CN107446571A - A kind of two-photon nitroreductase fluorescence probe of endoplasmic reticulum targeting and its synthetic method and application - Google Patents
A kind of two-photon nitroreductase fluorescence probe of endoplasmic reticulum targeting and its synthetic method and application Download PDFInfo
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Abstract
The invention provides a kind of two-photon nitroreductase fluorescence probe of endoplasmic reticulum targeting, its chemical name is：4 nitro N（2‑（4 Methyl benzenesulfonyl amidos）Second）Base naphthalimide, is synthesized by following steps：Ethylenediamine reacts with paratoluensulfonyl chloride obtains N completely（2 amino-ethyls）4 methyl this sulfonamide；The naphthalic anhydride of 4 nitro 1,8 and N（2 amino-ethyls）4 methyl benzenesulfonamides react to obtain.Present invention also offers a kind of fluorescence probe using single photon or two-photon fluorescence detection solution in and organism in Nitroreductase Activity application.The probe synthesis of the present invention is simple, high income, can be positioned at endoplasmic reticulum and carry out two-photon fluorescence detection to cell or tissue, high sensitivity, can resist the interference of a variety of interfering materials.
Description
Technical field
The present invention relates to a kind of quick response nitroreductase fluorescence probe, and in particular to a kind of double light of endoplasmic reticulum targeting
The fluorescence probe and its synthetic method of sub- effect and application, belong to organic molecule fluorescence probe field.
Background technology
Weary oxygen is a key character of malignant tumour occurrence and development, unrestrictedly increase mainly due to tumour cell,
Metabolism so that oxygen demand increase, caused by causing tumor region blood oxygen supply deficiency.Medical research shows, tumor hypoxia region
Generally refer to the cell mass in 10-20 μm of region of tumor vessel, the cell in this region is otherwise known as anoxic cell.Weary oxygen
Cell causes cell to be in the state that can not breed geo-stationary that also will not be dead because without sufficient oxygen supply, and
This state, generally lie in G1 and S phases border.The notable feature of anoxic cell is exactly that the cell of the type has very strong resist
The property of medicine and radioresistance, and if once having the oxygen supply of abundance, cell and can recovers normal vigor.Therefore, develop
Novel method, which carries out the treatment of development degree and cancer of weary oxygen detection to assessing tumour etc., has great directive significance.
In general, in anoxic cell, the bioactivity of some reproducibility biology enzymes can be strengthened to maintain weary oxygen
The basic survival condition of cell, these reproducibility biology enzymes mainly include nitroreductase（NTR）, azo reductase and quinone reduction
Enzyme.Wherein, in these reproducibility biology enzymes, nitroreductase is most representational.In the presence of weary oxygen and associated coenzymes
Under state, intracellular nitroaromatic can be gradually reduced to amino aromatic compound, this reduction by nitroreductase
The one-electron reduction of enzymatic reacts provides certain theoretical direction for the design of weary oxygen fluorescence probe.
Endoplasmic reticulum（ER）It is a kind of important monofilm organelle of generally existing in eukaryotic, there is tubulose and sheet
Two kinds of structures.Endoplasmic reticulum spreads all in whole cytoplasm, connects nucleus and cell membrane, carries the matter transportation between both
With signal transmission.The main Physiological Function of endoplasmic reticulum includes：Synthesis, processing and the modification of intracellular protein, it is stable intracellular
Calcium ion concentration etc..In addition to being related with nucleus, cell membrane, endoplasmic reticulum is also closely coupled with other multiple organelles,
Wherein mainly include：Golgiosome, lysosome, mitochondria etc..Found by electron microscope observation, endoplasmic reticulum and other cells
All there is the medium communicated with each other between device, the interaction between them maintains the normal metabolic mistake of cell
Journey.Therefore, studying the physiological function of endoplasmic reticulum has critically important medical value.
