WO2008025886A1

WO2008025886A1 - Metal chelates and chelating agents containing triazolyl subunits

Info

Publication number: WO2008025886A1
Application number: PCT/FI2007/050469
Authority: WO
Inventors: Jari Hovinen
Original assignee: Wallac Oy
Priority date: 2006-09-01
Filing date: 2007-08-31
Publication date: 2008-03-06

Abstract

The invention relates to novel chelating agents and metal chelates containing 1,2,3-triazolyl subunits, and their conjugates with biomolecules or particles. The invention concerns also a process for the preparation of the novel chelatingagents and chelates.

Description

1

METAL CHELATES AND CHELATING AGENTS CONTAINING TRIAZOLYL SUBUNITS

FIELD OF THE INVENTION

This invention relates to metal chelates, metal chelating agents, par- 5 tides and bioactive molecules containing 1 ,2,3-triazolyl subunits.

BACKGROUND OF THE INVENTION

The copper(l) catalyzed Huisgen's dipolar [2+3] cycloaddition of azide and alkynes is a powerful direct method to prepare 1 ,4-disubstituted 1 ,2,3-triazoles [KoIb. H.C.Sharpless, K.B., 2001 , Angew. Chem. Int. Ed. Engl.

10 40, 2004]. This reaction has been used increasingly in modular drug development and in the preparation of small-molecule radiopharmaceuticals, in DNA sequencing [KoIb, H.C., Sharpless, K.B., 2003, Drug Discov. Today, 8, 1128, Seo, T.S., Bai, X., Li, Z., Ruparel, H., Turro, N., Ju, 2004, Prog. Natl. Acad. Sci. USA, 101 , 5488] as well as for conjugation of various label molecules to

15 nanomaterials [Joralemon, M.J., O^'Reilly, R.K., Hawker, G.J., Wooley, K.L., 2005, J. Am. Chem. Soc, 127, 16892, Sun, E.Y., Josephson, L., Weissleder, R. 2006, MoI. Imaging 5, 122] and biomolecules [Agard, N.J., Prescher, J.A., Bertozzi, C.R., 2004, J.Am. Chem. Soc, 126, 15046]. The reaction has also been exploited in the preparation of peptidomimetics [Arosio, D., Bertoli, M.,

20 Manzoni, L., Scoiastico, C₁ 2006, Tetrahedron Lett., 47, 3697, and various organic molecules [Yap, A.H., Weinreb, S. M., 2006, Tetrahedron Lett., 47, 3035, Molander, G.A, Ham, J., 2006, Org. Lett, 8, 2767].

The labels used in works based on click chemistry are organic dyes. However, the organic fluorophores as labels have several drawbacks, such as

25 Raman and Raleygh scattering, low water solubility and concentration quenching. Thus multilabeling with organic fluorophores may not enhance detection sensitivity to the degree needed in several applications. Furthermore, they may decrease the the water solubility of the target molecules dramatically.

Time-resolved fluorometry exploits the unique fluorescence

30 properties of lanthanide(lll) chelates. The long fluorescence decay after excitation of these molecules allows time-delayed signal detection. This eliminates background signal originating e.g. from microplates or buffer components. The large Stokes shift (i.e. the difference in the chelate's excitation and emission lines), in turn, results in a high sϊgnal-to-background ratio. These unique prop-

35 erties of lanthanide(lll) chelates can be exploited particularity in homogenous assays, when the use of conventional chromophores causes very high background. The different photochemical properties of europium, terbium, dysprosium and samarium chelates enable even development of multi- parametric homogenous assays [Hemmila, L; Mukkala, V.-M. 2001 , Crit. Rev. 5 Clin. Lab. Sci. 441]. Furthermore, lanthanide(lll) chelates are in most cases readily soluble in water. Accordingly, a number of attempts have been made to develop highly luminescent chelate labels suitable for time-resolved fluoromet- ric applications. These include e.g. stabile chelates composed of derivatives of pyridines [US 4,920,195, US 4,801 ,722, US 4,761 ,481 , PCT/FI91 /00373,

10 US 4,459,186, EP A-0770610, Remuinan et al, J. Chem. Soc. Perkin Trans 2, 1993, 1099], bipyridines [US 5,216,134], terpyridines [US 4,859,777, US 5,202,423, US 5,324,825, Wang, Z., Yan, J., Matsumoto, K., 2005, Luminescence, 20, 347] or various phenolic compounds [US 4,670,572, US 4,794,191 , ltal Pat. 42508 A789] as the energy mediating groups and polycarboxylic acids

15 as chelating parts. In addition, various dicarboxylate derivatives [US 5,032,677, US 5,055,578, US 4,772,563] macrocyclic cryptates [US 4,927,923, WO 93/5049, EP-A-493745] and macrocyclic Schiff bases [EP-A-369-000] have been disclosed. Also a method for the labelling of biospecific binding reactant such as hapten, a peptide, a receptor ligand, a drug or PNA oligomer with luminescent

20 labels by using solid-phase synthesis has been published [US 6,080,839]. Similar strategy has also been exploited in multilabeling of oligonucleotides on solid phase [US 6,949,639].

Several beads containing lanthanide(NI) chelates as dye molecules have been prepared, most comonly simply by swolling chelates into the poly-

25 mer [Cummins, CM., Koivunen, M. E., Stephanian, A., Gee, S.J., Hammock, B. D., Kennedy, I. M., 2006, Biocensors and Bioelectronics, 21 , 1077]. Since the lanthanide(lll) chelates are not covalently bound to polymer particle, the signal obtained from the particle may decrease as the function of time because of leaking. This problem can be avoided by linking the chelate covalently to the

30 matrix. Indeed, this type of silica [Hai, X., Tan, M., Wang, G., Ye, Z., Yuan, J., Matsumoto, K., 2004, Anal. Sci., 20, 245] and polystyrene [Hakala, H., Sutela, T., Mukkala, V.-M., Hovinen, J., 2006, Org. Biomol. Chem., 1383] based nano- bead has recently been published. However, this strategy requires the use of hydrophilic polymerizable chelates. No methods to prepare particles covalently

35 bound to hydrophilic label molecules have been disclosed. OBJECTS AND SUMMARY OF THE INVENTION

The main object of the present invention is to provide chelating agents containing a 1 ,2,3-triazolyl subunit and metal chelates thereof, useful for labeling biomolecules and particles for use as probes in time resolved fluo- 5 rescence spectroscopy, magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT) or positron emission tomography (PET).

A particular object of this invention is to provide chelating agent containing a 1 ,2,3-triazolyl subunit which give a very strong fluorescense with dif- 10 ferent chelated lanthanide ions, particularly with europium (III), samarium (III), terbium (III) and dysprosium (III). Such lanthanide chelates are especially useful in multiparameter bioaffinity assays and in high-throughput screening of drug candidates.

A further object of this invention is to provide chelating agents con- 15 taining a 1 ,2,3-triazolyl subunit giving rise to metal chelates of high stability. A particular object is to achieve chelates with strong stability enough for use in in vivo applications, for example in MRI, SPECT or PET applications.

A further object is to provide chelates or chelating agents suitable for labeling of biomolecules or particles in solution exploiting Huisgen's 20 cycloaddition reaction.

Yet another object is to provide chelating agents containing a 1 ,2,3- triazolyl subunit suitable for labeling oligopeptides or oligonucleotides simultaneously with their synthesis on a solid phase.

Yet another object is to provide a solid support conjugated with che- 25 lates, chelating agents or biomolecules according to this invention.

