CN101962536B - Lysosome targeted fluorescent substance and synthesis method thereof - Google Patents
Lysosome targeted fluorescent substance and synthesis method thereof Download PDFInfo
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- CN101962536B CN101962536B CN 201010505360 CN201010505360A CN101962536B CN 101962536 B CN101962536 B CN 101962536B CN 201010505360 CN201010505360 CN 201010505360 CN 201010505360 A CN201010505360 A CN 201010505360A CN 101962536 B CN101962536 B CN 101962536B
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Abstract
The invention relates to a lysosome targeted fluorescent substance of which the structural formula is shown in the specifications. The synthesis method of the compound comprises the following steps: taking trifluoroacetic acid as a solvent and a reaction catalyst, enabling 4-bromaniline and paraformaldehyde to react to generate a bromine-substituted intermediate product, and then, carrying out Heck reaction to substitute bromine by 4-vinyl-N,N-dimethyl benzylamine. The method of the invention effectively applies the Heck reaction and has simple synthesis paths and higher yield, and the obtained product has stable properties and has the functions of fluorescence emission of visible light and lysosome targeting.
Description
Technical field
The present invention relates to a kind of fluorescent substance and synthetic method thereof of lysosome target, belong to the cell imaging field.
Background technology
The development of modern cell microoptic imaging technique and the meticulous functional study of organoid requires to have high-level efficiency, highly sensitive and have the development and application of the fluorescent reagent of organoid targeting.The micro-imaging of cell and organoid, even the Visual Dynamic of the meticulous function of organoid indicates, be the Focal point and difficult point of modern micro-imaging always, for mankind's various diseases and cell aging provide scientific basis intuitively, have extremely important biological significance and new drug development and be worth.
One not clear and definite effectively target spot of organoid can realize that the chemical small molecules of targeting of specific cells device is very rare.This field is just becoming the focus of domestic and international research at present, and has great academic significance and using value.So far, take the specific cells device as target spot, as plastosome, lysosome and golgi body etc. can act on these target spots in active somatic cell, and that uses comparative maturity has dyestuff and antibody two classes.One dyestuff all can have photobleaching and photic toxicity, namely loses fluorescence activity under the irradiation of ultraviolet-visible in the short period of time, perhaps produces singlet oxygen etc. very easily to the material of viable cell generation toxic action, is unfavorable for the Non-invasive of active somatic cell; Although the antibody low toxicity, its expensive price and easily inactivation drawbacks limit the cell imaging of antibody use.And dyestuff and the alternative scope of antibody itself also smaller, be no matter at fluorescence excitation and transmitting boundary or aspect the organoid targeting, all can't satisfy the needs of scientific research and practical application.
Summary of the invention
Technical problem to be solved by this invention is to provide fluorescent substance and the synthetic method thereof of a kind of efficient, low toxicity, lysosome target.
Technical scheme of the present invention is:
A kind of fluorescent substance of lysosome target, structural formula is as follows:
Above-mentioned substance can excite under UV-light, also can at the near-infrared region two-photon excitation, produce the visible fluorescence emission.By the viable cell experiment confirm, above-mentioned fluorescent substance can specificly act on lysosome, realizes lysosomal blur-free imaging.
The synthetic method of above-mentioned fluorescent substance: take 4-bromaniline and paraformaldehyde as raw material, with trifluoroacetic acid as solvent and catalysts, 0～25 ℃ of reaction obtains intermediate 5,11-two bromo-[1,5 diazocines] diazocine, again with Heck reaction with the bromine in intermediate with 4-vinyl-N, the N-dimethyl benzylamine replaces.
Reaction scheme is as follows:
N in paraformaldehyde is positive integer.
The synthetic method of above-mentioned fluorescent substance is more specifically:
In the 4-bromaniline: the paraformaldehyde mol ratio is 1: 2～2.5 ratio, and 4-bromaniline and paraformaldehyde are evenly mixed, and is added dropwise to excessive trifluoroacetic acid as solvent and catalysts, then 0～25 ℃ of reaction 70～160 hours;
It is 7～8 that reaction soln is neutralized to the pH value, uses organic solvent extraction, and the organic solvent evaporate to dryness is most of, and crystallization is intermediate 5,11-two bromo-[1,5 diazocine] diazocine;
Take the intermediate molar weight as benchmark; 4-vinyl-the N that adds 2.4～3.0 times; the N-dimethyl benzylamine; also add 5～10% palladium as catalyzer, 10～20% triphenyl phosphorus is as part, and the cesium carbonate of 4～6 times is as alkali; with anhydrous N; dinethylformamide is solvent, is heated to 137 ℃ of reactions 8～10 hours under nitrogen protection, separates obtaining fluorescent substance.
