CN108727265A - A kind of difunctional fluorescence probe and its preparation method and application of detection formaldehyde and pH - Google Patents
A kind of difunctional fluorescence probe and its preparation method and application of detection formaldehyde and pH Download PDFInfo
- Publication number
- CN108727265A CN108727265A CN201810581986.4A CN201810581986A CN108727265A CN 108727265 A CN108727265 A CN 108727265A CN 201810581986 A CN201810581986 A CN 201810581986A CN 108727265 A CN108727265 A CN 108727265A
- Authority
- CN
- China
- Prior art keywords
- compound
- formaldehyde
- concentration
- application
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 0 CCCC*(C(C1C=CC(OCCC(C)(*C)C(C)(C)C2CC2)=CC1C1=CC=C)=O)C1=O Chemical compound CCCC*(C(C1C=CC(OCCC(C)(*C)C(C)(C)C2CC2)=CC1C1=CC=C)=O)C1=O 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
- C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
- C07D221/04—Ortho- or peri-condensed ring systems
- C07D221/06—Ring systems of three rings
- C07D221/14—Aza-phenalenes, e.g. 1,8-naphthalimide
-
- 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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- 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
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Materials Engineering (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a kind of difunctional fluorescence probes and preparation method and application of detection formaldehyde and pH.Shown in the structure of the fluorescence probe such as formula (I), preparation method is：Compound (II) and compound (III) generate compound (IV) under the action of cesium carbonate；Nitrine under alkaline condition, is reduced to amino by compound (IV), ultimately generates compound (I).The present invention provides application of the fluorescence probe in detection formaldehyde and/or pH value.The present invention provides intracellular formaldehyde and the fluorescence probe of pH can be detected simultaneously, a kind of effective research tool is provided for the physiological action of formaldehyde and pH in research cell.
Description
Technical field
The present invention relates to a kind of difunctional fluorescence probes and its preparation method and application of detection formaldehyde and pH
Background technology
1, formaldehyde belongs to activated carbon cluster, is a kind of generally acknowledged carcinogen.Formaldehyde is derived from endo-oxidase and demethyl
The endogenous metabolism object that enzyme generates, in normal cell, the concentration of formaldehyde can reach 0.4mM or so.Excessively high formaldehyde in vivo
It can cause neurodegenerative disease, stages alzheimer's disease and various cancers etc..However current Analysis Methods for Formaldehyde is mostly not
Intracellular concentration of formaldehyde can be monitored in real time, therefore, develop a kind of novel fluorescence probe to detect intracellular formaldehyde gesture must
Row.
2, the concentration level of pH and formaldehyde is complementary under physiological environment, and pH is de- by changing lysine specificity
The activity of methylase, to influence the generation of formaldehyde, and FA and acidic micro-environment can be with co-induction Bone cancer pains.Therefore, multiple
PH and formaldehyde are imaged simultaneously in miscellaneous organism, it would be possible to help to further appreciate that mutual relationship.In recent years, glimmering
The development of light probe can be used for living cells or the pH in tissue or formaldehyde imaging.But not single probe can be right simultaneously
Two kinds of analytes imaging in same system.
3, fluorescence probe due to it is highly sensitive, detection in real time, biological damage is small the advantages that and it is interested to researchers.
Present invention aims at one novel formaldehyde of exploitation and pH fluorescence probes, can accurately detect intracellular concentration of formaldehyde and pH
Value.
Invention content
It is an object of the present invention to provide a kind of difunctional fluorescence probes that can detect concentration of formaldehyde and pH value.
Second purpose of the invention is to provide a kind of fluorescence probe preparation method.
Third mesh of the present invention is to provide application of the fluorescence probe in formaldehyde and pH detections.
The present invention uses following technical scheme to achieve the above object：
A kind of fluorescence probe, shown in structure such as formula (I)：
A kind of preparation method of the fluorescence probe, the preparation method is that：
Compound (II) and compound (III) generate compound (IV) under the action of cesium carbonate；Compound (IV) is in alkali
Under the conditions of property, nitrine is reduced to amino, ultimately generates compound (I)；
Reaction route is as follows：
Further, the preparation method specifically carries out in accordance with the following steps：
(1) by compound (III), Cs2CO3By the amount of substance than 1:1~1.5 (preferably 1:1) it, is blended in anhydrous DMF
Solution in compound (II) is added dropwise, and by the mixture in N2It is stirred overnight in 40 DEG C~50 DEG C under protection；Revolving removes molten
Agent, and by chromatography over silica (preferably with volume ratio for 1:10 ethyl acetate/petroleum ether is elution reagent) purifying
Gained residue is to get compound (IV)；
(2) stannous chloride, triethylamine, benzenethiol are pressed into the amount of substance than 1:2~3.5:2~3.5 (preferably 1:3:3) add
Enter into acetonitrile solvent, 15min~30min is stirred at 30 DEG C~40 DEG C, compound (IV), wherein compound is then added
(IV) it is 0.5~1 with the amount of the substance of stannous chloride dihydrate ratio：1 (preferably 0.7：1), and at 30 DEG C~40 DEG C continue
Stir 12h~14h；Reactant is concentrated under reduced pressure, by silica column chromatography (preferably with volume ratio 1：3 ethyl acetate/stone
Oily ether is as elution reagent) it is purified, obtain compound (I).
