CN109781678A - A kind of preparation and application of the ratio fluorescent probe detected applied to hypochlorous acid in mitochondria - Google Patents
A kind of preparation and application of the ratio fluorescent probe detected applied to hypochlorous acid in mitochondria Download PDFInfo
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- CN109781678A CN109781678A CN201711118691.5A CN201711118691A CN109781678A CN 109781678 A CN109781678 A CN 109781678A CN 201711118691 A CN201711118691 A CN 201711118691A CN 109781678 A CN109781678 A CN 109781678A
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- hypochlorous acid
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Abstract
The present invention relates to the preparations and application of a kind of ratio fluorescent probe detected applied to hypochlorous acid in mitochondria.Probe is with imidazo [1,5-a] pyridine fluorogen as energy donor, and for rhodamine fluorogen as energy acceptor, pyridiniujm structure is Mitochondrially targeted group.The probe can it is highly selective, with sensitivity with hypochlorous acid act on.In no hypochlorous acid, the fluorescence of 467 nm of probe emission；In the presence of having hypochlorous acid, probe emits the fluorescence of 467 nm and 575 nm simultaneously.With the increase of hypochlorous acid concentration, the fluorescence at 575 nm is gradually increased, and the fluorescence intensity at 467 nm gradually weakens, the ratio (I of the two575/I467) with hypochlorous acid concentration in a certain range (0.5-3.5 μM) be in good linear relationship.Probe can be used for hypochlorous imaging analysis in mitochondria, have broad application prospects.
Description
Technical field
The present invention relates to the preparation and application of a kind of hypochlorous acid ratio fluorescent probe, which can be used for secondary in mitochondria
The detection of chloric acid concentration.
Background technique
Hypochlorous acid is a kind of important signaling molecule, can regulate and control various kinds of cell physiological activity.The intracorporal hypochlorous acid of biology is
What hydrogen peroxide and chloride ion generated under the catalysis of myeloperoxidase.In immune system, hypochlorous acid can be to microorganism
Certain defense reaction is played in invasion.But if hypochlorous acid is horizontal excessive in vivo, it is easy to cause arthritis, atherosclerosis
The even diseases such as cancer.So the intracellular hypochlorous acid concentration of real-time monitoring has important biological significance.
Various analysis has been used for hypochlorous measurement at present, wherein fluorescence probe because high sensitivity, selectivity it is good,
Detection limits the features such as low, small to biological bulk damage and receives significant attention and obtained significant progress.Chlorine many times in document
Acid probe is that hypochlorous acid is measured by the change of single emission peak fluorescence intensity, and testing result is inevitably by the shadow of environmental factor
It rings.To reduce measurement error, we, which design, has synthesized Ratio-type hypochlorous acid fluorescence probe, realizes hypochlorous ratio measurement.
Mitochondria is the intracellular main portions for generating active oxygen, due to Intramitochondrial hypochlorous acid level and intracellular oxygen
It is closely related to change reduction balance, therefore it is most important to monitor hypochlorous concentration variation in mitochondria.
However, the report of the Ratio-type hypochlorous acid fluorescence probe in relation to Mitochondrially targeted positioning and few.
Based on this, inventor design with imidazo [1,5-a] pyridine structure be energy donor, rhodamine structure be energy by
Body, while Mitochondrially targeted group is introduced in the structure --- pyridiniujm structure constructs Ratio-type hypochlorous acid fluorescence probe.It visits
Needle set mitochondrial target function, and can apply to the measurement of hypochlorous acid concentration in mitochondria.
Summary of the invention
1. the purpose of the present invention is to provide a kind of preparation of Mitochondrially targeted Ratio-type hypochlorous acid fluorescence probe and answering
With.
2. the present invention discloses a kind of Mitochondrially targeted Ratio-type hypochlorous acid fluorescence probe, (IRP) is had the following structure:
3. the object of the invention, which also resides in, provides the fluorescence probe in detection mitochondria in terms of the fluorescence imaging of hypochlorous acid concentration
Application.
The preparation method of fluorescence probe of the present invention is (Fig. 1).
Prepare above-mentioned hypochlorous acid probe ethyl alcohol and PBS(0.01 M) buffer solution (V/V=3:7, pH 7.40) it is molten
Liquid.In the presence of no hypochlorous acid, 467 nm fluorescence of probe emission；In the presence of having hypochlorous acid, probe emits 467 nm simultaneously
With the fluorescence of 575 nm.With the increase of hypochlorous acid concentration, the fluorescence at 575 nm is gradually increased, and the fluorescence at 467 nm is strong
Degree weakens, the ratio (I of the two575/I467) with hypochlorous acid concentration in a certain range (0.5-3.5 μM) be in good linear pass
System, sees Fig. 2.
