WO2009115644A1

WO2009115644A1 - Lanthanide(iii) chelates comprising beta-diketones and conjugates derived thereof

Info

Publication number: WO2009115644A1
Application number: PCT/FI2009/050208
Authority: WO
Inventors: Jari Hovinen; Veli-Matti Mukkala
Original assignee: Wallac Oy
Priority date: 2008-03-20
Filing date: 2009-03-18
Publication date: 2009-09-24
Also published as: FI20080230A0

Abstract

This invention relates to luminescent lanthanide(III) chelates based on β-diketones comprising an azacycloalkane backbone and biomolecule conjugates and solid supports comprising these chelates. In contrast to lanthanide chelates based on monomeric β-diketones as well as lanthanide(III) chelates based on acyclic azaalkanes including several β-diketone subunits, the molecules of the present technology are luminescent even in the absence of additional chelators such as phosphine oxides.

Description

LANTHANIDE(III) CHELATES COMPRISING BETA-DIKETONES AND CONJUGATES DERIVED THEREOF

FIELD OF THE INVENTION

This invention relates to luminescent lanthanide(III) chelates based on β-diketones tethered to azacycloalkanes and biomolecule conjugates and solid supports based on these chelates.

BACKGROUND OF THE INVENTION

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

The commercially available DELFIA^® assay method uses a non-luminescent europium(III) chelate as the label [US 4,565,790; US 4,808,541]. After immunoreaction, europium(III) ion is dissociated from the non-luminescent chelate by lowering pH to 3.2 and the luminescence is enhanced with a mixture of β-diketone (4,4,4-trifluoro-l-(2-naphtyl)-butane-l,3-dione), detergent (Triton X-IOO) and chelator (trioctylphosphine oxide, TOPO). The new chelate formed has a very high luminescence, giving detection sensitivity of ca 5 ^• 10^"16 M. The trifluorinated methyl group of the β-diketone shifts the keto-enol equilibrium efficiently towards the strongly chelating enolic form, and thus enables chelate formation even in acidic solutions. More strongly acidic β-diketones allow the use of even more acidic measurement solutions [US 7,211,440].

However, the DELFIA^® technology cannot be used in applications requiring site-specificity. Furthermore, the use of the chelator TOPO is essential for high luminescence. To overcome these limitations, stable luminescent lanthanide(III) chelates have been developed. These chelates consist of a ligand with a reactive group for covalent conjugation to bioactive molecules, an aromatic structure, which absorbs the excitation energy and transfers it to the lanthanide ion and additional chelating groups, such as carboxylic acid moieties and amines. Unlike organic chromophores, these molecules do not suffer from Raman or Rayleigh scattering or concentration quenching. Although numerous stable luminescent lanthanide(III) chelates have been synthesized, none of them has as high quantum yield as the best β- diketone based chelates. Although β-diketones form relatively stable chelates with lanthanide(III) ions, these chelates are not stable enough for bioaffinity assays as such. However, derivatives containing a reactive group in the aromatic ring of the β-diketone have been proposed to be used as labels in immunoassays [DE 2628158]. β-diketones have also been used to enhance luminescence in a mixed ligand system with EDTA type ligands [US 4,374,120], macrocyclic complexes [US 6,750,005], as well as with chelates tethered to various aromatic ligands as luminescence sensitizers [US 6,344,360].

It has been shown that adding of several β-diketone structures to one backbone enhances the chelate stability and quantum yield [EP 0794174, Anal. Chem., 1998, 70, 596, Proc. SPIE 1995, 2388, 429, Clin. Biochem. 1988, 21, 173]. However, most of the structures disclosed in prior art have been used as ligands saturated in vitro with excess of metal ions. Furthermore, the remaining aqueous quenching has to be avoided by including TOPO in the measuring buffer [W.DeW.Horrocks, Jr., D.R.Sudnick, Ace. Chem. Res. 1981, 14, 384-392; W.DeW.Horrocks, Jr., D.R.Sudnick, /. Am. Chem. Soc. 1979, 101:2, 334-340 ].

SUMMARY OF THE INVENTION

It is the main object of the present invention to provide lanthanide(III) chelates and biomolecule labeling reactants, labeled biomolecules and solid supports based on β-diketones including an azacycloalkane backbone.

Thus the present technology concerns luminescent lanthanide(III) chelates comprising at least three β-diketone subunits, and an azacycloalkane backbone.

