CN105670608A - High-selectivity fluorescent probe capable of detecting nickel ions in mitochondria of living cells and preparation method thereof - Google Patents
High-selectivity fluorescent probe capable of detecting nickel ions in mitochondria of living cells and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a high-selectivity fluorescent probe capable of detecting nickel ions in mitochondria of living cells and a preparation method thereof. The probe is a pyridyl-containing triphenylamine-based amine derivative; in the preparation method, with triphenylamine as a starting material, an intermediate I 4-(N,N-diphenylamino) benzaldehyde and an intermediate II phosphonium salt of paranitrotoluene are firstly prepared, 4-paranitrostyryltriphenylamine intermediate is then prepared from the intermediate I and the intermediate II, 4-paraaminostyryltriphenylamine is then obtained by reduction under the action of Pd/C and hydrazine hydrate, and then condensation reaction with 2-pyridinebenzaldehyde is performed, so that Schiff base is obtained, and the Schiff base is reduced under the action of sodium borohydride, so that the target product is obtained. The probe disclosed by the invention has good single-photon fluorescent property at about 425nm. After HepG2 cells are dyed by the target product, the high imaging capability of the organic material on mitochondria in the cytoplasm of the HepG2 cells can be clearly observed, and nickel ions in the mitochondria can be reversibly detected. The probe disclosed by the invention has great significance for the design and preparation of organic dyes and life science research.
Description
One, technical field
The present invention relates to a kind of single photon optical material and its preparation method, specifically a kind of have hypotoxicity, the pyridyl triphenylamine class fluorescent probe that the nickel ion in viable cell plastosome can have recognition capability and its preparation method.
Two, background technology
Utilize viable cell imaging workstation to carry out the function research of cell and gene, it is the recent tendency of biomedical research. Fixed cell observes the static information that only can provide fixing cell instantaneously, the state viable cell observation of cell under normal physiological biochemical condition cannot be reflected, the cell being under normal physiological condition is carried out omnidistance scanning and record, obtain its continuously, comprehensively, dynamic process. Due to the dynamic activity process of normal cell of its display, it is very easy to find and determines the process of cell-cell interaction and intracellular signaling, and the interaction between biomolecules in viable cell level, it is possible not only to solve problem that is outstanding for a long time and that do not separate, more following researchs and proposes new problem, points out new direction.
Chemiluminescence probe shows good selectivity in the recognition process of object, thus, has potential using value in field exhibitions such as environmental monitoring, Journal of Molecular Catalysis and bioluminescence imagings, is subject to extensive concern. As compared to traditional detection method (such as atomic absorption spectrum and plasma emission spectrum, electrochemical process, colorimetry, biology and nano-sensor etc.), chemiluminescence probe mainly utilizes the fluorescent signal occurred in test process change (strengthen, weaken or emission wavelength displacement etc.) to be detected by object, have with low cost, simple to operate, detectability is low, susceptibility and selectivity advantages of higher. Viable cell imaging technique utilizes these fluorescent probes just, and such as small molecules organic dye or quantum dot carry out the mark molecule interested of specificity.
Triphenylamine is centered by nitrogen-atoms, has the compound of hub-and-spoke configuration.Owing to it has unique free radical character, bigger space steric hindrance, hyperconjugation electronic effect and higher hole mobility, thus have a wide range of applications in the field such as photoelectric material and hole mobile material. In addition, lone-pair electron and the phenyl ring of surrounding in atom N form big conjugated system, make the emission wavelength of molecule be positioned at blue green light region, show good optical characteristics, be widely used in the fields such as the storage of three-dimensional optical data, fluorescent probe and cell imaging.
Three, summary of the invention
There is the highly selective fluorescent probe and its preparation method that can detect nickel ion in viable cell plastosome it is desirable to provide a kind of, synthesized probe is for containing pyridyl triphenylamine base amino derivative, this probe possesses hypotoxicity and good optical property, in the imaging of cell mitochondrial position, and nickel ion can be had reversible detectivity.
