CN102757659B - Carbazole hemicyanine fluorescent dye and application thereof - Google Patents
Carbazole hemicyanine fluorescent dye and application thereof Download PDFInfo
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Abstract
The invention relates to a carbazole hemicyanine fluorescent dye and application thereof. The carbazole hemicyanine fluorescent dye has a structural general formula I, in which R1 and R2 are independently slected from hydrogen and C1-6 alkyl, C1-6 hydroxy instead of alkyl and C1-6 alkoxy; R3 is H or a formaldehyde radical; and Y is a halogen negative ion. The dye provided by the invention has sensitive response to the change of environment viscosity, the influence on the fluorescent property caused by solvent polarities is very little, and the dye has a two-photon property and can detect the environment viscosity by a ratio method. Therefore, the dye provided by the invention can be used for detecting the change of microenvironment viscosity of cells and tissues.
Description
Technical field
The present invention relates to a class based on carbazoles half cyanine dye, and this fluorescence dye is detected to the method for microenvironment viscosity in viable cell as the fluorescent probe of viscosity sensitivity.
Background technology
Viscosity is to weigh a kind of mobility and diffusible principal element of dense fluid, is the main reference index of diffuse fluid speed simultaneously.For the viscous fluid of macroscopical large volume, the method for mensuration fluid viscosity and the comparative maturity that instrument has developed.To micro, such as the viscosimetric analysis of tissue, cell levels, existing method often can not reach the object of test.And the viscosity of microenvironment has very important significance in Accurate Determining cell levels.
In cell, viscosity influence the transmission of material and signal, the interaction between biomacromolecule, and the diffusion of active metabolite (as ROS and RNS).The viscosity of normal cell endomembrane system microenvironment is the highest can reach 140cp (centipoise), and viscosity in tenuigenin is only 1 ~ 2cp, close with the viscosity in pure water; But under the condition of pathology, cell is apoptosis gradually, cause macromole coupling to be wound around and cause intracellular viscosity to increase greatly, the highest 300cp that may reach, can cause the generation of numerous disease.Therefore in living things system micro, most important to the accurate measurement of viscosity.The method of existing tested viscosity, as capillary viscosimeter, falling-sphere viscometer, rotational viscosimeters etc. all can not provide effective viscosity measurements on cell levels.And the small molecules fluorescent optical sensor with permeability of cell membrane effectively the small viscosity in reacting cells change, the visual small molecules fluorescent probe of therefore developing excellent property has great significance.
At present in the world the research of viscosity probe has been obtained to some outstanding achievements, but still in development initial stage.Take the previous work of this group as representative, developed a routine Cy5 derivative probe, and by fluorescence strengthen, the method for ratio fluorescence and life-span fluorescence imaging realized the mensuration to viable cell and sick cell viscosity preferably.But the synthetic work amount of this probe is larger, and it is good to meet spectrum property in medical diagnosis and bioluminescence imaging field still urgent demand simultaneously, has cell permeability, synthetic simple, is easy to the viscosity probe of industrialization.
Recently, Two Photon Fluorescence (TPM), due to its advantage unique in bio-imaging, has become and in biology laboratory, has become indispensable instrument.Compare traditional fluorescent microscope, TPM has increased the infiltration of sample, has reduced the interference of background fluorescence.TPM adopts two near infrared photons as excitaton source, and penetration depth increases (>500 micron).Due to exciting of long wavelength, it has avoided the background fluorescence in organism effectively.Although single photon microscope (OPM) should be many through what apply in viscosity measurements, as far as we know, report is not used for measuring viscosity by the method for TPM.
Carbazoles hemicyanine dye, owing to having multiple distortions site in its structure, in low viscous solution, fluorescence quantum yield is very low, and two-photon absorption interface is also smaller.Although the increase of viscosity, dye property is changed greatly.And up to the present also do not have bibliographical information, carbazoles hemicyanine dye to be used for being designed to the molecule rotor of environment viscosity sensitivity to detect the viscosity of solution or intracellular environment.
Summary of the invention
On the basis of existing technology, the present invention aims to provide a kind of novel carbazole class hemicyanine dye, this dyestuff should have very sensitive response to the variation of environment viscosity, fluorescence property is subject to the impact of solvent polarity very little, and itself has two-phpton property dyestuff, and can carry out testing environment viscosity by ratio approach.
