CN106977499A - A kind of fluorescence probe and its synthetic method and application that effect is directly targeted with mitochondria - Google Patents
A kind of fluorescence probe and its synthetic method and application that effect is directly targeted with mitochondria Download PDFInfo
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Abstract
The invention discloses a kind of fluorescence probe and its synthetic method and application that effect is directly targeted with mitochondria.Described fluorescence probe has the structure as shown in following formula (I), and its synthetic method is：Compound and glycine ethyl ester as shown in following formula (II) are taken, is dissolved in dimethyl sulfoxide (DMSO) or N, N dimethylformamide, in being reacted under heating condition, gained reactant is poured into frozen water, is filtered, is obtained object crude product.The experiment of applicant shows that the fluorescence probe has to mitochondria and is directly targeted effect but to cell without obvious inhibiting effect, with preferable potential value, is expected to as being directly targeted mitochondrial fluorescence probe.Structure shown in the formula (I), formula (II) is as follows：
Description
Technical field
The present invention relates to a kind of mitochondria fluorescence probe, and in particular to a kind of fluorescence that effect is directly targeted with mitochondria
Probe and its synthetic method and application.
Background technology
Mitochondria as intracellular crucial organelle, not only with for cell supply energy, participate in the important work(such as metabolism
Can, and participate in the important bioprocess such as cellular signal transduction and Apoptosis.Numerous studies show, mitochondrial quantity,
Distribution, structure, changes of function and nerve retrograde affection (such as parkinsonism, alzheimer's disease), metabolic pattern disease (such as II types
Diabetes, obesity), the illness such as cancer and angiocardiopathy it is closely related.Mitochondria is assigned " cellular signal transduction cell
The title such as device " and " motor of cell death ", associated " mitochondria " has become the fields such as life science and medical science
Study hotspot.In mechanism of drug action research, medicine plays medicine by being interacted with target spot and acted on, and mitochondria
Typically Principle Target device, therefore can be directly targeted mitochondrial small-molecule fluorescent probe particularly important for synthesis exploitation one kind.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of fluorescence probe that effect is directly targeted with mitochondria, and
Its synthetic method and application.
The fluorescence probe of the present invention for being directly targeted effect with mitochondria has the structure as shown in following formula (I)：
The synthetic method of compound is shown in above-mentioned formula (I)：Take compound and glycine ethyl ester as shown in following formula (II)
(C4H9NO2), it is dissolved in dimethyl sulfoxide (DMSO) or DMF, in being reacted under heating condition, gained reactant pours into ice
In water, filtering obtains object crude product；
The synthetic route of above-mentioned synthetic method is as follows：
Compound shown in involved formula (II) participates in reacting as reactant in above-mentioned synthetic method, and its chemical name is
The bromo- N- of 4- (3,4- methylene-dioxies phenethyl) -1,8- naphthalimides (4-Bromo-N- (3,4-methylenedioxy-
Phenethylamin) -1,8-naphthalimide), abbreviation NA-1a in this application.The compound can refer to Publication No.
Method disclosed in CN104557887A patent of invention is synthesized, also can designed, designed synthetic route synthesized.
In synthetic method of the present invention, the ratio between compound and the amount of material of glycine ethyl ester shown in formula (II) are usually
1：1~1：1.2, the preferably mole of glycine ethyl ester is slightly larger than the amount of the material of compound shown in formula (II), is easy to reaction to fill
Divide and carry out.
In synthetic method of the present invention, appropriate triethylamine can also be added before reactions as acid binding agent, to carry
The yield of high object.Under normal circumstances, the ratio between amount of material of triethylamine and glycine ethyl ester is 1：1~3：1.
In synthetic method of the present invention, reaction is preferably carried out under the conditions of 80~150 DEG C, more preferably 100~
Carried out under the conditions of 135 DEG C.Whether reaction can pass through TLC tracing detections completely.
