CN104448897B - The Two-photon fluorescent dye being parent with naphthalene of end-functionalization - Google Patents
The Two-photon fluorescent dye being parent with naphthalene of end-functionalization Download PDFInfo
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Abstract
The Two-photon fluorescent dye being parent with naphthalene of one class end-functionalization, has the structure of formula I, such as accompanying drawing 1. Many cells device can be carried out imaging at living cells by such Two-photon fluorescent dye, especially mitochondrion and lysosome, organelle at target imaging has relatively low fluorescence background with exterior domain, only there is in target many cells device region stronger fluorescence signal in living cells, and target many cells utensil is had very strong specificity labelling. This compounds has the water solublity of certain level, has good permeability of cell membrane simultaneously. And also there is bigger effective two photon absorption cross section. This compounds of the present invention also has relatively low bio-toxicity, phototoxicity, photobleaching simultaneously. The spectral region of its spectral region and biological sample has sufficiently large difference.
Description
Technical field
The present invention relates to a class end-functionalization with naphthalene be parent Two-photon fluorescent dye, its preparation method, and utilize this fluorochrome to the identification simultaneously of various kinds of cell device in living cells and imaging.
Background technology
Cell is the basic composition unit of all living things 26S Proteasome Structure and Function, the metabolism of biologic artifact, growth promoter, breeding, heredity, variation, irritability and to various biosiss such as the adaptabilities of environment, is all dependent on cell and shows and complete. In other words, disease is exactly the result of the cell generation pathological changes in the middle of human local tissue structure, as the diseases such as inflammation, canceration, hypertrophy are grown in the pathological changes of cell with regard to root. Such as: endoplasmic reticulum under pathological conditions, when sustaining damage or be subject to the effect of some factor, it may occur that swelling, hypertrophy and Cucumber accumulation, thus causing such as: the generation of metabolic disease diabetes, obesity etc.; Lysosome is abnormal, can cause a lot of disease, such as gout, pneumosilicosis etc. And these pathological processes, be often all attended by a certain kind or various kinds of cell device quantity, size, shape, result change. Therefore, as long as finding the method that can monitor these sick cells and activity thereof in real time, just can to a certain extent disease diagnosis and therapy be played very big help (VirchowR.L.K.DieCellularpathologie.NabuPress:USA, 2010:112-119).
The method kind of existing identification and detection organelle is a lot, and wherein fluorescent microscopic imaging becomes a kind of vision aid that can not be substituted of research organelle. Along with the development of micro-imaging technique, fluorescent dye has become as in micro-imaging is studied and most important depends on instrument. At present, Hoechst, DAPI, SYTO fluorochrome all has been applied in organelle correlational study. But there is following defect in actual many cells simultaneously imaging applications in the fluorescent microscopic imaging method of application fluorescent small molecule: dyestuff membrane passage is poor, many cells while imaging time to the non-endogenous damage of organelle etc. (Invitrogen:USA, 2010:499,516,525;JournalofmaterialschemistryB, 2013,1:438-442; JournaloftheAmericanChemicalSociety, 2010,132:12237-12239). In addition, they excite at near-infrared, dark-field imaging, avoid fluorescent bleach and light intoxicating, targeting to excite, high lateral resolution and longitudinal resolution, reduce biological tissue's specific absorbance and reduce tissue autofluorescence interference etc. in (Nature, 1999,2:989-996.; Science, 1997,275:530.; AngewandteChemieInternationalEdition, 2001,40:2098.) there is certain defect. Therefore design and develop a class two-photon probe molecule, utilize it to realize that to disease early diagnosis and etiologic etiological research, the work of multiple organelles realtime imaging simultaneously certainly will be had great potential significance.
Summary of the invention
The purpose of the present invention, first consists in the Two-photon fluorescent dye being parent with naphthalene providing a class end-functionalization, and described dyestuff has the structure (accompanying drawing 1) of formula I:
In formula I:
R1It is selected from :-H, halogen, C1-8Alkyl ,-(CH2)nO(CH2)mCH3��-(CH2)nS(CH2)mCH3With-(CH2)nNH(CH2)mCH3; Wherein, n and m is each independently selected from the integer of 1-8;
R2Selected from halogen, H and C1-8Alkyl;
L1And L2It is each independently selected from :-(CH2)p-��-S(CH2)p-��-(CH2)pS-��-S(CH2)pS-��-O(CH2)p-��-(CH2)pO-��-O(CH2)pO-��-NH(CH2)p-��-(CH2)pNH-��-NH(CH2)pNH-��-N(CH3)(CH2)p-��-(CH2)pN(CH3)-and-N (CH3)(CH2)pN(CH3)-; Wherein p is the integer of 1-8.
In the structural formula of above-mentioned group, "---" is in order to represent the free key that this group is connected with compound other parts.
