CN1181156C - Rare earth fluorescent marker and its application - Google Patents
Rare earth fluorescent marker and its application Download PDFInfo
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- CN1181156C CN1181156C CNB011280751A CN01128075A CN1181156C CN 1181156 C CN1181156 C CN 1181156C CN B011280751 A CNB011280751 A CN B011280751A CN 01128075 A CN01128075 A CN 01128075A CN 1181156 C CN1181156 C CN 1181156C
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Abstract
The present invention relates to a novel rare-earth fluorescent marker and application thereof. The novel rare-earth fluorescent marker is a fluorescent coordination compound of rare-earth ions of Eu<3+>, Sm<3+>, Gd<3+>, Tb<3+> and Dy<3+> and a ligand with a 2, 2': 6', 2'-bitripyridine skeleton structure or a 2, 6-dipyrazole pyridine skeleton structure. The coordination compound marks substances such as proteins, aminophenol, polypeptides, nucleic acid, ribonucleotide, organic compounds, etc. by the covalent combination of functional base groups contained in the coordination compound and the substances; further, the coordination compound is used for the fluorescent measurement of the substances.
Description
Technical field
The present invention relates to the determination techniques of micro-physiologically active substance, specifically a kind of rare-earth fluorescence labeling thing and application thereof.
Background technology
As the measuring method of giving birth to micro-physiologically active substance in the body test portion (cell tissue, blood, urine etc.), immunoassay, DNA hybridization assays method etc. has been widely used in the various clinical assays.In measuring, these all need use certain marker to come materials such as traget antibody, antigen, Nucleotide, nucleic acid.The material that the thing that serves as a mark at present uses comprises radioelement, enzyme, fluorescent chemicals, chemiluminescence compound etc.Their weak point is: the method for 1) using radioactively labelled substance, radioactively labelled substance and marked product thereof exist many inconvenience and trouble at aspects such as storage, transportation, use, liquid waste disposal, and can cause environmental pollution, and self attenuation problem of radioactively labelled substance, but cause its shelf time shorter.2) method of use enzyme labelling thing, the molecular weight of enzyme labelling thing is too big, and its activity very easily is subjected to the influence of temperature, preservation condition, acidity, hetero-ion, sanitas etc., causes this method reproducibility relatively poor.3) measuring method that uses the organic fluorescence marker because the influence of the strong background fluorescence that the light at random of exciting light and sample produce makes the sensitivity of this method lower, only is applicable to the mensuration of high density material when being used for giving birth to sample determination such as body tissue, serum.
The serve as a mark time-resolved fluorometry of thing of rare-earth fluorescent title complex has been widely used in the various mensuration in recent years.The rare-earth fluorescent title complex has characteristics such as fluorescence lifetime is extremely long, the Stokes displacement is big, the fluorescence radiation peak is sharp-pointed, by differentiating fluorometry duration of service, can eliminate background fluorescence that the light at random of exciting light and sample produce very effectively to fluorimetric influence, measure sensitivity thereby greatly improve.Present rare-earth fluorescence labeling thing and time-resolved fluorometry comprise:
(1) dissolving of Finland Wallac company exploitation strengthens time-resolved fluorometry (vehicle economy LFIA assay method), document 1:E.Soini and T.Lovgren, CRC.Crit.Rev.Anal.Chem., 1987,18,105-154. document 2:E.P.Diamandis and T.K.Christopoulos, Anal.Chem., 1990,62,1149A-1157A. document 3:I.Hemmila, J.Alloys Compd., 1995,225,480-485.The DELFIA assay method is used trivalent europium and N
1The title complex of-(the different sulphur itrile group of p-the phenyl)-diethyl triamine tetraacethyl thing that serves as a mark identifies materials such as antibody, antigen, after immune response finishes, because this marker is the non-fluorescent europium complex, so before carrying out time-resolved fluorometry, must in reaction system, add the weakly acidic so-called fluorescence that contains beta-diketon class ligand, trioctyl phosphine oxide and tensio-active agent and strengthen solution, just can measure after making europium ion be converted into the hyperfluorescence title complex.The sensitivity of DELFIA assay method is higher, its weak point is: contain big excessive beta-diketon class ligand and trioctyl phosphine oxide in the fluorimetric solution of this method, very easily be subjected to measuring environment, working sample and measure pollution, require extremely strict measurement operation environment and agents useful for same with the metal ion in the solution etc.; And this system can only carry out the liquid phase fluorometric assay, and its range of application is very limited.
(2) the FIAgen assay method of people such as Canadian Diamandis exploitation, document 4:E.P.Diamandis, Clin.Biochem., 1988,21,139-150. document 5:E.F.G.Dickson, A.Pollakand E.P.Diamandis, Pharmacol.Ther., 1995,66,207-235.This assay method is used fluorescence europium complex 4,7-two (chlorosulfonyl phenyl)-1,10-coffee sieve quinoline-2,9-dicarboxylic acid (being called for short BCPDA) and Eu
3+The title complex thing that serves as a mark identify materials such as antibody, antigen, after immune response finishes, need not add fluorescence and strengthen solution and can directly carry out time-resolved fluorometry immunocomplex.The shortcoming of this method be BCPDA europium complex fluorescence a little less than, it is lower to measure sensitivity.
(3) the TRACE assay method of the people such as Mathis of France exploitation, document 6:G.Mathis, Clin.Chem., 1995,41,1391-1397. document 7:G.Mathis, J.Clin. Ligand Assay, 1997,20,141-147.This method is to use three (bipyridine) pothole body (being called for short TBP) and Eu simultaneously
3+Fluorescence complex and cyanophycin (allophycocyanin) as the homogeneous phase time discrimination fluorescence assay method of fluorescent marker.Its shortcoming is to measure the lower and reagent of sensitivity to cost an arm and a leg.