It is the crude product of formula (I) compound made from above-mentioned synthetic method, can be using existing conventional purification process (as extracted
Take, silica gel column chromatography etc.) it is purified to improve the purity of formula (I) compound.Generally carried out using silica gel column chromatography
Purifying, it, through silica gel column chromatography, is 100 with by volume ratio to be specifically by obtained object crude product：1~1：1 petroleum ether and second
The eluent of acetoacetic ester composition, eluent is evaporated off solvent, obtains target product after purification.The stone of the composition eluant, eluent
The volume ratio of oily ether and ethyl acetate is preferably 20：1~5：1.It is preferred that first being carried out before silica gel column chromatography is carried out to crude product
Extract to reduce the burden of silica gel, specifically available such as ethyl acetate, dichloromethane or chloroform extractant are extracted, and collection has
Machine phase, silica gel column chromatography is carried out after removing solvent again.
Present invention additionally comprises the application in above-mentioned fluorescence probe in the cell mitochondrial fluorescent microscopic imaging.Described is thin
Born of the same parents are specially non-small cell lung cancer cell strain NCI-H460.
Compared with prior art, the invention provides a kind of new fluorescence probe that there is mitochondria to be directly targeted effect and
Its synthetic method.The experiment of applicant shows that the fluorescence probe has to mitochondria and is directly targeted effect but to cell without obvious
Inhibitory action, with preferable potential value, is expected to as being directly targeted mitochondrial fluorescence probe.
Brief description of the drawings
Fig. 1 is the electrospray ionization mass spectrum spectrogram of final product made from the embodiment of the present invention 6；
Fig. 2 is the mass spectrometry results of 5h plasmosins after cell incubation compound N A-2a in experimental example 1 of the present invention；
Fig. 3 is the mass spectrometry results of 5h mitochondrial proteins after cell incubation compound N A-2a in experimental example 1 of the present invention；
Fig. 4 is not incubated the mass spectrometry results of compound N A-2a plasmosins for cell in experimental example 1 of the present invention；
Fig. 5 is not incubated the mass spectrometry results of compound N A-2a mitochondrial proteins for cell in experimental example 1 of the present invention；
Fig. 6 is individually incubated 5h lines grain after compound N A-2a to extract mitochondria without administration processing in experimental example 1 of the present invention
Body protein mass spectrometry results；
Fig. 7 is negative control cell fluoroscopic examination result in experimental example 2 of the present invention；
Fig. 8 be experimental example 2 of the present invention in cell incubation 5h when fluoroscopic examination result.
Embodiment
With reference to specific embodiment, the present invention is described in further detail, to more fully understand present disclosure, but
The present invention is not limited to following examples.
Embodiment 1：NA-1a is the synthesis of compound shown in formula (II)
Weigh bromo- 1, the 8- naphthalic anhydrides of 4- to be dissolved in 150mL ethanol, stir and add 3,4- (methylenedioxy) phenyl ethylamines
(mol ratio of the bromo- 1,8- naphthalic anhydrides of 4- and 3,4- (methylenedioxy) phenyl ethylamines is 1：1.5), 70 DEG C of reaction temperature, react into
Journey is tracked monitoring using thin-layer chromatography.After reaction terminates, reaction system is cooled to room temperature, filters and retains filter cake, make
Use ethanol rinse filter cake, produce yellow solid (yield is about 90%).
Proton nmr spectra, carbon-13 nmr spectra analysis are carried out to gained yellow solid, specific spectral characteristic is as follows：
(1) proton nmr spectra, its spectrogram is as shown in Figure 1.
1H NMR(500MHz,DMSO-d6)δ:8.53 (ddd, J=9.5,7.9,1.0Hz, 2H), 8.31 (d, J=7.9Hz,
1H), 8.20 (d, J=7.9Hz, 1H), 7.99-7.96 (m, 1H), 6.85 (d, J=1.6Hz, 1H), 6.80 (s, 1H), 6.69
(dd, J=7.9,1.7Hz, 1H), 5.97 (s, 2H), 4.20-4.15 (m, 2H), 2.84 (t, J=7.5Hz, 2H).
