CN103044947B - Nile blue fluorescent dye, and preparation method and application of nile blue fluorescent dye - Google Patents

Nile blue fluorescent dye, and preparation method and application of nile blue fluorescent dye Download PDF

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CN103044947B
CN103044947B CN201310007599.7A CN201310007599A CN103044947B CN 103044947 B CN103044947 B CN 103044947B CN 201310007599 A CN201310007599 A CN 201310007599A CN 103044947 B CN103044947 B CN 103044947B
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nile blue
nbc
alkyl
fluorescent dye
nbb
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樊江莉
董惠娟
彭孝军
胡明明
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Dalian University of Technology
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Abstract

The invention discloses a nile blue fluorescent dye, a preparation method and an application of the nile blue fluorescent dye. The nile blue fluorescent dye has the following structure general formula I as shown in the specification. The nile blue fluorescent dye has the advantages of excellent photochemical and physical properties, such as high molar extinction coefficient, good photostability, and high fluorescent quantum yield. The dye is not sensitive to an internal environment of a cell, has good chemical stability and photostability, can solve the defects of high toxicity, severe photobleaching, and the like of an iysosome probe to the cell, and can be applied to iysosome fluorescence specificity markers.

Description

One class Nile blue fluorescence dye, its preparation method and application
Technical field
The present invention relates to fluorescent probe compounds and its production and use, this compound is applicable to the mark of Cytolysosome in field of fine chemical.
Background technology
Lysosome is the very important subcellular organelle of intracellular one, comprises the acid hydrolase that can decompose rejected material and cell debris in it, and they can digest organoid, food particles excessive or wearing and tearing, and can swallow up virus or bacterium.
Lysosomal Main Function is digestion, is intracellular digestion organs, aqtocytolysis, defence and all relevant with lysosomal digestion to the utilization of Cucumber., more and more fact proved, lysosome all plays an important role in much physiology, pharmacology and pathologic process meanwhile.Along with lysosomal research is subject to basic medical research worker and clinical scholar's attention day by day, therefore, develop molar absorptivity large, fluorescence quantum yield is high; Fluorescent emission wavelength is in long wave and have the fluorescent probe of larger Stokes shift, important in inhibiting in the application process of lysosome being carried out to mark, monitoring.
Fluorescent probe have Noninvasive, easy to operate, can realize the features such as visual detection, receive widespread attention.Many lysosomal location dyestuff all comprises a weakly alkaline chain at present, can make the lysosome organoid of probe oxytropism assemble.In recent years, software engineering researchers invent has gone out multiple lysosomal markup dyestuff, and a part is commercialization.A total shortcoming of these dyestuffs be excite, all shorter (～350-550nm) of emission wavelength, in the time carrying out viable cell co-focusing imaging, viable cell is caused to very large damage, simultaneously, easily there is photobleaching in this fluorochrome, and be vulnerable to the interference of autofluorescence, this series of deficiency all serious impact the application performance of dyestuff.Therefore develop that one excites, emission wavelength is long, good light stability, has hypotoxic lysosome probe to cell significant.
Nile blue fluorochrome has excellent photochemistry physical properties, such as high molar extinction coefficient, the good advantage such as light stability, high fluorescence quantum yield.Interior environment to cell is insensitive, has good chemical stability and light stability, and can solve the common lysosome probe drawback serious etc. to cell high toxicity and photobleaching.
A few class Nile blue dyestuffs of open report comprise (US Patent Number:6, on October 31st, 140,500,2000 such as 1SBPO, 2SBPO, Bona11, Bona12 at present; Development of water-soluble far-red fluorogenic dyes for enzyme sensing, Tetrahedron62 (2006) 578-585), but these published compounds are difficult for permeate through cell membranes enters viable cell inside, and cannot realize lysosome positioning dyeing.
Summary of the invention
The object of the present invention is to provide a class to there is the synthetic and application of the markup probe of lysosome of premium properties.