The above-mentioned fluorescent substance that the present invention relates to is a kind of axisymmetric compound molecule.Carbon-carbon double bond (CH=CH) in the compound molecule is as cut-point, carbon-carbon double bond near a side of molecule symmetry axis and carbon-carbon double bond away from the side transition of electron each other of molecule symmetry axis give body and acceptor, couple together by this conjugated structure of carbon-carbon double bond.Molecule under ultraviolet excitation or near-infrared region two-photon excitation, transition of electron delocalization range extension, molecule returns the process of ground state from excited state, launches macroscopic fluorescence.The bio-active group of this fluorescent substance molecule is N, N-dimethylbenzyl amine groups, namely in molecule at the group of carbon-carbon double bond away from symmetry axis one side.This bio-active group has the lysosomal effect of targeted cells.
In cell experiment, add the fluorescent substance solution of 2-10 μ mol/L concentration in the cell culture medium, and culturing cell 2～8 hours, adopt the LASER Light Source irradiating cell of 405nm wavelength, can see clearly lysosomal form in cell; Perhaps adopt 790nm wavelength laser light source irradiation cell, under the two-photon fluorescence imaging pattern, can observe lysosomal clear form in cell.
Fluorescent substance of the present invention is by bio-active group N, the targeting of N-dimethylbenzyl amido arrives in Cytolysosome, under ultraviolet excitation or near-infrared region two-photon excitation, emission naked eyes visible fluorescence, show clearly lysosomal form in cell, the position in cell except lysosome does not have fluorescent emission.Accordingly, fluorescent substance of the present invention can be used as the Cytolysosome preparation.
The inventive method is effectively used the Heck reaction, and synthesis path is simple, and productive rate is higher, and the product characteristics that obtains is stable, has visible fluorescence emission and lysosome targeting.The cell experiment confirmation, this compound can enter Cytolysosome by target, all obtains Cytolysosome image clearly under ultraviolet excitation or near-infrared region two-photon excitation.
Description of drawings
Fig. 1 Hela cell adds imaging after fluorescent substance;
Fig. 2 Hela cell adds imaging after lysosome staining agent LT Red;
Fig. 3 Hela cell is total to positioning and imaging after adding fluorescent substance and LT Red.
Embodiment
Below with specific examples, technical scheme of the present invention is described further.
Embodiment one: intermediate 5, and 11-two bromo-[1,5 diazocine] diazocine synthetic:
6.9g 4-bromaniline and 2.4g paraformaldehyde (n=30) mixing are placed in the 250mL Kjeldahl flask, add magnetic stir bar, reaction system is placed in cold-trap cools to-20 ℃.In the situation that magnetic agitation, slowly be added dropwise to the trifluoroacetic acid of 80mL in the system with the 100mL constant pressure funnel, trifluoroacetic acid drips about 50 minutes fully.4-bromaniline and paraformaldehyde in the question response system dissolve fully, and the temperature of system is elevated to 0 ℃, continue reaction 140 hours.Finish reaction, be made into the solution of 1mol/L with sodium hydroxide (NaOH) dissolution of solid.Reacted solution slowly is added dropwise in sodium hydroxide solution, stirs simultaneously, separate out a large amount of solids.After dripping, the pH of solution is 7 left and right, uses the 100mL chloroform extraction, and continuous extraction 3 times merges organic phase.To the 50mL left and right, standing 20 hours, there is bulk crystals to separate out in solution the organic phase solution rotary evaporation, take out bulk crystals with tweezers, obtain intermediate 5,11-two bromo-[1,5 diazocines] diazocine 5g, be faint yellow solid, productive rate is 53.8%.
1H?NMR(CDCl
3)δ4.12(d，J＝17.1Hz，2H)，4.25(s，2H)，4.64(d，J＝16.8Hz，2H)，7.00(d，J＝8.4Hz，2H)，7.05(s，2H)，7.27(d，J＝6.9Hz，2H)。
Embodiment two: intermediate 5,11-two bromo-[1,5 diazocine] diazocine synthetic
6.9g 4-bromaniline and 3.0g paraformaldehyde (n=30) mixing are placed in the 250mL Kjeldahl flask, add magnetic stir bar, react under room temperature.In the situation that magnetic agitation, slowly be added dropwise to the trifluoroacetic acid of 80mL in the system with the 100mL constant pressure funnel, trifluoroacetic acid drips about 50 minutes fully.4-bromaniline and paraformaldehyde in the question response system dissolve fully, continue reaction 140 hours.Finish reaction, be made into the solution of 1mol/L with sodium hydroxide (NaOH) dissolution of solid.Reacted solution slowly is added dropwise in sodium hydroxide solution, stirs simultaneously, separate out a large amount of solids.After dripping, the pH of solution is 8 left and right, uses the 100mL chloroform extraction, and continuous extraction 3 times merges organic phase.To the 50mL left and right, standing 20 hours, there is bulk crystals to separate out in solution the organic phase solution rotary evaporation, take out bulk crystals with tweezers, obtain intermediate 5,11-two bromo-[1,5 diazocines] diazocine 1.2g, be faint yellow solid, productive rate is 12.9%.