(2) carbon-13 nmr spectra, its spectrogram is as shown in Figure 2.
13C NMR(126MHz,DMSO-d6)δ:162.86,162.82,147.36,145.78,132.78,132.54,
131.72,131.50,131.11,129.90,129.30,128.94,128.36,122.79,122.01,121.61,109.09,
108.33,100.82,41.39,33.19。
Accordingly, it can be determined that above-mentioned yellow solid product is the bromo- N- of 4- (3,4- methylene-dioxy phenethyl) -1,8- naphthalenes two
Carboximide, shown in its chemical structural formula such as following formula (II)：
Embodiment 2：NA-1a synthesis
Embodiment 1 is repeated, unlike：
Reaction temperature is changed to 60 DEG C, and remaining reaction condition is constant (yield is about 75%).
Proton nmr spectra, carbon-13 nmr spectra analysis are carried out to gained yellow solid, target product is determined that it is.
Embodiment 3：NA-1a synthesis
Embodiment 1 is repeated, unlike：
Reaction temperature is changed to 80 DEG C, and remaining reaction condition is constant (yield is about 85%).
Proton nmr spectra, carbon-13 nmr spectra analysis are carried out to gained yellow solid, target product is determined that it is.
Embodiment 4：NA-1a synthesis
Embodiment 1 is repeated, unlike：
Reaction dissolvent is changed to methanol, and remaining reaction condition is constant (yield is about 85%).
Proton nmr spectra, carbon-13 nmr spectra analysis are carried out to gained yellow solid, target product is determined that it is.
Embodiment 5：NA-1a synthesis
Embodiment 1 is repeated, different conditions are：
The ratio between amount of material of the bromo- 1,8- naphthalic anhydrides of reaction raw materials 4- and 3,4- (methylenedioxy) phenyl ethylamines is changed to 1:1,
Remaining reaction condition is constant (yield is about 65%).
Proton nmr spectra, carbon-13 nmr spectra analysis are carried out to gained yellow solid, target product is determined that it is.
Embodiment 6：Compound shown in formula (I) is 4- (carbethoxyl group methylamino)-N- (3,4- (methylenedioxy) phenethyl) -1,
The synthesis of 8- naphthalimides (hereinafter referred to as NA-2a)
Weigh NA-1a to be dissolved in dimethyl sulfoxide (DMSO), add glycine ethyl ester and triethylamine (NA-1a, glycine ethyl ester and three
The mol ratio of ethamine is 1：1.2：2), 135 DEG C of reaction temperature, reaction process is tracked monitoring using thin-layer chromatography.Reaction knot
Shu Hou, reaction system is cooled to room temperature, pours into frozen water, there is yellow solid precipitation, makes to be extracted with ethyl acetate three times, collects and closes
And ester layer and use anhydrous MgSO4Dry, removal of solvent under reduced pressure, gained crude product carries out silica gel column chromatography separating purification (VPetroleum ether：
VEthyl acetate=20：1~5：1), obtain bright yellow solid (yield is 65%).
Proton nmr spectra, carbon-13 nmr spectra, Electrospray Ionization Mass Spectrometry, specific wave spectrum are carried out to gained bright yellow solid
Characteristic is as follows：
(1) proton nmr spectra, its spectral data is as follows.
1H NMR(400MHz,DMSO-d6)δ:8.66 (d, J=8.4Hz, 1H), 8.46 (d, J=7.2Hz, 1H), 8.26
(d, J=8.5Hz, 1H), 8.14 (s, 1H), 7.74 (t, J=7.8Hz, 1H), 6.90-6.75 (m, 2H), 6.67 (dd, J=
10.7,9.0Hz, 2H), 5.97 (s, 2H), 4.29 (d, J=5.5Hz, 2H), 4.17 (q, J=7.1Hz, 4H), 2.90-2.74
(m,2H),1.23(s,3H)。
(2) carbon-13 nmr spectra, its spectral data is as follows.
