CN109369569A - The fluorescence probe and its preparation method and application of one kind detection pyroracemic aldehyde - Google Patents
The fluorescence probe and its preparation method and application of one kind detection pyroracemic aldehyde Download PDFInfo
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Abstract
The invention discloses the fluorescence probes and its preparation method and application of a kind of detection pyroracemic aldehyde, synthesize a series of using o-phenylenediamine as the fluorescence probe with different electron donor-acceptor-donor (D-A-D) groups of recognition unit, the synthetic route is with a wide range of applications.The probe is very ideal to the linear response of pyroracemic aldehyde, and response range is 1-10 μM.The probe can be used for the fluorescence imaging of pyroracemic aldehyde in cell and organism, have the advantages that low, sensitive, the selective good, good light stability of background fluorescence.
Description
Technical field
The invention belongs to small organic molecule fluorescence probe fields, pass through the Intramolecular electron transfer sensitive to electrically regulation
(ICT) mechanism building it is a kind of have larger Stokes shift, pyroracemic aldehyde in organism is responded sensitive, detection limit it is low organic
Small-molecule fluorescent probe.
Background technique
Compared to other fluorescence probes, such as carbon nanotube, quantum dot, rare earth nanometer particle, small organic molecule fluorescence
Probe has the advantages that its own: having specific compound structure, can obtain the sample of purity is high；In addition, small organic molecule
The excellent reproducibility of fluorescence probe make its in preparation and application process with certain advantage (ACS Nano 2017,11:
7177-7188.).In the mechanism of building fluorescence probe, wherein faint variation is may be implemented in Intramolecular electron transfer (ICT)
Electrically regulation, can make the highest occupied molecular orbital energy (HOMO energy) and lowest unoccupied molecular orbital energy (LUMO of fluorescence probe
Energy) difference very little, i.e. the band-gap energy very little of molecule.This is mentioned to fluorescence probe of the launch wavelength in infrared window is designed
Possibility (Proc.Natl.Acad.Sci.USA.2017,114:962-967 is supplied；Adv.Mater.2017,29:1605497；
Adv.Mater.2016,32:6872-6879.)。
Pyroracemic aldehyde (MGO) is a kind of active small molecular with dicarbonyl structure, under normal level, the intracorporal acetone of people
Aldehyde is very low, but content is significant in some diseases such as blood or cerebrospinal fluid of diabetes, neurodegenerative disease patient
It increases.Pyroracemic aldehyde can react with amino acid, protein, nucleic acid etc. and modify (SEMIN CANCER BIOL.2018,49:64-
74.), so as to cause cell function exception.In addition to this, pyroracemic aldehyde, which also has, inhibits cell to grow, induce cell apoptosis, inhibit
The effects of activity and activation signal access of enzyme (Toxicol Lett.1999,110 (3): 145-75.).Therefore, for standard
Really understand effect of the pyroracemic aldehyde in physiology and pathologic process, it is necessary to develop highly sensitive fluorescence probe to biology intracorporal third
Keto-aldehyde in real time, dynamically, accurately detect.
Summary of the invention
The present invention is based on ICT mechanism to construct a series of o-phenylenediamine class fluorescence probes, uses electron donor-acceptor-
The structural model of donor obtains a series of full symmetric molecules with larger rigid structure by step double couple crosslinking reaction.Institute
A series of fluorescence probes obtained can generate good near-infrared fluorescent enhancing response to pyroracemic aldehyde, and response is sensitive, selective
It is good.
The present invention introduces the o-phenylenediamine group for having specific recognition to pyroracemic aldehyde on the phenyl ring as electron acceptor, leads to
Cross itself and acetone aldehyde reaction and generate piperazine structure, obtain the stronger tricyclic structure of electron-withdrawing ability, fluorescence probe launch wavelength with
Red shift, fluorescence intensity greatly enhances.Also, by carrying out different functionalization to both ends electron-donating group, weaken intermolecular
Pi-pi accumulation effect, improves fluorescent emission property.
The fluorescence probe of a kind of detection pyroracemic aldehyde provided by the invention, feature are that such fluorescence probe has following general formula I
Or general formula II:
In general formula I:
X is O, S or Se；
R is the sugar that H, C1-C8 alkyl, the substituted or non-substituted polyethylene glycol groups of different chain length or alkyl chain replace；
In general formula II:
X is O, S or Se；
R1And R2It is respectively independent are as follows: the substituted or non-substituted polyethylene glycol groups of H, C1-C8 alkyl, ester group, different chain length；R1With
R2Connection cyclization；R1Or R2Simultaneously five-membered ring or simultaneously hexatomic ring are formed with the connected phenyl ring of nitrogen-atoms.
