CN104479669A - Preparation method and applications of enhanced type fluorescent probe for detecting hydrogen sulfide - Google Patents
Preparation method and applications of enhanced type fluorescent probe for detecting hydrogen sulfide Download PDFInfo
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- CN104479669A CN104479669A CN201410657865.5A CN201410657865A CN104479669A CN 104479669 A CN104479669 A CN 104479669A CN 201410657865 A CN201410657865 A CN 201410657865A CN 104479669 A CN104479669 A CN 104479669A
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Abstract
The invention provides an enhanced type fluorescent probe for detecting hydrogen sulfide. The fluorescent probe is an NRS fluorescent probe, the structural general formula of the NRS fluorescent probe is shown as (I), the preparation method comprises the following steps: adding nile red, 2,4-dinitrofluorobenzene and potassium carbonate into dimethylformamide, heating to 70-80DEG C, performing reflux reaction for 3 hours, extracting the reaction liquor by dichloromethane and collecting an organic phase, drying the organic phase with anhydrous magnesium sulfate, and purifying through silica column chromatography to obtain a target product. The probe has a mitochondrial location function, can perform real-time and on-site online detection to mitochondria, and can help us to further master the physiological function of the hydrogen sulfide and promote the understanding of physiological action of H2S, and provide a visible detection tool for research and development of novel cardiovascular drugs.
Description
Technical field
The present invention relates to the fluorescent probe that hydrogen sulfide is detected, be specifically related to a kind ofly detect enhancement type fluorescent probe and the preparation method of hydrogen sulfide and apply in plastosome.
Background technology
Hydrogen sulfide (H
2s) being a kind of gas of irritant smell, is the gas molecules that after nitrogen protoxide (NO) and carbon monoxide (CO) the 3rd kind has information transfer function.Hydrogen sulfide in environment mainly comes from the release of hydrogen sulfide in industrial production, water treatment, penkeeping process.Hydrogen sulfide all plays an important role in a lot of biological physiological process, the adjustment of such as neurotransmission, the restraining effect etc. of insulin signaling.Hydrogen sulfide levels extremely can also may be closely related with diseases such as alzheimer's disease, Down's syndrome, diabetes and liver cirrhosis.The physiology of hydrogen sulfide and pathology functional study have become the focus of at present chemistry and biological study, and can in living biological systems one of the method key subject becoming this field of the detection hydrogen sulfide of accurate quick.
Find Endogenous Hydrogen Sulfide (hydrogen sulfide, H the nineties in last century
2s) important physiological action is played in vivo, such as cystathionine-B-synthetic enzyme (cystathionine-B-synthase, CBS), cystathione lyase (cystathionine-lyase, CSE) and in the physiological metabolism process of halfcystine transferring enzyme all can produce hydrogen sulfide, its place produced is in plastosome.The hydrogen sulfide derived from plastosome has very important regulating effect for cardio-cerebrovascular, immunity system and neural system.The fluorescent probe kind detected hydrogen sulfide is at present a lot, but the overwhelming majority does not have plastosome positioning function, and more the hydrogen sulfide that cannot realize producing in plastosome detects.
Summary of the invention
For the deficiencies in the prior art, the invention provides a kind of enhancement type fluorescent probe detecting hydrogen sulfide, this probe has plastosome positioning function, can carry out in real time, the on-line checkingi of original position to plastosome, to the physiological function that we understand hydrogen sulfide further be contributed to, and contribute to promoting H
2the understanding of S physiological action, the research and development for Novel cardiovascular medicine provide visual testing tool.
The technical solution used in the present invention is:
Detect an enhancement type fluorescent probe for hydrogen sulfide, described fluorescent probe is NRS fluorescent probe, and the general structure of described NRS fluorescent probe is as shown in (I):
A kind of preparation method detecting the enhancement type fluorescent probe of hydrogen sulfide, comprise the steps: Nile red, 2,4-dinitrofluorobenzene and salt of wormwood, join in dimethyl formamide, is heated to 70 DEG C-80 DEG C, back flow reaction is after 3 hours, reaction solution dichloromethane extraction is collected organic phase, by this organic phase anhydrous magnesium sulfate drying, purifies through silica gel column chromatography and obtain target product.Preparation feedback formula is as follows:
Described preparation method, described Nile red, the mol ratio of DNF and salt of wormwood is 1:1.2:1.2.