13C NMR(101MHz,DMSO-d6)δ:170.38,164.07,163.26,150.81,147.72,146.10,
134.38,133.18,131.25,130.11,129.68,128.86,125.23,122.45,121.93,120.68,109.44,
109.31,108.66,104.88,101.16,61.26,40.63,40.42,40.21,40.00,39.79,39.58,39.37,
33.84,14.59。
(3) electrospray ionization mass spectrum, its spectrogram is as shown in figure 1, ESI-MSm/z：445.14[M-H]-
Accordingly, it can be determined that above-mentioned bright yellow solid product is 4- (carbethoxyl group methylamino)-N- (3,4- (methylenedioxy) benzene
Ethyl) -1,8-naphthalimide, shown in its chemical structural formula such as following formula (I)：
Embodiment 7：NA-2a synthesis
Embodiment 6 is repeated, unlike：
Reaction temperature is changed to 80 DEG C, and remaining reaction condition is constant (yield is 25%).
Proton nmr spectra, carbon-13 nmr spectra, Electrospray Ionization Mass Spectrometry are carried out to gained bright yellow solid, determined that it is
Target product.
Embodiment 8：NA-2a synthesis
Embodiment 6 is repeated, unlike：
Reaction temperature is changed to 100 DEG C, and remaining reaction condition is constant (yield is 38%).
Proton nmr spectra, carbon-13 nmr spectra, Electrospray Ionization Mass Spectrometry are carried out to gained bright yellow solid, determined that it is
Target product.
Embodiment 9：NA-2a synthesis
Embodiment 6 is repeated, unlike：
The ratio between amount of material of reaction raw materials NA-2a and glycine ethyl ester is changed to 1:1, the constant (yield of remaining reaction condition
For 40%).
Proton nmr spectra, carbon-13 nmr spectra, Electrospray Ionization Mass Spectrometry are carried out to gained bright yellow solid, determined that it is
Target product.
Embodiment 10：NA-2a synthesis
Embodiment 6 is repeated, unlike：
The ratio between amount of material of reaction raw materials glycine ethyl ester and triethylamine is changed to 1:1, the constant (yield of remaining reaction condition
For 20%).
Proton nmr spectra, carbon-13 nmr spectra, Electrospray Ionization Mass Spectrometry are carried out to gained bright yellow solid, determined that it is
Target product.
Embodiment 11：NA-2a synthesis
Embodiment 6 is repeated, unlike：
Reaction dissolvent is changed to DMF, and remaining reaction condition is constant (yield is 60%).
Proton nmr spectra, carbon-13 nmr spectra, Electrospray Ionization Mass Spectrometry are carried out to gained bright yellow solid, determined that it is
Target product.
Embodiment 12：NA-2a synthesis
Embodiment 6 is repeated, unlike：
Reaction dissolvent is changed to glycol monoethyl ether, and remaining reaction condition is constant, and target compound is not generated.
Embodiment 13：NA-2a synthesis
Embodiment 6 is repeated, unlike：
Synthetic method is added without triethylamine, and remaining reaction condition is constant (yield is 15%).
Proton nmr spectra, carbon-13 nmr spectra, Electrospray Ionization Mass Spectrometry are carried out to gained bright yellow solid, determined that it is
Target product.
Mitochondrial characteristic is acted on to absolutely prove that fluorescence probe of the present invention has to be directly targeted, applicant is to it
Following experiments are carried out：
Experimental example 1：Whether mitochondria is directly targeted in the horizontal detection compound NA-2a of cell in-situ
A) compound incubation
(1) choose non-small cell lung cancer cell strain NCI-H460 to be inoculated in 70mm culture dishes, cell growth to logarithm is given birth to
For a long time when (cell attachment area is about 70%~75%), the nutrient solution in culture dish is carefully removed, using PBS twice,
Add the new DMEM nutrient solutions of 5mL.