Yet another object is to provide biomolecules and particles conjugated with chelates and chelating agents of this invention.

Thus, according to one aspect this invention concerns an A lanthanide chelate or chelating agent, comprising a 1 ,2,3-triazole subunit, wherein 30 said chelate or chelating agent is represented by formula (I)

( 1) wherein 4

A is a reactive group or not present, L is a linker or not present, group G comprises a chelating group having at least two carboxylic acid or phosphonic acid groups, or esters or salts of said acids, and a chromophoric moiety having one or more aromatic units, and G optionally includes a che-

5 lated lanthanide ion Ln³⁺, characterized in that

(i) in the group G

- the chelating part is attached to an aromatic unit of the chromophoric moiety either directly or via an Λ/-containing hydrocarbon chain,

- the aromatic units are tethered to each other either directly or via 10 N-containtng hydrocarbon chain and

- at least one of the aromatic units are directly tethered to the 1 ,2,3- triazole subunit,

(ii) the linker L is formed from one to ten moieties, each moiety being selected from the group consisting of phenylene, alkylene containing 1-12 15 carbon atoms, ethynydiyl (-C=C-), ethylenediyl (-C=C-), ether (-O-), thioether (-S-), amide (-CO-NH-, -CO-NR^'-, -NH-CO- and -N FT-CO-), carbonyl (-CO-), ester {-COO- and -OOC-), disulfide (-SS-), diaza (-N=N-), and tertiary amine, wherein R^' represents an alkyl group containing less than 5 carbon atoms,

(iii) the reactive group A is selected from the group consisting of 20 isothiocyanate, haloacetamido, maleimido, dichlorotriazinyl, pyridyldithio, thio- ester, aminooxy, hydrazide, amino, a polymerizing group, and a carboxylic acid or an acid halide or an active ester thereof,

(iv) the lanthanide ion is selected from europium(lll), terbium(lll), samarium(lll) and dysprosium(IM).

25 Another aspect of this inventions invention concerns chelating agent, comprising a 1 ,2,3-triazole subunit, wherein said chelate or chelating agent is represented by formula (I), characterized in that the reactive group A is an amino acid residue -CH(N HR¹ )R⁵ where R¹ is a transient protecting group and R⁵ is a carboxylic acid or its salt, acid halide or an ester. 30 Yet another aspect of this inventions invention concerns chelating agent, comprising a 1 ,2,3-triazole subunit, wherein said chelate or chelating agent is represented by formula (I), characterized in that the reactive group A is

-Y-O-PZ¹-O-R⁴

35 where one of the oxygen atoms optionally is replaced by sulfur,

Z¹ is chloro or NR²R³ R⁴ is a protecting group, R² and R³ are alkyl groups,

Y is absent or is a radical of a purine base or a pyrimidine base or any other modified base suitable for use in the synthesis of modified oligonu- 5 cleotides, said base being connected to the oxygen atom via either a) a hydrocarbon chain, which is substituted with a protected hy- droxyethyl group, or via b) a furan ring or pyrane ring or any modified furan or pyrane ring, suitable for use in the synthesis of modified oligonucleotides.

10 According to another aspect, this invention concerns a method for the preparation of the agent as defined above, wherein i) a group A-L¹= is reacted with a group G-l_²-N₃, where A, G, L¹ and L² are as defined above, to give a compound of formula (I), where A-L¹- is bound to a carbon atom and G-L²- is bound to the nitrogen atom in the triazole 15 subunit, or ii) a group A-L¹-N3 is reacted with a group G-L²=, where A, G, L¹ and L² are as defined above, to give a compound of formula (I), where A-L¹- is bound to the nitrogen atom and G-L²- is bound to a carbon atom in the triazole subunit.

20 BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows emission and excitation spectrum of a luminescent europium(lll) chelate 11a (A) and a luminescent terbium(lll) chelate 11b (B) synthesized according to this invention.

DETAILED DESCRIPTION OF THE INVENTION

25 According to one embodiment, the chelating part in G is attached to the said triazole subunit either (a) via a linker L² directly or via an /V-containing hydrocarbon chain, or (b) to an aromatic unit of the chromophoric moiety, either directly or via an Λ/-containing hydrocarbon chain.

According to another embodiment, the invention concerns a chelate

30 comprising

- a metal ion,

- a chelating part comprising at least two carboxylic acid or phos- phonic acid groups, or esters or salts of said acids, attached either to an aromatic unit of the chromophoric moiety, either directly or via an Λ/-containing 6

hydrocarbon chain, or where the chelating part is attached directly or via an N- containing hydrocarbon chain to the 1 ,2,3-triazole subunit and

- a group A, tethered to the triazole subunit optionally via a linker L¹, said group A being either a reactive group, enabling binding to a biomolecule

5 or to a functional group on a solid phase, or said group A being a radical of a a bioactive molecule or a particle, and

- an optional chromophoric moiety comprising one or more aromatic units.

According to a third embodiment, the invention concerns a particle 10 conjugated with a chelate or a labeled biomolecule according to this invention. According to a fourth embodiment, this invention concerns a labeled oligopeptide, obtained by synthesis on a solid phase, by introduction of an appropriate chelating agent according to this invention into the oligopeptide structure on an oligopeptide synthesizer, followed by deprotection and optionally al- 15 so introduction of a metal ion.

According to a fifth embodiment, this invention concerns a labeled oligonucleotide, obtained by synthesis on a solid phase, by introduction of an appropriate chelating agent according to this invention into the oligonucleotide structure on an oligonucleotide synthesizer, followed by deprotection and op-

20 tionally also introduction of a metal ion.

According to an sixth embodiment, this invention concerns a particle conjugated with the chelating agent according to this invention suitable for use in the synthesis of an oligonucleotide or an oligopeotide.

According to a seventh embodiment, this invention concerns a che- 25 lating agent containing a 1 ,2,3-triazolyl subunit and comprising

- an optional chromophoric moiety, and

- a chelating part comprising at least two carboxylic acid or phos- phonic acid groups, or esters or salts of said acids, attached to an aromatic unit of the chromophoric moiety, either directly or via an Λ/-containing hydro-

30 carbon chain, wherein the aromatic units are tethered to each other via N-containing hydrocarbon chains.

According to an eighth embodiment, this invention concerns bioactive molecules and particles conjugated with the chelating agents or chelates 35 of this invention. Finally, the invention concerns also a chelate comprising the aforementioned chelating agent and a metal ion.

Chelating agents

Chelating agents and metal chelates comprising a 1 ,2,3-triazole 5 subunit are new. Furthermore, no chelating agents, metal chelates, bϊoactive molecules or particles tethered to metal chelates containing triazole subunits have been disclosed.

The triazole ring can be conjugated directly without any linker L² to the chromophoric moiety, and thus it is part of the aromatic moiety capable of

10 absorbing light or energy and transferring the excitation energy to the chelated lanthanide ion. fn addition to the triazole subunit, the chromophoric unit may comprise unsubstituted pyridyl groups, pyridyl groups bearing other substitu- ents and/or other aromatic groups.

Alternatively, the 1 ,2,3-triazole subunit is tethered to a linker arm L².