The purpose of another aspect of the present invention, is in that the preparation method providing the Two-photon fluorescent dye being parent with naphthalene of the end-functionalization of the invention described above, and described method comprises the steps:
1) compound B1With R1-I 1:1-1:2 in molar ratio reacts, and prepares compound C1:
Reaction temperature is 20-80 DEG C, and the response time is 1-10 hour, and reaction dissolvent is selected from dichloromethane, ethanol, methanol, acetone or its mixture;
2) under DMAP (DMAP) catalysis, compound C1, compound B2React according to mol ratio 1:5:1-1:20:5 with EDCl [1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride], prepare compound C2:
Reaction temperature 30-180 DEG C, in 1-20 hour response time, reaction dissolvent is selected from dichloromethane, ethanol, methanol, acetone, glycerol, ethyl acetate, acetic acid or its mixture;
3) under DMAP (DMAP) catalysis, compound C2��H-L1-H and EDCl [1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride] reacts according to mol ratio 1:1:1-1:5:5, prepares compound C3:
Reaction temperature 25-120 DEG C, in 1-18 hour response time, reaction dissolvent is selected from dichloromethane, ethanol, methanol, acetone, glycerol, ethyl acetate, acetic acid or its mixture;
4) under DMAP (DMAP) catalysis, compound C3��R2-L2-COOH and EDCl [1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride] reacts according to mol ratio 1:5:1-1:20:5, prepares compound C4:
Reaction temperature 25-100 DEG C, in 1-10 hour response time, reaction dissolvent is selected from dichloromethane, ethanol, methanol, acetone, glycerol, ethyl acetate, acetic acid or its mixture,
5) compound C4Hydrolysis prepares compound I, and reaction system adds 1-10 times of compound C4The trifluoroacetic acid of molar equivalent, acetic acid or sodium hydroxide;
Reaction temperature is 25-50 DEG C, and the response time is 1-6 hour, and reaction dissolvent is selected from dichloromethane, ethanol, methanol, acetone, water or its mixture.
An above-mentioned class end-functionalization to the present invention with naphthalene be parent Two-photon fluorescent dye preparation method description in, the definition of each substituent group, namely to R1��R2��L1, and L2Definition, identical with the definition in the above-mentioned description to compound.
The naphthalene of end-functionalization provided by the present invention be parent Two-photon fluorescent dye can two-photon excitation, at living cells to target cell device, especially mitochondrion and lysosome, have stronger fluorescence signal. And this fluorochrome has the water solublity of certain level and good permeability of cell membrane, and has bigger effective two photon absorption cross section, also there is relatively low bio-toxicity, phototoxicity, photobleaching simultaneously. The spectral region of its spectral region and biological sample has sufficiently large difference.
Therefore, the purpose of further aspect of the present invention is in that the Two-photon fluorescent dye that naphthalene is parent providing above-mentioned end functionalized detects and the application in labelling at biology simultaneously. The especially preferred object that is applied to is the mitochondrion in living cells and lysosomal detection and labelling.
Accompanying drawing explanation
Accompanying drawing 9 width of the present invention:
Fig. 1 is the general structure I of the Two-photon fluorescent dye being parent with naphthalene of the end-functionalization of the present invention.
Fig. 2 is the fluorescent dye compound A of the present invention in embodiment 11Solvation effect characterization result. By compound A1In the different solvents being added separately to. Measure the ultra-violet absorption spectrum (a) in different solvents and fluorescence emission spectrum (b)
Fig. 3 is the fluorescent dye compound A of the present invention in embodiment 11The measurement result of the two photon absorption cross section in DMSO solvent. Mensuration solvent is: DMSO solvent. Assay method is: adopts femtosecond two-photoninduced fluorescence method, utilizes the NaOH solution (pH11) of fluorescein as reference, A used1Solution concentration is 1 �� 10-4M, laser pulse width 70fs, repetition rate 80MHz, the average output power 1.5W (780nm) of laser instrument, wavelengthtunable scope 700��980nm, femtosecond laser wavelength is adjusted to required test wavelength in an experiment.
Fig. 4 is the compound A of embodiment 4 synthesis2, with the A of final concentration of 2.0 ��Ms2Hatch Hela cell, at 37 DEG C, 5%CO2Hatch 30 minutes. Then, PBS concussion rinsing 5min �� 3, add cell culture medium, two-photon laser co-focusing imaging. Choose representative area, observe with oil mirror (100 ��), in triplicate. In imaging display Hela cell, endoplasmic reticulum has hyperfluorescence signal. The fluorescent collecting wave band of Fig. 4 is 490-550nm.
Fig. 5 is the fluorescent dye compound A of the present invention in embodiment 42Water-soluble characterization result. Use the compound A of variable concentrations2Aqueous solution, measure its absorbance under maximum absorption wavelength. In triplicate.
Fig. 6 is the compound A of embodiment 7 synthesis3, with the A of final concentration of 2.0 ��Ms3Hatch Hela cell, at 37 DEG C, 5%CO2Hatch 30 minutes. Then, PBS concussion rinsing 5min �� 3, add cell culture medium, two-photon laser co-focusing imaging. Choose representative area, observe with oil mirror (100 ��), in triplicate. Imaging display Hela Variations of Golgi Apparatus In The Cells have hyperfluorescence signal. The fluorescent collecting wave band of Fig. 6 is 490-550nm.
Fig. 7 is the fluorescent dye compound A of the present invention in embodiment 73The measurement result of the two photon absorption cross section in DMSO. Mensuration solvent is: DMSO. Assay method is: adopts femtosecond two-photoninduced fluorescence method, utilizes the NaOH solution (pH11) of fluorescein as reference, A used3Solution concentration is 1 �� 10-4M, laser pulse width 70fs, repetition rate 80MHz, the average output power 1.5W (780nm) of laser instrument, wavelengthtunable scope 700��980nm, femtosecond laser wavelength is adjusted to required test wavelength in an experiment.
