CN106977499B - A kind of fluorescence probe and its synthetic method and application being directly targeted effect with mitochondria - Google Patents
A kind of fluorescence probe and its synthetic method and application being directly targeted effect with mitochondria Download PDFInfo
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Abstract
The invention discloses a kind of fluorescence probe and its synthetic method and applications that effect is directly targeted with mitochondria.The fluorescence probe has the structure as shown in following formula (I), its synthetic method are as follows: take compound and glycine ethyl ester as shown in following formula (II), it is dissolved in dimethyl sulfoxide or N, in dinethylformamide, it is reacted under heating condition, gained reactant is poured into ice water, and filtering obtains object crude product.The experiment of applicant shows that the fluorescence probe has mitochondria and is directly targeted effect but to cell without obvious inhibiting effect, has preferable potential value, is expected to as the fluorescence probe for being directly targeted mitochondria.Structure shown in the formula (I), formula (II) is as follows:
Description
Technical field
The present invention relates to a kind of mitochondria fluorescence probes, and in particular to a kind of fluorescence that effect is directly targeted with mitochondria
Probe and its synthetic method and application.
Background technique
Mitochondria not only has as intracellular crucial organelle and supplies energy for cell, participates in the important function such as metabolism
Can, and participate in the important bioprocess such as cellular signal transduction and Apoptosis.A large number of studies show that the quantity of mitochondria,
Distribution, structure, changes of function and nerve retrograde affection (such as parkinsonism, alzheimer's disease), metabolic pattern disease (such as II type
Diabetes, obesity), the illnesss such as cancer and cardiovascular disease it is closely related.Mitochondria is assigned " cellular signal transduction cell
Titles, associated " mitochondria " such as device " and " motor of cell death " have become the fields such as life science and medicine
Research hotspot.In mechanism of drug action research, drug plays drug effect by interacting with target spot, and mitochondria
Usually Principle Target device, therefore to develop a kind of small-molecule fluorescent probe that can be directly targeted mitochondria particularly important for synthesis.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of fluorescence probes 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 shown in above-mentioned formula (I) are as follows: take compound and glycine ethyl ester as shown in following formula (II)
(C4H9NO2), it is dissolved in dimethyl sulfoxide or n,N-Dimethylformamide, is 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 formula involved in above-mentioned synthetic method (II) participates in reacting as reactant, its chemical name is
The bromo- N- of 4- (3,4- methylene-dioxy phenethyl) -1,8- naphthalimide (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 the patent of invention of CN104557887A is synthesized, can also designed, designed synthetic route synthesized.
In synthetic method of the present invention, the ratio between amount of substance of compound shown in formula (II) and glycine ethyl ester is usually
1:1~1:1.2, the preferably mole of glycine ethyl ester are filled slightly larger than the amount of the substance of compound shown in formula (II) convenient for reaction
Divide and carries out.
In synthetic method of the present invention, suitable triethylamine can also be added before reactions as acid binding agent, to mention
The yield of high object.Under normal conditions, the ratio between amount of substance 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~
It is carried out under the conditions of 135 DEG C.Whether reaction can pass through TLC tracing detection completely.
It is the crude product of formula (I) compound made from above-mentioned synthetic method, existing conventional purification process can be used (as extracted
Take, silica gel column chromatography etc.) it is purified with the purity of raising formula (I) compound.Silica gel column chromatography is generallyd use to carry out
Purifying, specifically by object crude product obtained through silica gel column chromatography, with the petroleum ether and second for being 100:1~1:1 by volume ratio
The eluent of acetoacetic ester composition, eluent are evaporated off solvent, obtain 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 to crude product before carrying out silica gel column chromatography
It extracts to reduce the burden of silica gel, specifically available such as ethyl acetate, methylene chloride or chloroform extractant are extracted, and collection has
Machine phase carries out silica gel column chromatography after removing solvent again.
