CN107382814A - It is a kind of based on small-molecule fluorescent probe and its preparation method and application - Google Patents
It is a kind of based on small-molecule fluorescent probe and its preparation method and application Download PDFInfo
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- CN107382814A CN107382814A CN201710797200.8A CN201710797200A CN107382814A CN 107382814 A CN107382814 A CN 107382814A CN 201710797200 A CN201710797200 A CN 201710797200A CN 107382814 A CN107382814 A CN 107382814A
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- IQEWYVIHLJJXKR-UHFFFAOYSA-N Nc1ccc-2c3c1cccc3-c1c3c-2cccc3ccc1 Chemical compound Nc1ccc-2c3c1cccc3-c1c3c-2cccc3ccc1 IQEWYVIHLJJXKR-UHFFFAOYSA-N 0.000 description 1
- FPYJFEHAWHCUMM-UHFFFAOYSA-N O=C(C=C1)OC1=O Chemical compound O=C(C=C1)OC1=O FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 description 1
- NUXKYBUGUFFOOT-UHFFFAOYSA-N O=C(C=CC1=O)N1c(c1ccc2)ccc-3c1c2-c1c2c-3cccc2ccc1 Chemical compound O=C(C=CC1=O)N1c(c1ccc2)ccc-3c1c2-c1c2c-3cccc2ccc1 NUXKYBUGUFFOOT-UHFFFAOYSA-N 0.000 description 1
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Abstract
The application belongs to sulfydryl biological micromolecule detection technique field, and in particular to it is a kind of based on small-molecule fluorescent probe and its preparation method and application.The present invention by elder generation 1 carbon or 3 carbon on occur nitration reaction connect a nitro, then nitro is reduced into amino, maleic anhydride substituted-amino is used again, so as to obtain the novel small-molecule fluorescent probe of two structures provided by the present invention, shown in its chemical constitution such as formula (I) or formula (II).The small-molecule fluorescent probe sends green glow when being combined with the sulfydryl biological micromolecule in biological cell, it is clearly distinguishable from the background blue light of biological cell, the advantages of with high sensitivity, selective good and biological hypotoxicity, and its preparation technology simple optimizing, greatly reduce the testing cost of sulfydryl biological micromolecule.
Description
Technical field
The invention belongs to sulfydryl biological micromolecule detection technique field, and in particular to it is a kind of based on small molecule fluorescent visit
Pin and its preparation method and application.
Background technology
Sulfydryl biological micromolecule includes cysteine, homocysteine and reduced glutathione etc., mainly passes through mercapto
Base-SH and play physiological action, sulfydryl (- SH) is chemism highest group in cell.Due to the strong configurational energy of sulfydryl
Power and reproducibility, they can be combined with heavy metal ion, or dispose the active oxygen in human body, in the antioxidant system of cell
In have the function that it is important, available for maintaining the normal physiologically active of biological cell and protect cells from foreign substance injury
Micromolecular compound.The sulfydryl biological micromolecule class of amino acid essential as cell, their physiology and disease in cell
Important role is all play during reason.For example, drowsiness, slow-growing, hepar damnification can be caused by lacking cysteine, it is high by half
Cystine level is too high may to induce alzheimer disease, angiocardiopathy and coronary heart disease etc..As many chemistry in cell
The precursor of material, glutathione are also essential.Therefore, by detecting containing for the sulfydryl biological micromolecule in biological cell
Amount, can obtain some physiology of biological cell and the information of pathology, be significant in terms of bioscience, medical science.
At present, the detection method of sulfydryl biological micromolecule is mainly electrochemical gaging, immunoassays, high performance liquid chromatography
Deng.But there is low, anti-light Bleachability poor, bio-toxicity height of testing cost height, poor selectivity, sensitivity etc. and lack in these methods
Fall into.
The content of the invention
In view of this, it is an object of the invention to provide it is a kind of based on small-molecule fluorescent probe and preparation method thereof.
The fluorescent probe molecule amount is small, and structure is novel, applied to the sulfydryl biological micromolecule in detection biological cell, has sensitivity
The advantages of high, selective good, preparation is simple and cost is cheap.
