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 as well as solid supports conjugated with said chelates, chelating agents or labeled biomolecules. Especially the invention relates to novel chelating agents useful in solid phase synthesis of oligonucleotides or oligopeptides and the oligonucleotides and oligopeptides so obtained.

Description

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

Invention field

The biomolecule that the present invention relates to one group of novel sequestrant, novel inner complex, makes marks with described inner complex or sequestrant and with described inner complex, sequestrant or label biomolecules conjugated solid carrier.

Background of invention

What be used to illustrate background of the present invention herein variously openly reaches other material, particularly provides the case of closing the subsidiary details that relates to enforcement and incorporate this paper by reference into.

Because various lanthanon (III) inner complex has unique luminescent properties, people usually in various routines and scientific research are used used as the non-radioactive marker.Because lanthanon (III) inner complex can send intensive, the aura of long period of decay, thereby they are ideal markers in the demanding analysis of susceptibility.The application of time resolved fluorescence analysis in diagnostics, scientific research and high flux screening based on lanthanide chelate is extensive day by day.Heterogeneous DELFIA

Technology is applied to require [Hemmil in the analysis of special susceptibility, weather resistance and multiple labeling approach

Deng, Anal.Biochem.1984,137,335-343].The succeeding in developing time-resolved purposes extended to based on fluorescence resonance of the stable chelate of highly luminescent can be transmitted the homogeneous analysis [Hemmil that the luminescent properties of inner complex changes during (TR-FRET), fluorescent quenching (TR-FQA) or the association reaction , I.; Mukkala, V.-M.Crit.Rev.Clin.Lab.Sci.2001,38,441-519].

Conjugation reaction is between the amino of bioactive molecules (as protein, peptide, nucleic acid, oligonucleotide or haptens) or different sulphur cyanato (isothiocyanato), the halo ethanoyl, 3 of sulfydryl and lanthanon (III) inner complex in solution in most cases; 5-two chloro-2; 4, carry out between 6-triazinyl derivative and the out of Memory group.Owing to all adopt excessive activation mark to carry out at labeled reactant described in all situations, therefore complicated purge process is inevasible.Particularly in the presence of the similar functional group of several reactivities in the time need adhering to the mark at several tagged molecule or special position, required biomolecule conjugated segregation with characterize extremely difficultly, and normally may accomplish hardly.The liquid phase mark of big biomolecule (as protein) can't be avoided naturally.In these situations, described labeled reactant must have selectivity and validity as much as possible.

Be suitable for the luminous inner complex mark of novel high that various time resolved fluorescences are used in order to develop, people have carried out a large amount of trials.These achievements comprise, stable chelate for example, and [US 4 by the various pyridine derivates as the capacity control group for they, 920,195, US4,801,722, 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 dipyridyl derivatives [US 5,216,134]; Various terpyridyl derivatives [US4,859,777, US 5,202,423, US 5,324, and 825] or various phenolic compound [US4,670,572, US 4,794,191, italian patent 42508 A789] and form as the polycarboxylic acid of chelating integral part.In addition, people also disclose various di-carboxylic acid salt derivatives [US 5,032,677, US 5,055,578, US 4,772,563] encircle kryptofix 222 [US4,927,923, WO 93/5049, EP-A-493745] and big ring Schiff's base [EP-A-369-000], greatly.People also disclose by solid phase synthesis process, with the method [US6,080,839] of luminescent marking substance markers biospecific conglutination reaction agent (as haptens, peptide, receptors ligand, medicine or PNA oligopolymer).People also develop the similar strategy [EP A 1152010, EP A 1308452] of multiple labelling oligonucleotide on solid phase.

Though disclosing, people comprised 2 of aryl-pyridine diacid and aryl replacement, the fluorescent rare earth inner complex [Hemmil of two [N, N-two (carboxyalkyl) aminoalkyl group] pyridine moieties of 6- Deng, J Biochem Biophys Methods 26; 283-90 (1993); US 4,761, and 481], but described herein inner complex of the present invention or sequestrant were not disclosing before this.

Goal of the invention and general introduction

Main purpose of the present invention is to provide the various label biomolecules that can be used for, and is used as the sequestrant and the metallo-chelate thereof of probe in time resolution fluorescence spectral, nuclear magnetic resonance (MRI) or positron emission tomography (PET).

Special purpose of the present invention is to provide the chelating lanthanide ion with different, particularly uses europium (III), samarium (III), terbium (III) and dysprosium (III) to send the very sequestrant of hyperfluorescence.These lanthanide chelates are particularly useful in the high flux screening of biological affinity analyzing of multiparameter and drug candidate.

Another object of the present invention is to provide various sequestrants, obtains the metallo-chelate of high stability.Special purpose be to obtain stability enough strong, can be used in the inner complex in the various application in the body (as in MRI or PET application).

Another object of the present invention is to provide inner complex or the sequestrant that is suitable for label biomolecules in solution.

Another object of the present invention be to provide be suitable for its on solid phase synthetic with the inner complex of tense marker oligopeptide or oligonucleotide.

Another object of the present invention is to provide and inner complex of the present invention, sequestrant or biomolecule conjugated solid carrier.

Therefore, first aspect of the present invention relates to sequestrant, and described sequestrant comprises:

-comprise the color development part of two or more aromatic units, wherein at least one aromatic units is a tri-alkoxy phenyl pyridyl group, wherein said alkoxyl group is identical or different, and described pyridyl is i) directly be tethered in together each other, form bipyridyl or terpyridyl base group respectively; Or ii) be tethered in together by the hydrocarbon chain that contains N each other;

-comprise at least two directly or be attached to carboxylic acid or the ester of phosphonyl group or described acid or the chelating moiety of salt on the aromatic units of color development part by the hydrocarbon chain that contains N; With

-optional directly or by linker x mooring in described color development part or mooring in the reactive group A of chelating moiety, described reactive group A can be bonded on the biomolecule or functional group of solid phase.

Another aspect of the present invention relates to inner complex, and described inner complex comprises:

-metal ion;

-comprise the color development part of two or more aromatic units, wherein at least one aromatic units is a tri-alkoxy phenyl pyridyl group, wherein said alkoxyl group is identical or different, and described pyridyl is i) directly be tethered in together each other, form bipyridyl or terpyridyl base group respectively; Or ii) be tethered in together by the hydrocarbon chain that contains N each other;

-comprise at least two directly or be attached to carboxylic acid or the ester of phosphonyl group or described acid or the chelating moiety of salt on the aromatic units of color development part by the hydrocarbon chain that contains N; With

-optional directly or by linker x mooring in described color development part or mooring in the reactive group A of chelating moiety, described reactive group A can be bonded on the biomolecule or functional group of solid phase.

The 3rd aspect of the present invention relates to and inner complex conjugated biomolecule of the present invention.

The 4th aspect of the present invention relates to and sequestrant conjugated biomolecule of the present invention.

The 5th aspect of the present invention relates to and inner complex of the present invention or label biomolecules conjugated solid carrier.