Fig. 8 is the compound A using embodiment 10 synthesis4, with the A of final concentration of 2.0 ��Ms4Hatch Hela cell, at 37 DEG C, 5%CO2Hatch 30 minutes.Then, PBS concussion rinsing 5min �� 3, add cell culture medium, two-photon laser co-focusing imaging. Choose representative area, observe with oil mirror (100 ��), in triplicate. Imaging display Hela cell there is hyperfluorescence signal in endoplasmic reticulum. The fluorescent collecting wave band of Fig. 8 is 490-550nm.
Fig. 9 is the fluorescent probe compounds A of the present invention in embodiment 104Water-soluble characterization result. Use compound A4The aqueous solution of variable concentrations, measures its absorbance under maximum absorption wavelength. In triplicate.
Detailed description of the invention
Unless otherwise indicated, term used herein has following implication.
Term used herein " alkyl " includes straight chained alkyl, branched alkyl and cycloalkyl. As mentioned by concrete alkyl title, such as " propyl group ", " isopropyl " or " cyclopropyl ", refer in particular to the concrete direct-connected alkyl of carbon number purpose, branched alkyl and cycloalkyl. Such as the description of general form, such as " C1-6Alkyl ", then include carbon number and meet all groups of requirement, " C1-6Alkyl " include but not limited to C1-4Alkyl, C1-3Alkyl, methyl, ethyl, n-pro-pyl, isopropyl, the tert-butyl group, cyclopropyl, cyclobutyl, methylcyclopropyl groups etc. Similar rule be also applied in this specification use other group.
The Two-photon fluorescent dye being parent with naphthalene of disclosure one class end-functionalization, has the structure of formula I:
In described formula I, L1, L2For linking arm; R1, R2For different substituents group.
In formula I, described R1It is selected from :-H, halogen, C1-8Alkyl ,-(CH2)nO(CH2)mCH3��-(CH2)nS(CH2)mCH3With-(CH2)nNH(CH2)mCH3; Wherein, n and m is each independently selected from the integer of 1-8;
It is further preferable that described R1It is selected from :-H, halogen, C1-8Alkyl ,-(CH2)nO(CH2)mCH3Or-(CH2)nNH(CH2)mCH3. Especially preferred R1It is-H or C1-8Alkyl.
One of detailed description of the invention, described R1It is-H.
One of detailed description of the invention, described R1Selected from C1-8Alkyl, is all direct-connected alkyl of 1-8, branched alkyl and cycloalkyl including carbon number, citing but be not limited to the group of following structure :-(CH2)n-��-(CHCH3)1-4-��-(CH3CCH3)1-2-or they combination in any after gained group. Preferred C1-4Alkyl, includes but not limited to-CH3,-CH2CH3,-CH2CH2CH3,-CH (CH3)CH3. Most preferably-CH3��
In formula I, described R2Selected from halogen, H and C1-8Alkyl.
One of detailed description of the invention, described R2It is H.
One of detailed description of the invention, described R2Selected from halogen, including F, Cl, Br, I; Preferably-Cl ,-Br and-I, it is most preferred that-Cl.
One of detailed description of the invention, described R2For C1-8Alkyl, is all direct-connected alkyl of 1-8, branched alkyl and cycloalkyl including carbon number, citing but be not limited to the group of following structure :-(CH2)n-��-(CHCH3)1-4-��-(CH3CCH3)1-2-or they combination in any after gained group. Preferred C1-4Alkyl, includes but not limited to-CH3,-CH2CH3,-CH2CH2CH3,-CH (CH3)CH3��
In formula I, described L1And L2It is each independently selected from :-(CH2)p-��-S(CH2)p-��-(CH2)pS-��-S(CH2)pS-��-O(CH2)p-��-(CH2)pO-��-O(CH2)pO-��-NH(CH2)p-��-(CH2)pNH-��-NH(CH2)pNH-��-N(CH3)(CH2)p-��-(CH2)pN(CH3)-and-N (CH3)(CH2)pN(CH3)-; Wherein p is the integer of 1-8.
As preferred embodiment, described L1It is selected from :-(CH2)p-��-NH(CH2)p-��-(CH2)pNH-��-NH(CH2)pNH-��-N(CH3)(CH2)p-��-(CH2)pN(CH3)-or-N (CH3)(CH2)pN(CH3)-; L1It is preferred that :-NH (CH2)pNH-or-N (CH3)(CH2)pN(CH3)-; Most preferably-NH (CH2)pNH-. In any detailed description of the invention, p is the integer of 1-8, it is preferable that p is the integer of 1-4, it is particularly preferred that p is 2 or 3.
As preferred embodiment, described L2It is selected from :-(CH2)p-��-(CH2)pS-or-(CH2)pO-; L2It is preferred that :-(CH2)p-. In any detailed description of the invention, p is the integer of 1-8, it is preferable that p is the integer of 1-4, it is particularly preferred that p is the integer of 1-3.
Each preferred feature described above can be mutually combined, gained technical scheme should be included in completely of the present invention and scope.