For achieving the above object, concrete technical scheme of the invention is as follows：
It is a kind of based on small-molecule fluorescent probe, its chemical structural formula is：
Present invention also offers a kind of preparation method of above-mentioned small-molecule fluorescent probe, comprise the following steps：
A) it will be dissolved in the first reaction dissolvent, and be then slowly added into concentrated nitric acid, and react, obtain the first intermediate product；
B) the first intermediate product, ferric trichloride and the activated carbon are dissolved in the second reaction dissolvent, then slowly added
Enter hydrazine hydrate, react, obtain the second intermediate product；
C) second intermediate product and maleic anhydride are reacted in the 3rd reaction dissolvent, obtains the small molecule fluorescent
Probe.
Preferably, the concentration of concentrated nitric acid described in step a) is 65%~68%；
Nitric acid and described mole addition in the concentrated nitric acid is than being 2:(14.4~15.2).
It is furthermore preferred that the temperature reacted described in step a) is 60 DEG C, the reaction time is 0.5~1h.
It is furthermore preferred that the first intermediate product described in step a) is 1- nitros or 3- nitros；
First reaction dissolvent is 1,4- dioxane.
Preferably, the first intermediate product described in step b), ferric trichloride and hydrazine hydrate mole addition than for (80~
90):(3~7):100；
Mole addition of the activated carbon and first intermediate product is than being 12:1~20:1.
It is furthermore preferred that the temperature reacted described in step b) is 90~100 DEG C, the time is 3~4h；
Second intermediate product is 1- amine or 3- amine；
It is furthermore preferred that the second reaction dissolvent described in step b) is the mixed solution of non-polar solven and alcohol；
The mixed volume ratio of the non-polar solven and alcoholic solution is 1:1~1:3.
Preferably, mole addition of the second intermediate product described in step c) and the maleic anhydride is than being 1:2~1:6；
The temperature of the back flow reaction is 70~90 DEG C, and the reaction time is 5~6h；
3rd reaction dissolvent is acetic acid.
The small-molecule fluorescent probe obtained present invention also offers foregoing small-molecule fluorescent probe or above-mentioned preparation method exists
Detect the application in intracellular sulfydryl biological micromolecule.
In summary, the present invention by elder generation 1 carbon or 3 carbon on occur nitration reaction connect a nitro, so
Nitro is reduced into amino afterwards, then using maleic anhydride substituted-amino, so as to obtain two structure novelties provided by the present invention
Small-molecule fluorescent probe, shown in its chemical constitution such as formula (I) or formula (II).In the small-molecule fluorescent probe and biological cell
Sulfydryl biological micromolecule sends green glow when combining, hence it is evident that is different from the background blue light of biological cell, has high sensitivity, selectivity
The advantages of good and biological hypotoxicity, and its preparation technology simple optimizing, greatly reduce the detection of sulfydryl biological micromolecule
Cost.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
The embodiment of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can also basis
The accompanying drawing of offer obtains other accompanying drawings.
Fig. 1 be in embodiment 2 fluorescence probe 1-NH-MA added in DMSO solution before and after cysteine (Cys) it is ultraviolet-
Visible absorption spectra figure；
Fig. 2 is the ultraviolet-ray visible absorbing light that fluorescence probe 3-NH-MA is added in DMSO solution before and after Cys in embodiment 2
Spectrogram；
Fig. 3 is the fluorescent emission spectrogram that fluorescence probe 1-NH-MA is added before and after Cys in DMSO solution in embodiment 3；
Fig. 4 is the fluorescent emission spectrogram that fluorescence probe 3-NH-MA is added before and after Cys in DMSO solution in embodiment 3；
Fig. 5 is fluorescence intensity block diagrams of the fluorescence probe 1-NH-MA under different substrates in embodiment 4；
Fig. 6 is fluorescence intensity block diagrams of the fluorescence probe 3-NH-MA under different substrates in embodiment 4；
Fig. 7 a are the cell that the cell of the biological micromolecule containing sulfydryl in embodiment 5 is added after fluorescence probe 1-NH-MA cultures
Image；
Fig. 7 b are thin after the cell addition fluorescence probe 1-NH-MA cultures without sulfydryl biological micromolecule in embodiment 5
Born of the same parents' image；
Fig. 7 c are the cell that the cell of the biological micromolecule containing sulfydryl in embodiment 5 is added after fluorescence probe 3-NH-MA cultures
Image；
Fig. 7 d are thin after the cell addition fluorescence probe 3-NH-MA cultures without sulfydryl biological micromolecule in embodiment 5
Born of the same parents' image.