Therefore, first the present invention provides class carbazoles half cyanine dye, has general structure I:
In formula I:
R
1and R
2be selected from independently of one another hydrogen, C
1-6alkyl, C
1-6hydroxyl substituted alkyl and C
1-6alkoxyl group;
R
3h or carboxaldehyde radicals;
Y
-for halogen anion.
In carbazoles half cyanine dye of general formula I of the present invention, on its precursor structure, substituent effect is to regulate the solvability of dyestuff in organic solvent or the aqueous solution, or the ability of cell transmembrane.In a specific embodiments of the present invention, the R in general formula I
1and R
2be selected from independently of one another C
1-6alkyl; Preferably C
1-3alkyl; Most preferably ethyl.
The present invention is again in a specific embodiments, described Y
-for Cl
-, Br
-or I
-; Most preferably I
-.
In the most preferred embodiment, carbazoles half cyanine dye of the present invention is selected from dyestuff Qcaz and dyestuff Hcaz.
The object of further aspect of the present invention is the preparation method of carbazoles half cyanine dye that the invention described above is provided, and comprises the steps:
(1) by 2,3,3-trimethylammonium-3H-indoles and R
2cH
2y reacts and within 6 ~ 24 hours, prepares quaternary ammonium salt II under 80～148 ℃ of conditions according to mol ratio 1:1 ~ 1:3, and reaction solvent is selected from toluene, orthodichlorobenzene, ethanol and acetonitrile;
(2) carbazole and XCH
2r
1under 25 ~ 50 ℃ of conditions, react 2 ~ 5 hours preparation intermediate compound III according to mol ratio 1:1 ~ 1:4, wherein X is halogen;
(3) compound III is reacted and is prepared midbody compound IV or V with trichlorine phosphine oxide in DMF solvent, wherein:
A. compound III is reacted and within 10 ~ 15 hours, is prepared midbody compound IV according to mol ratio 1:1.5 ~ 1:3 with phosphorus oxychloride under 80 ~ 95 ℃ of conditions;
B. compound III is reacted and within 15 ~ 24 hours, is prepared midbody compound V according to mol ratio 1:3 ~ 1:8 with phosphorus oxychloride under 90 ~ 110 ℃ of conditions;
(4) compound IV that the quaternary ammonium salt II that step (1) makes makes with step (3) or V react preparation Compound I under pyridine catalysis, and reaction solvent is ethanol, wherein:
A. quaternary ammonium salt II reacts 10 ~ 20 hours preparations Compound I, wherein R under 75 ~ 85 ℃ of conditions according to mol ratio 1.2:1 with compound IV
3for hydrogen;
B. quaternary ammonium salt II reacts 0.1 ~ 3 hour preparation Compound I, wherein R under 60 ~ 70 ℃ of conditions according to mol ratio 3:1 with compound V
3for carboxaldehyde radicals.
Carbazoles half cyanine dye disclosed in this invention has biabsorption and emission peak; The fluorescence property of this compounds is very responsive to soltion viscosity, and along with the increase of solvent viscosity, the emissive porwer of dyestuff increases sharply, and meets
– Hoffmann relational expression, is linear change in the ratio value of intensity and the logarithmic value of viscosity at each emission peak place, also meets
– Hoffmann relational expression, meets the performance of two-photon, and can use method test compounds solution environmental of living in or the bioenvironmental viscosity of ratio, and adoption rate imaging method detects the variation of viscosity in cell whereby.Based on this, still a further object of the present invention provides carbazoles half cyanine dye of the present invention in the application detecting in cell and the variation of tissue microenvironment viscosity.
Accompanying drawing explanation
Accompanying drawing 18 width of the present invention:
Fig. 1 compound Q caz(1 μ M) long wavelength's fluorescence quantum yield in different solvents.Wherein (a) methylene dichloride, (b) methyl alcohol, (c) ethanol, (d) water, (e) DMSO, (f) dioxane, (g) glycerine: water=2:8, (h) glycerine: water=4:6, (i) glycerine: water=6:4, (j) glycerine: water=8:2, (k) glycerine.
