CN104194773A - Cell-membrane target nitric oxide fluorescent probe as well as preparation method and application thereof - Google Patents
Cell-membrane target nitric oxide fluorescent probe as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a cell-membrane target nitric oxide fluorescent probe as well as preparation method and application thereof and belongs to the technical field of biological detection. The probe disclosed by the invention has prominent features that the probe has a capability capable of targeting and marking on the surface of the cell membrane, has a certain water solubility and cannot penetrate through the cell membrane to enter the inside of the cell. The fluorescent probe has very weak fluorescence and small biomedium background interference and can be selectively reacted with nitric oxide under physiological conditions to generate derivative products with strong fluorescence and does not cause interference to common active nitride oxides in a living body. Due to the cell-membrane target fluorescent probe disclosed by the invention, the process in which nitric oxide is released from the cell can be visually analyzed.
Description
Technical field
The present invention relates to a kind of cytolemma targeting fluorescent probe that detects nitrogen protoxide release in cell by fluorescence imaging and its preparation method and application, belong to nitric oxide production technical field of biological.
Background technology
Nitrogen protoxide (NO) is as one of signaling molecule important in physiological process, plays a part very importantly in cardiovascular systems, neural system and immunity system, is therefore significant for the analysis and research of NO content in organism.A lot of NO analytical procedures were developed in the last few years, comprise chemoluminescence method, colorimetry, electron spin resonance, electrochemical process and fluorometry, wherein fluorometry enjoys investigator's concern with its highly selective and highly sensitive advantage, produced a series of taking O-Phenylene Diamine as reactive group, the small molecules fluorescent probe taking rhodamine, fluorescein, Hua Jing, boron difluoride dipyrrylmethanes (BODIPY) etc. as fluorophore; Containing Cu
2+, Fe
3+deng the metal complex fluorescent probe of metal ion; Two-photon fluorescence probe based on FRET (fluorescence resonance energy transfer) etc.But all these probes are all marked at cell interior, provide the information of cell interior NO by the variation of fluorescence intensity.Can not despise but be discharged into NO outside the born of the same parents effect in the physiological processs such as intercellular signal transmission, immune defense and vasodilator by cytolemma, and existing fluorescent probe all can not be realized the detection to this part NO.Therefore, the synthetic a kind of cytolemma target NO fluorescent probe of design, detects for original position the NO discharging from cell in real time, and the research physiological function of NO and the appearance of relative disease are had to important directive significance with treatment.
Summary of the invention
The present invention is directed to the deficiency that prior art exists, a kind of cytolemma target nitric oxide fluorescent probe that can target be marked at cytolemma outside surface is provided, for cell being discharged into the nitric oxide production detection outside born of the same parents, the physiological process research and the disease research that participate in for nitrogen protoxide provide novel method.
The technical solution adopted in the present invention is specific as follows:
A kind of cytolemma target nitric oxide fluorescent probe, has the structure shown in general formula (I):
Wherein, R
1for C
8～C
16straight chained alkyl; R
2for-SO
3h or-SO
3na.
At R
1on position, introduce and the long-chain hydrophobic side of cell membrane phospholipid similar, can realize the targeting mark of fluorine boron tetramethyleneimine cell membrane; Meanwhile, at R
2on position, introduce water soluble group, can strengthen to a certain extent the water-soluble of fluorine boron tetramethyleneimine, stop its permeates cell membranes to enter cell interior, thereby realize, cell is discharged into extracellular nitric oxide production detection.
