CN1918149B - Novel chelating agents and highly luminescent and stable chelates and their use - Google Patents

Abstract

This invention relates to a group of novel chelating agents, novel chelates, biomolecules labeled with said chelates or chelating agents, and solid supports conjugated with said chelates, chelating agents, or labeled biomolecules. In particular, this invention relates to novel chelating agents that can be used for the solid-phase synthesis of oligonucleotides or oligopeptides, and the oligonucleotides and oligopeptides obtained therefrom.

Description

Novel chelating agents, highly luminescent and stable chelates and uses thereof

field of invention

The invention relates to a group of novel chelating agents, novel chelating compounds, biomolecules labeled with the chelating compounds or chelating agents and solid carriers conjugated with the chelating compounds, chelating agents or labeled biomolecules.

Background of the invention

The various publications and other materials used herein to illuminate the background of the invention, particularly the case which provides additional details concerning the practice, are incorporated herein by reference.

技术应用于要求特殊敏感度、耐久性和多标记途径的分析中[Hemmil

等，Anal.Biochem.1984，137，335-343]。高度发光的稳定螯合物的研制成功将时间分辨的用途扩展至基于荧光共振能传递(TR-FRET)、荧光淬灭(TR-FQA)或结合反应期间螯合物的发光性能发生改变的均相分析[Hemmil，I.；Mukkala，V.-M.Crit.Rev.Clin.Lab.Sci.2001，38，441-519]。Due to their unique luminescent properties, various lanthanide(III) chelates are commonly used as non-radioactive labels in a variety of routine and scientific research applications. Because of their intense, long-decay glow, lanthanide(III) chelates are ideal markers in sensitive assays. Time-resolved fluorescence assays based on lanthanide chelates are increasingly used in diagnostics, scientific research, and high-throughput screening. Heterogeneous DELFIA

The technique is used in assays requiring exceptional sensitivity, robustness, and multilabel pathways [Hemmil

et al., Anal. Biochem. 1984, 137, 335-343]. The successful development of highly luminescent stable chelates extends the use of time-resolved chelates based on fluorescence resonance energy transfer (TR-FRET), fluorescence quenching (TR-FQA), or uniformity of the chelate's luminescent properties during a conjugation reaction. Phase analysis [Hemmil , I.; Mukkala, V.-M. Crit. Rev. Clin. Lab. Sci. 2001, 38, 441-519].

In most cases, the conjugation reaction is between the amino group or sulfhydryl group of a biologically active molecule (such as a protein, peptide, nucleic acid, oligonucleotide or hapten) and the isosulfide of a lanthanide (III) chelate in solution. Between cyanoyl (isothiocyanato), haloacetyl, 3,5-dichloro-2,4,6-triazinyl derivatives and other information groups. Since the labeling reactions were carried out with an excess of activating label in all cases, laborious purification procedures were unavoidable. Especially in the presence of several functional groups with similar reactivity when it is necessary to attach several labeling molecules or labels at specific sites, the isolation and characterization of the desired biomolecular conjugates is extremely difficult and often nearly impossible . Liquid-phase labeling of large life molecules (such as proteins) is naturally unavoidable. In these cases, the labeling reaction must be as selective and efficient as possible.

Numerous attempts have been made to develop novel highly luminescent chelate labels suitable for various time-resolved fluorescence applications. These efforts include, for example, stable chelates composed of various pyridine derivatives as energy modulating groups [US 4,920,195, US 4,801,722, US 4,761,481, PCT/FI91/00373, US 4,459,186, EP A-0770610, Remuinan etc., J.Chem.Soc.Perkin Trans 2,1993,1099]; various bipyridine derivatives [US 5,216,134]; various terpyridine derivatives [US4,859,777, US 5,202,423, US 5,324,825] or various phenols Compound [US4,670,572, US 4,794,191, Italian patent 42508 A789] and polycarboxylic acid as a chelating component. In addition, various dicarboxylic acid salt derivatives [US 5,032,677, US 5,055,578, US 4,772,563], macrocyclic cryptate compounds [US 4,927,923, WO 93/5049, EP-A-493745] and macrocyclic Schiff bases [EP-A-369-000]. People have also disclosed a method of labeling biospecific adhesive reagents (such as haptens, peptides, receptor ligands, drugs or PNA oligomers) with luminescent markers by solid-phase synthesis [US6,080,839]. Similar strategies for multiple labeling of oligonucleotides on solid phases have also been developed [EP A 1152010, EP A 1308452].

Although fluorescent rare earth chelates comprising arylpyridinedioic acids and aryl-substituted 2,6-bis[N,N-di(carboxyalkyl)aminoalkyl]pyridine moieties have been disclosed [Hemmil et al., J Biochem Biophys Methods 26; 283-90 (1993); US 4,761,481], but the chelates or chelating agents described herein of the present invention have not been previously disclosed.

Purpose and summary of the invention

The main purpose of the present invention is to provide various chelating agents and their metal chelates that can be used to label life molecules and be used as probes in time-resolved fluorescence spectroscopy, magnetic resonance imaging (MRI) or positron emission tomography (PET).

A particular object of the present invention is to provide chelating agents which fluoresce strongly with different chelated lanthanide ions, in particular with europium(III), samarium(III), terbium(III) and dysprosium(III). These lanthanide chelates are especially useful in multiparametric bioaffinity assays and high-throughput screening of drug candidates.

Another object of the present invention is to provide various chelating agents to obtain highly stable metal chelates. A particular aim is to obtain chelates that are sufficiently stable to be useful in various applications in vivo, such as in MRI or PET applications.

Another object of the present invention is to provide chelates or chelating agents suitable for labeling biomolecules in solution.

Another object of the present invention is to provide chelates suitable for simultaneous labeling of oligopeptides or oligonucleotides with their synthesis on solid phase.

Another object of the present invention is to provide a solid carrier conjugated with the chelates, chelating agents or biomolecules of the present invention.

Accordingly, a first aspect of the present invention relates to a chelating agent comprising:

- a chromophoric moiety comprising two or more aromatic units, at least one of which is a trialkoxyphenylpyridyl group, wherein the alkoxy groups are the same or different, and the pyridyl groups are i) are tethered directly to each other, forming a bipyridyl or terpyridyl group, respectively; or ii) are tethered to each other via an N-containing hydrocarbon chain;

- a chelating moiety comprising at least two carboxylic or phosphonic acid groups or esters or salts of said acids attached directly or via an N-containing hydrocarbon chain to an aromatic unit of the chromophoric moiety; and

- Optionally tethered directly or via a linker x to said chromogenic moiety or to a reactive group A tethered to a chelating moiety, said reactive group A being capable of binding to a living molecule or functional group on a solid phase.

Another aspect of the present invention relates to a chelate comprising:

-Metal ion;

- a chromophoric moiety comprising two or more aromatic units, at least one of which is a trialkoxyphenylpyridyl group, wherein the alkoxy groups are the same or different, and the pyridyl groups are i) are tethered directly to each other, forming a bipyridyl or terpyridyl group, respectively; or ii) are tethered to each other via an N-containing hydrocarbon chain;

- a chelating moiety comprising at least two carboxylic or phosphonic acid groups or esters or salts of said acids attached directly or via an N-containing hydrocarbon chain to an aromatic unit of the chromophoric moiety; and

- Optionally tethered directly or via a linker x to said chromogenic moiety or to a reactive group A tethered to a chelating moiety, said reactive group A being capable of binding to a living molecule or functional group on a solid phase.

