CN108822031A - A kind of red transmitting fluorescence probe of two-photon detecting mitochondria - Google Patents
A kind of red transmitting fluorescence probe of two-photon detecting mitochondria Download PDFInfo
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- CN108822031A CN108822031A CN201810974343.6A CN201810974343A CN108822031A CN 108822031 A CN108822031 A CN 108822031A CN 201810974343 A CN201810974343 A CN 201810974343A CN 108822031 A CN108822031 A CN 108822031A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
- C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
- C07D215/04—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to the ring carbon atoms
- C07D215/06—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to the ring carbon atoms having only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, attached to the ring nitrogen atom
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
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- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
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Abstract
The present invention provides a kind of mitochondria fluorescence probe of two-photon red emission and its application in cell imaging, its chemical name is：The own yl-quinoline salt compounded of iodine of 2- (pyrene -1- vinyl)-N-；It can be synthesized by following steps：2- methylquinoline and iodohexane heat synthesis 1,2- dimethyl quinoline salt compounded of iodine first in ethanol solution；Then using nafoxidine as catalyst, pyrene -1- formaldehyde is added, and condensation reaction generates the own yl-quinoline salt compounded of iodine of 2- (pyrene -1- vinyl)-N- at room temperature.The present invention also provides the fluorescence probes in the application that mitochondria is imaged.Probe synthesis of the invention is simple, feux rouges can be issued under single Two Photon Fluorescence and mitochondria is imaged, had great application prospect.
Description
Technical field
The invention belongs to small organic molecule fluorescence probe fields, and in particular to a kind of fluorescence spy of two-photon detection mitochondria
Needle and its synthetic method.
Background technique
Mitochondria is a kind of coated organelle of double membrane structure, is present in the eukaryocyte of almost all kinds.Line
Plastochondria generates atriphos (ATP) by oxidation respiration and provides most energy for cell.In addition, mitochondria also with
Signal transduction, Apoptosis, the important pathological processes of cell differentiation are directly related.Mitochondrial Shape has variability,
Different types of cell, the different phase of cell, different metabolism states and cell state will affect the form of mitochondria.Cause
There is important physiology and pathology to be worth for this observation Mitochondrial Shape and dynamic.
Current scanline Electronic Speculum, transmission electron microscope and fluorescence microscope are the important tools for observing mitochondria.Wherein, fluorescence microscopy
Mirror and fluorescence imaging method can be used for dynamic, mitochondria in situ, being observed in living cells in real time changes, and have unique advantage.Cause
This, has obtained extensive development in recent years.And two-photon fluorescence imaging, as a subspecies of fluorescence imaging, even more because of its nothing
Analogous advantage, which receives, to be given more sustained attention.With the single photon fluorescence process for absorbing a ultraviolet-visible photon arrival excitation state
Difference, biphotonic process absorb two photons near infrared emission and realize molecule excitation.Distinctive two-photon absorption mode
And long excitation wavelength imparts the advantages that two photon imaging penetration depth is deep, photobleaching effect is low and light injury is small.In addition,
Red emitting molecule can effectively avoid the autofluorescence of zooblast, can more complement each other with two photon imaging.Therefore, it develops
The mitochondria fluorescence probe of two-photon red emission has important value.And up to the present, the report of such molecule still compared with
It is few, have to be developed.
Summary of the invention
This less status is reported for current two-photon feux rouges mitochondrial probe, the present invention provides a kind of two-photons to swash
Rubescent photoemissive mitochondria fluorescence probe, the probe is selectively good, high sensitivity.
It is a further object of the present invention to provide a kind of synthetic method of above-mentioned fluorescence probe, raw material is easy to get, synthesis step is simple
Single, high income.
Another object of the present invention is to provide the application in a kind of above-mentioned fluorescence probe cell imaging.
To achieve the above object, the present invention adopts the following technical scheme that.
A kind of mitochondria fluorescence probe of two-photon excitation red emission, chemical name be 2- (pyrene -1- vinyl)-N- oneself
Yl-quinoline salt compounded of iodine, abbreviation HVQ, chemical structural formula such as formula（I）It is shown：
Formula (I).
A kind of synthetic method of above-mentioned fluorescence probe, includes the following steps：
2- methylquinoline heats in ethanol with iodohexane to react, and end of reaction adds 1- pyrene formaldehyde and pyrrolidines, and room temperature is stirred
Reaction is mixed, product, the i.e. own yl-quinoline salt compounded of iodine of 2- (pyrene -1- vinyl)-N- are filtered to obtain.
In above-mentioned steps, the molar ratio of 2- methylquinoline, iodohexane and 1- pyrene formaldehyde is 1:1-2:0.8-1.2.2- methyl
The molar ratio of quinoline and pyrrolidines is 1:0.5-3.