Embodiment
The invention provides chemical constitution as shown in formula (I) or formula (II) it is a kind of based on small-molecule fluorescent probe：
The small-molecule fluorescent probe belongs to nucleophilic addition type structure, is a kind of higher micromolecular compound of conjugated degree.
Maleimide has stronger electron-withdrawing power in molecular structure, forms Photo-induced electron transfer (PET) effect, causes the spy
Pin fluorescent quenching, but when probe runs into the compound containing sulfydryl, with maleimide addition reaction, PET effects occur for sulfydryl
It is broken, the fluorescence of probe also recovers therewith.The small-molecule fluorescent probe is combined with the sulfydryl biological micromolecule in biological cell
When send green glow, hence it is evident that be different from the background blue light of biological cell, there is the excellent of the good and biological hypotoxicity of high sensitivity, selectivity
Point.
By the optimization of the synthesis technique to above-mentioned small-molecule fluorescent probe, the synthesis of above-mentioned small-molecule fluorescent probe includes
Following steps：
A) it will be dissolved in Isosorbide-5-Nitrae-dioxane, and be then slowly added into concentrated nitric acid, 0.5~1h is reacted at 60 DEG C, is obtained
To the first intermediate product；
B) the first intermediate product, ferric trichloride and the activated carbon are dissolved in reaction solution, are then slowly added into water
Hydrazine is closed, 3~4h is reacted at 90~100 DEG C, obtains 1- amine or 3- amine；
C) by second intermediate product and maleic anhydride in acetic acid 70~90 DEG C of 5~6h of back flow reaction, obtain described
Small-molecule fluorescent probe.
Further, the concentration of concentrated nitric acid described in step a) is 65%~68%；Nitric acid and institute in the concentrated nitric acid
The addition mol ratio stated is 2:(14.4~15.2)；
First intermediate product is 1- nitros and/or 3- nitros.
Further, mole addition of the first intermediate product described in step b), ferric trichloride and hydrazine hydrate is than being 84:5:
100；
Mole addition of the activated carbon and first intermediate product is than being 12:1~20:1.
The reaction solution is toluene and ethanol by volume 1:1~1:3 mixed solutions being mixed to get.
Further, mole addition of the second intermediate product described in step c) and the maleic anhydride is than being 1:4.
The building-up process of small-molecule fluorescent probe of the present invention can use formulas below to represent：
The small-molecule fluorescent probe obtained present invention also offers foregoing small-molecule fluorescent probe or above-mentioned preparation method exists
Application in sulfydryl biological micromolecule detection technique.
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other under the premise of creative work is not made
Embodiment, belong to the scope of protection of the invention.
Embodiment 1
The preparation process of the small-molecule fluorescent probe of the present embodiment mainly includes the following steps that：
1st, 0.5g (2mmol), 40mL Isosorbide-5-Nitrae-dioxane are placed in double-neck flask successively, stirred at 80 DEG C to
It is completely dissolved the yellow solution for clarification；Then 1mL concentrated nitric acids are slowly injected into syringe, concentrated nitric acid selection concentration is 65%
~68% concentrated nitric acid (14.4~15.2mol/L), after stirring 30min at 60 DEG C, obtain inclusion compound 1 and compound 2
Reaction solution；Then, reaction solution is poured into a large amount of frozen water while hot, cerise is obtained after being filtered under diminished pressure and being dried in vacuo 12h
Crude product；Finally, chromatography is used using volume ratio as 1.5:1 petroleum ether (PE)-dichloromethane (DCM) obtains as eluent
To kermesinus needle-like solid compound 1 (yield 20%) and brick-red solid compound 2 (yield 63%).