A third aspect of the invention relates to biomolecules conjugated to the chelates of the invention.

A fourth aspect of the invention relates to biomolecules conjugated to the chelating agents of the invention.

A fifth aspect of the present invention relates to a solid support conjugated to a chelate or labeled biomolecule of the present invention.

A sixth aspect of the present invention relates to labeled oligopeptides by introducing a suitable chelating agent of the present invention into the oligopeptide structure on the oligopeptide synthesizer, followed by deprotection and optionally introducing metal ions on a solid phase Synthesize above to obtain the labeled oligopeptide.

A seventh aspect of the invention relates to labeled oligonucleotides by introducing a suitable chelator of the invention into the oligonucleotide structure on an oligonucleotide synthesizer, followed by deprotection and optionally The labeled oligonucleotide is obtained by introducing metal ions and synthesizing on a solid phase.

An eighth aspect of the present invention relates to a solid support suitable for use in oligonucleotide synthesis, conjugated to the chelating agent of claim 1, wherein the reactive group A is connected to the chelating agent through a linker x, and A is :

-E-O-x'-

in:

x' is a linker connected to a solid carrier, which is the same as or different from linker x;

E can be absent, or be a purine or pyrimidine group or any other modified base suitable for use in the synthesis of modified oligonucleotides by:

i) a hydrocarbon chain substituted with a protected hydroxyethyl group is attached to an oxygen atom; or via

ii) A furan or pyran ring suitable for use in the synthesis of a modified oligonucleotide or any modified furan or pyran ring attached to an oxygen atom.

Detailed description of the invention

Chelating agent

Chelating agents and metal chelates based on a divalent aromatic structure in which the chromophoric moiety is predominantly comprised of one or more trialkoxyphenylpyridine groups are novel. The trialkoxyphenylpyridine group is capable of absorbing light or energy, and transferring the excitation energy to the chelated lanthanide ion, resulting in strong fluorescence regardless of the lanthanide ion used. In addition to the described trialkoxyphenylpyridine groups, the chromophoric units may also comprise unsubstituted pyridine groups, pyridine groups with further substituents and/or other aromatic groups.

In the compounds exemplified in the specific examples herein, the 4-position of the pyridyl group contains a trialkoxyphenyl substituent. While we believe this position is the most preferred, other positions on the pyridine ring are also available for substitution.

Alkoxy groups are preferably C1-C4 alkoxy groups.

According to a preferred embodiment, the chromophoric moiety comprises two or three pyridine groups, at least one of which is substituted by a trialkoxyphenyl group. These pyridyl groups can be tethered directly to each other to form bipyridyl or terpyridyl groups, respectively. Or more preferably said pyridyl groups are tethered to each other by N-containing hydrocarbon chains. N-containing hydrocarbon chains are understood to be chains which contain no other heteroatoms or aromatic groups other than N. In this case relatively stable chelates can be obtained. Chelating agents of this structure render the metal chelate sufficiently stable while also being suitable for in vivo use in various applications of MRI and/or PET.

Where the chelating moiety is attached to the aromatic unit of the chromophoric moiety, the chelating moiety may be attached to the pyridine ring or a substituent thereon (eg, phenyl).

In order for a chelating agent or chelate to covalently bind to a biomolecule or a solid support, the chelating agent or chelate must contain a reactive group A. But there are also applications where such covalent bonding is not essential. The chelating compounds of the present invention are also useful in applications where the chelating compounds do not require reactive groups. An example of this technique is illustrated, for example, in Blomberg et al., J. Immunological Methods, 1996, 193, 199. Another example where reactive group A is not required is the separation of eosinophils and basophils. In this application, positively and negatively charged chelates bind to negatively and positively charged cell surfaces, respectively.

Although in principle in many applications the reactive group A can be directly attached to the chromophore or the chelating moiety, it is ideal to separate the reactive group A from the chromophore or the reactive group A from the chelating moiety has a linker x (especially for steric reasons). Said linkers are particularly important where chelates have to be used in solid phase synthesis of oligopeptides and oligonucleotides, but are also desirable in solution labeling of biomolecules.

According to a preferred embodiment, the reactive group A is selected from the group consisting of isothiocyanate, haloacetamido, maleimide, dichlorotriazinyl, dichlorotriazinylamino, pyridinedithio, thioester group, oxygen-containing amino group, hydrazide group, amino group, polymer group, carboxylic acid or halogenated acyl group or its active ester group. Particularly in cases where the chelate or chelating agent has to be attached to microparticles or nanoparticles, reactive groups with polymeric groups are preferred. In this case the markers can be introduced into the particles during their manufacture.

The linker x is preferably formed by 1-10 moieties, each moiety selected from: phenylene, alkylene having 1-12 carbon atoms, acetylenediyl (-C≡C-), ethylenediyl (-C=C-), ether group (-O-), thioether group (-S-), amido group (-CO-NH-, -CO-NR'-, NH-CO and -NR'-CO -), carbonyl (-CO-), ester group (-COO- and -OOC-), disulfide (-SS-), diaza (-N=N-) and tertiary amine, where R' represents Alkyl groups of less than 5 carbon atoms.

According to a particularly preferred embodiment, the chelating agent is one of the following specific structures:

Among them: Z ¹ , Z ² and Z ³ are the same or different alkyl groups; R ⁶ is an alkyl ester or allyl ester; R ⁷ is an alkyl group, and n is 0 or 1.

Chelating agents used in peptide synthesis

According to a preferred embodiment, the chelating agent of the present invention is suitable for the synthesis of oligopeptides. In this application, the reactive group A is connected to the chelating agent through a linker x, A is an amino acid residue -CH(NHR ¹ )R ⁵ , wherein R ¹ is a transient protecting group, and R ⁵ is a carboxylic acid or a salt thereof , acid halides or esters. A particularly preferred chelating agent is the following structure:

Wherein: x is as previously defined, and the protecting group ^R is selected from Fmoc (fluorenylmethoxycarbonyl), Boc (tert-butoxycarbonyl) or Bsmoc (1,1-dioxobenzo[b]benzenethio- 2-ylmethoxycarbonyl), R ⁶ is an alkyl ester or allyl ester, R ⁷ is an alkyl group, Z ¹ , Z ² and Z ³ are the same or different alkyl groups, and n is 0 or 1.

Chelating agents can be introduced into living molecules with the aid of peptide synthesizers. For example, a chelating agent can be coupled to an amino-tethered solid support or an immobilized amino acid by carbodiimide chemistry (that is, in the presence of an activator, the carboxylic acid functional group of the labeling reagent interacts with the amino group of the solid support or amino acid). reaction). When the condensation step is complete, the transient amino protecting group of the labeling reagent is selectively removed while the material remains attached to the solid support (eg via piperidine in the case of the Fmoc-protecting group). A second coupling of chelators or other reagents (amino acids, haptens) is then performed as described above. When the synthesis of the desired molecule is complete, the material is removed from the solid support and deprotected. Purification can be performed by HPLC techniques. Finally the purified ligand is converted to the corresponding lanthanide(III) chelate by adding a known amount of lanthanide(III) ion.