A method of preparing above-mentioned cytolemma target nitric oxide fluorescent probe, comprises the following steps:
(1) under anhydrous and oxygen-free condition, 3,4-dinitrobenzoyl chloride is dissolved in methylene dichloride, splashes into C
8～C
16straight chained alkyl pyrroles's dichloromethane solution, stirring at room temperature 24～48 hours; Reaction finishes, after simple purification, purification thing is dissolved in anhydrous methylene chloride, be cooled to 0～-10 DEG C, under nitrogen protection, splash into 2,4-dimethyl pyrrole and phosphorus oxychloride, room temperature continues to stir 12～36 hours, then drips triethylamine, boron trifluoride diethyl etherate, and room temperature reaction to thin-layer chromatography shows that raw material reacts completely; Obtain red-purple powder compd A through purifying; Wherein, 3,4-dinitrobenzoyl chloride, straight chained alkyl pyrroles, 2, the mol ratio of 4-dimethyl pyrrole, phosphorus oxychloride, triethylamine, boron trifluoride diethyl etherate is 1.1～1.3:1:1～1.2:1～1.2:3～5:3～5;
(2) compd A is dissolved in to anhydrous methylene chloride, is cooled to-50 DEG C～-40 DEG C, under nitrogen protection, drip chlorsulfonic acid, dropwise, slowly rise to room temperature, treat that raw material reaction is complete, add weak base and regulate pH to 7～8, stopped reaction; Obtain garnet compd B through purifying; Wherein, the mol ratio of compd A, chlorsulfonic acid is 1:2～8;
(3) compd B is dissolved in to dehydrated alcohol, adds hydrazine hydrate and palladium carbon under nitrogen protection, reflux 3～5 hours, removes by filter palladium carbon after reacting completely, and after filtrate is spin-dried for, obtains above-mentioned cytolemma target nitric oxide fluorescent probe through purifying; Wherein, the mol ratio of compd B, hydrazine hydrate, palladium carbon is 1:5～10:0.5～1.5.
Described C
8～C
16straight chained alkyl pyrroles is 2-hexadecyl pyrroles, 2-dodecyl pyrroles or 2-eight alkyl pyrroles.
Described weak base is sodium bicarbonate.
Above-mentioned cytolemma target nitric oxide fluorescent probe is in the application of nitrogen protoxide detection field.
Cytolemma target nitric oxide fluorescent probe provided by the invention, its photoluminescent property is based on Photoinduced Electron metastasis (PET), the fluorescence intensity of this fluorescent probe own is very weak, under physiological condition, optionally react with NO, generation has the derivative of hyperfluorescenceZeng Yongminggaoyingguang, and is not subject to the interference of other common active nitrogen oxygen compound in organism.This probe synthetic method is simple, its saturated chain alkyl comprising can be inserted into and in cytolemma, realize non-covalent mark, water soluble group ensures that probe is only marked at the outside surface of cytolemma and can enter into cell interior simultaneously, is suitable for detecting being discharged into the nitrogen protoxide outside born of the same parents by cytolemma.
The present invention has the following advantages and beneficial effect:
1. cytolemma target nitric oxide fluorescent probe provided by the invention can be marked at cytolemma outside surface by target, and the original position that can realize be discharged into the NO outside born of the same parents by cytolemma detects in real time.
Cytolemma target nitric oxide fluorescent probe provided by the invention react with NO before and after fluorescence intensity change remarkable, and be not subject to the interference of other common active nitrogen oxygen compound in organism, for biological NO analyzing and testing efficient and sensible.
3. the preparation method of cytolemma target nitric oxide fluorescent probe provided by the invention is simple.
4. the labeling process of fine after birth target nitric oxide fluorescent probe cell membrane provided by the invention only needs several minutes.
Brief description of the drawings
Fig. 1 is the synthetic route chart of cytolemma target nitric oxide fluorescent probe.
Fig. 2 is cytolemma target nitric oxide fluorescent probe and the fluorescence spectrum figure with nitrogen protoxide derived products thereof; Wherein, the 1st, the excitation spectrum of probe itself, 1 ' is the emmission spectrum of probe itself; The 2nd, the excitation spectrum of probe and nitrogen protoxide derived products, 2 ' is the emmission spectrum of probe and nitrogen protoxide derived products.
Fig. 3 is cytolemma target nitric oxide fluorescent probe and the fluorescence intensity change figure of NO derivative in the 0.1M of different pH values phosphate buffer solution (containing 0.01% Triton X-100) thereof.
Fig. 4 is cytolemma target nitric oxide fluorescent probe and nitrogen protoxide and other active nitrogen oxygen compounds reacted fluorescence intensity comparison diagram in 0.1M phosphate buffer solution (containing 0.01% Triton X-100, pH7.4).