The compounds of this invention (II) be disclosed compound, preparation method can refer to document (K.J.Bruemmer,
R.R.Walvoord,T.F.Brewer,G.Burgos-Barragan,N.Wit,L.B.Pontel,K.J.Patel and
C.J.Chang,Development of a General Aza-Cope Reaction Trigger Applied to
Fluorescence Imaging of Formaldehyde in Living Cells,J.Am.Chem.Soc.,2017,139,
5338.)
The compounds of this invention (III) is disclosed compound, and preparation method can refer to document (2.H.
Park,S.-K.Chang,Signaling of water content in organic solvents by
solvatochromism of a hydroxynaphthalimide-based merocyanine dye.Dyes and
Pigments,2015,122,324.)
The present invention provides application of the fluorescence probe in detecting formaldehyde.
Further, the formaldehyde is preferably intracellular formaldehyde, and concentration is not higher than 1mmol/L.
Further, the cell is human cervical carcinoma cell Hela cells.
The present invention also provides application of the fluorescence probe in detecting pH value.
Further, the pH value is preferably the pH value of intracellular lysosome, a concentration of pH4.5-5.5.
Further, the cell is human cervical carcinoma cell Hela cells.
Invention further provides application of the fluorescence probe in detection formaldehyde and pH value.
Further, the formaldehyde is intracellular formaldehyde, and concentration is not higher than 1mmol/L；The pH value is intracellular lysosome
PH value, a concentration of pH4.5-5.5.
Further, the cell is human cervical carcinoma cell Hela cells.
Compound (I) of the present invention is used as a kind of fluorescence probe, can be applied to the fluorogenic quantitative detection of formaldehyde.Described
The fluoroscopic examination principle of Quant Formaldehyde concentration is：Compound (I) after being reacted with formaldehyde, slough by reactive group, generates glimmering
Stimulative substance compound (III) measures the fluorescence intensity change of the probe at launch wavelength 555nm when excitation is 455nm, to obtain
Obtain concentration of formaldehyde.The principle that concentration of formaldehyde is detected using the fluorescence probe of the present invention is as follows：
Compound (I) of the present invention is used as a kind of fluorescence probe, can be applied to the fluorogenic quantitative detection of pH.Described determines
Amount pH concentration fluoroscopic examination principle be：For compound (I) after being reacted with pH, amino obtains one as pH sensitive groups
A proton forms NH3 +Reactive group, fluorescence intensity increase, and measure when excitation is 365nm, probe at launch wavelength 455nm
Fluorescence intensity change, to obtain pH value.The principle that pH concentration is detected using the fluorescence probe of the present invention is as follows：
Confocal fluorescent microscopic imaging experiment has been well demonstrated that fluorescence probe of the present invention can be examined in cell
The variation of concentration of formaldehyde and intracellular acid or alkali environment are surveyed, the metabolic mechanism to study intracellular provides new tool,
Biological field has good foreground.
Compared with prior art, the beneficial effects of the present invention are：The present invention provides can detect intracellular formaldehyde simultaneously
With the fluorescence probe of pH, a kind of effective research tool is provided for the physiological action of formaldehyde and pH in research cell.
Description of the drawings
Fig. 1 is the nucleus magnetic hydrogen spectrum for the compound (I) that in the present invention prepared by embodiment 1.
Fig. 2 is the nuclear-magnetism carbon spectrum for the compound (I) that in the present invention prepared by embodiment 1.
Fig. 3 is the compound (I) that in the present invention prepared by embodiment 1, in DMSO/PBS buffer solutions (pH=7.4, v/v=1/
199) under the conditions of, the fluorescent absorption spectrogram when formalin of different equivalents is added.
Fig. 4 is the compound (I) that in the present invention prepared by embodiment 1, is respectively 3.5,7.5 DMSO/PBS buffer solutions in pH
(v/v=1/199) the fluorescent absorption spectrogram in.
Fig. 5 is the compound (I) that in the present invention prepared by embodiment 1, in DMSO/PBS buffer solutions (pH=7.4, v/v=1/
199) under the conditions of, the fluorescence emission spectrogram of compound when formalin of different equivalents is added.Fig. 5 excitation wavelength 455nm, launch wavelength
555nm。
Fig. 6 be the present invention in embodiment 1 prepare compound (I) (0.5 μM), DMSO/PBS buffer solutions (pH=7.4,
V/v=1/199 under the conditions of), with the fluorogram changed over time in formaldehyde (0.5mM) mechanism.Fig. 6 excitation wavelengths
455nm, launch wavelength 555nm.
Fig. 7 is the compound (I) that in the present invention prepared by embodiment 1, in DMSO/PBS buffer solutions (pH=7.4, v/v=1/
199) under the conditions of, the fluorogram of selective result.1-15 is respectively PBS, acetaldehyde, benzaldehyde, paranitrobenzaldehyde, para hydroxybenzene
Formaldehyde, acetone, formic acid, glucose, glutathione, homocysteine, cysteine, niter cake, hydrogen peroxide, tertiary butyl mistake
Hydrogen oxide, formaldehyde.Fig. 7 excitation wavelength 455nm, launch wavelength 555nm.