The 6th aspect of the present invention relates to the oligopeptide of mark; be incorporated in the oligopeptide structure on the oligopeptide synthesizer by the sequestrant that the present invention is suited, then carry out deprotection and the optional metal ion of introducing synthesizes the oligopeptide that obtains described mark on solid phase.

The 7th aspect of the present invention relates to the oligonucleotide of mark; be incorporated in the oligonucleotide structure on the oligonucleotide synthesizer by the sequestrant that the present invention is suited, then carry out deprotection and the optional metal ion of introducing synthesizes the oligonucleotide that obtains described mark on solid phase.

The 8th aspect of the present invention relate to be applicable to oligonucleotide in synthetic, with the sequestrant conjugated solid carrier of claim 1, wherein reactive group A is connected with described sequestrant by linker x, and A is:

-E-O-x’-

Wherein:

X ' is the linker that is connected with solid carrier, and x is identical or different with linker;

E can not exist, or for being applicable to modified oligonucleotides purine or pyrimidine group or any other modification base in synthetic, described base is passed through:

I) hydrocarbon chain that replaces with shielded hydroxyethyl is connected with Sauerstoffatom; Or pass through

Ii) be applicable to modified oligonucleotides in synthetic furan nucleus or the furans or the pyranoid ring of pyranoid ring or any modification be connected with Sauerstoffatom.

Detailed Description Of The Invention

Sequestrant

Based on most sequestrant and the metallo-chelates for the divalent aromatic structure that comprises one or more tri-alkoxy phenyl pyridine groups of the part of color development wherein is novel.Described tri-alkoxy phenyl pyridine group can absorb light or energy, and excitation energy is passed to the lanthanide ion that is chelated, thereby obtains strong fluorescence and irrelevant with used lanthanide ion.Except described tri-alkoxy phenyl pyridine group, chromophore elements also can comprise the unsubstituted pyridine group, contain other substituent pyridine group and/or other aromatic group.

In each illustrational compound of specific embodiment institute, the tri-alkoxy phenyl substituting group is contained in the 4-position of pyridine group herein.Though we believe that this position is most preferred, other position of described pyridine ring also can be used for replacing.

Alkoxy base is preferably the C1-C4 alkoxyl group.

According to embodiment preferred, described color development partly comprises two or three pyridine groups, and wherein at least one is replaced by tri-alkoxy phenyl.These pyridyl can directly be tethered in together each other, form bipyridyl or terpyridyl base group respectively.Perhaps more preferably described pyridyl is tethered in together by the hydrocarbon chain that contains N each other.The chain that the hydrocarbon chain that contains N is interpreted as not containing other heteroatoms except N or does not have aromatic group.Can obtain the good relatively inner complex of stability in this case.The sequestrant of this structure makes and enough the stablizing of metallo-chelate also is suitable for purposes in the body in MRI and/or the various application of PET simultaneously.

Be attached in the situation of color development aromatic units partly at chelating moiety, this chelating moiety can be attached to pyridine ring or the substituting group on it (as phenyl).

For sequestrant or inner complex covalency are bonded on biomolecule or the solid carrier, sequestrant or inner complex must contain reactive group A.But existing wherein this covalency bonding is not requisite application yet.Chelate compound of the present invention also can be used for wherein, and inner complex does not need in the application of reactive group.An example of this technology is at for example Blomberg etc., and J.Immunological Methods illustrates in 1996,193,199.Another example that does not need reactive group A is separating of eosinophil and basophilic cell.In this was used, the inner complex of positive charge and negative charge was bonded in respectively on the cell surface of negative charge and positive charge.

Though reactive group A can directly be attached on chromophoric group or the chelating moiety in many application in principle, it would be desirable to have linker x (particularly owing to the reason on the space) between reactive group A and the chromophoric group or between reactive group A and the chelating moiety respectively.Must be used for the situation of the solid phase synthesis of oligopeptide and oligonucleotide at inner complex, described linker particularly important, but in the solution mark of biomolecule, also be desirable.

According to embodiment preferred, described reactive group A is selected from isosulfocyanate radical, halo kharophen, maleimide amino, dichlorotriazine base, dichlorotriazine amino, pyridine dithio-, thioic acid sulfoacid ester group, contains oxygen amino, hydrazide group, amino, polymer-based group, carboxylic acid or acid halide or its active ester groups.Particularly must be attached in the situation of particulate or nanoparticle, preferably have the reactive group of polymer-based group at described inner complex or sequestrant.Can in the particulate manufacturing processed, marker be introduced in the particle in this case.

Described linker x is preferably formed by 1-10 part, each part is selected from: phenylene, the alkylidene group that contains 1-12 carbon atom, acetylene two bases (C ≡ C-), ethene two bases (C=C-), ether (O-), thioether group (S-), amide group (CO-NH-,-CO-NR '-, NH-CO and-NR '-CO-), carbonyl (CO-), ester group (COO-and-OOC-), (SS-), (N=N-) and tertiary amine, wherein R ' representative contains the alkyl that is less than 5 carbon atoms to diaza to disulphide.

According to particularly preferred embodiment, described sequestrant is a kind of in the following ad hoc structure:

Wherein: Z ¹, Z ²And Z ³Be identical or different alkyl; R ⁶Be alkyl ester or allyl ester; R ⁷Be alkyl, n is 0 or 1.

Be used for the synthetic sequestrant of peptide

According to an embodiment preferred, sequestrant of the present invention is applicable to the synthetic of oligopeptide.In this was used, reactive group A was connected with described sequestrant by linker x, and A is amino-acid residue-CH (NHR ¹) R ⁵, R wherein ¹Be protection from transients base, R ⁵Be carboxylic acid or its salt, acyl halide or ester.Particularly preferred sequestrant is following structure:

Wherein: x as defined above, protecting group R ¹Be selected from Fmoc (fluorenyl methoxy carbonyl), Boc (tert-butoxycarbonyl) or Bsmoc (1,1-two oxygenate benzo [b] benzene sulphur-2-ylmethoxy carbonyls), R ⁶Be alkyl ester or allyl ester, R ⁷Be alkyl, Z ¹, Z ²And Z ³Be identical or different alkyl, n is 0 or 1.

Sequestrant can be introduced in the biomolecule by means of peptide synthesizer.For example can make the solid carrier or the immobilization amino acid coupling (that is to say that in the presence of activator the carboxylic acid functional of labelled reagent and solid carrier or amino acid whose amino group react) of sequestrant and amino mooring by the carbodiimide chemistry.When condensation step was finished, the transition amino protecting group of labelled reagent was optionally shifted out, and described material still is attached to (as being attached on the solid carrier by piperidines) on the solid carrier in the situation of Fmoc-protecting group.Then as the above-mentioned coupling second time of carrying out sequestrant or other reagent (amino acid, haptens).After synthetic the finishing of desired molecule, material is removed and carries out deprotection from solid carrier.Can carry out purifying by the HPLC technology.The part of lanthanon (III) ion by adding known quantity after with purifying is converted into corresponding lanthanon (III) inner complex at last.