The example of the combination of preferred feature, one of is that the Two-photon fluorescent dye being parent with naphthalene of described end-functionalization, selected from compound A for specific embodiment of the present invention1-A4:
On the other hand, the preparation method that the invention provides the Two-photon fluorescent dye that naphthalene is parent of the invention described above one class end-functionalization, comprise the steps:
1) compound B1With R1-I 1:1-1:2 in molar ratio reacts, and prepares compound C1:
Reaction temperature is 20-80 DEG C, and the response time is 1-10 hour, and reaction dissolvent is selected from dichloromethane, ethanol, methanol, acetone or its mixture;
In detailed description of the invention, described step 1) preferred 20-70 DEG C of reaction temperature, more preferably 20-60 DEG C, it is most preferred that 20-50 DEG C; Preferred 1-9 hour of described response time, more preferably 1-8 hour, it is most preferred that 2-8 hour; The preferred dichloromethane of described reaction dissolvent, methanol, acetone or its mixture, more preferably dichloromethane, acetone or its mixture, it is most preferred that dichloromethane; Compound B1With R1-I reacts the preferred 1:1.1-1:2 of mol ratio, more preferably 1:1.2-1:2, it is most preferred that 1:1.25-1:2.
2) under DMAP (DMAP) catalysis, compound C1, compound B2React according to mol ratio 1:5:1-1:20:5 with EDCl [1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride], prepare compound C2:
Reaction temperature 30-180 DEG C, in 1-20 hour response time, reaction dissolvent is selected from dichloromethane, ethanol, methanol, acetone, glycerol, ethyl acetate, acetic acid or its mixture;
In detailed description of the invention, described step 2) preferred 30-170 DEG C of reaction temperature, more preferably 30-160 DEG C, it is most preferred that 30-150 DEG C; Preferred 1-19 hour of described response time, more preferably 1-18 hour, it is most preferred that 1-16 hour; The preferred dichloromethane of described reaction dissolvent, methanol, acetone, glycerol, ethyl acetate, acetic acid or its mixture, more preferably dichloromethane, methanol, acetone, ethyl acetate, acetic acid or its mixture, it is most preferred that dichloromethane, methanol, acetone, acetic acid or its mixture; Compound C1, compound B2The preferred 1:5:1-1:19:5 of mol ratio, more preferably 1:5:1-1:18:5, it is most preferred that 1:5:1-1:17:5 is reacted with EDCl [1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride].
3) under DMAP (DMAP) catalysis, compound C2��H-L1-H and EDCl [1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride] reacts according to mol ratio 1:1:1-1:5:5, prepares compound C3:
Reaction temperature 25-120 DEG C, in 1-18 hour response time, reaction dissolvent is selected from dichloromethane, ethanol, methanol, acetone, glycerol, ethyl acetate, acetic acid or its mixture;
In detailed description of the invention, described step 3) preferred 30-120 DEG C of reaction temperature, more preferably 30-110 DEG C, it is most preferred that 30-100 DEG C; Preferred 1-15 hour of described response time, more preferably 5-15 hour, it is most preferred that 5-14 hour; The preferred dichloromethane of described reaction dissolvent, ethanol, methanol, acetone, ethyl acetate, acetic acid or its mixture, more preferably dichloromethane, ethanol, methanol, ethyl acetate, acetic acid or its mixture, it is most preferred that dichloromethane, ethanol, ethyl acetate, acetic acid or its mixture; Compound C2��H-L1-H and EDCl [1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride] reacts the preferred 1:1.1:1-1:5:5 of mol ratio, more preferably 1:1.2:1-1:5:5, it is most preferred that 1:1.5:1-1:5:5.
4) under DMAP (DMAP) catalysis, compound C3��R2-L2-COOH and EDCl [1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride] reacts according to mol ratio 1:5:1-1:20:5, prepares compound C4:
Reaction temperature 25-100 DEG C, in 1-10 hour response time, reaction dissolvent is selected from dichloromethane, ethanol, methanol, acetone, glycerol, ethyl acetate, acetic acid or its mixture;
In detailed description of the invention, described step 4) preferred 30-100 DEG C of reaction temperature, more preferably 30-90 DEG C, it is most preferred that 30-80 DEG C; Preferred 1-9 hour of described response time, more preferably 5-9 hour, it is most preferred that 6-9 hour; The preferred dichloromethane of described reaction dissolvent, ethanol, methanol, acetone, ethyl acetate, acetic acid or its mixture, more preferably dichloromethane, ethanol, methanol, ethyl acetate, acetic acid or its mixture, it is most preferred that dichloromethane, ethyl acetate, acetic acid or its mixture; Compound C3��R2-L2-COOH and EDCl [1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride] reacts the preferred 1:5:1-1:19:5 of mol ratio, more preferably 1:5:1-1:18:5, it is most preferred that 1:5:1-1:17:5.
5) compound C4Hydrolysis prepares compound I, and reaction system adds 1-10 times of compound C4The trifluoroacetic acid of molar equivalent, acetic acid or sodium hydroxide;
Reaction temperature is 25-50 DEG C, and the response time is 1-6 hour, and reaction dissolvent is selected from dichloromethane, ethanol, methanol, acetone, water or its mixture.
In detailed description of the invention, described step 5) preferred 25-45 DEG C of reaction temperature, more preferably 25-40 DEG C, it is most preferred that 25-35 DEG C; Preferred 1-5 hour of described response time, more preferably 1.5-5 hour, it is most preferred that 2-5 hour; The preferred dichloromethane of described reaction dissolvent, ethanol, methanol, water or its mixture, more preferably dichloromethane, ethanol, water or its mixture, it is most preferred that dichloromethane, water or its mixture; Compound C4The preferred 1:1-1:9.5 of mol ratio, more preferably 1:1-1:9, it is most preferred that 1:1-1:8 is reacted with trifluoroacetic acid, acetic acid or sodium hydroxide.