Fig. 2 compound H caz(1 μ M) long wavelength's fluorescence quantum yield in different solvents.Wherein (a) methylene dichloride, (b) methyl alcohol, (c) ethanol, (d) water, (e) DMSO, (f) dioxane, (g) glycerine: water=2:8, (h) glycerine: water=4:6, (i) glycerine: water=6:4, (j) glycerine: water=8:2, (k) glycerine.
Fig. 3 compound Q caz(1 μ M) variation (λ of fluorescence spectrum in different viscosity solution
ex=375nm and 575nm).
Fig. 3 a be 340nm as exciting light, demonstration be compound Q caz(1 μ M) two emission band along with viscosity strengthen emission peak strengthen variation;
Fig. 3 b be 480nm as exciting light, demonstration be compound Q caz(1 μ M) variation that strengthens along with the increase emission peak of viscosity of long-wave band.
Fig. 4 compound H caz(1 μ M) variation (λ of fluorescence spectrum in different viscosity solution
ex=380nm and 580nm)
Fig. 4 a be 340nm as exciting light, demonstration be compound H caz(1 μ M) two emission band along with viscosity strengthen emission peak strengthen variation;
Fig. 4 b be 480nm as exciting light, demonstration be Hcaz(1 μ M) variation that strengthens along with the increase emission peak of viscosity of long-wave band.
The linear relationship of Fig. 5 compound Q caz between logarithm and the viscosity logarithm of two emission peak intensity place ratios.
The linear relationship of Fig. 6 compound H caz between logarithm and the viscosity logarithm of two emission peak intensity place ratios.
Fig. 7 compound Q caz(0.1mmol) in glycerine, although the increase of temperature, glycerine reduced viscosity, dye strength reduces gradually.
Fig. 8 compound H caz(0.1mmol) in glycerine, although the increase of temperature, glycerine reduced viscosity, dye strength reduces gradually.
Fig. 9 compound Q caz(0.1mmol) along with the increase of viscosity, the variation of dyestuff two-photon absorption boundary strength.
Figure 10 compound H caz(0.1mmol) along with the increase of viscosity, the variation of dyestuff two-photon absorption boundary strength.
Figure 11 compound Q caz(0.1mmol) in add different biomacromolecule fluorescence intensity changes: 1 parent dyestuff initial strength, 2 is halfcystine, 3 is bovine serum albumin, 4 is gsh, 5 is thymus nucleic acid, 6 is intensity in glycerine.
Figure 12 compound H caz(0.1mmol) in add different biomacromolecule fluorescence intensity changes: 1 parent dyestuff initial strength, 2 is halfcystine, 3 is bovine serum albumin, 4 is gsh, 5 is thymus nucleic acid, 6 is intensity in glycerine.
Figure 13 compound Q caz(5 μ M) fluorescence imaging in HeLa cell, Leica laser confocal fluorescence microscope × 100objective lens, excitation wavelength 800nm, wherein:
Figure 13 a is green channel fluorescence picture;
Figure 13 b is red channel fluorescence picture;
Figure 13 c is the Fluorescence Ratio picture of Figure 13 b and Figure 13 a.
Figure 14 compound Q caz(5 μ M) fluorescence imaging in HeLa cell, Leica laser confocal fluorescence microscope × 100objective lens, excitation wavelength 800nm, wherein:
Figure 14 a is green channel fluorescence picture;
Figure 14 b is red channel fluorescence picture;
Figure 14 c is the Fluorescence Ratio picture of Figure 14 b and Figure 14 a.
Figure 15 compound H caz(8 μ M) excite with 488nm, the single photon long wavelength fluorescence imaging in dead cell HeLa, collects POP scope and is: 555-595nm.
Figure 15 a is the fluorescence imaging of compound H caz in dead cell HeLa; Figure 15 b is the white light figure of compound H caz in dead HeLa.
Figure 16 compound H caz(8 μ M) excite with 488nm, the single photon long wavelength fluorescence imaging in HeLa cell, collects POP scope and is: 570-610nm.
Figure 16 a is the fluorescence imaging of compound H caz in viable cell HeLa; Figure 16 b is that compound H caz is at the white light figure living in HeLa.
Figure 17 compound Q caz(8 μ M) excite with 488nm, the single photon long wavelength fluorescence imaging in HeLa cell, collects POP scope and is: 555-595nm.
Figure 17 a is the fluorescence imaging of compound Q caz in viable cell HeLa; Figure 17 b is that compound Q caz is at the white light figure living in HeLa.
