CN104194773B - A kind of cell membrane targeting nitric oxide fluorescent probe and its preparation method and application - Google Patents
A kind of cell membrane targeting nitric oxide fluorescent probe and its preparation method and application Download PDFInfo
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Abstract
The present invention discloses a kind of for cell membrane targeting fluorescent probe detecting nitric oxide releasing in cell and preparation method thereof, belongs to technical field of biological.The outstanding feature of probe of the present invention is: has and can be marked at the ability of surface of cell membrane by targeting, have again certain water solublity simultaneously, will not enter into cell interior through cell membrane.The fluorescence of this fluorescent probe own is the faintest, and Biomedia ambient interferences is little, can the most optionally react with nitric oxide, generates the derivative products with hyperfluorescence, and active nitrogen oxygen compound common in organism does not produce interference.Cell membrane targeting fluorescent probe provided by the present invention can discharge nitric oxide production process to cell and carry out visual analyzing.
Description
Technical field
The present invention relates to a kind of cell membrane targeting fluorescent probe and system thereof being detected nitric oxide releasing in cell by fluorescence imaging
Preparation Method and application, belong to nitric oxide production technical field of biological.
Background technology
Nitric oxide (NO) is as one of signaling molecule important in physiological process, at cardiovascular system, nervous system and exempt from
Epidemic disease system plays very important effect, therefore the analysis and research of NO content in organism is significant.Near
Develop a lot of NO over Nian and analyzed method, including chemoluminescence method, colorimetry, electron spin resonance, electrochemical process and glimmering
Optical analysis, wherein fluorimetry enjoys the concern of researcher with its high selectivity and highly sensitive advantage, and creating one is
Row are with o-phenylenediamine as reactive group, with rhodamine, fluorescein, Hua Jing, boron difluoride dipyrrylmethanes (BODIPY) etc.
Small-molecule fluorescent probe for fluorogen；Containing Cu2+、Fe3+Metal complex fluorescent probe Deng metal ion；Based on fluorescence altogether
The two-photon fluorescence probe etc. of the energy that shakes transfer.But, all these probes are all marked at cell interior, by fluorescence intensity
Change provides the information of cell interior NO.But the NO being discharged into outside born of the same parents by cell membrane is transmitted at intercellular signal, exempts from
Effect in the physiological process such as epidemic disease defence and vasodilator can not be despised, and existing fluorescent probe all can not realize this portion
Divide the detection of NO.Therefore, a kind of cell membrane targeting NO fluorescent probe of design synthesis, for detection the most in real time from cell
The NO discharged, has important directive significance to the physiological function of research NO and the appearance of relevant disease with treatment.
Summary of the invention
The present invention is directed to the deficiency that prior art exists, it is provided that a kind of can be marked at the cell membrane targeting of membrane surface by targeting
Nitric oxide fluorescent probe, for the nitric oxide production detection being discharged into cell outside born of the same parents, the physiology mistake participated in for nitric oxide
Journey research and disease research provide new method.
The technical solution adopted in the present invention is specific as follows:
A kind of cell membrane targeting nitric oxide fluorescent probe, has a structure shown in logical formula (I):
Wherein, R1For C8～C16Straight chained alkyl；R2For-SO3H or-SO3Na。
At R1Introduce the chain hydrophobic end similar with cell membrane phospholipid structure on position, the fluorine boron pyrrolidine target to cell membrane can be realized
Tropism labelling；Meanwhile, at R2Introduce water soluble group on position, the water solublity of fluorine boron pyrrolidine can be strengthened to a certain extent, resistance
Only its permeates cell membranes enters cell interior, thus realizes being discharged into cell extracellular nitric oxide production detection.