15 The agent can be formed by a reaction between a chelating agent or a metal chelate tethered to an alkynyl function and a bioactive molecule, a linker molecule or a particle tethered to an azide function. Alternatively, the agent can be formed by a reaction between a chelating agent or a metal chelate tethered to an azide function and a bioactive molecule, a linker molecule

20 or a particle tethered to an alkynyl function. it is well established that Cu(I) catalyzed Huisgen's cycloaddition occurs readily only with primary alkynes. However, the reaction can be performed with non-terminal alkynes using certain organometallic catalysts such as Ru-complexes [Zhang, L., Chen, X., Xue, P., Sun, H.H.Y., Williams, I.D.,

25 Sharpless, K.B., Fokin, V. V., Jia, G., 2005, J. Am. Chem. Soc, 127, 15998]. Furthermore, if the non-terminal alkynes are sterically restricted, the reaction proceeds even without a catalyst [Agard, N.J., Prescher, J.A., Bertozzi, C.R., 2004, J.Am. Chem. Soc, 126, 15046].

The cycloaddition reaction between alkynes and azides catalyzed

30 by Cu⁺ and Ru(AcO)₂(PPh₃J₂ gives 1 ,4-dϊsubstituted 1 ,2,3-triazoles. The 1 ,5- disubstituted 1 ,2,3-triazoles, in turn, can be prepared using other type of Ru- based catalysts, such as Cp*RuCI(PPh₃)₂ [Zhang, L., Chen, X., Xue, P., Sun, H.H.Y., Williams, I.D., Sharpless, K.B, Fokin, V.V., Jia, G., 2005, J. Am. Chem. Soc, 127, 15998]. Uncatalyzed cycloaddition gives rise to isomeric mix-

35 tures. 8

In the compounds demonstrated by specific examples herein, the 1 ,4-substituted 1 ,2,3-triazole derivatives have been synthesized. Although this position is believed to be the most preferable, other positions may also be useful for substitution.

5 Although the reactive group A in principle in many applications could be attached directly to the chromophoric group or to the chelating part, it is highly desirable, especially for steric reasons, to have a linker L¹ between the reactive group A and the triazole subunit and chromophoric group or chelating part, respectively. The linker is especially important in case the chelate

10 shall be used in solid phase syntheses of oligopeptides and oligonucleotides, but it is desirable also in labelling biomolecules in solution.

According to a preferable embodiment, the reactive group A is selected from the group consisting of isothiocyanate, haloacetamido, maleimido, dichlorotriazinyl, pyridyldithio, thϊoester, aminooxy, hydrazide, amino, a po-

15 lymerizing group, a carboxylic acid or acid halide or an active ester thereof. Particularly in case the chelate or chelating agent shall be attached to mi- croparticle or nanoparticle it is preferable to have a reactive group which is a polymerizing group. In this case the label can be introduced in the particle during the manufacturing of the particles.

20 The linkers are preferably formed from 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 (-COO- and -OOC-), disulfide (-SS-), diaza (-N=N-), and tertiary

25 amine, wherein R^' represents an alkyl group containing [ess than 5 carbon atoms. In this case one of the linkers L¹ and L² can also be missing.

According to a particularly preferable embodiment, G is one of the following specific structures:

v wherein Z is independently selected from furyl, thienyl, phenyl or phenylethynyl, where phenyl is substituted or unsubstitued, or Z is not present, wherein G is a radical of any of the aforementioned structures and tethered to 5 the triazole subunit or to a linker L² which in turn is tethered to the triazole sub- unit.

In case Z is substituted phenyl, said phenyl is preferably trial koxy- substituted, most preferably trimethoxysubstituted. 10

Chelating agents for use in peptide synthesis

According to one preferred embodiment, the chelating agent according to this invention is suitable for use in the synthesis of an oligopeptide. In this application, the reactive group A is connected to the triazole subunit via 5 a linker L¹, and A is an amino acid residue -CH(NHR¹JR⁵ where R¹ is a transient protecting group and R⁵ is a carboxylic acid or its salt, acid halide or an ester. Particularly preferable chelating agents where G is selected from the structures

11

W

wherein Z is independently selected from furyl, thienyl, phenyl or phenylethynyf, where phenyl is substituted or unsubstitued or Z is not present, wherein G is a radical of any of the aforementioned structures and tethered to 5 the triazole subunit or to a linker L² which in turn is tethered to the triazole sub- unit.

Herein L² is as defined before and the protecting group R¹ is selected from a group consisting of Fmoc (fluorenylmethoxycarbonyl), Boc (tert- 12

butyloxycarbonyl), or Bsmoc (1 ,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbon- yl), and R" is an alkyl ester or an allyl ester.

The chelating agent can be introduced into biomolecules with the aid of peptide synthesizer. The chelating agent can be coupled to an amino 5 tethered solid support or immobilized amino acid e.g. by carbodiimide chemistry described in Jones, J., The Chemical Synthesis of Peptides, Oxford Unive- sity Press, Oxford, 1994, (i.e. the carboxylic acid function of the labeling reagent reacts with the amino group of the solid support or amino acid in the presence of an activator). When the condensation step is completed the tran-

10 sient amino protecting group of the labeling reagent is selectively removed while the material is still attached to the solid support (e.g with piperidine in the case of Fmoc-protecting group). Then second coupling of a chelating agent or other reagent (amino acid, hapten) is performed as above. When the synthesis of the desired molecule is completed, the material is detached from the solid

15 support and deprotected. Purification can be performed by HPLC techniques. Finally the purified ligand is converted to the corresponding metal chelate by addition of known amount of metal ion.

Chelating agents for use in oligonucleotide synthesis

According to another preferred embodiment, the chelating agent ac- 20 cording to this invention is suitable for use in the synthesis of an oligonucleotide. In this case the reactive group A is connected to the triazoie subunit via a linker L¹, and A is

-Y-O-PZ¹-O-R⁴ where one of the oxygen atoms optionally is replaced by sulfur, Z¹ is 25 chloro or NR²R³, R⁴ is a protecting group, R² and R³ are alkyl groups, and Y is absent or is a radical of a purine base or a pyrimidine base or any other modified base suitable for use in the synthesis of modified oligonucleotides. Said base is connected to the oxygen atom either via i) a hydrocarbon chain, which is substituted with a protected hydroxyethyl group, or via ii) a furan ring or 30 pyrane ring or any modified furan or pyrane ring, suitable for use in the synthesis of modified oligonucleotides.

The chelating agent can be introduced into oligonucleotides with the aid of oligonucleotide synthesizer. A useful method, based on a Mitsonobu al- kylation (J Org Chem, 1999, 64, 5083; Nucleosides, Nucleotides, 1999, 18,

35 1339) is disclosed in EP-A- 1152010. Said patent publication discloses a method for direct attachment of a desired number of conjugate groups to the 13

oligonucleotide structure during chain assembly. Thus solution phase labeling and laborious purification procedures are avoided. The key reaction in the synthesis strategy towards nucleosidic oligonucleotide building blocks is the aforementioned Mitsunobu alkylation which allows introduction of various chelating 5 agents to the nucleoside, and finally to the oligonucleotide structure. The chelating agents are introduced during the chain assembly. Conversion to the lan- thanide chelate takes place after the synthesis during the deprotection steps.

Normal, unmodified oligonucleotides have low stability under physiological conditions because of its degradation by enzymes present in the

10 living cell. It may therefore desirable to create a modified oligonucleotide according to known methods so as to enhance its stability against chemical and enzymatic degradation. Modifications of oligonucleotides are extensively disclosed in prior art. Reference is made to US 5,612,215. It is known that removal or replacement of the 2'-OH group from the ribose unit in an RNA chain

15 gives a better stability. WO 92/07065 and US 5,672,695 discloses the replacement of the ribose 2'-OH group with halo, amino, azido or sulfhydryl groups. US 5,334,711 discloses the replacement of hydrogen in the 2'-OH group by alkyl or alkenyl, preferably methyl or allyl groups. Furthermore, the in- ternucleotidic phosphodiester linkage can, for example, be modified so that

20 one ore more oxygen is replaced by sulfur, amino, alkyl or aikoxy groups. Preferable modification in the internucleotide linkages are phosphorothioate linkages. Also the base in the nucleotides can be modified.