The preparation method of the fluorescence probe of above-mentioned a kind of detection pyroracemic aldehyde, this method comprising the following specific steps
2.1, the preparation of general formula I
Molar ratio is two kinds of raw materials of 1:1-5: general formula compound AIAnd RY, nucleophilic displacement of fluorine is passed through under the action of inorganic base
Reaction, heating obtains general formula compound B at 30-100 DEG CI；Wherein, Y is halogen atom；R is H, C1-C8 alkyl, different chains
The sugar that long substituted or non-substituted polyethylene glycol groups or alkyl chain replace；X is O, S or Se；The halogen atom is Cl, Br, I；
General formula compound B obtained by upper stepIEtc. quality Fe as reducing agent, solvent be acetic acid under conditions of, 60-100
DEG C reaction 2-10 hours, obtain general formula compound I；
2.2, the preparation of general formula II
Molar ratio is the general formula compound A of 1:2-2.5II、BIIIt is dissolved in toluene and 1M wet chemical mixed solvent
In, with general formula compound AIIMolar ratio is nitrogen protection under the catalytic action of the tetrakis triphenylphosphine palladium of 1:0.04-0.20,
It is heated 10-20 hours at 110 DEG C；
General formula compound C obtained by upper stepIIEtc. quality Fe as reducing agent, solvent be acetic acid under conditions of, 60-100
DEG C reaction 2-10 hours, obtain general formula II class compound.
The fluorescence probe of above-mentioned a kind of detection pyroracemic aldehyde detects the application of pyroracemic aldehyde in the biological sample.
Above-mentioned application, the biological sample include but is not limited to tumour cell, nerve cell and living body zebra fish.
Since fluorescence probe of the present invention is based on ICT (Intramolecular electron transfer) mechanism to the response of pyroracemic aldehyde, pass through
Faint electrical regulation is so that obvious red shift occurs for wavelength of fluorescence, so fluorescence probe sensitivity with higher, fluorescence probe
It is also fairly obvious for cell and the intravital pyroracemic aldehyde imaging effect of zebra fish.Due to pyroracemic aldehyde in organism normal value compared with
Low, only several micromoles, this causes biggish obstruction to the detection for realizing endogenous pyroracemic aldehyde, and of the present invention
ICT type fluorescence probe then can for such issues that carry out deeper into research and exploration, and for using pyroracemic aldehyde as object
Disease early diagnosis research lays the foundation.
Detailed description of the invention
Fig. 1 is absorption and the fluorescence emission spectrum spectrogram of S1 probe of the present invention and pyroracemic aldehyde reaction product；
Fig. 2 is the S1 probe of the present invention image with various concentration acetone aldehyde reaction in the cell；
Fig. 3 is that S1 probe of the present invention is used for zebra fish in vivo pyroracemic aldehyde image.
Specific embodiment
An object of the present invention is to provide the pyroracemic aldehyde responsiveness fluorescence probe of general formula I:
Wherein, X O, S or Se.R is H, alkyl；The substituted or non-substituted polyethylene glycol groups of different chain length；What alkyl chain replaced
Sugared (such as glucose, pyranose, lactose, galactolipin).
Preferably, X S；R is
The second object of the present invention is to provide the pyroracemic aldehyde responsiveness fluorescence probe of general formula II:
Wherein, X O, S or Se.R1And R2Respectively stand alone as the substituted or non-substituted poly- second two of H, alkyl, ester group, different chain length
Alcohol radical；R1And R2Connection cyclization；R1Or R2Simultaneously five-membered ring or simultaneously hexatomic ring are formed with connected phenyl ring.
Preferably, X S；R1Or R2Pyrrolidinyl is formed with nitrogen-atoms for methyl, ethyl or connection cyclization；R1Or R2With nitrogen
The connected phenyl ring of atom is formed and hexatomic ring, i.e.,
The preferred pyroracemic aldehyde responsiveness fluorescence probe in part of the invention is as follows, these embodiments are just for the present invention
It is described further, it is not intended to limit the scope of the present invention in any way.