According to one aspect, this disclosure concerns lanthanide(III) chelates based on azacycloalkanes wherein three or four nitrogen atoms of the azacycloalkanes are substituted with a group R(C=O)CH₂(C=O)ArL¹-, and where one of the nitrogen or one of the carbon atoms is substituted with a group of G-L²- wherein

L¹ is a linker connecting Ar to the azacycloalkane, L² is a linker connecting G to the azacycloalkane or is not present, G is a reactive group or is not present,

Ar is an aryl group, optionally mono or multisubstituted,

R is a straight or branched alkyl chain with 1 to 9 carbon atoms substituted with three or more fluorine atoms and optionally substituted with other substituents than fluorine.

According to another aspect, this disclosure concerns a bioactive complex, i.e. biomolecule conjugated with a chelate according to this technology.

According to another aspect, this disclosure concerns a solid support conjugated with a chelate according to this technology.

According to another aspect, this disclosure concerns a solid support conjugated with a bioactive molecule labelled with a chelate according to this technology.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 shows an emission spectrum of a stable luminescent lanthanide chelate [an europium(III) chelate based on 6,6'-(lH-pyrazole-l,3-diyl)bis(pyridine)]. The emission spectrum is devided into several lines. λ_em (rel int %) 593 (14); 615 (50) 654 (4), 692 (32). Fig 2 shows an emission spectum of an europium(III) chelate according to this invention

(compound 7). The desired emission line at 615 nm is practically the only emission line detected.

DETAILED DESCRIPTION OF THE INVENTION

Lanthanide(III) chelates based on β-diketones including an azacycloalkane backbone are new. In contrast to lanthanide chelates based on monomeric β-diketones as well as lanthanide(III) chelates based on acyclic azaalkanes including several β-diketone subunits, the molecules of the present technology are luminescent even in the absence of additional chelators such as phosphine oxides. In addition, the chelates do not need to be saturated with an excess of metal ions.

According to one embodiment, the present technology concerns luminescent lanthanide(III) chelates including β-diketone subunits and a azacycloalkane backbone. According to one embodiment, the azacycloalkane backbone according to this technology includes a cyclic N-containing hydrocarbon chain with three or more nitrogen atoms. In a particular embodiment the azacycloalkanes are 1,4,7-triazacyclononane, 1,4,7-triazacyclodecane, 1,5,9-triazacyclododecane, cyclen and cyclam.

According to one embodiment the azacycloalkane backbone includes an aromatic residue. In a particular embodiment the aromatic residues is pyridine.

In the present technology, it was also observed that the excitation wavelength of the chelate can be tuned by modifying the structure (i.e. the chromophore or the alkyl substituent of the chromophore) of the β-diketone subunit. A high excitation wavelength is useful, since the choice of materials of the measuring equipments is more flexible and the background luminescence is less significant. For example, excitation wavelengths over 350 nm allow the use of cheap LED lasers as the light source.

According to a particular embodiment the present technology concerns lanthanide(III) chelates based on tri- or tetraazacycloalkanes, wherein three or four nitrogen atoms are substituted with a β-diketone group R(C=O)CH₂(C=O)ArL¹-, and wherein one of the nitrogen or one of the carbon atoms is optionally substituted with a group of G-L²-, and wherein

L¹ is a linker between Ar and the azacycloalkane,

L² is a linker between G and the azacycloalkane or is not present;

G is a reactive group or is not present, Ar is an aryl group, optionally mono or multisubstituted,

R is a straight or branched alkyl chain with 2 to 9 carbon atoms substituted with three or more fluorine atoms optionally substituted with other substituents than fluorine.

According to one embodiment, the linker L¹ includes one to three moieties each moiety being selected from an alkylene containing 1-3 carbon atoms, ether (-O-), thioether (-S-), amide (-CO-

NH-, -CO-NR -, -NH-CO- and -NR -CO-), carbonyl (-CO-), and ester (-COO- and -OOC-), wherein R' represents an alkyl group containing less than 5 carbon atoms. In a partucular embodiment the linker L¹ is selected from methylene and ethylene. An exeplary linker L¹ is methylene.

According to one embodiment, L² includes one to ten moieties, each moiety being selected from the group consisting of phenylene, alkylene containing 1-12 carbon atoms, ethynydiyl (-C≡C-), ethylenediyl (-C=C-), ether (-O-), thioether (-S-), amide (-CO-NH-, -CO-NR'-, -NH-CO- and - NR'-CO-), carbonyl (-CO-), ester (-C00- and -00C-), disulfide (-SS-), sulfonyl (-SO₂-), diaza (-N=N-), and tertiary amine, wherein R' represents an alkyl group containing less than 5 carbon atoms.