The a kind of of request of the present invention protection has the highly selective fluorescent probe that can detect nickel ion in viable cell plastosome, and its structural formula is as follows:
The present invention asks to protect the preparation method of above-mentioned highly selective fluorescent probe simultaneously, and described preparation method's step is as follows:
The preparation of A, intermediate I
Adding DMF, phosphorus oxychloride and triphenylamine under ice bath in container successively, be then warming up to 40-70 DEG C, reaction 14-18h, described reaction solution is through separation, washing dry faint yellow intermediate I;
The preparation of B, intermediate II
Nitrotoluene and N-bromine are dissolved in benzene for succimide (NBS), under the effect of catalyzer benzoyl peroxide (BPO), 7-10h is reacted at 60-100 DEG C, reaction is cooled to room temperature after terminating, filter and retain filtered liquid, then in filtered liquid, add triphenylphosphine, at 40-80 DEG C, react 2-5h, react and naturally cool to room temperature after terminating, filter, wash and obtain yellow intermediate II after vacuum-drying;
The preparation of C, intermediate III
T-BuOK, intermediate I and intermediate II are ground in mortar, after reacting completely, uses CH2Cl2By reactants dissolved, filter, concentrated, obtain the red intermediate III of dendroid;
The preparation of D, intermediate IV
By in molten for intermediate III ethanolic soln, then adding hydrazine hydrate, under the effect of catalyst P d/C, back flow reaction 1-3h at 50-80 DEG C, after reacting completely, takes out filter while hot, obtains milk yellow intermediate IV.
The preparation of E, intermediate V
The intermediate IV being dissolved in methanol solution being warming up to 50-80 DEG C, then adds glacial acetic acid, add 2-pyridylaldehyde, back flow reaction 4-6h again, reaction is taken out filter and is obtained yellow powder shape intermediate V after terminating.
The preparation of F, target product L
After reacting 0.5-2h at adding sodium borohydride, dissolving V, 10-40 DEG C, intermediate in ethanol in reaction vessel, take out filter, ethyl alcohol recrystallization, obtain brown target product L solid.
As preferably, in described steps A, the mole dosage of DMF, phosphorus oxychloride and triphenylamine is than being 1:1:1.
As preferably, in described step B, para-nitrotoluene, N-bromine are 1:12:12 for succimide (NBS), triphenylphosphine mole dosage ratio.
As preferably, in described step C, the mole dosage of t-BuOK, intermediate I and intermediate II is than being 5.4:1.8:2.2.
As preferably, in described step D, the mole dosage of intermediate III, hydrazine hydrate is than being 1:10.
As preferably, in described step e, intermediate IV, 2-pyridylaldehyde mole dosage are than being 7.2:9.4.
As preferably, in described step F, the mole dosage of sodium borohydride, intermediate V is than being 2:1.
Compared with the prior art, the useful effect of the present invention is embodied in:
1, the present invention using there is relatively high reaction activity and good biocompatibility trianilino group as main body, succinctly prepare triphenylamine nitro-derivative efficiently, by reduction reaction, condensation reaction, obtain pyridyl triphenylamine class fluorescent probe, finding through experiment, pyridyl triphenylamine class fluorescent probe prepared by the present invention has good single photon fluorescence character (Fig. 2) at about 425nm. After HepG2 cell is dyeed by target product, can clearly observe organic materials and the plastosome in HepG2 cell matter is had high imaging capability, and the reversibly nickel ion in detection line plastochondria. Probe of the present invention is significant for the design of organic dye, preparation and life science.
2, the pyridyl triphenylamine class fluorescent probe of the present invention's synthesis is the single photon optical material that a class has plastosome imaging function in tenuigenin. To cell not damaged, can be used for active somatic cell detection, there is obvious using value;
3, preparation method's raw material of the present invention is easy to get, and cost is low, and synthesis step is simple, is easy to operation.
Four, accompanying drawing explanation
Fig. 1 is preparation method's route map of the present invention.
Fig. 2 be the pyridyl triphenylamine class fluorescent probe of the present invention in acetonitrile under excitation wavelength 368nm to the fluorescence response figure of different metal ion.
Fig. 3 a-3c be pyridyl triphenylamine class fluorescent probe to HepG2 cell fluorescence confocal microscopic image figure,
Wherein Fig. 3 a is the fluorescence co-focusing Photomicrograph of the painted HepG2 cell of pyridyl triphenylamine class fluorescent probe; Fig. 3 b is bright field action figure; Fig. 3 c is superimposed photo.