In the ethyl alcohol and PBS(0.01 M of above-mentioned probe) in buffer solution, be separately added into a certain amount of active nitrogen, active oxygen and
Other common species, fluorometric investigation are shown in Fig. 3 the result shows that probe has good selectivity to hypochlorous acid.
It is thin that it is incubated for RAW264.7 respectively with probe of the present invention and mitochondrial dye Mito Tracker Deep Red
Then born of the same parents carry out imaging analysis, by the result of Fig. 4 it is found that probe being capable of targetted mitochondria.
It is incubated for RAW264.7 cell with probe of the present invention, is imaged after then further handling cell with LPS,
The ratio between blue red fluorescence intensity of probe is substantially reduced, therefore above-mentioned probe can be used for the hypochlorous detection of mitochondria, see Fig. 5.
Ratio-type hypochlorous acid fluorescence probe of the present invention can be applied not only to the hypochlorous acid detection in solution, and
And can be used for hypochlorous detection in cell mitochondrial, it has broad application prospects.
Detailed description of the invention
Fig. 1 is the synthetic route of probe of the present invention.
Fig. 2 is change in fluorescence when probe of the present invention changes with hypochlorous acid concentration.Fig. 2 (a) is fluorescent emission map；Fig. 2
It (b) is situation of change of the fluorescence intensity ratio in various concentration hypochlorous acid.
Fig. 3 is selectivity of the probe of the present invention to other common species in HOCl and organism.
Fig. 4 is the probe of the present invention distribution intracellular in RAW264.7.Fig. 4 (a) is probe blue channel (410-520
Nm the fluorescent image) being collected into；Fig. 4 (b) is the fluorescent image of Mito Tracker Deep Red；Fig. 4 (c) is (a) and (b)
Stacking chart；Fig. 4 (d) is bright-field image；Common location coefficient is 0.95.
Fig. 5 is fluorescence imaging of the probe of the present invention to HOCl in RAW264.7 cell mitochondrial.Fig. 5 (a) is before LPS is handled
The fluorescence of probe in the cell afterwards；Fig. 5 (b) is the ratio of blue light and feux rouges.
Specific implementation method
Embodiment 1: the synthetic route of probe is shown in Fig. 1:
Compound 1(1 mmol) and triethylamine (3 mmol) are dissolved in the anhydrous CH of 20 mL2Cl2In, it is stirred under the conditions of 0 DEG C, then
Chloracetyl chloride (2 mmol) is added in above-mentioned solution, stirring 4 hours is continued, vacuum distillation removes solvent, obtains red solid,
That is the non-separating-purifying of compound 2(is directly used in and reacts in next step).
Compound 2(1 mmol), pyridine (1 mL) are dissolved in 30 mL anhydrous acetonitriles, flowed back 12 hours.It is removed under reduced pressure molten
Agent, then column chromatography (methylene chloride: methanol=10:1) purifying, obtains yellow solid, is hypochlorous acid fluorescence of the present invention
Probe.