Chelating agents for oligonucleotide synthesis

According to another preferred embodiment, the chelating agent of the invention is suitable for the synthesis of oligonucleotides. In this case the reactive group A is attached to the chelating agent via a linker x, A being:

-EO-PZ-OR ⁴

in:

One of the oxygen atoms is optionally replaced by sulfur, Z is chlorine or NR ² R ³ , R ⁴ is a protecting group, R ² and R ³ are alkyl, E is absent or suitable for modified oligonucleotides Purine or pyrimidine bases or any other modified bases in synthesis. The base is attached to the oxygen atom through: i) a hydrocarbon chain substituted with a protected hydroxyethyl group; or through ii) a furan ring or a pyran ring or any modified The furan or pyran ring is attached to an oxygen atom.

Chelating agents can be introduced into oligonucleotides with the aid of an oligonucleotide synthesizer. A method based on Mitsonobu alkylation (J Org Chem, 1999, 64, 5083; Nucleosides, Nucleotides, 1999, 18, 1339) is disclosed in EP-A-1152010. Said patent discloses a method of directly attaching the desired number of conjugated groups to the oligonucleotide structure during strand formation. Thus avoiding liquid phase labeling and complicated purification process. The key reaction in the synthesis strategy of nucleoside oligonucleotide building blocks is the Mitsunobu alkylation reaction described above to introduce various chelating agents to nucleosides and finally to the oligonucleotide structure. Chelating agents are introduced during chain formation. The conversion to the lanthanide chelate occurred during the deprotection step after synthesis.

Unmodified oligonucleotides are normally less stable under physiological conditions because they are degraded by enzymes present in living cells. It would therefore be desirable to generate modified oligonucleotides according to known methods to enhance their resistance to chemical and enzymatic degradation. Various modifications of oligonucleotides have been extensively disclosed in the prior art. See US 5,612,215. We know that removing or replacing the 2'-OH group from the ribose sugar in the RNA strand will make it more stable. WO 92/07065 and US 5,672,695 disclose the replacement of the ribose 2'-OH group with a halogen, amino, azido or sulfhydryl group. US 5,334,711 discloses the replacement of a hydrogen in a 2'-OH group by an alkyl or alkenyl group (preferably methyl or allyl). In addition, for example, nucleotide phosphodiester linkages may be modified such that one or more oxygens are replaced by sulfur, amino, alkyl or alkoxy groups. A preferred modification in internucleotide linkages is phosphorothioate linkages. Bases in nucleotides can also be modified.

Preferably E is a group of any of the following bases: thymine, uracil, adenosine, guanine or cytosine, and said base is attached to the oxygen atom through: i) a hydrocarbon chain substituted with a protected hydroxyethyl group; or The oxygen atom is attached via ii) a furan ring having a protected hydroxyethyl group at its 4-position and optionally a hydroxyl, protected or modified hydroxyl at its 2-position.

Preferably the structure of the reactive group -EOP(NR ² R ³ )-OR ⁴ is selected from one of the following structures:

Wherein: - is the position of linker x, and DMTr is dimethoxytrityl.

Particularly preferred chelating agents are selected from one of the specific structures disclosed below:

Wherein: R ⁶ is alkyl ester or allyl ester, R ⁷ is alkyl, x is as defined above, A is -EOP(NR ² R ³ )-OR ⁴ as defined above, Z ¹ , Z ² and Z ³ are the same or different alkyl groups, and n is 0 or 1.

Chelate

The chelate comprises the above-mentioned chelating agent and chelated metal ions.

In the case of chelates for bioaffinity assays, the metal ion to be chelated is preferably a lanthanide, especially europium(III), samarium(III), terbium(III) or dysprosium(III). The chelating agent is preferably one of the above-mentioned preferred chelating agents.

Particularly preferred lanthanide chelates are:

Wherein: Z ¹ , Z ² and Z ³ are the same or different alkyl groups, and n is 0 or 1.

The chelates of the invention may also be used in in vivo MRI applications or PET applications. A preferred metal for MRI is gadolinium. In PET applications, radioactive metal isotopes are incorporated into the chelating agent just before use. Particularly suitable radioactive isotopes are Ga-66, Ga-67, Ga-68, Cr-51, In-111, Y-90, Ho-166, Sm-153, Lu-177, Er-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 fairly stable chelates, it is preferred to have a chromophoric moiety in which several pyridine groups are tethered to each other by an N-containing hydrocarbon chain.

Molecules of life

The biomolecules conjugated with the chelating agent or chelate of the present invention are preferably oligopeptides, oligonucleotides, DNA, RNA, modified oligonucleotides or polynucleotides (such as phosphorothioate, Phosphorodithioate, phosphoroamidate) and/or sugar-modified or base-modified oligonucleotides or polynucleotides, proteins, oligosaccharides, polysaccharides, phospholipids, PNA, LNA, antibodies, haptens, drugs , receptor binding ligands and lectins.

solid carrier conjugates

Chelates, chelating agents and biomolecules of the present invention can be conjugated to solid supports. The solid support is preferably particles (such as microparticles or nanoparticles), glass slides or thin plates.

In the case where the chelating compound or chelating agent has a polymeric group as a reactive group, the chelating compound or chelating agent can be introduced into a solid carrier such as a particle at the same time as it is prepared.

The life molecule covalently or non-covalently conjugated to the solid carrier is preferably a labeled oligopeptide, by introducing a chelating agent into the oligopeptide structure on the oligopeptide synthesizer, followed by deprotection and any The labeled oligopeptide is obtained by introducing metal ions and synthesizing on a solid phase. Alternatively, the biomolecules that are covalently or non-covalently conjugated to the solid carrier are preferably labeled oligonucleotides, and the oligonucleotide structure is introduced into the oligonucleotide synthesizer by introducing a chelating agent. , followed by deprotection and optional introduction of metal ions to obtain the labeled oligonucleotides by synthesis on a solid phase.

A solid support conjugated to a chelator having a reactive group A (A is -E-O-x'- as described above) attached to the chelator via a linker x is suitable for the synthesis of oligonucleotides.

The invention will be illustrated below by means of non-limiting examples.

Example

The invention will be illustrated by the following examples. The structures and synthetic routes used in the experimental part are described in Schemes 1-7. Scheme 1 illustrates the synthesis of oligopeptide-labeled reactant 4. Individual experimental details are given in Examples 1-4. Scheme 2 illustrates the synthesis of chelates 6-11. Individual experimental details are given in Examples 6-11. Scheme 3 illustrates the synthesis of chelates 20, 22 and 23. Individual experimental details are given in Examples 12-23. Scheme 4 illustrates the synthesis of building block 29 and the synthesis of chelates 30 and 31 for the introduction of lanthanide chelates to oligonucleotides on a solid phase. Experimental details are given in Examples 24-31. Schemes 5 and 6 respectively illustrate the use of structural units 4 and 29 in the preparation of synthetic oligopeptides and oligonucleotides on a solid phase. Experimental details are given in Examples 32 and 33. Scheme 7 illustrates the preparation of 1,4,7-triazecane-based oligonucleotide labeling reagents. Experimental details are given in Example 34.

