CN105524608A - Fluorescent probe AH and preparation and application thereof - Google Patents
Fluorescent probe AH and preparation and application thereof Download PDFInfo
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Abstract
The invention provides a fluorescent probe AH and preparation and application thereof and specifically provides a fluorescent probe capable of being used for selectively detecting intracellular alkaline phosphatase. The main synthesis method comprises the step of introducing 2',2-hydroxyphenyl benzothiazole (HBT) to an alkaline phosphatase similar substrate micromolecular 5'-adenosine monophosphate (AMP) so as to produce a compound with a structure represented by a formula defined in the description. HBT is produced under the action of the alkaline phosphatase, the selective detection on the alkaline phosphatase is achieved by using difference of a fluorescent property before and after a reaction, and a coloration action on the alkaline phosphatase of a zebra fish body can be exerted.
Description
Technical field
The present invention relates to fluorescent probe field, be specifically related to a kind of fluorescent probe that can be used for selective enumeration method alkaline phosphatase.Small molecules 5 ' ribose phosphoric acid adenosine (being called for short AMP) common in cell is upper introduces fluorophore 2 ' 2 hydroxyphenyl benzothiazole (HBT), after alkaline phosphatase effect, utilize the fluorescence difference of reactant and product to realize the alkaline phosphatase optionally detected in viable cell, and obtain good application on the living imaging of zebra fish.
Background technology
Alkaline phosphatase (ALP, ALKP, ALPase, AlkPhos) (EC3.1.3.1) is distributed widely in each internal organs organ of human body, and being wherein at most with liver, is secondly the tissues such as kidney, bone, intestines and placenta.ALP can mediating protein, nucleic acid, and the dephosphorylation of the biomacromolecule such as alkaloids.This dephosphorylation and kinase whose phosphorylation closely related, be regulate process necessary in the vital movements such as substance metabolism in organism, substance transportation, signal transduction.Higher and the hepatic diseases of the alkaline phosphatase of generally acknowledging at present, hepato-enteric circulation, skeletal diseases is closely related.The alkaline phosphatase of human serum is widely used in the instruction of disease, when ALP produces too much or excretion is obstructed, ALP level in blood all can be made to change.But the inherent mechanism of its level modulation is also unclear.Therefore, the probe that can detect cell alkaline phosphatase is in real time developed significant.
Fluorescent probe is one of means effectively detecting alkaline phosphatase in life entity, and phase absorbance-ratio method has the sensitive advantage of detection.A fluorescent probe with application prospect should have before and after effect that change in fluorescence obviously, fast to target molecule response, selectivity is good, synthesize the advantages such as simple.The method being applied to detection of alkaline phosphatase at present mainly concentrates on the detection of serum alkaline phosphatase level, and the importance detecting cell alkaline phosphatase is not subject to too many attention.The most widespread use of detection method or the absorption spectrum detecting the dephosphorylation product of pnpp (phosphoric acid p-nitrophenol) under 405nm, although the development that the fluorescent probe of detection of alkaline phosphatase has some new, the fluorescent probe that selectivity is good is still very few.Especially alkaline phosphatase, acid phosphatase, phosphodiesterase, adenylate cyclase has a lot of substrate crossover phenomenon for dephosphorylized effect, develop to its selectively probe there is very large challenge.
Summary of the invention
The present invention is exactly for the problems referred to above, provides a kind of fluorescent probe that can be used for alkaline phosphatase in selective enumeration method cell, this probe can in physiological conditions optionally with alkaline phosphatase effect, effect after fluorescence significantly strengthen.
The present invention adopts following technical scheme: adopt 2 ' 2 hydroxyphenyl benzothiazoles (HBT) as fluorescent parent, (being called for short AMP) introduces HBT to ubiquitous small molecules 5 ' ribose phosphoric acid adenosine in vivo, utilize the fluorescence difference of reactant and product to realize optionally detection of alkaline phosphatase, especially can be applied to the alkaline phosphatase detected in viable cell.The structure of the probe compound of synthesis represents with code name AH.