The invention also includes the applications in above-mentioned the fluorescence probe in the cell fluorescent microscopic imaging of mitochondria.Described is thin
Born of the same parents are specially non-small cell lung cancer cell strain NCI-H460.
Compared with prior art, the present invention provides it is a kind of new with mitochondria be directly targeted effect fluorescence probe and
Its synthetic method.The experiment of applicant shows that the fluorescence probe has mitochondria and is directly targeted effect but to cell without obvious
Inhibiting effect has preferable potential value, is expected to as the fluorescence probe for being directly targeted mitochondria.
Detailed description of the invention
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 plasmosin 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 protein after cell incubation compound N A-2a in experimental example 1 of the present invention；
Fig. 4 is the mass spectrometry results that cell is not incubated for compound N A-2a plasmosin in experimental example 1 of the present invention；
Fig. 5 is the mass spectrometry results that cell is not incubated for compound N A-2a mitochondrial protein in experimental example 1 of the present invention；
Fig. 6 is individually to be incubated for 5h line grain after compound N A-2a without drug treatment extraction mitochondria in experimental example 1 of the present invention
Body protein mass spectrometry results；
Fig. 7 is negative control cell fluorescence detection result in experimental example 2 of the present invention；
Fluorescence detection result when Fig. 8 is cell incubation 5h in experimental example 2 of the present invention.
Specific embodiment
The present invention is described in further detail combined with specific embodiments below, content to better understand the invention, but
The present invention is not limited to following embodiments.
The synthesis of compound shown in embodiment 1:NA-1a, that is, formula (II)
It weighs bromo- 1, the 8- naphthalic anhydride of 4- to be dissolved in 150mL ethyl alcohol, stirs and 3,4- (methylenedioxy) phenyl ethylamine is added
(bromo- 1, the 8- naphthalic anhydride of 4- and 3, the molar ratio of 4- (methylenedioxy) phenyl ethylamine are 1:1.5), 70 DEG C of reaction temperature, react into
Journey carries out tracking and monitoring using thin-layer chromatography.After reaction, reaction system is cooled to room temperature, filters and retain filter cake, makes
With ethanol rinse filter cake to get yellow solid (yield is about 90%).
Nuclear magnetic resonance spectroscopy, carbon-13 nmr spectra analysis are carried out to gained yellow solid, specific spectral characteristic is as follows:
(1) nuclear magnetic resonance spectroscopy.
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.
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- naphthalene two
Carboximide, shown in chemical structural formula such as following formula (II):
The synthesis of embodiment 2:NA-1a
Embodiment 1 is repeated, unlike:
Reaction temperature is changed to 60 DEG C, and remaining reaction condition is constant (yield is about 75%).
Nuclear magnetic resonance spectroscopy, carbon-13 nmr spectra analysis are carried out to gained yellow solid, determine that it is target product.
The synthesis of embodiment 3:NA-1a
Embodiment 1 is repeated, unlike:
Reaction temperature is changed to 80 DEG C, and remaining reaction condition is constant (yield is about 85%).
Nuclear magnetic resonance spectroscopy, carbon-13 nmr spectra analysis are carried out to gained yellow solid, determine that it is target product.
The synthesis of embodiment 4:NA-1a
Embodiment 1 is repeated, unlike:
Reaction dissolvent is changed to methanol, and remaining reaction condition is constant (yield is about 85%).
Nuclear magnetic resonance spectroscopy, carbon-13 nmr spectra analysis are carried out to gained yellow solid, determine that it is target product.
The synthesis of embodiment 5:NA-1a
Embodiment 1 is repeated, different conditions is:
Bromo- 1, the 8- naphthalic anhydride of reaction raw materials 4- and 3, the ratio between the amount of substance of 4- (methylenedioxy) phenyl ethylamine is changed to 1:1,
Remaining reaction condition is constant (yield is about 65%).