Figure 18 compound Q caz(8 μ M) excite with 488nm, the single photon long wavelength fluorescence imaging in dead cell HeLa, collects POP scope and is: 555-595nm.
Figure 18 a is the fluorescence imaging of compound Q caz in dead cell HeLa; Figure 18 b is the white light figure of compound Q caz in dead HeLa.
Embodiment
The following examples can make the present invention of those of ordinary skill in the art's comprehend, but do not limit the present invention in any way.
Compound H caz's is synthetic
The resultant current method of compound H caz is as follows:
(1) 2,3,3-trimethylammonium-3H-indoline (compound 1) synthetic according to fisher indoles synthetic method:
Weigh phenylhydrazine 54g(0.5mol) join in 250mL two-mouth bottle, stir lower 3-methyl-2-butanone 43g(0.5mol that slowly drips) be heated to 70 ~ 80 ℃, react 4 hours, branch vibration layer, water layer extracted with diethyl ether, with anhydrous magnesium sulfate drying filters after merging with ether layer, and decompression steams solvent, obtain rough hydrazone 70g, yield 80%.
By hydrazone 70g(0.4mol rough upper step) mix with 150mL Glacial acetic acid, in 90 ℃ of oil baths, react 3 hours, be cooled to room temperature, with extremely neutral with water layer in saturated aqueous sodium carbonate, water phase separated and organic phase, water extracted with diethyl ether, extraction liquid and organic phase merge, anhydrous sodium sulfate drying steams ether after filtering, then underpressure distillation, collects the cut of 130 ~ 140 ℃ of (0.08 ~ 0.09Mp) boiling ranges.Product is monodromy look oily liquids 52g(yield 82%).
(2) iodate N-ethyl-2,3,3-trimethylammonium-3H-indoline quaternary ammonium salt (compound 2) synthetic
By 3.2g(20mmol) 2; 3; 3-trimethylammonium-3H-indoline and 4.7g iodoethane are mixed in 100mL round-bottomed flask; add about 30mL toluene; reflux 7 hours under nitrogen protection; stop heating and be cooled to room temperature, filter the solid generating, wash and obtain pink solid quaternary ammonium salt 5.4g(yield 86% with ether).
(3) N-ethyl carbazole (compound 3)
With analytical balance accurately take carbazole (5.0g, 30mmol), sodium hydride (0.25g, 54mmol) is placed in the single port flask that 100mL is dry; and add iodoethane (5.1g; 33mmol) with 30mL DMF, the protection of vacuum nitrogen filling gas, back flow reaction 24h.After reaction finishes, solution is poured into water, solid is separated out, and filters, and then ethanol (2 × 300mL) recrystallization for excess, obtains white crystal, productive rate 76% (4.5g).
1H-NMR(400MHz,CDCl
3):1.31(t,3H,CH
3,J=13.2Hz),4.22(bp,2H,CH
2，J=7.2Hz),7.19(bp,2H,ArH,J=7.2Hz),7.31(d,2H,ArH,J=8.0Hz),7.41(bp,2H,ArH,J=8.0Hz),8.05(d,2H,ArH,J=8.0Hz)
(4) N-ethyl-3-aldehyde radical carbazole (compound 4) is synthetic:
Transfer pipet accurately measures the DMF that 9mL is dry and is placed in the single port flask that 100mL is dry, and single port flask is placed in to ice-water bath, and 5mL phosphorus oxychloride is splashed into single port flask.Analytical balance accurately takes N-ethyl carbazole (2.8g, 14.3mmol) and is dissolved in 40mL chlorobenzene and is placed in constant pressure funnel, loads onto constant pressure funnel.After 0.5h, the chlorobenzene solution of N-ethyl carbazole is slowly splashed into single port flask, stirring reaction 11h in the oil bath of 80 ~ 90 ℃.After completion of the reaction, cooling, adjust pH to neutral with 10% sodium hydrogen carbonate solution, with chloroform extraction, then use anhydrous sodium sulfate drying, solvent is sloughed in decompression, and dehydrated alcohol (2 × 200mL) recrystallization, obtains white powder 2, productive rate 80% (2.17g)
1H-NMR(400MHz,CDCl
3):1.34(t,3H,CH
3,J=8.0Hz),4.2(bp,2H,CH
2，J=8.0Hz),7.23(bp,H,ArH,J=6.8Hz),7.315(bp,2H,ArH),7.88(d,H,ArH,J=6.8Hz),8.02(d,H,ArH,J=8.0Hz),8.44(d,H,ArH),9.99(s,H,CHO)
(5) 3-[2-(1-N-ethyl-3,3-dimethyl-2-3 hydrogen-indoline base) vinyl]-9-ethyl-carbazole quaternary ammonium salt hemicyanine dye (compound H caz) synthetic:
Accurately weigh midbody compound 4(1.0g, 2mmol) and 2(0.75g, 3.0mmol), be placed in 100mL single necked round bottom flask, add 40mL ethanol, then add 0.5mL piperidines as catalyzer, stirring reaction 8h in the oil bath of 80 ℃.Solvent evaporated after completion of the reaction, obtains red oily liquids.Silica gel column chromatography is purified, and elutriant is methylene chloride/methanol (20:1), and obtaining product is brown color crystal, productive rate 64%(0.96g).