A kind of method preparing above-mentioned cell membrane targeting nitric oxide fluorescent probe, comprises the following steps:
(1) under the conditions of anhydrous and oxygen-free, by 3,4-dinitrobenzoyl chloride is dissolved in dichloromethane, instills C8～C16Straight chained alkyl pyrrole
The dichloromethane solution coughed up, is stirred at room temperature 24～48 hours；Reaction terminates, and after simple purification, purification thing is dissolved in anhydrous dichloromethane
In alkane, being cooled to 0～-10 DEG C, instill 2 under nitrogen protection, 4-dimethyl pyrrole and phosphorus oxychloride, room temperature continues stirring 12～36
Hour, then dripping triethylamine, boron trifluoride diethyl etherate, room temperature reaction to thin layer chromatography display raw material has reacted complete；Purified
Obtain aubergine powder compounds A；Wherein, 3,4-dinitrobenzoyl chlorides, straight chained alkyl pyrroles, 2,4-dimethyl pyrrole, three
Chlorethoxyfos, triethylamine, the mol ratio of boron trifluoride diethyl etherate are 1.1～1.3:1:1～1.2:1～1.2:3～5:3～5；
(2) compound A is dissolved in anhydrous methylene chloride, is cooled to-50 DEG C～-40 DEG C, drip chlorosulfonic acid under nitrogen protection, drip
Add complete, be slowly increased to room temperature, treat that raw material reaction is complete, add weak base regulation pH to 7～8, stopped reaction；Purified obtain dark
Red compound B；Wherein, compound A, the mol ratio of chlorosulfonic acid are 1:2～8；
(3) compound B being dissolved in dehydrated alcohol, nitrogen protection is lower adds hydrazine hydrate and palladium carbon, is heated to reflux 3～5 hours,
Being filtered to remove palladium carbon after reaction completely, filtrate obtains above-mentioned cell membrane targeting nitric oxide fluorescent probe through purification after being spin-dried for；Wherein,
Compound B, hydrazine hydrate, the mol ratio of palladium carbon are 1:5～10:0.5～1.5.
Described C8～C16Straight chained alkyl pyrroles is 2-cetyl pyrroles, 2-dodecyl pyrroles or 2-eight alkyl pyrroles.
Described weak base is sodium bicarbonate.
Above-mentioned cell membrane targeting nitric oxide fluorescent probe is in the application of nitric oxide detection field.
The cell membrane targeting nitric oxide fluorescent probe that the present invention provides, its photoluminescent property is based on Photo-induced electron transfer mechanism
(PET), the fluorescence intensity of this fluorescent probe own is the most weak, the most optionally reacts with NO, generates tool
There is the derivant of hyperfluorescence, and do not disturbed by other Typical reactive oxynitride in organism.This probe synthesis is simple,
Its saturated chain alkyl comprised can be plugged into realizing in cell membrane non-covalent labeling, and water soluble group ensures probe labelling simultaneously
At the outer surface of cell membrane without entering into cell interior, it is suitable for the nitric oxide that detection is discharged into outside born of the same parents by cell membrane.
The present invention has the following advantages and beneficial effect:
1. the cell membrane targeting nitric oxide fluorescent probe that the present invention provides can be marked at membrane surface by targeting, and it is right to realize
The original position of the NO being discharged into outside born of the same parents by cell membrane is detected in real time.
2. the present invention provides cell membrane targeting nitric oxide fluorescent probe and NO react before and after's fluorescence intensity change significantly, and not
Easily disturbed by other Typical reactive oxynitride in organism, analyzed detection efficient and sensible for biological NO.
3. the preparation method of the cell membrane targeting nitric oxide fluorescent probe that the present invention provides is simple.
4. the fine after birth targeting nitric oxide fluorescent probe that the present invention provides only needs a few minutes to the labeling process of cell membrane.
Accompanying drawing explanation
Fig. 1 is the synthetic route chart of cell membrane targeting nitric oxide fluorescent probe.
Fig. 2 is cell membrane targeting nitric oxide fluorescent probe and the fluorescence spectrum figure with nitric oxide derivative products thereof；Wherein, 1
Being the excitation spectrum of probe itself, 1 ' is the emission spectrum of probe itself；2 is the exciting light of probe and nitric oxide derivative products
Spectrum, 2 ' is the emission spectrum of probe and nitric oxide derivative products.
