CN1544944A - Fluorescent probe with rhodamine as parent substance and preparing process thereof - Google Patents

Fluorescent probe with rhodamine as parent substance and preparing process thereof Download PDF

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CN1544944A
CN1544944A CNA2003101089300A CN200310108930A CN1544944A CN 1544944 A CN1544944 A CN 1544944A CN A2003101089300 A CNA2003101089300 A CN A2003101089300A CN 200310108930 A CN200310108930 A CN 200310108930A CN 1544944 A CN1544944 A CN 1544944A
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carbon
alkyl
rhodamine
nitrogen
fluorescence probe
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CN1226627C (en
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孟庆华
郁美娟
任吉存
黄德音
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Shanghai Jiaotong University
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
    • C09B11/10—Amino derivatives of triarylmethanes
    • C09B11/24—Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label

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Abstract

The invention disclose a fluorescent probe using rhodamine as precursor and its preparing method. It uses 3-carboxyl rhodamine dye as precursor to react with N-hydroxysuccinimide and dicyclohexylcarbodiimide to obtain the fluorescent probe. It has higher yield of fluorescent quantum, and can be applied to labeling the amino-group in the molecules like amino acid, polypeptide, protein, etc, and also act as pH fluorescent indicator to determine pH values in cells and cell devices.

Description

A kind of is the fluorescence probe and the preparation method of parent with the rhodamine
Technical field: the present invention relates to a kind of is the fluorescence probe and preparation method thereof of parent with the rhodamine.
Background technology: rhodamine (Rhodamine) compounds is that a class has strong molar absorption coefficient and than the fluorescent dye of high-fluorescence quantum yield, Chang Zuowei fluorescence probe mark biomolecule.From at present existing rhodamine class fluorescence probe, this type of probe is many on the one hand connects reactive group in 5-, 6-position, the gained probe mostly is isomeric forms, 5 (6)-carboxyl rhodamine succinimide esters described in the European patent EP 0272007, in spectral quality, exist difference between the isomeride, when using isomer mixture mark biomolecule, utilize electrophoretic separation, band broadening appears in regular meeting, thereby therefore Interference Detection needs single isomeric forms is purified into the mark that is used further to biomolecule; On the other hand, common reactive group is sulphonyl halogen, isothiocyano in the probe, but to the equal mark of the multiple group in the biomolecule, thereby reaction selectivity is not strong.
Summary of the invention: the objective of the invention is to utilize the 3-position to contain the rhodamine of carboxyl, react under the dicyclohexylcarbodiimide effect with the N-hydroxy-succinamide that contains hydroxyl, change into and have the individual isomer form, can carry out the fluorescence probe of selected marker to amino-compound, also can consider fluorescence indicator according to the difference of gained derivant fluorescent characteristic under condition of different pH behind the fluorescence probe mark amino-compound, be used to measure the pH value in cell and the organelle used as pH.
The contained parent of the fluorescence probe that the present invention will prepare is a rhodamine, and reactive group is the N-hydroxy-succinamide base, and its structure is shown in (1):
Figure A20031010893000041
Wherein, R 1, R 2, R 3And R 4Respectively do for oneself hydrogen and replacing and alkyl, carboxyalkyl, aminoalkyl, naphthenic base, the aryl of not replacement; R 5, R 6, R 7, R 8Respectively do for oneself hydrogen, alkyl, carboxyalkyl, aminoalkyl, alkyl ether, alkyl thioether, halogen or alkoxy; R 1And R 7For having the alkyl chain of 2 to 5 carbon atoms, it is connected to 5 ' carbon on the nitrogen that is chained on the 6 ' carbon together; R 3And R 8For having the alkyl chain of 2 to 5 carbon atoms, it is connected to 4 ' carbon on the nitrogen that is chained on the 3 ' carbon together; R 2And R 5For having the alkyl chain of 2 to 5 carbon atoms, it is connected to 7 ' carbon on the nitrogen that is chained on the 6 ' carbon together; R 4And R 6For having the alkyl chain of 2 to 5 carbon atoms, it is connected to 2 ' carbon on the nitrogen that is chained on the 3 ' carbon together; R 1And R 2Form on the main chain alkyl or alkenyl chain of 5 atoms at the most together, it is made up of carbon and one or more heteroatoms that is selected from nitrogen or oxygen.R 3And R 4Form on the main chain alkyl or alkenyl chain of 5 atoms at the most together, it is made up of carbon and one or more heteroatoms that is selected from nitrogen or oxygen;