The third object of the present invention is to provide the preparation method for preparing pyroracemic aldehyde responsiveness fluorescence probe of the present invention:
The preparation method of 3.1 general formula I:
Molar ratio is two kinds of raw materials of 1:1-1:5: general formula compound AIAnd RY, it is taken under the action of inorganic base by nucleophilic
Generation reaction, heating obtains general formula compound B at 30-100 DEG CI；Wherein Y is halogen atom；R is C1-C8 alkyl, different chain length
Substituted or non-substituted polyethylene glycol groups；Wherein, the halogen atom is Cl, Br, I；
In another preferred example, the reaction includes: the compound A for being 1:4 by molar ratioI, 1- bromo- 2- (2- methoxyl group
Ethyoxyl) ethane is dissolved in DMF, heating reaction is carried out under alkali effect.
In another preferred example, inorganic base used is potassium carbonate.
In another preferred example, the SN2 elimination reaction times were 2-8 hours.
General formula compound B obtained by upper stepIIn Fe as reducing agent, solvent is 60-100 DEG C of reaction 2-under conditions of acetic acid
10 hours, obtain general formula compound I.
General formula I pyroracemic aldehyde response type fluorescence probe of the invention can the preparation of the reaction equation as shown in above formula.
Above-mentioned SN2 elimination reaction solvents can with but be not limited to DMF.
Above-mentioned SN2 elimination reaction alkali can with but be not limited to potassium carbonate.
In above-mentioned dinitro reduction reaction reducing agent can with but be not limited to Fe.
Above-mentioned dinitro reduction reaction solvent can with but be not limited to acetic acid.
The preparation method of 3.2 general formula II:
Molar ratio is the general formula compound A of 1:2-1:2.5II、BIIIt is dissolved in toluene and 1M wet chemical mixed solvent
In, with general formula compound AIIMolar ratio is that the Pd catalyst coupling reaction of 1:0.04-1:0.20 obtains general formula compound
CII。
In another preferred example, the reaction includes: the compound A for being 1:2.38:0.09 by molar ratioII、BII-5、Pd
(PPh3)4[tetrakis triphenylphosphine palladium] is dissolved in toluene and 1M wet chemical in the mixed solvent, is added under nitrogen protection
Thermal response.
In another preferred example, the reaction is heated at 110 DEG C carries out.
In another preferred example, the coupling reaction time is 10-20 hours.
General formula compound C obtained by upper stepIIIn Fe as reducing agent, solvent is 60-100 DEG C of reaction 2- under conditions of acetic acid
10 hours, obtain general formula II class compound.
General formula II pyroracemic aldehyde response type fluorescence probe of the invention can the preparation of the reaction equation as shown in above formula.X and R in formula1、R2
It is identical as the definition in general formula II.
In above-mentioned Pd catalyzed coupling reaction catalyst can with but be not limited to tetrakis triphenylphosphine palladium.
Above-mentioned Pd catalyzed coupling reaction solvent can with but be not limited to toluene and 1M wet chemical mixed solvent.
In above-mentioned dinitro reduction reaction reducing agent can with but be not limited to Fe.
Above-mentioned dinitro reduction reaction solvent can with but be not limited to acetic acid.
The fourth object of the present invention is that near infrared fluorescent probe of the present invention is answered what pyroracemic aldehyde in biological sample was detected
With
4.1, it the external spectral response of fluorescence probe and pyroracemic aldehyde and is tested with the spectral comparison of organic synthesis product
Three groups of experimental comparison groups are set, I or II fluorescence probe is respectively as follows:, I the or II fluorescence probe of pyroracemic aldehyde is added.It is molten
Agent condition is 40%DMAC, after 60%pH=7.4 10mM PBS, 37 DEG C of reaction 1h, surveys absorption and fluorescence emission spectrum respectively.
4.2, fluorescence imaging experiments of the fluorescence probe to pyroracemic aldehyde in nerve cell
To be added to SHSY5Y intracellular for the 1%DMSO solution of I or II fluorescence probe for being 10 μM by 5 μ L concentration, 37 DEG C,
5%CO2Under the conditions of be incubated for 1h, washed three times with PBS.It is separately added into the pyroracemic aldehyde that concentration is 0,5 μM, 10 μM, 15 μM concentration, and it is thin
Born of the same parents cultivate 1h, and PBS is flushed three times.Co-focusing imaging in PBS (excitation wavelength 488nm collects wavelength 600-900nm).