In recent years, the fluorescence probe for having many nitroreductases is reported, and these probes are all successfully applied
Cell imaging is technical, and still, these cells are generally at visible region, these probes tend to be carried on the back
Scape disturbs, and two-photon dyestuff has the characteristic that near-infrared excites so that ambient interferences are few, therefore have and preferably apply valency
Value.In addition, existing nitroreductase fluorescence probe for endoplasmic reticulum targeting still in open position, therefore, design synthesis
Two-photon nitroreductase fluorescence probe with endoplasmic reticulum targeting is very necessary.
The content of the invention
The problem of for the two-photon nitroreductase fluorescence probe of shortage endoplasmic reticulum targeting at present, the present invention provides a kind of
The two-photon nitroreductase fluorescence probe of endoplasmic reticulum targeting.
It is a further object of the present invention to provide a kind of synthetic method of easy above-mentioned fluorescence probe.
Another object of the present invention is to provide a kind of application of above-mentioned fluorescence probe on detection Nitroreductase Activity.
To achieve the above object, the present invention adopts the following technical scheme that.
A kind of two-photon nitroreductase fluorescence probe of endoplasmic reticulum targeting, its chemical name are：4- nitros-N-（2-（4-
Methyl benzenesulfonyl amido）Ethyl）- naphthalimide, referred to as Na-ER-NTR, its chemical structural formula such as formula（）It is shown：
Formula (I).
A kind of synthetic method of above-mentioned fluorescence probe, comprises the following steps：
（1）Ethylenediamine（Compound 1）Solvent is dissolved in, low temperature stirring is lower to add paratoluensulfonyl chloride（Compound 2）, then rise to room
Temperature stirring is complete to reaction, isolated N-（2- amino-ethyls）This sulfonamide of -4- methyl（Compound 3）.
As optimization, the mol ratio of the ethylenediamine and paratoluensulfonyl chloride is 1:1-1.5；
The concentration of ethylenediamine is the mol/L of 0.2- 0.3；
The low temperature is 0-4 DEG C；
The separating step is：HCl/water solution is added into reaction system, suction filtration removes filter residue, and filtrate is neutralized to analysis with alkali lye
Untill going out a large amount of white solids, filtered solid vacuum drying；
As optimization, HCl concentration is 2 mol/L；The alkali lye is 10%wt NaOH solutions.
（2）4- nitro -1,8- naphthalic anhydrides（Compound 4）It is dissolved in solvent, in the presence of triethylamine, stirs lower add
N-（2- amino-ethyls）- 4- methyl benzenesulfonamides（Compound 3）, then heating stirring backflow, separating-purifying after cooling
Compound 4- nitros-N-（2-（4- Methyl benzenesulfonyl amidos）Ethyl-naphthalimide（Compound 5）.
As optimization, 4- nitro-1,8-naphthalic acids acid anhydride and N-（2- amino-ethyls）The mol ratio of -4- methyl benzenesulfonamides
For 1:1.2-1.5；
The concentration of 4- nitro -1,8- naphthalic anhydrides is 0.05-0.06 mol/L；
Reaction temperature is 80-85 DEG C；
The separating-purifying step is：Remove organic reagent under reduced pressure, solid residue is eluent through post using dichloromethane and methanol
Chromatographic isolation；The volume ratio of dichloromethane and methanol is 20-10:1.
The synthetic route of above-mentioned fluorescence probe is as follows：
。
A kind of application of above-mentioned fluorescence probe Nitroreductase Activity in solution is detected, excitation wavelength are 440 nm, hair
The a length of 545nm of ejected wave, after detection time is effect 30-80min, preferably 60 min.
A kind of application of above-mentioned fluorescence probe Nitroreductase Activity in detection organism, one-photon excitation wavelength are
488 nm, emission band are 500-550 nm；Two-photon excitation wavelength is 760 nm, and emission band is 500-550 nm；
After detection time is effect 30-80 min, preferably 60 min.
The mechanism of above-mentioned fluorescence probe is as follows：
。
Endoplasmic reticulum of the present invention targets nitroreductase two-photon fluorescence probe, almost unstressed configuration, and in reduced form
DPN（NADH）In the presence of, after nitroreductase acts on, nitro is reduced to amino, sends intense fluorescence.