The photochemical properties of each chelate compound synthesized in the exemplary embodiment are summarized in Table 1.

experimental method

Various reagents for machine-assisted oligopeptide synthesis were purchased from Applied Biosystems (Foster City, CA). Adsorption column chromatography was performed on a column packed with silica gel 60 (Merck). NMR spectra were recorded on a Brucker 250 or Jeol LA-400 spectrometer operating at ¹ H 250.13 and 399.8 MHz, respectively. The internal standard used was _Me4Si . The unit of the coupling constant is Hz. IR spectra were recorded on a Perkin Elmer 2000 FT-IR spectrophotometer. Electrospray mass spectra were recorded on an Applied Biosystems Mariner ESI-TOF instrument. Using the recommended protocol, oligopeptides were synthesized on an Applied Biosystems 433A synthesizer, while oligonucleotides were synthesized on an Applied Biosystems Expedite instrument. Fluorescence spectra were recorded on a PerkinElmer LS 55 instrument.

The synthesis of each compound was performed as outlined in Schemes 1-7 below.

Example 1

-{[6-N-(4-甲氧基三苯甲基)氨基己基-亚氨基]双(亚甲基)双[4-(2，4，6-三甲氧基苯基)吡啶-6，2-一二基]双(亚甲基次氮基)}四(乙酸酯)1Synthesis of tetra(tert-butyl) 2, 2', 2", 2

-{[6-N-(4-methoxytrityl)aminohexyl-imino]bis(methylene)bis[4-(2,4,6-trimethoxyphenyl)pyridine-6 , 2-one dibase] bis(methylene nitrilo)} tetrakis(acetate) 1

-{[6-N-(4-甲氧基三苯甲基)己基亚氨基]双(亚甲基)双-(4-溴吡啶-6，2-二基)双(亚甲基次氮基)}四(乙酸酯)(4.0克，2.4毫摩尔)和三甲氧基苯基硼酸(1.1克，5.3毫摩尔)溶解于无水DMF(50毫升)中，加入Cs₂CO₃(2.0克，6.0毫摩尔)和Pd(PPh₃)₄(0.1克，96微摩尔)。在95℃下搅拌过夜后，加入三甲氧基苯基硼酸(0.5克，2.4毫摩尔)、Cs₂CO₃(0.79克，2毫摩尔)和Pd(PPh₃)₄(50毫克，43毫摩尔)。反应过夜后将混合物冷却至室温，过滤并蒸发。将混合物溶解于CH₂Cl₂中，用水(2·40毫升)洗涤。产物通过急骤层析法(硅胶，石油醚(40-60℃)/AcOEt/TEA 5∶2∶1，v/v/v)进行纯化。收率为3.1克(90％)。IR(薄膜)：1737(C＝O)，1128(C-O)。¹H MR(CDCl₃)：δ1.15-1.25(4H，m)；1.40-1.45(40H，m)；2.04(2H，t，J 6)；2.55(2H，t，J 7)；3.50(1H，s)；3.51(3H，s)。ESI-MS：[M+H]⁺1417.5，C₈₂H₁₀₉N₆O₁₅ ⁺的计算值为1417.8。Four (tert-butyl) 2, 2', 2", 2

-{[6-N-(4-methoxytrityl)hexylimino]bis(methylene)bis-(4-bromopyridine-6,2-diyl)bis(methylenenitrilo base)}tetrakis(acetate) (4.0 g, 2.4 mmol) and trimethoxyphenylboronic acid (1.1 g, 5.3 mmol) were dissolved in anhydrous DMF (50 mL), and Cs ₂ CO ₃ (2.0 g, 6.0 mmol) and Pd(PPh ₃ ) ₄ (0.1 g, 96 μmol). After stirring overnight at 95°C, trimethoxyphenylboronic acid (0.5 g, 2.4 mmol), Cs ₂ CO ₃ (0.79 g, 2 mmol) and Pd(PPh ₃ ) ₄ (50 mg, 43 mmol) were added ). After overnight reaction the mixture was cooled to room temperature, filtered and evaporated. The mixture was dissolved _{in CH2Cl2} and washed with water (2.40 mL). The product was purified by flash chromatography (silica gel, petroleum ether (40-60° C.)/AcOEt/TEA 5:2:1, v/v/v). Yield 3.1 g (90%). IR (thin film): 1737 (C=O), 1128 (CO). ¹ H MR (CDCl ₃ ): δ1.15-1.25 (4H, m); 1.40-1.45 (40H, m); 2.04 (2H, t, J 6); 2.55 (2H, t, J 7); 3.50 ( 1H, s); 3.51(3H, s). _ESI -MS: [M+H] ⁺ _1417.5 , _Calcd . for _C82H109N6O15 ⁺ 1417.8.

Example 2

-{(6-氨基己基亚氨基)双(亚甲基)-双[4-(2，4，6-三甲氧基苯基)吡啶-6，2-二基]双(亚甲基次氮基)}四(乙酸酯)2Synthesis of tetra(tert-butyl) 2, 2', 2", 2

-{(6-aminohexylimino)bis(methylene)-bis[4-(2,4,6-trimethoxyphenyl)pyridine-6,2-diyl]bis(methylene nitro base)} tetrakis (acetate) 2

Compound 1 (1.0 g, 0.7 mmol) was dissolved in dichloromethane (25 mL), and trifluoroacetic acid (0.25 mL) was added. After stirring at ambient temperature for 4 h, the mixture was washed with saturated NaHCO ₃ (2·50 mL). The organic phase was dried over _Na2SO4 , filtered and evaporated _. The product was purified by flash chromatography (silica gel, petroleum ether (40-60°C)/AcOEt/TEA 5:5:1, ₂ :5:1, finally 10% MeOH in _CH2Cl2 , 1% TEA) Purify. Yield 0.60 g (74%). IR (thin film): 1730 (C=O), 1128 (CO). ESI-MS: [M+H] ⁺ 1145.7, Calcd. for _C82H109N6O15 ⁺ _{1145.7 ,} [M+2H] ²⁺ _573.3 , Calcd. 573.3.

Example 3

Synthesis of allyl-protected oligopeptide-labeled reactants 3

Compound 2 (0.55 g, 0.48 mmol) was dissolved in anhydrous dichloromethane (5 mL). DCC (0.11 g, 0.53 mmol) and Fmoc-Glu-OAII (0.20 g, 0.48 mmol) were added, and the mixture was stirred at room temperature overnight. The resulting DCU was filtered off and the filtrate was concentrated under vacuum. Purification on silica gel (10% MeOH in dichloromethane) gave the title compound as a solid (300 mg). ESI-MS: [M+H] ⁺ _{1536.8 ,} Calcd. ^for _{C85H114N7O19 +} 1536.8.

Example 4

Synthetic oligopeptide labeling reactant 4

Compound 3 (157 mg, 0.1 mmol) was dissolved in anhydrous dichloromethane (2 mL). Pd(Ph ₃ P) ₄ (2.3 mg) and PhSiH ₃ (25 μl) were added and the mixture was stirred overnight at ambient temperature. The reaction mixture was then washed with 10% aqueous citric acid and dried over molecular sieves. Yield 95 mg (63%). ESI-MS: [M+H] ⁺ _{1496.8 ,} Calcd. for _C82H110N7O19 ⁺ _1496.8 .

