CN110156695A - It is a kind of detect cysteine fluorescence probe and its application - Google Patents

It is a kind of detect cysteine fluorescence probe and its application Download PDF

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Publication number
CN110156695A
CN110156695A CN201910408574.5A CN201910408574A CN110156695A CN 110156695 A CN110156695 A CN 110156695A CN 201910408574 A CN201910408574 A CN 201910408574A CN 110156695 A CN110156695 A CN 110156695A
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China
Prior art keywords
probe
cysteine
fluorescence probe
fluorescence
added
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CN201910408574.5A
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Inventor
林伟英
王利
任明光
李子红
代礼旋
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University of Jinan
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University of Jinan
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6447—Fluorescence; Phosphorescence by visual observation
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1003—Carbocyclic compounds
    • C09K2211/1011—Condensed systems
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms

Abstract

The present invention provides a kind of fluorescence probe for detecting cysteine, chemical structural formulas are as follows:.It is made after being reacted by the reaction product of 9,10- phenanthrenequione, 6- hydroxyl -2- naphthaldehyde and ammonium acetate with acryloyl chloride.The probe can be realized the high sensitivity of cysteine probe and highly selective, can be used as in display aqueous solution and in biological cell cysteine variation indicator, real-time qualitative and quantitative optical colorimetry detection can be carried out.The present invention is that one kind is simple, and quickly, sensitive cysteine specific detection agents have broad application prospects in biomolecule detection field.

Description

It is a kind of detect cysteine fluorescence probe and its application
Technical field
The invention belongs to technical field of analytical chemistry, and in particular to it is a kind of detect cysteine fluorescence probe and its answer With.
Background technique
Cysteine (Cysteine, Cys) is amino acid common in a kind of organism, in human normal physiological condition Under can synthesize.Cysteine is the amino acid in 20 kinds of natural amino acids uniquely containing reproducibility sulfydryl, is sulphur in cell The center material of metabolism.In zooblast, Cys takes part in synthesis and folding, redox equilibrium regulation, the cell letter of peptide chain A large amount of life processes such as number conduction, removing toxic substances, apoptosis, there is indispensable critical role in life system.Cysteine Lack the risk that can increase some diseases.Such as cancer, cutaneous lesions, hair decoloration, psychoneurosis and immune dysfunction. In addition, studies have shown that will lead to the generation of a variety of diseases when concentrations of mercaptans exception.Cys concentration is relatively low will lead to hypoevolutism, It pigmentation, skin injury, hepatic injury, oedema, lethargic sleep and has bad physiques.Therefore, the detection level of Cys is for academic research Have great importance with teaching.
So far, the method for detecting Cys has: spectrophotometry, liquid chromatography, fluorescent spectrometry, electrochemistry volt-ampere Method, colorimetric method.However, wherein most of require complicated instrument.Especially these technologies are generally not suitable in normal physiological Under the conditions of real-time detection Cys.It is at low cost currently, Imaging-PAM, because of its high sensitivity, detection limit is low, have real-time and The advantages that noninvasive imaging performance is a kind of method of detection small molecule biology to have a great attraction.Therefore, one kind is developed to be used for The fluorescence probe of selective imaging is still necessary in Cys body.
Summary of the invention
For problem of the prior art, the present invention provides a kind of fluorescence probe for detecting cysteine, and fast response time resists Interference performance is strong.
It is a further object of the present invention to provide a kind of above-mentioned fluorescence probe detection solution in or biological cell in half Guang ammonia The application of acid.
To achieve the above object, the present invention adopts the following technical scheme that.
A kind of fluorescence probe detecting cysteine, referred to as PIC, chemical structural formula are as shown in the formula (I):
Formula (I).
The preparation method of above-mentioned fluorescence probe, comprising the following steps:
(1) 9,10- phenanthrenequione, 6- hydroxyl -2- naphthaldehyde and the ammonium acetate back flow reaction in acetic acid, after reaction solution be added etc. bodies Ponding filters, and solid is using ethyl acetate: petroleum ether=1:2 v/v crosses silicagel column as leacheate, and eluent removal solvent obtains chemical combination Object 1:；
(2) in the presence of triethylamine, compound 1 and acryloyl chloride react in methylene chloride, and after reaction, decompression is spin-dried for solvent Crude product is obtained, using ethyl acetate: petroleum ether=1:5 v/v crosses silicagel column as leacheate, and eluent removal solvent obtains fluorescence probe.
Its synthetic route is as follows:
。
A kind of application of above-mentioned fluorescence probe in preparation detection solution, cell in cysteine reagent.
The invention has the following advantages that
The synthesis of fluorescence probe of the invention only needs two steps that can complete, and last handling process is relatively easy；The probe energy The high sensitivity of cysteine probe and highly selective is enough realized, can be used as in display aqueous solution and half Guang ammonia in biological cell The indicator of acid variation can carry out real-time qualitative and quantitative optical colorimetry detection.The present invention is that one kind is simple, quickly, spirit Quick cysteine specific detection agents have broad application prospects in biomolecule detection field.
Detailed description of the invention
Fig. 1 is one middle probe PIC of embodiment1H NMR spectra；
Fig. 2 is situation of change of the probe PIC with the increase fluorogram of cysteine；
Fig. 3 is selective histogram of the probe PIC to different ions and molecule；
Fig. 4 be probe PIC in cysteine solution with the variation of fluorescence color after ultraviolet light irradiation；
Fig. 5 is that probe PIC is applied to the cysteine fluorescence imaging carried out in cell.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention will be further described, but the present invention is not by the limit of following embodiments System.
The synthesis of 1 fluorescence probe of embodiment
It weighs respectively 9,10- phenanthrenequione (146 mg, 0.7mM, 1eq), 6- hydroxy benzaldehyde (362 mg, 2.1mM, 3 eq), second Sour ammonium (185 mg, 1.05 mM, 1.5 eq) is dissolved in 20 ml acetic acid, 110 DEG C of heating reflux reaction 10h.It is detected with TCL plate Reaction, after fully reacting, is cooled to room temperature, is poured into water suction filtration, gained crude product is separated with silicagel column, silica gel particle size For 200-300 mesh, leacheate is ethyl acetate/petroleum ether=1:2, and decompression is spin-dried for eluent and obtains compound 1:, yield 67%；
It is dissolved in compound 1 (169 mg, 0.47 mM, 1eq) in 5ml methylene chloride, 2 drop triethylamines is added, 0 DEG C is added dropwise dropwise Acryloyl chloride (72 mg, 0.7 mM, 1.5eq) reacts at room temperature 5h, is detected and is reacted with TCL plate, and after fully reacting, decompression is spin-dried for molten Agent obtains crude product, is then separated with silicagel column, silica gel particle size be 200-300 mesh, eluant, eluent proportion be ethyl acetate/ Petroleum ether=1:5, decompression are spin-dried for eluent, obtain fluorescence probe, yield 46%,1H NMR spectra such as Fig. 1.
Response of 2 fluorescence probe of embodiment to various concentration cysteine
Cysteine fluorescence probe prepared by Example 1 is dissolved in dimethyl sulfoxide (DMSO), and 1mmol/L stock solution is made. It is added in the centrifuge tube of 4 mL from 20 μ L are taken out in stock solution, the Cys standard that different equivalents (0-20 eq) are added is molten Liquid is diluted to 2 mL with the solution that PBS buffer solution (0.1 mol/L, pH=7.5) and DMF volume ratio are 9:1, measures its fluorescence Property.Fluorescence spectrum is as shown in Figure 2.From Figure 2 it can be seen that fluorescence intensity gradually increases at 414 nm with the addition of Cys concentration By force.
Selectivity of 3 fluorescence probe of embodiment to different ions
It is added in the centrifuge tube of 4 mL from 20 μ L are taken out in embodiment 2 in fluorescence probe stock solution, is separately added into equimolar The different competition molecular criteria solution of amount, one of them is added the Cys standard solution of equimolar amounts, detects solution after 30 min Fluorescence emission spectrum variation, Fig. 3 it can be found that the fluorescence of other ion-pair compounds has little effect, and Cys solution plus Enter to make the fluorescence significant changes of compound.
Visual retrieval of 4 fluorescence probe of embodiment to cysteine
It is added in two sample cells from 40 μ L respectively are taken out in embodiment 2 in fluorescence probe stock solution, an addition 3mL DMF, another adds 80 Μ m Cys after 3mL DMF is added, as shown in Figure 4: under ultraviolet light, it can be observed that solution is sent out Raw apparent color change: the solution colour of PIC becomes blue near colorless, illustrate PIC be it is a kind of have to add lustre to sense function The fluorescence probe of energy.
Imaging applications of 5 fluorescence probe of embodiment in living cells
35 mm of the Hela cell inoculation of suitable density to sterilizing are imaged in culture dish, in CO2Incubator (temperature 37 DEG C, 5 % CO2) in culture, after cell is adherent, 5 μM of probe DMSO solutions are added to the culture solution for giving birth to Hela cell In, it is imaged after 30 min are cultivated in carbon dioxide incubator with Laser Scanning Confocal Microscope；N- ethyl horse is added into system After carrying out the solution of 1 mM of acid imide (NEM), probe is added after waiting 30min, is excited with blue light；As a result as shown in figure 5, Wherein, a) be concentration and probe concentration be 5 μM be added in Hela cell cultivate 30min after light field figure, b) be NEM be added before blue channel it is glimmering Light image, c) be light field and fluorescence imaging figure coincidence pattern before NEM is added, d) it is to be added after 30 min are added in 1 m Μ NEM Probe is incubated for 30 min light field figures, e) it is glimmering to add blue channel after probe is incubated for 30 min after 1 m Μ NEM addition, 30 min Light image, f) it is to add light field and fluorescence imaging coincidence pattern after probe incubation 30min after 30 min are added in 1 m Μ NEM, G) added after probe is incubated for 30min after being 1 m Μ NEM addition, 30 min and 20 mM Cys light field figures, h be added) it is 1 m Μ Added after probe is incubated for 30min after 30 min of NEM addition and 20 mM Cys blue channel fluorescence imaging figures, i be added) it is 1 m Μ It is added after probe is incubated for 30min after 30 min of NEM addition and 20 mM Cys light fields and fluorescence imaging coincidence pattern is added.Light field at As can see the rough profile of cell, fluorescence imaging situation of the excitation observation when probe is only added then is carried out with blue light, Blue fluorescent emissions are observed at this time.After the aqueous solution of 1 mM of NEM is added into system, probe is added after waiting 30min, Excitation is carried out with blue light and is observed that blue-fluorescence weakens, while Cys fluorescence enhancement is added, and illustrates that this fluorescence probe can be right Endogenous cellular Cys carries out fluorescence imaging.

Claims (3)

1. a kind of fluorescence probe for detecting cysteine, chemical structural formula are as shown in the formula (I):
Formula (I).
2. a kind of preparation method of fluorescence probe as described in claim 1, which comprises the following steps:
(1) 9,10- phenanthrenequione, 6- hydroxyl -2- naphthaldehyde and the ammonium acetate back flow reaction in acetic acid, after reaction solution be added etc. bodies Ponding filters, and solid is using ethyl acetate: petroleum ether=1:2 v/v crosses silicagel column as leacheate, and eluent removal solvent obtains chemical combination Object 1:；
(2) in the presence of triethylamine, compound 1 and acryloyl chloride react in methylene chloride, and after reaction, decompression is spin-dried for solvent Crude product is obtained, using ethyl acetate: petroleum ether=1:5 v/v crosses silicagel column as leacheate, and eluent removal solvent obtains fluorescence probe.
3. a kind of application of fluorescence probe as described in claim 1 in preparation detection solution, cell in cysteine reagent.
CN201910408574.5A 2019-05-16 2019-05-16 It is a kind of detect cysteine fluorescence probe and its application Pending CN110156695A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115850203A (en) * 2022-12-30 2023-03-28 厦门大学附属心血管病医院 Cysteine selective probe and preparation method thereof

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CN106632212A (en) * 2016-12-19 2017-05-10 济南大学 Fluorescent probe for detecting cysteine in cell
CN108623533A (en) * 2018-07-13 2018-10-09 济南大学 A kind of fluorescence probe for detecting cysteine and application based on thiazole
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115850203A (en) * 2022-12-30 2023-03-28 厦门大学附属心血管病医院 Cysteine selective probe and preparation method thereof
CN115850203B (en) * 2022-12-30 2024-02-20 厦门大学附属心血管病医院 Cysteine selective probe and preparation method thereof

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