(2) appropriate compound N A-2a liquid storages (- 20 DEG C) are weighed, adding makes its final concentration of 5 μM in culture dish, act on 5h,
The control group of not dosing is set simultaneously.
B) mitochondrial separation
(1) after effect 5h, the culture medium in culture dish is carefully removed, is 0.25% pancreas with concentration using PBS twice
Protease digestion attached cell.5mL DMEM culture mediums are added, are dispelled and adherent cell collecting, with 2000rpm on centrifuge
Centrifuge 10min；
(2) abandoning supernatant, plus 1mL ice bath precoolings PBS, cleaning cell once, 2000rpm centrifugation 1min；
(3) supernatant discarding PBS, adds the appropriate mitochondria separation agent containing PMSF, 30min is incubated on ice, is regularly dispelled
Cell, prevents cell precipitation；
(4) cell suspension is transferred in appropriately sized glass homogenizer, be homogenized 40 times or so；
(5) cell homogenates is transferred in centrifuge tube, in 4 DEG C, 1000g centrifugations 10min；
(6) carefully supernatant is transferred in another centrifuge tube, in 4 DEG C, 3500g centrifugations 10min；
(7) part supernatant is collected, notes not touching precipitation when collecting.Careful to remove remaining supernatant, precipitation is to separate
Obtained mitochondria.
C) NA-2a is incubated with mitochondria
(1) mitochondria for extracting control group in above-mentioned steps (B) is divided into 2 parts, take wherein 1 part it is abundant with 5 μM of NA-2a
Mix after being incubated 5h in 37 DEG C of waters bath with thermostatic control；
(2) it is incubated and terminates after 4 DEG C, 9000g centrifugations 10min；
(3) supernatant is carefully removed, 100 μ L mitochondrias storing liquids is added and mixes, 4 DEG C of 9000g centrifuge 10min；
(4) supernatant is removed completely, and precipitation is the mitochondria for being incubated and being obtained after NA-2a；
D) the extraction of mitochondria and plasmosin matter
(1) amount according to cell cytosol adds 100-200 μ L RIPA lysates and PMSF is mixed, on ice ultrasonic degradation
30min, per 10min, vibration mixes cell pyrolysis liquid, it is fully cracked.RIPA lysates and PMSF ratio are 100:1；
(2) 40~80 μ L mitochondrias lysates and PMFS (mitochondria lysates are added according to mitochondria amount:PMSF=100:
1), mix, on ice ultrasonic degradation 20min, per 5min, concussion mixes cell pyrolysis liquid, mitochondria is fully cracked.
(3) endochylema and mitochondrial lysate are moved in 1.5mL EP pipes respectively, 4 DEG C of 12000rpm, centrifuges 10min；
(4) careful collection supernatant, gained protein sample after as cracking, for Mass Spectrometer Method.
E) NA-2a is incubated the Mass Spectrometer Method of the endochylema extracted and mitochondrial protein
Gained sample will be handled through step (D) and carries out Mass Spectrometer Method analysis immediately, as a result as shown in figs. 1 to 6.Wherein, Fig. 1
Final product NA-2a is dissolved in the mass spectrometry results in DMSO made from embodiment 6；Fig. 2 is cell incubation compound N A-
The mass spectrometry results of 5h plasmosins after 2a；Fig. 3 is the mass spectral analysis of 5h mitochondrial proteins after cell incubation compound N A-2a
As a result；Fig. 4 is the mass spectrometry results that cell is not incubated compound N A-2a plasmosins；Fig. 5 is that cell is not incubated compound
The mass spectrometry results of NA-2a mitochondrial proteins；Fig. 6 is to extract mitochondria without administration processing to be individually incubated compound N A-2a
5h mitochondrial protein mass spectral analysis result afterwards.Analysis result shows, the NA-2a in non-small cell lung cancer cell strain NCI-H460
It can be directly entered in mitochondria.