According to one embodiment the aryl group Ar is selected from the group consisting of phenyl, 9H-fluoren-2-yl, 1-naphthyl, 2-naphthyl, 2-phenanthroyl, 2-furyl, 3-furyl, 2-benzofuryl, 3- benzofuryl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-thiazolyl, 4-thiazolyl, 2- benzothiazolyl, 2-benzo[a]thienyl, 3-benzo[a]thienyl, 2-pyrimidyl, 4-pyrimidyl and 5-pyrimidyl.

According to one embodiment the aryl group Ar is mono- or multisubstituted. In a particular embodiment each substituent is independently selected from a group consisting of straight or branched alkyl, alkoxy, aryl, aroyl, aryloxy, nitro, amino, cyano, hydroxyl, carboxy, chloro, bromo, iodo, fluoro and acyl. If these substitutions include atoms that can be substituted these can in turn be substituted.

According to one embodiment the alkyl chain R is substituted with 3 to 9 fluorine atoms. In a particular embodiment R is substituted with 3 to 7 fluorine atoms.

According to one embodiment R is mono- or multisubstituted with other substituents than fluorine and each substituent is independently selected from a group consisting of straight or branched alkyl, alkoxy, aryl, aroyl, aryloxy, nitro, amino, cyano, hydroxyl, carboxy, chloro, bromo, iodo, fluoro or acyl. If these substitutions include atoms that can be substituted these can in turn be substituted.

According to a particular embodiment β-diketones are selected form a group consisting of 4,4,4- trifluoro- 1 -aryl- 1 ,3-butanedione, 4,4,5,5,5-pentafluoro- 1 -aryl- 1 ,3-pentanedione and 4,4,5,5,6,6,6-heptafluoro-l-aryl-l,3-hexanedione, wherein the aryl group is selected from a group consisting of thienyl, furyl, benzolfuryl, benzothienyl and phenyl.

The chelate must bear a reactive group G in order to enable covalent binding of the chelate to a bioactive molecule or to a solid support. According to one embodiment the reactive group G is selected from the group consisting of isothiocyanate, haloacetamido, maleimido, dichlorotriazinylamino, pyridyldithio, thioester, aminooxy, hydrazide, amino, a polymerizing group, and carboxylic acid, carboxylic acid halide and active ester of carboxylic acid. An exemplary active ester is N-hydroxysuccinimide.

In the case the chelate should be attached to a microparticle or nanoparticle during the manufacturing process of the particles, the reactive group G is a polymerizable group. An exemplary polymerizable group is methacroyl group.

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

In case the chelate is to be attached to solid supports including nanomaterials, biomolecules, and various organic molecules using copper-free Huisgen-Sharpless dipolar [2+3] cycloaddition reaction [US 2006/0110782], the reactive group G has to be a cycloalkylalkyne group.

It has been proposed [US 5,985,566] that oligonucleotides, DNA, RNA, oligopeptides, proteins and lipids can be transformed statistically by using label molecules tethered to platinum derivatives. In nucleic acids these molecules react predominantly at N7 of guanine residues. In case the reactive group G is

wherein A is cleaving ligand like Cl, (CH₃ )₂SO, H₂O, and NO₃ ^"

However, there exist applications where no such covalent binding is necessary. Chelating compounds of this invention can also be used in applications where no reactive groups in the chelate are needed. One example of this kind of technology is demonstrated in [J. Immunological Methods 1996, 193, 199]. Another example where no reactive group G is needed is the separation of eosinophilic and basophilic cells [WO2006/072668]. In this application positively and negatively charged chelates bind negatively and positively charged cell surfaces, respectively.

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

It is known that the presence of several chromophores decreases the solubility of lanthanide(III) chelates in H₂O. If needed this effect can be minimized by adding hydrophilic moieties to the ligands. These hydrophilic moieties can be e.g. carboxylic acids [HeIv. Chim. Acta, 1996, 79, 789], carbohydrates [Anal. Chem., 2003, 75, 3193], and sulfonic acids [Bioconjugate Chem. 2008, 19, 279].

According to one embodiment lanthanide(III) ions are selected from europium(III), terbium(III), samarium(III) and dysprosium(III). In a particular embodiment lantanide(III) ions are europium(III) and samarium(III).