Fig. 8 is the compound (I) that in the present invention prepared by embodiment 1, in DMSO/PBS buffer solutions (pH=7.4, v/v=1/
199) under the conditions of, high-efficient liquid phase chromatogram and mass spectrogram before and after formaldehyde is added.
Fig. 9 is the compound (I) of the preparation of embodiment 1 in the present invention to formaldehyde fluorescence imaging in cervical cancer cell.
Figure 10 is the compound (I) of the preparation of embodiment 1 in the present invention to endogenous formaldehyde fluorescence imaging in cervical cancer cell.
Figure 11 is the compound (I) that in the present invention prepared by embodiment 1, is added to the DMSO/PBS buffer solutions (v/v of different pH
=1/199) linear relationship of fluorescence emission spectrogram of compound and fluorescence intensity and pH in.Excitation wavelength 365nm, launch wavelength
455nm。
Figure 12 is the compound (I) that in the present invention prepared by embodiment 1, in DMSO/PBS buffer solutions (v/v=1/199), pH
Under conditions of difference 4,7.4, the fluorescence intensity of probe changes with time.Excitation wavelength 365nm, launch wavelength 455nm.
Figure 13 is the compound (IV) (0.5 μM) that in the present invention prepared by embodiment 1, in DMSO/PBS buffer solutions (pH=
7.4, v/v=1/199) under the conditions of, the value of different pH and the variation scattergram of fluorescence intensity.Excitation wavelength 365nm, launch wavelength
455nm。
Figure 14 is the compound (I) that in the present invention prepared by embodiment 1, in DMSO/PBS buffer solutions (pH=7.4, v/v=1/
199) under the conditions of, the fluorogram of selective result.1-15 is respectively PBS, acetaldehyde, benzaldehyde, paranitrobenzaldehyde, para hydroxybenzene
Formaldehyde, acetone, formic acid, glucose, glutathione, homocysteine, cysteine, niter cake, hydrogen peroxide, tertiary butyl mistake
Hydrogen oxide, pH.Fig. 7 excitation wavelength 365nm, launch wavelength 455nm.
Figure 15 is the cell of compound (I) and the Lyso-Tracker Red of commercialization that in the present invention prepared by embodiment 1
Image.
Figure 16 is the compound (IV) (0.5 μM) that in the present invention prepared by embodiment 1, in DMSO/PBS buffer solutions (pH=
7.4, v/v=1/199) under the conditions of, the value of different pH and the variation scattergram of fluorescence intensity.Excitation wavelength 365nm, launch wavelength
455nm。
Specific implementation mode
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This.
Embodiment 1
(1) preparation of compound (IV)
By compound (III) (0.65g, 2.4mmol) and Cs2CO3(0.78g, 2.4mmol) is blended in 10ml anhydrous DMFs
Solution in compound (II) (0.67g, 2mmol) is added dropwise, and by the mixture in N2Under be stirred overnight in 40 DEG C~50 DEG C.?
Remove solvent under high vacuum, and by using eluant ethyl acetate/petroleum ether (v/v, 1:10) chromatography over silica
Purifying gained residue is to get 0.57g compounds (IV) (yield 30%).1H NMR(500MHz,CDCl3)δ8.50–8.34(m,
3H), 7.59 (dd, J=8.3,7.4Hz, 1H), 6.94 (d, J=8.3Hz, 1H), 6.00 (dd, J=17.5,10.9Hz, 1H),
5.20-5.03 (m, 2H), 4.34 (t, J=6.7Hz, 2H), 4.17-4.02 (m, 2H), 2.22 (dd, J=13.8,6.9Hz,
1H), 2.10 (dt, J=14.2,7.0Hz, 1H), 1.67 (tt, J=7.7,6.6Hz, 2H), 1.45 (s, 3H), 1.41 (dt, J=
14.2,7.2Hz, 2H), 1.13 (s, 6H), 0.95 (t, J=7.4Hz, 3H)13C NMR(126MHz,CDCl3)δ164.18,
163.58,159.52,143.42,133.07,131.20,129.08,128.22,125.69,123.19,122.23,114.94,
114.06,105.61,67.10,65.70,45.53,39.89,34.57,30.13,22.31,22.13,20.29,18.01,
13.75.C25H30N4O3(M+H)435.2,found 435.2.
(2) preparation of compound (I)
Stannous chloride dihydrate (1g, 0.33mmol) is added in round-bottomed flask, then be added benzenethiol (0.11g,
It 1mmol) is mixed in 5ml acetonitrile solvents with triethylamine (0.10g, 1mmol), and mixture is stirred at 30-40 DEG C
Compound (IV) (0.1g, 0.23mmol) is then added in acetonitrile solvent, and is stirred for 12h at 30-40 DEG C by 15min.It will
Reactant be concentrated under reduced pressure, by using eluant ethyl acetate/petroleum ether (v/v, 1：3) silica column chromatography carries out pure
Change, obtains 120mg final compounds (I) probe DPFP.1H NMR(500MHz,CDCl3)δ8.47(s,3H),7.58(s,1H),
7.09 (d, J=8.3Hz, 1H), 6.07 (dd, J=17.4,10.8Hz, 1H), 5.29-5.06 (m, 2H), 4.56 (s, 1H),
4.45 (dd, J=15.8,8.2Hz, 1H), 4.30-4.00 (m, 2H), 2.21 (s, 1H), 1.71 (ddd, J=12.7,8.6,
6.7Hz, 2H), 1.47 (dt, J=15.1,7.4Hz, 2H), 1.35-1.16 (m, 9H), 0.99 (t, J=7.4Hz, 3H)13C
NMR(126MHz,CDCl3)δ164.38,163.89,133.31,131.35,128.30,125.69,122.35,115.06,
106.09,58.47,53.43,40.09,30.28,20.43,18.45,13.88.C25H30N4O3(M+H)409.2413,found
409.2492.