(4) with the serve as a mark time-resolved fluorometry of thing of the title complex of chlorosulphonation beta-diketon and europium, document 8:J.Yuan, K.Matsumoto and H Kimura, Anal.Chem., 1998,70,596-601. document 9: Matsumoto Kazuko, Yuan Jingli, day disclosure special permission communique, 1997, Te Kaiping 9-241233. document 10:K.Matsumoto and J.Yuan, United States Patent, 1999, Patent number5859297. document 11:J.Yuan and K.Matsumoto, Bunseki Kagaku, 1999,48,1077-1083.The mensuration of this method is highly sensitive, need not use fluorescence to strengthen solution, and its shortcoming is the marker poorly water-soluble, is not suitable for the mark of small-molecule substance.
Summary of the invention
The purpose of this invention is to provide a kind of rare-earth fluorescence labeling thing and application thereof highly sensitive, applied widely.
Technical solution of the present invention is: be with rare earth ion with contain 2,2 ': 6 ', 2 "-ter cycloheptapyridine skeleton structure or 2, the fluorescence complex that 6-two pyrazolyl pyridine skeleton structure class ligands form, its described ligand structure formula is:
Work as R
1=NH
2, NCS, SO
2Cl or
The time R
2=OH
Work as R
1During=H
When the skeleton structure bottom is the crown ether structure,
R
1=NH
2, NCS, SO
2Cl or
Fluorescence complex of the present invention can be widely used in preparation labelled protein, amino acid, polypeptide, nucleic acid, Nucleotide and other is marked with organic compounds; Concrete preparation method is as follows:
1) after thing to be marked being dissolved in the 0.1mol/L sodium bicarbonate buffer solution of pH value 9.1, add the ligand of fluorescent marker, stirring reaction is after 3 hours under the room temperature;
2) remove unreacted marker in the reaction solution by dialysis or column chromatography method;
3) collect the solution of mark, add rare-earth ion solution (marker: promptly make required marked product rare earth ion=1: 1～1.5);
The marked product low temperature that makes is preserved down; When marked product is protein such as traget antibody, should in solution, add sanitas (as NaN
3) and protein active stablizer (as BSA), low temperature is preserved down then;
Fluorescence complex of the present invention can widely be used in time-resolved fluorometry; Promptly utilize described rare-earth fluorescence labeling thing mark determinand in time-resolved fluorometry, by the fluorescence intensity of measuring fluorescent marker or the concentration that relative intensity of fluorescence ratio is measured determinand; At first utilize a kind of reaction raw materials of described rare-earth fluorescence labeling substance markers (as antibody or antigen), after labeled reactant raw material and determinand reaction, separate and remove unreacted labeled reactant raw material, measure the concentration of determinand then by the fluorescence intensity of time-resolved fluorometry assaying reaction resultant, or at first utilize determinand in the described rare-earth fluorescence labeling substance markers sample (as amino acid, polypeptide, medicine etc.), (utilize certain separation method such as liquid chromatography, electrophoresis etc.) with the mark determinand with after unreacted rare-earth fluorescence labeling thing separates, the fluorescence intensity of measuring the mark determinand by time-resolved fluorometry is measured the concentration of determinand;
Wherein said time-resolved fluorometry comprises time-resolved fluorescent immunoassay, time-resolved fluoroimmunoassay Histochemistry, time resolved fluorescence measurement microscope method, time resolved fluorescence cytoactive assay method, time resolved fluorescence liquid chromatogram measuring method and time resolved fluorescence capillary electrophoresis assay method;
Fluorescence complex of the present invention also can be used in nucleic acid (DNA and RNA) fluorometry; Promptly utilize described rare-earth fluorescence labeling thing mark determinand in nucleic acid (DNA and RNA) fluorometry, differentiate luminoscope with the time and measure its concentration; Promptly utilize described rare-earth fluorescence labeling substance markers nucleic acid to comprise nucleic acid probe, Nucleotide, oligonucleotide, after labeling nucleic acid and determined nucleic acid (comprising DNA and RNA) reaction, separate and remove unreacted labeling nucleic acid, measure the concentration of determined nucleic acid then by the fluorescence intensity of time-resolved fluorometry assaying reaction resultant;
Wherein said nucleic acid (DNA and RNA) fluorometry is nucleic acid hybridization assay method, fluorescent PCR assay method, gene chip assay method or molecular beacon assay method.
The present invention has following advantage:
1. rare-earth fluorescence labeling thing of the present invention is applied widely.Good water solubility; Such title complex can form covalent bonds and these materials of mark by materials such as its functional groups that contains and protein, amino acid, polypeptide, nucleic acid, Nucleotide, organic compound, and then is used for the time-resolved fluorometry of these materials; And such title complex also can be in nucleic acid (DNA and RNA) fluorometry.
2. rare-earth fluorescence labeling thing of the present invention is easy to use.The ligand marker that makes need not further be made with extra care, and can be directly used in the sign of protein etc.
3. rare-earth fluorescence labeling thing of the present invention is all very stable in solid-state or water-soluble attitude.Its photoluminescent property is not subjected to the influence of factors such as buffered soln composition.
4. the fluorescent quantum yield of rare-earth fluorescence labeling thing of the present invention is big.The maximum excitation optical wavelength of title complex is in the ultraviolet region, and its glow peak shape is the sharp-pointed shape glow peak of rare earth ion title complex feature.
Description of drawings
Fig. 1 is TTTA-Eu
3+Fluorescence spectrum in the 0.05mol/L of pH value 9.1 borate buffer solution, its concentration is 1.0 * 10
-6Mol/L.
Fig. 2 is BTTA-Eu
3+Fluorescence spectrum in the 0.05mol/L of pH value 9.1 borate buffer solution, its concentration is 1.0 * 10
-6Mol/L.
Fig. 3 is TTTA-Eu
3+And BTTA-Eu
3+The time-resolved fluorometry result of solution.Wherein solvent is the 0.05mol/L Tris-HCl buffered soln of pH value 7.8.
Fig. 4 is to use TTTA-Eu
3+The time-resolved fluorescent immunoassay of mark streptavidin is measured the working curve of TSH in the human serum.
Embodiment
The invention will be further described below by embodiment.