Fig. 5 is cytolemma target nitric oxide fluorescent probe and different concns nitrogen protoxide reacted fluorescence intensity change figure in 0.1M phosphate buffer solution (containing 0.01% Triton X-100, pH7.4).
Fig. 6 is the image of cytolemma target nitric oxide fluorescent probe for detection of nitrogen protoxide release in the mouse macrophage RAW264.7 activating.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described, is only the present invention is described and never limits the present invention.Embodiment 1
(1) by 308mg 3,4-dinitrobenzoyl chloride is dissolved in 150mL anhydrous methylene chloride, splashes into the 300mg 2-hexadecyl pyrroles who is dissolved in 30mL anhydrous methylene chloride, stirring at room temperature 48 hours under nitrogen protection, after reaction finishes, rotary evaporation is except desolventizing, the mixture purification by silica gel column chromatography of gained, the purification thing obtaining is dissolved in 100mL anhydrous methylene chloride again, be cooled to-10 DEG C, under nitrogen protection, splash into 0.12mL 2, 4-dimethyl pyrrole and 0.114mL phosphorus oxychloride, dropwise stirring at room temperature 36 hours, then add 0.72mL triethylamine, stirring at room temperature adds 0.646mL boron trifluoride diethyl etherate after 10 minutes, continue to stir 30 minutes, revolve and steam except desolventizing, gained mixture purification by silica gel column chromatography, eluent is methylene dichloride/normal hexane (50/50, v/v), obtain 176mg red-purple powder compd A (n=16), 1, 3-dimethyl-5-hexadecyl-8-(3 ', 4 '-dinitrobenzene)-boron difluoride-dipyrrylmethanes, productive rate is 28%.
(2) by gained 1; 3-dimethyl-5-hexadecyl-8-(3 '; 4 '-dinitrobenzene)-boron difluoride-dipyrrylmethanes 200mg is dissolved in 40mL anhydrous methylene chloride; be cooled to-50 DEG C; under nitrogen protection, drip the 0.17mL chlorsulfonic acid being dissolved in 10mL anhydrous methylene chloride; stir and slowly rise to room temperature after 10 minutes, rotary evaporation is concentrated.After concentrated, add the dilution of 10mL methyl alcohol, then add containing the aqueous solution (5mL) of 220mg sodium bicarbonate and be adjusted to pH 7.Remove by filter unnecessary sodium bicarbonate and revolve and steam except desolventizing, residuum purification by silica gel column chromatography, eluent is methylene chloride/methanol (70/30, v/v), obtains 195mg garnet solid B (n=16, R
2for sodium sulfonate), 1,3-dimethyl-2,6-sodium disulfonate-5-hexadecyl-8-(3 ', 4 '-dinitrobenzene)-boron difluoride-dipyrrylmethanes, productive rate is 73%.
(3) by gained 1; 3-dimethyl-2; 6-sodium disulfonate-5-hexadecyl-8-(3 '; 4 '-dinitrobenzene)-boron difluoride-dipyrrylmethanes 200mg is dissolved in 40mL ethanol; under nitrogen protection, add the palladium carbon of 0.12mL hydrazine hydrate and 39mg 10%; reflux 5 hours; after reacting completely; filtered while hot is removed palladium carbon, after filtrate is spin-dried for, and mixture purification by silica gel column chromatography; eluent is methylene chloride/methanol (65/35; v/v), obtain 67mg reddish-brown solid C (n=16, R
2for sodium sulfonate), 1,3-dimethyl-2,6-sodium disulfonate-5-hexadecyl-8-(3 ', 4 '-diaminobenzene)-boron difluoride-dipyrrylmethanes, productive rate is 36%.