1H-NMR(400MHz,CDCl
3):1.43(t,3H,CH
3,J=8.0Hz),1.62(t,3H,CH
3,J=7.6Hz),1.85(s,6H,CH
3),4.33(bp,2H,CH
2,J=8.0Hz),5.0(bp,2H,CH
2,J=7.6Hz),7.33(t,H,ArH，J=6.0Hz),7.42(t,2H,ArH,J=8.2Hz),7.51(bp,5H,ArH),7.89(d,H,CH，J=12.4Hz),8.39(d,H,CH,J=12.4Hz),8.46(d,H,ArH,J=8.2Hz),8.52(d,H,ArH,J=8.2Hz),9.07(s,H,ArH)
Implement 2
Compound Q caz's is synthetic
The synthetic method of compound Q caz is as follows:
(1) N-ethyl-3,6-dialdehyde-based carbazole (compound 5) synthetic:
Transfer pipet accurately measures the DMF that 18mL is dry and is placed in the single port flask that 100mL is dry, and single port flask is placed in to ice-water bath, and 15mL phosphorus oxychloride is splashed into single port flask.Analytical balance accurately takes N-ethyl carbazole (2.8g, 14.3mmol) and is dissolved in 40mL chlorobenzene and is placed in constant pressure funnel, loads onto constant pressure funnel.After 0.5h, the chlorobenzene solution of N-ethyl carbazole is slowly splashed into single port flask, stirring reaction 60h in the oil bath of 90 ~ 95 ℃.After completion of the reaction, cooling, adjust pH to neutral with 10% sodium hydrogen carbonate solution, with chloroform extraction, then use anhydrous sodium sulfate drying, solvent is sloughed in decompression, and dehydrated alcohol (2 × 200mL) recrystallization, obtains white powder 5, productive rate 90% (3.17g)
1H-NMR(400MHz,CDCl
3):1.34(t,3H,CH
3,J=8.0Hz),4.2(bp,2H,CH
2，J=8.0Hz),7.23(bp,H,ArH,J=6.8Hz),7.315(bp,2H,ArH),7.88(d,H,ArH,J=6.8Hz),8.02(d,H,ArH,J=8.0Hz),8.44(d,H,ArH),9.99(s,H,CHO)
(2) 3-[2-(1-N-ethyl-3,3-dimethyl-2-3 hydrogen-indoline base) vinyl]-6-aldehyde radical-9-ethyl-carbazole quaternary ammonium salt hemicyanine dye (compound Q caz) synthetic:
Accurately weigh midbody compound 5(1.1g, 2mmol) and 2(0.375g, 1.5mmol), be placed in 100mL single necked round bottom flask, add 40mL ethanol, then add 0.5mL piperidines as catalyzer, stirring reaction 4h in the oil bath of 75 ℃.Solvent evaporated after completion of the reaction, obtains red oily liquids.Silica gel column chromatography is purified, and elutriant is methylene chloride/methanol (18:1), and obtaining product is brown color crystal, productive rate 45%(0.54g).