Embodiment 1
Marker 4 '-(2 -thienyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine-6,6 "-dimethylamine tetraacethyl list N-hydroxyl succinic diamide ester (be called for short NHS-TTTA) synthetic.Marker NHS-TTTA is synthetic by synthetic route shown in following,
Specific operation process is as follows:
(1) 4 '-(2 -thienyl)-2,2 ': 6 ', 2 's-ter cycloheptapyridine (compound 1) is synthetic
In the dry methyl alcohol of 500ml, add 23.1 gram ammonium acetates, 16.3 gram N-[2-(pyrid-2 '-yl)-2-oxoethyl] pyridinium iodide (N-[2-(2 '-pyridyl)-the 2-oxoethyl] the pyridine iodide, 50mmol), with 10.76 gram (E)-3-(2 "-thenyl)-1-(pyrid-2 '-yl) prop-2-enone ((E)-3-(2 " thienyl)-1-(2 '-pyridyl)-2-acrylketone, 50mmol), solution stirring was heated up in a steamer 24 hours next time.After reaction solution is chilled to room temperature, placed 1 hour, filter collecting precipitation at-15 ℃, behind cold methanol (about 15 ℃) thorough washing, product acetonitrile recrystallization.Get target compound 6.91 grams (43.8% yield).
1H NMR (CDCl
3Be deuterochloroform) measurement result: 8.74 (d, J, 7.9Hz, 2H), 8.69 (s, 2H), 8.64 (d, J, 7.9Hz, 2H), 7.87 (t, J, 7.9Hz, 2H), 7.78 (d, J, 3.6Hz, 1H), 7.44 (d, J, 5.1Hz, 1H), 7.38-7.32 (m, 2H), 7.19-7.15 (m, 1H).
(2) 4 '-(2 -thienyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine-1,1 "-dioxide (compound 2) synthetic
Add 12.61 in the 500ml methylene dichloride and digest compound 1 (40mmol) and 40 gram m-chloro-benzoic acid peroxides, stirring reaction is after 20 hours under the room temperature, and reaction solution washs 4 times with 10% yellow soda ash of 200ml.Organic phase with anhydrous sodium sulfate drying after, methylene dichloride is removed in distillation.Product is dissolved in the 300ml methyl alcohol, removes by filter the insolubles of trace, behind the filtrate decompression distillation for removing methanol, product acetonitrile thorough washing and vacuum-drying.Get target compound 8.53 grams (61.4% yield).
1H NMR (CDCl
3) measurement result: 9.23 (s, 2H), 8.35 (d, J, 6.6Hz, 2H), 8.23 (d, J, 7.9Hz, 2H), 7.70 (d, J, 3.6Hz, 1H), 7.45-7.28 (m, 5H), 7.16-7.13 (m, 1H).
(3) 6,6 "-dinitrile-4 '-(2 -thienyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine (compound 3) synthetic
In the 300ml methylene dichloride, add 8.69 and digest compound 2 (25mmol) and 24.80 gram (CH
3)
3SiCN (250mmol), stirring at room is after 20 minutes, and stirring down slowly, (about 20 minutes) splash into 14.05 gram Benzoyl chlorides (100mmol).Stir after 20 hours under the reaction solution room temperature, solvent evaporated under reduced pressure to liquor capacity is 150m1, adds 10% wet chemical 600ml, continues to stir 1 hour under the room temperature.Filter collecting precipitation, behind the water thorough washing, use cold methylene dichloride (about 15 ℃) washing again, vacuum-drying then.Get target compound 9.0 grams (98.5% yield).
1H NMR (DMSO-d
6) measurement result: 8.95 (d, J, 7.9Hz, 2H), 8.62 (s, 2H), 8.32-8.26 (m, 2H), 8.19 (d, J, 7.6Hz, 2H), 8.07 (d, J, 3.6Hz, 1H), 7.86 (d, J, 5.1Hz, 1H), 7.28-7.31 (m, 1H).
(4) 4 '-(2 -thienyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine-6,6 "-dicarboxylic acid methyl esters (compound 4) synthetic
Digesting compound 3 with 4.40 joins in the solution of 45ml sulfuric acid-45ml acetic acid-12ml water.75-80 ℃ of following stirring reaction is after 48 hours, and reaction solution joins in the 300ml frozen water, filters collecting precipitation, behind the water thorough washing, uses absolute ethanol washing again, vacuum-drying (getting 4.85 gram hydrolysates).
In the dry methyl alcohol of 400ml, the outside cooling with frozen water adds 8 gram sulfur oxychlorides down, stirs after 15 minutes, adds 4.85 and restrains the said hydrolyzed products, after reaction solution stirs back and heats up in a steamer 8 hours, continues under the room temperature to stir 16 hours.After boiling off solvent, resultant is with chloroform extracting repeatedly.Chloroformic solution is used anhydrous sodium sulfate drying after washing with saturated sodium bicarbonate solution.After boiling off solvent, resultant separates with silica gel column chromatography, launches with methylene chloride-methanol (w/w, 99: 1), collects the-individual component that washes out at first.After boiling off solvent, product toluene recrystallization, vacuum-drying gets target compound 2.50 grams (48.1% yield).Results of elemental analyses (%) is pressed C
23H
17N
3O
4S calculated value: C=64.03, H=3.97, N=9.74; Measured value: C=63.76, H=3.83, N=9.52.
1H NMR (CDCl
3) measurement result: 8.16 (d, J, 7.8Hz, 2H), 8.78 (s, 2H), 8.20 (d, J, 7.6Hz, 2H), 8.03 (t, J, 7.8Hz, 2H), 7.82 (d, J, 3.6Hz, 1H), 7.49 (d, J, 5.1Hz, 1H), 7.22-7.19 (m, 1H), 4.08 (s, 6H).
(5) 6,6 "-dihydroxymethyl-4 '-(2 -thienyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine (compound 5) synthetic
In the dry ethanol of 200ml, add 2.89 and digest compound 4 (6.7mmol) and 1.05 gram NaBH
4, stir after 3 hours under the room temperature, stir back and heat up in a steamer 1 hour.Pressure reducing and steaming ethanol adds the 100ml saturated sodium bicarbonate aqueous solution in the product, be heated to boiling under stirring.Cooled and filtered is collected insolubles, washes the final vacuum drying with water.Product by heating is dissolved in the 200ml tetrahydrofuran (THF), removes by filter insolubles, behind the pressure reducing and steaming tetrahydrofuran (THF), product acetonitrile thorough washing, vacuum-drying.Get target compound 1.82 grams (72.2% yield).