The present invention is adopted the Two-photon fluorescent dye being parent with naphthalene of the end-functionalization that said method synthesizes, adopts nmr spectrum or mass spectrum to confirm its structure.
Fluorescent dye of the present invention possesses advantages below:
Described fluorescent dye compound introduces and different organelle-specificity binding sites, improves dyestuff to the detection of living cells difference organelle and recognition specificity, specificity;
Described fluorescent dye compound has the two-photon performance of excellence, has the bleaching of low bio-light, photic damage and bio-toxicity when being applied to biological sample imaging;
Described fluorescent dye compound side effect is little, and raw material is easy to get, simple in construction, it is easy to preparation, easy industrialization;
In consideration of it, Two-photon fluorescent dye of the present invention can be used for the same tense marker of living cells various kinds of cell device. Except being directly used in the same tense marker of living cells various kinds of cell device with form specifically described herein, the compositions of the Two-photon fluorescent dye compound containing the present invention can be used for the same tense marker of living cells various kinds of cell device. One of Two-photon fluorescent dye compound provided by the present invention that should comprise effective dose in described compositions. Furthermore it is also possible to comprise other component required for biological sample dyeing, for instance solvent, pH adjusting agent etc. These components are all that one's own profession is known in the art.Above-mentioned composition can exist as an aqueous solution, or can be formulated as other suitable form existence of solution before use with water.
The present invention also provides for the Two-photon fluorescent dye pin compound living cells various kinds of cell device the using the invention described above method with tense marker, and the method includes the step making described compound contact with biological sample. Term used herein " contact " may be included in solution or solid phase and contacts.
Following non-limiting example can make those of ordinary skill in the art more fully understand the present invention, but does not limit the present invention in any way.
Embodiment 1
Prepare fluorescent dye compound A1, adopt following synthetic route:
(1) synthesis of compound 1-1
By 20mmolB1And 100mmolB2Join in the round-bottomed flask containing 10ml dichloromethane solution, add 20mmolEDCl and a small amount of DMAP, nitrogen protection, react under room temperature. After reaction terminates, rotation, except solvent, column chromatography for separation, obtains faint yellow solid powder-product, for compound 1-1, yield 76%.
(2) synthesis of compound 1-2
20mmol compound 1-1 and 40mmol iodomethane are joined in the round-bottomed flask containing 10ml dichloromethane solution, nitrogen protection. Stop after room temperature reaction 8h. After reaction terminates, rotation, except solvent, column chromatography for separation, obtains faint yellow solid powder-product compound 1-2, yield 89%.
(3) synthesis of compound 1-3
20mmol compound 1-2 and 80mmol ethylenediamine are joined in the round-bottomed flask containing 20mmolEDCl and a small amount of DMAP20ml dichloromethane solution, nitrogen protection. Stop after reaction 5h. Decompression steams solvent, and pillar layer separation obtains yellow solid powder compounds 1-3, yield 65%.
(4) synthesis of compound 1-4
20mmol compound 1-3 and 100mmol butanoic acid are joined in the round-bottomed flask containing 20mmolEDCl and a small amount of DMAP20ml dichloromethane solution, nitrogen protection. Stop after reaction 7h. Decompression steams solvent, and pillar layer separation obtains yellow solid powder compounds 1-4, yield 65%.
(5) fluorescent dye compound A1Synthesis
It is 25 DEG C by compound 1-4 in reaction temperature, response time is 5 hours, and reaction dissolvent is selected from dichloromethane, adds the trifluoroacetic acid of 1.5 times of equivalents, after reaction terminates, with water (containing 1% trifluoroacetic acid): acetonitrile=10:1-1:10 prepares the fluorescent dye compound A of liquid phase for eluant1, productivity 21%.1HNMR (400MHz, DMSO) �� 10.11 (s, 1H), 7.97-7.93 (m, 2H), 7.63 (d, 2H), 6.74 (d, 2H), 6.43 (s, 2H), 4.31 (dd, 2H), 3.48 (m, 5H), 2.78 (s, 3H), 2.23-2.16 (m, 6H), 1.01-0.97 (dd, 5H).
Embodiment 2
Fluorescent dye compound A1Solvation effect detection test
Use the fluorescent dye compound A of above-described embodiment 1 synthesis1It is added separately in different solvents, measures the ultra-violet absorption spectrum in different solvents and fluorescence emission spectrum. Test result shows, along with the change of solvent polarity, the maximum absorption wavelength in ultra-violet absorption spectrum has corresponding movement, and fluorescence emission spectrum also exists the movement of maximum emission wavelength too. Fig. 2 (a) is probe A1Ultra-violet absorption spectrum in different solvents, Fig. 2 (b) is fluorescent dye compound A1Fluorescence emission spectrum in different solvents. Instrument is Agilent8453 ultraviolet spectrophotometer and AgilentCaryEclipse spectrofluorophotometer respectively.