1H-NMR(400MHz,CDCl
3):1.43(t,3H,CH
3,J=8.0Hz),1.69(t,3H,CH
3,J=7.6Hz),1.86(s,6H,CH
3),4.35(bp,2H,CH
2,J=8.0Hz),5.11(bp,2H,CH
2,J=7.6Hz),7.45(t,H,ArH，J=6.0Hz),7.53(bp,2H,ArH,J=8.2Hz),7.58(bp,3H,ArH),8.2(d,H,ArH,J=7.6Hz),8.05(d,H,CH，J=12.4Hz),8.39(d,H,CH,J=12.4Hz),8.43(d,H,ArH),9.17(s,H,ArH),9.35(s,H,ArH),10.12(s,H,CHO)
13C-NMR(100MHz,CDCl
3):13.95,14.46,18.42,27.42,30.93,38.60,43,73,52.03,58.37,109.67,110.7,114.0,122.7,123.17,124.32,126.32,127.68,129.32,131.65,140.42,143.22,144.04,156.78,180.68,191.95,207.03
HRMS-ESI:m/z?calcd?M
+for?C29H29N2O
+,421.2274;found,421.2285
The viscosity test of compound Q caz and Hcaz
Compound concentration is 1 × 10
-3the compound Q caz of M and the DMSO solution of Hcaz, accurate measuring 10 these solution of μ L join in 10mL Glycerine-Aqueous Solution respectively, ultrasonic 10 minutes, remove after bubble and leave standstill 1 hour, on uv-spectrophotometric instrument and fluorophotometric instrument, measure its Absorption and emission spectra.Selected excitation wavelength is 330nm.Instrument is ultraviolet-visible pectrophotometer, model: Hp8453; Spectrophotofluorometer, model: FP-6500, fluorescence lifetime tester: Horiba Jobin Yvon Fluoromax-4p.The excitaton source of two-photon fluorescence excitation spectrum is a stand lock mould femto second titanium sapphire excitor, and fluoroscopic examination utilizes Lycra Laser Scanning Confocal Microscope fluorescence detecting system to detect.
Wherein, glycerine-ethanolic soln comprises glycerine, ethanol and V
glycerine: V
waterrespectively 1:9,2:8,3:7,4:6,5:5,6:4,7:3,8:2, the two-phase mixing solutions of 9:1.
When the above-mentioned solution preparing leaves standstill for some time at a certain temperature after bubble is eliminated, on ultra-violet absorption spectrum instrument and luminoscope, test, calculate dyestuff in different solvents and under the various volume ratios fluorescence quantum yield of compound is (as Fig. 1,2), from figure, can know that the fluorescence quantum yield of dyestuff in different types of low viscosity solvent changes little, illustrate that dyestuff is insensitive to the polarity of solvent.But, in the time increasing gradually the viscosity of solvent, the ultra-violet absorption spectrum of dyestuff also changes not quite, but the intensity of fluorescent emission is but along with the increase of viscosity obviously increases, take compound Q caz as example, in the time that 330nm place excites, dyestuff strengthens 5 times in the fluorescence intensity of 375nm place emission peak, strengthens 50 times (as Fig. 3) in the fluorescence intensity of 575nm place emission peak; The logarithm of fluorescence intensity ratio at the fluorescence intensity at 575nm place and 375nm place and the logarithm of solvent viscosity have fine linear relationship (as Fig. 5), can be used for the viscosity of detection of biological environment (in cell).Meanwhile, we have tested the impact of temperature on dyestuff.Fluorescence intensity is along with the variation meeting of envrionment temperature changes (as Fig. 7) accordingly, dyestuff is in glycerine, in the time that from 1 to 49 ° of C of envrionment temperature changes, dyestuff all reduces in the emissive porwer of long wavelength and shortwave strong point, but the fluorescence intensity change of long wave strong point is more obvious, this is identical with the ratio test result that changes water and glycerine, illustrates that the change of temperature has caused the variation of viscosity, occurs identical fluorescent appear.The test result of compound H caz and compound Q caz phenomenon are similar.When 330nm place excites, dyestuff strengthens 7 times in the fluorescence intensity of 380nm place emission peak, in the emission peak fluorescence intensity at 580nm place up to 40 times (as Fig. 4); The logarithm of fluorescence intensity ratio at the fluorescence intensity at 580nm place and 380nm place and the logarithm of solvent viscosity have fine linear relationship (as Fig. 6), and fluorescence intensity is along with the variation meeting of envrionment temperature changes (as Fig. 8) accordingly.