1H NMR (DMSO-d
6) measurement result: 8.63 (s, 2H), 8.50 (d, J, 7.3Hz, 2H), 8.03 (t, J, 7.3Hz, 2H), 7.93 (d, J, 3.6Hz, 1H), 7.82 (d, J, 5.1Hz, 1H), 7.61 (d, J, 7.1Hz, 2H), 7.28-7.31 (m, 1H), 4.74 (s, 4H).
(6) 6,6 "-two brooethyls-4 '-(2 -thienyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine (compound 6) synthetic
At the dry N of 200ml dry tetrahydrofuran-30ml, add 2.17 in the dinethylformamide and digest compound 5 and 4.75 gram phosphorus tribromides, after reaction solution stirs back and heats up in a steamer 5 hours, the pressure reducing and steaming solvent.Resultant is dissolved in the 300ml chloroform, and chloroformic solution is with the 10%Na of 4 * 100ml (promptly four times each use 100 milliliters)
2CO
3Behind the solution washing 4 times, use anhydrous sodium sulfate drying.Remove by filter sodium sulfate, pressure reducing and steaming chloroformic solution, product normal hexane thorough washing, vacuum-drying gets target compound 2.02 grams (69.7% yield).
1H NMR (CDCl
3) measurement result: 8.71 (s, 2H), 8.54 (d, J, 7.8Hz, 2H), 7.87 (t, J, 7.8Hz, 2H), 7.78 (d, J, 3.6Hz, 1H), 7.52 (d, J, 7.8Hz, 2H), 7.48 (d, J, 5.1Hz, 1H), 7.21-7.19 (m, 1H), 4.70 (s, 4H).
(7) 4 '-(2 -thienyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine-6,6 "-dimethylamine tetraacetic acid tetraethyl ester (compound 7) synthetic
Add 2.04 and digest compound 6 (4mmol) in the dry acetonitrile of 200ml-50ml dry tetrahydrofuran, 1.53 gram ethyl diacetate base amine (8.1mmol) are dissolved in the solution and the 5.52 gram K of the dry acetonitrile of 30ml
2CO
3(40mmol), reaction solution removes by filter insolubles after stirring back and heating up in a steamer 24 hours.Behind the pressure reducing and steaming solvent, resultant is dissolved in the 200ml chloroform, chloroformic solution is used anhydrous sodium sulfate drying after washing with 4 * 100ml saturated aqueous sodium sulfate.Filter the back solution decompression and boil off solvent, with petroleum ether final vacuum drying.Gained oily matter separates with silica gel column chromatography, launches with ethyl acetate-methyl alcohol-tetrahydrofuran (THF) (w/w/w, 10: 3: 2), collects first component that washes out at first.After boiling off solvent, vacuum-drying.Get target compound 1.95 grams (67.9% yield).
1H NMR (CDCl
3) measurement result: 8.67 (s, 2H), 8.51 (d, J, 7.8Hz, 2H), 7.86 (t, J, 7.8Hz, 2H), 7.78 (d, J, 3.6Hz, 1H), 7.63 (d, J, 7.8Hz, 2H), 7.45 (d, J, 5.1Hz, 1H), 7.20-7.17 (m, 1H), 4.19 (q, J, 7.3Hz, 8H), 3.72 (s, 8H), 3.46 (s, 4H), 1.26 (t, J, 7.3Hz, 12H).
(8) 4 '-(2 -thienyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine-6,6 "-dimethylamine tetraacethyl (be called for short TTTA) synthetic
Digest compound 7 (2.7mmol) with 1.94 and join in the 120ml ethanol, add 4 gram potassium hydroxide and 10ml water then, reaction solution stirs back and heats up in a steamer 3 hours.The pressure reducing and steaming solvent is dissolved in resultant in the 150ml water, removes by filter micro-insolubles, and the very long trifluoroacetic acid aqueous solution that splashes into to the pH value of solution is about till 1 in the solution under stirring.Stir under the solution room temperature after 3 hours, filter collecting precipitation, the trifluoroacetic acid aqueous solution thorough washing with 1%, vacuum-drying.Resultant is joined in the 200ml acetonitrile, stir back and heat up in a steamer 2 hours, filter collecting precipitation, vacuum-drying.Get target compound 0.89 gram (51.4% yield).Results of elemental analyses (%) is pressed C
29H
31N
5O
9S (TTTA2H
2O) calculated value: C=54.29, H=4.87, N=10.91; Measured value: C=54.09, H=4.57, N=10.41.
1H NMR (DMSO-d
6) measurement result: 8.75 (s, 2H), 8.59 (d, J, 7.8Hz, 2H), 8.18 (t, J, 7.8Hz, 2H), 8.10 (d, J, 3.6Hz, 1H), 7.87 (d, J, 5.1Hz, 1H), 7.75 (d, J, 7.8Hz, 2H), 7.34-7.31 (m, 1H), 4.72 (s, 4H), 4.28 (s, 8H).
(9) NHS-TTTA's is synthetic
With TTTA2H
2O is at P
2O
5In the vacuum drier after the thorough drying, get 181.7mg (0.3mmol) and be dissolved in the dry N of 5ml, in the dinethylformamide, stir and add the N-hydroxyl succinic diamide (NHS) of 34.5mg (0.3mmol) and the N of 61.9mg (0.3mmol) down, N '-dicyclohexyl charing diimine (DCC), stir after 24 hours under the room temperature, remove by filter insolubles.Filtrate decompression concentrates except that after desolvating, a small amount of washed with isopropyl alcohol of resultant, vacuum-drying.Get target compound 180mg (85.4% yield).The NHS-TTTA that makes need not further make with extra care, and can be directly used in the sign of protein etc.