Example 5

Synthetic Free Acid 5

Compound 1 (0.40 g, 0.28 mmol) was dissolved in trifluoroacetic acid (10 mL), stirred at room temperature for 1 hour and then concentrated. The residue was triturated with diethyl ether. The product was collected by filtration and dried. Yield 260 mg (100%). ESI-MS: [M+H] ⁺ _921.42 , Calcd. for _C46H61N6O14 ⁺ _{921.4 .}

Example 6

Synthesis of Terbium Chelate 6

Compound 5 (78 mg, 0.085 mmol) was dissolved in water (2 mL), and terbium(III) chloride (35 mg, 0.093 mmol) was added within 15 minutes at pH 6.5. After 2 hours at room temperature the pH of the reaction mixture was raised to 8.5 by the addition of 1M NaOH. The resulting precipitate was removed by centrifugation, the aqueous phase was concentrated, and the product was precipitated with acetone. ESI-MS: [M+H] ⁺ _{1075.9 ,} ^Calcd . for _{C46H55N6O14Tb- 1075.3} .

Example 7

Synthesis of Dysprosium Chelate 7

The synthesis was carried out as in Example 6, but using dysprosium(III) chloride. _ESI -MS: [M+H] ⁺ _1080.3 , ^Calcd . for _{C46H55N6O14Dy- 1080.2} .

Example 8

Synthesis of Europium Chelate 8

The synthesis was carried out as in Example 6, but using europium(III) chloride. ESI-MS: [M+ _H ] ⁺ _1092.3 , ^Calcd . for _{C46H55N6O14Eu- 1092.3} .

Example 9

Synthesis of Dysprosium Chelate Activated by Iodoacetamido 9

Compound 7 (16 mg, 14.3 micromol) was dissolved in water. Iodoacetic anhydride (51.3 mg, 0.145 mmol; previously dissolved in 0.2 mL of chloroform) and DIPEA (25 μL) were added, and the mixture was stirred at room temperature for 1.5 hours. The organic phase was removed and the product was separated from the aqueous phase by precipitation from THF. _ESI -MS: [M+H] ⁺ _1248.2 , _Calcd ^. for _{C48H57N6O15IDy-} 1248.2.

Example 10

Synthesis of Iodoacetamido-Activated Terbium Chelate 10

Compound 6 was activated as described in Example 9 to afford compound 10. ESI-MS: [M+H] ⁺ _1243.8 , _Calcd ^. for _{C48H57N6O15ITb-} 1243.8 _.

Example 11

Synthesis of isothiocyanate-activated europium chelate 11

Compound 8 (15 mg, 0.014 mmol) was dissolved in a mixture of pyridine, water and triethylamine (200 μL; 9:1.5:0.1; v/v/v). 1,4-Phenylenediisothiocyanate (7.9 mg) was added, and the mixture was stirred at room temperature for 4 hours.

Example 12

Synthesis of diethyl 4-(2,4,6-trimethoxyphenyl)pyridine-2,6-dicarboxylate 12

2,4,6-Trimethoxyphenylboronic acid (2.12 g, 10.0 mmol) and diethyl 4-bromopyridine-2,6-dicarboxylate (3.33 g, 11.0 mmol) were dissolved in anhydrous DMF (50 ml). Cesium carbonate (4.56 g, 14.0 mmol) and tetrakis(triphenylphosphine)-palladium(0) (0.23 g, 0.20 mmol) were added, and the mixture was degassed with argon. The mixture was heated at 95°C for 48 hours. The mixture was cooled to room temperature, then filtered. The filtrate was concentrated in vacuo, the residue was dissolved in chloroform ₍ 60 mL), washed with 10% aqueous citric acid and water, dried over _Na2SO4 and concentrated. Purification was carried out on silica gel (petroleum ether, boiling point 40-60° C.; ethyl acetate, 5:3→2:5, v/v). Yield 2.09 g (54%). ¹ HNMR (CDCl ₃ ): δ1.45 (6H, t, J 7.1); 3.74 (6H, s); 3.90 (3H, s); 4, 49 (4H, q, J 7.1); ); 8.28(2H, s). IR (film)/cm ^-1 1743, 1610 (C=O); 1339, 1238, 1128 (CO). ESI-MS: [M+H] ⁺ _390.19 , calcd for _C20H24NO7 ⁺ _390.15 .

Example 13

Synthesis of ethyl 4-(2,4,6-trimethoxyphenyl)-6-(hydroxymethyl)pyridine-2-carboxylate 13

Compound 12 (2.83 g, 7.27 mmol) was suspended in ethanol (140 mL), and the mixture was heated to 45°C. Sodium borohydride (0.29 g) was added and the mixture was stirred for 1 hour and cooled to room temperature. The pH of the solution was adjusted to 3 with 6M HCl and concentrated. The residue was suspended in dichloromethane and washed with saturated NaHCO ₃ . The organic layer was dried over _Na2SO4 and purified on silica gel (eluent petroleum ether (boiling point 40-60 °C):ethyl acetate:triethylamine, 2:5: ₁ ; v/v/v) . ESI-MS _: [M+H] ⁺ _348.14 , calcd for _Ci8H22NO6 ⁺ 348.14.

Example 14

Synthesis of ethyl 4-(2,4,6-trimethoxyphenyl)-6-(bromomethyl)pyridine-2-carboxylate 14

Phosphorus trichloride (0.778 g, 2.87 mmol) was dissolved in anhydrous DMF (10 mL) at 0°C. Compound 13 (1.0 g, 2.8 mmol) was added, and the mixture was stirred at room temperature for 3.5 hours, then neutralized with saturated NaHCO ₃ . The mixture was extracted with dichloromethane. The organic phase was dried, concentrated and purified on silica gel (1% ethanol in dichloromethane as eluent). ESI-MS: [M+H] ⁺ _{410.10, calcd for Ci8H21BrNO5 +} ^410.05 _.

Example 15

Synthesis of N-(2-(2,2,2-trifluoroacetylamino)ethyl)-6-(hydroxymethyl)-4-(2,4,6-trimethoxyphenyl)pyridine-2-amide 15

Compound 13 (1.0 g, 2.8 mmol) was dissolved in ethylenediamine (10 mL), stirred at room temperature for 2.5 hours and concentrated (oil pump). The residue was dissolved in DMF (25 mL), and ethyl trifluoroacetate (5 mL) was added. After 2 hours at room temperature, all volatile components were removed in vacuo and the residue was purified on silica gel (10% MeOH in dichloromethane as eluent). ESI _- MS: [M+H] ⁺ _458.14 , _Calcd . ^for _C20H23F3N3O6 + _458.15 .

Example 16

Synthesis of N-(2-(2,2,2-trifluoroacetylamino)ethyl)-6-(bromomethyl)-4-(2,4,6-trimethoxyphenyl)pyridine-2-amide 16

Compound 15 was brominated as described in Example 14 to afford the title compound. ESI-MS: [M+H] ⁺ _520.06 , _Calcd . _{for C20H22BrF3N3O5} ⁺ _520.07 .