Described preparation method, the eluent in described silica gel column chromatography is CH
2cl
2: CH
3oH=40:1; SiO
2300-400 order.
The present invention also provides a kind of application detecting enhancement type fluorescent probe sulfonium ion or hydrogen sulfide in detection water body of hydrogen sulfide.
Above-mentioned application states fluorescent probe in the application detecting hydrogen sulfide in viable cell plastosome.
Viable cell described in above-mentioned application is Hela cell strain, MCF-7 cell strain or RAW264.7 cell strain.
The invention has the beneficial effects as follows:
Fluorescent probe molecule of the present invention can quick position in plastosome, and detection real-time online can be carried out to the sulfonium ion in plastosome, contribute to the physiological function that we understand hydrogen sulfide further, contribute to promoting H
2the understanding of S physiological action, the research and development for Novel cardiovascular medicine provide visual testing tool.This probe has good optical stability, solvability, and has higher selectivity, not by the interference of other species such as active oxygen and active nitrogen.Laser confocal imaging experiment shows that this kind of probe has good cell permeability, has no side effect to Cell and organism body.
Accompanying drawing explanation
Fig. 1 is that the NRS fluorescent probe of embodiment 1 preparation is to different concns Na
2the absorption spectrum response of the S aqueous solution.
Fig. 2 is that the NRS fluorescent probe of embodiment 1 preparation is to different concns Na
2the fluorescence spectrum response of the S aqueous solution.
Fig. 3 be embodiment 1 prepare NRS fluorescent probe at 642.89nm place to different concns Na
2the fluorescence intensity change trend of the S aqueous solution.
Fig. 4 is that the NRS fluorescent probe prepared of embodiment 1 is to the Na of 64 times
2the fluorescence response curve of the S aqueous solution.
Fig. 5 is that NRS fluorescent probe prepared by embodiment 1 responds the fluorescence intensity of different substances at 642.89nm place.
Fig. 6 is that the NRS fluorescent probe of embodiment 1 preparation is to the fluorescence co-focusing micro-imaging of hydrogen sulfide in plastosome.
Embodiment
Implement the preparation method that 1 one kinds are detected the enhancement type fluorescent probe of hydrogen sulfide
Preparation feedback formula is as follows:
Wherein: Nile red (Nile Red, 9-(dimethylamino)-2-hydroxy-5H-Benzo [a] phenoxazin-5-one) is commercially available in the U.S., also can synthesize.
By Nile red (0.0183g, 0.06mmol), DNF (0.0135g, 0.072mmol) and K
2cO
3(0.0099g, 0.072mmol) is dissolved in 8mL dimethyl formamide (DMF), is heated to 75 DEG C, back flow reaction 3h.Reaction solution is poured in 50mL water, with methylene dichloride (50mL × 3) extraction, collect organic phase, then use anhydrous magnesium sulfate drying, through silica gel column chromatography purification (eluent CH
2cl
2: CH
3oH=40:1; SiO
2300-400 order), obtain red-purple target product NRS fluorescent probe, H-NMR:(300MHz, DMSO-d6) δ 8.97 (d, J=2.7Hz, 1H), 8.52 (dd, J=9.3,2.7Hz, 1.6Hz, 1H), 8.27 (d, J=9.0Hz, 1H), 8.26 (s, 1H), 7.63 (m, J=9.0Hz, 2H), 7.40 (d, J=9.3Hz, 1H), 6.87 (dd, J=9.0,2.7Hz, 1H), 6.73 (dd, J=2.7Hz, 1H), 6.35 (s, 1H), 3.43 (s, 6H); HRMS:473.1079.