Embodiment 2
Marker N, N, N
1, N
1-[2,6-two (3 '-amine methyl isophthalic acid '-pyrazolyl)-4-(2 "-thienyl) pyridine] tetraacethyl list N-hydroxyl succinic diamide ester (being called for short NHS-BTTA) synthetic:
Marker NHS-BTTA is synthetic by synthetic route shown in following,
Specific operation process is as follows:
Synthesizing of (1) 2,6-two bromo-4-(2 '-thienyl) pyridines (Compound I)
Add 3.25 gram 4-amidos-2 in 250ml acetate, 6-dibromo pyridine (12.9mmol) and 12.9 gram thiophene after the stirring and dissolving, stir and are added dropwise to 1.93 down and restrain the nitrosyl isopentyl ester and be dissolved in solution in the 13ml acetate.Stir under the reaction solution room temperature after 24 hours, 50 ℃ were continued stirring reaction 3 hours down.The pressure reducing and steaming solvent, resultant is extracted resultant with the 10% salt of wormwood neutralization of 40ml out with the chloroform of 4 * 60ml.Chloroformic solution with anhydrous sodium sulfate drying after, the pressure reducing and steaming solvent.Resultant separates with silica gel column chromatography, launches with methylene chloride-methanol (w/w, 99: 1), collects first component that washes out at first.After boiling off solvent, product recrystallizing methanol twice, vacuum-drying.Get target compound 1.97 grams (47.9% yield).Results of elemental analyses (%) is pressed C
9H
5NBr
2S calculated value: C=33.88, H=1.58, N=4.39; Measured value: C=33.46, H=1.46, N=4.23.
1H NMR (CDCl
3) measurement result: 7.60 (s, 2H), 7.50-7.48 (m, 2H), 7.16-7.13 (m, 1H).
Synthesizing of (2) 2,6-two (3 '-carboxylate methyl ester-1 '-pyrazolyl)-4-(2 '-thienyl) pyridine (Compound I I)
In the 150ml dry tetrahydrofuran, add 10.1 gram 3-carboxylate methyl ester base pyrazoles (80mmol), the potassium metal that adds 3.12 gram fritters after the stirring and dissolving, solution adds the Compound I (20mmol) of 6.38 grams after 60 ℃ of following stirring reactions are intact to the potassium metal total overall reaction.After the reaction solution continuously stirring is returned and is heated up in a steamer 7 days, the pressure reducing and steaming solvent, resultant is with 6 * 150ml chloroform extracting solvend.The pressure reducing and steaming solvent, product separates with silica gel column chromatography, launches with methylene chloride-methanol (w/w, 99: 1), collects first component that washes out at first.After boiling off solvent, product benzene recrystallization, vacuum-drying.Get target compound 3.0 grams (36.7% yield).Results of elemental analyses (%) is pressed C
19H
15N
5O
4S calculated value: C=55.74, H=3.69, N=17.10; Measured value: C=55.47, H=3.62, N=16.82.
1H NMR (CDCl
3) measurement result: 8.60 (d, J, 2.7Hz, 2H), 8.22 (s, 2H), 7.79 (d, J, 3.6Hz, 1H), 7.53 (d, J, 5.0Hz, 1H), 7.20-7.18 (m, 1H), 7.03 (d, J, 2.7Hz, 2H), 4.01 (s, 6H).
Synthesizing of (3) 2,6-two (3 '-methylol-1 '-pyrazolyl)-4-(2 '-thienyl) pyridine (compound III)
In the 300ml dry tetrahydrofuran, add 1.30 gram LiAlH
4, add 2.72 then and digest compound II (6.64mmol), stir after 4 hours under the reaction solution room temperature, slowly splash into 1.1ml water, 15% sodium hydroxide of 1.1ml and 4.5ml water.Stir after 30 minutes under the room temperature, remove by filter precipitation, the solvend with in 3 * 100ml tetrahydrofuran (THF) extracting precipitation merges tetrahydrofuran solution.Behind the pressure reducing and steaming solvent, product acetonitrile thorough washing, vacuum-drying gets target compound 1.60 grams (68.2% yield).
1H NMR (DMSO-d
6) measurement result: 8.88 (d, J, 2.6Hz, 2H), 7.96 (d, J, 3.6Hz, 1H), 7.90 (s, 2H), 7.85 (d, J, 5.1Hz, 1H), 7.29-7.26 (m, 1H), 6.60 (d, J, 2.5Hz, 2H), 4.58 (s, 4H).
Synthesizing of (4) 2,6-two (3 '-brooethyl-1 '-pyrazolyl)-4-(2 '-thienyl) pyridine (compound IV)
In the 200ml dry tetrahydrofuran, add 1.59 and digest compound III (4.50mmol), add 3.65 gram PBr after the stirring and dissolving
3, after reaction solution stirs back and heats up in a steamer 4 hours, the pressure reducing and steaming solvent.10% sodium carbonate solution that adds 100ml in the resultant fully stirs the back and extracts resultant out with chloroform.Add 6 gram solid sodium bicarbonates in the chloroformic solution again, stir under the room temperature after 1 hour, add anhydrous sodium sulfate drying.Underpressure distillation removes and desolvates after filtering, product normal hexane thorough washing, and vacuum-drying gets target compound 2.03 grams (94.1% yield).
1H NMR (CDCl
3) measurement result: 8.50 (d, J, 2.7Hz, 2H), 8.02 (s, 2H), 7.72 (d, J, 3.6Hz, 1H), 7.50 (d, J, 5.1Hz, 1H), 7.19-7.16 (m, 1H), 6.56 (d, J, 2.7Hz, 2H), 4.59 (s, 4H).
(5) N, N, N
1, N
1-[2,6-two (3 '-amine methyl isophthalic acid '-pyrazolyl)-4-(2 "-thienyl) pyridine] tetraacetic acid tetraethyl ester (compound V) synthetic
Add 1.20 and digest compound IV (2.50mmol) in the dry acetonitrile of 100ml-30ml dry tetrahydrofuran, the ethyl diacetate base amine (5.2mmol) of 984mg is dissolved in the solution and the 3.45 gram K of the dry acetonitrile of 30ml
2CO
3(25mmol), reaction solution removes by filter insolubles after stirring back and heating up in a steamer 24 hours.The pressure reducing and steaming solvent is dissolved in resultant in the 200ml chloroform, chloroformic solution with 2 * 100ml water washing after, use anhydrous sodium sulfate drying.Filter the back solution decompression and boil off solvent, vacuum-drying.Gained oily matter separates with silica gel column chromatography, launches with ethyl acetate-methylene dichloride (w/w, 100: 10), collects first component that washes out at first.After boiling off solvent, vacuum-drying.Get target compound 0.87 gram (50.0% yield).
