Technical field

The present invention relates to the field of luminescent lanthanide chelates suitable as label for detection in bioassay applications.

Background of invention

Time-resolved fluorometry (TRF) 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. Moreover, lanthanide chelates have been used in magnetic resonance imaging (MRI) and position emission tomography (PET).

For TRF applications, an optimal label has to fulfill several requirements. First, it has to be photochemically stable both in the ground state and in the excited state and it has to be kinetically and chemically stable. 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. For the purpose of labeling, it should have a reactive group to allow covalent attachment to a biospecific binding reactant, and the affinity and

nonspecific binding properties of the labeled biomolecules have to be retained.

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. As a consequence hereof, a number of attempts (see e.g. the review in Bioconjugate Chem., 20 (2009) 404) 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. Based on chelate stability studies with cyclic azamacro cycles (such as DOTA), higher stabilities over open chain chelates (such as DPT A) has been observed, and thus, the main focus with disclosed chelates with three chromophores has been on azamacrocycles tethered to the various chromophores. Lately, high lanthanide chelate stabilities have been observed with open chain ligands utilizing several l-hydroxy-2-pyridinone and salisylamide groups although these disclosed chelates are normally eight dentate and do not contain carboxylic acids for coordination to a lanthanide ion, high luminescence and stabilities have been obtained. However, those 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.

Von Lode et al. (Anal. Chem. 75(2003) 3193) describe a europium chelate for quantitative point-of-care immunoassays using direct surface measurement, wherein the chelates either have a nonadentate or heptadentate structure. Further, antibodies coupled with said chelates are described.

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.

It is thus an object of the present invention to provide further chelates suitable for labeling biospecific binding reactants.

It is a further object of the present invention to provide further precursors of said chelates.

It is another object of the present invention to provide a detection agent comprising a biospecific binding reactant conjugated to a chelate of the present invention. Moreover, it is an object of the present invention to provide further methods for detecting an analyte in a bio specific binding assay.

In another aspect, it is also an object of the present invention to provide further methods of labelling a biospecific binding reactant.

Summary

The above objects are solved by a compound of formula (I)

or a salt thereof,

wherein

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 of Chi and Ch ₂ is a chelating group, wherein at least one of Chi and Ch ₂ is selected from -C(=0)NHR _A , -C(=0)NR _A RB, -C(=S)NHR _A or -C(=S)NR _A R _B ;

wherein each R _A and R _B is independently selected from

i) a reactive group Z;

ii) optionally substituted -Ci_i ₂ -alkyl, optionally substituted -Ci-i ₂ -alkyl-NH-C(=0)- Ci-i ₂ -alkyl, or optimally substituted -Ci-i ₂ - alkyl-NH-C(=0)-C i-i ₂ - alkyl- NHC(=0)CH ₃ ; or iii) -R _AI -R _A2 -NHC(=0)-C _{I -6} -alky l-(optionally substituted 6- to l2-membered aryl or optionally substituted 5- to l4-membered heteroaryl), and wherein R _AI is a -C _1-12 - alkyland R _A2 is a bond or -NHC(=0)-Ci-i ₂ -alkyl-,

wherein the 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);

wherein at least one of Ri, R2, R3, R _A , R _B , A _I or A2 is a reactive group Z,

and wherein Ln ³⁺ is a lanthanide ion.

In one aspect, the objects are solved by a compound of formula (II)

or a salt thereof, wherein 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.

In one aspect, 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.

In one aspect, the objects are solved by a method of detecting an analyte in a biospecific binding assay, said method comprising the steps of:

a) forming a complex between the analyte and the compound of the invention or the detection agent of the invention;

b) exciting said complex with a radiation having an excitation wavelength of the compound of the invention or the detection agent of the invention, thereby forming an excited complex; and

c) detecting emission radiation emitted from said excited complex. In another aspect, the objects are solved by a method of labelling a biospecific binding reactant with a compound of the invention, comprising the steps of

a) providing a biospecific binding reactant; and

b) conjugating the biospecific binding reactant with the compound of the invention.

In yet another aspect, 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.

In another aspect, 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.

In yet another aspect, the objects are solved by providing solid support material conjugated with a compound of the invention or the detection agent of the invention.

Detailed description

The present invention relates to compounds of formula (I)

or a salt thereof,

wherein

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 of Chi and Ch ₂ is a chelating group, wherein at least one of Chi and Ch ₂ is selected from -C(=0)NHR _A , -C(=0)NR _A RB, -C(=S)NHR _A or -C(=S)NR _A R _B ;

wherein each R _A and R _B is independently selected from

i) a reactive group Z;

ii) optionally substituted -Ci_i ₂ -alkyl, optionally substituted -Ci-i ₂ -alkyl-NH-C(=0)- Ci-i ₂ -alkyl, or optimally substituted -Ci-i ₂ - alkyl-NH-C(=0)-C i-i ₂ - alkyl- NHC(=0)CH ₃ ; or

iii) -R _Ai -R _A2 -NHC(=0)-Ci - ₆ -alky l-(optionally substituted 6- to l2-membered aryl or optionally substituted 5- to l4-membered heteroaryl), and wherein R _AI is a -Ci_i ₂ - alkyland R _A2 is a bond or -NHC(=0)-Ci-i ₂ -alkyl-,

wherein the 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);

wherein at least one of 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:

In a particularly preferred embodiment, at least one chelating group Chi and Ch ₂ is -C(=0)NHRA or -C(=0)NR _A RB.

In another preferred embodiment, the other chelating group of Chi or Ch ₂ is not -C(=0)NHRA, -C(=0)NRARB, -C(=S)NHR _A or -C(=S)NR _A RB but a chelating group selected from -COO , -PO3 ² , -P(CH ₃ )0 ₂ , -P(phenyl)0 ₂ , -CH ₂ P0 ₃ ² . It is preferred that this chelating group Chi or Ch ₂ is -COO , of course provided that the other chelating group is -C(=0)NHRA, -C(=0)NRARB, -C(=S)NHRA or - C(=S)NR _A RB and preferably C(=0)NHR _A or -C(=0)NR _A RB.

These groups may be substituted with various groups to improve water solubility and/or to shift excitation wavelength and/or to enhance the molar excitation coefficient. Any group of Gi, G ₂ and G3 not being a conjugating group simply represents a single bond between the pyridine and Ri, R ₂ or R3, respectively.

In one preferred embodiment, at least two of Gi, G ₂ and G3 are independently a conjugating group.

In a particularly preferred embodiment, each of Gi, G ₂ and G3 is selected as a conjugating group.

In some embodiments, the conjugating group consists of one, two or three moieties, each moiety being selected from ethenylene (-CH=CH-), ethynediyl (-CºC-), carbonyl (-C(=0)-), and biradicals of (hetero)aromatic ring or ring systems

(-Het/Ar), e.g. phenylene, biphenylene, naphthylene, pyridylene, pyrazinylene, pyrimidinylene, pyridazinylene, furylene, thienylene, pyrrolylene, imidazolylene, pyrazolylene, thiazolylene, isothiazolylene, oxazolylene, isoxazolylene,

fyrazanylene, l ,2,4-triazol-3,5-ylene, and oxadiazolylene.