First the present invention discloses a class Nile blue fluorescence dye, has following general structure I:
Figure GDA0000444438240000021
In general formula I:
R 1and R 2independently selected from H, C 1-9alkyl, by C 1-9phenyl that alkyl replaces arbitrarily, by C 1-9naphthyl, halogen, OR that alkyl replaces arbitrarily 7, N (R 7) 2, cyano group, (CH 2cH 2o) nh, (CH 2) mcOOM and (CH 2) msO 3m;
R 3and R 4be selected from independently of one another H, (CH 2) 2nH 2, 2-morpholine ethyl, C 1-6alkyl, C 1-6phenyl, C that alkyl replaces arbitrarily 1-6naphthyl, halogen, hydroxyl, sulfydryl, cyano group, nitro, haloalkyl, alkylamino, amido that alkyl replaces arbitrarily;
R 5and R 6be selected from independently of one another H, C 1-6alkyl, by C 1-9phenyl, C that alkyl replaces arbitrarily 1-9naphthyl, halogen, cyano group, (CH that alkyl replaces arbitrarily 2cH 2o) nh, (CH 2) mcOOM or (CH 2) msO 3m;
Respectively the do for oneself integer of 0-9 of n, m;
M is H, K, Na, Li, NH 4, NH 3r 7, NH 2(R 7) 2, NH (R 7) 3or N (R 7) 4;
R 7for H, C 1-9alkyl or CH 2cH 2oH.
The invention described above provide the maximum emission peak of Nile blue fluorescent probe of general structure I at 687nm place, be 4.5-6.5(lysosomal pH scope at pH) time, fluorescence remains unchanged substantially, therefore can be used for the lysosomal location of viable cell and monitoring.With the I of 1 μ M concentration 37 ℃ of hatchings after 12 hours cell survival rate can reach 98%, and good light stability.Structure I and LysoSensorTM Green DND-189(are below all referred to as LysoSensor Green) in viable cell, lysosomal dyeing site is basic identical, and Pearson coefficient is more than 0.95.In addition, the Nile blue fluorochrome of structure I also has high molar extinction coefficient, the good advantage such as light stability and high fluorescence quantum yield.
Therefore, the Nile blue dyestuff with general formula I structure of the present invention has solved common lysosome probe cell has been had to high toxicity and serious etc. the drawback of photobleaching on the accurate lysosomal basis of mark.With respect to exciting, all commercialization lysosome probes of shorter (～350-550nm) of emission wavelength, I is in the time carrying out viable cell co-focusing imaging, can reduce significantly the damage that viable cell is caused, thereby have good effect in the whole apoptotic process of mark and monitoring cell.
Given this, further aim of the present invention is to provide described Nile blue fluorescence dye in the application of preparing in lysosome fluorescent specific labelled reagent.Especially in the application for the preparation of in the apoptotic reagent of monitoring.
Accompanying drawing explanation
Accompanying drawing 6 width of the present invention:
Fig. 1 is the absorption in different solvents, the mensuration of emmission spectrum to fluorescent probe compounds NBB of the present invention and NBC.The concentration of probe compound NBB and NBC is 5 μ M, and test system is water, ethanol, methyl alcohol, acetone, DMSO etc.Fig. 1 a is the absorption spectrum of NBB in different test systems; Fig. 1 b is the emmission spectrum of NBB in different test systems; Fig. 1 c is the absorption spectrum of NBC in different test systems; Fig. 1 d is the emmission spectrum of NBC in different test systems;
Fig. 2 carries out pH fluorometric titration test to fluorescent probe compounds NBB of the present invention and NBC.The concentration of probe compound NBB and NBC is 5 μ M, and test system is HEPES buffered soln.Fig. 2 a and 2b are fluorometric titration spectrum and the titration curve of NBB to pH, and excitation wavelength is 649nm; Fig. 2 c and 2d are the titration curve of NBC to pH, and excitation wavelength is 649nm;
Fig. 3 is the light stability contrast test of probe compound NBB of the present invention and NBC and commercially available dyestuff LysoSensor Green.