Embodiment 2 one kinds detects the real-time detection of enhancement type fluorescent probe for hydrogen sulfide in water body of hydrogen sulfide
Be dissolved in dimethyl sulphoxide aqueous solution by the NRS fluorescent probe of preparation in embodiment 1, compound concentration is 5 × 10
-6the NRS dimethyl sulphoxide solution of M, wherein dimethyl sulfoxide (DMSO) DMSO and H
2the volume ratio 8:2 of O, successively adds the Na that molar equivalent is NRS fluorescent probe 3.2 times in NRS dimethyl sulphoxide solution
2s, is respectively 3.2 times, 6.4 times, 9.6 times, 12.8 times, 16 times after accumulation ... 51.2 doubly, the Na of 54.4 times
2s, the color of visible solution is along with Na
2the increase of S concentration becomes redness from purple.Each time solution carries out ultraviolet spectrophotometry and Tissue with Spectrofluorometric examination respectively, draws NRS fluorescent probe to the Na of different concns
2the S aqueous solution response absorption spectrum as shown in Figure 1 with fluorescence spectrum as shown in Figure 2.In Fig. 1 at the crest at 565nm place with Na
2the increase of S concentration and declining, the crest at 460nm place is with Na
2the increase of S concentration and raising, NRS fluorescent probe is for Na as shown in Figure 1
2s has response, Na in absorption
2s can make NRS color change.In Fig. 2 at the crest at 642nm place with Na
2the increase of S concentration and raising, NRS fluorescent probe is for Na as shown in Figure 2
2s has response on fluorescence, Na
2s can make NRS Fluorescence Increasing.
Fig. 3 be this NRS fluorescent probe at 642.89nm place to different concns Na
2the fluorescence intensity change trend of the S aqueous solution.As shown in Figure 3, the fluorescence intensity at 642nm crest place is with Na
2s concentration increases and strengthens.
Fig. 4 is this NRS fluorescent probe to the Na of 64 times
2the fluorescence response curve of the S aqueous solution.As seen from the figure, the Na of 64 times is added
2after S, NRS fluorescent probe and Na
2the time of response of S is that 1min, NRS fluorescent probe shows Na
2s fast spectrum responds.Experimental result shows that NRS can fast for Na
2the real time on-line monitoring of S.
Respectively to the NRS dimethyl sulphoxide solution (5 × 10 of above-mentioned 3ml
-6m) adding concentration in is 1 × 10
-2the NRS of M, Na
2s, Hg (AC)
2, Kl, NaNO
3, AlCl
3, H
2o
2, hydroxyl free radical, after the different ions 72uL such as NaClO, carries out fluorescence spectrophotometry test, and as shown in Figure 5, this NRS fluorescent probe is only to Na for result
2s has response, only has Na
2the change in fluorescence of S is very large, and the fluorescence intensity of other materials does not change substantially.Experiment shows that this NRS fluorescent probe has higher selectivity, not by the interference of other species such as active oxygen or active nitrogen.
Embodiment 3 one kinds detects the real-time detection of enhancement type fluorescent probe for hydrogen sulfide in viable cell plastosome of hydrogen sulfide
To containing viable cell be Hela cell strain culture dish in, adding concentration in embodiment 2 is 5 × 10
-5the NRS dimethyl sulphoxide solution of M, mix with cell culture fluid, after dyeing 5min, carry out cleaning three times with the phosphate buffer soln of pH=7.4, add the sodium sulfide solution of 50 times of molar equivalents, observe under finally this culture dish being placed in Laser Scanning Confocal Microscope.Experimental result finds, contaminates to have in the Hela cell mitochondrial of NRS to present obvious fluorescence, as shown in Figure 6: (1) Rh123 dyes, and it can be positioned in plastosome.(2) be NRS dyeing, it overlaps with Rh123, shows that NRS also can be positioned in plastosome.(3) be the stacking diagram of (1) and (2).Experimental result shows, NRS can enter cell, has good cell-membrane permeable, and NRS can be positioned plastosome, can detect the hydrogen sulfide in cell mitochondrial.