Its chemical equation is as follows：
The hydrogen nuclear magnetic resonance modal data of compound 1 and compound 2 is as follows：
Compound 1-1H NMR(400MHz,CDCl3)：δ8.28(m,2H),7.84(m,3H),7.74(m,2H),7.62(t,
1H, J=8Hz), 7.57 (m, 2H), 7.44 (t, 1H, J=8Hz)；
Compound 2-1H NMR(400MHz,CDCl3)：δ 8.48 (d, 1H, J=8Hz), 8.26 (m, 4H), 8.10 (d, 1H,
), J=8Hz 7.82 (d, 1H, J=8Hz), 7.76 (d, 1H, J=8Hz), 7.68 (t, 1H, J=8Hz), 7.55 (m, 2H).
2nd, by 0.1248g (0.42mmol) compound 1,0.004g (0.025mmol) FeCL3With 0.0083g work
Property charcoal add double-neck flask, then add 5mL toluene and 5mL ethanol dissolving；Then, it is past mixed after heating stirring to 98 DEG C of boilings
The hydrazine hydrate that 0.25mL (5mmol) is slowly added dropwise in conjunction liquid is reacted, and 0.0054g FeCl are added in centre3, 0.0049g activity
Charcoal, 125 μ L hydrazine hydrates, 2mL ethanol and 2mL toluene, it is filtered under diminished pressure after reacting 4h, filtrate is spin-dried for；Finally, use chromatography with
PE：DCM=1:1.25 eluent obtains the compound 3 of crocus through column chromatography.
Its chemical equation is as follows：
The hydrogen nuclear magnetic resonance modal data of compound 3 is as follows：
1H NMR(400MHz,DMSO-d6)：δ 8.42 (d, 1H, J=7.6Hz), 8.16 (t, 2H, J=6.8Hz), 7.69
(d, 1H, J=8.0Hz), 7.59 (q, 3H, J=9.2Hz), 7.5-7.4 (m, 2H), 7.27 (t, 1H, J=7.27Hz), 7.24
(d, 1H, J=8.8Hz), 6.05 (s, 2H).
3rd, the distinctive points of this step and step 2 are：Compound 1 replaces with compound 2, and remaining place and step 2 are identical,
No longer repeat one by one herein.
The chemical equation of this step is as follows：
The hydrogen nuclear magnetic resonance modal data of compound 4 is as follows：
1H NMR(400MHz,DMSO-d6)：δ 8.28 (d, 1H, J=4Hz), 8.22 (d, 1H, J=8Hz), 8.10 (d, 1H,
), J=12Hz 8.01~7.95 (m, 2H), 7.66 (d, 1H, J=8Hz), 7.53 (d, 1H, J=8Hz), 7.44~7.37 (m,
3H), 6.77 (d, 1H, J=12Hz), 6.12 (s, 2H).
4th, compound 3 is added in the acetic acid solution that 15mL contains 0.196g (2mmol) maleic anhydride, at 100~120 DEG C
Lower backflow 6h；After TLC detection reactions completely, solvent is removed with Rotary Evaporators, then with saturation NaHCO3Solution and DCM
It is extracted twice, and through anhydrous sodium sulfate drying；Finally, chromatography is used with n-hexane (HeX)：DCM=1:2 be eluent post
Chromatography obtains the compound 5 of crocus solid-state.
The chemical equation of this step is as follows：
The hydrogen nuclear magnetic resonance modal data of compound 5 is as follows：
1H NMR(400MHz,DMSO-d6)：δ 8.24 (t, 2H, J=8Hz), 7.78~7.68 (m, 5H), 7.60~7.51
(m, 2H), 7.33 (t, 1H, J=8Hz), 7.22 (d, 1H, J=8Hz), 6.97 (s, 2H).
5th, the distinctive points of this step and step 4 are：Compound 3 replaces with compound 4, finally use chromatography with just oneself
Alkane (HeX)：DCM=1:4.5 be that eluent obtains the compound 6 of golden yellow solid-state, yield about 83% with column chromatography.Its leeway
Side and step 4 are identical, no longer repeat one by one herein.