Preferably Y is a radical of any of the bases thymine, uracil, adenine, guanine, cytosine, 7-deazaadenine or 7-deazaguanine, and said base is

25 connected to the oxygen atom via i) a hydrocarbon chain, which is substituted with a protected hydroxyethyl group, or via ii) a furan ring having a protected hydroxyethyl group in its 4-position and optionally a hydroxy!, protected hydroxy! or modified hydroxyl group in its 2-position.

Preferably a reactive group -Y-O-P(NR²R³J-O-R⁴ has a structure se-

30 lected from one of the following structures: 14

O O

KJ ,JsJ

DMTrO. I DMTrα I

DMTrd O V^ W>

ay X⁾ JJ / ^CH3

^DMTrO 0 DMTrO ^ DMTrd ^ ^DMTr0 ?

(HTfeN-'V^™ (H^N-^α^^¹ (/-Pr)₂N'^O'^'^CN {^faN^'lV^^ΛN

where - is the position of the linker L¹ and DMTr is 4,4' dimethoxytrityl.

15

A particularly preferable chelating agent for this use is selected from one of the specific structures disclosed below

W ^W

R where R" is an alkyl ester or an ally! ester. 16

Chelates

The chelates comprise a chelating agent as describes above and a chelated metal ion.

In case the chelate is to be used in bioaffinity assays, the chelated metal ion M is preferably a lanthanide, especially europium(lll), samarium(lll), terbiυm(lll) or dysprosium(lll). G in the chelating agent is preferably one of the following:

17

wherein M is metal and Z is independently selected from furyl, thie- nyl, phenyl or phenylethynyl, where phenyl is substituted or unsubstitued or Z is not present, wherein G is a radical of any of the aforementioned structures and tethered to the triazole subunit or to a linker L² which in turn is tethered to

5 the triazole subunit.

In case the chelate is to be used in bioaffinity assays, the chelated metal ion M is preferably a lanthanide, especially europium(lll), samarium(lll), 10 terbium(lll) or dysprosium(lll). The chelating agent is preferably one of the preferable agents mentioned above.

The chelates according to this invention can also be used in vivo in MRI applications or in PET applications. A preferable metal to be used in MRI is gadolinium. However, also lanthanides, particularly europium (III), but also 15 other lanthanides such as samarium (III) and dysprosium (III) are useful in MRI appiications. In SPECT applications the most suitable isotyopes are Tc-98m and ln-111. In PET applications a radioactive metal isotope is introduced into the chelating agent just before use. Particularly suitable radioactive isotopes are Ga-66, Ga-67, Ga-68, Cr-51 , ln-111 , Y-90, Ho-166, Sm-153, Lu-177, Er- 20 169, Tb-161 , Dy-165, Ho-166, Ce-134, Nd-140, Eu-157, Er-165, Ho-161 , Eu- 147, Tm-167 and Co-57. In order to obtain very stable chelates, it is preferable to have a chromophoric moiety where there are several pyridyl groups tethered to each other via N-containing hydrocarbon chains.

According to a particularly preferable embodiment, the chelate is 25 one of the following specific structures:

18

Biomolecules

The biomolecule conjugated with a chelating agent or a chelate according to this invention is preferably an oligopeptide, oligonucleotide, nucleoside, nucleotide, nucleoside 5 ^'-triphosphate, DNA, RNA, modified oligo- or polynucleotide, such as phosphoromonothioate, phosphorodithioate, phos- 19

phoroamidate and/or sugar- or base modified oligo- or polynucleotide, protein, oligosaccaride, polysaccaride, phospholipide, PNA, LNA, antibody, hapten, drug, receptor binding ligand and lectine.

Particle conjugates

5 The chelates, chelating agents and biomolecules according to this invention may be conjugated on a particle. The particle is preferably a particle such as a microparticle or nanoparticle, a slide or a plate.

In case the chelate or chelating agent has a polymerizing group as reactive group, then the chelate or chelating agent may be introduced in the

10 particle simultaneously with the preparation of the particles.

The biomolecule conjugated with the particle, either covalently or noncovalently is preferable a labeled oligopeptide, obtained by synthesis on a solid phase, by introduction of a chelating agent into the oligopeptide structure on an oligopeptide synthesizer, followed by deprotection and optionally intro-

15 duction of a metal ion. Alternatively, the biomolecule conjugated with the particle, either covalently or noncovalently is preferable a labeled oligonucleotide, obtained by synthesis on a solid phase, by introduction of a chelating agent into the oligonucleotide structure on an oligonucleotide synthesizer, followed by deprotection and optionally introduction of a metal ion.

20 The invention will be illuminated by the following non-restrictive Examples.

EXAMPLES

The invention is further elucidated by the following examples. The structures and synthetic routes employed in the experimental part are depicted

25 in Schemes 1-7. Scheme 1 illustrates the synthesis of the chelating agents containing 4-((3-hydroxypropyl)-1 tf-1 ,2,3-triazolyI subunits. The experimental details are given in Examples 1-8. Scheme 2 illustrates the synthesis of various cheating agents with based on pyridine-2,6-diyl)bis(methylenenitrilo)]- tetrakis(acetate).

30 Experimental details are given in Examples 9-11. Scheme 3 illustrates the synthesis of the building block designed for the introduction of lan- thanide chelates to the oligonucleotide structure on solid phase. Experimental details are given in Examples 12-14. Scheme 4 illustrates the synthesis of the lanthanide chelates tethered to alkynyl or azido functions. Experimental detals

35 are given in Examples 15 and 16. Scheme 5 demonstrates the method for co- 20

valent conjugation of azido tethered lanthanide(lll) chelates to alkynyl derivat- ized particles exploiting Huisgen's cycloaddition reaction. Scheme 6 demonstrates the labeling of 3^'-O-{5-azidopentyloxymethyl)thymidine 5^"-triphosphate [Hovinen, J., Azhayev, A., Azhayeva, E., Guzaev, A, Lόnnberg, H., 1994, J. 5 Chem. Soc. Perkin Trans 1 , 211] with the luminescent europium(lll) chelate tethered to an alkynyl group. Scheme 7 illustrates synthesis of lanthanide(lll) chelates where the 1 ,2,3-triazole subunit is part of the chromophoric moiety. Experimental details are given in Examples 17-19.

Experimental procedures

10 Adsorption column chromatography was performed on columns packed with silica gel 60 (Merck). NMR spectra were recorded either on a Brucker 250 or a Jeol LA-400 spectrometers operating at 250.13 and 399.8 MHz for ¹H, respectively. Me₄Si was used as an internal reference. Coupling constants are given in Hz. IR spectra were recorded on a Perkin Elmer 2000

15 FT-IR spectrophotometer. Electrospray mass spectra were recorded on a Applied Biosystems Mariner ESI-TOF instrument. Fluorescence spectra were recorded on a PerkinElmer LS 55 instrument. HPLC purifications were performed using a Shimazu LC 10 AT instrument equipped with a diode array detector, a fraction collector and a reversed phase column (LiChrocart 125-3 Purospher

20 RP-18e 5 μm). Mobile phase: (Buffer A): 0.02 M triethylammonium acetate (pH 7.0); (Buffer B): A in 50 % (v/v) acetonitrile. Gradient: from 0 to 1 min 95% A, from 1 to 21 min from 95% A to 100% B. Flow rate was 0.6 mL min^'1.