Fig. 3 is cell membrane targeting nitric oxide fluorescent probe and NO derivant thereof the 0.1M phosphate buffer solution at different pH value
Fluorescence intensity change figure in (containing 0.01% Triton X-100).
Fig. 4 is that cell membrane targeting nitric oxide fluorescent probe delays at 0.1M phosphoric acid with nitric oxide and other active nitrogen oxygen compounds
Reacted fluorescence intensity comparison diagram in dissolved liquid (containing 0.01% Triton X-100, pH7.4).
Fig. 5 is that cell membrane targeting nitric oxide fluorescent probe (contains at 0.1M phosphate buffer solution with variable concentrations nitric oxide
0.01% Triton X-100, pH7.4) in reacted fluorescence intensity change figure.
Fig. 6 is the oxidation in the mouse macrophage RAW264.7 detect activation of cell membrane targeting nitric oxide fluorescent probe
The image of nitrogen release.
Detailed description of the invention
The invention will be further described with embodiment below in conjunction with the accompanying drawings, is only that the explanation present invention is in no way intended to limit the present invention.
Embodiment 1
(1) by 308mg 3,4-dinitrobenzoyl chloride is dissolved in 150mL anhydrous methylene chloride, and the lower instillation of nitrogen protection is dissolved in
The 300mg 2-cetyl pyrroles of 30mL anhydrous methylene chloride, is stirred at room temperature 48 hours；After reaction terminates, rotary evaporation
Removing solvent, the mixture of gained is purified with silica gel column chromatography, and the purification thing obtained is re-dissolved in 100mL anhydrous methylene chloride,
Being cooled to-10 DEG C, nitrogen protection is lower instills 0.12mL 2,4-dimethyl pyrrole and 0.114mL phosphorus oxychloride, drips complete room temperature
Stir 36 hours, then add 0.72mL triethylamine, add 0.646mL boron trifluoride diethyl etherate after being stirred at room temperature 10 minutes, continue
Continuous stirring 30 minutes, rotation is evaporated off solvent, and gained mixture silica gel column chromatography is purified, and eluent is dichloromethane/normal hexane
(50/50, v/v), obtains 176mg aubergine powder compounds A (n=16), 1,3-dimethyl-5-cetyl-8-(3 ', 4 '-dinitro
Base benzene)-boron difluoride-dipyrrylmethanes, productivity is 28%.
(2) by molten for gained 1,3-dimethyl-5-cetyl-8-(3 ', 4 '-dinitro benzene)-boron difluoride-dipyrrylmethanes 200mg
In 40mL anhydrous methylene chloride, being cooled to-50 DEG C, the lower dropping of nitrogen protection is dissolved in 0.17 in 10mL anhydrous methylene chloride
ML chlorosulfonic acid, is slowly increased to room temperature after stirring 10 minutes, rotary evaporation concentrates.10mL methanol dilution is added after concentration, then
Add the aqueous solution (5mL) containing 220mg sodium bicarbonate and be adjusted to pH 7.It is filtered to remove unnecessary sodium bicarbonate and rotation is evaporated off molten
Agent, residue silica gel column chromatography purifies, and eluent is methylene chloride/methanol (70/30, v/v), obtains 195mg dark red solid
B (n=16, R2For sodium sulfonate), 1,3-dimethyl-2,6-sodium disulfonate-5-cetyl-8-(3 ', 4 '-dinitro benzene)-boron difluoride-
Dipyrrylmethanes, productivity is 73%.
(3) by gained 1,3-dimethyl-2,6-sodium disulfonate-5-cetyl-8-(3 ', 4 '-dinitro benzene)-boron difluoride-two pyrroles's first
Alkane 200mg is dissolved in 40mL ethanol, adds 0.12mL hydrazine hydrate and the palladium carbon of 39mg 10% under nitrogen protection, heats back
Flowing 5 hours, after reaction completely, filtered while hot removes palladium carbon, and after filtrate is spin-dried for, mixture silica gel column chromatography is purified, eluent
For methylene chloride/methanol (65/35, v/v), obtain 67mg red brown solid C (n=16, R2For sodium sulfonate), 1,3-dimethyl-2,6-
Sodium disulfonate-5-cetyl-8-(3 ', 4 '-diaminobenzene)-boron difluoride-dipyrrylmethanes, productivity is 36%.