Be used for oligonucleotide synthetic sequestrant

According to another embodiment preferred, sequestrant of the present invention is applicable to the synthetic of oligonucleotide.Reactive group A is connected with sequestrant by linker x in this case, and A is:

-E-O-PZ-O-R ⁴

Wherein:

Choose wantonly for one in the Sauerstoffatom and replaced by sulphur, Z is chlorine or NR ²R ³, R ⁴Be protecting group, R ²And R ³Be alkyl, E do not exist or for be applicable to modified oligonucleotides in synthetic purine bases or the base of pyrimidine bases or any other modification.Described alkali passes through: i) hydrocarbon chain that replaces with shielded hydroxyethyl is connected with Sauerstoffatom; By ii) be applicable to modified oligonucleotides in synthetic furan nucleus or the furans or the pyranoid ring of pyranoid ring or any modification be connected with Sauerstoffatom.

Sequestrant can be introduced in the oligonucleotide by means of the oligonucleotide synthesizer.One based on Mitsonobu alkylated reaction (J Org Chem, 1999,64,5083; Nucleosides, Nucleotides, 1999,18,1339) method be disclosed among the EP-A-1152010.Described patent disclosure a kind ofly the conjugation group of desired number directly is attached to the structural method of oligonucleotide at the chain shaping.Thereby liquid phase mark and complicated purge process have been avoided.Committed step in the nucleosides oligonucleotide structural unit synthesis strategy is that various sequestrants are introduced into nucleosides, is introduced into the above-mentioned Mitsunobu alkylated reaction of described oligonucleotide structure at last.Between the chain shaping period, introduce sequestrant.The reaction of lanthanide chelate has taken place to be converted in synthetic back during deprotection steps.

Because the enzyme that unmodified oligonucleotide is present in the viable cell is degraded, therefore its stability is lower under physiological condition under the normal circumstances.Thereby the oligonucleotide that generates modification according to known method is to strengthen it and resist chemical degradation and enzyme liberating is that everybody is wished.Various modifications about oligonucleotide in prior art have a large amount of disclosing.Referring to US 5,612,215.We know that removing or replace 2 '-OH group in the RNA chain from ribose will make its stability better.WO 92/07065 and US 5,672,695 disclose employing halogen, amino, azido-or sulfydryl displacement ribose 2 '-OH group.US 5,334, and 711 disclose the hydrogen in alkyl or alkenyl (preferable methyl or allyl group) the displacement 2 '-OH group.In addition, for example can carry out modification, thereby one or more oxygen is replaced by sulphur, amino, alkyl or alkoxyl group bonding between the nucleotide phosphodiesterase diester.In internucleotide linkage, preferably be modified as the thiophosphoric acid bonding.Also can carry out modification to the alkali in the Nucleotide.

Preferred E is the group of any following alkali: thymus pyrimidine, uridylic, adenosine, guanine or cytosine(Cyt), and described alkali passes through: i) hydrocarbon chain that replaces with shielded hydroxyethyl is connected with Sauerstoffatom; Or by ii) having shielded hydroxyethyl on its 4-position and choosing the furan nucleus that on its 2-position, has the hydroxyl of hydroxyl, hydroxyl and protected or modification wantonly and be connected with Sauerstoffatom.

Preferred reaction base-E-O-P (NR ²R ³)-O-R ⁴Structure be selected from one of following structure:

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

Particularly preferred sequestrant is selected from a kind of in the ad hoc structure of following discloses:

Wherein: R ⁶Be alkyl ester or allyl ester, R ⁷Be alkyl, x as defined above, A for as defined above-E-O-P (NR ²R ³)-O-R ⁴, Z ¹, Z ²And Z ³Be identical or different alkyl, n is 0 or 1.

Inner complex

Described inner complex comprises above-mentioned sequestrant and the metal ion that is chelated.

Be used for the situation of biological affinity analyzing at inner complex, the metal ion that is chelated is preferably lanthanon, especially europium (III), samarium (III), terbium (III) or dysprosium (III).Sequestrant is preferably a kind of in the above-mentioned preferred sequestrant.

Particularly preferred lanthanide chelate is:

Wherein: Z ¹, Z ²And Z ³Be identical or different alkyl, n is 0 or 1.

Inner complex of the present invention also can be used in interior MRI application of body or the PET application.The preferable alloy that is used for MRI is a gadolinium.In PET uses, before being about to use, the radioactive metal isotropic substance is introduced in the sequestrant.Shi Yi radio isotope is 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 especially.In order to obtain the inner complex of quite stable, preferably have several pyridine groups wherein and be tethered in together color development part by the hydrocarbon chain that contains N each other.

Biomolecule

Be preferably the oligonucleotide of oligopeptide, oligonucleotide, DNA, RNA, modification or oligonucleotide or polynucleotide, protein, oligosaccharides, polysaccharide, phosphatide, PNA, LNA, antibody, haptens, medicine, the bonding part of acceptor and the Sugar receptors of polynucleotide (as monothio phosphoric acid ester, phosphorodithioate, phosphoramidate) and/or sugared modification or alkali modification with sequestrant of the present invention or inner complex conjugated biomolecule.

The solid carrier conjugate

But the equal conjugation of inner complex of the present invention, sequestrant and biomolecule is to solid carrier.Described solid carrier is preferably particle (as particulate or nanoparticle), slide or thin plate.

Have in the situation of polyradical as reactive group at inner complex or sequestrant, can in preparation solid carrier (as particle), inner complex or sequestrant be introduced wherein.

With described solid carrier covalently or non-covalently the conjugated biomolecule be preferably the oligopeptide that mark is crossed; by sequestrant being incorporated in the oligopeptide structure on the oligopeptide synthesizer, then carrying out the also optional introducing metal ion of deprotection and on solid phase, synthesize the oligopeptide that obtains described mark.Perhaps; with described solid carrier covalently or non-covalently the conjugated biomolecule be preferably the oligonucleotide that mark is crossed; by sequestrant being incorporated in the oligonucleotide structure on the oligonucleotide synthesizer, then carrying out the also optional introducing metal ion of deprotection and on solid phase, synthesize the oligonucleotide that obtains described mark.

Be applicable to the synthetic of oligonucleotide with sequestrant conjugated solid carrier with reactive group A of being connected with sequestrant by linker x (A be aforesaid-E-O-x '-).

Below will be illustrated the present invention by non-restrictive example.

Embodiment

The present invention will illustrate by each following embodiment.Used structure and synthetic route are seen and are set forth among the scheme 1-7 in experimental section.Synthesizing of scheme 1 explanation oligopeptide labeling reaction 4.In embodiment 1-4, provided each experimental detail.Scheme 2 explanation inner complex 6-11's is synthetic.In embodiment 6-11, provided each experimental detail.Scheme 3 illustrates the synthetic of inner complexs 20,22 and 23.In embodiment 12-23, provided each experimental detail.Scheme 4 explanations are used for lanthanide chelate is introduced into the synthesizing of structural unit 29 and the synthesizing of inner complex 30 and 31 of oligonucleotide on the solid phase.In embodiment 24-31, provided each experimental detail.Scheme 5 and 6 difference description architecture unit 4 and 29 prepare the purposes of synthesis of oligonucleotides peptide and oligonucleotide on solid phase.In embodiment 32 and 33, provided each experimental detail.Scheme 7 illustrates based on 1,4, the preparation of the oligonucleotide labelled reagent of 7-triazecane.In embodiment 34, provided each experimental detail.