1,3-dimethyl-2, the characterization data of 6-sodium disulfonate-5-hexadecyl-8-(3 ', 4 '-diaminobenzene)-boron difluoride-dipyrrylmethanes is:
1h NMR (300Mz, DMSO-d
6): δ 0.853 (3H, t, 6.9Hz), 1.243 (28H, s), 1.819 (3H, s), 2.648 (3H, s), 2.940 (2H, t), 4.788 (2H, t), 5.011 (2H, s), 6.412 (1H, d, J=7.5Hz), 6.525 (2H, s), 6.640 (1H, d, J=7.5Hz) .HRMS (ESI) calcd for C
33h
48bF
2n
4na
2o
6s
2 +([M+H]
+): 755.2872.Found:755.2845.
Embodiment 2
(1) by 355mg 3,4-dinitrobenzoyl chloride is dissolved in 150mL anhydrous methylene chloride, splashes into the 300mg 2-dodecyl pyrroles who is dissolved in 30mL anhydrous methylene chloride, stirring at room temperature 30 hours under nitrogen protection.After reaction finishes, rotary evaporation is except desolventizing, gained mixture purification by silica gel column chromatography; the purification thing obtaining is dissolved in 100mL anhydrous methylene chloride again; be cooled to-5 DEG C, under nitrogen protection, splash into 0.138mL 2,4-dimethyl pyrrole and 0.131mL phosphorus oxychloride; after dripping; stirring at room temperature 24 hours, then adds 0.72mL triethylamine, and stirring at room temperature adds 0.724mL boron trifluoride diethyl etherate after 10 minutes; continue to stir 30 minutes, revolve and steam except desolventizing.Gained mixture purification by silica gel column chromatography, eluent is methylene dichloride/normal hexane (50/50, v/v), obtain 292mg red-purple powder A (n=12), 1,3-dimethyl-5-dodecyl-8-(3 ', 4 '-dinitrobenzene)-boron difluoride-dipyrrylmethanes, productive rate is 41%.
(2) by gained 1; 3-dimethyl-5-dodecyl-8-(3 '; 4 '-dinitrobenzene)-boron difluoride-dipyrrylmethanes 200mg is dissolved in 40mL anhydrous methylene chloride; be cooled to-45 DEG C; under nitrogen protection, drip the 0.12mL chlorsulfonic acid being dissolved in 10mL anhydrous methylene chloride; stir and slowly rise to room temperature after 10 minutes, rotary evaporation is concentrated.After concentrated, add the dilution of 10mL methyl alcohol, then add containing the aqueous solution (5mL) of 152mg sodium bicarbonate and be adjusted to pH 7.5.Remove by filter unnecessary sodium bicarbonate and revolve and steam except desolventizing, residuum purification by silica gel column chromatography, eluent is methylene chloride/methanol (70/30, v/v), obtains 205mg garnet solid B (n=12, R
2for sodium sulfonate), 1,3-dimethyl-2,6-sodium disulfonate-5-dodecyl-8-(3 ', 4 '-dinitrobenzene)-boron difluoride-dipyrrylmethanes, productive rate is 75%.
(3) by gained 1; 3-dimethyl-2; 6-sodium disulfonate-5-dodecyl-8-(3 '; 4 '-dinitrobenzene)-boron difluoride-dipyrrylmethanes 200mg is dissolved in 40mL ethanol; under nitrogen protection, add the palladium carbon of 0.1mL hydrazine hydrate and 26mg 10%; reflux 4 hours; after reacting completely; filtered while hot is removed palladium carbon, after filtrate is spin-dried for, and mixture purification by silica gel column chromatography; eluent is methylene chloride/methanol (65/35; v/v), obtain 64mg reddish-brown solid C (n=12, R
2for sodium sulfonate), 1,3-dimethyl-2,6-sodium disulfonate-5-dodecyl-8-(3 ', 4 '-diaminobenzene)-boron difluoride-dipyrrylmethanes, productive rate is 35%.