The chemical equation of this step is as follows：
The hydrogen nuclear magnetic resonance modal data of compound 6 is as follows：
1H NMR(400MHz,CDCl3)：δ 8.48 (q, 4H, J=8Hz), 7.88~7.85 (m, 2H), 7.62~7.52 (m,
5H),7.31(s,2H)。
Embodiment 2
Take compound 5 and compound 6 to be respectively designated as fluorescence probe 1-NH-MA and 3-NH-MA, then use DMSO systems
It is 10 to take concentration-5Mol/L probe solution；Then, toward being separately added into and the Guang of concentration and probe concentration identical half in two kinds of probe solutions
Propylhomoserin solution (Cys)；Finally, its absorbance is detected using ultraviolet-uisible spectrophotometer.As a result as depicted in figs. 1 and 2, probe
1-NH-MA and 3-NH-MA absorption is respectively positioned between 350nm~480nm, probe before and after cysteine is added absorbing wavelength with
Absorption intensity has almost no change.
Embodiment 3
Take compound 5 and compound 6 to be respectively designated as fluorescence probe 1-NH-MA and 3-NH-MA, then use DMSO systems
It is 10 to take concentration-5Mol/L probe solution；Then, toward the cysteine being separately added into two kinds of probe solutions with various concentrations
Solution (Cys), influences of the test Cys to the fluorescence intensity of probe, find after tested when Cys concentration is 1 × 10-5During mol/L, visit
Pin luminous intensity is most strong.As shown in Figure 3 and Figure 4, probe 1-NH-MA and 3-NH-MA is not luminous or luminous weaker in itself, adds
Cys(10-5Mol/L occurs obvious Fluorescence Increasing after) immediately, fluorescence intensity is distinguished after 1-NH-MA and 3-NH-MA add Cys
71 times and 196 times are enhanced, illustrate that two kinds of fluorescence probes that the present embodiment provides have higher sensitivity.
Embodiment 4
Take compound 5 and compound 6 to be respectively designated as fluorescence probe 1-NH-MA and 3-NH-MA, then use DMSO systems
Take 15 groups of probe solutions (10-5Mol/L), wherein 10 groups of amino acid for being separately added into 1 times of equivalent fluorescence probe, the Amino acid score
Wei not cysteine (L-cysteine), glutamine (glutamine), homocysteine (DL-Homocysteine), paddy
Propylhomoserin (glutamic), amion acetic acid (aminoacetic), histidine (histidine), lysine (lysine), first sulphur ammonia
Sour (L-Methionine), phenylalanine (L-Phenylalanine), serine (L-Serine), threonine (L-
) and tryptophan (L-Tryptophan) Threonine；Other 2 groups are separately added into ascorbic acid (Ascorbic acid) and are passed through
Hydrogen peroxide (Hydrogen Peroxide)；Last group is used as blank control group.Then, fluorescence spectrum is used under 437nm
The luminous intensity of 15 groups of fluorescence probe 1-NH-MA solution is detected, as a result as shown in Figure 5；Detected under 439nm using fluorescence spectrum
The luminous intensity of 15 groups of fluorescence probe 3-NH-MA solution, as a result as shown in Figure 6.
As shown in Figure 5 and Figure 6, probe 1-NH-MA and 3-NH-MA fluorescence intensity is weaker close to zero, is adding non-mercapto
Fluorescence Increasing unobvious after base amino acid, ascorbic acid and hydrogen peroxide, without significant difference compared with blank control group.However,
Fluorescence Increasing is the most obvious after the mercaptoamino acids such as cysteine, glutamine and homocysteine are added.Illustrate this reality
The fluorescence probe 1-NH-MA and 3-NH-MA for applying example offer has very high selectivity to sulfydryl biological micromolecule.
Embodiment 5
Probe 1-NH-MA and 3-NH-MA are separately added into cultured attached cell nutrient solution, are placed in 37 DEG C of cell trainings
Support and a period of time is cultivated in case, then by cell with carrying out cell imaging after PBS.As a result as shown in Fig. 7 a and 7c, display
Cell sends stronger green fluorescence in addition probe 1-NH-MA and 3-NH-MA.
