CN108484622A - The synthesis of multi signal fluorescence probe and its application for distinguishing detection Hcy, Cys and GSH simultaneously - Google Patents
The synthesis of multi signal fluorescence probe and its application for distinguishing detection Hcy, Cys and GSH simultaneously Download PDFInfo
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Abstract
Distinguish the multi signal fluorescent molecular probe for detecting homocysteine (Hcy), cysteine (Cys) and glutathione (GSH) in cell simultaneously by blue, green, yellow three kinds of different fluorescent emission signals the invention discloses one kind, the chemical structure of general formula of such multi signal molecular probe is as follows：
Description
Technical field
The invention belongs to technical field of analytical chemistry, and in particular to the synthesis of a kind of multi signal fluorescence probe and such
Probe is distinguished quantitatively detect Hcy, Cys and GSH in the environment simultaneously, and triple channel fluorescence imaging intracellular Hcy, Cys and GSH's answers
With.
Background technology
Small molecule biological thiol such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) are in biology department
Play extremely important role in system.Hcy can be synthesized under a variety of enzymes auxiliary, by methionine in mammal in serum
The normal concentration of Hcy is 5-12 μM, and excessively high Hcy contents can cause folic acid and VB12The diseases such as shortage, senile dementia
(Chemical Communication,2014, 50, 6967-6969)；In addition, intracellular Cys can be synthesized by Hcy, just
Normal intracellular concentration is 30-200 μM, and Cys contents can lead to slow-growing, the diseases such as cardiovascular and hepatic injury extremely
(Chemical Communication, 2013, 49, 9176-9178).GSH is that low molecular weight most abundant in cell is biological
Mercaptan can be synthesized by Cys, glutamic acid and glycine in organism, and normal concentration is 0.5-10 mM, as intracellular anti-
Oxidant can scavenger-cell toxin and free radical, maintain vivo oxidation reduction reaction balance, while maintaining stem cell normal function
(Stem Cell Reports, 2018,10,600-614), but GSH contents can cause leucocyte content abnormal extremely,
HIV infection, the potential risks such as cancer (Biosensors and Bioelectronics, 2017,90,117-124).Due to
These three biological thiols have similar structure and reactivity, there is Hcy in the cell and are converted into Cys, Cys is converted into GSH
The variation of this bio-metabolic process, one of which content may cause the association of both other to change, and many diseases
Generation is all closely related with their changes of contents, therefore, develops efficient detection method/technology and is used for while distinguishing detection simultaneously
These three biological thiols of quantitative Hcy, Cys and GSH, especially quantitatively endogenous cellular Hcy, Cys and GSH is to understanding its work in depth
With mechanism and function, have very important significance to the early diagnosis of disease.
In recent years, multi signal fluorescence probe can monitor and visualize simultaneously two or more analyses in living cells because of it
Object and widely paid close attention to, because be provided for a kind of non-destructive, it is highly selective with sensitivity, it is visualization, easy to operate
Analysis method.Although small molecule biological thiol homocysteine (Hcy), cysteine (Cys) and glutathione (GSH)
Structure it is similar with reactivity, but have also been developed some mono-/bis-fluorescence channel fluorescence probes for detect Hcy, Cys and
1 kind or 2 kinds (such as Chinese patent CN105524055A, CN105588823A, CN105601658A, Journal of in GSH
the American Chemical Society, 2014, 136, 574-577、Chemical Science, 2016, 7,
256-260、Analytical Chemistry, 2015, 87, 3460–3466、Chemistry - An Asian
Journal, 2017,12,2098-2103 etc.).But the mercaptan fluorescence probe reported also cannot achieve same using triple channel
These three biological thiols of time zone sorting survey Hcy, Cys and GSH, especially Hcy, Cys and GSH tri- of endogenous cellular various concentration
Channel fluorescence imaging analysis detects.
Invention content
In view of the foregoing, overcome some the deficiencies in the prior art, the purpose of the present invention is to provide one kind to utilize threeway
Road fluorescence signal distinguishes the fluorescent molecular probe of detection Hcy, Cys and GSH simultaneously, can utilize different fluorescence signals（Color）Point
Hcy, Cys and GSH in imaging cells are analysed, some analyzing detecting methods are provided for fields such as analysis detection, medicine early diagnosis
And thinking.