Example 1

The synthesis of tetra-terf-butyl 2,2^',2^",2^'"-{[4^'-(4^"-ethynylphenyl)- 25 2,2^':6^',2^"-terpyridine-6,6^"-diyl]bis(methylenenitrilo)}tetrakis (acetate) (1c).

Tetra-terf-butyl 2,2^',2^",2^'"-{[4^'-(4^"-bromophenyl)-2,2^':6^',2^"-terpyri- dine-6,6^"-diyl]bis(methylenenitrilo)}tetrakis (acetate) (0.71 g, 0.77 mmol), synthesized as disclosed in Peuralahti, J. Hakala, H., Mukkala, V.-M., Hurskainen, P., Mulari, O., Hovinen, J. 2002, Bioconjugate Chem., 13, 870, (Ph₃P)₂PdCI₂ 30 (13 mg) and CuI (8 mg) were dissolved in the mixture of THF (3 mL) and TEA (2 mL) an deaerated with argon. Trimethylsilyl acetylene (165 μL, 1.16 mmol) was added , and the mixture was heated overnight at 55 ⁰C, before being cooled to RT and concentrated. The residue was dissolved in dichloro- methane, washed with water and dried over Na₂SO₄, and concentrated. The 35 residue was redissolved in dichloromethane (25 mL). Tetrabutylammonium 21

fluoride (1.5 eq) was added, and the mixture was stirred for 30 min at RT, before being washed with 10% aq. citric acid, dried over Na₂SC>₄, and concentrated. Purification on silica gel (eluent PE:EA: TEA 3:5:1 (WWv)) yielded the title compound as an oil. ESI TOF MS for C₄₉H^N₅O₈ (M+H)⁺: calcd, 848.46; 5 found, 848.56.

Example 2

The synthesis of tetra-ferf-butyl-2,2^',2^",2^'"-{[6,6^'-[(4-ethynylpyra- zole-1 ^",3^"-diyl)bis(pyridine)-2,2^'-diyl]bis(methylenenitrilo)}tetrakis(acetate) 1d.

Tetra-terf-butyl-2,2^',2^",2^'"-{[6,6^'-[(4-ethynylpyrazole-1 ^",3^"-diyl)bis-

10 (pyridine)-2,2^'-diyl]bis(methylenenitrilo)}tetrakis(acetate), synthesized as disclosed in Jaakkola, L., Peuralahti, J., Hakala, H., Kunttu, J., Tallqvist, P., Muk- kala, V.-M., Hovinen, J. 2005, Bioconjugate Chem., 16, 700, was converted to the titled compound using the method disclosed in Example 1 but the reaction was performed at ambient temperature. ¹H NMR (CDCI₃): δ 8.84 (1H, s); 8.06

15 (1 H, d, J 7.4); 8.00 (1 H, d, J 8.0); 7.82 (1 H, t, 7.9); 7.78 (1 H, t, J 7.7); 7.71 (1 H, d, J 7.7); 7.55 (1 H, d, J 7.3); 4.16 (2H, s); 4.05 (2H, s); 3.56 (4H, s); 3.53 (4H, s); 3.25 (1 H, s); 1.48 (18H, s); 1.46 (18H, s). ESI TOF MS for C₄IH₅₇N₆O₈ (M+H)⁺: calcd, 761.42; found, 761 .51.

Example 3

20 The synthesis of 2,2^'-2^",2^'"-{[4^'-(5-ethynyl-2-furyl)-2,2^':6^',2^"-ter- pyridine-6,6^'-diyl]bis(methylenenitrilo)}tetrakis(acetate) tetra-fe/f-butyl ester, 1e. 2,2^'-2^" _I2^'"-{[4^'-(5-bromo-2-furyl)-2,2^':6^',2^"-terpyridine-6,6^'-diyl]- bis(methylenenitrilo)}tetrakis(acetate) tetra-terf-butyl ester, synthesized as disclosed in US 2005/0084451 A1 , was converted to the titled compound as dis-

25 closed in Example 1. ¹H NMR (CDCI₃) 8.71 (2H, s); 8.51 (2H, d, J 7.7); 7.86 (2H, t, J 7.9); 7.72 (2H, d, J 7.7); 7.11 (1 H, d, J 3.5); 6.82 (1 H, d, J 3.5); 4.19 (4H, s); 3.56 (8H, s); 3.53 (1 H, s); 1 .48 (36H, s). ESI TOF MS for C₄IH₅₇N₆O₈ (M+H)⁺: calcd, 838.44; found, 838.45.

Example 4

30 The synthesis of diethyl 4-(1 -(3-hydroxypropyl)-1 H-1 ,2,3-triazol-4- yl )pyrid ine-2 ,6-d icarboxylate, 2a.

Diethyl 4-ethynylpyridine-2,6-dicarboxylate, 1a, (0.34 g, 0.54 mmol), synthesized as disclosed in Takalo, H., Hanninen, E., Kankare, J., 1993, HeIv. Chim. Acta, 76, 877, and 3-azidopropan-1-ol (55 mg, 0.54 mmol) were dis- 22

solved in DMSO (2 ml_). CuI (11 mg, 0.05 mmol) was added, and the mixture was stirred at 60 C for 1 h before being concentrated in vacuo (oii pump). Purification on silica gel {eluent MeOH: CH₂Cb, 5:95, v/v) yielded the title compound. ¹H NMR (CDCI₃): δ 8.63 (2H, s); 8.27 (1H, s); 4.63 (2H₁ t, J 6.8); 4.45 5 (4H, q, J 7.0 ); 1.79 (2H, m); 1.41 (6H, t, J 7.0). ESI TOF MS for Ci₆H_2IN₄O₅ (M+H)⁺: calcd, 349.15; found, 349.15.

Example 5

The synthesis of tetra-tert-butyl 2,2^',2^",2^'"-[4-((3-hydroxypropyl)- 1 HA ,2,3-triazol-4-yl)pyridine-2,6-diyl)bis(methylenenitrilo)]tetrakis(acetate), 2b.

10 Reaction of tetra-terf-butyl 2,2^',2^",2^'"-[4-(ethynylpyridine-2,6-diyl)- bis(methylenenitrilo)]tetrakis(acetate), 1b, synthesized as disclosed in Han- ninen, E., Takalo, H., Kankare, J., 1988, Acta Chem. Scand. Ser B, 42, 614, with 3-azidopropan-1-ol as disclosed in Example 4 yielded the title compound. 1H NMR (CDCI₃): δ 8.08 (1 H₁ s); 7.95 82H, s); 4.59 (2H₁ 1, J 6.8); 4.06 (4H, s);

15 3.67 (2H, t, J 5.9); 3.51 (8H₁ s); 2.18 (2H, p); 1.45 (36H, s). ESI TOF MS for C₃₆H₅₉N₆O₉ (M+H)⁺: calcd, 719.43; found, 719.44.

Example 6

The synthesis of tetra-terf-butyl 2,2^',2^",2^'"-{[4^'-(4^"-[(1-(3-hydroxy- propyl)-1H-1 ,2,3-triazol-4-yl]phenyl)-2,2^':6^',2^"-terpyridine-6,6^"-diyl]bis(methy- 20 lenenitrilo)}tetrakis (acetate) (2c).