Embodiment 3
Fluorescent dye compound A1Two-photon effective absorption cross-section detection test:
Adopt femtosecond two-photoninduced fluorescence method, utilize the NaOH solution (pH11) of fluorescein as reference, fluorescent dye compound A embodiment 1 synthesized1The test of two photon absorption cross section in the dimethyl sulfoxide solvent being added separately to, solution concentration used is all 1 �� 10-4M, as follows by computing formula:
C in formula is the concentration of solution, and n is the refractive index of solvent, can table look-up and obtain.F is up-conversion fluorescence intensity, experiment record. �� is two photon absorption cross section. The physical quantity of reference solution is used that subscript r represents.
Measure the two-photon effective absorption cross-section (�� ��, Fig. 3) in DMSO solvent. The excitaton source of two-photon fluorescence excitation spectrum is a stand lock mould femto second titanium sapphire laser, laser pulse width 70fs, repetition rate 80MHz, the average output power 1.5W (780nm) of laser instrument, wavelengthtunable scope 700��980nm, femtosecond laser wavelength is adjusted to required test wavelength in an experiment. Shown in experimental result (Fig. 3), fluorescent dye compound A1Two-photon effective absorption cross-section can reach 238GM at 770nm.
Embodiment 4
Prepare fluorescent dye compound A2:
(1) synthesis of compound 2-1
By 20mmolB1And 100mmolB2Join in the round-bottomed flask containing 10ml dichloromethane solution, add 20mmolEDCl and a small amount of DMAP, nitrogen protection, react under room temperature. After reaction terminates, rotation, except solvent, column chromatography for separation, obtains faint yellow solid powder-product, compound 2-1, yield 76%.
(2) synthesis of compound 2-2
20mmol compound 2-1 and 40mmol iodomethane are joined in the round-bottomed flask containing 10ml dichloromethane solution, nitrogen protection. Stop after room temperature reaction 8h. After reaction terminates, rotation, except solvent, column chromatography for separation, obtains faint yellow solid powder-product, compound 2-2, yield 89%.
(3) synthesis of compound 2-3
20mmol compound 2-2 and 80mmol ethylenediamine are joined in the round-bottomed flask containing 20mmolEDCl and a small amount of DMAP20ml dichloromethane solution, nitrogen protection. Stop after reaction 5h. Decompression steams solvent, and pillar layer separation obtains yellow solid powder compounds 2-3, yield 65%.
(4) synthesis of compound 2-4
20mmol compound 2-3 and 100mmol chloro butanoic acid are joined in the round-bottomed flask containing 20mmolEDCl and a small amount of DMAP20ml dichloromethane solution, nitrogen protection. Stop after reaction 7h. Decompression steams solvent, and pillar layer separation obtains yellow solid powder compounds 2-4, yield 61%.
(5) fluorescent dye compound A2Synthesis
It is 25 DEG C by upper step compound 2-4 in reaction temperature, response time is 5 hours, reaction dissolvent is selected from dichloromethane, add the trifluoroacetic acid of 1.5 times of equivalents, after reaction terminates, with water (containing 1% trifluoroacetic acid): acetonitrile=10:1-1:10 prepares the fluorescent dye compound A of liquid phase for eluant2, productivity 19%.1HNMR (400MHz, DMSO) �� 10.17 (s, 1H), 7.98-7.94 (m, 2H), 7.63-7.60 (d, 2H), 6.74-6.70 (d, 2H), 6.33 (s, 2H), 4.31 (dd, 2H), 3.48 (m, 7H), 2.78 (s, 3H), 2.23-2.16 (m, 6H), 1.91-0.97 (m, 2H).
Embodiment 5
Fluorescent dye compound A2To the identification of endoplasmic reticulum in tumor living cell and labelling
Use the fluorescent dye compound A of embodiment 4 synthesis2, with the A of final concentration of 2.0 ��Ms2Hatch Hela cell respectively, at 37 DEG C, 5%CO2Hatch 30 minutes. Then, PBS concussion rinsing 5min �� 3, add cell culture medium, two-photon laser co-focusing imaging. Choose representative area, observe with oil mirror (100 ��), in triplicate. In imaging display Hela cell, endoplasmic reticulum has hyperfluorescence signal. The fluorescent collecting wave band of Fig. 4 is 490-550nm. Test result (Fig. 4) shows fluorescent dye compound A2Obvious fluorescence signal is had at the endoplasmic reticulum place of tumor cell.
Embodiment 6
Fluorescent dye compound A2Water-soluble detection test
Use the fluorescent dye compound A of above-described embodiment 4 synthesis2It is added to the water, measures at variable concentrations A2Absorbance under the maximum absorption wavelength of aqueous solution.Test result (Fig. 5) display is as compound A2When concentration is 6.0 ��Ms, absorbance offsets, i.e. fluorescent dye compound A2Dissolubility in water is 6.0 ��Ms. Fig. 5 is different probe A2Absorbance under the maximum absorption wavelength of concentration. Instrument is Agilent8453 ultraviolet spectrophotometer respectively.
Embodiment 7
Prepare fluorescent dye compound A3
(1) synthesis of compound 3-1
By 20mmolB1And 100mmolB2Join in the round-bottomed flask containing 10ml dichloromethane solution, add 20mmolEDCl and a small amount of DMAP, nitrogen protection, react under room temperature. After reaction terminates, rotation, except solvent, column chromatography for separation, obtains faint yellow solid powder-product, compound 3-1, yield 76%.