The present invention has advantages below：
The probe synthesis of the present invention is simple, high income, can be positioned at endoplasmic reticulum and carry out two-photon fluorescence to cell or tissue
Detection, high sensitivity, can resist the interference of a variety of interfering materials.
Brief description of the drawings
Fig. 1 is fluorescence probe1H H NMR spectroscopies；
Fig. 2 is fluorescence probe13C H NMR spectroscopies；
Fig. 3 is abosrption spectrogram of the fluorescence probe in phosphate buffer；
Fig. 4 is the dynamics that fluorescence probe detects nitroreductase；
Fig. 5 for nitroreductase to fluorescence probe titer；
Fig. 6 is selectivity of the fluorescence probe in phosphate buffer；
Fig. 7 is single photon and the two-photon cell imaging application of fluorescence probe；
Fig. 8 is positioning of the fluorescence probe to endoplasmic reticulum；
Fig. 9 is the two-photon imaging of tissue application of fluorescence probe.
Embodiment
With reference to embodiment and accompanying drawing, the present invention will be further described, but the present invention is not limited by following embodiments
System；Compound number in example corresponds to the number in above-claimed cpd.
The synthesis of the fluorescence probe of embodiment 1.
In 25 mL round-bottomed flasks, to toluene under the conditions of 40 DEG C（5mL）In ethylenediamine is slowly added dropwise（Compound 1）1
Mmol, paratoluensulfonyl chloride is added in the case where being sufficiently stirred（Compound 2）1.0 mmol are cold after being heated to reflux the h of 3- 4
But to room temperature, 5ml 2M HCl/water solution is added into reaction, filters and removes filter residue, filtrate is neutralized to precipitation largely with NaOH
Untill white solid, decompression filters, and solid vacuum drying, obtains this sulphonyl of solid N-(2-amino-ethyl)-4-methyl
Amine（Compound 3）, yield：72 %.Product directly carries out next step reaction without purifying.
In 100 mL round-bottomed flasks, 243 mg 4- nitro naphthalic acid glycosides are added（Compound 4）（1 mmol）With 20
ML ethanol, stir 0.1 mL triethylamines of lower addition and 321 mg N-（2- amino-ethyls）- 4- methyl benzenesulfonamides（Compound 3）
（1.5 mmol）, after being then heated to reflux 6-8 hours, room temperature is cooled to, removes organic reagent under reduced pressure, solid residue is through column chromatography
Separation（Dichloromethane and methanol are eluent）, obtain light yellow solid 4- nitros-N-（2-（4- Methyl benzenesulfonyl amidos）Second
Base-naphthalimide（Compound 5）359 mg, yield 81.2%, its nucleus magnetic hydrogen spectrum are composed as depicted in figs. 1 and 2 with carbon.
1H NMR (400 MHz, CDCl3) δ 8.71 (dd, J 1 =8.7 Hz, J 2 =0.9 Hz, 1H), 8.60
(dd, J 1 =7.2 Hz, J 2 =0.8 Hz, 1H), 8.56 (m, 2H), 8.09 (td, J 1 =7.6 Hz, J 2 =1.3 Hz,
1H), 7.80 (t, J=5.8 Hz, 1H), 7.57 (d, J=8.2 Hz, 2H), 7.22 (d, J=8.2 Hz, 2H),
4.11 (t, J=6.4 Hz, 2H), 3.11 (q, J=6.4 Hz, 2H), 2.25 (s, 3H); 13C NMR (101
MHz, CDCl3) δ 163.48, 162.69, 149.60, 142.92, 138.16, 132.09, 130.55, 129.95,
129.17, 128.84, 127.12, 126.77, 124.65, 123.27, 123.14, 40.11, 21.30。
The fluorescence probe Na-ER-NTR absorption spectrums of embodiment 2 are tested
The fluorescence probe Na-ER-NTR in embodiment 1 is weighed, with dimethyl sulfoxide (DMSO)（DMSO）It is configured to 1 mM mother liquor.