Every photochemical properties of institute's each inner complex of synthetic is summarized in the table 1 in exemplary embodiment.

Experimental technique

Be used for the auxiliary oligopeptide synthetic of machine all ingredients available from AppliedBiosystems (Foster City, CA).The adsorpting column chromatogram method is carried out on the pillar that silica gel 60 (Merck) is housed.Exist respectively ¹Record NMR spectrum on the Brucker 250 of H 250.13 and the operation of 399.8MHz place or the Jeol LA-400 spectrometer.Be designated as Me in used ₄Si.The unit of coupling constant is Hz.Record IR spectrum on Perkin Elmer 2000 FT-IR spectrophotometers.Record electrospray mass spectrum on Applied Biosystems Mariner ESI-TOF instrument.The testing program of adopt recommending, synthesis of oligonucleotides peptide on Applied Biosystems 433A synthesizer, and on Applied Biosystems Expedite instrument synthesis of oligonucleotides Nucleotide.On PerkinElmer LS 55 instrument, write down fluorescence spectrum.

Carry out the synthetic of each compound as the general introduction in following scheme 1-7.

Embodiment 1

Synthetic four (tertiary butyl) 2,2 ', 2 ", 2

-{ [the amino hexyl-imino-of 6-N-(4-methoxyl group trityl)] two (methylene radical) two [4-(2,4, the 6-trimethoxyphenyl) pyridine-6,2-one or two bases] two (methylene radical nitrilos) } four (acetic ester) 1

With four (tertiary butyl) 2,2 ', 2 ", 2

-[6-N-(4-methoxyl group trityl) hexyl imino-] two (methylene radical) two-(4-bromopyridine-6,2-two bases) two (methylene radical nitrilos) } four (acetic ester) (4.0 grams, 2.4 mmole) and the trimethoxy-benzene ylboronic acid (1.1 the gram, 5.3 mmole) be dissolved in the dry DMF (50 milliliters), add Cs ₂CO ₃(2.0 grams, 6.0 mmoles) and Pd (PPh ₃) ₄(0.1 gram, 96 micromoles).After 95 ℃ stirring is spent the night down, add trimethoxy-benzene ylboronic acid (0.5 gram, 2.4 mmoles), Cs ₂CO ₃(0.79 gram, 2 mmoles) and Pd (PPh ₃) ₄(50 milligrams, 43 mmoles).After reaction is spent the night mixture is cooled to room temperature, filters and evaporation.Mixture is dissolved in CH ₂Cl ₂In, water (240 milliliters) washing.(silica gel, sherwood oil (40-60 ℃)/AcOEt/TEA 5: 2: 1 v/v/v) carries out purifying to product by flash chromatography.Yield is 3.1 grams (90%).IR (film): 1737 (C=O), 1128 (C-O). ¹H?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, C ₈₂H ₁₀₉N ₆O ₁₅ ⁺Calculated value be 1417.8.

Embodiment 2

Synthetic four (tertiary butyl) 2,2 ', 2 ", 2

-{ (the amino hexyl imino-of 6-) two (methylene radical)-two [4-(2,4, the 6-trimethoxyphenyl) pyridine-6,2-two bases] two (methylene radical nitrilos) } four (acetic ester) 2

Compound 1 (1.0 grams, 0.7 mmole) is dissolved in the methylene dichloride (25 milliliters), adds trifluoroacetic acid (0.25 milliliter).After stirring 4 hours at ambient temperature, use saturated NaHCO ₃(250 milliliters) purging compound.Organic phase is at Na ₂SO ₄Last drying, filtration and evaporation.(silica gel, sherwood oil (40-60 ℃)/AcOEt/TEA 5: 5: 1,2: 5: 1 is CH to product at last by flash chromatography ₂Cl ₂In 10%MeOH, 1% TEA) carry out purifying.Yield is 0.60 gram (74%).IR (film): 1730 (C=O), 1128 (C-O).ESI-MS:[M+H] ⁺1145.7, C ₈₂H ₁₀₉N ₆O ₁₅ ⁺Calculated value be 1145.7, [M+2H] ²⁺573.3 calculated value is 573.3.

Embodiment 3

The oligopeptide labeling reaction 3 of synthesis of allyl protection

Compound 2 (0.55 gram, 0.48 mmole) is dissolved in the anhydrous methylene chloride (5 milliliters).Add DCC (0.11 gram, 0.53 mmole) and Fmoc-Glu-OAII (0.20 gram, 0.48 mmole), mixture at room temperature stirs and spends the night.The DCU that elimination generates, concentrated filtrate under vacuum.On silica gel, carry out purifying (10%MeOH in the methylene dichloride), obtain the title compound (300 milligrams) of solid state.ESI-MS:[M+H] ⁺1536.8, C ₈₅H ₁₁₄N ₇O ₁₉ ⁺Calculated value be 1536.8.

Embodiment 4

The peptide-labeled reactant 4 of synthesis of oligonucleotides

Compound 3 (157 milligrams, 0.1 mmole) is dissolved in the anhydrous methylene chloride (2 milliliters).Add Pd (Ph ₃P) ₄(2.3 milligrams) and PhSiH ₃(25 microlitre), mixture stir at ambient temperature and spend the night.Reaction mixture is with the washing of 10% aqueous citric acid solution then, and dry on molecular sieve.Yield is 95 milligrams (63%).ESI-MS:[M+H] ⁺1496.8, C ₈₂H ₁₁₀N ₇O ₁₉ ⁺Calculated value be 1496.8.

Embodiment 5

Synthetic free acid 5

Compound 1 (0.40 gram, 0.28 mmole) is dissolved in the trifluoroacetic acid (10 milliliters), at room temperature stirs after 1 hour concentrated.Resistates is developed with diethyl ether.Filter and collect product and dry.Yield is 260 milligrams (100%).ESI-MS:[M+H] ⁺921.42, C ₄₆H ₆₁N ₆O ₁₄ ⁺Calculated value be 921.4.

Embodiment 6

Synthetic terbium inner complex 6

Compound 5 (78 milligrams, 0.085 mmole) is dissolved in the water (2 milliliters), is to add terbium chloride (III) (35 milligrams, 0.093 mmole) 6.5 times in 15 minutes at pH.Make the pH of reaction mixture rise to 8.5 by the NaOH that adds 1M after at room temperature 2 hours.The centrifugal precipitation that is generated of removing concentrates water, uses the acetone precipitation product.ESI-MS:[M+H] ⁺1075.9, C ₄₆H ₅₅N ₆O ₁₄Tb ^-Calculated value be 1075.3.

Embodiment 7

Synthetic dysprosium inner complex 7

Synthesize as embodiment 6, but that employing is Dysprosium trichloride (III).ESI-MS:[M+H] ⁺1080.3, C ₄₆H ₅₅N ₆O ₁₄Dy ^-Calculated value be 1080.2.