1,3-dimethyl-2, the characterization data of 6-sodium disulfonate-5-dodecyl-8-(3 ', 4 '-diaminobenzene)-boron difluoride-dipyrrylmethanes is:
1h NMR (300Mz, DMSO-d
6): δ 0.853 (3H, t, 6.9Hz), 1.245 (20H, s), 1.821 (3H, s), 2.648 (3H, s), 2.940 (2H, t), 4.791 (2H, t), 5.011 (2H, s), 6.413 (1H, d, J=7.5Hz), 6.528 (2H, s), 6.641 (1H, d, J=7.5Hz) .HRMS (ESI) calcd for C
29h
40bF
2n
4na
2o
6s
2 +([M+H]
+): 699.5683.Found:699.5656.
Embodiment 3
(1) by 429mg 3,4-dinitrobenzoyl chloride is dissolved in 150mL anhydrous methylene chloride, splashes into the 300mg 2-eight alkyl pyrroles that are dissolved in 30mL anhydrous methylene chloride, stirring at room temperature 24 hours under nitrogen protection.After reaction finishes, rotary evaporation is except desolventizing, gained mixture purification by silica gel column chromatography, the purification thing obtaining is dissolved in 100mL anhydrous methylene chloride again, be cooled to 0 DEG C, under nitrogen protection, splash into 0.165mL 2, 4-dimethyl pyrrole and 0.157mL phosphorus oxychloride, after dripping, stirring at room temperature 12 hours, then add 0.714mL triethylamine, stirring at room temperature adds 0.638mL boron trifluoride diethyl etherate after 10 minutes, continue to stir 30 minutes, revolve and steam except desolventizing, gained mixture purification by silica gel column chromatography, eluent is methylene dichloride/normal hexane (50/50, v/v), obtain 487mg red-purple powder A (n=8), 1, 3-dimethyl-5-eight alkyl-8-(3 ', 4 '-dinitrobenzene)-boron difluoride-dipyrrylmethanes, productive rate is 58%.
(2) by gained 1; 3-dimethyl-5-eight alkyl-8-(3 '; 4 '-dinitrobenzene)-boron difluoride-dipyrrylmethanes 200mg is dissolved in 40mL anhydrous methylene chloride; be cooled to-40 DEG C; under nitrogen protection, drip the 0.05mL chlorsulfonic acid being dissolved in 10mL anhydrous methylene chloride, stir and slowly rise to room temperature after 10 minutes, rotary evaporation is concentrated; after concentrated, add the dilution of 10mL methyl alcohol, then add containing the aqueous solution (5mL) of 68mg sodium bicarbonate and be adjusted to pH 8.Remove by filter unnecessary sodium bicarbonate and revolve and steam except desolventizing, residuum purification by silica gel column chromatography, eluent is methylene chloride/methanol (70/30, v/v), obtains 212mg garnet solid B (n=8, R
2for sodium sulfonate), 1,3-dimethyl-2,6-sodium disulfonate-5-eight alkyl-8-(3 ', 4 '-dinitrobenzene)-boron difluoride-dipyrrylmethanes, productive rate is 75%.
(3) by gained 1; 3-dimethyl-2; 6-sodium disulfonate-5-eight alkyl-8-(3 '; 4 '-dinitrobenzene)-boron difluoride-dipyrrylmethanes 200mg is dissolved in 40mL ethanol; under nitrogen protection, add the palladium carbon of 0.07mL hydrazine hydrate and 15mg 10%; reflux 3 hours; after reacting completely; filtered while hot is removed palladium carbon, after filtrate is spin-dried for, and mixture purification by silica gel column chromatography; eluent is methylene chloride/methanol (65/35; v/v), obtain 64mg reddish-brown solid C (n=8, R
2for sodium sulfonate), 1,3-dimethyl-2,6-sodium disulfonate-5-eight alkyl-8-(3 ', 4 '-diaminobenzene)-boron difluoride-dipyrrylmethanes, productive rate is 35%.
1,3-dimethyl-2, the characterization data of 6-sodium disulfonate-5-eight alkyl-8-(3 ', 4 '-diaminobenzene)-boron difluoride-dipyrrylmethanes is:
1h NMR (300Mz, DMSO-d
6): δ 0.853 (3H, t, 6.9Hz), 1.244 (12H, s), 1.821 (3H, s), 2.648 (3H, s), 2.940 (2H, t), 4.791 (2H, t), 5.011 (2H, s), 6.413 (1H, d, J=7.5Hz), 6.528 (2H, s), 6.641 (1H, d, J=7.5Hz) .HRMS (ESI) calcd for C
25h
32bF
2n
4na
2o
6s
2 +([M+H]
+): 643.4620.Found:643.4593.