The present invention also aims to provide a kind of preparation method is simple, synthesis of multi signal mercaptan fluorescent molecular probe with
Application process.
The present invention solves the problems, such as that the specific technical solution taken is, the synthesis of a kind of multi signal fluorescence probe and its same time zone
Sorting surveys the application of Hcy, Cys and GSH, and the chemical structure of general formula of probe is as follows：
, wherein R=hydrogen/alkyl/aromatic radical.
The synthesis of detection Hcy, Cys and GSH multi signal fluorescence probe is distinguished simultaneously, which is characterized in that the fluorescent molecular
The preparation method of probe includes the following steps：
Step 1. synthesizes 9- hydroxyls -2,3,6,7- tetrahydrochysene -1H, 5H, 11H pyrans simultaneously [2,3-F] pyrido [3,2,1-ij] quinoline
Quinoline -11- ketone
A. appropriate diphenyl malonate and 8- hydroxyl julolidines are added in dry toluene, 100 DEG C are reacted 8-12 hours,
B. it being filtered after reaction is cooled to room temperature, solid is dried in vacuo after being washed with ether, obtains hydroxyl -2 celadon solid 9-,
3,6,7- tetrahydrochysene -1H, 5H, 11H pyrans simultaneously [2,3-F] pyrido [3,2,1-ij] quinoline -11- ketone；
Step 2. synthesizes the chloro- 11- oxos -2,3 of 9-, 6,7- tetrahydrochysene -1H, 5H, 11H pyrans simultaneously [2,3-F] pyrido [3,2,1-
Ij] quinoline -10- formaldehyde
Under the conditions of nitrogen protection, by the appropriate dry n,N-Dimethylformamide steamed again（DMF）It is slowly added to isometric
Phosphorus oxychloride（POCl3）In, it is stirred 30 minutes at 20-50 DEG C, obtains red solution,
By 9- hydroxyls -2,3,6,7- tetrahydrochysene -1H, 5H, 11H pyrans simultaneously [2,3-F] pyrido [3,2,1-ij] quinoline -11- ketone
It is dissolved in appropriate n,N-Dimethylformamide, is added dropwise to stepMixed solution in, mixture continue under nitrogen protection
60 DEG C are stirred to react 12 hours；
By stepIn reaction solution pour into appropriate ice water, adjust pH to 5 ~ 6 with 20% NaOH solution, generate a large amount of heavy
It forms sediment, filtering, solid wash 3 times with appropriate amount of deionized water, vacuum drying, obtains the chloro- 11- oxos -2,3 of 9-, 6,7- tetrahydrochysene -1H, 5H,
11H pyrans simultaneously [2,3-F] pyrido [3,2,1-ij] quinoline -10- formaldehyde；
Step 3. synthesizes multi signal fluorescence probe（E）-3-（The chloro- 11- oxos -2,3 of 9-, 6,7- tetrahydrochysene -1H, 5H, 11H- pyrans
And [2,3-f] pyrido [3,2,1-ij] quinoline -10- bases）- 2- alpha-cyanoacrylates（Ester）
By the chloro- 11- oxos -2,3 of 9-, 6,7- tetrahydrochysene -1H, 5H, 11H pyrans simultaneously [2,3-F] pyrido [3,2,1-ij] quinoline
Quinoline -10- formaldehyde and cyanoacetic acid（Ester）It is added in appropriate anhydrous methylene chloride, appropriate triethylamine is added dropwise, reaction is stirred at room temperature；
By stepMiddle reaction solution is added dropwise in appropriate absolute ethyl alcohol, and obtained solid is dried in vacuo, obtains claim by filtering
Fluorescent molecular probe described in 1（E）-3-（The chloro- 11- oxos -2,3 of 9-, 6,7- tetrahydrochysene -1H, 5H, 11H- pyrans simultaneously [2,3-f] pyrrole
Pyridine simultaneously [3,2,1-ij] quinoline -10- bases）- 2- alpha-cyanoacrylates（Ester）.
The multi signal fluorescent molecular probe of the present invention distinguishes detection Hcy, Cys and GSH application method simultaneously：Without special theory
Bright, it is 4 that probe molecule, which is usually dissolved in organic phase and water phase volume ratio, at room temperature:For analyzing detection in the environment of 6, have
Machine is mutually dimethyl sulfoxide (DMSO) (DMSO), and water phase is the aqueous solution of phosphate buffer solution (PBS) and analyte of pH=7.4.