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 (-Het/Ar-) 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, -

(CH2CH ₂ 0)I- ₄ CH2CH ₂ 0H, -(CH2CH20)I- ₄ CH2CH ₂ 0CH ₃ , -Ci-e-alkyl-NCHsRn, - (CH ₂ ) 1 -6N(CH ₂ CH ₃ )RI 1 , -Ci-6-alkyl-N(Ri 1)2, -Ci-6-alkyl-N ⁺ (CH ₃ ) ₂ Rii, - NHC(=0)Rio, -NCH ₃ C(=0)RIO, -C(=O)NHRI ₀ , -C(=0)NCH ₃ RIO, -NHC(=O)NHRI ₀ , -NHC(=S)NHRIO, -C(=0)Rio, -F, -Cl, -Br, -I, -CF ₃ , -CN, hydroxyl (-OH), mercapto (-SH), -OR ₉ ,-SR ₉ , and a hydrophilic group, and wherein R ₉ is selected from - CF ₃ , -Ci-12-alkyl, -Ci- ₆ -alkyl-COOH, -Ci-e-alkyl-COO , -Ci- ₆ -alkyl-S0 ₃ H, -Ci_ ₆ - alkylS03 ,-Ci-6-alkyl-0-P03 ² , -Ci -6-alky I-PO3 ² , -Ci- ₆ -alklyl-0-P03H2, -Ci- ₆ -alkyl- PO3H2, -Ci- ₆ -alkyl-OH, -(CH ₂ CH ₂ 0) _I-4 CH ₂ CH ₂ 0H, -(CH ₂ CH ₂ 0)i- ₄ CH ₂ CH ₂ 0CH ₃ , - Ci- ₆ -alkyl--NCH ₃ Rii, -(CH ₂ )I-6N(CH ₂ CH3)RI I, -Ci-6-alkyl-N(Rii) ₂ , -Ci-e-alkyl- N ⁺ (CH ₃ ) ₂ RH, -NHC(=0)RIO, -NCH ₃ C(=0)RIO, -

C(=0)NHRio, -C(=0)NCH ₃ RIO, -NHC(=0)NHRIO, -NHC(=S)NHRIO, -C(=0)RIO and a hydrophilic group, wherein Rio is selected from hydrogen, -Ci-12-alkyl, -Ci- ₆ - alkyl-OH, -CH(CH ₂ OH) _2, -C(CH ₂ OH) ₃ , -Ci-e-alkyl-COOH, -Ci-e-alkyl-COO , -Ci_ ₆ - alkyl-S03H, -Ci- ₆ -alkylS03 ,-Ci- ₆ -alkyl-0-P03 ² , -Ci- ₆ -alkyl-P03 ² , -Ci-6-alkyl-O- PO3H2, -Ci- ₆ -alkyl-P03H2, -Ci- ₆ -alkyl-N ⁺ (CH3)2Rn, and a hydrophilic group; and wherein Rn is selected from hydrogen, -Ci-12-alkyl, preferably Ci- ₆ -alkyl, -Ci- ₆ - alkyl-COO , -Ci-e-alkylSOs , -Ci-e-alkyl-COOH, -Ci- ₆ -alkyl-S0 ₃ H, -Ci-e-alkyl N ⁺ (CH ₃ )2(Ci-6-alkyl)-S0 ₃ ^· , -Ci- ₆ -alkyl-0-P0 ₃ ^2· , -Ci- ₆ -alkyl-P0 ₃ ^2· , -Ci-e-alkyl-O- PO3H2, -Ci-6-alkyl-P0 ₃ H ₂ .

With different R groups, the excitation wavelength of the chelates may be shifted to a desired longer wavelength.

In a preferred embodiment, two of Gi, G ₂ and G ₃ , in particular all three, are independently selected as the conjugating groups phenylethynyl, phenyl, thienyl and furyl, which each are optionally substituted. In one embodiment, two of Gi, G ₂ and G ₃ , in particular all three, are independently selected phenylethynyl (-CºC-C ₆ H ₅ -).

It is particularly preferred that two or all three of Gi, G ₂ and G ₃ is each an optionally substituted phenylethynyl, which each may be substituted with the substituents described above.

In another preferred embodiment, 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.

In this embodiment, it is particularly preferred that 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 .

In another preferred embodiment, 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. In this embodiment, it is particularly preferred that 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.

In yet another preferred embodiment, Ri, R ₂ and/or R ₃ are hydrogen. As already described above, at least one reactive group Z is required in the chelate molecule of the invention. Typically, the number of Z groups in the chelate molecule is 1, 2, or 3, in particular 1 or 2, more preferably 1. In case 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. 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.

In a preferred embodiment, the reactive group Z comprises azido (- N ₃ ), -CºCH, -CH=CH ₂ , amino (-NH ₂ ), aminooxy (-0-NH ₂ ), carboxyl (-COOH), aldehyde (-CHO), mercapto (-SH), maleimido groups or activated derivatives thereof, including isocyanato (-NCO), isothiocyanato (-NCS), diazonium (-N ⁺ N), bromoacetamido, iodoacetamido, reactive esters, pyridyl-2-dithio, or 6-substituted 4- chloro- 1 ,3,5-triazin-2-ylamino.

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.

It follows that upon reaction with a biospecific binding reactant (see further below), the reactive group Z 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- CH ₂ -NH-), an amide (-NH-CO-, -CO-NH-, -NCH ₃ -CO- and -CO-NCH ₃ -), and aliphatic thioether (-S-), a disulfide (-S-S-), a 6-substituted- 1,3, 5-triazine-2, 4-

diamine, , wherein n = 1-6; and a triazole (e.g. formed by the so-called“click” chemistry).

It is particularly preferred that 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 (-

N3), -CºCH, -CH=CH ₂ , amino (-NH ₂ ), aminooxy (-0-NH ₂ ), carboxyl (-COOH), aldehyde (-CHO), mercapto (-SH), maleimido groups or activated derivatives thereof, including isocyanato (-NCO), isothiocyanato (-NCS), diazonium (-N ⁺ N), bromoacetamido, iodoacetamido, reactive esters, pyridyl-2-dithio, or 6-substituted 4- chloro-l,3,5-triazin-2-ylamino, wherein preferably the 6-substituted 4-chloro- 1,3,5- triazin-2-ylamino is a 4,6-dichloro-l,3,5-triazin-2-ylamino group.

Further, it is preferred that the reactive group Z comprises a spacer which is a distance-making biradical, wherein the said spacer consists of one to five moieties, each moiety is selected from the group consisting of phenylene, -(CH ₂ ) _I-IO -, an ethynediyl (-CºC-), an ether (-0-), a thioether (-S-), a disulfide (-S-S-), an amide (-C(=0)-NH-, -NH-C(=0)-, -C(=0)-NHCH ₂ - and -NHCH ₂ -C(=0)-), a thiourea (-NH-C(=S)-NH-) and a triazole.

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.