Take 25 μ L probes mother liquors to add in 3 mL volumetric flasks of identical 5 respectively, add the μ L of DMSO solution 225, wherein
Two addition reduced Coenzyme Is（NADH, 300 μM of final concentration）.The two add one of addition nitro of the volumetric flask of coenzyme
The phosphate buffer solution of reductase（The μ g/mL of final concentration 2.0）, another is not added with.3 volumetric flasks use phosphate buffer（0.01
M PBS, pH=7.4）Constant volume, 37 DEG C of 60 min of effect, then carries out absorption spectrum test, using wavelength as abscissa, to inhale
Luminosity is that ordinate makees Fig. 2.As shown in Figure 2, the absorption intensity very little of independent probe, faint suction peak occurs at 360 nm.
After adding NADH, occur strong absworption peak at 340 nm, be absorbed as NADH absworption peak herein.Add nitroreductase
Afterwards, a small absworption peak gradually is generated at 440 nm, is herein the absorption after probe and nitroreductase interaction
Peak.
The fluorescence probe Na-ER-NTR of embodiment 3 and the kinetic test of nitroreductase effect.
It is standby that fluorescence probe mother liquor in embodiment 2 is diluted to 5 μM of probe face liquid.
Nitroreductase compound concentration is 0 by PBS using the concentration of pH=7.4 as 0.01 M, 0.5,1.0,1.5,2.5,
5.0 μ g/mL serial solution.
Probe face liquid is mixed with the nitroreductase of various concentrations respectively, final concentration of 300 μM of wherein NADH, then
Carry out fluoroscopic examination（λex =440 nm, λem = 545 nm）, every 3 min tests once, 80 min are tested, record each system
In the fluorescence intensity that changes over time, the curve that fluorescence intensity changes over time is established, as shown in figure 4, reaction system fluorescence is strong
Degree gradually enhancing, and leveled off to steadily in nearly 60 min, reaction system fluorescence intensity reaches saturation state.
Titration detection of the nitroreductase of the various concentrations of embodiment 4 to fluorescence probe Na-ER-NTR.
The middle probe working solution of Example 3 and the PBS solution containing 5 % DMSO solutions, respectively with the nitros of various concentrations also
Protoenzyme（0,0.1,0.2,0.5,1.0,1.5,2.0,2.5,3.0,4.0,5.0 μ g/mL）Fully 1 h of effect, enters at 37 DEG C
Row fluoroscopic examination（λex =440 nm, λem = 545 nm）, fluorescence intensity and nitroreductase concentration curve are established, such as Fig. 5 institutes
Show, with the increase of nitroreductase concentration, reaction system fluorescence intensity gradually increases.
Fluorescence probe Na-ER-NTR is to different ions and the selectivity of active small molecular for embodiment 5.
Dose volume is 5 mL, and concentration is 40 mM various different ions, active oxygen and active nitrogen solution as standby.
Probe mother liquor, 225 μ L DMSO, final concentration of 300 μ added in 5 mL volumetric flask in 25 μ L embodiments 2
The active oxygen that M NADH and concentration is 2.5 mM zwitterion, concentration is 100 μM；Concentration is 1 mM active sulfur；Concentration
For 100 μM of active nitrogen；Concentration is 2.0 μ g/mL nitroreductase, with phosphate buffer constant volume, phase at 37 DEG C after shaking up
Fluoroscopic examination is carried out after the h of interaction 1（λex =440 nm, λem = 545 nm）, establish the column of fluorescence intensity and each ion
Figure, is shown in Fig. 6, wherein, 1-22 is respectively：Probe Na-ER-NTR, calcium chloride, copper chloride, ferrous sulfate, magnesium chloride, zinc chloride,
Potassium fluoride, KI, potassium rhodanate, sodium bromide, natrium nitrosum, sodium nitrate, sodium acetate, sodium sulphate, NaHS, half Guang ammonia
Acid, glutathione, homocysteine, hydroxyl radical free radical, sodium hypochlorite, hydrogen peroxide, tertbutanol peroxide, nitro reduction
Enzyme.The concentration of test ion is 2.5 mM, and activity keto concentration is 100 μM, and active sulfur active nitrogen concentration is 1 mM, and active nitrogen is dense
Spend for 100 μM, nitroreductase concentration is 2.0 μ g/mL.By Fig. 6 it can be found that other in addition to nitroreductase are analyzed
Thing has little to no effect to the fluorescence of fluorescence probe.