1H NMR (CDCl
3) measurement result: 8.51 (d, J, 2.7Hz, 2H), 8.00 (s, 2H), 7.72 (d, J, 3.6Hz, 1H), 7.49 (d, J, 5.1Hz, 1H), and 7.19-7.16 (m, 1H), 6.56 (d, J, 2.7Hz, 2H), 4.20 (q, J, 7.2Hz, 8H), 4.09 (s, 4H), 3.66 (s, 8H), 1.28 (t, J, 7.2Hz, 12H).
(6) N, N, N
1, N
1-[2,6-two (3 '-amine methyl isophthalic acid '-pyrazolyl)-4-2 "-thienyl) pyridine] tetraacethyl (being called for short BTTA) synthetic
The compound V (1.22mmol) of 850mg is joined in the 60ml ethanol, add 1.8 gram potassium hydroxide and 7ml water then, reaction solution stirs back and heats up in a steamer 3 hours.The pressure reducing and steaming solvent is dissolved in resultant in the 50ml water, removes by filter micro-insolubles, and the very long aqueous hydrochloric acid that splashes into to the pH value of solution is about till 1 in the solution under stirring.Stir under the solution room temperature after 3 hours, filter collecting precipitation, with the aqueous hydrochloric acid thorough washing of the concentration 37% of 100 times of dilutions, vacuum-drying.Get target compound 660mg (92.6% yield).Results of elemental analyses (%) is pressed C
25H
27N
7O
9S (BTTAH
2O) calculated value: C=49.92, H=4.52, N=16.29; Measured value: C=50.08, H=4.38, N=16.09.
1H NMR (DMSO-d
6) measurement result: 8.90 (d, J, 2.5Hz, 2H), 7.98 (d, J, 3.6Hz, 1H), 7.90 (s, 2H), 7.86 (d, J, 5.1Hz, 1H), 7.30-7.27 (m, 1H), 6.59 (d, J, 2.5Hz, 2H), 4.00 (s, 4H), 3.52 (s, 8H).
(7) NHS-BTTA's is synthetic
With BTTAH
2O is at P
2O
5In the vacuum drier after the thorough drying, get 175.0mg (0.3mmol) and be dissolved in the dry N of 5ml, in the dinethylformamide, stir and add the N-hydroxyl succinic diamide (NHS) of 34.5mg (0.3mmol) and the N of 61.9mg (0.3mmol) down, N '-dicyclohexyl charing diimine (DCC), stir after 24 hours under the room temperature, remove by filter insolubles.Filtrate decompression concentrates except that after desolvating, a small amount of washed with isopropyl alcohol of resultant, vacuum-drying.Get target compound 172mg (84.2% yield).The NHS-BTTA that makes need not further make with extra care, and can be directly used in the sign of protein etc.
Embodiment 3
TTTA and BTTA and rare earth ion (Eu
3+And Tb
3+) the complex fluorescent property testing
1. fluorescence spectrum, fluorescence intensity and fluorescence lifetime
In the aqueous solution TTTA and BTTA with can form stable title complex rapidly after rare earth ion mixes.0.05mol/L borate buffer solution with pH value 9.1 is that solvent has been measured TTTA-Eu
3+, TTTA-Tb
3+And BTTA-Eu
3+, BTTA-Tb
3+Uv-vis spectra in this solvent, fluorescence spectrum, molar absorptivity (ε), fluorescent quantum yield (φ) and fluorescence lifetime (τ).It is day beautiful UV 7500 spectrophotometers that uv-vis spectra is measured with instrument.The fluorometric assay instrument is a Perkin Elmer LS 50B spectrophotofluorometer.Quantum yield is measured and is used 4 '-phenyl-2,2 ': 6 ', 2 "-ter cycloheptapyridine-6,6 "-dimethylamine tetraacethyl and Eu
3+And Tb
3+Title complex record (its Eu as standard substance
3+The ε of title complex
335nm=14300, φ=0.160, Tb
3+The ε of title complex
337nm=14000, φ=0.100) document 12:M.Latva, H.Takalo, V.-M.Mukkala, C.Matachescu, J.C.Rodriguez-Ubis and J.Kankare, J.Lumin., 1997,75,149-169, calculating formula is φ
1=I
1ε
2C
2φ
2/ I
2ε
1C
1, I in the formula
2, ε
2, φ
2, C
2Be fluorescence intensity, molar absorptivity, quantum yield and the concentration of standard substance, I
1, ε
1, φ
1, C
1Fluorescence intensity, molar absorptivity, quantum yield and concentration for determinand.Table 1 is depicted as two kinds of ligands and absorption and the photoluminescent property of title complex in the 0.05mol/L of pH value 9.1 borate buffer solution thereof.
Absorption and the photoluminescent property of several compounds of table 1. in the 0.05mol/L of pH value 9.1 borate buffer solution
| Compound | Maximum absorption wavelength (nm) | Molar absorptivity (mol -1Lcm -1) | Maximum fluorescence emission wavelength (nm) | The fluorescent quantum yield | Fluorescence lifetime (ms) |
| TTTA TTTA-Eu 3+TTTA-Tb 3+BTTA BTTA-Eu 3+BTTA-Tb 3+ | 291 296，336 296，336 271，307 275，319 275，319 | 31200 26700，24600 26700，24300 33900，34100 33600，25200 33600，25000 | 615 545 620 545 | 0.150 0.002 0.114 0.001 | 1.284 1.352 |
As seen from Table 1, TTTA-Eu
3+And BTTA-Eu
3+Be the hyperfluorescence compound, and have very long fluorescence lifetime, and TTTA-Tb
3+And BTTA-Tb
3+It only is the hypofluorescence compound.
As shown in Figure 1, 2, the maximum excitation optical wavelength of two kinds of title complexs is all in the ultraviolet region, and its maximum fluorescence emission wavelength is respectively at 615nm and 620nm, and its glow peak shape is Eu
3+The sharp-pointed shape glow peak of title complex feature.