An θ is inorganic or organic anion, comprises chlorion, bromide ion, sulfate radical, bisulfate ion, pyrovinic acid root, sulfaminic acid root, perchlorate, benzene sulfonic acid root, oxalate, maleate, acetate, glycolic acid root, methoxyimino acetic acid root, propionate, lactate, amber acid radical, tartrate anion.
The preparation method of fluorescence probe of the present invention is: have the rhodamine of carboxyl to be dissolved in the dry organic solvent for 1 mole the 3-bit strip, add 0.5～5 mole of N-hydroxy-succinamide, be added dropwise to the dicyclohexylcarbodiimide (DCC) that is dissolved in dry organic solvent by constant pressure funnel while stirring, the amount of dicyclohexylcarbodiimide is 1～5 mole, at 0 ℃ of temperature stirring reaction 1h～120h that arrives between the organic solvent boiling point, with reacting liquid filtering, pressure reducing and steaming part filtrate, add the precipitation agent precipitation, filter, drying gets target product, promptly is the fluorescence probe of parent with the rhodamine.Its course of reaction is shown below:
Figure A20031010893000051
The dry organic solvent that the present invention adopts has acetonitrile, propionitrile, N, dinethylformamide, dimethyl sulfoxide (DMSO), perhaps their potpourri.
The precipitation agent that the present invention adopts has ether, ethyl acetate, perhaps their potpourri.
Fluorescence probe prepared in accordance with the present invention, there is not the separation of mixed isomers, contained reactive group is the N-hydroxy-succinamide base, not with reactions such as hydroxyl, sulfydryls, amino there is stronger selective reaction ability, can be used for the mark that amino acid, polypeptide, protein, biogenic amine etc. contain amino-compound.Also can consider the fluorescence indicator of this class probe according to the difference of gained derivant fluorescent characteristic under condition of different pH behind the fluorescence probe mark amino-compound, be used to measure the pH value in cell and the organelle as pH.
Embodiment: the following examples are to further specify of the present invention, rather than limit the scope of the invention.
Embodiment:
The preparation of rhodamine B-N-succinimide ester:
Rhodamine B 2.4g (0.005mol) is dissolved in the dry acetonitrile of 100ml, add N-hydroxy-succinamide 0.6g (0.0052mol), be added dropwise to the dry acetonitrile solution 50ml that contains dicyclohexylcarbodiimide 1.2g (0.0058mol) by constant pressure funnel while stirring, stirring at room reaction 20h.Use silica gel thin-layer plate,, carry out thin-layer chromatographic analysis (TLC) monitoring in the dinethylformamide, confirm reaction yield about 90% in N.Remove by filter the powder that reaction generates, pressure reducing and steaming part filtrate, adding dry ether precipitation is filtered, and the ether washing with acetonitrile/ether recrystallization, drying, gets the rhodamine B-N-succinimide crystalline esters of bright green, and structure is as the formula (2).Fusing point: 202～205 ℃; Ultraviolet-visible spectrum, fluorescence spectrometry are in methyl alcohol: absorb (exciting) maximal value: 565nm; Fluorescent emission maximal value: 587nm; Infrared spectrum (KBr) is measured: 1732.04, and 1763.11cm -1(v C=O); Nuclear magnetic resonance measuring, 1H-NMR (CDCl 3) δ/ppm:8.39 (1H, d, 4-position H), 7.95 (1H, t, 5-position H), 7.81 (1H, t, 6-position H), 7.44 (1H, d, 1-position H), 7.05 (2H, d, 1 ', 8 '-position H), 6.85 (4H, q, 2 ', 4 ', 5 ', 7 '-position H), 3.61 (8H, q ,-N-CH 2-CH 3CH 2), 2.75 (4H, s, the CH of succinimide 2), 1.30 (12H, t ,-N-CH 2-CH 3CH 3).
Figure A20031010893000061