Nuclear magnetic resonance spectroscopy, carbon-13 nmr spectra analysis are carried out to gained yellow solid, determine that it is target product.
Embodiment 6: compound, that is, 4- shown in formula (I) (carbethoxyl group methylamino)-N- (3,4- (methylenedioxy) phenethyl) -1,
The synthesis of 8- naphthalimide (hereinafter referred to as NA-2a)
It weighs NA-1a to be dissolved in dimethyl sulfoxide, glycine ethyl ester and triethylamine (NA-1a, glycine ethyl ester and three is added
The molar ratio of ethamine is 1:1.2:2), 135 DEG C of reaction temperature, reaction process carries out tracking and monitoring using thin-layer chromatography.Reaction knot
Shu Hou, reaction system are cooled to room temperature, and are poured into ice water, there is yellow solid precipitation, are made to be extracted with ethyl acetate three times, are collected and close
And ester layer and with anhydrous MgSO4It is dry, solvent is removed 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 65%).
Nuclear magnetic resonance spectroscopy, carbon-13 nmr spectra, Electrospray Ionization Mass Spectrometry, specific wave spectrum are carried out to gained bright yellow solid
Characteristic is as follows:
(1) nuclear magnetic resonance spectroscopy, spectral data are 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, spectral data are 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, 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 chemical structural formula such as following formula (I):
The synthesis of embodiment 7:NA-2a
Embodiment 6 is repeated, unlike:
Reaction temperature is changed to 80 DEG C, and remaining reaction condition is constant (yield 25%).
Nuclear magnetic resonance spectroscopy, carbon-13 nmr spectra, Electrospray Ionization Mass Spectrometry are carried out to gained bright yellow solid, determined that it is
Target product.
The synthesis of embodiment 8:NA-2a
Embodiment 6 is repeated, unlike:
Reaction temperature is changed to 100 DEG C, and remaining reaction condition is constant (yield 38%).
Nuclear magnetic resonance spectroscopy, carbon-13 nmr spectra, Electrospray Ionization Mass Spectrometry are carried out to gained bright yellow solid, determined that it is
Target product.
The synthesis of embodiment 9:NA-2a
Embodiment 6 is repeated, unlike:
The ratio between reaction raw materials NA-2a and the amount of substance of glycine ethyl ester are changed to 1:1, the constant (yield of remaining reaction condition
For 40%).
Nuclear magnetic resonance spectroscopy, carbon-13 nmr spectra, Electrospray Ionization Mass Spectrometry are carried out to gained bright yellow solid, determined that it is
Target product.
The synthesis of embodiment 10:NA-2a
Embodiment 6 is repeated, unlike:
The ratio between reaction raw materials glycine ethyl ester and the amount of substance of triethylamine are changed to 1:1, the constant (yield of remaining reaction condition
For 20%).
Nuclear magnetic resonance spectroscopy, carbon-13 nmr spectra, Electrospray Ionization Mass Spectrometry are carried out to gained bright yellow solid, determined that it is
Target product.
The synthesis of embodiment 11:NA-2a
Embodiment 6 is repeated, unlike:
Reaction dissolvent is changed to n,N-Dimethylformamide, and remaining reaction condition is constant (yield 60%).
Nuclear magnetic resonance spectroscopy, carbon-13 nmr spectra, Electrospray Ionization Mass Spectrometry are carried out to gained bright yellow solid, determined that it is
Target product.
The synthesis of embodiment 12:NA-2a
Embodiment 6 is repeated, unlike:
Reaction dissolvent is changed to glycol monoethyl ether, and remaining reaction condition is constant, does not generate target compound.
The synthesis of embodiment 13:NA-2a
Embodiment 6 is repeated, unlike:
Synthetic method is added without triethylamine, and remaining reaction condition is constant (yield 15%).
Nuclear magnetic resonance spectroscopy, carbon-13 nmr spectra, Electrospray Ionization Mass Spectrometry are carried out to gained bright yellow solid, determined that it is
Target product.