Embodiment 8

Synthetic europium inner complex 8

Synthesize as embodiment 6, but that employing is Europium trichloride (III).ESI-MS:[M+H] ⁺1092.3, C ₄₆H ₅₅N ₆O ₁₄Eu ^-Calculated value be 1092.3.

Embodiment 9

The amino activatory dysprosium of synthetic iodacetyl inner complex 9

Compound 7 (16 milligrams, 14.3 micromoles) is dissolved in the water.Adding iodoacetic acid acid anhydride (51.3 milligrams, 0.145 mmole; The chloroform that is dissolved in 0.2 milliliter in advance is interior) and DIPEA (25 microlitre), mixture at room temperature stirred 1.5 hours.Remove organic phase, make product and aqueous phase separation by being precipitated out from THF.ESI-MS:[M+H] ⁺1248.2, C ₄₈H ₅₇N ₆O ₁₅IDy ^-Calculated value be 1248.2.

Embodiment 10

The amino activatory terbium of synthetic iodacetyl inner complex 10

As described in embodiment 9, compound 6 is activated, obtain compound 10.ESI-MS:[M+H] ⁺1243.8, C ₄₈H ₅₇N ₆O ₁₅ITb ^-Calculated value be 1243.8.

Embodiment 11

Synthetic different sulphur cyanato activatory europium inner complex 11

Compound 8 (15 milligrams, 0.014 mmole) is dissolved in mixture (200 microlitres of pyridine, water and triethylamine; 9: 1.5: 0.1; V/v/v) in.Add 1,4-phenylene diisothio-cyanate (7.9 milligrams), mixture at room temperature stirred 4 hours.

Embodiment 12

Synthetic 4-(2,4, the 6-trimethoxyphenyl) pyridine-2,6-diethyl dicarboxylate 12

With 2,4,6-trimethoxy-benzene ylboronic acid (2.12 grams, 10.0 mmoles) and 4-bromopyridine-2,6-diethyl dicarboxylate (3.33 grams, 11.0 mmoles) is dissolved in the dry DMF (50 milliliters).Add cesium carbonate (4.56 grams, 14.0 mmoles) and four (triphenylphosphine)-palladiums (0) (0.23 gram, 0.20 mmole), mixture outgases with argon.Mixture heated 48 hours down at 95 ℃.Mixture is cooled to room temperature, filters then.Concentrated filtrate under the vacuum is dissolved in resistates in the chloroform (60 milliliters), and aqueous citric acid solution with 10% and water washing are at Na ₂SO ₄Last dry and concentrated.(elutriant is a sherwood oil, boiling point 40-60 ℃ to carry out purifying on silica gel; Ethyl acetate, 5: 3 → 2: 5, v/v).Yield is 2.09 grams (54%). ¹HNMR(CDCl ₃)：δ1.45(6H，t，J?7.1)；3.74(6H，s)；3.90(3H，s)；4，49(4H，q，J?7.1)；6.22(2H，s)；8.28(2H，s)。IR (film)/cm ^-11743,1610 (C=O); 1339,1238,1128 (C-O).ESI-MS:[M+H] ⁺390.19, C ₂₀H ₂₄NO ₇ ⁺Calculated value be 390.15.

Embodiment 13

Synthetic 4-(2,4, the 6-trimethoxyphenyl)-6-(hydroxymethyl) pyridine-2-carboxylic acids ethyl ester 13

Compound 12 (2.83 gram, 7.27 mmoles) is suspended in the ethanol (140 milliliters) mixture heating up to 45 ℃.Add sodium borohydride (0.29 gram), mixture stirred 1 hour, and was cooled to room temperature.HCl with 6M is also concentrated with the pH regulator to 3 of solution.Resistates is suspended in the methylene dichloride, with saturated NaHCO ₃Washing.Organic layer is at Na ₂SO ₄Last dry, and on silica gel purifying (elutriant is sherwood oil (boiling point 40-60 a ℃): ethyl acetate: triethylamine, 2: 5: 1; V/v/v).ESI-MS:[M+H] ⁺348.14, C ₁₈H ₂₂NO ₆ ⁺Calculated value be 348.14.

Embodiment 14

Synthetic 4-(2,4, the 6-trimethoxyphenyl)-6-(brooethyl) pyridine-2-carboxylic acids ethyl ester 14

Phosphorus trichloride (0.778 gram, 2.87 mmoles) is dissolved in 0 ℃ the dry DMF (10 milliliters).Add compound 13 (1.0 grams, 2.8 mmoles), mixture at room temperature stirred 3.5 hours, used saturated NaHCO then ₃Neutralization.Use the dichloromethane extraction mixture.Organic phase is carried out drying, is concentrated and purifying (elutriant adopts 1% ethanol in the methylene dichloride) on silica gel.ESI-MS:[M+H] ⁺410.10, C ₁₈H ₂₁BrNO ₅ ⁺Calculated value be 410.05.

Embodiment 15

Synthetic N-(2-(2,2, the 2-trifluoroacetamido) ethyl)-6-(hydroxymethyl)-4-(2,4, the 6-trimethoxyphenyl) pyridine-2-acid amides 15

Compound 13 (1.0 grams, 2.8 mmoles) is dissolved in the quadrol (10 milliliters), at room temperature stirred 2.5 hours and concentrated in (oil pump).Resistates is dissolved among the DMF (25 milliliters), adds Trifluoroacetic Acid Ethyl Ester (5 milliliters).After at room temperature 2 hours, remove all volatiless in a vacuum, resistates is purifying (elutriant is the 10%MeOH in the methylene dichloride) on silica gel.ESI-MS:[M+H] ⁺458.14, C ₂₀H ₂₃F ₃N ₃O ₆ ⁺Calculated value be 458.15.

Embodiment 16

Synthetic N-(2-(2,2, the 2-trifluoroacetamido) ethyl)-6-(brooethyl)-4-(2,4, the 6-trimethoxyphenyl) pyridine-2-acid amides 16

As described in embodiment 14, compound 15 is carried out the bromination processing, obtain title compound.ESI-MS:[M+H] ⁺520.06, C ₂₀H ₂₂BrF ₃N ₃O ₅ ⁺Calculated value be 520.07.

Embodiment 17

Synthetic 7-((6-(2-(2,2, the 2-trifluoroacetamido) ethylamino formyl radical)-4-(2,4, the 6-trimethoxyphenyl) pyridine-2-yl) methyl)-1,4,7-triazonane-1,4-dicarboxylic acid di-t-butyl ester 17

With [1,4,7] 7-triazacyclononane-1,4-dicarboxylic acid di-t-butyl ester (0.75 gram, 2.3 mmoles) and compound 16 (2.3 mmole) are dissolved in the dry DMF (60 milliliters).The DIPEA (11.4 mmole) that adds 2.0 milliliters, mixture at room temperature stirs and spends the night.To doing, product is purifying (elutriant is a diethyl ether) on silica gel with solvent evaporation.Yield is 1.20 grams.ESI-MS:[M+H] ⁺769.34, C ₃₆H ₅₂F ₃N ₆O ₉ ⁺Calculated value be 769.37.