(2) synthesis of compound 3-2
Upper step 20mmol compound 3-1 and 40mmol iodomethane are joined in the round-bottomed flask containing 10ml dichloromethane solution, nitrogen protection. Stop after room temperature reaction 8h. After reaction terminates, rotation, except solvent, column chromatography for separation, obtains faint yellow solid powder-product, compound 3-2, yield 89%.
(3) synthesis of compound 3-3
Upper step 20mmol compound 3-2 and 80mmol ethylenediamine are joined in the round-bottomed flask containing 20mmolEDCl and a small amount of DMAP20ml dichloromethane solution, nitrogen protection. Stop after reaction 5h. Decompression steams solvent, and pillar layer separation obtains yellow solid powder compounds 3-3, yield 65%.
(4) synthesis of compound 3-4
Upper step 20mmol compound 3-3 and 100mmol acetic acid are joined in the round-bottomed flask containing 20mmolEDCl and a small amount of DMAP20ml dichloromethane solution, nitrogen protection. Stop after reaction 7h. Decompression steams solvent, and pillar layer separation obtains yellow solid powder compounds 3-4, yield 55%.
(5) fluorescent dye compound A3Synthesis
It is 25 DEG C by upper step compound 3-4 in reaction temperature, response time is 5 hours, reaction dissolvent is selected from dichloromethane, add the trifluoroacetic acid of 1.5 times of equivalents, after reaction terminates, with water (containing 1% trifluoroacetic acid): acetonitrile=10:1-1:10 prepares the fluorescent dye compound A of liquid phase for eluant3, productivity 19%.1HNMR (400MHz, DMSO) �� 10.17 (s, 1H), 7.97-7.94 (m, 2H), 7.63-7.61 (d, 2H), 6.74-6.71 (d, 2H), 6.33 (s, 3H), 4.31 (dd, 2H), 3.48 (m, 5H), 2.23-2.16 (m, 7H).
Embodiment 8
Fluorescent dye compound A3To the labelling of Golgi body in living cells
Use the fluorescent dye compound A of embodiment 7 synthesis3, with the A of final concentration of 2.0 ��Ms3Hatch Hela cell respectively, at 37 DEG C, 5%CO2Hatch 30 minutes. Then, PBS concussion rinsing 5min �� 3, add cell culture medium, two-photon laser co-focusing imaging. Choose representative area, observe with oil mirror (100 ��), in triplicate. Test result (Fig. 6) shows fluorescent dye compound A3Hyperfluorescence signal is had at Hela Variations of Golgi Apparatus In The Cells. The fluorescent collecting wave band of Fig. 6 is 490-550nm.
Embodiment 9
Fluorescent dye compound A3Two-photon effective absorption cross-section detection test:
Adopt femtosecond two-photoninduced fluorescence method, utilize the NaOH solution (pH11) of fluorescein as reference, fluorescent dye compound A embodiment 7 synthesized3The test of two photon absorption cross section in the dimethyl sulfoxide solvent being added separately to, solution concentration used is all 1 �� 10-4M, as follows by computing formula:
C in formula is the concentration of solution, and n is the refractive index of solvent, can table look-up and obtain.F is up-conversion fluorescence intensity, experiment record. �� is two photon absorption cross section. The physical quantity of reference solution is used that subscript r represents.
Measure two-photon effective absorption cross-section (�� ��, Fig. 7) in DMSO. The excitaton source of two-photon fluorescence excitation spectrum is a stand lock mould femto second titanium sapphire laser, laser pulse width 70fs, repetition rate 80MHz, the average output power 1.5W (780nm) of laser instrument, wavelengthtunable scope 700��980nm, femtosecond laser wavelength is adjusted to required test wavelength in an experiment. Shown in experimental result (Fig. 7), fluorescent dye compound A3Two-photon effective absorption cross-section can reach 263GM at 770nm.
Embodiment 10
Prepare fluorescent dye compound A4
(1) synthesis of compound 4-1
By 20mmolB1And 100mmolB2Join in the round-bottomed flask containing 10ml dichloromethane solution, add 20mmolEDCl and a small amount of DMAP, nitrogen protection, react under room temperature. After reaction terminates, rotation, except solvent, column chromatography for separation, obtains faint yellow solid powder-product, compound 4-1, yield 76%.
(2) synthesis of compound 4-2
20mmol compound 4-1 and 40mmol iodomethane are joined in the round-bottomed flask containing 10ml dichloromethane solution, nitrogen protection. Stop after room temperature reaction 8h. After reaction terminates, rotation, except solvent, column chromatography for separation, obtains faint yellow solid powder-product, compound 4-2, yield 89%.
(3) synthesis of compound 4-3
20mmol compound 4-2 and 80mmol ethylenediamine are joined in the round-bottomed flask containing 20mmolEDCl and a small amount of DMAP20ml dichloromethane solution, nitrogen protection. Stop after reaction 5h. Decompression steams solvent, and pillar layer separation obtains yellow solid powder compounds 4-3, yield 65%.
(4) synthesis of compound 4-4
20mmol compound 4-3 and 100mmol monoxone are joined in the round-bottomed flask containing 20mmolEDCl and a small amount of DMAP20ml dichloromethane solution, nitrogen protection. Stop after reaction 7h. Decompression steams solvent, and pillar layer separation obtains yellow solid powder compounds 4-4, yield 53%.