Heating reaction temperature is 80 DEG C, and the time is 24-48 h.Being stirred to react the time is 12-24 h.
It further include purification step after filtering as optimization.After the purification step is product ethanol washing three times, in second
It is recrystallized in alcohol.
The synthetic route of above-mentioned fluorescence probe is as follows：
。
A kind of application of above-mentioned mitochondria fluorescence probe in detection cell mitochondrial.One-photon excitation wavelength is 405
Nm, detection wave band are 570-620 nm.Two-photon excitation wavelength is 800 nm, and detection wave band is 570-620 nm.Detection time
After 30 min of effect.
The working principle of above-mentioned fluorescence probe is as follows：
The present invention constructs fluorescence probe using pyrene as parent, by double bond bridging quinolinium.Planar structure and inner electron transition
It ensure that the two-phpton property of probe；The conjugated structure and strong electron-withdrawing group of suitable size ensure that red emission；Sun
Ion salt and fat-soluble it ensure that probe to the targeting of mitochondria.
Beneficial effects of the present invention are：
Fluorescence probe of the present invention can be obtained through one kettle way chemical synthesis, and synthesis step isolates and purifies simply.Probe has
Two-phpton property, in imaging applications, penetration depth is deep, photobleaching effect is low, light injury is small；Transmitting light is feux rouges, can be effective
The autofluorescence of zooblast is avoided, with good application prospect.
Detailed description of the invention
Fig. 1 is fluorescence probe HVQ's1H H NMR spectroscopy；
Fig. 2 is the high resolution mass spectrum of fluorescence probe HVQ；
Fig. 3 is single two photon imaging picture that probe colours mitochondria；
Fig. 4 is the probe common location imaging picture dark red with mitochondria.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention will be further described, but the present invention is not by the limit of following embodiments
System.
The synthesis of 1 fluorescence probe HVQ of embodiment
With ethanol as solvent, 2- methylquinoline is added, the iodohexane solution of 1.2 eq is added, is heated to 80 DEG C of 24 h of reaction, instead
Reaction system is cooled to room temperature after answering, the 1- pyrene formaldehyde of 1 eq is added, the pyrrolidines of 1 eq is added, is stirred at room temperature 12
H has a large amount of solids to be precipitated, and filtering can obtain crude product, then crude product is recrystallized in ethanol with dehydrated alcohol three times again,
The own yl-quinoline salt compounded of iodine of sterling 2- (pyrene -1- vinyl)-N- is obtained, yield is 48 %.Product1H NMR and HRMS spectrogram are respectively such as
Shown in Fig. 1 and Fig. 2：1H NMR (400 MHz, DMSO-d 6) δ 9.32 (d, J = 15.2 Hz, 1H), 9.19 (d, J
= 8.9 Hz, 1H), 9.03 (dd, J = 12.8, 9.1 Hz, 2H), 8.92 (d, J = 8.3 Hz, 1H),
8.63 (d, J = 9.1 Hz, 1H), 8.52 – 8.42 (m, 5H), 8.39 – 8.30 (m, 2H), 8.28 -
8.17 (m, 3H), 8.01 (t, J = 7.5 Hz, 1H), 5.24 (d, J = 8.4 Hz, 2H), 2.00 (d, J
= 7.5 Hz, 2H), 1.60 (q, J = 7.6 Hz, 2H), 1.45 - 1.21 (m, 4H), 0.84 (t, J =
7.2 Hz, 3H). HRMS (ESI): m/z calculated for C33H30N 443.2373 [M+], found:
443.2380。
The cell imaging of 2 fluorescence probe HVQ of embodiment
（1）Cell culture, processing and dyeing
It is 3 × 10 by density5The HeLa cell inoculation of a/mL is into the 35 mm imaging culture dish of sterilizing, in CO2Incubator
（Temperature is 37 DEG C, 5 % CO2）Keep cell adherent within culture 12 hours or more.It is obtained with 4% paraformaldehyde processing attached cell 30min
To dead cell sample.The DMSO solution of fluorescence probe prepared by the embodiment 1 that compound concentration is 2.5 mM is mother liquor, thin to life or death
Mother liquor is added in born of the same parents' culture dish, making its final concentration is 5 μM.Continue to continue to cultivate 1 h respectively under the same conditions, then will
Cell culture fluid siphons away, and is rinsed cell 3 times with culture medium, then carries out cell imaging experiment.
（2）Laser Scanning Confocal Microscope imaging
Respectively using 405 nm and 800 nm as excitation wavelength, it is 570-620 nm that red channel, which collects wavelength, obtains fluorescence picture
As shown in Figure 3, wherein DIC is light field image, and SPEF is single photon image figure, and TPEF is two photon imaging figure, DIC-SPEF
For the stacking chart of the two.As can be seen from the figure probe can provide strong fluorescence in red channel under single, double photon microscope,
And filigree shape is presented in fluorescence signal, meets the standard type of mitochondria.