2. pH value of buffer solution is to the influence of complex fluorescent character
Tris-HCl buffered soln with the 0.05mol/L of different pH values is solvent, has measured TTTA-Eu under the different pH values
3+Fluorescence intensity and fluorescence lifetime, it the results are shown in Table 2.Measurement result shows, in the pH value is the variation range of 9-5.8, and TTTA-Eu
3+Fluorescence intensity and fluorescence lifetime be subjected to the influence of pH value little.The TTTA-Eu that in Tris-HCl and borate buffer solution, records in addition
3+Fluorescence intensity and fluorescence lifetime much at one, TTTA-Eu is described
3+Very high stability is arranged, the influence that its photoluminescent property is not formed by buffered soln in these buffered soln.
Table 2 pH value changes TTTA-Eu
3+The influence of photoluminescent property
| The pH value | 10.0 | 9.0 | 8.5 | 8.0 | 7.5 | 6.8 | 5.8 | 5.4 |
| Fluorescence intensity (arb. unit) | 37.63 | 43.61 | 46.16 | 46.61 | 45.90 | 43.55 | 41.97 | 40.64 |
| Fluorescence lifetime (ms) | 1.103 | 1.072 | 1.163 | 1.217 | 1.229 | 1.209 | 1.182 | 1.157 |
3. use TTTA-Eu
3+And BTTA-Eu
3+Carry out the sensitivity of time-resolved fluorometry
0.05mol/L Tris-HCl buffered soln with pH value 8.0 is solvent, the TTTA-Eu of preparation series concentration
3+And BTTA-Eu
3+Solution has been determined use TTTA-Eu by time-resolved fluorometry
3+And BTTA-Eu
3+Carry out the sensitivity of time-resolved fluorometry.Measuring with instrument is WALLAC VICTOR 1420 multiple labeling calculating instruments (PerkinElmer Life Sciences company products), and condition determination is: excitation wavelength, 340nm; Detect wavelength, 615nm; Dead time (delay time), 0.2ms; The window time (window time), 0.4ms; Cycling time (cycling time): 1.0ms.
As shown in Figure 3,2 times of minimum lower limits that detect of calculating with the background signal standard deviation get TTTA-Eu
3+And BTTA-Eu
3+The minimum lower limit that detects of the time-resolved fluorometry of solution is respectively: TTTA-Eu
3+, 1.3 * 10
-12Mol/L, BTTA-Eu
3+, 1.4 * 10
-12Mol/L.Illustrate and use both time-resolved fluorometries all to have very high sensitivity.
Embodiment 4
Use NHS-TTTA-Eu
3+The mark streptavidin
After the streptavidin of 5mg being dissolved in the 0.1mol/L sodium bicarbonate buffer solution of pH value 9.1 of 10ml, add the NHS-TTTA of 10mg, stirring reaction is after 3 hours under the room temperature.Solution under 4 ℃ to 3 liters contain 0.25 the gram NaN
3The 0.1mol/L sodium hydrogen carbonate solution of (sodium azide) carries out three dialysis, each 24 hours, removes unreacted marker.Solution standard EuCl takes a morsel after the dialysis
3(concentration is 1.0 * 10 to solution
-5Mol/L) carry out the concentration that TTTA in the solution is determined in fluorometric titration, calculate mark rate (ratio of TTTA concentration and streptavidin concentration) then.The mark rate of the mark streptavidin that this method makes is 20.Add EuCl in the gained solution
3Solution (Eu
3+: TTTA=1.5: 1), promptly obtain TTTA-Eu
3+Mark streptavidin solution.Add the bovine serum albumin (BSA) of 20mg and the NaN of 25mg in this solution
3After ,-20 ℃ of freezing preservations down.
When above-mentioned solution is used for time resolved fluoro-immunoassay, with containing 0.2%BSA, 0.9%NaCl and 0.1%NaN
31000 times of the 0.05mol/L Tris-HCl buffered soln dilutions of pH value 7.8 after use.
Embodiment 5
Use NHS-BTTA-Eu
3+The mark streptavidin
After the streptavidin of 5mg being dissolved in the 0.1mol/L sodium bicarbonate buffer solution of pH value 9.1 of 10ml, add the NHS-BTTA of 10mg, stirring reaction is after 3 hours under the room temperature.Separate the streptavidin and the unreacted marker of mark with Sephadex G-50 post, with the NH of 0.05mol/L
4HCO
3Solution is as wash solution, and every 1.5ml divides receipts, measures each minute and receives the absorbancy of solution at 280nm, merges the labelled protein solution that washes out.Labelled protein solution standard EuCl takes a morsel
3(concentration is 1.0 * 10 to solution
-4Mol/L) carry out the concentration that BTTA in the solution is determined in fluorometric titration, calculate mark rate then.The mark rate of the mark streptavidin that this method makes is 18.Add EuCl in the gained solution
3Solution (Eu
3+: BTTA=1.5: 1), promptly obtain BTTA-Eu
3+Mark streptavidin solution.Add the BSA of 20mg and the NaN of 25mg in this solution
3After ,-20 ℃ of freezing preservations down.
Embodiment 6
Use TTTA-Eu
3+The time-resolved fluorescent immunoassay of mark streptavidin is measured the thyrotropin (TSH) in the human serum
1. the anti-people TSH α of biotin labeling-subunit MONOCLONAL ANTIBODIES SPECIFIC FOR
Press document 13:J.Yuan, G.Wang, H.Kimura and K.Matsumoto, Anal.Sci., 1998,14, method preparation among the 421-423.When being used for time resolved fluoro-immunoassay, with containing 0.2%BSA, 0.9%NaCl and 0.1%NaN
3The 0.05mol/L Tris-HCl buffered soln of pH value 7.8 use after being diluted to desired concn.
2.96 the bag quilt of microwell plate
Anti-people TSH β-subunit monoclonal antibody (10 mcg/ml that in each hole of 96 microwell plates (Fluoro Nunc microwell plate), add 60 microlitres, behind the 0.1mol/L sodium hydrogen carbonate solution of pH value 9.6 Mitsubishi Chem.Co. product), placed 24 hours down for 4 ℃.After discarding solution, each hole is with twice of the 0.05mol/L Tris-HCl solution washing of the pH value 7.8 that contains 0.05% polysorbas20, with the 0.05mol/L Tris-HCl solution washing of pH value 7.8 once, promptly obtain 96 microwell plates of anti-people TSH β-subunit monoclonal antibody bag quilt again.