Embodiment 18

Synthetic 6-((1,4, the 7-triazonan-1-yl) methyl)-N-(2-(2,2, the 2-trifluoroacetamido)-ethyl)-4-(2,4, the 6-trimethoxyphenyl) pyridine-2-acid amides 18

Compound 17 (1.0 grams, 1.3 mmoles) is dissolved in the trifluoroacetic acid (25 milliliters), and mixture at room temperature stirred 30 minutes.Solvent evaporation is extremely done.ESI-MS:[M+H] ⁺569.28, C ₂₆H ₃₆F ₃N ₆O ₅ ⁺Calculated value be 569.27.

Embodiment 19

(((((2-(2 for 6-for 4-for synthetic 6-; 2; the 2-trifluoroacetamido) ethylamino formyl radical)-and 4-(2,4, the 6-trimethoxyphenyl) pyridine-2-yl) methyl)-7-((6-(ethoxy carbonyl)-4-(2; 4; the 6-trimethoxyphenyl) methyl pyridine-2-yl))-1,4, the 7-triazonan-1-yl) methyl)-4-(2; 4, the 6-trimethoxyphenyl) pyridine-2-carboxylic acids ethyl ester 19

Compound 18 (0.39 gram, 0.7 mmole) and 14 (0.43 gram, 1.4 mmoles) are dissolved in the anhydrous acetonitrile (20 milliliters).Add K ₂CO ₃(0.48 gram, 3.5 mmoles), mixture refluxed 3 hours.The elimination precipitation, evaporating solvent.Product is purifying (10%EtOH/CH on silica gel ₂Cl ₂).ESI-MS:[M+H] ⁺1227.4, C ₆₂H ₇₄F ₃N ₈O ₁₅ ⁺Calculated value be 1227.5.

Embodiment 20

Synthetic 6-((4-((6-(2-aminoethylamino formyl radical)-4-(2; 4; the 6-trimethoxyphenyl)-and pyridine-2-yl) methyl)-7-((6-carboxyl-4-(2; 4; the 6-trimethoxyphenyl) methyl pyridine-2-yl))-1,4, the 7-triazonan-1-yl) methyl)-4-(2; 4, the 6-trimethoxyphenyl) pyridine-2-carboxylic acids dysprosium (III) 20

Compound 19 is dissolved in the potassium hydroxide methanol solution of 0.1M, and at room temperature stirred 4 hours.Remove all volatiless in a vacuum.Handle resistates with Dysprosium trichloride and obtain title compound.ESI-MS:[M+H] ⁺1239.1, C ₅₆H ₆₆DyN ₈O ₁₄ ⁺Calculated value be 1238.4.

Embodiment 21

Synthetic 6-((4, two ((6-(ethoxy carbonyl)-4-(2,4, the 6-trimethoxyphenyl)-pyridine-2-yl) methyl)-1,4 of 7-, 7-triazonan-1-yl) methyl)-4-(2,4, the 6-trimethoxyphenyl)-pyridine-2-carboxylic acids ethyl ester 21

With 1,4,7-7-triazacyclononane (31.5 milligrams) and compound 14 (0.3 gram, 0.76 mmole) are dissolved in the anhydrous acetonitrile (20 milliliters).Add salt of wormwood (0.17 gram), mixture refluxes and spends the night.Make mixture be cooled to room temperature, filter and concentrate.(the elutriant CH of purifying on silica gel ₂Cl ₂: EtOH: HOAc is 80: 20: 1, v/v/v), obtains title compound (0.17 gram, 62%).ESI-MS:[M+H] ⁺11 17.5, C ₆₀H ₇₃N ₆O ₁₅ ⁺Calculated value be 1117.5.

Embodiment 22

Synthetic 6-((4, two ((6-carboxyl-4-(2,4, the 6-trimethoxyphenyl) pyridine-2-yl) methyl)-1,4 of 7-, 7-triazonan-1-yl) methyl)-4-(2,4, the 6-trimethoxyphenyl) pyridine-2-carboxylic acids dysprosium (III) 22

Compound 21 is carried out deprotection, as described in embodiment 20, handle then, obtain title compound with Dysprosium trichloride.

Embodiment 23

Synthetic 6-((4, two ((6-carboxyl-4-(2,4, the 6-trimethoxyphenyl) pyridine-2-yl) methyl)-1,4 of 7-, 7-triazonan-1-yl) methyl)-4-(2,4, the 6-trimethoxyphenyl) pyridine-2-carboxylic acids terbium (III) 23

Compound 21 is carried out deprotection, as described in embodiment 20, handle then, obtain title compound with terbium chloride.

Embodiment 24

Synthetic 2-dimethyl-4-bromo-6-brooethyl-2-pyridylmethyl imino--(diacetate esters) 24

With 4-bromo-2,6-two (brooethyl) pyridine (2.66 gram, 7.7 mmoles) and imido acetyl dme (1.24 restrain 7.7 mmoles) are dissolved in 60 ℃ the anhydrous acetonitrile (60 milliliters).Add salt of wormwood (5.3 gram), mixture stirred 40 minutes, was cooled to room temperature then, filtered and concentrated.Resistates is dissolved in the methylene dichloride, washes with water twice and at Na ₂SO ₄Last dry.Purifying on silica gel (elutriant is sherwood oil (boiling point 40-60 a ℃): ethyl acetate, and 10: 1-5: 1, v/v), obtain title compound (1.45 gram).ESI-MS:[M+H] ⁺424.06, C ₁₃H ₁₇Br ₂N ₂O ₄ ⁺Calculated value be 424.09.

Embodiment 25

Synthetic 2,2 ', 2 ", 2

-[6-hydroxyl hexyl imino-]-two (methylene radical) two (4-bromine) pyridine-6,2-two bases) and two (methylene radical nitrilos) } four (acetate) four (methyl esters) 25

Compound 24 (2.8 grams, 6.6 mmoles) is dissolved in the dry DMF.Add DIPEA (6.0 milliliters, 34.0 mmoles) and 6-amino-1-hexanol (0.2 gram, 3.6 mmoles), reaction mixture stirred 4 hours down at 60 ℃, was evaporated to dried then.Resistates is dissolved in CH ₂Cl ₂In (30 milliliters), wash with water twice.Organic phase is at Na ₂SO ₄Last dry, and be evaporated to dried.Product is by silica gel chromatography purifying (CH ₂Cl ₂The MeOH of middle 0-3%), obtain the compound 25 of 2.4 grams (91%).ESI-MS:[M+H] ⁺802.16, C ₃₂H ₄₆Br ₂N ₅O ₉ ⁺Calculated value be 802.22.