The common location of 3 probe of embodiment and commercialization probe in living cells
In order to further confirm that coloration station of the probe HVQ in cell, we are dark red with commercial mitochondrial dye mitochondria
(MTDR) common location dyeing has been carried out with HVQ to be imaged.
In the experiment of living cells common location, first with 30 min of MTDR staining cell of 200 nM, 2 μM of HVQ dyeing is added
60 min of cell, then siphons away cell culture fluid, is rinsed cell 3 times with culture medium, carries out cell imaging.It is sharp with 405 nm
Wavelength is sent out, collects the fluorescence of 570-620 nm to acquire the fluorescence signal of HVQ；It is excitation wavelength with 647 nm, collects 663-738
The fluorescence of nm acquires the fluorescence signal of MTDR.Fluorescence picture is obtained as shown in figure 4, wherein, Red channel is HVQ imaging
Figure, DR channel are MTDR image, and Merged channel is the two stacking chart.Calculate the rates of redying of two kinds of dyestuffs is
90%, illustrate that probe HVQ dyes mitochondria in living cells.
Claims (8)
1. a kind of mitochondria fluorescence probe of two-photon excitation red emission, chemical name be 2- (pyrene -1- vinyl)-N- oneself
Yl-quinoline salt compounded of iodine, chemical structural formula such as formula（I）It is shown：
Formula (I).
2. the synthetic method of mitochondria fluorescence probe according to claim 1, which is characterized in that include the following steps：2-
Methylquinoline heats in ethanol with iodohexane to react, and end of reaction adds 1- pyrene formaldehyde and pyrrolidines, and reaction is stirred at room temperature,
Filter to obtain product, the i.e. own yl-quinoline salt compounded of iodine of 2- (pyrene -1- vinyl)-N-.
3. synthetic method according to claim 2, which is characterized in that 2- methylquinoline, iodohexane and 1- pyrene formaldehyde rub
You are than being 1:1-2:0.8-1.2.
4. synthetic method according to claim 2, which is characterized in that the molar ratio of 2- methylquinoline and pyrrolidines is 1:
0.5-3。
5. synthetic method according to claim 2, which is characterized in that heating reaction temperature is 80 DEG C, time 24-48
h；Being stirred to react the time is 12-24 h.
6. synthetic method according to claim 2, which is characterized in that further include purification step after filtering.
7. synthetic method according to claim 2, which is characterized in that the purification step is product ethanol washing three times
Afterwards, it recrystallizes in ethanol.
8. a kind of application of mitochondria fluorescence probe as described in claim 1 in detection cell mitochondrial.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109574922A (en) * | 2018-12-21 | 2019-04-05 | 济南大学 | A kind of mitochondrial membrane potential fluorescence probe and its synthetic method and application |
| CN109912559A (en) * | 2019-03-29 | 2019-06-21 | 济南大学 | A kind of fluorescence probe and its preparation method and application of reversible detection mitochondria |
| CN110776458A (en) * | 2019-11-01 | 2020-02-11 | 济南大学 | Fluorescent probe for detecting mitochondrial membrane potential and preparation method and application thereof |
| CN113004276A (en) * | 2021-03-15 | 2021-06-22 | 天津理工大学 | Fluorescent probe for positioning mitochondria and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7338428B2 (en) * | 2002-09-09 | 2008-03-04 | New York University | Combinatorial fluorescent library based on the styryl scaffold |
-
2018
- 2018-08-24 CN CN201810974343.6A patent/CN108822031A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7338428B2 (en) * | 2002-09-09 | 2008-03-04 | New York University | Combinatorial fluorescent library based on the styryl scaffold |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109574922A (en) * | 2018-12-21 | 2019-04-05 | 济南大学 | A kind of mitochondrial membrane potential fluorescence probe and its synthetic method and application |
| CN109912559A (en) * | 2019-03-29 | 2019-06-21 | 济南大学 | A kind of fluorescence probe and its preparation method and application of reversible detection mitochondria |
| CN110776458A (en) * | 2019-11-01 | 2020-02-11 | 济南大学 | Fluorescent probe for detecting mitochondrial membrane potential and preparation method and application thereof |
| CN110776458B (en) * | 2019-11-01 | 2021-10-26 | 济南大学 | Fluorescent probe for detecting mitochondrial membrane potential and preparation method and application thereof |
| CN113004276A (en) * | 2021-03-15 | 2021-06-22 | 天津理工大学 | Fluorescent probe for positioning mitochondria and preparation method and application thereof |
| CN113004276B (en) * | 2021-03-15 | 2022-06-03 | 天津理工大学 | Fluorescent probe for positioning mitochondria and preparation method and application thereof |
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Application publication date: 20181116 |