Example 17

Synthesis of 7-((6-(2-(2,2,2-trifluoroacetylamino)ethylcarbamoyl)-4-(2,4,6-trimethoxyphenyl)pyridin-2-yl) Di-tert-butyl methyl)-1,4,7-triazonane-1,4-dicarboxylate 17

[1,4,7]Di-tert-butyl triazacyclononane-1,4-dicarboxylate (0.75 g, 2.3 mmol) and compound 16 (2.3 mmol) were dissolved in anhydrous DMF (60 ml). 2.0 mL of DIPEA (11.4 mmol) was added, and the mixture was stirred overnight at room temperature. The solvent was evaporated to dryness and the product was purified on silica gel (diethyl ether as eluent). The yield was 1.20 g. ESI- _MS : [M+H] ⁺ _769.34 , _{Calcd. for C36H52F3N6O9} ⁺ _{769.37 .}

Example 18

Synthesis of 6-((1,4,7-triazonan-1-yl)methyl)-N-(2-(2,2,2-trifluoroacetylamino)-ethyl)-4-(2,4, 6-trimethoxyphenyl)pyridine-2-amide 18

Compound 17 (1.0 g, 1.3 mmol) was dissolved in trifluoroacetic acid (25 mL), and the mixture was stirred at room temperature for 30 min. The solvent was evaporated to dryness. ESI _- MS: [M+H] ⁺ _569.28 , _{Calcd. for C26H36F3N6O5} ⁺ _{569.27 .}

Example 19

Synthesis of 6-((4-((6-(2-(2,2,2-trifluoroacetylamino)ethylcarbamoyl)-4-(2,4,6-trimethoxyphenyl)pyridine- 2-yl)methyl)-7-((6-(ethoxycarbonyl)-4-(2,4,6-trimethoxyphenyl)pyridin-2-yl)methyl)-1,4, 7-triazonan-1-yl)methyl)-4-(2,4,6-trimethoxyphenyl)pyridine-2-carboxylic acid ethyl ester 19

Compounds 18 (0.39 g, 0.7 mmol) and 14 (0.43 g, 1.4 mmol) were dissolved in anhydrous acetonitrile (20 mL). K ₂ CO ₃ (0.48 g, 3.5 mmol) was added and the mixture was refluxed for 3 hours. The precipitate was filtered off and the solvent was evaporated. _The product was purified on silica gel (10% EtOH/ _CH2Cl2 ). _ESI -MS: [M+H] ⁺ _{1227.4 , Calcd. for C62H74F3N8O15 +} ^1227.5 .

Example 20

Synthesis of 6-((4-((6-(2-aminoethylcarbamoyl)-4-(2,4,6-trimethoxyphenyl)-pyridin-2-yl)methyl)-7- ((6-carboxy-4-(2,4,6-trimethoxyphenyl)pyridin-2-yl)methyl)-1,4,7-triazonan-1-yl)methyl)-4-( Dysprosium (III) 2,4,6-trimethoxyphenyl)pyridine-2-carboxylate 20

Compound 19 was dissolved in 0.1 M potassium hydroxide methanolic solution and stirred at room temperature for 4 hours. All volatile components were removed in vacuo. Treatment of the residue with dysprosium chloride gave the title compound. _ESI - ^MS : [M+H] ⁺ _{1239.1, Calcd. for C56H66DyN8O14 + 1238.4} .

Example 21

Synthesis of 6-((4,7-bis((6-(ethoxycarbonyl)-4-(2,4,6-trimethoxyphenyl)-pyridin-2-yl)methyl)-1,4 ,7-triazonan-1-yl)methyl)-4-(2,4,6-trimethoxyphenyl)-pyridine-2-carboxylic acid ethyl ester 21

1,4,7-Triazacyclononane (31.5 mg) and compound 14 (0.3 g, 0.76 mmol) were dissolved in anhydrous acetonitrile (20 mL). Potassium carbonate (0.17 g) was added and the mixture was refluxed overnight. The mixture was cooled to room temperature, filtered and concentrated. Purification on silica gel (eluent _{CH2Cl2 :} EtOH:HOAc 80:20:1, v/v/v) afforded the title compound (0.17 g, 62%). ESI _- MS: [M+H] ⁺¹¹ 17.5, Calcd. ^for _{C60H73N6O15 + 1117.5} .

Example 22

Synthesis of 6-((4,7-bis((6-carboxy-4-(2,4,6-trimethoxyphenyl)pyridin-2-yl)methyl)-1,4,7-triazonan-1 -yl)methyl)-4-(2,4,6-trimethoxyphenyl)pyridine-2-carboxylate dysprosium (III) 22

Deprotection of compound 21 followed by treatment with dysprosium chloride as described in Example 20 affords the title compound.

Example 23

Synthesis of 6-((4,7-bis((6-carboxy-4-(2,4,6-trimethoxyphenyl)pyridin-2-yl)methyl)-1,4,7-triazonan-1 -yl)methyl)-4-(2,4,6-trimethoxyphenyl)pyridine-2-carboxylic acid terbium (III) 23

Deprotection of compound 21 followed by treatment with terbium chloride as described in Example 20 afforded the title compound.

Example 24

Synthesis of 2-Dimethyl-4-bromo-6-bromomethyl-2-pyridylmethylimino-(diacetate) 24

4-Bromo-2,6-bis(bromomethyl)pyridine (2.66 g, 7.7 mmol) and iminoacetyl dimethyl ether (1.24 g, 7.7 mmol) were dissolved in anhydrous acetonitrile (60 ml )Inside. Potassium carbonate (5.3 g) was added and the mixture was stirred for 40 minutes, then cooled to room temperature, filtered and concentrated. The residue was dissolved in _{dichloromethane} , washed twice with water and dried over _Na2SO4 . Purification on silica gel (eluent petroleum ether (boiling point 40-60°C):ethyl acetate, 10:1-5:1, v/v) afforded the title compound (1.45 g). ESI-MS: [M+H] ⁺ _424.06 , ^calcd _{for C13H17Br2N2O4 + 424.09 .}

Example 25

-{[6-羟基己基亚氨基]-二(亚甲基)双(4-溴)吡啶-6，2-二基)二(亚甲基次氮基)}四(乙酸)四(甲酯)25Synthetic 2, 2', 2", 2

-{[6-Hydroxyhexylimino]-bis(methylene)bis(4-bromo)pyridine-6,2-diyl)bis(methylenenitrilo)}tetra(acetic acid)tetrakis(methyl ester )25

Compound 24 (2.8 g, 6.6 mmol) was dissolved in anhydrous DMF. DIPEA (6.0 mL, 34.0 mmol) and 6-amino-1-hexanol (0.2 g, 3.6 mmol) were added and the reaction mixture was stirred at 60°C for 4 hours and then evaporated to dryness. The residue was dissolved in _CH2Cl2 (30 mL) and washed twice with water _. The organic phase was dried over _Na2SO4 and evaporated to _dryness . The product _was purified by silica gel chromatography (0-3% MeOH in _CH2Cl2 ) to yield 2.4 g (91%) of compound 25. ESI _- MS: [M+H] ⁺ _802.16 , _Calcd . for _{C32H46Br2N5O9} + _802.22 ^.