In a particular embodiment the chelates are selected from the following structures:

wherein R(C=O)CH₂(C=O)Ar- is selected from a group consisting of 4,4,4-trifluoro-l-aryl-l,3- butanedione, 4,4,5, 5,5-pentafluoro-l-aryl-l,3-pentanedione and 4,4,5,5,6,6-heptafluoro-l-aryl- 1,3-hexanedione, wherein aryl is selected from thienyl, furyl and phenyl; L¹ selected from methylene and ethylene;

L² is a linker between G and the azacycloalkane, and includes one to ten moieties, each moiety being independently selected from the group consisting of phenylene, alkylene containing 1-12 carbon atoms, ethynydiyl (-C≡C-), ethylenediyl (-C=C-), ether (-0-), thioether (-S-), amide (- CO-NH-, -CO-NR -, NH-CO and -NR -CO-), carbonyl (-CO-), ester (-C00- and -00C-), disulfide (-SS-), sulfonyl (-SO₂-), diaza (-N=N-), and tertiary amine, wherein R' represents an alkyl group containing less than 5 carbon atoms;

G is a reactive group and is selected from the group consisting of isothiocyanate, bromoacetamido, iodoacetamido, maleimido, 4,6-dichloro-l,3,5-triazinyl-2-amino, pyridyldithio, thioester, aminooxy, hydrazide, amino, azido, alkyne, a polymerizing group, carboxylic acid, carboxylic acid halide, active ester of carboxylic acid, and

wherein A is cleaving ligand comprising Cl, (CH₃)₂SO, H₂O, and NO₃ ^" and wherein - is the position of linker L²;

Ln is lanthanide selected from a group consisting of europium, terbium, samarium and dysprosium; and n is 1 or 2.

According to one embodiment, this technology concerns a bioactive complex including a bioactive molecule conjugated with a chelate according to this technology. In a particular embodiment the bioactive molecule is selected from the group consisting of an oligopeptide, oligonucleotide, DNA, RNA, modified oligo- or polynucleotide, protein, oligosaccaride, polysaccaride, phospholipide, PNA, LNA, antibody, steroid, hapten, drug, receptor binding ligand and lectine.

According to another embodiment this technology concerns a solid support conjugated with a chelate according to this technology. In a particular embodiment the solid support is selected from the group consisting of a nanoparticle, a microparticle, a slide and a plate.

According to another embodiment this technology concerns a bioactive complex according to this technology conjugated to a solid support. In a particular embodiment the solid support is selected from the group consisting of nanoparticle, a microparticle, a slide and a plate.

EXAMPLES

The invention is further elucidated by the following examples. The structures and synthetic routes employed in the experimental part are depicted in Schemes 1-7. Scheme 1 illustrates the synthesis of the of (Z)-l-(3-(bromomethyl)phenyl)-4,4,4-trifluoro-3-hydroxybut-2-en-l-one, 2. The experimental details are given in Examples 1-2. Scheme 2 illustrates the synthesis the europium(II) chelate based on 1,5,9-triazacyclododecane 4. Experimental details are given in Examples 3 and 4. Schemes 3-5 illustrate the synthesis of the acyclic derivatives of β-diketones 7, 10 and 11. Experimental details are given in Examples 5-11. Scheme 6 illustrates the synthesis a thiophene derivatives 12-17. Experimental details are given in Examples 12-16. Schemes 7 and 8 illustrate the synthesis of a mercapto selective biomolecule labelling reactants (21, 27) based on a β-diketone according to this invention. 4,4,5, 5,5-pentafluoro-l-(thienyl)pentane-l,3-dione 28, 4,4,5,5,6,6,6-heptafluoro -l-(thienyl)pentane-l,3-dione 29, l-(5-cyano-2-thienyl)-4,4,5,5,5- pentrafluoro-l,3-pentadione 30, l-(5-carboxy-2-thienyl)-4,4,5,5,5-pentafluoro-l,3-pentadione 31, 4,4,5,5,6,6,6-heptafluoro-l-(2-furyl)-l,3-hexanedione 32, 4,4,5,5,5-pentafluoro-l-(2- naphthyl)-l,3-pentanedione 33, 4,4,5,5,6,6,6-heptafluoro-l-(2-naphthyl)-l,3-hexanedione 34, 1- (2-benzo[b]thienyl)-4,4,5,5,5-pentafluoro-l,3-pentanedione 35, l-(2-benzofuryl)-4,4,4-trifluoro- 1,3-butanedione 36, l-(2-benzofuryl)-4,4,5,5,5-pentafluoro-l,3-pentanedione 37 and l-(2- benzofuryl)-4,4,5,6,6,6-heptafluoro-l,3-hexanedione 38 were synthesized as disclosed in US 7,211,440.