The structural formula I of described fluorescent probe is as follows.
The preparation method of described fluorescent probe is: using 2 ' 2 hydroxyphenyl benzothiazoles (HBT) as fluorescent parent, and (being called for short AMP) introduces HBT to ubiquitous small molecules 5 ' ribose phosphoric acid adenosine in vivo; Concrete preparation process is as follows,
1) phosphorus oxychloride and HBT are in solvent after stirring reaction, and lower 70 DEG C of vacuum heats evaporates to dryness;
2) step 1) in obtain product cooling after, add solvent, after stirring, add 2 ' 3 '-O-isopropylideneadenosine, after stirred at ambient temperature reaction 10h, the lower 70 DEG C of heating evaporates to dryness of vacuum, add a small amount of water again, stir 1h, be spin-dried for propyl carbinol azeotropic;
3) step 2) in the product that obtains through purification by silica gel column chromatography (making eluent with methylene dichloride and methyl alcohol) compound 2 ' 3 '-O-isopropyadenosin5 ' (HBT-phosphate) (white solid);
4) step 3 is got) products therefrom is dissolved in acetic acid and water, and condensing reflux, be spin-dried for azeotropic at propyl carbinol 80 DEG C afterwards, methanol wash product three times, obtains final product AH.
Step 1) described in HBT and the add-on of phosphorus oxychloride be 1:3-10; Step 2) in the add-on of 2 ' 3 '-O-isopropylideneadenosine be the 1.2-1.5 of HBT add-on in the first step doubly; Step 3) in chromatographic rf be 0.1, described methylene dichloride and the ratio of methyl alcohol are 10:1; Step 4) in the amount of the acetic acid that adds and water be 4:1, the temperature of condensing reflux is 100 DEG C, and the time is 2h.
Step 1) and step 2) in, described solvent used is anhydrous pyridine; Step 1)-4) described in alr mode be magnetic agitation.
Described fluorescent probe may be used for the qualitative of alkaline phosphatase or detection by quantitative; Described fluorescent probe and the action model of alkaline phosphatase meet dibit point enzyme kinetics.
When described fluorescent probe is applied to detection of alkaline phosphatase, it generates the compound with structure I I, thus cause change in fluorescence, and the name of structure I I is called 2 ' 2 hydroxyphenyl benzothiazoles (HBT).
Described fluorescent probe is in detection by quantitative alkaline phosphatase, and after alkaline phosphatase effect, the fluorescence intensity that 512nm place is detected is proportional to the activity of alkaline phosphatase.
Described fluorescent probe can be used for the detection of alkaline phosphatase in cell, realizes the fluorescence imaging of alkaline phosphatase in cell; Described fluorescent probe can be used for the colour developing of zebra fish body alkaline phosphatase.
Beneficial effect of the present invention: this compound fluorescence under alkaline phosphatase exists occurs significantly to change, can be used for highly selective, high sensitivity ground detection of alkaline phosphatase.Especially, the alkaline phosphatase that this compound can be used in cell detects, and this kinetics mechanism for further investigation alkaline phosphatase physiology and pathologic process is in vivo significant.
Accompanying drawing explanation
The synthetic route chart of the fluorescent probe AH provided in Fig. 1 embodiment 1;
The principle schematic of Fig. 2 fluorescent probe AH provided by the invention detection of alkaline phosphatase;
The probe AH's that Fig. 3 the present invention synthesizes
1hNMR (a),
13cNMR (b),
32pNMR (c) spectrogram;
Ultraviolet-visible absorption spectroscopy (a), fluorescence excitation spectrum (b), the emmission spectrum (c) of fluorescent probe AH and the fluorophore HBT aqueous solution in Fig. 4 embodiment 2;
In Fig. 5 embodiment 3, fluorescent probe AH is to the selectivity schematic diagram of alkaline phosphatase;
Kinetics schematic diagram (a) that in Fig. 6 embodiment 4, fluorescent probe AH responds the ALP of different concns and the linear relationship (b) of speed of reaction and enzyme concn;
In Fig. 7 embodiment 5 fluorescent probe AH and alkaline phosphatase speed of reaction kinetics schematic diagram;
Fluorescent probe AH schematic diagram under the suppressed effect of alkaline phosphatase in Fig. 8 embodiment 6.