It is directly targeted the characteristic for acting on mitochondria to absolutely prove that fluorescence probe of the present invention has, applicant is to it
Carry out following tests:
Experimental example 1: whether mitochondria is directly targeted in the horizontal detection compound NA-2a of cell in-situ
A) compound incubation
(1) it chooses non-small cell lung cancer cell strain NCI-H460 to be inoculated in 70mm culture dish, it is raw that cell grows to logarithm
For a long time when (the adherent area of cell is about 70%~75%), the culture solution in culture dish is carefully removed, twice using PBS cleaning,
The new DMEM culture solution of 5mL is added.
(2) appropriate compound N A-2a liquid storage (- 20 DEG C) is weighed, being added in culture dish makes its final concentration of 5 μM, 5h is acted on,
The control group of not dosing is set simultaneously.
B) the separation of mitochondria
(1) after acting on 5h, the culture medium in culture dish is carefully removed, is 0.25% pancreas with concentration twice using PBS cleaning
Protease digestion attached cell.5mL DMEM culture medium is added, simultaneously adherent cell collecting is dispelled, with 2000rpm on centrifuge
It is centrifuged 10min；
(2) liquid is discarded supernatant, the PBS for adding 1mL ice bath to be pre-chilled, cleaning cell is primary, and 2000rpm is centrifuged 1min；
(3) PBS is discarded supernatant, the mitochondria separation agent containing PMSF in right amount is added, is incubated for 30min on ice, periodically dispels
Cell prevents cell precipitation；
(4) cell suspension is transferred in appropriately sized glass homogenizer, is homogenized 40 times or so；
(5) cell homogenates is transferred in centrifuge tube, at 4 DEG C, 1000g is centrifuged 10min；
(6) carefully supernatant is transferred in another centrifuge tube, at 4 DEG C, 3500g is centrifuged 10min；
(7) collection part supernatant pays attention to not touching precipitating when collecting.Careful to remove remaining supernatant, precipitating is to separate
Obtained mitochondria.
C) NA-2a and mitochondria are incubated for
(1) mitochondria that control group in above-mentioned steps (B) extracts is divided into 2 parts, take wherein 1 part it is abundant with 5 μM of NA-2a
5h is incubated in 37 DEG C of waters bath with thermostatic control after mixing；
(2) in 4 DEG C after being incubated for, 9000g is centrifuged 10min；
(3) supernatant is carefully removed, 100 μ L mitochondria storing liquids are added and mix, 4 DEG C of 9000g are centrifuged 10min；
(4) supernatant is completely removed, precipitating is the mitochondria for being incubated for and obtaining after NA-2a；
D) the extraction of mitochondria and plasmosin matter
(1) 100-200 μ L RIPA lysate is added according to the amount of cell cytosol and PMSF is mixed, on ice ultrasound cracking
30min, every 10min oscillation mix cell pyrolysis liquid, crack it sufficiently.The ratio of RIPA lysate and PMSF are 100:1；
(2) according to mitochondria amount be added 40~80 μ L mitochondria lysates and PMFS (mitochondria lysate: PMSF=100:
1) it, mixes, the 20min of ultrasound cracking on ice, every 5min concussion mixes cell pyrolysis liquid, cracks mitochondria sufficiently.
(3) lysate of endochylema and mitochondria is moved to respectively in 1.5mL EP pipe, 4 DEG C of 12000rpm, is centrifuged 10min；
(4) careful collection supernatant, gained protein sample, is used for Mass Spectrometer Method after as cracking.