Example 1

Synthesis of m-bromomethylacetophenone 1

m-Methylacetophenone (10.0 g, 74.5 mmol), iV-bromosuccinimide (13.93 g, 78.3 mmol) and dibenzoylperoxide (8 mg) were dissolved in carbon tetrachloride (70 mL) and the mixture was heated in reflux for 5 h, cooled to RT and filtered. The filtrate was concentrated and purified on silica gel (petroleum ether, bp 40-60 ⁰C / ethyl acetate, 10:1, v/v). Yield was 12.9 g. ¹H NMR (CDCl₃) δ 7.92 (IH, s); 7.89 (IH, d, J 7.8); 7.60 (IH, d, J 7.8); 7.46 (IH, t, J 7.8); 4.53 (2H, s); 2.62 (3H, s).

Example 2

Synthesis of (Z)-I -(3-(bromomethyl)phenyl)-4,4,4-trifluoro-3-hydroxybut-2-en-l -one 2 To a mixture of compound 1 (1.0 g, 4.69 mmol) and NaH (0.28 g, 60-65% dispersion in oil) in dry toluene (10 mL) was added ethyl trifluoroacetate. The reaction was initiated with gentle heating followed by heating at reflux for 2 h. To the cooled reaction mixture was then added 20% aq. H₂SO₄ (10 mL) and diethyl ether (10 mL). The organic phase was washed with water, dried over Na₂SO₄ and concentrated in vacuo. Purification was performed on silica gel (petroleum ether, bp 40-60 ⁰C / ethyl acetate, 5:2, v/v). Yield was 1.40 g. ¹H NMR (CDCl₃) δ 8.00 (IH, s); 7.91 (IH, d); 7.66 (IH, d); 7.52 (IH, t); 6.58 (IH, s); 4.54 (2H, s);

Example 3

Synthesis of l,5,9-tris[3-(4,4,4-trifluoro-3-hydroxybut-2-en-l-onyl)phenylmethyl]- 1,5,9- triazacyclododecane 3.

Compound 2 (0.28 g, 0.90 mmol) and 1,5,9-triazacyclododecane (0.05 g, 0.29 mmol) were dissolved in dry acetonitrile. Anhydrous K₂CO₃ (0.13 g) was added and the mixture was heated overnight at reflux, allowed to cool to RT, filtered and concentrated. Precipitation from diethyl ether yielded the title compound.

Example 4 Synthesis of l,5,9-tris[3-(4,4,4-trifluoro-3-hydroxybut-2-en-l-onyl)phenylmethyl]- 1,5,9- triazacyclodocecane europium(III) 4

A mixture of compound 3 (9 mg, 0.011 mmol) and EuCl₃ ^• 6H₂O (4 mg, 0.011 mmol) in acetonitrile (20 mL) was heated overnight at reflux before being concentrated in vacuo. The product was isolated by precipitation from diethyl ether.

Example 5

Synthesis of 3-acetylphenoxyacetic acid 5

m-Hydroxyacetophenone (4.08 g, 30 mmol) and bromoacetic acid (4.16 g, 30 mmol) were dissolved in 2 M NaOH (34 mL), and the mixture was heated at reflux for 1 d, cooled to RT and diluted with water (20 mL) and washed with dichloromethane (3 • 40 mL). pH was adjusted to 2 with 1 M hydrochloric acid, and the product was extracted with a mixture of dichloromethane and ethanol. The organic phase was dried over Na₂SO₄. Purification on silica gel (MeOHZCH₂Cl₂ 1: 9 + 1% triethylamine, v/v) yielded 2.3 g (39%) of the title compound. ¹H NMR (CDCl₃) δ 7.53 (tot 3 H, s and d); 7.35 (IH, t, J 7.8); 7.17 (IH, m); 4.61 (2H, s); 2.57 (3H, s). IR (KBr): 1682, 1064 cm^"1.

Example 6

Synthesis of the ketone 6

Compound 5 (0.12 g, 0.61 mmol) was dissolved in thionyl chloride (2 mL) and the mixture was heated at reflux for 1.5 h, concentrated in vacuo, and diluted with acetone (3 mL). This was then added dropwice into a ice cold solution of diethylene triamine (0.02 g, 0.20 mmol) and KOH (0.034 g, 3.1 mmol) in water (2 mL) during 30 min. After an additional ¹A h the product formed was isolated by filtration. The product was washed with water and dried in vacuo.

Example 7

Synthesis of the β-diketone 7

Compound 6 (0.035 g, 0.55 mmol) was converted to compound 7 using the method described in Example 2 except the reaction was performed in dry THF. Yield was 0.035 g. It was converted to the corresponding europium(III) chelate (Eu a 7) using the method described in Example 4.