Fluorescence spectrum detection of 2 compound of embodiment (I) (5 μM) under different concentration of formaldehyde.
The compound (I) for accurately weighing the preparation of a certain amount of embodiment 1, is configured to a concentration of 0.1mM's with dimethyl sulfoxide (DMSO)
Probe mother liquor, liquid-transfering gun are drawn 2 μ L and are added in 0.394mL PBS buffer solution (pH=7.4), and 4 μ L difference equivalents are separately added into
Formalin (make concentration of the final formaldehyde in mixed solution be respectively 0,0.0025,0.0075,0.01,0.025,0.04,
0.05,0.06,0.075,0.1,0.15,0.25,0.4,0.5,0.6,0.75,1,2,5mM), after reacting 3h at 37 DEG C, it is added
Into cuvette, the fluorescent absorption spectrum of compound (I) is then measured.
The experimental results showed that with the raising of concentration of formaldehyde, influx and translocation of the compound (I) at 455nm wavelength.It says
Bright, compound (I) reacts with formaldehyde, generates " turn-on " phenomenon, and fluorescence pattern is shown in Fig. 3.
The fluorescent absorption detection of 3 compound of embodiment (I) (5 μM) at various ph values.
The compound (I) for accurately weighing the preparation of a certain amount of embodiment 1, is configured to a concentration of 0.1mM's with dimethyl sulfoxide (DMSO)
Probe mother liquor, liquid-transfering gun, which draws 2 μ L and is added to the PBS buffer solution of 0.398mL difference pH value, (makes the final mixed solution pH value be
3.5,7.5)；2 μ L probe mother liquors separately are drawn using liquid-transfering gun, are added in 0.394mL PBS buffer solution (pH=7.4), then are added
Enter 4 μ L formalins (concentration of formaldehyde in final mixed solution is made to be 2mM) after reaction 3h, to be added in cuvette at 37 DEG C,
Then the fluorescent absorption spectrum of compound (I) is measured.
The experimental results showed that in pH=3.5, compound (I) enhances at 365nm wavelength, illustrates compound (I) in acid
Property environment in generate " turn on " phenomenon.After probe and formaldehyde react, absorption of the compound (I) at 455nm wavelength
Enhancing, generates " turn-on " phenomenon, and fluorescence pattern is shown in Fig. 4.
Compound (I) (5 μM) is in DMSO/PBS buffer solutions (pH=7.4, v/v=1/199) condition in 4 present invention of embodiment
The lower fluorescence spectrum detection being added under different equivalent of formaldehyde.
The compound (I) for accurately weighing the preparation of a certain amount of embodiment 1, is configured to a concentration of 0.1mM's with dimethyl sulfoxide (DMSO)
Probe mother liquor, liquid-transfering gun are drawn 2 μ L and are added in 0.394mL PBS buffer solution (pH=7.4), and 4 μ L difference equivalents are separately added into
Formalin (make the ultimate density of formaldehyde be respectively 0,0.0025,0.0075,0.01,0.025,0.04,0.05,0.06,
0.075,0.1,0.15,0.25,0.4,0.5,0.6,0.75,1,2,5mM), at 37 DEG C after reaction 3h, it is added in 96 orifice plates,
Then the fluorescence spectrum of compound (I) is measured.
The experimental results showed that when being excited with 455nm wavelength, with the raising of concentration of formaldehyde, compound (I) is in 555nm
The fluorescence intensity at place increases.Illustrate, compound (I) reacts with formaldehyde, the generation " turn- of more and more compounds (I)
On " phenomenons.The Monitoring lower-cut of formaldehyde fluorescence probe is 10 μM, R2=0.99, meet the requirement of intracellular formaldehyde examination, fluorogram
Spectrum is shown in Fig. 5.
Compound (I) (5 μM) is in DMSO/PBS buffer solutions (pH=7.4, v/v=1/199) condition in 5 present invention of embodiment
Lower addition 0.5mmol formaldehyde, the linear relationship that fluorescence intensity changes over time.
The compound (I) for accurately weighing the preparation of a certain amount of embodiment 1, is configured to a concentration of 0.1mM's with dimethyl sulfoxide (DMSO)
Probe mother liquor, liquid-transfering gun are drawn 2 μ L and are added in 0.394mL PBS buffer solution (pH=7.4), and 4 μ L formalins are added, make most
Whole formaldehyde is added in a concentration of 0.5mmol of buffer solution in 96 orifice plates, and 5min is often reacted at 37 DEG C, that is, surveys first order fluorescence
Intensity, coreaction 3h, statistical data, and do the correlation curve of linear relationship.Fluorescence exciting wavelength is 455nm, and launch wavelength is
555nm。
Statistics indicate that the fluorescence intensity of formaldehyde fluorescence probe, increase at any time and enhance.Fluorescence pattern is shown in Fig. 6.