In some preferred embodiments, the compound of the invention comprises a single reactive group Z, in particular as the substituent R _2, R _A or R _B .

Similarly, when any of Ri, R ₂ , R ₃ , Ai, and A ₂ is selected as a hydrophilic group, the hydrophilic group may include a spacer as described above.

In both instances, the spacer may be readily introduced in the course of the synthesis of the ligand or the chelate.

In a preferred embodiment, 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.

It is particularly preferred that the hydrophilic group comprises monosaccharides.

In another preferred embodiments, the hydrophilic group comprises monosaccharides or disaccharides.

In another preferred embodiment, Ai or A ₂ is hydrogen. It is particularly preferred that both Ai and A ₂ are hydrogen.

In yet another preferred embodiment, the lanthanide ion, Ln ³⁺ , is selected from europium(III), terbium(III), dysprosium(III) and samarium(III).

In a particularly preferred embodiment, the lanthanide ion is europium(III).

In another preferred embodiment, RA and/or RB is selected as a reactive group Z.

In this embodiment, it is particularly preferred that the reactive group Z comprises a spacer. The spacer may comprise one to five moieties wherein each moiety is preferably selected from -(CH ₂ ) _I-IO -, preferably -(CH ₂ ) _2-6 -, -C(=0)-NH-, -NH-C(=0), -C(=0)-NHCH ₂ - and phenylene. In a preferred embodiment, the spacer comprises three to five of the above moieties. In another preferred embodiment, 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. In another preferred embodiment, R _A and/ or R _B is selected as optionally

substituted -Ci-12-alkyl, optionally substituted -Ci-i ₂ -alkyl-NH-C(=0)-C 1-12-alkyl, or optionally substituted -Ci-i ₂ -alkyl-NH-C(=0)-Ci-i ₂ -alkyl-NHC(=0)CH ₃ . In this embodiment, it is particularly preferred that R _A and / or R _B is optionally substituted -

CH ₂ CH ₂ -NH-C(=0)-CH ₃ .

In an alternative embodiment, R _A and/ or R _B is selected as -R _AI -R _A2 -NHC(=0)-C _I-6 - alkyl-(optionally substituted 6 to l2-membered aryl or optionally substituted 5- to l4-membered heteroaryl), and wherein R _AI is a -C 1-12-alkyl and R _A 2 is a bond or -NHC(=0)-C i-12-alkyl-, wherein the aryl or heteroaryl is optionally substituted by one, two or three substituents selected from the group consisting of -NH2, -C1-12- alkyl, -C2-12-alkenyl, -C2-i2-alkinyl, halogen, -CF3, -Ci-i2-alkyl-NH2, -OH, -SH, - CN, -NCS, heteroaryl, and -NH-(optionally substituted 5- to l2-membered heteroaryl). In this embodiment, it is particularly preferred that 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. It is further preferred that 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.

In a preferred embodiment, R _AI is -Ci - ₆ -alkyl.

In another preferred embodiment, R _A 2 is a bond.

In yet another preferred embodiment, R _A , and optionally R _B , is

-C 1-12-alky l-NHC(=0)CH ₃ ; and preferably is -Ci- ₆ -alkyl-NHC(=0)CH ₃ .

In yet another preferred embodiment, R _A , and optionally R _B , is

wherein Li is selected from -Ci-12-alkyl or -Ci_i ₂ -alkyl-NHC(=0)-C 1-12-alkyl-. 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. Thus, Xi may comprise azido (- N ₃ ), -CºCH, -CH=CH ₂ , amino (-NH ₂ ), aminooxy (-0-NH ₂ ), carboxyl (-COOH), aldehyde (-CHO), mercapto (-SH), maleimido groups or activated derivatives thereof, including isocyanato (-NCO), isothiocyanato (-NCS), diazonium (-N ⁺ N), bromoacetamido, iodoacetamido, reactive esters, pyridyl-2-dithio, or 6-substituted 4- chloro-l,3,5-triazin-2-ylamino, wherein 6-substituted 4-chloro-l,3,5-triazin-2- ylamino is preferably a 4,6-dichloro-l,3,5-triazin-2-ylamino group.

In a preferred embodiment, 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.

In several particularly preferred embodiments, Ai, A ₂ , Ri, R ₂ and R ₃ are hydrogen. In this embodiment, it is also preferred that one of Chi and Ch ₂ is -C(=0)NHR _A and wherein the other chelating group is preferably -COO .

In one embodiment, the compound is

In one embodiment, the compound is

COO Na ⁺

NH ₂

(22) In another embodiment, the compound is

COO Na

NHCOChfe

(23)

In another embodiment, the compound is

In another embodiment, the compound is

COO Na ⁺

In another embodiment, the compound is

In another embodiment, the compound is

COO Na ⁺

In another embodiment, the compound is

In another embodiment, the compound is

(38) In another embodiment, the compound is

In another embodiment, the compound is

COO Na ⁺

In yet another embodiment, the compound is

COO Na ⁺

In another embodiment, the compound is

N H ₂

And in another embodiment, the compound is

Other salts of the abovementioned compounds are of course also preferred embodiments.

In another aspect, the present invention relates to compounds of formula (II)

or a salt thereof, wherein 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,

A 2, Chi or Ch ₂ as described in US 2005/0181393.

In yet another aspect, 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.

Examples of 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.

In a preferred embodiment, the biospecific binding reactant is selected from antibodies, e.g. Troponin I antibodies (anti-Tnl).

In another aspect, the present invention relates to a method of detecting an analyte in a biospecific binding assay, said method comprising the steps of:

a) forming a complex between the analyte and the compound of formula (I) or formula (II) or the detection agent of the invention;

b) exciting said complex with a radiation having an excitation wavelength of the compound of formula (I) or formula (II) or the detection agent of the invention, thereby forming an excited complex; and

c) detecting emission radiation emitted from said excited complex.

The method follows conventional assay steps as will be evident for the skilled person. In yet another aspect, the present invention relates to a method of labelling a biospecific binding reactant with a compound of the invention, comprising the steps of

a) providing a biospecific binding reactant; and

b) conjugating the biospecific binding reactant with the compound of formula (I) or formula (II).

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).

In another aspect, 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. 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 another aspect, 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. Such 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). This of course depends on the selection of the substituents. However, 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.

In some embodiments, the solid support material is selected from a nanoparticle, a microparticle, a slide, a plate, and a solid phase synthesis resin. As used herein the term“conjugating group” refers to a moiety connecting two other moieties by at least two covalent bonds. Therefore, a conjugating group is a biradical group. As used herein, 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. 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. Upon reaction with a biospecific binding reactant, the reactive group Z establishes a link to said biospecific binding reactant. Examples of such a link that is formed upon reaction of the reactive group and the biospecific binding reactant are moieties that include, but are not limited to, a thiourea (-NH-C(=S)-NH-), an aminoacetamide (-NH-CO-CH ₂ - NH-), an amide (-NH-CO-, -CO-NH-, -NCH ₃ -CO- and -CO-NCH ₃ -), and aliphatic thioether (-S-), a disulfide (-S-S-), a 6-substituted-l, 3, 5-triazine-2, 4-diamine, a

, wherein n = 1-6; and a triazole (e.g. formed by the so-called

“click” chemistry).