3. the mensuration of human serum TSH
In the hole of above-mentioned quilt back orifice plate, (the concrete concentration of TSH standardized solution is 100,10 to add the TSH standardized solution of 50 microlitres, 1,0.1,0.01,0.00mIU/L) series and human serum sample, place after 1 hour down for 37 ℃, discard solution, each hole is with the 0.05mol/LTris-HCl solution washing twice of the pH value 7.8 that contains 0.05% polysorbas20, again with the 0.05mol/L Tris-HCl solution washing of pH value 7.8 once.The anti-people TSH α of the biotin labeling-subunit monoclonal anti liquid solution (13 preparations of strength of solution reference literature get final product) that in each hole, adds 50 microlitres, place after 1 hour down for 37 ℃, discard solution, each hole is with the 0.05mol/L Tris-HCl solution washing twice of the pH value 7.8 that contains 0.05% polysorbas20, again with the 0.05mol/L Tris-HCl solution washing of pH value 7.8 once.The TTTA-Eu that in each hole, adds 50 microlitres
3+Mark streptavidin solution is placed after 1 hour down for 37 ℃, discards solution, each hole with the 0.05mol/L Tris-HCl solution washing of the pH value 7.8 that contains 0.05% polysorbas20 4 times after, carry out the solid phase time-resolved fluorometry.Measuring with instrument is WALLAC VICTOR 1420 multiple labeling calculating instruments, and condition determination is: excitation wavelength, 340nm; Detect wavelength, 615nm; Dead time (delay time), 0.2ms; The window time (window time), 0.4ms; Cycling time (cycling time): 1.0ms.
As shown in Figure 4,2 times of the standard deviation of the fluorescent signal during with zero-dose (background) are calculated the minimum lower limit that detects that TSII measures, and minimum the detecting down that gets this law is limited to 0.09mIU/L.
2,2 ': 6 ', 2 "-and ter cycloheptapyridine skeleton structure and 2, the synthetic route that 6-two pyrazolyl pyridine skeleton structures and crown ether form ligand is as follows:
Or
Claims (6)
1. a rare-earth fluorescence labeling thing is characterized in that: be with rare earth ion Eu
3+, Sm
3+, Gd
3+, Tb
3+, Dy
3+With contain 2,2 ': 6 ', 2 "-ter cycloheptapyridine skeleton structure or 2, the fluorescence complex that 6-two pyrazolyl pyridine skeleton structure class ligands form; Wherein said ligand structure formula is:
When
R
1=NH
2, NCS, SO
2Cl or
The time R
2=OH
Work as R
1During=H
Or
When the skeleton structure bottom is the crown ether structure,
R
1=NH
2, NCS, SO
2Cl or
2. application according to the described rare-earth fluorescence labeling thing of claim 1 is characterized in that: utilize labelled protein, amino acid, polypeptide, nucleic acid, the Nucleotide of described rare earth fluorescent marker preparation, its preparation method is as follows:
1) after thing to be marked being dissolved in the 0.1mol/L sodium bicarbonate buffer solution of pH value 9.1, add the ligand of fluorescent marker, stirring reaction is after 3 hours under the room temperature;
2) remove unreacted marker in the reaction solution by dialysis or column chromatography method;
3) collect the solution of mark, promptly make required marked product, its marker behind the adding rare-earth ion solution: rare earth ion=1: 1～1.5.
3. application according to the described rare-earth fluorescence labeling thing of claim 1, it is characterized in that: at first utilize a kind of reaction raw materials of described rare earth fluorescent marker mark, after labeled reactant raw material and determinand reaction, separate and remove unreacted labeled reactant raw material, measure the concentration of determinand then by the fluorescence intensity of time-resolved fluorometry assaying reaction resultant; Or at first utilize determinand in the described rare earth fluorescent marker mark sample, with after unreacted rare-earth fluorescence labeling thing separates, the fluorescence intensity of measuring the mark determinand by time-resolved fluorometry is measured the concentration of determinand with the mark determinand.
4. the application of marker according to claim 3 is characterized in that: wherein said time-resolved fluorometry is time-resolved fluorescent immunoassay, time-resolved fluoroimmunoassay Histochemistry, time resolved fluorescence measurement microscope method, time resolved fluorescence cytoactive assay method, time resolved fluorescence liquid chromatogram measuring method or time resolved fluorescence capillary electrophoresis assay method.
5. application according to the described rare-earth fluorescence labeling thing of claim 1, it is characterized in that: utilize described rare-earth fluorescence labeling substance markers nucleic acid, after labeling nucleic acid and determined nucleic acid reaction, separate and remove unreacted labeling nucleic acid, measure the concentration of determined nucleic acid then by the fluorescence intensity of time-resolved fluorometry assaying reaction resultant.
6. the application of marker according to claim 5 is characterized in that: wherein said nucleic acid fluorescent assay method is nucleic acid hybridization assay method, fluorescent PCR assay method, gene chip assay method or molecular beacon assay method.
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| CN102191041B (en) * | 2011-03-10 | 2014-04-16 | 江南大学 | Method for preparing bonded lignin-based terpyridine rare earth photoelectric material |
| CN102277155B (en) * | 2011-06-28 | 2014-10-15 | 中国科学院福建物质结构研究所 | Preparation method and application of organic white light-emitting material L-COOH |
| CN102329264B (en) * | 2011-06-28 | 2015-03-04 | 中国科学院福建物质结构研究所 | Preparation and application of organic white light emitting material L-Cl |
| CN102851025B (en) * | 2012-10-10 | 2014-01-22 | 南开大学 | Intelligent rare-earth luminescent supramolecular device with photocontrollability and preparation thereof |
| CN103554142B (en) * | 2013-11-06 | 2016-06-29 | 东南大学 | The preparation method of nucleotide rare earth coordination polymer luminescent material |
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