The sign number of 1,3-dimethyl-2,6-sodium disulfonate-5-cetyl-8-(3 ', 4 '-diaminobenzene)-boron difluoride-dipyrrylmethanes
According to for:1H NMR(300Mz,DMSO-d6):δ0.853(3H,t,6.9Hz),1.243(28H,s),1.819(3H,s),2.648
(3H, s), 2.940 (2H, t), 4.788 (2H, t), 5.011 (2H, s), 6.412 (1H, d, J=7.5Hz), 6.525 (2H, s), 6.640
(1H, d, J=7.5Hz) .HRMS (ESI) calcd for C33H48BF2N4Na2O6S2 +([M+H]+):755.2872.Found:
755.2845。
Embodiment 2
(1) by 355mg 3,4-dinitrobenzoyl chloride is dissolved in 150mL anhydrous methylene chloride, and the lower instillation of nitrogen protection is dissolved in
The 300mg 2-dodecyl pyrroles of 30mL anhydrous methylene chloride, is stirred at room temperature 30 hours.After reaction terminates, rotary evaporation
Removing solvent, gained mixture silica gel column chromatography is purified, and the purification thing obtained is re-dissolved in 100mL anhydrous methylene chloride, fall
Temperature is to-5 DEG C, and nitrogen protection is lower instills 0.138mL 2,4-dimethyl pyrrole and 0.131mL phosphorus oxychloride, after dripping, room
Temperature stirring 24 hours, then adds 0.72mL triethylamine, adds 0.724mL boron trifluoride diethyl etherate after being stirred at room temperature 10 minutes,
Continuing stirring 30 minutes, rotation is evaporated off solvent.Gained mixture silica gel column chromatography is purified, eluent be dichloromethane/just oneself
Alkane (50/50, v/v), obtains 292mg aubergine powders A (n=12), and 1,3-dimethyl-5-dodecyl-8-(3 ', 4 '-dinitro benzene)-
Boron difluoride-dipyrrylmethanes, productivity is 41%.
(2) by molten for gained 1,3-dimethyl-5-dodecyl-8-(3 ', 4 '-dinitro benzene)-boron difluoride-dipyrrylmethanes 200mg
In 40mL anhydrous methylene chloride, being cooled to-45 DEG C, the lower dropping of nitrogen protection is dissolved in 0.12 in 10mL anhydrous methylene chloride
ML chlorosulfonic acid, is slowly increased to room temperature after stirring 10 minutes, rotary evaporation concentrates.10mL methanol dilution is added after concentration, then
Add the aqueous solution (5mL) containing 152mg sodium bicarbonate and be adjusted to pH 7.5.It is filtered to remove unnecessary sodium bicarbonate and rotation is evaporated off molten
Agent, residue silica gel column chromatography purifies, and eluent is methylene chloride/methanol (70/30, v/v), obtains 205mg dark red solid
B (n=12, R2For sodium sulfonate), 1,3-dimethyl-2,6-sodium disulfonate-5-dodecyl-8-(3 ', 4 '-dinitro benzene)-boron difluoride-
Dipyrrylmethanes, productivity is 75%.
(3) by gained 1,3-dimethyl-2,6-sodium disulfonate-5-dodecyl-8-(3 ', 4 '-dinitro benzene)-boron difluoride-two pyrroles's first
Alkane 200mg is dissolved in 40mL ethanol, adds 0.1mL hydrazine hydrate and the palladium carbon of 26mg 10%, heating under nitrogen protection
Refluxing 4 hours, after reaction completely, filtered while hot removes palladium carbon, and after filtrate is spin-dried for, mixture silica gel column chromatography is purified, and drenches
Lotion is methylene chloride/methanol (65/35, v/v), obtains 64mg red brown solid C (n=12, R2For sodium sulfonate), 1,3-diformazan
Base-2,6-sodium disulfonate-5-dodecyl-8-(3 ', 4 '-diaminobenzene)-boron difluoride-dipyrrylmethanes, productivity is 35%.