Fluorescent probe AH imaging schematic diagram in Hela cell in Fig. 9 embodiment 7.
In Figure 10 embodiment 8, fluorescent probe AH is at zebra fish body living imaging schematic diagram.
Embodiment
Embodiment is used for further illustrating the present invention, but the invention is not restricted to embodiment.
Embodiment 1 (synthesis of probe):
As shown in Figure 1, the synthesis of AH: 50mL two mouthfuls of flask vacuum/nitrogen replace three times, add the dry pyridine of 20mL and the dry phosphorus oxychloride of 2mL, slowly add HBT0.82g, stirred at ambient temperature 1h.After 1h, the lower 70 DEG C of heating evaporates to dryness of vacuum, obtain light green solid.Stop heating after cooling, reconnect vacuum/nitrogen and replace three times, again add pyridine 20mL, make vortex, greenish precipitate is all dissolved, add 1.2g2', 3'-O-Isopropylideneadenosine (2', 3'-O-isopropylideneadenosine), stirs, the lower 70 DEG C of heating evaporates to dryness of room temperature reaction 10h, 10h final vacuum.Add a small amount of water again, stir 1h, be spin-dried for propyl carbinol azeotropic.Products therefrom is obtained compound 2 ' 3 '-O-isopropyadenosin5 ' (HBT-phosphate) (white solid) quality how many grams of 500mg through purification by silica gel column chromatography (making eluent with methylene dichloride and methyl alcohol).Get this compound 400mg, be dissolved in 4.8mL acetic acid and 1.2mL water, 100 DEG C of condensing reflux 2h.After reaction terminates, be spin-dried for 10mL propyl carbinol 80 DEG C of azeotropic vacuum.With methanol wash column three times, each 20mL.Finally obtain target compound AH (white solid).
1HNMR(400MHz,Dimethylsulfoxide-d6)δ(ppm):δ＝8.72(s,2H),8.61(s,1H),8.38(d,1H,J＝7.72Hz),8.28(s,1H),8.02(d,1H,J＝8.08Hz),7.98(d,1H,J＝7.88Hz),7.70(d,1H,J＝8.2Hz),7.48(t,1H,J＝7.32Hz),7.43(t,1H,J＝7.52Hz),7.35(t,1H,J＝7.2Hz),7.23(t,1H,J＝7.48Hz),5.95(d,1H,J＝5.2Hz),4.60(t,1H,J＝4.88Hz),4.1-4.3(m,4H).
13CNMR(Dimethylsulfoxide-d6,100MHz)δ(ppm):162.82,152.44,152.06,149.02,147.86,141.63,136.00,132.10,129.20,126.63,125.35,123.94,123.64,122.93,122.13,120.62,119.06,88.00,84.16,74.35,70.88,66.48.
32PNMR(Dimethylsulfoxide-d6,100MHz)δ＝0.04(s)。
Embodiment 2
As shown in Figure 4, (ultraviolet-visible absorption spectroscopy (a), fluorescence excitation spectrum (b), the emmission spectrum (c) of fluorescent probe AH and the fluorophore HBT aqueous solution):
AH is dissolved in the PB damping fluid of 100mM, is configured to the solution of 20uM.Get 3mL solution to add in the cuvette of 1cm X 1cm X 4cm, fluorescence excitation spectrum (b), the emmission spectrum (c) of the fluorophore HBT aqueous solution produced after measuring the ultraviolet-visible absorption spectroscopy (a) of this working fluid, AH and alkaline phosphatase effect); The maximum excitation wavelength of result display probe AH/HBT is 318nm/330nm.Probe AH maximum emission wavelength is 370nm, and fluorophore HBT is different with concentration in aqueous and have two emission peak 460nm/512nm.