Fig. 4 a is that the commodity staining agent Mito-Tracker of pyridyl triphenylamine class fluorescent probe and plastosome is to HepG2 cell fluorescence confocal microscopic image figure. In Fig. 4 a, the picture left above is that probe is to HepG2 cell fluorescence confocal microscopic image; Top right plot is that commodity staining agent Mito-Tracker is to HepG2 cell fluorescence confocal microscopic image; Lower-left figure is the bright field figure to HepG2 cell fluorescence confocal microscopic image; Bottom-right graph is that the commodity staining agent Mito-Tracker of pyridyl triphenylamine class fluorescent probe and plastosome is to the details in a play not acted out on stage, but told through dialogues stacking diagram of HepG2 cell fluorescence confocal microscopic image.
Fig. 4 b is the Pearson correlation coefficients between the commodity staining agent Mito-Tracker of pyridyl triphenylamine class fluorescent probe and plastosome.
Five, embodiment
By specific embodiment, technical solution of the present invention is further explained explanation below in conjunction with accompanying drawing.
1, the preparation of intermediate I
250mL flask adds 7.4mL (0.1mol) DMF, ice-water bath stirs, dropwise adds 9.2mL (0.1mol) phosphorus oxychloride subsequently, continue to stir, after 15min, in bottle, liquid becomes sticky thick, finally obtain white slightly red solid freeze salt. The chloroformic solution that 100ml contains 24.5g (0.1mol) triphenylamine adds in above-mentioned flask, intensification degree to 50 DEG C about 30min is entirely molten to freezing salt, back flow reaction 16h at 65 DEG C, tlc judgement is revolved after reacting completely and has been steamed chloroform, residuum is poured in 1000mL cold water, regulate solution ph to weakly alkaline with salt of wormwood, a large amount of yellow-green colour solid is had to precipitate out, take out filter, being spin-dried for solvent, resistates is purified through chromatographic column separation, and eluent is pure sherwood oil, obtain 7.81g product, product rate 71.5%.1HNMR:(DMSO-d6, 400Hz), δ (ppm): 6.89 (d, J=8.80Hz, 2H), 7.23 (q, 6H), 7.42 (t, J=7.80Hz, 4H), 7.72 (d, J=8.80Hz, 2H), 9.771 (s, 1H).
2, the preparation of intermediate II
In the round-bottomed flask of 500mL, add 13.7g (0.1mol) para-nitrotoluene respectively, 21.4g (0.12mol) N-bromine be dissolved in the benzene of 150mL for succimide (NBS), finally add 0.5g benzoyl peroxide (BPO) catalyzer, back flow reaction 8h at being warming up to 80 DEG C, after tlc judgement reacts completely, it is cooled to room temperature, adularescent solid (succinimide) precipitates out, take out filter, elimination white solid, filtrate adds 31.5g (0.12mol) triphenylphosphine, back flow reaction 3 hours at 50 DEG C; Being cooled to room temperature has a large amount of solids to precipitate out, and takes out filter, and solid benzene (5mL) washs three times, obtains 39.0g yellow solid. Product rate is 81.6%.1HNMR:((CD3)2CO, 400Hz), δ (ppm): 5.98 (d, J=8.00,2H), 7.47 (d, J=8.40Hz, 2H), 7.57-7.62 (m, 6H), 7.75 (t, J=7.60Hz, 3H), 7.82 (q, 8H).
3, the preparation of intermediate III
0.6g (5.4mmol) t-BuOK (potassium tert.-butoxide) is ground to form powder in mortar, then 1.0g (2.2mmol) bromination (4-oil of mirbane methyl) triphenylphosphine (intermediate II) is added, after grinding 5min, separately add 0.5g (1.8mmol) 4-(N, N-diphenyl amino) phenyl aldehyde (intermediate I) adds in mortar, grinding, after tlc judgement reacts completely. Use CH2Cl2Being dissolved, filter, filtrate is washed, anhydrous MgsO4G is dry, and concentrated, dehydrated alcohol recrystallization obtains 0.33g dendroid red solid. Product rate is: 46.5%.1HNMR:((CD3)2CO, 400Hz), δ (ppm): 7.03 (d, J=8.00Hz, 2H), 7.13 (d, J=7.20Hz, 6H), 7.38 (d, J=16.40,1H), 7.35 (d, J=7.80Hz, 4H), 7.51 (d, J=16.40Hz, 1H), 7.60 (d, J=8.00Hz, 2H), 7.85 (d, J=8.40Hz, 2H), 8.23 (d, J=8.00Hz, 2H).