The sign number of 1,3-dimethyl-2,6-sodium disulfonate-5-dodecyl-8-(3 ', 4 '-diaminobenzene)-boron difluoride-dipyrrylmethanes
According to for:1H NMR(300Mz,DMSO-d6):δ0.853(3H,t,6.9Hz),1.245(20H,s),1.821(3H,s),2.648
(3H, s), 2.940 (2H, t), 4.791 (2H, t), 5.011 (2H, s), 6.413 (1H, d, J=7.5Hz), 6.528 (2H, s), 6.641
(1H, d, J=7.5Hz) .HRMS (ESI) calcd for C29H40BF2N4Na2O6S2 +([M+H]+):699.5683.Found:
699.5656。
Embodiment 3
(1) by 429mg 3,4-dinitrobenzoyl chloride is dissolved in 150mL anhydrous methylene chloride, and the lower instillation of nitrogen protection is dissolved in
The 300mg 2-eight alkyl pyrroles of 30mL anhydrous methylene chloride, is stirred at room temperature 24 hours.After reaction terminates, rotary evaporation removes
Going solvent, gained mixture silica gel column chromatography to purify, the purification thing obtained is re-dissolved in 100mL anhydrous methylene chloride, cooling
To 0 DEG C, nitrogen protection is lower instills 0.165mL 2,4-dimethyl pyrrole and 0.157mL phosphorus oxychloride, after dripping, room temperature
Stir 12 hours, then add 0.714mL triethylamine, after being stirred at room temperature 10 minutes, add 0.638mL boron trifluoride diethyl etherate,
Continuing stirring 30 minutes, rotation is evaporated off solvent, and gained mixture silica gel column chromatography is purified, eluent be dichloromethane/just oneself
Alkane (50/50, v/v), obtain 487mg aubergine powders A (n=8), 1,3-dimethyl-5-eight alkyl-8-(3 ', 4 '-dinitro benzene)-
Boron difluoride-dipyrrylmethanes, productivity is 58%.
(2) gained 1,3-dimethyl-5-eight alkyl-8-(3 ', 4 '-dinitro benzene)-boron difluoride-dipyrrylmethanes 200mg is dissolved in
In 40mL anhydrous methylene chloride, being cooled to-40 DEG C, the lower dropping of nitrogen protection is dissolved in the 0.05mL in 10mL anhydrous methylene chloride
Chlorosulfonic acid, is slowly increased to room temperature after stirring 10 minutes, rotary evaporation concentrates, and adds 10mL methanol dilution, add after concentration
Aqueous solution (5mL) containing 68mg sodium bicarbonate is adjusted to pH 8.It is filtered to remove unnecessary sodium bicarbonate and rotation is evaporated off solvent, surplus
Excess silica gel column chromatography is purified, and eluent is methylene chloride/methanol (70/30, v/v), obtain 212mg dark red solid B (n=8,
R2For sodium sulfonate), 1,3-dimethyl-2,6-sodium disulfonate-5-eight alkyl-8-(3 ', 4 '-dinitro benzene)-boron difluoride-dipyrrylmethanes,
Productivity is 75%.
(3) by gained 1,3-dimethyl-2,6-sodium disulfonate-5-eight alkyl-8-(3 ', 4 '-dinitro benzene)-boron difluoride-dipyrrylmethanes
200mg is dissolved in 40mL ethanol, adds 0.07mL hydrazine hydrate and the palladium carbon of 15mg 10% under nitrogen protection, heats back
Flowing 3 hours, after reaction completely, filtered while hot removes palladium carbon, and after filtrate is spin-dried for, mixture silica gel column chromatography is purified, drip washing
Agent is methylene chloride/methanol (65/35, v/v), obtains 64mg red brown solid C (n=8, R2For sodium sulfonate), 1,3-dimethyl-2,6-
Sodium disulfonate-5-eight alkyl-8-(3 ', 4 '-diaminobenzene)-boron difluoride-dipyrrylmethanes, productivity is 35%.