Embodiment 3 (AH is to the selectivity of alkaline phosphatase):
Configuration alkaline phosphatase, acid phosphatase, adenylate cyclase, 3 '-5 ' aqueous solution of phosphodiesterase (four kinds of enzyme concns are 10uM), four kinds of enzymes are divided into some parts respectively, and the amount of every part is identical ,-20 DEG C of refrigerated storage.Slowly dissolve on ice before using, only use once for every part.The DMSO solution (important polar aprotic solvent) of configuration AH (20mM).Each reaction adds 2ulAH (20mM) and 2ul enzyme in the PB damping fluid of 196ul100mM, and use the multi-functional readout instrument of full wavelength scanner formula and 96 hole enzyme plates to measure, the final concentration of probe and enzyme is respectively 20uM and 100nM.Measure the fluorescence emission spectrum of this working fluid, λ ex=330nm, raster width is 5nm, λ em=512nm.
To the selectivity experimental result of alkaline phosphatase as shown in Figure 5, ordinate zou represents that wavelength is the fluorescence intensity at 512nm place to AH.Fig. 5 shows that AH has good selectivity to alkaline phosphatase, and system fluorescence significantly strengthens.Under condition determination, compared to alkaline phosphatase, the adenylate cyclase of same concentrations, 3 '-5 ' effect of phosphodiesterase can ignore, the catalytic efficiency of acid phosphatase be its 1/5.
Embodiment 4 (AH is to the detection by quantitative of alkaline phosphatase):
By the alkaline phosphatase stock concentrations gradient dilution of 10uM, each reaction adds the enzyme of 2ulAH (20mM) and 2ul different concns (10nM-250nM) in the PB damping fluid (100mM) of 196ul.The multi-functional readout instrument of full wavelength scanner formula and 96 hole enzyme plates are used to measure.Measure the fluorescence emission spectrum of this working fluid, λ ex=330nm, raster width is 5nm, λ em=512nm.Result shows: as Fig. 6, with the growth of enzyme concn, the generating rate of HBT grows proportionately.
Embodiment 5 (the zymetology parametric measurement of AH):
The DMSO solution (25mM) of configuration AH, and configure the liquid storage of gradient dilution.Each reaction adds the enzyme (100nM) of 2ulAH (10uM-25mM) and 2ul in the PB damping fluid (100mM) of 196ul.The multi-functional readout instrument of full wavelength scanner formula and 96 hole enzyme plates are used to measure.Measure the fluorescence emission spectrum of this working fluid, λ ex=330nm, raster width is 5nm, λ em=512nm.Result shows: as Fig. 7, probe AH is met the two-site model in enzyme kinetics by alkaline phosphatase enzyme catalysis, has two cover km/vmax values, wherein K
m1=1.89 μMs, V
max1=9.8*10
5pmol/min/nmol; K
m2=238.6 μMs, V
max2=1.64*10
7pmol/min/nmol.
Embodiment 6 (Inhibition test of AH):
The PB solution of configuration alkaline phosphatase enzyme specific inhibitor L-Phenylalanine (100mM).Each reaction adds the enzyme (100nM) of 2ulAH (20mM) and 2ul and 100ulL-Phenylalanine (100mM) or 100ul damping fluid (contrast) in the PB damping fluid (100mM) of 96ul.The multi-functional readout instrument of full wavelength scanner formula and 96 hole enzyme plates are used to measure.Measure the fluorescence emission spectrum of this working fluid, λ ex=330nm, raster width is 5nm, λ em=512nm.Result shows: as Fig. 8, AH can be suppressed by Inhibitors of Alkaline Phosphatase completely by the katalysis of alkaline phosphatase, again proves that AH is the probe substrate of alkaline phosphatase.