4.3, fluorescence probe is in living sample to the fluorescence imaging experiments of pyroracemic aldehyde
1) in E3 culture medium, by the zebra fish co-incubation 2h of 20 μM of I or II fluorescence probe and birth 3 days, E3 is used
Culture medium washes the pyroracemic aldehyde added three times such as various concentration: 0,5 μM, 10 μM, after living body culture 2h, then is washed three times with E3 culture medium.
Finally with fluorescence microscope, to the imaging of zebra fish living imaging fluorescence microscope, (excitation wavelength 488nm is received in E3 culture medium
Collect wavelength 600-900nm).
2) zebra fish first cultivates 1h with 50 μM of GSH, inhibits pyroracemic aldehyde activity.Again plus 20 μM I or II fluorescence probe be incubated for
2h finally adds 10 μM of pyroracemic aldehydes and is incubated for 2h jointly, and with fluorescence microscope imaging, (excitation wavelength 488nm collects wavelength 600-
900nm)。
Embodiment 1
The synthesis of compound S1
Compound AI- 1 prepares bibliography method (Anal.Chem.2018,90,7953-7962.) synthesis.Compound AI-
1 (100mg, 0.236mmol) and potassium carbonate (130mg, 0.944mmol) are added in 4mLDMF, stir 1h at 30 DEG C.It adds
The bromo- 2- of 1- (2- methoxy ethoxy) ethane (173mg, 0.944mmol) reacts 4h.TLC detection adds water and second after completion of the reaction
Acetoacetic ester extraction, organic layer successively use water, saturated common salt water washing, and anhydrous sodium sulfate is dry.It filters, filtrate decompression concentration, slightly
Product column chromatography for separation (methylene chloride: methanol=30:1v/v), obtains yellow solid 102mg, yield 70%.1H NMR
(300MHz,CDCl3) δ 7.50 (d, J=8.8Hz, 4H), 7.04 (t, J=19.9Hz, 4H), 4.32-4.10 (m, 4H), 3.77-
3.71(m,4H),3.62–3.56(m,4H),3.42–3.38(m,6H).13C NMR(126MHz,CDCl3)δ160.49(2C),
153.18(2C),142.38(2C),130.72(4C),128.23(2C),122.67(2C),115.21(4C),71.97(2C),
70.81(2C),69.59(2C),67.60(2C),59.10(2C)。
Compound BI- 1 (50.0mg, 0.081mmol), etc. the iron powders of quality be added in 2.0mL glacial acetic acid, nitrogen is protected
Shield, 100 DEG C of reaction 3h.TLC detection adds water and sodium bicarbonate to be neutralized to pH=7, ethyl acetate extraction, organic layer after completion of the reaction
Water, saturated common salt water washing are successively used, anhydrous sodium sulfate is dry.It filters, filtrate decompression concentration, crude product column chromatography for separation (two
Chloromethanes: methanol=30:1v/v), obtain orange/yellow solid 39.0mg, yield 87%.1H NMR(300MHz,CDCl3)δ7.49
(d, J=8.7Hz, 4H), 7.12 (d, J=8.7Hz, 4H), 4.26-4.20 (m, 4H), 4.05 (s, 4H), 3.93-3.88 (m,
4H),3.78–3.72(m,4H),3.63–3.57(m,4H),3.41(s,6H).13C NMR(126MHz,CDCl3)δ158.58
(2C),151.36(2C),138.15(2C),131.46(4C),127.16(2C),115.43(4C),113.42(2C),72.01
(2C),70.80(2C),69.78(2C),67.55(2C),59.12(2C).LR-ESI-MS:[M+H]+m/z 555.3。
Embodiment 2
The synthesis of compound S2
Compound AI- 1 (100mg, 0.236mmol) and potassium carbonate (130mg, 0.944mmol) are added in 4mLDMF, and 60
1h is stirred at DEG C.Add bromo- 1- propyl alcohol (132mg, 0.944mmol) the reaction 2h of 3-.TLC detection adds water and second after completion of the reaction
Acetoacetic ester extraction, organic layer successively use water, saturated common salt water washing, and anhydrous sodium sulfate is dry.It filters, filtrate decompression concentration, slightly
Product column chromatography for separation (methylene chloride: methanol=30:1v/v), obtains yellow solid 87mg, yield 70%.1H NMR(300MHz,
CDCl3) δ 7.52 (d, J=8.8Hz, 4H), 7.08 (d, J=8.9Hz, 4H), 4.21 (t, J=6.0Hz, 4H), 3.90 (t, J=
5.9Hz, 4H), 2.10 (p, J=6.0Hz, 4H)
Compound BI- 2 (50.0mg, 0.095mmol), etc. the iron powders of quality be added in 2.0mL glacial acetic acid, nitrogen is protected
Shield, 100 DEG C of reaction 3h.TLC detection adds water and sodium bicarbonate to be neutralized to pH=7, ethyl acetate extraction, organic layer after completion of the reaction
Water, saturated common salt water washing are successively used, anhydrous sodium sulfate is dry.It filters, filtrate decompression concentration, crude product column chromatography for separation (two
Chloromethanes: methanol=30:1v/v), obtain orange/yellow solid 20.2mg, yield 46%.1H NMR(300MHz,CDCl3)δ7.53
(d, J=8.7Hz, 4H), 7.10 (d, J=8.9Hz, 4H), 4.24 (t, J=5.8Hz, 4H), 4.05 (s, 4H), 3.88 (t, J=
5.9Hz, 4H), 2.11 (p, J=6.0Hz, 4H).