Fig. 4 is probe compound NBB of the present invention and NBC and the fluorescence imaging of LysoSensor Green in human breast cancer cell (MCF-7).The concentration of fluorescent probe compounds NBB and NBC is 50nM, and the concentration of LysoSensorGreen is 1 μ M.
Fig. 5 is that probe compound NBB of the present invention and NBC and LysoSensor Green redye fluorescence imaging in human breast cancer cell (MCF-7).The concentration of fluorescent probe compounds NBB and NBC is 2 μ M, and the concentration of LysoSensor Green is 1 μ M.
Fig. 6 is the MTT cytotoxicity test of probe compound NBB of the present invention and NBC.
Embodiment
Nile blue fluorescence dye of the present invention, has following general structure I:
Figure GDA0000444438240000031
One of embodiment, described R 3(or R 4) be 2-amino-ethyl or 2-morpholine ethyl.Preferably 2-amino-ethyl.
Two of embodiment, described R 1, R 2be selected from independently of one another hydrogen or methyl; Preferable methyl.
Further preferred specific embodiments, R 4(or R 3), R 5and R 6be selected from independently of one another hydrogen or methyl; Most preferably H.
The most preferably, rhodamine fluorescent dyes of the present invention comprises compound N BB and NBC:
The various raw materials that use in the present invention are all commercially available, or can be by well known to a person skilled in the art that in method or prior art, disclosed method is prepared simply by raw material well known in the art.
Will be appreciated that, after various ring substituents in the compounds of this invention have some to complete before above-mentioned steps is carried out or just, introduced or produced by conventional modified with functional group by the aromatics substitution reaction of standard, this is included in method steps of the present invention aspect.This reaction and modification comprise that for example substituting group is by introducing, substituent reduction, substituent alkylation and the substituent oxidation of aromatics substitution reaction.Reagent and reaction conditions for these processes are that chemical field is known.The specific examples of aromatics substitution reaction comprises introduces nitro with concentrated nitric acid; under Friedel Crafts condition, introduce acyl group with for example carboxylic acid halides and Lewis acid (as aluminum chloride); under Friedel Crafts condition, introduce alkyl with alkylogen and Lewis acid (as aluminum chloride), and introduce halogen group.Modify specific examples comprise by for example with nickel catalyzator carry out catalytic hydrogenation or with iron hydrochloric acid exist under carry out heat treated, by nitroreduction become amino; Alkylthio is oxidized to alkyl sulphinyl or alkyl sulphonyl.
Unless otherwise indicated, term used herein has following implication.
Term used herein " alkyl " comprises straight chained alkyl and branched-chain alkyl.As mentioned, single alkyl is as " propyl group ", only refers in particular to straight chained alkyl, as mentioned, single branched-chain alkyl is as " sec.-propyl ", only refers in particular to branched-chain alkyl.For example, " C 1-6alkyl " comprise C 1-4alkyl, C 1-3alkyl, methyl, ethyl, n-propyl, sec.-propyl and the tertiary butyl.Other group that similarly rule is also applicable to use in this specification sheets.
Term used herein " halogen " comprises fluorine, chlorine, bromine and iodine.
Following non-limiting example can make the present invention of those of ordinary skill in the art's comprehend, but does not limit the present invention in any way.