Utilize the scope of two-photon excitation wavelength from 720 to 920nm, tested the two-photon absorption interface (Φ δ) of dyestuff Qcaz and Hcaz.We can see, along with the increase of viscosity, two-photon absorption interface strengthens.Obtain the graph of a relation (Fig. 9,10) of two photon absorption cross section with two-photon excitation wavelength change according to test result.In the time increasing gradually the viscosity of solvent, the two-photon absorption interface of dyestuff increases gradually.At 720nm place, the two photon absorption cross section of Qcaz is along with the increase of viscosity is also to become gradually large, from the low viscous 10GM of 60% glycerine, increases viscosity in 99.9% glycerine time, and sectional area increases to 74GM.The two photon absorption cross section of Hcaz, from low viscous 5GM, is strengthened to 82GM.Find out thus, dyestuff is under high viscosity condition, and two-photon performance increases greatly.Thus, we can utilize two-photon long-wavelength excitation, the advantage of short wavelength's transmitting, reduce the interference of the spontaneous background fluorescence of organism, two emission ratios methods of combination dye, can well be applied to and in two-photon microcytoscope, carry out cell imaging experiment, the viscosity in sensitiveer detection cell biological body.
In living things system, the viscosity of (as cell) has very important significance to vital movement, and therefore the viscosity of detection of biological system (in cell) is a very important job.Due to the complicacy of intracellular environment, the biomacromolecule that cell contains, as the fluorescence property that protein, DNA etc. may affect probe, is caused interference to detected result, so need to investigate dyestuff and these macromolecular effects (as Figure 11,12).From test result, we can find out, protein, amino acid and DNA can ignore the impact of dyestuff, can think that this kind of dyestuff can be as the viscosity research of microenvironment in cell.
The cell experiment of compound Q caz and Hcaz
Under laser confocal scanning microscope, observe the dyeing of compound to viable cell HeLa:
Add respectively that to be furnished with compound Q caz and compound H caz concentration be that the PBS damping fluid 12 μ L of 5 μ M are in having cultivated six orifice plates of HeLa cell, at 37 ° of C, 5%CO
2cell culture incubator in hatch 30min.Then, PBS shakes rinsing 5min × 3, then adds cell culture medium, laser confocal scanning microscope (Leica, TCS-SP2, Germany) observation of cell form.Choose representative area, with oily mirror (100 ×) observation, in triplicate.
Under laser confocal scanning microscope, observe the dyeing of compound to viable cell HeLa:
Dyestuff of the present invention has the permeability of cytolemma, and small molecules dyestuff has membrane perviousness.After entering in cell, probe distributes at cell different positions, fluorescence intensity enhancing in various degree, and experimental result is shown in accompanying drawing 13, the intracellular imaging results of HeLa that compound Q caz shows under two-photon laser Laser Scanning Confocal Microscope, excitation wavelength 800nm.Lay respectively at 375nm and 575nm because dyestuff has two groups of emission wavelengths, so just adopt Dual channel detection, short wavelength's passage get (375 ± 20) nm blue channel (as Figure 13 a); Long wavelength be the red channel of getting (575 ± 20) nm (as Figure 13 b), and the fluorescence intensity of two passages is done after ratio, obtain compound Q caz the intracellular ratio image of HeLa (as Figure 13 c), can very clearly find out according to picture, in cell, the distribution of different viscosity position, reaches the object to viscosity profile ratio imaging in cell.In scale map, in cell, pseudo-colours is mainly green and blue portion, can know that dye fluorescence ratio is little, and intracellular viscosity is low.Only the color of a part reaches even redness of yellow, illustrates that in cell, viscosity is high at these location comparisons.In the cell tests of compound H caz, experimental result is shown in accompanying drawing 14, excitation wavelength 800nm.Lay respectively at 380nm and 575nm because dyestuff has two groups of emission wavelengths, so just adopt Dual channel detection, short wavelength's passage get (380 ± 20) nm blue channel (as Figure 14 a); Long wavelength be the red channel of getting (580 ± 20) nm (as Figure 14 b), both stack picture pinkiness (as Figure 14 c), and the fluorescence intensity of two passages is done after ratio, obtain compound H caz the intracellular ratio image of HeLa (as Figure 14 d).