The sign data of 1,3-dimethyl-2,6-sodium disulfonate-5-eight alkyl-8-(3 ', 4 '-diaminobenzene)-boron difluoride-dipyrrylmethanes
For:1H NMR(300Mz,DMSO-d6):δ0.853(3H,t,6.9Hz),1.244(12H,s),1.821(3H,s),2.648(3H,
S), 2.940 (2H, t), 4.791 (2H, t), 5.011 (2H, s), 6.413 (1H, d, J=7.5Hz), 6.528 (2H, s), 6.641 (1H, d,
J=7.5Hz) .HRMS (ESI) calcd for C25H32BF2N4Na2O6S2 +([M+H]+):643.4620.Found:
643.4593。
Embodiment 4
The cell membrane targeting nitric oxide fluorescent probe (hereinafter referred to as probe (I)) and the nitric oxide thereof that obtain with embodiment 1 spread out
Biological property testing: owing in probe (I), the length of saturated carbon chains is longer, for the labelling of cell membrane, effect compares embodiment
2 and the probe that obtains of embodiment 3 good, therefore have studied in detail probe (I) and the chemical property with nitric oxide derivative products thereof.
(1) spectral quality
It is solvent with 0.1M phosphate buffer solution (containing 0.01% Triton X-100, pH 7.4), determines probe
And the ultraviolet-visible absorption spectroscopy of NO derivant, fluorescence spectrum, molar absorption coefficient and fluorescence quantum yield (I).Fluorescence
Measuring with the fluorescein (fluorescence quantum yield is for 0.92) being dissolved in 0.1M sodium hydroxide solution as standard of quantum yield.Survey
Surely the results are shown in Table 1.
Table 1. probe (I) and the spectral quality of NO derivant thereof
Test result indicate that, the fluorescence of probe (I) itself is the most weak, and its excitation-emission is launched wavelength and is respectively 514nm, 526nm；
The derivative products fluorescence intensity obtained after reacting with nitric oxide is greatly enhanced, its excitation-emission wavelength be respectively 515nm,
529nm.The fluorescence spectrum of probe (I) and NO derivant thereof is as shown in Figure 2.
(2) pH value is on probe (I) and the impact of NO derivatives fluorescent character thereof
The phosphate buffer solution of storing solution 0.1M difference pH value (2.0-12.0) of probe (I) and NO derivant thereof (is contained
0.01% Triton X-100) dilution after, measure its fluorescence intensity, result is as shown in Figure 3.Probe (I) itself is glimmering
Light intensity is the most weak, does not changes with the change of pH value, reacts the derivative products generated with nitric oxide at acid weakly acidic pH
Under conditions of, fluorescence intensity is less with the change of pH value, in the basic conditions, due to triazole group proton dissociation, causes
Fluorescence intensity reduces.
(3) probe (I) selectivity to determination of nitric oxide
Probe (I) and NO and various active nitrogen oxygen compound NO are investigated respectively3 -、NO2 -、H2O2、O2 -、ONOO-、
ClO-、.OH、1O2, the response situation of AA (ascorbic acid).Reaction all (gathers containing 0.01% at 0.1M phosphate buffer solution
Ethylene glycol octyl phenyl ether, pH 7.4) in carry out, 37 DEG C are reacted 15 minutes, and the concentration of probe (I) is 5 μMs, and NO is
10 μMs, NO3 -For 1mM, NO2 -For 1mM, H2O2For 1mM, O2 -For 1mM, ONOO-For 1mM, ClO-
Being 500 μMs .OH is 500 μMs,1O2Being 500 μMs, AA is 1mM.Measurement result as shown in Figure 4, probe (I)
With NO3 -、NO2 -、H2O2、O2 -、ONOO-、ClO-、.OH、1O2, these active substances of AA reaction after, fluorescence intensity
Changing the faintest, generate triazole derivative products after reacting with NO, fluorescence intensity is significantly increased, and shows that probe (I) is to NO
Measure and there is good selectivity.