4, the preparation of intermediate IV
The two neck round-bottomed flasks of 250mL add the alcohol solvent of 3.0g (10mmol) intermediate III and 150mL, at being warming up to 80 DEG C, add 0.3gPd/C catalyzer, and dropwise drip the ethanolic soln adding 10mL containing 4.9mL85% (v/v) hydrazine hydrate, after dropwising, continue back flow reaction 2 hours, after tlc judgement reacts completely. Taking out filter while hot, have a large amount of solids to precipitate out, take out and filter to obtain milk yellow solid in filtrate, then screw out ethanol in filtrate, cooling, has a large amount of solid to precipitate out, again takes out filter, and the product taking out filter for twice merges, and vacuum-drying, obtains 2.56g milk yellow solid altogether. Product rate is 70.7%.1HNMR:((CD3)2CO, 400Hz), δ (ppm): 4.782 (s, 2H), 6.68 (d, J=8.40Hz, 2H), 6.93 (d, J=16.40Hz, 1H), 7.09-7.00 (m, 10H), 7.33-7.29 (m, 6H), 7.45 (d, J=8.80Hz, 2H) .MS (EI), m/z (%): 362.18 ([M]+,100)。
5, the preparation of intermediate V
By intermediate IV (2.6g in 250mL round-bottomed flask, 7.2mmol) it is dissolved in 120mL methyl alcohol, it is warming up to 70 DEG C, adds 3 glacial acetic acids (catalyzer) under backflow, more dropwise add 2-pyridylaldehyde (1.0g, 9.4mmol), react 5 hours at 70 DEG C, no longer precipitate out to solid, take out filter, vacuum-drying, obtains 3.1g yellow powder (product rate 88.4%).
m.p.189℃.1HNMR:(DMSO-d6, 400Hz), δ (ppm): 6.97 (d, J=8.0Hz, 3H), 7.03-7.07 (m, 6H), 7.19 (d, J=12.0Hz, 2H), 7.28-7.35 (m, 6H), 7.51-7.53 (m, 5H), 7.63 (d, J=8.0Hz, 2H), 7.95 (t, J=8.0Hz, 2H), 8.68 (s, 1H);13CNMR(100MHz,DMSO-d6, TMS, ppm): δ 160.02,151.13,147.89,147.78,136.67,136.14,131.89,131.78,129.67,129.22,128.14,127.70,127.50,126.72,124.93,123.78,123.45,121.81; MALDI-TOFm/z: calculated value, 450.210; Experimental value, 449.890.
6, the preparation of target product L
In 100mL round-bottomed flask by sodium borohydride (0.005g, 0.14mmol) in batches (average mark adds for four times, mild speed of response) be added to 25mL containing 0.15g (0.07mmol) intermediate V ethanolic soln in. Stirred at ambient temperature reacts 1 hour, has brown solid to precipitate out gradually, and TLC point plate spike takes out filter after reacting completely, the solid ethyl alcohol recrystallization obtained, and obtains 0.10 gram of brown solid (product rate 77.1%).1HNMR(400MHz,DMSO-d6), δ (ppm): 8.54 (J=4, s, 1H), 7.70 (J=8, t, 1H), 7.40 (J=8, d, 2H), 7.35 (J=8, d, 1H), 7.24-7.32 (m, 7H), 7.00-7.06 (m, 6H), 6.94 (J=8, t, 3H), 6.80 (J=20, d, 1H), 4.30 (J=4, d, 2H).13CNMR(100MHz,DMSO-d6), δ (ppm): 159.28,148.89,148.76,147.88,146.94,145.29,136.40,130.28,127.62,127.38,126.82,125.34,123.72,123.63,122.88,122.64,121.98,121.08,112.37,48.74.MS (APCI): theoretical value 453.22; Experimental value, 454.2283.