As used herein, the term“hydrophilic group” refers to a moiety that is present in order to improve the water solubility of the chelate. Thus, a compound comprising a hydrophilic group as a substituent has a higher solubility in water than the corresponding compound not comprising said hydrophilic group. Examples of hydrophilic groups are mono- and oligosaccharides, such as

monosaccharides and disaccharides, oligoalkylene glycols (e.g. those having 1-20 repeating units) such as oligoethylene glycol and oligopropylene glycol, and the like.

As used herein, the term“chelating group” refers to a moiety of a chelate that inter alia coordinates the metal ion of the chelate. Examples of chelating groups thus include, but are not limited to, groups comprising at least one of primary, secondary or tertiary amine, -C(=0)- or -C(=S)-group wherein the nitrogen, oxygen or the sulfur forms a coordination coordinate bond with the metal ion of the chelate. As used herein, 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. Representative

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.

As used herein, the term“-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.

As used herein, 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.

As used herein, the term“-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.

As used herein, "-(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. In one embodiment, one of the bicyclic -(5- to l2-membered)heteroaryl rings contains at least one carbon atom. In another embodiment, 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, quinazolinyl, and the like.

As used herein 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.

As used herein, the term“optionally substituted” refers to a group that is either unsubstituted or substituted.

Optional substituents on optionally substituted groups, when not otherwise indicated, 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, -

(CH ₂ CH ₂ 0) _I-4 CH ₂ CH ₂ 0H, -(CH ₂ CH ₂ 0) _I-4 CH ₂ CH ₂ 0CH ₃ , -Ci- ₆ -alkyl--NCH ₃ Rn, - (CH ₂ ) _I-6 N(CH ₂ CH ₃ )R _{I I} , -Ci- ₆ -alkyl-N(Rn) ₂ , -Ci- ₆ -alkyl-N ⁺ (CH ₃ ) ₂ Rn, - NHC(=0)Rio, -NCH ₃ C(=0)RIO, -C(=O)NHRI ₀ , -C(=0)NCH ₃ RIO, -NHC(=O)NHRI ₀ , -NHC(=S)NHRIO, -C(=0)Rio, -F, -Cl, -Br, -I, -CF ₃ , -CN, hydroxyl (-OH), mercapto (-SH), -OR9, -SR9, and a hydrophilic group, and wherein R9 is selected from - CF ₃ , -Ci-i ₂ -alkyl, -Ci-e-alkyl-COOH, -Ci-e-alkyl-COO-, -Ci- ₆ -alkyl-S0 ₃ H, -Ci-e- alkylS0 ₃ ,-Ci- ₆ -alkyl-0-P0 ₃ ² , -Ci - ₆ -alky l-P0 ₃ ² , -Ci- ₆ -alklyl-0-P0 ₃ H ₂ , -Ci- ₆ -alkyl- P0 ₃ H ₂ , -Ci- ₆ -alkyl-OH, -(CH ₂ CH ₂ 0)i_ ₄ CH ₂ CH ₂ 0H, -(CH ₂ CH ₂ 0)i_ ₄ CH ₂ CH ₂ 0CH ₃ , - Ci- ₆ -alkyl--NCH ₃ Rn, -(CH ₂ )i- ₆ N(CH ₂ CH ₃ )Rn, -Ci- ₆ -alkyl-N(Rn) ₂ , -Ci-e-alkyl- N ⁺ (CH ₃ ) ₂ RH, -NHC(=0)RIO, -NCH ₃ C(=0)RIO, -

C(=0)NHRio, -C(=0)NCH ₃ RIO, -NHC(=O)NHRI ₀ , -NHC(=S)NHRIO, -C(=0)RIO and a hydrophilic group, wherein Rio is selected from hydrogen, Ci_i ₂ -alkyl, -Ci- ₆ - alkyl-OH, -CH(CH ₂ OH) _2, -C(CH ₂ OH) ₃ , -Ci-e-alkyl-COOH, -Ci-e-alkyl-COO , -Ci_ ₆ - alkyl-S0 ₃ H, -Ci-e-alkylSOy, -Ci -e-alky l-0-P0 ₃ ² , -Ci -e-alky l-P0 ₃ ² , -Ci-e-alklyl-O- P0 ₃ H ₂ , -Ci- ₆ -alkyl-P0 ₃ H ₂ , -Ci- ₆ -alkyl-N ⁺ (CH ₃ ) ₂ Rn, and a hydrophilic group; and wherein Rn is selected from -hydrogen, -Ci-i ₂ -alkyl, -Ci - ₆ -alky l-COO , -Ci- ₆ - alkylS0 ₃ , -Ci-e-alkyl-COOH, -Ci_ ₆ -alkyl-S0 ₃ H, -Ci -e-alky l-N ⁺ (CH ₃ ) ₂ (Ci -e-alkyl)- S0 ₃ , -Ci - ₆ -alky l-0-P0 ₃ ² , -Ci- ₆ -alkyl-P0 ₃ ² , -Ci- ₆ -alkyl-0-P0 ₃ H ₂ , -Ci -e-alky l-P0 ₃ H ₂ .

As used herein, 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 . In many embodiments, europium(III) (Eu ³⁺ ) and terbium(III) (Tb ³⁺ ) are preferred.

It should be understood that 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). Hence, it should be understood that 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 ⁺ .

Preferably, 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.

As used herein, 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.,

Tetrahedron 61 (2005) 10827).

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 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 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 (-CºC-), an ether (-0-), a thioether (-S-), a disulfide (-S-S-), an amide (-C(=0)-NH-, -NH-C(=0)-, -C(=0)-NCH ₃ - and -NCH ₃ -C(=0)-), a thiourea (-NH-C(=S)-NH-) and a triazole.

As used herein, 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.

In the present context, the term“monosaccharide” is intended to mean C5-C7 carbohydrates being either in the acyclic or in cyclic form. Examples of

monosaccharides are C ₆ carbohydrates, e.g. those selected from

In the present context, the term“disaccharide” is intended to mean two

monosaccharides (cf. above) linked together, preferably via glycosidic bonds.

As used herein, the term“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.

As used herein the term“polysaccharide" refers to a saccharide polymer containing more than 10 units of monosaccharides, preferably linked together by glycosidic bonds. As used herein, the term“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).

As used herein, 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. However, 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. As used herein, an“antigen” is a molecule capable of inducing an immune response to produce an antibody. Thus, an antigen may be a molecule binding to an antibody.

As used herein, a“receptor ligand” is a molecule that is known to bind to a cell receptor. Examples of receptor ligands are neurotransmitters, hormones, growth factors or the like. Examples of corresponding receptors are G-protein coupled receptors, protein kinase receptors and the like.

As used herein, 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. Thus, the probe may comprise a complementary sequence to a target nucleic acid sequence.

As used herein, the term“protein” covers any type of protein, including enzymes or specific binding proteins that interact specifically with one or more target molecules. In the sense of the present invention, the term 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.

As used herein, the term“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.