Compound (I) (5 μM) is in DMSO/PBS buffer solutions (pH=7.4, v/v=1/199) condition in 6 present invention of embodiment
The fluorescence spectrum detection of lower selectivity result.
A certain amount of probe (I) is accurately weighed, the mother liquor of a concentration of 0.1mM is configured to dimethyl sulfoxide (DMSO), liquid-transfering gun is inhaled
2 μ L are taken to be added in 0.394mL PBS buffer solution (pH=7.4), being separately added into 4 μ L formalins, (final formaldehyde is in water
The equal 1mM of concentration) to biology related active small molecular aqueous solution (acetaldehyde, acetone, formic acid, 4- hydroxy benzaldehydes, 4- nitrobenzoyls
Aldehyde, benzaldehyde, hydrogen peroxide, tert-butyl hydroperoxide, NaHS, glutathione, cysteine, homocysteine, glucose,
Ultimate density is 1mM), 3h is reacted at 37 DEG C, measures its fluorescent value.Fluorescence exciting wavelength is 455nm, and launch wavelength is
555nm。
The experimental results showed that in addition to formaldehyde, compound (I) fluorescence intensity base in the presence of other relevant biological activity molecules
This does not change, and shows that its anti-interference ability is very good, i.e. the specificity of PARA FORMALDEHYDE PRILLS(91,95) is relatively good.Fluorescence pattern is shown in Fig. 7.
Compound (I) (5 μM) is in DMSO/PBS buffer solutions (pH=7.4, v/v=1/199) condition in 7 present invention of embodiment
Mechanism after being reacted with formaldehyde down proves.
A certain amount of probe (I) is accurately weighed, the mother liquor of a concentration of 0.1mM is configured to dimethyl sulfoxide (DMSO), liquid-transfering gun is inhaled
2 μ L are taken to be added in 0.394mL PBS buffer solution (pH=7.4), being separately added into 4 μ L formalins, (final formaldehyde is in water
The equal 1mM of concentration), reaction overnight, then utilize efficient liquid phase chromatographic analysis.Efficient liquid phase spectrogram is shown in Fig. 8.
It is demonstrated experimentally that the mechanism that the compound (I) that we describe is reacted with formaldehyde is correct.Compound (I) and formaldehyde
Intermediate is generated, and is eventually converted into compound (III), to the effect of signal turn-on.
Imaging analysis of the compound (I) in cervical cancer cell exogenous formaldehyde in 8 present invention of embodiment
A certain amount of probe (I) is accurately weighed, the mother liquor of a concentration of 10mM is configured to dimethyl sulfoxide (DMSO), liquid-transfering gun is drawn
2 μ L are added in 1.998mLDMEM culture mediums.It takes 1mL to be added in Hela cells, hatches 0.5h at 37 DEG C, with fresh DMEM
Culture medium washes twice, and is then incubated with different concentration of formaldehyde (final concentration of formaldehyde is respectively 0,0.3mM, 0.6mM, 1mM, 2mM)
Change 3h, fresh DMEM medium washes twice, and is finally imaged with 1200 Laser Scanning Confocal Microscopes of Olympus Fluoview FV.Figure
10 be cell confocal fluorescent imaging effect figure：(a) PBS (pH=7.4), (b) concentration of formaldehyde is 0.3mM, and (c) concentration of formaldehyde is
0.6mM, (d) concentration of formaldehyde is 1mM, and or (e) concentration of formaldehyde is 2mM, and fluorescence pattern is shown in Fig. 9
The imaging analysis of compound (I) endogenous formaldehyde in cervical cancer cell in 9 present invention of embodiment
A certain amount of probe (I) is accurately weighed, the mother liquor of a concentration of 0.1mM is configured to dimethyl sulfoxide (DMSO), liquid-transfering gun is inhaled
2 μ L are taken to be added in 0.198mLDMEM culture mediums.1mL is taken to contain inhibitor NaHSO3(final NaHSO3A concentration of 0.2mmol)
Or NAC (final a concentration of 0.2mmol of NAC) hatches 0.5h at 37 DEG C respectively.Then the culture solution of compound (I) is added to
In Hela cells, hatch 0.5h at 37 DEG C, washed twice with DMEM culture mediums, finally uses Olympus Fluoview FV 1200
Laser Scanning Confocal Microscope is imaged.Figure 10 is cell confocal fluorescent imaging effect figure：(a) the blank probe control of without inhibitor,
(b):NaHSO3A concentration of 0.2mmol；(c):A concentration of 0.2mmol of NAC；Station meter, 20 μm.Fluorescence pattern is shown in Figure 10.
The experimental results showed that being continuously increased with intracellular concentration of formaldehyde, it may be observed that intracellular fluorescence intensity is not
Disconnected enhancing, illustrates that compound (I) can detect the variation of intracellular concentration of formaldehyde.
Fluorescence emission spectrogram of compound of 10 compound of embodiment (I) (5 μM) at different pH.Excitation wavelength is 365nm, transmitting
Wavelength is 455nm.