Embodiment 4
The cytolemma target nitric oxide fluorescent probe (hereinafter to be referred as probe (I)) obtaining with embodiment 1 and the property testing of nitrogen protoxide derivative thereof: because the length of saturated carbon chains in probe (I) is longer, for the mark of cytolemma, the probe that effect obtains than embodiment 2 and embodiment 3 is good, has therefore studied probe (I) and the chemical property with nitrogen protoxide derived products thereof in great detail.(1) spectral quality
(contain 0.01% Triton X-100 with 0.1M phosphate buffer solution, pH 7.4) be solvent, measure ultraviolet-visible absorption spectroscopy, fluorescence spectrum, molar absorptivity and the fluorescence quantum yield of probe (I) and NO derivative thereof.The mensuration of fluorescence quantum yield is to be dissolved in fluorescein (fluorescence quantum yield is as 0.92) in 0.1M sodium hydroxide solution as standard.Measurement result is in table 1.
The spectral quality of table 1. probe (I) and NO derivative thereof
Experimental result shows, the fluorescence of probe (I) itself is very weak, and its excitation-emission emission wavelength is respectively 514nm, 526nm; The derived products fluorescence intensity obtaining after reacting with nitrogen protoxide has significantly enhancing, and its excitation-emission wavelength is respectively 515nm, 529nm.The fluorescence spectrum of probe (I) and NO derivative thereof as shown in Figure 2.
(2) impact of pH value on probe (I) and NO derivatives fluorescent character thereof
After phosphate buffer solution (containing 0.01% Triton X-100) dilution by the storing solution of probe (I) and NO derivative thereof by the different pH values of 0.1M (2.0-12.0), measure its fluorescence intensity, result as shown in Figure 3.The fluorescence intensity of probe (I) own is very weak, substantially do not change with the variation of pH value, react the derived products generating with nitrogen protoxide under the nearly neutral condition of acidity, fluorescence intensity is less with the variation of pH value, under alkaline condition, due to triazole group proton dissociation, cause fluorescence intensity to reduce.
(3) selectivity of probe (I) to determination of nitric oxide
Probe (I) and NO and various active nitrogen oxygen compound NO are investigated respectively
3 -, NO
2 -, H
2o
2, O
2 -, ONOO
-, ClO
-.OH,
1o
2, AA (xitix) response situation.Reaction is all carried out in 0.1M phosphate buffer solution (containing 0.01% Triton X-100, pH 7.4), and 37 DEG C are reacted 15 minutes, and the concentration of probe (I) is 5 μ M, and NO is 10 μ M, NO
3 -for 1mM, NO
2 -for 1mM, H
2o
2for 1mM, O
2 -for 1mM, ONOO
-for 1mM, ClO
-be 500 μ M .OH is 500 μ M,
1o
2be 500 μ M, AA is 1mM.Measurement result as shown in Figure 4, probe (I) and NO
3 -, NO
2 -, H
2o
2, O
2 -, ONOO
-, ClO
-.OH,
1o
2, after the reaction of these active substances of AA, fluorescence intensity change is very faint, generates triazole derived products with NO after react, fluorescence intensity significantly increases, and shows that probe (I) has good selectivity to NO mensuration.
Embodiment 5: probe (I) is for the nitric oxide production mensuration of the aqueous solution
(contain 0.01% Triton X-100 at 0.1M phosphate buffer solution, pH 7.4) in, add the storing solution of probe (I), making its concentration is 5 μ M, add the NO saturated solution of different concns, 37 DEG C of reactions were measured fluorescence intensity after 15 minutes again.As shown in Figure 5, along with the increase of NO concentration, fluorescence intensity strengthens result gradually.