1HNMR(CDCl
3)δ4.12(d，J＝17.1Hz，2H)，4.25(s，2H)，4.64(d，J＝16.8Hz，2H)，7.00(d，J＝8.4Hz，2H)，7.05(s，2H)，7.27(d，J＝6.9Hz，2H)。Embodiment three: intermediate 5,11-two bromo-[1,5 diazocine] diazocine synthetic
6.9g 4-bromaniline and 2.4g paraformaldehyde (n=30) mixing are placed in the 250mL Kjeldahl flask, add magnetic stir bar.In the situation that-20 ℃ and magnetic agitation, slowly be added dropwise to the trifluoroacetic acid of 80mL in the system with the 100mL constant pressure funnel, trifluoroacetic acid drips about 50 minutes fully.4-bromaniline and paraformaldehyde in the question response system dissolve fully, continue to react 70 hours under 0 ℃.Finish reaction, be made into the solution of 1mol/L with sodium hydroxide (NaOH) dissolution of solid.Reacted solution slowly is added dropwise in sodium hydroxide solution, stirs simultaneously, separate out a large amount of solids.After dripping, the pH of solution is 7 left and right, uses the 100mL chloroform extraction, and continuous extraction 3 times merges organic phase.To the 50mL left and right, standing 20 hours, there is bulk crystals to separate out in solution the organic phase solution rotary evaporation, take out bulk crystals with tweezers, obtain intermediate 5,11-two bromo-[1,5 diazocines] diazocine 2.6g, be faint yellow solid, productive rate is 27.9%.
1HNMR(CDCl
3)δ4.12(d，J＝17.1Hz，2H)，4.25(s，2H)，4.64(d，J＝16.8Hz，2H)，7.00(d，J＝8.4Hz，2H)，7.05(s，2H)，7.27(d，J＝6.9Hz，2H)。Embodiment four: fluorescent substance synthetic
Choose the there-necked flask of the 250mL of a drying, connect reflux condensing tube, logical nitrogen is got rid of air wherein, then vacuumizes, anhydrous and oxygen-free in triplicate, guarantee system.Add 5 of drying, 11-two bromo-[1,5 diazocine] diazocine 760mg (2m mol), 4-vinyl-N, N-dimethyl benzylamine 773mg (4.8m mol), catalyst acetic acid palladium Pd (OAc) in flask
210mg, the triphenyl phosphorus (PPh of 48mg
3) as part, and 2.6g cesium carbonate (Cs
2CO
3) as alkali, then add the anhydrous DMF of 150mL (DMF) as solvent.Under nitrogen protection, system temperature is elevated to 137 ℃, reaction 8h.Be cooled to room temperature after having reacted, remove by filter inorganic salt, the solvent evaporate to dryness.Use column chromatography product (hexanaphthene: chloroform=1: 1), collect second fluorescence band, obtain end product 810mg, productive rate is 57.7%, is faint yellow solid,
1H NMR (CDCl
3) δ 2.12 (s, 12H), 3.39 (s, 4H), 4.20 (d, J=16.5Hz, 2H), 4.32 (s, 2H), 4.71 (d, J=16.8Hz, 2H), 6.93 (s, 4H), 7.03 (s, 2H), 7.11 (d, J=8.4Hz, 2H), 7.24 (d, J=8.1Hz, 4H), 7.31 (d, J=8.4Hz, 2H), 7.39 (d, J=8.1Hz, 4H);
13C NMR (300MHz CDCl3) δ 44.63,58.80,63.39,67.02,125.11,125.32,125.64,126.41,127.52,128.04,128.11,129.87,133.37,136.71,147.68; FT-MS (ESI) C
37H
41N
4[M+H]
+: 541.33291.