Embodiment 26

Synthetic 2,2 ', 2 ", 2

-[6-(methoxyl group trityl oxygen base hexyl imino-] two (methylene radical) two (4-bromine) pyridine-6,2-two bases) two (methylene radical nitrilos) } four (acetate) four (methyl esters) 26

Compound 25 (1.0 grams, 1.24 mmoles) is dissolved in the pyridine (30 milliliters).Add chlorination MMTr (0.57 gram, 1.86 mmoles), reaction mixture at room temperature stirs and spends the night.Mixture is evaporated to dried, and resistates is dissolved in CH ₂Cl ₂, use saturated NaHCO ₃Washing.Organic phase is at Na ₂SO ₄Last dry, and be evaporated to dried.Product obtains the compound 26 of 1.0 grams (75%) by silica gel chromatography purifying (sherwood oil/AcOEt v/v, 5/1 → 5/1 → 1/1).ESI-MS:[M+H] ⁺1074.28, C ₅₂H ₆₁Br ₂N ₅O ₁₀ ⁺Calculated value be 1074.27.

Embodiment 27

Synthetic 2,2 ', 2 ", 2 -{ [6-(methoxyl group trityl) oxygen base hexyl imino-] two (methylene radical) two (4-(2,4, the 6-trimethoxyphenyl) pyridine-6,2-two bases) two (methylene radical nitrilos) } four (acetate) four (methyl esters) 27

As carrying out the reaction between compound 27 and the trimethoxy-benzene ylboronic acid as described in the embodiment 1, obtain title compound.Yield is 97%.ESI-MS:[M+H] ⁺1250.66, C ₇₀H ₈₄N ₅O ₁₆ ⁺Calculated value be 1250.59.

Embodiment 28

Synthetic 2,2 ', 2 ", 2 -[6-(hydroxyl hexyl imino-] two (methylene radical) two (4-(2,4, the 6-trimethoxyphenyl) pyridine-6,2-two bases), two (methylene radical nitrilos)) four (acetate) four (methyl esters) 28

Compound 27 (0.8 gram, 0.64 mmole) is dissolved in TFA in 5% (v/v) of methylene dichloride (16 milliliters) solution, and reaction mixture at room temperature stirred 3 hours.Add methyl alcohol (10 milliliters), mixture is evaporated to dried.Resistates is dissolved in the methylene dichloride, uses saturated NaHCO ₃Washing.Organic phase is at Na ₂SO ₄Last drying also is evaporated to dried.Product obtains the compound 28 of 0.4 gram (64%) by the silica gel chromatography purifying.ESI-MS:[M+H] ⁺978.53, C ₅₀H ₆₈N ₅O ₁₅ ⁺Calculated value be 978.46.

Embodiment 29

Synthetic phosphoramidite 29

Compound 28 (0.35 gram, 0.36 mmole) is evaporated to for three times dried from anhydrous acetonitrile, and is dissolved in the identical solvent.Add 2-cyanoethyl tetraisopropylphosph-ro phosphorus diamide (171 microlitres, 0.54 mmole) and tetrazolium (0.45M acetonitrile solution; 800 microlitres, 0.36 mmole), reaction mixture at room temperature vibrates 2 hours.Pour reaction mixture into saturated NaHCO ₃In (5 milliliters), and vigorous stirring.Add methylene dichloride, organic phase is at Na ₂SO ₄Last drying also is evaporated to dried.Product obtains the compound 29 of 0.20 gram (47%) by silica gel chromatography purifying (sherwood oil/AcOEt/ triethylamine v/v/v, 2/5/1).

Embodiment 30

Synthetic 2,2 ', 2 ", 2 -[6-(hydroxyl hexyl imino-] two (methylene radical) two (4-(2,4, the 6-trimethoxyphenyl) pyridine-6,2-two bases), two (methylene radical nitrilos) } four (acetate) terbium (III) 30

Compound 28 is carried out deprotection, as described in embodiment 20, obtain title compound then with the terbium chloride processing.ESI-MS:[M+H] ⁺1076.24, C ₄₆H ₅₅N ₅O ₁₅Tb ^-Calculated value be 1076.30.

Embodiment 31

Synthetic 2,2 ', 2 ", 2

-[6-(hydroxyl hexyl imino-] two (methylene radical) two (4-(2,4, the 6-trimethoxyphenyl) pyridine-6,2-two bases), two (methylene radical nitrilos) } four (acetate) dysprosium (III) 31

Compound 28 is carried out deprotection, as described in embodiment 20, obtain title compound then with the Dysprosium trichloride processing.ESI-MS:[M+H] ⁺1081.31, C ₄₆H ₅₅N ₅O ₁₅Dy ^-Calculated value be 1081.30.

Embodiment 32

Adopt block 4 synthesis of oligonucleotides peptide on solid phase

Employing Peuralahti etc. are at Bioconjugate Chem., and the method described in 13,2002,870 is incorporated into lanthanon (III) inner complex in the oligopeptide structure with compound 4.Therefore with the mode synthesis of oligonucleotides peptide of routine, reactant 4 is coupled to N-terminal.Carry out deprotection as mentioned above, be converted into corresponding lanthanon (III) inner complex and purification process.

Embodiment 33

Adopt block 29 synthesis of oligonucleotides Nucleotide on solid phase

Adopt Hovinen and Hakala at Org.Lett.3, the method described in 2001,2473 is incorporated into lanthanon (III) inner complex in the oligonucleotide structure with compound 29.Therefore with the mode synthesis of oligonucleotides Nucleotide of routine, reactant 50 is coupled to its 5 '-end.Carry out deprotection as mentioned above, be converted into corresponding lanthanon (III) inner complex and purification process.

Embodiment 34

Synthetic 9-[(trityl oxygen base) methyl]-1,4,7-triazecane 1,4,7-three-(2-oil of mirbane) sulphonamide 32

With 2-((trityl oxygen base) methyl) propane-1, the ethylidene triamine (1.0 mmole) and the triphenylphosphine (3.0 mmole) of 3-glycol (1.0 mmole), the protection of 2-oil of mirbane alkylsulfonyl are dissolved in the anhydrous THF (5 milliliters).Add DIAD (3.0 mmole) four times at 15 minutes branches, reaction is at room temperature spent the night.Remove all volatiless in a vacuum, resistates is precipitated out from diethyl ether.Throw out is dissolved in the methylene dichloride again, at silicagel column (elutriant CH ₂Cl ₂0.5%MeOH; V/v) isolate product on.ESI-MS:[M+H] ⁺971.21, C ₄₅H ₄₃N ₆O ₁₃S ₃ ⁺Calculated value be 971.20.