7, target product L is to the identification of nickel ion
Selective row is be one of the most important feature of fluorescent probe. As shown in Figure 2, when exciting with 368nm, fluorescence intensity is strong. When adding metal ion important on the environment widely with target product L equal quantities and physiology, as: Cu2+,Hg2+,Zn2+,Ni2+,Mg2+,Pb2+,Ca2+,Cd2+,Ag+,Fe3+,Al3+,Mn2+,Co2+,Cr3+,Li+,K+,Na+,La+,Ba2+Time, under room temperature, the fluorescence intensity of the target product L adding nickel ion in acetonitrile declines 14 times, and the obvious fluorescence intensity that do not have of other metal ions changes. Interference test show, other each metal ion species not jamming target product L to nickel ion identification. We use nickel ion sequestrant N, and N, N', N'-tetra-(2-pyridylmethyl) quadrol (TPEN) have detected target product L and identify Ni in acetonitrile2+Reversibility, result display-object product L has the recognition capability of good circulation.
8, the test of target product L single photon cell developing effect
The cover glass cleaning clean and sterilizing is put into 6 hole tissue culturing plates, liver cancer tissue cell (HepG2 cell) 5 × 105The density in individual/hole is seeded in the 6 orifice plate culture dish of diameter 35mm, and carry out cell cultures with DMEM as cell culture medium, containing foetal calf serum (10%), penicillin (100 μ g/mL) and Streptomycin sulphate (100ug/mL) in cell culture medium. Tissue Culture Dish is placed in containing 5%CO2And 95%O2Incubator in holding temperature 37 DEG C carry out cell cultures 24h, with PBS (phosphoric acid buffer, pH=7.4, Gibco Reagent Company produce) washing HepG2 cell three times, wash substratum. Then the DMSO solution (1mM) of 4 μ L target compound A or B is added respectively, cultivate 0.5h, with PBS buffered soln (pH=7.4) flush cover slide 6～7 times, drip 1mL4% paraformaldehyde/PBS solution fixed cell 10min, distilled water flushing cover glass 6～7 times. Cover glass is stuck in clean glass slide, is placed under laser confocal microscope (LSM-710, Zeiss, Germany) observation of cell form and fluorescence picked-up situation, the results are shown in Figure 3a-3c, 4a, 4b.
From Fig. 3 a-3c, 4a, 4b it will be clear that, target product L, all through the cytolemma of HepG2 cell, enters in the plastosome in tenuigenin, and it is painted completely uniformly, the uptake ratio of target product is very high, illustrate that the plastosome of HepG2 is had very high recognition capability by target product.The preparation of this kind of organic materials for the selecting of cell developing material, prepare, the aspect such as life science, Materials science all had great significance.
Claims (8)
1. one kind can be detected the highly selective fluorescent probe of nickel ion in viable cell plastosome, it is characterised in that:
The structural formula of described highly selective fluorescent probe is:
2. the preparation method of a kind of highly selective fluorescent probe detecting nickel ion in viable cell plastosome as claimed in claim 1, it is characterised in that operate according to the following steps:
The preparation of A, intermediate I
Adding DMF, phosphorus oxychloride and triphenylamine under ice bath in container successively, be then warming up to 40-70 DEG C, reaction 14-18h, described reaction solution is through separation, washing dry faint yellow intermediate I;
The preparation of B, intermediate II
Para-nitrotoluene and N-bromine are dissolved in benzene for succimide (NBS), under the effect of catalyzer benzoyl peroxide (BPO), 7-10h is reacted at 60-100 DEG C, reaction is cooled to room temperature after terminating, filter and retain filtered liquid, then in filtered liquid, add triphenylphosphine, at 40-80 DEG C, react 2-5h, react and naturally cool to room temperature after terminating, filter, wash and obtain yellow intermediate II after vacuum-drying;
The preparation of C, intermediate III
T-BuOK, intermediate I and intermediate II are ground in mortar, after reacting completely, uses CH2Cl2By reactants dissolved, filter, concentrated, obtain the red intermediate III of dendroid;
The preparation of D, intermediate IV
By in molten for intermediate III ethanolic soln, then adding hydrazine hydrate, under the effect of catalyst P d/C, back flow reaction 1-3h at 50-80 DEG C, after reacting completely, takes out filter while hot, obtains milk yellow intermediate IV.