Claims (7)
1. detect an enhancement type fluorescent probe for hydrogen sulfide, it is characterized in that, described fluorescent probe is NRS fluorescent probe, and the general structure of described NRS fluorescent probe is as shown in (I):
2. one kind is detected the preparation method of the enhancement type fluorescent probe of hydrogen sulfide as claimed in claim 1, it is characterized in that comprising the steps: Nile red, 2,4-dinitrofluorobenzene and salt of wormwood, join in dimethyl formamide, is heated to 70 DEG C-80 DEG C, back flow reaction is after 3 hours, reaction solution dichloromethane extraction is collected organic phase, by this organic phase anhydrous magnesium sulfate drying, purifies through silica gel column chromatography and obtain target product.
3. preparation method according to claim 2, is characterized in that, described Nile red, and the mol ratio of DNF and salt of wormwood is 1:1.2:1.2.
4. the preparation method according to Claims 2 or 3, is characterized in that, the eluent in described silica gel column chromatography is CH
2cl
2: CH
3oH=40:1; SiO
2300-400 order.
5. the application detecting enhancement type fluorescent probe sulfonium ion or hydrogen sulfide in detection water body of hydrogen sulfide as claimed in claim 1.
6. application according to claim 5, is characterized in that, described fluorescent probe is detecting the application of hydrogen sulfide in viable cell plastosome.
7. application according to claim 6, is characterized in that, described viable cell is Hela cell strain, MCF-7 cell strain or RAW264.7 cell strain.
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Cited By (5)
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| CN104860840A (en) * | 2015-04-27 | 2015-08-26 | 苏州罗兰生物科技有限公司 | Preparation and application of fluorescence enhancement type hydrogen sulfide fluorescent probe |
| CN108003866A (en) * | 2017-12-11 | 2018-05-08 | 济南大学 | A kind of dithiothreitol (DTT) fluorescence probe and its preparation method and application |
| CN110724524A (en) * | 2019-12-02 | 2020-01-24 | 济南大学 | Fluorescent probe for detecting polarity in cells and preparation method and application thereof |
| CN110804043A (en) * | 2018-08-04 | 2020-02-18 | 南京大学 | Application of merocyanine-coumarin fluorophore in mitochondrial hydrogen sulfide detection |
| CN112939887A (en) * | 2021-02-04 | 2021-06-11 | 山西大学 | Near-infrared fluorescent probe based on basic dye and preparation method and application thereof |
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| CN103937490A (en) * | 2014-04-24 | 2014-07-23 | 辽宁大学 | Fluorescent probe for detecting sulfur ions in mitochondria and application thereof |
| CN103923640A (en) * | 2014-04-30 | 2014-07-16 | 大连理工常熟研究院有限公司 | Benzothiazoles fluorescent probe for identifying hydrogen sulfide and application of fluorescent probe |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104860840A (en) * | 2015-04-27 | 2015-08-26 | 苏州罗兰生物科技有限公司 | Preparation and application of fluorescence enhancement type hydrogen sulfide fluorescent probe |
| CN108003866A (en) * | 2017-12-11 | 2018-05-08 | 济南大学 | A kind of dithiothreitol (DTT) fluorescence probe and its preparation method and application |
| CN110804043A (en) * | 2018-08-04 | 2020-02-18 | 南京大学 | Application of merocyanine-coumarin fluorophore in mitochondrial hydrogen sulfide detection |
| CN110804043B (en) * | 2018-08-04 | 2022-10-18 | 南京大学 | Application of merocyanine-coumarin fluorophore in mitochondrial hydrogen sulfide detection |
| CN110724524A (en) * | 2019-12-02 | 2020-01-24 | 济南大学 | Fluorescent probe for detecting polarity in cells and preparation method and application thereof |
| CN110724524B (en) * | 2019-12-02 | 2021-02-19 | 济南大学 | Fluorescent probe for detecting polarity in cells and preparation method and application thereof |
| CN112939887A (en) * | 2021-02-04 | 2021-06-11 | 山西大学 | Near-infrared fluorescent probe based on basic dye and preparation method and application thereof |
| CN112939887B (en) * | 2021-02-04 | 2022-05-27 | 山西大学 | Near-infrared fluorescent probe based on basic dye and preparation method and application thereof |
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Application publication date: 20150401 |