Reaction of compound 1c with 3-azidopropan-1-ol as disclosed in Example 4 yielded the title compound. ESI TOF MS for C₅₂H_6SN₈O₉ (M+H)⁺: calcd, 949.52; found, 949.55.

Example 7

25 The synthesis of tetra-terf-butyl-2,2^',2^",2^'"-{[6,6^'-[(4-(4-(1-(3-hyd- roxypropyl)-1H-1 ,2,3-triazol-4-yl)pyrazole-1 ^",3^"-diyl)bis(pyridine)-2,2^'-diyl]bis-

(methylenenitrilo)}tetrakis(acetate) 2d.

Reaction of compound 1d with 3-azidopropan-1-ol as disclosed in

Example 4 yielded the title compound. ESI TOF MS for C₄₄H₆₄N₉O₉ (M+H)⁺: 30 calcd, 862.48; found, 862.45. ¹H NMR (CDCI₃): δ 9.32 (1 H, s); 8.94 (1 H, s)

8.08 (1 H₁ d, J 8.0); 7.96 (1 H, d, J 7.9); 7.83 (1 H, t, J 7.4); 7.78 (1 H₁ t, J 7.7);

7.61 (1H, d, J 7.3); 7.53 (1H, d, J 7.6); 4.63 (2H, t, J 6.5); 4.22 (2H, s); 4.06

(2H, s); 3.70 (2H₁ 1, J 5.3); 3.53 (4H, s); 3.49 <4H, s); 2.22 (2H, m); 1.48 (18H, s);

1.46 (18H₁ S). 23

Example 8

The synthesis of tetra-terf-butyl 2,2^',2^",2^'"-{[4^'-(5-[(1-(3-hyciroxy- propyl)-1H-1 ,2,3-triazol-4-yl]-2-{furyl)-2_>2^':6^' _>2^"-terpyridine-6,6^"-diyl]bis- (methylenenitrilo)}tetrakis (acetate) (2e).

5 Reaction of compound 1e with 3-azidopropan-1-ol as disclosed in

Example 4 yielded the title compound. ¹H NMR (CDCI₃): 68.75 (2H, s); 8.53 (2H₁ d, J 7.9); 8.20 (1 H, s); 7.86 (2H, t, J 7.6); 7.67 (2H, d, J 7.6); 7.22 (1 H₁ d, J 3.5); 7.07 (1 H, d, J 3.5); 4.68 (2H₁ t, J 6.5); 4.21 (4H₁ s); 3.73 (2H, t, J 5.9); 3.59 {8H, s); 2.21 (2H₁ p); 1.46 (36H, s). ESI TOF MS for C₅₀H₆₇N₈Oi₀ (M+H)⁺: 10 calcd, 939.50; found, 939.49.

Example 9

The synthesis of tetra-ferf-butyl 2,2^',2^",2^'"-[4-((carboxyethyl)-1H- I ^.S-triazol^-yOJpyridine^.e-diylJbisfmethylenenitrilo^tetrakisfacetate), 3a.

Reaction of compound 1b with azidoacetic acid as disclosed in Ex- 15 ample 4 yielded the title compound. ¹H NMR (CDCI₃): δ 8.51 (1 H, s); 7.60 (2H, s); 4.76 (2H, s); 3.88 (4H, s); 3.39 (8H, s); 2.82 {2H, s); 1.44 (36 H, s). ESI TOF MS for C₃₅H₅₅N₆O₁₀ (M+H)⁺: calcd, 719.40; found, 719.48.

Example 10

The synthesis of tetra-terf-butyl 2,2^',2^",2^"'-[4-((3-aminopropyl)-1H- 20 1 ^.S-triazol^-yOJpyridine^.e-diyObistmethylenenitriloJltetrakisfacetate), 3b.

Reaction of compound 1b with 3-azidopropylamine as disclosed in Example 4 yielded the titfe compound. ESI TOF MS for C₃₆H_6ON₇O₈ (M+H)⁺: caicd, 718.45; found, 718.41.

Example 11

25 The synthesis of 4-{tetra-terf-butyl 2,2',2",2^'"-[4-(3-(1H-1 ,2,3-tri- azol-4-yl))pyridine-2,6-diyl)bis(methylenenitrilo)]tetrakis(acetato)} N-fluorenyl- methyloxycarbonyl phenylalanine, 3c.

Compound 1b was allowed to react with Λ/-fluorenylmethyl- oxycarbonyl 4-azidophenylalanine as disclosed in Example 4. When the reac-

30 tion was completed, all volatiles were removed in vacuo. The residue was dissolved in dichloromethane, washed with 10% aq. citric acid, dried over 4A molecular sieves and concentrated. ESI TOF MS for C₅₇H₇₂N₇Oi₂ (M+H)⁺: calcd, 1046.52; found, 1046.51. 24

Example 12

The synthesis of 2^'-deoxy-3-(3-azidopropyl)-5^'-O-(4,4^'-dimethoxy- trityl)uracil, 4.

2^'-Deoxy-5^'-O-(4,4^'-dimethoxytrityl)uracil (0.53 g, 1.0 mmol), 3-azido- 5 propan-1-ol (0.11 g, 1.1 mmol) and triphenylphosphine (0.29, g, 1.1 mmol) were dissolved in dry THF (20 ml_). Diisopropylazodicarboxylate {0.22 μl_, 1.1 mmol) was added in four portions, and the reaction was allowed to proceed overnight at RT. All volatiles were removed in vacuo. Purification on silica gel using diethyl ether as the eluent yielded the title compound. ESI TOF MS for 10 C₃₃H₃₅N₅ NaO₇ (M+Na)⁺: calcd, 636.24; found, 636.26. v_max 2098 crτT¹ (-N₃). 1H NMR (CDCI3): 7.76 (1 H, d, J 8.3); 7.39-7.26 (9H); 6.83 (4H₁ d, J 8.7); 6.31 (1 h, t, J 6.2); 5.55 (1 H, d, J 8.3); 4.54 81 H, m); 4.00 (2H, t J 6.1 ); 3.79 (6H₁ s); 3.50 (1 H₁ m); 3.45 (2H, m); 3.35 (2H, t, J 6.8); 2.44 (1H₁ m); 2.26 (1 H₁ m); 2.19 (1H, br s9; 1.91 (2H, p).

15 Example 13

The synthesis of 2^'-deoxy-5^'-O-(4,4^'-dimethoxytrityl)-3-{tetramethyl 2,2^",2^",2^'"-[4-(propyl-(1H-1 ₎2,3-triazol-4-yl)pyridine-2,6-diyl)bis(methyienenit- rilo)]tetrakis(acetato), uracil, 6.

Reaction of tetramethyl 2,2^',2^",2^'"-[4-ethynylpyridine-2,6-diyl)bis-

20 (methylenenitrilo)]tetrakis(acetate), 5, synthesized as disclosed in Kwiatkowski et al, 1994, Nucleic Acids Res., 22, 2604, with compound 4 yielded the title compound. ESI TOF MS for C₅₃H₆₁N₈O₁₅ (M+H)⁺: calcd, 1049.42; found,

1450.87.

Example 14

25 The synthesis of 2,2^',2^",2^'"-{4^'-[4-(propargylthioureidophenyl]-

2,2^':6^',2^"-terpyridine-6,6^"-diylbis(methylenenitrilo)}tetrakis(acetate) europium- (III), 8a.