Embodiment 6: probe (I) detects the release of cell NO in real time for original position
Taking mouse macrophage (RAW 264.7) as research object, investigate in detail the flag condition of probe cell membrane, comprise concentration and probe concentration, incubation time, toxicity etc.Experimental result shows, probe (I) can complete mark in the incubation time of 3～5 minutes, and the probe that concentration is 5 μ M can complete the detection of the NO that the scavenger cell to having activated discharges, as shown in Figure 6.
Claims (5)
1. a cytolemma target nitric oxide fluorescent probe, is characterized in that, has the structure shown in general formula (I):
Wherein, R
1for C
8～C
16straight chained alkyl, R
2for-SO
3h or-SO
3na.
2. a method of preparing cytolemma target nitric oxide fluorescent probe claimed in claim 1, comprises the following steps:
(1) under anhydrous and oxygen-free condition, 3,4-dinitrobenzoyl chloride is dissolved in methylene dichloride, splashes into C
8～C
16straight chained alkyl pyrroles's dichloromethane solution, stirring at room temperature 24～48 hours; Reaction finishes, after simple purification, purification thing is dissolved in anhydrous methylene chloride, be cooled to 0～-10 DEG C, under nitrogen protection, splash into 2,4-dimethyl pyrrole and phosphorus oxychloride, room temperature continues to stir 12～36 hours, then drips triethylamine, boron trifluoride diethyl etherate, and room temperature reaction to thin-layer chromatography shows that raw material reacts completely; Obtain red-purple powder compd A through purifying; Wherein, 3,4-dinitrobenzoyl chloride, straight chained alkyl pyrroles, 2, the mol ratio of 4-dimethyl pyrrole, phosphorus oxychloride, triethylamine, boron trifluoride diethyl etherate is 1.1～1.3:1:1～1.2:1～1.2:3～5:3～5;
(2) compd A is dissolved in to anhydrous methylene chloride, is cooled to-50 DEG C～-40 DEG C, under nitrogen protection, drip chlorsulfonic acid, dropwise, slowly rise to room temperature, treat that raw material reaction is complete, add weak base and regulate pH to 7～8, stopped reaction; Obtain garnet compd B through purifying; Wherein, the mol ratio of compd A, chlorsulfonic acid is 1:2～8;
(3) compd B is dissolved in to dehydrated alcohol, adds hydrazine hydrate and palladium carbon under nitrogen protection, reflux 3～5 hours, removes by filter palladium carbon after reacting completely, and after filtrate is spin-dried for, obtains above-mentioned cytolemma target nitric oxide fluorescent probe through purifying; Wherein, the mol ratio of compd B, hydrazine hydrate, palladium carbon is 1:5～10:0.5～1.5.
3. method according to claim 2, is characterized in that: described C
8～C
16straight chained alkyl pyrroles is 2-hexadecyl pyrroles, 2-dodecyl pyrroles or 2-eight alkyl pyrroles.
4. method according to claim 2, is characterized in that: described weak base is sodium bicarbonate.
5. the application of cytolemma target nitric oxide fluorescent probe claimed in claim 1 in nitrogen protoxide detection field.
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| CN106867513A (en) * | 2015-12-11 | 2017-06-20 | 中国科学院大连化学物理研究所 | A kind of cell membrane localization zinc ion fluorescent and its preparation method and application |
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| 侯风萍: "荧光素类荧光探针的合成、表征以及在生物成像上的应用", 《中国优秀硕士学位论文全文数据库 工程科技I辑》, no. 11, 15 November 2013 (2013-11-15), pages 3 - 4 * |
| 段二红: "罗丹明B类衍生物的合成及表征", 《中国优秀硕士学位论文全文数据库 工程科技I辑》, no. 01, 15 July 2007 (2007-07-15), pages 5 - 6 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106867513A (en) * | 2015-12-11 | 2017-06-20 | 中国科学院大连化学物理研究所 | A kind of cell membrane localization zinc ion fluorescent and its preparation method and application |
| CN106220664A (en) * | 2016-08-10 | 2016-12-14 | 浙江大学 | A kind of fluorescent probe detecting autophagy stream and preparation thereof and application |
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