As used herein, the term“PNA” or“peptide nucleic acid” refers to an artificially synthesized polymer similar to RNA or DNA. However, 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 various purine and pyrimidine bases are linked to the backbone by a methylene bridge (-CH ₂ -) and a -(C=0)- group.

As used herein, 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.

As used herein, the term“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.

As used herein, the term“drug” refers to a compound with pharmacological properties that may be used in the preventive or therapeutic treatment of a disease.

As used herein, the term“lectin” refers to carbohydrate-binding proteins, i.e.

macro molecules that are highly specific for sugar moieties.

As used herein, the term“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. As used herein, the term“analyte” refers to a target parameter of interest to be determined qualitatively and/or quantitatively in a sample. As used herein, 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. As used herein, 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.

As used herein, 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.

As used herein, 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.

As used herein, the term“luminescence” refers to the emission of light by a substance not resulting from heat. Examples of luminescence are

chemiluminescence, bioluminescence, photoluminescence, phosphorescence and the like.

As used herein, the term“activated derivative thereof’ refers to derivatives of azido (-N ₃ ), -CºCH, -CH=CH ₂ , amino (-NEE), aminooxy (-O-NH ₂ ), carboxyl (-COOH), aldehyde (-CHO), mercapto (-SH), maleimido groups which are reactive. The activated derivatives include, but are not limited to, isocyanato (-NCO),

isothiocyanato (-NCS), diazonium (-N ⁺ N), bromoacetamido, iodoacetamido, reactive esters, pyridyl-2-dithio, or 6-substituted 4-chloro-l,3,5-triazin-2-ylamino, wherein the 6-substituted 4-chloro-l,3,5-triazin-2-ylamino is preferably 4,6-dichloro-l,3,5- triazin-2-ylamino.

As used herein, the term“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. Examples

The following non-limiting examples are aimed to further demonstrate the invention. The structures and synthetic routes employed are presented in Schemes 1-8.

¹ H-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 a-cyano-4-cinnamic acid matrix. UV-Vis spectra were recorded on Pharmacia Ultrospec 3300 pro.

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.

Conditions for HPLC purification runs: Reversed phase HPLC (RP-18 column). The solvents were A: triethyl ammonium acetate buffer (20mM, pH7) and B: 50% acetonitrile in triethyl ammonium acetate buffer (20mM, 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. Example 1 Synthesis of 2-[4-(2, 2, 2-trifluoroacetamido)phenyl]acetic acid (2)

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 %). ¹ H- NMR (De-DMSO): 12.33 (1H, s), 11.22 (1H, s), 7.59 (2H, d, J=8.6 Hz), 7.29 (2H, d, J=8.5 Hz), 3.56 (2H, s). ¹³ C-NMR (D ₆ -DMSO): 171.50, 155.23, 154.94, 154.64, 154.35, 135.16, 132.86, 130.30, 121.39, 119.63, 117.34, 115.04, 112.75, 40.43. MALDI TOF-MS mass: calculated (M+2H ) 249.07; found 250.30.

Example 2 Synthesis of 6-{2-[4-(2,2,2-trifluoroacetamido)phenyl]acetamido}- hexanoic acid (3)

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. The residue was dissolved in dry DMF (20 ml) and after an addition of 6-amino hexanoic acid (0.78 g, 6.0 mmol), the mixture was stirred overnight at RT. After evaporation to dryness, the residue was suspended into H ₂ 0 (25 ml), the cooled mixture was filtrated and the product (2.00 g, 33 %) washed with cold H2O (3 x 10 ml). MALDI TOF-MS mass: calculated (M+1 H ) 361.14 (M+Na ⁺ ) 383.14; found 360.64 and 383.69.

Example 3 Synthesis of N-(2-aminoethyl)-4-bromo-6-(hydroxymethyl)picolinamide

(5)

A mixture of compound 4 (1.15 g, 4.42 mmol; Takalo, H., et al, Helv. Chim. Acta 79(1996)789) in diethylamine (14.7 ml, 0.22 mol) was stirred for 2.5 h at RT. The mixture was evaporated to dryness and co-evaporated from toluene (2 x 20 ml), and used for the next step without further purifications. Yield : 1.21 g (100%). MALDI TOF-MS mass: calculated (M+3FL) 276.04 and 278.04; found 276.58 and 278.58.

Example 4 Synthesis of N-(6-aminohexyl)-4-bromo-6-(hydroxymethyl)picolin- amide (6)

A mixture of compound 4 (0.26 g, 1 mmol), 1, 6-diamino hexane (2.3 g, 20 mol) in dry MeCN (5 ml) was stirred for overnight at 50 °C. After evaporation to dryness, the product (0.26 g, 80%) was purified by FC (silica gel, 10% EtOH/DCM/l5% TEA). 'H-NMR (D ₆ -DMSO): 8.81-8.75 (1H, m), 8.01 (1H, d, J=l.7 Hz), 7.83 (1H, d, J= 1.7 Hz), 4.64 (2H, s), 3.28 (2H, q, J=6.7 Hz), 2.52 (2H, q, J=6.7 Hz), 1.57-1.47 (2H, m), 1.38-1.32 (2H, m), 1.32-1.25 (4H, m). ¹³ C-NMR (D ₆ -DMSO): 163.33, 151.00, 145.27, 134.34, 125.82, 123.26, 63.75, 52.39, 41.64, 32.96, 29.55, 26.72, 26.44. MALDI TOF-MS mass: calculated (M+H ⁺ ) 330.08 and 332.08; found 330.66 and 332.67.

Example 5 Synthesis of compound 7

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. After evaporation to dryness, the residue was dissolved in DCM (100 ml) and EtOH (60 ml), washed with H ₂ 0 (3 x 20 ml) and dried with Na ₂ S0 ₄ . The crude material was suspended in DCM (20 ml), cooled at -20 C, filtrated and the product (1.24 g, 56%) was washed with cold DCM. 'H-NMR (D ₆ -DMSO): 11.17 (1H, s), 8.86 (1H, t, J=5.9 Hz); 8.18 (1H, t, J=5.7 Hz), 8.01 (1H, d, J=l.8 Hz), 7.84 (1H, t, J=l.8 Hz), 7.53 (2H, d, J=8.6Hz), 7.25 (2H, J=8.6 Hz), 5.59 (1H, bs), 4.64 (2H, s), 3.39 (2H, s), 3.38 (2H, q, J=6.2 Hz), 3.26 (2H, q, J=6.2 Hz). ¹³ C-NMR (D ₆ - DMSO): 170.68, 163.26, 163.19, 155.16, 154.87, 154.57, 154.28, 150.75, 134.94, 134.32, 134.14, 129.84, 125.93, 123.31, 121.28, 119.63, 117.34, 115.05, 112.76, 63.76, 42.15, 39.31, 38.71. MALDI TOF-MS mass: calculated (M+H ⁺ ) 503.06 and 505.06; found 503.64 and 505.71.