Dyestuff substantive dyeing:
The Hela cell that can go down to posterity is inoculated in 6 orifice plates, and 37 ℃, 5%CO
2under condition, cultivate 24 hours, add dyestuff, final concentration is 8 μ M, hatches 30min for 37 ℃, sops up substratum, and PBS washes 2 times, adds 1200 μ L fresh cultures, and 488nm excites according to fluorescence.
Ethanol is fixed: the Hela cell that can go down to posterity is inoculated in 6 orifice plates, and 37 ℃, 5%CO
2under condition, cultivate 24 hours, sop up substratum, wash 2 times with PBS, add appropriate 70% ethanol, hatch 30min for 37 ℃, sop up fixingly with ethanol, wash 2 times with PBS, add 1200 μ LPBS, add dyestuff, final concentration is 8 μ M, hatches 30min for 37 ℃, and 488nm excites according to fluorescence.
The further single photon fluorescence imaging picture of compound Q caz and Hcaz, dyestuff Qcaz(Figure 15, shown in 16) and dyestuff Hcaz can find out (Figure 17, shown in 18), for viable cell Figure 16, in 17, under identical test condition, fluorescence a little less than, show that intracellular ratio of viscosities is lower, intracellular viscosity maximum is about 100cP.After unicellular being fixed, (shown in 15,18) cell is determined death, and the intracellular fluorescence of observing obviously increases, and illustrates that k value raises, and k value maximum is more than 300cP.
Claims (1)
1.
oneclass carbazoles half cyanine dye, has general structure I:
In formula I:
R
1and R
2be selected from independently of one another C
1-6alkyl, C
1-6hydroxyl substituted alkyl and C
1-6alkoxyl group;
R
3it is carboxaldehyde radicals;
Y
-for halogen anion.
2. carbazoles half cyanine dye claimed in claim 1, is characterized in that described R
1and R
2be selected from independently of one another C
1-6alkyl.
3. carbazoles half cyanine dye claimed in claim 1, is characterized in that described R
1and R
2be selected from independently of one another C
1-3alkyl.
4. carbazoles half cyanine dye claimed in claim 1, is characterized in that described R
1and R
2it is ethyl.
5. carbazoles half cyanine dye claimed in claim 1, is characterized in that described Y
-for Cl
-, Br
-or I
-.
6. the preparation method of carbazoles half cyanine dye claimed in claim 1, comprises the steps:
(1) by 2,3,3-trimethylammonium-3H-indoles and R
2cH
2y reacts and within 6 ~ 24 hours, prepares quaternary ammonium salt II under 80 ~ 148 ℃ of conditions according to mol ratio 1:1 ~ 1:3, and reaction solvent is selected from toluene, orthodichlorobenzene, ethanol and acetonitrile;
(2) carbazole and XCH
2r
1under 25 ~ 50 ℃ of conditions, react 2 ~ 5 hours preparation intermediate compound III according to mol ratio 1:1 ~ 1:4, wherein X is halogen;
(3) compound III is reacted and within 15 ~ 24 hours, is prepared midbody compound V according to mol ratio 1:3 ~ 1:8 with phosphorus oxychloride under 90 ~ 110 ℃ of conditions;
(4), under pyridine catalysis, take ethanol as reaction solvent, quaternary ammonium salt II reacts 0.1 ~ 3 hour preparation Compound I under 60 ~ 70 ℃ of conditions according to mol ratio 3:1 with compound V.
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| CN104629754B (en) * | 2015-01-30 | 2016-08-03 | 山西大学 | Carbazoles Ratio-type pH fluorescent probe and its preparation method and application |
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| CN108484590B (en) * | 2018-05-18 | 2021-02-26 | 安徽大学 | Carbazole-based two-photon viscosity fluorescent probe and preparation method and application thereof |
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| CN111943948B (en) * | 2020-08-03 | 2021-11-12 | 南通大学 | Beta-carboline indolium salt and preparation method and application thereof |
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| CN115093358A (en) * | 2022-06-20 | 2022-09-23 | 遵义医科大学珠海校区 | Preparation method of 1-ethyl-2, 3, 3-trimethyl-3H-indole-1-halide |
| CN115650963B (en) * | 2022-09-09 | 2024-04-23 | 安徽大学 | Near-infrared two-photon fluorescent probe for measuring mitochondrial viscosity and application thereof |
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