 H, d, J=8.4 Hz), 7.81; (1 H, s), 7.80; (1H, s), 7.71; (2 H, d, J=8.4 Hz), 4.44; (2H, q, J=7.0 Hz), 4.17; (2H, s), 3.69; (4H, bs), 3.26; (4H, bs), 2.97; (4H, bs), 1.39; (3H, t, J=7.0 Hz). 13C NMR (D6-DMSO): 154.41, 160.60, 155.48, 155.18, 154.89, 154.59, 147.17, 138.30, 133.24, 133.06, 129.87, 128.40, 125.22, 121.41, 118,57, 116.19, 114.84, 112.54, 95.54, 86.36, 62.47, 57.68, 50.42, 45.90, 45.36, 14,45. MALDI TOF-MS mass: calculated (M+2H+) 505.54; found 505.31.
This compound 10 was synthesized from the compound 8 (WO2013026790) and 2 using a method analogous to the synthesis described in the Example 1. Yield: 76%. 1H-NMR (CDCl3): 8.11; (1H, d, J=0.5 Hz), 7.64; (1H, d, J=0.5 Hz), 6.09; (2H, s), 4.84; (2 H, s), 4.70; (4H, s), 4.58; (2H, s), 4.47; (2H, q, J=7.1 Hz), 4.29; (4H, q, J=7.1 Hz), 4.28; (2 H, q, J=7.1 Hz), 3.45; (1 H, bs), 1.44; (3H, t, J=7.1 Hz), 1.32; (3H, t, J=7.1 Hz), 1.31; (6H, t, J=7.1 Hz). 13C-NMR (CDCl3): 167.97, 167.89, 164.70, 161.04, 160.22, 158.87, 147.21, 134.13, 125.42, 125.17, 96.17, 94.27, 93.80, 87.78, 66.21, 65.42, 64.29, 61.86, 61.61, 61.49, 14.20, 14.08, 14.06. MALDI TOF-MS mass: calculated (M+H+) 588.56; found 589.03.
This compound 11 was synthesized from the compound 9 (WO2013092992) and 2 using a method analogous to the synthesis described in the Example 1. Yield: 80%. 1H-NMR (CDCl3): 8.08 (2H, s), 7.60; (2H, s), 7.45; (1H, d J=8.5 Hz), 6.50; (1H, dd, J=2.0 and 8.5 Hz), 6.45; (1 H, d, J=2.0 Hz), 4.85; (2H, s), 4.71; (2H, s), 4.63; (2H, s), 4.47; (2H, q, J=7.1 Hz), 4.30; (2H, q, J=7.1 Hz), 4.29; (2H, q, J=7.1 Hz), 1.44; (3H, t, J=7.1 Hz), 1.32; (3H, t, J=7.1 Hz), 1.31; (3H, t, J=7.1 Hz). 13C-NMR (CDCl3): 168.11, 168.00, 164.63, 161.71, 160.08, 159.95, 147.27, 134.81, 127.02, 125.50, 106.42, 105.38, 100.99, 91.30, 89.67, 65.91, 65.34, 64.32, 62.24, 61.93, 61.54, 14.20, 14.19, 14.07. MALDI TOF-MS mass: calculated (M+H+) 486.18; found 486.46.
A mixture of compound 10 (0.36 g, 0.61 mmol) and PBr3 (86 μl, 0.92 mmol) in dry CHCl3 (20 ml) was stirred for 2.5 h at RT, neutralized with 5% NaHCO3 solution (20 ml), the aqueous phase was extracted with CHCl3 (20 ml) and the combined organic phases were dried with Na2SO4. The product (0.33 g, 82%) was purified by FC (silica gel, 10% EtOH/DCM). 1H-NMR (CDCl3): 8.12; (1H, d, J=1.3 Hz), 7.79; (1H, d, J=1.3 Hz), 6.08; (2H, s), 4.71; (4H, s), 4.59; (2H, s), 4.51; (2H, s), 4.48; (2H, q, J=7.1 Hz), 4.30; (4H, q, J=7.1 Hz), 4.29; (2H, q, J=7.1 Hz), 1.43; (3H, t, J=7.1 Hz), 1.31; (9 H, t, J=7.1 Hz). 13C-NMR (CDCl3): 167.92, 167.79, 164.52, 156.29, 133.27, 125.98, 125.08, 96.09, 93.92, 93.76, 88.21, 66.22, 65.59, 65.43, 62.04, 61.62, 61.51, 32.96, 14.22, 14.07, 14.03. MALDI TOF-MS mass: calculated (M+H+) 650.13 and 652.13; found 651.08 and 653.02.