Embodiment 7 (probe AH is used for the detection of HELA cell alkaline phosphatase): HELA cell is cultivated according to AmericantypeTissueCultureCollection regulation.Be inoculated in the culture dish of 35mm × 12mm and cultivate 24h and make it adherent.Before using, nutrient solution is discarded, wash three times with 100mMPBS, add the PBS solution (PBS buffer concentration is 100mM, pH7.4,138mMNaCl) of 10uMAH, home position observation in 5min-30min.Inhibition test, will first with the PBS solution preincubate 15min of the levamisole (alkaline phosphatase enzyme specific inhibitor) of 10mM before adding probe.Laser Scanning Confocal Microscope OlympusFV1000 (Ex.405nm, Em.500-530nm) camera lens (× 40) is used to carry out observing and IMAQ.Control experiment, HT-29cells cultivates according to AmericantypeTissueCultureCollection regulation equally, adds probe in-situ and observes 30min.Result shows: as shown in Figure 9, in HELA cell, fluoroscopic image brightness is apparently higher than control group; And the gradually bright change of fluorescence is had at 5min, 10min, 30min.This experiment proves that probe AH may be used for the real-time detection of cell alkaline phosphatase.
Embodiment 8 (fluorescent probe AH is at zebra fish body living imaging schematic diagram):
Zebra fish is the mazarine longitudinal grin zebra fish adult of commercially available artificial breeding.Experiment fasted for one day prior.Experimental group is placed in the culture dish of 35mm × 12mm the 1h that suffocates to dead before using, wash three times, add the PBS solution of 10uMAH, home position observation in 5min-30min with 100mMPBS.Inhibition test, will first with the PBS solution preincubate 15min of the levamisole (alkaline phosphatase enzyme specific inhibitor) of 10mM before adding probe.Laser Scanning Confocal Microscope OlympusFV1000 (Ex.405nm, Em.500-530nm) camera lens (× 4) is used to carry out observing and IMAQ.Result such as Figure 10 shows, and when adding probe, zebra fish body has the change procedure from dark to bright, especially appears at its belly, fin, and gill portion, and in suppression group and blank group not this phenomenon.This experiment proves that probe AH can be used for the colour developing of zebra fish body alkaline phosphatase.
Claims (9)
1. a fluorescent probe AH, is characterized in that: the structure of described fluorescent probe for shown in structure I,
Structure code name: AH.
2. the preparation of a fluorescent probe AH according to claim 1, it is characterized in that: described fluorescent probe is using 2 ' 2 hydroxyphenyl benzothiazoles (HBT) as fluorescent parent, (being called for short AMP) introduces HBT to ubiquitous small molecules 5 ' ribose phosphoric acid adenosine in vivo; Concrete preparation process is as follows,
1) phosphorus oxychloride and HBT are in solvent after stirring reaction, and lower 70 DEG C of vacuum heats evaporates to dryness;
2) step 1) in obtain product cooling after, add solvent, after stirring, add 2 ' 3 '-O-isopropylideneadenosine, after stirred at ambient temperature reaction 10h, the lower 70 DEG C of heating evaporates to dryness of vacuum, add a small amount of water again, stir 1h, be spin-dried for propyl carbinol azeotropic;
3) step 2) in the product that obtains through purification by silica gel column chromatography (making eluent with methylene dichloride and methyl alcohol) compound 2 ' 3 '-O-isopropyadenosin5 ' (HBT-phosphate) (white solid);
4) step 3 is got) products therefrom is dissolved in acetic acid and water, and condensing reflux, be spin-dried for azeotropic at propyl carbinol 80 DEG C afterwards, methanol wash product three times, obtains final product AH.