Embodiment 5: probe (I) is nitric oxide production mensuration in aqueous solution
In 0.1M phosphate buffer solution (containing 0.01% Triton X-100, pH 7.4), add probe (I)
Storing solution so that it is concentration is 5 μMs, adds the NO saturated solution of variable concentrations, and 37 DEG C of reactions measured fluorescence after 15 minutes
Intensity.Result is as it is shown in figure 5, along with the increase of NO concentration, fluorescence intensity gradually strengthens.
Embodiment 6: probe (I) is the release of NO in detection cell the most in real time
With mouse macrophage (RAW 264.7) as object of study, the detailed examination probe flag condition to cell membrane, including visiting
Pin concentration, incubation time, toxicity etc..Test result indicate that, probe (I) can complete mark in the incubation time of 3～5 minutes
Note, the probe that concentration is 5 μMs can complete the detection to the NO that the macrophage that have activated is discharged, as shown in Figure 6.
Claims (5)
1. a cell membrane targeting nitric oxide fluorescent probe, it is characterised in that there is the structure shown in logical formula (I):
Wherein, R1For C8～C16Straight chained alkyl, R2For-SO3H or-SO3Na。
2. the method preparing cell membrane targeting nitric oxide fluorescent probe described in claim 1, comprises the following steps:
(1) under the conditions of anhydrous and oxygen-free, by 3,4-dinitrobenzoyl chloride is dissolved in dichloromethane, instills C8～C16Straight chained alkyl pyrrole
The dichloromethane solution coughed up, is stirred at room temperature 24～48 hours；Reaction terminates, and rotary evaporation removes solvent, and the mixture of gained is used
Silica gel column chromatography purification, is dissolved in purification thing in anhydrous methylene chloride, is cooled to 0～-10 DEG C, instills 2,4-under nitrogen protection
Dimethyl pyrrole and phosphorus oxychloride, room temperature continues stirring 12～36 hours, then drips triethylamine, boron trifluoride diethyl etherate, room temperature
React and show that raw material has reacted complete to thin layer chromatography；Purified obtain aubergine powder compounds A；Wherein, 3,4-dinitro benzenes
Formyl chloride, straight chained alkyl pyrroles, 2,4-dimethyl pyrrole, phosphorus oxychloride, triethylamine, the mol ratio of boron trifluoride diethyl etherate be 1.1～
1.3:1:1～1.2:1～1.2:3～5:3～5；
(2) compound A is dissolved in anhydrous methylene chloride, is cooled to-50 DEG C～-40 DEG C, drip chlorosulfonic acid under nitrogen protection, drip
Add complete, be slowly increased to room temperature, treat that raw material reaction is complete, add weak base regulation pH to 7～8, stopped reaction；Purified obtain dark
Red compound B；Wherein, compound A, the mol ratio of chlorosulfonic acid are 1:2～8；
(3) compound B being dissolved in dehydrated alcohol, nitrogen protection is lower adds hydrazine hydrate and palladium carbon, is heated to reflux 3～5 hours,
Being filtered to remove palladium carbon after reaction completely, filtrate obtains above-mentioned cell membrane targeting nitric oxide fluorescent probe through purification after being spin-dried for；Wherein,
Compound B, hydrazine hydrate, the mol ratio of palladium carbon are 1:5～10:0.5～1.5；
Described compound A, its structural formula isDescribed compound B, its structural formula is
Wherein, R1For C8～C16Straight chained alkyl, R2For-SO3H or-SO3Na。
Method the most according to claim 2, it is characterised in that: described C8～C16Straight chained alkyl pyrroles be 2-cetyl pyrroles,
2-dodecyl pyrroles or 2-eight alkyl pyrroles.
Method the most according to claim 2, it is characterised in that: described weak base is sodium bicarbonate.
5. the application in nitric oxide detection field of the cell membrane targeting nitric oxide fluorescent probe described in claim 1.
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