Claims (4)

1. one kind is the fluorescence probe of parent with the rhodamine, it is characterized in that it has structure shown in (1) formula:
Figure A2003101089300002C1
Wherein, R 1, R 2, R 3And R 4Respectively do for oneself hydrogen and replacing and alkyl, carboxyalkyl, aminoalkyl, naphthenic base, the aryl of not replacement; R 5, R 6, R 7, R 8Respectively do for oneself hydrogen, alkyl, carboxyalkyl, aminoalkyl, alkyl ether, alkyl thioether, halogen or alkoxy; R 1And R 7For having the alkyl chain of 2 to 5 carbon atoms, it is connected to 5 ' carbon on the nitrogen that is chained on the 6 ' carbon together; R 3And R 8For having the alkyl chain of 2 to 5 carbon atoms, it is connected to 4 ' carbon on the nitrogen that is chained on the 3 ' carbon together; R 2And R 5For having the alkyl chain of 2 to 5 carbon atoms, it is connected to 7 ' carbon on the nitrogen that is chained on the 6 ' carbon together; R 4And R 6For having the alkyl chain of 2 to 5 carbon atoms, it is connected to 2 ' carbon on the nitrogen that is chained on the 3 ' carbon together; R 1And R 2Form on the main chain alkyl or alkenyl chain of 5 atoms at the most together, it is made up of carbon and one or more heteroatoms that is selected from nitrogen or oxygen.R 3And R 4Form on the main chain alkyl or alkenyl chain of 5 atoms at the most together, it is made up of carbon and one or more heteroatoms that is selected from nitrogen or oxygen;
An is inorganic or organic anion, comprises chlorion, bromide ion, sulfate radical, bisulfate ion, pyrovinic acid root, sulfaminic acid root, perchlorate, benzene sulfonic acid root, oxalate, maleate, acetate, glycolic acid root, methoxyimino acetic acid root, propionate, lactate, amber acid radical, tartrate anion.
2. one kind is the preparation method of the fluorescence probe of parent with the rhodamine, it is characterized in that having the rhodamine of carboxyl to be dissolved in the dry organic solvent for 1 mole the 3-bit strip, add 0.5～5 mole of N-hydroxy-succinamide, be added dropwise to the dicyclohexylcarbodiimide (DCC) that is dissolved in dry organic solvent by constant pressure funnel while stirring, the amount of dicyclohexylcarbodiimide is 1～5 mole, at 0 ℃ of temperature stirring reaction 1h～120h that arrives between the organic solvent boiling point, with reacting liquid filtering, pressure reducing and steaming part filtrate, add the precipitation agent precipitation, filter drying, getting target product, promptly is the fluorescence probe of parent with the rhodamine.Its course of reaction is shown below:
Figure A2003101089300003C1
3. the preparation method of fluorescence probe according to claim 2 is characterized in that dry organic solvent has acetonitrile, propionitrile, N, dinethylformamide, dimethyl sulfoxide (DMSO), perhaps their potpourri.
4. the preparation method of fluorescence probe according to claim 2 is characterized in that precipitation agent has ether, ethyl acetate, perhaps their potpourri.
CNB2003101089300A 2003-11-27 2003-11-27 Fluorescent probe with rhodamine as parent substance and preparing process thereof Expired - Fee Related CN1226627C (en)

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Cited By (14)