Example 26

-{[6-(甲氧基三苯甲基氧基己基亚氨基]二(亚甲基)双(4-溴)吡啶-6，2-二基)二(亚甲基次氮基)}四(乙酸)四(甲酯)26Synthetic 2, 2', 2", 2

-{[6-(Methoxytrityloxyhexylimino]bis(methylene)bis(4-bromo)pyridine-6,2-diyl)bis(methylenenitrilo)} Tetra(acetic acid)tetra(methyl ester) 26

Compound 25 (1.0 g, 1.24 mmol) was dissolved in pyridine (30 mL). MMTr chloride (0.57 g, 1.86 mmol) was added and the reaction mixture was stirred at room temperature overnight. The mixture was evaporated to _dryness , the residue was dissolved in _CH2Cl2 and washed with saturated _NaHCO3 . The organic phase was dried over _Na2SO4 and evaporated to _dryness . The product was purified by silica gel chromatography (petroleum ether/AcOEt v/v, 5/1→5/1→1/1) to afford 1.0 g (75%) of compound 26. _ESI -MS: [M+H] ⁺ _1074.28 , Calcd. _{for C52H61Br2N5O10} ⁺ _1074.27 .

Example 27

Synthetic 2, 2', 2", 2 -{[6-(Methoxytrityl)oxyhexylimino]bis(methylene)bis(4-(2,4,6-trimethoxyphenyl)pyridine-6,2-di base) bis (methylene nitrilo)} tetrakis (acetate) tetrakis (methyl ester) 27

The reaction between compound 27 and trimethoxyphenylboronic acid was carried out as described in Example 1 to afford the title compound. The yield was 97%. _ESI -MS _: [M+H] ⁺ 1250.66, _Calcd . ^for _C70H84N5O16 + 1250.59.

Example 28

Synthetic 2, 2', 2", 2 -{[6-(Hydroxyhexylimino)bis(methylene)bis(4-(2,4,6-trimethoxyphenyl)pyridine-6,2-diyl)bis(methylene nitro base)) tetrakis (acetate) tetrakis (methyl ester) 28

Compound 27 (0.8 g, 0.64 mmol) was dissolved in a 5% (v/v) solution of TFA in dichloromethane (16 mL), and the reaction mixture was stirred at room temperature for 3 hours. Methanol (10 mL) was added and the mixture was evaporated to dryness. The residue was dissolved in dichloromethane and washed with saturated NaHCO ₃ . The organic phase was dried over _Na2SO4 and evaporated to _dryness . The product was purified by silica gel chromatography to afford 0.4 g (64%) of compound 28. ESI-MS: [M+H] ⁺ _{978.53 ,} Calcd. for _C50H68N5O15 ⁺ _978.46 .

Example 29

Synthesis of phosphoramidites 29

Compound 28 (0.35 g, 0.36 mmol) was evaporated three times to dryness from anhydrous acetonitrile and dissolved in the same solvent. 2-Cyanoethyltetraisopropylphosphorodiamide (171 μL, 0.54 mmol) and tetrazole (0.45M in acetonitrile; 800 μL, 0.36 mmol) were added and the reaction mixture was shaken at room temperature for 2 hours. The reaction mixture was poured into saturated NaHCO ₃ (5 mL) and stirred vigorously. Dichloromethane _was added, the organic phase was dried over _Na2SO4 and evaporated to dryness. The product was purified by silica gel chromatography (petroleum ether/AcOEt/triethylamine v/v/v, 2/5/1) to afford 0.20 g (47%) of compound 29.

Example 30

Synthetic 2, 2', 2", 2 -{[6-(Hydroxyhexylimino)bis(methylene)bis(4-(2,4,6-trimethoxyphenyl)pyridine-6,2-diyl)bis(methylene nitro base)} tetrakis (acetate) terbium (III) 30

Deprotection of compound 28 followed by treatment with terbium chloride as described in Example 20 gave the title compound. _ESI -MS: [M+H] ⁺ _1076.24 , _Calcd . ^for _{C46H55N5O15Tb-} 1076.30.

Example 31

-{[6-(羟基己基亚氨基]二(亚甲基)双(4-(2，4，6-三甲氧基苯基)吡啶-6，2-二基)二(亚甲基次氮基)}四(乙酸)镝(III)31Synthetic 2, 2', 2", 2

-{[6-(Hydroxyhexylimino)bis(methylene)bis(4-(2,4,6-trimethoxyphenyl)pyridine-6,2-diyl)bis(methylene nitro base)} tetrakis (acetate) dysprosium (III) 31

Deprotection of compound 28 followed by treatment with dysprosium chloride as described in Example 20 gave the title compound. ESI-MS _: [M+H] ⁺ _1081.31 , ^Calcd . for _{C46H55N5O15Dy- 1081.30} .

Example 32

Synthesis of oligopeptides on solid phase using block 4

Using the method described by Peuralahti et al. in Bioconjugate Chem., 13, 2002, 870, compound 4 was used to introduce the lanthanide (III) chelate into the oligopeptide structure. The oligopeptide was therefore synthesized in the conventional manner, reactant 4 being coupled to the amino terminus. Deprotection, conversion to the corresponding lanthanide(III) chelate and purification were performed as described above.

Example 33

Synthesis of oligonucleotides on solid phase using block 29

Compound 29 was used to introduce the lanthanide(III) chelate into the oligonucleotide structure following the method described by Hovinen and Hakala in Org. Lett. 3, 2001, 2473. The oligonucleotides were thus synthesized in a conventional manner, reactant 50 coupled to their 5'-ends. Deprotection, conversion to the corresponding lanthanide(III) chelate and purification were performed as described above.

Example 34

Synthesis of 9-[(trityloxy)methyl]-1,4,7-triazecane 1,4,7-tris-(2-nitrophenyl)sulfonamide 32

2-((trityloxy)methyl)propane-1,3-diol (1.0 mmol), 2-nitrobenzenesulfonyl protected ethylenetriamine (1.0 mmol) and tris Phenylphosphine (3.0 mmol) was dissolved in anhydrous THF (5 mL). DIAD (3.0 mmol) was added in four portions over 15 minutes and reacted overnight at room temperature. All volatile components were removed in vacuo and the residue was precipitated from diethyl ether. The precipitate was redissolved in dichloromethane and _the product was isolated on a silica gel column (0.5% MeOH in _CH2Cl2 as eluent; v/v). ESI-MS: _[ M+H] ⁺ _971.21 , _Calcd . ^for _{C45H43N6O13S3} + _971.20 .

plan 1

Scenario 2

Option 3

Option 4

Option 5

Option 6

Option 7

Table 1. Photochemical properties of some chelates synthesized

Claims (20)