The multi signal mercaptan fluorescence probe specific features of the present invention are as follows：The fluorescence probe dimethyl sulfoxide (DMSO)
(DMSO) it dissolves, probe molecule is dissolved in organic phase and water phase (4:6, v/v) in solution, after being reacted at room temperature 15 minutes with Hcy,
Emit the strong blue-fluorescence of 467 nm under 375 nm excitation wavelengths；After being reacted at room temperature 15 minutes with Cys, in 400 nm excitation wavelengths
The strong green fluorescence of 503 nm of lower transmitting；After being acted on 15 minutes with GSH, the yellow of 568 nm is emitted under 500 nm excitation wavelengths
Fluorescence.Probe itself just will produce strong blue-fluorescence when detection Hcy without apparent fluorescence with 375 nm excitations, with detection
Unstressed configuration or fluorescence are very weak when excitation wavelength (400/500 nm) excitation of Cys/GSH, and both other are also such.Therefore it realizes
Particular excitation and fluorescent emission signals detect specific analyte, in the presence of three kinds of biological thiols are equal, using different excitations and
Fluorescent emission signals also can well distinguish three.Above-mentioned fluorescent molecular probe realizes in same testing conditions similarly hereinafter time zone
Sorting surveys Hcy, Cys and GSH, to NAC, Gly, Ala, His, Met, Thr, Lys, Asp, Glu, Pro, Ser,
NaHS, NaHSO3, EtSH, PhSH, the amino acid such as n- Butylamine, aniline, containing sulfur derivatives, amine derive
Object nothing obvious responses to, and limits the detection of Hcy, Cys and GSH three respectively down to 0.2 nM, 0.7 nM and 1 nM.Therefore, originally
The multi signal fluorescent molecular probe of disclosure of the invention, which can be realized, quantitatively detects the highly sensitive differentiation of three.
Description of the drawings
Fig. 1 is that the multi signal fluorescence probe of the present invention is imaged L-02 by blue, green, red three color fluorescences simultaneously（Normal hepatocytes
Cell）The fluorescence imaging figure of endogenous cellular Hcy, Cys and GSH.
Fig. 2 is the hydrogen nuclear magnetic resonance spectrogram of the multi signal fluorescence probe of the present invention（R=ethyl）.
Specific implementation mode
Following further describes the present invention with reference to the drawings.
The general synthetic routes of multi signal fluorescent molecular probe of the present invention are shown below：
。
Embodiment 1. synthesizes 9- hydroxyls -2,3,6,7- tetrahydrochysene -1H, 5H, 11H pyrans simultaneously [2,3-F] pyrido [3,2,1-
Ij] quinoline -11- ketone
A. 16.3 g (63.4 mmol) diphenyl malonates and 10.0 g (52.8 mmol) 8- hydroxyl julolidines are added
Enter in 100 mL dry toluenes, be stirred to react at 100 DEG C 8-12 hours,
B. after complete reaction, filtered after reaction solution being cooled to room temperature, wash filter cake with ether, obtained solid vacuum drying obtains
To celadon solid 9- hydroxyls -2,3,6,7- tetrahydrochysene -1H, 5H, 11H pyrans simultaneously [2,3-F] pyrido [3,2,1-ij] quinoline -
11.0 g of 11- ketone, yield 80.9%.
Embodiment 2. synthesizes the chloro- 11- oxos -2,3 of 9-, 6,7- tetrahydrochysene -1H, 5H, 11H pyrans simultaneously [2,3-F] pyrido
[3,2,1-ij] quinoline -10- formaldehyde
Under the conditions of nitrogen protection, 18 mL are dried to the n,N-Dimethylformamide steamed again（DMF）It is slowly added to isometric
Phosphorus oxychloride（POCl3）In, it is stirred 30 minutes at 20-50 DEG C, obtains red solution,
By 15.0 g (58.3 mmol) 9- hydroxyls -2,3,6,7- tetrahydrochysene -1H, 5H, 11H pyrans simultaneously [2,3-F] pyrido
[3,2,1-ij] quinoline -11- ketone is dissolved in 70 mL n,N-Dimethylformamide, is added dropwise to stepMixed solution in,
Mixture continues to be stirred to react 12 hours for 60 DEG C under nitrogen protection；
After complete reaction, by stepIn reaction solution be poured slowly into 500 mL ice water, with 20% NaOH solution tune
PH to 6 is saved, a large amount of precipitations, filtering are generated, filter cake is washed 3 times with appropriate amount of deionized water, obtained solid is dried in vacuo, 9- is obtained
Chloro- 11- oxos -2,3,6,7- tetrahydrochysene -1H, 5H, 11H pyrans simultaneously [2,3-F] pyrido [3,2,1-ij] quinoline -10- formaldehyde
15.4 g, yield 86.9%.
Embodiment 3. synthesizes multi signal fluorescence probe（E）-3-（The chloro- 11- oxos -2,3 of 9-, 6,7- tetrahydrochysene -1H, 5H,
11H- pyrans simultaneously [2,3-f] pyrido [3,2,1-ij] quinoline -10- bases）- 2- cyanacrylates（R=ethyl）
By the chloro- 11- oxos -2,3 of 5.0 g (16.5 mmol) 9-, 6,7- tetrahydrochysene -1H, 5H, 11H pyrans simultaneously [2,3-F] pyrrole
Simultaneously 30 mL anhydrous two are added in [3,2,1-ij] quinoline -10- formaldehyde and the mixing of 2.79 g (24.7 mmol) ethyl cyanoacetate for pyridine
In chloromethanes, then 0.2 mL triethylamines are added dropwise, reaction is stirred at room temperature；
After complete reaction, by stepMiddle reaction solution is added dropwise in 300 mL absolute ethyl alcohols, filtering, obtained solid drying,
Obtain fluorescent molecular probe described in claim 1（E）-3-（The chloro- 11- oxos -2,3 of 9-, 6,7- tetrahydrochysene -1H, 5H, 11H- pyrans
And [2,3-f] pyrido [3,2,1-ij] quinoline -10- bases）4.5 g of -2- cyanacrylates, yield 68.5%.
Embodiment 4. synthesizes multi signal fluorescence probe（E）-3-（The chloro- 11- oxos -2,3 of 9-, 6,7- tetrahydrochysene -1H, 5H, 11H
Pyrans simultaneously [2,3-F] pyrido [3,2,1-ij] quinoline -10- bases）- 2- alpha-cyanoacrylates（R=hydrogen）
A. by the chloro- 11- oxos -2,3 of 1.0 g (3.29 mmol) 9-, 6,7- tetrahydrochysene -1H, 5H, 11H pyrans simultaneously [2,3-F] pyrrole
Simultaneously the anhydrous dichloromethanes of 10 mL are added in [3,2,1-ij] quinoline -10- formaldehyde and the mixing of 0.56 g (6.58 mmol) cyanoacetic acid for pyridine
In alkane, then 0.05 mL triethylamines are added dropwise, reaction is stirred at room temperature；
B. after complete reaction, reaction solution in step A is added dropwise in 50 mL absolute ethyl alcohols, is filtered, solid drying obtains right
It is required that the fluorescent molecular probe described in 1（R=hydrogen）（E）-3-（The chloro- 11- oxos -2,3 of 9-, 6,7- tetrahydrochysene -1H, 5H, 11H pyrans
And [2,3-F] pyrido [3,2,1-ij] quinoline -10- bases）0.7 g of -2- alpha-cyanoacrylates, yield 57.3%.
5. multi signal fluorescent molecular probe of embodiment（For this sentences the probe of R=ethyl, no specified otherwise, other
Probe operation step is similar）The application of detection Hcy, Cys and GSH is distinguished in an in vitro environment
Multi signal fluorescent molecular probe spectral quality experiment of the present invention：Probe is dissolved in dimethyl sulfoxide (DMSO) (DMSO)
It is configured to the probe solution of a concentration of 1 mM, Cys, Hcy and GSH aqueous solution of a concentration of 1mM is respectively configured.Specific test mode
For：Take the probe solution of 20 μ L, 1 mM, the Cys/Hcy/GSH aqueous solutions of the analysis pure DMSO, the desired amount of 1mM of 780 μ L and
For the desired amount of PBS buffered aqueous solutions in the sample cell of 2 mL, it is 4 that all tests, which keep the volume ratio of organic phase and water phase,:6
(each test sample total volume be 2 mL), such as after when requiring test Hcy 20 μM a concentration of, probe is reacted with Hcy
Fluorescence intensity, preparing sample situation is：Take the probe solution of 20 μ L, 1 mM, 1 mM of analysis pure DMSO, 40 μ L of 780 μ L
Hcy aqueous solutions and 1160 μ L PBS buffer solutions in the sample cell of 2 mL, at room temperature concussion shake up 15 minutes after
Its fluorescent emission intensity is measured with the excitation wavelength of 375 nm, other test operations are similar with above-mentioned steps.The multi signal probe
Molecule is realized distinguishes detection Hcy, Cys and GSH these three biological thiols, tool with different excitation wavelengths and fluorescent emission signals
There are very high sensitivity, detection limit to be very suitable for living cells endogenous Hcy, Cys respectively down to 0.2 nM, 0.7 nM and 1 nM
With imaging/quantitative analysis of GSH.
6. L-02 of embodiment（Normal liver cell）Endogenous Hcy, Cys and GSH triple channel fluorescence imaging analysis
L-02 cells are passaged in the burnt ware cell culture medium of copolymerization, after being cultivated 24 hours under standard growth conditions, are added suitable
Amount (5 μM) continuation of probe are cultivated 30 minutes under standard growth conditions, are then taken a picture under confocal fluorescent microscopic, respectively
Fluorescence imaging L-02 endogenous cellular Hcy, Cys and GSH are carried out with blue, green, red fluorescence channel, it will be seen from figure 1 that
The multi signal fluorescence probe of the present invention successfully realizes endogenic Hcy, Cys and GSH triple channel fluorescence imaging analysis in cell, and
Fluorescence power has reacted the height of Hcy, Cys and GSH content to a certain extent, has the energy of potential quantitative Hcy, Cys and GSH
Power, in great application values in fields such as biochemistry, analysis detection, the early diagnosis of disease.
The synthesis of the multi signal fluorescence probe of the present invention and its application for distinguishing detection Hcy, Cys and GSH simultaneously, development
The probe of a kind of efficiently simple multi-channel detection mercaptan, can be used for distinguishing detection Hcy, Cys and GSH simultaneously, is based on same spy
Different chemical reactions occur from Hcy, Cys and GSH using probe under needle, same testing conditions, generate different fluorescent materials,
To emit the fluorescence of blue, green, yellow three kinds of colors under particular excitation wavelength, achieve the purpose that while distinguishing detection, success is real
Endogenic Hcy, Cys and GSH in existing triple channel while fluorescence imaging cell, and with potential quantitatively Hcy, Cys and GSH
Ability.Wish that the development for multi signal biological thiol fluorescence probe from now on provides some thinkings.Although present disclosure is
Detailed introduction is made through passing through above preferred embodiment, but it should be appreciated that the description above is not considered as to the present invention
Limitation.After those skilled in the art have read the above, a variety of modifications and substitutions for the present invention all will be it is aobvious and
It is clear to.Therefore, there is the synthesis of the multi signal fluorescence probe of technical characteristic described herein and its distinguish detection Hcy, Cys simultaneously
With the application of GSH, the protection domain of this patent is each fallen within.
Claims (4)
1. multi signal fluorescence probe distinguishes detection Hcy, Cys and GSH simultaneously, which is characterized in that the fluorescent molecular probe
Chemical structure of general formula is such as shown in (1)：
（1）, wherein R=hydrogen/alkyl/aromatic radical.
2. the synthesis of multi signal fluorescence probe as described in claim 1, which is characterized in that the preparation of the fluorescent molecular probe
Method includes the following steps：
Step 1. synthesizes 9- hydroxyls -2,3,6,7- tetrahydrochysene -1H, 5H, 11H pyrans simultaneously [2,3-F] pyrido [3,2,1-ij] quinoline
Quinoline -11- ketone
A. appropriate diphenyl malonate and 8- hydroxyl julolidines are added in dry toluene, 100 DEG C are reacted 8-12 hours,
B. it being filtered after reaction is cooled to room temperature, solid is dried in vacuo after being washed with ether, obtains hydroxyl -2 celadon solid 9-,
3,6,7- tetrahydrochysene -1H, 5H, 11H pyrans simultaneously [2,3-F] pyrido [3,2,1-ij] quinoline -11- ketone；
Step 2. synthesizes the chloro- 11- oxos -2,3 of 9-, 6,7- tetrahydrochysene -1H, 5H, 11H pyrans simultaneously [2,3-F] pyrido [3,2,1-
Ij] quinoline -10- formaldehyde
Under the conditions of nitrogen protection, by the appropriate dry n,N-Dimethylformamide steamed again（DMF）It is slowly added to isometric three
Chlorethoxyfos（POCl3）In, it is stirred 30 minutes at 20-50 DEG C, obtains red solution,
By 9- hydroxyls -2,3,6,7- tetrahydrochysene -1H, 5H, 11H pyrans simultaneously [2,3-F] pyrido [3,2,1-ij] quinoline -11- ketone
It is dissolved in appropriate n,N-Dimethylformamide, is added dropwise to stepMixed solution in, mixture continue under nitrogen protection
60 DEG C are stirred to react 12 hours；
By stepIn reaction solution pour into appropriate ice water, adjust pH to 5 ~ 6 with 20% NaOH solution, generate a large amount of heavy
It forms sediment, filtering, solid wash 3 times with appropriate amount of deionized water, vacuum drying, obtains the chloro- 11- oxos -2,3 of 9-, 6,7- tetrahydrochysene -1H, 5H,
11H pyrans simultaneously [2,3-F] pyrido [3,2,1-ij] quinoline -10- formaldehyde；
Step 3. synthesizes multi signal fluorescence probe（E）-3-（The chloro- 11- oxos -2,3 of 9-, 6,7- tetrahydrochysene -1H, 5H, 11H- pyrans
And [2,3-f] pyrido [3,2,1-ij] quinoline -10- bases）- 2- alpha-cyanoacrylates（Ester）
By the chloro- 11- oxos -2,3 of 9-, 6,7- tetrahydrochysene -1H, 5H, 11H pyrans simultaneously [2,3-F] pyrido [3,2,1-ij] quinoline -
10- formaldehyde and cyanoacetic acid（Ester）It is added in appropriate anhydrous methylene chloride, appropriate triethylamine is added dropwise, reaction is stirred at room temperature；
By stepMiddle reaction solution is added dropwise in appropriate absolute ethyl alcohol, and obtained solid is dried in vacuo, obtains claim 1 by filtering
The fluorescent molecular probe（E）-3-（The chloro- 11- oxos -2,3 of 9-, 6,7- tetrahydrochysene -1H, 5H, 11H- pyrans simultaneously [2,3-f] pyrrole
Pyridine simultaneously [3,2,1-ij] quinoline -10- bases）- 2- alpha-cyanoacrylates（Ester）.
3. the synthesis of multi signal fluorescence probe as claimed in claim 2, which is characterized in that the stepThe chloro- 11- oxygen of 9-
Generation -2,3,6,7- tetrahydrochysene -1H, 5H, 11H pyrans simultaneously [2,3-F] pyrido [3,2,1-ij] quinoline -10- formaldehyde and cyanoacetic acid
（Ester）Molar ratio is 1:1.5-2.
4. multi signal fluorescence probe as described in claim 1 distinguishes the application of detection Hcy, Cys and GSH simultaneously, feature exists
In the fluorescent molecular probe can quantitatively detect Hcy, Cys and GSH in the environment, utilize the burnt microscopic imaging fluorescence of copolymerization
Technology can realize the application of Hcy, Cys and GSH in blue, green, red three kinds of colors difference imaging cells.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109912612A (en) * | 2019-04-04 | 2019-06-21 | 郑州大学 | A kind of synthesis, preparation and its application based on Molecular Logic Gates identification Cys, GSH and HOCl fluorescence probe |
| CN110894193A (en) * | 2018-09-13 | 2020-03-20 | 南京大学 | Synthesis of novel fluorescent probe and application of novel fluorescent probe in cysteine detection |
| CN111718319A (en) * | 2020-07-27 | 2020-09-29 | 山西大学 | Fluorescent probe for distinguishing and detecting mercaptan and monitoring Cys/GSH metabolism and preparation method thereof |
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