Example 6 Synthesis of compound 8

This compound 8 was synthesized from the compound 2 and 6 using a method analogous to the synthesis described in the Example 5. Yield: 100%. ¹ H-NMR (D ₆ - DMSO): 11.19 (1H, bs), 8.77 (1H, t, J=5.7 Hz), 8.09 (1H, d, J=l.8 Hz), 7.98 (1H, t, J=5.5 Hz), 7.83 (1H, d, J=l.8 Hz), 7.56 (2H, d, J=8.5 Hz), 7.26 (2H, d, J=8.5 Hz), 5.60 (1H, bs), 4.64 (2H, s), 3.37 (2H, s), 3.27 (2H, q, J=6.7 Hz), 3.03 (2H, q, J=6.8 Hz), 1.54-1.47 (2H, m), 1.42-1.35 (2H, m), 1.29-1.24 (4H, m). ¹³ C-NMR (D ₆ - DMSO):l70.08, 163.29, 162.79, 155.21, 154.91, 154.62, 154.33, 150.99, 135.11, 134.34, 129.77, 125.81, 123.29, 121.44, 119.68, 117.39, 115.10, 112.81, 63.76, 42.21, 39.23, 38.91, 29.48, 29.36, 26.49, 26.44. MALDI TOF-MS mass: calculated (M+H ⁺ ) 559.12 and 561.12; found 559.88 and 561.88.

Example 7 Synthesis of compound 9

This compound 9 was synthesized from the compound 3 and 6 using a method analogous to the synthesis described in the Example 5. Yield: 26%. MALDI TOF- MS mass: calculated (M+H ⁺ ) 618.16 and 620.16; found 616.99 and 618.99.

Example 8 Synthesis of compound 10

A mixture of compound 7 (0.54 g, 1.07 mmol) and PBn (121 mΐ, 1.29 mmol) in dry l,4-dioxane (150 ml) was stirred overnight at RT, concentrated, dissolved in DCM (60 ml) and neutralized with 5% NaHC0 ₃ (30 ml). The aqueous phase was extracted with DCM (20 ml) and DCM/EtOH (2 x 20/10 ml) and the combined organic phases were dried with Na ₂ S0 ₄ . After evaporation the product (0.53 g, 87%) was used in the next step without any further purifications. 'H-NMR (D ₆ -DMSO): 11.18 (1H, s),

8.78 (1H, t, J=5.9 Hz), 8.18 (1H, t, J=5.5 Hz), 8.08 (1H, d, J=l.8 Hz), 8.07 (1H, d, J=l.8 Hz), 7.53 (2H, d, J=8.5 Hz), 7.26 (2H, d, J=8.5 Hz), 4.71 (2H, s), 3.39 (2H, s), 3.38 (2H, q, J=6.l Hz), 3.26 (2H, q, J=6.l Hz). ¹³ C-NMR (D ₆ -DMSO): 170.68,

163.85, 158.04, 155.16, 154.86, 154.57, 154.27, 151.41, 134.93, 134.52, 134.15,

129.85, 129.51, 124.59, 121.27, 119.63, 117.33, 115.96, 112.76, 63.75, 42.14, 38.66, 33.31. MALDI TOF-MS mass: calculated (M+l ⁺ ) 566.99, 568.99 and 570.98; found 565.67, 567.67 and 569.68.

Example 9 Synthesis of compound 11

This compound 11 was synthesized from the compound 8 using a method analogous to the synthesis described in the Example 8. Yield: 84%. ¹ H-NMR (CDCh): 8.23 (1H, d, J=l .6 Hz), 7.78 (1H, d, J=l.6 Hz), 7.60 (2H, d, J=8.4 Hz), 7.27 (2H, d, J=8.4 Hz), 4.54 (2H, s), 3.52 (2H, s), 3.49 (3H, bs), 3.41 (2H, q, J=7.2 Hz), 3.19 (2H, q, J=7.0 Hz), 1.65-1.58 (2H, m), 1.51-1.44 (2H, m), 1.39-1.28 (4H, m). ¹³ C-NMR (CDCh): 171.49, 162.81, 156.81, 155.80, 155.55, 155.47, 155.25, 150.26, 135.11, 134.95, 132.58, 129.68, 124.89, 121.37, 118.94, 116.92, 114.62, 112.75, 42.68, 39.24, 39.12, 31.66, 29.10, 28.82, 26.13, 26.04. MALDI TOF-MS mass: calculated (M+l ⁺ ) 621.03, 623.03 and 625.03; found 621.78, 623.78 and 625.80.

Example 10 Synthesis of compound 12

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.

Example 11 Synthesis of compound 14

A mixture of compound 10 (0.20 g, 0.35 mmol), 13 (0.22 g, 0.35 mmol; Sund, H., et al, Molecules 22(2017)1807), dry K2CO3 (0.19 g, 1.40 mmol) and dry MeCN (10 ml) was stirred at 60 °C overnight. After filtration and washes with MeCN, the product (0.27 g, 69%) was purified by FC (silica gel, from 7.5% EtOH/DCM to 20% EtOH/DCM/2% TEA). MALDI TOF-MS mass: calculated (M+2H ) 1116.11 and 1118.10; found 1115.93 and 1118.06.

Example 12 Synthesis of compound 15

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

Example 14 Synthesis of compound 18

A mixture of the compound 17 (0.22 g, 0.55 mmol; Sund, H., et al., Molecules 22(2017)1807) and 14 (0.17 g, 0.15 mmol) in dry TEA (1 ml) and DMF (2 ml) was de-aerated with argon. After addition of bis(triphenylphosphine)palladium(II) chloride (10 mg, 14 umol) and Cul (5 mg, 28 umol), the mixture was stirred for 24 h at 55°C. After evaporation to dryness, the product (0.16 g, 51%) was purified by FC (silica gel, from 7.5% to 8% EtOH/DCM). MALDI TOF-MS mass: calculated (M+FF) 2098.75; found 2097.32.

Example 15 Synthesis of compound 19

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.

Example 16 Synthesis of compound 20

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 17 Synthesis of compound 21

Compound 18 (150 mg, 71 umol) in 0.5 M KOH/EtOH (12 ml) was stirred for 1 h at RT. After an addition of H ₂ 0 (7 ml), the mixture was further stirred at RT for 2 h. After EtOH evaporation and an additional overnight stirring at RT, the pH was adjusted to 6.5 by addition of 6 M HC1. EuCh (29 mg, 79 umol) in water (0.5 ml) was added within 5 min and the pH was maintained at 6.0-6.5 with suitable additions of solid NaHC0 ₃ . After stirring overnight at RT, the pH was adjusted to 8.5 with 1M NaOH. The precipitate was removed by centrifugation and the supernatant evaporated to dryness. The product was purified by HPLC. Yield: 113 mg (59%). MALDI TOF-MS mass: calculated (M+H ⁺ ) 1815.29; found 1815.49. R _f (HPLC): 12.65 min. UV: 358 nm. Example 18 Synthesis of compound 22

This compound 22 was synthesized from the compound 19 using a method analogous to the synthesis described in the Example 17. Yield: 45%. MALDI TOF- MS mass: calculated (M+H ⁺ ) 1871.35; found 1871.79. R _f (HPLC): 14.04 min. UV: 358 nm.