Experimental example 2：NA-2a is analyzed with mitochondria common location
1) with the alcohol-pickled processing cell climbing sheet special glass piece of 75% (volumetric concentration), the slide handled well is placed in 6
It is standby in each hole of orifice plate.
2) non-small cell lung cancer cell strain NCI-H460 is chosen to remove after original nutrient solution, using PBS attached cell,
PBS is removed, the trypsase of 1mL 0.25% is added, half a minute is stood, removes after pancreatin, culture is placed in incubator
Continue to digest 1min, add fresh medium and blow and beat attached cell to cell suspension.
3) it is separately added into by every μ L cell suspending liquids of hole 500 in each hole of 6 orifice plates, orifice plate is placed in incubator and cultivates number
Day, treat the complete adherent growth of cell.
4) using (5 μM) each Kong Zhongyu cell incubations 5h of addition of NA-2a dilutions, while setting negative control.
5) nutrient solution is removed, with PBS 2 times, each 5min.
6) add the advance 37 DEG C of incubations 30min's of 500 μ L per holeRed CMXRos mitochondrias are red
Fluorescence probe liquid storage is incubated 45min again in room temperature.
7) liquid in hole is removed, with PBS 2 times, each 5min.
8) the anti-fluorescent quenching mounting liquid of drop is added dropwise on slide, the slide for posting cell is covered, it is to avoid bubble occur,
Mounting is fixed with Instant cement.
9) fluoroscopic examination is carried out using laser confocal microscope.As a result as shown in Fig. 7~8, wherein Fig. 7 is negative control
Cell fluorescence testing result；Fluoroscopic examination result when Fig. 8 is cell incubation 5h.Analysis result is shown, thin in non-small cell lung cancer
NA-2a is same in born of the same parents' strain NCI-H460Red CMXRos mitochondria red fluorescence probes position phase in cell
Together, illustrate that NA-2a can be directly targeted mitochondria.
Claims (7)
1. a kind of fluorescence probe that effect is directly targeted with mitochondria, it is characterised in that：The fluorescence probe has such as following formula (I)
Shown structure：
2. the synthetic method of the fluorescence probe that effect is directly targeted with mitochondria described in claim 1, it is characterised in that：Take
Compound and glycine ethyl ester as shown in following formula (II), are dissolved in dimethyl sulfoxide (DMSO) or DMF, in fire-bar
Reacted under part, gained reactant is poured into frozen water, filter, obtain object crude product；
3. synthetic method according to claim 2, it is characterised in that：Triethylamine is added before reactions is used as acid binding agent.
4. the synthetic method according to Claims 2 or 3, it is characterised in that：Reaction is carried out under the conditions of 80~150 DEG C.
5. the synthetic method according to Claims 2 or 3, it is characterised in that：Also include purification step：Specifically will be obtained
Object crude product, through silica gel column chromatography, is 100 with by volume ratio：1~1：The eluant, eluent of 1 petroleum ether and ethyl acetate composition is washed
De-, solvent is evaporated off in eluent, obtains target product after purification.
6. the application of fluorescence probe described in claim 1 in the cell in mitochondrial fluorescent microscopic imaging.
7. application according to claim 6, it is characterised in that：Described cell is non-small cell lung cancer cell strain NCI-
H460。
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| CN108261414A (en) * | 2018-01-11 | 2018-07-10 | 广西师范大学 | A kind of pharmaceutical composition for treating lung cancer |
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| GUOHAI ZHANG.ET AL.: "A Novel Naphthalimide Compound Restores p53 Function in Non-small Cell Lung Cancer by Reorganizing the Bak·Bcl-xl Complex and Triggering Transcriptional Regulation", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108261414A (en) * | 2018-01-11 | 2018-07-10 | 广西师范大学 | A kind of pharmaceutical composition for treating lung cancer |
| CN108261414B (en) * | 2018-01-11 | 2019-09-06 | 广西师范大学 | A kind of pharmaceutical composition for treating lung cancer |
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| CN106977499B (en) | 2019-08-06 |
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