(5) fluorescent dye compound A4Synthesis
It is 25 DEG C by compound 4-4 in reaction temperature, response time is 5 hours, and reaction dissolvent is selected from dichloromethane, adds the trifluoroacetic acid of 1.5 times of equivalents, after reaction terminates, with water (containing 1% trifluoroacetic acid): acetonitrile=10:1-1:10 prepares liquid phase fluorescent dye compound A for eluant4, productivity 15%.1HNMR (400MHz, DMSO) �� 10.14 (s, 1H), 7.96-7.89 (m, 2H), 7.65-7.60 (d, 2H), 6.75-6.71 (d, 2H), 6.33 (s, 3H), 4.31 (dd, 2H), 4.22 (m, 2H), 3.48 (m, 5H), 2.23-2.16 (m, 4H).
Embodiment 11
Fluorescent dye compound A4To the labelling of endoplasmic reticulum in living cells
Use the fluorescent dye compound A of embodiment 10 synthesis4, with the A of final concentration of 2.0 ��Ms4Hatch Hela cell respectively, at 37 DEG C, 5%CO2Hatch 30 minutes. Then, PBS concussion rinsing 5min �� 3, add cell culture medium, two-photon laser co-focusing imaging. Choose representative area, observe with oil mirror (100 ��), in triplicate. Test result shows (Fig. 8) fluorescent dye compound A4Hela cell has in endoplasmic reticulum hyperfluorescence signal. The fluorescent collecting wave band of Fig. 8 is 490-550nm.
Embodiment 12
Fluorescent dye compound A4Water-soluble detection test
Use the fluorescent dye compound A of above-described embodiment 10 synthesis4It is added to the water, measures at variable concentrations A4Absorbance under the maximum absorption wavelength of aqueous solution.Test result shows as fluorescent dye compound A2When concentration is 7.0 ��Ms, absorbance offsets, and namely test result shows: fluorescent dye compound A4Dissolubility in water is 7.0 ��Ms. Fig. 9 is different probe A4Absorbance under the maximum absorption wavelength of concentration. Instrument is Agilent8453 ultraviolet spectrophotometer respectively.
Above content is in conjunction with concrete preferred implementation further description made for the present invention, it is impossible to assert that specific embodiment of the invention is confined to these explanations. For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, it is also possible to make some simple deduction or replace, protection scope of the present invention all should be considered as belonging to. As a kind of purposes that fluorescent dye is noval chemical compound of the present invention; it cannot be assumed that the compound of the present invention is only for fluorescent dye; for general technical staff of the technical field of the invention; under be used as the consideration of the identical mechanism of action of fluorescent dye based on the compounds of this invention; some simple inferences can also be made; draw other application purpose of the compound of the present invention, all should be considered as belonging to protection scope of the present invention.
Claims (5)
1. the Two-photon fluorescent dye being parent with naphthalene of end-functionalization, has the structure of formula I:
In formula I:
R1Selected from-H and C1-4Alkyl;
R2Selected from-Cl ,-Br ,-I ,-H or C1-4Alkyl;
L1Selected from-NH (CH2)pNH-, wherein p is the integer of 1-4;
L2Selected from-(CH2)p-, wherein p is the integer of 1-4.
2. the Two-photon fluorescent dye being parent with naphthalene of the end-functionalization described in claim 1, selected from compound A1-A4:
3. the preparation method of the Two-photon fluorescent dye being parent with naphthalene of the end-functionalization described in claim 1, comprises the steps:
1) compound B1With R1-I 1:1-1:2 in molar ratio reacts, and prepares compound C1:
Reaction temperature is 20-80 DEG C, and the response time is 1-10 hour, and reaction dissolvent is selected from dichloromethane, ethanol, methanol, acetone or its mixture;
2) under DMAP catalysis, compound C1, compound B2React according to mol ratio 1:5:1-1:20:5 with EDCl, prepare compound C2:
Reaction temperature 30-180 DEG C, in 1-20 hour response time, reaction dissolvent is selected from dichloromethane, ethanol, methanol, acetone, glycerol, ethyl acetate, acetic acid or its mixture;
3) under DMAP catalysis, compound C2��H-L1-H and EDCl reacts according to mol ratio 1:1:1-1:5:5, prepares compound C3:
Reaction temperature 25-120 DEG C, in 1-18 hour response time, reaction dissolvent is selected from dichloromethane, ethanol, methanol, acetone, glycerol, ethyl acetate, acetic acid or its mixture;
4) under DMAP catalysis, compound C3��R2-L2-COOH and EDCl reacts according to mol ratio 1:5:1-1:20:5, prepares compound C4:
Reaction temperature 25-100 DEG C, in 1-10 hour response time, reaction dissolvent is selected from dichloromethane, ethanol, methanol, acetone, glycerol, ethyl acetate, acetic acid or its mixture,
5) compound C4Hydrolysis prepares compound I, and reaction system adds 1-10 times of compound C4The trifluoroacetic acid of molar equivalent, acetic acid or sodium hydroxide;
Reaction temperature is 25-50 DEG C, and the response time is 1-6 hour, and reaction dissolvent is selected from dichloromethane, ethanol, methanol, acetone, water or its mixture.
4. in claim 1-2 end-functionalization described in any claim with naphthalene be parent Two-photon fluorescent dye detect and the application in labelling at biology simultaneously.
5. the application described in claim 4, it is characterised in that it is the mitochondrion in living cells and lysosome that described biology detects with marking target simultaneously.
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