Equimolar amounts of 2,2^',2^",2^'"-{4'-[4-isothiocyanatophenyl]- 2,2^':6^',2^"-terpyridine-6_I6^"-diylbis(methylenenitrilo)}tetrakis(acetate) europium-

30 (III), 7a synthesized as disclosed in US 4859,777, and propargylamine were dissolved in carbonate buffer, pH 9.3 and the reaction was allowed to proceed for 2 h at RT. The product was isolated by precipitation from acetone. 25

Example 15

The synthesis of 2,2^',2^",2^"*-{4^'-[4-(azidopropylthioureϊdophenyl]- 2,2^':6^',2^"-terpyridine-6,6"-diylbis(methylenenitrilo)}tetrakis(acetate) europium- (IH), 9a.

5 Equimolar amounts of 7a and azidoproylamine were dissolved in carbonate buffer, pH 9.3 and the reaction was allowed to proceed for 2 h at RT. The product was isolated by precipitation from acetone.

Example 16

The synthesis of 2,2^',2^",2^'"-{4'-[4-propargylthioureidoophenyl]- 10 ethynylpyridine-2,6-diylbis(methylenenitrilo) tetrakis(acetate) europium([ll), 9b.

Reaction of 2,2^',2^",2^'"-{4'-[4-isothiocyanatophenyl]ethynylpyridine- 2,6-diyEbis(methylenenitriIo) tetrakis(acetate) europium(lll), 7b with propargyl- amine as disclosed in Example 14 yielded the title compound.

Example 17

15 The synthesis of of 2,2^',2^",2^'"-[4-((3-hydroxypropyl)-1H-1 ,2,3- triazol-4-yl)pyridine-2,6-diyl)bis(methylenenitrilo)]tetrakis(acetate), europium(lll)

10.

Compound 2b was converted to the title compound using the method disclosed in Takalo, H., Mukkala, V.-M., Mikola, H., Liitti, P., Hemmila, 20 I₁ 1994, Bioconjugate Chem. 5, 278. Excitation: 277.06 nm; Emission 593.01

616.33 (max), 697.27 nm.

Example 18

The synthesis of 2_I2^',2^",2^{' "}-{[6,6^'-[(4-(4-(1-(3-hydroxypropyl)-1f/- 1 ,2,3-triazol-4-yl)pyrazole-1 ^",3^"-diyl)bis(pyridine)-2,2^'-diyI]bis(methylenenit-

25 rilo)}tetrakis(acetate) europium(lll) 11a, 2,2^',2^",2^'"-{[6,6^'-[(4-(4-(1-(3-hydroxy- propyl)-1 HA ,2,3-triazol-4-yl)pyrazole-1 ^",3^"-diyl)bis(pyridine)-2,2^'-diyl]bis(met- hylenenitrilo)}tetrakis(acetate) terbium(lll) 11 b and 2,2^',2^",2^{' "}-{[6,6^'-[(4-(4-{1- (3-hydroxypropyl)-1 HA ,2,3-triazol-4-yl)pyrazole-1 ^",3^"-diyl)bis(pyridine)-2,2^'- diyl]bis(methylenenitrilo)}tetrakis(acetate) dysprosium(lll) 11c.

30 The synthesis was preformed as disclosed in Example 17 Compound 11a t_R 10.01 min, ESI TOF MS for C₃₄H₄₄EuN₁₀O₉ (M+TEA)⁺: calcd, 889.25; found, 889.18. λ_max 324 nm (ε 14371). Excitation/nm 327; Emis- sion/nm, rel. int (%) 593.12 (14), 616.43 (50), 654.12 (3), 692.17 (32). εΦ 2310. τ 1.03 ms. 26

Compound 11b t_R 10.07 min, ESI TOF MS for C₃₄H₄₄EUN₁₀O₉ (M+ TEA)⁺: calcd, 895.25; found, 895.27. λ_max 324 nm (ε 14429). Excitation/nm 327; Emission/nm, rel. int (%) 491 (21), 544 (55), 587 (15), 623 (8), 647 (2), 669 (1 ) εΦ 10000. τ 2.61 ms. Compound 11c ESI TOF MS for C₃₄H₄₄DyNi₀O₉ 5 (M+TEA)⁺: calcd, 888.25; found, 899.31. Excitation/nm 328; Emission/nm, rel. int (%) 479 (37), 574 (53), 659 (1.5), 755 (2) εΦ 221. τ 0.01 ms.

Example 19

The synthesis of 2,2^' _I2^",2^'"-{[4^'-(5-[(1-(3-hydroxypropyl)-1 H-1 ,2,3- triazol-4-yl]-2-(furyl)-2,2^':6^',2^"-terpyridine-6,6^"- 10 diyl]bis(methylenenitrilo)}tetrakis (acetic acid) europium(lll) (12).

Compound 2e was converted to the title compound using the method disclosed in Example 17, but using cone, hydrochloric acid instead of

TFA. TOF MS for C₃₄H₃₀EUN₈OIO^" (M)^": calcd, 863.13; found, 863.07. λ_max 293 nm (ε 14722). Excitation/nm 327; Emission/nm, rel. int (%) 595 (13), 617 (50),

15 697 (37) εΦ 187; τ 0.71 ms.

69

27

Scheme 2

10 69

28 c

Scheme 4 29

Scheme 6 69

30

Scheme 7

Claims

31CLAIMS

1. A lanthanide chelate or chelating agent, comprising a 1 ,2,3- triazoie subunit, wherein said chelate or chelating agent is represented by formula (I)

(D wherein

A is a reactive group or not present, L is a linker or not present, group G comprises a chelating group having at least two carboxylic acid or 10 phosphonic acid groups, or esters or salts of said acids, and a chromophoric moiety having one or more aromatic units, and G optionally includes a chelated lanthanide ion Ln³⁺, characterized in that (i) in the group G

- the chelating part is attached to an aromatic unit of the chromo- 15 phoric moiety either directly or via an Λ/-containing hydrocarbon chain,

- the aromatic units are tethered to each other either directly or via N-containing hydrocarbon chain and

20 (ii) the linker L is formed from 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 (-COO- and -OOC-), disulfide (-SS-), diaza (-N=N-), and tertiary

25 amine, wherein R^' represents an alkyl group containing less than 5 carbon atoms,

(iii) the reactive group A is selected from the group consisting of isothiocyanate, haloacetamido, maleimido, dichlorotriazinyl, pyridyldithio, thio- ester, aminooxy, hydrazide, amino, a polymerizing group, and a carboxylic acid

30 or an acid halide or an active ester thereof,

(iv) the lanthanide ion is selected from europium(lll), terbium(lll), samarium(lll) and dysprosium(lll).

2. The lanthanide chelate or chelating agent according to claim 1 , characterized in that G is selected from the group consisting of 32

coo wherein Z is independently selected from furyl, thienyl, phenyl or phenylethynyl, where phenyl is substituted or unsubstitued, or Z is not present, wherein G is a radical of any of the aforementioned structures and tethered to the triazole subunit.

3. The lanthanide chelate or chelating agent according to claim 1 or 2 characterized in that G is represented by formula (II) 33

wherein Z is independently selected from furyl, thienyl, phenyl or phenyl- ethynyl, where phenyl is substituted or unsubstitued, or Z is not present. 5 4. The lanthanide chelate or chelating agent according to claim 3 characterized in that Z is not present.