In check experiment, by cell first using (elimination is into the cell containing sulfydryl after the pretreatment of N- methyl maleic anhydrides
Compound), then fluorescence probe 1-NH-MA and 3-NH-MA are separately added into, continue to cultivate one section in 37 DEG C of cell culture incubators afterwards
Time, carry out cell imaging.As shown in Fig. 7 b and 7d, cell does not launch fluorescence or fluorescence is very weak.
The present embodiment result illustrates that probe 1-NH-MA and 3-NH-MA have preferably selectivity in cell, furtherly
Understand that probe 1-NH-MA and 3-NH-MA can carry out sulfhydryl compound Visual retrieval in biological cell.
Claims (10)
1. it is a kind of based on small-molecule fluorescent probe, it is characterised in that its chemical structural formula is：
2. the preparation method of small-molecule fluorescent probe described in a kind of claim 1, it is characterised in that comprise the following steps：
A) it will be dissolved in the first reaction dissolvent, and be then slowly added into concentrated nitric acid, and react, obtain the first intermediate product；
B) the first intermediate product, ferric trichloride and the activated carbon are dissolved in the second reaction dissolvent, are then slowly added into water
Hydrazine is closed, reaction, obtains the second intermediate product；
C) second intermediate product and maleic anhydride are reacted in the 3rd reaction dissolvent, obtains the small molecule fluorescent and visit
Pin.
3. preparation method according to claim 2, it is characterised in that the concentration of concentrated nitric acid described in step a) be 65%~
68%；
Nitric acid and described mole addition in the concentrated nitric acid is than being 2:(14.4~15.2).
4. preparation method according to claim 2, it is characterised in that the temperature reacted described in step a) is 60 DEG C, instead
It is 0.5~1h between seasonable.
5. preparation method according to claim 2, it is characterised in that the first intermediate product described in step a) is 1- nitros
Or 3- nitros；
First reaction dissolvent is 1,4- dioxane.
6. preparation method according to claim 2, it is characterised in that the first intermediate product, tri-chlorination described in step b)
A mole addition ratio for iron and hydrazine hydrate is (80~90):(3~7):100；
Mole addition of the activated carbon and first intermediate product is than being 12:1~20:1.
7. preparation method according to claim 2, it is characterised in that the temperature reacted described in step b) is 90~100
DEG C time is 3~4h；
Second intermediate product is 1- amine or 3- amine.
8. preparation method according to claim 2, it is characterised in that the second reaction dissolvent described in step b) is nonpolar
The mixed solution of solvent and alcohol；
The mixed volume ratio of the non-polar solven and alcohol is 1:1~1:3.
9. preparation method according to claim 2, it is characterised in that the second intermediate product described in step c) and the horse
Carry out mole addition of acid anhydrides than being 1:2~1:6；
The temperature of the back flow reaction is 70~90 DEG C, and the reaction time is 5~6h；
3rd reaction dissolvent is acetic acid.
What 10. preparation method described in the small-molecule fluorescent probe or claim 2 to 9 any one described in claim 1 obtained
Application of the small-molecule fluorescent probe in intracellular sulfydryl biological micromolecule is detected.
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| CN112778137A (en) * | 2021-01-08 | 2021-05-11 | 山东师范大学 | End-position nitrated 3, 4-perylene dicarboxylic ester compound and synthetic method and application thereof |
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| CN101250405A (en) * | 2008-02-26 | 2008-08-27 | 南京大学 | Novel mercapto fluorescence probe and uses thereof |
| CN105712964A (en) * | 2016-01-18 | 2016-06-29 | 新乡医学院 | Preparation method and application of mercaptan fluorescence probe based on coumarin |
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| CN101250405A (en) * | 2008-02-26 | 2008-08-27 | 南京大学 | Novel mercapto fluorescence probe and uses thereof |
| CN105712964A (en) * | 2016-01-18 | 2016-06-29 | 新乡医学院 | Preparation method and application of mercaptan fluorescence probe based on coumarin |
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| YONGBIN ZHANG ET AL.: "An off–on fluorescent probe based on maleimide for detecting thiols and its application for bioimaging", 《SENSORS AND ACTUATORS B》 * |
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| CN112778137A (en) * | 2021-01-08 | 2021-05-11 | 山东师范大学 | End-position nitrated 3, 4-perylene dicarboxylic ester compound and synthetic method and application thereof |
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