Embodiment 1. fluorescent probe compounds NBB's is synthetic:
Figure GDA0000444438240000051
Fluorescent probe compounds NBB's is synthetic:
By compound 2(0.45g, 2.4mmol) add in the 100mL single port flask of 10mL ethanol, under ice bath, stir, measure 5-(dimethylamino)-2-nitrosophenol (0.29g, 2.0mmol), solid is joined in single port flask, drip 1 milliliter of concentrated hydrochloric acid, ice bath stirs 4 hours, removes solvent under reduced pressure.Recrystallization separates the solid NBB(92% obtaining with green metal gloss). 1H?NMR(400MHz,MeOD),δ:1.29(s,6H),3.38(q,J=8Hz,2H),3.98(t,J=8Hz,2H),6.89(d,1H),7.01(s,1H),7.26(q,1H),7.82(t,J=8Hz,1H),7.89(q,J=8Hz,2H),8.48(d,J=8Hz,1H),8.91(d,J=8Hz,1H), 13C?NMR(100MHz,MeOD),δ:39.68,48.44,48.30,47.08,95.79,96.38,114.88,124.38,124.39,129.64,132.00,132.32,132.45,133.80,148.55,152.92,156.84,157.62ppm;TOF?MS:m/z?calcd?for?C 20H 21N 4O +[M] +:333.17,found:333.1705
Embodiment 2. fluorescent probe compounds NBC's is synthetic:
Figure GDA0000444438240000052
Fluorescent probe compounds NBC's is synthetic:
By compound 3(0.61g, 2.4mmol) add in the 100mL single port flask of 10mL ethanol, under ice bath, stir, measure 5-(dimethylamino)-2-nitrosophenol (0.29g, 2.0mmol), solid is joined in single port flask, drip 1 milliliter of concentrated hydrochloric acid, ice bath stirs 4 hours, removes solvent under reduced pressure.Recrystallization separates the solid NBC(53% obtaining with green metal gloss). 1H?NMR(400MHz,MeOD),δ:3.33(m,J=4Hz,8H),3.63(s,3H),4.01(s,5H),4.23(d,J=4Hz,2H),7.00(d,J=4Hz,1H),7.18(s,1H),7.41(d,J=8Hz,1H),7.87(m,1H),7.96(t,J=8Hz,2H),8.54(d,J=8Hz,1H),8.97(d,J=8Hz,1H); 13C?NMR(100MHz,MeOD),δ:38.11,39.98,48.08,48.30,48.44,52.27,54.59,63.65,93.25,95.75,116.66,123.02,123.30,124.15,129.46,131.29,131.64,132.00,132.91,133.13,148.55,151.92,156.80,157.58ppm;TOF?MS:m/z?calcd?for?C 24H 27N 4O 2 +[M] +:403.21,found:403.2143
Embodiment 3. fluorescent probe compounds NBB and the absorption emmission spectrum of NBC in different solvents
Select respectively different solvents (as water, ethanol, methyl alcohol, acetone, DMSO etc.) to be made into the solution that concentration is 5 μ M fluorescent probe compounds NBB and NBC, and respectively its absorption, emmission spectrum are measured.Fig. 1 a is the absorption spectrum of NBA in different test systems; Fig. 1 a is the absorption spectrum of NBB in different test systems; Fig. 1 b is the emmission spectrum of NBB in different test systems; Fig. 1 c is the absorption spectrum of NBC in different test systems; Fig. 1 d is the emmission spectrum of NBC in different test systems; From figure, we can find out, in neutral and slant acidity solvent, the Absorption and emission spectra of compound N BB and NBC is all near infrared range, in the time that solvent presents weakly alkaline, due to the change of dye molecule conjugated structure, Absorption and emission spectra there will be blue shift to a certain degree.