Embodiment 3
The synthesis of compound S4
3- propargyl bromide (357mg, 3.0mmol), 2- [2- (2- nitrine ethyoxyl) ethyoxyl] ethyl -2,3,4,6- tetra--O-
Acetyl group-D- galactolipin pyranthrene (1940mg, 3.08mmol), CuI (300mg, 1.03mmol), DIPEA (400mg,
It 3.08mmol) is added in 20mL acetonitrile, is reacted overnight at 80 DEG C.TLC detection adds water and ethyl acetate extraction after completion of the reaction,
Organic layer successively uses water, saturated common salt water washing, and anhydrous sodium sulfate is dry.It filters, filtrate decompression concentration, crude product column chromatography point
From (methylene chloride: methanol=40:1v/v), white solid 1191mg, yield 74% are obtained.
Compound AI- 1 (150mg, 0.365mmol), potassium carbonate (5050mg, 36.5mmol) are added in 10mL acetone,
1h is stirred at 56 DEG C.Add previous step be made compound (536mg, 1.0mmol) the reaction was continued overnight.TLC detection has been reacted
It is filtered after finishing, filtrate decompression concentration.It obtains crude product column chromatography for separation (methylene chloride: methanol=50:1v/v), obtains bright yellow solid
328mg, yield 68%.1H NMR (300MHz, CDCl3) δ 7.76 (s, 2H), 7.55 (d, J=8.7Hz, 4H), 7.18 (d, J=
8.7Hz, 4H), 5.26 (s, 4H), 5.20 (t, J=8.9Hz, 2H), 5.11-4.96 (m, 5H), 4.75-4.50 (m, 6H),
4.33–4.21(m,4H),4.00–3.90(m,2H),3.85–3.65(m,3H),2.05(s,24H).
Compound BI- 4 (50.0mg, 0.038mmol), etc. the iron powders of quality be added in 2.0mL glacial acetic acid, nitrogen is protected
Shield, 100 DEG C of reaction 3h.TLC detection adds water and sodium bicarbonate to be neutralized to pH=7, ethyl acetate extraction, organic layer after completion of the reaction
Water, saturated common salt water washing are successively used, anhydrous sodium sulfate is dry.It filters, filtrate decompression concentration, crude product column chromatography for separation (two
Chloromethanes: methanol=20:1v/v), obtain orange/yellow solid 31.3mg, yield 66%.1H NMR(300MHz,CDCl3)δ7.74(s,
2H), 7.52 (d, J=8.5Hz, 4H), 7.20 (d, J=8.5Hz, 4H), 5.28 (s, 4H), 5.19 (t, J=9.4Hz, 2H),
5.12–4.97(m,5H),4.71–4.46(m,6H),4.31–4.20(m,4H),4.18–4.05(m,5H),4.00–3.88(m,
2H),3.75–3.65(m,2H),2.05(s,24H).LR-ESI-MS:[M+H]+m/z 1261.1.