With1H NMR、13C NMR and HRMS characterize it, and data are as follows:
1H NMR (400 MHz, d 6-DMSO), δ (ppm): 0.92 (t, 3H, J = 7.6 Hz, CH3), 1.09
(t, 6H, J = 6.8 Hz, CH3), 1.32 (q, 2H, J = 7.6 Hz, CH2), 1.68-1.73 (m, 2H,
CH2), 2.92 (t, 2H, J = 7.4 Hz, CH2), 3.26 (br, 4H, CH2), 3.32 (br, 4H, CH2 ),
3.68 (br, 4H, CH2), 5.48 (s, 2H, CH2), 6.34 (d, 2H, J = 9.2 Hz, ArH), 6.49
(d, 1H, J = 8.8 Hz, ArH), 6.56-6.61 (m, 2H, ArH), 6.68 (d, 1H, J = 2.0 Hz,
ArH), 6.75 (d, 1H, J = 7.2 Hz, ArH), 7.05 (d, 1H, J = 6.8 Hz, ArH), 7.52 (s,
1H, ArH), 7.54-7.62 (m, 2H, ArH), 7.85 (d, 1H, J = 6.8 Hz, ArH ), 8.14-8.17
(m, 2H, ArH), 8.29 (d, 1H, J = 7.2 Hz, ArH), 8.70 (t, 1H, J = 8.0 Hz, ArH),
8.74 (d, 2H, J = 5.6 Hz, ArH), 10.60 (s, 1H, NH)。
13C NMR (100 MHz, d 6-DMSO), δ (ppm): 167.6, 164.2, 163.8, 153.4,
153.1, 151.8, 151.7, 149.0, 146.9, 146.1, 138.9, 134.1, 129.5, 129.4, 129.2,
128.5, 128.2, 126.4, 124.3, 123.7, 123.3, 122.8, 118.7, 115.9, 112.4, 111.7,
108.5, 108.4, 104.1, 101.9, 97.5, 65.5, 60.6, 47.9, 44.1, 29.4, 29.1, 25.6,
22.1, 14.1, 12.9。
HRMS m/z: calcd for C47H48ClN8O4S [M]+: 823.3487; found: 823.3552. m/z:
calcd for C47H49ClN8O4S [M+H]2+/2: 412.1782; found: 412.1790。
Embodiment 2: change in fluorescence when probe of the present invention changes with hypochlorous acid concentration.
PBS buffer solution (containing 30% ethyl alcohol, pH 7.40) with manufacturing probe.It is added respectively into above-mentioned buffer solution different
Then the hypochlorite solution of concentration carries out fluorescence analysis, the nm of λ ex=370.As a result see Fig. 2.
Embodiment 3: the selectivity of probe of the present invention.
PBS buffer solution (containing 30% ethyl alcohol, pH 7.40) with manufacturing probe.It is separately added into certain a certain amount of chaff interferent
Then matter carries out fluorometric investigation analysis to above-mentioned sample, as a result sees Fig. 3.(1): blank, (2): H2O2, (3): t-
BuOOH, (4): t-BuO•, (5): 1O2, (6): -O2, (7): ON, (8): •OH, (9): ONOO-, (10):
NO2 -, (11): AcO-, (12): Br-, (13): Cl-, (14): I-, (15): CO3 2-, (16): SO4 2-, (17):-OCl.Wherein, hypochlorous acid concentration is 4 μM, and (2)-(9) concentration is 40 μM, other ion concentrations are 1 mM.
Embodiment 4: the distribution of probe of the present invention in the cell.
It is first probe culture RAW264.7 cell 30 minutes of the present invention with 1 μM, mitochondrial markers 0.2 are then added
μM Mito Tracker Deep Red continues to be incubated for 30 minutes.Fluorescence imaging is carried out using laser confocal microscope, respectively
Collect the fluorescent images of probe (410-520 nm) wave band and the fluorescence photo of Mito Tracker Deep Red.Then
It is overlapped analysis, common location coefficient is calculated, sees Fig. 4.
Embodiment 5: probe of the present invention is for hypochlorous imaging analysis in mitochondria.
Experimental group: first being stimulated RAW264.7 cell 6 hours with 1 μ g/mL LPS, and 1 μ g/mL PMA is then added and continues to train
It supports 30 minutes, 1 μM of probe of the present invention is then added in serum free medium, cultivate 30 minutes；Use laser co-focusing
Microscope carries out fluorescence imaging, collects the fluorescence of 410-520 nm and 560-700 nm, and count the ratio of two light；
Control group: being added 1 μM of probe in serum free medium, cultivates 30 minutes；Cell is washed 3 times with PBS respectively, then is carried out
Fluorescence imaging collects the fluorescence of 410-520 nm and 560-700 nm, and counts the ratio of two light, as a result sees Fig. 5.
Claims (3)
1. the chemical structural formula of hypochlorous acid fluorescence probe (IRP) is as follows:
2. application of the probe I PR described in claim 1 in detection sample containing hypochlorous acid.
3. sample containing hypochlorous acid as claimed in claim 2 is containing hypochlorous solution and biological cell.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117603203A (en) * | 2024-01-18 | 2024-02-27 | 德州学院 | Ratio fluorescent probe and preparation method and application thereof |
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Cited By (2)
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| CN117603203A (en) * | 2024-01-18 | 2024-02-27 | 德州学院 | Ratio fluorescent probe and preparation method and application thereof |
| CN117603203B (en) * | 2024-01-18 | 2024-04-12 | 德州学院 | Ratio fluorescent probe and preparation method and application thereof |
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