Example 19 Synthesis of compound 23

This compound 23 was synthesized from the compound 20 using a method analogous to the synthesis described in the Example 17. Yield: 47%. MALDI TOF- MS mass: calculated (M+EE) 1930.39; found 1929.35. R _f (HPLC): 12.43 min. Example 20 Synthesis of compound 24

Compound 21 (56 mg, 21 umol) in H ₂ 0 (0.5 ml) was added within 5 min to a mixture of CSCl ₂ (11 mΐ, 0.14 mmol) and NaHCCE (14 mg, 0.17 mmol) and CHCh (0.5 ml). After stirring for 30 min at RT, the aqueous phase was washed with CHCb (3x0.5 ml). The product was precipitated with acetone, centrifuged and washed with acetone. MALDI TOF-MS mass: calculated (M+EE) 1857.42 found 1857.50.

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 25 Synthesis of compound 30

A mixture of the compound 19 (0.33 g, 1.60 mmol; Takalo, H., et al Helv. Chim. Acta 79(1996)789) and 7 (0.67 g, 1.34 mmol) in dry TEA (4 ml) and DMF (6 ml) was de-aerated with argon. After addition of bis(triphenylphosphine)palladium(II) chloride (19 mg, 27 umol) and Cul (10 mg, 53 umol), the mixture was stirred for 24 h at 55°C. After evaporation to dryness, the residue was suspended in DCM (6 ml), cooled at -20 C, filtrated and the product (0.77 g, 92%) was washed with cold DCM. MALDI TOF-MS mass: calculated (M+2H ) 628.22; found 628.17.

Example 26 Synthesis of compound 31

This compound 31 was synthesized from the compound 19 and 8 using a method analogous to the synthesis described in the Example 25. The product (55%) was purified by FC (silica gel, 5% EtOH/DCM). MALDI TOF-MS mass: calculated (M+2H ⁺ ) 684.28; found 684.32.

Example 27 Synthesis of compound 32 A mixture of compound 30 (0.76 g, 1.21 mmol) and PBn (135 mΐ, 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

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%. MALDI TOF- MS mass: calculated (M+H ⁺ ) 745.19 and 747.18; found 745.31 and 747.44.

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.

Example 30 Synthesis of compound 35

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.

Example 31 Synthesis of compound 36

A mixture of the compound 17 (0.10 g, 0.25 mmol; Sund, H., et al, Molecules 22(2017)1807) and 34 (0.13 g, 0.10 mmol) in dry TEA (1 ml) and DMF (4 ml) was de-aerated with argon. After addition of bis(triphenylphosphine)palladium(II) chloride (10 mg, 14 umol) and Cul (5 mg, 28 umol), the mixture was stirred for 24 h at 55°C. After evaporation to dryness, the product (0.10 g, 52%) was purified by FC (silica gel, from 7.5% EtOH/DCM). MALDI TOF-MS mass: calculated (M+H ⁺ ) 1894.69; found 1894.98.

Example 32 Synthesis of compound 37

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.

Example 33 Synthesis of compound 38

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.

Example 34 Synthesis of compound 39

This compound 39 was synthesized from the compound 37 using a method analogous to the synthesis described in the Example 17. Yield: 77%. R _f (HPLC):

16.07 min. Example 35 Synthesis of compound 40

This compound 40 was synthesized from the compound 38 using a method analogous to the synthesis described in the Example 23. MALDI TOF-MS mass: calculated (M+H ⁺ ) 1836.21; found 1837.01. R _f (HPLC): 14.08 min.

Example 36 Synthesis of compound 41

This compound 41 was synthesized from the compound 39 using a method analogous to the synthesis described in the Example 20.

Example 37 Synthesis of N-(2-acetamidoethyl)-4-bromo-6- (hydroxymethyl)picolinamide 42 The mixture of acetic acid (0.23 g, 4.0 mmol), N-hydroxysuccinimimide (0.46 g, 4.0 mmol) and N,N-dicyclohexylcarbodiimide (0.83 g, 4.0 mmol) in dry l,4-dioxane (10 ml) was stirred for 2.5 h at RT, filtrated, the solid material was washed with 1,4- dioxane (3 x 2,5 ml) and the filtrate was evaporated to dryness. The residue was dissolved in dry DMF (10 ml) and added into the solution of N-(2-aminoethyl)-4- bromo-6-(hydroxymethyl)picolinamide (5) (1.10 g, 4.0 mmol) in dry DMF (10 ml). After stirring overnight at RT, the mixture was evaporated to dryness, The residue was dissolved in DCM (100 ml) and EtOH (60 ml), and washed with saturated aqueous NaCl solution (30 ml). The aqueous phase was extracted with DCM/EtOH (30/10 ml), the combined organic phase was washed with saturated aqueous NaCl solution (30 ml), dried with Na ₂ S0 ₄ and evaporated to dryness. The product (0.68 g, 54%) was used in the next step without any further purifications. ¹ H-NMR (D ₆ - DMSO: 8.89 (1H, t, J=5.8 Hz); 8.02 (1H, d, J=l.7 Hz), 7.97 (1H, t, J=5.4 Hz), 7.84 ( 1H, d, J=l.7 Hz), 5.57 (1H, bs), 4.65 (2H, s), 3.36 (2H, q, J=6.3 Hz), 3.22 (2H, q, J=6.3 Hz), 1.80 (3H, s). ¹³ C-NMR (D ₆ -DMSO): 169.87, 163.26, 163.17, 150.78, 134, 32, 125.91, 123.31, 63.75, 39.37, 38,65, 22.98. MAEDI TOF-MS mass: calculated

(M+H ⁺ ) 317.04 and 219.04; found 317.04 and 319.04.

Example 38 Synthesis of N-(2-acetamidoethyl)-4-bromo-6-(bromomethyl)picolin- amide 43

A mixture of compound 42 (0.73 g, 2.31 mmol) and PBn (260 mΐ, 2.77 mmol) in dry CHCh (150 ml) was stirred overnight at RT and neutralized with 5% NaHC0 ₃ (20 ml). The aqueous phase was extracted with CHCh/EtOH (40/20 ml) and the combined organic phases were dried with Na ₂ S0 ₄ . The product (0.43 g, 49%) was purified by FC (silica gel, 10% EtOH/DCM). 'H-NMR (CDCb): 8.26 (1H, d, J=l .6 Hz), 7.76 (1H, d, J=l.6 Hz), 6.23 (1H, bs), 4.51 (2H, s), 3.63 (2H, q, J=5.8 Hz), 3.51 (2H, q, J=5.8 Hz), 1.99 (3H, s). ¹³ C-NMR (CDCb): 170.67, 163.86, 156.87, 150.08, 135.20, 129.22, 125.05, 40.39, 39.41, 31.72, 23.24. MALDI TOF-MS mass:

calculated (M+H ⁺ ) 377.95, 379.95 and 380.85; found 377.56, 379.73 and 381.73.