Embodiment 4. fluorescent probe compounds NBB and NBC test pH fluorometric titration
The probe compound NBB of 5 μ M is joined in HEPES damping fluid (pH7.4), by adding salt slow acid to regulate pH to 3, the fluorescence intensity of recording responses, excitation wavelength is that 649nm test result is shown in Fig. 2 a and 2b figure.As can be seen from the figure, along with the decline of pH, the fluorescence intensity at probe compound maximum emission peak 665nm place strengthens gradually, pH be 3.5 o'clock substantially saturated.Under similarity condition, excitation wavelength is adjusted into 649nm, obtains the variation of relative intensity of fluorescence of NBB and NBC buffered soln respectively as Fig. 2 a and 2c by Sigmoidal, and pKa value is respectively 9.89, and ((Fig. 2 d) for Fig. 2 b) He 9.97.Be 4.5-6.5(lysosomal pH scope at pH) time, the fluorescence intensity of NBB and NBC does not have considerable change, shows that NBB and NBC all can be used for lysosomal mark and monitoring.The light stability contrast test of embodiment 5. fluorescent probe compounds NBB and NBC and LysoSensor Green
The HEPES buffered soln that compound N BB and NBC is made into respectively to 5uM packs in the cuvette that can seal, and the PBS buffered soln that commercialization lysosome location dyestuff LysoSensor Green is made into 50uM packs in the cuvette that can seal.The sodium nitrite solution of use 50g/L is contained in a rectangular parallelepiped glass jar and does cut-off filter, and elimination wavelength is less than the UV-light of 400nm.In addition, sodium nitrite solution also can play the effect of cold-trap, and the temperature remains within the normal range to make the temperature of sample.After the initial absorbance value of working sample, select 500W tungsten-iodine lamp as light source, apart from sample 30cm place, energising illumination, timing.Every 10 minutes, 30 minutes, after 1 hour, the fluorescence intensity after measure sample illumination.As shown in Figure 3, after illumination in 4 hours, compound N BB, NBC and LysoSensor Green fade respectively 2%, 3% and 5% in HEPES damping fluid and PBS damping fluid.Result demonstration, probe compound NBB and the NBC of the present invention's design have stronger light stability.
Embodiment 6. fluorescence microscopy Microscopic observation probe compound NBB and NBC are to dyeing in MCF-7 viable cell
MCF-7 cell is at DEME(invitrogen) in 10% FCS(invitrogen) cultivate.Confocal fluorescent imaging experiment the day before yesterday, cell kind is in the burnt culture dish of special cell copolymerization.Second day, add wherein the probe compound NBB/NBC of 50nM, remain on 37 ℃ and 5%CO 2under condition, hatch 30 minutes, then rinse after 3 times with phosphate buffer solution, carry out co-focusing imaging.
The culture density of cell is 2 × 10 5cells/mL.Imaging instrument is Olympus FV1000-IX81 inverted microscope, 100 times of oily mirrors.Exciting light is that 639nm excites, and collects 660-690nm wave band.
Fig. 4 a/d is white light figure, and Fig. 4 b/e is fluorescence imaging figure, and Fig. 4 c/f is respectively the stacking diagram of Fig. 4 a/d and Fig. 4 b/e.Lysosome is not that homogeneous distributes in cell, but the distribution of being scattered in tenuigenin, lysosomal pH is in the scope of 4.5-5.5.The pKa of NBB and NBC is all greater than 9.5, in the environment that therefore three is 4.5-5.5 at pH, can send more stable fluorescence.From Fig. 4, the luminous component of cell can be found out, probe compound NBB and NBC have entered into lysosome organoid.
Embodiment 7. fluorescence microscopy Microscopic observation probe compound NBB, NBC and LysoSensor Green redying MCF-7 viable cell
LysoSensor Green is a kind of commercialization plastosome green fluorescence probe, can be for lysosome specificity fluorescent dyeing in viable cell.Probe compound NBB and NBC and LysoSensor Green are carried out to viable cell dyeing to MCF-7, and relatively their dyeing sites to viable cell, can further determine that NBB and NBC are to lysosomal specificity fluorescent mark.
MCF-7 cell is at DEME(invitrogen) in 10% FCS(invitrogen) cultivate.Confocal fluorescent imaging experiment the day before yesterday, cell kind is in the burnt culture dish of special cell copolymerization.Second day, add respectively wherein probe compound NBB and the NBC of 2 μ M, remain on 37 ℃ and 5%CO 2under condition, hatch 30 minutes, then rinse 3 times with phosphate buffer solution.The commercially available dyestuff LysoSensor Green that adds again 1 μ M, remains on 37 ℃ and 5%CO 2under condition, hatch 15 minutes, then rinse after 3 times with phosphate buffer solution, carry out co-focusing imaging.