The preparation of E, intermediate V
The intermediate IV being dissolved in methanol solution being warming up to 50-80 DEG C, then adds glacial acetic acid, add 2-pyridylaldehyde, back flow reaction 4-6h again, reaction is taken out filter and is obtained yellow powder shape intermediate V after terminating;
The preparation of F, target product L
After reacting 0.5-2h at adding sodium borohydride, dissolving V, 10-40 DEG C, intermediate in ethanol in reaction vessel, take out filter, ethyl alcohol recrystallization, obtain brown target product L solid.
3. the preparation method of a kind of highly selective fluorescent probe detecting nickel ion in viable cell plastosome according to claim 2, it is characterised in that, in described steps A, the mole dosage of DMF, phosphorus oxychloride and triphenylamine is than being 1:1:1.
4. the preparation method of a kind of highly selective fluorescent probe detecting nickel ion in viable cell plastosome according to claim 2, it is characterized in that, in described step B, para-nitrotoluene, N-bromine are 1:12:12 for succimide (NBS), triphenylphosphine mole dosage ratio.
5. the preparation method of a kind of highly selective fluorescent probe detecting nickel ion in viable cell plastosome according to claim 2, it is characterised in that, in described step C, the mole dosage of t-BuOK, intermediate I and intermediate II is than being 5.4:1.8:2.2.
6. the preparation method of a kind of highly selective fluorescent probe detecting nickel ion in viable cell plastosome according to claim 2, it is characterised in that, in described step D, the mole dosage of intermediate III, hydrazine hydrate is than being 1:10.
7. the preparation method of a kind of highly selective fluorescent probe detecting nickel ion in viable cell plastosome according to claim 2, it is characterised in that, in described step e, intermediate IV, 2-pyridylaldehyde mole dosage are than being 7.2:9.4.
8. the preparation method of a kind of highly selective fluorescent probe detecting nickel ion in viable cell plastosome according to claim 2, it is characterised in that, in described step F, the mole dosage of sodium borohydride, intermediate V is than being 2:1.
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| CN106631998A (en) * | 2016-10-09 | 2017-05-10 | 安徽大学 | Copper ion biological probe capable of detecting living cell mitochondria and preparation method thereof |
| CN106977585A (en) * | 2017-03-24 | 2017-07-25 | 南京工业大学 | A kind of mitochondria positioning is used for two-photon fluorescence probe storehouse and its application of photodynamic therapy |
| CN114133413A (en) * | 2021-11-04 | 2022-03-04 | 广东工业大学 | Benzothiazole-triphenylamine compound and preparation method and application thereof |
| CN114436948A (en) * | 2022-01-19 | 2022-05-06 | 上海工程技术大学 | Dipyridyl triphenylamine aldehyde fluorescent material with aggregation-induced emission effect and preparation method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106631998A (en) * | 2016-10-09 | 2017-05-10 | 安徽大学 | Copper ion biological probe capable of detecting living cell mitochondria and preparation method thereof |
| CN106977585A (en) * | 2017-03-24 | 2017-07-25 | 南京工业大学 | A kind of mitochondria positioning is used for two-photon fluorescence probe storehouse and its application of photodynamic therapy |
| CN106977585B (en) * | 2017-03-24 | 2020-06-23 | 南京工业大学 | Two-photon fluorescence probe library for photodynamic therapy by mitochondria positioning and application thereof |
| CN114133413A (en) * | 2021-11-04 | 2022-03-04 | 广东工业大学 | Benzothiazole-triphenylamine compound and preparation method and application thereof |
| CN114436948A (en) * | 2022-01-19 | 2022-05-06 | 上海工程技术大学 | Dipyridyl triphenylamine aldehyde fluorescent material with aggregation-induced emission effect and preparation method and application thereof |
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| Publication number | Publication date |
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| CN105670608B (en) | 2017-09-26 |
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