* Cited by examiner, † Cited by third party
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CN100360932C (en) * 2005-09-16 2008-01-09 厦门大学 Reagent for detecting mercury ion in water and its preparation method
CN101135644B (en) * 2007-08-16 2010-05-19 南京大学 Mercury ion fluorescent color-developing agent and detecting method
CN101302220B (en) * 2007-05-08 2010-09-29 中国科学院化学研究所 N-benzoyl rhodamine B hydrazine, preparation and use thereof
CN101307226B (en) * 2008-07-14 2010-12-01 无锡中德美联生物技术有限公司 Fluorochrome for marking oligonucleotide and protein, method for preparing same and use
CN101921587A (en) * 2010-07-19 2010-12-22 西安交通大学 A kind of fluorescent probe and preparation method thereof with tumor cell proliferation inhibition activity
CN101093222B (en) * 2007-07-24 2011-11-23 山东师范大学 Fluorescent probe in use for detecting glutathion inside cell, synthesis method and application
CN102659744A (en) * 2012-05-09 2012-09-12 大连理工大学 Rhodamine fluorescent probe sensitive to pH change, synthetic method and application
CN101368945B (en) * 2007-08-15 2012-11-14 中国科学院理化技术研究所 fluorescent probe for detecting mercury ions in cells, synthetic method and application
CN103937490A (en) * 2014-04-24 2014-07-23 辽宁大学 Fluorescent probe for detecting sulfur ions in mitochondria and application thereof
CN105859705A (en) * 2016-06-01 2016-08-17 南开大学 Fluorescence labelled probe as well as preparation method and application thereof in protein labeling
CN105968105A (en) * 2016-06-01 2016-09-28 南开大学 Fluorescence labeling probe and preparation method thereof and labeling application to bacteria
CN107880009A (en) * 2017-11-28 2018-04-06 西安石油大学 A kind of rhodamine B fluorescence probe preparation method with surface-active
CN109161542A (en) * 2018-02-23 2019-01-08 广州简册生物技术有限公司 fluorescence in situ hybridization probe and its preparation method and application
CN110194901A (en) * 2019-05-14 2019-09-03 深圳市华星光电技术有限公司 A kind of preparation method and optical diaphragm of dyestuff

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CN100509817C (en) * 2006-04-11 2009-07-08 山东师范大学 Fluorescent probe for detecting superoxide anion free radical and synthesis method and use thereof

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100360932C (en) * 2005-09-16 2008-01-09 厦门大学 Reagent for detecting mercury ion in water and its preparation method
CN101302220B (en) * 2007-05-08 2010-09-29 中国科学院化学研究所 N-benzoyl rhodamine B hydrazine, preparation and use thereof
CN101093222B (en) * 2007-07-24 2011-11-23 山东师范大学 Fluorescent probe in use for detecting glutathion inside cell, synthesis method and application
CN101368945B (en) * 2007-08-15 2012-11-14 中国科学院理化技术研究所 fluorescent probe for detecting mercury ions in cells, synthetic method and application
CN101135644B (en) * 2007-08-16 2010-05-19 南京大学 Mercury ion fluorescent color-developing agent and detecting method
CN101307226B (en) * 2008-07-14 2010-12-01 无锡中德美联生物技术有限公司 Fluorochrome for marking oligonucleotide and protein, method for preparing same and use
CN101921587B (en) * 2010-07-19 2014-07-02 西安交通大学 Fluorescent probe having tumor cell proliferation inhibition activity and preparation method thereof
CN101921587A (en) * 2010-07-19 2010-12-22 西安交通大学 A kind of fluorescent probe and preparation method thereof with tumor cell proliferation inhibition activity
CN102659744A (en) * 2012-05-09 2012-09-12 大连理工大学 Rhodamine fluorescent probe sensitive to pH change, synthetic method and application
CN103937490A (en) * 2014-04-24 2014-07-23 辽宁大学 Fluorescent probe for detecting sulfur ions in mitochondria and application thereof
CN105859705A (en) * 2016-06-01 2016-08-17 南开大学 Fluorescence labelled probe as well as preparation method and application thereof in protein labeling
CN105968105A (en) * 2016-06-01 2016-09-28 南开大学 Fluorescence labeling probe and preparation method thereof and labeling application to bacteria
CN105968105B (en) * 2016-06-01 2019-04-05 南开大学 A kind of fluorescence labeling probe and preparation method thereof and the label application to bacterium
CN107880009A (en) * 2017-11-28 2018-04-06 西安石油大学 A kind of rhodamine B fluorescence probe preparation method with surface-active
CN109161542A (en) * 2018-02-23 2019-01-08 广州简册生物技术有限公司 fluorescence in situ hybridization probe and its preparation method and application
CN110194901A (en) * 2019-05-14 2019-09-03 深圳市华星光电技术有限公司 A kind of preparation method and optical diaphragm of dyestuff

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