Scheme 1

Scheme 2

Scheme 3

Scheme 4

Scheme 5

Scheme 6

Scheme 7

The photochemical properties of table 1. some inner complexs of synthetic

Claims (20)

1. sequestrant, described sequestrant comprises:

-comprise the color development part of two or three pyridyl, wherein at least one pyridyl is replaced by the phenyl that tri-alkoxy replaces, and wherein said alkoxyl group is identical or different, and described pyridyl is tethered in together by the hydrocarbon chain that contains N each other;

-comprise at least two directly or be attached to carboxylic acid or the ester of phosphonyl group or described acid or the chelating moiety of salt on the pyridyl of color development part by the hydrocarbon chain that contains N; With

-optional directly or by linker x mooring in described color development part or mooring in the reactive group A of chelating moiety, described reactive group A is selected from isosulfocyanate radical, the halo kharophen, maleimide amino, the dichlorotriazine base, dichlorotriazine amino, the pyridine dithio-, the thioic acid sulfoacid ester group, contain oxygen amino, hydrazide group, amino and carboxylic acid or its active ester groups, wherein said linker x is formed by 1-10 part, each part is selected from: phenylene, the alkylidene group that contains 1-12 carbon atom,-C ≡ C-,-C=C-,-O-,-S-,-CO-NH-,-CO-NR '-,-NH-CO-,-NR '-CO-,-CO-,-COO-,-OOC-,-SS-,-N=N-and tertiary amine, wherein R ' representative contains the alkyl that is less than 5 carbon atoms

Perhaps

When described sequestrant was applicable to that oligopeptide synthesizes, described reactive group A was connected with described sequestrant by linker x, and A is amino-acid residue-CH (NHR ¹) R ⁵, R wherein ¹Be fluorenyl methoxy carbonyl, tert-butoxycarbonyl or 1,1-two oxygenate benzo [b] benzene sulphur-2-ylmethoxy carbonyls, and R ⁵Be carboxylic acid or its salt, acyl halide or ester;

Perhaps

When described sequestrant was applicable to that oligonucleotide is synthesized, described reactive group A was connected with described sequestrant by linker x, and A is selected from:

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

2. the sequestrant of claim 1, wherein said reactive group A is connected on the sequestrant by linker x.

3. the sequestrant of claim 1, described sequestrant is selected from:

Wherein: Z ¹, Z ²And Z ³Be identical or different alkyl; R ⁶Be alkyl ester or allyl ester; R ⁷Be alkyl, n is 0 or 1.

4. the sequestrant of claim 1, described sequestrant are applicable to that oligopeptide is synthetic, and wherein said reactive group A is connected with described sequestrant by linker x, and A is amino-acid residue-CH (NHR ¹) R ⁵, R wherein ¹Be fluorenyl methoxy carbonyl, tert-butoxycarbonyl or 1,1-two oxygenate benzo [b] benzene sulphur-2-ylmethoxy carbonyls, and R ⁵Be carboxylic acid or its salt, acyl halide or ester.

5. the sequestrant of claim 4, described sequestrant is selected from:

Wherein: x as defined in claim 1, protecting group R ¹Be selected from fluorenyl methoxy carbonyl, tert-butoxycarbonyl or 1,1-two oxygenate benzo [b] benzene sulphur-2-ylmethoxy carbonyls, R ⁶Be alkyl ester or allyl ester, R ⁷Be alkyl, Z ¹, Z ²And Z ³Be identical or different alkyl, n is 0 or 1.

6. the sequestrant of claim 1, described sequestrant are applicable to that oligonucleotide is synthetic, and wherein said reactive group A is connected with described sequestrant by linker x, and A is selected from:

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

7. the sequestrant of claim 6, described sequestrant is selected from:

Wherein: R ⁶Be alkyl ester or allyl ester, R ⁷Be alkyl, x as defined in claim 1, A is defined in the claim 6, Z ¹, Z ²And Z ³Be identical or different alkyl, n is 0 or 1.

8. inner complex, described inner complex comprises:

-lanthanide ion;

-comprise the color development part of two or three pyridyl, wherein at least one pyridyl is replaced by the phenyl that tri-alkoxy replaces, and wherein said alkoxyl group is identical or different, and described pyridyl is tethered in together by the hydrocarbon chain that contains N each other;

-comprise at least two directly or be attached to carboxylic acid or the ester of phosphonyl group or described acid or the chelating moiety of salt on the pyridyl of color development part by the hydrocarbon chain that contains N; With

-optional directly or by linker x mooring in described color development part or mooring in the reactive group A of chelating moiety, described reactive group A is selected from isosulfocyanate radical, the halo kharophen, maleimide amino, the dichlorotriazine base, dichlorotriazine amino, the pyridine dithio-, the thioic acid sulfoacid ester group, contain oxygen amino, hydrazide group, amino and carboxylic acid or its active ester groups, wherein said linker x is formed by 1-10 part, each part is selected from: phenylene, the alkylidene group that contains 1-12 carbon atom,-C ≡ C-,-C=C-,-O-,-S-,-CO-NH-,-CO-NR '-,-NH-CO-,-NR '-CO-,-CO-,-COO-,-OOC-,-SS-,-N=N-and tertiary amine, wherein R ' representative contains the alkyl that is less than 5 carbon atoms.

9. the inner complex of claim 8, wherein reactive group A is connected on the sequestrant by linker x.

10. the inner complex of claim 8, described inner complex is selected from:

Wherein: Z ¹, Z ²And Z ³Be identical or different alkyl; N is 0 or 1; Described metal M is a lanthanon.

11. biomolecule, each inner complex conjugation among described biomolecule and the claim 8-10, wherein said biomolecule are selected from oligonucleotide or polynucleotide, protein, oligosaccharides, polysaccharide, phosphatide, PNA, LNA, antibody, haptens, medicine, the bonding part of acceptor and the Sugar receptors of oligopeptide, oligonucleotide, DNA, RNA, modification.

12. the biomolecule of claim 11, the oligonucleotide of wherein said modification or polynucleotide are the oligonucleotide or the polynucleotide of monothio phosphoric acid ester, phosphorodithioate, phosphoramidate and/or sugared modification or alkali modification.

13. biomolecule, each sequestrant conjugation among described biomolecule and the claim 1-7, wherein said biomolecule are selected from oligonucleotide or polynucleotide, protein, oligosaccharides, polysaccharide, phosphatide, PNA, LNA, antibody, haptens, medicine, the bonding part of acceptor and the Sugar receptors of oligopeptide, oligonucleotide, DNA, RNA, modification.

14. a solid carrier, each inner complex conjugation among described solid carrier and the claim 8-10, wherein said solid carrier is selected from particulate, slide or thin plate.

15. a solid carrier, each inner complex conjugation among described solid carrier and the claim 8-10, wherein said solid carrier is selected from nanoparticle.

16. the oligopeptide of a mark, described oligopeptide is incorporated in the oligopeptide structure on the oligopeptide synthesizer by the sequestrant with claim 4 or 5, then carries out deprotection and the optional metal ion of introducing synthesizes acquisition on solid phase.

17. the oligonucleotide of a mark; described oligonucleotide is then carried out the also optional introducing lanthanide ion of deprotection and synthesize acquisition on solid phase by each sequestrant among the claim 6-7 being incorporated in the oligonucleotide structure on the oligonucleotide synthesizer.

18. one kind with the mark oligopeptide of claim 16 or the mark oligonucleotide conjugated solid carrier of claim 17, wherein said oligopeptide or oligonucleotide covalently or non-covalently are fixed on the described solid carrier, and this carrier is selected from particulate, slide or thin plate.

19. one kind with the mark oligopeptide of claim 16 or the mark oligonucleotide conjugated solid carrier of claim 17, wherein said oligopeptide or oligonucleotide covalently or non-covalently are fixed on the described solid carrier, this carrier is selected from nanoparticle.

20. one kind with the sequestrant conjugated solid carrier that is applicable to oligonucleotide synthetic claim 1, wherein said reactive group A is connected with described sequestrant by linker x, A is selected from:

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