The culture density of cell is 2 × 10 5cells/mL.Imaging instrument is Olympus FV1000-IX81 inverted microscope, 100 times of oily mirrors.The exciting light of NBB and NBC is that 639nm excites, and collects 660-690nm wave band; The excitation wavelength of LysoSensor Green is 488nm, collects 495-515nm wave band.
As can be seen from Figure 5, in MCF-7 cell, the dyeing site of probe compound NBB, NBC and LysoSensor Green is substantially identical, and colocalization coefficient has reached more than 0.95.Show that NBB and NBC are positioned in lysosome organoid well.
The cytotoxicity test of embodiment 8. probe compound NBB and NBC
The HeLa that will detect and Raw264.7 cell 0.25% tryptic digestion, use containing the RPMI-1640 nutrient solution of 10% foetal calf serum and be made into individual cells suspension, with every hole 10 3～10 4individual cell is inoculated in 96 well culture plates, every pore volume 200 μ L; Culture plate is moved in incubator, 37 ℃, 5%CO 2and under saturated humidity, cultivate after 24 hours, adding respectively dye strength is 0.5 μ M and 1.0 μ M, continues to cultivate 12 hours; Every hole adds MTT solution (5mg/mL) 20 μ L, hatches 4 hours, stops cultivating, and carefully sops up culture supernatant in hole.Then, every hole adds the DMSO of 150 μ L, vibrates 10 minutes, and crystallisate is fully dissolved; In microplate reader, measure the absorbancy at 550nm place, each hole, calculate cell survival rate: test group optical absorbance/control group absorbance × 100%.
As can be seen from Figure 6, probe compound NBB and NBC, are hatched after 12 hours without obvious cytotoxicity Hela and Raw264.7 cell, and cell survival rate can reach more than 96%.

Claims (6)

1. a class Nile blue fluorescence dye, has following general structure I:
In general formula I:
R 1and R 2independently selected from H, C 1-9alkyl, by C 1-9phenyl that alkyl replaces arbitrarily, by C 1-9naphthyl, halogen, OR that alkyl replaces arbitrarily 7, N (R 7) 2, cyano group, (CH 2cH 2o) nh, (CH 2) mcOOM and (CH 2) msO 3m;
R 3and R 4in one be H, another is (CH 2) 2nH 2or 2-morpholine ethyl;
R 5and R 6be selected from independently of one another H, C 1-6alkyl, by C 1-9phenyl, C that alkyl replaces arbitrarily 1-9naphthyl, halogen, cyano group, (CH that alkyl replaces arbitrarily 2cH 2o) nh, (CH 2) mcOOM or (CH 2) msO 3m;
Respectively the do for oneself integer of 0-9 of n, m;
M is H, K, Na, Li, NH 4, NH 3r 7, NH 2(R 7) 2, NH (R 7) 3or N (R 7) 4;
R 7for H, C 1-9alkyl or CH 2cH 2oH.
2. fluorescence dye claimed in claim 1, is characterized in that, described R 1and R 2be selected from independently of one another H or C 1-9alkyl.
3. fluorescence dye claimed in claim 2, is characterized in that, described R 1and R 2be selected from independently of one another H or methyl.
4. fluorescence dye claimed in claim 3, is characterized in that, described R 1and R 2be methyl.
5. fluorescence dye claimed in claim 1, is characterized in that, described rhodamine fluorescent dyes comprises compound N BB and NBC:
Figure FDA0000444438230000012
6. Nile blue fluorescence dye claimed in claim 1 is in the application of preparing in lysosome fluorescent specific labelled reagent.
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