1. A chelating agent, said chelating agent comprising: - a chromophoric moiety comprising two or three pyridyl groups, wherein at least one of the pyridyl groups is substituted by a trialkoxy-substituted phenyl group, wherein the alkoxy groups are the same or different, and the pyridyl groups are separated from each other by containing The hydrocarbon chains of N are tethered together; - a chelating moiety comprising at least two carboxylic or phosphonic acid groups attached directly or via an N-containing hydrocarbon chain to the pyridyl group of the chromophoric moiety, or an ester or salt of said acid; and - a reactive group A selected from the group consisting of isothiocyanate, haloacetamido, maleimide, optionally tethered to the chromogenic moiety or to the chelating moiety directly or via a linker x Amino group, dichlorotriazine group, dichlorotriazine amino group, pyridine dithio group, thioester group, oxygen-containing amino group, hydrazide group, amino group and carboxylic acid or its active ester group, wherein the linker x consists of Formed by 1-10 moieties, each moiety is selected from: phenylene, alkylene group containing 1-12 carbon atoms, -C≡C-, -C=C-, -O-, -S-, -CO-NH-, -CO-NR'-, -NH-CO-, -NR'-CO-, -CO-, -COO-, -OOC-, -SS-, -N=N- and tertiary amines , where R' represents an alkyl group containing less than 5 carbon atoms, or When the chelating agent is suitable for oligopeptide synthesis, the reactive group A is connected to the chelating agent through a linker x, A is an amino acid residue -CH(NHR ¹ )R ⁵ , wherein R ¹ is fluorenyl methyl Oxycarbonyl, tert-butoxycarbonyl or 1,1-dioxobenzo[b]benzenethio-2-ylmethoxycarbonyl, and ^R is carboxylic acid or its salt, acid halide or ester; or When the chelating agent is suitable for oligonucleotide synthesis, the reactive group A is connected to the chelating agent through a linker x, and A is selected from:

Wherein: - is the position of linker x, and DMTr is dimethoxytrityl. 2. The chelating agent of claim 1, wherein the reactive group A is connected to the chelating agent through a linker x. 3. the chelating agent of claim 1, described chelating agent is selected from: Among them: Z ¹ , Z ² and Z ³ are the same or different alkyl groups; R ⁶ is an alkyl ester or allyl ester; R ⁷ is an alkyl group, and n is 0 or 1. 4. The chelating agent of claim 1, which is suitable for oligopeptide synthesis, wherein the reactive group A is connected to the chelating agent through a linker x, and A is an amino acid residue -CH(NHR ¹ ) R ⁵ , wherein ^R is fluorenylmethoxycarbonyl, tert-butoxycarbonyl or 1,1 ^- dioxobenzo[b]benzenethio-2-ylmethoxycarbonyl, and R is carboxylic acid or a salt thereof , acid halides or esters. 5. the chelating agent of claim 4, described chelating agent is selected from:

Wherein: x is as defined in claim ¹ , and the protecting group R is selected from fluorenylmethoxycarbonyl, tert-butoxycarbonyl or 1,1-dioxobenzo[b]phenylthio-2-ylmethoxy carbonyl group, R ⁶ is an alkyl ester or allyl ester, R ⁷ is an alkyl group, Z ¹ , Z ² and Z ³ are the same or different alkyl groups, and n is 0 or 1. 6. the chelating agent of claim 1, said chelating agent is suitable for oligonucleotide synthesis, wherein said reactive group A is connected with said chelating agent by linker x, and A is selected from:

Wherein: - is the position of linker x, and DMTr is dimethoxytrityl. 7. the chelating agent of claim 6, described chelating agent is selected from: Wherein: R ⁶ is alkyl ester or allyl ester, R ⁷ is alkyl, x is as defined in claim 1, A is as defined in claim 6, Z ¹ , Z ² and Z ³ are the same or different The alkyl group, n is 0 or 1. 8. A chelate, said chelate comprising: - lanthanide ions; - a chromophoric moiety comprising two or three pyridyl groups, wherein at least one of the pyridyl groups is substituted by a trialkoxy-substituted phenyl group, wherein the alkoxy groups are the same or different, and the pyridyl groups are separated from each other by containing The hydrocarbon chains of N are tethered together; - a chelating moiety comprising at least two carboxylic or phosphonic acid groups attached directly or via an N-containing hydrocarbon chain to the pyridyl group of the chromophoric moiety, or an ester or salt of said acid; and - a reactive group A selected from the group consisting of isothiocyanate, haloacetamido, maleimide, optionally tethered to the chromogenic moiety or to the chelating moiety directly or via a linker x Amino group, dichlorotriazine group, dichlorotriazine amino group, pyridine dithio group, thioester group, oxygen-containing amino group, hydrazide group, amino group and carboxylic acid or its active ester group, wherein the linker x consists of Formed by 1-10 moieties, each moiety is selected from: phenylene, alkylene group containing 1-12 carbon atoms, -C≡C-, -C=C-, -O-, -S-, -CO-NH-, -CO-NR'-, -NH-CO-, -NR'-CO-, -CO-, -COO-, -OOC-, -SS-, -N=N- and tertiary amines , where R' represents an alkyl group containing less than 5 carbon atoms. 9. The chelate of claim 8, wherein the reactive group A is connected to the chelating agent via a linker x. 10. the chelate of claim 8, described chelate is selected from: Wherein: Z ¹ , Z ² and Z ³ are the same or different alkyl groups; n is 0 or 1; the metal M is a lanthanide. 11. A life molecule, said life molecule is conjugated with any one of claims 8-10, wherein said life molecule is selected from the group consisting of oligopeptides, oligonucleotides, DNA, RNA, modified Sexual oligonucleotides or polynucleotides, proteins, oligosaccharides, polysaccharides, phospholipids, PNA, LNA, antibodies, haptens, drugs, receptor binding ligands and lectins. 12. The life molecule of claim 11, wherein the modified oligonucleotide or polynucleotide is a phosphorothioate, a phosphorodithioate, a phosphoroamidate and/or a sugar modification or an alkali modification oligonucleotides or polynucleotides. 13. A life molecule, said life molecule is conjugated with the chelating agent any one of claims 1-7, wherein said life molecule is selected from oligopeptide, oligonucleotide, DNA, RNA, modified oligonucleotides or polynucleotides, proteins, oligosaccharides, polysaccharides, phospholipids, PNA, LNA, antibodies, haptens, drugs, receptor binding ligands and lectins. 14. A solid support conjugated to the chelate according to any one of claims 8-10, wherein the solid support is selected from microparticles, glass slides or thin plates. 15. A solid carrier conjugated to the chelate according to any one of claims 8-10, wherein the solid carrier is selected from nanoparticles. 16. A labeled oligopeptide, said oligopeptide is introduced into the oligopeptide structure on the oligopeptide synthesizer by the chelating agent of claim 4 or 5, followed by deprotection and optional introduction of metal ions Synthesized on solid phase. 17. A labeled oligonucleotide, said oligonucleotide is introduced into the oligonucleotide structure on the oligonucleotide synthesizer by the chelating agent of any one of claims 6-7 , followed by deprotection and optional introduction of lanthanide ions for synthesis on solid phase. 18. A solid support conjugated with the labeled oligopeptide of claim 16 or the labeled oligonucleotide of claim 17, wherein the oligopeptide or oligonucleotide is covalently or non-covalently immobilized on On the solid carrier, the carrier is selected from particles, glass slides or thin plates. 19. A solid support conjugated with the labeled oligopeptide of claim 16 or the labeled oligonucleotide of claim 17, wherein the oligopeptide or oligonucleotide is covalently or non-covalently immobilized on On the solid carrier, the carrier is selected from nanoparticles. 20. A solid carrier conjugated with the chelating agent of claim 1 that is suitable for oligonucleotide synthesis, wherein the reactive group A is connected with the chelating agent through a linker x, and A is selected from: Wherein: - is the position of linker x, and DMTr is dimethoxytrityl.