E) NA-2a is incubated for the Mass Spectrometer Method of the endochylema and mitochondrial protein that extract
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
The mass spectrometry results in DMSO are dissolved in for final product NA-2a made from embodiment 6；Fig. 2 is cell incubation compound N A-
The mass spectrometry results of 5h plasmosin after 2a；Fig. 3 is the mass spectral analysis of 5h mitochondrial protein after cell incubation compound N A-2a
As a result；Fig. 4 is the mass spectrometry results that cell is not incubated for compound N A-2a plasmosin；Fig. 5 is that cell is not incubated for compound
The mass spectrometry results of NA-2a mitochondrial protein；Fig. 6 is to extract mitochondria without drug treatment to be individually incubated for compound N A-2a
The mitochondrial protein mass spectral analysis result of 5h afterwards.Analysis the results show that in non-small cell lung cancer cell strain NCI-H460 NA-2a
It can be directly entered in mitochondria.
Experimental example 2:NA-2a and mitochondria common location are analyzed
1) with 75% (volumetric concentration) alcohol immersion treatment cell climbing sheet special glass piece, the slide handled well is placed in 6
It is spare in each hole of orifice plate.
2) after choosing the original culture solution of non-small cell lung cancer cell strain NCI-H460 removing, attached cell is cleaned using PBS,
PBS buffer solution is removed, 0.25% trypsase of 1mL is added, stands half a minute, after removing pancreatin, culture is placed in incubator
Continue to digest 1min, fresh medium is added and blows and beats attached cell to cell suspension.
3) it is separately added into each hole of 6 orifice plates by every 500 μ L cell suspending liquid of hole, orifice plate is placed in incubator and cultivates number
Day, to the complete adherent growth of cell.
4) each Kong Zhongyu cell incubation 5h is added using (5 μM) of NA-2a dilution, while negative control is set.
5) culture solution is removed, is cleaned 2 times with PBS, each 5min.
6) every hole is added the preparatory 37 DEG C of incubations 30min's of 500 μ LRed CMXRos mitochondria is red
Fluorescence probe liquid storage is incubated for 45min in room temperature again.
7) liquid in hole is removed, is cleaned 2 times with PBS, each 5min.
8) the anti-fluorescent quenching mounting liquid of drop is added dropwise on glass slide, covers the slide for posting cell, avoids the occurrence of bubble,
Mounting is fixed with Instant cement.
9) fluorescence detection 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；Fluorescence detection result when Fig. 8 is cell incubation 5h.Analysis is the results show that thin in non-small cell lung cancer
NA-2a is same in born of the same parents' strain NCI-H460Red CMXRos mitochondria red fluorescence probe position phase in cell
Together, illustrate that NA-2a can be directly targeted mitochondria.
Claims (5)
1. a kind of fluorescence probe for being directly targeted effect 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 described in claim 1 for being directly targeted effect with mitochondria, it is characterised in that: take
Compound and glycine ethyl ester as shown in following formula (II), are dissolved in dimethyl sulfoxide or n,N-Dimethylformamide, in fire-bar
It is reacted under part, gained reactant is poured into ice water, and filtering obtains object crude product；
3. synthetic method according to claim 2, it is characterised in that: triethylamine is added before reactions as acid binding agent.
4. synthetic method according to claim 2 or 3, it is characterised in that: reaction carries out under the conditions of 80~150 DEG C.
5. synthetic method according to claim 2 or 3, it is characterised in that: further include purification step: being specifically will be obtained
Object crude product is washed through silica gel column chromatography with the eluant, eluent that the petroleum ether and ethyl acetate for being 100:1~1:1 by volume ratio form
De-, solvent is evaporated off in eluent, obtains target product after purification.
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| CN105777637A (en) * | 2014-12-22 | 2016-07-20 | 中国科学院化学研究所 | Water soluble mitochondrial targeting imaging probe and preparation method thereof |
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| Title |
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| A Novel Naphthalimide Compound Restores p53 Function in Non-small Cell Lung Cancer by Reorganizing the Bak·Bcl-xl Complex and Triggering Transcriptional Regulation;Guohai Zhang.et al.;《The Journal of Biological Chemistry》;20160219;第291卷(第08期);第4211-4225页 |
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