Embodiment five: fluorescent substance synthetic
There-necked flask with the 250mL of the anhydrous and oxygen-free of a drying connects reflux condensing tube, logical nitrogen protection.Add 5 of drying, 11-two bromo-[1,5 diazocine] diazocine TB1760mg (2m mol), 4-vinyl-N, N-dimethyl benzylamine 966mg (6m mol), catalyst acetic acid palladium Pd (OAc) in flask
210mg, the triphenyl phosphorus (PPh of 48mg
3) as part, and 2.6g cesium carbonate (Cs
2CO
3) as alkali, then add the anhydrous DMF of 120mL (DMF) as solvent.System temperature is elevated to 137 ℃, reaction 10h.Be cooled to room temperature after having reacted, remove by filter inorganic salt, the solvent evaporate to dryness.Use column chromatography product (hexanaphthene: chloroform=1: 1), collect second fluorescence band, obtain end product 1050mg, productive rate is 74.8%, is faint yellow solid,
1H NMR (CDCl
3) δ 2.12 (s, 12H), 3.39 (s, 4H), 4.20 (d, J=16.5Hz, 2H), 4.32 (s, 2H), 4.71 (d, J=16.8Hz, 2H), 6.93 (s, 4H), 7.03 (s, 2H), 7.11 (d, J=8.4Hz, 2H), 7.24 (d, J=8.1Hz, 4H), 7.31 (d, J=8.4Hz, 2H), 7.39 (d, J=8.1Hz, 4H);
13C NMR (300MHz CDCl3) δ 44.63,58.80,63.39,67.02,125.11,125.32,125.64,126.41,127.52,128.04,128.11,129.87,133.37,136.71,147.68; FT-MS (ESI) C
37H
41N
4[M+H]
+: 541.33291.
Embodiment six:
The application of fluorescent substance target lysosome of the present invention and imaging
Take synthetic fluorescent substance 1.7mg of the present invention, take dimethyl sulfoxide (DMSO) (DMSO) as solvent, be mixed with the solution of 10mmol/L.Adopt human hela cell (Hela) as target cell, add wherein the fluorescent substance solution of 10 μ mol/L concentration, cultivated 2 hours.Adopt the two-photon Laser Scanning Confocal Microscope imaging of 790nm LASER Light Source, can observe lysosomal clear form in cell, as shown in Figure 1.
Embodiment seven:
As target cell, add wherein Cytolysosome staining agent LT Red solution with mankind's cervical cancer cell (Hela), cultivated 8 hours.Adopt the Laser Scanning Confocal Microscope imaging of 405nm LASER Light Source, obtain Fig. 2.
Embodiment eight:
As target cell, add wherein the fluorescent substance solution of the present invention of 10 μ mol/L concentration with mankind's cervical cancer cell (Hela), and Cytolysosome staining agent LT Red solution as a comparison, cultivated 2 hours.Adopt the Laser Scanning Confocal Microscope imaging of 405nm LASER Light Source, obtain the highly lysosome image of coincidence, as shown in Figure 3.
Claims (4)
1. the fluorescent substance of a lysosome target, structural formula is as follows:
2. the preparation method of the fluorescent substance of lysosome target claimed in claim 1, it is characterized by: take 4-bromaniline and paraformaldehyde as raw material, with trifluoroacetic acid as solvent and catalysts, 0 ~ 25 ℃ of reaction obtains intermediate product 5,11-two bromo-[1,5 diazocines] diazocine, then with Heck reaction with the bromine in intermediate product with 4-vinyl-N, the N-dimethyl benzylamine replaces.
3. the preparation method of the fluorescent substance of lysosome target according to claim 2 is characterized by:
In the 4-bromaniline: the paraformaldehyde mol ratio is the ratio of 1:2 ~ 2.5, and 4-bromaniline and paraformaldehyde are evenly mixed, and is added dropwise to excessive trifluoroacetic acid as solvent and catalysts, then 0 ~ 25 ℃ of reaction 70 ~ 160 hours;
It is 7 ~ 8 that reaction soln is neutralized to the pH value, uses organic solvent extraction, and the organic solvent evaporate to dryness is most of, and crystallization is intermediate 5,11-two bromo-[1,5 diazocine] diazocine;
Take the intermediate molar weight as benchmark; 4-vinyl-the N that adds 2.4 ~ 3.0 times; the N-dimethyl benzylamine; also add 5 ~ 10% palladium as catalyzer; 10 ~ 20% triphenylphosphine is as part, and the cesium carbonate of 4 ~ 6 times is as alkali, with anhydrous N; dinethylformamide is solvent, is heated to 137 ℃ of reactions 8 ~ 10 hours under nitrogen protection.
4. the described fluorescent substance of claim 1 is as the purposes on the Cytolysosome preparation.
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| CZ307772B6 (en) * | 2017-09-26 | 2019-04-24 | Univerzita Karlova | The coumarin derivative of the Tröger base with cyanine substitution and its use |
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| CN102942559B (en) * | 2012-10-29 | 2014-08-06 | 安徽大学 | Flexible ether oxygen chain pyrimidine derivative, preparation method and application thereof |
| CN106770120A (en) * | 2016-12-29 | 2017-05-31 | 厦门生光生物科技有限公司 | A kind of lysosomal pH changes detection reagent and its application |
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| 分子靶向疗法及其药物在恶性胶质瘤治疗中的应用;周翔等;《现代诊断与治疗》;20070330;第18卷(第2期);106-109 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CZ307772B6 (en) * | 2017-09-26 | 2019-04-24 | Univerzita Karlova | The coumarin derivative of the Tröger base with cyanine substitution and its use |
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