5. A chelating agent, comprising a 1 ,2,3-triazole subunit, wherein said agent is represented by formula (I)

***% _(l)

10 wherein

A is a reactive group A, L is a linker L or not present, group G comprises a chelating part having at least two carboxylic acid or phosphonic acid esters, and a chromophoric moiety having one or more aromatic units, characterized in that

15 (i) in the group G

20 - at least one of the aromatic units are directly tethered to the 1 ,2,3- triazole subunit,

(ii) the linker L is formed from 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 (-0-), thioether

25 (-S-), amide (-CO-NH-, -CO-NR^'-, -NH-CO- and -NR^'-CO-}, carbonyl (-CO-), ester (-C00- and -00C-), disulfide (-SS-), diaza (-N=N-), and tertiary amine, wherein R^' represents an alkyl group containing less than 5 carbon atoms,

(iii) the reactive group A is an amino acid residue -CH(NHR¹)R⁵ where R¹ is a transient protecting group and R⁵ is a carboxylic acid or its salt,

30 acid halide or an ester. 34

6. The chelating agent according to claim 5, characterized in that G is selected from the group consisting of

V^{V ?}

5 wherein Z is independently selected from furyl, thienyl, phenyl or phenylethynyl, where phenyl is substituted or unsubstitued or Z is not present, wherein G is a radical of any of the aforementioned structures and tethered to the triazole subunit, and R¹ is selected from the group consisting of Fmoc, Boc or Bsmoc, and R^" is a an alkyl ester or an allyl ester.

10 7. The chelating agent according to claim 6 characterized in that G is represented by formula (Ha) 35

Ha

wherein Z is independently selected from furyl, thienyl, phenyl or phenylethynyl, where phenyl is substituted or unsubstitued or Z is not present, wherein G is a radical of any of the aforementioned structures and tethered to the triazole subunit, and R¹ is selected from the group consisting of Fmoc, Boc or Bsmoc, and R^" is a an afkyl ester or an allyl ester.

8. The chelating agent according to claim 7 characterized in that Z is not present.

10 9. A chelating agent, comprising a 1 ,2,3-triazole subunit, wherein said agent is represented by formula (I)

A-LT

0)

comprising

15 A is a reactive group, L is a linker L or not present, group G comprises a chelating part having at least two carboxylic acid or phosphonic acid esters, and a chromophoric moiety having one or more aromatic units, characterized in that

(i) in the group G

20 - the chelating part is attached to an aromatic unit of the chromophoric moiety either directly or via an Λ/-containing hydrocarbon chain,

- at least one of the aromatic units are directly tethered to the 1 ,2,3-triazole 25 subunit,

(ii) the linker L is formed from one to ten moieties, each moiety being selected from the group consisting of phenylene, alkylene containing 1-12 carbon atoms, ethynyldiyl (-C=C-), ethylenediyl (-C=C-), ether (-O-), thioether (-S-), amide (-CO-NH-, -CO-NR^'-, -NH-CO- and -NR'-CO-), carbonyl (-CO-), 30 ester {-COO- and -OOC-), disulfide (-SS-), diaza (-N=N-), and tertiary amine, wherein R^' represents an alkyl group containing less than 5 carbon atoms, 36

(iii) the reactive group A is

-Y-O-PZ¹ -O-R⁴ where one of the oxygen atoms optionally is replaced by sulfur,

Z¹ is chloro or NR²R³ 5 R⁴ is a protecting group,

R² and R³ are alkyl groups,

Y is absent or is a radical of a purine base or a pyrimidine base or any other modified base suitable for use in the synthesis of modified oligonucleotides, said base being connected to the oxygen atom via either 10 a) a hydrocarbon chain, which is substituted with a protected hy- droxyethyl group, or via b) a furan ring or pyrane ring or any modified furan or pyrane ring, suitable for use in the synthesis of modified oligonucleotides.

10. The chelating agent according to claim 9 characterized in that 15 Y is a radical of any of the bases thymine, uracil, adenine, guanine, cytosine,

7-deazaadenine or 7-deazaguanine, and said base is connected to the oxygen atom via i) a hydrocarbon chain substituted with a protected hydroxyethyl group, or

20 ii) a furan ring having a protected hydroxyethyl group in its 4-posi- tion and optionally a hydroxyl, protected hydroxyl or modified hydroxyl group in its 2-position.

11. The chelating agent according to claim 9, characterized in that -Y-O-P(NR²R³J-O-R⁴ is selected from the group consisting of

25 37

where - is the position of the linker L and DMTr is dimethoxytrityl.

12. The chelating agent according to claim 9, characterized in that

G is selected from the group consisting of

5 wherein Z is independently selected from furyl, thienyl, phenyl or phenylethynyl, where phenyl is substituted or unsubstitued or Z is not present, wherein G is a radical of any of the aforementioned structures and tethered to the triazole subunit, and R^" is an alkyl ester of an allyl ester.

13. The chelating agent according to claim 12 characterized in that

10 group G is represented by formula (lib) 38

14. The chelating agent according to cfaim 12 characterized in that Z is not present.

5 15. A biomolecule conjugate comprising a biomolecule conjugated to a lanthanide chelate or chelating agent according to claim 1.

16. The biomolecule conjugate according to claim 15 characterized in that the biomolecule is selected from the group consisting of an oligopeptide, oligonucleotide, nucleoside, nucleotide, nucleo- 10 side 5^'-triphosphate, DNA, RNA, modified olϊgo- or polynucleotide, protein, oli- gosaccaride, polysaccaride, phosphoiipide, PNA, LNA, antibody, hapten, drug, receptor binding ligand and lectine.

17. The biomolecule conjugate according to claim 15 characterized in that the modified oligo- or polynucleotide is a phosphoromonothioate, phos-

15 phorodithioate, phosphoroamidate and/or sugar- or basemodified oligo- or polynucleotide,

18. A conjugated particle comprising a particle conjugated to a lanthanide chelate or chelating agent according to any of the claims 1 - 13.

19. The conjugated particle according to claim 18, characterized in 20 that the particle is selected from the group consisting of a nanoparticle, a mi- croparticle, a slide or a plate.

20. A labeled oligopeptide, obtained by synthesis on a solid phase, by introduction of the chelating agent according to claims 5 - 8 into the oligopeptide structure on an oligopeptide synthesizer, followed by deprotection

25 and optionally also introduction of a metal ion.

21. A labeled oligonucleotide, obtained by synthesis on a solid phase, by introduction of the chelating agent according to any of the claims 9 - 13 into the oligonucleotide structure on an oligonucleotide synthesizer, followed by deprotection and optionally also introduction of a metal ion.

30 22. A particle conjugated with an agent according to claim 20 or 21 where the labeled oligopeptide or labeled oligonucleotide is covalently or non- covalently immobilized on said particle. 39

23. The conjugate according to claim 22, wherein the oligopeptide or oligonucleotide is covalently or noncovalently immobilized on the particle, which is selected from the group consisting of a nanoparticle, a microparticle, a slide or a plate.

5 24. A method for the preparation of the lanthanide chelate or chelating agent according to claim 1 or 5 characterized in that i) a group A-L= is reacted with a group G-N₃, where A, G, and L are as defined in any of the foregoing claims, to give a compound of formula (I) or (III) where A-L- is bound to a carbon atom and G is bound to the nitrogen 10 atom in the triazole subunit, or ii) a group A-L-N₃ is reacted with a group G-=, where A, G, and L are as defined in any of the foregoing claims, to give a compound of formula (I) or (III) where A-L- is bound to the nitrogen atom and G is bound to a carbon atom in the triazole subunit.

15 25. The method according to claim 24 characterized in that the reaction is catalysed by Cu⁺ and Ru(AcO)₂(PPh₃)2 to give an agent where the triazole is 1 ,4-disubstituted.