The compound (I) for accurately weighing the preparation of a certain amount of embodiment 1, is configured to a concentration of 0.1mM's with dimethyl sulfoxide (DMSO)
Probe mother liquor, liquid-transfering gun draw 2 μ L be added to 0.398mL difference pH value PBS buffer solution (finally pH value in water be 3.5
It in 10), after reacting 3h at 37 DEG C, is added in 96 orifice plates, then measures the fluorescence emission spectrum of compound (I).
The experimental results showed that when being excited with 365nm wavelength, when pH value is relatively low, compound (I) is glimmering at 455nm
Luminous intensity is stronger；When pH is in neutrality and is alkaline, fluorescence intensity of the compound (I) at 455nm is weaker.Illustrate that probe is quick to pH
Sense, fluorescence pattern are shown in Figure 11.
11 compound of embodiment (I) is visited under conditions of DMSO/PBS buffer solutions (v/v=1/199), pH difference 4,7.4
The fluorescence intensity of needle changes with time.
The compound (I) for accurately weighing the preparation of a certain amount of embodiment 1, the spy of a concentration of 1mM is configured to dimethyl sulfoxide (DMSO)
Needle mother liquor, liquid-transfering gun draw the PBS buffer solution (value of final pH is respectively 4,7.4) that 2 μ L are added to 0.398mL difference pH value,
It after reacting 0.5h at 37 DEG C, is added in 96 orifice plates, then measures the fluorescence spectrum of compound (I).
Statistics indicate that compound (I) is reacted completely in 25min or so.And in pH=4 and pH=7.4, have larger
The difference of fluorescence intensity.Fluorescence pattern is shown in Figure 12.
The compound (IV) (5 μM) that in 12 present invention of embodiment prepared by embodiment 1, in DMSO/PBS buffer solutions (pH=
7.4, v/v=1/199) under the conditions of, the value of different pH and the variation scattergram of fluorescence intensity.
The compound (IV) for accurately weighing the preparation of a certain amount of embodiment 1, the spy of a concentration of 1mM is configured to dimethyl sulfoxide (DMSO)
Needle mother liquor, the PBS buffer solution that 2 μ L of liquid-transfering gun absorption are added to 0.398mL difference pH value (make final pH distinguish in the value of buffer solution
From 3.5 to 9.5), it is added in 96 orifice plates, 3h, statistical data is reacted at 37 DEG C, and do the correlation curve of linear relationship.It is glimmering
Light excitation wavelength is 365nm, launch wavelength 455nm.
Statistics indicate that compound (IV) not to the sensibility of pH, can further illustrate be compound (I) amino base
Group is to the sensitivity of pH, " turn on " phenomenon just generated.Fluorescence pattern is shown in Figure 13.
Compound (I) (5 μM) is in DMSO/PBS buffer solutions (pH=7.4, v/v=1/199) item in 13 present invention of embodiment
The fluorescence spectrum detection of selective result under part.
The compound (IV) for accurately weighing the preparation of a certain amount of embodiment 1, the spy of a concentration of 1mM is configured to dimethyl sulfoxide (DMSO)
Needle mother liquor, liquid-transfering gun draw 2 μ L and are added to 0.394mL, are then separately added into the related active small molecular aqueous solution (second of 4 μ L biologies
Aldehyde, acetone, formic acid, 4- hydroxy benzaldehydes, 4- nitrobenzaldehydes, benzaldehyde, hydrogen peroxide, tert-butyl hydroperoxide, NaHS,
Glutathione, cysteine, homocysteine, glucose, ultimate density are 1mM), 0.5h is reacted at 37 DEG C, and it is glimmering to measure its
Light value.Fluorescence exciting wavelength is 365nm, launch wavelength 455nm.
The experimental results showed that in addition to pH, compound (I) fluorescence intensity in the presence of other relevant biological activity molecules is basic
There is no significant change, shows that its anti-interference ability is very good, i.e., it is relatively good to the specificity of pH.Fluorescence pattern is shown in Figure 14.
The cell imaging figure of compound (I) and the Lyso-Tracker Red of commercialization in 14 present invention of embodiment.
A certain amount of probe (I) is accurately weighed, the mother liquor of a concentration of 10mM is configured to dimethyl sulfoxide (DMSO), liquid-transfering gun is drawn
2 μ L are added in 1.998mLDMEM culture mediums.The culture solution that 1mL contains compound (I) is taken to be added in Hela cells, 37 DEG C
Lower hatching 0.5h, is washed twice with DMEM culture mediums, then the Lyso-Tracker Red (0.5 μm of ol) of use commercialization, 37 DEG C
Hatch 20min, is washed twice using 1ml PBS buffer solution (pH=7.4), it is finally total with Olympus Fluoview FV 1200
Focusing microscope carries out fluorescence imaging.Fig. 7 is cell confocal fluorescent imaging effect figure：(a) compound (I) excitation wavelength is
405nm, receiver wavelength range are 430-480nm, (b) Lyso-Tracker Red, excitation wavelength 543nm, receive wavelength model
It encloses for 590-640nm；(c) hybrid channel；Station meter, 20 μm.
The experimental results showed that compound (I) can detect the pH of intracellular lysosome, two groups of cells of Fiji softwares pair are used
Interior fluorescence intensity compares and analyzes, and show that Pearson correlation coefficient is 0.88, illustrates that compound (I) can be accurate
Detect lysosomal pH.Fluorescence pattern is shown in Figure 15.