The fluorescence probe Na-ER-NTR cell imagings of embodiment 6.
It is 3 × 10 by density5 Individual/mL HeLa cells are inoculated into the 35 mm imaging culture dishes of sterilizing, in CO2Training
Support case（Temperature is 37 DEG C, 5 % CO2）In after cell attachment, be divided into and being cultivated under different conditions in five groups of oxygen content：Temperature
For 37 DEG C, CO2Content is 5 %, and oxygen content is followed successively by 21 %, 15 %, 10 %, 5 %, 1 %, cultivates 12 h.Supported to five tissue cultures
The probe mother liquor added in ware in embodiment 2, it is 5 μM to make its final concentration.Continue to continue to cultivate respectively under the same conditions
0.5 h, then siphons away cell culture fluid, and cell 3 times is rinsed with PBS, shoot respectively the single photons of this five groups of cells/
Two-photon fluorescence image, one-photon excitation wavelength：488 nm, emission band：500 - 550 nm；Two-photon excitation wavelength：760
Nm, emission band：500 - 550 nm.As shown in fig. 7, wherein, (a-e) be Hela cells under different oxygen content light field into
Picture；(f-j) it is the single photon fluorescence imaging of Hela cells under different oxygen content；(k-o) it is Hela cells under different oxygen content
It is superimposed picture；(p-t) it is the two-photon fluorescence imaging of Hela cells under different oxygen content.As seen from Figure 7, under the conditions of weary oxygen
（The % of oxygen content 15,10 %, 5 %, 1 %）The fluorescence that the cancer cell of growth is sent is significantly stronger, and with the reduction of oxygen content, it is glimmering
Luminous intensity gradually strengthens.
The fluorescence probe Na-ER-NTR of embodiment 7 cellular localization imaging.
It is 3 × 10 by density5 Individual/mL HeLa cells are inoculated into the 35 mm imaging culture dishes of sterilizing, in CO2Training
Support case（Temperature is 37 DEG C, 5 % CO2）Middle culture, after cell attachment, cell is transferred to weary oxygen condition（Temperature is 37
DEG C, 5 % CO2, 1 % O2）Middle culture, cultivate 12 h.Then fluorescence probe mother liquor in embodiment 2 is added into culture dish, makes it
Final concentration of 5 μM.Continue to continue to cultivate 0.5 h respectively under the same conditions, then cell culture fluid is siphoned away, buffered with PBS
Liquid rinses cell 3 times, adds the red positioning dyestuff ER-Tracker Red of endoplasmic reticulum（Green skies biotech company）Continue
After being incubated 15 min, the fluoroscopic image of cell, excitation wavelength are shot：488 nm（Green channel）With 561 nm（Red channel）,
Emission band：500-550 nm and 625-675 nm, obtain Fig. 8, wherein, (a) is that the light field of Hela cells is imaged；(b) it is
Fluorescence imaging of the Hela cells in 500-550 nm green channels；(c) for Hela cells in 625-675 nm red channels
Fluorescence imaging；(d) the superposition picture of the red channel for Hela cells and green channel；(e) it is the common location of HeLa cells
Distribution map.It was found that the fluorescence probe is mainly enriched in the built-in online of cell, phase relationship number is 0.9.
The fluorescence probe Na-ER-NTR of embodiment 8 two-photon imaging of tissue test.
Prepare two mouse injection tumour cells（Mouse source breast cancer cell 4T-1）, mouse breast cancer tumour model is cultivated,
Solution takes tumour, and tumor biopsy is made.A part of tumor biopsy is immersed in tissue culture medium, CO2Incubator（Temperature is
37 DEG C, 5 % CO2）Cultivate；Another part tumor biopsy is immersed in containing the tissue culture medium that concentration is 5 μM of probe solutions
In, CO2Incubator（Temperature is 37 DEG C, 5 % CO2）Cultivate.After half an hour, tissue culture medium is siphoned away, rushed with PBS
Wash 3 times, by two groups of tumor tissues in two-photon condition（Excitation wavelength：760 nm, emission band：500 - 550 nm）Lower difference
Shoot 0-100nm depth fluoroscopic images, obtain Fig. 9, wherein, a) be immersed in tissue culture medium tumour two-photon fluorescence into
Picture；B) to be immersed in tumour two-photon fluorescence imaging in the tissue culture medium containing 20 μM of probe solutions.As shown in Figure 9, exist
It is significantly stronger that the fluorescence that the tissue soaked is sent is added in the nutrient solution of probe.