3. the preparation method of fluorescent probe according to claim 2, is characterized in that: step 1) described in HBT and the add-on of phosphorus oxychloride be 1:3-10; Step 2) in the add-on of 2 ' 3 '-O-isopropylideneadenosine be the 1.2-1.5 of HBT add-on in the first step doubly; Step 3) in chromatographic rf be 0.1, described methylene dichloride and the ratio of methyl alcohol are 10:1; Step 4) in the amount of the acetic acid that adds and water be 4:1, the temperature of condensing reflux is 100 DEG C, and the time is 2h; Described step 1) and step 2) in, solvent used is anhydrous pyridine; Described step 1)-4) in stir mode be magnetic agitation.
4. an application for fluorescent probe according to claim 1, is characterized in that: described fluorescent probe may be used for the qualitative of alkaline phosphatase or detection by quantitative.
5. the application of fluorescent probe according to claim 4, is characterized in that: described fluorescent probe and the action model of alkaline phosphatase meet dibit point enzyme kinetics.
6. the application of fluorescent probe according to claim 4, is characterized in that: when described fluorescent probe is applied to detection of alkaline phosphatase, and it generates the compound with structure I I, thus cause change in fluorescence;
Structure title: 2 ' 2 hydroxyphenyl benzothiazoles (HBT).
7. the application of fluorescent probe according to claim 4, is characterized in that: the application in detection by quantitative alkaline phosphatase, and after described fluorescent probe and alkaline phosphatase effect, the fluorescence intensity that 512nm place is detected is proportional to the activity of alkaline phosphatase.
8. the application of fluorescent probe according to claim 4, is characterized in that: described fluorescent probe can be used for the detection of alkaline phosphatase in cell, realizes the fluorescence imaging of alkaline phosphatase in cell.
9. the application of fluorescent probe according to claim 4, is characterized in that: described fluorescent probe can be used for the colour developing of zebra fish body alkaline phosphatase.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106589005A (en) * | 2016-11-01 | 2017-04-26 | 北京迪纳兴科生物科技有限公司 | Fluorescence signal amplification probe intermediate, fluorescent probe and preparation method thereof |
| CN108948081A (en) * | 2018-07-12 | 2018-12-07 | 青岛科技大学 | A kind of Ratiometric fluorescent probe measuring alkaline phosphatase and its synthetic method and application |
| CN112457303A (en) * | 2020-12-18 | 2021-03-09 | 浙江海洋大学 | Fluorescent compound and preparation method and application thereof |
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| CN103937488A (en) * | 2014-03-25 | 2014-07-23 | 中国科学院理化技术研究所 | Silicon-nanowire-based alkaline phosphatase fluorescent chemosensor, preparation method and application |
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| CN1180349A (en) * | 1996-01-16 | 1998-04-29 | 鲁米根公司 | Compounds, compositions and methods for generating chemiluminescence with phosphatase enzymes |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106589005A (en) * | 2016-11-01 | 2017-04-26 | 北京迪纳兴科生物科技有限公司 | Fluorescence signal amplification probe intermediate, fluorescent probe and preparation method thereof |
| CN106589005B (en) * | 2016-11-01 | 2019-08-06 | 北京擎科生物科技有限公司 | A kind of fluorescence signal amplifying probe intermediate, fluorescence probe and preparation method thereof |
| CN108948081A (en) * | 2018-07-12 | 2018-12-07 | 青岛科技大学 | A kind of Ratiometric fluorescent probe measuring alkaline phosphatase and its synthetic method and application |
| CN108948081B (en) * | 2018-07-12 | 2020-07-07 | 青岛科技大学 | Ratiometric fluorescent probe for determining alkaline phosphatase, and synthetic method and application thereof |
| CN112457303A (en) * | 2020-12-18 | 2021-03-09 | 浙江海洋大学 | Fluorescent compound and preparation method and application thereof |
| CN112457303B (en) * | 2020-12-18 | 2022-01-07 | 浙江海洋大学 | Fluorescent compound and preparation method and application thereof |
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