Comparative example 1
Bibliography (X.F.Wu, L.H.Li, W.Shi, Q.Y.Gong, X.H.Li, and H.M.Ma, Sensitive and
Selective Ratiometric Fluorescence Probes for Detection of Intracellular
Endogenous Monoamine Oxidase A, Anal.Chem., 2016,88,1440) disclose following compounds (1)：
Compound (1) is used as fluorescence probe, can only realize single monoamine oxidase detection in the cell, cannot achieve pair
The difunctional detection of pH and formaldehyde.
Comparative example 2：The compound (IV) (5 μM) that in the present invention prepared by embodiment 1, in DMSO/PBS buffer solutions (pH=
7.4, v/v=1/199) under the conditions of, the value of different pH and the variation scattergram of fluorescence intensity.
The compound (IV) for accurately weighing the preparation of a certain amount of embodiment 1, the spy of a concentration of 1mM is configured to dimethyl sulfoxide (DMSO)
Needle mother liquor, the PBS buffer solution that 2 μ L of liquid-transfering gun absorption are added to 0.398mL difference pH value (make final pH distinguish in the value of buffer solution
From 3.5 to 9.5), it is added in 96 orifice plates, 3h, statistical data is reacted at 37 DEG C, and do the correlation curve of linear relationship.It is glimmering
Light excitation wavelength is 365nm, launch wavelength 455nm.
Statistics indicate that compound (IV) not to the sensibility of pH, can further illustrate be compound (I) amino base
Group is to the sensitivity of pH, " turn on " phenomenon just generated.Fluorescence pattern is shown in Figure 16.
Comparative example 3
To investigate the uniqueness of probe of the present invention, can the present invention has also been investigated use with probe structure analogue compounds (2)
It is detected in pH：
The compound (IV) in comparative example 2 is replaced with compound (2), the results showed that, compound (2) is in same testing conditions
Under it is insensitive to pH.
Claims (10)
1. a kind of fluorescence probe, shown in structure such as formula (I)：
2. a kind of preparation method of fluorescence probe as described in claim 1, the preparation method is that：
Compound (II) and compound (III) generate compound (IV) under the action of cesium carbonate；Compound (IV) is in alkaline item
Under part, nitrine is reduced to amino, ultimately generates compound (I)；
Reaction route is as follows：
3. preparation method as claimed in claim 2, it is characterised in that：The preparation method specifically carries out in accordance with the following steps：
(1) by compound (III), Cs2CO3By the amount 1 of substance:1~1.5, it is blended in the solution in anhydrous DMF and chemical combination is added dropwise
Object (II), and by the mixture in N2It is stirred overnight in 40 DEG C~50 DEG C under protection；Revolving removes solvent, and passes through silica
Column chromatography eluting gained residue is to get compound (IV)；
(2) stannous chloride, triethylamine, benzenethiol are pressed into the amount of substance than 1:2~3.5:2~3.5 are added in acetonitrile solvent,
15min~30min is stirred at 30 DEG C~40 DEG C, and compound (IV), wherein compound (IV) and two water of stannous chloride is then added
The amount ratio for closing the substance of object is 0.5~1：1, and continue stirring 12h~14h at 30 DEG C~40 DEG C；Reactant is concentrated under reduced pressure,
It is purified by silica column chromatography, obtains compound (I).
4. application of the fluorescence probe as described in claim 1 in detecting formaldehyde.
5. application as claimed in claim 4, it is characterised in that：The formaldehyde is intracellular formaldehyde, and concentration is not higher than 1mmol/
L。
6. application of the fluorescence probe as described in claim 1 in detecting pH value.
7. application as claimed in claim 6, it is characterised in that：The pH value is the pH value of intracellular lysosome, a concentration of
pH4.5-5.5。
8. application of the fluorescence probe as described in claim 1 in detection formaldehyde and pH value.
9. application as claimed in claim 8, it is characterised in that：The formaldehyde is intracellular formaldehyde, and concentration is not higher than 1mmol/
L；The pH value is the pH value of intracellular lysosome, a concentration of pH4.5-5.5.