Example 39 Synthesis of ethyl 2-{{{6-[(2-acetamidoethyl)carbamoyl]-4-bromo- pyridin-2-yl}methyl} -amino} acetate 44

A mixture of compound 43 (0.42 g, 1.10 mmol), ethyl glysinate x HC1 (0.77 g, 5.54 mmol), DIPEA (2 ml) and dry MeCN (60 ml) was stirred for 3 d at RT. The mixture was evaporated to dryness and the product (0.l4g, 32%) was purified by FC (silica gel, from 5% to 10% EtOH/DCM. MALDI TOF-MS mass: calculated (M+H ⁺ ) 401.08 and 403.08; found 401.73 and 403.78.

Example 40 Synthesis of compound 46

A mixture of compound 44 (135 mg, 0.336 mmol), 45 (80 mg, 0.168 mmol; Sund,

H., et al., Molecules 22(2017)1807) and dry K ₂ CO ₃ (93 mg, 0.672 mmol) in dry MeCN (7 ml) was stirred for overnight at 55 °C. The mixture was filtrated, the solid material washed with MeCN (3 x 1.5 ml) and the filtrate evaporated to dryness. The product (0.19 g, 100%) was used for the next step without any further purifications. ‘H-NMR (CDCh): 8.52 (2H, bs), 8.15 (2H, d, J=l.5 Hz), 7.79 (2H, s), 7.60 (2H, d, J=8.5 Hz), 7.46 (2H, d, J=8.5 Hz). 7.35 (2H, s), 6.82 (2H, bs), 4.19 (4H, q, J=7.2 Hz), 3.94 (4H, s), 3.92 (4H, s), 3.59-3.53 (4H, m), 3.45 (4H, s), 3.47-3.41 (2H, m),

I.93 (6H, s), 1.25 (6H, t, J=7.2 Hz). ¹³ C-NMR (CDCh): 171.12, 170.91, 164.28, 159.03, 158.12, 156.11, 155.82, 155.53, 155.21, 150.17, 134.88, 132.61, 128.97, 128.86, 121.30, 121.23, 119.64, 117.49, 115.20, 113.40, 93.99, 86.72, 60.83, 60.08, 55.54, 40.11, 39.49, 23.02, 14.16. MALDI TOF-MS mass: calculated (M+H ⁺ ) 1115.21, 1117.21 and 1119.21; found 1115.29, 1117.17 and 1119.15.

Example 40 Synthesis of compound 47

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). MALDI TOF-MS mass: calculated (M+H ⁺ ) 1771.66; found 1772.67.

Example 40 Synthesis of compound 48

This compound 48 was synthesized from the compound 47 using a method analogous to the synthesis described in the Example 17. Yield: 92%. MALDI TOF- MS mass: calculated (M+H ⁺ ) 1601.92; found 1600.65. R _f (HPLC): 16.18 min. UV: 363 nm.

Example 41 Synthesis of compound 49

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

The conjugation was performed by using 10 fold excess taurine in 50 mM Na ₂ C0 ₃ buffer (pH 9.8) by stirring overnight at RT. The products were purified by using HPLC. The observed R _f (HPLC) values and UV absorption maxima are shown in Table 1.

The fluorescence parameters (in Table 1) for the taurine derivative of chelates 24, 25, 27, 40, 41, 49 and the reference chelate [structure 1 in von Lode P. et al., Anal.

Chem. 75(2003)] were measured in 50 mM TRIS buffer (pH 9.8). After the HPLC product fractions were evaporated, the residue was dissolved in 50 mM TRIS buffer (1 ml). As shown in Table 1, the absorption maximum (l _;i i, _¾ ), extinction coefficient (e), the brightness (ef), the quantum yield (f), the lifetime (t) for a corresponding excitation wavelength (Uxc). 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 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).

Table 1. Summary of characteristics of chelates

*) - means“not determined"

Despite the long organic side chain used for the biomolecule labelling, 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.

Also 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.,

Eur. J. Inorg. Chem. (2009)4357). Chromophores, which have Eu(III) ion complexing amide function at the pyridine chromophore, have been reported to have so low CT state that those chelates are luminescent only in organic solution, not in water (see Picot, A. et al., Inorg Chem. 46(2007)2659). Evidence for the energy back-transfer due to the low lying ILCT state is indicated by the luminescence lifetime measurements of the chelates in D ₂ 0, as no increase of life-times were observed (e.g. 0.37, 0.61 and 0.34 qs for chelates 27, 40 and 49, respectively). Thus, it is surprising that the novel chelates show luminescence in aqueous solutions.

Example 43 Labeling of antibody with labelling reagents 25, 28, 41 and 49

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.

Example 44 Troponin I immunoassay

The Tnl antibody labeled with the chelate 25, 28, 41 and 49 was tested in sandwich immunoassay for cardiac troponin I. As a reference compound a Tnl antibody labelled with a-gal-9-D Eu (von Lode P. et al., Anal. Chem. 75(2003)3193) was used. 10 mΐ of diluted tracer antibody f 5 ng/mΐ) and 20 mΐ 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 Victor™ Plate fluorometer. The results are summarized in Table 2.

The conventional 9-dentate a-galactose Eu chelate as a reference (Ref in Table 2) was prepared according to von Lode P. et al. Anal. Chem. 75(2003)3193 (structure

1)·

Table 2. Brightness of Tn-antibody labelled with chelates

* The value was calculated from the data in example 42 (Table 1) by applying the luminescence enhancing effect of drying (ca. 60% increase) mentioned in von Lode P. et al. Anal. Chem. 75(2003)3193.

Based on the brightness measurements in Table 1, it was assumed that the brightness of the same labels conjugated in biomolecules would also be at the same levels. However, the observed brightnesses of the conjugates of the invention in Table 2 were surprisingly high being over 10 fold higher compared to the taurine derivatives mentioned in example 42 (see Table 1). Based on the prior-art (Andraud, C., et al., Eur. J. Inorg. Chem. (2009)4357; and Picot, A. et al., Inorg. Chem. 2007, 46, 2659- 2665), it was contrastingly assumed that the use of Eu(III) complexing amide function at pyridine chromophores, e.g. for the labeling side chain, would not show significant luminescence in aqueous solution based applications, and thus, those chelate designs could have been useless for sensitive bio-affinity assays. In that respect, the observed luminescence intensities of the present chelates were surprisingly remarkable.

Scheme 1

21: L = (CH ₂ ) ₂

22: L = (CH ₂ ) ₆

Scheme 2 23: L = (CH ₂ ) ₂ NHCO(CH ₂ ) ₅ 26: L = (CH ₂ ) ₂ NHCO(CH ₂ ) ₅

21-23

27: L = (CH ₂ ) ₂

Scheme 3 28: L = (CH ₂ ) ₆

32: L = (CH ₂ ) ₂ 33: L = (CH ₂ ) ₆

Scheme 4

36: L = (CH ₂ ) ₂

Scheme 5 37: L = (CH ₂ ) ₆

40: L = (CH ₂ ) ₂ ; R ¹ = (Cl ₂ C ₃ N ₃ )NH

Scheme 6 41: L = (CH ₂ ) ₆ ; R ¹ =NCS

Scheme 7 46