Compound CI- 4 (17.0mg, 0.0135mmol) and LiOH (2.0mg, 0.0675mmol) are dissolved in 1.0mL mixing
In solvent (tetrahydrofuran: water=3:2v/v), 5h is reacted at room temperature.TLC detection is concentrated under reduced pressure after completion of the reaction, and C18 filler fills column
It separates (methanol: water=1:1v/v), obtains yellow compound 12.3mg, yield 99%.1H NMR(300MHz,CD3OD)δ8.27
(s, 2H), 7.46 (d, J=8.7Hz, 4H), 7.21 (d, J=8.8Hz, 4H), 5.27 (s, 4H), 4.69 (t, J=4.9Hz,
4H), 4.36-4.21 (m, 4H), 4.08-3.99 (m, 4H), 3.86 (d, J=11.6Hz, 3H), 3.70-3.61 (m, 4H),
3.23–3.14(m,3H).LR-ESI-MS:[M+H]+m/z 925.2。
Embodiment 4
The synthesis of compound S5
Compound AIIPrepare bibliography method (ACS Applied Materials&Interfaces, 2017,9 (37):
31352-31356.) synthesis.Compound AII(323mg, 0.84mmol), 4- (N, N- dimethylamino) phenyl boric acid pinacol ester
(500mg, 2mmol) and tetrakis triphenylphosphine palladium (93mg, 0.077mmol) are added to 20mL and steam toluene and 6.4mL 1M carbonic acid again
In aqueous solutions of potassium, nitrogen protection, back flow reaction 18h.TLC detection after completion of the reaction plus water and ethyl acetate extraction, organic layer according to
Secondary to use water, saturated common salt water washing, anhydrous sodium sulfate is dry.It filters, filtrate decompression concentration, crude product column chromatography for separation obtains purple
Black solid B5281mg, yield 30.2%.1H NMR(400MHz,CDCl3) δ 7.49 (d, J=8.6Hz, 4H), 6.82 (d, J=
8.6Hz,4H),3.06(s,12H).13C NMR(126MHz,CDCl3)δ153.56(2C),151.42(2C),142.02(2C),
130.43(4C),127.73(2C),117.50(2C),112.02(4C),40.10(4C).
Compound BII- 5 (150.0mg, 0.038mmol), etc. the iron powders of quality be added in 2.0mL glacial acetic acid, nitrogen is protected
Shield, 100 DEG C of reaction 4h.TLC detection adds water and sodium bicarbonate to be neutralized to pH=7, ethyl acetate extraction, organic layer after completion of the reaction
Water, saturated common salt water washing are successively used, anhydrous sodium sulfate is dry.It filters, filtrate decompression concentration, crude product column chromatography for separation (two
Chloromethanes: methanol=20:1v/v), obtain dark brown solid S5 73mg, yield 55.9%.1H NMR(300MHz,CDCl3)δ7.47
(d, J=8.6Hz, 4H), 6.92 (d, J=8.6Hz, 4H), 4.08 (s, 4H), 3.03 (s, 12H)13C NMR(101MHz,
CDCl3)δ151.67(2C),150.12(2C),138.02(2C),130.94(4C),122.40(2C),113.71(2C),
112.96(4C),40.53(4C)。
Embodiment 5
The performance measurement of near-infrared pyroracemic aldehyde fluorescence probe
5.1, the external spectral response of S1 probe and pyroracemic aldehyde
50 μM of S1 probes or 50 μM of S1 probes and 100 μM of pyroracemic aldehydes in 10mM PBS (40%DMAC, pH=7.4),
After 37 DEG C of reaction 1h, absorption and fluorescence emission spectrum are surveyed respectively.(excitation wavelength 488nm, launch wavelength 500-900nm).S1 is visited
Needle has intense UV absorption at 375nm certainly, and unstressed configuration emits in the case where changing wavelength light source excitation.S1 probe and pyroracemic aldehyde
After reacting 1h, maximum absorption wavelength red shift to 500nm, maximum emission wavelength 650nm.From the point of view of photoluminescent property, at this
It invents under illustrated experiment condition, which realizes fluorescence from " turn off " to " turn in infrared window really
The conversion of on "；As shown in Figure 1.
5.2, fluorescence imaging experiments of the S1 probe to pyroracemic aldehyde in nerve cell
10 μM of S1 probes and SHSY5Y cell are in 37 DEG C, 5%CO2Under the conditions of be incubated for 1h, washed three times with PBS, then plus not
With the pyroracemic aldehyde of concentration: 0,5 μM, 10 μM, 15 μM are washed three times with cell culture 1h, PBS, the co-focusing imaging (excitation in PBS
Wavelength 488nm collects wavelength 600-900nm).Figure it is seen that with the increase that acetone aldehyde concentration is added, S1 probe exists
Intracellular fluorescence gradually increases.Furthermore it is possible to find that the pyroracemic aldehyde variable gradient of experimental setup is smaller, but still it is available compared with
For apparent change in fluorescence comparison diagram, further illustrate that synthesising probing needle of the present invention responds pyroracemic aldehyde very sensitive in the cell.