Example 8

Synthesis of the nitro ketone 8

M-(4-nitrophenylethyl)ethane-l,2-diamine dihydrochloride (0.33 g, 1.15 mmol), synthesized as disclosed in [Bioconjugate Chem. 2002,13, 870], was suspended in dry acetonitrile (15 mL). DIPEA (2.4 mL) was added, and the mixure was allowed to stir for 20 min at RT. To the resulting clear solution compound 1 (0.86 g, 3.6 mmol) was added, and the mixture was heated at reflux for 5.5 h and then overnight at RT. All volatiles were removed in vacuo, the residue was dissolved in methylene chloride, washed with water and dried. Purification on silica gel (petroleum ether, bp 40-60 ⁰C / ethyl acetate, stepwise gradient 5:2, 1 :1, 2:5, v/v) yielded 0.44 g of compound 8. Example 9

Synthesis of the amino ketone 9

Compound 8 (0.41 g, 0.68 mmol) was dissolved in ethanol (25 mL). Tin dichloride (0.36 g, 1.91 mmol) was added, and the mixture was heated at reflux for 2.5 h, allowed to cool to RT and concentrated. Purification on silica gel ((petroleum ether, bp 40-60 ⁰C / ethyl acetate/triethylamine, 10:1 :1, v/v/v) yielded 0.17 g of the title compound.

Example 10 Synthesis of the amino β-diketone 10

Compound 9 (0.17 g, 0.30 mmol) was dissolved in dry DMF (1.5 mL). Sodium hydride (71 mg, 60% dispersion in oil) was added, and the mixture was stirred for 15 min at RT. Ethyl trifluoroacetate (1.3 mL) was added, and reaction was allowed to proceed overnight at RT. The reaction mixture was diluted with dichloromethane (40 mL), washed with 0.5 M acetic acid (25 mL) and twice with water (25 mL), dried over Na₂SO₄ and concentrated. Purification was performed on silica gel using ethyl acetate as the eluent. Yield was 0.18 g. It was converted to the corresponding europium(III) chelate (Eu c: 10) using the method described in Example 4.

Example 11

Synthesis of tetra β-diketone 11

The title compound was synthesized from ethane- 1,2-diamine and compound 2 as disclosed in Example 3. It was converted to the corresponding europium(III) chelate (Eu c 11) using the method described in Example 4.

Example 12

Synthesis of 2-(5-acetyl-2-thienyl)ethyl acetate 12

A mixture of thienylethanol (25.6 g, 0.20 mol) and acetic anhydride (57.2 g; 0.56 mol) were heated to 55 ⁰C. Phosphoric acid (5 mL; 85%) was slowly added, and the mixture was heated for 1 ¹A h at 70 ⁰C. The mixture was cooled to RT and concentrated to ca half volume. The residue was diluted with dichloromethane, washed with sat NaHCC>₃ and concentrated. The residue was dissolved in 0.2 M KOH in methanol, and stirred for 2 h at RT, diluted with water and extracted with dichloromethane. The organic layer was dried over Na₂SO₄ and concentrated. Purification on silica gel (eluent first CH₂Cl₂, then 5% MeOH in CH₂Cl₂) yielded 23 g of the title compound. ¹H NMR (CDCl₃): δ 7.55 (IH, d J 3.8); 6.90 (IH, d, J 3.7); 4.31 (2H, t, J 6.4); 3.17 (2H, t, J 6.4); 2.52 (3H, s); 2.07 (3H, s).

Example 13

Synthesis of 2-(5-acetyl-2-thienyl)ethanol 13

Compound 12 (23 g, 0.11 mol) was dissolved in 0.2 M KOH/MeOH (55 mL) and stirred for 2 h at RT. All volatiles were removed in vacuo. The residue was redissolved in water and extracted to dichloromethane. The organic phase was dried over Na₂SO₄ and concentrated. Purification on silica gel (eluent 10% MeOH in CH₂Cl₂ , v/v) yielded the title compound. ¹H NMR (CDCl₃): δ 7.55 (IH, d, J 3.8); 6.90 (IH, d, J 3.8); 3.91 (2H, br q); 3.11 (2H, t, J 6.0); 2.52 (3H, s); 1.82 (lH, br t, / 5.7).

Example 14

Synthesis of 2-(5-acetyl-2-thienyl)ethyl 4-methylbenzenesulfonate 14

Compound 13 (1.0 g, 5.87 mmol) was dissolved in dry pyridine (10 mL) at 0 ⁰C. tosyl chloride (1.68 g, 8,81 mmol) was added in small portions during 10 min, and the reaction was allowed to proceed in the same temperature for 2 h. The mixture was poured into 20 mL of ice-water, and allowed to stirr for 1.5 h. The product was extracted to ethyl acetate. The organic layer was dried over Na₂SO₄ and concentrated. Purification on silica gel (eluent 10% MeOH in CH₂Cl₂ , v/v) yielded 1.5 g of the title compound ¹H NMR (CDCl₃): δ 7.74 (2H, d, J 8.3); 7.50 (IH, d, J 3.8); 7.31 (2H, d, J 8.3); 6.86 (IH, d J 3.8); 4.24 (2H, t, J 6.4); 3.18 (2H, t, J 6.4); 2.51 (3H, s); 2.44 (3H, s).

Example 15 Synthesis of the β-diketone 15

Compound 14 (1.2 g, 3.7 mmol) was dissolved in dry toluene (40 mL). Sodium hydride (0.18 g, 40% dispersion in oil) was added, and the mixture was stirred for 15 min at rt. Ethyl pentafluoropropionate (1.1 niL, 7.4 mmol) was added, and the mixture was stirred overnight at rt. Hydrochloric acid (IM, 40 mL) and dichloromethane were added, and the mixure was stirred for 10 min.The organic phase was separated, and the aqueus phase was extracted twice with dichloromethane. The combined organic phases were dried and concentrated. . Purification on silica gel (eluent 10% MeOH in CH₂Cl₂, v/v) yielded the title compound. ¹H NMR (CDCl₃): δ 7.71 (2H, d, J 7.9); 7.36 (IH, d, J 3.8); 7.30 (2H, d, J 7.9); 6.73 (IH, d, J 3.4); 6.06 (IH, s); 4.21 (2H, t, / 6.5); 3.12 (2H, m); 2.42 (3H, s).

Example 16 Synthesis of the ligand 16

Compound 16 (60 mg, 0.22 mmol) and 1,4,7-triazacyclononane (9.6 mg, 0.07 mmol) were disscolved in dry acetonitrile (5 mL). K₂CO₃ was added, and the mixture was stirred overnight at reflux under argon. All volatiles were removed in vacuo. The residue was suspended in ethyl acetate and washed with 0.1 M aqueous HCl. The organic layer was dried over molecular sieves and concentrated to give the title compound ESI-TOF MS: found, 1024.21; calcd for C₃₉H₃₇N₃O₆S₃ ⁺ 1024.15.

Example 17 Synthesis of the europium(III) chelate 17

Compound 16 (6 mg, 5.9 μmol) was dissolved in the mixture of DMF and water. EuCl₃ ^• 6H₂O (2.2 mg, 6.0 μmol) was added, and the mixture was stirred for 2 h at rt and concentrated. Ex_max 345 nm

Photophysical properties of chelates according to this invention are shown in Table 1. The photophysical properties of the chelates were determined by measuring excitation and emission spectra and fluorescence lifetime in TS buffer (50 mM tris, 150 mM NaCl, pH 7.75) with LS-55 luminescence spectrometer (PerkinElmer Instruments, Connecticut, USA). Table 1. Photophysical properties of the chelates synthesized

n.d. = not determined The results can be summarized as follows: the acyclic analogues comprising several β-diketone subunits are practically nonluminescent in the absence of an additional chelator. the cyclic analogues comprising several β-diketone subunits (i.e. those based on azacycloalkane backbone) are luminescent even in the absense of an additional chelator. - the excitation maximum can be adjusted by changing the structure of the β-diketone subunit.

It will be appreciated that the methods of the present invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. It will be apparent for the expert skilled in the field that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive. It is known for a skilled person that β-diketones exhibit keto-enol tautomerism. Accordingly, it is obvious that the present disclosure includes both tautomers although only enol forms are generally presented in formulas and schemes.

Scheme 1

Scheme 2

Scheme 3

DIPEA EtOH

Scheme 4

Scheme 5

Scheme 6

Scheme 7

Scheme 8

Claims

1. A luminescent lanthanide(III) chelate comprising at least three β-diketone subunits, and an azacycloalkane backbone.

2. The luminescent lanthanide(III) chelate according to claim 1 wherein said azacycloalkane comprises at least three nitrogen atoms wherein said nitrogen atoms being substituted with a β- diketone group R(C=O)CH₂(C=O)ArL¹-, and wherein one of the nitrogen atoms or one of the carbon atoms of said azacycloalkane is substituted with a group of G-L²-, and wherein (i) L¹ is a linker between Ar and said azacycloalkane, and comprises one to three moieties, each moiety being selected from an alkylene containing 1-3 carbon atoms, ether (-O-), thioether (-S-), amide (-C0-NH-, -CO-NR -, NH-CO and -NR -CO-), carbonyl (-CO-), and ester (-C00- and -

0OC-), wherein R' represents an alkyl group containing less than 5 carbon atoms,

(ii) L² is a linker between G and said azacycloalkane, and comprises one to ten moieties, each moiety being independently selected from the group consisting of phenylene, alkylene containing 1-12 carbon atoms, ethynydiyl (-C≡C-), ethylenediyl (-C=C-), ether (-0-), thioether (- S-), amide (-C0-NH-, -CO-NR -, NH-CO and -NR -CO-), carbonyl (-CO-), ester (-COO- and - 0OC-), disulfide (-SS-), sulfonyl (-SO₂-), diaza (-N=N-), and tertiary amine, wherein R' represents an alkyl group containing less than 5 carbon atoms, (iii) G is a reactive group and is selected from the group consisting of isothiocyanate, bromoacetamido, iodoacetamido, maleimido, 4,6-dichloro-l,3,5-triazinyl-2-amino, pyridyldithio, thioester, aminooxy, hydrazide, amino, azido, alkyne, a polymerizing group, carboxylic acid, carboxylic acid halide, active ester of carboxylic acid, and

wherein A is cleaving ligand comprising Cl, (CHs)₂SO, H₂O, and NO3^" and wherein - is the position of linker L²,

(iv) Ar is an aryl group, optionally mono or multisubstituted,

(v) R is a straight or branched alkyl chain with 1 to 9 carbon atoms substituted with at least three fluoride atoms and optionally with other substituents than fluorine.

3. The luminescent lanthanide chelate according to claim 2 wherein said Ar is selected from the group consisting of phenyl, 9H-fluoren-2-yl, 1-naphthyl, 2-naphthyl, 2-phenanthroyl, 2-furyl, 3- furyl, 2-benzofuryl, 3-benzofuryl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2- thiazolyl, 4-thiazolyl, 2-benzothiazolyl, 2-benzo[a]thienyl, 3-benzo[a]thienyl, 2-pyrimidyl, A- pyrimidyl and 5-pyrimidyl.

4. The luminescent lanthanide chelate according to claims 2 and 3 wherein said Ar is mono- or multisubstituted, and wherein each substituent independently selected from alkyl, alkoxy, aryl, aroyl, aryloxy, nitro, amino, cyano, hydroxyl, carboxy, chloro, bromo, fluoro and acyl.

5. The luminescent lanthanide chelate according to claim 2 wherein R is substituted with 3 to 9 fluorine atoms.

6. The luminescent lanthanide chelate according to claim 2 wherein R is mono- or multisubstituted with other substituents than fluorine and each substituent is independently selected from a group consisting of alkyl, alkoxy, aryl, aroyl, aryloxy, nitro, amino, cyano, hydroxyl, carboxy, chloro, bromo, fluoro and acyl.

7. The luminescent lanthanide chelate according to claim 2, wherein R(C=O)CH₂(C=O)Ar- is selected from a group consisting of 4,4,4-trifluoro-l-aryl-l,3-butanedione, 4,4,5, 5,5-pentafluoro- l-aryl-l,3-pentanedione and 4,4,5,5,6,6-heptafluoro-l-aryl-l,3-hexanedione, wherein aryl is selected from thienyl, furyl and phenyl.

8. The luminescent lanthanide chelate according to claim 1 wherein said azacycloalkane is selected from a group consisting of 1,4,7-triazacyclononane, 1,4,7-triazacyclodecane, 1,5,9- triazacyclododecane, cyclen and cyclam.

9. The luminescent lanthanide chelate according claim 2 wherein the chelate is selected from a group consisting of

L² comprises one to ten moieties, each moiety being independently selected from the group consisting of phenylene, alkylene containing 1-12 carbon atoms, ethynydiyl (-C≡C-), ethylenediyl (-C=C-), ether (-0-), thioether (-S-), amide (-C0-NH-, -CO-NR'-, NH-CO and - NR'-CO-), carbonyl (-CO-), ester (-C00- and -00C-), disulfide (-SS-), sulfonyl (-SO₂-), diaza (-N=N-), and tertiary amine, wherein R' represents an alkyl group containing less than 5 carbon atoms;

10. A bioactive complex comprising a bioactive molecule conjugated with a chelate according to any of the claims 1-9.

11. The bioactive complex according to claim 10, wherein said bioactive molecule is selected from the group consisting of an oligopeptide, oligonucleotide, DNA, RNA, modified oligo- or polynucleotide, protein, oligosaccaride, polysaccaride, phospholipide, PNA, LNA, antibody, steroid, hapten, drug, receptor binding ligand and lectine.

12. The bioactive complex according to claim 10 conjugated to a solid support.

13. A solid support conjugated with a chelate according to any of the claims 1-9.