This compound 13 was synthesized from the compound 11 using a method analogous to the synthesis described in the Example 7. Yield: 89%. 1H-NMR (CDCl3): 8.10; (1 H, d, J=1.2 Hz), 7.75; (1H, d, J=1.2 Hz), 7.46; (1 H, d, J=8.5 Hz), 6.50; (1H, dd, J=2.3 and 8.5 Hz), 6.46; (1H, d, J=2.3 Hz), 4.72; (2H, s), 4.63; (2H, s), 4.60; (2H, s), 4.49; (2H, q, J=7.1 Hz), 4.30; (2H, q, J=7.1 Hz), 4.29; (2H, q, J=7.1 Hz), 1.45; (3H, t, J=7.1 Hz), 1.32; (3H, t, J=7.1 Hz), 1.31; (3H, t, J=7.1 Hz). 13C-NMR (CDCl3): 168.10, 167.97, 164.43, 160.05, 160.03, 157.41, 147.95, 134.88, 127.65, 126.00, 106.42, 105.28, 100.97, 91.97, 89.34, 65.92, 65.35, 62.40, 62.09, 61.54, 61.49, 32.88, 14. 22, 14.09, 14.97. MALDI TOF-MS mass: calculated (M+H+) 548.09 and 550.09; found 548.83 and 550.80.
A mixture of compound 7 (0.12 g, 0.15 mmol), 12 (0.21 g, 0.32 g), DIPEA (0.4 ml) and dry MeCN (3 ml) was stirred for 5.5 h at RT and evaporated to dryness. The product (0.19 g, 79%) was purified by FC (silica gel, first from 10% EtOH/DCM to 15% EtOH/DCM, then 15% EtOH/DCM/5% TEA). As the product contains 2-3 rigid isomers, the NMR spectra were too complicated to assigned the isomers. MALDI TOF-MS mass: calculated (M+H±) 1642.60; found 1643.57.
This compound 15 was synthesized from the compound 13 using a method analogous to the synthesis described in the Example 9. The product was purified by FC (silica gel, from 2% EtOH/DCM/1% TEA). Yield: 84%. As the product contains 2-3 rigid isomers, the NMR spectra were too complicated to assigned the isomers. MALDI TOF-MS mass: calculated (M+H+) 1438.54; found 1439.41.
A mixture of the compound 14 (92 mg, 64 μmol) and 0.5M KOH in EtOH (6.5 ml) was stirred for 1 h at RT and H2O (3 ml) was added. After stirring for 4 hours at RT, EtOH was evaporated, some H2O (2 ml) added and the residue was stirred for 24 h at RT. After addition of citric acid (41 mg, 0.21 mmol) in H2O (0.25 ml), the pH was adjusted to ca. 6.5 with 6M HCl. Europium(III) chloride (26 mg, 71 μmol) in H2O (0.25 ml) was added within 10 minutes and the pH was adjusted to ca. 9.5 with 1M NaOH. The mixture was stirred for 4-6 weeks at 95° C. (after the analytical HPLC chromatogram showed completed complexation), the pH was adjusted to ca. 7.0 with 1M HCl, evaporated to dryness, dissolved in 20 mmol TEAA buffer (1 ml) and purified with semi-preparative HPLC. Rf(HPLC)=16.0 min. UV=360 nm. MALDI TOF-MS mass: calculated (M+6H+) 1443.23; found 1443.96.
Ligand isomers shown in HPLC during Eu(III)-loading: 1) Rf(HPLC)=18.5 min, UV=345 nm; 2) Rf(HPLC)=20.4 min, UV=347 nm; 3) Rf(HPLC)=21.7 min, UV=347 nm. All this peaks finally gave the product peak at Rf=16.0 min. and UV=360 nm. The Eu complex formation caused the observed bathochromic shift of 13-15 nm at UV. This was separately secured by additional HPLC purification of the ligand isomers and loading of Eu(III) ion to each isomers. All those loadings gave finally the same product at Rf(HPLC)=16.0 min.
It is noted herein that the general Eu-loading methods disclosed in literature, patents and patents applications with similar macrocyclic ligands did not give the wanted chelates and only non-complexed ligands were obtained. After extensive experimentation it was determined that Eu loading required high pH (>9), high temperatures of around 80-90° C., and long incubation times of around two weeks. The difficulty of loading the Eu is indicating the high chelating stability once the chelates are formed.
This compound 17 was synthesized from the compound 15 using a method analogous to the synthesis described in the Example 11. Rf(HPLC)=19.2 min. UV=350nm. MALDI TOF-MS mass: calculated (M+4H+) 1295.23; found 1295.85.
Ligand isomers shown in the HPLC during Eu(III) loading: 1) Rf(HPLC)=21.7 min, UV=347 nm; 2) Rf(HPLC)=22.3 min, UV=339 nm; 3) Rf(HPLC)=23.6 min, UV=343 nm.
Compound 16 (43 mg, 21 μmol) in H2O (1 ml) was added within 5 min to a mixture of CSCl2 (22 μl, 0.29 mmol) and NaHCO3 (28 mg, 0.33 mmol) and CHCl3 (1 ml). After stirring for 40 min at RT, the aqueous phase was washed with CHCl3 (3×1 ml). The product was precipitated with acetone, centrifuged and washed with acetone.
This compound 19 was synthesized from the compound 17 using a method analogous to the synthesis described in the Example 13.
A mixture of compound 18 (2 mg) and taurine (2 mg) in 50 mM Na2CO3 buffer (300 μl, pH 9.8) was stirred for o/n at RT. The product was purified by using semi-preparative HPLC. Rf(HPLC)=15.2 min. UV=354 nm.
After the product fractions were evaporated and the residue was dissolved in 50 mM TRIS buffer (1 ml). The Eu concentration was measured by ICP-MS. The analyzing parameters were: the Peak Hopping mode, 20 sweeps/reading, 7 replicates, the Dwell time and the integration time was 50 ms and 1000 ms, respectively. Rhodium was used as the internal standard and the Europium was measured on Mass 152.929. 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 done 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, HNO3 (5 ml) and H2O2 (1 ml). Afterwards the sample was diluted with deionized water (100 ml).
This compound 21 was synthesized from the compound 19 using a method analogous to the synthesis described in the Example 15. Rf(HPLC)=17.2 min. UV=348 nm. After the product fractions were evaporated and the residue was dissolved in 50 mM TRIS buffer (1 ml). The Eu concentration was measured by IPC-MS using a method analogous in the Example 15.
Labeling of an Tnl antibody was performed as described in von Lode P. et al., Anal. Chem., 2003, 75, 3193-3201 by using 300 fold excess of the labelling reagents 18 or 19. The reactions were carried out overnight at RT. Labeled antibody was separated from the excess of chelates on Superdex 200 HR 10/30 gel filtration column (GE healthcare) by using Tris-saline-azide buffer (Tris 50 mM, NaCl 0.9%, pH 7.75) as an eluent. The fractions containing the antibody were pooled and the Eu concentration was measured by UV and secured by IPC-MS described in the Example 15.
The TnI antibody labeled with the chelate 18 or 19 was tested in sandwich immuno-assay for cardiac troponin I. As a reference compound a TnI antibody labelled with α-gal-9-D Eu (von Lode P. et al., Anal. Chem., 2003, 75, 3193-3201) was used. 10 μl of diluted tracer antibody (5 ng/μl) and 20 μl of TnI 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 TnI, Innotrac Diagnostics). The reaction mixtures were incubated 20 min at 36° C. with shaking. The wells were washed 6 times and dried prior to measurement with Victor™ Plate fluorometer.
The conventional 9-dentate α-galactose Eu chelate (Ref in Table 1) was prepared according to von Lode P. et al., Anal. Chem., 2003, 75, 3193-3201.
The results are summarized in Table 1. Both A and B standards were measured in 12 replicates and other standards C-F in 6 replicates.
Example 19. Photo-physical properties of novel chelates conjugated to taurine (chelates 20 and 21) and the labelled cTnl antibodies with chelates 18 and 19.
The measured photo-physical properties excitation wavelengths (λexc) luminescence decay times (τ), molar absorptivities (ε), estimated luminescence yields (εΦ) of the novel chelates (20 and 21) and the labelled cTnI antibodies with chelates 18 and 19 in 50 mM TRIS buffer (pH 7.75) are in the Table 2.
Dry measurements (18 (dry) and 19 (dry)) represents estimated luminescence yields based on the signal measurements after dry immunoassay done as described in the Example 18.
a)Coupled to protein
As it can be seen from the results in the Tabel 2, the taurine derivatives and labeled IgG has rather low luminescence (below 1000 M−1 cm−1) when measured in aqueous buffer, but the signals are much enhanced in dry format i.e. approximately 80-100 fold increase of brightness. This surprising improvement in luminescence in the dry format means that the skilled person can significantly improve the sensitivity of the assay—if necessary—by simply adding a drying step.
Without wishing to be bound by theory, it is hypothesised that the low luminescence intensities in aqueous buffer can be explained by the low-lying CT-state of the ligand, as has been published with similar type of pyridine dicarboxylic acids (see: Andraud, C., et al. in Eur. J. Inorg. Chem 2009, 4357; Inorg. Chem. 2011, 4987 and results of Takalo, H., et al., 2010, a poster presentation in the 1st International Conference on luminescence of Lanthanides Odessa, Ukraine). Moreover, such low luminescence has not previously been shown to be enhanced so significantly in dry measurement format.
| Number | Date | Country | Kind |
|---|---|---|---|
| PA 2014 00627 | Oct 2014 | DK | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15523292 | Apr 2017 | US |
| Child | 17361457 | US |