Claims (10)
1. a kind of two-photon nitroreductase fluorescence probe of endoplasmic reticulum targeting, its chemical name are：4- nitros-N-（2-（4- first
Base benzene sulfonamido）Second）Base-naphthalimide, its chemical structural formula such as formula（）It is shown：
Formula (I).
2. a kind of synthetic method of fluorescence probe as claimed in claim 1, it is characterised in that comprise the following steps：
（1）Ethylenediamine is dissolved in solvent, and low temperature stirring is lower to add paratoluensulfonyl chloride, is then warmed to room temperature stirring and extremely reacts complete,
Isolated N-（2- amino-ethyls）This sulfonamide of -4- methyl；
（2）4- nitro-1,8-naphthalic acid acid anhydrides are dissolved in solvent, in the presence of triethylamine, stir lower addition N-（2- amino second
Base）- 4- methyl benzenesulfonamides, then heating stirring backflow, separating-purifying obtains compound 4- nitros-N- after cooling（2-（4- first
Base benzene sulfonamido）Ethyl-naphthalimide.
3. synthetic method according to claim 2, it is characterised in that step（1）In, the ethylenediamine with to toluene sulphur
The mol ratio of acyl chlorides is 1:1-1.5；The concentration of ethylenediamine is the mol/L of 0.2- 0.3；The low temperature is 0-4 DEG C.
4. synthetic method according to claim 2, it is characterised in that step（1）In, separating step is：To reaction system
Middle addition HCl/water solution, suction filtration remove filter residue, and filtrate is neutralized to untill separating out a large amount of white solids with alkali lye, filtered solid
Body is dried in vacuo.
5. synthetic method according to claim 4, it is characterised in that HCl concentration is 2 mol/L；The alkali lye is
10%wt NaOH solutions.
6. synthetic method according to claim 2, it is characterised in that step（2）In, 4- nitro-1,8-naphthalic acid acid anhydrides
With N-（2- amino-ethyls）The mol ratio of -4- methyl benzenesulfonamides is 1:1.2-1.5；4- nitro -1,8- naphthalic anhydrides it is dense
Spend for 0.05-0.06 mol/L；Reaction temperature is 80-85 DEG C.
7. synthetic method according to claim 2, it is characterised in that step（2）In, separating-purifying step is：Decompression is steamed
Except organic reagent, solid residue is eluent through pillar layer separation using dichloromethane and methanol；The volume of dichloromethane and methanol
Than for 20-10:1.
8. a kind of application of fluorescence probe Nitroreductase Activity in solution is detected as claimed in claim 1, its feature exist
In excitation wavelength is 440 nm, and launch wavelength is 545 nm, after detection time is effect 30-80min.
9. a kind of application of fluorescence probe as claimed in claim 1 Nitroreductase Activity in detection organism, its feature
It is, one-photon excitation wavelength is 488 nm, and emission band is 500-550 nm；Two-photon excitation wavelength is 760 nm, hair
Ejected wave section is 500-550 nm；After detection time is effect 30-80 min.
10. according to any described application of claim 8 or 9, it is characterised in that after detection time is effect 60min.
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| CN110669821A (en) * | 2019-10-28 | 2020-01-10 | 郑州大学 | Preparation method and application of two-photon deoxyribozyme metal organic framework probe |
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| CN112472822A (en) * | 2020-12-02 | 2021-03-12 | 浙江大学 | Construction and application of endoplasmic reticulum targeted nano drug delivery system |
| CN113354583A (en) * | 2021-06-15 | 2021-09-07 | 上海大学 | Fluorescent probe for detecting hypoxic level, preparation method and application thereof |
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