5.3, S1 probe is in living sample to the fluorescence imaging experiments of pyroracemic aldehyde
20 μM of S1 probes cultivate 2h with the zebra fish of birth 3 days in E3 culture medium, are washed three times, are added not with E3 culture medium
With the pyroracemic aldehyde (A-C) of concentration: 0,5 μM, 10 μM, living body culture 2h, E3 culture medium are washed three times, aobvious with fluorescence in E3 culture medium
Micro mirror imaging；(D) zebra fish first cultivates 1h with 50 μM of GSH, adds 20 μM of S1 probes to be incubated for 2h, finally plus 10 μM of pyroracemic aldehydes are common
2h is incubated for, with fluorescence microscope imaging (excitation wavelength 488nm collects wavelength 600-900nm).It, still can be in this experiment
It is apparent from the increase that acetone aldehyde concentration is added in zebra fish body, the corresponding enhancing of fluorescence.It is living that pyroracemic aldehyde is set simultaneously
Property inhibit control group, zebra fish is first incubated for acetone aldehyde inhibitor GSH, adds pyroracemic aldehyde culture, fluorescence intensity substantially under
Drop, as shown in Figure 3.The above results explanation, fluorescence probe of the invention can be used for the internal acetone using zebra fish as living sample
Aldehyde detection.
Claims (4)
1. the fluorescence probe of one kind detection pyroracemic aldehyde, which is characterized in that the fluorescence probe has following general formula I or general formula II:
In general formula I:
X is O, S or Se；
R is the sugar that H, C1-C8 alkyl, the substituted or non-substituted polyethylene glycol groups of different chain length or alkyl chain replace；
In general formula II:
X is O, S or Se；
R1And R2It is respectively independent are as follows: the substituted or non-substituted polyethylene glycol groups of H, C1-C8 alkyl, ester group, different chain length；R1And R2Connection
Cyclization；R1Or R2Simultaneously five-membered ring or simultaneously hexatomic ring are formed with the connected phenyl ring of nitrogen-atoms.
2. a kind of preparation method of the fluorescence probe of a kind of detection pyroracemic aldehyde described in claim 1, which is characterized in that this method
Comprising the following specific steps
2.1, the preparation of general formula I
Molar ratio is two kinds of raw materials of 1:1-5: general formula compound AIAnd RY, nucleophilic substitution is passed through under the action of inorganic base,
Heating obtains general formula compound B at 30-100 DEG CI；Wherein, Y is halogen atom；R is H, C1-C8 alkyl, different chain length substitution
Or the sugar that non-substituted polyethylene glycol groups or alkyl chain replace；X is O, S or Se；The halogen atom is Cl, Br, I；
General formula compound B obtained by upper stepIEtc. quality Fe as reducing agent, solvent be acetic acid under conditions of, 60-100 DEG C reaction
2-10 hours, obtain general formula compound I；
2.2, the preparation of general formula II
Molar ratio is the general formula compound A of 1:2-2.5II、BIIBe dissolved in toluene and 1M wet chemical in the mixed solvent, with
General formula compound AIIMolar ratio is nitrogen protection, at 110 DEG C under the catalytic action of the tetrakis triphenylphosphine palladium of 1:0.04-0.20
Heating 10-20 hours；
General formula compound C obtained by upper stepIIEtc. the Fe of quality as reducing agent, solvent be acetic acid under conditions of, 60-100 DEG C is anti-
It answers 2-10 hours, obtains general formula II class compound.
3. a kind of fluorescence probe of a kind of detection pyroracemic aldehyde described in claim 1 detects answering for pyroracemic aldehyde in the biological sample
With.
4. application according to claim 3, which is characterized in that the biological sample includes but is not limited to that tumour is thin
Born of the same parents, nerve cell and living body zebra fish.
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| CN113387865B (en) * | 2021-06-15 | 2022-06-07 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Fluorescent probe for detecting methylglyoxal and preparation method and application thereof |
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