10. the application as described in claim 5 or 7 or 9, it is characterised in that：The cell is human cervical carcinoma cell Hela cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810581986.4A CN108727265B (en) | 2018-06-07 | 2018-06-07 | Dual-functional fluorescent probe for detecting formaldehyde and pH (potential of hydrogen), and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810581986.4A CN108727265B (en) | 2018-06-07 | 2018-06-07 | Dual-functional fluorescent probe for detecting formaldehyde and pH (potential of hydrogen), and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108727265A true CN108727265A (en) | 2018-11-02 |
| CN108727265B CN108727265B (en) | 2020-08-21 |
Family
ID=63932283
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810581986.4A Active CN108727265B (en) | 2018-06-07 | 2018-06-07 | Dual-functional fluorescent probe for detecting formaldehyde and pH (potential of hydrogen), and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108727265B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108752275A (en) * | 2018-06-07 | 2018-11-06 | 浙江工业大学 | A kind of pH fluorescence probes and its preparation method and application |
| CN110372590A (en) * | 2019-07-29 | 2019-10-25 | 济南大学 | A kind of fluorescence probe and its preparation method and application detecting lysosomal pH |
| CN113896700A (en) * | 2021-07-02 | 2022-01-07 | 北京大学深圳研究生院 | Micromolecular formaldehyde fluorescent probe and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105924394A (en) * | 2016-05-20 | 2016-09-07 | 浙江工业大学 | Two-photon formaldehyde fluorescent probe and preparation and application thereof |
| CN108752275A (en) * | 2018-06-07 | 2018-11-06 | 浙江工业大学 | A kind of pH fluorescence probes and its preparation method and application |
-
2018
- 2018-06-07 CN CN201810581986.4A patent/CN108727265B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105924394A (en) * | 2016-05-20 | 2016-09-07 | 浙江工业大学 | Two-photon formaldehyde fluorescent probe and preparation and application thereof |
| CN108752275A (en) * | 2018-06-07 | 2018-11-06 | 浙江工业大学 | A kind of pH fluorescence probes and its preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| KEVIN J. BRUEMMER ET AL.,: "Development of a General Aza-Cope Reaction Trigger Applied to Fluorescence Imaging of Formaldehyde in Living Cells", 《J. AM. CHEM. SOC.》 * |
| NIKOLAI I. GEORGIEV ET AL.,: "A novel water-soluble 1,8-naphthalimide as a fluorescent pH-probe and a molecular logic circuit", 《JOURNAL OF LUMINESCENCE》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108752275A (en) * | 2018-06-07 | 2018-11-06 | 浙江工业大学 | A kind of pH fluorescence probes and its preparation method and application |
| CN108752275B (en) * | 2018-06-07 | 2020-08-21 | 浙江工业大学 | pH fluorescent probe and preparation method and application thereof |
| CN110372590A (en) * | 2019-07-29 | 2019-10-25 | 济南大学 | A kind of fluorescence probe and its preparation method and application detecting lysosomal pH |
| CN110372590B (en) * | 2019-07-29 | 2021-10-26 | 济南大学 | Fluorescent probe for detecting pH of lysosome and preparation method and application thereof |
| CN113896700A (en) * | 2021-07-02 | 2022-01-07 | 北京大学深圳研究生院 | Micromolecular formaldehyde fluorescent probe and application thereof |
| CN113896700B (en) * | 2021-07-02 | 2023-08-15 | 北京大学深圳研究生院 | Small molecular formaldehyde fluorescent probe and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108727265B (en) | 2020-08-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108398409B (en) | Method for detecting hypochlorite by fluorescence ratio | |
| CN108727265A (en) | A kind of difunctional fluorescence probe and its preparation method and application of detection formaldehyde and pH | |
| CN113801105B (en) | Mitochondrion targeted peroxynitrite/bisulfite dual-response fluorescent probe | |
| Li et al. | A novel dual-channel fluorescent probe for nitroxyl detection and its application in HepG-2 cells | |
| CN109535147A (en) | A kind of formaldehyde fluorescence probe of quick response and its preparation method and application | |
| CN108047060A (en) | A kind of pyrene derivatives fluorescent probe molecule of recognition detection formaldehyde and its preparation method and application | |
| CN114181204B (en) | Near infrared fluorescent probe for detecting viscosity and preparation and application thereof | |
| CN108003869A (en) | A kind of fluorescence probe of highly sensitive detection hypochlorite and its synthetic method and application | |
| CN109608474B (en) | Compound for detecting tyrosinase and preparation method and application thereof | |
| CN110078665A (en) | A kind of hypochlorous fluorescence probe of detection of endoplasmic reticulum targeting and application | |
| Qiu et al. | A rapid-response near-infrared fluorescent probe with a large Stokes shift for senescence-associated β-galactosidase activity detection and imaging of senescent cells | |
| CN106967102A (en) | A kind of enhanced fluorescence probe of hydrogen peroxide based on Rhodamine Derivatives | |
| CN108752275A (en) | A kind of pH fluorescence probes and its preparation method and application | |
| CN108689933A (en) | A kind of hypochlorous fluorescence probe of quick high-selectivity analysis | |
| Cao et al. | A two-photon lysosome-targeted probe for endogenous formaldehyde in living cells | |
| CN109180716B (en) | Multi-signal ratio type distinguishing detection H2O2And H2Design, synthesis and application of fluorescent probe of S | |
| CN109824592A (en) | A kind of detection formaldehyde and the difunctional fluorescence probe intermediate of pH and its preparation method and application | |
| Zhang et al. | A lysosome-targeted fluorescent probe based on a BODIPY structure for Cys/Hcy detection | |
| CN110172070A (en) | A kind of fluorescence probe and its synthetic method and application detecting viscosity and hydrogen peroxide | |
| CN114105927B (en) | Construction of benzopyran nitrile fluorescent molecular probe and in-vitro diagnosis application thereof | |
| CN108752373A (en) | A kind of fluorescence probe identifying hydrogen peroxide based on benzene boron ester | |
| CN110642857B (en) | Difunctional fluorescent probe for detecting viscosity and pH, and preparation and application thereof | |
| CN108774226A (en) | It is a kind of to be used to detect fluorescence probe of silver ion and the preparation method and application thereof | |
| CN108409655A (en) | A kind of two-photon methyl-glyoxal fluorescence probe and its preparation method and application | |
| CN109734710A (en) | A kind of fluorescence probe detecting cysteine and its synthetic method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |